UNIT 
4: CELL COMMUNICATION AND CELL SIGNALING 
A. Host parasite interaction:
Bacteria are consistently associated with the body
surfaces of animals. There are many more bacterial cells
on the surface of a human (including the gastrointestinal
tract) than there are human cells that make up the animal.
The bacteria and other microbes that are consistently
associated with an animal are called the normal flora, or
more properly the "indigenous microbiota", of the
animal. These bacteria have a full range of symbiotic
interactions with their animal hosts.
Why is it called the normal flora?
The term flora is used because the majority of the
organisms concerned are bacteria. It has been estimated
that humans have approximately 1013 cells in the body
and something like 1014 bacteria associated with them,
the majority in the large bowel. Members of groups such
as viruses, fungi and protozoa are also regularly found in
healthy individuals, but form only a minor component of
the total population of resident organisms.
The organisms occur in those parts of the body that are
exposed to, or communicate with, the external
environment, namely the skin, nose and mouth, and
intestinal and urinogenital tracts. Internal organs and
tissues are normally sterile. The main organisms found in
these sites are shown in Figure 1.
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  1
The normal flora is acquired rapidly during and shortly
after birth and changes continuously throughout life
The organisms present at any given time reflect the age,
nutrition and environment of the individual. It is
therefore difficult to define the normal flora very
precisely because it is to a large extent environmentally
determined. This is well illustrated by data from NASA
astronauts who were rendered relatively bacteriologically
sterile by antibiotic treatment before their space flights. It
took only 6 weeks after the flight for their flora to
repopulate, and the repopulating species were precisely
those of their immediate neighbors. The bowel flora of
children in developing countries is quite different from
that of children in developed countries. In addition,
breastfed infants have lactic acid streptococci and
lactobacilli in their gastrointestinal tract, whereas
bottlefed children show a much greater variety of
organisms.
Different regions of the skin support different flora
Exposed dry areas have relatively few resident organisms
on the surface, whereas moister areas (axillae, perineum,
between the toes, scalp) support much larger populations.
Staphylococcus epidermidis is one of the commonest
species, making up some 90% of the aerobes and
occurring in densities of 103-104/cm2; S. aureus may be
present in the moister regions.
Anaerobic diphtheroids occur below the skin surface in
hair follicles, sweat and sebaceous glands,
Propionibacterium acnes being a familiar example.
Changes in the skin occurring during puberty often lead
to increased numbers of this species, which can be
associated with acne.
A number of fungi, including Candida, occur on the scalp
and around the nails. They are infrequent on dry skin, but
can cause infection in moist skin folds (intertrigo).
Both the nose and mouth can be heavily colonized by
bacteria
The majority of bacteria here are anaerobes. Common
species colonizing these areas include streptococci,
staphylococci, diphtheroids and Gram-negative cocci.
Some of the aerobic bacteria found in healthy individuals
are potentially pathogenic (e.g. Staph. aureus,
Streptococcus pneumoniae, Strep. pyogenes, Neisseria
meningitidis); Candida is also a potential pathogen.
The mucous membranes of the mouth can have the same
microbial density as the large intestine, numbers
approaching 1011/g wet weight of tissue.
Dental caries is one of the commonest infectious
diseases in developed countries
The surfaces of the teeth and the gingival crevices carry
large numbers of anaerobic bacteria. Plaque is a film of
bacterial cells anchored in a polysaccharide matrix,
which the organisms secrete. When teeth are not cleaned
regularly, plaque can accumulate rapidly and the
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
activities of certain bacteria, notably Streptococcus
mutans, can lead to dental decay (caries), as acid
fermented from carbohydrates can attack dental enamel.
The prevalence of dental decay is linked to diet.
The pharynx and trachea carry their own normal flora
The flora of the pharynx and trachea may include both α-
and β-hemolytic streptococci as well as a number of
anaerobes, staphylococci (including Staph. aureus),
Neisseria and diphtheroids. The respiratory tract is
normally quite sterile, despite the regular intake of
organisms by breathing. However substantial numbers of
clinically normal people may carry the fungus
Pneumocystis jiroveci (previously known as P. carinii) in
their lungs.
In the gut the density of microorganisms increases from
the stomach to the large intestine
Stomach contents harbor only transient organisms, the
acidic pH providing an effective barrier. However, the
gastric mucosa may be colonized by acid-tolerant
lactobacilli and streptococci. The upper intestine is only
lightly colonized (104 organisms/g), but populations
increase markedly in the ileum, where streptococci,
lactobacilli, enterobacteriaceae and Bacteroides may all
be present. Bacterial numbers are very high (estimated at
1011/g) in the large bowel, and many species can be
found (Fig. 2).
2 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
The vast majority (95-99%) are anaerobes, Bacteroides
being especially common and a major component of
fecal material. Harmless protozoans can also occur in the
intestine (e.g. Entamoeba coli) and these can be
considered as part of the normal flora, despite being
animals.
The urethra is lightly colonized in both sexes, but the
vagina supports an extensive flora of bacteria and fungi
The urethra in both sexes is relatively lightly colonized,
although Staph. epidermidis, Strep. faecalis and
diphtheroids may be present. In the vagina the
composition of the bacterial and fungal flora undergoes
age-related changes: Before puberty the predominant
organisms are staphylococci, streptococci, diphtheroids
and Escherichia coli.
Subsequently, Lactobacillus aerophilus predominates, its
fermentation of glycogen being responsible for the
maintenance of an acid pH, which prevents overgrowth
by other vaginal organisms.
A number of fungi occur, including Candida, which can
overgrow to cause the pathogenic condition 'thrush' if the
vaginal pH rises and competing bacteria diminish. The
protozoan Trichomonas vaginalis may also be present in
healthy individuals.
Advantages and disadvantages of the normal flora
Some of the species of the normal flora are positively
beneficial to the host
The importance of these species for health is sometimes
revealed quite dramatically under stringent antibiotic
therapy. This can drastically reduce their numbers to a
minimum, and the host may then be overrun by
introduced pathogens or by overgrowth of organisms
normally present in small numbers.
After treatment with clindamycin, overgrowth by
Clostridium difficile, which survives treatment, can give
rise to antibiotic-associated diarrhea or, more seriously,
pseudomembranous colitis.
Ways in which the normal flora prevents colonization by
potential pathogens include the following.
ƒ Skin bacteria produce fatty acids, which
discourage other species from invading.
ƒ Gut bacteria release a number of factors with
antibacterial activity (bacteriocins, colicins) as
well as metabolic waste products that help
prevent the establishment of other species.
ƒ Vaginal lactobacilli maintain an acid
environment, which suppresses growth of other
organisms.
ƒ The sheer number of bacteria present in the
normal flora of the intestine means that almost
all of the available ecologic niches become
occupied; these species therefore outcompete
others for living space.
Gut bacteria also release organic acids, which may have
some metabolic value to the host; they also produce B
vitamins and vitamin K in amounts that are large enough
to be valuable if the diet is deficient. The antigenic
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

stimulation provided by the intestinal flora helps to
ensure the normal development of the immune system.
What happens when the normal flora is absent?
Germ-free animals tend to live longer, presumably
because of the complete absence of pathogens, and
develop no caries. However, their immune system is less
well developed and they are vulnerable to introduced
microbial pathogens. At the time of birth, humans are
germ free, but acquire the normal flora during and
immediately after birth, with the accompaniment of
intense immunologic activity.
'symbiosis' (literally 'living together'). Symbiosis has no
overtones of benefit or harm and includes a wide
diversity of associations. Attempts have been made to
categorize types of association very specifically, but
these have failed because all associations form part of a
continuum (Fig.3). Three broad categories of symbiosis-
commensalism, mutualism and parasitism-can be
identified on the basis of the relative benefit obtained by
each partner. None of these categories of association is
restricted to any particular taxonomic group. Indeed
some organisms fit into each category depending upon
the circumstances in which they live (Fig. 4).

The disadvantages of the normal flora lie in the potential

for spread into previously sterile parts of the body

This may happen:

ƒ when the intestine is perforated or the skin is
broken;
ƒ during extraction of teeth (when viridans
streptococci may enter the bloodstream);
ƒ when organisms from the perianal skin ascend
the urethra and cause urinary tract infection.
Members of the normal flora are important causes of
hospital-acquired infection when patients are exposed to
invasive treatments. Patients suffering burns are also at
risk.

Overgrowth by potentially pathogenic members of the

normal flora can occur when the composition of the flora
changes (e.g. after antibiotics) or when:
ƒ the local environment changes (e.g. increases
in stomach or vaginal pH);
ƒ the immune system becomes ineffective (e.g.
AIDS, clinical immunosuppression).

Under these conditions, the potential pathogens take the

opportunity to increase their population size or invade
tissues, so becoming harmful to the host.

SYMBIOTIC ASSOCIATIONS

All living animals are used as habitats by other

organisms; none is exempt from such invasion-even
protozoans have their own flora and fauna. As evolution
has produced larger, more complex and better regulated
bodies, it has increased the number and variety of
habitats for other organisms to colonize. The most
complex bodies, those of birds and mammals (including
humans), provide the most diverse environments, and are
the most heavily colonized.

As the normal flora demonstrates, pathogenesis is not the

inevitable consequence of host-microbe associations.
Many factors influence the outcome of a particular
association, and organisms may be pathogenic in one
situation but harmless in another. To understand the
microbiologic basis of infectious disease, host-microbe
associations that can be pathogenic need to be placed
firmly in the context of other symbiotic (interspecies)
associations, such as commensalism or mutualism, where
the outcome for the host does not normally involve any
damage or disadvantage.

All associations in which one species lives in or on the

body of another can be grouped under the general term

INSTITUTE FOR ADVANCED STUDIES, JODHPUR  3
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
Commensalism: In commensalism one species of
organism uses the body of a larger species
At its simplest, a commensal association is one in which
one species of organism uses the body of a larger species
as its physical environment and may make use of that
environment to acquire nutrients.
Like all animals, humans support an extensive
commensal microbial flora on the skin, in the mouth and
in the alimentary tract. The majority of these microbes
are bacteria, and their relationship with the host may be
highly specialized, with specific attachment mechanisms
and precise environmental requirements. Normally such
microbes are harmless, but they can become harmful if
their environmental conditions change in some way (e.g.
Bacteroides, Escherichia coli, Staphylococcus aureus).
Conversely, commensal microbes can benefit the host:
ƒ by preventing colonization by more pathogenic
species (e.g. the intestinal flora);
ƒ by producing metabolites that are used by the
host (e.g. the bacteria and protozoa in the
ruminant stomach).
It follows that the normal definition of commensalism is
merely one of convenience, as the association can merge
into either mutualism or parasitism.
Mutualism: Mutualistic relationships provide reciprocal
benefits for the two organisms involved
Frequently the relationship is obligatory for at least one
member, and may be for both. Good examples are the
bacteria and protozoa living in the stomachs of domestic
ruminants, which play an essential role in the digestion
and utilization of cellulose, receiving in return both the
environment and the nutrition essential for their survival.
The dividing line between commensalism and mutualism
can be hard to draw. In humans, good health and
resistance to colonization by pathogens can depend upon
the integrity of the normal commensal enteric bacteria,
many of which are highly specialized for life in the
human intestine, but there is certainly no strict mutual
dependence in this relationship.
Parasitism: In parasitism the symbiotic relationship
benefits only the parasite.
The terms 'parasites' and 'parasitism' are sometimes
thought to apply only to protozoans and worms, but all
pathogens are parasites. Parasitism is a one-sided
relationship in which the benefits go only to the parasite,
the host providing parasites with their physicochemical
environment, their food, respiratory and other metabolic
needs, and even the signals that regulate their
development. Although parasites are thought of as
necessarily harmful, this is a view colored by human and
veterinary clinical medicine, and by the results of
laboratory experimentation. In fact many 'parasites'
establish quite innocuous associations with their natural
hosts and are not at all pathogenic under normal
circumstances (e.g. when their natural host is in good
health); the rabies virus, for example, coexists with many
wild mammals but can cause fatal disease in humans.
This state of 'balanced pathogenicity' is sometimes
explained as the outcome of selective pressures acting
upon a relationship over a long period of evolutionary
4 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
time. It may reflect selection of an increased level of
genetically determined resistance in the host population
and decreased pathogenicity in the parasite (as has
happened with myxomatosis in rabbits). Alternatively, it
may be the evolutionary norm, and 'unbalanced
pathogenicity' may simply be the consequence of
organisms becoming established in 'unnatural' (i.e. new)
hosts. So, like the other categories of symbiosis,
parasitism is impossible to define exclusively except in
the context of clearcut and highly pathogenic organisms.
The belief that 'harmfulness' is a necessary characteristic
of a parasite is difficult to sustain in any broader view,
and the reasons for this are discussed in more detail
below.
THE CHARACTERISTICS OF PARASITISM
Many different groups of organisms are parasitic, and
all animals are parasitized
Parasitism as a way of life has been adopted by many
different groups of organisms. Some groups, such as
viruses, are exclusively parasitic, but the majority include
both parasitic and free-living representatives. Parasites
occur in all animals, from the simplest to the most
complex, and are an almost inevitable accompaniment of
organized animal existence. We can see, then, that
parasitism has been an evolutionary success; as a way of
life it must confer very considerable advantages.
Parasitism has metabolic, nutritional and reproductive
advantages
The most obvious advantage of parasitism is metabolic.
The parasite is provided with a variety of metabolic
requirements by the host, at no energy cost to itself, so it
can devote a large proportion of its own resources to
replication or reproduction. This one-sided metabolic
relationship shows a broad spectrum of dependence, both
within and between the various groups of parasites.
Some parasites are totally dependent upon the host, while
others are only partly dependent.
Viruses are completely dependent upon the host for all
their metabolic needs
Viruses are at one extreme of the 'parasite dependency'
spectrum. They are obligate parasites, possessing the
genetic information required for production of new
viruses, but none of the cellular machinery necessary to
transcribe or translate this information, to assemble new
virus particles or to produce the energy for these
processes. The host provides not only the basic building
blocks for the production of new viruses, but also the
synthetic machinery and the energy required (Fig. 5).
Retroviruses go one stage further in dependence,
inserting their own genetic information into the host's
DNA in order to parasitize the transcription process.
Viruses therefore represent the ultimate parasitic
condition and are qualitatively different from all other
parasites in the nature of their relationship with the host.
The basis for the fundamental difference between viruses
and other parasites is the difference between virus
organization and the cellular organization of prokaryotic
and eukaryotic parasites. Non-viral parasites have their
own genetic and cellular machinery, and multi-enzyme
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
systems for independent metabolic activity and
macromolecular synthesis. The degree of reliance on the
host for nutritional requirements varies considerably and
follows no consistent pattern between the various groups,
nor does it follow that smaller parasites tend to be more
dependent; for example, some of the largest parasites, the
tapeworms, are wholly reliant upon the host's digestive
machinery to provide their nutritional needs. All, of
course, receive nutrition from the host, but whereas some
use macromolecular material (proteins, polysaccharides)
of host origin and digest it using their own enzyme
systems, others rely on the host for the process of
digestion as well, being able to take up only low
molecular weight materials (amino acids,
monosaccharides). Nutritional dependence may also
include host provision of growth factors that the parasite
is unable to synthesize itself. All internal parasites rely
upon the host's respiratory and transport systems to
provide oxygen, although some respire anaerobically in
either a facultative or obligate manner.
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  5
Parasite development can be controlled by the host
The advantage that parasitism confers in reproductive
terms makes it vital to coordinate parasite development
with the availability of suitable hosts. Indeed, one of the
characteristic features of parasites is that their
development may be controlled partly or completely by
the host, the parasite having lost the ability to initiate or
to regulate its own development. At its simplest, host
control is limited to providing the cell surface molecules
necessary for parasite attachment and internalization.
Many parasites, from viruses to protozoa, rely on the
recognition of such molecular signals for their entry into
host cells, and this process provides the trigger for their
replicative or reproductive cycles.
Other parasites, primarily the eukaryotes, require more
comprehensive and sophisticated signals, often a
complex of signals, to initiate and regulate their entire
developmental cycle. The complexity of the signal
required for development is one of the factors
determining the specificity of the host-parasite
relationship. Where the availability of one of the signals
entails that parasite development can occur in only one
species, host specificity is high. Where many host
species are capable of providing the necessary signals for
a parasite, specificity is low.
Disadvantages of parasitism
The most obvious disadvantage of parasitism arises from
the fact that the host controls the development of the
parasite. No development is possible without a suitable
host, and many parasites will die if no host becomes
available. For this reason, several adaptations have
evolved to promote prolonged survival in the outside
world and so maximize the chances of successful host
contact (e.g. virus particles, bacterial spores, protozoan
cysts and worm eggs). The prolific replication of
parasites is another device to achieve the same end.
Nevertheless, where parasites fail to make contact with a
host, their powers of survival are ultimately limited.
Adaptation to host signals can therefore have a
reproductive cost (i.e. the loss of many potential
parasites).
THE EVOLUTION OF PARASITISM
As so many organisms are parasitic and every group of
animals is subject to invasion by parasites, the
development of parasitism as a way of life must have
occurred at an early stage in evolution and at frequent
intervals thereafter. How this occurred is not fully
understood, and it may well have been different in
different groups of organisms. In many, parasitism most
probably arose as a consequence of accidental contacts
between organism and host. Of many such contacts some
would have resulted in prolonged survival, and under
favorable nutritional circumstances prolonged survival
would have been associated with enhanced replication,
giving the organism a selective advantage within the
environment.
Bacterial parasites evolved through accidental contact
In the case of bacteria, it is easy to see how accidental
contact in environments rich in free-living bacteria could
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
lead to successful colonization of the gastrointestinal
tract and external orifices. Initially the organisms
concerned would have had to be facultative parasites,
capable of life both within or outside host organisms
(many pathogenic bacteria still have this property, e.g.
Legionella, Vibrio), but selective pressures would have
forced others into obligatory parasitism. Such events are
of course speculative, but are supported by the close
relationship of enteric bacteria such as E. coli with free-
living photosynthetic purple bacteria.
Many bacteria and related parasites of humans and other
mammals may have originated via the route of accidental
contact, but it is clear that others have become adapted to
these hosts after initially becoming parasitic in other
species. Blood-feeding arthropods provide an example of
the most obvious route for this, as their parasites have
ready access to the tissues of the animals on which the
arthropods feed.
The pathway of virus evolution is uncertain
Clearly, parasitism by bacteria, which are undoubtedly
ancient organisms (they can be traced back 3-5 billion
years in the fossil record), depended upon the evolution
of higher organisms to act as hosts. Whether the same is
true of viruses is open to question, and depends upon
whether viruses are considered primarily or secondarily
simple. If viruses evolved from cellular ancestors by a
6 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
Many bacterial parasites have evolved to live inside host
cells
Bacteria that became parasitic by accidental contact
would have lived outside host cells at first and would not
have had the advantages of being intracellular. The
evolution of the intracellular habit required further
modifications to allow survival within host cells, but
could easily have been initiated by passive phagocytic
uptake. Subsequent survival of the microbe would
depend upon the possession of surface or metabolic
properties that prevented digestion and destruction by the
host cell. The success of intracellular life can be
measured not only by the large number of bacteria that
have adopted this habit, but also by the extent to which
some organisms have integrated their biology with that
of the host cell. The endpoint of such integration is
perhaps to be seen in the evolution of the eukaryote
mitochondrion, which is widely considered to be the
product of symbiotically associated heterotrophic purple
bacteria (Fig. 6).
process of secondary simplification, then parasitism must
have evolved long after the evolution of prokaryotes and
eukaryotes. If viruses are primitively non-cellular then it
is possible that they became parasitic at a very early
stage in the evolution of cellular life, at some point when,
because of environmental change, independent existence
became impossible. A third alternative is that viruses
were never anything other than fragments of the nuclear
material of other organisms and have in effect always
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

been parasitic. Modern viruses may, in fact, have arisen
by all three pathways.
Eukaryote parasites have evolved through accidental
contact
of the continuing battle between host and parasite-the
former attempting to contain or destroy, the latter
attempting to evade or suppress-and why the emergence
of new, and the return of old, infectious diseases are a
constant threat.

The evolution of parasitism by eukaryotes is likely to
have arisen much as it may have done in prokaryotes (i.e.
through accidental contact and via blood-feeding
arthropods). Examples can be found among both
protozoan and worm parasites to support this view:
ƒ There are protozoa such as the free-living ameba
Naegleria, which can opportunistically invade the
human body and cause severe and sometimes fatal
disease.
ƒ There are several species of nematode worms that
can live either as parasites or as free-living
organisms, Strongyloides stercoralis being the most
important in humans.
ƒ It is likely that trypanosomes (the protozoans
responsible for sleeping sickness) were primarily
adapted as parasites of blood-feeding flies and only
secondarily became established as parasites of
mammals.
Parasites are faced not only with the problems of
surviving within the environment they experience
initially, but also of surviving in that environment as it
changes in ways that are likely to be harmful to them.
The inflammatory and immune responses that follow the
establishment of infection are the most important means
by which the host can control infections by those
organisms able to penetrate its natural barriers and
survive within its body. These responses represent
formidable obstacles to the continued survival of
parasites, forcing them to evolve strategies to cope with
harmful changes in their environment. The successful
parasite is therefore one that can cope with, or evade, the
host's response in one of the ways shown in Figure 7.

Parasite adaptations to overcome host inflammatory and

immune responses

We can view the evolution of parasitism and the

adaptations necessary for life within another animal as
being exactly analogous to the adaptations necessary for
life within any other specialized habitat: the environment
in which parasites live is merely one of the many to
which organisms have become adapted in evolution
(comparable with life in soil, freshwater, salt water,
decaying material and so on). However, it is always
necessary to remember that in one major respect
parasitism is quite different from any other specialist
mode of life. This difference is that the environment in
which a parasite lives, the body of the host, is not
passive; on the contrary it is capable of an active
response to the presence of the parasite.
The attractiveness of animal bodies as environments for
parasites means that hosts are under continual pressures
from infection, and these pressures are increased when
hosts live:
ƒ close together;
ƒ in insanitary conditions;
ƒ in climates that favor the survival of parasite
stages in the external world.

Pressure of infection has been a major influence in

evolution

Pressure of infection has been a major selective influence

in evolution, and there is little doubt that it has been
largely responsible for the development of the
sophisticated inflammatory and immune responses we
All of these adaptations are known to exist within
see in humans and other mammals. In evolutionary terms
different groups of parasites and they are well
all infection has its costs to the host because it diverts
documented in the case of some of the major human
valuable resources from the activities of survival and
pathogens. Indeed they are often the very reason why
reproduction; there has therefore been pressure to
such organisms are major pathogens. Nevertheless,
develop means of overcoming infection whether or not it
transmission and survival of many parasites depends
causes disease. Of course, this is not the focus of clinical
upon the existence of particularly susceptible host
microbiology, which legitimately places emphasis on the
individuals (e.g. children) to provide a continuing
costs of infection in terms of frank disease, but it should
reservoir of infective stages.
be remembered because it explains more fully the nature

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UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
Changes in parasites create new problems for hosts
From what has been said above, it can be appreciated that
there is no such thing as a static host-parasite
relationship, and that concepts of unchanging
'pathogenicity' or 'harmlessness' cannot be justified. Each
relationship is an 'arms race', changes in one member
being countered by changes in the other. Quite subtle
changes in either can completely change the balance of
the relationship, towards greater or lesser pathogenicity
for example.
Perhaps the most important contemporary illustration of
this situation is the dramatic and explosive appearance of
HIV infections. This group of viruses was originally
restricted to non-human primates, but changes in the
Although the underlying genetic and metabolic changes
do not by themselves influence pathogenicity, the
expression of such changes in the face of intense and
selective chemotherapy certainly does so.
Host adaptations to overcome changes in parasites
Changes in the host can also alter the balance of a host-
parasite relationship. A particularly dramatic example is
the intense selection for resistant genotypes in rabbit
populations exposed to the myxomatosis virus, which
took place concurrently with selection for reduced
pathogenicity in the virus itself. There are no exactly
equivalent examples in humans, but in evolutionary time
there have been major selective influences on
populations prompting changes to permit survival in the
face of life-threatening infections. A good example is the
selective pressure exerted by falciparum malaria, which
has been responsible for the persistence of many alleles
associated with hemoglobinopathies (e.g. sickle cell
hemoglobin).
Although these abnormalities are detrimental to a varying
degree, they persist because they are (or were) associated
with resistance to malarial infection. Malaria has also
8 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
virus have permitted extensive infections in humans. Of a
different nature, but relevant to the general theme, is the
acquisition of drug resistance in bacteria and protozoa
(Fig. 8).
The activity of many antibiotics can be blocked by
bacterial enzymes coded for by genes located on
cytoplasmic DNA in plasmids. The ability of bacteria to
transfer plasmids between individual organisms means
that strains or species previously susceptible to an
antibiotic can acquire the ability to produce such
enzymes and so gain antibiotic resistance directly from
resistant organisms. These newly resistant forms are then
differentially selected under antibiotic treatment, the
susceptible individuals being deleted from the
population.
changed the frequency of certain HLA antigens in areas
where infection was severe.
Social and behavioral changes can be as important as
genetic changes in altering host-parasite relations
Social and behavioral changes can alter host-parasite
relations both positively and negatively (Fig. 9).
Although many bacterial infections of the intestine have
declined in importance with changes in human lifestyle,
there are other contemporary microbiologic problems in
the developed world whose onset can be traced directly
to sociologic, environmental and even medical change
(Fig. 9).
A particularly good example is disease arising from
domestication of pets (e.g. toxoplasmosis) because it
illustrates that human freedom from some infections
arises primarily because of lack of contact with the
organisms and not from any innate resistance to the
establishment of the infection itself. Diseases arising
from contact with infected animals or animal products
(zoonotic infections) constitute a constant threat that can
be realized by behavioral or environmental changes that
alter established patterns of human-animal contact.
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
Host-Parasite Relationships
If a symbiont either harms or lives at the expense of
another organism (the host), it is a parasitic organism,
and the relationship is called parasitism. In this
relationship the body of the host can be viewed as a
microenvironment that shelters and supports the growth
and multiplication of the parasitic organism. The
parasitic organism is usually the smaller of the two
partners and is metabolically dependent on the host.
There are many parasitic agents or organisms among the
viruses, bacteria, fungi, plants, and animals (Table 1).
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  9
By convention, when the word parasite is used without
qualification, it refers specifically to a protozoan or
helminthic (nematode, trematode, cestode) organism.
Several types of parasitism are recognized. If an
organism lives on the surface of its host, it is an
ectoparasite; if it lives in- ternally, it is an
endoparasite. The host on or in which the parasitic
organism either attains sexual maturity or reproduces is
the final host. A host that serves as a temporary but
essential environment for some stages of development is
an intermediate host.
In contrast, a transfer host is not necessary for the
completion of the organism’s life cycle but is used as a
vehicle for reaching a final host. A host infected with a
parasitic organism that also can infect humans is called a
reservoir host.
Because, by definition, parasitic organisms are dependent
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
on their hosts, the symbiotic relationship between the
host and parasite is a dynamic one (figure 1). When a
parasite is growing and multiplying within or on a host,
the host is said to have an infection. The nature of an
infection can vary widely with respect to severity,
location, and number of organisms involved (table 2).
An infection may or may not result in overt disease.
An infectious disease is any change from a state of
health in which part or all of the host body is not capable
of carrying on its normal functions due to the presence of
At times an infectious organism can enter a latent state in
which there is no shedding of the organism and no
symptoms present within the host. This latency can be
either intermittent or quiescent. Intermittent latency is
exemplified by the herpes virus that causes cold sores
(fever blisters). After an initial infection, the symptoms
subside. However, the virus remains in nerve tissue and
can be activated weeks or years later by factors such as
stress or sunlight. In a quiescent latency the organism
persists but remains inactive for long periods of time,
usually for years. For example, the varicella-zoster virus
causes chickenpox in children and remains after the
disease has subsided. In adulthood, under certain
conditions, the same virus may erupt into a disease called
shingles. Cold sores; Chickenpox (varicella) and shingles
(herpes zoster).
The outcome of most host-parasite relationships is
dependent on three main factors: (1) the number of
organisms present in or on the host, (2) the virulence of
the organism, and (3) the host’s defenses or degree of
10 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
an organism or its products. Any organism or agent that
produces such a disease is a pathogen [Greek patho,
disease, and gennan, to produce]. Its ability to cause
disease is called pathogenicity. A primary (frank)
pathogen is any organism that causes disease in a
healthy host by a direct interaction. Conversely, an
opportunistic pathogen is an organism that is either
normally free-living, or a part of the host’s normal
microbiota, but which may adopt a pathogenic role under
certain circumstances, such as when the immune system
is compromised.
resistance. Usually the greater the number of organisms
within a given host, the greater the likelihood of disease.
However, a few organisms can cause disease if they are
extremely virulent or if the host’s resistance is low. A
host’s resistance can drop so much that its own
microbiota may cause disease.
Such a disease is sometimes called an endogenous
disease because the agent originally comes from within
the host’s own body. Endogenous diseases can be a
serious problem among hospitalized patients with very
low resistance.
The term virulence [Latin virulentia, from virus, poison]
refers to the degree or intensity of pathogenicity. It is
determined by three characteristics of the pathogen:
invasiveness, infectivity, and pathogenic potential.
Invasiveness is the ability of the organism to spread to
adjacent or other tissues. Infectivity is the ability of the
organism to establish a focal point of infection.
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
Pathogenic potential refers to the degree that the
pathogen causes damage. A major aspect of pathogenic
potential is toxigenicity.
Toxigenicity is the pathogen’s ability to produce toxins,
chemical substances that will damage the host and
produce disease.
Virulence is often measured experimentally by
determining the lethal dose 50 (LD50) or the infectious
dose 50 (ID50). These values refer to the dose or number
of pathogens that will either kill or infect, respectively,
50% of an experimental group of hosts within a specified
period (figure 2).
It should be noted that disease can result from causes
other than toxin production. Sometimes a host will
trigger exaggerated immunological responses
(immunopathology) upon a second exposure or chronic
exposure to a microbial antigen. These hypersensitivity
reactions damage the host even though the pathogen
doesn’t produce a toxin. Tuberculosis is a good example
of the involvement of hypersensitivity reactions in
disease.
Some diseases also might be due to autoimmune
responses. For instance, a viral or bacterial pathogen may
stimulate the immune system to attack host tissues
because it carries antigens that resembled those of the
host, a phenomenon known as molecular mimicry.
Streptococcal infections may cause rheumatic fever in
this way.
Pathogenesis of Viral Diseases
The fundamental process of viral infection is the
expression of the viral replicative cycle in a host cell.
The steps for the infectious process involving viruses are
that a virus must
1. Enter a host
2. Contact and enter susceptible cells
3. Replicate within the cells
4. Spread to adjacent cells
5. Cause cellular injury
6. Engender a host immune response
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  11
7. Be either cleared from the body of the host, establish a
persistent infection, or kill the host
8. Be shed back into the environment
The determinants of pathogenicity are now discussed in
more detail.
Entry, Contact, and Primary Replication
The first step in the infectious process is the attachment
and entrance of the virus into a susceptible host and the
host’s cells. Entrance may be accomplished through one
of the body surfaces (skin, respiratory system,
gastrointestinal system, urogenital system, or the
conjunctiva of the eye). Other viruses enter the host by
needle sticks, blood transfusions and organ transplants,
or by insect vectors (organisms that transmit the
pathogen from one host to another). Some viruses
replicate at the site of entry, cause disease at the same
site (e.g., respiratory and gastrointestinal infections), and
do not spread throughout the body. Others spread to sites
distant from the point of entry and replicate at these sites.
For example, the enteroviruses enter through the
gastrointestinal tract but produce disease in the central
nervous system. Mucous membranes.
Viral Spread and Cell Tropism
Mechanisms of viral spread vary, but the most common
routes are the bloodstream and lymphatic system. The
presence of viruses in the blood is called viremia. In
some instances, spread is by way of nerves (e.g., rabies
virus, herpes simplex, and varicellazoster viruses).
Viruses exhibit cell, tissue, and organ specificities. These
specificities are called tropisms (Greek trope, turning).
A tropism by a specific virus usually reflects the
presence of specific cell surface receptors on the
eucaryotic host cell for that virus.
Cell Injury and Clinical Illness: Destruction of the
virus-infected cells in the target tissues and alterations in
host physiology are responsible for the development of
viral disease and clinical illness. Some tissue (e.g.,
intestinal epithelium) can rapidly regenerate after a viral
attack and withstand extensive damage. Other tissues,
such as nervous system tissues, are not able to regenerate
and may never resume normal functioning after damage
has occurred.
The potential effects of viruses on individual host cells
are the result of a complex series of events. There are
four generally accepted patterns of a viral infection. (1)
In lytic infections the virus multiplies and kills the host
cell immediately and new virions are released. (2) In
persistent viral infections the virus lives in the host cell
and releases small numbers of virions over a long period
of time. This causes little damage to the host cell. (3) In
latent infections, the virus resides in the cell but produces
no virions. At some later time the virus can be activated
and a lytic infection occurs. (4) Some viruses can
transform the host cell into a cancer cell that becomes the
focal point for a tumor.
Host Immune Response: Both humoral and cellular
components of the immune response are involved in the
control of viral infections.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
Recovery from Infection: The host will either succumb
or recover from a viral infection. Recovery mechanisms
involve nonspecific defense mechanisms and specific
humoral and cellular immunity. The relative importance
of each of these factors varies with the virus and the
disease.
Virus Shedding: The last step in the infectious process
is shedding of the infectious virus back into the
environment. This is necessary to maintain a source of
viruses in a population of hosts. Shedding often occurs
from the same body surface used for entry. During this
period, an infected host is infectious and can spread the
virus. In some viral infections, such as a rabies infection,
humans are dead-end hosts because virus shedding does
not occur.
Pathogenesis of Bacterial Diseases
The steps for infections by pathogenic bacteria include
1. Maintain a reservoir. A reservoir is a place to live
before and after causing an infection.
2. Initially be transported to the host.
3. Adhere to, colonize, and/or invade the host.
4. Multiply (grow) or complete its life cycle on or in the
host or the host’s cells.
5. Initially evade host defense mechanisms.
6. Possess the ability to damage the host.
7. Leave the host and return to the reservoir or enter a
new host.
The first five factors influence the degree of infectivity
and invasiveness. Toxigenicity plays a major role in the
sixth.
Maintaining a Reservoir of the Bacterial Pathogen
All bacterial pathogens must have at least one reservoir.
The most common reservoirs for human pathogens are
other humans, animals, and the environment. Since the
source and/or reservoir of the pathogen is part of the
infectious disease cycle.
Transport of the Bacterial Pathogen to the Host
An essential feature in the development of an infectious
disease is the initial transport of the bacterial pathogen to
the host. The most obvious means is direct contact—
from host to host (coughing, sneezing, body contact).
Bacteria also are transmitted indirectly in a variety of
ways. Infected hosts shed bacteria into their
surroundings.
Once in the environment bacteria can be deposited on
various surfaces, from which they can be either
resuspended into the air or indirectly transmitted to a host
later. Soil, water, and food are indirect vehicles that
harbor and transmit bacteria to hosts. Vectors and
fomites (inanimate objects that harbor and transmit
pathogens) also are involved in the spread of many
bacteria.
Attachment and Colonization by the Bacterial
Pathogen
After being transmitted to an appropriate host, the
bacterial pathogen must be able to adhere to and colonize
host cells and tissues.
12 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
In this context colonization means the establishment of a
site of microbial reproduction on or within a host. It does
not necessarily result in tissue invasion or damage.
Colonization depends on the ability of the bacteria to
compete successfully with the host’s normal microbiota
for essential nutrients. Specialized structures that allow
bacteria to compete for surface attachment sites also are
necessary for colonization.
Bacterial pathogens and many nonpathogens adhere with
a high degree of specificity to particular tissues.
Adherence factors called adhesins are one reason for this
specificity. Adhesins are specialized molecules or
structures on the bacteria’s cell surface that bind to
complementary receptor sites on the host cell surface.
They are one type of virulence factor.
Virulence factors are bacterial products or structural
components (e.g., capsules and adhesins) that contribute
to virulence or pathogenicity.
Invasion of the Bacterial Pathogen
Entry into host cells and tissues is a specialized strategy
used by many bacterial pathogens for survival and
multiplication. Pathogens often actively penetrate the
host’s mucous membranes and epithelium after
attachment to the epithelial surface. This may be
accomplished through production of lytic substances that
alter the host tissue by (1) attacking the ground substance
and basement membranes of integuments and intestinal
linings, (2) degrading carbohydrate protein complexes
between cells or on the cell surface (the glycocalyx), or
(3) disrupting the cell surface.
At times a bacterial pathogen can penetrate the epithelial
surface by passive mechanisms not related to the
pathogen itself. Examples include (1) small breaks,
lesions, or ulcers in a mucous membrane that permit
initial entry; (2) wounds, abrasions, or burns on the
skin’s surface; (3) arthropod vectors that create small
wounds while feeding; (4) tissue damage caused by other
organisms; and (5) existing eucaryotic internalization
pathways (e.g., endocytosis and phagocytoses.
Once under the mucous membrane, the bacterial
pathogen may penetrate to deeper tissues and continue
disseminating throughout the body of the host. One way
the pathogen accomplishes this is by producing specific
products and/or enzymes that promote spreading. These
products are virulence factors. Bacteria may also enter
the small terminal lymphatic capillaries that surround
epithelial cells. These capillaries merge into large
lymphatic vessels that eventually drain into the
circulatory system. Once the circulatory system is
reached, the bacteria have access to all organs and
systems of the host.
Growth and Multiplication of the Bacterial Pathogen
For a bacterial pathogen to be successful in growth and
reproduction (colonization), it must find an appropriate
environment (nutrients, pH, temperature, redox potential)
within the host. Those areas of the host’s body that
provide the most favorable conditions will harbor the
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
pathogen and allow it to grow and multiply to produce an
infection.
Some bacteria invade specific cells in which they grow
and multiply. Many of these intracellular pathogens have
evolved such elaborate nutrient-gathering mechanisms
that they have become totally dependent on the host’s
cells. Finally, some bacteria can actively grow and
multiply in the blood plasma. The presence of viable
bacteria in the bloodstream is called bacteremia. The
presence of bacteria or their toxins in the blood often is
termed septicemia [Greek septikos, produced by
putrefaction, and haima, blood].
Leaving the Host
The last determinant of a successful bacterial pathogen is
its ability to leave the host and enter either a new host or
a reservoir. Unless a successful escape occurs, the
disease cycle will be interrupted and the microorganism
will not be perpetuated. Most bacteria employ passive
escape mechanisms. Passive escape occurs when a
pathogen or its progeny leave the host in feces, urine,
droplets, saliva, or desquamated cells.
The Clonal Nature of Bacterial Pathogens
The main mechanism bacteria use to exchange genetic
information is the transfer of extrachromosomal genetic
elements, plasmids, and phages. Many genes that code
for bacterial virulence factors are found on plasmids or
phages. These mobile genetic elements can transfer
virulence factors between members of the same species
or different species by horizontal gene transfer. At times
the genetic elements are part of highly mobile DNA
(transposons), and there is a recombination between the
extrachromosomal DNA and the chromosome. When this
recombination occurs, the genes coding for virulence
may become chromosomal.
One important result of the conservation of these
chromosomal genes is that the bacteria are clonal. Some
bacterial pathogens have only one or a few clonal types
existing in the environment. For example, the bacterium
that causes typhoid fever (Salmonella typhi) has two
clonal types, whereas there are over 100 clonal types of
Haemophilus influenzae, but only a small number are
associated with bacterial pneumonia.
Regulation of Bacterial Virulence Factors
Some pathogenic bacteria have adapted to both the free-
living state and to an environment within a human host.
In the adaptive process, these pathogens have evolved
complex signal transduction pathways to regulate the
genes necessary for virulence. A virulence factor may be
present simply because the bacterium has been infected
by a phage. Often environmental factors control the
expression of the virulence genes. Common signals
include temperature, osmolality, available iron, pH,
specific ions, and other nutrient factors. Several
examples are now presented.
The gene for diphtheria toxin from Corynebacterium
diphtheriae (the pathogen that causes diphtheria) is
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  13
carried on the temperate bacteriophage λ, and its
expression is regulated by iron. The toxin is produced
only by strains lysogenized by the phages. Expression of
the virulence gene of Bordetella pertussis (the pathogen
that causes whooping cough) is enhanced when the
bacteria grow at body temperature (37°C) and suppressed
when grown at a lower temperature. Finally, the
virulence factors of Vibrio cholerae (the pathogen that
causes cholera) are regulated at various levels by many
environmental factors. Expression of the cholera toxin is
higher at pH 6 than at pH 8 and higher at 30 than at
37°C. Osmolality and available amino acids are also
important.
Pathogenicity Islands
Many bacteria (Yersinia spp., Pseudomonas aeruginosa,
Shigella flexneri, Salmonella typhimurium,
enteropathogenic E. coli) are pathogenic because they
have large segments of DNA, called pathogenicity
islands, that carry genes responsible for virulence. These
pathogenicity islands have been acquired during
evolution by horizontal gene transfer. A pathogen may
have more than one pathogenicity island. An excellent
example of virulence genes carried in a pathogenicity
island are those involved in protein secretion. So far, five
pathways of protein secretion (types I to V) have been
described in gram-negative bacteria. A set of
approximately 20 genes encode a pathogenicity
mechanism termed the type III secretion system that
enables gram-negative bacteria to secrete and inject
virulence proteins into the cytoplasm of eucaryotic host
cells.
Unlike other bacterial secretory systems, the type III
system is triggered specifically by contact with host cells,
which helps avoid inappropriate activation of host
defenses. Secretion of these virulence proteins into a host
cell initiates sophisticated “biochemical crosstalk”
between the pathogen and the host. The injected proteins
resemble eucaryotic factors that signal transduction
functions and they are capable of interfering with
eucaryotic signaling pathways.
Redirection of cellular signaling transduction may disarm
host immune responses or reorganize the cytoskeleton,
thus establishing subcellular niches for bacterial
colonization and facilitating “stealth and interdiction” of
host defense communication lines.
Pathogenicity islands generally increase microbial
virulence and are not present in nonpathogenic members
of the same genus or species. One specific example is
found in E. coli. The enteropathogenic E. coli possesses
large DNA fragments, 35 to 170 kilobases in size, that
contain several virulence genes absent from commensal
E. coli. Some of these genes code for proteins that alter
actin microfilaments within a host intestinal cell. As a
consequence, the host cell surface bulges and develops
into a cuplike pedestal to which the bacterium tightly
binds.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
A2. Cell fusion in both normal and abnormal cells
Cell fusion is a process in which two or more cells
become one by merging their plasma membranes. The
ability of a cell to fuse to other cells is referred to as
fusogenicity. The progeny of cell fusion are known as
hybrids. Perhaps the best-known hybrids are hybridomas,
which are made by fusing myeloma cells with
lymphocytes to produce monoclonal antibodies.
Although cells can be easily fused in the laboratory using
readily available chemicals, cell fusion in live organisms
appears to be a complex, poorly understood, multistep
process that involves cell-cell recognition, cell adhesion,
and membrane fusion.
Cell fusion is a part of normal development and tissue
homeostasis
Our life begins with fusion of our parents’ gametes. A
pregnancy depends on normal functioning of the
placental barrier, the main part of which is the
syncytiotrophoblast, a gigantic syncytium (a cell
resulting from fusion of numerous cells) whose surface
area can reach 10 square meters. As the embryo
develops, its muscles are formed by fusion of myoblasts
into syncytia through a multistep process that involves
products of multiple genes.
In the adult body, the maintenance of the bones is
controlled in part by osteoclasts, which are multinuclear
cells formed by the fusion of mononuclear progenitors.
Macroscopic foreign objects, such as a splinter or an
implanted device, are encapsulated by foreign body giant
cells (FBGC) that attempt to dissolve the intruder and are
thought to be formed by fusion of mononuclear
precursors. Langhans cells, a variation of FBGC, are
found in tuberculosis patients at the sites of local
inflammation.
The observations that embryonic stem cells may
differentiate into multiple cell types through cell fusion
suggested a new role of cell fusion in mammalian
development, although alternative explanations for these
observations have not been ruled out. Studies in the
nematode C. elegans provide evidence that cell fusion
can be a major part of development, at least in
invertebrates. About one-third of the cells that are born in
this organism are subsequently fused into 44 syncytia in
a reproducible and stereotypic way.
Remarkably, even the side of a cell that will fuse is
predetermined. In summary, cell fusion of normal
somatic cells is a tightly controlled process that is
restricted to only a few cell types in humans, and results
in terminally differentiated multinuclear cells incapable
of proliferation. Intriguingly, tumor cells appear to
violate strict rules of cell fusion.
Cell fusion and cancer
The idea that cell fusion contributes to cancer
progression was introduced almost 100 years ago with a
proposal that malignancy is a consequence of
14 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
hybridization between leukocytes and somatic cells.
Sixty years later, this idea was expanded by proposing
that hybridization of tumor cells with lymphocytes
results in metastatic cells, and that cell fusion promotes
the phenotypic and genotypic diversity of tumors.
Several lines of evidence support at least some of these
notions.
Many tumor cells are fusogenic
The propensity of various tumor cells to fuse
spontaneously in tissue culture has been a known, though
unexplained, phenomenon for many decades. In fact, this
property of tumor cells was used to make somatic cell
hybrids before fusion with polyethylene glycol (PEG) or
viruses had been developed. Some tumor cell lines are so
fusogenic that they fuse spontaneously more efficiently
than in the presence of PEG, a difference explainable by
the toxicity of this agent. The ability to fuse is not limited
to a particular cell or tumor type and can occur between
tumor cells as well as between a tumor and a normal cell.
Tumor cell fusion is not limited to tissue culture. Human
tumor cells injected into hamsters produced metastases
formed by hybrids of human and hamster cells. In
another study, treatment of chimeric mice with a
carcinogen resulted in tumors, in which about 1% of cells
had marker alleles from both parental strains, an
observation explainable by cell fusion.
Interestingly, despite these observations, cell fusion is
rarely, if at all, considered in contemporary studies that
use experimental models of cancer. Because the evidence
for cell fusion in human cancers is indirect, it is not as
compelling as that for experimental tumors. One
observation is that premature chromosome condensation
(PCC) is observed in tumor cells. PCC is a typical result
of a fusion between cells in different stages of the cell
cycle, although PCC can be also induced in mononuclear
cells, for example by caffeine.
Cell fusion could also explain the origin of multinuclear
tumor cells in which nuclei undergo asynchronous DNA
synthesis or mitosis, even though alternative
explanations of this phenomenon are as plausible. It is
unclear whether the scarcity of reports on cell fusion in
human tumors is due to rarity of this event, to a difficulty
to detect it, or to insufficient interest in this subject. The
last possibility should not be underestimated. For
example, the number of reports on apoptosis that were
published during the last ten years is about 100 times
higher than that published in the preceding twenty. With
all likelihood, this difference is due to a change in the
subject’s popularity rather than a change in incidence or
role of apoptosis in studied organisms.
Hybrids can be more malignant than the parental
cells
Importantly, while physiological fusion of normal
somatic cells produces non-proliferating differentiated
multinuclear cells, fusion of tumor cells results in
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
proliferating hybrids. The survival rate of these hybrids
in tissue culture can be as high as 1%. Considering that
the rate of cell fusion in experimental tumors was also
estimated at 1%, a 1 cm3 tumor of about 109 cells may
harbor 105 proliferating hybrid cells. The question is, can
any of these cells be more malignant than their parents?
In fact, one of the early reports on spontaneous cell
fusion came from an observation that cultures containing
a mixture of two mouse cell lines were occasionally
overgrown by a new cell line, whose karyotype was the
sum of the parental karyotypes. This observation
indicated that the new cell line is a hybrid, and that
hybrids can grow faster than their parents. The
subsequent studies indicated that, at least in experimental
systems, hybrids can be more drug-resistant and more
metastatic than the parental cells (Figure 1).
Cell fusion can contribute to drug resistance
Cell fusion can consolidate resistance to various drugs by
combining genes responsible for resistance to various
agents. For example, fusion of tumor cells that were
resistant to 5-fluorouracil with tumor cells resistant to
methotrexate produced hybrids that were resistant to both
drugs. An unexpected observation was that the hybrids
were resistant to mephalan, a drug to which both parental
lines were sensitive. This observation emphasized the
difficulty in predicting the diversity that cell fusion can
create. Our own experiments indicated that fusion of
drug-sensitive transformed cells to primary cells results
in heterokaryons that are resistant to apoptosis. Although
this effect lasted only a few days, it is not unreasonable
to speculate that even a temporary resistance may allow a
small fraction of tumor cells to survive a session of
chemotherapy.
Cell fusion can promote the ability to metastasize
The ability to metastasize is, arguably, the deadliest
property of cancers. A proposed link between cell fusion
and metastasis can be described as the “wolf in sheep’s
clothing” model. The model suggests that a tumor cell
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  15
becomes metastatic by fusion to normal cells that travel
throughout the body freely, such as lymphocytes or
macrophages. Several studies support this idea. One
study found that a parental cell line, when injected in a
mouse, produced hundreds of colonies in the liver, while
a derivative line produced only a few. Surprisingly, when
a colony formed by the derivative line was injected into a
new host, it produced as many colonies as did the
parental cell line.
The analysis of histocompatibility markers and
karyotypes of the host mice, the injected cells, and the
resulting metastases led to the conclusion that the
increase in the metastatic potential was due to a fusion
with the host cells. This conclusion was supported by the
finding that the high metastatic potential can be restored
in the derivative cells by fusing them with mouse bone
marrow cells before injection.
Other studies discovered that cell fusion can determine
the tissues into which tumor cells metastasize. For
example, fusion of nonmetastatic mouse plasmacytoma
or myeloma cells with lymphocytes or splenic dendritic
cells not only resulted in metastatic hybrids, but the
target tissues of metastases varied with the type of
normal cells used as well . One can only wonder whether
implications of this finding are considered in developing
cancer vaccines, which involves injecting patients with
hybrids between tumor cells and normal dendritic cells.
Because tissues that are common sites of metastases are
normally rich in macrophages, the “wolf in sheep’s
clothing” model argues that tumor cells gravitate to these
sites because, as macrophage hybrids, they acquire
macrophage’s tropism.
Another possibility is that tumor cells can acquire the
“sheep’s clothing” by fusing to a normal cell of a tissue,
thus producing a tumor cell that can grow in the new
environment. This mechanism is not unlike that proposed
for the stem cell differentiation, where a stem cell
differentiates by fusing to a resident cell from a host
tissue.
Cell fusion can increase tumor cell diversity
Perhaps the main property of cancer cells that makes
them malignant is the ability to produce diverse progeny.
Indeed, as an oncology textbook states, “…by the time of
initial diagnosis, cancers consist of multiple
subpopulations of cells with diverse genetic,
biochemical, immunologic, and biologic characteristics”.
How this diversity emerges and how it is maintained is
not clear. The evidence that cell fusion can contribute to
tumor heterogeneity came from using cell fusion as a tool
for somatic cell genetics, in particular to investigate
whether malignancy is a dominant or recessive trait.
Because hybrids between a highly and a weakly
malignant tumor cell line were highly malignant, the
initial conclusion was that malignancy is a dominant
trait. However, the subsequent studies found that hybrids
between tumor cells and normal cells were not
tumorigenic. The controversy was resolved by realizing
that hybrids become tumorigenic if they lose certain
“normal” chromosomes, an observation that eventually
led to the discovery of tumor suppressor genes.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
A less appreciated outcome of these studies was the
conclusion that chromosomal aberrations are hallmarks
of hybrids. These aberrations include chromosome
nondisjunction, mitotic recombination, translocations,
deletions, insertions, and inversions, a list that is
remarkably similar to that observed in tumor cells.
Although how exactly cell fusion causes these
abnormalities is not clear, the result is hardly surprising
considering that cell fusion produces cells with a sum of
parental chromosomes and more than two centrosomes.
The aberrant chromosome segregation associated with
cell fusion was proposed to explain aneuploidy as a
hallmark of cancer cells. Although aneuplody is a feature
of nearly all of more than 20,000 solid tumors analyzed
in human, and it was even proposed to be a cause rather
than a consequence of cancer, how and why tumor cells
become aneuploid is not clear. The observation that
tumor cells are prone to cell fusion, the fact that fusion
results in polyploidy cells, and the finding that cell fusion
is followed by abnormal chromosome segregation,
together argue that if cell fusion is a part of cancer
progression, then tumor cells should eventually become
aneuploid. However, this explanation of aneuploidy by
no means excludes other mechanisms, such as mutations
that cause abnormal mitosis.
Changes in epigenetic regulation that follow cell fusion
are another factor that can contribute to tumor diversity.
These changes appear to be unpredictable, and gene
expression can be selectively silenced, activated, or
unchanged. Considering that the human body has about
200 cell types that are different because of their gene
expression pattern, it is easy to imagine how cell fusion
can produce monsters. In summary, cell fusion can be an
engine of genomic and epigenetic variability that has a
potential to make cells with new properties at a rate
exceeding that achievable by random mutagenesis.
Why are tumor cells fusogenic?
There is no definitive answer to this question, which is
not surprising considering that the phenomenon lost
whatever popularity it had before modern experimental
approaches capable of dissecting its mechanism were
developed. Several factors were proposed to explain
formation of hybrids in tumors: viruses, cholesterol
crystals, and the natural fusogenicity of macrophages.
The ability of viruses to induce cell fusion is mediated by
the viral proteins that mediate entry of enveloped viruses
into the cell. These proteins are members of the fusion
B1.  CELL SIGNALLING: 
1. Cell-to-Cell Signaling: Hormones and Receptors:
No cell lives in isolation. In all multi cellular organisms,
survival depends on an elaborate intercellular
communication network that coordinates the growth,
differentiation, and metabolism of the multitude of cells
in diverse tissues and organs. Cells within small groups
often communicate by direct cell-cell contact. We will
examine how cells communicate by means of extra
cellular signaling molecules. These substances are
synthesized and released by signaling cells and produce a
specific response only in target cells that have receptors
for the signaling molecules. An enormous variety of
16 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
protein family, which includes both viral and cellular
proteins. The fusion proteins are identified by a
hydrophobic motif, known as the fusion peptide, which is
required for the fusogenicity of these proteins.
Perhaps any fusion protein or any other agent that
induces cell fusion should be investigated for its
carcinogenicity. For example, HIV is known to induce
syncytia, which suggests that in addition to its known
role in disease, it might be useful to investigate whether
this ability contributes to malignancies associated with
this virus, such as Kaposi’s sarcoma. Remarkably, the
genes that encode human endogenous retroviruses
(HERV) or their individual proteins, including the fusion
proteins, comprise at least eight percent of the human
genome. In fact, syncytin, the fusion protein that is
thought to mediate formation of the syncytiotrophoblast
and is specifically expressed in placenta, is the envelope
fusion protein of HERV-W. This finding indicates that
expression of HERV proteins can determine cell fate,
and, incidentally, gives support to the hypothesis that
placental mammals evolved because our ancestor had the
germ line infected with a retrovirus.
The sheer abundance of HERV genes in the human
genome provides an ample opportunity for their
deregulated expression. Indeed, HERV particles were
found in both normal and tumor tissues. Production of
HERV has been associated with multiple sclerosis and
cancer, although whether this association is causative is
unclear, in part because HERV particles do not appear to
be infectious. Perhaps a causal link between HERVs and
these diseases can be found by investigating whether
these particles induce cell fusion. Cell death is a
predominant outcome of cell fusion, which might explain
how expression of HERV can lead to multiple sclerosis,
while the ability of some cells to survive cell fusion can
contribute to cancer, as we discussed.
Overall, at least in experimental systems, cell fusion has
been linked to several fundamental features of cancer,
even though molecular mechanisms that cause this fusion
remain obscure. Although one can argue that this link is a
peculiarity of experimental tumors, the hypothesis that
cell fusion contributes to cancer in humans seems to be
equally plausible. Considering that modern tools of
experimental biology have not been applied to studies of
cell fusion in tumors, there is an abundance of
possibilities that are ready to be explored.
chemicals, including small molecules (e.g., amino acid
derivatives, acetylcholine), peptides, and proteins, are
used in this type of cell-to-cell communication. The extra
cellular products synthesized by signaling cells can
diffuse away or be transported in the blood, thus
providing a means for cells to communicate over longer
distances than is possible by chains of direct cell-cell
contacts.
2. Overview of Extracellular Signaling
Communication by extracellular signals usually involves
six steps: (1) synthesis and (2) release of the signaling
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
molecule by the signaling cell; (3) transport of the signal
to the target cell; (4) detection of the signal by a specific
receptor protein; (5) a change in cellular metabolism,
function, or development triggered by the receptor-signal
complex; and (6) removal of the signal, which often
terminates the cellular response.
In many eukaryotic microorganisms (e.g., yeast, slime
molds, and protozoans), secreted molecules coordinate
the aggregation of free-living cells for sexual mating or
differentiation under certain environmental conditions.
Chemicals released by one organism that can alter the
behavior or gene expression of other organisms of the
same species are called pheromones.
3. Signaling Molecules Operate over Various
Distances in Animals
In animals, signaling by extracellular, secreted molecules
can be classified into three types endocrine, paracrine,
or autocrine based on the distance over which the
signal acts. In addition, certain membrane-bound proteins
on one cell can directly signal an adjacent cell.
In endocrine signaling, signaling molecules, called
hormones, act on target cells distant from their site of
synthesis by cells of endocrine organs. In animals, an
endocrine hormone usually is carried by the blood from
its site of release to its target.
In paracrine signaling, the signaling molecules released
by a cell only affect target cells in close proximity to it.
The conduction of an electric impulse from one nerve
cell to another or from a nerve cell to a muscle cell
(inducing or inhibiting muscle contraction) occurs via
paracrine signaling. The role of this type of signaling,
mediated by neurotransmitters, in transmitting nerve
impulses. Many signaling molecules regulating
development in multicellular organisms also act at short
range.
In autocrine signaling, cells respond to substances that
they themselves release. Many growth factors act in this
fashion, and cultured cells often secrete growth factors
that stimulate their own growth and proliferation. This
type of signaling is particularly common in tumor cells,
many of which overproduce and release growth factors
that stimulate inappropriate, unregulated proliferation of
themselves as well as adjacent nontumor cells; this
process may lead to formation of tumor mass.
Some compounds can act in two or even three types of
cell-to-cell signaling. Certain small amino acid
derivatives, such as epinephrine, function both as
neurotransmitters (paracrine signaling) and as systemic
hormones (endocrine signaling). Some protein hormones,
such as epidermal growth factor (EGF), are synthesized
as the exoplasmic part of a plasma- membrane protein;
membrane-bound EGF can bind to and signal an adjacent
cell by direct contact. Cleavage by a protease releases
secreted EGF, which acts as an endocrine signal on
distant cells.
4. The Three stages of cell signaling are Reception,
transduction, and Response
Our current understanding that how chemical massager
act via signal transduction pathways had its origin in
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  17
pioneering work of Earl W. Sutherland, whose research
lead to a Noble prize in 1971. Sutherland and his
colleagues were investigating how the animal hormone
epinephrine stimulates the breakdown
(depolymerization) of the storage polysaccharide
glycogen with liver cells and skeletal muscle cells.
Glycogen depolymerization releases the sugar glucose-1-
phosphate, which the cell converts to glucose-6-
phosphate. The cell can then use this compound, an early
intermediate in glycolysis, for energy production. Alter-
natively, the compound can be stripped of phosphate and
released into the blood as glucose that can fuel cells
throughout the body. Thus one effect of epinephrine,
which is secreted from the adrenal gland during times of
physical or mental stress, is the mobilization of fuel
reserves.
Sutherland's research team discovered that epinephrine
stimulates glycogen breakdown by somehow activating a
cytosolic enzyme, glycogen phosphorylase. However,
when epinephrine was added to a test-tube mixture
containing the phosphorylase and its substrate, glycogen,
no depolymerization occurred.
Epinephrine could activate glycogen phosphorylase only
when the hormone was added to a solution containing
intact cells. This result told Sutherland two things. First,
epinephrine does not interact directly with the enzyme
responsible for glycogen breakdown; an intermediate
step or series of steps must be occurring inside the cell.
Second, the plasma membrane is somehow involved in
transmitting the epinephrine signal.
Thus Sutherland's early work suggested that the process
going on at the receiving end of a cellular conversation
can be dissected into three stages: reception,
transduction, and response (FIGURE 1):
1. Reception is the target cell's detection of a signal
coming from outside the cell. A chemical signal is
"detected" when it binds to a cellular protein, usually at
the cell's surface.
2. The binding of the signal molecule changes the
receptor protein in some way, thus initiating the process
of transduction. The transduction stage converts the
signal to a form that can bring about a specific cellular
response. In Sutherland's system, the binding of
epinephrine to the outside of a receptor protein in a liver
cell's plasma membrane leads via a series of steps to
activation of glycogen phosphorylase. Transduction
sometimes occurs in a single step but more often requires
a sequence of changes in a series of different molecules-a
signal-transduction pathway. The molecules in the
pathway are often called relay molecules.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
3. In the third stage of cell signaling, the transduced
signal finally triggers a specific cellular response. The
response may be almost any imaginable cellular activity-
such as catalysis by an enzyme (such as glycogen
phosphorylase), rearrangement of the cytoskeleton, or
activation of specific genes in the nucleus. The cell-
signaling process helps ensure that crucial activities like
these occur in the right cells, at the right time, and in
proper coordination with the other cells of the organism.
5. Signal Reception and the Initiation of Transduction
When we speak to someone, others nearby may hear our
message, sometimes with unfortunate consequences.
However, errors of this kind rarely occur between cells.
The signals emitted by α yeast cell are "heard" only by
its prospective mates, β cells. Similarly, although
epinephrine encounters many types of cells as it
circulates in the blood, only certain target cells detect and
react to the hormone. The signal receptor is the identity
tag on the target cell.
a. A signal molecule binds to a receptor protein,
causing the protein to change shape
A cell targeted by a particular chemical signal has
molecules of a receptor protein that recognizes the signal
molecule. The signal molecule is complementary in
shape to a specific site on the receptor and attaches there,
like a key in a lock-or like a substrate in the catalytic site
of an enzyme. The signal molecule behaves as a ligand,
the term for a small molecule that specifically binds to a
larger one. Ligand binding generally causes a receptor
protein to undergo a change in conformation-that is, to
change shape. For many receptors, this shape change
directly activates the receptor so that it can interact with
another cellular molecule. For other kinds of receptors,
as we'll see shortly, the immediate effect of ligand
binding is more limited, mainly causing the aggregation
of two or more receptor molecules.
b. Most signal receptors are plasma-membrane
proteins
18 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
Most signal molecules are water-soluble and too large to
pass freely through the plasma membrane. But, as
Sutherland learned for epinephrine, they still influence
cellular activity in major ways. Like yeast mating factors,
most water-soluble signal molecules bind to specific sites
on receptor proteins embedded in the cell's plasma
membrane. Such a receptor transmits information from
the extracellular environment to the inside of the cell by
changing shape or aggregating when a specific ligand
binds to it. We'll see how membrane receptors work by
looking at three major types: G-protein-linked receptors,
tyrosine kinase receptors, and ion-channel receptors.
G-Protein-Linked Receptors
A G-protein-linked receptor is a plasma-membrane
receptor that works with the help of a protein called a G
protein (FIGURE 2a).
Many different signal molecules use G-protein and many
other hormones, and neurotransmitters. These receptors
vary in their binding sites for recognizing signal
molecules and for recognizing different G proteins inside
the cell. Nevertheless, G-protein-linked receptor proteins
are all remarkably similar in structure. They each have
seven a-helices spanning the membrane, as shown in
FIGURE 3.
Loosely attached to the cytoplasmic side of the
membrane, the G protein functions as a switch that is on
or off depending on which of two guanine nucleotides is
attached, GDP or GTP. When GDP is bound, the G
protein is inactive; when GTP is bound, it is active.
(GTP, or guanosine triphosphate, is similar to ATP.)
When the appropriate chemical signal binds as a ligand
to the extracellular side of a G-protein-linked receptor,
the receptor is activated, changing conformation in such
a way that it, in turn, can activate a G protein: The
receptor binds a specific, inactive G protein and causes a
GTP to displace the GDP (FIGURE 2b). The activated G
protein then binds to another protein, usually an enzyme,
and alters its activity. These changes are only temporary,
however, for the G protein also functions as a GTPase
enzyme and soon hydrolyzes its bound GTP to GDP
(FIGURE 2c). Now inactive again, the G protein leaves
the enzyme. The GTPase function of the G protein
allows the pathway to shut down rapidly when the extra-
cellular signal molecule is no longer present.
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

G-protein receptor systems are extremely widespread and
diverse in their functions. In addition to the functions
already mentioned, they are important in embryonic
development, as indicated by genetic studies. For
instance, mutant mouse embryos lacking a certain G
protein do not develop normal blood vessels and die in
utero. Furthermore, G proteins ate involved in sensory
reception; in humans, for example, both vision and smell
depend on such proteins. Similarities in structure among
G proteins and G-protein-linked receptors of modern
organisms suggest that G proteins and G-protein linked
receptors evolved very early, possibly as sensory recep-
tors of ancient microbes.
The receptor protein is now recognized by specific relay
proteins inside the cell. Each such protein binds to a
specific phosphorylated tyrosine, undergoing a structural
change that activates it (the relay protein may or may not
be phosphorylated by the tyrosine kinase). One tyrosine-
kinase receptor dimer may activate ten or more different
intracellular protein simultaneously, triggering as many
different transduction pathways and particular cellular
responses. The ability of a single ligand-binding event to
trigger so many pathway is a key difference between
these receptors and G-protein receptors. Abnormal
tyrosine-kinase receptors that aggregate even without
ligand cause some kinds of cancer.

G-protein systems are involved in many human diseases,

including bacterial infections. The bacteria that cause
cholera, pertussis (whooping cough), and botulism,
among others, make their victims ill by producing toxins
that interfere with G-protein function. Although drugs for
treating infections and other kinds of diseases have often
been discovered by trial and error, pharmacologists now
realize that up to 60% of all medicines used today exert
their effects by influencing G-protein pathways.

Tyrosine-Kinase Receptors

Among the chemical signals impinging on cells in an

animal's body are growth factors, the local regulators that
stimulate cells to grow and reproduce. Cell reproduction
involves a variety of activities of different parts of the
cell, including protein synthesis in the cytoplasm,
chromosome duplication in the nucleus, and the
rearrangement of elements of the cytoskeleton. Helping
the cell regulate and coordinate these activities is a type
of receptor specialized for triggering more than one
signal-transduction pathway at once.

The receptor for a growth factor is often a tyrosine-

kinase receptor, one of a major class of plasma-
membrane receptors characterized by having enzymatic
activity. Part of the receptor protein on the cytoplasmic
side of the membrane functions as an enzyme, called a
tyrosine kinase, that catalyzes the transfer of phosphate
groups from ATP to the amino acid tyrosine on a Ion-Channel Receptors
substrate protein. Thus tyrosine-kinase receptors are
membrane receptors that attach phosphates to protein Some membrane receptors of chemical signals are
tyrosines. Tyrosine-kinase receptors often have the Ligand-gated ion channels. These channels are protein
structure shown in rough schematic form in FIGURE 4. pores in plasma membrane that open or close in response
Before the signal molecule binds, the receptors exist as to chemical signal, allowing or blocking the flow of
individual polypeptides. Notice that each has an specific ions such as Na + or Ca2+. Like the other
extracellular signal binding site, an intracellular tail receptors we have discussed these channel proteins bind
containing a number of tyrosines, and a single ex helix a signal molecule as a ligand at the specific site on their
spanning the membrane. The binding of a signal extracellular side (FIGURE 5).
molecule to such a receptor does not cause enough of a
conformational change to activate the cytoplasmic side of The shape change
the protein directly. produced in the channel
protein immediately leads
Instead, the activation occurs in three steps: (1) The to a change in the
ligand binding causes two receptor polypeptides to concentration of a
aggregate, forming a dimer (a protein consisting of two particular ion inside the
polypeptides). (2) This aggregation activates the cell. Often this change
tyrosine-kinase parts of both polypeptides, each of which directly affects cell
then (3) phosphorylates the tyrosines on the tail of the functioning in some way.
other polypeptide. In summary, the effect of the signal At a synapse between
molecule a tyrosine-kinase receptor is polypeptide nerve cells, for example, it
aggregation and phosphorylation of the receptor. may trigger an electrical
signal that propagates

INSTITUTE FOR ADVANCED STUDIES, JODHPUR  19
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
down the length of the receiving cell. Ligand-gated ion
channels are very important in the nervous system, as are
gated ion channels that are controlled by electrical
signals .
Intracellular Receptors:
Not all signal receptors are membrane proteins. Some are
proteins located in the cytoplasm or nucleus of target
cells. To reach such a receptor, a chemical messenger
must be able to pass through the target cell's plasma
membrane. A number of important signaling molecules
can do just that, either because they are small enough to
pass between the membrane phospholipids or because
they are themselves lipids and therefore soluble in the
membrane. Chemical messengers with intercellular
receptors include the steroid hormones and thyroid
hormones of animals, which are lipids; the small gaseous
molecule nitric oxide (NO)(which do not have known
receptor and directly activate guanlate cyclase to produce
c-GMP as secondary messanger); and certain small
signal molecules used by bacteria.
The behavior of testosterone is representative of steroid
hormones: Secreted by cells of the testis, the hormone
travels through the blood and enters cells all over the
body. In target cells-those with testosterone receptor
molecule in their cytosol – the hormone binds to the
receptor, activating it. The active form of the receptor
protein can then bind to and turn on genes in the nucleus
that control male sex characteristics. Thus, the receptor
carries out the complete transduction of the signal.
Prostaglandins, thrombaxones, prostacyclins are non-
polar but have membrane receptors.
c. Signal Transduction Pathways:
When signal receptors are plasma-membrane proteins,
like most of those we have discussed, the transduction
stage of cell signaling is usually a multistep pathway.
One benefit of such Pathways is signal amplification. If
some of the molecules in a Pathway transmit the signal to
multiple molecules of the next components of in the
series, the result can be a large number of activated
molecules at the end of the pathway. In other words, a
very small number of extracellular signal molecules can
produce a major cellular response. Moreover, multistep
pathways provide more opportunities for coordination
and regulation than simples systems do.
d. Pathway rely signals form receptor to cellular
responses:
The binding of a specific extracellular signal molecule to
receptor in the plasma membrane triggers the first step in
the chain of molecular interactions - the signal
transduction pathway-that leads to a particular response
within the cell. Like falling dominoes, the signal-
activated receptor activates another protein, which
activates another molecule and so on until the protein
that produces the final cellular response is activated. The
molecules that relay a signal from receptor to response,
sometimes called relay molecules, are mostly proteins.
The interaction of proteins is a major theme of cell
signaling. Indeed, protein interaction is a unifying theme
of all regulation at the cellular level.
20 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
Keep in mind that the original signal molecule is not
physically passed along a signaling pathway; in most
cases, it never even enters the cell. When we say that the
signal is relayed along a pathway, we mean that certain
information is passed on. At each step the signal is
transduced into a different form, commonly a
conformational change in a protein. Very often, the
conformational change is brought about by
phosphorylation.
e. Protein phosphorylation, a common mode of
regulation in cells, is a major mechanism of signal
transduction
We have already seen how phosphorylation is involved
in the activation of tyrosine kinase receptors. In fact, the
phosphorylation of proteins is a widespread cellular
mechanism for regulating protein activity. The general
name for an enzyme that transfers phosphate groups from
ATP to a protein is protein kinase. Unlike receptor
tyrosine kinases, most cytoplasmic protein kinases act
not on themselves, but on other substrate proteins; also,
they phosphorylate their substrates on either of two
amino acids, serine or threonine. Such serine/threonine
kinases are widely involved in signaling pathways in
animals, fungi, and plants.
Many of the relay molecules in signal-transduction path-
ways are protein kinases, and they often act on each
other. FIGURE 6, shows a hypothetical pathway contain-
ing three different protein kinases. This sequence is
similar to many known pathways, including those
triggered in yeast by mating factors and in animal cells
by many growth factors.
A cascade of protein phosphorylation, each, transmits the
signal bringing with it a conformational change. Each
shape change results from the interaction of the charged
phosphate groups with charged and polar amino acids.
The addition of phosphates often changes a protein from
an inactive form to an active form (although in other
cases phosphorylation decreases the activity of the
protein).
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
The importance of protein kinases can hardly be over-
stated. Fully 1 % of our own genes are thought to code
for protein kinases. A single cell may have hundreds of
different kinds, each with specificity for a different
substrate protein Together they probably regulate a large
proportion of thousands of the proteins in a cell. Among
these are most of the proteins that, in turn, regulate cell
reproduction. Abnormal activity of such a kinase
frequently causes abnormal cell growth and contributes
to the development of cancer.
For a cell to respond normally to an extracellular signal it
must have mechanisms for turning off the signal-
transduction pathway when the initial signal is no longer
present. The effects of protein kinases are rapidly
reversed in the cell by protein phosphatases, enzymes
that remove phosphate groups from proteins. At any
given moment, the activity of a protein regulated by
phosphorylation depends on the balance in the cell
between active kinase and active phosphatase molecules.
When the extracellular signal molecule is not present,
active phosphatase molecules predominate, and the
signaling pathway and cellular response shut down.
f. Certain small molecules and ions are key
components of signaling pathways (second
messengers)
Not all components of signal-transduction pathways are
proteins. Many signaling pathways also involve small,
non-protein, water-soluble molecules or ions, called
second messengers. (The extracellular signal molecule
that binds to the membrane receptor is a pathway's “first
messenger”) Because second messengers are both small
and water-soluble, they can readily spread throughout the
cell by diffusion. For example, as we'll see shortly, it is a
second messenger called cyclic AMP that carries the
signal initiated by epinephrine from the plasma
membrane of a liver or muscle cell into the cell's interior,
where it brings about glycogen breakdown. Second mes-
sengers participate in pathways initiated by both G-
protein linked receptors and tyrosine-kinase receptors.
The two most widely used second messengers are cyclic
AMP and calcium ions, Ca2+. A large variety of relay
proteins are sensitive to the cytosolic concentration of
one or the other of these second messengers.
Cyclic AMP
Once Earl Sutherland had established that epinephrine
somehow causes glycogen breakdown without passing
through the plasma membrane, the search began for the
second messenger (he coined the term) that transmits the
signal from the plasma membrane to the metabolic
machinery in the cytoplasm.
Sutherland found that the binding of epinephrine to the
plasma membrane of a liver cell elevates the cytoplasmic
concentration of a compound called cyclic adenosine
monophosphate, abbreviated cyclic AMP or cAMP. An
enzyme built into the plasma membrane, adenylyl
cyclase, converts ATP to cAMP in response to an
extracellular signalin this case, epinephrine. Adenylyl
cyclase becomes active only after epinephrine binds to a
specific receptor protein. Thus the first messenger, the
hormone, causes a membrane enzyme to synthesize
cAMP, which broadcasts the signal to the cytoplasm. The
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  21
cAMP does not persist for long in the absence of the
hormone, because another enzyme converts the cAMP to
an inactive product, AMP. Another surge of epinephrine
is needed to boost the cytosolic concentration of cAMP
again.
Subsequent research revealed that epinephrine is only
one of many hormones and other signal molecules that
trigger pathways involving cAMP. It also brought to
light the other components of cAMP pathways, including
G proteins, G-protein-linked receptors, and protein
kinases (FIGURE 7). The relay molecule immediately
after cAMP in a signaling pathway is usually protein
kinase A, a serine/threonine kinase. Cyclic AMP
activates this kinase. The active kinase then
phosphorylates various other proteins, depending on the
cell.
Further fine-tuning of cell metabolism is provided by
other G-protein systems that inhibit adenylyl cyclase. In
these systems, a different signal molecule activates a
different receptor, which activates an inhibitory G
protein.
Now that we know about the role of cAMP in G-protein
signaling pathways, we can explain in molecular detail
how certain microbes cause disease. Consider cholera, a
disease that is frequently epidemic in places where the
water supply is contaminated with human feces. People
acquire the cholera bacterium, Vibrio cholerae, by
drinking contaminated water. The bacteria colonize the
lining of the small intestine and produce a toxin, which
in this case is an enzyme that chernically modifies a G
protein involved in regulating salt and water secretion.
Because the modified G protein is unable to hydrolyze
GTP to GDP, it remains stuck in its active form,
continuously stimulating adenylyl cyclase to make
cAMP. The resulting high concentration of cAMP causes
the intestine cells to secrete large amounts of water and
salts into the intestines. An infected person quickly
develops profuse diarrhea and if left untreated can easily
die from the loss of water and salts.
Calcium Ions and Inositol Trisphosphate
Many signal molecules in animals, including
neurotransmitters, growth factors, and some hormones,
induce responses in their target cells via signal-
transduction pathways that; increase the cytosolic
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
concentration of calcium ions (Ca2+). Calcium is even
more widely used than cAMP as a second messenger.
Increasing the cytosolic concentration of Ca2++ causes
many responses in animal cells, including muscle cells to
contraction, secretion of certain substances, and cell
division. In plant cells, calcium functions as a second
messenger in signaling pathways plants have evolved for
coping with environmental stresses, such as drought or
cold. Cells use Ca2+ as a second messenger in both G-
protein pathways and tyrosine kinase receptor pathways.
Although cell always contain Ca2+, this ion can function
as a second messanger because its concentration in the
cytosol is normally much lower than the concentration
outside the cell. In fact, the level of Ca2+ in the blood
and extracellular fluid of an animal often exceeds that in
the cytosol by more than 10,000 times. Calcium ions are
actively transported out of cell and are actively imported
from the cytosol into the endoplasmic reticulum (and,
under some conditions, into mitochondria and
chloroplasts). As a result, the calcium concentration in
the ER is usually much higher than in the cytosol
(FIGURE 8). Because the cytosolic calcium level is low,
a small change in absolute numbers of ions represents a
relatively large percentage change in calcium
concentration.
In response to a signal relayed by a signal-transduction
Pathway, the cytosolic calcium level may rise, usually by
a mechanism that release Ca2+ from the cell’s ER. The
pathway leading to calcium release involve still other
second messengers, Diacyl glycerol (DAG) and inositol
triphosphate (IP3). These two messengers are produced
by cleavage of a certain kind of phospholipid in the
plasma membrane. FIGURE shows how this occurs and
how IP3 stimulates the release of calcium from the ER.
Because IP3 acts before calcium in the pathway, calcium
could be considered a "third messenger:' However,
scientists use the term second messenger for all small,
non-protein components of signal-transduction pathways.
In some cases, calcium ions activate a signal-
transduction protein directly, but often they function by
means of calmodulin, a Ca2+ -binding protein present at
high levels in eukaryotic cells (in an animal cell, for
example, calmodulin may represent as much as 1% of the
22 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
total protein). Calmodulin mediates many calcium-
regulated processes in cells. When calcium ions bind to
it, calmodulin changes conformation and then binds to
other proteins, activating or inactivating them. The
proteins most often regulated by calmodulin are protein
kinases and phosphatases-the most common relay
proteins in signaling pathways.
6. Cellular Responses to Signals
We now take a closer look at the cell's eventual response
to an extracellular signal-what some researchers call the
"output response: What is the nature of the final step in a
signaling pathway?
a. In response to a signal, a cell may regulate activities
in the cytoplasm or transcription in the nucleus
Ultimately, a signal-transduction pathway leads to the
regulation of one or more cellular activities. The
regulated activities may occur in the cytoplasm, such as a
rearrangement of the cytoskeleton, the opening or closing
of an ion channel in the plasma membrane, or some
aspect of cell metabolism. As we have discussed already,
the response of liver cells to signaling by the hormone
epinephrine helps regulate cellular energy metabolism.
The final step in the signaling pathway activates the
enzyme that catalyzes the breakdown of glycogen.
Many other signaling pathways ultimately regulate not
the activity of an enzyme but the synthesis of enzymes or
other proteins, usually by turning specific genes on or
off. Recall that the genes in a cell's DNA function by
being transcribe into an RNA version called messenger
RNA (mRNA), which leaves the nucleus and is
translated into a specific protein by ribosomes in the
cytoplasm. Special protein called transcription factors
control which genes are turned on-that is, transcribed into
mRNA-in a particular cell at a particular time. The
activity of a transcription factor may, itself be regulated
by a signaling pathway that extends into the cell nucleus.
All the different kinds of signal receptors and relay
molecules introduced in this chapter participate in gene-
regulating pathways, as well as in pathway leading to
other kinds of responses. The molecular messengers that
produce gene-regulation responses include grow factors
and certain plant and animal hormones.
b. Elaborate Pathways amplify and specify the cell’s
response to signals:
Why are there often so many steps between the original
signaling event at the cell surface and the cell’s
response? As Mentioned earlier, signaling pathways with
a multiplicity of steps have to important benefits: They
amplify the signal (and thus response), and they
contribute to the specificity of response.
c. Signal Amplification
Elaborate enzyme cascade amplifies the cell’s response
to signal. At each catalytic step in the cascade, the
number of activated products is much greater then in the
preceding step. For example, in the epinephrine-
triggered pathway in Figure, each adenylate cyclase
molecule catalyzes the formation of many c-AMP
molecules, each molecule of protein kinase A
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

phosophorylate many molecule of next kinase in the
pathway, and soon. The amplification effects depends the
fact that these protein persists in active form long enough
to process numerous molecules of the substrate before
they become inactive again. As a result signal
amplification, a small number of epinephrine molecule
binding to receptors on the surface of liver cell or
skeletal muscle can lead to a release of hundreds of
millions of glucose molecule from glycogen.
d. The specificity of cell signaling:
protein interacts both with microfilaments of the
cytoskeleton and with several different components of
signaling pathways that relay information from the cell
surface, including pathways regulating immune cell
proliferation. This multifunctional relay protein is thus
both a branch point and an important intersection point in
a complex signal transduction network that controls
immune cell behavior. When the WAS protein is absent,
the cytoskeleton is not properly organized and signaling
pathways are disrupted, leading to the WAS symptoms.

Consider two different cells in your body-a liver cell and

heart muscle cell, for example. Both are in contact with
your bloodstream and are therefore constantly exposed to
many different hormone molecules, as well as to local
regulators secreted by nearby cells. Yet the liver cell
responds to some signals but ignores others, and the
same is true for the heart cell. And some kinds of signals
trigger responses in both cells but different responses.
For instance, epinephrine stimulates the liver cell to
break down glycogen, but the main response of the heart
cell to epinephrine is contraction, leading to a more rapid
heartbeat. How do we account for this difference?

The explanation for the specificity exhibited in cellular

responses to signals is the same as the basic explanation
for virtually all differences between cells: Different kinds
of cells have different collections of proteins (FIGURE
9).

The response of a particular cell to a signal depends on

its particular collection of signal receptor proteins, relay
proteins, and proteins needed to carry out the response. A
liver cell, for example, is poised to respond appropriately
to epinephrine by having the proteins, as well as those
needed to manufacture glycogen.
Thus two cells that respond differently to the same signal
differ in one or more of the proteins that handle and
respond to the signal. Notice in FIGURE 9 that pathways
may some molecules in common. For example, cells A,
B, C all use the same receptor protein for the triangular
signal molecule; differences in other proteins account for
their differing responses.
Also note that in cell B, a pathway triggered by a single
kind of signal diverges to produce two responses, and
that in cell C, two pathways triggered by separate signals
converge to modulate a single response. Branching of
pathways and "cross-talk" between pathways are
important in regulating and coordinating a cell's
responses to incoming information. Moreover, the use of
some of the same proteins in more. than one pathway
allows the cell to recognize on number of different
protein it must make. The importance of relay proteins
that serve as points of branching or intersection in
signaling pathways is highlighted by the problems
arising when these proteins are defective or missing. For
instance, in an inherited disorder called Wiskott Aldrich
syndrome (WAS), the absence of a single relay protein
leads to such diverse effects as abnormal bleeding,
eczema, and a predisposition to infections and leukemia.
These symptoms are thought to arise primarily from the
absence of the protein in cells of the immune system. By
studying normal cells, scientists found that the WAS
protein is located just beneath the cell surface. The
To keep FIGURE 9 simple, we have not indicated the
inactivation mechanisms that are an essential aspect of
cell signaling. For a cell of a multicellular organism to
remain alert and capable of responding to incoming
signals, each molecular change in its signaling pathways
must last only a short time. As we saw in the cholera
example, if a signaling pathway components become
locked into one state, whether active or inactive, dire
consequences for the organism can result.
Thus a key to a cell’s continuing receptiveness to
regulation is the reversibility of the changes that signals
produce. The binding of signal molecules to receptors is
reversible, which means that the lower the concentration
of signal molecules the fewer will be bound at any given
moment. When they leave the receptor, the receptor
reverts to its inactive form Then, by a variety of means,
the relay molecules return to their inactive forms: The
GTPase activity intrinsic to a G protein hydrolyzes its
bound GTP; the enzyme phosphodiesterase converts
cAMP to AMP; protein phosphatases inactivates
phosphorylated kinases and other proteins; and so forth.
As a result, the cell is soon ready to respond to a fresh
signal.
 
 

INSTITUTE FOR ADVANCED STUDIES, JODHPUR  23
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
A3. SIGNAL TRANSDUCTION IN BACTERIA & BACTERIAL CHEMOTAXIS
Most prokaryotic signal-transduction systems and a few
eukaryotic pathways use phosphotransfer schemes
involving two conserved components, a histidine protein
kinase and a response regulator protein. The histidine
protein kinase, which is regulated by environmental
stimuli, autophosphorylates at a histidine residue,
creating a high-energy phosphoryl group that is
subsequently transferred to an aspartate residue in the
response regulator protein. Phosphorylation induces a
conformational change in the regulatory domain that
results in activation of an associated domain that effects
the response. The basic scheme is highly adaptable, and
numerous variations have provided optimization within
specific signaling systems. The domains of two-
component proteins are modular and can be integrated
into proteins and pathways in a variety of ways, but the
core structures and activities are maintained. Thus
detailed analyses of a relatively small number of
representative proteins provide a foundation for
understanding this large family of signaling proteins.
Chemotaxis
Chemoreceptor on bacteria detects attractant and
repellent and send signal to flagella. Some of these
molecules as aspartate, bind directly to chemoreceptors,
whereas others are carried by binding proteins.
The direction of rotation of flagella determines whether
bacteria swim smooth or tumble. Bacterium flagella is an
extra cellular appendage that is rotated by a motor
located at the junction of flagellum and cell envelope. It
is powered by the proton-motive force across the plasma
membrane. An intriguing feature of this motor is that it
can rotate clockwise or counterclockwise, and switch
between them almost instantaneously.
When the flagella rotate counterclockwise, the helical
filaments form a coherent bundle and cell swims
smoothly. When the flagella rotate clockwise, in contrast,
the bundle flies apart because the screw sense of the
helical flagella does not match the direction of rotation.
Each flagellum then pulls in a different direction, and so
the bacterium tumbles.
When a bacterium moves toward an increasing
concentration of an attractant, tumbling becomes less
frequent. In contrast, when it moves away from the
attractant, tumbling becomes more frequent. Repellent
have opposite effect on tumbling frequency. Tumbling
serves as reorient a misdirected bacterium. Bacteria
possesses a temporal sensing mechanism i.e a bacterium
detects a spatial gradient of attractant not by comparing
the concentration at its head and tail, but by traveling
through space and comparing its observations through
time.
Chemotaxis begins with the binding of molecules to four
kinds of receptors in the plasma membrane. In E.coli and
other gram negative bacteria, the PM is surrounded by an
outer membrane that is permeable to small molecules; a
periplasmic space separates the PM from the outer
membranes. The chemoreceptors are encoded by the tsr,
tar, trg and tap genes and were originally called Methyl
24 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
Accepting Chemotaxis Proteins (MCPs), because they
are reversibly methylated. Repellents and some
attractants bind to periplasmic proteins, which then
interact with the receptors. For eg. the attractant aspartate
binds directly to tar protein, which is also known as
aspartate receptor.
The four chemoreceptors have the same design. Each
~60 KD protein contains four regions:
1) A periplasmic domain that binds an attractant or a
repellent,
2) A transmembrane segment consisting of two α helices,
3) A cytosolic system and
4) A transmembrane region that can be reversibly
methylated at several sites.
Their cytosolic domains are highly conserved because
they interact with the same components of the processing
system. In fact, the cytosolic domains of tsr and tar
contain an identical 48 residue sequence. In contrast,
their periplasmic domains are distinctive because they
bind different attractants, repellents, and attractant-
protein complexes. A chimeric receptor formed from the
amino-terminal half of tar and the carboxy-terminal half
of tsr is triggered by aspartate and other attractant that
are normally sensed by tar. Thus, these chemoreceptors
transmit signals across the membrane in essentially same
way.
The three dimensional structure of periplasmic domain of
aspartate receptor dimerizes to form a highly elongated
structure. Each subunit is four helix bundle that is 20 Ao
in diameter and more than 70 Ao long. The binding sites
for aspartate are at the interface between subunits, more
than 60 Ao from the membrane surface.
A receptor triggered phosphorylation cascade controls
the direction of flagellar movement: Genetic analysis
revealed the existence of common processing genes and
provided valuable clues concerning its molecular
circuitry. Eight che loci have been identified: A, B, C, D,
W, R, Y and Z. mutation in these genes impair
chemotaxis to all attractant and repellent, in contrast with
mutation in a particular kind of receptor will block
responses to a subset of these agents. Bacteria devoid of
all cytosolic che proteins (known as gutted strains)
swims smoothly all the time; because their flagella rotate
counterclockwise(CCW) continuously. The addition of
cheY proteins to gutted stains, by controlling expression
of cheY gene, lead to increased clockwide(CW) rotation.
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
Furthermore, flagella rotates exclusively CW in mutant
that express abnormally high levels of cheY protein in
the presence of normal amounts of the other che proteins.
These finding indicates that the direction of flagellar
rotation is controlled by the che Y protein.
The essentiality of ATP for both the excitation and
adaptation phases of chemotaxis provides a tantalizing
hint as to how che Y might be controlled. A specific
aspatrate of che Y can become phosphorylated. Most
significant, phosphorylated cheY induces tumbling,
whereas dephosphorylated cheY has virtually no effect
on flagellar switch. Phosphorylated cheY is transient
species. These attached phosphoryl group is
simultaneously hydrolyzed in a reaction that is increased
100 fold by che Z.
How does cheY acquire phosphate group and how its
phosphorylation controlled by chemoreceptor? Melsimon
found that purified cheA protein undergoes a slow auto
phosphorylation. The site of phosphorylation is N-3 of
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  25
histidine residue. This auto phosphorylation is greatly
accelerated by the binding of cheA, together with che W,
to an unoccupied receptor or a repellent receptor
complex but not to an attractant receptor complex. The
phosphoryl group attached to cheA can then be
transferred to cheY. Hence, repellent increase the level
of phosphorylated cheA and, in turn, of phosphorylated
cheY. Attractant have the opposite effect. In this way,
repellent increase CW rotation and tumbling, whereas
attractants increase CCW rotation and smooth
swimming.
It is interesting to note that cheA belongs to a family of
regulatory protein that enables the bacteria to sense their
environment and internal metabolic state. For eg. NtrB,
which is a homologue of cheA, controls the expression
of genes that respond to nitrogen balance in cell. All
homologues of cheA are auto phosphorylating kinases
that regulate the activity of target proteins by
phosphorylating them. These sensor effector pairs are
called two component regulatory systems.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
Quorum sensing in Bacteria and other Organisms:
Quorum sensing is a type of decision-making process
used by decentralized groups to coordinate behavior.
Many species of bacteria use quorum sensing to
coordinate their gene expression according to the local
density of their population. Similarly, some social insects
use quorum sensing to make collective decisions about
where to nest. In addition to its function in biological
systems, quorum sensing has several useful applications
for computing and robotics.
Quorum sensing can function as a decision-making
process in any decentralized system, as long as individual
components have (a) a means of assessing the number of
other components they interact with and (b) a standard
response once a threshold number of components is
detected.
Bacteria
Some of the best-known examples of quorum sensing
come from studies of bacteria. Bacteria use quorum
sensing to coordinate certain behaviors based on the local
density of the bacterial population. Quorum sensing can
occur within a single bacterial species as well as between
diverse species, and can regulate a host of different
processes, essentially serving as a simple communication
network. A variety of different molecules can be used as
signals.
Mechanism
Bacteria that use quorum sensing constantly produce and
secrete certain signaling molecules (called autoinducers
or pheromones). These bacteria also have a receptor that
can specifically detect the signaling molecule (inducer).
When the inducer binds the receptor, it activates
transcription of certain genes, including those for inducer
synthesis. There is a low likelihood of a bacterium
detecting its own secreted Acetyl Homoserine Lactone
(AHL) . Thus, in order for gene transcription to be
activated, the cell must encounter signaling molecules
secreted by other cells in its environment. When only a
few other bacteria of the same kind are in the vicinity,
diffusion reduces the concentration of the inducer in the
surrounding medium to almost zero, so the bacteria
produce little inducer. However, as the population grows
the concentration of the inducer passes a threshold,
causing more inducer to be synthesized. This forms a
positive feedback loop, and the receptor becomes fully
activated. Activation of the receptor induces the up
regulation of other specific genes, causing all of the cells
to begin transcription at approximately the same time.
This coordinated behavior of bacterial cells can be useful
in a variety of situations. For instance, the
bioluminescent luciferase produced by V. fischeri would
not be visible if it were produced by a single cell. By
using quorum sensing to limit the production of
luciferase to situations when cell populations are large,
V. fischeri cells are able to avoid wasting energy on the
production of useless products.
Examples
Vibrio fischeri: Quorum sensing was first observed in
Vibrio fischeri, a bioluminiscent bacterium that lives as a
26 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
mutualistic symbiont in the photophore (or light-
producing organ) of the Hawaiian bobtail squid. When V.
fischeri cells are free-living (or planktonic), the
autoinducer is at low concentration and thus cells do not
luminesce. However, when they are highly concentrated
in the photophore (about 1011 cells/ml) transcription of
luciferase is induced, leading to bioluminescence.
Escherichia coli: In the Gram-negative bacteria
Escherichia coli, cell division may be partially regulated
by Autoinducer (AI-2)-mediated quorum sensing. This
species uses AI-2, which is produced and processed by
the lsr operon. Part of it encodes an ABC transporter
which imports AI-2 into the cells during the early
stationary (latent) phase of growth. AI-2 is then
phosphorylated by the LsrK kinase, and the newly
produced phospho-AI-2 can either be internalized or used
to suppress LsrR, a repressor of the lsr operon (thereby
activating the operon). Transcription of the lsr operon is
also thought to be inhibited by dihydroxyacetone
phosphate (DHAP) through its competitive binding to
LsrR. Glyceraldehyde 3-phosphate has also been shown
to inhibit the lsr operon through cAMP-CAPK-mediated
inhibition. This explains why when grown with glucose
E. coli will lose the ability to internalize AI-2 (because of
catabolite repression). When grown normally, AI-2
presence is transient.
Pseudomonas aeruginosa: The opportunistic bacteria
Pseudomonas aeruginosa uses quorum sensing to
coordinate the formation of biofilms, swarming motility,
exopolysaccharide production, and cell aggregation.
These bacteria can grow within a host without harming
it, until they reach a certain concentration. Then they
become aggressive, their numbers sufficient to overcome
the host's immune system and form a biofilm, leading to
disease. In this species, AI-2 was found to increase
expression of sdiA, a transcriptional regulator of
promoters which promote ftsQ, part of the ftsQAZ
operon essential for cell division. Another form of gene
regulation which allows the bacteria to rapidly adapt to
surrounding changes is through environmental signaling.
Recent studies have discovered that anaerobiosis can
significantly impact the major regulatory circuit of QS.
This important link between QS and anaerobiosis has a
significant impact on production of virulence factors of
this organism. It is hoped that the therapeutic enzymatic
degradation of the signaling molecules will prevent the
formation of such biofilms and possibly weaken
established biofilms. Disrupting the signalling process in
this way is called quorum quenching.
Social Insects
Social insect colonies are an excellent example of a
decentralized system, because no individual is in charge
of directing or making decisions for the colony. Several
groups of social insects have been shown to use quorum
sensing when making collective decisions.
Examples
Ants: Colonies of the ant Temnothorax albipennis nest in
small crevices between rocks. When the rocks shift and
the nest is broken open, these ants must quickly choose a
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
new nest to move into. During the first phase of the
decision-making process, a small portion of the workers
leave the destroyed nest and search for new crevices.
When one of these scout ants finds a potential nest, she
assesses the quality of the crevice based on a variety of
factors including the size of the interior, the number of
openings (based on light level), and the presence or
absence of dead ants. The worker then returns to the
destroyed nest, where she will wait for a short period
before recruiting other workers to follow her to the nest
she found using a process called tandem running. The
waiting period is inversely related to the quality of the
site- for instance a worker that has found a poor site will
wait longer than a worker that encountered a good site.
As the new recruits visit the potential nest site and make
their own assessment of its quality, the number of ants
visiting the crevice increases. During this stage ants may
be visiting many different potential nests. However,
because of the differences in the waiting period the
number of ants in the best nest will tend to increase at the
greatest rate. Eventually, the ants in this nest will sense
that the rate at which they encounter other ants has
exceeded a particular threshold, indicating that the
quorum number has been reached. Once the ants sense a
quorum, they return to the destroyed nest and begin
rapidly carrying the brood, queen, and fellow workers to
the new nest. Scouts that are still tandem-running to
other potential sites are also recruited to the new nest and
the entire colony moves.Thus although no single worker
may have visited and compared all of the available
Plant Two Component Signaling system
The "Prokaryotic" Lifestyle of Plants
At first glance, a 100-year-old tree and a bacterium do
not have much in common. However, both plants and
bacteria face a similar problem: They are confined to
their environment and have to endure and adapt to a
broad array of environmental conditions. To manage this
complex task, prokaryotic organisms rely on the two-
component signaling system, which is involved in such
diverse processes as chemotaxis, osmotic sensing, and
light perception (Stock et al., 2000). As shown in recent
years, all canonical elements of two-component signaling
systems are also found in plants (Schaller et al., 2001).
This Update focuses on recent advances in our
understanding of plant two-component systems, with
particular emphasis on the function of plant response
regulators.
Protein Elements and Biochemistry of Two-
Component Signaling Systems
In its simplest form, the two-component signaling system
is composed of two elements: a His kinase and a
response regulator (Fig. 1A). Although the majority of
prokaryotic two-component signaling systems are
composed of just these two proteins, there also exist
more complex signaling systems that include additional
elements, which are referred to as multistep, two-
component systems (Fig. 1B). The biochemistry of two-
component signal transduction is a phosphorelay: After a
signal-induced activation by the input-domain, the
transmitter of a His kinase autophosphorylates at a
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  27
options, quorum sensing enables the colony as a whole to
quickly make good decisions about where to move.
Honeybees: Honey bees (Apis mellifera) also use
quorum sensing to make decisions about new nest sites.
Large colonies reproduce through a process called
budding, in which the queen leaves the hive with a
portion of the workers to form a new nest elsewhere.
After leaving the nest, the workers form a swarm that
hangs from a branch or overhanging structure. This
swarm persists during the decision-making phase until a
new nest site is chosen.
The quorum sensing process in honey bees is similar to
the method used by Temnothorax ants in several ways. A
small portion of the workers leave the swarm to search
out new nest sites, and each worker assesses the quality
of the cavity she finds. The worker then returns to the
swarm and recruits other workers to her cavity using the
honey bee waggle dance. However, instead of using a
time delay, the number of dance repetitions the worker
performs is dependent on the quality of the site. Workers
that found poor nests stop dancing sooner, and can
therefore be recruited to the better sites. Once the visitors
to a new site sense that a quorum number (usually 10 to
20 bees) has been reached, they return to the swarm and
begin using a new recruitment method called piping. This
vibration signal causes the swarm to take off and fly to
the new nest location. In an experimental test this
decision-making process enabled honey bee swarms to
choose the best nest site in four out of five trials.
conserved His residue (Fig. 1A). The phophoryl group is
then transferred to an invariant Asp within a conserved
domain of the receiver (Fig. 1A). This His-to-Asp
phosphorelay controls the activity of the output domain
of the response regulator, which results, for example, in
interaction with other proteins or binding to DNA.
Figure 1. Schematic representation and basic features
of the simple (A) and the multistep (B) two-component
signaling systems. Signal perception by the input domain
of the His or hybrid kinase induces autophosphorylation
of the transmitter at a conserved His residue (H). The
phosphate (P) is relayed to a conserved Asp residue (D)
that is localized either in the receiver of the cognate
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
response regulator (A) or, in the case of hybrid kinases,
in an attached receiver domain (B). In the multistep, two-
component signaling system, His-containing
phosphotransfer (HPt) domain proteins serve as
phospho-His intermediates between the hybrid kinase
and the response regulator.
The outlined one-step, two-component signaling system
can be extended into a multistep system by including
additional protein modules containing phosphorylatable
Asp or His residues. The second Asp is again included in
a receiver module. In plants, the additional receiver
module is often carboxyterminally fused to the His
kinase, thereby creating a hybrid type of sensor kinase
(Fig. 1B). The second His is embedded in a domain
known as the HPt domain. The HPt domain enables the
transfer of a phosphoryl group between two receiver
modules. Many of the prokaryotic HPt domains are
physically linked to other two-component signaling
elements, but the Arabidopsis HPt domains are all
individual proteins.
Why are some two-component systems of the simple and
others of the complex type? The reason for extending the
complexity of two-component systems does not
apparently lie in signal amplification. Within a two-
component system, the signal is relayed as a phosphoryl
group in a strict one-to-one manner. Thus, the signaling
output of the simple and the multistep, two-component
system is the same. If it is not the gain in signal
amplification, what could be the reason behind the
apparent preference of multistep systems in plants? The
answer to this question will become clear in subsequent
sections.
Two-Component Signaling Elements of Arabidopsis
In the early 1990s, the cloning of three genes led to the
breakdown of a dogma. Until then, it was believed that
two-component signaling systems were restricted to
prokaryotes. However, with the realization that the
Arabidopsis ethylene receptor ETR1, the phytochrome
photoreceptors, and the yeast (Saccharomyces
cerevisiae) osmonsensor SLN1 were all related to His
kinases, it became clear that some genera of eukaryotes
depended on two-component systems as well. With
sequencing of the Arabidopsis genome now completed,
17 His and hybrid kinase-like proteins (AHK), five HPt
proteins (AHP), and 23 response regulators (ARR) have
been identified in Arabidopsis. Although analysis has
just begun, it is evident that, with the exception of
phytochromes, most AHKs, AHPs, and ARRs in
principle function as phosphotransfer proteins.
Ethylene Perception And Signaling
The ethylene perception and transduction system is one
of the best understood plant signaling pathways. The
initial identification of ETR1 (Fig. 2A) was followed by
the description of four additional ethylene receptors
either belonging to the hybrid kinase type (ETR2 and
EIN4) or the His kinase type (ERS1 and ERS2). By
generating ethylene-binding sites in a heterologous yeast
system and in genetic studies, it was elegantly shown that
ETR1 is a membrane-associated ethylene receptor (Fig.
2A). ETR1 exhibits His autophosphorylation activity in
vitro and interacts with several AHP proteins in the yeast
28 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
two-hybrid system. However, it has not yet been
demonstrated that ETR1 and the other members of the
ethylene receptor family act as ethylene-triggered hybrid
kinases in plants.
Figure 2. Structure of representative members of two-
component signaling elements from Arabidopsis. A,
Hybrid kinases: CRE1 and ETR1. The function of the
GAF-related domain of ETR1 is unknown. B, ARR:
ARR2, type-B response regulator; ARR4, type-A
response regulator. The GARP motif of type-B response
regulators is responsible for DNA binding and the
nuclear localization sequences (NLS) for nuclear
targeting. The Pro- and Gln-rich (P/Q-rich) domain
serves as transactivation domain, which may interact
with the basal transcription machinery. C, AHP: AHP1
and AHP2. The conserved amino acid residues (H, His;
D, Asp) of the different two-component elements are
indicated.
If ethylene is signaling through a two-component system,
one would expect to identify mutants affected in genes
coding for HPt domain proteins and response regulators.
Such mutants have not been reported, potentially because
of functional redundancy within these signaling
elements. The element shown to act downstream of the
ethylene receptors and found capable of physically
interacting with ETR1and ERS1 is CTR1 (constitutive
triple response 1), which is thought to function as a RAS-
like mitogen-activated protein kinase kinase kinase.
Thus, it appears that the hybrid kinase-like ethylene
receptors can function in a unique way linking the
perception of the hormone to the activity of a mitogen-
activated protein kinase cascade. However, although
CTR1 is a central component in ethylene signal
transduction, there are several indications for the
existence of additional signaling elements: (a) ctr1-null
mutants are still slightly responsive to ethylene, and (b)
the phenotype of quadruple etr1-etr2-ein4-ers2 receptor
mutants is more pronounced in comparison with that of
ctr1. With AHP and ARR knockout lines now in hand, it
should soon be possible to determine whether two-
component proteins represent these additional ethylene
signaling components.
The recent advances in our understanding of cytokinin
signaling offer important insights into plant two-
component systems. In 1996, a putative hybrid kinase
(CKI1) was described by Kakimoto, which, when
overexpressed, resulted in cytokinin-independent growth
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
of calli. However, most of the important advances came
just recently. By screening for the loss of cytokinin-
dependent greening and shoot formation of hypocotyl
explants from Arabidopsis, the cre1 (cytokinin response
1) mutant was identified. The CRE1 gene codes for a
hybrid kinase with an aminoterminal cytokinin-binding
domain flanked by two transmembrane stretches (Fig.
2A). In contrast to ETR1, CRE1 contains a second
receiver-like domain downstream of the transmitter (Fig.
2A). When expressed in yeast or fission yeast
(Schizosaccharomyces pombe) strains, in which the
endogenous hybrid kinases were knocked out, the CRE1
gene product rescued the altered growth phenotypes of
these mutants in the presence of cytokinin. These results
indicate that (a) CRE1 can bind cytokinin, (b) cytokinin
binding induces the His kinase activity of CRE 1, and (c)
CRE1 can transfer the phosphoryl group to the intrinsic
HPt domain proteins of yeast and fission yeast initiating
multistep, two-component signaling cascades. The
function of CRE1 as a sensor His kinase in planta is also
suggested by recent studies performed in Arabidopsis
protoplasts. However, although CRE1 is a cytokinin
receptor with His kinase activity, it is probably not
responsible for mediating all of the plant cytokinin
responses. Two additional CRE1-like His kinases in
Arabidopsis, AHK2 and AHK3, presumably also
function as cytokinin receptors.
Arabidopsis Response Regulators And Hpt Domain
Proteins
Although His kinase-like proteins were described in the
early 1990s, it required almost a decade for other two-
component signaling elements to be identified in higher
plants, namely response regulators and HPt domain
proteins. According to sequence homology, domain
composition, and transcriptional regulation, most
Arabidopsis ARRs can be described as either type A or
B. The type-A response regulators are relatively small
and contain a receiver domain along with short C-
terminal extensions (Fig. 2B). The mRNA of type-A
ARRs is rapidly accumulating to varying extent after
cytokinin treatment. The type-B ARRs have receiver
domains along with an extended C-terminal output
domain and are not induced by cytokinin (Fig. 2B).
Recent microarray studies, however, indicate that the
expression of the type-B response regulator ARR2 is
down-regulated by the phytochrome-A photoreceptor in
seedlings. We will now highlight recent insights into the
function of type-A and type-B ARRs.
Type-B ARRs: Response Regulators With
Transcription Factor Properties
The comparison of the amino acid sequences of the
output domains of type-B ARRs revealed a highly
conserved stretch of about 60 amino acids. This stretch
was given the name GARP motif according to the
founding members Golden2, ARR, and Psr1 (Fig. 2B).
The GARP motif is predicted to fold into an -helical
structure and is related to the DNA-binding motifs of the
basic helix-loop-helix and the MYB classes of
transcription factors. Thus, it was proposed that the
GARP motif represented a novel DNA-binding domain,
and that type-B ARRs might function as transcription
factors. By studying the subcellular distribution and
transactivation capability in vivo, it became evident that
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  29
type-B ARRs localized to the nucleus and were
transcriptionally active. The first hint pointing to
potential target promoters came with the histochemical
analysis of the expression pattern of an ARR2
promoter::uidA construct in transgenic Arabidopsis
plants. Although the ARR2 gene was expressed in almost
all Arabidopsis organs, particularly high -glucuronidase
activity was observed in pollen grains. A similar
expression pattern is described for the nuclear genes for
components of the mitochondrial respiratory complex I
(nCI genes). The pollen-specific activity of the nCI genes
is mediated by a conserved promoter element called the
pollen box. Lohrmann and colleagues (2001)
demonstrated sequence-specific binding of ARR2 to the
pollen box element and narrowed down the binding
sequence to a 5'-W/GAT/W-3' core, where W is either A
or T. This sequence matches the consensus binding site
identified independently by Sakai et al. (2000) using the
GARP domains of ARR1 and ARR2 in an oligo-
selection approach. With the identification of the nCI
genes as targets and with the demonstration of
transactivation capability, it is evident that ARR1,
ARR2, and presumably the other type-B ARRs function
as transcription factors inside the nucleus (Fig. 3).
Figure 3. Model of an Arabidopsis multistep
phosphorelay, demonstrating signal transduction from
the extranuclear membranes to the nucleus. Signal
perception by the input domain of a membrane-bound
sensor His kinase (e.g. CRE1) induces
autophosphorylation of the transmitter. The phosphate
(P) is relayed to the cognate AHP (e.g. AHP2) interacting
with the cytosolic receiver module of the kinase. The
phosphorylated AHP shuttles to the nucleus and transfers
the phosphate to the receiver domain of its cognate type-
B response regulator (e.g. ARR1 and ARR2). The
dephosphorylated AHP shuttles back to the cytosol
where it can be rephosphorylated. Phosphorylation of the
receiver domain of the response regulator results in a
conformational change in its output domain. This
conformational change may either induce binding of
type-B ARRs to their target promoter sequence (e.g. the
GAT box in the case of ARR1 and ARR2) and/or
activation of their transcriptional capacity. The promoter-
associated, active response regulators triggers the
transcription of its target genes (e.g. nCI genes, type-A
ARR genes). The conserved amino acid residues (H, His;
D, Asp) of the different two-component moduls are
indicated. POLII, RNA polymerase II-dependent basal
transcription machinery.
Recent evidence suggests that the type-B response
regulators ARR1 and ARR2 are involved in cytokinin
signaling. An arr1-null mutant was found to be partially
resistant to cytokinin in shoot regeneration and root
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
elongation, whereas ARR1-overexpressing lines
displayed the opposite, hypersensitive phenotype.
Similar alterations in sensitivity to cytokinin were
observed when the expression of several type-A ARR
genes including ARR4 were analyzed in the arr1 mutant
and ARR1-overexpressing lines. A comparable
cytokinin-inducible effect of ARR1 and ARR2 on the
expression of an ARR6 (type-A ARR)::reporter construct
was observed in Arabidopsis mesophyl protoplasts.
These results suggest that ARR1 and ARR2 mediate the
cytokinin-induced expression of type-A ARRs (Fig. 4). It
is of interest to note that ARR2 very strongly induced the
ARR6::reporter gene expression in Arabidopsis
protoplasts even in the absence of cytokinin. This
observation is indicative for the presence of a further
stimulus that enables the activation of ARR2 and, in turn,
ARR2-regulated target genes in protoplasts.
Figure 4. Model for the action of an ARR4-comprising
two-component signaling system on phytochrome B
signal transduction. Red light (R) induces the
photoconversion of the inactive Pr form of phytochrome
B (phyB) in the physiologically active Pfr form.
Phosphorylated ARR4 then stabilizes the active Pfr form
by inhibition of phyB dark reversion (dark rev.). Pfr
stabilization enhances the sensitivity of plants to red light
and induces more intense photomorphogenic responses
(phyB signaling). The phosphorylation state and the
activity of ARR4 is potentially regulated by a multistep
phosphorelay comprising of the HPt protein AHP1 and
presumably CRE1-like hybrid kinases (two-component
signaling). Therefore, ARR4 enables cross talk and thus
integration of signal information provided by hormone
(cytokinin) and the red-light signaling cascades (cross
talk). The expression of ARR4 and the protein
composition of the two-component system are regulated
by cytokinin (transcriptional regulation). The conserved
amino acid residues (H, His; D, Asp) of the different
two-component modules are indicated. FR, Far-red light;
P, phosphate residue.
From the study in Arabidopsis protoplasts, it is not clear
whether the activities of ARR1 and ARR2 are regulated
by Asp phosphorylation. This raises the question of
whether cytokinin signals through a canonical
phosphorelay to regulate the transcription factor
properties of type-B response regulators. However, the
conservation of the canonical phosphorylation sites in the
type-B ARRs (and also the type-A ARRs) are strongly
indicative of the functional relevance of these sites.
Moreover, Sakai and colleagues (2001) provided in
planta evidence that the phospho-accepting receiver
domain suppresses the function of ARR1 in the absence
of cytokinin and that the cytokinin signal releases this
30 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
suppression. This release could potentially be induced by
phosphorylation of the highly conserved Asp in the
receiver domain of ARR1.
The finding that type-B ARRs are localized to the
nucleus raises the question as to how the signal
information perceived by the extranuclear and
membrane-bound sensor His kinases is transduced to the
nuclear compartment. For instance, one of the cognate
sensor His kinases of ARR1 and ARR2 is very likely the
cytokinin receptor CRE1, which is predicted to be
present on an extranuclear membrane. Moreover,
protein-protein interaction studies revealed that the
sensor His kinases CKI1, CRE1, and ETR1 cannot
directly interact with ARR1 and ARR2. As outlined in
Figure 1B, the Arabidopsis HPt domain proteins could
perform the information transfer as part of a multistep,
two-component phosphorelay. His kinases (ETR1, CKI1,
and CRE1) as well as response regulators (ARR2) can
interact with AHPs, indicating that HPt domain proteins
may serve as molecular adaptors. AHPs are localized to
the cytosol as well as the nucleus of the plant cell, and
thus have the ability to perform functions in both
compartments. Although the AHP proteins have a size of
around 12 kD and may enter the nucleus by diffusion, at
least some AHPs transiently accumulate in the nucleus of
Arabidopsis protoplasts in response to cytokinin
application. These observations support the following
model of AHP action (Fig. 3). After activation by the
specific signal the sensor His-kinase autophosphorylates
and then transfers the phosphoryl group to the cognate
AHP. The AHP then shuttles into the nucleus and relays
the phosphate to nuclear type-B ARRs. Phosphorylation
of the receiver module triggers the transcription factor
properties of type-B ARRs, which results in an altered
activity of their target genes. The dephosphorylated AHP
then moves back to the cytoplasmic compartment to be
rephosphorylated. This model would indicate that higher
plants have adapted the ancestral two-component
systems to a novel type of eukaryotic intracellular
signaling mechanism. Because of the necessity for a
shuttling HPt domain protein, this adaptation could only
be achieved by use of multistep, two-component
systems.
TYPE-A ARRs: Response Regulators Modulating
Other Signal Transduction Pathways
As mentioned above, the transcription of type-A
response regulator genes like ARR4 is rapidly induced
by cytokinin. This and some additional criteria
established the hypothesis that type-A ARRs are primary
response genes of cytokinin. Recent studies suggest that
the cytokinin-regulated transcriptional control of type-A
ARR genes is mediated by type-B response regulators
Fig. 4). An effector protein recently was identified for a
type-A response regulator, thereby lending insight into
the signaling mechanisms that operate downstream of
these response regulators. Sweere and colleagues (2001)
demonstrated that ARR4 specifically interacts with the
plant photoreceptor phytochrome B. It is interesting that
ARR4 did not interact with the COOH terminus of
phytochrome B, which is related to transmitter domains
of His kinases, but with the extreme NH2-terminus.
Phytochrome B is a red/far-red reversible chromoprotein,
which regulates red light-specific photomorphogenetic
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
responses in plants. It is synthesized in the inactive Pr
form and photoconverted by red light into the
physiologically active Pfr form. Far-red light is able to
induce the photoconversion of the active Pfr form back to
the inactive Pr form. In addition, active Pfr converts back
to inactive Pr in a light-independent manner known as
dark reversion, which is thought to be an additional
switch-off mechanism for phytochromes. Independent
approaches in yeast and in Arabidopsis indicated that the
interaction of ARR4 with phytochrome B resulted in the
stabilization of the physiologically active Pfr form by
inhibition of Pfr-to-Pr dark reversion (Fig. 4).
The functional relevance of the phytochrome B:ARR4
interaction and Pfr stabilization was examined in
transgenic plants overexpressing ARR4. These plants
displayed a hypersensitivity to red light in respect to all
photomorphogenic responses tested (e.g. hypocotyl
elongation, root growth, and flowering time). Moreover,
transgenic Arabidopsis plants expressing a mutant form
of ARR4, which was not phosphorylated at the
conserved Asp residue in a plant cell-free phosphorelay
system, displayed hyposensitive phenotypes to red light
in hypocotyl elongation and flowering time. These data
suggests that the mutant protein exerts a dominant-
negative effect on the function of endogenous ARR4 and
that Asp phosphorylation of ARR4 plays an important
role in modulating phytochrome B activity. It is
interesting that the above-mentioned wild-type and
mutant ARR4-overexpressing lines also displayed weak
hyposensitive and hypersensitive, respectively,
phenotypes in response to cytokinin treatment. Recent
studies in protoplasts have shown that the overexpression
of type-A ARRs represses their own expression,
suggesting that type-A ARRs may also provide a
negative feedback regulation in cytokinin signaling.
Because no His kinase activity of phytochrome B has
been reported and because of the primary cytokinin
response properties of ARR4, the photoreceptor may be a
target of a cytokinin-modulated two-component signaling
system (Fig. 4). CRE1-like cytokinin receptors might
regulate the activity of ARR4 by a His-Asp phosphorelay
and thereby modulate phytochrome-B signaling. Such a
phosphorelay may involve the HPt domain protein
AHP1, which has the ability to function in a
phosphorelay with CRE1 and also acts upstream of
ARR4. AHP1 and ARR4 are found in both the cytosol
and nucleus. Thus, the putative AHK-AHP1-ARR4
phosphorelay, as well as the interaction of ARR4 with
phytochrome B, may occur in the cytosol to modulate
cytosolic, phytochrome B-dependent processes. On the
other hand, AHP1 may also be able to transfer the
phosphoryl group from the cytosol to the nucleus, where
ARR4 may then modulate nuclear, phytochrome B-
dependent responses. Regardless of which sensor His
kinase regulates ARR4 activity, the studies by Sweere
and colleagues suggest a novel type of cross talk between
an ancestral two-component system and the phytochrome
B-dependent light-signaling pathway (Fig. 4). This cross
talk may enable the integration of signal information
provided by hormone (cytokinin) and red light
(phytochrome B) signal transduction cascades. It is now
of major interest to identify the effector proteins of the
remaining type-A response regulators and to clarify the
underlying cross talk mechanisms.
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  31
A Complex Plant Two-Component Signaling Network
Emerges: Conclusions And Perspectives
As described above, there is now the first experimental
evidence that hybrid kinases like CRE1, AHK2, and
AHK3 signal through multistep phosphorelays
containing AHPs and ARRs. In addition, a wealth of
yeast two-hybrid and in vitro protein-protein interaction
studies indicate the possibility of a complex two-
component signaling network in cells of higher plants. A
detail of such a potential network is shown in Figure 5.
Specificity to some of these interactions can be
recognized. For example, ETR1 and CRE1 interact with
both AHP1 and AHP2, whereas CKI1 interacts with only
AHP2 (Fig. 5). Specificity is also apparent for the
interaction of ARRs with AHPs. ARR2 interacts with
AHP1 and AHP2, whereas the interaction of ARR4 is
restricted to AHP1 (Fig. 5). For instance, as indicated in
previous sections, two-component signaling elements
from Arabidopsis can participate in phosphorelays from
biologically irrelevant fungal systems. However, even
given the preliminary nature of this emerging picture,
cross talk between different two-component signaling
pathways would have major functional implications.
Two-component systems may form a complex signaling
network capable of sensing, computing, and integrating a
broad range of signals. The sessile lifestyle of plants
makes it necessary that every cell monitors and integrates
a multitude of inputs to reach a finely balanced decision
on how to react at the cellular and the whole-plant level.
The increased potential for cross talk potentially
represents an additional reason as to why multistep two-
component systems rather than simple two-component
systems were utilized in plants. We have already
received our first glance at this complexity from studies
revealing cross talk between the ARR4-comprising two-
component system with phytochrome B-dependent signal
transduction. Overlapping sets of synergistic and
antagonistic physiological responses are also described
for many other stimuli such as ethylene and cytokinin.
Complexly interconnected two-component systems could
participate in the generation of these overlapping
physiological responses.
Figure 5. Detail from the emerging network of two-
component signaling systems in Arabidopsis. Lines
indicate direct protein-protein interactions detected in the
yeast two-hybrid system and in vitro. Proteins that are
not connected do not display physical interaction. ETR1,
CRE1, and CKI hybrid kinase suggested to be involved
cytokinin signaling. AHP1 and AHP2, HPt domain
proteins. ARR2 and ARR4, Response regulators. The
conserved amino acid residues (H, His; D, Asp) of the
different two-component moduls are indicated.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
C. Cell-Cell Interactions
The Extracellular Matrix
Although animal cells are not surrounded by cell walls,
many of the cells in tissues of multicellular organisms are
embedded in an extracellular matrix consisting of
secreted proteins and polysaccharides. The extracellular
matrix fills the spaces between cells and binds cells and
tissues together. One type of extracellular matrix is
exemplified by the thin, sheetlike basal laminae, or
basement membranes, upon which layers of epithelial
cells rest (Figure.). In addition to supporting sheets of
epithelial cells, basal laminae surround muscle cells,
adipose cells, and peripheral nerves. Extracellular matrix,
however, is most abundant in connective tissues. For
example, the loose connective tissue beneath epithelial
cell layers consists predominantly of an extracellular
matrix in which fibroblasts are distributed. Other types of
connective tissue, such as bone, tendon, and cartilage,
similarly consist largely of extracellular matrix, which is
principally responsible for their structure and function.
32 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
Extracellular matrices are composed of tough fibrous
proteins embedded in a gel-like polysaccharide ground
substance. In addition to fibrous structural proteins and
polysaccharides, the extracellular matrix contains
adhesion proteins that link components of the matrix
both to one another and to attached cells
The major structural protein of the extracellular matrix is
collagen, which is the single most abundant protein in
animal tissues. The collagens are a large family of
proteins, containing at least 19 different members. They
are characterized by the formation of triplet alpha helices
in which three polypeptide chains are wound tightly
around one another in a ropelike structure. Collagen
molecules assemble in a regular staggered array to form
fibrils. The molecules overlap by one-fourth of their
length, and there is a short gap between the N terminus
of one molecule and the C terminus of the next. The
assembly is strengthened by covalent cross-links between
side chains of lysine or hydroxylysine residues, primarily
at the ends of the molecules
Connective tissues also contain elastic fibers, which are
particularly abundant in organs that regularly stretch and
then return to their original shape eg. Lungs. Elastic
fibers are composed principally of a protein called
elastin, which is crosslinked into a network by covalent
bonds formed between the side chains of lysine residues
(similar to those found in collagen).
The fibrous structural proteins of the extracellular matrix
are embedded in gels formed from polysaccharides called
glycosaminoglycans, or GAGs, which consist of
repeating units of disaccharides. One sugar of the
disaccharide is either N-acetylglucosamine or N-
acetylgalactosamine and the second is usually acidic
(either glucuronic acid or iduronic acid). Hyaluronan is
the only GAG that occurs as a single long polysaccharide
chain. All of the other GAGs are linked to proteins to
form proteoglycans, which can consist of up to 95%
polysaccharide by weight. In addition to being
components of the extracellular matrix, some
proteoglycans are cell surface proteins that function in
cell adhesion.
Adhesion proteins, the third class of extracellular matrix
constituents, are responsible for linking the components
of the matrix both to one another and to the surfaces of
cells. The prototype of these molecules is fibronectin,
which is the principal adhesion protein of connective
tissues. Fibronectin is a dimeric glycoprotein consisting
of two polypeptide chains, each containing nearly 2500
amino acids. In the extracellular matrix, fibronectin is
further crosslinked into fibrils by disulfide bonds.
Fibronectin has binding sites for both collagen and
GAGs, so it crosslinks these matrix components. A
distinct site on the fibronectin molecule is recognized by
cell surface receptors and is thus responsible for the
attachment of cells to the extracellular matrix.
Basal laminae contain a distinct adhesion protein called
laminin. Laminins can self-assemble into meshlike
polymers. Such laminin networks are the major structural
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

components of the basal laminae synthesized in very

early embryos, which do not contain collagen.

The major cell surface receptors responsible for the

attachment of cells to the extracellular matrix are the
integrins. The integrins bind to short amino acid
sequences present in multiple components of the
extracellular matrix, including collagen, fibronectin, and
laminin.
Figure: Stable cell-cell junctions mediated by the
Direct Cell-Cell Interactions cadherins Homophilic interactions between
cadherins mediate two types of stable cell-cell
Direct interactions between cells, as well as between adhesions. In adherens junctions, the cadherins are
cells and the extracellular matrix, are critical to the linked to bundles of actin filaments via the catenins.
development and function of multicellular organisms. In desmosomes, desmoplakin links members of the
Some cell-cell interactions are transient, such as the cadherin superfamily (desmogleins and
interactions between cells of the immune system and the desmocollins) to intermediate filaments.
interactions that direct white blood cells to sites of tissue
inflammation. In other cases, stable cell-cell junctions
play a key role in the organization of cells in tissues. For
example, several different types of stable cell-cell
junctions are critical to the maintenance and function of
epithelial cell sheets. Plant cells also associate with their
neighbors not only by interactions between their cell
walls, but also by specialized junctions between their
plasma membranes.

Cell Adhesion Proteins: Cell-cell adhesion is a selective

process, such that cells adhere only to other cells of
specific types. Such selective cell-cell adhesion is
mediated by transmembrane proteins called cell adhesion
molecules, which can be divided into four major groups:
the selectins, the integrins, the immunoglobulin (Ig)
superfamily (so named because they contain structural
domains similar to immunoglobulins), and the cadherins.
The selectins mediate transient interactions between
leukocytes and endothelial cells or blood platelets. The
selectins recognize cell surface carbohydrates. One of
their critical roles is to initiate the interactions between
leukocytes and endothelial cells during the migration of
leukocytes from the circulation to sites of tissue
inflammation. The selectins mediate the initial adhesion
of leukocytes to endothelial cells. This is followed by the
formation of more stable adhesions, in which integrins
on the surface of leukocytes bind to intercellular
adhesion molecules (ICAMs), which are members of the
Ig superfamily expressed on the surface of endothelial
cells. The firmly attached leukocytes are then able to
penetrate the walls of capillaries and enter the underlying
tissue by migrating between endothelial cells.
The fourth group of cell adhesion molecules, the
cadherins, also display homophilic binding specificities.
They are not only involved in selective adhesion between
embryonic cells but are also primarily responsible for the
formation of stable junctions between cells in tissues. For
example, E-cadherin is expressed on epithelial cells, so
homophilic interactions between E-cadherins lead to the
selective adhesion of epithelial cells to one another. It is
noteworthy that loss of E-cadherin can lead to the
development of cancers arising from epithelial cells,
illustrating the importance of cell-cell interactions in
controlling cell behavior. Different members of the
cadherin family, such as N-cadherin (neural cadherin)
and P-cadherin (placental cadherin), mediate selective
adhesion of other cell types.
Tight Junctions: Tight junctions, which are usually
associated with adherens junctions and desmosomes
in a junctional complex, are critically important to
the function of epithelial cell sheets as barriers
between fluid compartments. Tight junctions play
two roles-- First, tight junctions form seals that
prevent the free passage of molecules (including
ions) between the cells of epithelial sheets. Second,
tight junctions separate the apical and basolateral
domains of the plasma membrane by preventing the
free diffusion of lipids and membrane proteins
between them. Consequently, specialized transport
systems in the apical and basolateral domains are
able to control the traffic of molecules between
distinct extracellular compartments. Tight junctions
are the closest known contacts between adjacent
cells. They were originally described as sites of
apparent fusion between the outer leaflets of the
plasma membranes, although it is now clear that the
membranes do not fuse. Instead, tight junctions
appear to be formed by a network of protein strands
that continues around the entire circumference of
the cell. Each strand in these networks is thought to
be composed of transmembrane proteins (claudins
and occludin) that bind to similar proteins on
adjacent cells, thereby sealing the space between
their plasma membranes.

INSTITUTE FOR ADVANCED STUDIES, JODHPUR  33
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

Gap Junctions: Gap junctions, which are found in connexin extracellular domains hence the term
most animal tissues, serve as direct connections "gap junction," which was coined by electron
between the cytoplasms of adjacent cells. They microscopists.
provide open channels through the plasma  
membrane, allowing ions and small molecules (less
than approximately a thousand daltons) to diffuse
freely between neighboring cells, but preventing the
passage of proteins and nucleic acids. Consequently,
gap junctions couple both the metabolic activities
and the electric responses of the cells they connect.
Most cells in animal tissues including epithelial
cells, endothelial cells, and the cells of cardiac and
smooth muscle communicate by gap junctions. In
electrically excitable cells, such as heart muscle
cells, the direct passage of ions through gap
junctions couples and synchronizes the contractions
of neighboring cells. Gap junctions also allow the
passage of some intracellular signaling molecules,
such as cAMP and Ca2+, between adjacent cells,
potentially coordinating the responses of cells in
tissues.

Gap junctions are constructed of transmembrane

proteins called connexins. Six connexins assemble
to form a cylinder with an open aqueous pore in its
center. Such an assembly of connexins in the plasma
membrane of one cell then aligns with the
connexins of an adjacent cell, forming an open
channel between the two cytoplasms. The plasma
membranes of the two cells are separated by a gap
corresponding to the space occupied by the
 
 
D1. Uncontrolled Cell division leads to Cancer:
 

In the normal circumstances, the proliferation of body
cells is under strict control. The cells differentiate, divide
and die in a sequential manner in a healthy organism.
Cancer is characterized by loss of control of cellular
growth and development leading to excessive
proliferation and spread of cells.
Cancer is derived from a Latin word meaning crab. It is
presumed that the word cancer originated from the
character of cancerous cells which can migrate and
adhere and cause pain (like a crab) to any part of the
body.
Neoplasia literally means new growth. Uncontrolled
growth of cells results in tumors (a word originally used
to represent swelling). Oncology (Greek: oncos-tumor)
deals with the study of tumors.
The tumors are of two types.
a. Benign tumors: They usually grow by expansion and
remain encapsulated in a layer of connective tissue.
Normally benign tumors are not Life threatening e.g.
moles, warts. These types of benign tumors are not
considered as cancers.
b. Malignant tumors or cancers: They are
characterized by uncontrolled proliferation and spread of
cells to various parts of the body, a process referred to as
metastasis. Malignant tumors are invariably life
threatening e.g. lung cancer, leukemia.
About 100 different types of human cancers have been
recognized. Cancers arising from epithelial cells are
referred to as carcinomas while that from connective
tissues are known as sarcomas. Methods for the early
detection and treatment of cancers have been developed.
However, little is known about the biochemical basis of
cancer.
Metastasis: Metastasis refers to the spread of cancer
cells from the primary site of origin to other tissues of
the body where they get deposited and grow as secondary
tumors. Metastasis is the major cause of cancer related
morbidity and mortality. The biochemical basis of
metastasis is not clearly known. It is believed that the
morphological changes in tumor cells, loss of contact
inhibition, loss of anchorage dependence and alterations
in the structure of certain macromolecules are among the
important factors responsible for metastasis.
Metastasis is Genetically Controlled: Cancer cells
cannot regulate their growth and division. As a result,
they develop into malignant tumors that may potentially
invade neighboring tissue. Sometimes, cancer cells de-
tach from the primary tumor and settle elsewhere in the
body, where they grow and divide, producing secondary
tumors. This process, called metastasis, is often the cause
of death in cancer patients.
A metastatic cancer cell spreads from the primary tumor
by entering the blood or lymphatic circulatory system.

34 INSTITUTE FOR ADVANCED STUDIES, JODHPUR 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
These cells are carried in the circulation until they lodge
in a capillary bed. Normally, more than 99 percent of the
cells die. The surviving cells invade tissue adjacent to the
capillary bed and begin dividing to fom1 a secondary
tumor. To reach a new site, the tumor cells pass through
the layer of epithelial cells lining the interior wall of the
capillary (or lymph vessel) and penetrate the surrounding
extracellular matrices. The extracellular matrix is a
meshwork of proteins and carbohydrate molecules
separating tissues; it is a scaffold for tissue growth and
inhibits the migration of cells. To establish a secondary
tumor, metastatic cells secrete enzymes that digest this
meshwork, creating holes through which they can
migrate.
The ability to invade extracellular matrix is also a proper-
ty of some normal cell types. Implantation of the embryo
in the uterine wall during pregnancy, for example,
requires cell migration across the extracellular matrix.
White blood cells reach the site of infection by
penetrating capillary walls. The mechanism of invasion
is probably the same in these cells as in cancer cells. The
difference is that in normal cells, the invasive ability is
controlled and tightly regulated, while in tumor cells,
regulation has been lost.
Metastasis is regulated by genes that encode or regulate
matrix-cutting enzymes. Protein-cutting enzymes called
metallo-proteinases are necessary for cell invasion.
Tumor cell lines with high metastatic capacity produce
more metallo-proteinases than do tumor cell lines with
low invasive properties. Activated metallo-proteinases
are inhibited by the presence of a tissue inhibitor of
metalloproteinase (TIMP). Normal cells produce TIMP
and thereby suppress inappropriate activity of
metalloproteinase. In tumor cells, TIMP inhibits
metalloproteinase only when the number of TIMP
molecules is greater than the number of
metalloproteinase molecules. On the basis of its action
and regulation, TIMP can be considered as a protein that
suppresses metastasis.
Incidence: Cancer is the second largest killer disease
(the first being coronary heart disease) in the developed
countries. It is estimated that cancer accounts for more
than 20% of the deaths in United States. Based on the
current rate of incidence, it is believed that one in every 3
persons will develop cancer at sometime during his life.
Although humans of all age develop cancer, the
incidence increases with advancement of age. More than
70% of the new cancer cases occur in persons over 60
years. Surprisingly; cancer is a leading cause of death in
children in the age group 3-13 years, half of them die due
to leukemia.
Etiology: In general, cancers are multifactorial in origin.
The causative agents include physical, chemical, genetic
and environmental factors. A survey in USA has shown
that about 90% of all cancer deaths are due to avoidable
factors such as tobacco, pollution, occupation, alcohol
and diet.
Most of the cancers are caused by chemical carcinogens,
radiation energy and viruses. These agents may damage
DNA or interfere with its replication or repair.
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  35
1. Chemical Carcinogens: It is estimated that almost
80% of the human cancers are caused by chemical
carcinogens in nature. The chemicals may be organic
(e.g. dimethyl benzanthracene, benzo (a) pyrene,
dimethyl nitrosamine) or inorganic (arsenic, cadmium) in
nature. Entry of the chemicals into the body may occur
by one of the following mechanisms.
1. Occupation e.g. asbestos, benzene.
2. Diet e.g. Aflatoxin B produced by fungus
(Aspergillus flavus) contamination of
foodstuffs, particularly peanuts.
3. Drugs-certain therapeutic drugs can be
carcinogenic e.g. diethylstibesterol.
4. Life style e.g. cigarette smoking (includes
benzo(a)pyrene and polonium).
a. Mechanism of action: Although a few of the
chemicals are directly carcinogenic, majority of them
require prior metabolism to become carcinogenic. The
enzymes such as cytochrome P450 responsible for the
metabolism of xenobiotics are involved in dealing with
the chemical carcinogens. In general, a chemically non-
reactive procarcinogen is converted to an ultimate
carcinogen by a series of reactions.
The carcinogens can covalently bind to purines,
pyrimidines and phosphodiester bonds of DNA, often
causing unrepairable damage. The chemical carcinogens
frequently cause mutations (a change in the nucleotide
sequence of DNA), which may finally lead to the
development of cancer; hence they are regarded as
mutagens.
b. Ames assay: This is a laboratory test to check the
carcinogenecity of chemicals. Ames assay employs the
use of a special mutant strain of bacterium, namely
Salmonella typhimurium (His-). This organism cannot
synthesize histidine; hence the same should be supplied
in the medium for its growth. Addition of chemical
carcinogens causes mutations (reverse mutation)
restoring the ability of the bacteria to synthesize histidine
(His+). By detecting the strain of Salmonella (His+) in
the colonies of agar plates, the chemical mutagens can be
identified. The Ames assay can detect about 90% of the
chemical carcinogens. This test is regarded as a
preliminary screening procedure. Animal experiments
are conducted for the final assessment of
carcinogenecity.
c. Promoters of carcinogenesis: Some of the chemicals
on their own are not carcinogenic. Certain substances
known as promoting agents make them carcinogenic.
The application of benzo (a) pyrene to the skin, as such,
does not cause tumor development. However, if this is
followed by the application of croton oil, tumors will
develop. In this case, benzo (a) pyrene is the initiating
agent while croton oil acts as a promoting agent or
promoter. Several compounds that act as promoting
agents in various organs of the body have been
identified. These include saccharin and phenobarbital.
2. Radiation energy: Ultraviolet rays, X-rays and y-rays
have been proved to be mutagenic in nature causing
cancers. These rays damage DNA, which is the basic
mechanism to explain the carcinogenecity of radiation
energy. For instance, exposure to UV rays results in the
formation of pyrimidine dimers in DNA while X-rays
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

cause the production of free radicals. These types of
molecular damages are responsible for the carcinogenic
effects of radiations.
3. Carcinogenic Viruses: The involvement of viruses in
the etiology of cancer was first reported by Rouse in
1911. He demonstrated that the cell-free filtrates from
certain chicken sarcomas (tumors of connective tissues)
promote new sarcomas in chickens. Unfortunately, this
epoch-making discovery of Rouse was ignored for
several years. This is evident from the fact that Rous was
awarded the Nobel Prize in 1966 at the age of 85 for his
discovery in 1911.
Table 1: Selected tumor viruses
Class Members
DNA Viruses
Adenovirus Adenovirus 12 and 18
Herpesvirus Epstein-Barr virus, Herpes Simplex virus
Papovirus Papilloma virus, Polyoma Virus
RNA Viruses
Reterovirus type B Mammary tumor virus of mouse
Reterovirus type C Leukemia, Sarcoma
The presence of viral particles and the enzyme reverse
transcriptase, besides the occurrence of base sequence
in the DNA of malignant cells, complementary to tumor
viruses indicate the involvement of viruses in cancer. The
viruses involved in the development of cancer,
commonly known as oncogenic viruses, may contain
either DNA or RNA. A selected list of tumor viruses is
as follows:
DNA-the ultimate in carcinogenesis: DNA is the
ultimate critical macromolecule in carcinogenesis.
Several evidences support this fact.
1. Cancers are transmitted from mother to
daughter cells. In other words, cancer cells
beget cancer cells.
2. Chromosomal abnormalities are observed in
many tumor cells.
3. Damage to DNA caused by mutations often
results in carcinogenesis.
4. Laboratory experiments have proved that
purified oncogenes can transform normal cells
into cancer cells.

D3. Molecular Basis of Cancer

It is clear that the two main properties of cancer,

uncontrolled cell division and metastasis, result from
mutations. As mentioned earlier, these mutations can
involve large-scale genomic instability, chromosome
loss, chromosome rearrangement, or the insertion of
foreign (often viral) DNA sequences into loci. Smaller
scale mutations, including nucleotide substitutions or
deletions, or more subtle modifications that alter only the
amount of a gene product or the time over which it is
active may also be involved.
In general, control of cell division is regulated in two
ways: (l) by genes that normally function to suppress cell
division and (2) by genes that normally function to
promote cell division. The first group of regulatory genes
is called tumor suppressor genes. When expressed, these
genes halt passage through the cell cycle and prevent
mitosis. For cell division to occur, these genes or their
gene products (or both) must be inactive or absent. If
tumor suppressor genes become permanently inactivated
or deleted through mutation, control over cell division is
lost, and the mutant cells begin to proliferate in an
uncontrolled fashion.
31.3.1. Tumor-Suppressor Genes Normally Suppress
Cell Division
Studies have identified a number of genes that when mu-
tated, confer a predisposition to specific cancers (Table
2). One example of an inherited predisposition to cancer
is retinoblastoma, an inherited cancer of the retinal cells
of the eye.
Retinoblastoma: Retinoblastoma (RB) occurs with a
frequency that ranges from 1 in 14,000 to 1 in 20,000,
and most often appears between the ages of 1 and 3
years. Two forms of retinoblastoma are known. In the
familial form (about 40 percent of all cases), individuals
who carry one mutant RB allele are far more susceptible
to developing retinoblastoma than those with two normal
wild-type RB alleles.

Genes that normally promote cell division are called

protooncogenes. These genes can be "on" or "off," and
when they are "on," they promote cell division. To stop
mitosis, these genes or their gene products (or both) must
be inactivated. If these genes become permanently
switched on, then uncontrolled cell division occurs,
leading to tumor formation. Mutant forms of
protooncogenes are called oncogenes. In the next
sections, we examine how mutations in tumor suppressor
genes can lead to a loss of cell cycle control and lead to
cancer formation. Then, we consider the role of
Predisposition to this form of cancer is inherited as an
protooncogenes and oncogenes. In recent years, third
autosomal dominant trait although the mutation itself is
categories of genes that control the cell death or
actually recessive, as we will explain later. About 90
apoptosis are also believed to be involved in
percent of those who inherit the mutant RB allele will
carcinogenesis.

36 INSTITUTE FOR ADVANCED STUDIES, JODHPUR 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
develop retinal tumors, usually in both eyes. In addition,
these individuals are predisposed to other forms of
cancer, including osteosarcoma, a bone cancer, even if
they do not develop retinoblastoma.
The second form of retinoblastoma (the remaining 60
percent) is not familial, and tumors develop
spontaneously. This sporadic form is characterized by the
appearance of tumors only in one eye, and onset occurs
at a much later age than in the familial form.
By studying both types of retinoblastoma, Alfred
Knudson and his colleagues developed a model that
requires the presence of two mutated copies of the RB
gene in the same retinal cell for tumor formation; in other
words, tumor development is a recessive trait. In the
familial form, one mutant RB allele is inherited and is
carried by all cells of the body, including cells of the
The Retinoblastoma (RB) Gene: The retinoblastoma
(RB) gene, located on chromosome 13, encodes a protein
designated as pRB. This protein is found in the nuclei of
all cell and tissue types examined so far and also is found
in both resting (GO) cells and actively dividing cells.
Moreover, pRB is present at all stages of the cell cycle
and is part of a regulatory pathway that is important in
cell cycle progression. It acts as a molecular switch that
controls the passage of cells from G1 into S phase.
Because the normal gene is expressed ubiquitously in the
body, cells can progress through the G/S transition only
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  37
retina [Fig 4 (a)]. If the second RB allele becomes
mutated in a retinal cell, retinoblastoma will develop.
Therefore, individuals carrying an inherited mutation of
the RB gene are predisposed to develop retinoblastoma,
as only one additional mutational event is required to
cause tumor formation. This does not happen in all cases.
About 10 percent of those inheriting a mutant RB allele
do not develop cancer. Presumably, the wild-type allele
of the RB gene does not mutate in any retinal cells.
In the nonfamilial sporadic cases [Fig 4 (b)], inde-
pendent mutations in both normal RB alleles must occur
in the same retinal cell for a tumor to develop. As might
be expected, these events are far less frequent and occur
at a much later age. As predicted by Knudson's model,
such sporadic forms of retinoblastoma are more likely to
occur in a single eye.
when pRB is inactivated by phosphorylation. pRB is
regulated by the action of CDKs. When CDK4 binds to
cyclin D, the kinase phosphorylates pRB. pRB is
phosphorylated in the S and the G2/M stages of the cell
cycle, but is not phosphorylated in GO and G1. When
pRB is unphosphorylated (its active state), it binds to
members of the E2F family of transcription factors.
Transcription factors are proteins that bind to the
promoter region of genes and regulate transcription. E2F
transcription factors control the expression of some 20-
30 genes required to move the cell from G1 to S. In its
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

unphosphorylated form, pRB binds to E2F, blocking
transcription, and keeping the cell in G1. (Fig 5) The
phosphorylation of pRB by CDK4 takes place in late G1.
When phosphate groups are added, pRB releases E2F,
allowing transcription of genes that move the cell from G
1 into S.
Knudson's model, familial cases inherit one mutant allele
through the germ line and develop a mutation in the
remaining normal allele in a somatic cell. Sporadic cases,
on the other hand, require two independent mutations of
the WT gene within the same cell, and tumors more often
involve only one kidney.

The WT gene is on the short arm of chromosome 11 and

encodes a transcription factor. This protein has four
contiguous zinc finger domains, motifs characteristic of
DNA-binding proteins that regulate transcription. WT has
a restricted pattern of expression; it is normally active
only in cells of the fetal kidney and only during the brief
time when the nephron (the basic filtration unit of the
kidney) is being formed. The structure of the WT protein,
its pattern and time of expression, and the resultant
mutant phenotype have led to the hypothesis that the WT
Direct evidence for the role of pRB in regulating the cell
gene encodes a nuclear protein which turns off genes that
cycle comes from several lines of experimental work.
maintain cell division. Alternatively, the gene product
Osteosarcoma (bone cancer) cells are homozygous for
may switch on genes that begin differentiation of the
mutant RB alleles and do not produce pRB. When
fetal cells into components of the kidney. In tumor cells,
osteosarcoma cells are injected into a cancer-prone strain
the mutant gene is switched on at the appropriate time in
of mice, tumors are formed. If a normal RB gene is
the appropriate cells, but the altered gene product is
transferred to the cancer cells, pRE is produced and no
unable to regulate its target genes, resulting in continued
tumors are formed when these genetically modified cells
cell division, aberrant differentiation, and tumor
are injected into mice.
formation.
In a separate two-step experiment, a normal RB gene was
Although both RB and WT genes encode nuclear proteins
transferred into osteosarcoma cells. These cells produced
that suppress tumor formation, there are some differences
pRB and stopped cell division. When D or E cyclins
in their properties and modes of action. pRb does not
were added to these pRB-blocked cells, cell division
bind to DNA; instead, it interacts with E2F, as described
resumed. The addition of cyclins resulted in
in the previous section. WT has all the characteristics of
phosphorylation of pRb, presumably by activating CDK4
a DNA-binding transcription factor. pRb is expressed in
or another kinase. The results demonstrate that active
all dividing cells, whereas the WT protein is found only
pRb stops cell division, that pRB is a target of a G1
in a restricted period of kidney development. pRb is a
CDK/cyclin complex, and that inactivating pRB allows
general regulator of cell division; the WT protein is a
passage through the cell cycle and subsequent division,
cell- or tissue-specific regulator of gene activity during
confirming the role of pRB in G1 control.
fetal or neonatal development.
In normal retinal cells, pRB is active and prevents
p53: Guardian of the Genome
passage into S by interacting with the transcription factor
E2F. In retinoblastoma cells, both copies of the RB gene
Another tumor suppressor gene p53, has been implicated
are mutated, and no functional pRB is present. As a
in human cancer. p53 encodes a nuclear transcription
result, E2F activates the genes required for passage
factor. p53 mutations are found in a wide range of
through the G/S checkpoint into the S phase. This
cancers, including breast, lung, bladder, and colon
important checkpoint is continually overridden in these
cancers. It is estimated that 50-60 percent of all cancers
cells, resulting in uncontrolled growth and tumor
are associated with mutations in the p53 gene, suggesting
formation.
that it controls one or more key events in the proliferation
of all cells and is not involved in a cell- or tissue-specific
The Wilms Tumor Gene: Wilms tumor (WT) is a
form of regulation. Inherited mutations of p53 are
kidney cancer found primarily in children. It has a
associated with the Li-Fraumeni syndrome, an autosomal
frequency of about 1 in 10,000 births, and, like
dominant condition with a predisposition to develop
retinoblastoma, it is found in two forms, a noninherited
cancers in several tissues at a high frequency.
sporadic form and a familial form conferring a
predisposition to WT. The familial predisposition is
Normally the p53 protein is present in cells at a low
inherited as an autosomal dominant trait. According to
level, in a rapidly degraded, inactive form. Several types

38 INSTITUTE FOR ADVANCED STUDIES, JODHPUR 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
of signals can shut down p53 degradation, causing a
rapid increase in p53 protein levels. Chemical damage to
DNA, double-stranded breaks in DNA induced by
ionizing radiation, or the presence of DNA repair
intermediates generated by exposure to ultraviolet light
all cause a shutdown ofp53 turnover. Activation of p53
protein can lead to several responses, including (1) DNA
repair; (2) cell cycle arrest; and (3) apoptosis, a
genetically programmed pathway of cell death. These
tasks are accomplished by target genes activated by p53
in its role as a transcription factor. The expression of
more than 20 target genes are controlled by the action of
p53.
Cell cycle arrest by p53 can occur at several points in the
cycle, including G1. To arrest the cycle at this phase, p53
activates transcription of a CDK inhibitor called p21. The
p21 protein targets a number of CDK/cyclin complexes,
including the CDK4/cyclin D complex (which was
discussed earlier).
Inhibiting the CDK4/cyclin D complex keeps pRB in its
active configuration, repressing the transcription of genes
needed to move the cell from G 1 into S phase. Cells
without functional p53 protein cannot arrest in G 1
following irradiation and move immediately from G 1
into S. These cells do not repair the DNA damage caused
by the radiation and, as a result, have a high rate of
mutation. Thus, the p53 gene is often referred to as the
"guardian of the genome."
As just mentioned, more than 50 percent of all human
cancers carry a mutation in the p53 gene. The active p53
protein is a tetramer, so mutation of one p53 allele
usually abolishes all p53 activity, because almost all
tetramers will contain at least one defective subunit. As a
result, p53 mutations act as dominant negative mutations,
and individuals heterozygous for a p53 mutation will
develop cancer with a frequency of 90-95 percent. The
central role of the p53 gene in cell cycle control
emphasizes the relationships between cancer and the cell
cycle and between genes that regulate cell growth and
cancer.
Breast Cancer Genes: Mutations in BRCAl, a gene on
the long arm of chromosome 17, are associated with a
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  39
predisposition to breast cancer; this predisposition is
inherited as an autosomal dominant trait. About 85
percent of women carrying one mutant BRCA1 allele will
develop a mutation in the second allele and get breast
cancer. These women also have an increased risk of
ovarian cancer.
A second breast cancer gene, BRCA2, on the long arm of
chromosome 13, is also inherited as an autosomal
dominant predisposition to breast cancer, but is not
associated with an increased risk of ovarian cancer.
Together, these genes account for a large majority of
breast cancer associated with a genetic predisposition
(about 10 percent of all cases of breast cancer). The
mutant forms of these genes play no role in sporadic
cases of breast cancer, which make up 90 percent of all
cases.
Both BRCA1 and BRCA2 encode large proteins that are
confined to the nucleus and expressed in many tissues.
Expression is at its highest during S phase of the cell
cycle. Circumstantial evidence, including their common
pattern of expression, similar phenotype of the mutant
alleles (breast cancer), cellular localization, and
experimental evidence from studies in mice, suggests that
both genes have similar functions and are involved in
DNA repair. Direct evidence for the role of the BRCAI
protein in DNA repair also exists. In cells exposed to
ionizing radiation (which produces double stranded DNA
breaks), BRCAI is phosphorylated (a sign of molecular
activation). Current efforts center on identifying the
kinase proteins that carry out this phosphorylation, in an
attempt to place the BRCA 1 and the BRCAl proteins in
a pathway associated with DNA repair.
Recent evidence suggests that the BRCAI protein can be
phosphorylated by two different kinases and that either
kinase is activated by double-stranded DNA breaks (Fig
7). In this model, DNA damage activates the ATM
kinase or the Chkl kinase (or both). These kinases
phosphorylate the BRCA1 and p53 proteins. The
activated p53 protein arrests replication during S phase to
allow DNA repair. The activated BRCA 1 protein
participates in DNA repair with the BRCA2 protein, the
mRAD51 protein, and other nuclear proteins involved in
DNA repair.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

The role of BRCA1 and BRCA2 proteins in DNA repair

may also explain why they are not associated with
sporadic cases of breast cancer. In sporadic cases, at least
three mutations would have to accumulate in a breast cell
for it to become cancerous: Both copies of the BRCAl or
BRCA2 genes would have to be mutant, and at least one
other mutation in a cell-cycle regulation gene would have
to occur. On the other hand, those who inherit a mutant
copy of either the BRCAl or BRCA2 gene already have
one mutation and need only two more to trigger breast
cancer.

31.3.2. Oncogenes induce uncontrolled cell division:

We now discuss the second category of genes involved in

cell cycle regulation: those that normally promote cell
division, called protooncogenes. When expressed, these
genes promote or maintain cell division. To stop
division, these genes or their gene products (or both)
must be inactivated. As mutant alleles called oncogenes,
they induce or maintain the uncontrolled cell division ii. Chromosomal translocation: Some of the tumors
associated with cancer. The protein products of exhibit chromosomal abnormalities. This is due to the
protooncogenes are found throughout the cell, including rearrangement of genetic material (DNA) by
the plasma membrane, cytoplasm, and nucleus. In spite chromosomal translocation i.e. splitting off a small
of their wide-ranging locations within the cell, fragment of chromosome which is joined to another
suggesting varying functions, all protooncogene proteins chromosome. Chromosomal translocation usually results
characterized to date alter gene expression either directly in over-expression of protooncogenes.
or indirectly.

Unlike tumor suppressor genes, where mutations in both

alleles of a gene are necessary to promote the
development of cancer, only one of the two copies of a
protooncogene needs to mutate to induce malignancy,
resulting in a dominant phenotype.

Thus genes capable of causing cancer are known as

oncogenes (Greek : oncos-tumor or mass). Oncogenes
were originally discovered in tumor causing viruses.
These viral oncogenes were found to be closely similar to
certain genes present in the normal host cells, which are
referred to as protooncogenes. Now, about 40 viral and
cellular protooncogenes have been identified.
Protooncogenes encode for growth-regulating proteins.
The activation of protooncogenes to oncogenes is an
important step in the causation of cancer.

a. Activation of protooncogenes to oncogenes:

There are several mechanisms for converting the
protooncogenes to oncogenes; some of the important
ones are described here under.
i. Viral insertion into chromosome: When certain
retroviruses (genetic material RNA) infect cells, a
complementary DNA (cDNA) is made from their RNA
by the enzyme reverse transcriptase. The cDNA so
produced gets inserted into the host genome (Fig.8). The
integrated double-stranded cDNA is referred to as
provirus. This pro-viral DNA takes over the control of
the transcription of cellular chromosomal DNA and
transforms the cells. Activation of protooncogene myc to
oncogene by viral insertion ultimately causing
carcinogenesis is well known (e.g. avian leukemia).
Some DNA viruses also get inserted into the host
chromosome and activate the protooncogenes.
Burkitt's lymphoma, a cancer of human B-lymphocytes,
is a good example of chromosomal translocation. In this
case, a fragment from chromosome 8 is split off and
joined to chromosome 14 containing myc gene (Fig. 9).
This results in the activation of inactive myc gene leading
to the increased synthesis of certain proteins, which make
the cell malignant.
iii. Gene amplification: Several-fold amplifications of
certain DNA sequences are observed in some cancers.
Administration of anticancer drugs Methotrexate (an
inhibitor of the enzyme dihydrofolate reductase) is
associated with gene amplification. The drug becomes
inactive due to gene amplification resulting in a several-
fold (about 400) increase in the activity of dihydrofolate
reductase.
iv. Point mutation: The ras protooncogene is the best
example of activation by point mutation (change in a
single base in the DNA). The mutated ras oncogene

40 INSTITUTE FOR ADVANCED STUDIES, JODHPUR 
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produces a protein (GTPase), which differs in structure oncoproteins. These proteins are the altered version of
by a single amino acid. This alteration diminishes the their normal counterparts and are involved in the
activity of GTPase, a key enzyme involved in the control transformation and multiplication of cells. Some of the
of cell growth. products of oncogenes are below discussed below.

The presence of ras mutations is detected in several
human tumors-90% of pancreatic, 50% of colon and 30%
of lung. However, ras mutations have not been detected
in the breast cancer.
b. Classification and Mechanism of oncogenes:
Oncogenes encode for certain proteins, namely
i. Growth factors: Several growth factors stimulating
the proliferation of normal cells are know. They regulate
cell division by transmitting the message across the
plasma membrane to the interior of the cell
(transmembrane signal transduction). It is believed that
growth factors play key role in carcinogenesis.

A selected list of polypeptide growth factors, and their sources and major functions are:

Epidermal growth factor (EGF) Salivary gland, Fibrobast Stimulates the growth of Epidermal & Epithelial cells
Platelet derived growth factor (PDGF) Platelets Stimulates growth of mesenchymal cells,
promotes wound healing
Transforming Growth factor (TGF-α) Epithelial cells Similar to EGF
Transforming Growth factor (TGF-β) Platelets, Kidney, placenta Inhibitory effect on tumor cells
Tumor Necrosis factor (TNF-α) Monocytes Necrosis of tumor cells
Interleukin-2 Helper T-Lymphocytes Stimulates growth & maturation of T-Cells

The cell proliferation is stimulated by growth factor. In activating proteins (GAP) which accelerate the
general, a growth factor binds to a protein receptor on the hydrolysis of GTP of ras P21. Thus, in normal cells, the
plasma membrane. This binding activates cytoplasmic activity of ras P21 is well regulated.
protein kinases Ieading phosphorylation of intracellular
target protein. The Phosphorylated proteins in turn, act as
intracellular messengers to stimulate cell division, the
mechanism of which is not clearly known.

Transforming growth factor (TGF-α) is a protein

synthesized and required for the growth of epithelial
cells. TGF-α is produced in high concentration in
individuals suffering from psoriasis, a disease
characterized by excessive proliferation of epidermal
cells.

ii. Growth factor receptors: Some oncogenes encoding

growth factor receptors have been identified. Over
expression and/or structural alterations in growth factor
receptors are associated with carcinogenesis. For
instance, the over expression of gene erb-B, encoding
EGF-receptor is observed in lung cancer.

iii. GTP-binding proteins: These are a group of signal

transducing proteins. Guanosine triphosphate (GTP)-
binding proteins are found in about 30% of human
cancers. The mutation of ras protooncogene is the single-
most dominant cause of many human tumors.

The involvement of ras protein (product of ras Gene)

with molecular weight 21 Kd (P21) in multiplication is
illustrated in Fig. 10. The inactive ras is in a bound state
with GDP. When the cells are stimulated by growth
factors, ras P21 gets activated by exchanging GDP for
GTP.

This exchange process is catalysed by guanine nucleotide

releasing factor (GRF). The active ras P21 stimulates
regulators such as cytoplasmic kinases, ultimately
causing DNA replication and cell division. In normal Point mutations in ras gene result in the production of
cells, the activity of ras P21 is short-lived. The GTPase altered ras P21, lacking GTPase activity. This leads to the
activity, which is an integral part (intrinsic) of ras P21, occurrence of ras P21 in a permanently activated state,
hydrolyses GTP to GDP, reverting ras 21 to the original causing uncontrolled multiplication of cells.
state. There are certain proteins, namely GTPase

INSTITUTE FOR ADVANCED STUDIES, JODHPUR  41
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
iv. Non-receptor tyrosine kinases: These proteins are
found on the interior of the inner plasma membrane.
They phosphorylate the cellular target proteins (involved
in cell division) in response to external growth stimuli.
Mutations in the protooncogenes (e.g. ab/) encoding non-
receptor tyrosine kinases increase the kinase activity and,
in turn, phosphorylation of target proteins causing
unlimited cell multiplication.
Colon Cancer Results from a Series of Mutations:
Cancer is clearly a multistep process resulting from a
number of specific mutational events. Studies of tumors
such as retinoblastoma have established that in some
cases only a few steps are required to transform a normal
cell into a malignant one. However, most cancers
develop in several steps, with intermediate levels of
genetic and cellular transformation.
Colorectal cancer has been used to obtain detailed infor-
mation about the nature and order of genetic and cellular
events that result in a malignant growth. The malignant
form of colorectal tumors develops from preexisting
benign tumors. Several discrete precancerous stages
occur, and these stages can be isolated and studied.
Furthermore, both hereditary and sporadic forms of
colorectal cancer exist. As a result, colorectal cancer is a
useful model for studying the interaction of genetic and
environmental factors in tumor formation.
Subsequent mutations in polyp cells cause intermediate
stages of tumor formation. Mutations in the ras oncogene
on chromosome 12 (combined with a mutant APC allele)
cause the polyp to grow larger and to develop fingerlike
(villous) outgrowths. This is the intermediate adenoma
stage. To progress further, a polyp cell must acquire
mutations in the DCC (deleted in colon cancer) gene on
chromosome 18. Mutations in both DCC alleles results in
late-stage adenomas. Finally, mutation of the p53 gene
causes the transition to a cancerous cell. As described
earlier, mutations in p53 are pivotal to the development
of a number of cancers, including lung, brain, and breast
cancers as well as colon cancer. Metastasis occurs after
the formation of colon cancer, and it involves an
unknown number of mutational steps.
HNPCC and Colon Cancer: HNPCC-associated colon
cancer develops after a small number of polyps are
formed, rather than the hundreds or thousands associated
with FAP. Genes associated with HNPCC have been
mapped to loci at 2p 16 and 3p21. Mutations at these loci
generate a cascade of mutations in short, tandemly
repeated microsatellite sequences located throughout the
42 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
Two forms of genetic predisposition to colon cancer are
known: (1) an autosomal dominant trait, known as
familial adenomatous polyposis (FAP) and (2) a
genetically complex trait, known as hereditary
nonpolyposis colorectal cancer (HNPCC). FAP is present
in about 1 percent of all cases, and HNPCC is
responsible for 2-4 percent of all colon cancers. The
remaining 95 percent of all cases are sporadic.
FAP and Colon Cancer: In the FAP-associated model,
spontaneous cases of colon cancer begin with a mutation
in the APC gene, which maps to the long arm of
chromosome 5. This mutation occurs in a normal
epithelial cell in the lining of the colon. In spontaneous
cases, an APC mutation causes epithelial cells to partially
escape cell cycle control. These cells divide to form a
small cluster called a polyp. Those who inherit a mutant
APC allele have the mutation in all cells of their body. As
a result, APC heterozygotes form hundreds or thousands
of polyps. In either case, polyps are clones, all of which
carry an APC mutation. The loss of the corresponding
allele on the homologous copy of chromosome 5 is not
necessary for polyp formation. The first step and the
relative order of subsequent mutations are shown in
Figure 11.
genome. The gene on chromosome 2 is a DNA repair
gene called MSH2. Inactivation of this gene causes the
rapid accumulation of mutations and the development of
colorectal cancer. A second DNA repair gene, MLH 1,
associated with HNPCC has been mapped to
chromosome 3. At least two other DNA repair genes
related to MLH 1 and MSH2 have been identified and
remain to be mapped. These genes are called mismatch
repair genes (MMR) and generate a condition called
microsatellite instability. Mutations in any MMR genes
promote genome wide genetic instability, accelerating
the rate at which mutations accumulate, with colon
cancer. as one outcome. It may turn out that mutations in
anyone of these genes is enough to cause HNPCC.
Mutations leading to HNPCC are similar to FAP-
associated colon cancer, but have several important
differences. FAP begins with a mutation in APC, and the
formation of hundreds or thousands of polyps. Each
polyp progresses slowly toward cancer by accumulating
mutations in other genes (ras, DCC, etc.) However,
because there are so many polyps, there is a high
probability that at least one polyp will accumulate all the
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
mutations necessary to cause colon cancer. HNPCC
begins with mutation in DNA repair genes in an
epithelial cell of the colon, generating many genome
wide mutations, one of which may include APC.
Mutation of APC is a relatively rare event, so only a few
polyps will form. However, even though there are few
polyps, the continuing high mutation rate ensures that at
least one polyp will acquire all the mutations necessary
to become cancerous. FAP-associated colon cancer is
therefore a disease of rapid tumor initiation, with slow
progression to cancer, while HNPCC is a disease with
slow initiation of tumors, but rapid progression to cancer.
Both forms of colon cancer are mediated by mutations in
the APC gene.
Gatekeeper and Caretaker Genes: The two pathways
to colon cancer in FAP and HNPCC offer an insight into
the nature of genes that control cancer predisposition.
These differences indicate that mutations in two types of
genes cause predisposition to cancer: gatekeeper genes
and caretaker genes. The FAP gene is a gatekeeper; it
normally inhibits cell growth. In general, tumor suppres-
sor genes are gatekeepers. In different cell types, only
one or a few genes serve as gatekeepers, and if both
copies of the gatekeeper mutate, a specific cancer, such
as retinoblastoma or colon cancer, develops. Individuals
D4 Viruses and Cancer
Tumor cells also can arise by non-genetic means through
the actions of specific tumor viruses. Tumor viruses are
of two distinct types. There are viruses with DNA
genomes (e.g. papilloma and adenoviruses) and those
with RNA genomes (termed retroviruses). RNA tumor
viruses are common in chickens, mice and cats but rare
in humans. The only currently known human retroviruses
are the human T-cell leukemia viruses (HTLVs) and the
related retrovirus, human immunodeficiency virus (HIV).
Retroviruses can induce the transformed state within the
cells they infect by two mechanisms. Both of these
mechanisms are related to the life cycle of these viruses.
When a retrovirus infects a cell its RNA genome is
converted into DNA by the viral encoded RNA-
dependent DNA polymerase (reverse transcriptase). The
DNA then integrates into the genome of the host cell
where it can remain being copied as the host genome is
duplicated during the process of cellular division.
Contained within the sequences at the ends of the
retroviral genome are powerful transcriptional promoter
sequences termed long terminal repeats (LTRs). The
LTRs promote the transcription of the viral DNA leading
to the production of new virus particles.
At some frequency the integration process leads to
rearrangement of the viral genome and the consequent
incorporation of a portion of the host genome into the
viral genome. This process is termed transduction.
Occasionally this transduction process leads to the virus
acquiring a gene from the host that is normally involved
in cellular growth control. Because of the alteration of
the host gene during the transduction process as well as
the gene being transcribed at a higher rate due to its
association with the retroviral LTRs the transduced gene
confers a growth advantage to the infected cell. The end
result of this process is unrestricted cellular proliferation
leading to tumorigenesis. The transduced genes are
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  43
predisposed to a specific form of cancer inherit one
mutant copy of such a gatekeeper and need only one
additional mutation to initiate tumor formation. In
spontaneous cases, both copies of the gatekeeper must
mutate for cancer to develop.
Caretaker genes maintain the integrity of the genome, in-
cluding DNA repair genes such as MSH2 and MLH 1.
These genes repair DNA damage caused by
environmental agents such as ultraviolet light, or
mismatches that occur during DNA replication. Mutation
of a caretaker gene does not promote tumor formation
directly, but leads to genetic instability that increases the
mutation rate of all genes, including gatekeeper genes. If
a gatekeeper gene mutates and begins tumor formation,
the process is accelerated by the high rate of mutation in
the cell.
3. Genes that regulate apoptosis
A new category of genes that regulate programmed cell
death (apoptosis) have been discovered. These genes are
also important in the development of tumors.The gene,
namely bcl-2, causes B-cell lymphoma by preventing
programmed cell death. It is believed that over-
expression of bcl-2 allows other mutations of
protooncogenes that, ultimately, leads to cancer.
termed oncogenes. The normal cellular gene in its
unmodified, non-transduced form is termed a proto-
oncogene since it has the capacity to transform cells if
altered in some way or expressed in an uncontrolled
manner. Numerous oncogenes have been discovered in
the genomes of transforming retroviruses.
The second mechanism by which retroviruses can
transform cells relates to the powerful transcription
promoting effect of the LTRs. When a retrovirus genome
integrates into a host genome it does so randomly. At
some frequency this integration process leads to the
placement of the LTRs close to a gene that encodes a
growth regulating protein. If the protein is expressed at
an abnormally elevated level it can result in cellular
transformation. This is termed retroviral integration
induced transformation. It has recently been shown that
HIV induces certain forms of cancers in infected
individuals by this integration induced transformation
process.
Cellular transformation by DNA tumor viruses, in most
cases, has been shown to be the result of protein-protein
interaction. Proteins encoded by the DNA tumor viruses,
termed tumor antigens or T antigens, can interact with
cellular proteins. This interaction effectively sequesters
the cellular proteins away from their normal functional
locations within the cell. The predominant types of
proteins that are sequestered by viral T antigens have
been shown to be of the tumor suppressor type. It is the
loss of their normal suppressor functions that results in
cellular transformation.
Unified hypothesis of carcinogenesis: The
multifactorial origin of cancer can be suitably explained
by oncogenes. The physical and chemical agents, viruses
and mutations all lead to the activation of oncogenes
causing carcinogenesis. The antioncogenes and the genes
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

regulating apoptosis are intimately involved in of a protein, namely vinculin, is said to be

development of cancer. A simplification of a unified
hypothesis of carcinogenesis is depicted in fig 12.
e)
responsible for the loss of anchorage property in
cancer cells.
Alteration in permeability properties: The
tumor cells have altered permeability and
transport across the membranes.

2. Biochemical changes

a) Increased replication and transcription: The

Characteristics of growing tumor cells:
The morphological and biochemical changes in the
growing tumor cells are briefly described here. These
observations are mostly based on the in vitro culture
studies. Knowledge on the alterations in the biochemical
profile of tumor cells guides in the selection of
chemotherapy of cancers.
1. General and morphological changes
a) Shape of cells: The tumor cells are much rounder
in shape compared to normal cells.
b) Alterations in cell structures: The cytoskeletal
structure of the tumor cells with regard to actin
filaments is different.
c) Loss of contact inhibition: The normal cells are
characterized by contact inhibition i.e. they form
monolayers. Further, they cannot move away
from each other. The cancer cells form multi
layers due to loss of contact inhibition. As a
result, the cancer cells freely move and get
deposited in any part of the body, a property
referred to as metastasis.
d) Loss of anchorage dependence: The cancer cells
can grow without attachment to the surface. This
is in contrast to the normal cells, which firmly
adhere to the surface. Alteration in the structure
synthesis of DNA and RNA is increased in cancer
cells, indicating an increase in anabolic processes.
b) Increased glycolysis: The fast growing tumor
cells are characterized by elevation in aerobic and
anaerobic glycolysis. This truly reflects the
increased energy demands multiplying cells.
c) Increased pyrimidine metabolism: A reduction
in the catabolic reactions such as degradation of
pyrimidines is observed in tumor cells.
d) Enzyme alterations: The activities of certain
enzymes are changed e.g. proteases.
e) Reduced requirement of growth factors: The
tumor cells require much less quantities of
growth factors. Despite this fact, there is an
increased production of growth factors by these
cells.
f) Synthesis of fetal proteins: During fetal life,
certain genes are active, leading to the synthesis
of specific proteins. These genes are suppressed
in adult cells. However, the tumor cells
synthesize the fetal proteins e.g.:
carcinoembryonic antigen, α-fetoprotein.
g) Alterations in the structure of molecules:
Changes in the structure of glycoproteins and
glycolipids are observed
h) Reduced synthesis of certain molecules: A
diminished synthesis of specialized proteins is
seen in tumor cells.
i) Changes in isoenzymes: The isoenzyme profile
of cancer cells is close to the fetal pattern.
j) Alterations in antigens: A loss of regularly
occurring antigens coupled with the appearance
of new antigens in tumor cells is reported.

EXTRA CONCEPTS
 

• The incidence of cancer increases as age advances, more than 70% of the new cases occurring in persons
over 60.
• Chemical carcinogens cause about 80% of the human cancers.
• The products of oncogenes (growth factors, GTP-binding proteins) have been implicated in the development
of cancer. Antioncogenes apply breaks and regulate the cell proliferation.
• The physical and chemical agents, viruses and mutations result in the activation of oncogenes causing
carcinogenesis.
• The abnormal products of tumor cells, referred to as tumor markers, (CEA AFP, PSA) are useful for the
diagnosis and prognosis of cancer.
• Knowledge on the alterations in the biochemical profile of tumor cells is helpful for the chemotherapy of
cancers.

44 INSTITUTE FOR ADVANCED STUDIES, JODHPUR 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

  
E1. Cells, Tissues, and Organs of the Immune System

The immune system is an organization of cells, tissues, more organelles (especially lysosomes and
organs, and molecules with specialized roles in phagolysosomes), and have a plasma membrane covered
defending against viruses, microorganisms, cancer cells, with ruffles or microvilli. Macrophages have receptors
and nonself proteins (e.g., organ transplants). for antibodies and complement; these can coat
microorganisms or foreign material and enhance
Cells of the Immune System phagocytosis. This enhancement is termed opsonization.
Macrophages spread throughout the animal body and
The cells responsible for both nonspecific and specific take up residence in specific tissues where they are given
immunity are the white blood cells called leukocytes. special names. Since macrophages are highly phagocytic,
[Greek leukos, white, and kytos, cell]. All of the their function in nonspecific resistance will be discussed
leukocytes originate from pluripotent stem cells in the in more detail in the context of phagocytosis.
fetal liver and in the bone marrow of the animal host
(figure 1), from which they migrate to other body sites,
undergo further development, and perform their various
functions. These cells of the immune system are present
throughout the host’s body. Some become residents
within tissues, where they respond to local trauma and
sound the alarm; others circulate in body fluids and are
recruited to the sites of infection. In defending the host
against pathogenic microorganisms, leukocytes cooperate
with each other first to recognize the pathogen as an
invader and then to destroy it. These different leukocytes
are now briefly examined.

Lymphoid Cells

Lymphocytes [Latin lympha, water, and cyte, cell] are

the major cells of the specific immune system.
Lymphocytes can be divided into three populations: T
cells, B cells, and natural killer cells. B cells or B
lymphocytes reach maturity within the bone marrow,
circulate in the blood, and also settle in various lymphoid
organs. T cells or T lymphocytes mature in the thymus
gland; they can remain in the thymus, circulate in the
blood, or reside in lymphoid organs such as the lymph
nodes and spleen. B cells and T cells will be discussed
further along with their roles in specific immunity.
Natural killer cells are important in killing cells infected
with either viruses or intracellular bacteria and
destroying cancer cells.

Mononuclear Cells

There are two types of mononuclear (i.e., a single large Granulocytes

nucleus) cells—monocytes and macrophages. Both types
are highly phagocytic and make up the monocyte-
macrophage system. Recall that during phagocytosis
large particles and even other microorganisms are
engulfed and enclosed in a phagocytic vacuole or
phagosome.
Monocytes [Greek monos, single, and cyte, cell] are
mononuclear phagocytic leukocytes with an ovoid or
kidney-shaped nucleus and granules in the cytoplasm that
stain gray-blue (figure 1). They are produced in the bone
marrow and enter the blood, circulate for about eight
hours, enlarge, migrate to the tissues, and mature into
macrophages.
As just noted, macrophages [Greek macros, large, and
phagein, to eat] are derived from monocytes and are also
classified as mononuclear phagocytic leukocytes.
However, they may be larger than monocytes, contain
Granulocytes have irregular-shaped nuclei with two to
five lobes, and the cytoplasmic matrix has granules
(figure 1) that contain reactive substances that kill
microorganisms and enhance inflammation. Because of
the irregular-shaped nuclei, granulocytes are also called
polymorphonuclear leukocytes or PMNs. Three types
of granulocytes exist: basophils, eosinophils, and
neutrophils.
Basophils [Greek basis, base, and philein, to love] have
an irregular-shaped nucleus with two lobes, and the
granules stain bluish-black with basic dyes. Basophils are
nonphagocytic cells that function by releasing histamine,
prostaglandins, serotonin, and leukotrienes from their
granules upon appropriate stimulation. Because these
physiological mediators influence the tone and diameter
of blood vessels, they are termed vasoactive agents.
Basophils (and mast cells) possess high-affinity receptors
for an immunoglobulin(IgE) and thereby become coated

INSTITUTE FOR ADVANCED STUDIES, JODHPUR  45
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

with IgE antibodies. Once coated, antigens can trigger
the cell to secrete vasoactive mediators. These
inflammatory mediators play a major role in certain
allergic responses such as eczema, hay fever, and asthma.
Eosinophils [Greek eos, dawn, and philien] have a two-
lobed nucleus connected by a slender thread of
chromatin, and the granules stain red with acidic dyes.
Unlike basophils, eosinophils are mobile cells that can
migrate from the bloodstream into tissue spaces. Their
role is important only in the defense against protozoan
and helminth parasites, mainly by releasing cationic
proteins and reactive oxygen metabolites into the
extracellular fluid to damage the parasite’s plasma
membranes.

Neutrophils [Latin neuter, neither, and philien] stain substances and defensins to kill ingested
readily at a neutral pH, have a nucleus with three to five microorganisms. Neutrophils are described in more detail
lobes connected by slender threads of chromatin, and in the contexts of the inflammatory response and
contain fine primary and secondary inconspicuous phagocytosis.
granules. Like macrophages, neutrophils have receptors
for antibodies and complement
proteins and are highly phagocytic
cells. However, unlike the
macrophage, neutrophils do not
reside in healthy tissue but rapidly
migrate to the site of tissue damage
and infection where they are the
principal phagocytic and
microbicidal cells. The lytic
enzymes and bactericidal
substances in neutrophils are contained within large Mast Cells
primary and smaller secondary granules. Primary
granules contain peroxidase, lysozyme, and various Mast cells are bone marrow–derived cells found in
hydrolytic enzymes, whereas secondary granules have connective tissue. They contain granules with histamine
collagenase, lactoferrin, and lysozyme. Both of these and other pharmacologically active substances that
granules help accomplish intracellular digestion. contribute to the inflammatory response. Mast cells,
Neutrophils also use oxygen-dependent and oxygen- along with basophils, play an important role in the
independent pathways that generate antimicrobial development of allergies and hypersensitivities.

46 INSTITUTE FOR ADVANCED STUDIES, JODHPUR 
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

Dendritic Cells

Dendritic cells can recognize specific pathogen-

associated molecular patterns on microorganisms and
play an important role in nonspecific resistance. They
can differentiate between potentially harmful
microorganisms and “self” molecules. After the pathogen
is recognized, it binds to the dendritic cell’s pattern
recognition receptors and then is phagocytosed. These
cells are also stimulated by endogenous activators such
as interferon-γ, heat-shock proteins, and tumor necrosis
factor that are released in response to microbial infection.
After stimulation, dendritic cells migrate to the
bloodstream or lymphatic system and present antigens to
T cells. Thus dendritic cells also play an important role in
the specific immune response.

Organs and Tissues of the Immune System

The spleen is the large secondary lymphoid organ
Based on function, the organs and tissues of the immune located in the abdominal cavity. Whereas lymph nodes
system can be divided into primary or secondary are specialized for trapping microorganisms and antigens
lymphoid organs or tissues. The primary organs or from local tissues, the spleen specializes in filtering the
tissues are where immature lymphocytes mature and blood and trapping blood-borne microorganisms and
differentiate into antigen-sensitive mature B and T cells. antigens. Once trapped by macrophages and dendritic
The thymus is the primary lymphoid organ and bone cells, the pathogen is phagocytosed and antigens are
marrow is the primary lymphoid tissue. The secondary presented to B and T cells, which become activated to
organs and tissues serve as areas where lymphocytes may carry out their immune functions.
encounter and bind antigen, whereupon they proliferate
and differentiate into fully mature, antigen-specific
effector cells. The spleen is a secondary lymphoid organ
and the lymph nodes and mucosal-associated tissues
(GALT, gut-associated lymphoid tissue and SALT, skin
associated lymphoid tissues) are the secondary lymphoid
tissues.

Primary Lymphoid Organs and Tissues

Immature undifferentiated lymphocytes are generated in

the bone marrow, and mature and become committed to a
particular antigenic specificity within the primary
lymphoid organ/tissues. The two most important of these
in mammals are the thymus and bone marrow. Lymph nodes lie at the junctions of lymphatic vessels
where they filter out harmful microorganisms and
The thymus is a lymphoid organ located above the heart. antigens from the lymph; pathogens and antigens are
Precursor cells from the bone marrow migrate into the trapped by phagocytic and dendritic cells. Fixed
thymus to the outer cortex where they proliferate. As macrophages then phagocytose the foreign material.
they mature and acquire T cell surface markers, they
move to the inner cortex where approximately 90% die, It is within the lymph nodes that B cells proliferate into
possibly as part of the acquisition of immune tolerance. antibody- secreting plasma cells. Dendritic and T cells
The other 10% move into the medulla, become mature T are also found here; dendritic cells serve as antigen-
cells, and enter the bloodstream (figure). In birds, presenting cells and T helper cells promote the B-cell
undifferentiated lymphocytes move from the bone immune response.
marrow to the bursa of Fabricius where B cells mature.
In mammals, the bone marrow is the site of B-cell
maturation. Like thymic selection during T-cell
maturation, a selection process within the bone marrow
eliminates B cells with self-reactive antibody receptors
(the acquisition of tolerance).

Secondary Lymphoid Organ/Tissue

The spleen is the most highly organized secondary

lymphoid organ and the lymph nodes the most highly
organized tissue.

INSTITUTE FOR ADVANCED STUDIES, JODHPUR  47
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

E2. Antigens

The immune system distinguishes between “self” and Most antigens, however, have more than one determinant
“nonself” through an elaborate recognition process. Prior site or more than one copy of the same epitope, and are
to birth the body inventories the proteins and various termed multivalent. Multivalent antigens generally elicit
other large molecules present (self) and removes most T a stronger immune response than do monovalent
cells specific for self-determinants. ubsequently self- antigens.
substances can be distinguished from nonself substances,
and lymphocytes can produce specific immunologic
reactions against the latter, leading to their removal.
Substances, such as proteins, nucleoproteins,
polysaccharides, and some glycolipids, that elicit an
immune response and react with the products of that
response are called antigens (antibody generator). Most
antigens are large, complex molecules with a molecular
weight generally greater than about 10,000 dalton. The
ability of a molecule to function as an antigen depends
on its size and structural complexity.

Antigenicity and Immunogenicity

A. Antigenicity is defined as the property of a substance

(antigen) that allows it to react with the products of a
specific immune response (antibody or T-cell receptor).

B. Immunogenicity is defined as the property of a

substance (immunogen) that endows it with the capacity
to provoke a specific immune response.

C. All immunogens are antigens, but the reverse is

not true.
1. Immunogens are usually complex, large molecules,
that are able to induce an immune response by
themselves. Immunogens are also antigens in that
specific antibodies or specific T cells recognizing them
will be formed as a consequence of the immune response
they elicit.
2. Certain low molecular weight substances, known as
haptens, are unable to induce an immune response by
themselves. However, if haptens are coupled to an
immunogenic carrier molecule, the immune system will
recognize them as separate epitopes and produce
antihapten antibodies that react with soluble hapten
molecules, free of carrier protein. Thus, a hapten is an
antigen, but not an immunogen.
3. Unfortunately, the terms antigen and immunogen are
often interchanged.
While B lymphocytes recognize epitopes expressed by
Antigenic Determinants native antigens, T lymphocytes recognize small peptides
generated during antigen processing or derived from
Each antigen can have several antigenic determinant newly synthesized proteins. Studies with x-ray
sites or epitopes (figure 1). Epitopes are the regions or crystallography and two-dimensional nuclear magnetic
sites in the antigen that bind to the antigen-binding site of resonance imaging have resulted in the detailed
a specific antibody or with a T cell receptor. Antibodies characterization of epitopes presented by some small
are formed most readily in response to determinants that proteins, such as lysozyme, in their native configuration.
project from the foreign molecule or to terminal residues
of a specific polymer chain. Chemically, determinants From such studies the following rules have been derived
include sugars, organic acids and bases, amino acid side for antibody-antigen recognition:
chains, hydrocarbons, and aromatic groups.
1. Most epitopes are composed of a series of 15 to 22
The number of antigenic determinant sites on the surface amino acids located on discontinuous segments of the
of an antigen is its valence. The valence determines the polypeptide chain, forming a roughly flat area with peaks
number of antibody molecules that can combine with the and valleys that establish contact with the folded
antigen at one time. If one determinant site is present, the hypervariable regions of the antibody heavy and light
antigen is monovalent. chains.

48 INSTITUTE FOR ADVANCED STUDIES, JODHPUR 
 
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2. Specific regions of the epitope constituted by a few
amino acids bind with greater affinity to specific areas of
the antibody binding site, and thus are primarily
responsible for the specificity of antigen-antibody
interaction. On the other hand, the antibody binding site
has some degree of flexibility, which contributes to the
good fit with the corresponding epitope.
3. A polypeptide with 100 amino acids may have as
many as 14 to 20 non-overlapping determinants.
However, a typical 100 amino acid globular protein is
folded over itself, and most of its structure is hidden
from the outside. Only surface determinants on
molecules will usually be accessible for recognition by B
lymphocytes and for interactions with antibodies.
Characteristics of Immunogenicity
Many different substances can induce immune responses.
The following characteristics have an important
influence in the ability that a substance has to behave as
an immunogen.
A. Foreigness. As a rule, only substances recognized as
“non-self” will trigger the immune response. Microbial
antigens and heterologous proteins are obviously “non-
self” and are strongly immunogenic.
coupled to a protein to form a nucleoprotein. The
autoimmune responses characteristic of some of the so-
called autoimmune diseases (e.g., systemic lupus
erythematosus) are often directed to DNA and RNA that
may have stimulated the immune system as
nucleoproteins.
4. Polypeptides. Hormones such as insulin and other
polypeptides, although relatively small in size (M.W.
1500), are usually immunogenic when isolated from one
species and administered over long periods of time to an
individual of a different species.
Haptens and Carriers
A. Landsteiner, Pauling, and others discovered in the
1930s and 1940s that small aromatic groups, such as
aminobenzene sulphonate, amino-benzene arsenate, and
amino-benzene carboxylate could be chemically coupled
to immunogenic proteins (carriers) and induce
antihapten antibodies in this form. These authors used
hapten-carrier conjugates to study the specificity of the
immune response.
Many small organic molecules are not antigenic by
themselves but can become antigenic if they bond to a
larger carrier molecule such as a protein (figure 2).

B. Molecular Size. The most potent immunogens are

macromolecular proteins [molecular weight (M.W.) >
100,000]. Molecules smaller than 10,000 daltons are
weakly immunogenic.

C. Chemical Structure and Complexity. Proteins and

polysaccharides are among the most potent immunogens,
although relatively small polypeptide chains, nucleic
acids, and even lipids can, given the right circumstances,
be immunogenic.
1. Proteins. Large heterologous proteins expressing a
wide diversity of antigenic determinants are potent
immunogens.
a. The immunogenicity of a protein is strongly
influenced by its chemical composition. Positively
charged (basic) amino acids, such as lysine, arginine,
and histidine are repeatedly present in the antigenic
sites of lysozyme and myoglobin, while aromatic
amino acids (such as tyrosine) are found in two of
albumin's six antigenic sites. Therefore, it appears that
basic and aromatic amino acids may contribute more
strongly to immunogenicity than other amino acids;
basic proteins with clusters of positively charged
amino acids are strong immunogens.
b. There appears to be a direct relationship between
antigenicity and chemical complexity: aggregated or
chemically polymerized proteins are much stronger
immunogens than their soluble monomeric
counterparts.
2. Polysaccharides. Polysaccharides are among the most
important natural antigens, since either pure
polysaccharides or the sugar moieties of glycoproteins,
lipopolysaccharides, glycolipid-protein complexes, etc.,
are immunogenic. Many microorganisms have
polysaccharide-rich capsules or cell walls, and a variety
of mammalian antigens, such as the erythrocyte antigens
(A, B, Le, H), are short-chain polysaccharides They cannot stimulate antibody formation or T-cell
(oligosaccharides). responses by themselves, but can react with antibodies
3. Nucleic acids. Nucleic acids usually are not once formed. Such small molecules are termed haptens
immunogenic, but can induce antibody formation if [Latin haptein, to grasp]. When lymphocytes are

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UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
stimulated by the combined molecule, they can react to
either the hapten or the larger carrier molecule.
Conjugation of a hapten to a carrier protein makes the
hapten immunogenic because the carrier protein can be
processed and presented to specific T cells. As a result,
both hapten-specific and carrier-specific antibodies can
be made. One example of a hapten is penicillin. By itself
penicillin is not antigenic. However, when it combines
with certain serum proteins of sensitive individuals, the
resulting molecule does initiate a severe and sometimes
fatal allergic immune reaction. In these instances the
hapten is acting as an antigenic determinant on the carrier
molecule.
B. In the last two decades the hapten-carrier concept has
found significant applications in medicine, particularly to
enhance immunization protocols, and has also been
demonstrated to be the pathological basis for some
abnormal immune reactions:
1. Poorly immunogenic polysaccharides have been
shown to induce strong immune responses when
conjugated to immunogenic proteins, and this
observation has resulted in vaccine development for
immunoprophylaxis of infectious diseases.
2. Using drug-protein conjugates it has been possible to
produce antibodies to a wide variety of drugs, which are
used in numerous drug immunoassays (e.g., plasma
digoxin levels).
3. Some hypersensitivity reactions to some drugs,
chemicals, and metals is believed to result from
spontaneous coupling of these compounds to endogenous
proteins, creating hapten-carrier combinations. One
example of this mechanism is the spontaneous coupling
of the penicilloyl derivative of penicillin to a host
protein, believed to be the first step toward developing
hypersensitivity to penicillin.
Factors Associated with the Induction of an Immune
Response
Besides the chemical nature of the immunogen, other
factors strongly influence the development of an immune
response.
A. Genetic Constitution of the Animal. Different
animal species or different strains of one given species
show different degrees of responsiveness to a given
antigen. In humans, different individuals can behave as
“high responders” or “low responders” to any given
antigen. The genetic control of the immune response
seems mainly related to the repertoire of MHC
molecules, which bind antigen fragments and present
them to the immune system, as we will discuss later. The
animal will respond well to those antigens that are
processed into peptides or oligopeptides with high
affinity for the binding sites of the MHC molecules.
B. Method of Antigen Administration. A given dose of
antigen may elicit no detectable response when injected
intravenously, but may elicit a strong immune response if
injected intradermally. This last route of administration
results in slow removal from the site of injection and
prolonged antigenic stimulation. The presence of
dendritic cells in the dermis (where they are known as
Langerhans cells) is believed to be a significant factor
determining the vigorous immune responses obtained
when antigens are injected intradermally. The injected
50 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
antigen is trapped on the surface of those dendritic cells,
which migrate to the lymph node follicles, where the
initial stages of the immune response take place.
C. Use of Adjuvants. Adjuvants are agents that, when
administered along with an antigen, enhance the specific
response. Several factors seem to contribute to this
enhancement, including delayed release of antigen,
nonspecific inflammatory effects, and the activation of
monocytes and macrophages.
1. One of the most effective adjuvants is Complete
Freund's Adjuvant (CFA), a water-in-oil emulsion with
killed mycobacteria in the oil phase. Bacillus Calmette-
Guérin (BCG) is an attenuated strain of Mycobacterium
bovis used as vaccine against tuberculosis and is also an
effective adjuvant. Muramyl-dipeptide (MDP), the
active moiety of Mycobacterium tuberculosis and of
BCG, also has adjuvant properties.
2. Several other microbial and inorganic compounds
have been used as adjuvants. Some of these adjuvants
have been used therapeutically with the aim of boosting
the immune response of compromised patients. Many of
these compounds cause intense inflammatory reactions
and discomfort.
3. Aluminum hydroxide, an inert compound that
absorbs the immunogen, stimulates phagocytosis, and
delays removal from the inoculation site, is the adjuvant
most frequently used with human vaccines. Aluminum
hydroxide is not as effective as most of the adjuvants
listed above, but is considerably less toxic.
Exogenous and Endogenous Antigens
A. Most of the antigens to which we react are of
exogenous origin, and include microbial antigens,
environmental antigens (such as pollens and pollutants),
and medications. The objective of the immune response
is the elimination of foreign antigens, but, in some
instances, the immune response itself may have a
deleterious effect (hypersensitivity states).
B. “Alloantigens,” i.e. antigenic determinants that
distinguish one individual from another within the same
species, are unique exogenous antigens. These are alleles
of highly polymorphic systems, which define the
antigenic makeup of the cells and tissues of an
individual. A classic example is the A, B, O blood group
antigens: some individuals carry the A specificity, some
are B positive, some are AB positive, and some express
neither A nor B (O). Other alloantigenic systems are the
histocompatibility (MHC or in the human, HLA)
antigens of nucleated cells and tissues, the platelet (P1)
antigens, and the immunoglobulin allotypes. Examples of
sensitization to exogenous alloantigens include:
1. Women who may become sensitized to fetal red cell
antigens or immunoglobulin alloantigens during
pregnancy.
2. Repeated blood transfusions that can induce
sensitization against cellular or immunoglobulin
alloantigens from the donor(s).
3. Organ transplantation that usually results in
sensitization against histocompatibility alloantigens
expressed in the transplanted organ.
C. Endogenous antigens, by definition, are part of self,
and the immune system should not react against them.
The response to self antigens can be the cause of severe
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

pathological situations (autoimmune diseases), although delivers strong activating signals that apparently override
it may also have an important role in normal catabolic the need for co-stimulatory signals.
processes (i.e., antibodies to denatured IgG may help in
eliminating antigen-antibody complexes from C. Special Characteristics of the Immune Response to
circulation; antibodies to oxidized LDL may help in T-Independent Antigens. The antibody produced in
eliminating a potentially toxic lipid). response to stimulation with T-independent antigens is
predominantly IgM. The switch to IgG production
T-Dependent and T-Independent Antigens requires T-cell help or cytokine production; this switch
does not occur, therefore, with T-independent antigens.
A. Functional Definition. Early studies on the Immunological memory for T independent antigens is
physiological role of T lymphocytes included either not evident or very weak.
experiments in which inbred rodents were sublethally
irradiated to render them immunoincompetent, and their
immune systems were then reconstituted with T
lymphocytes, B lymphocytes, or mixtures of T and B
lymphocytes obtained from normal animals of the same
strain. After reconstitution of the immune system, the
animals were challenged with a variety of antigens, and
their antibody responses were measured.

1. For most antigens, including complex proteins,

heterologous cells, and viruses, a measurable antibody
response was only observed in animals reconstituted with
mixtures of T and B lymphocytes. Since T lymphocytes
do not synthesize antibody, their role must be one of
assisting the proliferation and/or differentiation of B
lymphocytes. The antigens that can elicit antibody
responses exclusively in animals reconstituted with both
T and B cells are known as T-dependent antigens.
Structurally, T-dependent antigens are usually complex
proteins with large numbers of different antigenic
determinants with little repetition among themselves.
2. Other antigens, particularly polysaccharides, can
induce antibody synthesis in animals depleted of T
lymphocytes, and are known as T-independent antigens.
3. It should be noted that there appears to be a continuous
gradation from T-dependence to T-independence, rather
than two discrete groups of antigens in many species.
However, this differentiation is useful as a “working
classification” of antigens.

B. Biological Basis of T-Independence. Two theories

Superantigens
concerning the signaling by T-independent antigens have
been proposed:
Certain antigens provoke such a drastic immune response
that they are termed superantigens. Superantigens are
1. Some T-independent antigens, such as bacterial
bacterial proteins. These superantigens nonspecifically
lipopolysaccharides (LPS), have mitogenic properties
stimulate T cells to proliferate by interacting with both
and can deliver dual signals to B cells, one by occupying
class II major histocompatibility complex products on
the antigen-specific receptor (membrane
antigen-presenting cells and the T-cell receptor (figure
immunoglobulin), and the other by a nonspecific
3). Good examples of superantigens are the
mitogenic signal involving the lipid moiety of LPS,
staphylococcal enterotoxins that cause food poisoning
whose nature has not been well characterized. The
and the toxin that causes toxic shock syndrome.
association of these two signals would be sufficient to
Superantigens cause symptoms by stimulating the release
stimulate B cells and promote their differentiation into
of massive quantities of cytokines from T cells and
antibody-producing cells.
should be considered possible chronic associates in such
2. Other T-independent antigens (such as
diseases as rheumatic fever, arthritis, Kawasaki
polysaccharides) do not have mitogenic properties, but
syndrome, atopic dermatitis, and one type of psoriasis.
are constituted by multiple repeats of a limited number of
sugar molecules, allowing extensive cross-linking of
membrane immunoglobulins. Receptor cross-linking

INSTITUTE FOR ADVANCED STUDIES, JODHPUR  51
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

E3. Immunoglobulin Structure

I. General Structure of Immunoglobulins technique that separates proteins by charge in a first step,
allowing their antigenic characterization in a second step.
A. Information concerning the precise structure of the
antibody molecule started to accumulate as technological
developments were applied to the study of the general
characteristics of antibodies. By the early 1940s
antibodies had been characterized electrophoretically as
gamma globulins (Fig. 1) and also classified into large
families by their sedimentation coefficient determined
by analytical ultracentrifugation (7S and 19S antibodies).
It also became evident that plasma cells were
responsible for immunoglobulin synthesis and that a
malignancy known as multiple myeloma was a
malignancy of immunoglobulin-producing plasma cells.
B. As protein fractionation techniques became available, 2. Reduction of disulfide bonds. If the IgG molecule is
complete immunoglobulins and their fragments were incubated with a reducing agent containing free SH
isolated in large amounts, particularly from the serum groups and fractionated by gel filtration (a technique that
and urine of patients with multiple myeloma. These separates proteins by size) in conditions able to
proteins were used both for studies of chemical structure dissociate noncovalent interactions, two fractions are
and for immunological studies that led to the definition obtained. The first fraction corresponds to polypeptide
of antigenic differences between proteins from different chains of M.W. 55,00 (heavy chains); the second
patients; this was the basis for the initial identification of corresponds to polypeptide chains of M.W. 23,000 (light
the different classes and subclasses of immunoglobulins chains) (Fig. 3).
and the different types of light chains.

B. The IgG Structural Model. The sum of data

II. Immunoglobulin G (IgG):
Immunoglobulin Molecule
obtained by proteolysis and reduction experiments
resulted in the conception of a diagrammatic two-
dimensional model for the IgG molecule (Fig. 4).
The Prototype

A. General Considerations. IgG, a 7S immunoglobulin,

is the most abundant immunoglobulin in human serum
and in the serum of most mammalian species. It is also
the immunoglobulin most frequently detected in large
concentrations in multiple myeloma patients. For this
reason, it was the first immunoglobulin to be purified in
large quantities and to be extensively studied from the
structural point of view. The basic knowledge about the
structure of the IgG molecule was obtained from two
types of experiments:

1. Proteolytic digestion. The incubation of purified IgG

with papain, a proteolytic enzyme extracted from the
latex of Carica papaya, results in the splitting of the
molecule into two fragments that differ both in charge
and antigenicity. These fragments can be easily
demonstrated by immuno-electrophoresis (Fig. 2), a

52 INSTITUTE FOR ADVANCED STUDIES, JODHPUR 
 
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C. Proteolytic Fragments and Functional Topography
of the Molecule
1. Papain digestion, splitting the heavy chains in the
hinge region (so designated because this region of the
molecule appears to be stereoflexible) results in the
separation of two Fab fragments and one Fc fragment
per IgG molecule (Fig. 5).
a. The Fab fragments are so designated because they
contain the antigen binding site.
b. The Fc fragment is so designated because it can be
easily crystallized.
c. If the disulfide bond joining heavy and light chains
in the Fab fragments is split, a complete light chain
can be separated from a fragment that comprises about
half of one of the heavy chains, the NH2 terminal half.
This portion of the heavy chain contained in each
Fab fragment has been designated the Fd fragment.
2. A second proteolytic enzyme, pepsin, splits the heavy
chains at the carboxyl side of the disulfide bonds that
join them at the hinge region, producing a double Fab
fragment or F(ab')2 (Fig. 6), while the Fc portion of the
molecule is digested into peptides.
3. The comparison of Fc, Fab, F(ab')2, and whole IgG
molecules shows both important similarities and
differences between the whole molecule and its
fragments.
a. Both Fab and F(ab')2 contain antibody binding sites,
but while the intact IgG molecule and the F(ab')2 are
bivalent, the Fab fragment is monovalent. Therefore,
a Fab fragment can bind to an antigen, but cannot
cross-link two antigen molecules.
b. An antiserum raised against the Fab fragment reacts
mostly against light-chain determinants; the
immunodominant antigenic markers for the heavy
chain are located in the Fc fragment.
c. The F(ab')2 fragment is identical to the intact
molecule as far as antigen-binding properties, but
lacks the ability to fix complement, bind to cell
membranes, etc., which are determined by the Fc
region of the molecule.
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  53
III. The Structural and Antigenic Heterogeneity of
Heavy and Light Chains
A. Immunoglobulin Classes. Five classes of
immunoglobulins were identified due to antigenic
differences of the heavy chains and designated as IgG
(the classic 7S immunoglobulin), IgA, IgM (the classic
19S immunoglobulin), IgD, and IgE. IgG, IgA, and IgM
together constitute over 95% of the whole
immunoglobulin pool in a normal human being and are
designated as major immunoglobulin classes. Because
they are common to all humans, the immunoglobulin
classes can also be designated as isotypes. The major
characteristics of the five immunoglobulin classes are
summarized in Table 1.
B. Light-Chain Isotypes. The light chains also proved to
be antigenically heterogeneous and two main isotypes
were defined: kappa and lambda. Each immunoglobulin
molecule is constituted by a pair of identical heavy
chains and a pair of identical light chains; hence, a given
immunoglobulin molecule can have either kappa or
lambda chains but not both. A normal individual will
have a mixture of immunoglobulin molecules in his
serum, some with kappa chains (e.g., IgGk), and others
with lambda chains (e.g., IgGλ). Normal serum IgG has a
2:1 ratio of kappa chain- over lambda chain-bearing IgG
molecules. In contrast, monoclonal immunoglobulins,
the results of the synthetic activity of malignant
proliferations of plasma cells, such as multiple myeloma,
have one single heavy-chain isotype and one single light-
chain isotype, since they are the product of large number
of cells all derived from a single mutant, constituting one
large clone of identical cells.
C. Immunoglobulin Subclasses. Antigenic differences
between the heavy chains of IgG and IgA exist and
define subclasses of those immunoglobulins. The most
important structural and biological characteristics of IgG
and IgA subclasses are listed in Tables 2 and 3.
1. IgG subclasses. Some interesting biological and
structural differences have been demonstrated for IgG
proteins of different subclasses.
a. From the functional point of view IgG1 and IgG3 are
more efficient in terms of complement fixation and have
greater affinity for monocyte receptors. Those properties
can be correlated with a greater degree of biological
activity, both in normal antimicrobial responses, in
which these properties have direct consequences in
opsonization and bacterial killing, and in pathological
conditions, in which the formation of immune complexes
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
containing IgG1 and IgG3 antibodies is more likely to
have pathogenic consequences.
b. From the structural point of view the IgG3 subclass
has the greatest number of structural and biological
differences relative to the remaining IgG subclasses.
Most differences appear to result from the existence of an
extended hinge region (which accounts for the greater
M.W.), with a large number of disulfide bonds linking
the heavy chains together (estimates of their number vary
between 5 and 15). This extended hinge region seems to
be easily accessible to proteolytic enzymes, and this
lability of the molecule is likely to account for its
considerably shorter half-life.
2. IgA subclasses. Of the two subclasses known, it is
interesting to note that a subpopulation of IgA2
molecules carrying the A2m(1) allotype is the only
example of a human immunoglobulin molecule lacking
the disulfide bond joining heavy and light chains. The
IgA2 A2m(1) molecule is held together through
noncovalent interactions between heavy and light chains.
IV. Immunoglobulin Regions and Domains
A. Constant and Variable Regions. The light chains of
human immunoglobulins are composed of 211 to 217
amino acids. As mentioned above, there are two major
antigenic types of light chains (k and λ); when the amino
acid sequences of light chains of the same type were
compared, it became evident that two regions could be
distinguished in the light-chain molecules: a variable
region, comprising the portion between the amino
terminal end of the chain and residues of 107 to 115, and
a constant region, extending from the end of the
variable region to the carboxyl terminus (Fig. 7).
1. The light-chain constant regions were found to be
almost identical in light chains of the same type, but
differ markedly in k and λ chains. It is assumed that the
difference in antigenicity between the two types of light
chains is directly correlated with the structural
differences in constant regions.
54 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
2. The amino acid sequence of the light-chain variable
regions is different even in proteins of the same
antigenic type, and early workers thought that this
sequence would be totally individual to any single
protein. With increasing data, it became evident that
some proteins shared similarities in their variable
regions, and it has been possible to classify variable
regions into three groups, Vk, Vλ, and VH. Each group
has been further subdivided into several subgroups.
3. The light-chain V-region subgroups (Vk, Vλ) are
“type” specific (i.e., Vk subgroups are only found in k
proteins and Vλ subgroups are always associated with λ
chains). In contrast, the heavy-chain V-region subgroups
(VH) are not “class” specific. Thus, any given VH
subgroup can be found in association with the heavy
chains of any of the known immunoglobulin classes and
subclasses.
4. The heavy chain of IgG is about twice as large as a
light chain; it is composed of approximately 450 amino
acids, and a variable and a constant region can also be
identified. The variable region is composed of the first
113 to 121 amino acids (counted from the amino terminal
end), and subgroups of these regions can also be
identified. The constant region is almost three times
larger; for most of the heavy chains, it starts at residue
116 and ends at the carboxyl terminus (Fig. 7). The
maximal degree of homology is found between constant
regions of IgG proteins of the same subclass.
B. Immunoglobulin Domains. The immunoglobulin
molecule contains several disulfide bonds formed
between contiguous cysteine residues. Some of them join
two different polypeptide chains (interchain disulfide
bonds), keeping the molecule together. Others
(intrachain bonds) join different areas of the same
polypeptide chain, leading to the formation of “loops.”
These “loops” and adjacent amino acids constitute the
immunoglobulin domains, which are folded in a
characteristic β-pleated sheet structure (Fig. 8).
1. Variable regions of both heavy and light chains have a
single domain, which is involved in antigen binding.
2. Light chains have one single constant region domain
(CL), while heavy chains have several constant region
domains (three in the case of IgG, IgA, and IgD; four in
the case of IgM and IgE). The constant region domains
are generically designated as CH1, CH2, and CH3, or, if
one wishes to be more specific, they can be identified by
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
the class of immunoglobulins to which they belong by
adding the symbol for each heavy chain class (γ, α, µ, δ,
ε). For example, the constant region domains of the IgG
molecule can be designated as Cγ1, Cγ2, and Cγ3.
3. Different functions have been assigned to the different
domains and regions of the heavy chains. For instance,
Cγ2 is the domain involved in complement fixation,
while both Cγ2 and Cγ3 are believed to be involved in
the binding to phagocytic cell membranes.
4. The “hinge region” is located between CH1 and CH2,
and its name is derived from the fact that studies by a
variety of techniques, including fluorescence
polarization, spin-labeling, electron microscopy, and x-
ray crystallography, have shown that the Fab fragments
can rotate and waggle, coming together or moving apart.
As a consequence, IgG molecules can change their shape
from a “Y” to a “T” and vice versa, using the region
intercalated between Cγ1 and Cγ2 as a “hinge.” The
length and primary sequence of the hinge regions play an
important role in determining the segmental flexibility of
IgG molecules. For example, IgG3 has a 12 amino acid
hinge amino terminal segment and has the highest
segmental flexibility. The hinge region is also the most
frequent point of attack by proteolytic enzymes. In
general, the resistance to proteolysis of the different IgG
subclasses is inversely related to the length of the hinge
amino terminal segments—IgG3 proteins are the most
easily digestible, while IgG2 proteins, with the shortest
hinge region, are the most resistant to proteolytic
enzymes.
V. The Immunoglobulin Superfamily of Proteins
The existence of globular “domains” (Fig. 8) is
considered as the structural hallmark of immunoglobulin
structure. A variety of other proteins that exhibit amino
acid sequence homology with immunoglobulins also
contain Ig-like domains (Fig. 9). Such proteins are
considered as members of the immunoglobulin
superfamily, based on the assumption that the genes
which encode them must have evolved from a common
ancestor gene coding for a single domain, much likely
the gene coding for the Thy-1 molecule found on murine
lymphocytes and brain cells.
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  55
1. The T-cell antigen receptor molecule, the major
histocompatibility antigens, the polyimmunoglobulin
receptor on mucosal cells (see below), and the CD2
molecule on T lymphocytes are some examples of
proteins included in the immunoglobulin superfamily.
2. The majority of the membrane proteins of the
immunoglobulin superfamily seem to be functionally
involved in recognition of specific ligands that may
determine cell-cell contact phenomena and/or cell
activation.
VI. The Antibody Combining Site
The binding of antigens by antibody molecules takes
place in the Fab region, and is basically a noncovalent
interaction that requires a good fit between the antigenic
determinant and the antigen binding site on the
immunoglobulin molecule. The antigen binding site
appears to be formed by the variable regions of both
heavy and light chains folded in close proximity, forming
a pouch where an antigenic determinant or epitope will
fit.
A. Hypervariable Regions. Certain sequence stretches
of the variable regions vary widely from protein to
protein, even among proteins sharing the same type of
variable regions. For this reason, these highly variable
stretches have been designated as hypervariable
regions.
B. The structure of hypervariable regions is believed to
play a critical role in determining antibody specificity
since these regions are believed to be folded in such a
way that they form a “pouch” where a given epitope of
an antigen will fit. In other words, the hypervariable
regions will interact to create a binding site whose
configuration is complementary to that of a given
epitope. Thus, these regions can be also designated as
complementarity-determining regions.
VII. Immunoglobulin M: A Polymeric Molecule
A. The Pentameric Nature of IgM. Serum IgM is
basically composed of five subunits (monomeric
subunits, IgMs), each one of them composed of two light
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

chains (k or λ) and two heavy chains (µ). The heavy
chains are larger than those of IgG by about 20,000
daltons, corresponding to an extra domain on the
constant region (CH4).
B. The J chain, a third polypeptide chain, can be
revealed by adequate methodology in IgM molecules.
This is a small polypeptide chain of 15,000 daltons, also
found in polymeric IgA molecules. One single J chain is
found in any polymeric IgM or IgA molecule, regardless
of how many monomeric subunits are involved in the
polymerization. It has been postulated that this chain
plays some role in the polymerization process.

VIII. Immunoglobulin A: A Molecularly

Heterogeneous Immunoglobulin

A. Serum IgA is molecularly heterogeneous, composed

of a mixture of monomeric, dimeric, and larger
polymeric molecules. In a normal individual, over 70–
90% of serum IgA is monomeric. Monomeric IgA is
similar to IgG, and composed of two heavy chains (α)
and two light chains (k or λ). The dimeric and polymeric
forms of IgA found in circulation are covalently bonded
synthetic products containing J chains.
B. IgA is the predominant immunoglobulin in secretions.
Secretory IgA molecules are most frequently dimeric,
contain J chains as do all polymeric immunoglobulin
molecules, and, in addition, contain a unique polypeptide
chain, designated as secretory component (SC) (Fig.11).
C. Secretory component is constituted by a single
polypeptide chain of approximately 70,000 daltons with
five homologous immunoglobulin-like domains. It is
synthesized by epithelial cells in the mucosa and by
hepatocytes, initially as a larger membrane molecule
known as a polyimmunoglobulin receptor, from which
SC is derived by proteolytic cleavage separating SC from
the intramembrane and cytoplasmic segments of its
membrane form.

56 INSTITUTE FOR ADVANCED STUDIES, JODHPUR 
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

IX. The Minor Immunoglobulin Classes: IgD and IgE
A. General Concepts. IgD and IgE were the last
immunoglobulins to be identified due to their low
concentrations in serum. Both are monomeric
immunoglobulins, similar to IgG, but their heavy chains
are larger than γ chains. IgE has five domains in the
heavy chain (one variable and four constant); IgD has
four heavy-chain domains (as most other monomeric
immunoglobulins).
B. IgD and IgM are the predominant immunoglobulin
classes in the B lymphocyte membrane, where they are
the antigen-binding molecules in the antigen-receptor
complex. The B-cell antigen complex is composed of
membrane Ig and several other membrane proteins
including Igα and Igβ, which have sequence motifs in
their cytoplasmic portions that are required for signal
transduction.
1. Membrane IgD and IgM are monomeric. The heavy
chains of membrane IgD and IgM (δm, µm) differ from
that of the secreted forms at their carboxyl termini, where
the membrane forms have a hydrophobic transmembrane
section and a short cytoplasmic tail that are lacking in the
secreted forms. In contrast, a hydrophilic section is found
at the carboxyl termini of heavy chains of secreted Igs.
2. The biological role of circulating IgD is not clear.
C. IgE is an extremely important immunoglobulin
because of its biological properties. Its biological role
appears to be predominantly related to antiparasitic
responses, but its main clinical relevance is related to
allergic reactions.
1. IgE has the unique property of binding to Fcε
receptors on the membranes of mast cells and basophils.
The binding of IgE to those receptors has an extremely
high affinity (7.7 × 109 1/M-1), about 100-fold greater
than the affinity of IgG binding to monocyte receptors.
2. The high affinity binding of IgE to basophil membrane
receptors is the basis for its designation as
homocytotropic antibody, and is responsible for its role
in allergic reactions. In allergic individuals, if those IgE
molecules have a given antibody specificity and react
with the antigen while attached to the basophil or mast
cell membranes, they will trigger the release of histamine
and other substances that cause the symptoms of allergic
reactions.
3. IgE is the most thermolabile immunoglobulin and
loses biological activity (i.e., the ability to bind to high
affinity Fcε receptors) after heating at 56°C for 30
minutes. This binding depends on the tertiary structure of
the C-terminal portion of Cε2 and the N-terminal portion
of the Cε3 domain. Circular dichroism studies
demonstrated that heating changes the configuration of
Cε3 and Cε4 domains, and the changes in configuration
of Cε3 are likely to be critical in preventing proper
binding to the receptor.

E4. Diversity of Antibodies

One unique property of antibodies is their remarkable

diversity. According to current estimates each human can
synthesize more than 1011 (100 billion) different kinds of
antibodies. How is this diversity generated? The answer
is threefold: rearrangement of antibody gene segments,
somatic mutations, and generation of different codons
during antibody gene splicing.

Immunoglobulin genes are split or interrupted genes with

many gene segments. Embryonic B cells contain a small
number of gene segments, close together on the same
chromosome, that determine the constant (C) region of
the light chains (figure 1).

Separated from them, but on the same chromosome, is a

larger cluster of segments that determines the variable
(V) region of the light chains. During B-cell
differentiation, one segment for the constant region is
joined by a process of recombination mediated by
specific proteins to one segment for the variable region.
This splicing produces a complete light-chain antibody
gene. A similar splicing mechanism also occurs to join
the constant and variable segments of the heavy chains.
Because the light-chain genes actually consist of three
parts, and the heavy-chain genes consist of four, the
formation of a finished antibody molecule is slightly
more complicated than previously outlined (figure 2).

INSTITUTE FOR ADVANCED STUDIES, JODHPUR  57
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
The germ line DNA for the light chain gene contains
multiple coding sequences called V and J (joining)
regions. During the differentiation of a B cell, a deletion
The amount of antibody diversity in the mouse that can
be generated by combinatorial joining is shown in table
1. In this animal the k light chains can be formed from
any combination of about 250 to 350 Vk and 4 Jk regions
giving a maximum of approximately 1,400 different k
chains. The λ chains have their own Vλ and Jλ regions
but smaller in number than their k counterparts (6
58 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
(which is variable in length) occurs that joins one V gene
segment with one J segment. This DNA joining process
is termed combinatorial joining because it can create
many combinations of the V and J regions. When the
light-chain gene is transcribed, transcription continues
through the DNA region that encodes for the constant
portion of the gene. RNA splicing subsequently joins the
V J and C regions, creating mRNA. Combinatorial
joining in the formation of a heavy-chain gene occurs by
means of DNA splicing of the heavy-chain counterparts
of V and J along with a third set of D (diversity)
sequences (figure 3a). Initially, all heavy chains have the
µ type of constant region. This corresponds to antibody
class IgM (figure 3b). Another DNA splice joins the VDJ
region with a different constant region that can
subsequently change the class of antibody produced by
the B cell (figure 3c).
different λ chains). The heavy chains have approximately
250 to 1,000 VH, 10 to 30 D, and 4 JH regions, giving a
maximum of 120,000 different combinations. Because
any light chain can combine with any heavy chain, there
will be a maximum of 2x108 possible k chain antibody
types.
The value of 2x 108 different antibodies is actually an
underestimate because antibody diversity is further
augmented by two processes:
1. The V regions of germ line DNA are susceptible to a
high rate of somatic mutation during B-cell development
in response to an antigen challenge. These mutations
allow B-cell clones to produce antibodies with somewhat
different polypeptide sequences.
2. The junction for either VJ or VDJ splicing in
combinatorial joining can occur between different
nucleotides and thus generate different codons in the
spliced gene. For example, one VJ splicing event can join
the V sequence CCTCCC with the J sequence TGGTGG
in two ways: CCTCCC TGGTGG --- CCGTGG, which
codes for the amino acids proline and tryptophan; and
CCTCCC TGGTGG --- CCTCGG, which codes for
proline and arginine. Thus the same VJ joining could
produce polypeptides differing in a single amino acid.
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
E.5 Specific and Non-Specific Immune Response
The vertebrate body possesses two mechanisms which
protect it from potentially dangerous viruses, bacteria,
other pathogens, and abnormal cells which could develop
into cancer.
1. One of these mechanisms is nonspecific, that is, it does
not distinguish between infective agents.
2. The second mechanism is specific in that it responds in
a very specific manner (e.g., production of antibodies) to
the particular type of infective agent.
I. Nonspecific Defense against Infection
Nonspecific defense mechanisms help prevent entry and
spread of invading microbes in an animal's body.
⇒ An invading microbe must cross the external
barrier formed by the skin and
mucous membranes.
⇒ If the external barrier is penetrated, the microbe
encounters a second line of defense: interacting
mechanisms of phagocytic white blood cells,
antimicrobial proteins, and the inflammatory
response.
A. The skin and mucous membranes provide first-line
barriers to infection
The skin and mucous membranes act as physical barriers
preventing entry of pathogens, and as chemical barriers of
anti-pathogen secretions.
⇒ In humans, oil and sweat gland secretions acidify the
skin (pH 3 to 5), which discourages microbial
growth.
⇒ Saliva, tears, and mucous secretions contain
antimicrobial proteins and wash away potential
invading microbes.
⇒ An enzyme (lysozyme) in perspiration, tears, and
saliva attacks the cell walls of many bacteria and
destroys other microbes entering the respiratory
system and eyes.
⇒ In the respiratory tract, nostril hairs filter inhaled
particles and mucus traps microorganisms that are
then swept out of the upper respiratory tract by cilia,
thus preventing their entrance into the lungs.
⇒ In the digestive tract, stomach acid kills many
bacteria that enter with foods or those trapped in
swallowed mucus from the upper respiratory system.
B. Phagocytic cells, inflammation, and antimicrobial
proteins function early in infection
1. Microbes that penetrate the skin or mucous membranes
encounter amoeboid white blood cells capable of
phagocytosis or cell lysis.
Neutrophils are cells that become phagocytic in infected
tissue. Neutrophils are produced in the bone marrow from
the granulocyte-monocyte stem cell. These cells are often
called poly morpho nuclear cells (PMN's). This is because
of the polymorphic shape of the nucleus.
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  59
⇒ Comprise 60% - 70% of total white cells plays
main role is in inflammation..
⇒ They are the first cells to arrive at the site of
inflammation by leaving the blood, through the
endothelium into the tissue (extravasation).
⇒ Attracted by chemical signals (chemotaxis), they
enter infected tissues by amoeboid movement
⇒ Only live a few days as they destroy themselves
when destroying pathogens
⇒ Neutrophils are attracted into the tissue by
chemo tactic factors that include complement
proteins, clotting proteins (stimulated to be
produced by tissue damage) and T cell derived
cytokines. In the tissues, neutrophils are active
phagocytic cells, like macrophages. Neutrophils,
however, do not act as antigen presenting cells.
Neutrophils, instead, are most effective at killing
ingested microorganisms and can do this by
oxygen dependent or independent pathways.
Monocytes comprise only about 5% of the white blood
cells, but they provide an even more effective phagocytic
defense. They mature, circulate for a few hours, then
migrate to the tissues where they enlarge and become
macrophages.
Macrophages are large amoeboid cells that use
pseudopodia to phagocytize microbes, which are then
destroyed by digestive enzymes and reactive forms of
oxygen within the cell. Macrophages are long lived.
⇒ Most wander through interstitial fluid
phagocytosing bacteria, viruses, and cell debris
⇒ Some reside permanently in organs and
connective tissues
⇒ Fixed macrophages are especially numerous in
the lymph nodes and spleen.
Eosinophils represent about 1.5% of the total white cell
count and have limited phagocytic activity.
⇒ Contain destructive enzymes which are
discharged against the outer covering of the
invading pathogen. Eosinophils are granulocytes
named because of their intense staining with
'eosin'.
⇒ Under the microscope, eosinophils typically
have a bilobed nucleus and contain many basic
crystal granules in their cytoplasm. The granules
are eosinophil mediators that are toxic to many
organisms and also to tissues as in asthma
Asthma.
⇒ Eosinophils are motile and phagocytic and are
particularly active in parasitic infection. In fact,
blood work showing an increase in eosinophils
is often caused by parastic diseases.
⇒ Main contribution is defense against larger
invaders such as parasitic worms.
Mast cells are formed in the tissue from undifferentiated
precursor cells released into the blood from the bone
marrow. They are not the tissue counterparts of basophils
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
but they have a similar importance in allergic reactions
and are only found in tissues.
⇒ There are two types of mast cells found either in the
connective tissues or in mucosal sites. Both types
contain numerous granules with preformed mediators
which can be released from mast cells after
stimulation. The preformed mediators include
histamine and other pharmacologically active
substances.
⇒ Stimulation also results in the production of newly
formed mediators by mast cells such as
prostaglandins and leukotrienes. Stimulation of mast
cells occurs in several ways such as by the
anaphylatoxins (C3a, and C5a) of the complement
system or by the crosslinking of surface IgE. Mast
cells have high affinity Fc receptors for the IgE that
is produced against an allergen. As a result, mast cell
release is most significant in either acute
inflammation or in allergic responses.
Natural killer (NK) cells destroy the body's own infected
cells, especially those harboring viruses. These cells are
sometimes called large granular lymphocytes (LGL's). NK
cells have some surface markers in common with T cells,
and they are also functionally similar to cytotoxic T
lymphocytes (CTL's). Like CTL's, NK cells are
particularly important in the killing of cellular targets
(usually tumor cells). Unlike CTL's, however, the killing
by NK cells is nonspecific, they do not need to recognize
antigen/MHC on the target cell. NK cells do not have a T
cell receptor and are not T cells. An NK cell kills a target
cell by releasing perforin (and other molecules) which
damages the target cell membrane leading to death. NK
cells also cause death by inducing apoptosis in the target.
The cytokine TNF alpha is released by the NK cells and
may be involved in this process.
⇒ Also assault abnormal cells that could form
tumors
⇒ Are not phagocytic, but attack the membrane,
causing cell lysis
2. The inflammatory response
A localized inflammatory response occurs when there is
damage to a tissue due to physical injury or entry of
microorganisms.
⇒ Vasodilation of small vessels near the injury
increases the blood supply to the area, which
produces the characteristic redness.
⇒ The dilated vessels become more permeable
allowing fluids to move into surrounding tissues
resulting in localized edema (swelling).
Chemical signals are important in initiating an
inflammatory response
⇒ Histamine is released from injured circulating
basophils (type of white blood cell) and mast
cells (type of cell found in connective tissue).
⇒ Released histamine causes localized
vasodilation, and the capillaries in the area
become leakier.
60 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
⇒ Increased blood flow to the site of injury
delivers clotting elements that help block the
spread of pathogenic microbes and begin the
repair process.
Migration of phagocytic cells into the injured area is also a
result of increased blood flow and increased leakage from
the capillaries.
⇒ Phagocytes are attracted to the damaged tissues
by several chemicals given off.
⇒ Neutrophils arrive first, followed closely by
monocytes which develop into macrophages.
⇒ The neutrophils eliminate microorganisms and
then die.
⇒ Macrophages destroy pathogens and clean up
the remains of damaged tissue cells and dead
neutrophils.
⇒ Dead cells and fluid leaked from the capillaries
may accumulate as pus in the area before it is
absorbed by the body.
3. Antimicrobial proteins
A number of proteins function in nonspecific defense by
either directly attacking microorganisms or impeding their
reproduction. The three most important nonspecific
protein groups are complement proteins, interferon and
the Cytokines.
The complement system is a group of at least 20 serum
proteins that interact with other defense mechanisms.
These proteins interact in a series of steps that result in
lysis of the invading microbes. Complement activation is a
cascade of events which resembles in some ways the
coagulation pathway. It consists of a series of enzymes
that become activated by each other in a cascade fashion.
It's confusing and hard to remember, but still important
because complement has an important role in clearing
antigen. This is done by the four major functions of
complement activation:
⇒ local vasodilation
⇒ attraction of immune cells, especially
phagocytes (chemotaxis)
⇒ opsonization (or tagging) of foreign organisms
for phagocytosis
⇒ destruction of invading organisms by the
membrane attack complex (MAC attack)
The interferons are substances produced by virus-
infected cells that help other cells resist infection by the
virus.
⇒ They are secreted by infected cells as a
nonspecific defense earlier than specific
antibodies appear.
⇒ Cannot save the infected cell, but their diffusion
to neighboring cells stimulates production of
proteins in those cells that inhibit viral
replication.
⇒ Not a virus-specific defense; interferon
produced to infection by one strain of virus
produces resistance in cells to other unrelated
viruses.
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
⇒ Most effective against short-term infections
(colds and influenza).
⇒ Interferons are now being mass produced using
recombinant DNA technology and are being
tested as treatments for viral infections and
cancer.
Cytokines are the chemicals produced by these cells in
order to communicate and orchestrate the attack. Just as
hormones in the endocrine system can produce an effect
on other cells, so cytokines can act on other immune cells,
especially cells that are close by.Cytokines have several
important characteristics:
⇒ The same cytokine may be made by a number of
different cells.
⇒ The same cytokine may have different effects in
different circumstances. (This is called
'pleotropy')
⇒ Different cytokines may have the same activity
depending on the situation ('redundancy').
⇒ Cytokines often act together and increase the
effects of one another ('synergy').
⇒ They may also act as antagonists.
⇒ Most cytokines have either paracrine or
autocrine effects. Paracrine means they act on
cells near to them or that they are actually
touching. The autocrine function of IL-2 is well
known because, when a T cell is stimulated to
make IL-2, it stimulates itself via the IL-2
receptor to proliferate. An example of an
uncommon endocrine function for cytokines is
IL-1 which can cause fever by stimulating the
hypothalamus.
II. How Specific Immunity Arises
The Adaptive immune system is the body's third line of
defense and is very specific in its response.
It is distinguished from nonspecific defenses by:
¾ Specificity and Diversity
¾ Self/non-self recognition
¾ Memory
Specificity refers to the immune system's ability to
recognize and eliminate particular microorganisms and
foreign molecules Lymphocytes provide the specificity
and diversity of the immune system. Lymphocytes (a type
of white blood cell) are responsible for both humoral and
cell-mediated immunity. The different responses are due
to the two main classes of lymphocytes in the body: B
cells and T cells.
⇒ Early B and T cells (as well as other
lymphocytes) develop from stem cells in the
bone marrow and are very much alike. They
only differentiate after reaching their site of
maturation.
⇒ B cells (B lymphocytes) are responsible for the
humoral immune response.
⇒ They form in the bone marrow and remain there
to complete their maturation.
⇒ T cells (T lymphocytes) are responsible for the
cell-mediated immune response.
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  61
⇒ They also form in the bone marrow, and then
migrate to the thymus gland to mature.
Diversity refers to the immune system's ability to respond
to numerous kinds (millions) of invaders, which are
recognized by their antigenic markers. Each population of
antibody-producing lymphocytes is stimulated by a
specific antigen; the stimulated lymphocytes synthesize
and secrete the appropriate antibody.
Antigens interact with specific lymphocytes, inducing
immune responses and immunological memory. The
ability of the immune system to respond to the wide
variety of antigens which enter the body is based in the
enormous diversity of antigen-specific lymphocytes
present in the system. Each lymphocyte will recognize and
respond to only one antigen. This specificity is determined
during embryonic development before any antigens are
encountered, and is the consequence of the antigen
receptor on the lymphocyte's surface.
When an antigen enters the body and binds to receptors on
the specific lymphocytes, those lymphocytes are activated
and begin to divide and to differentiate.
⇒ The divisions produce a large number of
identical effector cells (clones), which bind to
the antigen that stimulated the response.
⇒ If, for example, a B cell is activated, it will
proliferate to produce a large number of plasma
cells that will each secrete an antibody that
functions as an antigen receptor for the specific
antigen that activated the original B cell.
Thus, each antigen activates only a small number of the
diverse group of lymphocytes. The activated cells
proliferate to produce a clone of millions of effector cells
which are specific for the original antigen (clonal
selection).
⇒ Clonal selection = Antigenic-specific selection
of a lymphocyte that activates it to produce
clones of effector cells dedicated to eliminating
the antigen that provoked the initial immune
response. One clone of cells consists of effector
cells. Another clone of cells consists of memory
cells.
⇒ Effector cells are the short-lived cells that
actually defend the body during an immune
response.
⇒ Effector cells are populations of cells resulting
from division of lymphocytes that were
activated by the binding of antigens to their
antigen receptors.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
E6. INTERACTION RESPONSIBLE FOR ANTIGEN-ANTIBODY INTERACTION:
Strength of Antigen-Antibody Interactions
The noncovalent interactions that form the basis of
antigenantibody (Ag-Ab) binding include hydrogen
bonds, ionic bonds, hydrophobic interactions, and van
der Waals interactions (Figure 1). Because these
interactions are individually weak (compared with a
covalent bond), a large number of such interactions are
required to form a strong Ag-Ab interaction.
Furthermore, each of these noncovalent interactions
operates over a very short distance, generally about 1 x
10 -7 mm (1 angstrom, Å); consequently, a strong Ag- Ab
interaction depends on a very close fit between the
antigen and antibody. Such fits require a high degree of
complementarity between antigen and antibody, a
requirement that underlies the exquisite specificity that
characterizes antigen-antibody interactions.
62 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
Antibody Affinity Is a Quantitative Measure of
Binding Strength
The combined strength of the noncovalent interactions
between a single antigen-binding site on an antibody and
a single epitope is the affinity of the antibody for that
epitope. Low-affinity antibodies bind antigen weakly and
tend to dissociate readily, whereas high-affinity
antibodies bind antigen more tightly and remain bound
longer. The association between a binding site on an
antibody (Ab) with a monovalent antigen (Ag) can be
described by the equation
Antibody Avidity
The affinity at one binding site does not
always reflect the true strength of the
antibody-antigen interaction. When
complex antigens containing multiple,
repeating antigenic determinants are
mixed with antibodies containing
multiple binding sites, the interaction of
an antibody molecule with an antigen
molecule at one site will increase the
probability of reaction between those
two molecules at a second site. The
strength of such multiple interactions
between a multivalent antibody and
antigen is called the avidity. The
avidity of an antibody is a better
measure of its binding capacity within
biological systems (e.g., the reaction of
an antibody with antigenic determinants
on a virus or bacterial cell) than the
affinity of its individual binding sites.
High avidity can compensate for low
affinity. For example, secreted
pentameric IgM often has a lower affinity than IgG, but
the high avidity of IgM, resulting from its higher
valence, enables it to bind antigen effectively.
Cross-Reactivity
Although Ag-Ab reactions are highly specific, in some
cases antibody elicited by one antigen can cross-react
with an unrelated antigen. Such cross-reactivity occurs
if two different antigens share an identical or very similar
epitope. In the latter case, the antibody’s affinity for the
cross-reacting epitope is usually less than that for the
original epitope.
Cross-reactivity is often observed among polysaccharide
antigens that contain similar oligosaccharide residues.
The ABO blood-group antigens, for example, are
glycoproteins expressed on red blood cells. Subtle
differences in the terminal residues of the sugars attached
to these surface proteins distinguish the A and B blood-
group antigens.An individual lacking one or both of
these antigens will have serum antibodies to the missing
antigen(s). The antibodies are induced not by exposure to
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
red blood cell antigens but by exposure to cross-reacting
microbial antigens present on common intestinal
bacteria. These microbial antigens induce the formation
of antibodies in individuals lacking the similar blood-
group antigens on their red blood cells. (In individuals
possessing these antigens, complementary antibodies
would be eliminated during the developmental stage in
which antibodies that recognize self epitopes are weeded
out.) The blood-group antibodies, although elicited by
microbial antigens, will cross-react with similar
oligosaccharides on foreign red blood cells, providing the
basis for blood typing tests and accounting for the
necessity of compatible blood types during blood
transfusions. A type A individual has anti-B antibodies; a
E 7. PRIMARY AND SECONDARY IMMUNE RESPONE:
The primary immune response is the proliferation of
lymphocytes to form clones of effector cells specific to an
antigen during the body's first exposure to the antigen.
⇒ There is a 10- to 17-day lag period between
initial exposure and maximum production of
effector cells.
⇒ The lymphocytes selected by the antigen are
differentiating into effector B cells and T cells
during the lag period.
⇒ Activated B cells give rise to effector cells
called plasma cells, which secrete antibodies
(humoral response) that eliminate the activating
antigen.
A secondary immune response occurs when the body is
exposed to a previously encountered antigen.
⇒ The response is faster (2 to 7 days) and more
prolonged than a primary response.
⇒ The antibodies produced are more numerous,
and they are more effective at binding the
antigen.
This ability to recognize a previously encountered antigen
is known as immunological memory.
⇒ Based on memory cells, which are produced
during clonal selection for effectors in a
primary immune response.
⇒ Memory cells are not active during the primary
response and survive in the system for long
periods. (Effector cells produced in the primary
response are active, and thus, short-lived.)
⇒ When the same antigen that caused a primary
immune response again enters the body, the
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  63
type B individual has anti-A; and a type O individual
thus has anti-A and anti-B.
A number of viruses and bacteria have antigenic
determinants identical or similar to normal host-cell
components. In some cases, these microbial antigens
have been shown to elicit antibody that cross-reacts with
the host-cell components, resulting in a tissue-damaging
autoimmune reaction. The bacterium Streptococcus
pyogenes, for example, expresses cell-wall proteins
called M antigens. Antibodies produced to streptococcal
M antigens have been shown to cross-react with several
myocardial and skeletal muscle proteins and have been
implicated in heart and kidney damage following
streptococcal infections.
memory cells are activated and rapidly
proliferate to form a new clone of effector cells
and memory cells.
⇒ These new clones of effector and memory cells
are the secondary immune response.
⇒ This acquired immunity has long been
recognized as a resistance to some infections
encountered earlier in life (e.g., chicken pox).
Lymphocyte development gives rise to an immune system
that distinguishes self from non-self
⇒ Like all blood cells, lymphocytes derive from
stem cells in bone marrow or liver of a
developing embryo. Initially, all lymphocytes
are alike, but depending on the site of
maturation, they develop into T cells or B cells.
⇒ Cells that remain in the bone marrow develop
into B cells The "B" stands for bursa of
Fabricius an organ found in birds where they
were first discovered. However, you can equate
the "B" with bone.
⇒ Cells that migrate to the thymus
develop/matures into T cells. The "T" stands for
thymus.
Immune tolerance for self
Self/non-self recognition is the ability of the immune
system to distinguish between the body's own molecules
and foreign molecules (antigens).
⇒ Develops before birth when T and B
lymphocytes begin to mature in the thymus and
bone marrow of the embryo.
⇒ Antigen receptors on the surfaces of
lymphocytes are responsible for detecting
foreign molecules that enter the body. There are
no lymphocytes reactive against the body's own
molecules under normal conditions.
⇒ Self-tolerance = the lack of a destructive
immune response to the body's own cells.
⇒ Failure of this system leads to autoimmune
disorders that destroy the body's own tissues.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
E8. The Complement Components
The proteins and glycoproteins that compose the
complement system are synthesized mainly by liver
hepatocytes, although significant amounts are also
produced by blood monocytes, tissue macrophages, and
epithelial cells of the gastrointestinal and genitourinary
tracts. These components constitute 5% (by weight) of
the serum globulin fraction.
Most circulate in the serum in functionally inactive forms
as proenzymes, or zymogens, which are inactive until
proteolytic cleavage, which removes an inhibitory
fragment and exposes the active site.
The complement-reaction sequence starts with an
enzyme cascade. Complement components are
designated by numerals (C1–C9), by letter symbols (e.g.,
factor D), or by trivial names (e.g., homologous
restriction factor).
Peptide fragments formed by activation of a component
are denoted by small letters. In most cases, the smaller
fragment resulting from cleavage of a component is
designated “a” and the larger fragment designated “b”
(e.g., C3a, C3b; note that C2 is an exception: C2a is the
larger cleavage fragment).
The larger fragments bind to the target near the site of
activation, and the smaller fragments diffuse from the
site and can initiate localized inflammatory responses by
binding to specific receptors.
The complement fragments interact with one another to
form functional complexes. Those complexes that have
enzymatic activity are designated by a bar over the
number or symbol (e.g., C4b2a, C3bBb).
Complement Activation
Figure 1 outlines the pathways of complement activation.
The early steps, culminating in formation of C5b, can
occur by the classical pathway, the alternative
pathway, or the lectin pathway. The final steps that lead
to a membrane attack are the same in all pathways.
The Classical Complement Pathway
Activation of the classical complement pathway
requires initiation by the interaction of antibodies with an
antigen that is usually cell bound (figure 1).
The order of effectiveness in activating complement is as
follows: IgM Æ IgG3 Æ IgG1Æ IgG2.
However, some microbial products (lipid A of endotoxin
and staphylococcal protein A) or plasmin (a proteolytic
64 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
enzyme that dissolves the fibrin of blood clots) may
activate C1 directly without antibody participation.
Following binding of antigen to antibody, the C1
complement component, which is composed of three
proteins (q, r, and s), attaches to the Fc portion of the
antibody molecule through its C1q subcomponent.
In the presence of calcium ions, a trimolecular complex
(C1qrs - Ag - Ab) that has esterase activity is rapidly
formed.
The activated C1’s subcomponent attacks and cleaves its
natural substrates in serum (C2 and C4).
This leads to binding of a portion of each molecule (C2b
and C4b) to the antigen-antibody-complement complex
with the release of small C4a and C2a fragments. (The
released C2 fragment traditionally has been called C2a.
For consistency, this text will label all larger complement
fragments that are bound to the target cell as b
fragments.) With the binding of C2b to C4b, an enzyme
with trypsinlike proteolytic activity is generated. The
natural substrate for this enzyme is C3; thus it is termed a
C3 convertase.
Through the activity of C 4b2b (the bar indicates an
active enzyme complex), C3 is cleaved into a bound
subcomponent C3b and a C3a soluble component.
C3b then absorbs to bound C4b2b, forming the complex
C 4b2b 3b, which acts as a C5 convertase and cleaves
C5 into fragments C5a and C5b. C6 and C7 rapidly bind
to C5b, forming a C 5b67 complex that possesses an
unstable membrane-binding site; once bound to a
membrane, this complex is stable.
C8 and C9 then bind, forming the membrane attack
complex (C 5b67 89) that creates a pore in the plasma
membrane of the target cell. It is believed that the actual
pore is a doughnut-shaped polymer of C9. (The perforin
pores generated by cytotoxic T lymphocytes are
somewhat similar to complement pores.)
If the cell is eucaryotic, Na and H2O enter through the
pore leading to osmotic lysis of the cell. If the cell is a
gram-negative bacterium, lysozyme from the blood
enters through the pore and digests the peptidoglycan cell
wall causing the bacterium to lyse osmotically. In
contrast, gram-positive bacteria are resistant to the
cytolytic action of the membrane attack complex because
they lack an exposed outer membrane and the thick
peptidoglycan prevents an attack on the plasma
membrane.
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
E9. MAJOR HISTOCOMPATIBILITY COMPLEX (HLA in HUMANS):
The major histocompatibility complex (MHC;
sometimes referred to as human leukocyte antigens or
HLA in humans) is a group of glycoproteins (proteins
with attached sugar chains) embedded in the plasma
membranes of cells.
As T cells interacts with the antigen that come form the
inside of the cells they use an antigen recognition system
which is distinct from the antigen recognition
system.The T cell receptor (TCR) for antigen interacts
with the antigen almost exclusively only when fragment
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  65
of antigen is on surface of cell and, bound to a molecule
known as MHC gene product.
Thus the role of MHC molecules coded by the MHC is
to bind to peptide fragments derived from the protein
antigens and interact with TCR. The Binding of MHC
molecule with peptide is selective.
⇒ MHC molecules may be viewed as third set of
recognition molecules for the antigen in the
immune response, in addition to antigen
specific T cells and B cells.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

⇒ Every vertebrate species has MHC genes and

products which are cause of rejection of tissues
between different members of same species
and are dominated by T cells.
⇒ These important "self-markers" are coded for
by a family of genes
⇒ There are at least 20 MHC genes and at least
100 alleles for each gene.
⇒ This results in millions of different MHC
molecules.
⇒ The probability that two individuals will have
matching MHC sets is virtually zero unless
they are identical twins.

There are two main classes of MHC molecules in the

body:
¾ Class I MHC molecules are located on all
nucleated cells of the body.
¾ Class II MHC molecules are found only
on specialized cells, such as
macrophages, B cells, and activated T Although MHC molecules give identity to the cell as
cells. being "self" the major function of MHC molecules is to
bind to an antigen, then present this antigen on the
surface of the cell, thereby facilitating antigen binding to
a T cell
⇒ Class MHC I molecules facilitate antigen
binding to cytotoxic T
cells (CD 8 Cells).
⇒ Class MHC II molecules facilitate antigen
binding to helper T cells
(CD 4 cells).

Each MHC-antigen combination forms a unique

complex that is recognized by specific antigen receptors
on certain T cells

E10. HUMORAL AND CELL MEDIATED Immune Responses

The body will mount either a humoral response or a cell-
mediated response, depending on the antigen which
stimulates the system.
⇒ Humoral immunity produces antibodies in
response to toxins (ex bee venom), free
bacteria, and viruses present in the body fluids.
⇒ "Humor" is medieval term for body fluids.
Here it refers to the fluid of the blood and the
lymph.
⇒ Antibodies to these types of antigens are
synthesized by certain lymphocytes and then
secreted as soluble proteins which circulate
through the body in blood plasma and lymph.
⇒ Cell-mediated immunity is the response to
intracellular bacteria and viruses, fungi,
protozoans, worms, transplanted tissues, and
cancer cells.
⇒ Depends on the direct action of certain types
of T- lymphocytes rather than antibodies.
In humoral immunity or cell-mediated immunity, there
are several general steps involved in the immune
responses.
1. The antigen must encounter the B-lymphocytes, T-
lymphocytes, and antigen-presenting cells (APCs)
capable of carrying out an adaptive immune
response.
2. Naive lymphocytes must recognize epitopes of an
antigen by means of antigen-specific receptor
molecules on their surface and become activated.
This is known as clonal selection.
3. The large clones of identical B-lymphocytes, T4-
lymphocytes, and T8-lymphocytes now differentiate
into effector cells capable of directing body
defenses against the original antigen resulting in its
destruction or neutralization.
4. Some of the B-lymphocytes, T4-lymphocytes, and
T8-lymphocytes differentiate into long-lived,
circulating memory cells.

66 INSTITUTE FOR ADVANCED STUDIES, JODHPUR 
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

In the humoral response, B cells produce antibodies
against extracellular pathogens
⇒ The humoral response occurs when an antigen
binds to B cell receptors that are specific for
the antigen.
⇒ The B cells differentiate into a clone of plasma
cells which begin to secrete antibodies that are
most effective against pathogens circulating in
the blood or lymph.
⇒ Memory cells are also produced and form the
basis for secondary immune responses.
The antibodies produced during humoral immunity
ultimately defend the body through a variety of different
means. These include:
1. Opsonization
Opsonization, or enhanced attachment, refers to the
antibody molecules IgG, IgE and the complement
proteins C3b and C4b attaching antigens to phagocytes.
This results in much more efficient phagocytosis.

The selective activation of a B cell results from one of

two mechanisms:

1. T-dependent antigens: Antigens that evoke the

cooperative response of macrophages, helper T cells, and
B cells
o These antigens cannot stimulate antibody
production without helper T cell
involvement.
o Most antigens are T-dependent. Memory
cells are produced in T-dependent
responses.

2. T-independent antigens: Antigens that trigger

humoral immune responses without macrophages or T
cell involvement
o These antigens usually are long chains of
repeating units, such as polysaccharides or
protein subunits often found in bacterial
capsules and flagella.
o B cells are stimulated directly by the
antigen, which probably binds
simultaneously to several antigen receptors
on the B cell surface.
o The antibody production (humoral response)
is usually much weaker than that of T-
dependent antigens.
o No memory cells are generated in T-
independent responses.
Whether activated by T-dependent or T-independent
antigens, a B cell gives rise to a clone of plasma cells.
⇒ Each of these effector cells secretes up to 2000
antibodies per second into the body fluids for
its 4- to 5-day lifespan.
⇒ The specific antibodies help eliminate the
foreign invader from the body.
2. Antibody-Dependent Cellular Cytotoxicity
(ADCC) by NK cells
NK cells are capable of antibody-dependent cellular
cytotoxicity or ADCC. NK cells have receptors on their
surface for the Fc portion of IgG. When IgG is made
against epitopes on "foreign" membrane-bound cells,
e.g., virus-infected cells and cancer cells, the Fab
portions of the antibodies react with the "foreign" cell.
The NK cells then bind to the Fc portion of the antibody.
The NK cell then releases pore-forming proteins called
perforins, proteolytic enzymes called granzymes, and
chemokines. Granzymes pass through the pores and
activate the enzymes that lead to apoptosis of the
infected cell by means of destruction of its structural
cytoskeleton proteins and by chromosomal degradation.
As a result, the cell breaks into fragments that are
subsequently removed by phagocytes. Perforins can also
sometimes result in cell lysis. (When NK cells are
carrying out ADCC, they are sometimes also referred to
as killer cells.)

INSTITUTE FOR ADVANCED STUDIES, JODHPUR  67
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

3. Neutralization of Exotoxins

For an exotoxin to cause harm it must first bind to

receptors on a susceptible host cell. Antitoxin antibodies,
mainly IgG, are made against protein exotoxins. They
combine with the exotoxin molecules before they can
interact with host target cells and thus neutralize the
toxin.

IN THE CELL-MEDIATED RESPONSE,

CYTOTOXIC-T CELLS DEFEND AGAINST
INTRA-CELLULAR PATHOGENS

⇒ Antigen-activated cytotoxic T cells kills cells

4. Neutralization of Viruses
In order for viruses to infect a cell and replicate, they
must first adsorb to receptors on the host cell's plasma
membrane. Antibodies made against the epitopes on
viral capsids or on viral envelope glycoproteins that
function in the attachment of the virus to the host cell
receptor prevent viral adsorption (see Fig). Neutralizing
antibodies are especially important in preventing viral
reinfection.
that are infected by pathogens (e.g., viruses,
bacteria).
⇒ Host cells infected by viruses and other
pathogens display antigens complexed with
class I MHC molecules on their surfaces.
⇒ Cytotoxic T cells have specific receptors that
recognize and bind to antigen-class I MHC
markers. (Note that this differs from Helper T
cells which bind to antigen-class II MHC
complexes.)
⇒ The cytotoxic T receptor can bind to any cell
in the body displaying the antigen-class I
MHC marker since class I MHC is present on
all nucleated cells.
⇒ When a cytotoxic T cell binds to an infected
cell (target cell), it releases chemicals that
forms a lesion in the infected cell's membrane.
⇒ Cytoplasm escapes through the lesion and
eventually cell lysis occurs.
⇒ Destruction of the host cell not only removes
the site where pathogens can reproduce, but
also exposes the pathogens to circulating
antibodies from the humoral response.
⇒ Cytotoxic cells continue to live after
destroying the infected cell and may kill many
others displaying the same antigen-class I
MHC marker.

6. Agglutination of Microorganisms

Agglutination is mainly a function of antibodies with

multiple reactive Fab sites such as IgM and IgA. The
antibodies link microorganisms together (cause them to
agglutinate) so they can be filtered out of the lymph and
blood and be phagocytosed more effectively by the fixed
macrophages of the mononuclear phagocytic
(reticuloendothelial) system (see Fig)

68 INSTITUTE FOR ADVANCED STUDIES, JODHPUR 
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
Helper T lymphocytes function in both humoral and
cell-mediated immunity
Cells that take up antigens, such as B cells and
macrophages alert the immune system, via helper T
cells, of the presence of a foreign antigen.
⇒ Such cells engulf foreign material (e.g.,
bacteria)
⇒ The foreign protein (e.g., from broken down
bacteria) binds to a class II MHC molecule and
is conveyed to the outside of these cells
⇒ The class II MHC molecule with bound
foreign antigen is recognized by a helper T
cell.
E11. IMMUNITY AND HEALTH AND DISEASE
A. Immunity can be achieved naturally or artificially
Active immunity is the immunity conferred by recovery
from an infectious disease.
⇒ Depends on response by the person's own
immune system
⇒ May be acquired naturally from an infection to
the body or artificially by immunization, also
known as vaccination
⇒ Vaccines may be inactivated bacterial toxins,
killed microbes, parts of microbes, or viable but
weakened microbes.
⇒ In all cases the organisms can no longer cause
the disease but can act as antigens and stimulate
an immune response.
⇒ A person vaccinated against an infectious agent
who encounters the pathogen will show the
same rapid, memory-based secondary response
as someone who has had the disease.
Passive immunity is immunity which has been transferred
from one individual to another by the transfer of
antibodies.
⇒ Naturally occurs when IgG antibodies cross the
placenta from a pregnant woman to her fetus.
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  69
Helper T cells that bind to a class II MHC-antigen
complex on these cells are induced to differentiate into
either of two clones of cells:
1. Activated helper T cells: Secrete chemicals that
stimulate other lymphocytes (e.g., stimulates
differentiation of B cells into antibody-secreting plasma
cells and induces cytotoxic T cells to become active
killers).
2. Memory helper T cells: These cells become
important when there is a second exposure to the antigen
⇒ Some antibodies (IgA) are transferred to nursing
infants though breast milk.
⇒ Provides temporary protection to newborns
whose immune systems are not fully operational
at birth. - - Persists as long as the antibodies last
(a few weeks or months), but it can provide
protection from infections until a baby's own
immune system has matured.
B. The immune system's capacity to distinguish self
from non-self limits blood transfusion and tissue
transplantation
The body's immune system distinguishes between self (the
body's own cells) and nonself (foreign cells). Nonself
includes pathogens and cells from other individuals of the
same species.
1. Blood groups and blood transfusion
The human ABO blood groups provide a good example of
nonself recognition. The antigen present on the surface of
the erythrocytes is not antigenic to that person but may be
recognized as foreign if placed in the body of another
individual.
⇒ Individuals of blood type A have the A antigen
and make anti-B antibodies.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
⇒ Individuals of blood type B have the B antigen
and make anti-A antibodies.
⇒ Individuals of blood type AB have the A and B
antigen and make no antibodies.
⇒ Individuals of blood type O have neither the A
nor B antigen and make anti-A and anti-B
antibodies.
Blood group antibodies can cause blood of a different
antigenic type to agglutinate, resulting in a life-threatening
transfusion reaction.
⇒ Type O individuals are universal donors since
their blood has neither antigen.
⇒ Type AB individuals are universal recipients
since they produce neither antibody A or
antibody B.
⇒ The blood group antibodies are present in the
body before a transfusion occurs because they
form in response to the body's normal bacterial
flora that have epitopes very similar to blood
group antigens. This phenonomenon is termed
as cross reactivity.
Usually IgM class antibodies do not cross the placenta,
thus they present no harm to a developing fetus with a
blood type different from the mother.
⇒ The Rh factor is another blood group antigen.
Rh factor causes problems when a mother is Rh
negative and her fetus is Rh positive (inherited
from the father).
⇒ When small amounts of fetal blood cross the
placenta and come into contact with the mother's
lymphocytes, the mother develops antibodies
against the Rh factor.
⇒ Usually only a problem in the second child since
the response will be quick due to sensitization
and formation of memory cells during the first
baby's gestation.
⇒ Unlike blood group antibodies, Rh antibodies
are IgG class, which can cross the placenta.
⇒ The mother's antibodies cross the placenta and
destroy the red blood cells of the Rh positive
fetus.
2. Tissue grafts and organ transplantation
The MHC is a biochemical fingerprint unique to each
individual.
⇒ Complicates tissue grafts and organ transplants
since foreign MHC molecules are antigens and
cause cytotoxic T cells to mount a cell-mediated
response. In the case of bone marrow
transplants, which are used to treat leukemia and
other cancers, the graft rather than the host is the
source of immune rejection
⇒ The donated bone marrow contains lymphocytes
that will react against the recipient.
⇒ This graft versus host rejection is limited if the
MHC molecules of the donor and recipient are
well matched.
The reactions of the immune system to transfusions, tissue
grafts, and organ transplants are normal reactions of a
healthy immune system, not disorders of the system.
70 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
C. Abnormal immune function can lead to disease
1. Allergies (Hypersensitivity): Allergy = A
hypersensitivity of the body's defense system to an
environmental antigen called an allergen. Some believe
these reactions to be evolutionary remnants to infection by
parasitic worms due to similarities in the responses. IgE
class antibodies are commonly involved in allergic
reactions; these antibodies recognize pollen as allergens
⇒ IgE antibodies attach by their tails to
noncirculating mast cells found in connective
tissues. (Remember mast cells are involved in
the inflammatory response.)
⇒ When a pollen grain bridges the gap between
two adjacent IgE monomers, the mast cell
responds with a reaction called degranulation.
⇒ Degranulation involves the release of histamine
and other inflammatory agents.
⇒ Histamine causes dilation and increased
permeability of small blood vessels, which
results in the common symptoms of an allergy.
⇒ Antihistamines are drugs used to treat allergies
since they interfere with the action of histamine.
⇒ Anaphylactic shock is a life-threatening reaction
to injected or ingested antigens; it is the most
serious type of acute allergic response.
⇒ Occurs when mast cell degranulation causes a
sudden dilation of peripheral blood vessels and a
drastic drop in blood pressure.
⇒ Death may occur in a few minutes.
⇒ This hypersensitivity may be associated with
foods (peanuts, fish) or insect venoms (wasp or
bee stings).
⇒ Epinephrine may be injected to counteract the
allergic response.
2. Autoimmune diseases
Autoimmune disease = An immune system reaction
against self Examples:
⇒ Some cases involve immune reactions against
components of the body's own cells, which are
released by the normal breakdown of skin and
other tissues, especially nucleic
acids(DNA/RNA) in Systemic Lupus
Erythematous(SLE).
⇒ Rheumatoid arthritis is an autoimmune disease
in which inflammation damages cartilage and
bones in joints.
⇒ Destruction of insulin-producing pancreas cells
by an autoimmune reaction appears to cause
insulin-dependent diabetes.
⇒ In multiple sclerosis, T cells reactive against
myelin infiltrate the central nervous system and
destroy the myelin of neurons.
⇒ Antibodies produced to repeated streptococcal
infections may react with heart tissues and cause
valve damage in some people.
⇒ Other autoimmune diseases are Grave's disease
and Rheumatic fever.
 
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INSTITUTE FOR ADVANCED STUDIES, JODHPUR  71
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
3. Immunodeficiency diseases
Immunodeficiency refers to a condition where an
individual is inherently deficient in either humoral or cell-
mediated immune defenses.
™ Severe combined immunodeficiency (SCID) is a
congenital disorder in which both the humoral and
cell-mediated immune defenses fail to function.
Not all cases of immunodeficiency are inborn conditions.
⇒ Some cancers, like Hodgkin's disease, damage
the lymphatic system and make the individual
susceptible to infection.
⇒ Some viral infections cause depression of the
immune system (e.g., AIDS).
™ Acquired immunodeficiency syndrome (AIDS):
Acquired immunodeficiency syndrome is a severe
immune system disorder caused by infection with the
human immunodeficiency virus (HIV). Individuals
with AIDS are highly susceptible to opportunistic
diseases, infections, and cancers that take advantage
of a deficient immune system. Mortality rate
approaches 100%. HIV probably evolved from
another virus in central Africa and may have gone
unrecognized for many years. There are two major
72 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
strains: HIV-1 and HIV-2. HIV infects cells,
including helper T cells.
⇒ After entry, HIV RNA is reverse-transcribed and
the product DNA is integrated into the host cell
genome.
⇒ In this provirus form, the viral genome directs
the production of new virus particles.
⇒ HIV is not eliminated from the body by
antibodies for several reasons:
⇒ The latent provirus is invisible to the immune
system.
⇒ The virus undergoes rapid mutational changes in
antigens during replication which eventually
overwhelms the immune system.
⇒ The population of helper T-cells eventually
declines to the point where cell-mediated
immunity collapses. Researchers are not entirely
sure why this occurs.
⇒ Secondary infections characteristic of HIV
infection develop (Pneumocystis pneumonia and
Kaposi's sarcoma).
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
E12. IMMUNIZATION AND VACCINES:
Immunization is the means of providing specific
protection against most common and damaging pathogens.
Specific immunity can be acquired either by passive or by
active immunization and both modes of immunization can
occur by natural or artificial means.
Passive Immunity: Immunity can be acquired, without
the immune system being challenged with an antigen. This
is done by transfer of serum or gamma-globulins from an
immune donor to a non-immune individual. Alternatively,
immune cells from an immunized individual may be used
to transfer immunity. Passive immunity may be acquired
naturally or artificially.
Naturally acquired passive immunity: Immunity is
transferred from mother to fetus through placental transfer
of IgG or colostral transfer of IgA.
Artificially acquired passive immunity: Immunity is
often artificially transferred by injection with γ-globulins
from other individuals or γ-globulin from an immune
animal. Passive transfer of immunity with immune
globulins or γ-globulins is practiced in numerous acute
situations of infections (diphtheria, tetanus, measles,
rabies, etc.), poisoning (insects, reptiles, botulism), and as
a prophylactic measure (hypogammaglobulinemia). In
these situations, γ-globulins of human origin are preferable
although specific antibodies raised in other species are
effective and used in some cases (poisoning, diphtheria,
tetanus, gas gangrene, botulism). While this form of
immunization has the advantage of providing immediate
protection, heterologous γ-globulins are effective for only
a short duration and often result in pathological
complications (serum sickness) and anaphylaxis.
Active Immunity: This refers to immunity produced by
the body following exposure to antigens.
Naturally acquired active immunity: Exposure to
different pathogens leads to sub-clinical or clinical
infections which result in a protective immune response
against these pathogens.
Artificially acquired active immunity: Immunization
may be achieved by administering live or dead pathogens
or their components. Vaccines used for active
immunization consist of live (attenuated) organisms, killed
whole organisms, microbial components or secreted toxins
(which have been detoxified).
⇒ The first live vaccine was cowpox virus introduced
by Edward Jenner as a vaccine for smallpox;
however, variolation, innoculation using pus from a
patient with a mild case of smallpox has been in use
for over a thousand years.
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  73
⇒ Live vaccines are used against a number of viral
infections (polio (Sabin vaccine), measles, mumps,
rubella, chicken pox, hepatitis A, yellow fever, etc.).
The only example of live bacterial vaccine is one
against tuberculosis (Mycobacterium bovis: BCG).
While live vaccines normally produce only self-
limiting non-clinical infections and subsequent
immunity, they carry a serious risk of causing overt
disease in immunocompromised individuals.
⇒ Killed (heat, chemical or UV irradiation) viral
vaccines include those for polio (Salk vaccine),
influenza, rabies, influenza, rabies, etc. Most
bacterial vaccines are killed organisms ( typhoid,
cholera, plague, pertussis, etc.).
⇒ Other bacterial vaccines utilize their cell wall
components (haemophilus, pertussis, meningococcus,
pneumococcus, etc.). Some viral vaccines (hepatitis-
B, rabies, etc.) consist of antigenic proteins cloned
into a suitable vector (e.g., yeast). When the
pathogenic mechanism of an agent involves a toxin, a
modified form of the toxin (toxoid) is used as a
vaccine (e.g., diphtheria, tetanus, cholera). These
subunit vaccines are designed to reduce the toxicity
problems. Each type of vaccine has its own
advantages and disadvantages.
⇒ Anti-idiotype antibodies are also under trial.
Similarly, DNA vaccines and immunodominant
peptides (recognized by the MHC molecules) are
under investigation, particularly for protection
against viral diseases.
⇒ The protective immunity conferred by a vaccine may
be lifelong (measles, mumps, rubella, small pox,
tuberculosis, yellow fever, etc.) or may last as little as
six months (cholera).
Application of immunological techniques:
1. Monoclonal Antibodies with Virtually Any
Desired Specificity Can Be Readily Prepared
2. Proteins Can Be Detected and Quantitatively
determined by Using an Enzyme-Linked
Immunosorbent Assay (ELISA).
3. Western Blotting Permits the Detection of
Proteins Separated by Gel Electrophoresis
4. Immune-Fluorescent Markers Make Possible the
Visualization of Proteins in the Cell using
fluorescence microscopy
5. Gold (heavy metal) tagged antibodies make
Possible the Visualization of Proteins in the Cell
using electron microscopy
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
E13. IMMUNITY AGAINST INFECTIOUS PATHOGENS
I. Introduction
During evolution, an extremely complex system of anti-
infections defenses has emerged. But at the same time as
vertebrates and mammals developed their defenses,
microbes continued evolving as well, and many became
adept at avoiding the consequences of the anti-infections
defense mechanisms. The interplay between host
defenses, microbial virulence, and microbial evasion
mechanisms determines the outcome of the constant
encounters between humans and pathogenic organisms.
II. Nonspecific Anti-Infectious Defense Mechanisms
Nonspecific defense mechanisms play a most important
role as a first line of defense, preventing penetration of
microorganisms beyond the outer exposed surfaces of the
body. The following is a brief description of the most
important nonspecific defense mechanisms.
A. Physical and Chemical Barriers, including the
integrity of the epithelial and mucosal surfaces, the flow
of mucosal secretions in the respiratory tract, the acidity
of the gastric contents, and the secretion of lysozyme in
tears, saliva, and most secretions. The importance of
these barriers is apparent from the prevalence of
infections when their integrity is compromised.
B. Inducible Nonspecific Responses, including fever
and release of interferons, activated when infectious
agents manage to invade, particularly effective in
preventing viral replication.
C. Phagocytosis. As a microbe penetrates beyond the
skin or mucosal surface, it will encounter cells able to
ingest it. Two types of cells are particularly adept at
nonimmune phagocytosis: tissue macrophages and
granulocytes (particularly neutrophils). This nonimmune
phagocytosis involves a variety of recognition systems
(Fig. 1).
74 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
1. CR1 and CR3 receptors are able to interact with C3b
and iC3b on the microbial membrane, generated as a
consequence of complement activation by the alternative
pathway, a property common to many bacteria (see
below).
2. Mannose receptors on phagocytic cells may mediate
ingestion of organisms with mannose-rich
polysaccharides, such as Candida albicans. Mannose
mediated phagocytosis is amplified by a mannose-
binding protein that promotes phagocytosis through
complement activation and direct interaction with the
C1q (collectin) receptor on phagocytic cells.
3. C-reactive protein binds to certain bacterial
polysaccharides and has very similar effects to the
mannosebinding protein, activating complement and
promoting phagocytosis, both through CR1 and CR3, as
well as by other receptors, including the FcγRI and the
C1q receptor, both of which bind this protein.
D. Activation of the Complement System via the
Alternative Pathway. A variety of microorganisms
(bacteria, fungi, viruses, and parasites) can activate
complement by the alternative pathway (see Table 1). In
most cases where adequate studies have been carried out,
polysaccharidic structures have been proven to be
responsible for complement activation of the alternative
pathway. This activation will lead to phagocytosis,
through the generation of C3b, and to chemotaxis,
through the release of C3a.
E. Acute Phase Reaction and Leukocyte Chemotaxis.
The initial recognition by phagocytes and the activation
of the complement system by the alternative pathway, by
themselves, may not be sufficient to eradicate the
invading microorganism, but the response is quickly
amplified by a multitude of cytokines released by
macrophages activated as a consequence of phagocytosis,
including IL-1, IL-8, and TNF-α.
1. IL-1 and TNF- α cause an increase in body
temperature, mobilize neutrophils from the bone marrow,
and upregulate the synthesis of a variety of proteins
known as acute phase reactants, including C-reactive
protein and the mannose-binding protein mentioned
above.
2. TNF α and IL-1 up-regulate the expression of cell
adhesion molecules in the endothelial cells of
neighboring endothelial cells, thus promoting adherence
of leukocytes, and increase vascular permeability. Both
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
factors facilitate the migration of leukocytes off the
vessels, toward the focus of infection.
3. IL-8 has chemotactic properties. Together with other
chemotactants, such as C5a and bacterial peptides, it
attracts neutrophils toward the focus of infection.
F. Natural Killer (NK) Cells are able to destroy viral
infected cells as a consequence of the delivery of an
activating signal in the absence of an inhibitory signal.
1. The activating signal is delivered as a consequence of
the interaction between a recognition molecule (NKR-
P1) on the NK cells and glycoproteins expressed on the
membrane of the infected cells.
2. The inhibiting signal is usually delivered as a
consequence of the interaction between a membrane
protein in the NK cell membrane and MHC-I molecules
on the membrane of normal cells. Viral infected cells
often have a reduced expression of MHC-I molecules
due either to a down-regulation of cellular protein
synthesis or a specific inhibition of MHC-I transport to
the cell membrane, discussed later in this chapter.
B. Other Mechanisms for the Generation of Natural
Antibodies. Cross-reaction is probably the most
common explanation for the emergence of “natural”
antibodies, but other mechanisms, such as the mitogenic
effects of Tindependent antigens and the nonspecific
stimulatory effects of lymphokines released by antigen-
stimulated T lymphocytes, which could activate B cells
responding to other antigens, could explain the rise of
“nonspecific” immunoglobulins that is observed in the
early stages of the humoral response to many different
antigens. It is only a matter of random probability that
some of those “nonspecific” immunoglobulins may play
the role of “natural antibody” relative to an unrelated
antigen.
C. The Significance of “Natural” Antibodies.
“Natural” antibodies may play an important protective
role. Antibodies elicited to E. coli K100 cross-react with
the polyribophosphate of Haemophilus influenzae and
can protect experimental animals against infection with
the latter organism. It is logical to assume that such
cross-immunizations may be rather common and play an
important protective role against a variety of infectious
agents.
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  75
G. g/D T Lymphocytes are predominantly localized to
the mucosal epithelia, where they appear to recognize
infected epithelial cells by a nonimmunological
mechanisms (i.e., not involving the T-cell receptors),
which are subsequently immunization. It was eventually
demonstrated that the cell wall polysaccharides of several
strains of enterobacteriaceae and the AB
oligosaccharides of human erythrocytes are structurally
similar. Thus, crossreactive antibodies to
enterobacteriaceae are responsible for the “spontaneous”
development of antibodies to human red cell antigens in
chickens.
3. Newborn babies of blood groups A, B, or O do not
have either anti-A or anti-B isohemagglutinins, but will
develop them during the first months of life, as they get
exposed to common bacteria with polysaccharide
capsules. However, newborns are tolerant to their own
blood group substance, so they will only make antibodies
against the blood group substance that they do not
express. Blood group AB individuals never produce AB
isoagglutinins.
IV. The Protective Role of Specific Antibodies
A. The Humoral Immune Response. If a pathogen is
not eliminated by nonimmunological means and
continues to replicate, it will eventually spread through
the blood and lymph, and will usually be trapped by
macrophages and follicular interdigitating cells in the
lymph nodes and spleen. Those cells are able to
internalize antigens interacting with receptors such as the
mannose receptor (see above), process the antigen in the
endosomic compartment, and express MHC-II-associated
antigen-derived peptides. This creates the ideal
conditions for the onset of an immune response: B
lymphocytes can interact with membrane-bound antigen
while helper T cells recognize MHC-II with antigen-
derived peptides presented by the same APC. The
antigen-recognizing cells will interact and costimulate
each other and, after a time lag necessary for
proliferation and differentiation of B cells into antibody-
producing cells, circulating antibody will become
detectable.
1. A primary immune response will take 5 to 7 days
(some times as long as 2 to 3 weeks) to be detected. The
predominating isotype of the antibodies made early in a
primary immune response is IgM.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
2. A secondary immune response has a shorter lag
phase (as short as 3–4 days) and the predominant isotype
of the antibodies is IgG.
3. The differentiation between IgM and IgG antibodies is
a useful index for discriminating between recent and past
infections, particularly in the case of viral infections.
B. Antibody-Dependent Anti-Infectious Effector
Mechanisms. As soon as specific antibodies become
available, they can protect the organism against infection
by several different mechanisms.
1. Complement-mediated lysis of a microorganism or
virus-infected cell can result from the activation of the
complete sequence of complement. However, as
discussed below, both mammalian cells and most
pathogenic microorganisms have developed mechanisms
that allow them to resist complement-mediated lysis.
2. Opsonization and phagocytosis are, in contrast, of
extreme physiological significance. Several proteins can
opsonize and promote phagocytosis, as discussed above,
but IgG antibodies are the most efficient opsonins,
particularly when complement is activated and C3b joins
IgG on the microbial cell membrane.
a. Opsonins. Because of their role in promoting
opsonization, IgG antibodies and C3b are considered
“opsonins.” Both C3b and IgG alone are efficient
opsonins, but their effects are synergistic.
b. Fc and CR1 receptors, expressed on the membranes
of all phagocytic cells, mediate phagocytosis and also
deliver activating signals to the cells, inducing release of
cytokines, as mentioned above.
c. Killing through opsonization has been demonstrated
for bacteria, fungi, and viruses, while phagocytosis of
antibody/complement-coated unicellular parasites has not
been clearly demonstrated.
d. The biological significance of phagocytic cells as
ultimate mediators of the effects of opsonizing antibodies
is obvious; the protective effects of antibodies are lost in
patients with severe neutropenia or with severe
functional defects of their phagocytic cells, and those
patients have increased incidence of infections with a
variety of opportunistic organisms.
3. Antibody-dependent cell-mediated cytotoxicity
(ADCC). Cells with Fc receptors may be able to
participate in killing reactions that target antibody-coated
cells. IgG1, IgG3, and IgE antibodies and cells with
FcγR or FcεR are usually involved. Large granular
lymphocytes or monocytes are the most common effector
cells in ADCC, but in the case of parasitic infections,
eosinophils play the principal role in cytotoxic reactions.
Different effector mechanisms are responsible for killing
by different types of cells: large granular lymphocytes
kill through the release of granzymes and signaling for
apoptosis; monocytes kill by releasing oxygen active
radicals and nitric oxide; eosinophil killing is mostly
mediated by the release of a “major basic protein,”
which is toxic for parasites.
4. Toxin neutralization. Many bacteria release toxins,
which are often the major virulence factors responsible
for severe clinical symptoms. Antibodies to these toxins
prevent their binding to cellular receptors and promote
their elimination by phagocytosis.
5. Virus neutralization. Most viruses spread from an
initial focus of infection to a target tissue via the blood
stream. Antibodies binding to the circulating virus
change its external configuration and prevent either its
76 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
binding to cell receptors or its ability to release nucleic
acid into the cell.
6. Mucosal protection. Secretory antibodies seem to
play their protective role by preventing the attachment
and penetration of microbial agents through mucosal
surfaces.
C. Factors Influencing the Effectiveness of an Anti-
Infectious Humoral Response. The effectiveness of the
humoral immune response in eliminating or preventing
the proliferation of an infectious agent or in neutralizing
a toxin before it causes severe disease depends on
whether or not antibodies can be available in time to
prevent the infection to develop or the toxin to reach its
cellular target.
1. If the relevant antibodies are present in circulation, as
a result of vaccination, previous infection, or
crossreaction between different microorganisms,
protection is most effective, the microorganism or its
toxin(s) will be almost immediately neutralized, and the
infection will remain subclinical.
2. If preformed antibodies are not available, protection
will depend on whether antibody synthesis can take place
before the “incubation period” (period of time during
which the infectious agent is multiplying but has not yet
reached sufficient mass to cause clinical disease) is over.
a. Some infectious agents, such as the influenza virus,
have very short incubation periods (about 2 to 3 days)
and, in such cases, not even a secondary immune
response can be protective.
b. In most infections, the duration of the incubation
period is sufficiently long as to allow a secondary
immune response to provide protective antibodies. Thus,
for many infections, particularly the common viral
diseases of childhood, previous exposure and acquisition
of memory ensure that antibody will be produced in time
to maintain subsequent exposures, which play the role of
natural “booster” doses, probably explaining the
“immunity for life” associated with them.
c. The goal of prophylactic immunization may vary from
case to case. In diseases with very short incubation
periods, it is essential to maintain the levels of
neutralizing antibody in circulation necessary to
immediately abort infection. In most other diseases, it
may be sufficient to induce immunological memory,
since once memory has been induced, the immune
system will be able to respond in time to prevent the
development of clinical infection.
d. Protection by humoral immunity is only possible if the
infectious agent is easily available to the antibodies
produced against it. Thus, organisms that grow
intracellularly are not easy to eliminate by antibodies. In
addition, as discussed in detail later in this chapter, those
organisms able to change their antigenic make-up during
the course of an infection can persist in spite of a
vigorous humoral response.
V. The Protective Role of Cell-Mediated Immunity
Many organisms have the ability to grow and replicate
intracellularly, some as an absolute requirement, others
as an option that allows them to survive after
phagocytosis. Antibodies are largely ineffective against
those organisms, and T lymphocytes play a major role in
their elimination, as reflected by the finding of
lymphocytic infiltrates in tissues infected by intracellular
infectious agents, such as viruses. The immune system
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
has two basic options to eliminate those organisms: to
kill the infected cell, or to enhance the infected cell's
ability to kill intracellular organisms. Either option
requires the persistent activation of the TH1
subpopulation of helper cells.
A. Lymphocyte-Mediated Cytotoxicity. It can be easily
demonstrated that viral-infected cells are lysed as a
consequence of their incubation with “immune”
lymphocytes, obtained from an animal previously
exposed to the same virus.
1. Recognition of infected cells
a. Virus-infected cells that express MHC-I molecules
with associated viral peptides can be easily recognized
by CD8+ cytotoxic T cells. While CD8+ lymphocytes,
when strongly stimulated, are able to release IL-2 and
differentiate into effector cytotoxic T cells, usually that
differentiation requires the collaboration of TH1 helper T
cells. The activation of CD4+ TH0 cells and their
differentiation into TH1 helper T cells is likely to take
place in lymphoid tissues, since macrophages are often
infected by viruses and virus-derived peptides are likely
to be presented by those cells both in association with
MHC-I and MHC-II molecules.
b. Macrophages are often infected by intracellular
organisms of all kinds, including bacteria, fungi, and
protozoa. Thus, recognition by CD4+ and CD8+ T
lymphocytes is likely to take place, resulting in the
differentiation of effector CD8+ cytotoxic T cells.
2. Killing involves recognition of the target cell, release
of perforins and esterases, and delivery of apoptosis
signals through the fas-fas ligand interaction.
B. Lymphocyte-Mediated Activation of Macrophages
and Other Inflammatory Cells. In many cases, the cells
infected by intracellular organisms are the tissue
macrophages, and the persistence of the infection
depends on a delicate balance between a state of relative
inactivity by the macrophage and mechanisms that allow
the infectious agent to escape proteolytic digestion once
inside the cytoplasm (see below).
1. Macrophage activation
a. CD4+ TH1 lymphocytes are activated as a
consequence of their interaction with infected
macrophages expressing MHC-II-associated peptides and
releasing IL-12. Once activated, TH1 lymphocytes
release a variety of lymphokines, particularly interferon-
g, which activates macrophages, enhancing their ability
to kill intracellular organisms and GM-CSF, which
promotes differentiation and release of granulocytes and
monocytes from the bone marrow and delivers
costimulatory signals to B cells.
b. Once the initial activation signals are delivered,
macrophages and lymphocytes enter in a complex cycle
of self and mutual activation involving a variety of
cytokines. In addition, several of these cytokines may
activate other types of cells and have chemotactic
properties, for which reason they are known as
chemotactic cytokines or chemokines. This group of
cytokines includes interleukin-8 (IL- 8), RANTES,
macrophage inflammatory proteins (MIP),
macrophage chemotactic proteins (MCP), and
migration-inhibition factor (MIF). Collectively the
chemokines attract, activate, and retain leukocytes to the
area where a cell-mediated immune reaction is taking
place.
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  77
2. Inflammatory reaction. In concert with the release of
chemotactic cytokines, the expression of CAMs in
neighboring microvessels is up-regulated, favoring
adherence and migration of monocytes and granulocytes
to the extravascular space. Inflammatory cells
accumulate in the area of infections and, as a
consequence of crossactivation circuits involving
phagocytes and TH1 lymphocytes, the localized
macrophages and granulocytes become activated.
Consequently, a. the enzymatic contents and respiratory
burst of the cells become more intense, making them
better suited for killing intracellular organisms.
b. Phagocytosis is enhanced.
c. Activated macrophages release higher levels of
cytokines, including IL-12, which continues to promote
the differentiation of TH1 cells, as well as IL-1, IL-6, and
TNF-α, which in association with IL-8, play the major
role in inducing the synthesis of reactive proteins,
increasing body temperature, and several other metabolic
effects characteristic of the inflammatory reaction.
VI. The Immune Deficiency Syndromes as Models for
the Study of the Importance of Immune Defenses
against Infections in Humans
Most of our information about the immune system in
humans has been learned from the study of patients with
immunodeficiency diseases. The most characteristic
clinical features of immunocompromised patients are the
repeated or chronic infections, often caused by
opportunistic agents. There are some characteristic
associations between specific types of infections and
generic types of immune deficiency, which provide the
best data about the role of the different components of
the immune system.
A. Patients with antibody deficiencies and conserved
cell-mediated immunity suffer from repeated and chronic
infections with pyogenic bacteria.
B. Patients with primary deficiencies of cell-mediated
immunity usually suffer from chronic or recurrent fungal,
parasitic, and viral infections.
C. Neutrophil deficiencies are usually associated with
bacterial and fungal infections caused by common
organisms of low virulence, usually kept in check
through nonimmune phagocytosis and other inate
resistance mechanisms.
D. Isolated complement component deficiencies are also
associated with bacterial infections, most frequently
involving Neisseria gonorrhoeae and N. meningitidis,
whose elimination appears to require the activation of the
membrane attack complex.
VII. Escape from the Immune Response
Many infectious agents have developed the capacity to
avoid the immune response. Several mechanisms are
involved.
A. Anti-Complementary Activity has been
characterized for bacterial capsules and outer proteins of
some bacteria. For example, the M protein of group A
Streptococcus inactivates complement convertases,
preventing activation of the alternative pathway. The
anti-complementary activity of bacterial outer
components has as a net result a decreased level of
opsonization by C3b and other complement fragments.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
B. Resistance to Phagocytosis can be evident at
different levels of the sequence of events associated with
ingestion and digestion of microorganisms:
1. Resistance to ingestion is usually associated with
polysaccharide capsules, which repeal and inhibit the
function of phagocytic cells. Haemophilus, Pasteurella,
Klebsiella, Pseudomonas, and some yeasts (e.g.,
Cryptococcus neoformans) have such capsules.
2. Ability to survive after ingestion is characteristic of
the group of bacteria known as facultative intracellular
(Mycobacteria, Brucella, Listeria, and Salmonella), as
well as of some fungi and protozoa (Toxoplasma,
Trypanosoma cruzi, and Leishmania). These
microorganisms survive inside phagocytosis by several
different strategies.
a. Secretion of molecules that prevent the formation of
phagolysosomes; these bacteria survive inside
phagosomes that are relatively devoid of toxic
compounds (Mycobacterium tuberculosis, Legionella
pneumophila, and Toxoplasma gondii).
b. Synthesis of outer coats that protect the bacteria
against proteolytic enzymes and free toxic radicals (such
as the superoxide radical).
c. Depression of the response of the infected phagocytic
cells to cytokines which usually activate their killing
functions, such as interferon-γ.
d. Exit from the phagosome into the cytoplasm, where
the bacteria can live and multiply unharmed
(Trypanosoma cruzi).
e. The resistance to phagocytosis of several organisms
seems to involve a combination of these strategies.
i. Mycobacterium leprae is coated with a phenolic
glycolipid layer that scavenges free radicals and releases
a compound that inhibits the effects of interferon-γ. In
addition, the release of IL-4 and IL-10 is enhanced,
contributing to the down-regulation of TH1 lymphocytes.
ii. Two glycolipids present in the membrane of
Leishmania parasites inhibit the response of monocytes
and neutrophils to cytokines and a variety of stimuli; in
addition, Leishmania seems to resist the effects of
proteolytic enzymes in the phagolysosome.
f. Viruses taken up by phagocytes as antigen-antibody
complexes often infect the phagocytic cells. The
infection of phagocytic cells is frequent when viruses
disseminate through the blood stream.
g. Phagocytic cells are also infected by the HIV virus,
either as a consequence of ingestion of virus-IgG
antibody complexes or as a consequence of interactions
with a CD4+ related molecule which is expressed in their
membrane. In either case, the infection of the
monocyte/macrophage is not cytotoxic, but the virus
continues to replicate in small amounts and these cells
become permanent sources of reinfection.
C. Ineffective Immune Responses
1. Polysaccharide capsules are not only antiphagocytic,
but poorly immunogenic and elicit predominantly IgM
and IgG2 antibodies, which are inefficient as opsonins
(the FcγR of phagocytic cells recognize preferentially
IgG1 and IgG3 antibodies).
2. Neisseria meningitidis often induces the synthesis of
IgA antibodies. In vitro data suggest that IgA can act as a
weak opsonin or induce ADCC (monocytes/macrophages
and other leukocytes express Fcα receptors on their
membranes), but the physiological protective role of IgA
antibodies is questionable. Patient sera with high titers of
IgA antibodies to N. meningitidis failed to show
78 INSTITUTE FOR ADVANCED STUDIES, JODHPUR
bactericidal activity until IgA-specific anti-Neisseria
meningitidis antibodies were removed. This observation
suggests that the IgA antibodies acted as a “blocking
factor,” preventing opsonizing IgG antibodies from
binding to the same epitopes.
3. Antiviral antibodies are not always neutralizing and
protective.
a. Retroviruses induce both neutralizing and non-
neutralizing (blocking) antibodies. Both seem to
recognize the same epitopes; when non-neutralizing
antibodies predominate, they block the binding of
neutralizing antibodies.
b. Hepatitis B virus induces the synthesis of large
quantities of soluble antigenic proteins by the infected
host cells. These circulating antigens will block the
antiviral antibodies before they reach the infected cells,
in a sense, acting as a “deflector shield” that protects the
infected tissues from “antibody aggression.”
D. Loss and Masking of Antigens have been
demonstrated with schistosomula (the larval forms of
schistosoma).
1. In the few hours after hatching, there is a rapid
decrease in the surface antigens, as reflected by a
progressive decrease in the binding of purified and
fluorescein-labeled heterologous antischistosomular
antibodies to the parasite membrane. This is observed
even in parasites cultured in serumfree medium.
2. Schistosomula recovered from infected animals have
host proteins bound to the outer layers, masking the
remaining parasite antigens.
E. Antigenic Variation has been characterized in
bacteria (Borrelia recurrentis), parasites (trypanosomes,
the agents of African sleeping sickness; Giardia
lamblia), and viruses (human immunodeficiency virus,
HIV).
1. African trypanosomes have a surface coat constituted
mainly of a single glycoprotein (variant-specific surface
glycoprotein or VSG), for which there are about 103
genes in the chromosome. At any given time, only one of
those genes is expressed, the others remain silent. For
every 106 or 107 trypanosome divisions, a mutation
occurs that replaces the active VSG gene on the
expression site by a previously silent VSG gene. The
previously expressed gene is destroyed, and a new VSG
protein is coded, which is antigenically different. The
emergence of a new antigenic coat allows the parasite to
multiply unchecked. As antibodies emerge to the newly
expressed VSG protein, parasitemia will decline, only to
increase as soon as a new mutation occurs and a different
VSG protein is synthesized.
2. Giardia lamblia has a similar mechanism of variation
but the rate of surface antigen replacement is even faster
(once every 103 divisions).
3. Borrelia recurrentis, the agent of relapsing fever
carries genes for at least 26 different variable major
proteins (VMP) which are sequentially activated by
duplicative transposition to an expression site. The
successive waves of bacteremia and fever correspond to
the emergence of new mutants which, for a while, can
proliferate unchecked until antibodies are formed.
4. HIV exhibits a high degree of antigenic variation,
which seems to be the result of errors introduced by the
reverse transcriptase when synthesizing viral DNA from
the RNA template. The mutation rate is relatively high
(one in every 103 progeny particles), and the immune
 
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 
response selects the mutant strains that present new
configurations in the outer envelope proteins, allowing
the mutant to proliferate unchecked by preexisting
neutralizing antibodies.
F. Cell-to-Cell Spread allows infectious agents to
propagate without being exposed to specific antibodies or
phagocytic cells.
1. Listeria monocytogenes, after becoming intracellular,
can travel along the cytoskeleton and promote the fusion
of the membrane of an infected cell with the membrane
of a neighboring noninfected cell, which is subsequently
invaded.
2. Herpes viruses, retroviruses, and paramyxoviruses
cause the fusion of infected cells with noninfected cells
allowing viral particles to pass from cell to cell.
G. Integration of Microbial Genomes is a tactic
exclusive of viruses, particularly DNA viruses and
retroviruses. The integration of viral genomes in the host
genome allows the virus to cause persistent or latent
infections, with minimal replication of the integrated
virus and minimal expression of viral proteins in the
membranes of infected cells.
H. Immunosuppressive Effects of Infection. Although
immunosuppressive effects have been described in
association with bacteria and parasitic infections, the best
documented examples of infection-associated
immunosuppression are those described in viral
infections.
1. Measles. Patients in the acute phase of measles are
more susceptible to bacterial infections, such as
pneumonia. Both delayed hypersensitivity responses and
then in vitro lymphocyte proliferation in response to
mitogens and antigens are significantly depressed during
the acute phase of measles and the immediate
convalescence period, usually returning to normal after 4
weeks. Recent investigations suggest that infection of
monocytes/macrophages with the measles virus is
associated with a down-regulation of interleukin-12
synthesis, which can explain the depression of cell-
mediated immunity associated with measles.
2. Cytomegalovirus. Mothers and infants infected with
cytomegalovirus show depressed responses to CMV
virus, but normal responses to T-cell mitogens,
suggesting that, in some cases, the immunosuppression
may be antigenspecific, while in measles it is obviously
nonspecific.
3. Influenza virus has been found to depress CMI in
mice, apparently due to an increase in the suppressor
activity of T lymphocytes.
4. Epstein-Barr virus releases a specific protein that has
extensive sequence homology with interleukin-10. The
biological properties of this viral protein are also
analogous to those of interleukin-10; both are able to
inhibit lymphokine synthesis by T-cell clones.
5. Human immunodeficiency virus. HIV infection is
associated with depletion of CD4+ cells, which are the
primary target of the virus. The depletion of CD4 cells
results mostly from viral replication itself and by the
priming of infected cells for apoptosis, which is triggered
by stimuli that normally would cause T-cell proliferation.
In addition, FcR-dependent phagocytosis is depressed in
HIV-infected monocyte-derived macrophages, further
contributing to the immunological compromise of the
infected patients.
INSTITUTE FOR ADVANCED STUDIES, JODHPUR  79
X. Abnormal Consequences of the Immune Response
A. The Activation of T lymphocytes by Bacterial
Superantigens
1. A variety of bacterial exotoxins, such as
staphylococcal enterotoxins-A and -B (SE-A and SE-
B), staphylococcal toxic shock syndrome toxin-1
(TSST-1), exfoliating toxin, and streptococcal exotoxin
A, as well as other unrelated bacterial proteins (such as
streptococcal M proteins) have been characterized as
“superantigens.”
2. Superantigens are defined by their ability to stimulate
T cells without being processed. The stimulation of T
cells is polyclonal; thus the designation of “superantigen”
is a misnomer, but it has gained popularity and is widely
used in the literature.
3. The best studied superantigens are the staphylococcal
enterotoxins, which are potent polyclonal activators of
murine and human T lymphocytes, inducing T-cell
proliferation and cytokine release. TSST-1 also appears
to activate monocytes and is a potent B-cell mitogen,
inducing B-cell proliferation and differentiation.
4. Mechanism of action. Superantigens bind directly
and simultaneously to the nonpolymorphic area of class
II MHC on professional accessory cells (macrophages
and related cells) and to the Vβ chain of the α/β TcR
(Fig.).
a. For example, staphylococcal enterotoxins bind
exclusively to specific subfamilies of Vβ chains that are
expressed only by certain individuals. When expressed,
these Vβ chain regions can be found on 2–20% of a
positive individual's T cells, and the cross-linking of the
TcR2 and of the APC by the enterotoxin activates all T
cells (both CD4 and CD8+) expressing the specific Vβ
region recognized by the enterotoxin.
b. The massive T-cell activation induced by
superantigens results in the release of large amounts of
IL-2, interferon-γ, lymphotoxin (TNF-β), and TNF-α.
c. Patients infected by bacteria able to release large
amounts of superantigens (e.g., S. aureus-releasing
enterotoxins or TSST-1 and Group A Streptococcus-
releasing) may develop septic shock as a consequence of
the systemic effects of these cytokines, which include
fever, endothelial damage, profound hypotension,
disseminated intravascular coagulation, multiorgan
failure, and death.
d. After the initial burst of cytokine release, the
stimulated T cells either undergo apoptosis or become
anergic. This effect could severely disturb the ability of
the immune system to adequately respond to
superantigen-releasing baceria.
UNIT 4: CELL COMMUNICATION AND CELL SIGNALING 

B. Infection as a Consequence of the Uptake of the infection on the immune system, and the possibility
Antigen-Antibody Complexes. that the immune response may be more of a problem than
The immune response, in some cases, facilitates the the infection itself.
access of infectious agents to cells in which they will be
able to proliferate.
1. Macrophages are often infected by intracellular
organisms that are ingested as a consequence of
opsonization.
2. Babesia rodhaini, a bovine intraerythrocytic parasite,
penetrates the host's cells after it has bound complement,
particularly C3. Absorption of C3b-containing
circulating antigen-antibody complexes to the CR1
expressed by red cells allows the parasite to gain access
to the red cell, which becomes its permanent location.

C. Post-Infectious Tissue Damage. Several examples of

deleterious consequences of the immune response have
to do with the predominant role of the immune response
in the pathogenesis of the disease.

1. Immune-complex-induced inflammation. Antigen-

antibody complexes, if formed in large amounts, can
cause disease by being trapped in different capillary
networks and leading to inflammation. The clinical
expression of immune complex-related inflammation
depends on the localization of the trapped complexes:
vasculitis and purpura, when the skin is predominantly
affected, glomerulonephritis if trapping takes place on
the glomerular capillaries, arthritis when the joints are
affected. Viruses are often involved in the formation of
circulating antigen-antibody complexes.

2. Immune destruction of infected cells and tissues.

An immune response directed against an infectious agent
may be the main cause of damage to the infected tissue.
a. In subacute sclerosing panencephalitis, a
degenerative disease of the nervous system associated
with persistent infection with the measles virus, the
response against viral epitopes expressed in infected
neurons is believed to be the primary mechanism of
disease.
b. In some forms of chronic active hepatitis, the
immune response directed against viral epitopes
expressed by infected hepatocytes seems to cause more
tissue damage than the infection itself.
c. Cross-reactions with tissue antigens have been
proposed as the basis for the association of streptococcal
infections with rheumatic carditis and
glomerulonephritis. Antibodies to type 1 streptococcal M
protein crossreact with epitopes of myocardium and
kidney mesangial cells and cause inflammatory changes
in the heart and glomeruli, respectively.

X. Epilogue

The outcome of an infectious process depends on a very

complex set of interactions with the immune system. A
successful pathogen has developed mechanisms that
avoid fast elimination by an immunocompetent host.
These mechanisms allow the infectious agent to
replicate, cause disease, and spread to other individuals
before the immune response is induced. The immune
response, on the other hand, is a powerful weapon that,
once set in motion, may destroy friendly targets. Thus,
the therapeutic strategies in infectious diseases have to
consider all these questions, such as the particular
survival strategy of the infectious agents, the effects of

80 INSTITUTE FOR ADVANCED STUDIES, JODHPUR