CYTOKINES

PRESENTED BY Md.Akmal Yazdani M.PHARM(PHARMACOLOGY) 2nd Semester

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Development of effective immune response involves- Lymphoid cell interaction among - Inflammatory cell these mediated by - Hematopoitic cell group of protein

Cytokines - cytokines have role in cell-cell communication

INTRODUCTION
Cytokines are low M.W protein or glycoprotein. Secreted by- W.B.C and other cell in response to stimuli. Assist in regulating development of IMMUNE EFFECTOR cell. Cytokines secreted by lymphocyte- Lymphokines Cytokine secreted by Monocyte and macrophages are Monokines. Lymphokine and Monokines- misleading term, because these are not only secreted by lymphocyte ,Monocyte and macrophages other cell may secrete it.

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CELLS SECRETING CYTOKINES

Principle cell secreting CK are

TH cell  Dendritic cell  Macrophages


Ck secreted from these cells activate entire network of interacting cells Physiological involvement that requires CK involvement are--------------y Development of Cellular immunity y Induction of inflammatory response y Regulation of hemetopoisis y Control of cellular proliferation,diff, wound healing.

TYPES OF CYTOKINES

A.Interleukins (cytokine secreted by lymphocyte &act on other interleukins) Eg- IL-1 to IL-29 B. Tumor necrosis factor(TNF) C. Interferon D. Chemokines(Low mol.wt cytokines that affect chemotaxis and other aspect of leucocytes behavior)

PROPERTIES OF CYTOKINES

CK binds to specific receptors present on cell membrane of target cell

Triggering signal transduction mechanism Alter gene expression on target cells. Target cell have different susceptibility for different cytokines.
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INDUCTION AND FUNCTION OF CK

CONTD..
CK

receptor made up of several different chain & receptor for given CK may exist in various combination of these chains. Chain combination varies in affinity for different cytokines ,also vary in their ability to initiate the signal transduction pathway. CK & their fully assembled receptor have high affinitywith dissociation constant 10-10 to 10-12M high affinity so mediate biological response even at picomolar concentration.
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ACTION OF CYTOKINES

CONTD.

CK regulate the intensity and duration by- stimulating or inhibiting action - Proliferation and/or differentiation of various cells. - regulating the secretion of antibodies or other CK. Target cell + cytokine response of CK receptors and secretion of other CK which affect target cells. CK secreted by even small no.of lymphocyte activated by antigen affect many target cell.
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ATTRIBUTES OF CK

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CONTD..

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CONTD.

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CONTD..

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Activity of CK recognized in 1960 , in supernatant derived from in-vitro culture of lymphocytes were found to contain soluble protein/factor that regulate proliferation, differentiation and maturation of immune system cells. Biochemical isolation and purification hampered due to low conc. In supernatant and absence of well defined assay system. Greater advancement with gene cloning techniques during1970s and 1980swhich made it possible to produce pure CK by expressing the proteins from cloned gene. Derivation of monoclonal antibodies specific for each of these CK has made it possible to develop rapid quantitative immunoassay for each of them.

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CYTOKINE SUPERFAMILY
Mol.wt of CK is less than 30kDa. However long list of molecules all share common properties and nearly all of the recently described CK act in concert with others. Once the gene encoding various CK were cloned, sufficient quantity of purified preparation becomes available for detailed study of structure and function. Structural study shows that CK belongs to one of the four groups.

Hematopoitic family y Interferon family y Chemokine family y Tumor necrosis factor family
y

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CONTD..
Structure of two member of hamatopoitin family IL-2 and IL-4 are depicted as. Left fig- is topographical representation of pr.str.of IL-2 showing -helical region ( and A-D) containing chains of molecules. Right fig- 3-D structure of IL-2 Structure other CK belonging to hamatopoitin family considered to be similar

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STRUCTURE OF IL-2

Fig-a

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Ribbon model of IL-4deduced from X-ray crystallographic analysis of the molecule. - helices in red helices in blue

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Fig-b

CYTOKINES RECEPTORS
CK exerts their biological effect by binding to specific receptors CK receptors fall in five categories y a. Immunoglobin suprfamily y b. Class-iCK receptor family(Hematopoitin receptor family,act in immune and hemetopoitic system) y Class-ii CK receptor family(Interferon recptor family) y TNF receptor family y Chemokine receptor family

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IMMUNOGLOBIN RECEPTOR SUPERFAMILY

Large no. of cellsurface and secreted proteins,all member atlest have one common domain in protein structure

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CONTD

Ig superfamily includes receptor for IL-1

Two receptors for IL-1, IL- ,IL-1 . y Both binds to same receptor and produce same response. y Two difft receptor for IL-1 are known
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Type-1-IL-1R Expressed on many cell type

Type-2 -IL-1R Expressed only on B-cells

IL-1 binding to type 2 receptor does not response of typical type-1; i.e,type 2 IL1R is a decoy means to remove IL-1thereby preventing it to bind to type-1 receptor.

IL-1 play key role in inflammation and designed as proinflammatory cells.

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CLASS-I CYTOKINE RECEPTORS/HEMATOPOITIC

FAMILY

Mainly function in the immune and hematopoitic system. Members of this family consist of aa sequence motifs in extracellular domain consisting four positionally conserved cysteine residue(CCCC),and conserved tryp-ser(WSXWS) where X is conserved

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CLASS-II CYTOKINE /INTERFERON FAMILY

Posses conserved CCCC motifs but lacks WSXWS (C refers to conserved cysteine)

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Common feature of class-I and class-ii CK receptor is multiple subunit.

One subunit binds to specific CK and, y other subunit mediate signal transduction.
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Engagement of class-I and ii receptor family induce tyrosine phosphorylation of the receptor through activity of protein tyrosin kinase closely associated with cytosolic domain of this receptor.

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TNF RECEPTOR FAMILY

Extracellular domaincomposed of 2-6 repeat cytosine rich motifs. Member of this family contain P-55,P-75 receptor of TNF(TNF-R1, and TNF-R2 respectively)

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CHEMOKINE RECEPTOR FAMILY

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Chemokine receptors associate with G-proteins to transmit cell signals following ligand binding. Activation of G proteins, by chemokine receptors, causes the subsequent activation of an enzyme known as phospholipase C (PLC) PLC cleaves a molecule called phosphatidylinositol (4,5)-bisphosphate (PIP2) into two second messenger (IP3) and (DAG) that trigger intracellular signaling events.
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DAG activates another enzyme (PKC), and IP3 triggers the release of calcium from intracellular stores. These events promote many signaling cascades (such as the MAP kinase pathway), that generate responses like chemotaxis, degranulation, release of superoxide anions and changes in the avidity of cell adhesion molecules called integrins within the cell harbouring the chemokine receptor

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SUBFAMILIES OF CLASS-I CK RECEPTOR HAVING

SIGNALING SUBUNIT COMMON

All subfamilies have identical signal transducing subunit. GM-CSF family includes receptors of IL-3 and IL5 Sharing of signal transducing subunit among receptor explain redundancy and antagonism GM-CSF, IL-3 and IL-5, all binds with low affinity to alpha subunit and all 3 subunit associate noncovalently with common signal transducing beta Subunit.The resulting dimeric receptor exhibit increased affinity for CK and capable of transducing a signal across the membrane after CK binds.

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Alpha subunit-CK specific receptor protein Beta subunitcommon signal transducing unit

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CONTD.

-Can not transduce activation signal

Noncovalent association of beta subunit yield high affinity dimeric receptor transduce signal.

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CONTD..

Association CK specific subunit with common signaling beta subunit allow generation of identical signal by difft CK

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CONTD.

IL-3 and GM-CSF exhibit antagonism, binding of IL-3 is inhibited by GM-CSF ;and binding of GM-CSF is inhibited by IL-3.this antagonism is caused by competition limited number of beta subunit available to associate with CK specific alpha subunit of dimeric receptor

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Similar situation found among IL-6 receptor subfamily; which include the receptor of IL-6,IL11 LIF(leukemia inhibitinf factor), oncostatinM(OSM) and ciliary neurotropic factor (CNTF) Common signal transducing subunit gp130associates with one/two CK specific subunit LIF and and OSM share common structural both binds to same structural subunit alpha. CK binds to receptor of this family display overlapping biological activity IL-6,OSM,LIF induce synthesis of acute phase protein(Acute-phase proteins are a class of proteins whose plasma concentrations increase (positive acute-phase proteins) or decrease (negative acute-phase proteins) in response to inflammation), by hepatocytes and diff of myeloid leukemia cells into macrophage.

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IL6,LIF and CNTF affect neuronal development, IL6,IL11,OSM stimulate megakaryocyte maturation and platelate production. In IL2 receptor subfamily describe 3rd signal transducing subunit

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IL-6 SUBFAMILY OF CLASS I CK RECEPTOR

FAMILY

Gp130 common signaling pathway

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IL-2 RFECEPTOR SUBFAMILY OF CLASS I FAMILY CK

3rd Signal transducing pathway defines the receptor of IL-2 family Includes IL 2,15,7,9,4.In IL2 and other gamma chain acts as signal transducer.whic are all dimer.
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IL-2 R RECEPTOR
Most thoroughly studied receptor, because of central role in the clonal proliferationof T- cells IL2 receptor occurs in three form hat exhibit difft affinity for IL2;

Low affinity- IL2R monomeric y intermediate affinity dimeric IL 2R and y High affinity IL2R , , , as shown in figy

Because IL2R- chain expressed only by activated T- cell , sometime called TAC antigen (T-cell activation)or CD25(surface marker in the maturation of T- cell) Monoclonal antibody or,anti TAC or anti CD25 used to identify IL2R- .on the cell.

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Gamma chain continuously expressed on most lymphoid cells , Alpha and beta chain more restricted and enhanced after antigen activate resting lymphocyte.only antigen activated CD4+ and CD8+ will express high affinity IL2 receptor and proliferate in response to physiologic level.

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CYTOKINE RECEPTOR INITIATE SIGNALING

Binding of CK induce dimerization of the receptor subunit which leads to the activation of receptor subunit associated JAK tyrosine kinase by reciprocal phosphorylation, subsequently the activated JAKs phosphorylate various tyrosine residue ,resulting in creation of docking site for STATs on the receptor and the activation of one/more STAT transcription factors. phosphorylated STAT dimerize and translocate to nucleus where they 41 activate specific genes.

CYTOKINE RECEPTOR INITIATE SIGNALING

Majority of CK receptor are included under class I and class ii receptors. class I and class ii receptors lacks signaling motifs(intrinsic tyrosine kinase domain) First events after interaction of CK with one of these receptor is protein tyrosine phosphorylation. Discovery of major signaling pathway invoked by interaction of IFN- with its receptor leads to realization that signal transduction through most,if not all classi and classii CK receptors involves following steps. Which are the basic unifying CK signaling model.

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CONTD.
i) The CK receptors composed of separate subunits One chain for CK binding and transduction ,other chain for signaling Ii) Different inactive protein tyrosine kinase are assiociated with difft subunit of receptor; alpaha chain associated with protein tyrosine kinase JAKs kinase family, association of JAK and receptor occurs spontaneously not require binding of CK,however in absence of CK JAK lacks protein tyrosine kinase activity. Iii) CK binding induce association of two separate receptor associated JAKs. Activated JAKs create docking site for the STATtranscription factor by phosphorylatoin of specific tyrosine residue on CK receptr subunit.

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CONTD.
Once receptor associated JAKs activated they activate specific tyrosine in the receptor subunit of complex; STAT binds to these phosphorylated tyrosine .specific STAT play essential role in signaling pathway of difft CK shown in fig-12-2. Iv) After undergoing JAK mediated phosphorylation STAT translocate from receptor docking site to nucleus.

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CYTOKINE ANTAGONIST

They inhibit biological activity of CK. Act in two way---y Bind directly to CK receptor but fails to activate the cell y Bond directly to CK inhibit activity. Best characterized inhibitor is the IL-1 receptor antagonist IL-1Ra(play a role in regulating intensity of inflammatory response, potential treatment for inflammatory disease.) CK inhibitor are found in blood stream and ECF, arise from enzymatic cleavage of ECF domain of CK receptor.soluble cytokine receptor detected are-IL1,2,4,6,7;IFNSoluble IL-2 receptor released from chronic T-cell activation is best characterizated. Presence of sIL-2R used as clinical marker of chronic T-cell activation observed in no. of disease; autoimmunity, transplant rejection and AIDS.

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CYTOKINE SECRETION BY TH1 AND TH2

SUBSETS

The immune response to particular pathogen must induce particular set effectors function that can eliminate disease or toxic products. Diff in CK secretion pattern among TH cell subset as determinant of immune response made to particular antigenic challenge. CD4+ exerts most of their helper fn through secreted CK which either act in autocrine pattern or modulate the response in paracrine fashion. CD8+ also secrete CK but restricted than CD4+ Two CD4+ TH cell subpopulation designed as TH1 and TH2 .

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CONTD..

Characterization of TH1 and TH2

TH1 responsible for CMI, delayed hypersensitivity and activation of Tc cell for the opsonization promoting Ig-G antibodies,this subst also associated with promotion of excessive inflammation and tissue injury. y TH2 subset stimulate eosinophil differentiation, provide help to B cell and promote production of large amount of IgM,IgE,.TH2 also support allergic reactions.
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Difference in the CK secreted by TH1 and TH2 cell determine the biological function of these subsets.
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CK ,MEDIATED GENERATION AND CROSS REG OF TH1 SUBSETS

Antigen activated native CD4+ cell produce IL-2 and proliferate If it proliferate in IL12dominated Environment, it generates TH1 cells that secrete char.profile of CK including IFN . A positve

feedback loop establish when IFN secretes by

expanding TH1 population stimulate dendritic cell or macrophage to produce more IL-12 if environment dominated by IL-4 TH2 population emerges,secrete a CK that promote eosinophil activation and synthesis of certain antibody class .

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CYTOKINE PROFILE ARE CROSS REGULATED

Cross-regulation at intracellular level

Signal through the TCR and CK receptor determine whether the cell will produce the TH1 promoting transcription factor,Tbet,or the TH2 promoting GATA3.experimantal evidence supports a model in which exposure of cell bearing receptor for IFN to IFN induce the formation of T-Bet which upregulate the synthesis of IFN and repress the synthesis of GATA-3.exposure of IL-4 to IL-4 induce the formation of GATA-3 which upregulate the synthesis of IL-4 and IL-5but repress the expression of T-Bet

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TH! AND TH2 BALANCE DETERMINE DISEASE

OUTCOME

m-RNA isolated from lesion from tuberculoid and lepromatous leprosy patient was analysed by southern blotting tech. using CK probe indicated CK produced by TH1 cell predominate in tuberculoid patient where as CK produced by TH2 cell predominate in the lepromatous patients.

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CYTOKINE RELATED DISEASE

Defects in complex regulatory network governing expression of CK and CK receptor implicated in many disease. XSCIDS(X-linked severe combined immunodeficiency syndrome) result from defect in chain gene which maps to X- chromosome, immunodeficiency due to loss of T-cell and NK cell activity, due to loss of all CK fn mediated by IL-2 subfamily. People defective in IFN are susceptible to mycobacterial infection. Number of disease due to over /under expression of CK/CK receptors

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SEPTIC SHOCK
The symptoms of bacterial septic shock is , which is often fatal, include drop in B.P, fever,diarrhoea, and widespread blood clotting In various organ. Develops because bacterial cell wall endotoxin binds on TLRs on dendritic cell and macrophages, causing them to overproduce IL-1 and TNF- to a level that cause septic shock. Neutralization of IL-1 and TNFby monoclonal antibodies or antagonist will prevent fatal shock from developing. Inj. of recombinant-IL-1 receptor antagonist prevent result 3 fold decrease in mortality. Common cause of sepsis is overwhelming production of proinflammatory CK as TNF- , IL1

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CYTOKINE ACTIVITY IMPLICATION IN LYMPHOID

AND MYELOID CANCER

Abnormality in CK/CK receptor associated with some types of cancer. Abnormally high level of IL-6 are secreted by cardiac myxoma cell(benign heart tumor),myeloma and plasmacytoma cells cervical and bladder cancer cells. In myeloma and plasmacytoma cells ,IL-6 appears to operate in autocrine manner to stimulate cell proliferation, monoclonal antibodies to IL-6 inhibit their growth.

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CHAGAS DISEASE Caused by protozoan Trypanosoma cruzi causes severe immune suppression. Antigen ,mitogen,Cd3 monoclonal antibody normally can activate peripheral T-cells, but in presence of T.cruzi , T-cell not activated by any of these.

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CYTOKINE BASED THERAPY

Availability of purified cloned CK, monoclonal antibody directed against CK, and soluble CK receptor offer specific clinical therapy to modulate immune response. Soluble TNF- receptor (Enbrel), and monoclonal antibody against TNF- (remicade and humiral), used to treat rheumatoid arthritis in more than million patients. Problems arising in adapting the CK for safe and routine medical use,one problems in maintaining effective dose over clinically sig.period. Since during immune response high conc . Of CK in vicinity of target cell for this CK should be administerd systematically.

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CK RELATED THERAPEUTIC AGENT SELECTIVELY

MODULATE THE IMMUNE SYSTEM
Fig-a

fig-b

a.Anti IL-2R monoclonal antibody binds to CK Anti IL-2R monoclonalreceptor on the cell surface and antibody binds to CK preventing interaction of CK receptor on the cell surface with its receptor. and preventing b. Conjugation of toxin with CK result in destruction of cell expressing CK receptor.
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CYTOKINE IN HEMATOPOIESIS

SCF- stem cell factor Myelod progenitor in presence of erythropoitin proceed,suitabl e conc.of IL-3, GM-CSF,IL1,IL-6 cause it ti entr diffrntn pathway to generate constituent
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HEMATOPOIETIC CYTOKINES
HEMATOPOITIC GROWTH FACTOR

ERYTHROPOITIN G-CSF

THROMBOPOIETIN M-CSF

SITES OF PRODUCTION KIDNEY, LEVER ENDOTHELIAL CALL,FIBROBLAST,M ACROPHAGES LIVER, KIDNEY ENDOTHELIAL CALL,FIBROBLAST,M ACROPHAGES
BONEMARROW STROMAL CELL CONSTITUTIVELY T-CELL(TH1 AND TH2) MACROPHAGE ,MAST CELL

MAIN FUNCTION ERYTHROCYTE PRODUCTION NEUTROPHIL PRODUCTION

PLATELATE PRODUCTION MACROPHAGE AND OSTEOCLAST PRODUCTION

STEM CELL, PROGENITOR CELL SURVIVAL/DIVISION MAST CELL DIFF MACROPHAGE ,GRANULOCYTE PRODUCTION,DENDRITIC CELL MATURATION AND ACTIVATION

SCF/c-kit LIGAND GM-CSF IL-3 ILIL-6

T-CELL(TH1 AND TH2) STEM CELL, MYELOID PROGENITOR CELL GROWTH 5ACTIVATED HELPER T-C EOSINOPHIL PRODUCTION ACTIVATED TCELL,MONOCYTE FIBROBLAST, ENDOTHELIAL CELL BONEMARROW AND LYMPHOID STEM CELL BONEMARROW STOMAL CELL PLATALETS PRODUCTION;Ig PRODUCTION IN B-CELL

IL-7 IL-11

T-CELL SURVIVAL

GROWTH FACTOR FOR MEGAKARYOCYTE

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REFERENCES
a)

b)

c) d) e) f) g) h)

KUBY IMMUNOLOGY 6th edition page(302-324) RANG AND DALES PHARMACOLOGY 6th edition(222-224) www.ncbi.nlm.nih.gov/entrez/Abstract www.jem.org www.pubmedcentral.nih.gov www.muliplemyeloma.org www.nature.com/nature/journal www.gdb.org

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THANK YOU
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