UNIVERSITÄT KONSTANZ
FACHBEREICH BIOLOGIE
Role of cellular and molecular factors involved in the K/BxN sera induced arthritis
Dissertation
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr.rer.nat) an der Universität Konstanz
(Fachbereich Biologie)
Vorgelegt von
Narendiran Rajasekaran
Tag der mündlichen Prüfung: 7th November 2005 1. Referent: Prof. Harald Illges (Immunology)
2. Referent: Prof. Albrecht Wendel (Biochemical Pharmacology)
i
Dedicated to:
My Parents & Teachers
ii
“Everything must be transformed by the Knowledge of the truth.”
The Mother
“Impossibility is only the sum of greater unrealized possibilities.
It veils an advanced stage and a yet unaccomplished journey.”
Sri Aurobindo
“Whoso forsaketh all desires and goeth onwards free from all yearnings, selfless and without egosim he goeth to peace”
The Bhagavad Gita, Second discourse, Verse 71
iii
Acknowledgements
Thank you!
Prof. Harald Illges for providing me this opportunity to work in Germany for my doctoral thesis and for being my doctoral father. I thank you for the productive discussions and suggestions during my PhD work, and for evaluating my thesis.
Prof. Steffen Gay for the lively discussions we had on histology and on the blood monocyte study. I am also grateful for your consent to be my examiner.
Prof. Albrecht Wendel for accepting to be my examiner.
Prof. Rolf Brauer and Dr. Marion Huckel for your help with mice techniques.
Prof. Rolf Knippers for all your advice and constructive criticisms that helped me improve my oral presentations.
Elvira Jeisy Walder for doing all the histology in this study. I thank you for being a wonderful friend and for all your crucial support during this work.
Urlike Beck and Elizabeth Naidoo for your assistance in my lab work. Thank you Ulli for always being there for me.
Prof. Sameh Basta for your valuable suggestions during this work and for the lively discussions we had in immunology. I especially thank you for your patience in teaching me Flow Cytometry and for all your encouragement.
Heidi Henseliet and Andrea for your help in the animal house. I thank Heidi for being my translator in the animal house. I also extend my thanks to all the people working in the animal house for their day-to-day assistance in my animal experiments.
Melanie, Vera, Anette, Eva, Danny and Sarah for being great friends and colleagues. I also thank Melanie for helping me write the German version of thesis summary.
All the members of the Department of Immunology, floor M10 and P11 for your support and co- operation.
Fr.Dreher, Karim, Judith and Mayo for making me feel at home in Konstanz.
All my Indian friends in the University of Konstanz for being good company.
Lawrence, Subhasis and Sam, my former lab mates, for making my initial days in Konstanz comfortable.
Brigette Schanze for the secretarial assistance.
Mom, Dad and Maheswari for all your love and support.
Thanks to my best friend Madhan who was instrumental in beginning my research career in Germany. You have been a great motivation to me ever since our acquaintance in JIPMER and you continue to be so.
Your positive criticisms during my PhD did help to bring the best out of me.
Last but not the least
Prof. Marcus Groettrup for your timely help and inspiration during the final stages of my PhD.
iv
Abbreviations
Ab Antibody Ag Antigen
DAF Decay Accelerating Factor DC Dendritic Cell
EDTA EthyleneDiamineTetraacetic Acid FACS Fluorescence Activated Cell Sorter FGF Fibroblast Growth Factor FITC Fluorescein Isothiocyanate GC Germinal Center
GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor GPI Glucose-6-Phosphate-Isomerase
i.p Intra peritoneal IC Immune Complex Ig Immunoglobulin IL Interleukin k Da Kilo Dalton
l, ml, µl Liter, Milliliter, Micro liter M, mM Molar, Millimolar
MA Milli ampere
MAC Membrane Attack Complex MBL Mannose Binding Lectin
MCP Monocyte Chemoattractant Protein MIP Macrophage Inflammatory Protein NK cells Natural Killer cells
NOD Non-obese Diabetic O.D Optical Density PBL Peripheral Blood Lymphocytes PBS Phosphate-Buffered Saline PDGF Platelet-Derived Growth Factor PE Phycoerythrin RA Rheumatoid Arthritis
RANKL Receptor Activator of Nuclear-κβ Ligand RF Rheumatoid Factor
SCR Short Consensus Repeat SLE Systemic Lupus Erythematosus TCR T Cell Receptor
TGF Transforming Growth Factor TNF Tumor Necrosis Factor
TRANCE TNF-related Activation-induced Cytokine Receptor v/v, w/v volume / volume, weight / volume
VCAM Vascular Cell Adhesion Molecule VEGF Vascular Endothelial Growth Factor WASP Wiskott Aldrich Syndrome Protein
v
CONTENTS
1 INTRODUCTION ... 1
1.1 AUTOIMMUNITY... 1
1.2 RHEUMATOID ARTHRITIS... 4
1.2.1 Clinical and pathological manifestations of rheumatoid arthritis ... 4
1.2.2 Autoantibodies in rheumatoid arthritis... 8
1.2.3 Genetic factors ... 9
1.2.4 Role of T and B lymphocytes in rheumatoid arthritis ... 10
1.2.5 Role of Monocytes/Macrophages... 11
1.2.6 Cytokines in rheumatoid arthritis ... 11
1.2.7 Animal models of rheumatoid arthritis ... 15
1.3 THE LY6C LOW SUBSET OF BLOOD MONOCYTES INVOLVED IN INITIATION OF K/BXN SERA INDUCED ARTHRITIS... 19
1.3.1 Macrophages in rheumatoid arthritis... 19
1.3.2 Monocytes and macrophages... 20
1.3.3 Macrophages in inflammation ... 22
1.3.4 Why study macrophages in rheumatoid arthritis?... 23
1.3.5 Monocytes in K/BxN sera induced arthritis... 26
1.3.6 Defining blood monocytes... 29 1.4 INTRA-ARTICULAR INJECTION OF K/BXN SERA INDUCES ARTHRITIS IN BALB/C MICE: A NEW MODEL TO STUDY THE ACUTE PHASE OF DISEASE INDEPENDENT OF
vi
SYSTEMIC ACTIVATION... 31
1.5 ROLE OF TRANSCRIPTIONAL FACTOR STAT1 IN THE SYNOVIUM OF K/BXN SERA INDUCED ARTHRITIS... 32
1.6 ROLE OF MMP-9 IN K/BXN SERA INDUCED ARTHRITIS... 35
1.7 ROLE OF WISKOTT ALDRICH SYNDROME PROTEIN AND CD40 IN K/BXN SERA INDUCED ARTHRITIS... 38
1.7.1 CD40 ... 38
1.7.2 Wiskott Aldrich Syndrome protein ... 40
1.8 ROLE OF IL-12 REQUIRED FOR K/BXN SERA INDUCED ARTHRITIS... 41
1.9 ROLE OF HISTAMINE RECEPTORS IN K/BXN SERA INDUCED ARTHRITIS... 45
1.10 PERIARTICULAR MANIFESTATIONS OF K/BXN MURINE MODEL OF RHEUMATOID ARTHRITIS: DO ANTI–GPI ANTIBODIES MEDIATE PATHOGENESIS INDEPENDENT OF INFLAMMATION?... 49
2 AIMS OF THE PRESENT INVESTIGATION... 50
3 MATERIALS AND METHODS ... 52
3.1 MOUSE STRAINS... 52
3.1.1 KRN transgenic mouse strain ... 52
3.1.2 K/BxN mouse strain ... 52
3.1.3 Knock out mice... 53
3.1.4 BALB/c ... 53
3.2 INDUCTION OF ARTHRITIS IN MICE USING K/BXN SERA TRANSFER... 53
3.2.1 Preparation of sera for K/BxN sera transfer ... 53 vii
3.2.2 Induction of arthritis by K/BxN serum transfer ... 54
3.2.3 Intra-articular knee injections to induce arthritis ... 54
3.2.4 Measurement of knee thickness for intraaricularly induced arthritis ... 55
3.3 HISTOLOGY... 55
3.3.1 Tissue fixation ... 55
3.3.2 Decalcification... 56
3.3.3 Hematoxylin and eosin staining... 56
3.3.4 Toludine blue staining... 57
3.4 GELATIN ZYMOGRAPHY... 57
3.5 MACROPHAGE DEPLETION... 58
3.6 FLOW CYTOMETRY... 59
3.6.1 Preparation of cell suspensions from the blood and synovium for flow cytometry 59 3.6.2 Fluorescent staining of cells ... 60
4 RESULTS ... 61
4.1 THE LY6C LOW SUBSET OF BLOOD MONOCYTES INVOLVED IN INITIATION OF K/BXN SERA INDUCED ARTHRITIS... 61
4.1.1 Intra-peritoneal administration of liposomal clodronate does not deplete the synovial lining of the joints. ... 61
4.1.2 Defining subpopulations in blood monocytes ... 63
4.1.3 Status of blood monocytes on clodronate treatment ... 63
4.1.4 Onset of arthritis associated with an increase in Ly6Clow monocytes ... 66
viii
4.1.5 Blood monocytes migrate into the synovial joint during arthritis. ... 68
4.1.6 A single treatment of liposomal clodronate failed to protect mice from arthritis. 71 4.1.7 Clodronate treatment after disease onset does not prevent sera induced arthritis. 73 4.2 INTRA-ARTICULAR INJECTION OF K/BXN SERA INDUCES ARTHRITIS IN BALB/C MICE 76 4.3 TRANSCRIPTIONAL FACTOR STAT1 IS NOT ESSENTIAL IN THE SYNOVIUM OF K/BXN SERA INDUCED ARTHRITIS... 81
4.4 MMP-9 ASSOCIATED WITH INFLAMMATORY PHASE OF K/BXN SERA INDUCED ARTHRITIS... 82
4.4.1 Zymography on ankle protein extracts in arthritic mice to analyze gelatinolytic activity of MMP-9 and MMP-2... 82
4.4.2 A reduced arthritis in MMP-9-/- mice ... 84
4.5 WASP AND CD40 ARE NOT ESSENTIAL IN SERA-INDUCED ARTHRITIS... 89
4.6 IL-12 NOT REQUIRED FOR K/BXN SERA INDUCED ARTHRITIS... 92
4.7 ROLE OF HISTAMINE RECEPTORS IN K/BXN SERA INDUCED ARTHRITIS... 96
4.8 PERIARTICULAR MANIFESTATIONS OF K/BXN MURINE MODEL OF RHEUMATOID ARTHRITIS:... 96
4.8.1 Cartilage and bone ... 99
4.8.2 Muscle and tendons... 99
4.8.3 Periosteum ... 103
ix
5 DISCUSSION ... 106
5.1 THE LY6C LOW SUBSET OF BLOOD MONOCYTES INVOLVED IN INITIATION OF K/BXN SERA INDUCED ARTHRITIS... 106
5.2 INTRA-ARTICULAR INJECTION OF K/BXN SERA INDUCES ARTHRITIS IN BALB/C MICE 111 5.3 TRANSCRIPTIONAL FACTOR STAT1 IS NOT ESSENTIAL IN THE SYNOVIUM OF K/BXN SERA INDUCED ARTHRITIS... 113
5.4 MMP-9 ASSOCIATED WITH INFLAMMATORY PHASE OF K/BXN SERA INDUCED ARTHRITIS... 114
5.5 WISKOTT ALDRICH SYNDROME PROTEIN AND CD40 ARE NOT INVOLVED IN K/BXN SERA INDUCED ARTHRITIS... 117
5.6 IL-12 NOT REQUIRED FOR K/BXN SERA INDUCED ARTHRITIS... 118
5.7 HISTAMINE RECEPTORS H1 AND H2 ARE NOT ESSENTIAL IN K/BXN SERA INDUCED ARTHRITIS... 119
5.8 PERIARTICULAR MANIFESTATIONS OF K/BXN MURINE MODEL OF RHEUMATOID ARTHRITIS... 120
6 SUMMARY ... 126
7 ZUSAMMENFASSUNG ... 129
8 REFERENCES... 133
x
1 Introduction
1.1 Autoimmunity
Autoimmune disease occurs when a specific adaptive immune response is mounted against self antigens. Normally an adaptive immune response results in the clearance of the antigen from the body. Virus infected cells are removed by cytotoxic T cells and soluble antigens are cleared by the formation of immune complexes, which are subsequently cleared from the system by the macrophages. However, when an adaptive immune response develops against a self antigen the response is sustained since the antigen cannot be cleared completely. This results in the effector pathways of the immune response to cause a chronic inflammatory injury to the tissues.
The immune system normally acquires self-tolerance by clonal deletion of autoreactive T cells in the thymus in the prenatal period and by clonal deletion or functional suppression of autoreactive T and B cells at later stages of the development. In autoimmunity there is a failure in the maintenance of self-tolerance, a failure to distinguish between self and non-self antigens, and an autoimmune response, characterized by the activation and clonal expansion of autoreactive lymphocytes and production of autoantibodies, is produced against the autologous antigens of the normal body tissues.
Studies with twins and families have shown an important role for both the environmental and genetic factors in the induction of autoimmune diseases. Consistent association of 1
autoimmunity is with the MHC genotype particularly the MHC class II alleles but in some cases there is a strong association event with MHC class I alleles. The association of MHC with autoimmune disease is not surprising since autoimmune diseases involve T cells and the ability of T cells to respond to a particular antigen depends on the MHC genotype. Thus the susceptibility to autoimmune disease is determined by differences in the ability of different allelic variants of the MHC molecules to present autoantigenic peptides to autoreactive T cells. Another hypothesis for this association is that self peptides associated with certain MHC molecules may drive positive selection of developing thymocytes that are specific for particular autoantigens. Such autoantigens may be present at too low levels to drive a negative thymic selection but be present at sufficient levels to drive positive intrathymic selection. Alternatively, the presence of two different TCR on a single T cell may cause autoimmunity.
Several other families of genes may be important in increasing the susceptibility to autoimmune disease. In humans the inherited deficiency of members of the classical complement pathway is strongly associated with the development of systemic lupus erythematosus (SLE). In mice and humans abnormalities in apoptosis due to defects in genes regulating apoptosis like Fas (CD95) and Fas ligand (CD95 ligand) are strongly associated with SLE. Inherited variations in secretion of certain cytokines are also associated with autoimmune disease.
The mechanism of tissue injury in autoimmunity is similar to that taking place in hypersensitivity reactions. As in case of hypersensitivity tissue damage is mediated by
2
both T cells and antibodies. The tissue injury can be due to IgG or IgM responses to autoantigens located on cell surfaces or the extracellular matrix or can be mediated by immune complexes containing autoantibodies to soluble autoantigens. The latter autoimmune responses are systemic and are characterized by autoimmune vasculitis.
Activated effector T cells specific for the self peptide: self MHC complexes can cause local inflammation by activation of macrophages or may even directly damage the tissue cells. Rheumatoid arthritis, insulin-dependent diabetes mellitus and multiple sclerosis are some such examples.
Autoimmune diseases can be classified broadly into two categories: organ specific and non-organ specific. In organ specific autoimmunity inflammation or dysfunction is produced by the autoantibody or cell mediated reactions against a specific target antigen located in a specialized tissue or organ. Examples are Autoimmune hemolytic anemia (erythrocyte specific autoantibodies), Hashimoto’s thyroditis (thyroid autoantibodies and autoreactive T cells), Myasthenia gravis (acetylcholine receptor autoantibodies), Grave’s disease (thyrotropin receptor autoantibodies) and Type I insulin-dependent diabetes (pancreatic beta-cell autoreactive T cells and autoantibodies). In systemic autoimmune disease tissue injury and inflammation occur in multiple sites in organs without relation to their antigenic makeup and are usually initiated by the vascular leakage and tissue deposition of circulating autologous immune complexes. The autoantibodies are produced against ubiquitous nuclear or cytoplasmic antigens. Systemic lupus erythematosus (SLE) is a good example where multiple autoantibodies are produced
3
particularly anti-nuclear and anti-DNA antibodies. A classification of common autoimmune diseases is given in Table-1.
1.2 Rheumatoid arthritis
Rheumatoid arthritis (RA) is a common autoimmune disease with a prevalence of 1% .Its etiology is complex with immunological, genetic and hormonal factors determining its development. Inflammatory and degenerative lesions of the distal joints, frequently associated with multiorgan involvement, characterize rheumatoid arthritis. The disease waxes and wanes for many years followed by a chronic stage of the disease, which is associated with deformity and functional impairment.
1.2.1 Clinical and pathological manifestations of rheumatoid arthritis
The most common clinical manifestations of rheumatoid arthritis is the association of pain, swelling and stiffness of the metacarpo-phalangeal (sole of the foot) and wrist joints. The disease is initially limited to the small distal joints but with time it progresses from the distal to the proximal joints and in the late stages the ankles, knees and elbows are also involved.
4
Table-1
Some common autoimmune diseases
Syndrome Autoantigen Consequence Antibody to cell surface or matrix antigens
Autoimmune hemolytic
anemia Rh blood group antigen,
I antigen Destruction of red blood cells by the complement and
phagocytes, anemia Autoimmune
thrombocytopenic
Platelet integrin GpIIb:IIIa
Abnormal bleeding Goodpasture syndrome Non-collagenous domain of
basement membrane collagen type IV
Glomerulonephritis Pulmonary hemorrhage Pemphigus vulgaris Epidermal cadherin Blistering of skin Acute rheumatic fever Streptococcal cell wall
antigens. Antibodies cross react with the cardiac
muscle
Arthritis, myocarditis, late scarring of heart valves Immune complex disease
Mixed essential cryoglobulinemia
Rhematoid factor IgG complexes (with or without
hepatitis C antigens)
Systemic vasculitis
Systemic lupus
erythematosus DNA, histones, ribosomes,
snRNP, scRNP Glomerulonephritis, vasculitis, arthritis T cell mediated
Insulin-dependent
diabetes melitus Pancreatic β-cell antigen β-cell destruction Rheumatoid arthritis Unknown synovial joint
antigen
Joint inflammation and destruction Experimental
autoimmune encephalomyelitits
(EAE), Multiple sclerosis
Myelin basic protein, proteolipid protein, myelin
oligodendrocyte glycoprotein
Brain invasion by CD4 T cells, paralysis
5
In the early stages of the disease the inflammatory lesion is limited to the lining of the normal diarthrodial joint. The normal synovial lining is a thin membrane composed of two types of synoviocytes, type A synoviocyte and the type B synoviocyte (Figure 1).
The type A synoviocyte is a phagocytic cell of the monocytes/macrophage lineage and has a rapid turn over. The type B synoviocyte is a specialized fibroblast. This cellular lining is located on top of a loose acellular stroma that contains numerous capillaries.
Under normal conditions, the synovial lining provides nutrients for the maintenance of the healthy cartilage and the proteoglycans in the synovial fluid provide lubrication for the articular surface. But in RA deformative tissue destruction is due to abnormal functioning of these cells (Figure 2). The earliest pathological changes, seen with the onset of the first symptoms, affect the endothelium of the microvasculature, whose permeability is increased, causing edema and a sparse inflammatory infiltrate, consisting predominantly of neutrophils, into the subsynovial space
In the chronic stage, the size and number of the synovial lining cells increases and synovial membrane takes a villous appearance. There is a massive infiltration of joint cavity by lymphocytes, plasmablasts, and granulation tissue forming what is known as pannus. The pannus tissue behaves to some extent like a tumor and continues to grow in the following months and invades into the joint. The synovial space is filled by the exudative fluid. The invasive pannus erodes the cartilage and there is a progressive destruction of the bones and tendons leading to severe pain and limitation of movement.
6
Figure 1. The structural organization of cells in a normal synovium
Figure 2. Rheumatoid synovium showing major cell types involved and sites of joint destruction (Adapted from Feldman M et al)
7
In RA there are frequent signs and symptoms of a systemic disease like vasculitis. The most frequent sign is the formation of rheumatoid nodules over pressure areas like the elbows.
1.2.2 Autoantibodies in rheumatoid arthritis
Rheumatoid arthritis is associated with rheumatoid factor (RF) and other anti- immunoglobulin antibodies. RF is an IgM antibody against autologous IgG. The affinity of IgM rheumatoid factor is relatively low. The antigenic determinants recognized by RF are located on the Cγ2 and Cγ3 domains of IgG and RF mostly reacts with IgG1, IgG2 and IgG4. RF is neither specific nor diagnostic for RA. It is found in only 70-80% of RA cases while it can be found in other conditions like Sjorgens syndrome. Also RF antibodies are present in normal individuals. However, high titers of RF seem to be associated with more rapid progression of articular disease. The primary pathogenic role of RF was described by Zvaifler et al (Matsumoto et al. 2003). In this model immune complexes formed by RF and other autoantibodies fix complement and release chemotoactic factors like C5a. Inflammatory cells are subsequently recruited to the rheumatoid joint along a chemotactic gradient where they are activated and contribute to local destruction. Neutrophils, in particular, accumulate in synovial fluid where they engulf immune complexes and release proteolytic enzymes. Considerable data is available to support this hypothesis. Other autoantibodies have also been associated with RA. Anticollagen antibodies reacting with different types of collagen have been detected in 15-20% of patients. This low frequency may indicate that they may not have a pathogenic role but arise as a response to the degradation of articular collagen, which
8
could yield immunogenic peptides. Antibodies against single-stranded DNA can be detected in about one-third of RA patients. The epitopes recognized by anti-ssDNA antibodies correspond to DNA associated proteins. But these antibodies are not involved in immune complex formation. Recently antibodies against the ubiquitous glycolytic enzyme Glucose-6-phosphate isomerase have been detected in humans. But they are neither specific nor sensitive for RA (Kassahn et al. 2002; Matsumoto et al. 2003; van Gaalen et al. 2004) .
1.2.3 Genetic factors
A number of autoimmune diseases have been linked to one or more alleles of the major histocompatibility complex (MHC). In 1970, investigators noted that RA patients of northern European origin exhibited an increased prevalence of the MHC Class II gene, DR4, compared to control subjects. In southern European patients with RA, DR1 was the predominant HLA haplotype. Only specific subtypes of DR4 and DR1 were associated with the development (or severity) of RA, however. For example, the specific DR4 subtypes DRB1*0401 (Dw4), DRB1*0404 and *0408 (Dw14), and DRB1*0405 (Dw15) appeared to convey susceptibility to development of RA, while subtypes (e.g., DRB1*0402 (Dw10) and DRB1*0403 (Dw13) did not. This led to the search for a common sequence among the different HLA DRB1 alleles that were associated with RA.
Susceptible alleles bind a negatively charged amino acid at the p4 pocket of the binding groove (Woulfe et al. 1995). Mutation analysis revealed that position 71 of the DRb chain in particular correlates with the genetic linkage of RA susceptibility (Hammer et al.
1995). How this linkage leads to a proinflammatory response needs to be determined.
9
1.2.4 Role of T and B lymphocytes in rheumatoid arthritis
The prominent T cell infiltrate in the inflamed rheumatoid synovium suggests that they are important factors in this disease. A major genetic predisposition of the disease lies in the HLA-DR locus. As the only known function of DR is to present peptides to CD4+ Tcells this genetic association clearly indicates a role for T cells in some stage of the disease. It could be in defining the T cell repertoire of antigen receptors or in presentation of autoantigens. However, the autoantigen recognized by autoreactive T cells is not known. Many studies suggest that the TCR Vβ chain repertoire is skewed in both the joint and peripheral blood of RA patients (Goronzy et al. 1994; Gonzalez-Quintial et al.
1996). The role of T cells in the inflammatory process of the disease is still not clear.
Firestein and Zvaifler (Firestein and Zvaifler 1990) suggest that T cells may not be important in perpetuating the disease at late stages based on the lack of any evidence of T cell proliferation in the synovium and the low levels of T cell derived cytokines in the inflamed joint. This is further supported by failed therapeutic trials targeting T cell functions. Further isolated synovial fibroblasts can exert their invasive and destructive capacities in the absence of T cells and T cell derived cytokines.
Takameura et al suggest that B cells could be important driving factors in RA and T cell activation in RA synovium is B cell dependent. Their conclusions were based on the fact that T cells derived from germinal centers of tertiary lymphoid follicles showed enhanced pro-inflammatory cytokine production and presence of B cells in the germinal centers was required for this. A significant proportion of B cell clones also produce rheumatoid factors (RF).
10
1.2.5 Role of Monocytes/Macrophages
The synovial infiltrates are rich in activated monocytes and macrophages that play a critical role in the pathogenesis. (Figure 3) One of the significant roles of macrophages is antigen presentation to CD4+ lymphocytes. Macrophage lymphocyte clusters are commonly seen in the inflamed synovium and the macrophages expressing high levels of MHC class II antigens are in close association with CD4+ lymphocytes. The other critical role of synovial macrophages is in the induction and perpetuation of local inflammatory changes in the joint. They occur at the cartilage pannus junction and known to mediate cartilage and bone destruction through secretion of matrixmetalloproteases. They also secrete proinflammatory cytokines like IL-1, IL-6, IL-10, IL-13, IL-15, IL-18, tumour necrosis factor (TNF)-α and Granulocyte–Macrophage Colony-Stimulating Factor (GM- CSF), chemokines and chemoattractants [eg IL-8, macrophage inflammatory protein (MIP)-1 and monocyte chemoattractant protein (MCP)-1], and neopterin (Burmester et al. 1997).
1.2.6 Cytokines in rheumatoid arthritis
Analysis of cytokine mRNA and protein in rheumatoid arthritis tissue revealed that many proinflammatory cytokines such as TNF-α, IL-1, IL-6, GM-CSF, and chemokines such as IL-8 are abundant. This is compensated to some degree by the increased production of
11
Figure 3. A schematic diagram of pathogenesis of rheumatoid arthritis
12
anti-inflammatory cytokines like IL-10 and TGF-β and cytokine inhibitors such as IL1-ra and soluble TNF-R. However this upregulation of the anti-inflammatory cytokines is not sufficient to neutralize all the TNF-α and IL-1 that are produced. Rheumatoid joint cellcultures spontaneously produce TNF-α and IL-1 with out need for any extrinsic stimulation. Neutralizing TNF-α with anti- TNF-α antibodies it was found that production of cytokines like IL-1, GM-CSF, IL-6 and IL-8 were greatly reduced (Brennan et al. 1989). Blocking IL-1 using IL-1 receptor antagonist reduced IL-6 and IL- 8 production, but not that of TNF-α. These results suggested that the cytokines in RA were involved in a network and blocking TNF-α can block the production of other cytokines, making it a good candidate for therapy. A balance of proinflammatory and inflammatory cytokines exists in the synovium and disturbance in this balance contributes to the pathogenesis in the synovium (Figure 4).
Finally putting together the pathogenic aspect of RA into a perspective, two theories are suggested regarding the pathogenesis of rheumatoid arthritis (RA). The first holds that the T cell, through interaction with an - as yet unidentified - antigen, is the primary cell responsible for initiating the disease as well as for driving the chronic inflammatory process. This theory is based upon the known association of RA with class II major histocompatability antigens, the large number of CD4+ T cells and skewed T cell receptor gene usage in the RA synovium. The second theory holds that, while T cells may be important in initiating the disease, chronic inflammation is self-perpetuated by macrophages and fibroblasts in a T-cell independent manner. This theory is based upon
13
Figure 4. The key cytokines in rheumatoid arthritis
14
the relative absence of activated T cells phenotypes in chronic RA and the preponderance of activated macrophage and fibroblast phenotypes. There is also a controversy as to whether the process of joint destruction is directly driven by the inflammatory process or the basic process of joint destruction with fibroblast proliferation and activation of other cells is driven independently of the inflammatory process. Unraveling the pathogenesis of RA is thus necessary to evolve therapeutic approaches.
1.2.7 Animal models of rheumatoid arthritis
Animal models are necessary to study the pathogenic mechanisms of disease that occur in humans. Many elegant models of transgenic and gene disrupted mice serve as tools to decipher the pathogenic mechanisms of rheumatoid arthritis. But no animal model can fully resemble RA in humans since most animal models run for weeks to months but human disease takes years to develop and shows heterogeneity between patients. Since RA is a very complex disease with multiple factors involved in disease it is difficult to search for a dominant arthritogen and animal models can only be used to find putative antigens. However they can be used to study common and selective pathways that cause inflammation and chronic bone and cartilage destruction. Animal models are important for screening novel treatment agents that can be subsequently used in trials with RA patients.
The earliest model of arthritis was the rat adjuvant induced arthritis. In 1954 Stoerk et al observed that rats injected with extracts of spleen as an emulsion in Freund’s complete adjuvant developed a chronic polyarthritis. Pearson and Wood found that by just
15
injecting Freund’s complete adjuvant into the skin arthritis can be induced but adding macerated tissue enhanced arthritis. They further modified this procedure by injecting heat killed tubercle bacteria in mineral oil into footpad of rats to induce arthritis. This has been adopted as the standard procedure for rat Adjuvant Induced Arthritis (AIA).
Streptococcal cell wall induced arthritis is another model of rat arthritis where injection of complexes of peptidoglycan and polysaccharide from group A streptococci called 10S PG-PS is injected intraperitoneally. The most important mice models are Collagen induced arthritis (CIA) and adjuvant induced arthritis using methylated bovine serum albumin. The murine collagen induced arthritis model has been extensively used to study RA. Unlike the adjuvant induced arthritis models, where the pathology involves T cells and no immune complex formation occurs, the CIA is mixture of immune complex mediated disease and delayed type hypersensitivity reaction in the joint. It is induced by immunizing mice with type II collagen in Freund’s complete adjuvant. The anti-CII antibodies are also able to induce arthritis by passive transfer. Passive transfer with T cells from disease mice also yielded disease. This model is highly suitable and widely used to try to understand the immunoregulation of autoimmune arthritis and to identify ways to induce tolerance or to selectively target T cell receptors involved in collagen epitope recognition.
The recent K/BxN model is a spontaneous model of arthritis wherein arthritis occurs in mice that express both the transgene encoded KRN T cell receptor and the IAg7 MHC class II allele (Kouskoff et al. 1996; Mangialaio et al. 1999). These transgenic T cells have specificity for a self-peptide derived from the glycolytic enzyme glucose-6-
16
phosphate isomerase (GPI) and are able to breach tolerance in the B cell compartment resulting in the production of autoantibodies to the same antigen (Matsumoto et al. 1999;
Basu et al. 2000). Affinity-purified anti-GPI Ig from these mice can transfer joint specific inflammation to healthy recipients (Matsumoto et al. 1999). The mechanism for joint- specific disease arising from autoimmunity to a ubiquitious autoantigen has been extensively studied. This model is unique in that antibodies against a ubiquitous enzyme are capable of initiating a tissue specific disease. It has been shown that GPI bound to the surface of cartilage acts as a target for the immunogloblin binding and subsequent complement mediated damage. Antibodies to the IgG1 isotype are predominantly responsible for the disease and monoclonal antibodies can transfer disease only in pairs that bind specific epitopes. Arthritis is dependent on the alternate complement cascade and Fc receptors are essential for the initiation of the disease. This gives an explanation for the joint specificity since the cartilage surface, unlike other cells, is devoid of alternate complement pathway regulatory proteins like decay accelerating factor (DAF/CD55) and membrane cofactor of proteolysis (MCP/CD46). Arthritis can be transferred to mice lacking B cells and T cells. This is a unique feature of this model since autoreactive T cells are required for inducing autoimmunity and antibody production but joint destruction is mediated by the adaptive response to the innate immune system players. Thus the passive transfer model can be used to study the pathology of joint inflammation independent of the factors causing autoimmunity.
Neutrophils and mast cells were found to be essential for joint destruction. Mice treated with neutrophils depleting monoclonal antibodies were protected from disease. Mast cell deficient mice, W/Wv and S1/S1d were resistant to arthritis, and transfer of mast cells
17
in to the W/Wv mice restored disease. Using various knock out mice the role some of the genes involved in the disease has been elucidated (Table 1). IL-1 has a critical role in arthritis induction while TNF-α has a strong but not absolute requirement (Ji et al. 2002).
A recent study by Kim et al (Kim et al. 2005) has shown an indispensable role for NK cells. NK cells act by suppressing the anti-inflammatory properties of TGF-β through IL- 4 and IFNγ secretion. Thus a role for IL-4 and IFNγ has also been suggested. However sera transfer has not been done in IFNγ and IL-4 knockout mice, which may definitely prove their role. A very important player in RA is the macrophage and we have shown that macrophages are absolutely essential for K/BxN sera induced arthritis. Susceptible mice treated with liposomal clodronate, which specifically eliminates macrophages by apoptosis, failed to develop disease on administration of K/BxN sera (Solomon et al submitted). The K/BxN model of rheumatoid arthritis is unique since it depicts another aspect of autoimmune disease. The organ specificity of the disease is determined by the type of innate immune response that occurs in the organ in response to the adaptive immunity rather than on an organ specific antigen. GPI can thus be one of the several molecules that can migrate to the joint and induce disease in a similar mechanism. This has assumed more significance since it has been shown that antibodies against GPI do exist in a subset of RA patients and may be involved in disease induction (Schaller et al.
2001; Kassahn et al. 2002). This model is also different from the commonly used CIA model where IFNγ and NK cells (Chiba et al. 2004) play an anti-inflammatory role. Thus the K/BxN model may represent the heterogeneity of RA that exists in humans and can be used to study pathways and drug targets aimed at specific subsets of patients.
18
1.3 The Ly6C low subset of blood monocytes involved in initiation of K/BxN sera induced arthritis
1.3.1 Macrophages in rheumatoid arthritis
Rheumatoid arthritis is characterized by chronic joint inflammation primarily involving the synovial membrane. The rheumatoid synovial lining layer is characterized by vascular proliferation, the accumulation of T cells and macrophages, synovial lining thickening. In humans the events responsible for these changes are due to stimulation of T cells by unknown antigens presented in the groove of HLA- DR4, leading to macrophage activation and production of chemokines like IL-1 and TNF-α. The release of these and other inflammatory mediators and activation of synoviocytes, chondrocytes and osteoclasts, bring about the inflammatory changes in the joint. Monocytes play a crucial role in this inflammatory process. The synovial lining layer consists of two types of cells, the synovial A cells, made of macrophages (Edwards and Willoughby 1982) and synovial B cells, made of fibroblast like cells. Bone marrow grafting experiments in the mice have shown that the resident synovial type A cells are of the bone marrow origin. Monocytes migrate from the blood stream through the high endothelial venules in the synovial membrane into the lining layer. After entry in to the synovial layer the monocytes mature into macrophages with distinct phenotypic and functional characteristics (Ridley et al.
1990).
19
1.3.2 Monocytes and macrophages
Originally, monocytes and macrophages were classified as cells of the reticulo- endothelial system - RES. Van Furth et al. (1972) proposed the mononuclear phagocyte system -- MPS, and monocytes and macrophages became basic cell types of this system.
Their development takes in the bone marrow and passes through the following steps:
stem cell - committed stem cell - monoblast - promonocyte - monocyte (bone marrow) - monocyte (peripheral blood) - macrophage (tissues). Monocyte differention in the bone marrow proceeds rapidly within 3 days. During differentation, granules are formed in monocyte cytoplasm.
The process of haematopoiesis is controlled by a group of at least 11 growth factors.
Three of these glycoproteins initiate the differentiation of macrophages from uni- and bipotential progenitor cells in the bone marrow. The progression from pluripotential stem cell to myeloid-restricted progenitor is controlled by IL-3, which generates differentiated progeny of all myeloid lineages. As IL-3 responsive progenitors differentiate, they became responsive to GM-CSF and M-CSF, the two growth factors giving rise to monocyte/macrophage-restricted progeny. After lineage commitment, cells are completely dependent on these growth factors for continued proliferation and viability.
The blood monocytes are young cells that already possess migratory, chemotactic, pinocytic and phagocytic activities, as well as receptors for IgG Fc-domains (FcγR) and iC3b complement. Under migration into tissues, monocytes undergo further differentiation to become multifunctional tissue macrophages. Monocytes are generally,
20
therefore, considered to be immature macrophages. However, it can be argued that monocytes represent the circulating macrophage population and should be considered fully functional for their location, changing phenotype in response to factors encountered in specific tissue after migration.
Macrophages can be divided into normal and inflammatory macrophages. Normal macrophages include macrophages in connective tissue (histiocytes), liver (Kupffer's cells), lung (alveolar macrophages), lymph nodes, spleen, bone marrow, serous fluids (pleural and peritoneal macrophages), skin (histiocytes, Langerhans's cell) and in other tissues.
The macrophage population in a particular tissue may be maintained by three mechanisms: influx of monocytes from the circulating blood, local proliferation and biological turnover. Under normal steady-state conditions, the renewal of tissue macrophages occurs through local proliferation of progenitor cells and not via monocyte influx. Originally, it was thought that tissue macrophages were long-living cells. More recently, however, it has been shown that depending on the type of tissue, their viability ranges between 6 and 16 days.
Inflammatory macrophages are present in various exudates. They may be characterized by various specific markers, e.g. peroxidase activity, and since they are derived exclusively from monocytes they share similar properties.
21
1.3.3 Macrophages in inflammation
Monocytes begin to emigrate into the extravascular tissue, the site of infammation, quite early in infammation and within 48 hrs they may constitute the predominant cell type at the inflammatory site. Adhesion molecules and chemotactic molecules mediate this extravasation of monocytes. When the monocyte reaches the extravascular site it undergoes transformation into the macrophage. The macrophage can be activated by a variety of stimuli like IFNγ secreted by T cells and NK cells and bacterial endotoxins.
Activation results in increased cell size, increased levels of lysosomal enzymes, higher metabolic rate and greater ability to phagocytose and kill ingested microorganisms.
Macrophages are involved in both adaptive and immune responses. They act as professional antigen presenting cells and present ingested antigens to T cells. They also participate in the innate immunity by acting as professional phagocytes and thus forming the first line of defense in the body’s immune system. However, activated macrophages secrete a wide variety of inflammatory mediators which when left unchecked may lead to tissue injury and fibrosis characteristic of chronic inflammation.
If the inflammation is short lived and the irritant is eliminated macrophages eventually disappear by death (apoptosis) or they may move into the lymph nodes. However if the irritant is persistent it leads to chronic inflammation and the macrophage persists at the site of inflammation and this is mediated by different mechanisms.
1) Recruitment of monocytes from the circulation
This is mediated by adhesion molecules and chemotatic factors. Most of the macrophages 22
present in the focus of chronic inflammation are recruited from the circulating monocytes. Chemotactic stimuli for monocytes include chemokines produced by activated macrophages, lymphocytes and mast cells e.g MCP-1, C5a, growth factors such as platelet derived growth factor and transforming growth factor-α (TGF-α), fragments from breakdown of collagen and fibronectin, and fibrinopeptides.
2) Local proliferation of macrophages after their emigration from the blood stream also contributes to the macrophage population in chronic inflammation.
3) Immobilization of macrophages within the site of inflammation by cytokines and oxidized lipids.
The products of activated macrophages serve to eliminate injurious agents such as microbes and to initiate the process of repair. Products such as reactive oxygen and nitrogen intermediates help in the killing of host cells and extracellular matrix proteases like MMPs help in tissue repair. Other cytokines like TNF-α cause influx of more inflammatory cells to the site of infection. But these same beneficial factors in the body’s defense can induce considerable tissue destruction when activated inappropriately causing a persistent inflammatory response.
1.3.4 Why study macrophages in rheumatoid arthritis?
Rheumatoid arthritis is one such disease where the macrophages are inappropriately activated and contribute to both inflammation and tissue destruction. Macrophages are found in abundance in the inflamed synovial membrane particularly in the cartilage-
23
pannus junction. They are activated as is evident by their expression of MHC class II molecules. They secrete proinflammatory and regulatory cytokines like IL-1, IL-6, IL-15, IL-18, TNF-α and granulocyte macrophage colony stimulating factor (GM-CSF). They also secrete chemokines and chemoattractants like IL-8, macrophage inflammatory protein (MIP-1) and monocytes chemoattractant proein (MCP-1).
Matrixmetalloproteinases and neopterin are also over expressed by the macrophages in the rheumatoid synovium (Burmester et al. 1997; Bresnihan 1999). Activation of macrophages is not only restricted to the rheumatoid synovium, but also extends to the peripheral blood monocytes and the precursors of myelomonocytic lineage in the bone marrow.
In the RA synovial membrane, the recently immigrated monocytes mature into macrophages subsets that differentially colonize the synovial sublining and lining layers (Hogg et al. 1985; Mulherin et al. 1996). The functional diversity of the macrophages in these areas may differentially contribute to the disease progression. Synovial macrophages can also differentiate into stimulatory or inhibitory macrophages capable of influencing T cell reactivity (Klareskog et al. 1982). This has also been demonstrated in a model of experimental arthritis (Kinne et al. 1995; van den Berg et al. 1996). The macrophage subpopulations may be responsible for the separate synthesis of proinflammatory (IL-1 and TNF-α) and regulatory cytokines (IL-10) and a shift in this balance leads to disease (Miossec and van den Berg 1997). Further a subset of synovial macrophages also has an effect on the angiogenic process (Koch 1998). The degree of macrophage infiltration and activation correlates with joint pain and inflammatory status 24
of the patient (Tak et al. 1997) and also with the radiological progression of permanent joint damage (Mulherin et al. 1996).
At the cartilage-pannus junction, the site of tissue destruction, macrophages express IL-1 and TNF-α significantly. They also secrete the proteases collagenases, stromelysin, gelatinase B and leucocyte elastase (Tetlow et al. 1993). At the bone-pannus junction osteoclasts also derived from the myelomonocytic lineage cause bone erosion through cytokines and cell-cell contact (Gravallese et al. 1998).
The activation of circulating monocytes in RA has been shown by the production of prostanoids and prostaglandin E2 (Highton et al. 1995) (Bomalaski et al. 1986), cytokines (e.g IL-1, IL-8, IL-10, IL-6) (Hahn et al. 1993; Liote et al. 1996; Schulze- Koops et al. 1997), soluble CD14 and neopterin. Neopterin is exclusively produced by monocytes in relation to disease activity. An increased production of gelatinase B and Tissue inhibitor of metalloproteinase (TIMP-1) has also been shown (Ahrens et al. 1996;
Gruber et al. 1996). Increased integrin expression and monocytes adhesiveness is found in the RA blood monocytes (Mazure et al. 1995; Liote et al. 1996). Surprisingly monocytes also exhibit a suppressor function in the production of RF (Okawa-Takatsuji et al. 1988). Therefore monocytes seem to be preshaped before their entry into the inflamed synovium. This has been further demonstrated in the gene expression studies done on RA monocytes collected from patients undergoing leukapheresis. Apart from the normal cytokines involved in inflammation other novel genes like growth-related oncogene protein (GRO)-a/ melanoma growth-stimulatory activity, MIP-2/GRO-b, ferritin, a1-antitrypsin, lysozyme, transaldolase, Epstein-Barr virus encoded small
25
nuclear RNA (EBER)-1/EBER-2-associated protein, thrombospondin-1, an angiotensin receptor-II carboxyl-terminal homologue, the RNA polymerase-II elongation factor, and for five unknown and functionally undefined genes are known to be upregulated in the RA blood monocytes (Stuhlmuller et al. 2000). Thus the extent of the monocyte activation in RA and the effect it has on the disease remains to be investigated.
1.3.5 Monocytes in K/BxN sera induced arthritis
In the K/BxN sera transfer model of rheumatoid arthritis T cells are not required in the inflammatory phase of the disease (Ji et al. 2002). The macrophages are thus activated independent of T cells interaction and mediators released by T cells. In order to elucidate the role of macrophages in the K/BxN sera induced model of arthritis we administered Liposome-encapsulated Clodronate (Liposomal clodronate) intra-peritoneally to deplete mice of macrophages and then induced arthritis. Mice were completely protected against arthritis with no signs of clinical or histological manifestations (Solomon et al submitted). Clodronate (dichloromethylene bisphosphonate) that is encapsulated within liposomes is phagocytosed by macrophages and subsequently the liposome bilayers are disrupted by the lysosomal phospholipases releasing the clodronate into the cell. The clodronate is accumulated intracellularly and when its concentration reaches threshold levels it kills the cell by apoptosis (Van Rooijen and Sanders 1994; van Rooijen et al.
1996). This was intriguing since an intra-peritoneal administration of Liposomal clodronate caused the depletion of macrophages in the blood, liver, spleen and peritoneum but not from the synovium since the liposomes used for encapsulation were multilammelar vesicles whose large size prevented them from accessing the synovium.
26
Previously a local depletion of the synovial lining layer by intra-articular administration of Liposomal Clodronate has been shown to ameliorate arthritis in mice models like collagen type II arthritis, antigen induced arthritis (Kinne et al. 1995; van Lent et al.
1996; Richards et al. 1999). A systemic administration of clodronate however failed to give a complete protection against the disease (Osterman et al. 1994; Kinne et al. 1995).
The protection obtained in our model must be due to the depletion of monocytes/macrophages from the blood.
It has been now shown in our model that cytokines IL-1 and TNF-α are essential for disease development (Ji et al. 2002; Choe et al. 2003), as are mast cells and neutrophils (Lee et al. 2002; Wipke et al. 2004). FcγRIII, mast cells, neutrophils have been shown to be critical for the initial localization of anti-GPI IgG into the joints (Allen and Nilsen- Hamilton 1998). A four staged model has been proposed to explain how autoantibody induced arthritis occurs in this sera induced arthritis. In the first stage the injected Ab encounters Ag in the circulation, forms soluble ICs and engages the FcγRIII on neutrophils in the blood stream, thus activating the neutrophils. The neutrophils, through the release of cytokines cause a small change in the local vasculature of the synovium, allowing the ICs to access the mast cells found in close proximity to the microvasulature of the synovium. The mast cells get activated through the FcγRIII receptors and release vasoactive mediators like histamine, prostaglandins and TNF-α that further amplify the vascular change allowing more antibodies and ICs to enter the joint. Thus during the first stage ICs allow the rapid influx of Abs above a threshold level into joint through a
27
sequential interaction with neutrophils and mast cells. The second stage involves the binding of the anti-GPI antibodies to the GPI on the articular cartilage surface. In the third stage the Abs that are bound to the antigen serve as substrates for activation of innate immune system. The alternate complement pathway is activated, and the PMNs, NK cells, mast cells and macrophages through their FcRs are also activated. The activation of these pathways results in production of inflammatory cytokines IL-1β, TNF- α and chemokines like macrophage inflammatory protein-1 and monocyte chemoattractant protein-1. A strong inflammatory response ensues in the joint and this occurs during 24-48 hrs when joint swelling is already observed and neutrophils are detectable histologically. The arthritis at this stage is reversible and in the anti-GPI serum transfer model the acute arthritis that develops with a single injection of anti-GPI Abs resolves over time and by day 14 no inflammation is evident. The fourth stage is the chronic phase involving macrophages, synoviocytes. It is characterized by synovial hyperplasia, pannus formation and bone and cartilage erosion.
But in these studies the role of blood monocytes has largely been neglected. During inflammation monocytes migrate to sites of inflammation from the blood and differentiate into mature macrophages where they contribute to disease progression.
Monocytes can get activated to mature monocytes/macrophages in the blood due to activation by immune complexes or LPS. A systemic activation of macrophages due to local inflammation leading to elevated levels of IL-1 and IL-6 has also been shown in AIA (Simon et al. 2001). Further the TLR4 receptor has been shown to be essential for this disease (Choe et al. 2003) and LPS activation of blood monocytes through the
28
TLR4 receptors may contribute to disease initiation and progress. So in this context we found blood monocytes as most appropriate candidates of systemic activation that may contribute to the disease.
1.3.6 Defining blood monocytes
Monocytes are defined as blood mononuclear cells with bean shaped nuclei, expression of CD11b, CD1c, and CD14 in humans and CD11b and F4/80 in mice, and lack B, T, NK and DC markers. Recent studies have shown that blood monocytes are a heterogeneous population that function differentially in the steady state and inflammation (Drevets et al.
2004; Sunderkotter et al. 2004). Passilick et al have demonstrated that human monocytes can be divided into two populations by the presence or absence of the Fc receptor CD16 (Passlick et al. 1989). The CD16+ monocytes do not express the chemokine receptor CCR2 and have an enhanced capacity for transendothelial migration (Weber et al. 2000;
Randolph et al. 2002). In mice Geissmann et al (Geissmann et al. 2003) have reported the existence of two distinct monocyte subsets that differ in their expression of chemokine receptors and adhesion molecule. The first subset is characterized as GR1+CXC3CR1lowCCR2+CD62L+ phenotype and corresponds to the CD14+CD16+ monocytes of humans. These CCR2+ monocytes are short lived and capable of differentiating into both macrophages and dendritic cells. These monocytes are classified as “inflammatory macrophages”. The other subset consists of GR1-CXC3CR1highCCR2- CD62L- phenotype and resembles the CCR2+CD16- phenotype of human monocytes.
This subpopulation is called the “resident macrophages” and represents the precursors of tissue resident macrophages and dendritic cells.
29
In another study Sunderkotter et al have differentiated monocytes on the basis of the expression of Ly6C, a hemopoietic cell differentiation antigen. They provide a new paradigm to study blood monocytes in different mice models of inflammation. Blood monocytes can be distinguished from peripheral neutrophils and lymphocytes flow cytometrically by their low granularity, as reflected by their low SSC, and their high level expression of CD11b/Mac1. The SSClow CD11bhi monocytes can further be distinguished phenotypically based on the surface expression of Ly6C (ER-MP20) into Ly6C -/low, Ly6Cmed and Ly6Chi cells. The Ly6C expression on the Ly6Chi cells resembles that on the bone marrow monocytes. All three subsets showed monoytic morphology and phagocytic capability. All three subpopulations had the capacity to proliferate but the highest proliferative capability was seen in the Ly6Chi population. The monocytes that appear from the bone marrow are of the Ly6Chi phenotype that loses Ly6C expression as they mature into macrophages. And it’s the Ly6Cmed/high phenotype that is involved in inflammatory conditions and is capable of migrating to the site of inflammation. Using this paradigm we analyzed mouse monocytes and their subsets in K/BxN sera induced arthritis. Our results showed that the Ly6Clow monocyte subset is essential for the initiation of arthritis while the Ly6Chi subset is involved in chronic phase of the disease.
30
1.4
Intra-articular injection of K/BxN sera induces arthritis in Balb/c mice: A new model to study the acute phase of disease independent of systemic activation.
An intraperitoneal injection of 200µl of K/BxN sera in susceptible mice like Balb/c results in arthritis on day 3. The disease then peaks on day 6 reaching a plateau till day 10 and resolves completely by day 15. On injecting mice with K/BxN sera intraperitoneally anti-GPI antibodies that enter the blood stream have to be transported into the synovium to reach threshold levels to induce disease in the joints. A systemic activation of macrophages and neutrophils (Wipke et al. 2004) by the anti-GPI-GPI immune complexes in the blood is required to secrete vasodilatory cytokines like TNF-α which then act locally on the joint blood vessels facilitating the transport of the Abs into the synovium. The antibodies that enter the synovium initiate inflammation once they have reached threshold levels. So arthritis in this model is initiated by activation of neutrophils and monocytes in the blood and mediators secreted by them. But resident macrophages and fibroblasts within the joint are bound to have an important role in the initiation of the disease. We wanted to create a model where we can study the cellular and cytokine mediators involved in arthritic induction locally in the synovium with out the involvement of the systemically produced mediators. To achieve this we carried out an intra-articular transfer of the K/BxN sera into the knee joints of mice.
31
1.5
Role of transcriptional factor STAT1 in the synovium of K/BxN sera induced arthritis
Recently it was shown by Kim et al (Kim et al. 2005) that NKT cells play a role in the K/BxN sera induced arthritis. NKT cell deficient mice are protected against arthritis.
Their results suggest that NKT cells induce arthritis by suppressing TGFβ1 by producing IFNγ and IL-4. In animal models it has been demonstrated that TGFβ1down regulates proinflammatory cytokine production and exerts protective effects on collagen-induced arthritis in mice (Kuruvilla et al. 1991). It has also been demonstrated that TGFβ is capable of promoting inflammation by enhancing neutrophillic infiltration and the induction of angiogenesis in the joint tissue (van Beuningen et al. 1994). A balance between the pro-inflammatory and anti-inflammatory effects of TGFβ1 seems to be crucial in determining the outcome of RA. Several experiments demonstrated that IFNγ is a negative regulator of TGFβ on immune cells in vivo and in vitro (Marth et al. 1997;
Seder et al. 1998).
In human RA a FcγR-mediated activation of NK cells has been shown to be a disease- determining factor (Stewart-Akers et al. 2004). IFNγ is very important activator of macrophages and since we know that macrophages are essential for K/BxN sera induced arthritis it possible that the interaction of NK cells and macrophages through secretion of IFNγ may play a important role in this model. Macrophages are primed by IFNγ via JAK1/2-STAT1 pathway so that they can subsequently undergo a classical type 1
32
activation upon exposure to T helper (Th1) cytokines such as IFN-γ or other activators, including tumor necrosis factor and lipopolysaccharide (Dalton et al. 1993) (Nacy et al.
1985). Further an inhibition of IFN-γ induced activation of macrophages through the LPS induced production of suppressor molecule, suppressor of cytokine signaling 3, has been also demonstrated. So IFN-γ mediated responses through the JAK/STAT1 pathway may be important in regulating proinflammatory and suppressive activities of macrophages.
We therefore sought to investigate the role down stream signaling of the IFN-γ pathway.
The JAK-STAT (Janus Kinase-Signal Transducer and Activator of Transcription) signaling pathway mediates the IFN response. Binding of the IFN molecules to their cognate receptor induces receptor homo or heterodimerization, resulting in autophosporylation and activation of receptor associated JAK kinases. Upon activation, the JAK kinases phosporylate conserved tyrosine residues in the cytoplasmic tail of the receptors, thereby creating docking sites for the SH2-domain of the STAT proteins. The receptor bound STAT proteins are also phosporylated on a conserved tyrosine residue by JAK kinases. This allows homo or heterodimerization of STAT proteins via reciprocal SH2-phosphotyrosine. The STAT dimers translocate to the nucleus where they bind their response elements in the promoter of target genes. They modulate transcription through recruitment of transcriptional coactivators or repressors to the promoter. Seven mammalian STATs have been identified and the STATs involved in IFN signaling are STAT1 and STAT2. STAT2 mediates IFN type I (IFN-α, -β) signaling, whereas STAT1 is involved in signaling by both type I as well as type II IFN (IFN-γ), which are the
33
mediators of inflammatory reaction after viral infection and host defense to bacteria and parasites and have important functions in the innate immune system. IFN-γ is made mainly by activated dendritic cells (DC), macrophages, natural killer cells, Th1 cells and CD8+ cytotoxic T cells. Activation of IFN-γ receptor 1 and 2 by IFN-γ, leads to phosphorylation of STAT1 that bind as homodimers to the IFN response element known as γ-IFN-activated sequence (GAS) on the IFN-responsive genes.
In this study we interfered with the STAT1 mediated transcriptional regulation and studied its effect in K/BxN sera induced arthritis. Several strategies have been used to block the action of STAT proteins, including antisense methods, ectopic expression of dominant negative mutants, inhibition of upstream kinases and phosphotyrosyl peptides.
An alternative approach involves the use of double-stranded ‘decoy’ oligonucleotides.
The double–stranded DNA decoy closely corresponds to the responsive element within the promoter region of a responsive gene. When a sufficient concentration of decoy in the target cells is achieved the authentic interaction between a transcription factor and its endogenous response element in the genomic DNA is impaired, with subsequent modulation of gene expression (Bennett et al. 1992). Here we used STAT1 decoy ODN directed against the acute phase response element. The STAT decoys ODN are encapsulated into liposomes and delivered intra-articularly. Since we are targeting the macrophages and fibroblasts in the synovium in order to study the effect of IFN-γ signaling as well as that of other cytokines which meditate their effects through JAK/STAT pathway we did this experiment using the intra-articular injection model of
34
K/BxN sera transfer. We were also able to study the role STAT1 in these cells in the synovium independent of the effect of cytokines secreted by the systemic activation of macrophages and neutrophils.
1.6
Role of MMP-9 in K/BxN sera induced arthritis
Rheumatoid arthritis involves chronic joint inflammation with erosive synovitis and joint destruction. Enzymes involved in the breakdown of the matrix elements, allowing inflammatory cells to migrate into the site of inflammation may induce longterm irreversible damage to the joints. Matrix Metallo Proteinases (MMPs) play an important role in this scenario. These enzymes are secreted by both the resident cells of joint tissues as well as by invading cells, they are active around neutral values of pH, and they have the combined ability to degrade all the components of the Extra Cellular Matrix ECM.
MMPs play significant roles in both developmental and repair processes, and it appears that aberrant regulation, which can occur at many levels, leads to their hyperactivity in diseases such as rheumatoid arthritis (RA).
There is now significant evidence for the over expression of MMPs in tissues derived from patients with arthritic disease. Cultures of cells derived from rheumatoid synovia secreted a collagenolytic activity into the medium (Dayer et al. 1976), and stromelysin-1 (Sirum and Brinckerhoff 1989) and collagenase-1 were detected by immunolocalisation at sites of cartilage erosion in rheumatoid joints (Woolley and Evanson 1977). Both of
35
these enzymes, as well as the Tissue Inhibitor of MetalloProteinase (TIMP)-1, were immunolocalised in synovial samples from both RA and OA patients (Hembry et al.
1995). Further MMP-1, MMP-3 and MMP-10 have been shown to associate with the invasive behavior of synovial fibroblasts (Tolboom et al. 2002).
The MMP family consists of 25 zinc-dependent and calcium-dependent proteinases in mammalian systems, and MMPs are now thought to be the major proteolytic enzymes that facilitate tissue remodeling in both physiological and pathological situations [21–23].
MMPs can be classified into at least five main groups, according to their substrate specificity, primary structure and cellular localization; namely, the collagenases, gelatinases, stromelysins, matrilysins and MT-MMPs [25]. There are some MMPs, however, such as macrophage elastase (MMP-12), stromelysin-3 (MMP-11), MMP-19, enamelysin (MMP-20), CA-MMP (MMP-23) and epilysin (MMP-28), that apparently do not fall into any of these categories. In addition, some enzymes, such as MT1-MMP (MMP-14), which displays collagenolytic activity and is membrane associated, may be classified into more than one group.
Because MMPs can degrade almost all components of the ECM their main function is presumed to be ECM remodeling e.g. during embryonic development, tissue growth, and morphogenesis. Ongoing research has shown that MMPs not only remodel the ECM but may also be important in a number of other physiologic processes (McCawley and Matrisian 2001): a. MMPs may affect cell migration by changing cells from an adhesive to a non-adhesive phenotype and/or by ECM degradation, b. MMPs may alter the
36
