Tchetverikov, I.
Citation
Tchetverikov, I. (2005, February 17). Matrix metalloproteinases involvement in rheumatoid
arthritis. Retrieved from https://hdl.handle.net/1887/625
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in theInstitutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/625
Chapter 1: General introduction
I. RA, definition and classification criteria II. Pathogenesis and pathophysiology of RA
- Etiological and predisposing factors - Cells involved
- Cytokines involved - Enzymes involved III. M atrix M etalloproteinases
IV. Involvement of the M M Ps in joint pathology in general and in RA in particular - Tissue level
- Synovial fluid - Systemic circulation - MMP/TIMP imbalance V. Aims of the project
I. Rheumatoid arthritis, definition and classification criteria
Rheumatoid arthritis (RA) is a chronic inflammatory disease, which primarily affects tissue of multiple joints.1 Extra-articular manifestations such as subcutaneous nodules, vasculitis and serositis emphasize the systemic nature of RA. Joint destruction is the major factor contributing to the physical disability in RA patients, who are presenting themselves initially with swollen and painful joints, often resulting in impaired joint motion.2 Later in the course of the disease articular cartilage and subchondral bone destruction lead to joint deformities and as a consequence, social and occupational life of the patients suffers and the quality of life of the patients becomes severely impaired. W orldwide, 0.5-1% of the general population is affected by the disease, with a female to male ratio of 3:1. The incidence of the disease is estimated between 100 to 300 new disease cases per 100,000 individuals.1;3
Historically, no specific tests are available for diagnosing RA. Most commonly used, Rheumatoid Factors (antibodies to the patients own immunoglobulins) are increased in the systemic circulation of approximately 80% of the RA patients. Also the presence of autoantibodies to cyclic citrullinated peptides (CCPs) is shown to be highly specific for RA. The diagnosis of RA is based on clinical grounds in combination with laboratory and radiological findings. In order to standardize epidemiological studies, classification criteria for RA were developed in 1958 and revised in 1987,4Table 1.
Table 1. The 1987 revised criteria for the classification of rheumatoid arthritis.
For classification purposes, a patient shall be said to have rheumatoid arthritis if he/she has satisfied at least 4 of these 7 criteria. Criteria I - IV must have been present for at least 6 weeks. Criteria II - V must be observed by a physician. Patients with 2 clinical diagnoses are not excluded.
I. Morning stiffness Morning stiffness in and around the joints, lasting at least 1 hour before maximal improvement.
II. Arthritis of 3 or more joint areas
At least 3 joint areas simultaneously have had soft tissue swelling or fluid (not bony overgrowth alone). The 14 possible areas are right or left PIP, MCP, wrist, elbow, knee, ankle, and MTP joints.
III. Arthritis of hand joints At least 1 area swollen (as defined above) in a wrist, MCP, or PIP joint.
IV. Symmetric arthritis Simultaneous involvement of the same joint areas (as defined in II) on both sides of the body (bilateral involvement of PIPs, MCPs, or MTPs is acceptable without absolute symmetry). V. Rheumatoid nodules Subcutaneous nodules, over bony prominences, or extensor
surfaces, or in juxtaarticular regions. VI. Serum rheumatoid
factor
Demonstration of abnormal amounts of serum rheumatoid factor by any method for which the result has been positive in <5% of normal control subjects.
Once the diagnosis RA is made, the disease activity is assessed and carefully monitored.5 The goal of treating RA patients is to prevent structural joint damage and maintain joint function. Not long ago, the initial drug treatment for RA involved mainly nonsteroidal anti- inflammatory drugs6 (NSAID’s). This concept has changed in the last decennia. It has been shown that approximately 75% of the patients with disease of recent onset have joint erosions or develop erosions shortly after the onset of symptoms.7 Several studies indicate that early pharmacological interventions using disease-modifying antirheumatic drugs (DMARD) may be crucial in preventing irreversible joint damage and thus maintaining quality of life of RA patients.8 Moreover, encouraging results are achieved using new agents, such as TNF antagonists and an IL-1 receptor antagonist, which are specifically designed to target pro-inflammatory cytokines TNF-Į and IL-1 respectively.9 Thus, our improved understanding of the disease, successes of early DMARD treatment and the use of newly developed biologic agents have changed treatment protocols and improved care for RA patients.5 However, while new therapies and approaches are being evaluated, diagnosis RA remains associated with increased long-term morbidity and early mortality.10
II. Pathogenesis and pathophysiology of RA Etiological and predisposing factors
Although it is generally accepted that immune-mediated mechanisms play a crucial role in RA, the factors initiating the synovitis in RA remain unknown. Twin and family studies provide indications that both genetics and environmental factors induce RA,11 whereas genetic and hormonal factors are thought to determine the individual susceptibility to RA. The importance of genetic factors is shown by the association between the expression of major histocompability class II antigens (HLA-DR4) and the increased risk not only to develop RA, but also to develop a more severe disease.12Since women are affected more often than men and pregnancy has an ameliorating effect on RA,13 hormonal factors are also suggested to be important for a person’s susceptibility to RA.
The current hypothesis of the pathogenesis of the disease is an autoimmune mediated process in which several cells play a role.
Cells involved
peptides do not necessarily undergo pinocytosis and may directly bind to the MHC class II molecules on cell surface.
Next, macrophages interaction with CD4+ T-cells commences. CD4+ T-cells are thought of as an important link in the chain of events from the initiation and to the perpetuation of the inflammation. T-cells produce adhesion molecules which allow the traffic through the synovial membrane into the joints.19 CD4+ T-cells that bear an appropriate receptor, to which the antigen presented by the macrophages “fits”, interact with the macrophage via molecules such as LFA-1 (lymphocyte function antigen), ICAM-1 (intercellular adhesion molecule) and co-stimulatory molecules B7/CD28.20 During interaction macrophages and T-cells are activated and produce a variety of cytokines. These cytokines induce production of proteolytic enzymes by fibroblast-like synoviocytes (FLS). Moreover, other lymphocytes such as neutrophils are attracted to the joints during inflammation.
The cells of the cartilage itself, chondrocytes, also contribute to the cartilage degradation in RA. Once activated, these cells are capable of producing substantial amounts of proteolytic enzymes,21;22 which also may contribute to the degradation of the components of the articular cartilage such as proteoglycans and collagen.23 Moreover, the surface of cartilage contains embedded immune complexes, which facilitate activation of the neutrophils attracted to inflamed joints.10
Cytokines involved
Fibroblasts-like synoviocytes are shown to produce a variety of cytokines such as vascular endothelial growth factor (VEGF)24 and transforming growth factor (TGF)-ß.25;26 Autostimulation of FLS by TGF-ß results in increased production of IL-1ß, TNF-Į, IL-827 and several chemokines such as Monocyte Chemotactic Protein (MCP)-1, -2, -3, -4.28;29 Whereas IL-8 is likely to attract neutrophils to the inflamed joints,30;31 MCP’s specifically attract monocytes to the joints.28
Macrophages, abundantly present in the joints of RA patients, also produce pro-inflammatory cytokines (IL-1, -2, -6, -15, -18, GM-CSF, TNF-Į).32;33 Especially IL-1 and TNF-Į are thought of as cytokines responsible for the perpetuation of the synovitis by further increasing cytokine production, induction of adhesion molecules and as a consequence accumulation of more inflammatory cells in the joint.
T cells are other cells which are attracted to the joints. These T cells are abundantly present in the inflamed synovium and consist mainly of CD4+ T-helper cells, which can be divided in TH1 and TH2 helper cells. TH1 produces pro-inflammatory cytokines (IL-2, INFȖ),
whereas the latter group may be immunosuppressive by producing IL-4, -5 and -10.34;35 The production of macrophages derived cytokines is high in RA synovium and only relatively low levels of T-cells cytokines are found.36-38 However, there may also be an imbalance between TH1 and TH2 populations in favor of TH139-41 and the albeit low
Enzymes involved
During the inflammatory process, joint degradation is mediated by proteolytic enzymes. Due to the diversity of the tissue, multiple enzyme systems are likely to be involved. Based on the structure of the catalytic domain, four types of enzymes are distinguished. Table 2 shows these enzyme groups (serine, cysteine, metallo- and aspartic proteases) together with representative proteases of each group.
Table 2. Proteases involved in turnover of the extracellular matrix 1. Aspartic proteinases Cathepsin D
2. Cysteine proteinases Cathepsin B; Cathepsin K; Cathepsin L; Cathepsin S; Cathepsin V; Calpain
3. Serine protease Neutrophil elastase; Cathepsin G; Proteinase 3; Plasmin; Plasma kallikrein; Tissue kallikrein; tPA; uPA; Tryptase; Chymase; Granzyme A; Granzyme B
4. Zn M etalloproteinases
M atrix M etalloproteinases Collagenase 1, 2, 3 (MMP-1, -8, -13); Gelatinase A and B (MMP2 and 9); Stromelysin 1, 2, 3 (MMP3, 10, 11); MT1, 2, 3, 4, 5, 6MMP (MMP14, 15, 16, -17, -24, -25); Matrilysin-1 (MMP-7); Metalloelastase (MMP-12)
ADAM s family members Aggrecanases (ADAMTS-1, -4, -5) III. M atrix M etallopreoteases
In 1962 Gross and Lapière decided to investigate the nature of the enzymes involved in the process of a tadpole of a frog losing its tail. Apparently, there had to be an enzyme, which could facilitate the degradation of the collagenous component of the tail. Results of the culture of explants of the tail on reconstituted collagen fibrils revealed a clearing under the explants after a few days.44 The enzyme responsible for collagen degradation was recovered in the medium and was shown to be able to cleave triple-helical part of the collagen molecule into ¼ and ¾ fragments. Collagenase was discovered which marked the beginning of the MMP era.
Table 3. Matrix Metalloproteinases
MW, kDa
Matrix Metalloproteinases latent active Examples substrates
Collagenase-1 MMP-1 55 45 Collagens I, II, III, VII, VIII and X, gelatin,
Collagenase-2 MMP-8 75 58 Collagens I, II, III, V, VII, VIII and X, gelatin, Į1-proteinase inhibitor, fibronectin
Collagenase-3 MMP-13 60 48 Collagens I, II, III and IV, gelatin, aggrecan, plasminogen activator inhibitor 2, tenascin
Gelatinase A MMP-2 72 66 Collagens I, IV, V, VII, X, XI and XIV, gelatin, elastin, aggrecan, proTNF, roMMP-9 and -13
Gelatinase B MMP-9 92 86 Collagens IV, V, VII, X and XIV, gelatin, elastin, proteoglycan link protein, fibronectin, proTNF
Stromelysin-1 MMP-3 57 45 Collagens III, IV, IX and X, gelatin, aggrecan, proteoglycan link protein, fibronectin, fibrinogen, antithrombin-III, proTNF, proMMP-1, -7, -8, -9, -13 Stromelysin-2 MMP-10 57 44 Collagens III, IV and V,
gelatin,aggrecan, elastin, fibronectin, proMMP-1and -8
Stromelysin-3 MMP-11 51 44 Į1-proteinase inhibitor
MT1-MMP MMP-14 66 56 Collagens I, II and III, gelatin, casein, proTNF, dermatan sulfate proteoglycan, MMP-2, and -13,
MT2-MMP MMP-15 72 60 proMMP-2, gelatin, fibronectin, tenascin, nidogen, laminin
MT3-MMP MMP-16 64 52 MT4-MMP MMP-17 57 53 MT5-MMP MMP-24 63 45 MT6-MMP MMP-25 56 Matrilysin-1 MMP-7 28 19 Matrilysin-2 MMP-26 28 Metalloelastase MMP-12 54 45 Xenopus collagenase MMP-18 55 42 MMP-19 MMP-19 54 45 Enamelysin MMP-20 54 22 XMMP MMP-21 70 53 CMMP MMP-22 52 43 CA-MMP MMP-23 Epilysin MMP-28 59
(macrophage elastase, stromelysin-3, MMP-19, enamelysin, CA-MMP) are still unclassified according to the above mentioned criteria. Collagenases, as first shown by Gross and Lapière, can cleave triple-helical collagen. Gelatinases have broad substrate specificity and can further degrade collagenase-generated collagen fragments. Stromelysins can degrade most of the ECM components but not the triple-helical collagen. Matrilysins are also shown to have broad substrate specificity. The expression of the MMPs is regulated through growth factors, hormones, and cytokines and during oncogenic cell transformation.49 MMPs are produced as pre-pro-enzymes and in most cases are secreted in pro-form to be activated extracellularly, whereas some MMP (e.g. MT-MMPs, stromelysin-3 and epilysin) are secreted as active enzyme after they have been activated intracellularly by a furin-type proprotein convertase.50 Action of activated MMPs are controlled by the endogenous inhibitor Į2Macroglobulin (Į2M)51-53 and by specific Tissue
Inhibitors of Matrix Metalloproteinases (TIMPs).54
Į2M is considered the major inhibitor of activated MMPs in body fluids, e.g. in the
systemic circulation, synovial fluid and wound fluid.52;55 TIMPs are likely to be more important in the regulation of the MMP activity at tissue level.54 Up to date, there are four known TIMP subclasses, namely TIMP-1, -2, -3 and -4. All of them bind tightly to most MMPs.56;57 However, there are differences in the properties of the four TIMPs. For example, TIMP-2 and -3 can effectively inhibit MT-MMP, whereas TIMP-1 does not. TIMP-2 complexe formation with proMMP-2, seems to be important for the activation of the latter.58 TIMP-1, on the other hand, forms complexes with proMMP-9.59 TIMP-3, in contrast to other TIMPs, can effectively inhibit tumor necrosis factor-Į converting enzyme.60
Furthermore, TIMPs also have a variety of biological properties of which some are likely to be independent from MMP inhibitory activity. For instance, overexpression of TIMP-1, -2 and -3 suppresses tumor cell growth61;62 and TIMP-1 has an anti-apoptotic effect.63 While MMPs play an important role in normal tissue remodeling, they are also indicated to be involved in a great number of pathological conditions, e.g. tumor progression, neurological diseases and joint pathology.47;64-66
IV. Matrix Metalloproteases involvement in arthritic diseases
A great number of small MMP inhibitors has been developed and examined. The results obtained in the animal models and in vitro experiments provided sufficient basis to advance into the clinical trials. However, only one inhibitor so far was fully tested in the clinic (Ro-32-3555)68 in RA patients and it failed to show prevention of joint damage progression. Also the results of the early clinical trials aimed at tumor metastasis control in cancer patients were extremely disappointing until recently when Marimastat (BB-2516) was shown to have beneficial effects in gastric cancer. Moreover, use of broad spectrum MMP inhibitors in oncology patients was associated with dose dependant musculoskeletal toxicity.69 It can be concluded that a lot of knowledge has been accumulated in past decades regarding Matrix Metalloproteases but the understanding of this complex system is still limited. Thus, what evidence of MMP involvement in joint pathology is available? Tissue level
Multiple studies have shown the presence of MMPs mRNA in synovial tissue of RA patients.70-75 Koshy and colleagues showed that production of different MMPs (e.g. MMP-1, -3, -8, -13 and -14) was induced by pro-inflammatory factors such as interleukin-1 (IL-1) and oncostatin M (OSM).76 Konttinen and co-workers77provided an extensive mRNA analysis of different MMPs in synovial tissue of RA and trauma patients. According to their results, several MMPs (MMP-2, -3, -11 and -19) were expressed in both conditions. MMP-1, -9 and -14 were always expressed in rheumatoid synovial tissue but to a lower extent in synovial tissue of trauma patients. MMP-13 and -15 were expressed in RA only, whereas MMP-20 was not found at all and MMP-8 was rarely found in both conditions. This study implies that there are clear differences in mRNA expression between inflamed and “non-inflammatory” synovium and between different MMPs within the RA population. Except for MMP-20, all studied MMPs are expressed in synovial tissue and thus are likely to be involved in the process of joint tissue remodeling and destruction. Recently, Tolboom et al.78 showed that mRNA of 13 different MMPs is expressed by RA fibroblast-like synoviocytes. Moreover, the results showed that high expression levels of MMP-1, -3, -9 and -10 are correlated with an increase in invasive growth of these cells in an in vitro invasion model. In addition to synovial fibroblast, chondrocytes show high MMP production levels. Since chondrocytes are part of the articular cartilage and are embedded in extra cellular matrix (ECM), only a small proportion is exposed on the cartilage surface. It is therefore likely that high levels of proMMPs found in synovial fluid of inflamed joints mainly originate from synovial cells such as fibroblast-like synoviocytes or from inflammatory cells such as neutrophils and macrophages, which migrate to the joint during inflammation.
Synovial Fluid
compared to controls.79-83 Also proMMP-3 levels found in other arthritides such as osteoarthritis or crystal-induced arthritis were increased albeit not as extremely as in RA patients.82;84 These findings indicated that high proMMP-3 levels are likely to indicate the presence of synovitis since the highest proMMP-3 concentrations were found in inflammatory joint conditions. Moreover, several studies showed a correlation between SF proMMP-3 levels and markers of systemic inflammation such as C-reactive protein (CRP) or Erythrocyte Sedimentation Rate (ESR), suggesting that MMP-3 levels are closely correlated to systemic inflammation.82;83 Studies performed to investigate changes in synovial fluid composition upon knee injuries showed long lasting increases not only in cartilage degradation products but also in levels of proMMP-3, implying that persistent increases in SF levels of these molecules may be associated with development of posttraumatic osteoarthritis.80;85-87 Interestingly, Dahlberg et al. showed that proMMP-3 levels were also increased in the contralateral, uninjured knee following unilateral knee injury.88 The mechanism for this remains unclear, but the results imply a systemic effect on cartilage metabolism following knee injury.
In addition to MMP-3, also other MMPs were found in synovial fluid in joint diseases: gelatinases (MMP-2 and -9) and collagenases (MMP-1, -8 and -13) were found to be highly increased in SF of RA patients.89-94
Systemic circulation
that proMMP-3 levels in serum of RA patients are correlated with the degree of joint damage progression, which is supported by the fact that MMP-3 can degrade multiple components of ECM, e.g. collagens III, IV, IX and X, gelatin, aggrecan, proteoglycan link protein or fibronectin and is also capable of activation of other MMPs (proMMP1, 7, 8, -9, -13). Others argued that proMMP-3 levels are tightly correlated with CRP levels and not with a number of new joint erosions, and therefore are likely to reflect the inflammatory component of the RA disease process,99 rather than the joint destruction.
Also gelatinases were implicated in joint pathology. Gruber et al. showed that proMMP-9 levels are increased in the systemic circulation of RA patients and suggested that inflamed synovium may be the source of the MMPs found in the circulation.100 Goldbach-Mansky and colleagues also found increased concentrations of proMMP-9 in serum of RA patients but suggested that another gelatinase, proMMP-2, is likely to be involved in the development of joint erosions, since high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. However, they also indicated that serum levels of measured proMMP-2 had no prognostic value for development of joint erosions.90
On the contrary, serum levels of collagenase 1 (proMMP-1) were shown to be indicative of the process of joint degradation. Cunnane et al. showed that cumulative levels of circulating proMMP-1 found in serum of RA patients were correlated with a number of new joint erosion as documented by serial radiographs.99 They also argued that the pathological processes of joint inflammation and the development of joint erosions may be uncoupled in RA, since proMMP-1 levels and not proMMP-3 were correlated with the rate of erosion formation whereas proMMP-3 and not proMMP-1 levels were correlated with CRP levels measured in the study. Another collagenase, proMMP-8, was shown to be increased in serologically active RA and was correlated with other markers of systemic inflammation.101
MMP/TIMP imbalance
together with an increase, although to a lesser extend, in TIMP levels.85;103 Thus, based on molar ratios, it was calculated that protein levels of TIMPs would be insufficient to counteract increased proMMP production levels in joint pathology and that it could contribute to excessive cartilage degradation in RA. However, the net activity of the proteolytic system also depends on the activation of the produced proMMP in vivo, which still remains an unknown variable. Also determination of the proportion of the activated MMPs in complex with TIMPs remains a challenge. Furthermore, it has also been shown that activated MMPs can easily form complexes with a general protease scavenger D2Macroglobulin (D2M), which is present in high concentrations in SF of RA patients53and also in affected joint cartilage.104
V. Aims of the project
The goal of this project was to investigate which MMPs (and in what form) are present in synovial fluid and in the systemic circulation of patients with arthritic diseases in order to identify candidate markers to describe the disease process, to predict the course of the disease and/or to monitor patients treatment. To this end the new techniques such as MMP activity measurements in serum were developed.
VI. Outline of this thesis
Chapter 2 of this thesis is designed to investigate whether activated MMPs form complexes with Į2Macroglobulin in the systemic circulation of rheumatoid arthritis
patients and to show feasibility of MMP activity measurements as a marker of disease activity in RA.
Chapter 3 provides the basic characteristics of the assay used to measure MMP activity in the systemic circulation using fluorogenic substrates. Age, gender and time of sampling related differences are investigated. MMP activity in diseases involving joint pathology is compared to other systemic conditions.
Chapter 4 describes the study aimed on identifications of MMPs, which are likely to be involved in joint pathology and could be used as disease specific markers in synovial fluid and systemic circulation.
Chapter 5 illustrates how the markers identified in the previous chapter are used to predict the development of joint destruction in an Early Arthritis Clinic cohort and shows which markers can be used to describe the disease process.
Chapter 6 indicates MMP/TIMP imbalance as shown by an increased MMP/TIMP molar ratio and high levels of MMP/Į2Macroglobulin complexes in synovial fluid of injury
patients. It also suggests a disease specific pattern of MMP activation in joint diseases. Chapter 7 shows that MMP/Į2Macroglobulin complex levels in the systemic circulation
of RA patients are reduced upon treatment with disease modifying drugs such as leflunomide or methotrexate.
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