CHAPTER 5: CYTOKINES
dysregulation that leads to:
 CYTOKINES
 Are small soluble proteins
that regulate the immune
system.
 Have activity-modulating
effects on the hematopoietic
and immune system through
activation of cell bound
receptor proteins in
response to specific stimuli
such as:
a. Bacterial
lipopolysaccharides
b. Flagellum
.. through the ligation of cell
adhesion molecules or through
the recognition of foreign
antigens by host lymphocytes.
The effects of cytokine in vivo
include:
a. Regulation of cell growth
b. Regulation of cell differentiation
c. Regulation of gene expression
These effects are achieved
through both:
a. Autocrine stimulation:
(within the cell)
b. Paracrine stimulation
 Cytokines do not act alone
but in conjunction with
many other cytokines that
are induced during the
process of immune
activation.
Cytokine Cascade: produces
spectrum of activities that lead
to rapid generation of:
A. Innate immune response
B. Adaptive immune response
Cytokine Storm: caused by
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massive overproduction and
a. Shock
b. Multi-organ failure
c. Death
 Initially, cytokines were
named based on their
activities and the type of
cells from which they were
first isolated.
 Pleiotropic: having many
different effects; nature of
cytokine activity that relates
to the widespread
distribution of cytokine
receptors on many cell types
and the ability of cytokines
to alter the expression of
numerous genes.
 Redundancy: many
different cytokines may
share properties; acitvate
some of the same pathway
and genes; many cytokines
share receptor subunits.
Example:
a. IL-6
b. IL-11
c. Leukemia inhibitory
factor
d. Oncostatin M
e. Ciliary neutrophilic factor
f. Cardiolipin
.. all of which are seen in gp130
subunit.
 The increasing clinical usage
of cytokines, cytokine
antagonist, cytokine
receptor antagonist are seen
in the conditions such as:
a) Rheumatoid Arthritis
b) Psoriasis
c) Asthma
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d) Crohn’s disease
e) Transplantation
f) Cancer treatment
 Pattern of cytokine
expressions can determine
wether a host will be able to
mount an effective defense
against and survive certain
infections.
 Numerous immunodeficiency
syndromes and leukemias
are caused by defects in
cytokines or their signal or
receptor transduction unit.
 CYTOKINES OF THE
INNATE IMMUNE
RESPONSE:
 Responsible for many of the
physical and symptoms
attributed to inflammation
such as fever, swelling, pain
and cellular infiltrates into
damage tissues.
a. Interleukin 1
b. Tumor necrosis factor
alpha
c. IL-6
d. Chemokines
e. Transforming growth
factor beta
f. Interferon alpha
g. Interferon-beta
 Interleukins- these are
unrelated cytokines that
must satisfy the three
criteria in order to be
classified as IL:
i. Must have their gene
cloned.
ii. Must be inducible in
leukocytes
iii. biological activities in
inflammatory processes
must be catalogued.
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 INTERLEUKIN 1
 Mediator of host
inflammatory response to
natural immunity.
 Major cellular source:
activated mononuclear
phagocytes
 Has the ability to cause:
a) Fever - because IL1 is
an endogenous pyrogen
which gives signal to the
hypothalamus to set the
the body temperature to
a high level.
b) He synthesis of acute
phase plasma proteins
by the liver.
c) Induces the production
of vascular cell-
adhesion molecules as
well as chemokines and
IL-6.
d) Induces the production
of colony forming
factor in Bone Marrow.
 Interleukin 1 is induced by:
i. Cytokines
ii. Bacterial antigens
iii. Bacterial
lipopolysaccharides.
 Interleukin 1 is consist of :
a. IL 1 alpha
b. IL 1 beta
c. IL receptor antagonist
 Interleukin 1 alpha
-Found intracellular within:
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 A. Monocytes
B. Macrophages
-Can be released after cell
death and can help attract
inflammatory cells to the areas
where cells and tissues are
being damaged.
 Interleukin 1 beta
-Have systemic activity
-Cleaved intracellularly to active
form that is then secreted by
monocytes.
 Interleukin 1 Receptor
Antagonist
-Produced by:
A. Monocytes
B. Macrophages
-Acts as antagonist to IL-1 by
blocking the IL-1 receptor and
limiting the availability of the
receptor for IL-1.
-Regulate the physiologica
response to IL-1 and turn off
the response when no longer
needed.
 TUMOR NECROSIS
FACTOR
 First isolated from tumor
cells and were so named
because they induced lysis
in these cells.
 Principal mediator of host
response to gram negative
bacteria.
 Mediator of both natural and
acquired immunity and an
important link between
specific immune response
and acute inflammation.
 Major source:
Lipopolysacchardie activated
mononuclear phagocytes
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 TNF alpha
-most prominent member of
TNF supefamily.
Exist in both:
a. Soluble form
b. Membrane bound form
Causes:
a. Vasodilation
b. Increased vascular
permeability
 TNF alpha secreted by
activated monocytes and
macrophages can activate T
cells through it’s ability to
induced the expressionof:
A. Chemokines
B. MHC II molecules
C. Vascular Adhesion
Molecules
* these actions enhance antigen
presentation and activate T cells
to respond to pathogens that
triggered the initial
inflammatory response.
 Secreted at higher levels can
have deleterious systemic
effects:
I. Septic Shock
II. Decreased blood
pressure
III. Decresae tissue
perfusion
IV. DIC
 TNF Receptor 1
-expressed in most tissues
-binds to soluble TNF alpha
-It is the primary mediator of
TNF-alpha signa transduction.
 TNF Receptor 2
- Expressed in epithelial cells
and cells of the immune system
-Acitvated by membrane bound
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form of TNF alpha.
 TNF alpha activity at least
partially regulated by soluble
forms of both TNF receptors
acts to:
a. Binds to excess TNF alpha
b. Combine the short life of
TNF apha
c. Serves to limit the cytokines
signaling activity.
 INTERLEUKIN-6
 Secretion is primarilly
triggered by IL-1.
 Synthesized by mononuclear
phagocytes, vascular
endothelial cells, fibroblast
and other cells in response
to IL-1 , TNF and activated T
cells.
 Pleiotrophic cytokine
affects:
A. Inflammation
B. Acute phase reactions
C. Immunoglobulin
synthesis
D. Activation states of T
and B cells.
 IL-6 stimulates B cells to
proliferate and differentiate
into plasma cells and
induces CD4 + T cells to
produce greater quantities of
both pro and
antiinflammatory cytokines.
 Only one Il-6 receptor has
been identified
 CHEMOKINES
 Family of cytokines that
enhance motility nad
promote migration of many
IMMUNOLOGY AND SEROLOGY
types of WBCs toward the
source of
chemokine( chemotaxis)
 Four Families (based on the
position of N terminal
cysteine residues)
1. CXC
2. C3XC
3. CC
4. C
 Chemotactic production in
inflammation is induced by:
A. IL-6
B. TNF alpha
 Chemokines plus cell
adhesion facilitates the
extravasation of leukocytes
into the damage tissues.
 Leukocytes rolling in the
endothelial cells activate
their chemokine receptors in
the presence of chemokines
(activates integrin)
Intergrin is a cell adhesion
molecule that gives leukocytes
firm adhesion.
 Shared expression of
chemokine receptors among
different types of leukocytes
allows colocalization of
multiple cell types to the
damaged tissues and helps
broadens the response to
tissue damage.
 TRANSFORMING GROWTH
FACTOR BETA
 Before, this was known to be
a factor that induced the
growth arrest in tumor cells.
 Now, it is known to induced
antiproliferative acitivity in
wide variety of cell types.
 It has three isoforms:
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 A. TGF B1
B. TGF B2
C. TGF B3
 Active TGF serves as
regulator of:
A. Cell growth
B. Cell differentiation
C. Apoptosis
D. Migration
E. Inflammatory response
( it downregulate
inflammation when no
longer needed)
 In immune response, TGF
beta acts as :
1. Acivator of proliferations
2. Inhibitor of proliferation
depending on the
developmental stage of the
affected cell.
 INTERFERON
 It is a glycoprotein that
exert virus specific but host
specific antiviral activity by
inducing the transcription of
cellular genes coding for
antiviral proteins that
selectively inhibit the
synthesis of viral RNA and
proteins.
 Immunoregulatory
functions:
A. Inhibits B cell
activation and antibody
production enhancement
by T cell acitivity
B. Enhancement of
activity of NK cells.
 Inhibit the growth of
nonviral intracellular
parasites
 The followinf stimulates
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inteferon production:
A. Viral infection
B. Intracellular parasites
C. Protozoa
D. Bacteria
E. Bacterial products
TYPE I INTERFERON
A. Interferon alpha
B. Interferon beta
TYPE II INTERFERON
A. Interferon gamma
 Interferon alpha
 It is a major interferon
produced by virus induced
leukocyte cultures.
 Primary producer cells:
null lymphocytes
 Other name: leukocyte
interferon
 MAJOR ACTIVITIES:
A. Antiviral activity
B. Activation of NK cell
 Used to treat hepatitis C and
Kaposi Sarcoma, as well as
certain leukemias and
lymphomas.
 Interferon beta
 Major interferon produced
by double stranded RNA
induced fibroblast cultures.
 Primary producer cells:
A. Fibroblast
B. Eputhelial cells
C. Macrophage
 Other names:
A. Fibroblast interferon
B. Epithelial interferon
C. Fibroepithelial
interferon
 MAJOR ACTIVITY: antiviral
 Used in treating patients
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 with multiple sclerosis.
 Interferon gamma
( CYTOKINE THAT IS
INVOLVED IN ADAPTIVE
IMMUNE RESPONSE)
 Major interferon produced
by stimulated lymphocyte
cultures
 Primary producer cells: B
lymphocytes
 MAJOR ACTIVITY:
Immunoregulation
 CYTOKINES IN THE
ADAPTIVE IMMUNE
RESPONSE
 Cytokines in adaptive
immune response are mainly
secreted by T cells especially
T helper cells and it affects T
and B cell function more
directly.
 There are 3 subclasses of T
helper cells:
a) Th1 cells- driven by the
expression of IL12 by
dendritic cells; primarily
responsible for cell
mediated immunity.
b) Th2 cells-
developmentally
regulated by IL-4 ;
drives antibody mediated
immunity.
c) T regulatory cells
IMMUNOLOGY AND SEROLOGY
(Treg)- derived from IL-
10 responsive naive T
cells; regulate the
activities of Th1 and Th2
cells.
 Each subclass has specific
functions and produces a
dfferent cytokines.
 TH1 CYTOKINES
 It is driven by IL-12
 IL-12 is produced by
dendritic cells in damaged
tissues in response to cetain
stimuli such as
mycobacteria, intracellular
bacteria and viruses. It
increases the cytolytic
activity of NK cells.
 Activation of Th1 cells
induces high level
expression of IFN gamma.
a) INTERFERON GAMMA
 It is the principal cytokine
produced by Th1 cells and it
affects the RNA expression
of more than 200 genes.
 FUNCTION:
A. It regulates the genes
involved in regulation and
activation of CD4 + T cells,
CD8 + T cells, NK cells etc.
B. Stimulates antigen
presentation in MHC I and
MHC II molecules.
C. Strong stimulator of
macrophages and boost
theor tumoricidal activity.
b) INTERLEUKIN-2
 Also secreted by Th1 cells.
 Also known as T cell growth
factor.
 FUNCTION:
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 A. it drives the growth and
differentiation of both T and
B cells and induces lytic
activity in NK cells.
B. It can also activate the
proliferation of Th2 cells and
heps generate IgG1 and IgE
producing cells.
 TH2 CYTOKINES
Th2 cells are primarily
responsible for antibody
mediated immunity.
a. INTERLEUKIN -4
 It is one of the key cytokines
regulating Th2 immune
activities and helps drives
antibody response in variety
of diseases.
 IL4 activity on naive T cells
turns on the genes that
generate Th2 cells and turns
off the genes that promotes
Th1 cells.
b. INTERLEUKIN-10
 Has anti-inflammatory and
suppresive effects on Th1
cells.
 Cell that produces it:
monocytes, macrophage,
CD8 + T cells and Th2 CD4+
Tcells.
 FUNCTION:
A. It inhibits antigen
presentation by macrophage
and dendritic cells
B. Stimulates CD8 + T cells.
C. Induces the production of
MHC II on B cells.
D. Serves as antagonist to
IFN gamma.
 CYTOKINES associated
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with Tregs Cells
 Tregs cells are CD4 and
CD25 + T cells that are
selected in the thymus.
 Role: Establish peripheral
tolerance to wide variety of
self antigens, allergens,
tumor antigens, transplant
antigens and infectious
agents.
 It express their suppresive
activities on Th1 and Th2
cells by producing more IL-
10 and TGF beta or Il-5.
 ERYTHROPOIETIN AND
COLONY STIMULATING
FACTORS
COLONY STIMULATING
FACTORS include:
a) Interleukin 3
b) Erythropoietin (EPO)
c) Granulocyte CSF
d) Macrophage CSF
e) Granulocyte-Macrophage
CSF
 In response to IL-1, the
different colony forming
stimulating factors acts on
bone marrow cells resulting
to net effect of increase in
white blood cells to respond
to ongoing inflammatory
responses.
 Interleukin 3
 Is a multilineage colony
stimulating factor that
induces CD34 + bone
marrow cells to form T and
B cells.
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 GM-CSF
 Acts to drive differentiation
toward other white cell
types.
 If M-CSF is activated: the
cells becomes macrophage
 If G-CSF is activated: the
cells become neutrophils.
 IL3 + GM-CSF = Drives the
development of basophils
and mast cells.
 IL3+ GM-CSF+ IL-5
=drives the development of
eosinophils.
 ERYTHROPOIETIN(EPO)
 Regulates the RBC
production on bone
marrow but it is primarily
produced by the kidneys.
 RBC proliferation induced by
EPO improves oxgenation
of the tissues and
eventually switches off EPO
production.
 CYTOKINES AND ANTI-
CYTOKINE THERAPIES
 Cytokine inhibiting biologics
disrupts the interaction
between cytokines and their
receptors.
 Monoclonal antibodies that
are less immunogenic
function as receptor
antagonists.
Example:
 Inflixmab (Remicade)-
IMMUNOLOGY AND SEROLOGY
blocks the activity of TNF
alpha in rheumatoid arthritis
and Chron’s disease.
 Etanercept (Enbrel)- bind to
TNF alpha and block it’s
activity.
CLINICAL ASSAY FOR
CYTOKINES
 Clinical evaluation of
cytokine profile could be
prognostic and diagnostic
value to the physician.
 For and instance:
Tuberculosis is controlled by
cell mediated immunity not
antibody mediated immunity
so immunity is therefore
driven by Th1 cells not Th2
cells.
 ELISpot- is the most basic
test for cytokine which
employs Enzyme Linked
Immunosorbent Assay
(ELISA) technqiue on invitro
activated peripheral white
blood cells.
PROCEDURE:
1. A monoclonal or polyclonal
antibody specific for cytokine is
precoated onto a microplate.
2. Antigen stimulated white
blood cells will be used as
positive control and saline
stimulated white blood cells are
used as negative controls.
Both are pipetted into the wells.
3. Placed the microplate in
humidifiec C02 incubator at
37C fro a specified period of
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time.
Note: During the incubation the
immobilized antibody binds to
the secreted cytokines.

4. Wash to remove any cells

and unbound sunstances.

5. Biotinylated polyclonal
antibody (enzyme labeled
secondary antibody) is added.

6. Wash to remove unbound

biotinylated polyclonal antibody.

7. Alkaline phosphatase
conjugates to streptavidin is
added.

8. Unbound enzyme is washed

away and substrate solution
is added.

9. Substrate changes color in

the presence of the enzyme
labeled antibody bound to
cytokine. (colored precipitate
will form at the sites of cytokine
localization and with each spot
representing and individual
cytokine secreting cells.)

10. Spots are counted using

steomicroscope or automated
ELISpot reader.

IMMUNOLOGY AND SEROLOGY 9