1.1 INTRODUCTION AND LITERATURE REVIEW Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease. The main characteristic is a persistent synovitis of diarthrodial joints, often symmetrical in distribution, resulting in pain, stiffness, and loss of function. The disease is often characterized by morning stiffness of the distal joints that improves with mobilization, and swelling of the soft tissues. The disease can occur at any age, but the peak incidence of disease onset is between the ages of 25 and 55. Women are affected more commonly than men. The incidence of the disease increases with age. RA is one of the commonest inflammatory diseases worldwide with significant morbidity and a higher risk of mortality. There is no single marker to identify the disease process. Classification criteria were developed (1956, 1958) and later revised in 1987 by the American College of Rheumatology (ACR) (Table 1.1) (Arnett et al., 1988). The patient is diagnosed to have RA if he or she satisfied at least four of the seven stated criteria. Criteria through must be present for at least weeks. To date it has not been possible to prevent or to completely arrest the disease process through medical therapy. At the cellular level, activated T-lymphocytes, B-lymphocytes as well as tissue macrophages and dendritic cells migrate to synovial tissue, and polymorphonuclear cells accumulate in synovial fluid and on cartilage surfaces. Persistent joint inflammation leads to articular pain, swelling, cartilage erosion, and eventual joint deformity (Figure1.1) (Ernest et al., 1996) The inflammatory processes seen in RA patients are regulated by mediators (e.g. cytokines) which are elevated in synovial fluid as well as in the blood (Feldmann et al., 1996). Cytokines are extracellular signalling molecules that coordinate the inflammatory and immune responses in the host defence against infections and injuries. Cytokines produced by T cells and macrophages have many different effects on their target cells mediated through specific receptors. Cytokines regulate the activation of inflammatory effector cells involved in processes such as: proliferation, cartilage and bone destruction, and systemic disease involvement. Inflammation in cartilage and bone, involved both destructive as well as protective cytokines. The destructive cytokines (or pro-inflammatory) are tumour necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8) (Brennan et al., 1990; 1992) interleukin-18 (IL-18), interferon-γ (IFN-γ),(Dayer et al., 1999; McInnes et al., 2001) and the protective cytokines (or anti-inflammatory) consist of interleukin-4 (IL-4), interleukin-10 (IL-10) (Husby et al., 1988; Chu et al., 1991), interleukin-13 (IL-13), interleukin-16 (IL-16) (Klimiuk et al., 1999) and transforming growth factor- β (TGF-β) (Wahl et al., 1989) In RA, the production of pro-inflammatory cytokines is prolonged resulting in intense inflammation. Table1.1 American College of Rheumatology (ACR) criteria for classification of rheumatoid arthritis No Criterion Morning stiffness Definition Morning stiffness in and around joints, lasting at least hour before maximal improvement Arthritis of or more joint At least joint areas simultaneously have had areas soft tissue swelling or fluid (not bony overgrowth alone) observed by a physician Arthritis of hand joints At least area swollen as above in wrist, MCP, or PIP joint Symmetric arthritis Simultaneous involvement of the same joint areas on both sides of the body (bilateral involvement of PIPs, MCPs, or MTPs is acceptable without absolute symmetry) Rheumatoid nodules Subcutaneous nodules, over bony prominences or extensor surfaces or in juxta-articular regions, observed by a physician Serum Rheumatoid Factor Demonstration of abnormal amounts of serum (IgM) rheumatoid factor by any method that has been positive in < 5% of normal control subjects Radiographic changes Radiographic changes typical of RA on PA hand and wrist x-rays, which must include erosions or unequivocal bony decalcification localized to or most marked adjacent to the involved joints Figure 1.1 Pathogenesis of Rheumatoid Arthritis. In the normal knee joint, the synovium consists of a synovial membrane (usually one or two cells thick) and underlying loose connective tissue. Synoviallining cells are designated type A (macrophage-like synoviocytes) or type B (fibroblast – like synoviocytes). In early RA, the synovial membrane becomes thickened because of hyperplasia and hypertrophy of the synovial lining cells. An extensive network of new blood vessels is formed in the synovium. T cells (predominantly CD4 +) and B cells (some of which become plasma cells) infiltrate the synovial membrane. These cells are also found in the synovial fluid, along with large numbers of neutrophils. In the early stages of RA, the synovial membrane begins to invade the cartilage. In established RA, the synovial membrane becomes transformed into inflammatory tissue, the pannus. This tissue invades and destroys adjacent cartilage and bone. The pannus consists of synoviocytes and plasma cells (Figure taken from N Engl J Med 2001:344:911). 1.1.1. A role for HLA-DRB1 in rheumatoid arthritis The aetiology of RA is multi-factorial, involving genetic and environmental factors. One of the key genetic factors is linked to the human leukocyte antigen-DR (HLADR), which is located on human chromosome within the Major Histocompatibility Complex (MHC). MHC genes are divided into three regions: HLA class I, II and Class III. RA has been linked to HLA class II molecules. These genes are divided into three major sub regions: HLA-DP, HLA-DQ and HLA-DR. Each sub region contains at least functional α and β chain pair. The α and β chain products from the same sub region associate non-covalently to form the membrane heterodimers. The HLA-DR sub region contains functional β chain genes, designated DRβI and DRβIII. These β chains are highly polymorphic. In the DRβ chain, there are three major regions of variability. These regions are designated hyper variable region-1 (HV1), hyper variable region-2 (HV2) and hyper variable region-3 (HV3) (Gregersen et al., 1987). The DNA sequence of amino acids (QKRAA or QRRAA) between positions 70 to74 in the HV3 molecules of HLA-DRB1 locus alleles is thought to be responsible for susceptibility to RA (Mattey et al., 2001). This region is now called the “shared epitope” (SE) (Table 1.2). According to this hypothesis, the role of HLA-DR antigens as a marker of disease susceptibility is well known. In addition to their role in RA susceptibility, DRB1 alleles that contain the shared sequences are associated with more severe extra-articular manifestations and radiographic erosions (Weyand et al., 1992; Del Rincon et al., 1999). Although it is not known precisely how HLA class II genes contribute to disease susceptibility, the studies have documented a close association of HLA class II genes markers particularly HLA-DRB1 *04 patients with RA. Table 1.2 Amino acid sequences in DRB1 “shared epitope” HLA-DRB1* Allele Amino Acid Position 70 71 72 73 74 0101 Q R R A A 0102 Q R R A A 0401 Q K R A A 0404 Q R R A A 0405 Q R R A A 0408 Q R R A A 1001 R R R A A 1402 Q R R A A A= L-Alanine; Q=L-Glutamine; R= L-Arginine; K=L-Lysine However, this association varied between different ethnic groups. In the Caucasians, the (DR4 β-chain allele) DRB1*04 subtypes associated with RA are: HLADRB1*0401 and HLA-DRB1*0404 (Nepom et al., 1986; Zosch et al., 1986; Ollier et al., 1988). In Japanese, Taiwanese, Koreans and Polynesians, the DR4 association was with DRB1*0405 (Edmonds et al., 1998). Functionally, synovial tissue and synovial fluid macrophages exhibit an activated phenotype in RA, and express high levels of surface human leukocyte antigen-DR (HLA-DR). HLA-DR facilitates antigen presentation in the form of peptide fragments to T cells in the synovium. Once the T cells within the synovium are activated by a binding event between their receptor (specifically the α/β chains of the CD3 complex) and its cognate antigen of HLA class II molecules, the T-helper cells are committed to proliferate and produce cytokine signals. With the added presence of cytokine signals, the T cells are further stimulated to proliferate. These observations were used to support the concept of T cells playing a central role in the production of cytokines in RA. Any immune response involves the interaction of many different cell types, and it is not possible to consider cell-mediated responses and antibody-mediated responses separately. However, T cells play an important role in the regulation of virtually all immune responses, providing help for antibody production by B-lymphocytes (B cell), and providing growth factors for B cells, T cells, and several other cell types. 1.1.2. Cytokines in rheumatoid arthritis Cytokines produced by T cells, such as IFN-γ, IL-2 and IL-4 have been demonstrated in rheumatoid joints (Simon et al., 1994). Based on the pattern of cytokines production, the T lymphocytes are classified into four subsets, T-helper (Th1), Thelper (Th2), T-helper (Th3) and T-helper (Th0). Th1 cells produce IL-2, and IFN-γ that executes cell-mediated immune responses, whereas Th2 cells produce IL-4 and IL-10 and assist in humoral immunity (Abbas et al., 1996). However, the divisions are not absolute and there is considerable overlap or redundancy in function between the cells that are assigned to the different subsets. T cells expressing cytokines of both patterns have been designated as "Th0". The two subsets (Th1 and Th2) produce cytokines that cross-regulate development and activities of each other (Figure 1.2). For instance, IFN-γ produced by Th1 cells amplifies production of Th1 and inhibits proliferation of Th2 cells (Fitch et al., 1993) whereas IL-10 produced by Th2 cells block activation of Th1 cells. Recently, TGF-β has been classified into Th3 producing cytokine, which is immunoregulatory and inhibit the Th1 mediated cytokines. Th1, Th2 and Th3 differentiation and the equilibrium of these three subsets of helper T cells are responsible for the nature of any immune response. Th1associated cytokines, such as IL-2 and IFN-γ, are considered macrophage activators (Bellosevic et al., 1990), whereas cytokines secreted by Th2 and Th3 lymphocytes, such as IL-4, IL-10, IL-13 and TGF-β, exhibit suppressive activities on macrophage functions and antagonize the effect of Th1 secreted cytokines (Bogdan et al., 1993). The path of differentiation by activated T cells is dependent on antigen presentation, the type of antigen presenting cell (APCs), and the elements within the microenvironment where the T cells are activated. Published data suggest that rheumatoid arthritis is a “Th1-mediated” disease (Ronnelid et al., 1998). TH ⊕ IL-2 IL-4 IL-13 IL-10 IL-2 IFN-γ TNF-α TGF-β IL-10 ⊕ IL-4 TH TH Inflammation Tissue injury Figure 1.2 The regulatory function of Th1, Th2 and Th3 cells: The equilibrium of these three subsets of helper T cells is responsible for the nature of any immune response. Cytokines produced by Th2 and Th3 lymphocytes (IL-4, IL-10, IL-13 & TGF-β) antagonize the effect of Th1– secreted cytokines (IL-2, IFN-γ and TNF-α). IFN- γ produced by Th1 cells amplifies production of Th1 cytokines and inhibits proliferation of Th2 cells. IL-2 and IL-4 are shown to be autocrine growth factors for Th1 and Th2 cells, respectively. 1.1.3. Pro- and anti-inflammatory cytokines Cytokines refer to a broad class of regulatory proteins secreted by lymphocytes, macrophages and variety of other cells. The production of cytokines is regulated by various stimuli acting at the level of transcription or translation. Cytokines act by binding to corresponding receptors on the surface of target cells to influence cell behaviour. The resulting effects within the target cells are brought about by signal transduction across the cell membrane. Unlike classical hormones, which are generally produced by one type of specialized glands, cytokines tend to be produced by more than one cell type and by a number of tissues. Structural analysis has enabled grouping of cytokines into families. There are six major families of cytokines: interferons, tumour necrosis factors, transforming growth factors, interleukins, chemokines (chemotactic polypeptide) and colony stimulating factors (Table 1.3). Cytokines are polypeptides or glycoprotein with a molecular weight of 30 kDa or less. Some cytokines are synthesized as larger precursors that are later cleaved by proteases, leading to biological active molecules. Cytokines act by binding to specific receptors and induce each other’s production in an autocrine (cytokine that binds to a receptor of the same cell) and/or paracrine (cytokine from one cell type that binds to another cell type) fashion, often initiating cytokine cascades. The result is a system that is involved in almost all biological processes, including mediating the pathophysiological events in autoimmunity and inflammatory diseases. Of significance to RA is the fact that many inflammatory cytokines have been described to be present in increased quantities in the micro environment of RA joints. 10 region of HLA-DRB1 and disease severity in rheumatoid arthritis. J Rheumatol 2002; 29: 1358-1365. Kim HY, Kim TG, Park SH, Lee SH, Cho CS, Han H. Predominance of HLADRB1*0405 in Korean patients with rheumatoid arthritis. Ann Rheum Dis 1995: 54 (12): 988-990. Kim HY, Min JK, Yang HI, Park SH, Hong YS, Jee WH, Lee SH, Cho CS, Kim TG, Han H. The impact of HLA-DRB1*0405 on disease severity in Korean patients with seropositive rheumatoid arthritis. Br J Rheumatol 1997; 36: 440-443. Kim SH, Eisenstein M, Reznikov L, Fantuzzi G, Novick D, Rubinstein M, Dinarello CA Structural requirements of six naturally occurring isoforms of the IL-18 binding protein to inhibit IL-18. Proc Natl Acad Sci USA 2000; 97: 1190-1195. Kinikli G, Ates A, Turgay M, Akay G, Kinikli S, Tokgoz G. HLA-DRB1 genes and disease severity in rheumatoid arthritis in Turkey.Scand J Rheumatol. 2003; 32: 277280. Klimiuk PA, Goronzy JJ, Weyand CM. IL-16 as an anti-inflammatory cytokine in rheumatoid synovitis. J Immunol 1999; 1; 162(7):4293-9. Koch AE, Kunkel SL, Strieter RM. Cytokines in rheumatoid arthritis. J Investig Med 1995; 43:28-38. Koh WH, Chan SH, Lin YN, Boey ML. Association of HLA-DRB1*0405 with extraarticular manifestations and erosions in Singaporean Chinese with rheumatoid arthritis. J Rheumatol 1997; 24: 629–632. Kong KF, Yeap SS, Chow SK, Phipps ME. HLA-DRB1 genes and susceptibility to rheumatoid arthritis in three ethnic groups from Malaysia. Autoimmunity 2002; 35: 235-239. 172 Kretowski A, Mironczuk K, Karpinska A et al. Interleukin-18 promoter polymorphisms in type diabetes. Diabetes 2002: 51 (11): 3347-3349. La Cava A, Balasa B, Good A, van Gunst K, Jung N, Sarvetnick N. H-2D end confers dominant protection from IL-10-mediated acceleration of autoimmune diabetes in the nonobese diabetic mouse. J Immunol 2001; 167 (2): 1066-1071. Lacki JK, Moser R, Korczowska I, Mackiewicz S, Muller W. TNF-alpha gene polymorphism does not affect the clinical and radiological outcome of rheumatoid arthritis. Rheumatol Int 2000; 19: 137–140. Larsen BA, Alderdice CA, Hawkins D, Martin JR, Mitchell DM, Sheridan DP. Protective HLA-DR phenotypes in rheumatoid arthritis. J Rheumatol 1989: 16 (4): 455-458. Lee HS, Lee KW, Song GG, Kim HA, Kim SY, Bae SC. Increased susceptibility to rheumatoid arthritis in Koreans heterozygous for HLA-DRB1*0405 and *0901. Arthritis Rheum 2004; 50: 3468-3475. Lee YH, Kim HJ, Rho YH, Choi SJ, Ji JD, Song GG. Interleukin-1 receptor antagonist gene polymorphism and rheumatoid arthritis. Rheumatol Int 2004; 24(3):133-6. Leung BP, McInnes IB, Esfandiari E, Wei XQ, Liew FY. Interleukin-18 can promote synovial inflammation through activation of peripheral blood and synovial neutrophils. Arthritis Rheum 2000; 43:1253 MacGregor A, Ollier W, Thomson W, Jawaheer D, Silman A. HLADRB1*0401/0404 genotype and rheumatoid arthritis: increased association in men, young age at onset, and disease severity. J Rheumatol 1995; 22: 1032-1036. 173 Maini RN, Taylor PC, Paleolog E, Charles P, Ballara S, Brennan FM, Feldmann M.Anti-tumour necrosis factor specific antibody (infliximab) treatment provides insights into the pathophysiology of rheumatoid arthritis.Ann Rheum Dis. 1999; 58 Suppl 1:I56-60. Marshall JD, Aste-Amezaga M, Chehimi SS, Murphy M, Olsen H, Trinchieri G. Regulation of human IL-18 mRNA expression. Clin Immunol 1999; 90 (1): 15-21. Mattey DL, Dawes PT, Gonzalez-Gay MA, Garcia-Porrua C, Thomson W, Hajeer AH, Ollier WE. HLA-DRB1 alleles encoding an aspartic acid at position 70 protect against development of rheumatoid arthritis. J Rheumatol 2001; 28: 232-239. McInnes IB, Gracie JA, Liew FY. Interleukin-18: a novel cytokine in inflammatory rheumatic disease. Arthritis Rheum 2001; 44: 1481–1483. Medzhitov R, Preston-Hurlburt P, Kopp E, Stadlen A, Chen C, Ghosh S, Janeway CA Jr. MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways. Mol Cell 1998; 2(2):253-258. Montminy M. Transcriptional regulation by cyclic AMP. Annu Rev Biochem 1997: 66: 807-822. Moran H, Chen SL, Muirden KD, Jiang SJ, Gu YY, Hopper J, Jiang PL, Lawler G, Chen RB. A comparison of rheumatoid arthritis in Australia and China. Ann Rheum Dis 1986;45(7):572-8. Muneta Y, Shimoji Y, Yokomizo Y, Mori Y. Molecular cloning of porcine interleukin-1beta converting enzyme and differential gene expression of IL-1beta converting enzyme, IL-1beta, and IL-18 in porcine alveolar macrophages. J Interferon Cytokine Res 1999: 19 (11): 1289-1296. 174 Nakamura K, Okamura H, Wada M, Nagata K, and Tamura T. Endotoxin-induced serum factor that stimulates gamma interferon production. Infect Immun 1989; 57: 590-595. Nepom GT, Seyfried CE, Holbeck SL, Wilske KR, Nepom BS. Identification of HLA-Dw14 genes in DR4+ rheumatoid arthritis.Lancet 1986;2(8514):1002-1005. Nepom GT. Major histocompatibility complex-directed susceptibility to rheumatoid arthritis. Adv Immunol 1998: 68: 315-332. Nepon G, Byers P, Seyfried C, Healey LA, Wilski KR, Stage D, Nepon BS. HLA genes associated with rheumatoid arthritis: identification of susceptibility alleles using specific oligonucleotide probes. Arthritis Rheum 1989; 32: 15 Novick D, Kim SH, Fantuzzi G, Reznikov LL, Dinarello CA, Rubinstein M Interleukin-18 binding protein: a novel modulator of the Th1 cytokine response. Immunity 1999; 10: 127-36 Novick D, Schwartsburd B, Pinkus R, Suissa D, Belzer I, Sthoeger Z, et al. A novel IL-18BP ELISA shows elevated serum IL-18BP in sepsis and extensive decrease of free IL-18. Cytokine 2001; 14: 334–342. Okamura H, Tsutsi H, Komatsu T et al. Cloning of a new cytokine that induces IFNgamma production by T cells. Nature 1995; 378 (6552): 88-91. Okamura H, Tsutsui H, Kashiwamura S, Yoshimoto T, and Nakanishi K. Interleukin-18: a novel cytokine that augments both innate and acquired immunity. Adv Immunol 1998; 70: 281-312. Ollier W, Carthy D, Cutbush S, Okoye R, Awad J, Fielder A, Silman A, Festenstein H. HLA-DR4 associated Dw types in rheumatoid arthritis.Tissue Antigens1989;33(1):30-37. 175 Ollier W, Venables PJ, Mumford PA, Maini RN, Awad J, Jaraquemada D, D'Amaro J, Festenstein H. HLA antigen associations with extra-articular rheumatoid arthritis. Tissue Antigens 1984; 24: 279–291. O'Neill L. How Toll-like receptors signal: what we know and what we don't know. Curr Opin Immunol 2005; 17:1-7. O'Neill L. The Toll/interleukin-1 receptor domain: a molecular switch for inflammation and host defence. Biochem Soc Trans 2000; 28(5):557-563. Ota M, Seki T, Fukushima H, Tsuji K, Inoko H. HLA-DRB1 genotyping by modified PCR-RFLP method combined with group-specific primers. Tissue Antigens 1992; 39: 187-202. Overbergh L, Giulietti A, Valckx D, Decallonne R, Bouillon R, Mathieu C. The use of real-time reverse transcriptase PCR for the quantification of cytokine gene expression. J Biomol Tech 2003; 14(1):33-43. Pages F, Berger A, Lebel-Binay S et al. Proinflammatory and antitumor properties of interleukin-18 in the gastrointestinal tract. Immunol Lett 2000; 75 (1): 9-14. Pawlik A, Kurzawski M, Czerny B, Gawronska-Szklarz B, Drozdzik M, Herczynska M. Interleukin-18 promoter polymorphism in patients with rheumatoid arthritis. Tissue Antigens. 2006; 67(5):415-418. Perdriger A, Chales G, Semana G, Guggenbuhl P, Meyer O, Quillivic F, Pawlotsky Y. Role of HLA-DR-DR and DR-DQ associations in the expression of extraarticular manifestations and rheumatoid factor in rheumatoid arthritis. J Rheumatol 1997; 24: 1272–1276. Pizarro TT, Michie MH, Bentz M, Woraratanadharm J, Smith MF Jr, Foley E, Moskaluk CA, Bickston SJ, Cominelli F. IL-18, a novel immunoregulatory cytokine, 176 is up-regulated in Crohn's disease: expression and localization in intestinal mucosal cells.J Immunol 1999;162(11):6829-35. Plater-Zyberk C, Joosten LAB, Helsen MMA, Sattonnet-Roche P, Siegfried C, Alouani S, et al. Therapeutic effect of neutralizing endogenous IL-18 activity in the collagen-induced model of arthritis. J Clin Invest 2001; 108: 1825–1832. Pociot F, Molvig J, Wogensen L, Worsaae H, Nerup JA TaqI polymorphism in the human interleukin-1 beta (IL-1 beta) gene correlates with IL-1 beta secretion in vitro. Eur J Clin Invest 1992; 22: 396-402. Prevoo ML, van 't Hof MA, Kuper HH, van Leeuwen MA, van de Putte LB, van Riel PL: Modified disease activity scores that include twenty-eight-joint counts. Development and validation in a prospective longitudinal study of patients with rheumatoid arthritis. Arthritis Rheum 1995, 38:44-48. Puren AJ, Fantuzzi G, Gu Y, Su MS, and Dinarello CA. Interleukin-18 (IFN-γinducing factor) induces IL-8 and IL-1ß via TNF-α production from non-CD14+ human blood mononuclear cells. J Clin Invest 1998; 101: 711-721. Robak E, Robak T, Wozniacka A, Zak-Prelich M, Sysa-Jedrzejowska A, Stepien H. Proinflammatory interferon-gamma-inducing monokines (interleukin-12, interleukin18, interleukin-15)-serum profile in patients with systemic lupus erythematosus. Eur Cytokine Netw 2002; 13 (3): 364-368. Ronnelid J, Berg L, Rogberg S, Nilsson A, Albertsson K, Klareskog L. Production of T-cell cytokines at the single-cell level in patients with inflammatory arthritides: enhanced activity in synovial fluid compared to blood.Br J Rheumatol1998;37(1):714. 177 Rueda B, Gonzalez-Gay MA, Mataran L, Lopez-Nevot MA, Martin J. Interleukin-18promoter polymorphisms are not relevant in rheumatoid arthritis. Tissue Antigens. 2005; 65(6):544-8. Ruiz-Morales JA, Vargas-Alarcon G, Flores-Villanueva PO, Villarreal-Garza C, Hernandez-Pacheco G, Yamamoto-Furusho JK, Rodriguez-Perez JM, PerezHernandez N, Rull M, Cardiel MH, Granados J. HLA-DRB1 alleles encoding the "shared epitope" are associated with susceptibility to developing rheumatoid arthritis whereas HLA-DRB1 alleles encoding an aspartic acid at position 70 of the beta-chain are protective in Mexican Mestizos. Hum Immunol 2004; 65: 262-269. Sayer D, Whidborne R, Brestovac B, Trimboli F, Witt C, Christiansen F. HLA-DRB1 DNA sequencing based typing: an approach suitable for high throughput typing including unrelated bone marrow registry donors. Tissue Antigens 2001; 57: 46-54. Scott P. IL-12: initiation cytokine for cell-mediated immunity. Science1993;23;260(5107):496-7. Seki E, Tsutsui H, Nakano H, Tsuji N, Hoshino K, Adachi O, Adachi K, Futatsugi S, Kuida K, Takeuchi O, Okamura K.Lipopolysaccharide-induced IL-18 H, Fujimoto secretion from J, Akira murine S, Nakanishi Kupffer cells independently of myeloid differentiation factor 88 that is critically involved in induction of production of IL-12 and IL-1beta. J Immunol 2001; 15:166(4):2651-7. Sharp JT. Radiologic assessment as an outcome measure in rheumatoid arthritis. Arthritis Rheum 1989; 32: 221–229. Simon AK, Seipelt E, Sieper J. Divergent T-cell cytokine patterns in inflammatory arthritis.Proc Natl Acad Sci U S A 1994 ;30;91(18):8562-6. 178 Sivalingam SP, Yoon KH, Koh DR, Fong KY. Single-nucleotide polymorphisms of the interleukin-18 gene promoter region in rheumatoid arthritis patients: protective effect of AA genotype. Tissue Antigens 2003; 62 (6):498-504. Stastny P. Association of the B-cell alloantigen DRw4 with rheumatoid arthritis. N Engl J Med 1978; 298(16):869-871. Stastny P. Mixed lymphocyte cultures in rheumatoid arthritis. J Clin Invest 1976; 57: 1148-1157. Sugiura T, Kawaguchi Y, Harigai M et al. Association between adult-onset Still's disease and interleukin-18 gene polymorphisms. Genes Immun 2002: (7): 394-399. Takada T, Suzuki E, morohashi K, Gejyo. Association of single nucleotide polymorphisms in the IL-18 gene with sarcoidosis in a Japanese population. Tissue Antigens 2002; 60: 36-42. Takeba Y, Suzuki N, Wakisaka S, Takeno M, Kaneko A, Asai T, Sakane T. Involvement of cAMP responsive element binding protein (CREB) in the synovial cell hyperfunction in patients with rheumatoid arthritis. Clin Exp Rheumatol 2000: 18 (1): 47-55. Takeda K, Tsutsui H, Yoshimoto T, Adachi O, Yoshida N, Kishimoto T, Okamura H, Nakanishi K, Akira S. Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity 1998; 8: 383-390. Tone M, Thompson SA, Tone Y, Fairchild PJ, Waldmann H. Regulation of IL-18 (IFN-gamma-inducing factor) gene expression. J Immunol 1997; 159 (12): 6156-6163. Turesson C, Schaid DJ, Weyand CM, Jacobsson LT, Goronzy JJ, Petersson IF, Sturfelt G, Nyhall-Wahlin BM, Truedsson L, Dechant SA, Matteson EL. The impact of HLA-DRB1 genes on extra-articular disease manifestations in rheumatoid arthritis. Arthritis Res Ther 2005; 7: R1386-93. 179 Turesson C, Schaid DJ, Weyand CM, Jacobsson LT, Goronzy JJ, Petersson IF, Sturfelt G, Nyhall-Wahlin BM, Truedsson L, Dechant SA, Matteson EL. The impact of HLA-DRB1 genes on extra-articular disease manifestations in rheumatoid arthritis. Arthritis Res Ther2005; 7(6):R1386-93. Ushio S, Namba M, Okura T, Hattori K, Nukada Y, Akita K, Tanabe F, Konishi K, Micallef M, Fujii M, Torigoe K, Tanimoto T, Fukuda S, Ikeda M, Okamura H, Kurimoto M Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J Immunol 1996; 156: 4274-4279. van der Heijde DM. Joint erosions and patients with early rheumatoid arthritis. Br J Rheumatol 1995; 34: 74–78. van der Heijde DMFM, van Leeuwen MA, van Riel PLCM, Koster AM, van t'Hof MA, van Rijswijk MH, et al. Biannual radiographic assessments of hands and feet in a three-year prospective followup of patients with early rheumatoid arthritis. Arthritis Rheum 1992; 35: 26–34. Veale D, Rogers S, FitzGerald O. Classification of clinical subsets in psoriatic arthritis. Br J Rheumatol 1994; 33: 133–138. Verweij CL. Tumour necrosis factor gene polymorphisms as severity markers in rheumatoid arthritis.Ann Rheum Dis 1999;58 Suppl 1:I20-6. Wakitani S, Imoto K, Murata N, Oonishi H, Ochi T, Yoneda M. An association between the natural course of shoulder joint destruction in rheumatoid arthritis and HLA-DRB1*0405 in Japanese patients. Scand J Rheumatol 1998; 27: 146-148. Wakitani S, Murata N, Toda Y, Ogawa R, Kaneshige T, Nishimura Y, Ochi T. The relationship between HLA-DRB1 alleles and disease subsets of rheumatoid arthritis in Japanese. Br J Rheumatol 1997; 36: 630-636. 180 Waldron-Lynch F, Adams C, Amos C, Zhu DK, McDermott MF, Shanahan F, et al. Tumour necrosis factor 5' promoter single nucleotide polymorphisms influence susceptibility to rheumatoid arthritis (RA) in immunogenetically defined multiplex RA families. Genes Immun 2001; 2: 82–87. Wesche H, Henzel WJ, Shillinglaw W, Li S, and Cao Z. MyD88: an adapter that recruits IRAK to the IL-1 receptor complex. Immunity 1997: 837-847 Wesche H, Korherr C, Kracht M, Falk W, Resch K, and Martin MU. The Interleukin1 Receptor Accessory Protein (IL-1RAcP) Is Essential for IL-1-induced Activation of Interleukin-1 Receptor-associated Kinase (IRAK) and Stress-activated Protein Kinases (SAP Kinases). J Biol Chem 1997; 272:7727–7731. Weyand CM, Hicok KC, Conn DL, Goronzy JJ. The influence of HLA-DRB1 genes on disease severity in rheumatoid arthritis. Ann Intern Med 1992: 117 (10): 801-806. Weyand CM, McCarthy TG, Goronzy JJ. Correlation between disease phenotype and genetic heterogeneity in rheumatoid arthritis. J Clin Invest 1995; 95: 2120-2126. Weyand CM, Xie C, Goronzy JJ. Homozygosity for the HLA-DRB1 allele selects for extraarticular manifestations in rheumatoid arthritis. J Clin Invest 1992; 89: 2033– 2039. Wilson AG, de Vries N, van de Putte LB, Duff GW. A tumour necrosis factor alpha polymorphism is not associated with rheumatoid arthritis. Ann Rheum Dis 1995; 54: 601-603. Wilson AG, Symons JA, McDowell TL, McDevitt HO, Duff GW. Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci USA 1997; 94: 3195–3199. 181 Wordsworth BP, Allsopp CE, Young RP, Bell JI. HLA-DR typing using DNA amplification by the polymerase chain reaction and sequential hybridization to sequence-specific oligonucleotide probes. Immunogenetics 1990; 32: 413-418. Wordsworth BP, Lanchbury JS, Sakkas LI, Welsh KI, Panayi GS, Bell JI. HLA-DR4 subtype frequencies in rheumatoid arthritis indicate that DRB1 is the major susceptibility locus within the HLA class II region. Proc Natl Acad Sci USA 1989; 86: 10049–10053. Yamamura M, Kawashima M, Taniai M, Yamauchi H, Tanimoto T, Kurimoto M, et al. Interferon-γ-inducing activity of interleukin-18 in the joint with rheumatoid arthritis. Arthritis Rheum 2001; 44: 275–285. Yen JH, Chen CJ, Tsai WC, Lin CH, Ou TT, Wu CC, et al. Tumor necrosis factor promoter polymorphisms in patients with rheumatoid arthritis in Taiwan. J Rheumatol 2001; 28: 1788–1792. Yen JH, Chen JR, Tsai WJ, Tsai JJ, Liu HW. HLA-DRB1 genotyping in patients with rheumatoid arthritis in Taiwan. J Rheumatol 1995; 22: 1450-1454. Zetterquist H, Olerup O. Identification of the HLA-DRB1*04, -DRB1*07, and DRB1*09 alleles by PCR amplification with sequence-specific primers (PCR-SSP) in hours. Hum Immunol 1992; 34: 64-74. Zhang X, Llamado L, Pillay I, Price P, Will R.Interleukin-1 gene polymorphism disease activity and bone mineral metabolism in rheumatoid arthritis. Chin Med J (Engl). 2002; 115(1):46-49. Ziolkowska M, Koc A, Luszczykiewicz G, Ksiezopolska-Pietrzak K, Klimczak E, Chwalinska-Sadowska H, Maslinski W. High levels of IL-17 in rheumatoid arthritis patients: IL-15 triggers in vitro IL-17 production via cyclosporin A-sensitive mechanism. Immunol. 2000; 164(5):2832-8. 182 Zoschke D, Segall M. Dw subtypes of DR4 in rheumatoid arthritis: evidence for a preferential association with Dw4. Hum Immunol 1986; 15(1):118-124. 183 APPENDIX A PREPARATION OF STOCK SOLUTIONS Buffers and reagents Phosphate-buffer saline (PBS) Working solution, pH=7.4 To make 10U stock solution, 1liter: Sodium chloride, NaCl (Merck, Germany) 80.0 g Potassium chloride, KCl (Merck) 2.0 g Potassium phosphate dibasic, KH2PO4 (BDH Labs, England) 2.0 g Sodium phosphate monobasic, Na2HPO4.7H2O (Sigma, USA) 11.5 g De-ionized water 900 ml Dissolve and adjust to pH 7.4 by addition of 1M NaOH solution. TAE (Tris-acetate-EDTA) buffer, pH 7.6 To make 1000 ml (10x) Tris base-40 mM 48.4 g 11.42 ml glacial acetic acid 20 ml 0.5 M EDTA (pH 8.0) Dissolve and make up the volume to liter with distilled water. Dilute stock 10 times to obtain 1x-working solution. TBE (Tris/borate/EDTA) electrophoresis buffer To make 10x stock solution, 1liter: 108 g Tris base-890 mM 55g boric acid-890 mM 40 ml 0.5 M EDTA (pH 8.0) Dissolve and make up the volume to liter with distilled water. Dilute stock 10 times to obtain 1x-working solution. 184 Reagents used for DNA EXTRACTION (Prepared in the Laboratory) To make 10x stock solution To make 500 ml,1.44M Ammonium chloride (MW 53.49) 38.51gram of NH4Cl dissolved in 500ml of purified water To make 500 ml,100mM Sodium Bicarbonate (MW 84.01) 42.00gram of NaHCO3 dissolved in 500ml of purified water To make 500 ml, 4M Sodium chloride (MW 58.44) 116.88gram of NaCl dissolved in 500ml of purified water To make 250 ml, 5M Sodium chloride (MW 58.44) 73.05gram of NaCl dissolved in 250ml of purified water To make 500 ml, 20mM Disodium EDTA (MW 372.24) 3.72gram of Na2EDTA dissolved in 500ml of purified water To make 500 ml, 10% Sodium duode sulphate 50.0gram of SDS dissolved in 500ml of purified water To make 250 ml, 20% Sodium duode sulphate 50.0gram of SDS dissolved in 250ml of purified water To make 250 ml, 1M Tris-hydro chloric acid (MW157.60),pH7.6 39.40gram of TRIS-HCl dissolved in 250ml of purified water Red cell lysis buffer (RCLB ) To make 1x working solution 100 ml of NH4Cl (1.44M Ammonium chloride) and 100ml of NaHCO3 (100mM Sodium Bicarbonate) make up the volume to liter with distilled water. It was stored at room temperature. 185 Nuclei lysis buffer (NLB) To make 1x working solution 100 ml of NaCl (4M Sodium chloride), 100ml of Tris-HCl (1M Tris-hydro chloric acid) and Na2EDTA (20mM Disodium EDTA), make up the volume to liter with distilled water. It was stored at room temperature Proteinase K buffer To make 1x stock solution 10ml of Na2EDTA (20mM Disodium EDTA) and 10ml of SDS (10% Sodium duode sulphate), make up the volume to 100ml with distilled water. It was stored at room temperature Proteinase K 10mg/ml in Proteinase K buffer Dissolved 100mg Proteinase K in 10ml Proteinase K buffer (5M NaCl & 20% w/v SDS) It was stored at -20°C Paraformaldehyde (4%) To make 1000 ml, Paraformaldehyde 4.0 g PBS, pH 7.4 1000 ml o Stir and heat at 56 C for 30-60 minutes until the powder dissolves. Preparation of diethyl-pyrocarbonate (DEPC)-treated water DEPC-treated water was prepared by collecting water into RNase-free glass bottle, adding DEPC (Sigma) to 0.01% (v/v) and allowed to standing overnight. The water was then autoclaved to inactivate DEPC. 186 Formaldehyde gel electrophoresis buffers DEPC-treated water and sterile plastic ware or baked glassware were used to prepare the following solutions: a. 10 x MOPS 3-[N-Morpholino) propanesulfonic acid] Buffer To make 1000 mL, 41.2 g MOPS 200 mM, pH 7.0 26.6 ml 3M NaOAc [8 mM Final concentration] 10 ml 0.5 M EDTA, pH 8.0 [1 mM Final concentration] Dissolve in 600 ml DEPC-treated distilled water and make up the volume to 1000 mL. b. RNA Sample Buffer 50% Formamide (from 100% stock solution) x MOPS Buffer 7% Formaldehyde (from 37% stock solution) 0.04% Bromophenol Blue (BPB) DNA and RNA sample loading dye A 10 x sample buffer stock consists of 50% glycerol 0.25% bromophenol blue 0.25% xylene cyanole FF in TAE buffer. A commercially available dye was used in our experiments (Promega). 187 [...]... precursors Interleukin- 18 acts via an interleukin- 18 receptor (IL-18R) complex (Torigoe et al., 1997) The IL-18R complex is made of two non identical chains: a ligand binding chain termed IL-18Rα and a non-ligand binding chain termed IL-18Rβ IL-18Rα, characterized previously as IL-1R related protein (IRrP) (Parnet et al., 1996), binds IL -18 at a relatively low affinity Generation of IL-18Rα deficient... phosphorylation of NF-IL6 prior to association with the CCAAT/enhancer binding protein (C/ERP) binding site Interleukin- 18 has been found in synovial tissue, and enhanced levels of IL -18 were measured in the joint and in the serum of rheumatoid arthritis patients (RA) The role of IL -18 in the pathogenesis of RA remains poorly understood However, recent evidence suggests that IL -18 enhances the infiltration of inflammatory... Chinese patients with RA 2 the serum profiles of both pro-inflammatory (TNF-α, IL-1, IL-6, IL-8, IL-12 and IL -18) and anti-inflammatory (IL-4, IL-10, TGF-β) cytokines in Chinese patients with RA 3 the single nucleotide polymorphisms of IL -18 gene promoter region 4 the gene expression (mRNA) and cytokines secretion profiles of IL -18, IL-18BP and TNF-α in peripheral blood mononuclear cells in normal individuals... cytokine profiles (IL-12, IL-15 and IL -18) would give us useful insight into the pathogenesis of RA patients We hypothesized that genetic polymorphisms of HLA-DRB1*04 genes and cytokine genes can modify the disease pattern in rheumatoid arthritis We also postulate that this may be due to genetic polymorphisms resulting in differential gene expression 34 and cytokine profiles As IL -18 is important in the... IL-18BP likely represents the extra cellular domain of the IL -18 receptor ligand binding chain, which lost its transmembrane domain and is now a secreted protein There are four isoforms of human IL-18BP (IL-18BPa, -BPb, -BPc and -BPd) IL-18BPa has the greatest affinity for IL -18, with a rapid on-rate, a slow off-rate, and a Kd of 399 pM (KIM et al., 2000) IL-18BPb and IL-18BPd lack a complete Ig domain... 23 1.1.10 IL -18 binding protein (IL-18BP) Interleukin- 18 binding protein (IL-18BP), a constitutively secreted protein that binds mature IL -18 with high affinity, provides a potential mechanism to regulate IL -18 activity A soluble IL-18BP (38 kDa) has been isolated from human urine (Novick et al., 1989 and 1990; Engelmann et al., 1990) IL-18BP belongs to the immunoglobulin superfamily, and has limited... (Jovanovic et al., 1998) In arthritis, the effects of IL-17 on cartilage were associated with destruction and lack of repair, including activation of nitric oxide and catabolic enzymes, with a decrease of chondrocyte proliferation and proteoglycan synthesis 1.1.8 Interleukin- 18 (IL -18) (IFN-γ inducing factor) Recently another cytokine, Interleukin -18 (IL -18) has been added to the list of potential contributors... normal individuals and RA patients following in- vitro stimulation by LPS and PHA 5 the IL -18 protein profile following treatment with monoclonal antibody therapy against TNF-α (infliximab) in RA patients 35 2.1 INTRODUCTION Rheumatoid arthritis (RA) is a chronic inflammatory disease of unknown etiology Genetic and environmental factors play an important role in its pathogenesis Genetically, the most... homology to a number of other regulatory proteins including inhibins, activins and bone morphogenetic proteins The TGF-β cytokines are produced by every leukocyte lineage, including lymphocytes (T-helper 3 cell), macrophages, and DCs TGF-β is highly pleiotropic in their biological effects, which include involvement in wound healing, tissue repair, 25 development and haemopoiesis It was originally described... IL -18 increases the release of glycosaminoglycans (GAG), which are by-products of its degradation Synovial tissue explanted from patients with rheumatoid arthritis 18 exhibits elevated levels of IL -18 mRNA as well as more intense staining of IL -18 protein in lymphocytes and macrophages compared to that obtained from patients with osteoarthritis Fibroblast- like synovial cells isolated from the joint . found in synovial tissue, and enhanced levels of IL-18 were measured in the joint and in the serum of rheumatoid arthritis patients (RA). The role of IL-18 in the pathogenesis of RA remains. elevated levels of IL-18 are present in the synovium of RA patients but not in patients with osteoarthritis (OA). Interleukin-18, together with IL-12 and IL-15 induced, and sustained articular. the binding of IL-18 would trigger the activation of Ras, with the aid of non-receptor protein tyrosine kinases such as LCK, resulting in the cascade through Raf to MEK (MAP kinase or ERK kinase).