INTRODUCTION TO IMMUNOLOGY AND SEROLOGY- LABORATORY

OUTLINE
I Immunology
∗ Serological Test
II The Immune System
A Immunity
i. Two Classification of Immunity
III Functions and Mechanisms of Immune System
A Innate or natural Immunity
B Adaptive or Acquired Immunity
THE IMMUNE SYSTEM
IV. Natural or Innate Immunity
The immune system is a complex system of
V. Biochemical factors
structures and processes that has evolved to protect
VI. Components of the natural immunity
us from disease.
A. Physical and Anatomical barriers
B. Phagocytic Barriers • Molecular and cellular components make up the
VII. Cellular Components of Natural Immunity immune system.
VIII. Steps in Phagocytosis • The function of these components is divided up into
IX. Main function of the Monocyte, Macrophage, and non-specific mechanisms, those which are innate to
Dendritic cell an organism, and responsive responses, which are
X. Process of Phagocytosis adaptive to specific pathogens.
XI. Cells of the Immune System • Fundamentals or classical immunology involves
- Eosinophils, Basophils, Mast cells, Dendritic cells studying the components that make up the innate and
XII. Acute Phase reactants adaptive immune system.
XIII. Toll-like receptors
XIV. Inflammation IMMUNITY
• Summary Acute Phase Reactants • The way in which the body can protect itself from
• Additional Notes About Three Pathways invasion by pathogenic microorganism and provide a
IMMUNOLOGY defense against their harmful effect

• Study of the molecules, cells, organs, and systems
responsible for the recognition and disposal of foreign
materials (non-self) in our body.
• Study of the body’s response to infectious diseases
• Study the desirable and undesirable consequences of
immune interactions.
• Study of the ways in which the immune system can be
advantageously manipulated to protect against or treat
disease.
• Immunology – study of the Immune system.
• Serology- study of serum and its immune
components
• refers to the diagnostic identification of antibodies in
the serum.
TWO MAJOR CLASSIFICATION OF IMMUNITY
a. Non-specific immunity
• are things that protect the body from various bacteria’s,
viruses, and pathogens. These include the first and
second line of defense, such as the skin, fever (body
gets hot as an attempt to kill the pathogen).
b. Specific immunity
• are things that protect the body from specific
pathogens. It includes the third line of defense. They
include the lymphocytes (white blood cells) such as the
macrophages, t cells, and memory b cells.

• Serological tests - are diagnostic methods that are used
to identify antibodies and antigens in a patient's sample.
 Serological test is performed to diagnose infections
and autoimmune diseases, to check if a person has
immunity to certain diseases, and in many other
situations, such as determining an individual’s blood
type.
FUNCTIONS AND MECHANISMS OF IMMUNE
SYSTEM
INNATE OR NATURAL IMMUNITY
• Natural immunity
• The first line of defense
• Non-specific
o 1. Physical barriers (skin, saliva, etc.)
o 2. Cellular components (macrophages,
neutrophils, basophils, mast cells)

LEYBA, DIMAPILIS | MMLS 3-1 1

INTRODUCTION TO IMMUNOLOGY AND SEROLOGY- LABORATORY

ADAPTIVE OR ACQUIRED IMMUNITY Difference of Gastro-Intestinal Tract and

Genitourinary Tract
The second line of defense
• Involves building up memory of encountered GASTRO-INTESTINAL GENITOURINARY TRACT
infections TRACT
• Enhanced response specific to the pathogen or foreign Contains residential The flow of the urine and low pH
pathogens roaming free in the bloodstream. bacteria, which not only provides sufficient protection.
• Cellular components are lymphocytes such as T cells, help in digestion of
which are directed especially towards pathogens that certain polysaccharides
have colonized cells and directly kill infected cells or but also play an
help control the antibody responses. important role in the
control of potential
THE NATURAL OR INNATE IMMUNITY pathogens.
Low pH which The vaginal wall is lined by
 How does the body know? maintained within the squamous epithelium which is
stomach by gastric composed of rich amount of
 Self refers to particles, such as proteins and other juices has bactericidal collagen. In response to estrogen,
molecules, that are a part of, or made by, your body. They and viricidal actions. In glycogen is deposited upon the
can be found circulating in your blood or attached to the intestine, low pH epithelial surface just prior to
and anaerobic condition ovulation. This is degraded
different tissues. Something that is self should not be
are maintained. anaerobically by the lactobacilli to
targeted and destroyed by the immune system. The non- produce lactic acid which act as a
reactivity of the immune system to self-particles is called deterrent of pathogenic infection.
tolerance. MAMMARY GLAND
 Non-self refers to particles that are not made by your body, • Milk contains bacterial inhibitors called lactenius that
and are recognized as potentially harmful. These are include complement, lysozymes. Lactoferrin and an
sometimes called foreign bodies. These can be bacteria, enzyme called lacto-peroxidase.
viruses, parasites, pollen, dust, and toxic chemicals. The
non-self-particles and foreign bodies that are infectious or RESPIRATORY TRACT
pathogenic make proteins called antigens that allow our • The walls of the respiratory tract are mucous covered
body to know that they intend to cause damage. when the suspended particles (-5 um) present in the air
enter the respiratory tract, they get stuck to these
 Antigens are anything that causes an immune response. mucous covered walls. The mucous layer of the upper
respiratory tract is provided with antiseptic properties
Antigens can be entire pathogens, like bacteria, viruses,
by virtue of the presence of the lysosomes and IgA in
fungi, and parasites, or smaller proteins that pathogens it.
express. Some pathogens are general, whereas others are BIOCHEMICAL FACTORS
very specific. ∗ Chemical secretions produced by the body that inhibit
microbial growth.
 Cytokines are molecules that are used for cell signaling, or
cell-to-cell communication. Cytokines are similar to • KERATIN
chemokines, wherein they can be used to communicate  Skin protein that has very little water.
with neighboring or distant cells about initiating an immune
• LYSOZYME
response. Cytokines are also used to trigger cell trafficking,
 Enzyme found in many body fluids and secretions such
or movement, to a specific area of the body. as tears. It can break down the cell wall of gram-
positive bacteria and few gram-negative bacteria by
 Chemokines are a type of cytokines that are released by hydrolysing the peptidoglycan layer.
infected cells. Infected host cells release chemokines in
order to initiate an immune response, and to warn • HYDROCHLORIC ACID AND BILE SALT IN THE
neighboring cells of the threat. GASTROINTESTINAL TRACT
 Bile is secreted into the small intestine where it has two
effects: it neutralizes the acid - providing the alkaline
conditions needed in the small intestine. it emulsifies
fats - providing a larger surface area over which the
COMPONENTS OF THE NATURAL IMMUNITY lipase enzymes can work.

1. Physical / Anatomical Barriers • COMPLEMENT

 Proteins in serum that act as a non-specific against
• Skin and mucous membranes- major barrier to a infection.
various invading microorganism
o Defensins- most abundant antimicrobial peptides • Interferons are small proteins produced in response to
in the mucus membranes. viral infection.

LEYBA, DIMAPILIS | MMLS 3-1 2

INTRODUCTION TO IMMUNOLOGY AND SEROLOGY- LABORATORY

ROLE OF NORMAL FLORA

• Commensal organisms that are harmless inhabitants
of our body.
• Occupies the attachment sites for potentially
pathogenic organisms.
• Produces substance against pathogenic organisms
• Complete for essential nutrients for the growth of
pathogenic microorganisms.

THE INFLAMMATORY REACTIONS

• A vascular and cellular reaction to the presence of
invading microorganisms or injury.
• One of the most effective defense mechanisms in
human and other animals.  Neutrophil migrates from the blood vessel to the matrix,
• The damage cells at the site of injury initiate the tissue secreting proteolytic enzymes, in order to dissolve
response by releasing chemical factors such as intercellular connections (to the improvement of its mobility)
histamine which in turn trigger vasodilation and and envelop bacteria through phagocytosis.
increased permeability of capillaries, permitting influx
of fluids and blood cells into the site.

STAGES OF INFLAMMATORY REACTIONS

 Initiation (damage to tissue)

 Tissue response
 Leukocyte response
 Tissue repair (resolution)
 Cure

2. Phagocytic Barriers
PHAGOCYTOSIS
• Process of endocytosis by which certain cells
• The function of ingesting and destroying
(phagocytes) ingest or engulf other cells or particles
antigens is mediated by phagocytes
• The major mechanism used to remove pathogens and cell
CELLULAR COMPONENTS OF NATURAL debris.
IMMUNITY
PHAGOSOME
• Internal compartment containing the ingested particles or
NEUTROPHILS (PMN CELLS) antigens
• The structure where the phagocytes release its enzymes
• The most abundant population of circulating white blood to digest the ingested particles.
cells (40-70 %)
• Neutrophils are spherical cells of about 12-15 um diameter
and with numerous ciliary projections.
• Multilobulated nucleus
• Granular cytoplasm
• The first responders of inflammatory cells to migrate
towards the site of inflammation or injury.
• The hallmark of acute inflammation
• Acute phase of bacterial infection
• Chemotaxis

Neutrophils have at least three distinct granules subsets:

1. Primary or azurophilic granules
 which contain potent hydrophilic enzymes (e.g
elastate) and myeloperoxidases (MPO).

2. Secondary or Specific granules

 which contain high levels of the iron-binding protein
lactoferrin.

3. Tertiary or gelatinase granules

 which contain matrix metalloproteinases.

LEYBA, DIMAPILIS | MMLS 3-1 3

INTRODUCTION TO IMMUNOLOGY AND SEROLOGY- LABORATORY

• 4 - 10 percent of total circulating white blood cells

• Phagocytes
• They do not remain in the circulation for long. They
stay in peripheral blood for up to 70 hours, and then
they migrate to the tissues and become known as
STEPS IN PHAGOCYTOSIS macrophages.
• Differentiate into Macrophage and Dendritic cells in
1. Chemotaxis the tissue.
o movement in response to chemical stimulation • Migrate within approximately 8–12 hours in response
o pseudopods move toward microorganisms at site of to inflammation.
infection. • Digestive vacuoles may also be observed in the
cytoplasm.
2. Adherence
o attachment to a microbe. Granules are present at the early stage of cell maturation.
3. Ingestion 2 types
o engulfing pathogen with pseudopodia wrapping
around pathogen 1. Azurophil granules, may contains peroxidase, acid
o pathogen is placed into a vesicle (phagosome) that phosphatase, and arylsulfatase; this indicates that these
contains lysozyme. granules are similar to the lysosomes of neutrophils.
4. Digestion 2. may contain, -glucuronidase, lysozyme, and lipase, but no
o phagosome maturation alkaline phosphatase.
o lysozyme merges with phagosome and releases
digestive enzymes>becomes a phagolysosome
o microbes are usually killed at this point.

5. Elimination
o phagocytes eliminate remaining pieces of microbe
via exocytosis.

MONOCYTES

• Mononuclear cells, are the largest cells in the

peripheral blood, with a diameter that can vary from
12 to 22 μm (average size of 18 μm)
• A distinguishing feature is an irregularly folded or
horseshoe-shaped nucleus that occupies almost one- MAIN FUNCTION OF THE MONOCYTE,
half of the entire cells. MACROPHAGE, & DENDRITIC CELLS
• Ameboid cytoplasm and non-granulated
• The abundant cytoplasm stains a dull grayish blue 1. Phagocytosis
and has a ground-glass appearance due to the
• using intermediary (opsonising or opsonization)
presence of fine dust like granules.
proteins such as antibodies or complement that coat
the pathogen, as well as by binding to the microbe
directly via pattern-recognition receptors that
recognize pathogens.

2. Antigen presentation

3. Cytokine production

4. Monocytes are also capable of killing infected host cells

via antibody-dependent cell-mediated cytotoxicity.

LEYBA, DIMAPILIS | MMLS 3-1 4

INTRODUCTION TO IMMUNOLOGY AND SEROLOGY- LABORATORY
MACROPHAGES
• Macrophages and their precursors, called the
monocytes play a central role in both innate and
acquired immunity.
• Responsible for detecting, engulfing and destroying
pathogens and apoptotic cells.
• Mononuclear phagocytes.
• May assume different morphologic forms, some
develop abundant cytoplasm and are called epitheloid
cells (activated macrophages).
• Macrophages are found in sub-epithelial
connective tissue, in the interstice of parenchymal
organs, in the lining of the vascular sinusoids in
the liver and spleen and in the lymphatic sinuses
of lymph nodes, alveoli.
ADDITIONAL NOTES
• Tolerance- ability of the body to recognize the self and not
attack.
• Autoimmune disorders- abnormalities of the immune
system that attack and targets the self (e.g RA and SLE)
• Biochemical reactions are part of the innate immunity.
• Normal flora is non-self because they can be
opportunistic in some point.
• Normal flora or the commensals are microorganisms
that do not affect nor interfere the body’s system.
• Sterile sites do not have microorganisms and if it does It is
called pathogens (e.g blood infected with E. coli).
• Inflammation is an immune response.
• Granules observed in PMN’s are the lysosomes
• PMN’s Primary granules: myeloperoxidase and
lysosomes.
• PMN’s Secondary granules: Lactoferrin iron binding
enzyme)
• Hematoxylin (basic) and Eosin (acidic) stain reacts equally
at Neutrophil (neutral)
• Hematoxylin reacts in basophil
• Eosin reacts with eosinophil or the formerly called
acidophil.
EOSINOPHILS
• Eosinophils are always present in parasitic infections.
• Bi-lobed nucleus, pink staining with eosin and
characteristic cytoplasmic granules.
• Make up 0.5-1% of the White blood cells (WBC)
• Considered to be a homeostatic regulator of
inflammation. Functionally, the eosinophil attempts to
suppress an inflammatory reaction to prevent the
excessive spread of the inflammation.
• May also play a role in the host defense mechanism
because of its ability to kill certain parasites.
LEYBA, DIMAPILIS | MMLS 3-1
BASOPHILS
• Less than 1% of all circulating white blood cells.
• The smallest of the granulocytes, they are between 10-
15 um in diameter and contain coarse, densely training
deep-bluish purple granules that often obscure the
nucleus.
• Granules contains histamine, a small amount of
heparin, and eosinophil chemotactic factor-A, all of
which have an important function in inducing and
maintaining immediate hypersensitivity reactions.
• Granules lack hydrolytic enzymes, although
peroxidase is present.
• Exist for only a few hours in the blood stream.
BASOPHILIC GRANULES
• Have high concentrations of heparin and histamine
• Play an important role in acute, systemic,
hypersensitivity reactions.
• Degranulation occurs when an antigen binds to two
adjacent IgE antibody molecules located on the
surface of mast cells.
• Release of the contents of these granules results to
increased vascular permeability, smooth muscle
spasm, and vasodilation.
• If severe, this reaction can result in anaphylactic shock.
MAST CELLS
• Resemble basophils, but they are connective tissue
cells of mesenchymal origin, widely distributed
throughout the body.
• Larger than basophils, with a small round nucleus and
more granules.
• Unlike basophils, they have a long-life span of between
9-18 months.
• The enzyme content of the granules helps to
distinguish them from basophils, as they contain acid
phosphatase, alkaline phosphatase, and protease.
• Play a role in hypersensitivity reactions by binding IgE
• Mast cells are bind in Immunoglobulin A.
5
INTRODUCTION TO IMMUNOLOGY AND SEROLOGY- LABORATORY
DENDRITIC CELLS
• Are also named because they are covered with long
membranous extensions that make them resemble
nerve cell dendrites.
• Their main function is to phagocyte antigen and
present it to helper T lymphocytes.
• After capturing antigen in the tissue by phagocytosis or
endocytosis, they migrate to the blood and to lymphoid
organs, where they present antigen to T lymphocytes
to initiate the acquired immune response.
• The most potent phagocytic cell in the tissue.
Dendritic cells are classified according to their tissue
location:
• Langerhans cells are found on the skin and mucous
membranes
• Interstitial dendritic cells populate the major organs
such as the heart, lungs, liver, kidney, and the
gastrointestinal tract
• Interdigitating dendritic cells are present in the T
lymphocyte areas of secondary lymphoid tissue and
the thymus.
NATURAL KILLER CELLS
• NK cells play a major role in the host-rejection of both
tumours and virally infected cells.
• Cytotoxic; small granules in their cytoplasm contain
special proteins such as perforin and proteases known
as granzymes.
• When in close proximity to a cell, perforin forms pores in
the cell membrane of the target cell through which the
LEYBA, DIMAPILIS | MMLS 3-1
granzymes and associated molecules can enter,
inducing apoptosis.
• Are activated in response to interferons or macrophage-
derived cytokines.
• They serve to contain viral infections while the adaptive
immune response is generating antigen-specific
cytotoxic T cells that can clear the infection.
• NK cells have the ability to recognize any damaged
cell and to eliminate such target cells without prior
exposure to them.
• By quickly engaging infected target cells, NK cells
will give the immune system tie to activate the
adaptive response of specific T and B cells.
PROCESS OF PHAGOCYTOSIS
• Attachment and adherence- (enhance by opsonins)
• Internalization -formation of phagosome
• Degradation- fusion with cytoplasmic granules to form
phagolysosome.
NOTE: Engulfment of material is facilitated by the actin-
myosin contractile system. The phagosome is the
organelle formed by phagocytosis of material. It then
moves toward the centrosome of the phagocyte and is
fused with lysosomes, forming a phagolysosome and
leading to degradation.
• Exocytosis- release of antigenic debris to the outside
ENDOCYTOSIS AND KILLING OF MICROBES
• Innate immunity is the defense mechanism which ingest
extracellular macromolecules via endocytosis and
particulate material via phagocytosis. In endocytosis,
macromolecules within the extracellular tissue fluid are
taken by cell via the invagination and pinching off a small
regions of the plasma membrane.
• Endocytosis occurs through either pinocytosis or receptor-
mediated endocytosis, both help to internalize extracellular
macromolecules either by nonspecific membrane
invagination or by binding to specific membrane receptors
Figure 6.1 ENDOCYTOSIS- The internalization of
macromolecules within the extracellular fluid- occurs by
pinocytosis or receptor mediated endocytosis. In both
processes, the ingested material is degraded via the
endocytic processing pathway.
6
INTRODUCTION TO IMMUNOLOGY AND SEROLOGY- LABORATORY

TOLL-LIKE RECEPTORS (TLR) • Opsonization- binds to phosphocholine, phospholipids,

• Pattern-recognition receptors (PRR) present in highest peptidoglycan, ribonuclear proteins (bacteria, fungi,
concentration on monocytes, macrophages, and parasites)
neutrophils • Promotes phagocytosis- binds to specific receptors on
• They recognize pathogen-associated molecules monocytes, macrophages, and neutrophils.
derived from various microorganisms
• Stimulates phagocytosis by binding its specific
receptor (TLR) to a particular substance or ligand
• Activates the production of proinflammatory cytokines
and type 1 Interferon
• Each of these receptors recognizes a different
microbial product.
 TLR 1 recognizes lipoprotein found in
mycobacteria
 TLR 2 recognizes teichoic acid and
peptidoglycan in gram positive bacteria.
 TLR 4 recognizes lipopolysaccharide in gram
negative bacteria.
• Overactivation of TLRs can ultimately lead to disruption
of immune homeostasis and thus increase the risk for
inflammatory diseases and autoimmune disorders.

ACUTE- PHASE REACTANTS

• All acute phase reactants increase the likelihood of
phagocytosis of pathogens and help healing
occurs.
• Normal serum constituents that increase rapidly by at
least 25% due to infection, injury or tissue trauma.
• Many act by binding to microorganisms and promoting
adherence, the first step in phagocytosis.
• Produced by hepatocytes (liver parenchymal cells)
within 12-24 hours in response to an increased
cytokine (IL6- IL-1,TNF-a, a macrophage inhibitory
protein.
• Enhance the phagocytosis of antigen.
• Biomarkers for the diagnosis of inflammatory disease.
• Indicators of successful organ transplant..
• Predicts the ameliorative effect of cancer therapy.
C- REACTIVE PROTEIN (CRP)
• <10 mg/L, Increases rapidly within 4-6 hours following
infection, surgery, or other trauma of the body, levels
peak value within 48 hours.
• Decline rapidly with cessation of stimulus.
• Indicator of an acute inflammation
ACUTE PHASE-REACTANTS
• Agglutination
• Precipitation
• Activation of complement by classical pathway
• Used to monitor the treatment of inflammation
and infection.
• Used to follow the course of organ transplant
(increased level may indicate an organ rejection)
SERUM AMYLOID A
• An apolipoprotein synthesized in the liver.
• Associated with HDL-cholesterol
• Cleans up the area of tissue injury by removing
cholesterol from cholesterol-filled macrophages.
• Facilitates recycling of cell membrane cholesterol
and phospholipids for reuse.
• Significantly increased in bacterial infections than
in viral infections.

LEYBA, DIMAPILIS | MMLS 3-1 7

INTRODUCTION TO IMMUNOLOGY AND SEROLOGY- LABORATORY

MANNOSE- BINDING PROTEIN/ MANNOSE-BINDING
LECTIN
• Calcium-dependent opsonin
• Recognizes mannose and other carbohydrates
found primarily in bacteria, some yeast, viruses
and several parasites.
• Widely distributed to mucosal surfaces
• Similar to complement protein C1q, binding
activates complement cascade and promote
phagocytosis.
• Major functions are opsonization and lysis of bacterial
cells.
• Enhance chemotaxis
• Modulates the activity of T and B cells
• Contributes in the clearance of immune complexes and
pathogen elimination.
SUMMARY
CHARACTERISTIC OF ACUTE PHASE REACTANTS
(SOURCE: CLINICAL IMMUNOLOGY AND SEROLOGY: STEVENS)

ALPHA 1- ANTITRYPSIN PROTEIN FUNCTION

• Plasma inhibitor of proteases released from WBC
especially Elastase (elastin and collagen). C-reactive protein Opsonization, complement activation
• Counteract the effects of neutrophil invasion Serum amyloid A Activates monocytes and
during inflammation. macrophages
• Regulates expression of proinflammatory
cytokines (TNF-a, IL-1, IL-6). Alpha1-antitrypsin Protease inhibitor
• Reacts with serine protease triggering of the
complement cascade or fibrinolysis. Fibrinogen Clot formation

HAPTOGLOBIN Haptoglobin Binds hemoglobin

• An alpha2-globulin that primarily bind irreversibly
to free hemoglobin- released by intravascular
hemolysis.
• Once bound, the complex is cleared rapidly by
Kupffer and parenchymal cells in the liver.
• Protects the kidney from damage and prevents
the loss of Iron by urinary excretion.
• Provide protection against oxidative damage
mediated by free hemoglobin.
• •
FIBRINOGEN
• The most abundant of the coagulation factors,
forms the fibrin clot.
• Fibrin stimulates endothelial adhesion and
proliferation critical to the healing process.
• Fibrin clots creates barrier that prevent the
spread of microorganisms
• Promote aggregation of RBC
• Increased levels contribute to an increased risk
for developing coronary artery disease.
(especially in women).
• Fibrinogen makes blood more viscous and
serves to promote aggregation of red blood cells
(RBCs) and platelets.
• Increased levels may contribute to an
increased risk for developing coronary artery
disease.
Ceruloplasmin Binds copper and oxidizes iron
Complement C3 Opsonization, lysis
ADDITIONAL NOTES:
INFLAMMATION
Can defined as the body’s overall reaction to injury or
invasion by an infectious agent.
• The four cardinal symptoms or clinical symptoms of
inflammation are: redness (erythema), swelling (edema),
heat, and pain.
• MAJOR EVENTS THAT OCCUR RAPIDLY AFTER
TISSUE INJURY ARE:
1. Increased blood supply to the affected area.
2. Increased capillary permeability caused by
contraction of endothelial cells lining the blood vessels.
3. Migration of WBC’s mainly neutrophils, from the
capillaries to the surrounding tissue in a process called
diapedesis.
4. Migration of macrophage to the injured area.
5. Acute phase reactants stimulates phagocytosis of
microorganisms.

CERULOPLASMIN
• The principal copper- transporting protein in plasma
• Act as ferroxidase, oxidizing iron from toxic ferrous iron
(Fe2+) to nontoxic ferric form (Fe3+)
• Serves as means of releasing iron from ferritin for
binding to transferrin
• Wilsons disease- a genetic disorder characterized by
massive increase of copper in the tissue due to a
depletion of ceruloplasmin.
 In Wilsons disease, copper accumulates in the liver
and subsequently in other tissues such as the
brain, corneas, kidneys, and bones.

COMPLEMENT
• Series of serum proteins whose overall function is
mediation of inflammation.

LEYBA, DIMAPILIS | MMLS 3-1 8

INTRODUCTION TO IMMUNOLOGY AND SEROLOGY- LABORATORY
PATHWAYS OF THE COMPLEMENT SYSTEM
 The complement system can be activated in three different
ways.
 The first pathway described, the classical pathway,
involves nine proteins that are triggered primarily by
antigen–antibody combination. Pillemer and colleagues
discovered an antibody independent pathway in the 1950s
that plays a major role as a natural defense system.
 Second pathway, the alternative pathway, was originally
called the properdin system because the protein properdin
was thought to be the main initiator of this pathway. Now it
is known that properdin’s major function is to stabilize a key
enzyme complex formed along the pathway and that the
other forms of activation are more prominent.
 The third pathway, likely the most ancient of the three, is
the lectin pathway, another antibody-independent means
of activating complement proteins. Its prototypic
constituent, mannose- (or mannan-) binding lectin (MBL),
adheres to mannose found mainly in the cell walls or outer
coating of bacteria, viruses, yeast, and protozoa. Although
each of these pathways will be considered separately,
activation seldom involves only one pathway.
• Most plasma complement proteins are synthesized in
the liver with the exception of C1 components; these
are mainly produced by intestinal epithelial cells and
Factor D, which is made in adipose tissue.1,6 Other
cells, such as monocytes and macrophages, are
additional sources of early complement components,
including C1, C2, C3, and C4.6,7 Most of these
proteins are inactive precursors, or zymogens, which
are converted to active enzymes in a very precise
order.
THE CLASSICAL PATHWAY
• The classical pathway, the first activation cascade
described, is the main antibody-directed mechanism for
triggering complement activation. However, not all
immunoglobulins are able to activate this pathway.
• The immunoglobulin classes that can
activate the classical pathway include IgM, IgG1, IgG2,
and IgG3, but not IgG4, IgA, or IgE. IgM is the most
efficient of the activating immunoglobulins because it has
multiple binding sites; thus, it takes only one molecule
attached to two adjacent antigenic determinants to initiate
the cascade.
• Two IgG molecules must attach to antigen within 30 to 40
nm of each other before complement can bind; it may take
at least 1,000 IgG molecules to ensure that there are two
close enough to initiate such binding. Some epitopes,
notably the Rh group are too far apart on the cell for this
to occur; therefore, they are unable to fix complement.
Within the IgG group, IgG3 is the most effective, followed
by IgG1 and then IgG2.
LEYBA, DIMAPILIS | MMLS 3-1
• Complement activation can be divided into three main
stages, each of which is dependent on the grouping
of certain reactants as a unit. The first stage involves
the recognition unit, which in the case of the
classical pathway is C1. Once C1 is fixed, the next
components activated are C4, C2, and C3, known
collectively as the activation unit of the classical
pathway (and the lectin pathway). C5 through C9
comprise the membrane attack complex (MAC);
this last unit complete the lysis of foreign particles.
THE ALTERNATIVE PATHWAY
• The alternative pathway was originally named for the
protein properdin, a constituent of normal serum with a
concentration of approximately 5 to 15 μg/mL.
• Although the alternative pathway can be activated on its
own, it appears that it functions mainly as an amplification
loop for activation started from the classical or lectin
pathways. Although properdin has been confirmed to bind
and initiate activation, the primary function of properdin is
to stabilize the C3 convertase formed from activation of
other factors. In addition to properdin, the serum proteins
Factor B and Factor D are unique to this pathway. C3 is a
key component of this pathway as well as the two other
pathways.
• Triggering substances for the alternative pathway include
bacterial cell walls, especially those containing
lipopolysaccharide, fungal cell walls, yeast, viruses, virally
infected cells, tumor cell lines, and some parasites,
especially trypanosomes. All of these can serve as sites
for binding the complex C3bBb, one of the end products of
this pathway. The conversion of C3 is the first step in this
pathway.
STEPS IN ALTERNATIVE PATHWAY
1. C3 is hydrolyzed by water to produce C3b, which binds
Factor B and together they attach to target cell surface.
2. B is cleaved by Factor D into the fragment’s Ba and Bb. Bb
combines with C3b to form C3bBb, an enzyme with C3
convertase activity.
3. More C3 is cleaved, forming more C3bBb. This enzyme is
stabilized by properdin, and it continues to cleave additional
C3.
4. If a molecule of C3 remains attached to the C3bBbP
enzyme, the convertase now has the capability to cleave C5.
The C5 convertase thus consists of C3bBbP3b. After C5 is
cleaved, the pathway is identical to the classical pathway.
9
INTRODUCTION TO IMMUNOLOGY AND SEROLOGY- LABORATORY

THE LECTIN PATHWAY References:

• The lectin pathway represents another means of activating Notes from the discussion by: Sir Jason Biraquit
complement. Instead of activation through antibody binding,
the lectin pathway is activated by recognition of surface Book: Clinical Immunology and Serology A Laboratory
moieties that are found on pathogens Perspective (Stevens)
• This pathway provides an additional link between the innate
and acquired immune response because it involves
nonspecific recognition of carbohydrates that are common EMILIO AGUINALDO COLLEGE-CAVITE
constituents of microbial cell walls and that are distinct from
those found on human cell surfaces. Although this pathway
is the most recently described of the three activation
pathways of complement, it is probably the most ancient.
• The lectin pathway molecules are structurally similar to
those of the classical; the classical and lectin pathways
even share the components C4 and C2. Once C4 and C2
are cleaved, the rest of the pathway is identical to the
classical pathway. The role C1q serves in the classical
pathway is filled by three classes of recognition molecules
in the lectin pathway: lectins, ficolins, and CL-K.
• The structure of all three classes of recognition molecules
is similar to that of C1q because they are all classed as
collectins. One key lectin, called mannosebinding, or
mannan-binding, lectin (MBL), binds to mannose or related
sugars in a calcium-dependent manner to initiate this
pathway.
• These sugars are found in glycoproteins or carbohydrates
of a wide variety of microorganisms such as bacteria,
yeasts, viruses, and some parasites. MBL is considered an
acute phase protein because it is produced in the liver and
is normally present in the serum but increases during an
initial inflammatory response.
• The enzymatic role played by C1r and C1s in the classical
pathway is played in the lectin pathway by serine proteases
called MBL-associated serine proteases (MASPs). There
are currently three MASPs identified, labeled MASP-1,
MASP-2, and MASP-3.
• The lectin pathway plays an important role as a defense
mechanism in infancy, during the interval between the loss
of maternal antibody and the acquisition of a full-fledged
antibody response to pathogens.

LEYBA, DIMAPILIS | MMLS 3-1 10