Cytokines profiles in patients with rheumatoid arthritis genetic polymorphisms, protein and gene expressions of tumour necrosis factor x interleukin 18 and interleukin 18 binding proteins

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 molecu

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 1 through 4 must be present for at least 6 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

1 Morning stiffness Morning stiffness in and around joints, lasting

at least 1 hour before maximal improvement

2 Arthritis of 3 or more joint

areas

At least 3 joint areas simultaneously have had soft tissue swelling or fluid (not bony overgrowth alone) observed by a physician

3 Arthritis of hand joints At least 1 area swollen as above in wrist, MCP,

or PIP joint

4 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)

5 Rheumatoid nodules Subcutaneous nodules, over bony prominences

or extensor surfaces or in juxta-articular regions, observed by a physician

6 Serum Rheumatoid Factor

(IgM) Demonstration of abnormal amounts of serum rheumatoid factor by any method that has been

positive in < 5% of normal control subjects

7 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 lining 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

Synovial-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 (HLA-DR), which is located on human chromosome 6 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 1 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 2 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

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, helper 1 (Th1), helper 2 (Th2), T-helper 3 (Th3) and T-helper 0 (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

T-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 associated cytokines, such as IL-2 and IFN-γ, are considered macrophage activators

Th1-(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).

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

TH 2

TH 3

⊕ IL-2 IL-4

IL-13

IL-10

IL-2 IFN- γ TNF-α

Inflammation Tissue injury

⊕ IL-4

TH 1

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

The role of cytokines in the regulation of immune response and inflammation are well recognized(Brennan et al., 1996) Pertaining to the inflammatory process, cytokines

are designated into pro-inflammatory cytokines (e.g TNF-α, IFN-γ, IL-1, IL-2, IL-6,

IL-8, IL-12, IL-15, IL-17 and IL-18) (Arend et al., 2004) and anti-inflammatory cytokines (e.g IL-4, IL-10 (Chomarat et al., 1994; Katsikis et al., 1994),IL-13, IL-16

(Klimiuk et al., 1999) and TGF-β) (Lafyatis et al., 1989; Brennan et al., 1990; Lotz et

al., 1990) In RA, the balance between pro-inflammatory and anti-inflammatory

cytokines determines the degree and extent of inflammation which can lead to major clinical effects Anti-inflammatory cytokines and cytokine antagonists counteract the effects of pro-inflammatory cytokines Hence the relative concentrations of a cytokine

to its inhibitor or antagonist will determine its final effect

Table 1.3 Cytokine families

Group Cytokines

Interleukins IL-1: IL-1α, IL-1β, IL-Ra, IL-18

IL-2/IL-4: IL-5, IL-15, IL-13 IL-6/IL-12

Interferons IFN-α, IFN-β, IFN-γ

Tumour necrosis factor TNF-α, TNF-β (LT-α), LT-β

Growth factors TGF-β, EGF, PDGF, FGF AND Vascular endothelial

growth factors (VEGF) Chemokines C-X-C subgroup (IL-8), C-C subgroup (MIP-1α), C

subgroup (lymphotactin) Colony stimulating

factors Granulocyte colony-stimulating factor (G-CSF)

1.1.4 Interferon-γ (IFN-γ)

The interferons (IFNs) comprise a family of secreted glycoprotein Isaacs and Lindenmann first coined the term in 1957 as a result of their studies on the phenomenon of viral interference (Isaacs and Lindenmann 1957) Three major classes

of IFNs (IFN-α, IFN-β, IFN-γ) have been identified, and classified according to their biochemical and antigenic properties IFN-α and IFN-β are structurally related, bind

to the same cellular receptor, and have pronounced antiviral effect IFN-γ is mainly produced by T lymphocytes and larger granular lymphocytes (LGL) from blood or lymphoid tissues upon stimulation by specific antigens, mitogens or alloantigens It is noted for its immunomodulatory rather than antiviral effects

IFN-γ is primarily an up-regulator of the immune system Both CD4+ and CD8+ T lymphocytes can produce IFN-γ, but the former is considered a major producer in response to antigen stimulation One of the most striking effects of IFN-γ is its ability

to activate macrophages IFN-γ is modulating the expression of MHC class I and MHC class II antigens in a variety of cell types Short peptide fragments from intracellular antigens degraded in cytosol are bound to MHC class I molecules and the complex recognized by CD8+ T cells On the other hand, peptides from exogenous antigen degraded in endosomal cellular vesicle bind to MHC II molecules and the complex is then recognized by CD4+ T helper cells This critical step in the immune response is affected by IFN-γ, which induces or enhances the expression of MHC II

antigen on macrophages (Amadi et al., 1989) In an inflammatory response, the

accumulation of large numbers of activated macrophages is responsible for much of the tissue damage These cells release inflammatory cytokines (e.g TNF-α, IL-12 and

IL-15), hydrolytic enzymes, reactive oxygen and nitrogen intermediates, which damage the surrounding tissues

1.1.5 Tumour necrosis factors-α (TNF-α)

Tumour necrosis factors (TNF) are primary mediators of immune and inflammatory response There are two forms of TNF namely TNF-α and TNF-β (lymphotoxin -α) that show considerable homology in their amino acid sequences TNF-α is produced

by activated macrophages, T cells, as well as by many other cells including NK cells, B-lymphocytes and fibroblasts, whereas TNF-β is a different molecule, which is produced by activated T-lymphocytes only TNF-α and TNF-β are encoded in two closely linked genes within the MHC-HLA class III region on the short arm of human chromosome 6 (6q21.3) Similar to many other cytokines that are processed from the cell membrane, human TNF-α is synthesized as a pro-protein, which needs to be activated in order to bind to cell surface receptors The active form of TNF-α consists

of three non-covalently bound monomers packed together around a three- fold axis to form a cone-like trimeric molecule TNF-α and LT-α are very similar in their biological activities A potent stimulator of TNF-α production is the lipopolysaccharide (LPS), the cell-wall constituent of Gram-negative bacteria Manifestations such as fever, shock and activation of neutrophils, which accompany gram-negative bacteria infections, are directly or indirectly caused by TNF-α The LPS of the bacteria cell wall stimulates macrophages to produce TNF-α, which

subsequently stimulates the production of IL-1 (Dinarello et al., 1986), in addition to

having a direct effect on tissues Activated macrophages have long been known to be able to inhibit the growth of tumour cells and protect the host from infection

TNF-α and IL-1 have been studied most extensively because of their pathophysiological role in joint destruction Both cytokines are found at high levels in

the synovial fluid as well as in the serum of patients with RA (Feldmann et al., 1996)

TNF-α has certain effects on lymphocytes, neutrophils and the vascular endothelium

In vitro studies have indicated that TNF-α play a primary role in the cytokine cascade

in RA, controlling the production of IL-1 and other pro-inflammatory cytokines,

including IL-6 & IL-8 (Kishimoto et al., 1992; Rosenbaum et al., 1992; Kontny et al.,

2000) An anti-TNF antibody added to cultures of rheumatoid synoviocytes result in

decreased bioactivity and mRNA expression of IL-1 (Brennan et al., 1989)

Anti-TNF-α antibodies also inhibit the production of other pro-inflammatory cytokines

such as IL-6 and IL-8 (Butler et al., 1995) TNF-α and IL-1 mediate joint

inflammation and destruction by inducing the synthesis and release of inflammatory matrix metalloproteinases (MMP), prostaglandins and nitric oxide (NO) in a variety

of cell types This indicates that TNF-α is the key pro-inflammatory cytokine in the rheumatoid joint through its paracrine actions However, it is unclear which factors up-regulate TNF-α production in the joints, particularly in the relative absence of IFN-γ

Biological responses to TNF-α are mediated by ligand binding via two structurally distinct receptors: type 1 (TNFR1) and Type II (TNFR2) Both receptors are transmembrane glycoproteins that have multiple cysteine-rich repeats in exctracellular N-terminal domain TNFR1 and TNFR2 are present on all cell types except erythrocytes Both TNF receptors are subject to proteolytic cleavage by members of the matrix metalloprotease family and are shed from the surface of cells in response to inflammatory signals such as TNF-α ligand-receptor binding The shed extracellular

domains of both receptors retain their ability to bind TNF-α and therefore act as natural inhibitors of TNF-α bioactivity

1.1.6 Interleukin-1 (lymphocyte activating factor)

Interleukin-1 (IL-1), earlier known as lymphocyte activating factor (LAF) is a

multifunctional cytokine with most of its biological activities relevant to the inflammatory process and joint destruction in RA IL-1 is a 17-kd protein that is mostly produced by monocytes and macrophages but is also produced by endothelial

cells, B cells, and activated T cells (Durum et al., 1985) Together with TNF-α it is

detectable in large amounts in rheumatoid synovium and is produced by synovium derived mononuclear cells, both constitutively and after LPS stimulation There are three members of IL-1: IL-α, IL-β and IL-Ra Of these IL-1α and IL-1β function as agonist through one of the two IL-1 receptor family: IL-1 receptor I (IL-1RI)

(Greenfeder et al., 1995; Yoon et al., 1998) The IL-1 receptor antagonist (IL-1Ra) is

a specific, high affinity antagonist that inhibits IL-1α and IL-1β and thus controls the final effect exerted by IL-1 IL-1β is the predominant form of IL-1, released by human monocytes This cytokine is involved in the pathogenesis of RA synovitis because it stimulates synoviocytes and monocytes, and promotes bone and cartilage degradation Similar to many enzymes and other cytokines, it is synthesized in a “pro” form that must be enzymatically (IL-β converting enzyme) cleaved for full expression

of activity Many of the IL-1 features are shared by TNF-α and the two cytokines often augment each other’s activity

1.1.7 Interleukin-12 (IL-12), Interleukin-15 (IL-15) and Interleukin-17 (IL-17)

In addition to cell-cell contact (macrophages-HLA-DRB1*04 – T cell receptor interaction) of T cells with macrophages, there are other co-stimulators: IL-12, IL-15 and IL-17 resulting in T cell driven joint inflammation in RA IL-12 is released by Dentritic cells (DCs) and macrophages through innate immune mechanisms, and induces IFN-γ production from natural killer cells and T cells; this leads to enhanced

Th1 cell development (Caspi et al., 1998) IL-12 levels are elevated in the sera and

synovial fluids (SF) of patients who have RA, although in paired samples, higher

IL-12 levels were found in SF than in blood which suggested local synthesis in the joint

(Kim et al., 2000) Recently, McInnes et al have reported a potential role for interleukin-15 (IL-15) in the pathogenesis of RA (Mclnnes et al., 1997) IL-15 is a

pleiotropic cytokine derived from several cell types including macrophages and fibroblasts IL-15 mediates its activity through a heterotrimeric receptor consisting of

a unique 15Rα chain, in combination with β and γ chains of the 2 receptor

IL-15 was identified as a T cell proliferating factor with biological activities similar to

that of IL-2 (Grabstein et al., 1994; Giri et al., 1995) In spite of its functional

similarities with IL-2, IL-15 is not produced by T-cells Furthermore, IL-15 could stimulate the induction of TNF-α production in RA through activation of synovial T

cells (Mclnnes et al., 1996) Ziolkowska et al., (2000) demonstrated that IL-15

exerted its pro-inflammatory properties via the induction of IL-17 They found changes in levels of IL-15 and IL-17 in synovial fluids of RA patients were correlated

Human IL-17 is a newly identified 20-30 kd glycosylated cytokine that is secreted by

CD4+ activated memory T cells (Yao et al., 1995) IL-17 up-regulates the production

of pro-inflammatory cytokines including IL-β and TNF-α in human peripheral blood

macrophages (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 to the pathogenesis of RA IL-18 was first identified as a factor

capable of inducing IFN-γ production in mice with endotoxic shock (Nakamura et al.,

1989) and was known as IFN-γ inducing factor (IGIF) In 1995, the cytokine was

purified from mouse liver, and the molecule was subsequently cloned (Okamura et al., 1995; Okamura et al., 1995) IL-18 is an 18-kDa glycoprotein derived by enzymatic

cleavage of a 23 kDa precursor (pro-IL-18), by IL-1ßconverting enzyme (ICE) (Yong

Gu et al., 1997; Ghayur et al., 1997; Dinarello et al., 1998) Pro-IL-18 expression is

widespread, including monocyte/macrophages, dendritic cells, Kupffer cells, keratinocytes, articular chondrocytes, synovial fibroblasts and osteoblasts (Udagawa

et al., 1997; McInnes et al., 2000) ICE regulates processing and secretion of IL-18

from intracellular 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 mice confirmed that this receptor is nevertheless essential for signaling IL-18Rβ chain, initially

termed IL-1 receptor accessory protein-like (IL-1 RAcPL), is related and similar to IL-1RacP in that it does not bind to the ligand directly, but rather binds to the complex formed by the IL-18 and IL-18Rα chain generating a high affinity complex

The IL-18R complex recruits the signaling molecules, IL-1 receptor-associated kinase

(IRAK) and TNF-R-associated factor-6 (TRAF-6) (Kojima et al., 1998), which can

phosphorylate nuclear factor kappaB (NF-kB)-inducing kinase (NIK) with subsequent

activation of transcription factor NF-kB (Matsimoto et al., 1997) Alternatively, 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) This kinase (MAPK) thus allows nuclear translocation and subsequent phosphorylation of NF-IL6 prior to association with the CCAAT/enhancerbinding protein (C/ERP) binding site

Interleukin-18 has been found in synovialtissue, and enhanced levels of IL-18 were measured in the jointand in the serum of rheumatoid arthritis patients (RA) The role

of IL-18 in the pathogenesis of RA remains poorly understood However, recentevidence suggests that IL-18 enhances the infiltration of inflammatorycells into the

synovial tissue (Leung et al., 2000) The role of IL-18 in arthritis can be summarized

by the following observation In chondrocytes, IL-18 increases gene expression for inducible nitric oxide synthetase (iNOS), cyclooxygenase 2 (COX-2), and stromelysin

(Olee et al., 1999) Thus, in addition to mononuclear phagocytes, chondrocytesare potential sources of IL-18 in the joint Exposure of normal human articular cartilage

to IL-18 increases the release of glycosaminoglycans (GAG), which are by-products

of its degradation Synovial tissue explanted from patients with rheumatoid arthritis

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 fluid of patients with RA constitutively secretes IL-18 when cultured In patients with active

RA, synovial levels of IL-18 are elevated, unlike elevated serum levels the synovial

levels of IL-18 do not correlate with the C-reactive protein level Gracie and colleagues (1999) reported that significantly elevated levels of IL-18are present in the synoviumof RA patients but not in patients with osteoarthritis(OA)

Interleukin-18, together with IL-12 and IL-15 induced, and sustained articular Th1

cell responses, with conspicuous IFN-γ production (Gracie et al., 1999) IL-18 also

increased NO production from these cells independent of other cytokines Similar to other cytokine networks, TNF-α and IL-1β also induced IL-18 Significantly higher levels of IL-18 receptor gene expression have been observed in synovial tissue from

RA patients than in OA patients (Gracie et al., 1999) In vitro, synovial tissue was

shown to produce IFN-γ following stimulation by IL-18; however, IL-18-induced NO production was independent of IFN-γ As initially reported in studies by Puren and

colleagues (Puren et al., 1998) using PBMCs, TNF-α synthesis by macrophages in

synovial cultures was induced by IL-18

Several arthritis models have studied IL-18 in vivo (Leung et al., 2000; Wei et al., 2001; Ye et al., 2004) Co-administration of IL-18 in the collagen-induced arthritis

(CIA) mice, facilitates the development of an erosive, inflammatory arthritis compared with controls treated with phosphate buffered saline Importantly, the reduced CIA in IL-18 knockout mice can be reversed by the administration of

recombinant murine IL-18 (Liew et al., 2003) These data strongly suggest a proinflammatory role for IL-18 in RA (Liew et al., 2003; Gracie et al., 2004)

In addition to its proinflammatory activity in arthritis, IL-18 contributes to other

human diseases such as cancer (Pages et al., 1999; Dayer et al., 1999), diabetes (Nolsoe et al., 2003), Crohn’s disease (Pizarro et al., 1999) Figure 1.4 summarizes

several of IL-18’s functions in promoting or suppressing diseases In infectious diseases, IL-18 plays a crucial part in promoting the inflammatory response during bacterial sepsis, a response that can lead to hepatic injury and multiple organ failures IL-18 is important for host defense against some types of pathogens Up-regulation of Fas ligand in Th1 cells by IL-18 may increase apoptosis of Fas receptor expressing cells that interact with the activated Th1 cells This induction of apoptosis allows IL-

18 to have anti-tumor activity

Figure 1.3 Proposed model for IL-18 signal transduction based on IL-1-initiated pathways Following stimulation with IL-18, NF-kB is activated depending on the

recruitment and activation of the 18 signalling molecules, IRAK and TRAF6

IL-18 binding protein is a natural IL-IL-18 inhibitor

NFkB/IkB

Anti-IL-18 antibodies IL-18 BP ICE inhibitors

IL-18 IL-18Rα & β

NFkB

Gene expression

Ras Raf

MEK MAPK

MAPK

NF-IL6

IRAK TRAF6

NIK IKK-α

Cell membrane

Figure 1.4 Potential roles for IL-18 in various pathological conditions Green highlighting indicates a potentially beneficial effect of IL-18

1.1.9 IL-18 promoter gene polymorphisms

Recently, it was reported that IL-18 protein expression is regulated by the IL-18

promoter gene (Tone et al., 1997; Marshall et al., 1999) Two single nucleotide

polymorphisms (SNPs) at positions -607 and -137 in the promoter region are found to

be associated with diabetes (Kretowski et al., 2002), necrotizing enterocolitis (Heninger et al., 2002) and sarcoidosis (Takada et al., 2002) These promoter region

polymorphisms are predicted to be the binding sites for cyclic AMP-responsive

element-binding protein (CREB) (Haus-Seuffert et al., 2000) and human histone H4 gene-specific transcription factor-1 (H4TF-1) (Dailey et al., 1988; Giedraitis et al.,

2001) respectively At position -607 the change from C (cytosine) to A (adenosine) nucleotide disrupts a potential CREB binding site and at position -137 the change

from G (guanine) to C (cytosine) nucleotide affects the H4TF-1 binding site In the

IL-18 gene promoter transcription activity assay following stimulation, low promoter activity was observed for A and C alleles at positions -607 and -137 respectively In contrast, higher promoter activity was observed for C and G alleles at similar

positions (Giedraitis et al., 2001) We therefore hypothesised that RA patients may

have higher IL-18 gene promoter transcription activity as a contributing factor to their disease pathogenesis It is postulated that RA patients would have higher frequencies

of C alleles at position -607 and/or higher frequencies of G alleles at position -137 of the IL-18 promoter gene Alternatively, higher presence of A alleles at position -607 and/or presence of C alleles at position -137 would confer some protective effect against the development of RA

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 homology to the IL-1 type II receptor 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 and do not bind or

neutralize IL-18

Similar to neutralizing antibodies of IL-18, the IL-18BP inhibits IL-18 induction of

IFN-γ (Novick et al., 1999), IL-8, and activation of NF-kB in vitro However, unlike

antibodies to IL-18, the IL-18BP does not exhibit species specificity, and hence

human IL-18BP neutralizes both human and murine IL-18 (KIM et al., 2000)

Administration of IL-18BP to mice abrogated circulating IFN-γ following LPS stimulation Thus, IL-18BP functions as an inhibitor of the early Th1 cytokine response Serum IL-18BP is significantly elevated during sepsis, indicatingit’s in- vivo role in regulating immune responses

1.1.11 Transforming growth factor-beta (TGF-β)

Several established cytokine families are recognized for their roles in regulating the immune response With these groups of families, the members of the immune regulatory family of transforming growth factor-beta (TGF-β) is viewed as having a

unique and essential role in regulating immune function (Letterio et al., 1998) These

molecules are now recognized to have profound effects on the immune cells,

including all classes of lymphocytes, macrophages (Tsunawaki et al., 1988) and

dendritic cells The name transforming growth factor (TGF) has been given to two different kinds of molecules, now differentiated by the designations α and β The TGF-α molecule is a member of the epidermal growth factor (EGF) family with which it shares biological and structural properties The TGF-β molecules form a family of their own (TGF-β superfamily) Human TGF-β molecules occur in five different but related (66-80% sequence identity) forms designated TGF-β1 through TGF-β5, all encoded by different genes on distinct chromosomes TGF-β1, TGF-β2 and TGF-β3 products are most common in humans and they have overlapping and distinct functional properties TGF-β1, TGF-β2 and TGF-β3 genes are about 100kb (seven exons) and located on the short arm of human chromosome-19 (19q13), chromosomes-1 (1q41) and chromosome-14 (14q24) respectively

Members of the TGF-β subgroup show more distant structural 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,

development and haemopoiesis It was originally described as a promoter of cell growth in soft agar, but many experiments have demonstrated that it is bifunctional and can be either a growth promoter or a growth inhibitor TGF-β tends to inhibit the growth of lymphoid, epithelial and endothelial cells TGF-β is produced and secreted

as latent forms, which need to be activated in order to bind to cell surface receptors

TGF-β is a potent regulator of pro-inflammatory cytokines and it is stored within

human platelets (Assoian et al., 1983) It exerts its anti-inflammatory effect by

inhibiting the synthesis of pro-inflammatory cytokines such as IL-1α, IL-1β, and TNF-α The action of TGF-β on cells is dependent on the cell type, its state of differentiation and the total milieu of cytokines present T lymphocytes are clearly influenced by TGF-β at all stages of development, from their differentiation to their activation and proliferation, and serve as a paradigm for the pleiotropic nature of this cytokine Early studies of the effects of TGF-β on human lymphocyte functions revealed that activated T cells self synthesize and secrete TGF-β upon re-stimulation Exogenous TGF-β favours the Th2 differentiation Conditions favouring Th2 differentiation were also found to induce the subsequent production of high levels of TGF-β (Seder et al., 1998) Cytokines inducing Th2 and Th3 differentiation may be useful in the treatment of patients with RA

1.1.12 Interleukin 4 (IL-4)

IL-4 is an anti-inflammatory cytokine produced by cells of the T-lymphoid lineage, principally by activated Th2 cells, basophils and mast cells It was originally discovered through its action on B-lymphocytes IL-4 is known as B cell growth

factor or B-cell stimulating factor (Defrance et al., 1987) IL-4 can induce many

effects that are beneficial in RA including: inhibition of cytokine secretion in

monocytes and synovial mononuclear cells (Vannier et al., 1992), induction of

IL-1Ra , limitation of the induction of monocyte-derived hydrogen peroxidase and prevention of macrophage colony formation IL-4 also inhibits spontaneous formation

of TNF-α, IL-β and IL-6 (Bonder et al., 1999)

1.1.13 Interleukin-10 (IL-10)

Interleukin-10 (IL-10) is produced by monocytes, macrophages and lymphocytes

(T-cell and B (T-cell) It is a potent inhibitor of the expression of several pro-inflammatory cytokines Similar to IL-4, IL-10 can also inhibit the synthesis and secretion of

cytokines, but in contrast, IL-10 inhibits class II MHC expression (Waal Malefyt et al., 1991), T cell proliferation (Taga et al., 1992), cytokine secretion from monocytes and IL-8 chemotaxis of CD4+ T cells (Waal Malefyt et al., 1991) The ability of IL-10 to

inhibit cytokine synthesis by both T cells and natural killer cells was soon found to be due to its inhibitory effects on the macrophage-monocyte accessory cell This inhibition of activation of cells of the macrophage- monocyte and dendritic cell lineages led to IL-10 being termed a ‘macrophage-deactivating factor’ (Bogdan and Nathan 1993) Finally, IL-10 inhibits inflammatory metalloproteinases (MMP) secretion and induces the MMP’s inhibitor (TIMP-1) on monocytes

Previous studies found a low level of T cell derived cytokines present in the synovium This point to factors other than antigen dependent response as the aetiopathogen of

RA The study of these newer cytokines IL-15, IL-17 and IL-18 might shed further light on the aetiopathogenesis of RA

Figure 1.5 The role of cytokines released by cells that subserve “natural immunity” in

promoting the development of Th1 cells Antigen presenting cell (APC) present antigenic peptides on their HLA class II molecules to T helper cells, the APC also delivers co-stimulatory signals that are required for IL-2 production and activation of the Th cells The presence of IL-12, IL-15, IL-17 and IL-18 in addition to INF-γ favors the development Th cells into Th1 cells Th1 cytokines causing inflammation, tissue damage and up- regulates the inducible nitric oxide synthase (iNOS) and matrix metalloproteinases (MMPs) production TGF-β, IL-10 and IL-4 suppress the Th1 mediated cytokines

APS

IL-10 IL-4

Inflammation and joint destruction

Peptide

Macrophages

⊕ IL-12 IL-15 IL-18

Th 1

1.2 THERAPEUTIC POSSIBILITIES FOR RHEUMATOID ARTHRITIS

1.2.1 Cytokine as therapeutic targets

TNF-α and IL-1 mediate joint inflammation and destruction of bone as well as cartilage by inducing the synthesis and release of other inflammatory mediators (MMPs, prostaglandins & NOS) Studies indicate that TNF-α is the principal pro-

inflammatory cytokine in the rheumatoid joint (Feldmann et al., 1996; Drynda et al.,

2002) Both cytokines TNF-α and IL-1 are found at high levels in the synovial fluids

as well as in the blood of RA patients Consequently targeting the pro-inflammatory cytokines would appear to be a rational strategy in the therapy of arthritis Several experimental therapeutic approaches for the treatment of arthritis have targeted the pro-inflammatory cytokines through the use of anti-cytokine antibodies (monoclonal antibodies to TNF-α), soluble cytokine receptors (soluble TNF receptors and soluble Type I IL-1 receptors), natural cytokine inhibitors (IL-1 receptor antagonists) and anti-cytokine proteins (IL-10 and IL-4)

1.2.2 Anti-cytokine antibodies in rheumatoid arthritis treatment

Anti-cytokine monoclonal antibodies (MAb) can be effectively used in the management of RA It functions as a neutralizing factor at the receptor level, thereby inhibiting the action-targeted cytokine In addition, MAbs has several distinctive advantages when used as therapeutic agents: (1) they can be artificially produced to any cytokine target, (2) they have long circulating half lives and (3) their physical properties of size and affinity can be modified to meet the therapeutic demand MAbs are also modified to avoid immunogenicity response

1.2.3 Anti-TNF-α monoclonal antibody

Three MAb directed against TNF-α have been developed for clinical use: a chimeric (mouse Fc and human IgG1) monoclonal anti- TNF-α antibody (infliximab), a humanized (both the Fc and non-antigen-binding regions of the Fab segment are

replaced) MAb (CDP571) (Knight et al., 1993; Harriman et al., 1999) and a fully

human MAb (D2E7) Administration of infliximab has shown clinical improvement

of RA symptoms (Elliott et al., 1994: Rankin et al., 1995; Moreland et al., 1997; Feldmann et al., 2005) Although re-treatment with the infliximab has been effective,

the development of anti-infliximab limits re-treatment efficacy Treatment with CDP571 resulted in significant improvements in measures of disease activity, including: pain score, tender and swollen joint counts and C-reactive protein levels

1.2.4 Soluble cytokine receptors as therapy

Cytokine receptors bind their ligands with high affinity and specificity Soluble versions of these receptors inhibit the bioactivity of excess cytokines in the extracellular milieu Such receptors retain their binding specificity of membrane-bound forms and can efficiently neutralize the cytokine The binding of soluble receptors to their natural ligands prevents the ligands from participating in cell-surface receptor-mediated signal transduction Physiologically soluble receptors play

an important role in regulating cytokine activity

1.2.5 Soluble TNF-receptor

Two distinct TNF receptors, type I (p55) and type II (p75), are found on the surface of

a wide variety of cells including synovial fibroblast cells Both p55 and p75 receptor participate in the signal transduction but appear to use different pathways, and in

some cases, mediate different effects Both receptors are involved in TNF-α stimulated DNA synthesis and the release of other pro-inflammatory cytokines in synovium

Soluble receptors are synthetically fused with an antibody Fc region to extend their half-life The p75-based molecule (Etanercept) consists of two p75 soluble TNF receptors (p75 sTNFR) joined to the Fc portion of a human immunoglobulin -G1 (IgG1) molecule Analogously, the p55-based molecule (Lenercept) is composed of two p55 soluble TNF receptors (p55 sTNFR) linked to a human IgG1-Fc Patients receiving Etanercept by subcutaneous administration experienced significant improvement in all measures of disease activity, including swollen joints count, pain

score and C-reactive protein levels (Moreland et al., 1999)

1.2.6 Soluble receptors for Interlukin-1

IL-1 binds to specific membrane proteins or receptors that are composed of an extracellular cytokine-binding, membrane-spanning domain and an intracellular signal transuding domain Two types of receptors (type I and type II) have been detected on the cell surface However, type II receptor does not function in signal transduction,

and its major function is to serve as a decoy molecule to absorb IL-1 (Bresnihan et al.,

1998) Soluble forms of receptors have been detected in human body fluids (Jouvenne

et al., 1996) A soluble recombinant human IL-1R type I (rHuIL-1R1) has been tested

in patients with RA Only one out of 23 patients demonstrated relevant clinical

improvement with rHuIL-1R1 (Gampion et al., 1996)

1.2.7 Recombinant human Interleukin-1 Receptor antagonist (IL-1Ra)

Interleukin-1 Receptor antagonist (lacking the agonist activity) is a naturally occurring protein that binds to the cell surface IL-1R, blocking its interaction with IL-

1, and inhibiting IL-1- mediated signal transduction A recombinant version of human

IL-1Ra (rHuIL-1Ra) has been tested in patients with RA (Jiang et al., 2000) The

observed clinical response to rHuIL-1Ra was not very impressive However, treatments of RA patients with neutralizing TNF-α (using either neutralizing Abs or

soluble receptors) or IL-1 receptor antagonist (IL-Ra) yielded promising results in

controlling chronic inflammation and cartilage degradation respectively Despite these encouraging results, none of these treatments are “cure” for RA patients

1.2.8 Methotrexate in the treatment of rheumatoid arthritis

Methotrexate (MTX), an immunomodulatory agent, acts by inhibiting dihydrofolate reductase that in turn reduces the production of thymidylate and hence the synthesis

of DNA (Bressolle et al., 2000) It was initially developed for the treatment of malignancies (Gaubner et al., 1951) and is currently used for treatment of RA In RA

the effect may be partly due to the modulation of immunologic or inflammatory

reactions by some cytokines Genestier et al., (1998) has proved that a low

concentration of MTX is effective in controlling the inflammatory manifestations of

RA in short-term and long-term prospective studies (Kremer et al., 1988) They also

reported that MTX selectively induces apoptosis of activated, but not resting lymphocytes Alarcon and colleagues (1990) have studied serum samples obtained from RA patients treated with either MTX or placebo in order to determine the effect

of MTX on the production of rheumatoid factor- IgM (RF-IgM) They observed a significant decrease in RF- IgM levels in the MTX-treated patients, whereas there

were no significant changes in the placebo group Besides modulating the B cell immunity, studies have shown that MTX also invokes T cell immunity in RA

(Thomas et al., 1993) The association of HLA-DRB1 and the role of T cell in the

development of RA have been well studied The inhibiting effect of MTX on IL-1, TNF-α, IL-6 and IL-8 has been studied in animal models, in RA patients (Chang et al.,

1992; Olsen et al., 1987; Wluka et al., 2000) and their isolated cells (Constantin et al

1998)

1.2.9 Limitations in the management of RA

Currently there are many new therapeutic agents that are successful in the management of RA, however; complete remission of the disease is still rare It is well established that RA is a polygenic disease with multiple aetiological factors Therefore the conventional drugs that help in controlling the symptoms are mostly non-specific, target broad biological pathways and mechansisms which will affect multiple cytokines On the other hand cytokine therapy will help in targeting specific cytokine or members of the same family thereby limiting the disease It is quite effective in most cases, but not all RA patients show similar response This is due to the fact that multiple cytokine polymorphism or ethnic differences exists Therefore for effective management of RA, it would be necessary to subdivide the patients based on their cytokine profile, genetic background and clinical status

1.3 OBJECTIVE OF THE PRESENT PROJECT

Numerous studies indicate that polarized cytokine responses as a consequence of deficiencies or imbalance in the cytokine network might determine disease susceptibility and severity Polarized cytokine profiles may not only play a part in immunopathological reactions but can also confer protection

The molecular mechanisms that underlie the difference in pro- and anti-inflammatory activities in inflammatory disease remains unclear It is possible to derive clinically useful information by understanding the molecular regulatory steps underlying the difference This in turn will help in the identification of persons displaying specific cytokine profiles and enabling clinicians to prognosticate or decide a most appropriate treatment regimen

RA patients on the whole are being treated effectively However to date, there is no study directed towards identification of the roles of genetic polymorphisms of both HLA DRB1 and cytokines genes in local RA patients An approach, involving comparison of the baseline patterns (such as demography, clinical features and radiological changes), laboratory investigations, genetic factors (HLA-DRB1*04 positivity status) and 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

and cytokine profiles As IL-18 is important in the chronic inflammatory process, we studied this cytokine in greater depth

In order to evaluate the above hypothesis the following studies were undertaken:

1 the association of HLA-DRB1*04 subtypes and genetic polymorphisms of cytokine genes-tumour necrosis factor-α (TNF-α), interleukin 1-α (IL-1α), interleukin 1-β (IL-1β) and IL-1 receptor antagonist (IL-Ra) with disease susceptibility and severity in 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 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

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 thoroughly studied genes are those located on the major histocompatibility complex (MHC) class II region MHC genes play crucial roles in the immune responses, and are important in tissue transplantation, autoimmunity and inflammatory diseases HLA-DRB1 is the most polymorphic locus All HLA-DRB1 alleles associated with RA encode a conserved amino acid sequence (QKRAA, QRRAA, OR RRRAA) at position 70-74 in the third hypervariable region (HVR3) of

the DRβ1 chain, which is commonly called the shared epitope (SE) (Gregersen et al., 1987; Wordsworth et al., 1989; Auger et al., 1997; Ruiz-Morales et al., 2003) The

association between HLA-DRB1*04 and RA has been reported in many studies

(Stasny et al., 1978; Mattey et al., 2001; Gonzalez-Gay et al., 2002; Khani-Hanjani et al., 2002) However, the association of disease severity and presence of the SE varies among different populations (Gao et al., 1991; Kong et al., 2002; Kinikli et al., 2003; Ruiz-Morales et al., 2003) In Caucasians, the DRB1*04 allele associated are HLA- DRB1*0401 and HLA-DRB1*0404 (MacGregor et al., 1995) In Japanese,

Taiwanese, Koreans and Polynesians, the DR4 association was with DRB1*0405

(Wakitani et al., 1997; 1998; Yen et al., 1995; Kim et al., 1997; Lee et al., 2004; Helen et al., 1986)

The contribution of the HLA genes represents about 40% of the total genetic

background in RA (Deighton et al., 1989; Wordsworth et al., 1989) Therefore,

identification of other genetic markers including cytokine genes-tumour necrosis

factor-α (TNF-α), interleukin-α (IL-1α), interleukin-β (IL-1β) and IL-1 receptor

antagonist (IL-Ra) is an important challenge (Cantagrel et al., 1999) Cytokines with

polymorphic gene sequences are potential markers of disease severity since their gene products are involved in the pathogenesis of RA Differences in severity between individuals could be related to different levels of cytokine production or functional differences resulting from polymorphisms in their genes

Use of anti-cytokine agents in the clinical settings to counteract the inflammatory processes, by acting upon IL-1 and TNF-α further explains their roles in the

rheumatoid disease process (Feldmann et al., 1996) The TNF locus has been mapped

to the short arm of chromosome 6 It lies in the class III region of the MHC, about 250

kb centromeric of the HLA-B locus and850 kb telomeric of HLA-DR In view of its biological effectsand gene location it has been speculated that polymorphisms withinthis locus might contribute to MHC associations with autoimmune and infectious

diseases (Jacob et al., 1992) This is particularly those in which TNF-α has been implicated in the initiations or perpetration of the inflammatoryresponse, such as in rheumatoid arthritis Increased production of TNF-α has been demonstrated in individuals with HLA-DRB1*04 alleles, as compared to those with HLA-DRB1*02 allele, suggesting that TNF-α production is under genetic control and is linked to the

MHC (Jacob et al., 1990; Abraham et al., 1993)

Various methods have been used to type the polymorphisms in the HLA-class II loci

These methods range from traditional serology (Stasny et al., 1978) to DNA-based

methods such as polymerase chain reaction (PCR)-restriction fragment length

polymorphism (RFLP) (Ota et al., 1992), PCR sequence-specific oligonucleotides

(SSO) (Wordsworth et al., 1990) and PCR sequence-specific primers (SSP) (Zetterquist et al., 1992) Recently, various HLA-DRB1 sequencing based typing

(SBT) methods have been developed to further increase the accuracy of the HLA genotyping and facilitate the identification of novel HLA-DRB1 polymorphisms

(Sayer et al., 2001) Our laboratory has standardized the PCR-SSP and PCR-RFLP for

the high resolution genotyping of the HLA-DRB1 Thus PCR-RFLP a technically simple, practical and inexpensive method for accurate determination of the HLA-DRB1 alleles can be used for routine HLA typing work

We genotyped the HLA-DRB1*04 polymorphisms and cytokine genes polymorphisms of TNF-α, IL-1α, IL-1β and IL-Ra and analyse them as potential markers of disease susceptibility and severity in local of Chinese RA patients

2.2 MATERIALS AND METHOD

2.2.1 Subjects recruitment and sample collection

A total of sixty-four adult Chinese patients with RA, followed-up at the rheumatology clinic, National University Hospital, Singapore were recruited for the study Informed consent was obtained from the patients recruited for the study There were fifty-five females and nine males All patients satisfied the American College of Rheumatology

(ACR) 1987 revised criteria for classification of RA (Arnett et al., 1988) Ninety

unrelated healthy individuals were recruited as controls for the study Clinical data and laboratory investigation results were obtained through chart review

Peripheral venous blood samples were collected during routine venepuncture Data collected include:

1 Co-morbid conditions,

2 Disease onset, American College of Rheumatology (ACR) criteria, mode of onset, pattern of onset,

3 Disease duration,

4 Disease activity according to DAS 28 scoring,

5 Joint erosions and deformities, and Extra-articular manifestations,

6 Therapeutic data,

7 Surgical intervention data,

The HLA Class II DR genetic typing was performed on genomic DNA using polymerase chain reaction restriction fragment length polymorphisms (PCR-RFLP)

method Rheumatoid factors (IgM) in the serum were quantitated using commercially available ELISA kits

2.2.2 Genomic DNA extraction (salting out method)

Ten milliliters of peripheral venous whole blood was obtained under aseptic technique into sodium citrated (Na-Citrate) tubes and the genomic DNA was extracted using a

modified overnight salting-out method (Miller et al., 1988,) Briefly, the

anti-coagulated blood was centrifuged for 15 min at 2600 rpm and the buffy coat layer (about 1ml) transferred into a new 15ml centrifuge-tube (This was done by removing the plasma to a waste container and then carefully pipetting off the white cells that form a layer on top of the red cells.) Red cell lysis buffer (0.144 M NH4Cl, 10mM NaHCO3) was added and the tube left at room temperature for 5 min This was then centrifuged again for 15 min at 2600 rpm and the white cell pellet resuspended in the

3 ml nuclei lysis buffer (10mM Tris-HCL pH 8.2, 0.4 M NaCl and 2 mM Na2EDTA

pH 8.0) The cell lysates were incubated overnight at 550C with 100 μl proteinase K (1 mg proteinase K in 1% SDS solution and 2mM Na2EDTA) and 100μl 20% sodiumdodecyl sulphate solution The following day, 1ml of 5M NaCl was added and the mixture shaken vigorously (15 sec) before centrifuging for 25 min at 2800 rpm The supernatant containing DNA was then transferred into a clean 15 ml tube and exactly 2 volumes of absolute ethanol were added with gentle inversion The precipitated genomic DNA strands were removed and transferred to a 1.5ml microcentrifuge tube containing 100 μl TE buffer (10mM Tris-EDTA, 0.2mM

Na2EDTA pH 7.5) The purity and concentration of DNA was determined by spectrophotometer (1 O.D at 260nm = 50μg/ml) A ratio of 1.8 to 2.0 (the O.D value