Cytokines Cytokines are low M.wt. glycoproteins which are produced by cells of the innate and adaptive immune systems and, in particular by CD4+ T cells, which orchestrate many effector mechanisms. Cytokines serve as chemical messengers within the immune system. Cells regulated by a particular cytokine must express a receptor for that factor. Cytokines produced by lymphocytes were collectively called lymphokines. Cytokines produced by monocytes and macrophages were called monokines.

Chemokines are closely related chemotactic cytokines, they are potent attractors for various leukocytes. These factors were not the exclusive products of lymphocytes and monocytes/ macrophages, thus the more appropriate term cytokine was coined as a generic name for these glycoprotein mediators. Since these factors mediate signals between leukocytes the term interleukin "IL" was coined.

The macrophage-derived "LAF" and T cell-derived growth factors were given the names IL-1 and IL-2 respectively. Currently numbers have been assigned to more than 30 ILs,. Many ILs have biologic effects on cell types outside the immune system e.g IL-2 not only acts to promote T cell proliferation but also stimulates osteoblasts. Tumor growth factor  "TGF-" similarly acts on many cells including connective tissue fibroblasts as well as T cells and B cells. Thus cytokines have pleiotropic properties, since they can affect the activity of many different cell types.

There is a functional redundancy among cytokines, e There is a functional redundancy among cytokines, e.g IL-2 and IL-4 can both function as T cell growth factors. Cytokines act in vivo. Cytokines often exhibit additive, synergistic or antagonistic properties.

General properties of Cytokines : A- Common Functional Properties Some Cytokines e.g IFN and IL-2 are synthesized by cells and rapidly secreted. Others e.g TNF- and TNF- may be secreted or expressed as membrane-associated proteins. Most cytokines have very short-half lives. Synthesis and function typically occur in a burst. Cytokines facilitate communication between cells and do so at very low concentrations "10–10 – 10–15".

Cytokines may act locally either on the same cell that secreted it "autocrine" or on other cells "paracrine", like hormones, they may act systemically "endocrine". Cytokines exert their functional effects by binding to specific receptors on target cells. They may stimulate or inhibit target cells e.g TH1 cells secrete IFN, which activates macrophages, inhibits B cells and is directly toxic for certain cells. TH2 cells secrete IL-4 and IL-5, which activate B cells and IL-10, which inhibits macrophage activation. "Fig 11.2 "

B- Common Systemic Activities Cytokines play a crucial role in the amplification of the immune response. The release of cytokines from antigen-activated cells results in the activation of multiple different cell types. The production of high levels of cytokines by a powerful stimulus can trigger deleterious systemic effects such as toxic shock syndrome. Therapeutic manipulation of the immune system using recombinant cytokines or cytokine antagonists can affect multiple physiologic systems.

C- Common Cell Sources and Cascading Events A given cell may make many different cytokines. One cell may be the target of many cytokines each binding to its own specific cell-surface receptor. One cytokine may affect the action of another, which may lead to an additive, synergistic or antagonistic effect on the target cell. Interactions of the multiple cytokines produced during a typical immune response are often referred to as the cytokine cascade. This cascade largely determines whether a response to an antigen will be primarily Ab-mediated or cell-mediated. The antigenic stimulus appears to play a key role in the initiation of cytokine responses by these cells.

D- Common Receptor Molecules It is common for cytokines to have overlapping "redundant" functions e.g both IL-1 and IL-6 induce fever and several other common biologic phenomena. Several cytokines use multichain receptors to mediate their effects on target cells, some of these receptors share at least one common receptor molecule called common  chain, it is an intracellular signaling molecule, this explains the functional overlap among different cytokines. "Fig 11.6"

Functional Categories of Cytokines A- Cytokines That Regulate Immune Response B and T cell activation in response to antigen stimulation is regulated by cytokines. Cytokines act solely on antigen-activated lymphocytes. CD4+ T cells are the major source of immunoregulatory cytokines. Naïve CD4+ T cells differentiate into subpopulations of TH1 and TH2 cells each of which possesses characteristic cytokine profiles. Fig. "11.2 "

Many intracellular bacteria (e Many intracellular bacteria (e.g Listeria) and viruses, activate dendritic cells, macrophages and NK cells to produce IL-12 and IFN-, these cytokines produce TH1 from TH0. Other pathogens "e.g worms" cause release if IL-5 by other cells "e.g mast cells", IL-4 promote the development of TH0 into TH2 cells. Low levels of processed antigen bind poorly to TCR of TH0 cells, they differentiate into TH2 cells to produce IL-4 and IL-5.

Increased levels of processed antigen, TH0 cells tend to differentiate into TH1 cells to produce IL-2, IFN and TNF. TH1 pathway facilitates cell-mediated immunity with activation of macrophages, NK cells, and cytotoxic T cells, whereas the TH2 pathway is essential for humoral immunity. TH subsets can also regulate the growth and effector functions of each other as a result of the activity of cytokines.

IL-10, TGF- produced by TH2 cells inhibit the activation and growth of TH1 cells. Production of IFN by TH1 cells inhibits proliferation of TH2 cells. Cytokines provide the regulatory signals needed for activation and then cause cells to return to quiescent state.

B- Cytokines That Facilitate Innate Immune Responses and Activate Inflammatory Responses Innate Immunre Responses Cytokines that facilitate innate immune responses stimulated by viruses and microbial pathoges e.g IL-1, IL-6, TNF, IFN, IFN. IL-1, IL-6, TNF are produced largely by phagocytes, their actions include: they are endogenous pyrogens causing fever. They stimulate the production of acute phase protein produced by hepatocytes e.g c-reactive protein "CRP" which binds to phosphoryl choline on bacterial surfaces and acts like opsonin and activates the classical complement pathway.

Another acute phase protein is the mannan-binding lectin "MBL" which binds mannose residues on bacteria and acts as opsonin and activates complement. They increase the number of circulating neutrophils. They activate NK cells. IFN, IFN are synthesized by many cell types after viral infection, they block the spread of viruses to uninfected cells. IFN, IFN induce increased MHC class I expression on most uninfected cells making newly infected cells susceptible to killing of CD8+ cytotoxic T cells. IFN is produced by activated NK cells and effector T cells and appears after the induction of adaptive immune responses.

2. Inflammatory responses Many cytokines activate inflammatory cells, they are known as proinflammatory cytokines e.g IL-1, IL-6, TNF, their action includes: They increase vascular permeability. Vascular endothelial cells increase their expression of adhesion molecules e.g E and P-selection and ICAM-1. Neutrophils attach to endothelial cells.

IFN attract macrophages to the site of antigen localization. TGF- plays a role in terminating the inflammatory response by promoting the accumulation and proliferation of fibroblasts and the deposition of extracellular matrix proteins required for tissue repair.

C- Chemokines: Cytokines that affect Leukocyte movement Chemokines are closely related low M.wt. chemotactic cytokines containing 70-80 residues with conserved sequences and they are potent attractors for various leukocytes subsets, such as neutrophils, monocytes and lymphocytes. Examples of chemokines include; IL-8 which is produced by monocytes, mobilize and activate neutrophils and promote angiogenesis, RANTES; produced by T cells, degranulates basophils and activates T cells, MCP-1;

produced by monocytes and activates macrophages, stimulate basophil histamine release and promotes TH2 immunity, MIP-1 alpha produced by monocytes and promotes TH1 immunity, MIP-1 beta produced by monocytes, neutrophils and endothelial cells, it competes with HIV-1 for chemokine receptor binding. MCP, monocyte chemotactic protein, MIP, macrophage inflammatory protein, RNTES, Regulated upon Activation Normal T Expressed and Secreted

D- Cytokines that stimulate hematopoiesis Cytokines capable of inducing growth of hematopoietic cells in vitro were initially characterized using cultures of bone marrow cells grown in soft agar and thus are referred to as colony-stimulating factors "CSFs". Several CSFs are known e.g Macrophage CSF "M-CSF". Granulacyte CSF "G-CSF".

GM-CSF. IL-3 is capable of stimulating clonal growth of a large number of cell population. IL-3 in presence of erythropoietin stimulates the development of normoblasts and RBCs. IL-7 produced by bone marrow and thymic stromal cells induces differentiation of lymphoid stem cells into progenitor B and T cells. G.CSF is used to reverse the neutropenia caused by high dose chemotherapy. GM.CSF is used to treat patients undergoing bone marrow transplantation to boost clonal expansion of the granulocyte and macrophage population.

Cytokine Receptors Cytokines can act only on target cells that express receptors for that cytokine. Resting cells do not express a given receptor or express low affinity receptor e.g IL-2 receptor is expressed as intermediate affinity dimmer " and  chains" or as a high affinity trimer ", ,  chains", the later responds to IL-2. Knowledge about cytokine-cytokine receptor interactions may be useful in devising strategies to prevent the action of cytokines involved in inflammatory responses such as rheumatoid arthritis, or in transplantation rejection.

Families of Cytokine Receptors Immunoglobulin superfamily receptors. Class I cytokine receptor family "hematopoietin receptor family". Class II cytokine receptor family "interferon receptor family". TNF receptor superfamily. Chemokine receptor family. Fig 11.6

Ig super family receptors have at least one Ig-like domain, e Ig super family receptors have at least one Ig-like domain, e.g IL-1 and M-CSF. Class I cytokine receptors "hematopoietin receptor family" are composed of two polypeptide chains; a cytokine-specific subunit " chain" and signal transducing subunit " or  chain". Most cytokines use the class I family of cytokine receptors e.g- IL-2, IL-3, IL-5, IL-15, GM-CSF, G-CSF. Class II cytokine receptors "interferon receptor family", their lignads are interferons or have biologic activities that overlap with those associated with some interferons "e.g IL-10".

TNF receptor "TNFR" superfamily activation is mediated by intracellular signals through a set of adaptor proteins called TNF receptor-associated factor "TRAFs". TNFR and TNF superfamilies regulate the life and death of activated cells of the immune system. Chemokine receptors belong to a super family of serpentine G Protein coupled receptors " snake_like extracellular cytoplasmic structural configuration and their association with G proteins, which mediate signal transduction". Some chemokine receptors bind a diverse set of pathogens e.g S. pneumoniae which binds to platelet activating factor receptor, plasmodium vivax which binds to the Duffy blood group antigen, HIV-1 binds to chemokine receptors "CXCR4, CCR5" for viral entry into T cells and macrophages respectively.

Cytokine Receptor-Mediated Signal Transduction Fig 11.8

Cytokines must generate intracellular signals that result in the production of active transcription factors and, ultimately, gene expression. Binding of a cytokine to its cellular receptor induces dimerization or polymerization of receptor polypeptides at the cell surface. Signaling is intiated by the activation of JAK kinases, a family of cytosolic protein tyrosine kinases that interact with the cytoplasmic domains of the receptor. This results in the phosphorylation of tyrosine residues present on the cytoplasmic domain of the receptor and on a family of transcription factors know as signal transduction and activators of transcription "STATs".

Once phosphorylated, the STAT transcription factors dimerize and is translocated to the nucleus, where they bind to enhancer regions of genes induced by the cytokine. Downregulation of cytokine-mediated singaling is accomplished via cytoplasmic proteins, these suppressors of cytokine. Signaling "SOCS" proteins regulate signal transduction by direct interactions with cytokine receptors and signaling proteins with a generic mechanism of targeting associated proteins for degradation. The deregulation of normal SOCS may play a causal role in some diseases. The manipulation of SOCS function might provide potent therapeutic options in the future.

Role of Cytokines and Cytokine Receptors in Disease Toxic Shock syndrome It is initiated by the release of superantigen. It causes a burst of cytokines from T cells after activation. Excessive production of cytokines causes dysregulation of the cytokine network leading to high levels of IL-1 and TNF. These cytokines induce systemic reactions including fever, blood clotting, diarrhea, a drop in blood pressure, and shock, sometimes these reactions are fatal. 2. Bacterial Septic shock Overproduction of cytokines is also associated with gram-negative bacterial infection. Endotoxins produced by these bacteria stimulate macrophages to produce IL-1 and TNF causing fatal form of bacterial septic shock.

3. Cancers Several lymphoid and myeloid cancers have been shown to be associated with abnormally high levels of cytokines and/or cytokines receptor expression. Adult T cell leukemia disease which is strongly associated with T cell leukemia virus 1 "HILV-1". T cells infected with HTLV-1 constitutively produce IL-2 and express the high affinity IL-2 R in the absence of activation by antigen, this results in autocrine stimulation of infected T cells, leading to their uncontrolled growth. In neoplastic B cells, large amounts of IL-6 is produced. In Hodgkin's disease, IL-5 is produced in large amount.

Autoimmunity and immune-based diseases T cells exert a controlling influence on the generation of autoantibodies and on the regulation of autoimmunity. Some are due to the action of TH subset-derived cytokines including IL-10, IFN, IL-4. Several cytokine and cytokine receptor abnormalities have been shown to be associated with systemic autoimmune diseases. Systemic lupus erythematosus "SLE, an autoimmune disease has been shown to be associated with elevated levels of IL-10. Cytokines also play an important role in the pathophysiology of other immune-based diseases including allergy, asthma and inflammatory diseases e.g rheumatoid arthritis.

Therapeutic and diagnostic exploitation of cytokines and cytokine receptors Cytokine inhibitors/antagonists Circulating soluble cytokine receptors maintain their ability to bind to the cytokine for which the receptor is specific, thus neutralizing their activity. Examples of such inhibitors are those that bind to IL-2, IL-4, IL-6, IL-7, IFN, and TNF. TNF inhibitors are used to treat rheumatoid arthritis as there is increased levels of TNF and IL-1 in joints. IL-1 receptor antagonist "IL-1Ra" binds to IL-1 receptor on CD4+ T cells, thus preventing their activation. IL-1Ra may be used to treat chronic inflammatory diseases.

2. Reversing cellular deficiencies Cellular deficiency arising from chemotherapy or radiotherapy is treated by administration of growth factors e.g G-CSF, GM-CSF. 3. Treatment of immunodeficiencies Patients with X-linked agammaglobulinemia have been treated with G-CSF to reverse neutropenia. IFN reduces the severity of infections probably by increasing nonoxidative antimicrobial activity.

4. Treatment of Cancer and transplant patients Culturing populations of NK cells or CTL cells in the presence of high concentrations of IL-2 generates effector cells with potent antitumor activity, IFN augments the killing of tumor cells as it enhances the expression of MHC II molecules. Cytokine receptor-specific antibodies have also proven useful in the treatment of certain cancers. In adult T cell leukemia lymphoma "ATLL" leukemic cells express the IL-2R  chain "CD25", anti CD25 antibodies "anti-Tac antibodies" induce response in one third of the patients. Anti CD25 therapy is used as part of regimen of immunosuppressive therapy to treat patients receiving organ transplants. Anti CD25 reduces the response to alloantigens.

5. Treatment of allergies and asthma Injection of antibodies specific for IL-4 "the major cytokine responsible for promoting B cell isotype class switching to IgE" decrease IL-4 production in mice. Another approach is to use soluble IL-4 receptors. N:B  TH17 T Cells and the IL-17 family of Cytokines is filling the gaps of how immune responses are regulated. IL-17 stimulates the mobilization and generation of neutrophils, thereby bridging the gap between innate and adaptive immunity.

TH17 cells represent an arm of the adaptive immune system specialized for enhanced host protection against extracellular bacteria and some fungi, microbes probably not well covered by TH1 or TH2 immunity. IL17 stimulates the mobilization and de novo generation of neutrophils by granulocyte- colony stimulating facter (G-CSF), thereby bridging innate and adaptive immunity. IL-17 cytokines are key mediators in a divrse range of autoinflammatory disorders.