What are Cytokines?
" Many cells are involved in immune responses
" Rely heavily on each other, must coordinate
Cytokines
functions to thwart infections
" Signals are transmitted from one cell to others
by secretion of CYTOKINES
Lectures 30-31
 Small proteins or glycoproteins
 Direct development of WBC
 Execute effector functions
Properties of Cytokines Types of Cytokine Signaling
" Involved in signal transduction " Cytokine may be secreted from cell and signal cell that
secreted it  AUTOCRINE action (e.g. IL-2)
" Bind to cell surface receptors
" Cytokine may be secreted
" Trigger cascades, similar in
from cell and signal second
function to BCR and TCR
cell in close proximity 
" Receptors have extremely high PARACRINE action
affinity (e.g. IL-1, IL-2)
" KD ca. 10-10 to 10-12 M " Cytokine may be secreted
by cell and signal other cells
" Allow cytokines to exert activity at
in distant parts of body 
extremely low concentrations
ENDOCRINE action
Roles of Cytokines Cytokines are  Schizophrenic
" Stimulate or inhibit many functions critical to immunity " Cytokine signaling is complex
and much is not understood
 Cellular activation, proliferation, and differentiation
 Secretion of antibodies and cytokines
" May be PLEIOTROPIC 
single cytokine, many effects
" Cytokine signaling often up-regulates cytokine receptor
expression
" Sometimes REDUNDANT 
multiple cytokines, same effect
" Signal may stimulate secretion of other cytokines
" May produce SYNERGISTIC
" Diffuse to other cells; many types potentially affected
effects   the whole is greater
" Cytokines produced by TH affect B cells, TC cells, MĆ,
than the sum
granulocytes, hematopoietic stem cells (HSCs)
" Effects of cytokines may be
" Entire network potentially activated by a single cell
ANTAGONISTIC
Attributes of Cytokines Cytokine Nomenclature
" Encompass two classes of secreted signaling molecules
" May trigger CASCADE affect
 Identified as lymphocyte products  LYMPHOKINES
 Cytokine produced by one cell signals
 Identified as monocyte and MĆ products  MONOKINES
another cell
" Old terminology misleading; molecules are secreted by a
 Second cell produces new cytokine(s),
broad spectrum of cells  collectively termed  cytokines
signals third cell
 Many are INTERLEUKINS - secreted by and affect leukocytes
 Third cell produces numerous cytokines
 Others have chemoattractant role - CHEMOKINES
that signal multiple cells
" Four structural families of cytokines
" Signal passed on is amplified in much
 Hematopoietin family
the same way as in kinase or other
 Interferon family
signal transduction cascades  Chemokine family
 Tumor necrosis factor (TNF) family
Signaling Molecules with Related
Biological Functions of Cytokines
Functions
" GROWTH FACTORS and HORMONES are similar in " May be produced by many cells;
principally TH and MĆ
structure and function to cytokines
 All are secreted low molecular weight signaling molecules
" Capable of regulating immune
 All exert effects by binding to receptors on target cells
system in its entirety
 All require minute amounts to exert effects
" Activate and repress innate
" Growth factors are produced constitutively, hormones
immunity, adaptive immunity,
and cytokines are produced in response to stimuli inflammation, hematopoiesis
" Hormones are produced by GLANDS and affect " Over 100 cytokines known
particular cell type(s) in distant locations in body
" Rarely act independently
" Cytokines may function in either an autocrine or
" Often stimulate production of
paracrine fashion
other cytokines  CASCADES
Conferring Specificity to Cytokines Cytokine Receptor Families
" Some cytokines are functionally redundant; actions are
" Four families of cytokines, five cytokine receptor
non-specific in many ways
families
" Production of cytokines usually results in a specific
" Receptor families based on structure, not
response
necessarily the family of cytokine recognized
" How is specificity of response controlled?
 Immunoglobulin superfamily receptor
" Cells can t respond to cytokine if they don t have receptor
 Class I cytokine (hematopoietin) receptor family
 Receptor expression may be dependent on interaction with
 Class II cytokine (interferon) receptor family
antigen (TH , TC , B cells)
 TNF receptor family
 May require direct interaction with cell producing cytokine
 Cytokine accumulates in junction between cells; only site where  Chemokine receptor family
cytokine concentration is high enough to elicit effect
Cytokine Receptor Families Cytokine Receptor Families
" Immunoglobulin superfamily " Class II (interferon) receptor
family
 Characteristic immunoglobulin
fold domains
 Originally thought to bind only
 Typically contain 3 Ig folds interferons; recently disproved
 Bind to IL-1, M-CSF, and C-Kit  Similar in structure to class I;
contain CCCC motif
" Class I (hematopoietin)
" TNF receptor family
receptors
 Repeating domain structure
 Encompass most IL receptors
 Multiple conserved cys residues in
 Intimately associated with
each domain
hematopoiesis
 Binds TNF and other cytokines
 Two conserved motifs - CCCC and
WSXWS  Binds certain CELL-SURFACE
ligands (CD40/CD40L, Fas/FasL)
Cytokine Receptor Families Cytokine Receptor Subfamilies
" Chemokine receptor family " Many cytokine receptors have
two different subunits
 Seven transmembrane
spanning domains
 May have unique cytokine
 Usually coupled to G PROTEIN specific subunit combined
with common subunit
" Interaction with chemokine
 Cytokines sharing a common
activates G protein
subunit comprise a SUBFAMILY
 Transduces signal
" GM-CSF, IL-3, and IL-5
 Activates multiple
receptors have unique Ä… chains
target proteins
 All share same ² chain - signal transducing subunit
 Results in production
of second messengers,  Complete receptor does not assemble until cytokine contacts Ä…
including IP3/DAG, Ca++, chain - cytokine specificity subunit
cAMP
 Explains redundancy and antagonism (how?)
Redundancy and Antagonism Among
Cytokine-Receptor Interactions
Cytokines
" Cytokine initially associates " Same ² chain shared by 3 receptors
with specific Ä… chain
 ² chain transduces signal
 All 3 receptors transduce same signal;
" Affinity for partial receptor
explains redundancy
is extremely low
" Prior binding of IL-3 prevents
" Binding allows interaction
binding of GM-CSF and vice versa
with ² chain
 Number of common ² chains is limiting
" Two changes occur  Binding of first cytokine to Ä… chain
sequesters ² chains in high-affinity
 Affinity for cytokine increase substantially
complexes
 Receptor becomes competent to transduce signal
 ² chains unavailable for association with
" Signal transduction domain resides in ² subunit, but only second cytokine/Ä… complex
dimeric receptor can transduce signal  Explains antagonism
The IL-2 Receptor Roles of Subunits in IL-2 Receptor
" Among best-characterized
" ² and Å‚ chains involved in signal transduction
cytokine receptors
" Ä… chain confers high affinity binding
 Monomeric, dimeric, and
heterotrimeric forms  Ä…, ², Å‚
" Activation of TH and TC cells by antigen induces
 Class I receptor; CCCC and
WSXWS motifs on ² and Å‚ expression of Ä… chain
" Three structural forms with
" Results in production of high affinity receptor
different IL-2 affinities
 High affinity: all 3 chains " Cells producing only ² and Å‚ chains are capable
 Intermediate affinity: ² and Å‚
of responding to IL2
chains only
 Require higher levels of cytokine
 Low affinity: Ä… chain only
 Provided by activated TH cells
 Ä… chain expressed only on activated T cells;  T-cell activation Ag
Mechanism of Cytokine Receptor
The Cytokine Receptor Enigma
Signal Transduction
" Observations: " Ä… subunit and ²(Å‚)-subunit(s)
both associated with JANUS
 Addition of cytokines evokes changes in immune cells
KINASE (JAK)
 Changes mediated through tyrosine phosphorylation
 BUT& cytosolic domains of cytokine receptors have no tyrosine
" Cytokine associates with ²(Å‚)
kinase activity
subunit(s), allowing Ä… to bind
" How does interaction between cytokine and receptor
 Induces conformational change in
JAKs, low level of activation
result in tyrosine phosphorylation events?
 Two JAKs phosphorylate each
" Riddle was solved when proteins associated with
other, high level of activation
cytosolic domain of receptor were identified
 JAKs phosphorylate tyrosine on
 Each component of receptor is associated with a tyrosine kinase
cytokine receptor (P-Tyr)
 Kinases not activated unless high affinity receptor assembles
 Creates docking point for proteins
involved in signal transduction
Mechanism of Cytokine Receptor
Cytokine Antagonism
Signal Transduction
" Target proteins  STATs,  signal " Cytokines are essential for proper immune response,
transducers and activators of necessary for activation of immune system
transcription
" Clearance of antigen represents  successful response
" STATs contain Src homology 2
" Immune response must be shut off; disorders ensue if it
(SH2) domain that binds P-Tyr s
remains active
on cytokine receptor
" Host produces CYTOKINE ANTAGONISTS to assist in
 Two STAT molecules brought into
proximity with JAK
turning off immune response
 Specific Tyr adjacent to STAT SH2
" Two mechanisms of action:
domain is phosphorylated
 Bind cytokine; prevents interaction with receptor
 Phosphorylated STATs dimerize
 Binds receptor; prevents interaction with cytokine
 Competent to translocate to
nucleus and activate transcription
Host-derived Cytokine Antagonists Virus-derived Cytokine Inhibitors
" RECEPTOR ANTAGONISTS  interfere with cytokine
" Viruses produce several inhibitors of cytokine
binding; IL-1 receptor antagonist (IL-1Ra) is best
action, allows evasion of immune response
understood
 Binds IL-1 receptor, does not activate, prohibits binding of IL-1 " Some produce proteins that bind and sequester
 Mechanism of action unclear, may bind Ä… or ² chain and
cytokines
prohibit Ä…/² interaction
 Include TNF- and IL-1-binding proteins
" CYTOKINE INHIBITORS  bind and sequester soluble
 Cytokines with central role in immune response
cytokines; several known, sIL-2R best characterized
 Derived from enzymatic cleavage of extracellular domain of " Others produce cytokine-like molecules
cytokine receptor, usually the Ä… chain
 IL-10 specialized cytokine, represses TH1 response
 Cleavage dissociates binding domain from cell surface receptor
(CMI)
 Capable of binding soluble cytokine, prevents association with
 Essential for clearance of intracellular pathogens
cell surface receptors
Subpopulations of TH Cells
Cytokines Tailor the Immune Response
Dictate Response
" Some pathogens are efficiently cleared by humoral " Two types of TH cells are involved in activation of
immune response immune system; differ in cytokines secreted
 Antibodies best method for clearance of extracellular soluble
" TH1 cells are involved in activation of CMI
antigens (e.g. toxins) and bacterial cells
 Promote secretion of opsonizing IgG antibodies - complement
 Usually (not always) effective for eukaryotic parasites
activating and Fc receptor binding
" Other infections are more adequately dealt with by cell  Promotes activation of TC cells and inflammatory response
mediated immunity
" TH2 cells involved in activation of humoral response
 Required for intracellular pathogens - some bacteria, all viruses
 Stimulates eosinophil and B-cell activation and differentiation
 Infected cells must be destroyed to contain infection and
 Stimulates production of IgM, IgE, and noncomplement -
phagocytose extracellular pathogens
activating IgG subtypes (Fc receptor binding only)
" Cytokines mediate which type of response is initiated
TH2 Activation
" Causes production of GM-CSF, IL-3, IL-4, IL-5, IL-10, and IL-13
TH1 Activation
" Stimulates total antibody production
" Triggers secretion of IL-2, IFN-Å‚,
TNF-², GM-CSF, and IL-3
" Stimulates class switching in B-cells
" Boosts production of opsonizing
IgG, activates MĆ, triggers
" Stimulates eosinophil and mast-cell production
inflammatory response, activates TC
" Components act coordinately to
" Antibodies and immune cells produced
clear intracellular infections and
particularly suited for attack of large pathogens
extracellular pathogens
" Inflammatory response contains
" IgE facilitates interaction between parasite and
infection (hopefully)
eosinophils/mast cells
" Antibodies provide attachment
point for TC and phagocytic cells
" Causes degranulation, parasite cell membranes
" TC lyses infected cells, MĆ clears
damaged
pathogen released from lysed cells
Generation of TH1 Response Generation of TH2 Response
" Specific cytokines required - IFN-Å‚, IL-12, and IL-18
" Critical cytokine is IL-4, will induce TH2 response even if IL-12 is
present
" APC encounter LPS and other bacterial/viral antigens, secrete IL-12 -
central in TH1 response
" Source of IL-4 unclear, may be produced by TH cells if not
stimulated by IL-12
" IL-12 stimulates TH1 to produce IFN-Å‚,
synergistic with IL-12, results in
" Triggers TH2 production of IL-4
production of IL-12 ²-chain receptor
(autocrine loop), IL-5, IL-10, and IL-13
" Activated TH1 cells secrete IL-18,
stimulates IFN-Å‚ production, promotes " IL-4 and IL-10 work synergistically to
proliferation of TH1 cell subpopulation suppress TH1 response
" Knockout mice reveal importance of
" Knockout mice revealed production of IL-
cytokines
4 essential for production of TH2 response
" Failure to produce any of theses 3 results in
failure to generate TH1 response
TH1 and TH2 Suppress Each Other Type of Response and Disease Outcome
" Cytokines produced by TH1 response suppress TH2 response, due to " Two types of leprosy - TUBERCULOID and LEPROMATOUS
differential action of cytokines
" TH1 response results in tuberculoid leprosy
" IFN-Å‚ activates STAT1, results in production of T-Bet - transcription factor
" CMI response kills most bacteria
" Promotes production of IFN-Å‚, suppresses
" Infection contained to skin, progresses slowly, patient survives
production of IL-4 and IL-5
" TH2 response results in lepromatous leprosy
" Promotes stimulation of TH1 cell population
" IL-4 activates STAT6, results in production of GATA- " Bacteria not killed effectively, disseminate
3-transcription factor
" System wide infection, severely damages bone marrow and
" Suppresses production of INF-Å‚,
cartilage
" Supports expression of IL-4 and IL-5, stimulates
" Not clear why one response predominates over other
TH2 population
Overproduction of Cytokines  Too Much Other Mechanisms of Overproduction
" Some bacteria produce SUPER ANTIGENS
of a Good Thing!
" Crosslink MHC class II and V² chain of TCR non-specifically
" Septic shock - drop in blood pressure, systemic clotting, fever, diarrhea
" Results in activation of T-cell, secretes cytokines
" Accompanies certain Gram (-) bacterial infections
" May non-specifically activate 5% to 25% of total T-cell population
" LPS causes overproduction of TNF-Ä… and IL-1
" Normal antigens only activate 0.01% of T-cell population
" Responsible for septic shock
" Mass activation of T-cells causes over production of cytokines
" Injection of recombinant TNF-Ä… into mice causes similar symptoms
" Responsible for toxic shock
" Addition of monoclonal antibodies prevents effect
" Several Gram (+) organisms possess super antigens (Staphylococcus
aureus, Streptococcus pyogenes)