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Inflammatory Mediators, Role in Periodontal Pathogenesis,

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  1. 1. CYTOKINES INTRODUCTION Periodontitis is a chronic inflammatory disease characterized by infiltration of leukocytes, loss of connective tissue, resorption of alveolar bone and formation of periodontal pockets. Periodontal diseases are caused by accumulation of bacteria and their products on the tooth surface and under the gingiva; recent efforts have focused on the contribution of the host cell response to these bacteria in the pathogenesis of the lesions. Bacteria are known to induce recruitment and activation of leukocytes, which, inturn, release inflammatory mediators and cytokines DEFINITIONS OF CYTOKINES: Steve offenbacher 1996 defined cytokines as mediator molecules, which direct and regulate inflammation and wound healing. The term cytokine meaning the cell protein is reserved for molecules, which transmit information or signals from one cell to another. It is part of a fundamental cell-to-cell communication network. Okada and S. Murakami 1998 cytokines are small soluble proteins produced by a cell that alters the behavior or properties of another cell, locally or systemically. Cytokines previously called as Lymphokines (cytokines produced by lymphocytes) or Monokines (cytokines produced by Monocytes). However it is now known that there is considerable overlap, e.g. TNF- a, IL-6 are made by both cell types and so this nomenclature is not in wide use.  Interleukins describes molecular messengers acting between leukocytes. The term dates from 1979. 1
  2. 2.  Two other related groups of molecules have been described; they are Interferons and Growth factors.  Interferons are molecules first described in the 1960s with non-specific antiviral effects.  Growth factors are molecules, which stimulate the growth of variety of cells. The cytokines are a diverse group of intercellular signaling proteins that regulate not only local and systemic immune and inflammatory response but also  Wound healing  Hematopoiesis and many biologic processes.  Over 100 structurally dissimilar and genetically unrelated cytokines have been identified to date.  Most are peptides/glycoproteins with molecular wt ranging from 6000 to 60,000.  They are extremely potent compounds that act at concentration of 10 -10 to 10-15 M by binding to specific surface receptors on target cells.  These cytokines are not only released from lymphocytes and monocytes but also from variety of other cells like fibroblasts and endothelium that interact with the cells of the immune system and participate in inflammatory reaction. 2
  3. 3.  Cytokines act as an Autocrine hormone, stimulating the same cell from which it was secreted.  Cytokines also may stimulate cells in close proximity to the cells, which secreted them (Paracrine effect).  These protein hormones may enter the circulation, where they can interact with immune cells at some distance from their point of origin ( Endocrine effect)  Intracrine a mechanism involving the direct action of a cytokine within a cell. CLASSIFICATION JAN LINDHE 1. Proinflammatory cytokines E.g. IL-1, IL-6 and TNF 2. Chemotactic cytokines E.g. IL-8 3. Lymphocytes signaling cytokines E.g. Cytokines released by Th1- IL-2, IFN. 3
  4. 4. Cytokines released by Th2- IL-4,IL-5,IL-10 and IL-13. NIESENGARD AND NEWMAN First group: Cytokines, which serve as mediators of innate immunity. E.g. Interferon a and b, TNF and IL-6. Second group: Cytokines that regulate the growth and differentiation of lymphocytes. E.g. IL-2, IL-4 AND TGF-BETA Third group: These cytokines regulate the hematopoietic activity in the bone marrow and collectively referred to as Colony Stimulating Factors or CSF’s produced by stromal cells or antigen stimulated T- lymphocytes. E.g. GM-CSF, G-CSF, M-CSF, MULTI- CSF, IL-3, IL-7. Fourth group: Cytokines which share the common property of being activators of inflammatory cell function. E.g. Interferon α CYTOKINE RECEPTORS AND RELATED MOLECULES: 4
  5. 5.  Cytokines require specific cell surface receptors through which to mediate their rage of actions on different cells.  Frequently the action of a cytokines on a cell wall, include the up regulation of surface expression of its receptor as well as release of the molecule itself.  Receptors vary in their form, some being single and other multiple chain complexes.  The largest family of cytokine receptors is called the cytokine receptor super family and it is characterized by an extra cellular region of structural homology approximately 200 amino acids long.  Receptors for cytokines such as IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-12, granulocyte stimulating factor and granulocyte- macrophage colony stimulating factor belong to this family.  A second family of related cytokine receptors can be considered part of the immunoglobulin super family and contains receptors for all IFN types in addition to the two IL-1 receptors, and M-CSF.  TNF-α, TNF- β and a number of related cytokines bind to another family of receptors, including the nerve growth factor receptor and Fas which signals cell death. 5
  6. 6.  Most cytokine receptors are type 1-membrane glycoproteins containing a single transmembrane domain.  However, the actual functional receptor often consists of two or more subunits, which can be shared among different cytokine receptor complexes.  Chemokines all bind to a distinct class of receptors called Seven transmembrane glycoproteins.  A few of these receptors are specific, with only one chemokine known to bind; others are shared with several Chemokines.  There is also one promiscuous receptor, known as the Duffy antigen, on red blood cells, which binds many different Chemokines. Basic model for cytokine action A simple model for cytokine activation of a cell is shown (The structures illustrated are based on those of the IL-6 receptor) cytokine binds to its receptor on the cells and induces dimerization or polymerization of receptor polypeptides of the cell surface. This causes activation of intracellular signaling pathways 6
  7. 7. (e.g. kinase cascades) resulting in the production of active transcription factors which migrate to the nucleus and bind to the enhancer region of gene, induced by that cytokine. ROLE OF CYTOKINES IN THE EARLY PHASES OF AN IMMUNE RESPONSE Among the cytokines released by macrophages in response to microbial components, TNF- α and IL-2 are particularly important. This early phase of mediator release has three fundamental functions: 1. Supply signals to endothelial cells that initiate recruitment of leukocytes. 2. Activate Phagocytic cells within the tissues and so provide innate resistance while the T-cell mediated response is developing. 3. Provide some of the signals that determine the type of T-cell response that will develop. 7
  8. 8. Role of cytokines in leukocyte recruitment TNF-α, IL-1 and lipopolysaccharides (LPS) induce expression of E- selectin and P-selectn on endothelium. These interact with oligosaccharides or circulating leukocytes, causing them to slow and roll along the endothelial surface. The cytokines also increase expression of ICAM-1. Chemokines, including MCP-1, RANTES and MIP-1α released from macrophages, tissue cells and endothelium respectively attach to the endothelium, when they trigger tethered leukocytes, enhancing the functional affinity of leukocyte integrins. The integrins interact with their ligand, ICAM-1 causing firm adhesion of the leukocytes to the endothelium. Finally, the cells migrate through the endothelium and can move cup gradients of chemotactic mediators. The details of the recruitment and the precise sets of adhesion molecules and chemokines vary between different leukocyte populations. Two human syndromes illustrate these phases. 8
  9. 9.  In patients with leukocyte adhesion deficiency type II, sialyl Lewis X, the ligand for E-selectin, is absent and neutrophil rolling does not occur.  Similarly, in LAD-1 the beta sub unit of the leukocyte Integrins are absent-neutrophils can take part in the rolling phase but do not adhere, and transmigrate. In both syndromes bacterial infections are common. Interleukin-1:  Interleukin –1 is a very potent multifunctional cytokine that appears to be a central regulator of the inflammatory and immune responses.  The term Interleukin-1 was introduced in 1979.  IL-1 is a Pleotropic cytokine with a variety of activities.  It includes osteoclast activating factor (OAF) because of stimulation of osteoclasts and lymphocyte activating factor (LAF) because of its ability to stimulate proliferation of phytohemagglutination-treated T-cells.  It is secreted by monocytes, macrophages, B-cells, fibroblasts, neutrophils, and epithelial cells.  Bacterial Lipopolysaccharide is a potent commonly used stimulus for IL-1 production.  IL-1 synthesis is suppressed by several endogenous factors, such as Corticosteroids, Prostaglandins, Cytokines like IFN-α, IL-4.  Some of the other cytokines share biologic activities with IL-1, most importantly IL-6 and TNF factor.  There are 2 principal forms of IL-1 that have agonist activity, IL-1 α, IL- 1β, with a third ligand, IL-1 receptor agonist (IL-1ra) that functions as a competitive inhibitor.  Two IL-1 receptors are found on the surface of the target cells, designated IL-1 receptor-1 and IL-1 receptor 2.  IL-1R1 is generally thought to mediate most of the responses to IL-1.  IL-1R2 has been reported to function principally as a decoy receptor. 9
  10. 10. Biologic effects of IL-1 include: Lymphocyte activation, macrophage activation, Natural killer stimulation, Prostaglandin formation, Fever induction, Anorexia, Acute phase protein release, Adrenocorticotophin release, Corticosteroid release, Cytokine gene expression, Plasminogen activator, Endothelial cell activation, Tumor cell growth inhibition, and suppression of lipoprotein lipase gene expression. Interleukin-2: IL-1 Induce matrix metalloproteinase expression Enhance Osteoclast formation and activity Stimulate adhesion molecule and chemokine expression Stimulate apoptosis of matrix producing cells Inflammation Bone loss Limit repair of periodontium Mechanism by which IL-1 could contribute to the net loss of periodontal tissues Connective tissue breakdown Stimulate production of inflammatory mediators(PGE 2) 10
  11. 11.  IL-2 (α andβ) was originally called T-cell growth factor because of its effect on mitogen or antigen activating T-cells and is known to play a general role in immune responses.  IL-2 also stimulates macrophage functional activity, modulates natural killer function and induces natural killer proliferation.  It is secreted by Th cells and NK cells.  The molecular wt of IL-2 is approximately 20,000 KD. Interleukin-2 receptors:  Three different forms of the human IL-2 receptors have been identified. These receptors interact with IL-2 with characteristically different affinities, specifically high, intermediate and low affinities.  The high affinity IL-2 receptors comprise at least two different IL-2 binding subunits termed IL-2Rα and IL-2Rβ. Clinical aspects:  Used in Tumors immunotherapy, either using IL-2 alone or in combination with in-vitro activated lymphokine-activated killer cells.  The potential for IL-2 as a cancer treatment is based on activation of cells, which are cytotoxic to the tumor.  Adverse effects: fever, chills, fatigue, nausea, vomiting, capillary leakage syndrome or vascular leakage syndrome, characterized by the accumulation of edema fluid in pleural cavities. Interleukin-3:  IL-3 is a distinct hematopoietic factor affecting multiple hematopoietic cell lineages. 11
  12. 12.  IL-3 also known as burst-promoting factor, B- cells stimulating factor, hemopoietic cell growth factor and multi colony stimulating factor is produced primarily by activated helper T type I and II cells.  This molecule stimulates the growth of colonies of mast cells, neutrophils, macrophages, eosinophils, and megakaryocytes.  It is secreted by activated helper T cells, NK cells.  IL-3 acts as a link between the T- lymphocytes and mast cells of the immune system, and the hematopoietic system, which generates the accessory cells granulocytes, phagocytes, and platelets, which carry out repair and defense responses. Interleukin-4  IL-4 originally called T-cell-derived B-cell growth factor (BCGF-1) because of its activation of B cells.  Also called as migration inhibition factor.  It is also play a role in the activation, proliferation and differentiation of B cells, T-cell growth, macrophage function, and growth of mast cells.  IgE synthesis by B cells is also induced by IL-4.  It is secreted by helper Tcells.  Mwt 15000 to 20000.  Receptors for this cytokine found on T-cells, B-cells, mast cells, myeloid cells, fibroblasts, neuroblasts, stromal cells, endothelial cells and monocytes. Effects on macrophages:  It can activate macrophage cytocidal function and increase macrophage expression of class II MHC proteins.  It suppresses the synthesis of proinflammatory cytokines, such as IL-1, IL-6, IL-8 and TNF- α and activated monocytes. 12
  13. 13. Interleukin-5:  Interleukin-5 is the name given to a lymphokine (a cytokine produced by lymphocytes).  Coffman and colleges found that IgA enhancing factor was IL-5 when the protein is sequenced.  Metcalf used the term eosinophil colony stimulatory factor to describe this cytokine.  Thus initially known as B- cells growth and differentiation factor, IgA enhancing factor, eosinophil colony stimulating factor.  IL-5 is heterogeneous glycoprotein with a molecular weight of 40000- 50000.  The major function of IL-5 in humans is to stimulate the production of eosinophils.  IL-5 not only increases the number of eosinophils but also has been reported to increase their function. Interleukin-6:  Interleukin-6 is a multifunctional cytokine produced by various cells such as activated monocytes or macrophages, endothelial cells, activated T- cells, and fibroblasts.  Formerly these molecules were known as B- cells stimulatory factor II, interferon B2 and plasmacytoma growth factor.  Its effect on B cells is to promote growth and facilitate maturation of the B cells causing immunoglobulin secretion.  IL-6 increases in sites of gingival inflammation and plays a role in bone resorption. 13
  14. 14. SOME OF THE EFFECTS OF INTERLEUKIN-6 (IL-6) IL-6 is a typical cytokine, in that it has a range of effects on many organ systems. In addition, IL-6 stimulates osteoclast formation and activity, particularly other osteogen depletion Interleukin-7:  Secreted by thymus, spleen and bone marrow stromal cells that functions as a growth factor for T and B cells precursors.  It was formerly known as lymphopoitin 1 based on its capacity to influence early lymphopoiesis.  IL-7 enhances the function of mature activated lymphocytic cells, particularly those with cytotoxic activity. At higher concentrations, IL-7 also increases macrophage cytotoxic activity and induces cytokine secretion by monocytes. Interleukin-8: Interleukin-8 is Chemotactic for neutrophils, increases their adherence to the endothelium. In general these cytokines are produced by: 1. Antigen stimulated T lymphocytes 14
  15. 15. 2. Mononuclear phagocytes, endothelial and epithelial cells, and fibroblasts that have been activated by other cytokines or LPS. 3. Platelets. All members of this family stimulate leukocytes and contribute to inflammatory responses. Interleukin-9:  Interleukin-9 is a heavily glycosylated polypeptide lymphokine with an apparent MW of 30000 to 40000.  It is secreted by IL-2 activated T-cells and Hodgkin's lymphoma cells.  It is a T cell growth factor, which acts in synergy with other cytokines. Interleukin-10:  IL-10 is an 18000 MW protein that is produced late in the activation process by Th2 cells, CD8 T cells, monocytes, keratinocytes and activated B cells.  It was originally called cytokine synthesis inhibitory factor because of its ability to inhibit cytokine production by activated T-lymphocytes i.e., Th1 cells and NK cells.  IL-10 inhibits the antigen presenting capacity of monocytes.  IL-10 also synergies with other cytokines to stimulate proliferation of B cells and mucosal mast cells.  Together with TGF beta it causes IgA production by B cells. Interleukin-11: Biologic activities of IL-11: Growth promotion of a plasmacytoma cell line: 15
  16. 16.  Originally IL-11 was detected based on its ability to stimulate the proliferation of an IL-6 dependent mouse plasmacytoma cell line.  The ability of IL-11 to support the growth of such a plasmacytoma cell line suggest that cytokine may be removed in the establishment and maintenance of plasmacytomas in vivo and may play an important role in the tumourogenesis. Hematopoietic colony stimulating activity:  Alone, IL-11 cannot support the growth of megakaryocyte colonies but stimulates and increases in numbers, size of megakaryocyte colonies in combination with IL3.  Therefore IL-11 is not a megakaryocyte colony stimulating factor but rather acts like a megakaryocyte potentiator, their results suggest that IL- 11 may play an important role in megakaryocytopoiesis and possibly in vitro platelet production. Interleukin-12:  It was originally called cytotoxic lymphocytes maturation factor (CLMF) or NK cell stimulatory factors.  Its molecular wt about 35000 to 40000.  It is produced predominantly on activation by B cells and macrophages.  It acts synergistically with IL-2 to induce IFN-γ by T-cells and NK cells  It is a key factor in the development of Th1 cells, stimulating both their proliferation and differentiation.  It suppresses Th2 dependent functions, such as the production of IL-4, IL- 10, IgE antibodies.  IL-12 also induces the production of GM-CSF, TNF, IL-16, IL-2. Interleukin-13: 16
  17. 17.  The marked structural homology between IL-4 and IL-13 and the close juxtaposition of their genes on the chromosome suggest that gene duplication occurred.  IL-13 is predominantly expressed in activated Th2 cells and regulates human B cell and monocytic activity. Interleukin-14:  IL-14 is a 50-60 KD glycosylated cytokine otherwise known as the high mol wt B cell growth factor.  IL-14 is thought to play a role in the development of B cell memory.  It enhances the proliferation of activated B cells and inhibits the synthesis of immunoglobulin.  It is produced by follicular dendritic cells and activated T cells.  IL-14 receptors are found only in cells of the B cells lineage.  IL-14, participates mainly in secondary humoral immune responses. Interleukin-15:  IL-15 is widely expressed in kidney, lung, liver, heart and bone marrow stroma.  It is produced most abundantly by epithelial cells and monocytes, but not by T lymphocytes.  It functions as a signal from non lymphoid cells for generating T cell dependent immune responses. Interleukin-16:  IL-16 which was previously known as lymphocyte chemoattractant factor (LCF) is produced by lymphocyte and induces the directional migration of CD4+ T cells, eosinophils and monocytes. 17
  18. 18.  The chemoattractant effect of LCF is blocked by anti CD4 Fab fragments suggesting that CD4 or CD4 related molecules are required for the effects of LCF on target cells. Interferons:  Interferons have been divided into two types: Type I an viral interferon has been further divided into alpha and beta subcategories. Type II or immune interferon is referred to as gamma interferon.  IFN-alpha is leukocyte derived where as IFN-beta is derived from fibroblasts. IFN-gamma is however derived from stimulated T cells of both CD4+ and CD8+ lineage.  Both IFN- α and β are characterized by their antiviral activity, IFN γ appears to be more integrated part of the immune system.  IFN γ is stimulated by IL-2 has a molecular wt of 35 –70 KD and in addition to its antiviral activity appears to have an important role in the stimulation of cytotoxic T cells and NK cells activity.  It also plays an important role in B-cells differentiation and in production of Igs under some conditions. Tumor necrosis factor:  TNF is a principal mediator in the host inflammatory response.  The main cell type secreting TNF is the mononuclear phagocyte.  The main stimulus for release is the Lipopolysaccharide of bacterial cell walls.  There are two structurally and functionally similar forms of TNF α and β but they differ biochemically.  TNF α is 17 KD is derived from stimulated macrophages and appears to have significant stimulatory activity on the cytoxic T lymphocytes (CTL) responsible for lysing tumor or virally infected cells. 18
  19. 19.  TNF-β is a 25 KD glycoprotein derived from activated T cells with a 28% homology to TNF-α  β Form is occasionally known as lymphotoxin these virtually have similar actions, which includes CTL stimulation, osteoclast activation of PMNLs and antiviral activity.  TNF also appears however to act synergistically with cytokines and induces release of IL-1. Transforming growth factorβ :  TGF β was initially discovered as a growth factor for fibroblasts and promotes wound healing.  T lymphocytes produce TGFβ.  Humans express at least three forms of TGF β called TGF β -1, TGF β -2, TGF β -3.  These are products of separate genes, but they all bind to 5 types of high affinity cell surface receptors. 19
  20. 20.  TGF β has anti proliferative effects on a wide variety of cell types including macrophages, endothelial cells and T and B-lymphocytes.  TGF is a chemoattractant and promotes many functional activities of fibroblasts.  TGF β is a potential mediator of inflammation because it is a product of activated macrophages, a potent chemoattractant for macrophages and can activate macrophages to produce IL-1.  It is chemo attractive for neutrophils and monocytes and it stimulates monocyte expression of adhesion proteins Chemokines:  They generally have low molecular weights, ranging from 7-14 KDa, and stimulate recruitment of leukocytes.  Chemokines are secondary proinflammatory mediators, i.e., they are typically induced by primary proinflammatory mediators such as Interleukin –1 or TNF.  By recruitment of leukocytes, chemokine activity leads to activation of host defense mechanisms and stimulates the early events of wound healing.  There are two major chemokine subfamilies based upon the portion of cystine residues i.e. CXC and CC.  The CXC family members also known as the alpha Chemokines. They primarily stimulate neutrophils.  C Chemokines are also known as the beta Chemokines. They stimulate Basophiles, eosinophils, T-lymphocytes and NK cells. Conclusion Cytokines are a significant and integral component of the host response to periodontal infection. Additionally, these molecules are important as physiologic mediators in the periodontium, serving in both normal processes and 20
  21. 21. pathogenic mediators. A therapeutic goal in clinical Periodontics can be aimed at maintaining a physiological role for the cytokines while recognizing that their over production results in pathologic changes. 21