IMMUNOLOGY AND SEROLOGY
CYTOKINES
LECTURE
Introduction to Cytokines
➢ Cytokines are small soluble proteins that
regulates the immune system, orchestrating
both innate and adaptive immunity to
infection.
➢ These are produced by different cell types
and have modulating effects on the
hematopoietic and immune system through
activation of cell -bound receptor.
➢ Cytokines cascade produces a spectrum of
activities that lead to the rapid generation of
innate and adaptive immune responses. In
CYTOKINES of the INNATE IMMUNE RESPONSE
fact, the ability or inability to generate certain
cytokine patterns often determines the
clinical course of infection.
➢ In extreme cases, massive production and
dysregulation produces a “cytokine storm”
that leads to shock, multiorgan failure or
even death.
➢ Initially cytokines are named based on their
activities and types of cells from which they
were first isolated. The major cytokine
families include:
✓ Tumor necrosis factor (TNF)
170170 ✓ Interferons (IFN)
✓ Chemokines
✓ Transforming growth factor (TGF)
✓ Colony-stimulating factor (CSF)
✓ Interleukins (IL)
➢ Interleukins are unrelated cytokines that
must satisfy 3 criteria to be classified as
interleukins: they must have their genes
cloned; they must be inducible in leukocytes;
their biological activities in inflammation must
be catalogued.
➢ The pleiotropic (having many different
effects) of cytokines relates to the
widespread distribution of cytokine receptors
on many cell types and ability of cytokines to
alter expression of numerous genes. Thus,
some cytokines may have overlapping
effects and may alter the activity of many of
the same genes.
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➢ The increasing clinical usage of cytokines,
cytokine antagonist, and cytokine receptor
antagonists in conditions such as rheumatoid
arthritis, psoriasis, asthma, transplantation,
and cancer treatments increases the
demand of testing cytokine activity in
laboratories. The patter of cytokine expression
can also determine whether the host will be
able to mount an effective defense against
certain infections.
➢ Cytokines involved in the innate immune
response are responsible for many of the
physical symptoms attributed to
inflammation, such as fever, swelling, pain
and cellular infiltrates into the damaged
tissues.
➢ The innate immune response is non-specific
but occurs within the first hours of contact
with microorganisms. The main function of
the innate immune response is to recruit
effector cells to the area, and this is where
the cytokines are involved.
➢ Cytokines of the innate immune response
includes: Keke ni a & B
✓ Chemokines Mtgtransform sa tumor
✓ Interferon alpha and beta I -1
✓ Transforming Growth Factor beta
I -6
✓ Tumor necrosis factor alpha
✓ Interleukin-1
✓ Interleukin-6
Chemokines
➢ Chemokines are family of cytokines that
enhance motility and promote migration of
many types of white blood cells toward the
source of chemokine (chemotaxis).
➢ Chemokines are classified into four families
based on the position of N-terminal cysteine
residues:
• CXC – contains single amino acid
between 1st and 2nd cysteines
 IMMUNOLOGY AND SEROLOGY
CYTOKINES
LECTURE

• CC – has adjacent cysteine residues. Chemokine Chemokine Chemokine

• C – lacks one of the cysteines Group names receptors
• CX3C – has 3 amino acids between CC Chemokines MCP-1, MCAF, CCR2
the cysteines. JE
➢ Chemokines plays a key role in initiation and MIP-1α CCR1, CCR5
development of inflammatory responses in MIP-1β CCR5
numerous disease processes. RANTES CCR1, CCR3,
CCR5
Eotaxin CCR3

CXC CROα, MGSA, CXCR2

Chemokines MIP-2, KC
IL-8 CXCR1, CXCR2
IP-10, CRG-2 CXCR3
SDF-1 CXCR4

➢ CXCR4 and CCR5 serves as a receptor for

and HIV in CD4+ cells thus absence of these
receptors presents an immunity to HIV.
➢ RANTES and SDF-1 is highly polymorphic
ligands that blocks virus ability to bind and
delays the progression to full-blown AIDS.

➢ TNF-α and IL-6 are among the many
cytokines that induces a chemokine
production in the inflammatory response.
Combined with integrins, a cell adhesion
molecule, chemokines facilitate the
extravasation of WBC into the tissues.
➢ The spectrum of chemokines and cytokines
expressed in the inflammatory response
determines the type of cells that respond and
the genes that are turned on in response to
the stimuli.
➢ The types of cell surface receptors
expressed by WBCs are often
developmentally regulated – for example,
immature T cells possess only the
chemokine receptors related to lymphoid
tissue homing. Only mature T cells
expresses that allows them to participate in
an ongoing immune reaction
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INTERFERONS
➢ Interferons were originally so named
because they interfere with viral replications.
However, it is the Type I interferons
consisting of IFN-α and IFN-β that functions
primarily in this manner.
➢ These interferons are produced by dendritic
cells and induce the production of proteins
and pathways that directly interfere with viral
replication and cell division.
➢ Type I IFN activates NK cells and enhances
the expression of MHC Class I proteins, thus
increasing the recognition and killing of virus-
infected cells.
➢ It is also active against certain malignancies
and other inflammatory processes.
o IFN-β for treatment of multiple
sclerosis
o IFN-α for treatment of Hepatitis C,
Kaposi’s sarcoma, certain leukemias
and lymphomas
 IMMUNOLOGY AND SEROLOGY
CYTOKINES
LECTURE

(down-regulation of immune
response)
• Serves as both activator and inhibitor
of proliferation depending on the
developmental stage of the affected
cells.

Functions of TGF-B
Regulates expression of CD8 in double negative
thymocytes
Inhibits immature thymocytes by being an autocrine
inhibitory factor
Inhibits activation of macrophage
Blocks the production of IL-12 (differentiation of naïve
T cell to Th1 cells)
Inhibits the proliferation of activated B cells.

➢ Type 2 IFN
TNF-α (Cachectin)
• Interferon gamma (Immune
interferon) – secreted by T cells and
NK cells. ➢ Tumor necrosis factor were first isolated from
• This is the principal molecule tumor cells and were so named because they
produced by Th1 cells, which affects induced lysis in these cells.
the RNA expression levels of more ➢ TNF-α is most prominent member of the
than 200 genes. TNF, which consists of at least 19 different
• Most potent activator of peptides.
macrophages and boosts their ➢ It exists in both membrane-bound and
tumoricidal activity. soluble forms and causes vasodilation and
• Involved in regulation and activation increases vasopermeability.
of CD4+ Th1 cells, CD8+ cytotoxic ➢ The soluble form is derived from the
lymphocytes, and NK cells membrane bound form by proteolytic
• Function: Major macrophage cleavage with TNF-α-converting enzyme.
activator, and increases expression Membrane bound TNF-α can mediate all
of MHC class 1 and 2 cytotoxic and inflammatory effects of TNF
through cell-to-cell contact.
➢ The main trigger of TNF-α production is
➢ TGF-β the presence of lipopolysaccharide found in
• The transforming growth factor beta gram-negative bacteria.
is composed of 3 isoforms: TGF-β1, ➢ It is secreted by activated monocytes and
TGF- β2 and TGF- β3. macrophage.
• It induces antiproliferative activity in ➢ TNF has a deleterious effect, leading to
wide variety of cell types. It is also the septic shock, sudden drop in blood pressure,
primary regulator of cell growth, disseminated intravascular coagulation and
differentiation, apoptosis, migration tissue fluid infiltration.
and the inflammatory response

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 IMMUNOLOGY AND SEROLOGY
CYTOKINES
LECTURE

➢ TNF-B - Known as lymphotoxin, produced by

T cells.
➢ For killing and endothelial activation

INTERLEUKIN-1

➢ The IL-1 family consists of IL-1α, IL-1β and

IL-1RA (receptor antagonist).
➢ IL-1α and IL-1β are proinflammatory
cytokines produced by monocytes and
macrophage and is produced in the presence
of microbial pathogens, bacterial
lipopolysaccharides or other cytokines.
➢ IL-1α are mostly found as intracellular,
whereas IL1β is responsible for systemic
activity.
➢ IL-1 acts as endogenous pyrogen and
induces fever in acute phase response INTERLEUKIN-6
through its actions on the hypothalamus,
which acts as the body’s thermostat that sets ➢ It is a single protein produced by lymphoid
the body temperature to high levels. and nonlymphoid cell types and is primarily
➢ IL-1 also induces the production of cell triggered its secretion by IL-1.
adhesion molecules and assists in ➢ This pleiotrohic cytokine affects
diapedesis. inflammation, acute phase reactions,
➢ IL-1RA blocks the receptor of IL-1, limiting immunoglobulin synthesis, and activation of
the activity of the IL-1 T and B cells. IL-6 stimulates B cells to
proliferate and differentiate into plasma cells.
➢ IL-6 binds to IL-6Rα (IL-6 specific receptor)
and gp130 and is activated by janus kinase
(JAK). The binding of IL-6 causes the genes

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 IMMUNOLOGY AND SEROLOGY
CYTOKINES
LECTURE

for producing CRP, complement C3 and

fibrinogen are activated.
IFN-γ

➢ Interferon gamma is the principal molecule

CYTOKINES of the ADAPTIVE IMMUNE produced by Th1 cells. Genes involved in
RESPONSE regulation and activation of CD4+ Th1 cells,
CD8+ cytotoxic cells, NK cells, IL-12R and
IL-18R are all regulated by IFN-γ.
➢ Cytokines involved in adaptive immune
➢ It also stimulates antigen presentation by
response are mainly secreted by T-helper
MHC Class I and II molecules.
cells (Th cells) and affects T and B cell
➢ In addition, IFN-γ also strongly stimulates
function more directly.
macrophages and boosts their tumoricidal
➢ There are 3 main subclasses of Th cells:
activity.
Th1, Th2 and T Regulatory cells (Treg
➢ It is produced by:
cell). Each has a specific function and
o ligation of TCR by MHC peptide
produces a different set of cytokines.
o Cytokine stimulation by IL-12 and IL-
➢ Once T cell receptor (TCR) captures antigen,
18
clonal expansion of those particular CD4+ T
helper cells occurs. Differentiation into Th1,
Th2 or Treg cell lineage is influenced by
spectrum of cytokines expressed in initial
response.
o Th1 cells is driven by IL-12 produced
by dendritic cells,
o Th2 cells is regulated by IL-4,
derived from antibody-mediated
immunity.
o Treg cells are derived from IL-10-
responsive naïve cells and TGF-β

IL-2

➢ Also known as T cell growth factor

➢ Secreted by Th1 cells in addition to IFN-γ. It
drives the growth and differentiation of both
T and B cells and induces lytic activity in NK
cells.
➢ IL-2 alone can activate proliferation of Th2
cells and helps to generate IgG1 and IgE-
producing cells (which activates delayed
hypersensitivity).
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 IMMUNOLOGY AND SEROLOGY
CYTOKINES
LECTURE

➢ The clonal expansion of activated T helper

cells is necessary in mounting an adequate
immune response to any immunologic
challenge.

Cytokines associated with Treg Cells

➢ Treg cells are CD4+ and CD25+ T cells and

plays a key role in establishing peripheral
tolerance to a wide variety of self-antigens,
allergens, tumor antigens, transplant
antigens and infectious agents.
➢ TGF-β regulates the activity of T cells by
IL-4 inducing expression of Foxp3, a transcription
factor that causes Treg cells to suppress the
activity of other cells.
➢ IL-4 is one of the key cytokines in regulating ➢ Tregs are also responsible for inducing IL-10
Th2 immune activities and helps drive and TGF-β in adaptive T regulatory 1 cells
antibody responses in variety of diseases. (Tr1 cells). Tr1 cells are CD4+ T cells that are
➢ IL-4 turns on the genes that generates Th2 induced from antigen activated naïve T cells
cells and turns off the genes that generates in the presence of IL-10.
Th1 cells, such as genes that produces IFN-
γ and IFN-γ R subunits. The MCP-1
enhances IL-4 production by naïve T cells
and plays a role in moving naïve T cells
towards the Th2 pathway.
➢ Th2 cells functions includes regulation of
immune response including those of allergic
reaction, autoimmune diseases, and fighting
off parasites.
➢ IL-4 stimulates genes that codes for MHC
Class I, IL5, IL-13, and promotes production
of IgG2a and IgE.

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 IMMUNOLOGY AND SEROLOGY
CYTOKINES
LECTURE

T cell Th1 cell Th2 cell Treg cell ➢ G-CSF enhances the function of neutrophils
subset and decrease the IFN-γ production and
increases IL-4 production in T cells.
➢ IL-3 in conjunction with GM-CSF, drives the
Cytokine IFN-γ IL-4 IL-10, TGF-β development towards basophil, and addition
produces: of IL-5 will develop into eosinophils.

Drives MHC-II, MHC-I, IL-6, IL-2, CTLA-4,

cytokines: ICAM, IL-10, IL-13, IL-2Rα, Foxp3
VCAM, IL2, IL-4R, B7.1,
Hematopoietic
TNF-α, TNF- B7.2, M-CSF stem cell
β, IFN-γR, IL- IL-3
12R, IL-18R IL-3
Multipotential
Acts on: Enhances Enhances Establishment hematopoietic T and B cell
the activity the activity of peripheral stem cell

of: Cytotoxic of: Antibody tolerance

T cells formation inhibition of: Granulocyte, Granulocyte, Granulocyte,
Natural Allergic Th1 cells Th2 macrophage stem macrophage stem macrophage stem
cell cell cell
Killer cells response cells Antigen- GM-CSF GM-CSF, IL-3, & IL-5 GM-CSF, IL-3
Antigen Antiparasitic presenting
presentation response cells Monocyte and
PMNs Eosinophils Basophils
Macrophage
Antigen M-CSF
presentation

ERYTHROPOIETIN & COLONY-STIMULATING

FACTOR
Study Guide: Cytokines Associated With Innate
➢ The CSFs includes: Immunity
o Erythropoietin (EPO) CYTOKINE SECRETED BY ACTIONS
o Granulocyte-colony stimulating Interleukin-1β Monocytes, Inflammation,
factor (G-CSF) (IL-1β) macrophages, fever, initiation
o Macrophage-colony stimulating dendritic cells of the
factor (M-CSF) acute-phase
o Granulocyte-macrophage-colony response
stimulating factor (GM-CSF) Tumor necrosis Monocytes, Inflammation,
➢ In response to inflammatory cytokines such factor-α (TNF-α) macrophages, initiation of the
as IL-1, the different CSFs acts on bone neutrophils, acute-phase
marrow cells at different developmental NK cells, response, death
stages and promote specific colony activated T cells of tumor cells
formation for various cell lineages. Interleukin-6 Monocytes, Initiation of the
➢ IL-3 is a multilineage CSF that induces (IL-6) macrophages, acute-phase
CD34+ bone marrow stem cells to form T endothelial response,
and B cells during the differentiation cycle. cells, activation of B
➢ GM-CSF acts to drive toward other WBCs. If Th2 cells and T cells
M-CSF is activated, the cells become
macrophage, and so on.
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 IMMUNOLOGY AND SEROLOGY
CYTOKINES
LECTURE

Transforming T cells, Inhibition of Interferon-γ Th1 cells, CD8+ Activation of

growth macrophages, both T- and B- (IFN-γ) T cells, NK cells macrophages,
factor-β (TGF-β) other cell types cell increased
proliferation, expression of
induction of IgA, class
inhibition of I and II MHC
macrophages molecules,
Interferon-α Macrophages, Protects cells increased
(IFN-α) dendritic cells, against viruses, antigen
Interferon-β virally infected increases presentation
(IFN-β) cells class I MHC
expression,
activates NK
cells

Study Guide: Cytokines Associated With Adaptive

Immunity
CYTOKINE SECRETED BY ACTIONS
Interleukin-2 T cells Growth and
(IL-2) proliferation of
T and B cells
NK activation
and
proliferation
Interleukin-4 Th2 cells, mast Promotion of
(IL-4) cells Th2
differentiation,
stimulation of B
cells to
switch to IgE
production
Interleukin-5 Th2 cells Eosinophil
(IL-5) generation and
activation, B-
cell
differentiation
Interleukin-10 Th2 cells, Suppression of
(IL-10) monocytes, Th2 cells,
macrophages inhibition of
antigen
presentation,
inhibition of
interferon-
gamma

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 IMMUNOLOGY AND SEROLOGY
CYTOKINES
LECTURE

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 IMMUNOLOGY AND SEROLOGY
CYTOKINES
LECTURE

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 IMMUNOLOGY AND SEROLOGY
CYTOKINES
LECTURE

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 IMMUNOLOGY AND SEROLOGY
CYTOKINES
LECTURE

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 IMMUNOLOGY AND SEROLOGY
CYTOKINES
LECTURE

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THE COMPLEMENT SYSTEM

• A humoral mechanism of nonspecific immune response consisting of 14 distinct serum

proteins (nine components) that proceed in a cascading sequence of activation, resulting
in cell lysis.
NOTE
• 1. Complement promotes opsonization and lysis of foreign cells and immune complexes
• 2. Chronic activation of these systems can lead to inflammation and tissue damage
• 3. Most plasma complement proteins are synthesized in the liver, with the exception of C1 components, which are mainly
produced by intestinal epithelial cells, and factor D, which is made in adipose tissue.
• 4. Order of discovery = C 1 2 3 4 5 6 7 8 9
• 5. Order of activation = C 1 4 2 3 5 6 7 8 9
• 6. Acts as powerful opsonin = C3b
• 7. Acts as chemotaxin = C5a
• 8. Acts as anaphylatoxin = C3a, C4a, C5a
• 9. Ions involved = calcium and magnesium
• 10. C3 convertase
• a. Classical and Lectin pathway → C4b2a or C4b2b b. Alternative pathway → C3bBb
• 11. C5 convertase
• a. Classical and Lectin: C4b2a3b or C4b2b3b b. alternative pathway → iC3bBb3b
• An anaphylatoxin is a small peptide that causes increased vascular permeability,
contraction of smooth muscle, and release of histamine from basophils and mast cells
I. PROTEINS OF THE COMPLEMENT SYSTEM
 Bacterium
Catt

CLASSICAL PATHWAY
Bacterium
Yours 't recognition
unit
042
YCIqrs Y Catt
C2 ~
. . .
a

-
- -

[ 2b

{
¥ Recognition •
Cola
- - -

C4.b2ac.cz convertase )
- - -

czb Unit
↳~ Activation
_ . -
Cga
unit

C4b2a Cos convertase 'J ☆

C4b2a3b Ccs convertase )

(3 Activation
C1q Binds to Fc of IgM and IgG
C3a
- - -
-

( 52 - - - .

Csa
←

☆ ianaphytatoxin
C4b2a3b
C5b

C1r Activates C1s ↳ ~

ta
-
. . .
cga
Ccs convertase ) Ca
C1
Coo
Ca
Membrane

C1s Cleaves C4 and C2

C5b -

anaphylatoxin * Attack
(56678g complex
C. 6

a Membrane
Coo
Attack
C4 Part of C3 convertase (C4b)
ca
*
complex
0566789

C2 Binds to C4b – form C3 convertase

pie
C3 qaetor KEY INTERMEDIATE IN ALL PATHWAY, most concentrated complement
ingrownwettpaitoi component BACTERIUM

C5 Initiates membrane attack complex Y

Clqrs
Y
Catt

642 . . _
c4a
Recognition

C6 Binds to C5b in MAC

unit
C2~ . . -
Cab

C4b2a -03 convertase

C7 Binds to C5bC6 in MAC 632 -

-
.

C3a

Activation
C4b2a3b Unit

C8 Starts pore formation on membrane C5

T
/ C. b- a _
anaphylatoxin

Cbib

C9 Polymerizes to cause cell lysis

CG convertase
-

he
Membrane attack
performed
"
.

'M
"
Coo
[9
'

≠
/ complex

polymer C5b 6789

ALTERNATIVE PATHWAY
Factor B Binds C3b to form C3 convertase
Factor D Cleaves factor B
Properdin Stabilizes C3 convertase and C5 convertase in the presence of
magnesium
MBL PATHWAY
MBL Binds to mannose
MASP-1 Helps cleave C4 and C2
MASP-2 Cleaves C4 and C2
II.PLASMA REGULATORY PROTEINS FOR
COMPLEMENT
C1 Inhibitor (C1INH) Dissociates C1r and C1s from C1q
Factor I Cleaves C3b and C4b
Factor H Cofactor with I to inactivate C3b, prevents binding of B to C3b
The principal soluble regulator of the alternative pathway
C4- binding protein Acts as a cofactor with I to inactivate C4b
S protein (Vitronectin) Prevents attachment of the C5B67 complex to cell membrane
DAF(CD55) Dissociates C2b or Bb from binding sites, thus preventing formation of
C3 convertase
MIRL(CD59) Prevents insertion of C9 into cell membrane
CR1 (CD 35) Cofactor with I; mediates transport of immune complexes
It is found mainly on peripheral blood cells
It binds C3b and C4b but has the greatest affinity for C3b.Once bound to
CR1, both C4b and C3b can then be degraded by Factor I.
MCP (CD 46) Found on virtually all epithelial and endothelial cells except erythrocytes.
MCP is the most efficient cofactor for Factor I-mediated cleavage of C3b.
It also helps to control the alternative pathway because binding of Factor
B to C3b is inhibited
III. DEFICIENCIES OF COMPLEMENT COMPONENTS
DEFICIENT COMPONENT ASSOCIATED DISEASE
C1 (q, r,or s) Lupus-like syndrome; recurrent infections
C2 Lupus-like syndrome; recurrent infections, atherosclerosis
*most common deficiency
C3 Severe recurrent infections, glomerulonephritis (S.pyogenes
infection) *most severe deficiency
C4 Lupus-like syndrome
C5-C8 Neisseria infections
C9 No known disease association
C1 INH Hereditary angioedema
DAF Paroxysmal nocturnal hemoglobinuria
PNH
MIRL Paroxysmal nocturnal hemoglobinuria
FACTOR H OR FACTOR I Recurrent pyogenic infections
MBL Pneumococcal diseases, sepsis, Neisseria infections
PROPERDIN Neisseria infections
MASP – 2 Pneumococcal diseases
THE CLASSICAL PATHWAY
• It was the first complement pathway being studied, hence, named as classical
• It is initiated by Ag-Ab complex/binding (1 molecule of IgM, and 2 molecules of IgG for
activation)
• Recognition Units: C1q, C1r, C1s - which require the presence of calcium to maintain
structure
• C1q has a molecular weight of 410,000 and is composed of six strands that form
six globular heads with a collagen-like tail portion. C1q “recognizes” the fragment
crystallizable (Fc) region of two adjacent antibody molecules, but at least two of
the globular heads of C1q must be bound to initiate the classical pathway.
• Activation Units: C4, C2, C3
• Membrane attack complex (MAC) : C5,C6,C7,C8 ,and C9
THE PROPERDIN/ ALTERNATIVE PATHWAY
• It was the first complement pathway discovered
• Originally thought to be initiated/activated by Properdin or Factor P
• Factor P acts as a stabilizer for C3 convertase and C5 convertase in the presence of
Magnesium
• It is Initiated or activated by cobra venom factor, Lipopolysaccharide, bacterial cell wall,
yeast, virally infected cells, tumor cell lines, some parasites (e.g., trypanosomes) , and
aggregation of IgA
• It bypasses C1q,C1r, and C1s
• Activation starts with native C3 (iC3) through water hydrolysis, or from classical and
lectin pathways.
THE LECTIN /MBL PATHWAY
• The most recently described complement pathway
• The lectin pathway represents another means of activating complement without antibody
being present. Lectins ,which are calcium dependent, are proteins that bind to carbohydrates
• The lectin pathway molecules are structurally similar to those of the classical
• MBL - binds to mannose or related sugars in a calcium-dependent manner to initiate
this pathway

MBL (Mannan Binding Lectin) Homologous to C1q

MASP-1 (Mannose-binding-lectin-associated serine Homologous to C1r
protease)
MASP-2 Homologous to C1s
NOTE
• C3 - All complement pathways converge at the point of cleavage of ___
63 to C3b

• Hemolytic uremic syndrome (HUS) is the most common cause of renal failure in children
and is characterized by hemolytic anemia, low platelet count, and acute renal failure.
The atypical form of HUS (aHUS) occurs because of complement dysregulation caused
by genetic polymorphisms. The genetic mutations associated with aHUS include
those of Factor H, MCP, Factor I, Factor B, and thrombomodulin, as well as
autoantibodies to Factor H and Factor I.
LABORATORY DETECTION OF COMPLEMENTS
• 1. Techniques to determine complement abnormalities generally fall into two categories:
(a) measurement of components as antigens in serum and (b) measurement of
functional activity
• 2. The methods most frequently used to measure individual components include radial
immunodiffusion (RID) and nephelometry.
• 3. ELISA can be used to measure individual complement components
• 4. The hemolytic titration (CH50) assay is most commonly used assay for classical
pathway This assay measures the amount of patient serum required to lyse 50% of a
standardized concentration of antibody-sensitized sheep erythrocytes. The titer is
expressed in CH50 units, which is the reciprocal of the dilution that is able to lyse 50% of
the sensitized cells.
TWO TYPES OF HEMOLYTIC TITRATION ASSAY

• CH50 - for classical pathway

• AH50 - for alternative pathway. Magnesium chloride and ethylene glycol tetra-acetic acid
(EGTA) are added to the buffer and calcium is left out. This buffer chelates calcium,
which blocks classical pathway activation.
INTERPRETATION OF LABORATORY
FINDINGS
• 1. Decreased levels of complement components or activity may be caused by decreased
production, consumption, or in vitro consumption
• 2. Specimen handling is extremely important. Blood should be collected in a clot tube
with no serum separator
• 3. The tube should be spun down and the serum should be frozen or placed on dry ice if
it is not tested within 1 to 2 hours
COMPLEMENT FIXATION TEST

• 1. Indicator cells = sheep RBC coated with anti-sheep antibody / amboceptor/ hemolysin
• 2. Source of complement = guinea pig
• 3. Positive result = no hemolysis
• 4. Negative result = with hemolysis
COMPLEMENT FIXATION TEST

• 1. Indicator cells = sheep RBC coated with anti-sheep antibody / amboceptor/ hemolysin
• 2. Source of complement = guinea pig
• 3. Positive result = no hemolysis
• 4. Negative result = with hemolysis
 IMMUNOLOGY AND SEROLOGY LECTURE
WEEK 9: HYPERSENSITIVITY REACTIONS

INTRODUCTION HYPERSENSITIVITY

➢ Immune response is a defense ➢ Portier and Richet suggested a toxin to

mechanism by which the body rids itself be responsible for these effects coined
of potentially harmful antigens. In some the term anaphylaxis.
cases, however, this process can end up ➢ Hypersensitivity reactions has two
causing damage to the host. components: the priming dose of
➢ When this type of reaction occurs, it is antigen is essential which is required to
termed as hypersensitivity, which can be stimulate the immune system, followed
defined as a heightened state of immune by a shocking dose of the same antigen
responsiveness. Typically, it can be that results in the deleterious
exaggerated response to a harmless consequences.
antigen that results in injury to the tissue, ➢ It is initially based on the time it takes for
disease or even death. the reactions to cause tissue damage,
➢ There are 4 major categories of mainly immediate and delayed
hypersensitivity reactions depending on hypersensitivity.
what type of antibody and antigen does it ➢ British immunologists P.G.H. Gell and
trigger, what cell initiates it, the time it R.R.A. Coombs devised a classification
takes to manifest and the type of damage system for such reactions based on four
it does to the individual. different categories.

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 IMMUNOLOGY AND SEROLOGY LECTURE
WEEK 9: HYPERSENSITIVITY REACTIONS
r

amount of IgE in response to small

amounts of allergen.
Type I Hypersensitivity

➢ Type I Hypersensitivity – also known as

the anaphylactic reaction occurs within
a very short time frame, usually within
seconds to minutes between exposure to
antigen and the onset of the clinical
symptoms.
➢ The main reactant of the hypersensitivity
reaction is the IgE. The trigger for the
release of the IgE are known as the
atopic antigens or allergen.
➢ Atopy refers to an inherited tendency to
respond to naturally occurring inhaled
and ingested allergens with continued
production of IgE. Typically, patients who
exhibit allergic reactions produce a large

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 IMMUNOLOGY AND SEROLOGY LECTURE
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➢ IgE is primarily synthesized in the
lymphoid tissue of the respiratory and GI
tracts.
➢ Antigens processed by the tissue
macrophages is mediated by the IL-4 to
be recognized by Naïve T cell
transforming into Th2 cell.
➢ Th2 cells produces IL-3, IL-4, IL-5, IL-9,
and IL-13.
➢ IL-4 and IL-13 is used to activate B cells
to switch to plasma cells which further
increases the production of IgE.
➢ IL-3, IL-5 and IL-9 are involved in
eosinophils while IL-4, IL-9, and IL-13
acts to stimulate the overproduction of
mucus, a characteristic of most allergic
response.
➢ Plasma cells releases IgE and binds
with Fc receptors on the cell membranes
of mast cells and basophils
(sensitization). IgE molecules cross
links with the allergen, activating the
degranulation of these effector cells.
➢ These cells contain numerous
cytoplasmic membranes. Histamine,
which compromises approximately 10%
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of total weight of granular constituents, is
found 10x greater supply per mast cell
than in basophils.
➢ Unlike mast cells which are tissue
residents, basophils tend to respond to
chemotactic factors and accumulate to
tissues during inflammatory reactions.
➢ In response to the presence of IgE, the
number of receptors on the surface
increases, indicating a possible
mechanism of upregulation during an
allergic reaction.
➢ Crosslinking of surface-bound IgE, on
basophils causes the release of the
chemical mediators from the cytoplasmic
granules. The primary mediators include
histamine, heparin, ECF-A, neutrophil,
chemotactic factor, and proteases.
 Mediators Function
Histamine • H1 receptor on smooth muscle of bronchioles and small intestines - Increases
capillary permeability, altered cardiac contractility, and increased mucus secretion.
IMMUNOLOGY
• H2 receptor AND SEROLOGY
– increases LECTURE
gastric acid secretion, increases vascular permeability of
capillaries
WEEK and venules.
9: HYPERSENSITIVITY REACTIONS
• H3 receptor on nervous tissues – increased stimuli to heat and cold.
• H4 receptor found on effector cells – involved in immune regulation, chemotactic
factor

Eosinophil Chemotactic Attracts eosinophils to the area and induces expression of eosinophils to the area and
Factor of Anaphylaxis induces expression of eosinophil receptors for C3b.
ECF -

Prostaglandins Mimics the effect histamine causing bronchoconstriction and vasodilation

Leukotriene Responsible for late-phase symptoms of immediate sensitivity. 1000x more potent than
histamine in causing increased vascular permeability, bronchoconstriction, hypotension,
chemotactic factor for neutrophils and eosinophils

Platelet activating Factor Causes aggregation of platelets, chemotactic factor for neutrophils and eosinophils

Heparin Makes the blood less viscous

Mediators of Type 1 Hypersensitivity

MEDIATOR ACTIONS
Primary (Performed) Histamine Smooth muscle contraction, vasodilation, increased
vascular permeability

Heparin Smooth muscle contraction, vasodilation, increased

Hep HEP na vascular permeability
Eosinophil chemotactic factor of Chemotactic for eosinophils
anaphylaxis (ECF-A)

Neutrophil chemotactic factor of Chemotactic for neutrophils

anaphylaxis (NCF-A)

Proteases (e.g., tryptase, Convert C3 to C3b, stimulate mucus production,

chymase) activate cytokines

Secondary (Newly Prostaglandin PGD2 Vasodilation, increased vascular permeability

synthesized)
Leukotriene LTB4 Chemotactic for neutrophils, eosinophils

Leukotrienes LTC4, LTD4, Increased vascular permeability, bronchoconstriction,

LOKO -

LOKO LTE4 mucus secretion

C Papa .

Platelet activating factor (PAF) Platelet aggregation

Cytokines IL-1, IL-3, IL-4, IL-5, Increase inflammatory cells in area, and increase IgE
IL-6, IL-9, IL-13, IL-14, IL-16, production
TNF-α, GM-CSF

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 IMMUNOLOGY AND SEROLOGY LECTURE
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Clinical Manifestations of Type I

Hypersensitivity

➢ The clinical manifestations caused by

release of both preformed and newly
synthesized mediators from mast cells
and basophils vary from a localized skin
reaction to a systemic response known
as anaphylaxis.
➢ Symptoms depends on variables such as
route of exposure, dosage, frequency
of exposure. Test for Type I Hypersensitivity
➢ Anaphylaxis is the most severe type of
allergic response because of its acute ➢ Skin test is an introduction of small
reaction that simultaneously involve amount of different antigens either by
multiple organs and can be fatal if not cutaneous (prick test) or intradermal
promptly treated. injection (tuberculin, diluted 100-1000x).
➢ Examples of allergic agents includes Reactions such as scratching and
venoms from bees, snakes; drugs such reddening is observed within the
as penicillin, ibuprofen; foods such as localized site.
nuts, seafoods, eggs, dairy products; ➢ Resident mast cells degranulate and
latex, pollen, dust, animal hair. produces a wheal after 15-20 minutes. A
➢ Clinical anaphylaxis begins within wheal of >3mm than the negative
minutes after antigenic challenge and control is considered positive.
may include Respiratory:
bronchospasm and laryngeal edema;
Skin: vascular congestion, flushing,
pruritus, urticaria and angioedema; Gl
tract: diarrhea, vomiting: Cardiovascular:
intractable shock on smooth muscles of
circulatory system, hypotension,
arrythmias. The severity depends on the
consequent buildup of IgE on mast cells
and basophils.
➢ Avoidance of known allergen is the best
way to prevent immediate
hypersensitivity reactions. Drugs to treat
manifestations from Type I
hypersensitivity includes antihistamines,
antileukotrienes, bronchodilators,
decongestants, and corticosteroids.
Another approach is the ➢ In vitro test such as the
hyposensitization by introducing very Radioimmunosorbent test (RIST) that
small quantities of sensitizing antigen uses radiolabeled IgE to compete with
with the idea of IgG buildup, blocking the patient IgE for binding sites on a solid
IgE antibodies before it gets contact with phase coated with anti-IgE. It is now
the antigen. replaced by noncompetitive solid-phase
immunoassays.

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➢ Radioallergosorbent test (RAST) 2. complement generated lysis if the

follows the same principle but involves complement cascades goes to
the use of fluorescent labels rather than completion.
radioactivity. Allergen-specific IgE
testing is a non-competitive solid-phase
immunoassay in which solid-phase
(cellulose, agarose) is coated with
specific allergen and reacted with
patient serum. A labeled Anti-IgE is
added.

Type II Hypersensitivity

➢ Also known as the cytotoxic sensitivity

or antibody-dependent cell
cytotoxicity. The main reactants for this
type of hypersensitivity includes IgG and
IgM.
➢ These are triggered by the antigens
found on cell surfaces, maybe
heteroantigens or self-antigens.
➢ Antibody coats the cell surface, promotes
phagocytosis, and complement
activation. Macrophage, neutrophils, and
eosinophils have Fc receptors that bind
to the Fc region of the antibody on target
cells.
➢ Membrane attack complex acts on self-
cells as seen in transfusion reaction,
hemolytic disease of the newborn and
hemolytic anemia.
➢ ADCC or antibody-dependent cellular
cytotoxicity involves the binding of
cytotoxic cells with Fc receptors in the Fc
region of the antibodies that is coating the
target cell.
➢ Cell cytotoxicity usually involves
individual cells, but aggregation of tissue
damage is usually organ-specific
including Goodpasteur syndrome,
Hashimoto's thyroiditis, myasthenia
gravis, insulin-dependent diabetes
mellitus

➢ NK cells also contain Fc receptors thus

triggering the cytotoxicity.
➢ If complement is activated, complement
can trigger cellular destruction in two
ways:
1. coating cells with C3b, thus
facilitating the phagocytosis or

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 IMMUNOLOGY AND SEROLOGY LECTURE
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➢ Autoimmune hemolytic anemia is

another example of type lI
hypersensitivity that is directed against
self-antigens, because individuals with
this disease attacks their own RBCs. It is
categorized at which temperature the
antibody reacts - either warm at 37°C
(WAIHA) and cold below 30°C (CAIHA).
➢ WAIHA is believed to be developed from
exposure to vial or respiratory infections
and the antibody produced against them
autoreacts with the self-antigens, in this
case the RBCs.
➢ CAIHA have been to occur following
Mycoplasma pneumonia infection and
other cold agglutinin-producing disease
➢ ABO blood group is of primary such as chronic lymphocytic leukemia,
importance in considering transfusion. lymphoma, myelodysplastic syndrome,
The body does not produce antibody SLE.
against the self-antigens present in the
red blood cells. In incompatible blood
transfusion reactions, antibody coats
the donor cells causing the activation of
the complement pathway, lysing the cell
in the process. Clinical manifestation of
incompatible blood transfusion includes
massive intravascular hemolysis.
➢ Erythroblastosis fetalis, also known as
the hemolytic disease of the newborn
(HDFN) develops when the mother's
antibody crosses the placenta and
attacks the fetal RBCs. This condition
occurs when a pre-sensitized mother (Rh
negative) triggers an immune response
Rh-positive fetus. which results to severe
hemolysis, anemia and
hyperbilirubinemia which can be fatal.

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 IMMUNOLOGY AND SEROLOGY LECTURE
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Test for Type II Hypersensitivity
➢ Coombs test made it possible for the
detection of coated antibody or
complement on the surface of red blood
cells. It is initially tested by using by
polyspecific antihuman globulin,
which is a mixture of IgG, C3b and C3d.
➢ If the test is positive, then it is treated
again with a monospecific Anti-IgG and
Anti-C3b and Anti-C3d to determine
which of these is present. If the hemolytic
anemia is caused by IgM, it will only react
with the complement.
Type III Hypersensitivity
➢ It is also known as the immune-complex
hypersensitivity or complement-
mediated hypersensitivity. The
combination of antigen and antibody
reaction that accumulates in a particular
tissue or organ involves the type III
hypersensitivity.
➢ In a normal course, immune complexes
are removed by phagocytes, however in
response to excess antigen, too much
immune complexes are produced in
cases of autoimmunity, severe recurrent
infections, and contact with outside
environment. Deposition occurs when
the clearance is exceeded.
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➢ The deposition of immune complexes still
triggers the activation of complement
which provokes inflammation.
➢ Depositions occurs depending on the
concentration of antigen or antibody. If
antigen is excess, the sites on antibody
molecules become filled before cross-
links can be formed. If antibody is excess,
a lattice cannot be formed due to lack of
antigenic determinant sites.
➢ Activation of the complements leads to
the production of anaphylatoxins and
opsonin, hence causing a further
damage on the tissue due to aggregation
of effector cells.
➢ Formation of C5a, C3a and C2a triggers
the degranulation of mast cells that
causes the release of histamine, dilating
the blood vessel walls.
➢ Formation of C3b and C4b stimulates
the phagocytic activity of macrophages
and neutrophils.
➢ Arthus reaction is a classic example of
localized type III reaction demonstrated
by Maurice Arthus.
➢ An immunized rabbit that produces
circulating antibodies were injected with
a localized inflammatory antigen result to
a necrotic lesion that may ulcerate due to
subsequent formation and accumulation
 IMMUNOLOGY AND SEROLOGY LECTURE
WEEK 9: HYPERSENSITIVITY REACTIONS
of immune complexes that deposit to
dermal blood vessels.
➢ Serum sickness is a generalized
type III reaction causes by deposition of
immune complexes in different sites of
the body.
Passive immunizations such as vaccines
used to treat diphtheria, tetanus and
gangrene triggers this type of reaction.
➢ The single dose triggers a manifestation
like both priming and shocking dose,
but usually self-limiting diseases and
does not develop sequelaes. Symptoms
includes typical inflammatory response
together with lymphadenopathy.
➢ Autoimmune diseases fall into this type
of hypersensitivity reactions such as
✓ Rheumathoid arthritis
✓ Systemic lupus
erythematosus
✓ Goodpasteur disease
➢ Drugs also triggers the accumulation of
immune complexes.
✓ Penicillin
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Ready Get set
PS : HMMM
✓ Sulfonamides
➢ Infectious diseases that overwhelms
the system.
✓ Hepatitis
✓ Meningitis
✓ Mononucleosis
✓ Malaria
Tests for Type III Hypersensitivity
➢ In specific diseases such as SLE and
rheumatoid arthritis, the presence of
antibody can be detected by
agglutination reactions using antigen-
coated carrier particles, including red
blood cells, latex, or enzymes.
Fluorescent staining is also used to
determine the deposition of immune
complexes in tissues in vivo.
Type IV Hypersensitivity
➢ It is also known as delayed
hypersensitivity because its
response is delayed. It starts with
sensitization phase of about 1-2
weeks after exposure to the antigen.
Subsequent exposure to antigen
takes several hours for the symptoms
to develop and reach a peak of 48-72
hours.
➢ It was first discovered by Robert
Koch who observed that individuals
with Mycobacterium tuberculosis
develop a localized inflammatory
response when injected intradermally
with a filtrate from the organism.
➢ The reaction is usually characterized
by the action of the Langerhan cells
on the skin. It differs from other
hypersensitivity reactions due to it
 IMMUNOLOGY AND SEROLOGY LECTURE
WEEK 9: HYPERSENSITIVITY REACTIONS

being a cell-mediated type, without ivy, soaps, strong chemicals, nickel

involvement of antibody or which causes allergic dermatitis.
complement. The antigen will be ➢ What happens is the compounds
transported through the lymphatics serves as a hapten but then
where transported by the plasma proteins
➢ T cells are activated by the antigen. present, which makes them a true
➢ Th1 cells play a major role in its immunologic antigen.
manifestations as these cells takes ➢ Dermatitis usually last for 3-4 weeks
time to be sensitized and releases after the antigen has been removed.
cytokines that causes an
inflammatory response. ➢ Tuberculin hypersensitivity uses a
purified protein derivative (PPD) of
the Mycobacterium tuberculosis and
injected intradermally and is read
after 48-72 hours. A positive test
indicates that the person has been
exposed to MTB or related organism,
but not necessarily mean there is a
presently active case.

➢ Test for Type IV Hypersensitivity

➢ Other tuberculin test such as
lepromin test is useful for leprosy.
➢ Fungal test such as for
coccidiomycosis, trichophyton and
histoplasmosis.

➢ Contact dermatitis is due to contact

with low molecular weight
compounds that touches the skin.
Common examples include poison

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