Riikka Nissinen

IMMUNOLOGICAL FEATURES OF CHRONIC ACTIVE RHEUMATOID ARTHRITIS

CHEMOKINE RECEPTOR AND CYTOKINE PROFILE IN SYNOVIAL FLUID, PERIPHERAL BLOOD AND

GUT IN PATIENTS WITH RHEUMATOID ARTHRITIS

Immunological features of chronic active rheumatoid arthritis.Chemokine receptor and cytokine profile in synovial fluid, peripheral blood

and gut in patients with rheumatoid arthritis.

Riikka Nissinen

Department of Molecular MedicineNational Public Health Institute, Helsinki

andDepartment of BiosciencesDivision of Biochemistry

Faculty of ScienceUniversity of Helsinki

Finland

Academic dissertation

To be publicly discussed, with the permission of the Faculty of Science in the University of Helsinki, in the Niilo Hallman Auditorium of the Hospital for Children and Adolescents,

on December 11th 2003, at 12 o’clock.

Helsinki 2003

National Public Health Institute

Mannerheimintie 166

00300 Helsinki, Finland

Phone +358-9-47441

Publications of the National Public Health Institute, KTL A22 / 2003

ISBN 951-740-398-4 (printed version) ISSN 0359-3584 (printed version)

ISBN 951-740-399-2 (pdf version) ISSN 1458-6290 (pdf version)

Copyright National Public Health Institute

Yliopistopaino

Supervised by:

Professor Outi Vaarala

Laboratory of Immunological Diseases Department of Molecular Medicine National Public Health InstituteHelsinki, Finland

Professor Marjatta Leirisalo-Repo

Division of RheumatologyDepartment of MedicineHelsinki University Central HospitalHelsinki Finland

Professor Timo Palosuo

Laboratory of ImmunobiologyDepartment of Health and Functional CapacityNational Public Health InstituteHelsinki, Finland

Reviewed by:

Docent Kaisa Granfors

Department of Human Microbial Ecology and Inflammation National Public Health Institute Turku, Finland

Opponent:

Professor Ronald F van Vollenhoven

Rheumatology Unit, Karolinska Hospital and Karolinska InstituteStockholm, Sweden

Professor Jukka Pelkonen

Division of Clinical MicrobiologyUniversity of Kuopio Kuopio, Finland

Kansanterveyslaitos (KTL)

Mannerheimintie 166

00300 Helsinki, Finland

puh. 09-47441

Folkhälsoinstitutet

Mannerheimvägen 166

00300 Helsingfors

tel. 09-47441

JULKAISIJA-UTGIVARE-PUBLISHER

To my family

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CONTENTS

ABSTRACT ......................................................................................8

ORIGINAL PUBLICATIONS ...................................................... 10

ABBREVIATIONS ........................................................................ 11

INTRODUCTION ........................................................................ 12

REVIEW OF LITERATURE ...................................................... 131.1 Genetic factors ........................................................................................................131.2 Environmental factors ...........................................................................................14

2. Clinical manifestations of RA ..............................................................152.1 Joints ........................................................................................................................152.2 Systemic manifestations.........................................................................................17

3. Pathogenesis of RA ..............................................................................183.1 Macrophages ..........................................................................................................183.1.1 Macrophages in RA.............................................................................................203.2 T-cells........................................................................................................................203.2.1 T-cells in RA.........................................................................................................223.3 B-cells ......................................................................................................................233.3.1 B-cells in RA .......................................................................................................23

4. Mediators of inflammation ..................................................................264.1 Cytokines..................................................................................................................274.1.1 Pro-inflammatory cytokines...............................................................................294.1.2 Anti-inflammatory cytokines .............................................................................334.1.3 Regulatory cytokines ...........................................................................................344.2 Chemokines and chemokine receptors ..............................................................35

5. Gut immune system...............................................................................405.1 Gut associated immune activation in RA ..........................................................42

6. TNF-α blockade as a biological treatment for RA............................42

AIMS OF THE STUDY .................................................................45

PATIENTS AND METHODS ......................................................461. Patients .....................................................................................................462. Methods ...................................................................................................48

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RESULTS AND DISCUSSION.....................................................491. Autoantibodies in patients with chronic active RA (Publications II and IV) .........................................................................492. Expression of chemokine receptors and cytokines in SF and PB of patients with chronic active RA (Publications I and II) .............513. Expression of chemokine receptors and cytokines in the gut of patients with chronic active RA (Publication III) .............................564. Expression of chemokine receptors and cytokines in patients with chronic active RA during anti-TNF-α treatment (Publication II) ..595. Expression of chemokine receptors and cytokines, and the number of T-cells in patients with chronic active RA responding and not responding to anti-TNF-α treatment (Publication II)...............63

GENERAL DISCUSSION AND FUTURE ASPECTS ................65

SUMMARY AND CONCLUSIONS..............................................68

REFERENCES ..............................................................................70

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ABSTRACT

Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown aetiology characterized by chronic inflammation of joints. RA cannot be cured by currently available therapies and has substantial personal, social and economic costs. The prevalence of RA in Finland is about 1%.

In patients with RA the synovial tissue and fluid is invaded by inflammatory cells. These cells, called leukocytes, and their products, play essential roles in the chronic inflammatory process. Leukocytes produce cytokines and chemokines which orchestrate the inflammatory events that eventually cause synovitis and destruction of cartilage and bone.

In this study we examined the functional differences of leukocytes in synovial fluid (SF), in peripheral blood (PB) and in gut biopsies of patients with RA, in patients with other arthritides, such as chronic reactive arthritis, spondylarthropathy, ankylosing spondylitis, acute gout, psoriatic arthritis and juvenile idiopathic arthritis, and in control subjects. We also studied the effect of anti- tumor necrosis factor-α (TNF-α) treatment on the leukocytes of patients with arthritides.

Our results demonstrated that T-cells and monocytes infiltrating the joints of patients with RA and other arthritides show an increased activation of both Type 1 and Type 2 immune markers. A more pronounced Type 1 immune response in joints, as demonstrated by an increased chemokine receptor CCR5/CCR3 ratio, was found in patients with RA than in patients with other arthritides. Monocytes but not T-cells in PB showed an increased activation in patients with RA.

After treatment with anti-TNF-α antibody an increase was seen in the secretion of Type 1 and Type 2 cytokines and in the expression of chemokine receptors on T-cells and monocytes both in patients with RA and in patients with other arthritides. Because T-cells are shown to be hyporesponsive in patients with RA, these observations suggest a restoration of peripheral cell-mediated immunity associated functions and/or a blockade of the accumulation of inflammatory cells into joints as a response to TNF-α blockade.

In the intestinal biopsy samples from RA patients, increased mRNA expression of IL-10, CCR5 and CCR4 was detected. This suggests that patients with RA have activation of gut-associated immune cells.

Finally, the arginine-citrulline converting enzyme peptidylarginine deiminase (PAD) was recognized as a new antigen against which patients with inflammatory rheumatic diseases frequently show immunoglobulin (Ig) G class antibodies.

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In conclusion, the present study demonstrates that chronic inflammation in patients with RA, characterized by infiltration of T-cells and monocytes expressing Type 1 and Type 2 chemokine receptors and cytokines, is rebalanced by the treatment with infliximab, an anti-TNF-α monoclonal antibody. The hyporesponsive state of T-cells was shown to normalize as a consequence of removal of excess TNF-α and, interestingly, patients not responding to the treatment were shown to have higher chemokine receptor levels before treatment with infliximab. The study also showed that the gut immune system is activated in patients with RA and suggests that this activation might contribute to the pathogenesis of the disease.

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ORIGINAL PUBLICATIONS

This thesis is based on the following articles, which are referred to in the text by their Roman numerals.

I Nissinen R, Leirisalo-Repo M, Tiittanen M, Julkunen H, Hirvonen H, Palosuo T and Vaarala O. CCR3, CCR5, IL-4 and IFN-γ expression on synovial and peripheral T-cells and monocytes in patients with rheumatoid arthritis. The Journal of Rheumatology 2003, 30(9): 1928-34.

II Nissinen R, Leirisalo-Repo M, Peltomaa R, Palosuo T, and Vaarala O. Cytokine and chemokine receptor profile of peripheral blood mononuclear cells during treatment with infliximab in patients with active rheumatoid arthritis. In press in Annals of the Rheumatic Diseases.

III Nissinen R, Leirisalo-Repo M, Nieminen AM, Halme L, Färkkilä M, Palosuo T and Vaarala O. Immune activation in the small intestine in patients with rheumatoid arthritis. In press in Annals of the Rheumatic Diseases.

IV Nissinen R, Paimela L, Julkunen H, Tienari PJ, Leirisalo-Repo M, Palosuo T and Vaarala O. Peptidylarginine deiminase, the arginine to citrulline converting enzyme, as an autoantigen in patients with rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren syndrome. In press in Scandinavian Journal of Rheumatology. Scandinavian Journal of Rheumatology 2003, 32.

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ABBREVIATIONS

aCCP anti-cyclic citrullinated peptide AFA anti-filaggrin antibodyAIDS acquired immunodeficiency syndromeCCR chemokine receptor, CCCCR5∆32 mutated CCR5 gene lacking 32 basepair sequenceCIA collagen-induced arthritisCRP C-reactive proteinCXCR chemokine receptor, CXCELISA enzyme linked immunosorbent assayGM-CSF granulocyte macrophage colony stimulating factor HIV human immunodeficiency virusHLA human leukocyte antigenICAM-1 intracellular adhesion molecule-1IFN interferonIg immunoglobulinIL interleukinIL-1R interleukin-1 receptorIL-1ra interleukin-1 receptor antagonistIL-2R interleukin-2 receptorIP-10 interferon-γ inducible protein MCP chemokine, monocyte chemoattractant proteinMDC chemokine, macrophage derived chemokine MHC major histocompatibility complexMIP chemokine, macrophage inflammatory proteinMMP matrix metalloprotein MS multiple sclerosisNK natural killer cellPAD peptidylarginine deiminasePB peripheral bloodPBMC peripheral blood mononuclear cellsPGE2 prostaglandin E2RA rheumatoid arthritisRANTES chemokine, regulated upon activation T-cell expressed and secretedRF rheumatoid factorSF synovial fluidTARC chemokine, thymus and activation regulated chemokine TCR T-cell receptorTGF tumor growth factorTNF tumor necrosis factorTNF-R tumor necrosis factor-α receptorVCAM-1 vascular cell adhesion molecule-1

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INTRODUCTION

Rheumatoid arthritis (RA) is a systemic autoimmune disease of unknown aetiology. The disease is characterised by articular inflammation and by the formation of an inflammatory and invasive tissue, rheumatoid pannus that eventually leads to the destruction of joints. The genetic background predisposes to the disease, but also person-related and environmental factors, such as age, gender, infectious agents, smoking and dietary factors are thought to play a role in the disease pathogenesis.

The prevalence of RA globally is around 1 %. However, the occurrence of the disease varies among populations: RA is rare in less developed rural parts of the world and more common in the industrialized countries. A fall in the incidence, especially in women, and a rise in the peak age of onset of RA has been observed over the last few decades. Possible explanations for these phenomena are an increased use of oral contraceptive pill among women and higher life expectancy.

The most characteristic immunological aberration of RA and one classification criterion for the disease is the presence of rheumatoid factor (RF) in the serum of RA patients. Current therapy for RA is directed primarily toward diminishing inflammation present in joints rather than to prevent or completely arrest the progression of the disease. In patients with RA activated T- and B-cells as well as tissue macrophages and dentritic cells migrate to synovial tissue. Polymorphonuclear cells accumulate in the synovial fluid (SF) and on cartilage surfaces. Persistent joint inflammation leads to articular pain, swelling, cartilage erosion and eventually to joint deformity. In many cases organ systems other than joints also become inflamed.

In the present study the features of cell mediated and humoral immunity were investigated in the PB, SF and in the gut of patients with RA. Furthermore, the effect of a novel biological treatment that blocks tumor necrosis factor (TNF)-α with a monoclonal antibody (infliximab) was also studied in patients with RA.

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REVIEW OF LITERATURE

1. Epidemiology

RA is currently described to occur worldwide in about 1 % of the adult popu-lation. However, the occurrence of the disease is not the same throughout the world: in North American Indians the prevalence of RA is five times as high as in the Caucasian population and in Asian and African populations the prevalence is lower than in Caucasians. The prevalence of RA in the Finnish population is 0.8 %. (Figure 1) (reviewed in Silman et al., 2002).

Over the last few decades a fall in the incidence and severity of RA has been reported (Doran et al., 2002, Kaipiainen-Seppanen et al., 1996). Also, the peak age of onset of the disease has risen in recent years. In Finland, the incidence of RA diminished during the 1980s by about 15 % and the mean onset of the disease rose by five years to around 55-60 years (Kaipiainen-Seppanen et al, 1996).

Figure 1. Prevalence of RA (modified from Silman et al, 2002).

1.1 Genetic factors

Human leukocyte antigen (HLA) -genes located within the major histocompat-ibility complex (MHC) on chromosome 6p have been found to have a strong association with RA (Stastny, 1978). Individuals carrying HLA-DR4 and HLA-DR1 alleles in particular have been shown to possess a higher risk of the disease.

0

1

2

3

4

5

6

Niger

ia

SouthAfri

ca, r

ural

Pakist

an

HongKong

Greec

e

Japan

China

Brazil

Finlan

d

Sweden

USA

England

Jam

aica

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ndians,

USA

Yakim

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s, USA

Pre

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nce

(%

)

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It has been suggested that the susceptibility alleles all share a single epitope that is responsible for the predisposition of homozygote individuals to RA. It has also been suggested that the relationship of HLA and RA may be related more to the severity of the disease, rather than to the tendency to acquire RA (Thomson et al., 1999).

In the short arm of chromosome 6 resides several genes that have an influ-ence on the immune defence. Although the data is inconsistent, some studies have shown associations between TNF alleles, such as TNF-c1 and TNF-b3, and RA (Hajeer et al., 2000). Other genes also linked to RA include those that code for corticotrophin releasing hormone, oestrogen synthase, interferon (IFN)-γ and interleukin (IL)-10 (reviewed in Silman et al, 2002). The overall magnitude of the genetic component is, however, only modest (Jarvinen et al., 1994).

1.2 Environmental factors

Since the concordance of RA in identical twins is not 100% other non-genetic factors also play a role in the disease aetiology (reviewed in Silman et al, 2002).

Throughout the world RA is more common in women than it is in men. Observations, such as the levels of male sex hormones, especially testosterone, are reported to be lower in men with RA, or that women who take the oral con-traceptive pill are at a reduced risk of developing RA, suggest that hormonal factors may play a role in the development of the disease. Pregnancy has also been considered as a risk factor for RA, but the studies show that the onset of RA is rare during pregnancy, yet increases after delivery (Brennan et al., 1994).

Smoking is associated with increased production of RF, especially in men, and an increased risk of developing RA (Tuomi et al., 1990, Heliovaara et al., 1993). On the contrary, populations consuming a diet high in omega-3 fatty acids have been reported to be protected from RA (Ariza-Ariza et al., 1998).

A large number of infectious agents such as viruses and bacteria have also been suggested to trigger RA. However, no relationship between infectious agents and the development of RA has been found.

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2. Clinical manifestations of RA

The onset of RA can be either insidious and slow or acute and abrupt. In either case, usual symptoms are symmetric joint pain and swelling, generalized fatigue and morning stiffness. In most patients RA proceeds into a chronic disease, which progresses perpetually. Articular symptoms of RA are characterised by symmetric inflammatory polyarthritis affecting primarily the small joints in the beginning of the disease. The most frequently affected joints are those of fingers and toes, wrists, knees and ankles. Extra-articular manifestations may occur and are frequently present in patients with severe disease and include ocular, pulmonar, hematologic, vascular, cardiac, neurologic and mucosal tissues.

2.1 Joints

The joint space, consisting of SF, cartilage and bone, is surrounded by a capsule. The joint capsule is further composed of outer collagen consisting of synovial sublining and inner synovial membrane or synovium (Figure 2).

In the normal joint the synovial membrane surrounds the articular spaces and tendon sheats, leaving the cartilage unattached, and is covered with a lining cell layer consisting of synoviocytes with the thickness of one to two cells. The lining layer cells are categorized as type A and type B synoviocytes. Type A cells comprise 25% of the synovial lining cells and express CD14, CD68 and type I complement receptor, and thus are similar to tissue macrophages. Type B cells are fibroblast-like synoviocytes in structure and produce glycosaminoglycans, such as hyaluronan, found in synovial tissue and SF.

At the microscopic level, below the lining layer cells lies the stromal layer, which in normal synovium is quite acellular. However some fibroblasts, mast cells, dentritic cells, and tissue macrophages interspersed with venules have been found. The extracellular matrix of the superficial synovial stroma is composed of hyaluronan, chondroitin-6-sulfate proteoglycan and collagen. The abundance of collagen increases and that of hyaluronan decreases in the deeper synovial stroma (Figure 3a).

Figure 2. Diagrammatic picture of the syno-

vial joint; normal joint (left) and rheumatoid arthritis (right) (modified from Feldmann et al., 1996).

inflamedsynovialmembrane

pannus

synovial fluid:neutrophils,macrophages,T-cells

capsule

synovialmembrane

cartilage

T-cells,macro-phages

Normal RA

16 17

In patients with RA the inflamed synovial membrane expands on the surface of the cartilage leading to cartilage degradation, bony erosions and destruction of ligaments and tendons (Figure 2). The continuous inflammation thickens the synovial membrane lining layer, consisting of activated macrophages (type A synoviocytes) and fibroblast-like synoviocytes (type B synoviocytes) (Figure 3b). In the sublining stroma, increased neovascularization facilitates the mas-sive influx of leukocytes. The most abundant cells in the RA synovial sublining are macrophages and helper T-cells, but also cytotoxic T-cells, B-cells, dentritic cells and fibroblasts are found. Polymorphonuclear cells are rare in the synovial membrane, but are abundant in the SF of patients with RA. While in the RA synovial membrane the number of helper T-cells predominate over cytotoxic T-cells, in RA SF they are equally represented (Forre et al., 1982) (Figure 3b).

Figure 3. Schematic illustration of normal synovium and RA

synovium. (a) Structure of normal synovium characterized by a thin synovial lining layer, sparse cellularity of the synovial tissue stroma and rare inflammatory cells in the synovial fluid. (b) Histologic changes in the synovium associated with RA. The synovial lining layer thickens, vascular proliferation occurs in the synovial tissue stroma, marked by formation of high endothelial venules. Inflammatory cell migration into RA synovium then forms a perivascular infiltrate of lymphocytes and macrophages, germinal center formation and neutrophil accumulation in synovial fluid (modified from Sundy et al., 1998).

Normal synovium

Tissue macrophage

Lymphocyte

Vessel

SynovialMembrane

Synovial Lining Layer

Synovial FluidType B synoviocyteType A synoviocyte

RA synovium

High Endothelial

Venule

Germinal Center

Neutrophil

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2.2 Systemic manifestations

Although RA is characterized by chronic synovial inflammation, the disease process is not restricted entirely to the joints. Extra-articular manifestations occur, particularly in RF-positive disease, noteably in males and in the elderly (reviewed in O’Gradaigh et al., 2001).

Rheumatoid nodules are found in at least 25 % of patients with RA (Ziff, 1990). The nodules are lymphoid aggregates that are created in the subdermal tissue, usually in RF- positive patients.

Systemic rheumatoid vasculitis is also common in patients with severe RF-positive RA. The probable cause of systemic rheumatoid vasculitis is the aggre-gation of immune complexes in the endothelium of blood vessels. Patients with RA also have an increased risk of death from coronary artery disease without an association of systemic rheumatoid vasculitis. However, clinical heart disease is rare in patients with RA.

The most common manifestations affecting the eye are keratoconjunctivitis sicca and secondary Sjögren’s syndrome, which occur in 17 % of RA patients.

Pulmonary involvement includes pleuritis and pleural effusions, nodules, interstitial involvement and airway disease. Pulmonary diseases are one of the leading causes of death in patients with RA.

Peripheral neuropathies can be the result of the pressure caused by synovitis or scar tissue on neurons, by medication or by vasculitis.

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3. Pathogenesis of RA

The immune system consists of leukocytes (white blood cells) that are derived from a common pluripotent hematopoietic stem cell in the bone marrow. These pluripotent cells divide further into two more specialized stem cell types: the common myeloid progenitor and the common lymphoid progenitor. The former gives rise to granulocytes, including neutrophils, basophils, eosinophils, monocytes, macrophages, mast cells and dentritic cells, and the latter to B-cells, T-cells and natural killer (NK)-cells, commonly known as lymphocytes. Granulocytes are involved in innate immunity, in which the features that are common to many pathogens are recognized by germ-line receptors. The identifica-tion of a pathogen leads to its immediate destruction and removal from the tissue. Furthermore, granulocytes act as antigen presenting cells for cells involved in adaptive immunity mediated by lymphocytes. B-cells recognize antigens with their immunoglobulin surface receptor, whereas T-cells recognize antigen peptides on antigen presenting cells in association with MHC-protein with their T-cell recep-tor (TCR). When foreign antigen is recognized by an immunoglobulin receptor on a B-cell or as a peptide-MHC complex by TCR on a T-cell, the lymphocyte will clonally expand and differentiate into effector cells that further stimulate the immune system. While T-cells identify intracellular or ingested pathogens presented by antigen presenting cells, B-cells recognize pathogens, such as bacteria, residing in the extracellular fluid.

3.1 Macrophages

Monocytes are derived from myeloid progenitor cells in the bone marrow. Macrophages are usually the first immune cells to encounter pathogens, and act primarily as phagocytic cells that engulf microorganisms to destroy them in intracellular vesicles. Macrophages recognize pathogens with receptors that display conserved structures on microorganisms, present antigens to T-cells and also activate them.

Monocytes are circulating precursors of tissue macrophages. They lack phagocytic capacity and are considered inert while in the blood compartment. When recruited they rapidly cross the endothelium, migrate to the site of inflammation and mature to macrophages. Naïve macrophages develop into different phenotypic forms depending on the environmental factors present in the tissue. Macrophages can be activated through the classical pathway or through the alternative pathway (Duffield, 2003) (Figure 4). The classical pathway of differentiation can be triggered by bacterial lipoproteins, bacterial DNA, parasitic proteins or carbohydrates, opsonized particles, hypoxia, abnormal matrix or IFN-γ acting in concert with pro-inflammatory cytokines. IFN-γ enhances the response of activated macrophages by promoting the release of cytokines and toxic compounds, such as free oxygen radicals and nitric oxide. Classically activated macrophages also show antimicrobial activity, phagocytosis

18 19

of opsonized particles and immune complexes and are involved in matrix degradation. Also, the amount of pro-inflammatory cytokines and chemokines produced by activated macrophages is upregulated.

Macrophages activated through the alternative pathway have been exposed to IL-4, IL-10, IL-13, tumor growth factor (TGF)-β or glucocorticoids. These macrophages produce anti-inflammatory cytokines and are resistant to re-activa-tion. Alternatively activated macrophages are also involved in matrix synthesis and stabilization, enhacement of cell survival and proliferation, angiogenesis and antigen presentation. Macrophages activated by either pathway induce the phagocytosis of debris and apoptotic cells (Opferman et al., 2003).

Figure 4. The function of classically and alternatively activated mac-

rophages. Monocytes can differentiate into classically or alternatively activated macrophages. Their respective and overlapping functions are indicated in the boxes (modified from Duffield, 2003).

Classically activatedmacrophage

Alternatively activatedmacrophage

Monocyte

•Generation of anti-inflammatorycytokines and chemokines

•Matrix synthesis and stabilization•Inducing cell survival and proliferation•Angiogenesis•Presentation of antigen

•Anti-microbial activity•Phagocytosis of opsonized particles•Matrix degradation•Phagocytosis of immune complexes•Generation of pro-inflammatory

cytokines and chemokines

•Phagocytosis of debris•Phagocytosis of apoptotic cells

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3.1.1 Macrophages in RA

Macrophages play a central role in the inflammation of the synovial membrane and the cartilage-pannus junction in patients with RA. The abundance and activa-tion of macrophages at the inflamed synovial membrane correlates significantly with the severity of the disease (Mulherin et al., 1996). Activated macrophages overexpress MHC class II molecules and produce pro-inflammatory or regulatory cytokines and growth factors (IL-1, IL-6, IL-10, IL-13, IL-15, IL-18, TNF-α and granulocyte macrophage colony stimulating factor (GM-CSF)), chemokines (IL-8, macrophage inflammatory protein (MIP)-1, monocyte chemoattractant protein (MCP)-1), metalloproteinases and neopterin, that are detected in the inflamed joint (Kinne et al., 2000). The activation of the monocyte-lineage cells is not restricted to synovial macrophages as it extends also to circulating monocytes and polymorphonuclear cells (Kinne et al, 2000).

3.2 T-cells

T-cells develop from bone marrow derived common lymphoid progenitors. The progenitor cells migrate to the thymus where the actual maturation of T-cells occurs. In the thymus, the T-cell precursor rearranges its T-cell receptor genes and goes through a selection process in which survival of the developing T-cell depends on the relative affinity of its receptor towards self-proteins. T-cells bearing receptors, that have a strong affinity towards the body’s own proteins, are removed from the repertoire in a process called negative selection. T-cells that do not show self-reactivity transmit a survival signal that leads to positive selection of these T-cells. At the time of positive selection, thymocytes express both CD4 and CD8 co-receptor molecules and are referred to as double positive cells. At the end of the selection process, mature thymocytes express only one of these co-receptors thus having matured into either helper (CD4) or cytotoxic (CD8) T-cells. As T-cells leave the thymus they circulate in the blood and tissues as naïve T-cells. When encountering an antigen in tissue, T-cells become activated and further differentiate into one of the subtypes of effector T-cells according to the local cytokine environment (Figure 5).

T-cells are divided into Type 1, Type 2 and Type 3 effector cells. Type 1 T-cells are characterized by the expression of chemokine receptors CCR5 and CXCR3 and by the secretion of IFN-γ and IL-2. These cells help cytotoxic T-cells in the clearance of intracellular pathogens, they activate macrophages and are considered to be effector cells in several cell-mediated autoimmune diseases. Type 2 T-cells express CCR3, CCR4 and CCR8 and secrete IL-4, IL-5, IL-6, IL-10 and IL-13 cytokines. Type 2 cells activate B-cells to produce antibodies, which bind to and help destroy extracellular pathogens. Type 2 T-cells also have a central role in allergic reactions (Figure 5). Type 3 CD4 T-cells originate from the mucosa, are activated by mucosal antigens and secrete TGF-β. These cells provide assistance for IgA production and can enhance their own differentiation by secreting

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TGF-β. The chemokine receptor expression of type 3 T-cells is not known. Type 2 and 3 cytokines play anti-inflammatory roles in suppressing type 1 responses. An additional type of regulatory T-cell, which is driven by IL-10 and secretes both IL-10 and TGF-β, has been termed as Tr1 cell (Groux et al., 1997).

Autoimmune diseases are believed to arise when cells of specific tissues become targets of T-lymphocytes and/ or autoantibodies. T-cells are important in promoting and initiating the tissue damage seen in these disorders. The mecha-nisms by which T-cells cause the destruction of tissues include direct damage through cell-mediated cytotoxicity processes or indirect damage mediated by non-lymphocyte mechanisms.

Figure 5. Differentiation of a T helper cell into Type 1 and Type 2 cells.

T-cells migrate to the thymus from the bone marrow and express a double positive phenotype before entering the blood circulation as naive T-cells. Environmental factors determine which type of T-cell the circulating naïve T-cell will differentiate into. Chemokine receptor (CCR or CXCR) and cy-tokine profile and the type of immune response characteristic of each cell type is indicated on the right.

Cell-mediated cytotoxicity involves mechanisms such as perforin- and Fas-mediated lysis of the cell. In perforin mediated lysis, the target cell is killed by cytotoxic T-cells that secrete perforin directly into the target cell. The released perforin creates pores in the plasma membrane of the target cell and causes an osmotic imbalance between the cell and its surroundings. The solutes that flow

cellmediated

CD4

CD4

CD8

Environmental factors

Doublepositive

TCR

CD4

CD3

Naive Th

TCR

CD3

Type 1 ThIL-2

IFN-�

CD4

IL-4IL-5IL-6

IL-10IL-13

CCR5CXCR3

CCR3

Type 2 Th

CCR4 CCR8

Thymus

CD3TCR

antibodymediated

22 23

into the cell across the cell membrane eventually result in bursting of the target cell. Another central mechanism in cell death is apoptosis or programmed cell death (reviewed in Opferman et al, 2003). In this process, the Fas-ligand, present in the membranes of activated cytotoxic and helper T-cells, binds to the Fas-protein in the target cell membrane, which leads to activation of caspases that induce apoptosis of the target cell.

T-cells can also kill by secreting cytokines that can ligate pro-apoptotic receptors on the target cells. Furthermore, autoreactive T-cells can indirectly kill their targets by enchancing their susceptibility to death effector mechanisms mediated by other leukocytes, by promoting the recruitment of inflammatory cells to the target tissue or by causing the autoreactive B-cells to differentiate into autoanti-body secreting plasma cells.

3.2.1 T-cells in RA

The role of T-cells in the pathogenesis of RA is still not completely understood. In favour of the assumption that T-cells play a role in RA is the association of disease susceptibility and outcome with HLA-DR antigens (van Zeben et al., 1991). Also, a high number of CD4 T-cells has been found in the synovial mem-branes of patients with RA (Iannone et al., 1994). Further evidence in favour of T-cells in the pathogenesis of RA has been described from animal models in which a single T-cell clone was shown to transfer the disease (Klareskog et al., 1983, Taurog et al., 1983). In addition, the depletion of T-cells in non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mice, that are engrafted with synovial tissue isolated from patients with active disease, show a decreased production of macrophage derived cytokines (IL-1β, TNF-α and IL-15). This suggests that the decrease in cytokine production is due to the disappearance of synovial CD68+ macrophages, which depend on T-cells for their survival (Weyand et al., 2000). Also the beneficial effects of CD4 blocking agent on disease symptoms in patients further support the role of T-cells in RA (Choy et al., 2002).

However, since the most striking feature of RA T-cells is hyporesponsiveness, the primacy of T-cells in early and chronic RA pathogenesis has been questioned(Smeets et al., 1998). The hyporesponsiveness of RA T-cells is reflected by a reduced response to mitogenic stimulation and decreased calcium-influxes (Kingsley et al., 1987, Allen et al., 1995b), defective TCR mediated signalling (Maurice et al., 1997), lowered T-cell proliferation rate (Cush et al., 1991) and reduced expression of T-cell derived cytokines, such as IFN-γ and IL-2 (Firestein et al., 1988, Dolhain et al., 1996a).

Most of the T-cells infiltrating the rheumatoid synovium express CD45RO and CD4, indicating that they are of helper and of memory subset T-cells.

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Instead only ten to fifteen percent of T-cells in the RA synovium contain granzyme A and perforin, the molecules specific for cytotoxic T-cells. This suggests that T-cells expressing CD8 are infrequent in the RA synovium, whereas in RA SF CD4 and CD8 T-cells are equally represented (Forre et al, 1982). TCR α/β is expressed on most of the T-cells in RA synovium and only a minority show TCR γ/δ expression, although it has been found that expression of TCR γ/δ is increased in the synovium and PB of patients with active RA (Jacobs et al., 1992).

3.3 B-cells

B-cells develop from the same bone marrow derived common lymphoid progenitors as T-cells and mature in the bone marrow before entering the circulation.

In the first phase of development, progenitor B-cells in the bone marrow rearrange their immunoglobulin genes. This stage is independent of antigen, but dependent on the interaction with stromal cells. Stromal cells contribute to B-cell development in two ways: by providing specific adhesive contacts through cell-adhesion molecules to the developing B-cells and by providing growth factors that stimulate B-cell differentiation and proliferation. When the rearrangement of Ig genes is complete an immature B-cell carrying an IgM antigen receptor is formed. B-cells can now interact with antigens in their surroundings, and at this stage cells that are strongly stimulated by self-antigen either die or are inactivated in a process of negative selection. The surviving immature B-cells enter the cir-culation where they mature to express IgD along with IgM. B-cells are activated with their specific antigen in a secondary lymphoid organ, after which they proliferate and differentiate into antibody-secreting plasma cells and part of them further differentiate into long-lived memory cells.

Besides acting as antibody producing cells, B-cells that secrete lymphotoxin drive the maturation of follicular dentritic cells and the organization of B-cell follicles (Gonzalez et al., 1998). B-cells also play a role in the development of T-cells and mucosal M-cells (Golovkina et al., 1999, Flynn et al., 1998). B-cells themselves can differentiate into polarized cytokine-producing effector cells that can influence the differentiation of CD4+ T-cells into Th1 or Th2 subsets (Harris et al., 2000). Furthermore, IL-10 positive B-cells with immunoregulatory functions have been recently identified (Fillatreau et al., 2002).

3.3.1 B-cells in RA

RA was originally considered as an antibody-driven disease (Vaughan, 1973, Johnson et al., 1973), but the precise role of B-cells in RA is not well understood. Nevertheless, the role of B-cells appears to be significant; B-cells act as potential links between the cells of adaptive and innate immune systems, and direct the cellular components, such as cytokines and chemokines, in inflammation.

The indicators proposing an enchanced B-cell activation in patients with RA include formation of T-cell and B-cell aggregates in the synovium, expression of

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co-stimulatory molecules, such as CD40 ligand, enhanced production of autocrine cytokines for B-cells (IL-6 and IL-10) and abnormalities in the negative selection of B-cells in RA (Schroder et al., 1996, Weyand et al., 2001).

In PB, an overall high activation of B-cells and enchanced frequency of memory B-cells has been reported in patients with RA (Lindenau et al., 2003, Bohnhorst et al., 2001). In the synovium a reduced proliferative capacity and an enhanced receptor revision of the B-cell receptor have been found to occur (Reparon-Schuijt et al., 2001, Itoh et al., 2000).

3.3.1.1 Auto-antibodies and peptidylarginine deiminase

A basic requirement for an autoimmune disease to occur is the appearance of autoreactive lymphocytes. These cells have to be activated and develop into effector cells in order for them to have a damaging effect on the target tissue of the host. In general, autoimmune reactions are quite common since almost all healthy people produce some amount of low affinity autoantibodies, yet the incidence of severe autoimmune diseases remains very low. In general, it is believed that a defect in the immune mechanisms, most likely in the regulatory functions, is the basis for the development of autoimmune diseases. However, knowledge about these mechanisms is very scarce and environmental and genetic factors might also contribute to the incidence of autoimmune disorders.

In RA, there is little evidence concerning the existence of autoreactive T-cells. Despite the fact that many autoantibodies, produced by B-cells, have been detected in RA patients. The most common immunological aberration in RA is the occurrence of RF. RF is typically the IgM autoantibody that reacts against the Fc-portion of IgG and is found in 70-80 % of patients with RA. Also antiperinuclear factor (APF) and “antikeratin” antibodies (AKA) are characteristic of RA (Young et al., 1979, Nienhuis et al., 1964). They recognize partially overlapping epitopes in human epidermal filaggrin and are therefore renamed collectively as antifilaggrin autoantibodies (AFA) (Sebbag et al., 1995). The epitopes on the targets of AFAs have recently been shown to be citrullinated peptides (Schellekens et al., 1998).

Protein bound citrulline residues are formed by post translational modification of arginine residues by the enzyme PAD. PAD is expressed in many types of cells and is hormonally controlled (Nagata et al., 1990, Takahara et al., 1992). For the time being, relatively few proteins, including filaggrin, fibrin, trichohyalin, vimentin and myelin basic protein, have been shown to be targets of deimination and contain citrulline residues (reviewed in Utz et al., 2000). Also, the physiological role of these modifications remains to be explored. In attempts to develop better tools for the diagnosis of RA, measuring antibodies by enzyme-linked immunosorbent assay (ELISA) to filaggrin-derived citrullinated peptides (anti-cyclic citrullinated peptide antibodies, aCCP) and to deiminated recombinant filaggrins has shown great promise, with a sensitivity of about 70 % and a specificity of at least 96% (Schellekens et al, 1998, Vincent et al., 2002).

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Moreover, AFA, along with RF, have been shown to precede the onset of clinical RA, usually by several years (Aho et al., 2000).

Four different types of PADs have been found in mammalian tissues, including the epidermal type (Type I), muscle type (Type II), and hair follicle type (Type III) (Guerrin et al., 2003, Rus’d et al., 1999, Terakawa et al., 1991, Akiyama et al., 1999, Sugawara et al., 1982, Nishijyo et al., 1997). Type IV (also known as Type V) is expressed in several types of blood cells (Asaga et al., 2001). Types II and IV appear to be more widely expressed, detectable, e.g. in epidermis, stomach, skeletal muscle, brain, ovary and uterus (Ishigami et al., 1996, Ishigami et al., 1998, Yamakoshi et al., 1998, Sugawara et al, 1982, Kubilus et al., 1983). Antibodies to PAD type II were immunologically highly crossreactive to type III enzyme (Terakawa et al, 1991). Based on cloning studies of PAD genes, the sequences indicate that the enzymes from different species are highly homologous to each other (Watanabe et al., 1989, Tsuchida et al., 1993).

Other autoantibodies detected in RA include anti-RA33 antibodies, which bind to the A2-protein of the nuclear ribonucleoprotein complex (Hassfeld et al., 1989), anti-p68 antibodies, which bind to the carbohydrate epitopes of BIP heat shock protein (Blass et al., 1998), and anti-Sa antibodies, the target of which is not known (Despres et al., 1994). However, none of these are competitive with regard to the specificity and sensitivity of RF, AFA or aCCP.

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4. Mediators of inflammation

The inflammatory mediators are responsible for the development of clinical symptoms of inflammation. They cause vasodilation, increased permeability of blood vessels and migration of leukocytes to the site of inflammation. They are released from precursors residing in the plasma or are synthesized in the active inflammatory or in tissue cells. To date, hundreds of different mediators are known, some with very specific and others with broad-spectrum effects on the factors of inflammation.

The mediators derived from the precursors in the plasma include complement and the quinine system and those derived from cells include eicosanoids, reac-tive oxygen radicals, NO, degradative enzymes, cytokines and chemokines and chemokine receptors (reviewed in Moilanen, 2002).

The complement is composed of 20 different proteins that in a chain reac-tion create a structure called the membrane attack complex. The membrane attack complex creates a pore in the membrane of micro-organisms, allowing a free influx of solutes and water across the lipid bilayer. The disruption of the lipid bilayer leads to the loss of cellular homeostasis, disruption of the proton gradient across the membrane, penetration of degradative enzymes into the cell and eventual destruction of the target cell.

The kinin system is activated in association with tissue damage, virus infection or allergic reaction. Kinins are formed from a plasma-derived precursor called kininogen in a reaction catalysed by kallikrein. Further, kallikrein is formed from an inactive precursor, prekallikrein, which is activated by a Hageman factor. The Hageman factor in part is activated when it encounters a negatively charged surface structure, such as collagen, the basement membrane of a blood vessel, bacterial lipopolysaccharide or urate crystal. Perhaps the most important kinin is bradykinin. Bradykinin is a short-lived and locally effective mediator that causes vasodilation and reduction of blood pressure.

Eicosanoids are local inflammatory mediators which regulate the tonus of blood vessels, platelet aggregation and the activity of the kidneys. Eicosanoids are divided into prostacyclins (PGI2), prostaglandins (PGD2, PGE2, PGF2a), tromboxans (TXA2) and leukotriens (LTA4, LTB4, LTC4, LTD4, LTE4). One of the most important eicosanoids in inflammation is PGE2. During the acute phase of inflammation PGE2 is released from intracellular granules in mast cells, whereas in chronic inflammation PGE2 is produced by monocytes and macrophages. PGE2 causes vasodilation and enhances the effect of histamine, bradykinin and other mediators of inflammation to increase vascular permeability PGE2 also sensitizes the neurons to transmit pain and might also have immunosuppressive effects.

Reactive oxygen radicals and degradative enzymes are produced by phagocytic cells. The oxygen radicals are toxic compounds that break down enzymes, lipids, structural proteins and components of the cell membrane. The NAPDH oxidase present in the membrane of phagosomes reduces oxygen to

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superoxydianion (O2-) and in some cases further to hydrogen peroxide (H2O2). The degradative enzymes are bactericides that act in the phagolysosomes of phagocytic cells. They catalyze the degradation of tissue components and are stored in preformed granules. In RA, macrophages produce and secrete matrix metalloproteinases, which in part are responsible for the destruction of cartilage.

Nitric oxide (NO) is a gaseous signal molecule that participates in the regula-tion of blood pressure and circulation. NO is formed in a reaction catalysed by nitric oxide synthase. It activates neutrophils, NK-cells and T- and B-lympho-cytes, affects the degranulation of eosinophils and the infiltration of neutrophils and is produced by macrophages, endothelial cells, neurons, chondrocytes and hepatocytes. In the inflammatory process of the joint, NO enhances inflammation and tissue destruction.

Since chronic inflammation of the joints occurs in patients with RA, many inflammatory mediators have been shown to play roles in prolonging the inflam-matory process. Cytokines and chemokine attract lymphocytes to the inflammed tissue and thereby contribute to chronic inflammation present in the RA joint.

4.1 Cytokines

Cytokines are locally acting protein mediators that are involved in almost all bio-logical processes, including cell growth and activation, inflammation, immunity and differentiation. These proteins form a network of synergistic, complementary, antagonistic and inhibitory factors in a biological response in a tissue that depends on the balance between such molecules present. Cytokines induce the activation of a signaling cascade inside the cell after binding specific receptors. They are mostly produced by activated inflammatory cells, although epithelial cells, chondrocytes and hepatocytes are also known to secrete cytokines. The role of cytokines in the pathogenesis of autoimmune diseases has been the object of intensivestudy in recent years. For example, cytokines play a key role in the regulation of autoreactive T-cells. So called Type 1 responses have been implicated in many autoimmune diseases as evidenced by elevated levels of IFN-γ in tissues, by the amelioration of disease with anti-cytokine treatment and in studies of IFN-γ and IL-12 knockout mice. On the contrary, Type 2 T-cells are associated in particular with allergic responses and inhibit cell-mediated immunity, thus Type 2 cytokines can theoretically inhibit Type 1 mediated autoimmune diseases.

Analysis of the expression of cytokines at mRNA and protein levels in patients with RA has revealed that many proinflammatory cytokines are abundant in synovial tissue (Vervoordeldonk et al., 2002). These proinflammatory cytokines such as TNF-α, IL-1, IL-6 and GM-CSF, and chemokines such as IL-8, are mainly secreted by cells of the monocyte/ macrophage lineage.

Rheumatoid synovium is not a rich source of T-cell cytokines, however several studies have found a preferential activation of Type 1 cells in the target tissue, sug-gesting that Type 1 rather than Type 2 cytokines are involved in the pathogenesis

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of the disease (Dolhain et al, 1996a, Canete et al., 2000, Isomaki et al., 1999, Morita et al., 1998, Ronnelid et al., 1998, van der Graaff et al., 1999). It has been shown that the absolute number of cytokine producing T-cells in rheumatoid SF is low, yet the spontaneous production of cytokines at the level of a single T-cell is increased in the arthritic joint when compared to PB T-cells (Ronnelid et al, 1998). The increased expression of inflammatory mediators in rheuma-toid joints is counteracted to some degree by the production of anti-inflam-matory cytokines such as IL-10 and TGF-β and by cytokine inhibitors such as the IL-1 receptor antagonist (IL-1ra) and the soluble TNF-α receptor (TNF-R) (Cope et al., 1992, Firestein et al., 1994, Lotz et al., 1990, van Roon et al., 2001). Despite the anti-inflammatory mediators, a pronounced chronic inflammation is characteristic of the RA joint. Cytokines found in increased concentrations in RA synovium are listed in Table 1. The classification into pro-inflammatory, anti-inflammatory and regulatory cytokines is made based on the standard practice used in the study of RA.

Table 1. Cytokines expressed in RA synovium. The classification of cytokines is made based on the standard practice used in the study of RA (modified from Ver-voordeldonk et al., 2002 and Feldmann et al, 1996).

Cytokine Main producing cells in RA Synovialmembrane

Pro-inflammatory cytokinesTNF-� macrophages +IFN-� T-cells + GM-CSF macrophages +LT T-cells +/�IL-1 � ��� macrophages + IL-2 T-cells +/�IL-6 FLS, macrophages +IL-12 macrophages , dendritic cell +IL-15 FLS, macrophages +IL-17 Activated memory CD4+ T-cells +IL-18 macrophages +

Anti-inflammatorycytokinesIFN-� FLS ?IL-4 Th2 cells �IL-11 FLS +IL-13 Th2 cells +

Regulatory cytokinesIL-10 Th2 cells, macrophages, B-cells +TGF-� macrophages +

Abbreviations:TNF�tumor necrosis factor, IFN�interferon, IL�interleukin, LT�lymphotoxin,TGF�transforming growth factor, Th�T helper cell, FLS�fibroblast-like synoviocyte

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4.1.1 Pro-inflammatory cytokines

TNF-α TNF-α is a 17 kD protein composed of three identical subunits. It is produced mainly by monocytes and macrophages, but also by activated helper T-, B- and NK-cells, mast cells polymorphonuclear leukocytes, astrocytes and smooth muscle cells. Its main function appears to promote inflammation by inducing fever, shock, tissue injury, energy substrate mobilization, cachexia, bone resorp-tion, differentiation and proliferation of hematopoietic cells and production of acute-phase proteins. It is the most rapidly released cytokine under stress and has the ability to stimulate the production of other pro-inflammatory cytokines, such as IL-1, IL-6, and chemokines such as IL-8 (Tracey et al., 1987, Butler et al., 1995). The primary sources of TNF-α, monocytes and macrophages, are also one of its main targets. TNF-α causes the activation and differentiation of monocytes and macrophages, acts as a chemotactic agent for monocytes and induces the expression of adhesion molecules on endothelial cells, which further induces the migration of leukocytes to the site of TNF-α release. TNF-α also promotes inflam-mation by upregulating the production of matrix metalloprotein-1 (MMP-1) and PGE2 (Dayer et al., 1985). TNF-α reduces the expression of MHC class II proteins on macrophages and acts as a growth factor for monocytes by preventing programmed cell death (Mangan et al., 1991, Watanabe et al., 1991).

Resting T-cells do not respond to TNF-α as they do not express the receptor, TNF-R. Instead, activated T-cells increase the expression of the IL-2 receptor (IL-2R) and thus the proliferative response to IL-2 as well as the production of IL-2 in response to TNF-α. IL-2R expression is also increased on NK-cells upon exposure to TNF-α (Ostensen et al., 1987).

Endothelial cells, which are also major targets of TNF-α, increase their expression of adhesion molecules, such as intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, when responding to TNF-α (Osborn, 1990). In addition to this, TNF-α recruits leukocytes to the site of inflammation by inducing angiogenesis and the release of chemokines produced by a variety of cells.

TNF-α appears to inhibit collagen synthesis by fibroblasts and to stimulate the resorption of proteoglycans in cartilage and inhibit their synthesis (Osborn, 1990). TNF-α has also the ability to kill cells infected with viruses and to inhibit viral replication (Wong et al., 1991, Feduchi et al., 1991). On the other hand, TNF-α has been shown to have an anti-inflammatory role by stimulating the release of corticotrophin, a hormone that stimulates the release of cortisol from the adrenal cortex, which indirectly downregulates inflammation (Tilders et al., 1994).

Practically all cells have TNF-R and therefore respond to TNF-α. Two different forms of TNF-R, p55 and p75, have been described (Tartaglia et al., 1992). TNF-Rp55 is ubiquitously expressed on all cells, whereas TNF-Rp75 is mainly found on hemopoietic and endothelial cells (Tartaglia et al, 1992). Signaling through

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TNF-Rp55 has an effect on antiviral activity, fibroblast proliferation and induc-tion of superoxide dismutase, whereas signaling through TNF-Rp75 affects the proliferation of thymocytes and cytotoxic T-cells (Tartaglia et al, 1992). The receptors are also produced naturally as soluble molecules, which inhibit the action of TNF-α by competition of binding with cell surface receptors (Aderka et al., 1992) (reviewed in Vassalli, 1992).

IL-1IL-1 exists in partially homologous 17 kD IL-1α and IL-1β forms that are derived from 31 kD precursors synthesized in the cytoplasm of cells (Dinarello, 1991). Both the mature α and β forms and the pre-IL-1α precursor are biologically active, whereas pre-IL-β lacks biological activity. IL-1 is mostly expressed by monocytes and macrophages but is also produced by endothelial cells, B-cells, activated T-cells, fibroblasts and neutrophils. There are two specific IL-1 recep-tors (IL-1R), the biologically active IL-1R type I and inert IL-1R type II (Sims et al., 1994). IL-1RII acts as a decoy receptor, binding IL-1 both on the plasma membrane and in the fluid phase as a soluble receptor. This prevents IL-1 from interacting with the functional IL-1RI thus inhibiting the intracellular signaling pathway (Colotta et al., 1994). Also another molecule, IL-1ra, is a natural inhibi-tor of IL-1 as it downregulates IL-1 function through competitive ligation of the IL-1 receptor (Arend et al., 1990).

IL-1 is important in the host defence against micro-organisms that reside preferentially inside cells, such as Listeria monocytogenes and Mycobacteria tuberculosis (Denis et al., 1994, Rogers et al., 1994). The beneficial effects of IL-1 seen in Listeria infection include increased numbers of leucocytes in the PB, enchanced migration of leucocytes and enchanced macrophage responsiveness to IFN-γ that has an increased MHC class II expression (Rogers et al, 1994). IL-1 induces destruction of cartilage by the release of MMPs and other proteolytic enzymes that degrade proteoglycans and type II collagen (Dayer et al., 1986). IL-1 also stimulates the production of other pro-inflammatory cytokines, except TNF-α which resides upstream from IL-1 in the pro-inflammatory cytokine cascade (Butler et al, 1995). IL-1 also affects acute phase protein production via IL-6 (Moshage et al., 1988).

TNF-α and IL-1 in RATNF-α and IL-1 are the most abundant pro-inflammatory cytokines produced in the rheumatoid joint. These proteins have been detected in SF and synovium (Vervoordeldonk et al, 2002) and are immunohistologically localized predomi-nantly in macrophages (Chu et al., 1991). TNF-α and IL-1 synergize in numerous biological functions, both in vitro and in vivo, but when TNF-α appears to play a more important role in the initiation of inflammation both locally and systemati-cally, IL-1 appears to be more involved in local cartilage and bone destruction.

The importance of TNF-α in RA is evidenced by several experimental and clinical observations. In vitro, neutralization of TNF-α by anti-TNF-α antibodies

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resulted in a pronounced reduction in the expression of pro-inflammatory cytokines, IL-1 and IL-6, IL-8 and GM-CSF in synovial cell cultures (Brennan et al., 1989a). In vivo, the neutralization of TNF-α before onset of the disease suppressed collagen-induced arthritis (CIA) in mice and the blockade of TNF-αafter the onset of disease resulted in amelioration of clinical symptoms and prevention of the disease (Williams et al., 1992, Thorbecke et al., 1992). In humans, pro-inflammatory cytokine levels, angiogenesis, leukocyte migration and serum MMP levels diminished after anti-TNF-α antibody was administered (Maini et al., 1999). TNF-α contributes to the erosive changes in the RA joint via osteoclast activation (Roux et al., 2000).

TNF-R p55 and p75 are upregulated in active RA tissues both at mRNA and protein levels (Brennan et al., 1992). Elevated levels of soluble TNF-R have been found in the serum and SF of patients with RA, with the observation that an increased concentration of soluble TNF-R found in plasma correlates with disease activity (Cope et al, 1992).

In patients with RA, IL-1 plays an important role in cartilage and bone erosion. In vitro studies suggest that IL-1 can cause cartilage destruction by stimulating the production of degrative enzymes (MMPs) (Krane et al., 1990) and by block-ing the repair processes in cartilage (Dayer, 2003). IL-1 also downregulates Fas antigen expression on synoviocytes, which implies that impaired apoptosis may contribute to the proliferation of the synovial lining cell layer in patients with RA (Tsuboi et al., 1996). Serum levels of IL-1 are found to correlate with disease activity in patients with RA (Dayer, 2003).

IL-1ra is mainly produced by synovial lining cells in RA (Deleuran et al., 1992). However, since a ten-fold concentration of IL-1ra is needed to block the bioactivity of IL-1, the molar ratio detected in RA synovium, is suggested to be insufficient to block the action of IL-1 (Firestein et al, 1994).

IFN-γBiologically active IFN-γ is a 34 kD homodimer glycoprotein and a Type 1 cytokine that has receptors on virtually all cells of the body. Activated T-cells and NK-cells are the main producers of IFN-γ, but macrophages also secrete IFN-γ. IFN-γ is the major activator of macrophages and it stimulates several macrophage functions including tumor cell cytotoxicity (Le et al., 1983, Pace et al., 1983), antimicrobial activity (Nathan et al., 1983), increased killing of intra-cellular pathogens (Torrico et al., 1991) and induction of MHC class II proteins (Basham et al., 1983, Keller et al., 1988). IFN-γ stimulates the production of IFN-β, IL-1α, IL-1β, TNF-α, interferon-γ inducible protein (IP-10) and IL-12 in human and murine systems and inhibit the expression of some cytokines and chemokines, such as IL-8, IL-10 and MCP-1 in human monocytes (Chomarat et al., 1993, Ohmori et al., 1994, Gusella et al., 1993). It also upregulates the Fcγ receptors on phagocytes (Guyre et al., 1983, Fertsch et al., 1984) and adhesion molecules, such as ICAM-1, on endothelial cells (Dustin et al., 1986). IFN-γ sup-presses Type 2 responses by inhibiting IL-4, which mediates the antibody isotype

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switch (favoring IgE) and by inhibiting macrophage derived production of IL-10 (Pene et al., 1988, Chomarat et al, 1993). IFN-γ promotes T-cell proliferation in low concentrations, T-cell apoptosis in high concentrations and can augment the development of cytotoxic lymphocyte activity in vitro (Siegel, 1988). IFN-γ is mainly induced by IL-12 and IL-18, which are secreted by monocyte lineage cells. The pro-inflammatory actions of IFN-γ synergise with TNF-α in many in vitro test systems. However, this might critically depend on the state of differentia-tion of the cells, as seen when TNF-α was found to enchance IFN-γ−triggered MHC class II expression in undifferentiated macrophages, yet it inhibited such enhancement in mature macrophages (Watanabe et al, 1991).

IL-2IL-2, originally known as “T-cell growth factor”, has both immunostimulatory and immunosuppressive effects on the immune system. IL-2 promotes the proliferation of naïve T-cells and their maturation into Type 1 deviated lym-phocytes, it enhances the cytotoxicity of T-cells and promotes the production of pro-inflammatory cytokines. Furthermore, IL-2 promotes the proliferation of NK-cells and of γ/δ subsets of T-cells (Robertson et al., 1990, Toribio et al., 1988). IL-2 can also synergize with IL-12 to facilitate NK-cells to produce IFN-γ and TNF-α (Carson et al., 1994). However, IL-2 suppresses the immune response by promoting programmed cell death, differentation of Th2 cells and IL-10 and TGF-β secreting immunoregulatory T-cells (Treg).

IFN-γ and IL-2 in RAPro-inflammatory cytokines produced by T-cells, such as IFN-γ and IL-2, are found in the joints of patients with RA, although their detection has been controversial due to the method used.

In RA, SF-derived T-cells have been shown to express increased levels of IFN-γ when compared to PB (Kusaba et al., 1998, Ronnelid et al, 1998), and the expression of IFN-γ mRNA in RA synovial membrane has been shown to be increased when compared to the synovial membrane of patients with spondyloarthropathies (Canete et al, 2000).

Locally administered IFN-γ was found to promote development of CIA in mice, whereas systemically administered IFN-γ exerted a protective effect (Mauritz et al., 1988, Nakajima et al., 1990). On the contrary, in patients with RA, the production of IL-1 by synovial macrophages and further the IL-1 and TNF-α induced matrix metalloproteinase production and glycosaminoglycan release by cultured cartilage fragments were found to be inhibited by IFN-γ (Ruschen et al., 1989, Andrews et al., 1989). In cultured human articular chondrocytes, IFN-γ synergizes with TNF-α in prostaglandin E2 production, but decreases TNF-α−induced production of caseinase, an indicator of the proteoglycan deg-radation enzyme, stromelomysin (Bunning et al., 1989). IFN-γ also inhibits bone

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resorption in an in vitro system (Gowen et al., 1986). In CIA, IFN-γ production declines with time (Mussener et al., 1997), whereas

in man no difference in the expression of IFN-γ can be found in synovial membranes between patients with recent-onset RA and RA of long duration (Smeets et al, 1998).

4.1.2 Anti-inflammatory cytokines

IL-4IL-4 is a 20 kD anti-inflammatory cytokine produced by activated T-cells, mast cells, NK-cells, basophils and eosinophils. IL-4 is characteristic of Type 2 T-cells and its main function is to direct T-cell differentiation towards Type 2, while suppressing the Type 1 immune response. IL-4 promotes the growth and differentiation of cytotoxic T-cells and mast cells, but acts as a growth inhibitor for immature thymocytes and as a maturation promoting factor for CD4+/CD8 cells in the thymus (Widmer et al., 1987, Trenn et al., 1988). On B-cells, IL-4 acts as an isotype switch factor in favour of IgE and IgG4 (IgG1 in mouse). Inflammatory functions, such as H2O2 production, intracellular antimicrobial activity and induction of IFN-γ responsive genes, are downregulated by IL-4 in monocytes and macrophages. Furthermore, IL-4 increases the expression of MHC class II molecules, IL-1ra and TNF-R, while downregulating the production of pro-inflammatory cytokines IL-1, TNF-α, IL-6, IL-8 and IL-12. In vivo the overproduction of IL-4 has been associated with elevated IgE and allergic diseases, where IL-4 induces the rolling on and adhesion of circulating eosinophils to endothelial cells (Bochner et al., 1994). In asthma, IL-4 seems to be important in inducing the Type 2 deviated T-cell population, while IL-5 mediated effects appear to be more important at effector levels.

IL-5IL-5 is a homodimeric glycoprotein secreted mostly by Type 2 cells. In the mouse IL-5 induces the differentiation of activated conventional B cells into Ig-secreting cells, as well as the growth of progenitors of CD5+ B-cells and the production of IgM by B-cells. In man, the biologic effects of IL-5 are best characterized in eosinophils. IL-5 induces terminal maturation of eosinophils, prolongs eosinophil survival by delaying apoptotic death, possesses chemotactic activity for eosinophils, increases eosinophil adhesion to endothelial cells and enhances eosinophil effector functions. Of the type 2 cytokines, IL-5 has one of the most important roles in the hyper-reactivity of the airways in patients with asthma (reviewed in Foster et al., 2002).

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IL-13IL-13 has a low degree of sequence homology with IL-4, despite sharing most of its biological functions. This includes induction of IgG4 and IgE production by B cells and the upregulation of CD23, CD71 and MHC class II expression. It also induces the downregulation of NK and monocyte function, yet distinct from IL-4 synergism only IL-13 is required for worm removal. While being produced at high levels by Type 2 T-cells, IL-13 can also be expressed by both Type 1 and naive T-cells. IL-13 has a critical role as an effector molecule during established allergic inflammation, whereas it is responsible for the hypersecretion of mucus by mucus cells (reviewed in Foster et al, 2002).

IL-4, IL-13 and IL-5 in RAIL-4 is believed to play a protective role in arthritis, although its virtual absence in synovial samples from patients with RA points to its lack of protective mecha-nisms rather than to its active regulation (Miossec et al., 1990, Cohen et al., 1995). In the CIA mouse, IL-4 delays the onset and diminishes the clinical symptoms of CIA, as well as preventing joint damage and bone erosion (Horsfall et al., 1997). In patients with RA, IL-4 has been shown to inhibit the production of pro-inflammatory cytokines and to induce the production of the natural anti-inflammatory cytokine IL-1ra in ex vivo synovial tissue specimens (Miossec et al., 1992, Chomarat et al., 1995). In vitro, IL-4 inhibits the activation of Type 1 helper T-cells and in this way decreases the production of IL-1, TNF-α and TNF-R, and it inhibits cartilage damage (van Roon et al., 1997, Hart et al., 1996). Furthermore, recombinant IL-4 has been shown to inhibit the spontaneous production of total IgG and IgM and IgM RF in non-stimulated T plus B cell cultures from patients with RA (Hidaka et al., 1992). IL-4 also inhibits bone resorption through an effect on osteoclast activity and survival in patients with RA (Miossec et al., 1994).

Reports exist stating the absence of IL-5 expression in SF and IL-5 mRNA in the rheumatoid nodule (Bakakos et al., 2002, Hessian et al., 2003), however the role of IL-5 in RA remains virtually unknown.

IL-13 has been detected in the SF of patients with RA and recombinant IL-13 has been shown to reduce the expression of pro-inflammatory cytokines produced by SF macrophages (Isomaki et al., 1996). In a recent study, Relic et al. showed that IL-13 and IL-4 have a protective role on human synoviocytes against apoptosis (Relic et al., 2001).

4.1.3 Regulatory cytokines

IL-10IL-10 has a dual role in the inflammatory process since it has both anti-inflam-matory and proinflammatory potential. IL-10 is a Type 2 cytokine in mice, yet both Type 1 and Type 2 cells can produce it in humans (Fiorentino et al., 1989, Yssel et al., 1992). It is also produced by B-lymphocytes, mast cells, eosinophils,

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monocytes, keratinocytes and a variety of tumor cells. IL-10 inhibits the production of pro-inflammatory cytokines secreted by macrophages and upregulates the production of anti-inflammatory molecules, such as IL-1ra and soluble p55 and p75 TNF-receptors. IL-10 also downregulates the production of activator and co-stimulatory molecules on monocytes and dentritic cells, such as ICAM-1, CD80 and CD86, and it downregulates macrophage production of toxic oxygen radicals, nitric oxide synthase and prostaglandin synthesis (Bogdan et al., 1991, Mertz et al., 1994). In T-cells, IL-10 inhibits T-cell proliferation by inhibiting the production of IL-2 and IFN-γ (de Waal Malefyt et al., 1993). IL-10 may also contribute to the induction of T-cell anergy by downregulating the ligand-receptor co-stimulatory interaction between antigen presenting cells and T-cells (Schwartz, 1996). On the other hand, IL-10 enhances immune activity by stimu-lating the proliferation and activation of NK-cells, B-cells and IL-2-activated cytotoxic T-cells (Carson et al., 1995, Chen et al., 1991, Rousset et al., 1992) and by upregulating Fc receptors on monocytes which enchances antibody mediated cytotoxicity (te Velde et al., 1992).

In asthma IL-10, has varying effects on the production of IgE. IgE synthesis in peripheral mononuclear cells is enchanced by IL-10 in individuals sensitive to mite (Kawano et al., 1995), and it has been demonstrated that IL-10 expression in asthmatic patients is reduced (Borish et al., 1996). Conversely, it has been demonstrated that IL-10 reduces IL-4-induced IgE production in nonatopic individuals (Punnonen et al., 1993).

IL-10 in RAIn patients with RA no difference has been seen in the number of cells sponta-neously producing IL-6 or IL-10 in PB when compared to controls (Berg et al., 2001). Instead, high IL-10 concentrations have been detected in the serum and SF of patients with RA (Cush et al., 1995, Moller et al., 2002). IL-10 levels have also been shown to correlate with serum RF titers and in vitro levels of spontaneous IgM RF production (Cush et al, 1995), thus suggesting that in RA activation of IL-10 secretion is linked to inflammatory activity. In patients with RA, IL-10 also plays a part in cartilage degradation that is mediated by antigen-stimulated mono-nuclear cells (van Roon et al, 1997). In CIA, systematically administered IL-10 reduces joint swelling, cellular infiltration, pro-inflammatory cytokine production and cartilage degradation (Persson et al., 1996).

4.2 Chemokines and chemokine receptors

Chemokines are chemotactic cytokines that signal through seven transmembrane G-protein coupled receptors (Murphy, 1994). To date over 50 different chemo-kines have been identified, each with different binding properties to one or more receptors. Chemokines and their receptors are divided into four different families, CC-, CXC-, CX3C- and XC-, according to the position of conserved cysteine residues (C) that lie close to the N-terminus region of the protein. Chemokines

36 37

and chemokine receptors are involved in the migration of leukocytes from the blood to the lymph nodes, Peyer’s patches and to sites of inflammation and are, in general, classified into “inflammatory” and “lymphoid” categories based on the site of production and the eliciting stimuli. In inflammatory responses, when the extravasation of leukocytes occurs from the vasculature to the site of inflam-mation, selectins located on endothelial cells bind to carbohydrates on leucocytes and allow the rolling of cells onto the vascular endothelium. Chemokine receptors on the surface of leucocytes then adhere to their ligands immobilized on the endothelial layer and trigger a firm adhesion and transmigration of leukocytes through the endothelium. In tissue, chemokines form an immobilized concentra-tion gradient along which the leucocytes migrate sequentially to the site of inflam-mation (Foxman et al., 1997). In this process, the chemokine receptor on the leukocyte undergoes ligand-induced desensitisation, internalisation and recycling thus allowing the cell to move forward by ligation of another receptor on the leukocyte surface (Premont et al., 1995, Daaka et al., 1998). Since chemokine receptors are required to perform distinct steps during extravasation and the positioning of cells within tissues, many different types of chemokine receptors are expressed on the leukocyte surface (Table 2a, b).

CCR3CCR3 is an eosinophil chemoattractant receptor for multiple inflammatory/inducible chemokines (Heath et al., 1997). The receptor is most selective for the binding of eotaxin, eotaxin-2, eotaxin-3, and also for other ligands and agonists that exist. This includes the regulated upon activation T-cell expressed and secreted (RANTES), MCP-3, MCP-4, MIP-5/leucotactin-1 and human immunodeficiency virus (HIV) Tat (Daugherty et al., 1996, Garcia-Zepeda et al., 1996, Kitaura et al., 1996, Ponath et al., 1996, Forssmann et al., 1997, Youn et al., 1997, Albini et al., 1998, Shinkai et al., 1999). In humans, CCR3 is expressed on eosinophils (Daughertyet al, 1996), basophils (Uguccioni et al., 1997), mast cells (Ochi et al., 1999), Type 2 T-cells (Gerber et al., 1997, Sallusto et al., 1997), dentritic cells (Rubbert et al., 1998) and microglial cells of the brain (He et al., 1997). In vitro activities of CCR3 include the arrest of eosinophils under flow conditions (Kitayama et al., 1998), eosinophil and Th2 cell chemotaxis (Sallusto et al, 1997), degranulation of eosinophils and basophils (Uguccioni et al, 1997), HIV-1 entry into microglial cells (He et al, 1997) and HIV-specific T-cell cytotoxicity (Hadida et al., 1998).

CCR3 is found to be involved in the early recruitment of Th2 cells during antigen presentation, however repeated rounds of antigen stimulation involves the predominant use of CCR4 (Lloyd et al., 2000). Furthermore, CCR3 is shown to be important in allergic inflammations, including asthma (Ying et al., 1997, Cosmi et al., 2001). In intradermal allergic inflammation, the CCR3 expressing cells have been found to be non-lymphoid cells (Ying et al, 1997), whereas in asthmatic patients, the cells expressing CCR3 have been eosinophils rather than T-cells (Cosmi et al, 2001).

36 37

Table 2a, 2b (next page). Chemokine receptors (modified from Onuffer et al., 2002).

CCR4CCR4 was originally reported to be a selective marker for Th2 lymphocytes, but it has recently been shown to be expressed also on circulating Th1 cells (Bonecchi et al., 1998, Sallusto et al., 1998). In addition to T-cells, CCR4 is also expressed on NK-cells, thymocytes and immature dendritic cells (Murphy et al., 2000). CCR4 is a receptor for thymus and activation regulated chemokine (TARC), macrophage derived chemokine (MDC) and MIP-1β (Bonecchi et al, 1998, Inngjerdingen et al., 2000) and is involved in dendritic cell trafficking, T-cell recirculation from tissue to lymph node, T-cell migration through thymus during maturation and homing of memory T-cells to inflamed skin (Sozzani et al., 1999, Campbell et al., 1999). As evidence of CCR4 involvement in homing of T-cells to the skin, an increased number of circulating CCR4 positive lymphocytes was detected in a Th2 dominated disorder, atopic dermatitis (Nakatani et al., 2001). Further, it is also implied that CCR4 is involved in the process of negative selection of potentially autoreactive thymocytes in human thymus (Annunziato et al., 2001).

Chemo-kinereceptor

Exression Ligand Presumedfunction

Proposed therapeutic indications

CXCR1 N, M, M�, T, DC,astrocyte,endothelium

CXCL6,8

neutrophil recruitment

gout, psoriasis, cancer, lung reperfusion injury

CXCR2 N, T, M, M�,DC, Eo, endothelium

CXCL1,2, 3, 5, 6, 7, 8

neutrophil recruitment, angiogenesis

lung reperfusion injury, gout, psoriasis, cancer, atherosclerosis

CXCR3 N, T (Th1), B, DC, Eo, P, smooth muscle cells

CXCL9,10, 11

Th1 response, angiostasis

MS, RA, sacroidosis, allograft rejection, cancer

CXCR4 N, M, M�, T, B, DC, P, astrocyte

organogenesis HIV infection, cancer

CXCR5 M, M�, T, B, astrocyte,neuron

CXCL13 B-cellmigration

Not identified

CXCR6 T, DC CXCL16 Not known Not identified

CX3CR1 N, M M�,neuron

CX3CL1 Cell adhesion to endothelia and neurons

Allograft rejection, glomerulonephrisis, CNS inflammation

XCR1 T XCL1, 2 Not known Not identified

Abbreviations:B=B-cell, Ba=basophil, DC=dendritic cell, iDC= immature dendritic cell, Endo= endothelium, Eo=eosinophil, Fibro=fibroblast, M=monocyte, M�=macrophages, MC=mast cell, N=neutrophil, NK=natural killer cell, P=platelet, T=T-cell, Th= T-helper cell.

38 39

CCR5CCR5 is a Type 1 chemokine receptor expressed on activated T-cells (Bleul et al., 1997), mature and immature dendritic cells (Granelli-Piperno et al., 1996), monocytes (Alkhatib et al., 1996), thymocytes (Zaitseva et al., 1998) and CD34+ hematopoietic progenitor cells (Ruiz et al., 1998). High –affinity ligands and high-potency agonists for CCR5 include MIP-1α, RANTES, MIP-1β, MCP-2 none of which is selective for CCR5. Additional ligands include MCP-1, MCP-4, eotaxin and MCP-3. MCP-3 appears to act as an antagonist (Blanpain et al., 1999). CCR5 is constitutively expressed with CD4 (Xiao et al., 1999), but the physiological role of CCR5 is unclear. However, the studies done with CCR5-deficient mice show reduced efficancy in the clearance of Listeria infection, resistance to lipopolysaccaride (LPS)-induced endotoxemia, increased susceptibility to Cryptococcus infection (Huffnagle et al., 1999), enchanced delayed-type hypersensitivity reaction and

Chemo-kinereceptor

Exression Ligand Presumed function Proposed therapeutic indications

CCR1 N, M, M�, T, B, Eo, Ba, P, iDC,

CCL3, 5, 7, 14, 15, 16, 23

Th1 response MS, RA, allograft rejection, viral myocarditis

CCR2 M, M, M�, T, Ba, NK, iDC, endo, fib

CCL2, 7, 13, 16

Inflammation MS, arthritis, asthma, atherosclerosis

CCR3 T (Th2), Ba, Eo, MC, P, DC

CCL5, 7, 8, 11, 13, 15, 24, 26, 28

Th2 response asthma, contact dermatitis

CCR4 T, Ba, P, iDC CCL17, 22 Th2 response sepsis, asthma, contact dermatitis

CCR5 M, M�, T, NK, thymocyte, DC

CCL3, 4, 5, 8, 14

Th1 response MS, RA, intestinal inflammtion, allograft rejection, HIV infection

CCR6 M, M�, T, B, iDC CCL20 DC function psoriasis

CCR7 T, B, DC CCL19, 21 lymphocyte and DC migration to LN

cancer

CCR8 N, M, T, B, thymocyte CCL1 Th2 response atopic dermatitis, asthma

CCR9 T, thymocyte CCL25 lymphocyte trafficking in thymus and small intestine

intestinal inflammation

CCR10 T, melanocyte, dermal endo and fib, Langerhans cell

CCL27, 28 lymphocyte trafficking in skin and colon

skin inflammation, ulcerative colitis

Abbreviations:B=B-cell, Ba=basophil, DC=dendritic cell, iDC= immature dendritic cell, Endo= endothelium, Eo=eosinophil, Fibro=fibroblast, M=monocyte, M�=macrophages, MC=mast cell, N=neutrophil, NK=natural killer cell, P=platelet, T=T-cell, Th= T-helper cell.

Table 2b.

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increased humoral responses to T-cell dependent antigenic challenge (Zhou et al., 1998). However, in CCR5 deficient mice the increase in cytokine production was observed both in Type 1 (IFN-γ) and in Type 2 (IL-4) cytokines, and therefore is different from CCR1 deficient mice in which a difference in Type 1 /Type 2 balance was observed (Gao et al., 1997).

CCR5 is a major HIV co-receptor that controls the susceptibility of individuals to HIV-1 infection and acquired immunodeficiency syndrome (AIDS) (Samson et al., 1996). HIV is blocked naturally by the inactive mutated CCR5 molecule, a result of inheriting a 32-base pair deletion in the CCR5 gene (CCR5Δ32) (Dean et al., 1996, Huang et al., 1996, Samson et al, 1996). The CCR5Δ32 mutant allele is common in the caucasian population and high resistance to HIV infection appears in otherwise healthy CCRΔ32 homozygotes. CCR5Δ32 heterozygotes have reduced expression of normal CCR5 on cells, due to the mutated CCR5Δ32 allele (Benkirane et al., 1997) and if infected with HIV, progress less rapidly to AIDS (Murphy et al, 2000).

CCR5, CCR3 and CCR4 in RAWhile chemokines in RA have been under extensive investigation, relative few studies regarding the chemokine receptor distribution and expression currently exist. It has been shown that T-cells in the synovial membrane and SF of patients with RA produce high levels of inflammatory chemokine receptor, CCR5 (Qin et al., 1998, Loetscher et al., 1998, Suzuki et al., 1999, Ruth et al., 2001). Supporting this, RANTES and MIP-1α, the ligands for CCR5, have been detected at high levels in the SF of patients with RA, suggesting that CCR5 positive cells migrate to the joints in response to these chemokines and take part in inflammatory reactions (Robinson et al., 1995, Suzuki et al, 1999). The production of IFN-γ and IL-2 cytokines has been shown to occur in CCR5 positive SF T-cells in patients with RA (Suzukiet al, 1999, Dolhain et al., 1996b). This suggests that the predominant immune response occurring in RA joints is Type 1 dominated.

CCR3 is another inflammatory chemokine receptor expressed at elevated levels on SF derived T-cells and monocytes in patients with RA (Ruth et al, 2001, Katschke Jr et al., 2001). It has been suggested that CCR5 and CCR3 may be important in monocyte retention in the joint (Katschke Jr et al, 2001), while other chemokine receptors such as CCR1, CCR2 and CCR4 could be involved in the homing of leukocytes to the joints (Katschke Jr et al, 2001, Ruth et al, 2001).

It has also been found that people with a 32 nucleotide deletion in the CCR5 gene (CCR5Δ32) show a decreased prevalence to RA and in the case of a disease have a less severe form of the disease than individuals with normal CCR5 gene (Zapico et al., 2000)

40 41

5. Gut immune system

The gastrointestinal tract consists of the esophagus, ventricle, small intestine and large intestine. The small intestine begins from the end of the esophagus, called the pylorus, and extends to the cecum, is from 5 to 10 m long and is divided into duodenum, jejunum and ileum. Correspondingly, the large intestine begins from the end of the small intestine, extends to the anal canal and is divided into the caecum, colon and rectum.

The immune system in the gastrointestinal tract consists of organized lymphoid tissue in the form of Peyers’ patches, found mainly in the small intestine and appendix, solitary lymph nodules present in the colon and ileum and mesenteric lymph nodes. Overlying Peyers’ patches and between the enterocytes (gut epithelial cells) lay M-cells, on the basolateral side of enterocytes reside intraepithelial lymphocytes, and in the mucosa of the gut reside lamina propria lymphocytes (Figure 6). Lymphocytes of each compartment have a distinct phenotype and function. Intraepithelial lymphocytes are predominatly cytotoxic T-cells, that express increased amounts of TCR γ/δ (10-15 % in humans) and CD8αα homodimer.

Figure 6. Schematic diagram of the gut immune system. Overlying Peyers’ patches and between the enterocytes lay M-cells. On the basolateral side of enterocytes reside intraepithelial lymphocytes (IELs), and in the mucosa of gut reside lamina propria lymphocytes (LPLs).

Enterocytes

BasementMembrane

Food and Bacterial Antigens

c c c c c c c c c

M cell

IELs

Gut Lumen

Lamina Propria

Peyer’s patch

LPLs

40 41

Although it is known, that when CD8αα T-cells mature they do not transit through the double positive selection process in the thymus (see section 3.2), the precise maturation pathway and site is under debate. Two possible sites have been suggested: the intestine and the thymus. However, almost all lamina propria lymphocytes mature in the thymus, express TCR α/β and are mostly helper T-cells (65 %) (reviewed in Guy-Grand et al., 2002 and Abreu-Martin et al., 1996).

While virtually all intraepithelial lymphocytes express the gut-homing integrin, αEβ7, only 30-40% of lamina propria lymphocytes show positivity for the antigen. However, the majority of lamina propria lymphocytes and intraepithelial lymphocytes are activated memory cells expressing CD45RO and to a lesser extent CD25. The CD4+ TCRα/β+ expressing intraepithelial lymphocytes express mainly IL-4, IL-5 and IL-6, while the main cytokine expressed by CD8+ TCRα/β+ intraepithelial lymphocytes is IFN-γ. Instead TCR γ/δ intraepithelial lymphocytes show a cytokine production pattern that includes IFN-γ, TGF-β, TNF-α and IL-5. The most characteristic feature of intraepithelial lymphocytes is their rapid cytolytic response to the limited repertoire of TCR molecules that contain evolutionary conserved sequences, such as heat shock proteins (Abreu-Martin et al, 1996).

The cytokine expression of lamina propria lymphocytes is Type 2 deviated and includes the prominent expression of IL-4 and IL-5 with low amounts of IFN-γ. Neither intraepithelial lymphocytes nor lamina propria lymphocytes proliferate well in response to stimulation via the TCR/CD3 complex or conventional mitogens (Mosley et al., 1991, Mowat et al., 1989), instead they express cytokines when stimulated via CD2 and CD58.

Activation of the gut-associated immune system has been noted in many autoimmune diseases. Patients with spondyloarthropathies, a group of related disorders with common clinical and genetic characteristics that include ankylosing spondylitis, reactive arthritis, psoriatic arthritis and arthritis in patients with inflammatory bowel disease, show histologic signs of gut inflammation and a fraction of these patients go on to develop clinical Crohn’s disease. The environmental trigger in spondyloarthropathies is thought to be an infection in the gut, which leads to chronic inflammation in the intestine.

Coeliac disease is a gluten-triggered autoimmune disorder characterized by small-bowel mucosal lesions in genetically susceptible individuals. Villous athrophy with crypt hyperplasia and lymphocytic infiltration are features common in the biopsy specimens (Maki et al., 1997).

Immune aberrations related to the gut, such as an enhanced immune response to cow milk proteins and an increased number of IL-1α and IL-4 positive lamina propria cells, have also been noted in type 1 diabetes (Westerholm-Ormio et al., in press, Karjalainen et al., 1992, Vaarala et al., 1996, Cavallo et al., 1996).

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5.1 Gut associated immune activation in RA

The relationship between diseases of the gastrointestinal tract and arthritis is well established. Gut lesions have been found in patients with rheumatic diseases, while peripheral arthritis resembling RA is occasionally found in patients with gastrointestinal diseases (Wands et al., 1976, Hazenberg et al., 1992). Gut abnormalities such as increased permeability, elevated numbers of inflammatory cells and villous atrophy have been reported in patients with RA (Marcolongo et al., 1979, O’Farrelly et al., 1988). Occult intestinal inflammation, which may be related to non-steroidal anti-inflammatory drug therapy or be disease associated, occurs in approximately 70 % of patients with RA according to Sartor (Sartor, 1989). Supporting the role of the gut-associated immune system in the pathogenesis of RA, several studies report beneficial effects from dietary manipulation in patients with RA (Muller et al., 2001).

An increased number of TCR γ/δ expressing T-cells and the expression of gut associated surface molecule αEβ7, which is expressed on >95 % of intestinal intraepithelial lymphocytes and on 40 % of lamina propria lymphocytes, are found on SF derived T-cells when compared to PB lymphocytes in patients with RA (Baumgart et al., 1996). It has also been reported that the number of TCR γ/δ expressing T-cells is elevated in the synovium of patients with RF-positive RA when compared to PB (Andreu et al., 1991). These studies suggest an accumulation of gut-derived lymphocytes into the inflamed joints. Also, the observation that sulfasalazine, a drug known for its efficacy in the treatment of ulcerative colitis and Crohn’s disease, has the ability to induce remission in patients with RA, suggest that inflammatory events occurring in the synovium and gut are related.

6. TNF-α blockade as a biological treatment for RA

The first clue that TNF-α might be an effective therapeutic target for RA came from a study where antibodies against TNF-α were added to the cultures of dissociated rheumatoid synovial membranes (Brennan et al., 1989b). A signifi-cant decrease in the production of IL-1 was detected after TNF-α was blokaded. Further studies reported a downregulation of synovial GM-CSF (Haworth et al., 1991), IL-6 and IL-8 (Butler et al, 1995), suggesting that TNF-α coordinates the production of other pro-inflammatory cytokines.

Treatment with agents blocking the function of TNF-α has proved to be highly effective in RA, Crohn’s disease and psoriatic arthritis. In addition to showing promising activity in psoriasis, spondyloarthropathies and graft-versus-host disease (Feldmann et al., 2001, Reimold, 2003). However, the mechanisms behind the clinical effect of the TNF-α blocking treatment are not fully understood. TNF-α blocking by antibodies or soluble receptors (Figure 7) reduces the expression of vascular adhesion molecules (Paleolog et al., 1996) and inhibits the spontaneous production of IL-1 and IL-6 in an animal model (Fong et al., 1989). Within

42 43

days after starting TNF-α blocking therapy serum IL-6 levels and C-reactive protein (CRP) normalize (Charles et al., 1999, Elliott et al., 1993). In patients with RA an increase in the production of IFN-γ by lymphocytes has also been reported following treatment with soluble TNF-α receptor (Berg et al, 2001). At the same time, reduction in the homing of lymphocytes to the joints can be seen. Blocking TNF-α reduces the raised concentrations of serum vascular endothelial growth factor (VEGF) (Paleolog et al, 1996) detected in patients with RA and reduces angiogenesis. However, the immediate immunological mechanisms resulting from the blocking of TNF-α appear to be crucial since clinical improvement is seen within days after the onset of treatment.

To date there are two blocking agents in clinical use, a soluble TNF-α receptor, etanercept, and a monoclonal antibody, infliximab (Maini et al., 1998, Weinblatt et al., 1999).

Etanercept is a dimeric fusion protein consisting of an extracellular ligand-binding portion of the human p75 TNF-α cellular receptor that is lined to the Fc portion of human IgG1. The binding of etanercept to circulating TNF-α inhibits the binding of TNF-α to the p55 and p75 cell surface receptors and in this way prevents the activation of cells.

Figure 7. Principle of TNF-α blockade. Soluble TNF-α is prevented from binding to its surface receptor by soluble receptor or monoclonal antibody.

TNF-�

soluble TNF-�receptor

anti-TNF-�antibody

surface TNF-� receptor

cytosol

44 45

Infliximab is a chimeric IgG1κ monoclonal antibody composed of human constant and murine variable regions. It binds with high affinity to both soluble and transmembrane forms of TNF-α. Binding to soluble TNF-α results in loss of bioactivity, while binding to membrane bound TNF-α leads to cytotoxicity by complement- or antibody-dependent mechanisms. Proof in defining which mechanism is relevant for infliximab is still missing (Feldmann et al., 1997).

Interestingly, different TNF-α blocking agents show different, although rare, side effects. Infliximab can exacerbate latent tuberculosis and etarnecept has been reported to induce neurological symptoms (Mikuls et al., 2003, Fleischmann et al., 2002). In addition, the development of IgM class antibodies against double stranded DNA has been observed during treatment with TNF-α blockers and, although rare, even the development of clinical systemic lupus erythematosus has been reported (Weinblatt et al, 1999, Charles et al, 1999, Maini et al, 1998). Exacerbation of an existing congestive heart failure (Feldman et al., 2003) and other yet minor side effects such as headache, nausea and upper respiratory tract infections have also been described (Hanauer, 1999).

44 45

AIMS OF THE STUDY

The aim of this study was to characterize the immunological features of patients with RA using several different approaches. The detailed aims of the individual studies are listed below.

Publication IIn the first study the aim was to characterize the cytokine and chemokine-receptor profiles of T-cells and monocytes in the PB and SF of patients with RA and other arthritides.

Publication IIThe aim of the second study was to analyze immunological changes in patients with RA during treatment with a monoclonal anti-tumor necrosis factor-α (TNF-α) antibody, infliximab.

Publication IIIIn the third study, the aim was to analyze whether inflammation within the gut-associated immune system is associated with RA. The expression of chemokine receptor (CCR4, CCR5) and cytokine (IL-2, IL-10, IFN-γ, TNF-α and TGF-β) specific mRNA in intestinal biopsies from patients with RA was studied.

Publication IVSince antibodies to citrulline-containing epitopes of filaggrin are highly specific to RA, the aim of the fourth study was to determine whether the enzyme PAD, responsible for the post- translational modification of peptide-bound arginine residues to citrulline, constitutes an autoantigen for patients with RA.

46 47

PATIENTS AND METHODS

1. Patients

The demographics of patients used in this study are described in the Table 3.

Patients in the publication IThe study included 20 patients with RA who presented with acute swollen knee. They all fulfilled the 1987 criteria for RA (Arnett et al., 1988). Of the patients, 85% were seropositive and 90% had erosions. Five of the patients were not on disease modifying drugs (DMARDs) at the time of the study. The others were receiving methotrexate either as a single drug (6 patients) or in combination (5 patients, including 1 with infliximab), one was treated with leflunomide and infliximab, and two patients were receiving sulphasalazine (one as single DMARD and one 1 in combination with auranofin). Low dose prednisone was used by nine patients. For the control patients we included nine patients with other arthritides who also presented with acute synovitis in the knee. Three patients had chronic reactive arthritis, two had spondylarthropathy (ESSG criteria (Dougados et al., 1991)), two had ankylosing spondylitis (modified New York criteria (van der Linden et al., 1984)), and one had acute gout. Two patients (22%) were seropositive and 4 (44%) had erosions. Four patients were receiving sulphasalazine and two patients methotrexate. Low dose prednisone was used by 3 patients.

Patients in the publication IIWe studied 25 patients with active RA not responding to conventional therapy with DMARDs including methotrexate, and five patients with other chronic active joint diseases (two patients with psoriatic arthritis, one patient with chronic reactive arthritis, one patient with spondyloarthropathy and one with juvenile idiopathic arthritis) in whom therapy with infliximab had begun. At the time of examination, all patients were using varying anti-rheumatic drugs either as single DMARD or in different combinations. Twenty-one patients with RA and four patients with other arthritides were on low dose (<10 mg/ day) prednisone.

In RA patients the response to infliximab was evaluated at week two by calculating the ACR response (Felson et al., 1995) (in two patients the information was available only at week six). Patients showing an improvement of at least 20% (ACR20) were considered to be responders. Therefore fifteen patients with RA were responders and ten were non-responders.

EDTA-blood was collected from the patients immediately before the infusions of infliximab (3 mg/kg) at the beginning and at two and six weeks during therapy. EDTA blood was taken at two weeks intervals from healthy controls.

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Table 3. Patients

Patients in the publication IIIDuodenal gut biopsies were obtained from 13 patients with RA and from 15 control subjects in whom gastroscopy was performed with clinical indications. In patients with RA the endoscopy was performed in the presence of abdominal pain or if bleeding was suspected. In the controls the endoscopy was performed in the presence of abdominal pain or symptoms of reflux. In addition to routine samples, three additional biopsies of duodenum were obtained in 0.9 % NaCl. In all of the cases, the duodenum was normal both by inspection and by histology.

Patients in the publication IVSerum samples from 57 recent-onset RA patients were obtained before the institution of antirheumatic drug therapy and three years later. All patients with recent-onset RA fulfilled the criteria for RA as established by the American College of Rheumatology (Felson et al, 1995).

Patients Publication Number ofpatients

Age, years, mean (range)

Genderfemale/ male

Markersstudied

RA I 20 55 (29-69) 16/ 4 Intracellularcytokines and

Otherarthritides I 9 44 (31-64) 2/7

CCRs in PB and SF

RA II 25 55 (34-76) 19/ 6 CRP, SAA, RF AFA, aCCP,

CCRs and Other

arthritides II 5 38 (22-56) 0/ 5 cytokines in PB before and after

infliximab treatment

Controls I, II 9 54 (51-65) 9/0 See I and II

RA III 13 59 (39-83) 12/ 1 Cytokines and Controls III 15 50 (30-77) 10/ 5 CCRs in gut

RA IV 57 42 (19-64) 47/ 10 Anti-MS IV 20 33 (17-44) 14/6 PAD

Controls IV 90 40 (36-46) 41/ 49 antibodies

48 49

2. Methods

Methods used in this thesis are listed in Table 4. The detailed description of the method can be found in the respective publication indicated for each method.

Table 4. Methods

Method Original publication

Cell separation from PB I, II

Cell separation from SF I

Stimulation of cells

1. with PHA II

2. with PMA +ionomycin I

Flow cytometry

1. Surface marker staining I, II, III

2. Intracellular staining I

Quantitative real-time RT-PCR II, III

ELISA for anti-PAD IV

ELISA for IFN-� and IL-5 II

ELISA for IL-4 and TNF-� II

ELISA for AFA II

ELISA for CRP, SAA and RF II

Table 4

48 49

RESULTS AND DISCUSSION

1. Autoantibodies in patients with chronic active RA (Publications II and IV)

Patients with RA had elevated levels of anti-PAD antibodies when compared to patients with multiple sclerosis (MS) or healthy controls (for medians and range see Table 5).

The levels of RF were elevated in patients with RA treated with infliximab when compared to controls (p<0.0001) however, they decreased significantly two weeks after the start of treatment with infliximab (Table 5).

Table 5. RF values of patients with RA and in patients with other arthritides before treatment with infliximab and two and six weeks after. Control samples were taken two weeks apart. Anti-PAD levels in patients with RA and MS and healthy controls are indicated at the bottom.

PAD, the enzyme responsible for the conversion of arginine-residues to citrulline and the subsequent formation of RA-specific epitopes on filaggrin (Girbal-Neuhauser et al., 1999, Schellekens et al, 1998), was shown to be an autoantigen in RA (publication IV). The expression of PAD enzymes type II and IV was recently demonstrated in PB and the SF cells of patients with RA, suggesting that PAD type II is involved in the citrullination of SF proteins during inflammation (Vossenaar et al., 2002).

Patients Publication n RFIU/ ml median, (range)

anti-PADOD 405nm median, (range)

RABefore treatmentwith infliximab

II 25 157 (3-437) ND

Two weeks after II 25 114 (4-406) NDSix weeks after II 20 267 (5-443) ND

Other arthritidesBefore treatmentwith infliximab

II 5 6 (3-12) ND

Two weeks after II 5 5 (3-12) NDSix weeks after II 5 5 (4-9) ND

Control subjects0 weeks I, II 9 4 (2-17) NDAt two weeks II 9 4 (2-18) ND

Early RA IV 57 ND 0.374 (0.1-2.7)MS IV 20 ND 0.042 (0.0-0.11)Control subjects IV 90 ND 0.094 (0.00-0.31) p<0.0001, p<0.005, ND= not done

50 51

Schellekens et al (Schellekens et al, 1998) and Girbal-Neuhauser et al (Girbal-Neuhauser et al, 1999) recently presented evidence that RA patients have autoantibodies against the modified filaggrin epitopes in synthetic peptides containing citrulline instead of arginine, but not against the unmodified peptides. Since antibodies recognise their epitopes based on their 3-dimensional structure the true nature of the disease-specific epitopes in natural filaggrin molecules still remains to be established. Our results show that the great majority of RA patients with recent-onset disease also have a humoral immune response against the filaggrin-modifying enzyme PAD. Whether the PAD-enzyme alone or as a complex with filaggrin forms the relevant antigen(s), is not known.

Interestingly, no anti-PAD antibodies could be detected in MS, a disease where the PAD-enzyme has been shown to play a role in the disease specific lesions (Moscarello et al., 1994). It may also be noted that antibodies to citrullinated peptides derived from myelin basic protein were not elevated in a recent study of MS patients (de Seze et al., 2001).

As a conclusion, the enzyme PAD, responsible for the formation of RA-specific citrulline-containing epitopes in filaggrin, was recognised as an autoantigen against which a high proportion of patients with RA showed IgG class antibodies. The immune response to PAD was not specific to RA and is therefore likely to have little if any diagnostic or clinical applicability. The observation is, however, of interest as it adds a new element to the chain of immune aberrations in inflammatory rheumatic diseases.

We saw a reduction of IgM RF levels in infliximab treated patients with RA. The present study is in line with an earlier report whereby a reduction in IgM-RF titers was seen following infliximab therapy in an open-labelled, randomized, placebo-controlled trial (Charles et al., 2000). In their study, the reduction was seen in patients who simultaneously developed anti-dsDNA antibodies.

50 51

2. Expression of chemokine receptors and cytokines in SF and PB of patients with chronic active RA (Publications I and II)

The number of SF -derived CD4 and CD8 T-cells and CD14 monocytes expressing CCR5 and CCR3 was increased when compared with the PB cell populations in patients with RA and in patients with other arthritides (Figure 8).

Figure 8. The percentage of CD3-gated CD8 and CD4 T-lymphocytes and CD14-gated monocytes expressing CCR5 (a) and CCR3 (b) in synovial fluid (SF) and peripheral blood (PB) from patients with RA. PB and SF cells were compared by paired t-test.

The CCR5/ CCR3 ratio of SF-derived CD4 T-cells was increased in the patients with RA when compared to patients with other arthritides (median 5.5 vs 2.4, p=0.039) (Figure 9a). The CCR5/CCR3 ratio of PB monocytes and CD8 T-cells differed significantly between patients with RA, patients with other arthritides and healthy controls (p= 0.013 and p=0.033 in Kruskal-Wallis test). When combined data was analysed, the ratio of CCR5+/ CCR3+ monocytes was higher in patients with RA than in patients with other arthritides or healthy controls (median 1.0 vs 0.5, p=0.013 and median 1.0 vs 0.3, p=0.004, respectively) (Figure 9b). In CD8 T-cells the ratio of CCR5+/ CCR3+ cells was higher in patients with RA and in patients with other arthritides than in healthy controls (median 0.5 vs 0.2, p=0.016 and median 0.8 vs 0.2, p=0.025, respectively).

The number of PB-derived monocytes expressing CCR3 in patients with RA and patients with other arthritides was decreased when compared to controls (median 0.8 vs 2.4, p=0.001 for RA, median 2.4 vs 0.75, p=0.010 for other arthritides).

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Figure 9. The ratio of CCR5- and CCR3-expressing CD3-gated CD4 T-cells a) in synovial fluid (SF) from patients with RA and patients with other arthritides and b) peripheral blood (PB) CD14+ monocytes from patients with RA, patients with other arthritides and healthy controls. The median values are indicated by horizontal lines and p-values from Mann-Whitney test are shown in the figure.

In patients with RA (Figure 10a), the number of CD8 T-cells spontaneously expressing IL-4 was higher in SF than in PB (median 0.2 vs 0.1, p=0.006). In patients with other arthritides (Figure 10b), the number of CD8 T-cells spontaneously expressing IFN-γ was also higher in SF than in PB (median 0.2 vs 0.1, p=0.030).

Figure 10. Percentage of cells with spontaneous cytokine (IL-4 and IFN-γ) production in synovial fluid (SF) and peripheral blood (PB) derived CD3-gated CD8+ T-cells in patients with RA (a) and with other arthritides (b). Peripheral blood (PB) and synovial fluid (SF) derived cells were compared by paired t-test. The median values are indicated by horizontal lines.

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When stimulated with mitogen (PHA), the PB mononuclear cells of patients with RA and of patients with other arthritides showed a decrease in the secretion of IFN-γ and also in the ratio of IFN-γ/ IL-4 mRNA when compared to controls (Table 6). Furthermore, the number of mitogen (PMA+ionomycin) stimulated PB CD4 T-cells expressing IFN-γ had decreased in patients with RA and in patients with other arthritides when compared to healthy controls (Table 6).

A decreased number of IFN-γ−expressing CD8 T-cells in PB was seen in patients with other arthritides after PMA +ionomycin stimulation when compared to healthy controls or to patients with RA (Table 6).

In patients with other arthritides, but not those with RA, a decrease in the expression of TNF-α mRNA as compared to controls was also noted (Table 6).

Spontaneous CCR5 expression correlated positively with spontaneous IFN-γ expression in CD8 T-cells from SF-derived cells of patients with RA (r=0.604, p=0.005) (Figure 11). No such correlations were seen in the patients with other arthritides.

Table 6. Cytokine expression in mitogen stimulated peripheral blood mononuclear cells (PBMC). The result from a comparison of patient groups to controls using the Mann-Whitney test is shown.Cytokine RA

median, (range) Other arthritides median, (range)

Healthy controls median, (range)

IFN-� (PHA) 1513 (0-18703)* 3753 (0-7370)* 11970 (6788-32082)

IFN-�/ IL-4 mRNA (PHA) 0.2 (0-13)* 0 (0-0.32)* 1.7 (0.3-6.7)

CD4+ IFN-�+ (PMA) 8.6 (2.0-21.8)* 6.7 (2.6-11.3)* 16.9 (8.0-22.8)

CD8+ IFN-�+ (PMA) 7.8 (1.2-25.8)� 3.1 (0.9-9.0)* 11.7 (6.9-30.5)

TNF-� mRNA (PHA) 277 (0-8324) 222 (0-338)* 645 (270-1701)

*= p<0.005, � = p=0.0025

Ficoll extracted PBMC was incubated with mitogen as follows:PHA= stimulation with 5 �g/ml phytohaemagglutinin (PHA) for 24h at 37�C, PMA= stimulated with 25 ng/ml PMA, 1�g/ml ionomycin and 10 �g/ml Brefeldin A (BFA) for 16-18 hours at 37�C. The result of comparison of patient groups to controls with Mann-Whitney test is shown.

54 55

Figure 11. Correlation between spontane-ous CCR5 and IFN-γ expression in syno-vial fluid (SF) derived CD8+ cells in patients with RA. The result from the Spearman’s correlation test is shown.

We studied here SF-derived immune cells, which are considered to reflect target tissue infiltrating cells. Although the synovial tissue infiltrating cells are the effector cells in the joint it has been shown that SF T-cells from RA patients can be pathogenic in transfer experiments and therefore represent the cell population present in the target tissue (Mima et al., 1995).

Recently many groups have reported an accumulation of CCR3 and CCR5 expressing T-cells and monocytes in the joints of patients with RA (Ruth et al, 2001, Katschke Jr et al, 2001, Patel et al., 2001, Suzuki et al, 1999). We also found CCR3 and CCR5 expressing T-cells and monocytes infiltrating the joints of patients with RA, but in our study this was also true for patients with other arthritides, as recently suggested by Bruhl et al. (Bruhl et al., 2001). The accumulation of both CCR5 and CCR3 positive cells in SF indicates the presence of Type 1 and Type 2 cells in the target tissue. Supporting this, we found that the intracellular levels of both IL-4 and IFN-γ were higher in target tissue-derived CD8 T-cells than in PB derived CD8 T-cells in both patient groups. The fact that CCR5/ CCR3 ratio of SF CD4 T-cells was higher in patients with RA than in patients with other arthritides, refers to a more vigorous Type 1 immune response in the inflamed tissue of the patients with RA when compared to the patients with other arthritides. Supporting the view that a Type 1 immune response is present in target tissue T-cells, we found ex vivo that the Type 1 chemokine receptor CCR5 correlated with the Type 1 cytokine IFN-γ in patients with RA. This observation is supported by an in vitro study by Suzuki and co-workers (Suzuki et al, 1999), who showed that CCR5 positive CD4 T-cells isolated from the SF of patients with RA produced IFN-γ upon stimulation with mitogen.

We saw also an increase in the CCR5/ CCR3 ratio of monocytes and a decrease in the number of CCR3 expressing monocytes in PB of patients with chronic active RA. This might indicate that the balance between the phenotypes of activated monocytes in PB reflects the type of immune response present in the target tissue. Several factors support the view that arthritis severity may be

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54 55

associated with the degree of systemic activation of monocytes/ macrophages. The appearance of highly proliferative colony-forming cells in PB, a high incidence of interstitial pulmonary involvement, overexpression of bone marrow stromal antigen-1 in serum and terminal differentiation of macrophages in rheumatoid nodules are all characteristic features of patients with severe RA (Santiago-Schwarz et al., 1996, Horie et al., 1997, Lee et al., 1996, Klimiuk et al., 1997, Palmer et al., 1987). The activation of peripheral monocytes, seen as an increased expression of CCR3, CCR4 and CCR5, has also been reported in patients with RA by Katchke and coworkers (Katschke Jr et al, 2001).

The present study detected poor responsiveness of PB cells to mitogen stimulation in patients with arthritides. This was evidenced by a decreased secretion of IFN-γ and a decrease in the ratio of IFN-γ/ IL-4 mRNA. Furthermore, we saw an exhaustion in both CD4 and CD8 T-cells. It has been reported that poor proliferation capacity and cytokine production upon mitogen and antigenic stimulation exists in PB T-cells in patients with RA (Allen et al., 1995a, Kitas et al., 1988). This could be explained by an impaired signal transduction via the CD3/ TCR complex in the SF cells of patients with RA (Maurice et al, 1997). Studies on signalling cascades have shown defects in overall phosphorylation, especially in the phosphorylation of a tyrosine kinase p38, and in protein folding of tyrosine kinase p56lck in patients with RA (Maurice et al, 1997, Romagnoli et al., 2001). Defects in the signalling cascade have also been demonstrated in peripheral mononuclear cells of patients with type 1 diabetes. The expression of tyrosine kinase p56lck has been reported to be very low and the absence of sufficient expression of the protein may be the cause of the hyporesponsive state of peripheral T-cells in diabetic patients (Nervi et al., 2000). In our study the hyporesponsiveness of T-cells was restricted to PB.

56 57

3. Expression of chemokine receptors and cytokines in the gut of patients with chronic active RA (Publication III)

In patients with RA, CCR4 mRNA levels in intestinal biopsies were higher than in control subjects (median 1 vs 0.2, p=0.001 for patients with RA) (Figure 12a).

The levels of CCR5 and IL-10 mRNA in intestinal biopsies were higher in patients with RA when compared to control subjects (median 61 vs 15, p=0.046 and median 1 vs 0.3, p=0.019) (Figure 12b, c). No differences were seen when compared to patients with other arthritides and to control subjects.

Figure 12. Chemokine receptor CCR4 (a) and CCR5 (b) and cytokine IL-10 (c) specific mRNA was detected by quantitative RT-PCR in duodenal bi-opsies from patients with RA and from healthy controls. Individual results are shown as a relative amount (2-ΔΔCt) of target the gene compared to the calibrator, both normalized to an endogenous reference (18S). The median values are indicated by horizontal lines and p-values of the Mann-Whitney test are shown in the pictures.

We saw an increased expression of CCR5 and CCR4 mRNA in the duodenal biopsies of patients with arthritides when compared to healthy controls, thus suggesting an altered immune response in the gut. Normal human small intestinal lymphocytes express CCR5 and CXCR3, the so called Type 1 immune response associated chemokine receptors, and lack the expression of CXCR1, CXCR2,

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CCR1, CCR3, CCR4 and CCR7 (Agace et al., 2000). A role for CCR5 as a receptor on gut-homing PB lymphocytes has been

suggested by Agace et al. (Agace et al, 2000). They showed that enhanced expression of CCR5 is associated with peripheral lymphocytes expressing the gut-associated homing marker β7.

Furthermore, the expression of RANTES, a ligand of CCR5, is increased in the inflamed intestinum (McCormack et al., 2001). The expression of CCR5 has been associated with a Type 1 immune response as well as with secretion of IFN-γ , but, despite findings related to CCR5 expression, we did not find increased expression of IFN-γ or IL-2 mRNA levels in our RA patients. We observed the highest levels of IFN-γ mRNA expression in RA, yet there was great variation in both patient and control groups.

CCR4 has been associated with a Type 2 immune response, but CCR4 positive cells are not exclusively Type 2 restricted and are also expressed on target tissues in immunological disease with Type 1 deviation (Andrew et al., 2001). Expression of CCR4 is found in the inflamed intestinum, but not on intestinal T-cells of healthy individuals (Agace et al, 2000). In an animal model of chronic intestinal inflammation, IL-10 deficient mice expressed CCR4, CCR2 and CCR6 mRNA locally in the inflamed mucosa (Scheerens et al., 2001). Another study showed that CCR4 was absent in the lung and skin of normal subjects, but was present in the lung of atopic patients (Nouri-Aria et al., 2002), hence supporting the notion that CCR4 is induced in inflamed mucosa. The increased CCR4 mRNA expression in the intestine of patients with RA can be considered as a marker of inflammation, however it is too speculative to draw conclusions on the T1/T2 polarisation of the mucosal immune response in RA.

IL-10 has a dual role in the inflammatory process as it displays both anti-inflammatory and pro-inflammatory potential. The role of IL-10 in intestinal homeostasis is evidenced by studies on IL-10 deficient mice, who develop chronic inflammation in the intestine with no obvious inflammatory lesions elsewhere (Kuhn et al., 1993). However, the anti-inflammatory role of IL-10 in RA has recently been questioned, since the detection of high IL-10 concentrations in the serum and SF (Cush et al, 1995, Moller et al, 2002). IL-10 has also been shown to correlate with serum RF titers and in vitro levels of spontaneous IgM RF production from PBMC (Cush et al, 1995) suggesting that the activation of IL-10 secretion is linked to inflammatory activity in RA. An increased number of IL-10-expressing CD3+ CD8+ T-cells in the ileal lamina propria lymphocytes has been reported in patients with spondyloarthropathy (Van Damme et al., 2001).

It has been proposed that many of the abnormalities seen in the gut of patients with RA may be the result of therapy with anti-rheumatic drugs, since the use of non-steroidal anti-inflammatory drugs (NSAID) can induce small intestinal damage. However, no correlation between the type, dose and duration of NSAID therapy and the severity of inflammation of the gut has been reported. Instead, an increase in the expression of HLA-DR, CD44 and TCR

58 59

γ/δ on mucosal lymphocytes was reported in patients with RA not treated with NSAIDs (Abuzakouk et al., 1999). The increase was significant when compared to osteoarthritis patients, who were treated with NSAIDs at least for six months prior to endoscopy.

In conclusion, patients with RA display evidence of inflammatory activation in the gut, which may play a role in the pathogenesis of the disease.

58 59

4. Expression of chemokine receptors and cytokines in patients with chronic active RA during anti-TNF-α treatment (Publication II)

In patients with RA, the number of CD4 T-cells and CD14 monocytes expressing CCR3 increased during the treatment. The number of CD4 T-cells expressing CCR3 increased between 0 and 2 weeks (median 0.4 vs 0.8, p= 0.028) and between 0 and 6 weeks (median 0.4 vs 0.6, p=0.013) (Figure 13a). The number of CD14 monocytes expressing CCR3 increased between 0 and 6 weeks (median 0.9 vs 2.4, p=0.009) (Figure 13b). No changes in the expression of CCR3 on CD8 T-cells were seen in patients with RA during the follow-up period.

Furthermore the expression of CCR5 on CD8 T-cells increased in patients with RA during the treatment, with the increase between 2 and 6 weeks being significant (median 0.13 vs 0.2, p=0.040) (Figure 13c). No changes were seen in the expression of CCR5 on CD4 T-cells or monocytes during the follow-up period.

Figure 13. Effect of infliximab treatment on the percentage of a) CD4 T-cells expressing CCR3 and c) CD8 T-cells expressing CCR5 collected from CD3-gate and b) CD14-gated monocytes expressing CCR3 studied with flow cytometry in patients with RA. The median values are indicated with horizontal lines.

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PHA-stimulated IFN-γ and IL-4 secretion from PBMC’s increased in the patients with RA during the six weeks period of treatment (medians 1513 vs 14190 pg/ml, p< 0.0001 for IFN-γ and medians 23 vs 42 pg/ml, p=0.044 for IL-4) (Figure 14a, b). This was especially noted in RA patients who showed an increase in IFN-γ by PBMC’s already after two weeks of treatment (medians 1513 vs 10780 pg/ml, p= 0.001) and this further increased between two and six weeks of follow-up times (median 10780 vs 14190 pg/ml, p= 0.022) (Figure 14a).

The IFN-γ and IL-4 secretion by PHA stimulated PBMC increased also in the patients with other arthritides during the first two weeks of treatment (median 3753 vs 4473 pg/ml, p=0.043 for IFN-γ and median 27 vs 54 pg/ml, p=0.043 for IL-4).

In addition, the ratio of IFN-γ/ IL-4 mRNA in PHA stimulated PBMC increased in both patient groups during the treatment. The increase between 0 and 6 weeks being significant in patients with RA (median 0.2 vs 1.5, p<0.0001) and for patients with other arthritides (median 0.1 vs 1.0, p=0.043). No changes were seen in the control subjects.

In patients with RA, TNF-α mRNA levels increased during treatment, whereby the increase between 2 and 6 weeks was significant (median 260 vs 752, p=0.009).

Figure 14. The effect of infliximab on the secretion of IFN-γ (a) and IL-4 (b) studied by ELISA from PHA stimulated peripheral blood mononuclear cells in patients with RA. The median values are indicated with horizontal lines.

The blockade of excess TNF-α by infliximab normalized the mitogen response of PBMC in patients with arthritides. This was evidenced by normal level production of both, IFN-γ and IL-4, after the first infusion of infliximab. It has been reported that in patients with RA the activation of Type 1 immune response, described as an increase in the number of PBMC spontaneously producing IFN-γ, occurs after four weeks of treatment with, etanercept, a soluble TNF receptor (Berg et al, 2001). The IFN-γ production by PBMC stimulated with microbial antigens (purified protein derivative, influenza virus) or autoantigen

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(collagen type II) was shown to increase in the RA patients during treatment. No increase in mitogen-stimulated IFN-γ production was detected, but instead, an increase in the secretion of IL-2 was observed. Increased secretion of IL-2 and IFN-γ has also been reported in patients with spondyloarthropathy receiving infliximab (Baeten et al., 2001). As a conclusion, our study together with those mentioned above, suggests that a recovery of an impaired Type 1 response is associated with TNF-α blockade. In our study we also found an increase in Type 2 associated cytokine secretion after PHA stimulation in patients receiving infliximab. Supporting this observation, Schuerwegh et al (Schuerwegh et al., 2003) reported an increase in the number of Type 2 T-cells twenty-four hours after treatment with infliximab in patients with RA.

Our observation that PHA stimulation increases the IFN-γ/IL-4 mRNA ratio suggests that there is a more pronounced effect on Type 1 immunity. This is supported by a study where the number of memory CD4 T-cells expressing IFN-γ increased after treatment with anti-TNF-α antibodies as compared to the number of IL-4 expressing CD4 T-cells (Maurice et al., 1999). However, also contradicting results exist. The rise in the number of IL-4-positive PB CD4 T-cells was more pronounced than the rise in IFN-γ or IL-2 positive CD4 T-cells, thus resulting in a significant increase in the Th2/ Th1 cytokine ratio in patients with RA treated with anti-TNF-α antibodies (Schuerwegh et al, 2003).

It has been reported that the chronic overproduction of TNF-α suppresses T-cell functions by impairing the activation of T-cells via the CD3/TCR complex, and that the treatment of patients with TNF-α blocking agent results in the recovery of T-cell responses (Cope et al., 1994). These findings of enhanced Type 1 immunity as a response to the blocking of TNF-α challenges the para-digm of pathogenicity of Type 1 cells in RA, as discussed also in the context of spondyloarthropathy (Baeten et al, 2001). It is possible that the recovery of T-cell function is associated with the recovery of regulatory mechanisms and that an impaired peripheral T-cell function may fundamentally be associated with autoimmunity.

We found that the number of T-cells and monocytes expressing CCR3 and CCR5 increased as a response to treatment for RA patients. This indicates activa-tion of peripheral T-cells and is in accordance with the increase in the mitogen stimulated cytokine response observed. It is also possible that the increase in the number of CCR3 and CCR5 expressing cells reflects the decreased accumulation of inflammatory cells from periphery to inflamed joints as suggested previously (Paleolog et al, 1996). In patients with other arthritides, we did not see a change in the expression of chemokine receptors during treatment. This might be due to the small number of and the heterogenous disease pattern among the patients studied.

It has been reported that TNF-α decreases CCR5 expression in PB mono-cytes and alveolar macrophages by the production of the CCR5 ligand RAN-TES (Lane et al., 1999). Downregulation of CCR5 as a response to TNF-α is also detected on dentritic cells (Sallusto et al., 1999). Hornung et al (Hornung

62 63

et al., 2000) reported a functional role for TNF-α in CCR5 expression on PB lymphocytes. They showed that TNF-α downregulated the expression of CCR5 on previously activated PB lymphocytes and delayed the expression of CCR5 on the cells that were early in the activation process. Also, CCR3 is downregulated by TNF-α as shown by Sato and co-workers (Sato et al., 2000). Thus, the increase in CCR3 and CCR5 expression on T-cells and monocytes can be explained by a rebound effect due to the blocking of TNF-α.

In our study we saw an increase in mitogen stimulated mRNA synthesis of TNF-α but no differences were seen at the protein level in PBMC. It has been reported that in PB, the number of cells producing TNF-α is increased in patients with RA both before and after the TNF-α blockade (Berg et al, 2001). However, observations on synovial tissue biopsies of patients with RA indicate a reduction in the number of TNF-α positive areas two weeks after treatment with infliximab (Ulfgren et al., 2000). In line with our study, they found that the treatment did not cease TNF-α production, yet the increase of TNF-α mRNA, seen in our study, was probably a result of the positive feedback from blocking soluble TNF-α.

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5. Expression of chemokine receptors and cytokines, and the number of T-cells in patients with chronic active RA responding and not responding to anti-TNF-α treatment (Publication II)

The effect of treatment was determined at two weeks follow-up before the second infusion of the TNF-α blocking agent.

The number of CD8 T-cells expressing CCR3 was higher before initiating treatment in non-responders as compared to responders (median 0.3 vs 0.1, p=0.022) (Figure 15a).

The number of CD4 T-cells expressing CCR3 and CCR5 were higher in patients not responding to the treatment as compared to the responders throughout the follow-up period. The difference between non-responders and responders for CCR5 was statistically significant before start of treatment (median 0.2 vs 0.1, p=0.013) (Figure 15b) and for CCR3 and CCR5 after 6 weeks of treat-ment (median 1.5 vs 0.3, p=0.020 and median 0.3 vs 0.1, p=0.029, respectively). A similar trend was also observed for CCR3 before start of treatment (median 0.5 vs 0.3, p=0.053) (Figure 15c).

In patients with RA, the number of CD3 T-cells increased in those responding to treatment and this kind of change was not seen in non-responders. When the change in the number of CD3 T-cells during treatment was compared, the responders and non-responders differed (p=0.001).

No statistically significant differences in other immunological parameters between responders and non-responders were seen [data not shown].

Studies investigating the biological differences between patients responding and not responding to the TNF-α blockade are few (van Vollenhoven et al, 2003).

In our study, we saw that when patients with RA were divided into responding and non-responding groups according to ACR 20% criteria, the patients respond-ing to the treatment had lower numbers of T-cells expressing CCR3 and CCR5 before the start of infusions when compared to the non-responders. High levels of CCR3 and CCR5 on CD4 and CD8 positive T-cells were thus predictive markers of a poor effect of treatment. This suggests that the activation of peripheral T-cells is an important mechanism for TNF-α blocking and it is possible that this treatment is most effective in those patients with an impaired peripheral T-cell function, due to excess TNF-α that downregulates chemokine receptors. The high levels of CCR3 and CCR5 on T-cells of the patients not responding to infliximab might indicate that in these patients the activity of RA is more depen-dent on other inflammatory mediators and less on TNF-α.

Some reports show that the number of circulating lymphocytes transiently increased in a dose-dependent fashion after monoclonal anti-TNF-α antibody treatment (Lorenz et al., 1996). In our study, the number of T-cells increased in the patients who showed good clinical response to the treatment, but no overall

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changes in T-cells were seen in the patient group as a whole. Our results suggest that change in T-cell number is related to the effect of treatment.

Figure 15. The percentage of CD8 (a) and CD4 (b, c) T-cells expressing CCR3 (a, c) and CCR5 (b), studied by flow cytometry, before the start of treatment in patients with RA responding and not responding to the treatment. The median values are indicated with horizontal lines.

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64 65

GENERAL DISCUSSION AND FUTURE ASPECTS

As RA is an autoimmune disease characterized by chronic inflammation of the joint, inflammatory mediators play an important role in the disease course and its severity. The chronically inflamed synovial membrane is characterized by leukocytic infiltration where monocytes and macrophages are especially abundant, yet T-cells are also found. In patients with RA, inflammatory mediators such as cytokines, chemokines and chemokine receptors, are currently under intensive investigation.

The precise role of chemokine receptors in the pathogenesis of autoimmune diseases is not known. Several lines of evidence show that Type 1-associated chemokine receptors are found in the target tissue of patients with Type 1-deviated autoimmune diseases, while Type 2-associated chemokine receptors are thought to play an important role in allergy.

In autoimmune disorders, such as MS and RA, an increased number of CCR5 and CXCR3 expressing cells have been found in the target tissue. It has been speculated that infiltration of Type 1 cells into inflammatory sites of these disorders is promoted by an enhanced and localized expression of chemokines that preferentially bind to Type 1 chemokine receptors. Evidence of selective production of CXCR3 and CCR5 ligands has been reported in MS and RA. While CXCR3 and CCR5 ligands, IP-10 and MIP-1α respectively, have been found in the brain lesions of patients with MS, no production of Type 2-associated chemokines have been detected (Balashov et al., 1999). In patients with RA, the expression of CCR5 ligands, MIP-1α and RANTES, occur at high levels in SF. However, since RANTES is also a ligand for CCR3, the accumulation of CCR3 expressing cells in the joints of patients with RA, as observed by us and others, may likely be due to chemoattraction by RANTES.

Chemokine receptor expression on peripheral blood-derived cells may reflect the type of immune reaction present in the target tissue of patients with autoimmune disorders. Supporting this view, elevated expression of CCR5 on CD3 T-cells was found in the PB from patients with progressive MS. In patients with RA, an increased expression of chemokine receptors and gp39, a late macrophage differentiation marker, on circulating monocytes, support the observation that monocyte/ macrophage lineage cells have an activated phenotype before entering the target tissue. Our results suggest that an elevated peripheral CCR5/ CCR3 ratio on monocytes might reflect the type of immune reaction present in the inflamed joint of patients with RA.

Besides TNF-α blockade, other biological treatments have recently been addressed in many studies (Onuffer et al., 2002, Adorini, 2003). These include chemokine receptor antagonists, antibodies to chemokines and soluble receptors against cytokines developed to treat autoimmune disorders and infectious diseases. Some of these molecules have been reported in clinical trials (Onuffer et al, 2002, Adorini, 2003).

66 67

Since chemokine receptors have a presumed role in acute and chronic inflammation, and in angiogenesis and angiostasis, they provide an attractive target for immune intervention. The major chemokine receptors targeted by current drug development programs include CCR1, CCR2, CCR3, CCR5, CXCR2, CXCR3 and CXCR4. The main indications for chemokine receptor antagonists at present are HIV infection, allergic diseases and MS. A MIP-1α antagonist is reported to be in clinical development for patients with RA (Onuffer et al, 2002). Since the SF has an elevated number of cells expressing several chemokine receptors, it would be of interest to treat RA patients with combined antagonist therapy to block the function of more than one chemokine receptor.

In recent years, there has been a resurgence in studies on the role of B-cells, in addition to T-cells, in rheumatic inflammation (Edwards et al., 2001, Takemura et al., 2001, De Vita et al., 2002). During the development of B-cell depleting agents, an interesting study was performed on patients with RA who did not respond to conventional treatment with DMARDs or to treatment with anti-TNF-α antibodies. In these patients, the depletion of B-cells with an anti-CD20 agent (rituximab) resulted in a marked and long lasting clinical improvement (De Vita et al, 2002). We found that high chemokine receptor expression was associated with a poor response to TNF-α blockade. It would be interesting to study if these patients with RA who responded to anti-CD20 treatment belonged to the subgroup of patients who had high chemokine receptor expression. The effect of anti-CD20 treatment in a subgroup of patients not responding to TNF-α blockade might indicate that in these patients the activity of RA is more dependent on other inflammatory mechanisms than TNF-α alone.

Studies on the effect of the TNF-α blockade to the cytokine environment in the target tissue are scarce. A recent report noted a decrease in TNF-α and IL-1 expression in the synovial membrane biopsies from patients with RA after a single infliximab infusion (Ulfgren et al, 2000), while another study did not find any effect of humanized D2E7 anti-TNF-α antibody on the local production of IL-1β or TNF-α in the synovium (Barrera et al., 2001). To our knowledge there are no studies to date on the effect of a TNF-α blockade on T-cell derived cytokine profiles in the target tissue. It would be of considerable interest to study if the rebalancing effect of the TNF-α blockade on immune mechanisms can also be seen in target tissue, such as that detected by us and others as normalized production of Type 1 and Type 2 cytokines and chemokine receptors in PB (Berg et al, 2001, Baeten et al, 2001, Nissinen et al., in press).

As TNF-α plays a central role in mucosal inflammation, the effect of a TNF-α blockade on gut-associated inflammatory cells in patients with Crohn’s disease has recently been the focus of many studies. Infliximab-induced apoptosis is seen in lamina propria T-cells and further, in circulating monocytes in patients with Crohn’s disease (Lugering et al., 2001, ten Hove et al., 2002). Also, an increase in the phosphorylation of tyrosine kinase p38, a protein involved in the intracellular signalling cascade leading to gene transcription, was seen when excess TNF-α was blocked. A single infusion of infliximab resulted in a significant

66 67

increase in the activity of mitogen-stimulated p38 in the mucosal macrophages of patients with inflammatory bowel disease (Waetzig et al., 2002). It has also been reported that infliximab downregulates IFN-γ production in activated gut T-lymphocytes from patients with Crohn’s disease (Agnholt et al., 2001). In patients with RA, decreased overall phosphorylation and conformational changes of tyrosine kinases have been reported in SF T-cells, resulting in defective T-cell signaling (Maurice et al, 1997, Romagnani, 2000). We saw in our study activation of gut-associated cells in patients with RA and it would thus be of interest to see the effect of a TNF-α blockade on signaling cascades, cytokine and chemokine receptor profiles in the gut of patients with RA after treatment with infliximab.

68 69

SUMMARY AND CONCLUSIONS

Patients with RA displayed both Type 1 and Type 2 immune cell accumulation in the inflamed tissue, an activation of the gut-associated immune cells and systemic humoral immune activation, as evidenced by a marked auto-antibody formation.

Higher concentrations of antibodies to PAD were detected in patients with RA when compared to patients with MS or healthy controls. Eventhough citrullinated myelin basic proteins are a characteristic feature of disease specific brain lesions in patients with MS, no autoantibodies to citrullinated peptides derived from myelin basic protein have been detected (de Seze et al, 2001). Neither, did we see any PAD specific auto-antibodies in patients with MS. On the contrary, in the majority of patients with RA in whom autoantibodies to citrullinated filaggrin and to filaggrin derived cyclic citrullinated peptides are seen, elevated levels of auto-antibodies to PAD were also detected.

Type 1 and Type 2 associated cytokines and chemokine receptors were expressed at elevated levels in the joints of patients with various arthritides. These observations further confirm previous findings of the nonspecific accumulation of inflammatory cells in the joints of patients with RA. Moreover, IFN-γ−expressing T-cells were shown to associate with the expression of CCR5 on T-cells in the SF of patients with RA, thus suggesting that Type 1 immune response takes place in RA joints. In addition, the increased ratio of CCR5/ CCR3 monocytes in the PB of patients with RA might reflect the type of immune response present in the target tissue.

The recovery of hyporesponsive peripheral T-cells in patients treated with infliximab was detected by an increased cytokine secretion from and an increased mRNA expression in mitogen stimulated cells as well as elevated expression of CCR3 and CCR5 on peripheral T-cells and monocytes. Since T-cells are hyporesponsive in patients with RA, perhaps due to excessive monokine expression, blocking TNF-α, the cytokine responsible for the production of other monocyte derived pro-inflammatory mediators, relieves the suppression seen on T-cells. Conversely, the increase in peripheral chemokine receptor expression might be due to a decrease in the accumulation of inflammatory cells from the periphery to the inflamed joints.

Before the start of infliximab treatment, we saw a higher chemokine receptor expression on T-cells of patients who did not respond during the follow-up period as opposed to patients responding to the treatment. This suggests that in patients not responding to treatment the activity of RA is more dependent on other inflammatory mediators and less on TNF-α. As a consequence of the treatment, the number of peripheral T-cells increased in patients responding to the treatment. This might be due to the blockade of the extravasation of these cells from the blood vessels into the inflamed tissues.

We also saw an increase in the mRNA expression of CCR4, CCR5 and IL-10 in the intestine of patients with RA. This suggests that an activation of the gut immune

68 69

system is related to the disease. However, these results should be confirmed at the protein level.

In conclusion, the present study demonstrates that chronic inflammation in patients with RA, which is characterized by infiltration of T-cells and monocytes expressing Type 1 and Type 2 chemokine receptors and cytokines, is rebalanced by the treatment with infliximab, an anti-TNF-α monoclonal antibody. The hyporesponsive state of T-cells was shown to normalize as a consequence of removal of excess TNF-α and, interestingly, patients not responding to the treatment were shown to have higher chemokine receptor levels before treatment with infliximab. The study also showed that the gut immune system is activated in patients with RA and suggests that this activation might contribute to the pathogenesis of the disease.

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