Involvement of matrix metalloproteinases and their inhibitors in peripheral synovitis and down-regulation by tumor necrosis factor ╨Ю┬▒ blockade in spondylarthropathy.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 50, No. 9, September 2004, pp 2942–2953 DOI 10.1002/art.20477 © 2004, American College of Rheumatology Involvement of Matrix Metalloproteinases and Their Inhibitors in Peripheral Synovitis and Down-Regulation by Tumor Necrosis Factor ␣ Blockade in Spondylarthropathy Bernard Vandooren,1 Elli Kruithof,1 David T. Y. Yu,2 Markus Rihl,3 Jieruo Gu,2 Leen De Rycke,1 Filip Van den Bosch,1 Eric M. Veys,1 Filip De Keyser,1 and Dominique Baeten1 and SpA patients. Involvement of MMPs and TIMPs in SpA synovitis was suggested by the correlation with cellular infiltration, vascularization, and cartilage degradation. Higher serum levels of MMPs 3 and 9 were revealed in SpA and RA patients as compared with healthy controls. Production of MMP-3, but not MMP-9, in the serum reflected the presence of peripheral synovitis, as indicated by 1) the correlation between serum levels, SF levels (which were 1,000-fold higher than the serum levels), and synovial expression of MMP-3, 2) the increased levels of MMP-3 in AS patients with peripheral disease and not exclusively axial involvement, and 3) the correlation of serum and SF MMP-3 with parameters of synovial, but not systemic, inflammation. The modulation of the MMP/ TIMP system by tumor necrosis factor ␣ (TNF␣) blockade was confirmed by the down-regulation of all MMPs and TIMPs in the synovium and a pronounced and rapid decrease of serum MMP-3. Conclusion. MMPs and TIMPs are highly expressed in SpA synovitis and mirror both the inflammatory and tissue-remodeling aspects of the local disease process. Serum MMP-3, originating from the inflamed joint, represents a valuable biomarker for peripheral synovitis. Modulation of the MMP/TIMP system by infliximab could contribute to the antiinflammatory and tissue-remodeling effects of TNF␣ blockade in SpA. Objective. To investigate the role of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in spondylarthropathy (SpA) synovitis. Methods. Paired samples of synovial biopsy tissue as well as serum and synovial fluid (SF) from 41 patients with SpA and 20 patients with rheumatoid arthritis (RA) and serum samples from 20 healthy controls were analyzed by immunohistochemistry and enzyme-linked immunosorbent assay for the presence of MMPs 1, 2, 3, and 9 and TIMPs 1 and 2. In addition, sera from 16 patients with ankylosing spondylitis (AS) and peripheral synovitis and 17 patients with AS and exclusively axial involvement were analyzed. An additional cohort of SpA patients was analyzed at baseline and after 12 weeks of infliximab treatment. Results. Staining for MMPs and TIMPs showed a cellular and interstitial pattern in the synovial lining and sublining layers that was similar between the RA Dr. Rihl’s work was supported by the Deutsche Forschungsgemeinschaft (RI 119/1-1) and the Rheumatology Competence Network. Dr. De Rycke’s work was supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT/SB/11127). Dr. Baeten’s work was supported by the Fund for Scientific Research–Vlaanderen (FWO-Vlaanderen). 1 Bernard Vandooren, MD, Elli Kruithof, MD, Leen De Rycke, MD, Filip Van den Bosch, MD, PhD, Eric M. Veys, MD, PhD, Filip De Keyser, MD, PhD, Dominique Baeten, MD, PhD: Ghent University Hospital, Ghent, Belgium; 2David T. Y. Yu, MD, PhD, Jieruo Gu, MD: University of California at Los Angeles; 3Markus Rihl, MD: Hannover Medical School, Hannover, Germany. Drs. Vandooren and Kruithof contributed equally to this article. Address correspondence and reprint requests to Elli Kruithof, MD, Department of Rheumatology, 0K12IB, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. E-mail: elli.kruithof@ ugent.be. Submitted for publication December 6, 2003; accepted in revised form May 20, 2004. Inflammation and structural damage of the joint are 2 major hallmarks of autoimmune arthritides such as rheumatoid arthritis (RA) and spondylarthropathy (SpA). In RA, several pivotal mediators involved in disease mechanisms have been identified, including interleukin-1 (IL-1) and tumor necrosis factor ␣ (TNF␣) 2942 INVOLVEMENT AND BLOCKADE OF MMPs/TIMPs IN SpA SYNOVITIS as proinflammatory mediators (1), along with RANK ligand (2) and matrix metalloproteinases (MMPs) as central mediators of joint destruction. MMPs participate in extracellular matrix (ECM) degradation by cleavage of ECM constituents such as collagens and proteoglycans. The MMPs exert physiologic (e.g., wound healing, embryogenesis) and pathologic (e.g., cancer, atherosclerosis) functions that are dependent on activation by precursor zymogens and inhibition by binding to specific inhibitors (␣2-macroglobulin, tissue inhibitors of matrix metalloproteinases [TIMPs]). To date, more than 20 MMPs and 4 TIMPs have been recognized (3). Expression of the MMPs and TIMPs has been studied extensively in the synovial tissue (4–7), synovial fluid (SF) (8–10), and serum (11–13) of patients with RA. In the synovial compartment, they are produced by macrophages, synovial fibroblasts, endothelial cells, neutrophils, and chondrocytes (14). Active up-regulation of MMPs is observed after stimulation with proinflammatory cytokines such as IL-1␤ (15) and TNF␣ (15–18). Their biologic relevance has been linked to joint destruction (19,20), angiogenesis (21), and cell trafficking (22). MMP-1 (collagenase 1) cleaves, inter alia, type II collagen, leading to irreversible cartilage destruction, whereas MMP-3 (stromelysin 1) cleaves proteoglycans, fibronectin, and the smaller collagens and also activates proMMP-1 (3). Accordingly, MMPs 1 and 3 are considered to play a pivotal role in joint destruction (14). MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are mediators of joint destruction (23), but additionally have a particular role in angiogenesis (21). Moreover, in the joint, the function of MMPs is regulated by the presence of specific inhibitors such as TIMPs 1 and 2. Whereas these molecular pathways have been well analyzed in RA, the mechanisms of joint inflammation and destruction are poorly understood in SpA. Diffuse cartilage destruction is found in both diseases. However, focal bone erosions are far more common in RA than in SpA, with the exception of psoriatic arthritis (PsA) which might also manifest marked focal bone destruction (24). In contrast, hypervascularity is a hallmark of SpA rather than RA (25,26). Using microarray as a screening strategy for the identification of important mediators in SpA synovitis (27), we found a profound down-regulation of MMP-3 in SpA synovium after infliximab treatment (data not shown). Based on these initial results, we analyzed the involvement of MMPs and TIMPs in SpA synovitis by addressing the following questions. 1) What is the baseline expression of MMPs and TIMPs in the inflamed peripheral joint? 2) Does the expression of these 2943 mediators correlate with parameters of inflammation and/or tissue remodeling (synovial vascularization, matrix degradation)? 3) Are serum MMP levels a good reflection of the MMP/TIMP activity in the joint? 4) Can the MMP/TIMP system be modulated by therapies such as TNF␣ blockade? PATIENTS AND METHODS Patients. To study the synovial expression of MMPs and TIMPs in SpA, we obtained paired synovium, SF, and serum samples from 41 patients with SpA fulfilling the European Spondylarthropathy Study Group (ESSG) criteria (28). All patients had peripheral synovitis with involvement of at least 1 knee joint and underwent a needle arthroscopy for biopsy sampling. This cohort comprised patients with PsA (defined as SpA with skin psoriasis) (n ⫽ 19), patients with ankylosing spondylitis (AS) fulfilling the New York criteria for AS (29) (n ⫽ 10), and patients with undifferentiated SpA (USpA) (n ⫽ 12). As control groups, we obtained paired synovium, SF, and serum samples from 20 patients with RA fulfilling the American College of Rheumatology (formerly, the American Rheumatism Association) criteria (30), as well as serum from 20 healthy controls (10 men and 10 women). To further analyze the relationship between MMP/TIMP expression and peripheral joint disease in SpA, we additionally obtained serum samples from 33 patients with AS fulfilling the New York criteria (29), of whom 17 had no peripheral joint involvement but exhibited exclusively axial symptoms, whereas the other 16 had at least 1 swollen joint. Table 1 summarizes the demographic and clinical characteristics of the different patient groups. Finally, to analyze the effect of TNF␣ blockade on the MMP/TIMP system, we obtained serum samples from 12 infliximab-treated and 10 placebo-treated patients with SpA and peripheral joint involvement, at different time points between baseline and week 12 after initiation of treatment (31). Furthermore, in 9 of the infliximab-treated patients with peripheral synovitis, synovial tissue samples were obtained at baseline and week 12, as described before (32). The demographics and clinical characteristics of this cohort at baseline and week 12 are given in Table 1. All patients provided their written informed consent prior to inclusion in the study. The study was approved by the ethics committee of the local faculty of medicine. Immunohistochemistry of synovial biopsy tissue. Synovial tissue biopsy samples (16 from each individual patient) were obtained by needle arthroscopy of the knee as described previously (32). Eight of the biopsy samples from each patient were stored in formaldehyde and embedded in paraffin, and the other 8 were snap frozen and mounted in Jung tissuefreezing medium (Leica Instruments, Nussloch, Germany) and utilized for immunohistochemistry. Paraffin-embedded biopsy tissues were stained with hematoxylin and eosin for histologic analysis, which involved determination of the mean thickness of the synovial lining layer, vascularity of the sublining layer, cellular infiltration of the sublining layer, and presence of lymphoid aggregates, plasma cells, and polymorphonuclear cells. Frozen sections of 58.3 ⫾ 16.8 12/8 NA 6.4 ⫾ 7.8 NA 9.0 ⫾ 7.7 5.3 ⫾ 4.5 42 ⫾ 27 14/6 7/13 8/12 42.8 ⫾ 9.8 12/29 19/10/12 6.5 ⫾ 7.9 NA 3.2 ⫾ 3.8 3.4 ⫾ 4.2 26 ⫾ 26 36/5 12/29 1/40 15/2 0/17 0/17 0 2.9 ⫾ 1.9 28 ⫾ 13 43.7 ⫾ 8.3 2/15 0/17/0 17.5 ⫾ 8.6 NA 15/1 2/14 1/15 4.6 ⫾ 4.9 4.9 ⫾ 4.1 43 ⫾ 33 48.1 ⫾ 14.3 3/13 0/16/0 16.7 ⫾ 14.0 NA AS with peripheral AS without peripheral joint disease joint disease (n ⫽ 17) (n ⫽ 16) 11/1 0/12 0/12 7.3 ⫾ 5.3 2.5 ⫾ 2.2 20 ⫾ 16 11/1 0/12 0/12 1.1 ⫾ 1.2 1.2 ⫾ 2.4 8⫾7 9/1 0/10 0/10 6.6 ⫾ 5.2 2.3 ⫾ 2.4 24 ⫾ 30 9/1 0/10 0/10 10.9 ⫾ 11.0 1.8 ⫾ 2.2 23 ⫾ 29 49.1 ⫾ 11.0 NA 51.0 ⫾ 10.9 NA 3/9 NA 2/8 NA 6/5/1 NA 6/3/1 NA 8.2 ⫾ 7.1 NA 11.2 ⫾ 10.1 NA 59.8 ⫾ 20.2 16.8 ⫾ 13.1 64.7 ⫾ 22.9 68.1 ⫾ 27.9 Week 12 Baseline Baseline Week 12 Placebo-treated SpA (n ⫽ 10) Infliximab-treated SpA (n ⫽ 12) * Except where indicated otherwise, values are the mean ⫾ SD. SpA ⫽ spondylarthropathy; RA ⫽ rheumatoid arthritis; AS ⫽ ankylosing spondylitis; PsA ⫽ psoriatic arthritis; USpA ⫽ undifferentiated SpA; NA ⫽ not applicable; VAS ⫽ visual analog scale; CRP ⫽ C-reactive protein; ESR ⫽ erythrocyte sedimentation rate; NSAID ⫽ nonsteroidal antiinflammatory drug; DMARD ⫽ disease-modifying antirheumatic drug. RA (n ⫽ 20) SpA with peripheral joint disease (n ⫽ 41) Clinical and descriptive features of the patients* Age, years Sex, no. female/male Subtype, no. with PsA/AS/USpA Disease duration, years Patient’s global disease activity assessment, mm VAS Number of swollen joints Serum CRP, mg/dl ESR, mm/hour Medication, no. taking/not taking NSAID DMARD Corticosteroids Table 1. 2944 VANDOOREN ET AL INVOLVEMENT AND BLOCKADE OF MMPs/TIMPs IN SpA SYNOVITIS the synovial biopsy tissue were stained and scored as described previously (25,33,34). The following mouse monoclonal antibodies (mAb) were used: anti–MMP-1 mAb (5 g/ml) (collagenase, IgG2a, clone 41-1E5; Oncogene, Cambridge, MA), anti–MMP-2 mAb (4 g/ml) (gelatinase A, IgG1, clone 757F7; Oncogene), anti–MMP-3 mAb (0.5 g/ml) (stromelysin 1, IgG1, clone SL-1 IIIC4; Oncogene), anti–MMP-9 mAb (5 g/ml) (gelatinase B, type IV collagenase, IgG1, clone 36020.111; R&D Systems, Abingdon, UK), anti–TIMP-1 mAb (2 g/ml) (IgG1, clone 102D1; Oncogene), and anti–TIMP-2 mAb (2 g/ml) (IgG1, clone T2-101; Oncogene). After incubation with the primary antibody, sections were sequentially incubated with a biotinylated second antibody, with a streptavidin–horseradish peroxidase link, and finally with amino-ethyl-carbazole substrate as chromogen. Parallel sections were incubated with irrelevant isotype and concentration-matched mAb as negative controls. Sections were coded and analyzed semiquantitatively on a 4-point scale (ranging 0–3) by 2 independent observers (BV and EK) who were blinded to the diagnosis and clinical data (25,33,34). Global interobserver agreement on the different markers was 80% for the lining layer (mean correlation coefficient 0.675, P ⬍ 0.001) and 70% for the sublining layer (mean correlation coefficient 0.760, P ⬍ 0.001). Of particular interest in this study was MMP-3, and the interobserver agreement on MMP-3 expression in the lining and sublining layers was a mean 0.718 (P ⬍ 0.001) and 0.830 (P ⬍ 0.001), respectively. Moreover, MMP-3 samples were scored twice by the same observer, resulting in a global intraobserver mean correlation coefficient of 0.672 (P ⬍ 0.001) for the lining layer and 0.686 (P ⬍ 0.001) for the sublining layer. Immunoassays. Serum and SF levels of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 were measured by enzyme-linked immunosorbent assay (ELISA) (Biotrak; Amersham Pharmacia Biotech, Buckinghamshire, UK) in accordance with the manufacturer’s instructions. Dilution levels for the serum samples were as follows: 1:1 for MMP-1, 1:250 for MMP-2, 1:1 for MMP-3, 1:20 for MMP-9, 1:150 for TIMP-1, and 1:4 for TIMP-2. Dilution levels for the SF samples were as follows: 1:400 for MMP-3 and 1:40 for MMP-9. Levels of cartilage oligomeric matrix protein (COMP) in the SF were measured by ELISA (AnaMar Medical, Uppsala, Sweden) in accordance with the manufacturer’s instructions. SF samples were diluted 1:40. Duplicate samples from each individual were assayed. Statistical analysis. Differences between groups were analyzed using the Mann-Whitney U test, and analysis of the matched pairs was performed using Wilcoxon’s signed-rank test. Correlations between variables were assessed by Spearman’s rank test. Bonferroni correction for multiple testing was applied where indicated. A P value of less than 0.05 after Bonferroni correction was considered to be statistically significant. 2945 Figure 1. Immunohistochemical staining for matrix metalloproteinase 1 (MMP-1) (A), MMP-2 (B), MMP-3 (C), MMP-9 (D), tissue inhibitor of matrix metalloproteinases 1 (TIMP-1) (E), and TIMP-2 (F) in the lining layer as well as in the sublining layer. Synovial expression of MMP-3 is characterized by cellular and diffuse interstitial expression in the sublining layer and a less extensive expression in the lining layer. In some patients, a characteristic distribution of interstitial expression of MMP-3 adjacent to the lining layer is observed (G and H). MMP-9 is expressed primarily in the sublining layer of synovium, with a patchy aspect that is especially pronounced in the perivascular regions, and there is also staining of the intravascular cells (I and J). Original magnification ⫻ 640 in A–F, H, and J, and ⫻ 320 in G and I. RESULTS Equal expression of MMPs and TIMPs in SpA and RA synovium. Immunohistochemical staining of synovial tissue samples from 41 patients with SpA showed clear expression of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 in both the lining layer and the sublining layer. As illustrated in Figure 1, the different MMPs and TIMPs showed both a cellular and an interstitial staining pattern, a more pronounced, but 2946 VANDOOREN ET AL Table 2. Correlation between synovial MMP and TIMP expression and local disease features in the synovium and synovial fluid COMP levels in patients with spondylarthropathy* MMP-1 Lining Sublining MMP-2 Lining Sublining MMP-3 Lining Sublining MMP-9 Lining Sublining TIMP-1 Lining Sublining TIMP-2 Lining Sublining Vascularity Cellular infiltration Polymorphonuclear cells present Synovial fluid COMP levels NS NS NS NS NS NS NS NS 0.490† NS NS NS NS NS NS NS NS 0.450† 0.345‡ NS 0.375‡ NS NS NS NS 0.409‡ 0.354‡ 0.457† 0.541† 0.582† NS NS NS NS 0.449† 0.458† NS 0.463† NS NS NS NS NS 0.380‡ NS 0.336‡ ⫺0.474† ⫺0.613† * Values are Spearman’s rho correlation coefficients. Expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) was assessed by immunohistochemistry (semiquantitative scores on a 0–3 scale) and correlated with local disease features (semiquantitative scores on a 0–3 scale). COMP ⫽ cartilage oligomeric matrix protein; NS ⫽ not significant. † P ⬍ 0.01. ‡ P ⬍ 0.05. certainly not exclusive, staining for MMP-3 in the sublining layer than in the lining layer, and a pronounced peri- and intravascular staining for MMP-9. When both the degree and the pattern of immunoreactivity in the SpA samples were compared with those from similar stainings of the 20 RA samples, we found no significant differences for all of the investigated molecules (data not shown). However, within the SpA group, the expression of MMP-1 was higher in the AS and USpA synovial tissue samples (n ⫽ 22) than in the PsA samples (n ⫽ 19) (P ⫽ 0.027 for the lining layer and P ⫽ 0.010 for the sublining layer), and a similar difference was observed within the SpA group in the levels of MMP-2 in the sublining layer (P ⫽ 0.042). The pattern of expression, however, was similar between the AS/USpA patients and PsA patients. Correlation between synovial MMP/TIMP expression and local features of synovitis in SpA. Because of the strong expression of MMPs and TIMPs in SpA synovitis and the potential role of this molecular system in tissue remodeling, we investigated the relationship between the expression levels of MMPs and TIMPs and the features of local disease in SpA synovitis, such as inflammatory cell infiltration (global number of inflammatory cells and number of polymorphonuclear cells in the synovial membrane), extent of vascularization (num- ber of blood vessels in the synovium), and degree of cartilage breakdown (COMP levels in SF) (25,33). As shown in more detail in Table 2, the expression of MMPs 3 and 9 and TIMPs 1 and 2 correlated with the level of global inflammatory cell infiltration as well as with the presence of polymorphonuclear cells. Moreover, the expression of MMPs 2, 3, and 9 correlated with the extent of vascularity, confirming the role of these MMPs in vascular remodeling. Finally, SF levels of COMP, a marker of collagen degradation, showed a strong inverse correlation with the synovial expression of TIMP-2, suggesting that high TIMP-2 levels are associated with decreased cartilage breakdown in SpA. Of interest, the COMP levels were equally elevated in the SF from RA patients and the SF from SpA patients (median 26.2 units/liter, range 8.8–75.2 and median 26.4 units/liter, range 12.4–90.0, respectively; P not significant). Origination of serum MMP-3 from the inflamed joint, reflecting peripheral synovitis in SpA. Since we clearly demonstrated the expression of the MMPs and TIMPs in inflamed SpA synovium, and since previous reports have indicated that serum MMP levels are elevated in SpA, we further investigated whether the serum levels of MMPs and TIMPs were a reflection of their expression in the peripheral joint. First, we evaluated the serum levels of MMPs and TIMPs in SpA 3.79 (1.90–53.59) 1,401.96 (325.59–2,933.64) 25.53 (0.08–1,190.64)† 447.36 (2.12–2,259.95)§ 1,680.75 (972.95–2,892.36) 78.01 (7.20–541.65) SpA patients (n ⫽ 41) 4.35 (2.33–25.42) 1,791.19 (2.50–3,741.72) 44.59 (0.42–224.92)‡ 570.15 (110.92–2,157.38)¶ 1,774.44 (742.91–4,254.35) 79.89 (5.26–582.95) RA patients (n ⫽ 20) 5.23 (2.18–8.69) 1,149.53 (725.55–3,807.65) 10.61 (5.44–21.06) 237.92 (109.43–2,335.55) 1,642.88 (832.99–3,175.93) 51.22 (25.63–199.86) Healthy controls (n ⫽ 20) 4.3 (3.0–29.2) 1,336.6 (903.3–2,646.9) 89.4 (2.0–277.5) 301.2 (124.9–819.5) 1,474.1 (1,076.6–2,622.2) 57.2 (7.2–328.8) Baseline 4.6 (3.6–13.8) 1,545.2 (997.8–2,178.1) 9.8 (2.5–8.3) 383.6 (54.5–575.2) 1,609.4 (971.7–2,621.6) 31.3 (20.1–193.6) After infliximab SpA patients treated with infliximab (n ⫽ 12) Levels of serum MMPs and TIMPs in SpA patients, RA patients, and healthy controls and in SpA patients before and after treatment with infliximab* 0.386 0.721 0.007 0.878 0.959 0.169 P * Values for levels of biomarkers are the median (range) ng/ml. P values are by Mann-Whitney U test for comparisons between SpA patients, RA patients, and healthy controls, and by paired Wilcoxon signed-rank test for comparisons before and after treatment with infliximab. See Tables 1 and 2 for definitions. † P ⫽ 0.004 versus healthy controls. ‡ P ⬍ 0.001 versus healthy controls. § P ⫽ 0.090 versus healthy controls. ¶ P ⫽ 0.030 versus healthy controls. MMP-1 MMP-2 MMP-3 MMP-9 TIMP-1 TIMP-2 Table 3. INVOLVEMENT AND BLOCKADE OF MMPs/TIMPs IN SpA SYNOVITIS 2947 2948 VANDOOREN ET AL Figure 2. Correlations between MMP-3 expression in the synovium, synovial fluid (SF), and serum of patients with spondylarthropathy (SpA). A correlation is evident between MMP-3 levels in the SF (in ng/ml) and the expression of MMP-3 in the lining layer (semiquantitative score on a 0–3 scale) in SpA patients (n ⫽ 29; r ⫽ 0.497 by Spearman’s rank test, P ⬍ 0.01) (A), and also between SF MMP-3 and serum MMP-3 levels (both in ng/ml) in SpA patients (n ⫽ 29; r ⫽ 0.567 by Spearman’s rank test, P ⬍ 0.01) (B). Serum MMP-3 levels were compared between ankylosing spondylitis (AS) patients with exclusively axial involvement (n ⫽ 16) and AS patients with peripheral joint disease (n ⫽ 17) (C); significantly (P ⫽ 0.009) higher serum MMP-3 levels are seen in AS patients with peripheral joint disease compared with those with exclusively axial symptoms. Boxes and whiskers show the median and range. See Figure 1 for other definitions. patients (n ⫽ 41) in comparison with RA patients (n ⫽ 20) and healthy controls (n ⫽ 20). As shown in Table 3, MMP-3 and MMP-9 were increased in the serum of SpA and RA patients compared with healthy controls, whereas there were no differences in the expression levels of the other mediators. Furthermore, there were no significant differences in the serum levels of MMPs and TIMPs between the RA and SpA patients or between the subtypes of SpA (data not shown). On the basis of these results, we further analyzed whether the expression of serum MMP-3 and MMP-9 originated from the inflamed peripheral joints of patients with SpA. ELISA analysis of SF from 41 SpA patients revealed detectable levels of both MMP-3 (median 30,244.0 ng/ml, range 0–90,780.0) and MMP-9 (median 182.8 ng/ml, range 0–2,742.0). The SF levels of MMP-3 correlated strongly with the expression of MMP-3 in the lining layer (r ⫽ 0.497, P ⬍ 0.01) (Figure 2A), suggesting that secretion of MMP-3 occurs locally. Moreover, the SF levels of MMP-3 were ⬃1,000-fold higher than the MMP-3 levels in paired serum samples (P ⬍ 0.001) and showed a strong correlation with the serum MMP-3 levels (r ⫽ 0.567, P ⬍ 0.01) (Figure 2B). Remarkably, we found significantly higher serum MMP-3 levels in the AS patients who had peripheral joint involvement (n ⫽ 16) (median 64.2 ng/ml, range 1.4–1,155.2) as compared with the AS patients who had exclusively axial involvement (n ⫽ 17) (median 17.0 ng/ml, range 0–54.9) (P ⬍ 0.01) (Figure 2C). The specificity of this finding was emphasized by the fact that the parameters of systemic disease activity (C-reactive protein [CRP] levels and erythrocyte sedimentation rate [ESR]) did not differ between these 2 groups of AS patients. In the same manner, serum MMP-3 levels in the AS patients with exclusively axial involvement were not significantly increased in comparison with those in the healthy controls, and 6 of the 11 patients who had an elevated ESR and increased CRP level demonstrated serum MMP-3 levels in the normal range. In contrast, in the AS patients with peripheral involvement, elevated serum MMP-3 levels were observed in 10 of the 18 patients who had a normal ESR and in 4 of the 5 patients with CRP values in the normal range (data not shown). In contrast to the MMP-3 findings, SF levels of MMP-9 were not significantly correlated with either the synovial expression of MMP-9 or the serum levels of MMP-9 (data not shown). Moreover, MMP-9 levels were 3 times more elevated in the serum (median 447.4 ng/ml, range 2.1–2,260.0) than in the SF (median 182.8, range 0–2,742.0) (P ⬍ 0.05). Finally, no differences in serum MMP-9 levels could be demonstrated between the AS patients with peripheral joint involvement (623.2 ng/ml, range 124.9–1,452.0) and those without peripheral joint involvement (953.8 ng/ml, range 556.0– 1,970.0; P not significant). The suggestion that the expression of serum MMP-3, but not serum MMP-9, was INVOLVEMENT AND BLOCKADE OF MMPs/TIMPs IN SpA SYNOVITIS a reflection of peripheral joint inflammation rather than global inflammation was further supported by the much stronger correlation of SF and serum MMP-3 levels with synovial inflammatory infiltration than with systemic inflammatory parameters such as the CRP level and ESR (data not shown). Down-regulation of the MMP/TIMP system in inflamed synovium by infliximab treatment. Since we demonstrated the expression and involvement of the MMPs/TIMPs in peripheral synovitis in SpA, we aimed to confirm that this system was not constitutively expressed, but could be modulated by therapy, as suggested by microarray experiments during infliximab treatment (data not shown). As shown in Table 1, there was a significant and consistent improvement in the parameters of global and peripheral joint disease in the infliximab-treated group as compared with the placebotreated group. Immunohistochemical analysis of synovial biopsy tissue showed a significant down-regulation of MMP-3 (P ⫽ 0.017) and TIMP-1 (P ⫽ 0.026) and showed a similar trend for MMP-9 (P ⫽ 0.059) in the lining layer following treatment with infliximab. There was no treatment effect on MMP-1, MMP-2, and TIMP-2 within the lining layer, which might be attributable to their low baseline expression. Within the sublining layer, a significant down-regulation of synovial expression was observed for MMP-1 (P ⫽ 0.030), MMP-2 (P ⫽ 0.038), MMP-3 (P ⫽ 0.023), MMP-9 (P ⫽ 0.011), TIMP-1 (P ⫽ 0.011), and TIMP-2 (P ⫽ 0.010) following treatment with infliximab (Figure 3). Serum levels of MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 from patients with peripheral synovitis were not elevated at baseline as compared with the baseline levels in healthy controls, and did not change significantly after 12 weeks of treatment (Table 3). In contrast, serum levels of MMP-3 were elevated prior to initiation of treatment (median 89.4 ng/ml, range 2.0–277.5) and decreased substantially over 12 weeks (median 9.8 ng/ml, range 2.5–18.3) (P ⫽ 0.007) in the infliximab-treated group (Figures 4A and B). In comparison, the placebo-treated group showed a significant increase in serum TIMP-2 levels only (data not shown). As demonstrated by sequential analysis at various time points between baseline and week 12, the maximum down-regulation of serum MMP-3 levels in patients with SpA and peripheral synovitis was achieved within the first week of treatment and was sustained during the whole followup period (Figure 4C). 2949 Figure 3. Effect of infliximab (5 mg/kg intravenously at week 0, week 2, and week 6) on synovial expression of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 in patients with SpA, as assessed by immunohistochemistry. Representative sections from the evaluations done at baseline (left) and week 12 (12 we) (right) are shown (original magnification ⫻ 320), and the corresponding semiquantitative scores on a 0–3 scale are as follows: MMP-1, in the lining layer score 1 versus 0, in the sublining layer score 2 versus 0; MMP-2, in the lining layer score 2 versus 1, in the sublining layer score 2 versus 0; MMP-3, in the lining layer score 0 versus 0, in the sublining layer score 3 versus 0; MMP-9, in the lining layer score 1 versus 0, in the sublining layer score 2 versus 0; TIMP-1, in the lining layer score 3 versus 0, in the sublining layer score 2 versus 0; TIMP-2, in the lining layer score 1 versus 1, in the sublining layer score 1 versus 0. P values are by paired Wilcoxon signed-rank test, comparing week 12 with baseline values. See Figures 1 and 2 for definitions. DISCUSSION On the basis of preliminary findings obtained by microarray, which revealed a decrease in synovial 2950 Figure 4. Effect of infliximab (5 mg/kg intravenously at week 0, week 2, and week 6) on serum levels of MMP-3 and MMP-9 in patients with SpA. Serum levels of MMP-3 (A) and MMP-9 (B) for each individual patient are illustrated at baseline (week [we] 0) and at week 12 after initiation of therapy. In the placebo-treated patient cohort, no significant changes were noted (data not shown). In addition, serum levels of MMP-3 were evaluated at baseline, week 1, week 2, week 6, and week 12 after initiation of therapy in 10 patients with SpA (C). A significant down-regulation of serum MMP-3 is already evident at 1 week after initiation of the infliximab treatment (ⴱ ⫽ P ⬍ 0.05). P values are by paired Wilcoxon signed-rank test, comparing each time point with baseline. ns ⫽ not significant (see Figures 1 and 2 for other definitions). MMP-3 in SpA patients during infliximab treatment (data not shown), the aim of the present study was to investigate the MMP/TIMP system in SpA synovitis. The VANDOOREN ET AL present study demonstrated the presence of all investigated MMPs and TIMPs in both the lining and the sublining layer of SpA synovium. Although both the degree and the pattern of expression were specific to each of the investigated MMPs and TIMPs, we found no significant difference in the staining pattern between SpA patients and a RA control group. Moreover, both the level of expression and the staining pattern were similar to those observed in previous studies in RA (35–37). Although the present study was not designed to assess the complex functional interactions between proenzymes, activated MMP forms, and inhibitory TIMPs (38), the similar expression of the MMP/TIMP system in SpA and RA might suggest that it is equally involved in local disease features in both diseases. Indeed, the demonstration of MMP-9 in intravascular cells, in the vessel wall, and around the vessels, as well as the correlations between the expression of MMPs 2, 3, and 9 and the degree of vascularity in the present study suggest that the previously demonstrated involvement of these mediators in angiogenesis (39–42) also applies to inflamed synovium in SpA. These findings are consistent with those from a study indicating that elevated SF MMP-9 concentrations in PsA were positively correlated with the degree of synovial vascularization (43). Another functional aspect of interest with regard to MMPs and TIMPs is their role in cartilage and bone degradation. Although it was not the primary aim of this study to investigate this aspect in detail, it is interesting to note that expression of TIMP-2 showed a strong inverse correlation with the SF levels of COMP, a marker of cartilage degradation. Nevertheless, the similar expression of all investigated MMPs and TIMPs in SpA patients and RA patients might suggest that the differences in destructive progression between these 2 diseases are not directly related to the MMP system, although the complex functional regulation of the system precludes making strong conclusions from descriptive studies. It should be noted further that the PsA patients included in the present study all fulfilled the ESSG criteria for SpA and therefore might not be representative of patients with polyarticular, erosive PsA. These findings on the involvement of the MMP/ TIMP system in SpA synovitis and a previous report that described elevated serum levels of MMP-3 in PsA patients (44) raise the question as to whether serum MMPs reflect the presence of peripheral joint disease in SpA and thus might be useful biomarkers. When we compared the serum concentrations in SpA patients who INVOLVEMENT AND BLOCKADE OF MMPs/TIMPs IN SpA SYNOVITIS had active peripheral joint disease with those in healthy controls, we demonstrated elevated levels of MMP-3 with a similar trend for MMP-9 in the SpA patients, but this was not found for the other investigated mediators. Paralleling the data on synovial expression, the serum levels of the different MMPs and TIMPs were not different between SpA and RA patients, nor were they different between the various SpA subgroups. Further experiments involving the SpA samples indicated that serum MMP-3, but not MMP-9, originated from the inflamed joint, since the serum concentrations were 1,000-fold lower than the SF levels and correlated with the SF levels, which in turn correlated with the synovial expression of MMP-3. This was further confirmed by increased serum MMP-3 concentrations in the AS patients with peripheral synovitis compared with the AS patients with exclusively axial inflammation, whereas the serum levels of MMP-9 as well as the CRP levels and ESR were not different between the 2 AS groups. Moreover, expression of serum, but not SF, MMP-3 showed a weak correlation with the CRP levels, but not with the ESR, whereas both the serum and the SF MMP-3 levels correlated well with the degree of inflammation of the synovium. Similarly, half of the AS patients with exclusive axial involvement who had elevated CRP levels and an increased ESR demonstrated MMP-3 serum levels in the normal range, whereas many of the SpA patients with peripheral joint involvement showed normal-range CRP levels and normal ESR values but elevated levels of serum MMP-3. These data are consistent with findings of increased serum MMP-3 levels in PsA patients but not AS patients (44) and with a decrease in serum MMP-3 after total joint replacement (45). Taken together, these findings indicate that serum MMP-3 reflects peripheral joint disease, rather than global inflammation, in SpA patients. Thus, serum MMP-3 warrants further evaluation for its value as a biomarker of peripheral synovitis in clinical followup and therapeutic trials. Possible explanations as to why expression of serum MMP-3, but not MMP-9 or other mediators, is a reflection of local inflammation remain purely speculative. As a way of approaching the value of these biomarkers and to evaluate whether the MMP/TIMP system in SpA is constitutive or can be modulated, we investigated the effect of TNF␣ blockade by infliximab on MMPs and TIMPs in synovium and serum. Previous studies in RA indicated that synovial MMPs can be down-regulated by methylprednisolone, methotrexate, leflunomide, and interferon-␤ (46–48), while serum 2951 MMPs are also decreased by TNF␣ blockade (49–51). However, no significant effect of TNF␣ blockade has been demonstrated on synovial MMP-1, MMP-3, and TIMP-1 in RA (51) nor on serum MMP-1 and MMP-3 in AS (52), although the latest study might be biased by the small number of patients with peripheral joint disease. The present study clearly indicated a rapid and sustained down-regulation of serum MMP-3 after infliximab therapy, whereas there were no changes in any of the other MMPs and TIMPs in either the infliximabtreated group or the placebo-treated group. This highly significant decrease in serum MMP-3, which reflects the clinical effect of infliximab on peripheral synovitis in SpA (31,33), confirms the potential use of this agent for monitoring synovial inflammation in clinical trials in SpA. Moreover, the decrease in serum MMP-3 paralleled a strong down-regulation of the expression of MMP-3 as well as the other MMPs and TIMPs in the synovial membrane. This study is the first to demonstrate the effect of TNF␣ blockade on the MMP/TIMP system in the synovial membrane, and the findings are in accordance with the stimulatory effect of TNF␣ in vitro on MMP and TIMP production by synovial fibroblast and monocytes/ macrophages (16–18). However, it provides additional evidence that whereas MMP production can by induced by TNF␣ as well as numerous other inflammatory stimulators in vitro, targeted blockade of TNF␣ is sufficient to profoundly down-regulate the MMP/TIMP system in inflamed SpA synovium in vivo. The major impact of infliximab on the synovial expression of MMPs in SpA supports the hypothesis that infliximab not only interferes with inflammation, but could also influence tissue remodeling (vascularization, cartilage degradation) in the joints of SpA patients (ref. 33, and Kruithof E, et al: unpublished observations). In conclusion, the present study indicates the involvement of the MMP/TIMP system in the biologic processes of peripheral SpA synovitis. Although synovial tissue analysis is superior to serum analysis for the evaluation of the MMP/TIMP system in inflammatory arthritis, serum MMP-3 appears to be a specific biomarker for peripheral joint inflammation in SpA. 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