Lecture # : CYTOKINES

Cytokines are small glycoproteins produced by a number of cell types, predominantly leukocytes that regulate immunity, inflammation and hematopoiesis.

INTERLEUKIN

1.      Interleukin–1

Interleukin–1 was originally discovered as a factor that induces fever, cause damage to joints and regulate bone marrow cells and lymphocytes. Later, the presence of two distinct proteins, IL–1α and IL–1β, was confirmed, which belong to a family of cytokines, the IL–1 superfamily. Ten ligands of IL–1 have been identified, termed IL–1F1 to IL–1F10. With the exception of IL–1F4, all of their genes map to the region of chromosome 2. The major cell source of IL–1 is the activated mononuclear phagocyte. Other sources include dendritic cells, epithelial cells, B cells, astrocytes, fibroblasts and large granular lymphocytes (LGL).

a.      Endotoxins, macrophage–derived cytokines such as TNF or IL–1 itself, and contact with CD4⁺ cells trigger IL–1 production.

b.      IL–1 can be found in circulation following Gram–negative bacterial sepsis. It produces the acute–phase response in response to infection. IL–1 induces fever as a result of bacterial and viral infections.

c.       It suppresses the appetite and induces muscle proteolysis, which may cause severe muscle “wasting” in patients with chronic infection.

d.     IL–1β causes the destruction of β cells leading to type 1 diabetes mellitus.

e.      IL–1 serves as mediator for activation of T–helper cells, resulting in IL–2 secretion as well as activation of B–cell

f.        It is a stimulator of fibroblast proliferation, which causes wound healing.

g.      Autoimmune diseases exhibit increased IL–1 concentration.

h.      It suppresses further IL–1 production via increase in the synthesis of PGE₂.

Kineret (Anakinra)

a.      Kineret is a human IL–1 receptor antagonist and is produced by recombinant DNA technology. It is non–glycosylated and is made up of 153 amino acids. With the exception of an additional methionine residue, it is similar to native human IL–1Ra.

b.      Human IL–1Ra is a naturally occurring IL–1 receptor antagonist, a 17–kDa protein, which competes with IL–1 for receptor binding and blocks the activity of IL–1.

c.       Kineret is recommended for the treatment of severely active rheumatoid arthritis for patients 18 years of age or older. It reduces inflammation, decreases bone and cartilage damage.

d.     Kineret also improves glycemia and β–cell secretory function in type 2 diabetes mellitus. It is administered daily at a dose rate of 100 mg/day by subcutaneous injection.

2.      Interleukin–2

a.      IL–2 is a single polypeptide chain of 133 amino acid residues, is produced by immune regulatory cells that are principally T cells. When a helper T cell binds to an APC using CD28 and B7, CD4⁺ cells produce IL–2.

b.      IL–2 supports the proliferation and differentiation of any cell that has high –affinity IL–2 receptors. It is necessary for the activation of T cells.

c.       Resting T lymphocytes (unstimulated) belonging to either the CD4⁺ or the CD8⁺ subsets possess few high–affinity IL–2 receptors, but following stimulation with specific antigen, there is a substantial increase in their numbers.

d.     IL–2 is the major growth factor for T lymphocytes, and the binding of IL–2 to its specific receptors on TH cells stimulates the proliferation of these cells and the release of a number of cytokines from these cells.

e.      IL–2 is required for the generation of CD8⁺ cytolytic T cells, which are important in antiviral responses. It increases the effector function of NK cells.

f.        When peripheral blood lymphocytes are treated with IL–2 for 48–72 hours, lymphokine–activated killer (LAK) cells are generated, which can kill a much wider range of targets including the tumor cells.

g.      Receptors: IL–2Rα (low affinity); IL–2RβƔ (intermediate affinity); IL–2RαβƔ (high affinity)

Clinical uses of Interleukin–2

a.      Proleukin (Aldesleukin) is a recombinant human IL–2 that received approval for the treatment of renal cell carcinoma in 1992 and for the treatment of metastatic melanoma in 1998. It is also being evaluated for the treatment of non–Hodgkin’s lymphoma (NHL).

b.      IL–2 has been tested for antitumor effects in cancer patients as part of LAK (Lymphokine–activated killer) therapy. LAK cell therapy involves infusion into cancer patients of their own (autologous) lymphocytes after they have been treated in vitro with IL–2 for a minimum of 48 hours to generate LAK cells.

c.       HIV is a retrovirus that infects CD4⁺ cells. After HIV becomes integrated into the genome of the CD4⁺ cells, activation of these cells results in the replication of virus, which causes lysis of the host cells. Patients infected with HIV, and with AIDS, generally have reduced numbers of helper T cells and fhte CD4:CD8 ratio may be as low as 0.5:1 instead of the normal 2:1. As a consequence, very little IL–2 is available to support the growth and proliferation of CD4⁺ cells despite the presence of effector cells, B cells and cytolytic T cells. Proleukin has not been approved for the treatment of HIV; however, studies show that proleukin in combination with antiretroviral therapy significantly increases the number of CD4⁺ cells levels. Low frequency doses of subcutaneous proleukin at maintained intervals increased CD4⁺ cell levels.

3.      Interleukin–4

a.      IL–4 is a pleiotropic cytokine produced by TH₂ cells, mast cells and NK cells. Other specialized subsets of T cells, basophils and eosinophils also produce IL–4. It regulates the differentiation of antigen–activated naïve T cells. These cells then develop to produce IL–4 and a number of other TH₂ –type cytokines including IL–5, IL–10 and IL–13.

b.      IL–4 suppresses the production of TH₁ cells. It is required for the production of IgE and is the principal cytokine that causes isotype switching of B cells from IgG expression to IgE and IgG4. As a consequence, it regulates allergic disease. IL–4 leads to a protective immunity against helminths and other extracellular parasites. The expression of MHC class II molecules on B cells and the expression of IL–4 receptors are upregulated by IL–4. In combination with TNF, IL–4 increases the expression of VCAM–1 and decreases the expression of E– selectin, which results in eosinophil recruitment in lung inflammation.

c.       Receptors: Insulin receptor substrate (IRS–1/2); Janus family tyrosine kinases–signal transducers and activators of transcription (JAK–STAT)

4.      Interleukin–5

a.      IL–5 is secreted predominantly by TH₂ lymphocytes. However, it can also be found in mast cells and eosinophils. It regulates the growth, differentiation, activation and survival of eosinophils.

b.      IL–5 contributes to eosinophil migration, tissue localization and function, and blocks their apoptosis. Eosinophils play a seminal role in the pathogenesis of allergic disease and asthma and in the defense against helminths and arthropods.

c.       The proliferation and differentiation of antigen–induced B lymphocytes and the production of IgA are also stimulated by IL–5. TH₂ cytokines IL–4 and IL–5 play a central role in the induction of airway eosinophilia and AHR. It is a main player in inducing and sustaining the eosinophilic airway inflammation.

d.     IL–5 is usually not present in high levels in humans. However, in a number of disease states where the number of eosinophils is elevated, high levels of IL–5 and its mRNA can be found in the circulation, tissue and bone marrow. These conditions include the diseases of the respiratory tract, hematopoietic system, gut and skin. Some other examples include food and drug allergies, atopic dermatitis, aspirin sensitivity and allergic or non–allergic respiratory disease.

e.      Receptor: IL – 5R

5.      Interleukin–6

a.      IL–6 is a proinflammatory cytokine, which is a member of the family of cytokines termed “the IL–6 type cytokines.” The cytokine affects various processes including the immune response, reproduction, bone metabolism and aging. IL–6 is synthesized by mononuclear phagocytes, vascular endothelial cells, fibroblasts and other cells in response to trauma, burns, tissue damage, inflammation, IL–1 and, to a lesser extent, TNF–α.

b.      IL–6 is elevated in patients with retroviral infection, autoimmune diseases and certain types of benign or malignant tumors. It stimulates energy mobilization in the muscle and fatty tissue, resulting in an increase in body temperature.

c.       IL–6 acts a myokine – a cytokine produced by muscles – and muscle contraction occurs as a result of elevated IL–6 concentrations.

d.     Receptor: IL–6R

6.      Interleukin–9

a.      IL–9 stimulates the release of a number of mediators of mast cells and promotes the expression of the high–affinity IgE receptors (FcεR1α).

b.      IL–9 augments TH₂–induced inflammation and enhances mucus hypersecretion and the expression of its receptors is increased in asthmatic airways.

c.       It also promotes eosinophil maturation in synergy with IL–5.

d.     IL–9 activates airway epithelial cells by stimulating the production of several chemokines, proteases, mucin genes and ion channels.

e.      It is an essential cytokine for asthmatic disease as biopsies from asthmatic patients show an increase in the expression of IL–9 compared to healthy individuals, and therefore it is an important therapeutic target for clinical intervention.

7.      Interleukin–10

a.      It is an anti–inflammatory cytokine that was first called human cytokine synthesis inhibitory factor.

b.      The major biological effect of IL–10 is the regulation of the TH₁/TH₂ balance. TH₁ cells are involved in cytotoxic T–cell responses whereas TH₂ cells regulate B–cell activity and function. IL–10 is a promoter of TH₂ response by inhibiting IFN–Ɣ production form TH1 cells. This effect is mediated via suppression of IL–12 synthesis in accessory cells. IL–10 is involved in assisting against intestinal parasitic infection, local mucosal infection by costimulating the proliferation and differentiation of B cells. Its indirect effects also include the neutralization of bacterial toxins.

c.       IL–10 is a potent inhibitor of IL–1, IL–6, IL–10 itself, IL–12, IL–18, CSF and TNF. It not only inhibits the production of proinflammatory mediators but also augments the production of anti–inflammatory factors including soluble TNF–α receptors and IL–1RA.

d.     IL–10 downregulates the expression of MHC class II molecules (both constitutive and IFN–Ɣ induced), as well as that of costimulatory molecule, CD86, and adhesion molecule, CD58.

e.      It is a stimulator of NK cells, enhances their cytotoxic activity, and also augments the ability of IL–18 to stimulate NK cells.  

8.      Interleukin–11

a.      IL–11 is produced by bone marrow stroma and activates B cells, plasmacytomas, hepatocytes and megakaryocytes.

b.      IL–11 induces acute–phase proteins, plays a role in bone cell proliferation and differentiation, increases platelet levels after chemotherapy and modulates antigen–antibody response.

c.       It promotes differentiation of progenitor B cells and megakaryocytes.

d.     The recovery of neutrophils is accelerated by IL–11 after myelosuppressive therapy.

e.      IL–11 also possesses potent anti–inflammatory effects due to its ability to inhibit nuclear translocation of NF–κB.

f.        Also play a role in epithelial cell growth, osteoclastogenesis and inhibition of adipogenesis.

Oprelvekin (Neumega)

a.      Oprelvekin is a recombinant human IL–11 but differs due to lack of glycosylation and the amino–terminal proline residue.

b.      It is used to stimulate bone marrow to induce platelet production in nonmyeloid malignancies in patients undergoing chemotherapy.  

9.      Interleukin–13

a.      IL–13 appeared similar to IL–4 until their unique effector functions were recognized. Nevertheless, IL–13 and IL–4 have a number of overlapping effects. IL–13 also plays an essential role in resistance to most GI nematodes.

b.      It regulates mucus production, inflammation, fibrosis and tissue remodeling. IL–13 is a therapeutic target for a number of disease states including asthma, idiopathic pulmonary fibrosis, ulcerative colitis, cancer and others.

c.       IL–13 induces physiological changes in organs infected with parasites that are essential for eliminating the invading pathogen. In the gut, it induces a number of changes that make the surrounding environment of the parasites less hospitable, such as increasing contractions and hypersecretion of glycoproteins from gut epithelial cells. This results in the detachment of the parasites from the wall of the gut and their subsequent removal.

d.     IL–13 is believed to inhibit TH1 responses, which will inhibit the ability of the host to eliminate the invading pathogens.

e.      IL–13 induces the expression of eotaxins. These chemokines recruit eosinophils into the site of inflammation in synergy with IL–5. Eosinophils release IL–13 and induce the production of IL–13 from TH₂ cells, which is mediated via IL–18.

f.        Receptor: IL–4Rα, IL–13Rα1, IL–13Rα2

10.  Interleukin–18

a.      IL–18 augments T–cell and NK–cell maturation, cytotoxicity and cytokine production. It stimulates TH differentiation, promotes secretion of TNF– α, IFN–Ɣ and GM–CSF and enhances NK cell cytotoxicity by increasing FasL expression. IL–8–mediated neutrophil chemotaxis is promoted by IL –18 via its effects on TNF–α and IFN–Ɣ, which are stimulatory in action. It plays an important role in maintaining synovial inflammation and inducing joint destruction in rheumatoid arthritis. In synovium of patients with rheumatoid arthritis, enhanced levels of TNF–α and IL–1 are associated with augmented expression of IL–18.

b.      IL–18 also induces IL–4, IL–10 and IL–13 production, increases IgE expression on B cells and in association with IL–2, it enhances stimulus– induced IL–4 production from TH₂ cells. 

c.       Bone marrow–derived basophils produce IL–4 and IL–13 in response to a stimulus from IL–18 and IL–3. IL–18 in combination with IL–12 induces IFN–Ɣ from dendritic cells and bone marrow–derived macrophages.

d.     IL–18 plays a critical role in host defense against bacterial, viral, fungal and protozoan infection.

e.      Receptors: IL–18Rα and IL–18Rβ

INTERFERON

1.      Interferon I

The principal biological actions of type I IFNs include inhibition of viral replication, inhibition of cell proliferation, increase in the lytic potential of NK cells and the modulation of MHC molecule expression. They increase the expression of MHC class I molecules and decrease the expression of MHC class II molecules.

Type I IFNs exerts their biological effects after binding to distinct heterodimeric cell surface receptors on the target cells. Binding of the agonist to the cell surface receptors results in activation of the Janus–activated kinase (JAK)–STAT signaling pathway.

Viral infection is the most potent natural signal for the synthesis of type I IFNs.

Subgroups:

a.      IFN–α (18 kDa) – subdivided into IFN–α1 and IFN–α2/IFN–ω

b.      IFN–β (20 kDa)

Clinical utility

a.      Interferon–α

IFN–α may be used for the treatment of condylomata acuminata (venereal or genital warts), malignant melanoma, hairy cell leukemia and hepatitis B and C, and other types of cancer including skin, kidney and bone cancers.

Click here for full discussion of Condylomata acuminate

b.     Interferon–α–2a (Roferon–A)

This is used for the treatment of chronic myeloid leukemia, Kaposi sarcoma, lymphoma, hairy cell leukemia, hepatitis B and C and cancer of the skin and kidney. It can only be administered by injection or into the bloodstream, and the most common method is subcutaneous injection.

The antiviral or antitumor activity of IFN–α–2a is mediated via inhibition of viral replication and modulation of host immune response as well as its antiproliferative activity. It is filtered through the glomeruli, and its proteolytic degradation takes place during tubular reabsorption. The common side effects include flu–like symptoms of fever, fatigue, chills, dry mouth, GI disorders, changes in mood and temporary effects on the bone marrow. The occasional side effects may include skin rash, hair thinning, loss of appetite and loss of fertility.

c.       Peginterferon–α–2a

Pegylated α–IFN is made by attaching polyethylene glycol (PEG) to the α–IFN. PEG is a large water–soluble molecule that decreases the clearance of α–IFN and also increases the duration of its activity. This modified cytokine is used to treat chronic hepatitis C. however; it is rarely used as a single therapeutic agent for hepatitis C because of its low response rate.

Click here for use of polyethylene glycol (PEG) in Blood Banking

d.     Interferon–α–2b

Interferon–α–2b is a soluble α–IFN protein produced by recombinant DNA technology. Both IFN–α–2b and –α–2a are pure clones of single IFN subspecies, but they differ by virtue of two amino acids. The potencies of both α–2a and α–2b IFNs are similar. IFN–α–2b is also available in pegylated form. All IFN–α cytokines augment the killing of target cells by lymphocytes and inhibit the replication of virus in infected cells.

e.      Interferon–β

Natural IFN–β is predominantly synthesized by fibroblasts. Its sequence is 30% homologous to that of IFN–α.

IFN–β–1a is used to treat patients with a relapsing form of MS. It is not a cure for MS; however, it may slow some disabling effect of the disease. IFN–β–1a may also decrease the number of relapses of MS. The possible mechanism of action for the treatment of MS includes the antagonism of IL –4 and IFN–Ɣ. It also modifies the mechanics of blood barrier since it inhibits cell adhesion, cell migration and metalloproteinase activity. IFN – β induces IL–10 and TGF–β, which is anti–inflammatory cytokines. It is also used for the treatment of genital warts.   

2.      Interferon II

Interferon–Ɣ

a.      This is the only type II IFN whereas there are more than 20 types of type I IFNs.

b.      IFN–Ɣ is produced by activated T lymphocyte (TH₁ and CD8⁺ cells), NK cells, B cells, NKT cells and professional APCs. It promotes the activity of cytolytic T lymphocytes, macrophages and NK cells. The self–activation and activation of nearby cells in part may result from IFN–Ɣ production by professional APCs, which include monocyte/macrophages and dendritic cells. The early host defense against infection is likely to utilize IFN–Ɣ secreted by NK and professional APCs. In acquired immune responses, T lymphocytes are the major source of IFN–Ɣ.

c.       IFN–Ɣ production is regulated by IL–12 and IL–18, both cytokines secreted by APCs. In the innate immune response, a link is established between infection and IFN–Ɣ by these cytokines.

d.     The production IFN–Ɣ is inhibited by IL–4, IL–10 and TGF–β.

e.      IFN–Ɣ is a potent activator of mononuclear phagocytes. The expression of both MHC class I and class II molecules is augmented by IFN–Ɣ as IFN– Ɣ–induced upregulation of MHC class I molecules is pivotal for host defense against intracellular pathogens, resulting in an increased susceptibility to cytolytic T cells for recognition and consequent promotion of cell–mediated immune response.

f.        IFN–Ɣ is produced by TH₁ cells and shifts the response toward TH₁ phenotype. This is accomplished by activation of NK cells that promotes innate immunity, augmenting specific cytolytic response and induction of macrophages. The induction of cytotoxic immunity can be direct or indirect via suppression of TH₂ response. Another direct effect of IFN–Ɣ is the differentiation of naïve CD4⁺ lymphocytes toward a TH₁ phenotype. The cytokines present are very important in this differentiation process. Furthermore, induction of IL–12 and suppression of IL–4 by IFN result in differentiation toward a TH₁ phenotype.

g.      IFN–Ɣ is an inhibitor of cell growth and proliferation. The proliferation is inhibited by augmenting the levels of Cip/Kip, CKIs and Ink4. It increases p21 and p27 CKIs, which inhibit the function of cyclin E:CDK2 and cyclin D:DCK4, respectively. This results in stopping the cell cycle at G1/S interphase. IFN–Ɣ induces apoptosis via activation of STAT–1, which results in the production of large amounts of IRF–1 (IFN regulatory factors). Apoptosis may be needed to kill the invading pathogen–infected macrophages.

h.      IFN–Ɣ also induces the costimulatory molecules on the macrophages, which increases cell–mediated immunity. As a consequence, there is an activation and increase in the tumoricidal and antimicrobial activity of mononuclear phagocytes, granulocytes and NK cells.

COLONY–STIMULATING FACTORS (CSF)

CSFs are glycoproteins that support hematopoietic colony formation. They influence the survival, proliferation and maturation of hematopoietic progenitor cells and regulate the activities of the mature effector cells. There are three lineage–specific CSFs, granulocyte colony–stimulating factor (G–CSF), monocyte–macrophage colony –stimulating factor (M–CSF) and erythropoietin, and two multi–potential CSFs, IL–3 and GM–CSF.

Clinical utility

1.      Granulocyte Colony–Stimulating Factor

a.      G–CSF is a glycoprotein produced by macrophages, endothelium and various leukocytes. It stimulates the bone marrow to produce granulocytes and stem cells and then directs their migration from the bone marrow to the peripheral blood. G–CSF is a growth factor for the proliferation, differentiation, effector function and survival of neutrophils.

b.      G–CSF mobilizes bone marrow–derived cells into the bloodstream. These stem cells can migrate to ischemic myocardium and differentiate into cardiomyocytes, smooth muscle cells and endothelial cells. They may also induce metalloproteinases and vascular endothelial growth factor and thus play a role in tissue healing. Furthermore, G–CSF induces proliferation and enhanced survival of cardiomyocytes. This accomplished via activation of G–CSF receptors in myocardium. G–CSF in association with TGF–β and collagen enhances ventricular expansion in the infarcted area.

c.       G–CSF activates neutrophils, transforming them into cells capable of respiratory burst and release of secretory granules. It also modulates the expression of adhesion molecules on neutrophils as well as CD11b/CD18 and plasma elastase antigen levels. G–CSF induces proliferation of endothelial cells, phagocytic activity of neutrophils, and reactive oxygen intermediate production by neutrophils and antibody–dependent cellular toxicity by neutrophils.

Filgrastim (Neupogen)

a.      Filgrastim is a 175–amino–acid glycoprotein. It differs from natural G– CSF due to lack of glycosylation and has an extra N–terminal methionine.

b.      Filgrastim administered to patients receiving cytotoxic chemotherapy for advanced cancer has resulted in a dose–dependent amelioration of neutropenia associated with cancer chemotherapy. It is well tolerated and may reduce the morbidity and mortality rate associated with chemotherapy, possibly permitting higher doses and a greater antitumor response.

c.       Filgrastim is also used after autologous stem cell transplantation to treat neutropenia. It reduces the duration of neutropenia and lessens morbidity secondary to bacterial and fungal infections. Additional use of this drug includes the treatment of severe congenital neutropenia, of neutropenia in patients with AIDS resulting from with zidovudine and of patients with donating peripheral blood for stem cell transplantation

2.      Granulocyte–Macrophage Colony–Stimulating Factor

GM–CSF is a glycoprotein produced by macrophages, T cells, mast cells, fibroblasts and endothelial cells. It stimulates stem cells to produce neutrophils, monocytes, eosinophils and basophils. Monocytes migrating into tissue from the circulating blood differentiate into macrophages and undergo maturation.

Sargramostim (Leukine)

a.      Sargramostim is a 127–amino–acid glycoprotein, which is similar to natural GM–CSF except for variation in glycosylation and presence of a leucine in position 23.

b.      It has beneficial effects on bone marrow function in patients receiving high –dose chemotherapy in the setting of autologous bone marrow transplantation as well as for the treatment of advanced cancers.

c.       Sargramostim is used in AIDS, myelodysplastic syndrome and aplastic anemia where it stimulates bone marrow function. It has not shown beneficial effects in graft–versus–host disease but may be of value in patients with early graft failure. It has been used in patient donating peripheral blood stem cells because it mobilizes CD34⁺ progenitor cells.

TUMOR NECROSIS FACTOR–α

TNF–α is a 185 amino–acid glycoprotein, which is cleaved from a 212–amino–acid peptide, and the cleavage occurs on the cell surface of mononuclear phagocytes. In humans, the genes for TNF–α is present on chromosome 7p21. The major cell source of TNF–α is the macrophage, specifically the endotoxin–activated mononuclear phagocyte. Other sources include endothelium after tissue damage, antigen–stimulated T cells, activated NK cells and activated mast cells.

1.      Local increasing concentration of TNF–α cause heat, swelling, redness and pain.

2.      TNF–α cause vascular endothelial cell to express new adhesion molecules.

3.      It increases the mobilization and effector function of neutrophils and their adhesiveness for endothelial cells.

4.      TNF–α induces the production of IL–1, IL–6, TNF–α itself and chemokines via stimulation of macrophages.

5.      It exerts an IFN–like protective effect against viruses and augments expression of MHC class I molecules.

6.      TNF–α is an endogenous pyrogen that acts on cells in hypothalamic regulatory regions of the brain to induce fever.

7.      It suppresses appetite.

8.      The hypothalamic–pituitary–adrenal axis is stimulated via the release of corticotropin–releasing hormone by TNF–α.

9.      TNF–α induces acute–phase responses by activating hepatocytes.

10.  Acute–phase proteins including C–reactive protein and mannose–binding protein (MBP) are detected in blood in response to an infection.

11.  TNF–α suppresses bone marrow stem cell division and reduces tissue perfusion by depressing myocardial contractility.

Tumor Necrosis Factor Receptors

There are two distinct types of TNF receptors, TNF–R1 (Cd120a or P55) and TNF–R2 (CD120b or P75). They are both implicated in inflammatory processes. TNF receptors are transmembrane proteins with intracellular domains that lack intrinsic enzymatic activity, and consequently, they require cytoplasmic proteins that help initiate the receptor–induced signaling pathways.

Etanercept (Enebrel)

Etanercept is a genetically engineered protein that is soluble TNF–α receptor. Its molecular weight is 75 kDa. It binds to TNF–α. It is used for the treatment of rheumatoid arthritis, juvenile rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis.

Structurally, two TNF–α receptors are linked to an Fc portion of an IgG1 molecule. Consequently, an artificial antibody is constituted with two Fab sites, which are soluble human 75–kDa TNF–α receptors. It competitively inhibits the binding of TNF molecules to the TNF receptor sites. The binding of etanercept to TNF renders the bound TNF biologically inactive, resulting in the reduction of the inflammatory activity.

CHEMOKINES

Chemokines are a large family of small heparin–binding chemotactic cytokines released by many cell types. They are composed of four groups called CXC, CC, C and CX3C. The designation and classification is based on the spacing of conserved cysteines and X is an amino acid. Many members constitute the CXC and CC groups, which is not the case of C and CX3C chemokines. Neutrophils and lymphocytes are the target of CXC chemokines. The targets of CC chemokines are diverse, including basophils, dendritic cells, macrophages and eosinophils. The CXC family includes chemokines CXCL1 – CXCL17, the CC family includes CCL1 – CCL28, the C family includes XCL1 – XCL2 and the CX3C family includes only CX3CL1.

The early signals produced during innate immune responses are the main stimuli for the secretion of chemokines. Various chemokines are secreted by a stimulus resulting from viral infection, bacterial products and proinflammatory cytokines including IL–1 and TNF–α. Consequently, some chemokines are proinflammatory in nature and are produced during an immune response to direct leukocytes to the site of injury/infection, whereas others are homeostatic in nature and control the migration of cells during routine tissue maintenance or development.

1.      CXC (α–chemokines) – composed of two N–terminal cysteines, separated by one amino acid designated with “X” in the name.

Two groups:

a.      ELR positive group – specific to neutrophils (e.g. IL – 8)

b.      ELR negative group – chemoattractants for lymphocytes

ELR refers to the presence or absence of Glutamic acid–leucine–arginine

2.      CC (β–chemokines) – regulate the migration of monocytes, dendritic cells and NK cells. It is composed of two adjacent cysteines near their amino–terminus.

3.      C (Ɣ–chemokines) – composed of two cysteines, one on the N–terminus and the other a downstream cysteine. The two chemokines in this group functions to attract T–cell precursors to the thymus:

a.      Lymphotactin–α (XCL1)

b.      Lymphotactin–β (XCL2)

4.      CX3C (δ–chemokines) – composed of three amino acids between two cysteines. These are secreted and present on cell surface and serve both as chemoattractant and as an adhesion molecule. There is only one member: fractalkine (CX3CL1).

Function of Chemokines

1.      Induce the migration of leukocytes.

2.      Directs lymphocytes towards the lymph nodes, which allows them to interact with the APCs and detect any invading pathogens. Such chemokines are called homeostatic chemokines and do not require a stimulus for their secretion.

3.      Capable of activating leukocytes to initiate an immune response and are involved in both innate and acquired immunity.

4.      Other chemokines play a role in development and are involved in angiogenesis and cell maturation.

Application of Chemokines

1.      In HIV infection

Human immunodeficiency virus requires CD4 and either CXCR4 or CCR5 to enter target cells. This allows the entry of HIV into CD4⁺ T cells or macrophages, which eventually leads to the destruction of CD4⁺ T cells and almost total inhibition of antiviral activity. Individuals who possess a nonfunctional variant of CCR5 and are homozygous for this gene remain uninfected despite multiple exposures to HIV.

2.      Diabetes with Insulin Resistance

Cytokines and chemokines have been implicated in insulin resistance. The cytokines which may play a role include IL–6 and TNF–α. CCR2 are present on adipocytes, and activation of inflammatory genes by the interaction of CCR2 with the ligand CCL2 results in impaired uptake of insulin–dependent glucose. Adipocyte also synthesize CCL2, resulting in the recruitment of macrophages. CCL3 may also be involved in insulin resistance.

3.      Atherosclerosis

CCL2 is present in lipid–laden macrophages and atherosclerotic plaques that are rich in these macrophages. The production of CCL2 in endothelial and smooth muscle cells is stimulated by minimally oxidized low–density lipoprotein. As a consequence, CCL2 is involved in the recruitment of foam cells to the vessel wall. Patients who are homozygous for the polymorphism in the promoter of CCL2 appear to have a high risk for developing coronary artery disease as opposed to patients who are heterozygous. CXCR2 and CX3CR1 are also implicated in cardiovascular disease.