cytokines

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CYTOKINES

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INTRODUCTIONThe development of an effective

immune response involves lymphoid cells, inflammatory cells, and hematopoietic cells.

The complex interactions among these cells are mediated by a group of proteins collectively designated cytokines to denote their role in cell-to-cell communication.

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Introduction Cytokines are low molecular weight

regulatory proteins or glycoproteins secreted by white blood cells and various

other cells in the body in response to a number of stimuli.

These proteins assist in regulating the development of immune effector cells

And some cytokines possess direct effector functions of their own.

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IntroductionMany referred as

interleukinsSecreted by

leukocytes and act on other leukocytes

IL-1 through IL 29 have been described

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Chemokines Group of low

molecular weight cytokines

Affect chemotaxis and other aspects of leukocyte behaviour

Play important role in inflammatory response

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PROPERTIES1. Bind to specific receptors on the membrane

of target cell2. Cytokine receptors may be made up from

several different chains 3. Cytokines & their fully assembled receptors

exhibit very high affinity for each other & deliver intracellular signals

4. Particular cytokine bind to receptors on the membrane

i. Autocrine actionii. Paracrine actioniii. Endocrine action

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PROPERTIES

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PROPERTIES

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PROPERTIES5. Cytokines regulate the intensity &

duration of immune response6. Binding of a given cytokine to

responsive target cells generally stimulates increased expression of cytokine receptors and secretion of other cytokines

7. Exhibit attributes of pleiotropy, redundancy, synergy, antagonism and cascade induction

8. Share many properties with hormones

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ELISA assay of cytokine

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Cytokines belong to four families

• Falls in 4 familiesi. Hematopoietin familyii. Interferon familyiii. Interleukin familyiv. Tumor necrosis factor family

• All have molecular mass less than 30kDa

• All have similarities and few rarely act alone

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Cytokines belong to four families• The amino acid sequences of these

family members differ considerably• All have high degree of α helical

structure and little or no β sheet structure

• Molecules have similar polypeptide fold, with for α helical regions (A-D)

• In which the 1st and 2nd helices & the 3rd and 4th helices run roughly parallel to one another & are connected by loops

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INTERLEUKIN 4

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Cytokines have numerous biological functions

• Although a variety of cells can secrete cytokines, the principal producers are Tн cells, dendritic cells, and macrophages

• Cytokines released from these cell types activate an entire network of interacting cells

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• Among numerous physiological responses that require cytokine involvement areDevelopment of cellular and humoral

immune responsesInduction of inflammatory responseRegulation of hematopoiesisControl of cellular proliferationDifferentiationHealing of wounds

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What keeps cytokines from activating cells in a non specific fashion during the immune response?

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What keeps cytokines from activating cells in a non specific fashion during the immune response?

Specificity is maintained by careful regulation of the expression of cytokine receptors on cells

Cytokine receptors are expressed on a cell only after that cell has interacted with antigen, limiting cytokine response to antigen activated lymphocytes

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Specificity maintained if cytokine secretion occurs only when the cytokine-producing cell interacts directly with target cell, thus ensuring that effective concentrations of the cytokine occur in the vicinity of the intended target.

In case of Tн cell, a major producer of cytokines, cellular interactions occurs when the T-cell receptor recognizes an antigen-MHC complex on an appropriate antigen-presenting cell, such as a macrophage, dendritic cell, or B lymphocyte.

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• The concentration of cytokines secreted at the junction of these interacting cells reaches high enough local concentration to affect the target APC, but not more distant cells.

• Half-life of cytokines in the blood stream or other extracellular fluids into which they are secreted is usually very short, ensuring that they act for only a limited period and thus over a short distance.

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Cytokine Receptors• Cytokine receptors fall into 5 families

Immunoglobulin superfamily receptorsClass I cytokine receptor family (also

known as hematopoietin receptor family)

Class II cytokine receptor family (also known as interferon receptor family)

TNF receptor familyChemokine receptor family

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Immunoglobulin superfamily

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Class I cytokine receptor family

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Class II cytokine receptor family

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TNF receptors

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Chemokine receptors

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Cytokine receptors initiate signaling

• Although some cytokine receptors lie outside the class I and class II families, majority are included within these two families.

• Class I and class II cytokine receptors lack signaling motifs

• Unifying model emerged from studies of the molecular events triggered by binding of IFN-γ to its receptor, a member of the Class II family

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Interferon gamma • Originally discovered because of its

ability to induce cells to block or inhibit the replication of a wide variety of viruses

• Antiviral activity is a property it shares with IFN-α and IFN-β

• IFN-γ plays a central role in many immunoregulatory proteins including i. Regulation of mononuclear phagocytesii. B cell switching to certain IgG classesiii. Support or inhibition of the

development of Tн cell subsets

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• The cytokine receptor is composed of separate subunits

• Different inactive protein kinases are associated with different subunits of the receptor.

• Cytokine binding induces the association of the two separate cytokine receptors subunits and activation of the receptor-associated JAKs.

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• Activated JAKs create docking sites for the STAT transcription factors by phosphorylation of specific tyrosine residues on cytokine receptor subunits.

• After undergoing JAK-mediated phosphorylation, STAT transcription factors translocate from receptor docking sites at the membrane to the nucleus, where they initiate the transcription of specific genes.

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• In addition to IFN-γ, a number of other class I and class II ligands have been shown to cause dimerization of their receptors.

• An important element of cytokine specificity derives from the exquisite specificity of the match between cytokine and their receptors.

• Another aspect of cytokine specificity is that each particular cytokine induces transcription of a specific subset of genes in a given cell type; the resulting gene products then mediate the various effects typical of that cytokine.

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• Specificity is traceable to three factorsi. Particular cytokine receptors start particular

JAK-STAT pathwaysii. Transcriptional activity of activated STATs is

specific because a particular STAT homodimer/heterodimer will only recognize certain sequence motifs & thus can interact only with the promoters of certain genes.

iii. Only those target genes whose expression is permitted by a particular cell type can be activated within that variety of cell

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• i.e., in any given cell type only subset of the potential target genes of a particular STAT may be permitted expression.

• For eg., IL-4 induces one set of genes in T cells, another in B cells and third in eosinophils.

• IL-1 does not signal via the JAK-STAT pathway but utilizes a kinase designated IL-1 receptor-associated kinase, or IRAK.

• IRAK proteins also utilized by TLRs for signal transduction

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Cytokine antagonists Number of proteins can inhibit cytokine

activity○ Can bind to receptor, fail to activate the cell, OR○ Can bind directly to cytokine, inhibiting it

Enzymatic cleavage of receptors and release of these can bind cytokines in the blood- Marker of chronic T cell activation (transplant rejection, AIDS)

Viruses have developed strategies○ Cytokine homologs○ Soluble cytokine binding proteins○ Homologs of cytokine receptors○ Interference with intracellular signaling○ Interference with cytokine secretion○ Induction of cytokine inhibitors in the host cell

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Cytokine antagonists• Epstein-Barr virus (EBV) produces an

IL-10-like molecule that binds to the IL-10 receptor and like cellular IL-10, suppresses Tн 1-type cell-mediated responses which are effective against many intracellular parasites such as viruses.

• Molecules produced by viruses that mimic cytokines allow the virus to manipulate the immune response in ways that aid the survival of the pathogen.

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Cytokine antagonistsEBV also produce an inducer of IL-1 Ra, the

host antagonist of IL-1.The pox viruses have been shown to encode a

soluble TNF-binding protein and a soluble IL-1 binding protein.

Since both TNF and IL-1 exhibit a broad spectrum of activities in the inflammatory response, these soluble cytokine-binding proteins may prohibit or diminish the inflammatory effects of the cytokines, thereby conferring on the virus a selective advantage.

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Cytokine antagonists

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Cytokine secretion by Tн1 and Tн2 subsets

• CD4⁺ TH cells exert most of helper functions through secreted cytokines, which either act on the cells that produce them in an autocrine fashion or modulate the responses of other cells through paracrine pathways.

• Although CD8⁺ CTLs also secrete cytokines, their array of cytokines generally is more restricted than that of CD4⁺ Tн cells.

• Two CD4⁺ Tн cell subpopulations designated Tн1 and Tн2 can be distinguished in vitro by the cytokines they secrete.

• Both subsets secrete IL-3 and GM-CSF but differ in the other cytokines they produce.

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TH1 and Tн2 cells are characterized by the following functional differences:•TH1 subset is responsible for many cell-mediated functions, such as delayed-type hypersensitivity & activation of TC cells, & for the production of opsonization-promoting IgG antibodies, that is, Ab that bind to the high-affinity Fc receptors of phagocytes and interact with the complement system.– Associated with promotion of excessive

inflammation & tissue injury.•TH2 subset stimulates eosinophil activation, and differentiation, provides help to B cells, promotes production of large amounts of IgM, IgE, and non-complement activating IgG isotypes

• Supports allergic reactions

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• Differences in the cytokine secreted by Tн1 and Tн2 cells determine the different biological functions of these subsets.

• A defining cytokine of the Tн1 subset, INF-γ, activates macrophages, stimulating these cells to increase microbicidal activity, up-regulate the level of class II MHC, secrete cytokines such as IL-12, which induces Tн cells to differentiate into the Tн1 subset.

• IFN-γ secretion by Tн1 cells also induces antibody class switching to IgG classes that support phagocytosis and fixation of complement.

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• TNF-β and IFN-γ are cytokines that mediate inflammation, and it is their secretion that accounts for the association of Tн1 cells with inflammatory phenomena such as delayed hypersensitivity.

• Tн1 cells produce IL-2 and IFN-γ cytokines that promote the differentiation of fully cytotoxic Tс cells from CD8+ precursors.

• This pattern of cytokine production makes the Tн1 subset particularly suited to respond to viral infections and intracellular pathogens.

• Finally, IFN-γ inhibits the expansion of the Tн2 population.

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Development of Tн1 and Tн2 subsets determination

• The cytokine environment in which antigen-primed Tн cells differentiate determines the subset that develops.

• IL-4 is essential for the development of a Tн2 response, and IFN-γ, IL-12, and IL-18 all are important in the physiology of the development of Tн1 cells.

• Tн1 development is also critically dependent on IFN-γ, which induces a number of changes, including the up-regulation of IL-12 production by macrophages and dendritic cells, and the activation of the IL-12 receptor on activated T cells, which it accomplishes by up-regulating expression of the chain of the IL-12 receptor.

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• IL-18, promotes proliferation and IFN-γ production by both developing and fully differentiated Tн1 cells and by NK cells.

• So a regulatory network of cytokines positively controls the generation of Tн1 cells.

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• The generation of Tн2 cells depends critically on IL-4.

• Exposing naive helper cells to IL-4 at the beginning of an immune response causes them to differentiate into Tн2 cells.

• This influence of IL-4 is predominant in directing Tн cells to the Tн2 route.

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Cytokine profiles are cross regulated

• The critical cytokines produced by Tн1 and Tн2 subsets have two characteristic effects on subset development.

1. promote the growth of the subset that produces them

2. inhibit the development and activity of the opposite subset, an effect known as cross-regulation

• IFN- γ (secreted by the Tн1 subset) preferentially inhibits proliferation of the Tн2 subset, and IL-4 and IL-10 (secreted by the Tн2 subset) down-regulate secretion of IL-12, one of the critical cytokines for Tн1 differentiation, by both macrophages and dendritic cells.

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• Cross regulation- when antibody production is high, cell-mediated immunity is low, and vice versa.

• Two transcription factors, T-Bet and GATA-3, are key elements in determining subset commitment and cross-regulation.

• The expression of T-Bet drives cells to differentiate into Tн1 cells and suppresses their differentiation along the Tн2 pathway.

• Expression of GATA-3 does the opposite, promoting the development of naive T cells into Tн2 cells while suppressing their differentiation into Tн cells.

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TH1/TH2 balance determines disease outcomes

• The progression of some diseases may depend on the balance between the Tн1 and Tн2 subsets.

• In humans, a well-studied example of this phenomenon is leprosy, which is caused by Mycobacterium leprae, an intracellular pathogen that can survive within the phagosomes of macrophages.

• In tuberculoid leprosy, a cell-mediated immune response forms granulomas, resulting in the destruction of most of the mycobacteria, so that only a few organisms remain in the tissues.

• Although skin and peripheral nerves are damaged, tuberculoid leprosy progresses slowly and patients usually survive.

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TH1/TH2 balance determines disease outcomes

• In lepromatous leprosy, the cell-mediated response is depressed and, instead, humoral antibodies are formed, sometimes resulting in hypergammaglobulinemia.

• The mycobacteria are widely disseminated in macrophages, often reaching numbers as high as 1010 per gram of tissue.

• Lepromatous leprosy progresses into disseminated infection of the bone and cartilage with extensive nerve damage.

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Cytokine Related DiseasesDefects in the complex regulatory networks

governing the expression of cytokines and cytokine receptors have been implicated in a number of diseases.

Genetic defects in cytokines, their receptors, or the molecules involved in signal transduction following receptor-cytokine interaction lead to immunodeficiencies such as severe combined immunodeficiency (SCID).

Other defects in the cytokine network can cause inability to defend against specific families of pathogens.

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Cytokine Related Diseases• For eg., people with a defective receptor

for INF-γ are susceptible to mycobacterial infections that rarely occur in the normal population.

• In addition to the diseases rooted in the genetic defects in cytokine activity, a number of disease conditions result from overexpression or underexpression of the cytokine or cytokine receptors.

• Therapies aimed at preventing the potential harm caused by cytokine activity

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1. Septic shock• Bacterial infections remain a major

cause of septic shock, which may develop a few hours after infection by certain Gram negative bacteria including E.coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter aerogenes, and Neisseria meningitidis.

• Symptom- drop of blood pressure, fever, diarrhea and wide spread clotting of blood in various organs.

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Septic shock• Bacterial septic shock apparently develops

because bacterial cell wall endotoxins bind TLRs on dendritic cellls and macrophages, causing them to overproduce IL-1 and TNF-α to levels that cause septic shock.

• A common feature of sepsis is an overwhelming production of proinflammatory cytokines such as TNF-α and IL-1β.

• The cytokine imbalance often causes very abnormal body temperature and respiratory rate and high white blood cell counts, followed by capillary leakage, tissue injury, and lethal organ failure

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Septic shock• The increases in TNF-α and IL-1 occur

rapidly in early sepsis, so neutralizing these cytokines is most beneficial early in the process.

• Appr. 24 hrs following onset of sepsis, the levels of TNF-α and IL-1 fall dramatically, and other factors become more important.

• Cytokines critical in the later stages may include IL-6, MIF, and IL-8.

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2. Bacterial toxic shock is caused by superantigens

• A variety of MO produce toxins that act as superantigens.

• Superantigens bind simultaneously to class II MHC molecule and to the variable domain Vβ domain of the T-cell receptor, activating a particular Vβ domain.

• Because of their unique binding ability, superantigens can activate large numbers of T cells irrespective of their antigenic specificity.

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Bacterial toxic shock is caused by superantigens

• Bacterial superantigens have been implicated as the causative agent of several diseases, such as bacterial toxic shock and food poisoning.

• Included among these bacterial superantigens are several enterotoxins, exfoliating toxins, and toxic shock syndrome toxin (TSST) from S aureus and Mycoplasma arthritidis supernatant (MAS).

• The large no of T cells activated by these superantigens results in excessive production of cytokines.

• TSST, for eg, shown to induce extremely high levels of TNF-α & IL-1

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3.Lymphoid and myeloid cancers• Abnormalities in the production of cytokines or

their receptors have been associated with some types of cancer.

• For eg, abnormally high levels of IL-6 are secreted by cardiac myxoma cells, myeloma and plastocystoma cells, and cervical and bladder cancer cells.

• In myeloma & plastocytoma cells, IL-6 appears to operate in an autocrine manner to stimulate cell proliferation.

• When Mabs to IL-6 are added to invitro cultures of myeloma cells, their growth is inhibited.

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4. Chaga’s disease• Causative agent- Trypanosoma cruzi,

characterised by severe immune suppression

• Evidence that soluble factor produced by T. cruzi leads to reduction in T cell IL-2 (CD25) receptor

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Cytokine-based Therapies• Problems with cytokine therapies:

o Effective dose levelso Short half-lifeo Potent biological response modifiers• Can cause unpredictable side effects

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Cytokine-based Therapies

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Cytokines in hematopoiesis• Many cytokines have been shown to play

essential roles in hematopoiesis.• During hematopoiesis, cytokines act as

developmental signals that direct commitment of progenitor cells into and through particular lineages.

• Suitable concentrations of a group of cytokines including IL-3, GM-CSF, IL-1 and IL-6 will cause it to enter differentiation pathways that lead to the generation of monocytes, neutrophils and other leukocytes of the myeloid group

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Cytokines in hematopoiesis• The participation f leukocytes in

immune response often results in their death and removal.

• Hematopoetic cytokines that stimulate production of neutrophils (G-CSF), myleoid cells (GM-CSF), platelets (IL-11), and RBCs (erythropoietin) have been used in clinical applications, most often as supportive therapy for patients with immunodeficiency resulting from a genetic defect or from cancer chemotherapy.

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Cytokines in hematopoiesis

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Classification • Major cytokines include:

Lymphokines Interleukins (IL) Monokines Interferons (IFN) colony stimulating factors (CSF) Tumor Necrosis Factors-Alpha and

Beta (TNF)

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Lymphokines • Lymphokines include:

Colony-stimulating factors (csfs), including GM-CSF.

Interferons (ifns) – IFNγ.Interleukins IL-1 to IL-8, IL-10, IL-13. Macrophage inflammatory protein-1 beta

(mip-1β).Neuroleukin (lymphokine product of lectin-

stimulated T cells).Osteoclast-activating factor.Platelet-derived growth factor (PDGF).Transforming growth factor beta (tgfβ).Tumour necrosis factor-alpha (cachectin)

(TNFα).Tumour necrosis factor-beta

(tnfβ, lymphotoxin α, LT).

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Monokines • A monokine is a type

of cytokine produced primarily by monocytes and macrophages.

• Examples include interleukin 1 and tumor necrosis factor-alpha.

• Other monokines include alpha and beta interferon, and colony stimulating factors.

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Interleukeins • They are secreted

regulatory proteins produced by lymphocytes, monocytes and various other cell types and are released by cells in response to antigenic and non-antigenic stimuli. Consist of IL1 to IL37.

• IL-1 activates Antigen Presenting Cells and CD4+ lymphocytes; affects the differentiation of the B-Cells and T-Cells and other immunocompetent cells and takes part in the regulation of production of other cytokines and GM-CSF (Granulocyte-Macrophage Colony-Stimulating Factor).

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Interleukeins • IL-2 stimulates the proliferation and

activation of B-Cells and T-Cells. IL-4 plays a role in the differentiation of TH2, in allergic responses, and in the switching of antibody types.

• IL-3 is a potent activator of hemopoietic cells. It stimulates NK-Cells and acts as a synergist with IL-4 during the induction of CD4+ lymphocyte activation process.

• IL-5 stimulates the production and maturation of eosinophils during inflammation.

• IL-7 is known as the growth factor of the immature B-Cells and T-Cells. It induces apoptosis of tumor cells and causes differentiation of cells from a subgroup of acute myeloblastic leukemia.

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Interleukeins • IL-8 acts as a chemotactic factor that attracts

neutrophils, basophils and T-Cells to sites of inflammation.

• IL-9 stimulates the excretion of IL-2, IL-4, IL-6, IL-11, and takes part in a stimulation of cytotoxicity of T-killers and NK-Cells, inducing apoptosis.

• IL-10 acts to repress secretion of pro-inflammatory cytokines.

• IL-11 is a pro-inflammative factor, which regulates the functions of B-Cells and T-Cells. It also takes part in the induction of various killer cells activities and acts as an autocrine factor for the proliferation of megacaryocytes.

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Interleukeins • IL-12 is a critical linker between the innate

immunity and adaptive immunity, capable of TH1 (T Helper Type-1) differentiation and IFN-Gamma release by T-Cells and NK cells.

• IL-13 is very sensitive to monocytes and B-Cells. IL-13 does not act on T-Cells but inhibits the proliferation of leukemic pro-B-Cells.

• IL-14 is a BCGF (B-Cell Growth Factor) and the hyper production of this interleukin enables the progression of NHL-B (B-cell Type Non Hodgkin's lymphoma).

• IL-15 is analogous to IL-2 and increases the anti-tumor activities of T-killers and NK-Cells, and the production of cytokines CD4+ lymphocytes.

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Interleukeins • IL-17 is principally produced by CD4+

T-Cells, which induces granulopoiesis via GMCSF. It takes part in the regulation of many cytokines and can reinforce the antibody dependant tumor cell destruction.

• IL-18 acts as a synergist with IL-12, especially in the induction of IFN-Gamma production and inhibition of angiogenesis.

• IL-19 is produced mainly by monocytes and is similar to IL-10 in its function. It is stimulated by GM-CSF and regulates the functions of macrophages, and also suppresses the activities of TH1 and TH2.

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Interleukeins • IL-21 executes an important role in

the regulation of hematopoiesis and immune response. It promotes a high production of T-Cells, fast growth and maturation of NK-Cells and B-Cells population.

• IL-22 is produced by activated T-Cells in acute inflammation. It is similar to IL-10 in function, but does not prohibit the production of pro-inflammatory cytokines through monocytes.

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Interferons • Based on the type of receptor through which

they signal, human interferons have been classified into three major types.1. Interferon type I:

All type I IFNs bind to a specific cell surface receptor complex known as the IFN α receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains. The type I interferons present in humans are IFN-α, IFN-β and IFN-ω.

2. Interferon type II: Binds to IFNGR that consists of IFNGR1 and IFNGR2 chains. In humans this is IFN-γ.

3. Interferon type III: Signal through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12).

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Chemokines • Chemokines have been classified into four

main subfamilies : 1. CXC Chemokines (contain CXL1 to

CXL17) 2. CC Chemokines (contain CCL1 to

CCL28) 3. CX3C Chemokines (contain CX3CL1) 4. XC Chemokines (contain XCL1 & XCL2)

• All of these proteins exert their biological effects by interacting with G protein-linked transmembrane receptors called chemokine receptors, that are selectively found on the surfaces of their target cells.

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Colony stimulating factors• Colony-stimulating factors (CSFs) are

secreted glycoproteins that bind to receptor proteins on the surfaces of hemotopoietic stem cells, thereby activating intracellular signaling pathways that can cause the cells to proliferate and differentiate into a specific kind of blood cell.

• The colony-stimulating factors are soluble, in contrast to other, membrane-bound substances of the hematopoietic microenvironment.

• They transduce by paracrine, endocrine, or autocrine signaling.

• Colony-stimulating factors include:– CSF1 - Macrophage colony-stimulating

factor(MCSF)– CSF2 - Granulocyte macrophage colony

stimulating factors(GMCSF).– CSF3 - Granulocyte colony-stimulating

factors(GCSF)

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Tumor necrosis factors• Tumor necrosis factors (or the TNF family)

refer to a group of cytokines that can cause cell death (apoptosis).

• Nineteen cytokines have been identified as part of the TNF family on the basis of sequence, functional, and structural similarities. They include:

• Tumor necrosis factor (TNF), formerly known as TNFα or TNF alpha, is the best-known member of this class. TNF is a monocyte-derived cytotoxin that has been implicated in tumor regression, septic shock, and cachexia.

• Lymphotoxin-alpha, formerly known as Tumor necrosis factor-beta (TNF-β), is a cytokine that is inhibited by interleukin 10.

• Lymphotoxin-alpha (LT-alpha) and lymphotoxin-beta (LT-beta), two related cytokines produced by lymphocytes that are cytotoxic for a wide range of tumor cells in vitro and in vivo.

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Tumor necrosis factors• T cell antigen gp39 (CD40L), a cytokine that seems to

be important in B-cell development and activation.• CD27L, a cytokine that plays a role in T-cell activation.

It induces the proliferation of co-stimulated T cells and enhances the generation of cytolytic T cells.

• CD30L, a cytokine that induces proliferation of T cells.• FASL, a cytokine involved in cell death.• 4-1BBL, an inducible T cell surface molecule that

contributes to T-cell stimulation.• OX40L, a cytokine that co-stimulates T cell

proliferation and cytokine production.• TNF-related apoptosis inducing ligand (TRAIL), a

cytokine that induces apoptosis.

cytokines

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