Comparison of Synovial Tissues From the Knee Joints and the Small Joints of Rheumatoid Arthritis Patients

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1 ARTHRITIS & RHEUMATISM Vol. 46, No. 8, August 2002, pp DOI /art , American College of Rheumatology Comparison of Synovial Tissues From the Knee Joints and the Small Joints of Rheumatoid Arthritis Patients Implications for Pathogenesis and Evaluation of Treatment Maarten C. Kraan, 1 Richard J. Reece, 2 Tom J. M. Smeets, 1 Douglas J. Veale, 2 Paul Emery, 2 and Paul P. Tak 1 1 Maarten C. Kraan, MD, PhD, Tom J. M. Smeets, BSc, Paul P. Tak, MD, PhD: Academic Medical Center/University of Amsterdam, Amsterdam, The Netherlands; 2 Richard J. Reece, MD, Douglas J. Veale, MD, PhD, Paul Emery, MD, PhD, FRCP: University of Leeds, Leeds, UK. Address correspondence and reprint requests to Maarten C. Kraan, MD, PhD, Division of Clinical Immunology and Rheumatology, F4-218, Department of Internal Medicine, Academic Medical Center/University of Amsterdam, Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands. m.c.kraan@amc.uva.nl. Submitted for publication January 23, 2002; accepted in revised form April 8, Objective. Serial synovial biopsy samples are increasingly being used for the evaluation of novel therapies for rheumatoid arthritis (RA). Most studies have used tissues from knee biopsies, but technical improvements have made serial small joint arthroscopy feasible as well. Theoretically, there could be differences in the features of synovial inflammation between various joints as a result of mechanical factors, differences in innervation, and other factors. We therefore undertook this study to compare the cell infiltrate in paired synovial biopsy samples from inflamed knee joints and paired inflamed small joints of patients with RA. Methods. Nine RA patients with both an inflamed knee joint and an inflamed small joint (wrist or metacarpophalangeal joint) underwent an arthroscopic synovial biopsy of both joints on the same day. Multiple biopsy specimens were collected and stained for macrophages, T cells, plasma cells, fibroblast-like synoviocytes, and interleukin-6 (IL-6) by immunohistochemistry. Sections were evaluated by digital image analysis. Results. There were no significant differences in mean cell numbers for all markers investigated in samples from the knee joint compared with samples from the small joints. We detected statistically significant correlations for the numbers of sublining macrophages, T cells, and plasma cells, as well as for IL-6 expression, between the knee joint and the small joints. However, there was no significant correlation between different joints for the numbers of intimal macrophages or fibroblast-like synoviocytes. Conclusion. The results of this study show that the inflammation in one inflamed joint is generally representative of that in other inflamed joints. Therefore, it is possible to use serial samples from the same joint, selecting either large or small joints, for the evaluation of antirheumatic therapies. It can be anticipated that rapid developments in immunology, molecular biology, and biotechnology will lead to a dramatic increase in the number of new, targeted therapies for arthritis. Therefore, there is a clear need for surrogate markers that could be used for selection purposes during the process of developing these new therapies. Examination of serial synovial samples has been proposed as a method that can be used to predict a potential effect of novel antirheumatic interventions (1). All previous studies have used serial synovial samples from the knee joint for technical reasons. However, clinically detectable arthritis of the knee joint is present in only 30 40% of patients with rheumatoid arthritis (RA). Therefore, new techniques have been developed to obtain serial synovial biopsy samples from small joints, as well (2). It is not known, however, if the features of the synovium in the large and small joints of RA patients are comparable. Factors such as differences in innervation (3), vascularity (4), and mechanical strain and stress (5) could theoretically influence the synovial infiltrate. We therefore compared the cell infiltrate in paired synovial biopsy samples from inflamed knee 2034

2 KNEE JOINTS VERSUS SMALL JOINTS IN RA 2035 joints and inflamed small joints in the same patients with RA. PATIENTS AND METHODS Patients. We evaluated 9 patients with RA (according to the 1987 criteria of the American college of Rheumatology [formerly, the American Rheumatism Association] [6]) who had both a clinically inflamed knee joint and wrist (n 5 patients) or metacarpophalangeal (MCP; n 4 patients) joint. Clinical inflammation was defined as both joint swelling and pain at physical examination. Six patients were recruited at the Division of Clinical Immunology and Rheumatology, Academic Medical Center/University of Amsterdam and 3 patients at the Department of Rheumatology, University of Leeds. All patients gave their informed consent, and the Medical Ethics Committee at each center approved the study protocol. Clinical assessment of the RA patients was performed on the day of the arthroscopy and included the number of tender and swollen joints (28 joints counted, including both knee joints). Serum levels of C-reactive protein (CRP) were also measured. Knee arthroscopy. In all 9 patients, arthroscopy was performed on the inflamed knee under local anesthesia, using a small-bore, 2.7-mm arthroscope (Storz, Tuttlingen, Germany) and utilizing an infrapatellar skin portal for macroscopic examination of the synovium and a second suprapatellar portal for the biopsy procedure (7). At each arthroscopy, synovial biopsy samples were obtained from the suprapatellar pouch, the cartilage pannus junction, the patellar gutters, and the tibiofemoral junction using a grasping forceps (Storz). Small joint arthroscopy. The arthroscopy of the wrist joint was performed under local anesthesia using a small-bore, 1.9-mm arthroscope (Storz) through a radial and an ulnar skin portal at the dorsum of the wrist at the proximal and/or distal carpal row. Both portals were used for macroscopic examination of the synovium and for the biopsy procedure. At each arthroscopy, synovial biopsy samples were obtained with a grasping forceps (Storz). Arthroscopy of the MCP joint was performed under local anesthesia using a small-bore, 1.9-mm arthroscope (Storz) through a medial and a lateral skin portal at the dorsum of the finger. Both portals were used for macroscopic examination of the synovium and for the biopsy procedure. At each arthroscopy, synovial biopsy samples were obtained with a grasping forceps (Storz). If there was macroscopic variation of the synovitis, samples were obtained from both macroscopically inflamed and macroscopically noninflamed regions (7). To minimize sampling error, a minimum of 6 tissue specimens were embedded en bloc in TissueTek OCT compound and stored in liquid nitrogen for immunohistochemistry; another 6 specimens were fixed in formalin and embedded in paraffin for hematoxylin and eosin staining. Immunohistochemical analysis. Serial sections were stained with the following mouse monoclonal antibodies (mab): anti-cd68 (EBM11; Dako, Glostrup, Denmark), anti- CD3 (SK7; Becton Dickinson, San Jose, CA), anti-cd138 (B-B4; Beckman Coulter, Mijdrecht, The Netherlands), anti- CD55 (Mab67; Serotec, Oxford, UK), and anti interleukin-6 (anti IL-6) (B-E8; BioSource, Nivelles, Belgium). Staining for cell markers and cytokines was performed as described previously (8). Following a primary step of incubation with mab, bound antibody was detected according to a 3-step immunoperoxidase method. Horseradish peroxidase activity was detected using hydrogen peroxide as substrate and aminoethylcarbazole (Sigma, St. Louis, MO) as dye. Slides were counterstained with Mayer s hematoxylin (Sigma) and mounted in Kaiser s glycerol gelatin (Merck, Darmstadt, Germany). Evaluation of the slides. All sections were coded and analyzed in a random order by an independent observer who was blinded to the clinical data (MCK), as described previously (9). Briefly, 3 separate representative regions, including the intimal lining layer and the synovial sublining, were selected for the evaluation of each section. From each region, 6 consecutive high-power fields were captured and digitized, resulting in a total of 18 high-power fields (surface area of 2.1 mm 2 ). The cell count was corrected for the tissue area analyzed and presented as the number of cells per mm 2. For the evaluation of IL-6, the number of pixels was corrected for the mean optical density of these pixels, resulting in the integrated optical density. Statistical analysis. Wilcoxon signed rank tests were used to compare staining in knee joints with that in paired wrist/mcp joints. Correlations between the knee and wrist/ MCP biopsy samples were calculated using Spearman s rho. P values less than 0.05 were considered statistically significant. RESULTS Clinical features. Of the 9 patients studied, 5 underwent arthroscopic synovial biopsies of the wrist and knee joints, and 4 underwent these biopsies of the MCP and knee joints. In the latter group, the second MCP joint was studied in 2 patients and the third MCP joint in the other 2 patients. Seven patients were women and 2 were men; their mean age was 55 years (range years). The mean disease duration was 37 months (range months). All patients had active arthritis in multiple joints. The mean tender joint count was 18 (range 6 28), the mean swollen joint count was 17 (range 6 28), and the mean serum CRP level was 33 mg/liter (range 4 120). Immunohistologic features. Staining was negative in control sections, where the primary antibody was omitted or irrelevant antibodies were applied. Rheumatoid synovium was characterized by edema, hypervascularity, and increased cellularity of the synovial sublining, as well as by hyperplasia of the intimal lining layer, which was consistent with previous observations. Increased synovial tissue mass was especially due to hyperplasia of the synovial sublining or subsynovium, which merges with the joint capsule. In this region, large numbers of

3 2036 KRAAN ET AL Figure 1. Representative microscopic images of rheumatoid synovial tissue from a paired knee joint and small (wrist) joint, depicting staining for CD68 macrophages, CD55 fibroblast-like synoviocytes, and CD3 T cells (original magnification 400). macrophages, T cells, and plasma cells were found (Figure 1). In absolute numbers, the macrophage was, on average, the predominant cell type by far. The thickened intimal lining layer, which varied focally, mainly comprised macrophages and fibroblast-like synoviocytes. There was also abundant expression of IL-6. Synovial inflammation was present in all samples. However, marked variation between patients was ob- served for each cell marker as well as for cytokine expression, confirming the heterogeneity of rheumatoid synovitis. We examined the features of inflammation in synovial tissue from knee joints compared with tissue from paired small joints. Paired, nonparametric analysis revealed no significant differences in infiltration of the synovial sublining by macrophages, T cells, and plasma

4 KNEE JOINTS VERSUS SMALL JOINTS IN RA 2037 Table 1. Cell numbers (per mm 2 ) and IL-6 expression (integrated optical density) in paired synovial samples obtained on the same day from the knee joint and small joints of patients with rheumatoid arthritis* Knee joint Small joint P Intimal lining layer Macrophages Fibroblast-like synoviocytes Synovial sublining Macrophages T cells Plasma cells IL-6 32,167 23,016 30,083 30, * Values are the mean SD. IL-6 interleukin-6. cells (Table 1). In addition, there was no significant difference in IL-6 expression between the knee joints and the small joints of the same patients. Comparing knee joints with small joints, we observed statistically significant correlations for all inflammatory cells in the synovial sublining as well as for IL-6 expression (Table 2). The correlations presented here support the view that synovial sublining infiltration in one joint represents the systemic process in RA. In contrast, there was no significant correlation between different joints when intimal macrophages or fibroblast-like synoviocytes were examined (Table 2). The numbers of intimal macrophages and fibroblast-like synoviocytes tended to be higher in knee joints, but this difference did not reach statistical significance. These data suggest that intimal lining layer hyperplasia depends in part on local factors. Table 2. Correlations (calculated by Spearman s rho) between paired synovial samples obtained on the same day from the knee joint and small joints of patients with rheumatoid arthritis, for each marker investigated* Spearman s P Intimal lining layer Macrophages NS Fibroblast-like synoviocytes NS Synovial sublining Macrophages T cells Plasma cells IL *NS not significant; IL-6 interleukin-6. DISCUSSION The results presented here show comparable signs of synovial inflammation in paired samples from the knee joint and small joints of RA patients, particularly with regard to the numbers of sublining macrophages, T cells, B cells, and plasma cells, as well as with regard to IL-6 expression. In contrast, we did not observe a trend toward a positive correlation between different joints for both major cell populations (i.e., the macrophages and the fibroblast-like synoviocytes) in the intimal lining layer. Previous methodologic studies have addressed questions regarding the optimal biopsy technique, the characteristics of the cartilage pannus junction versus those of the suprapatellar pouch, the reduction of sampling error, and different systems for the evaluation of microscopic slides. The present study is the first to demonstrate the immunohistologic features of paired synovial biopsy samples from different clinically involved joints of RA patients. A potential limitation of the study is the relatively small number of patients evaluated. This is due to the challenging study design. It is obviously difficult to justify performing arthroscopy in 2 joints of the same patient on the same day strictly for research purposes. However, the characteristic feature of paired samples is that each observation in one sample has a matching observation in the other sample; thus, the influence of specific patient characteristics is minimized. This is a major advantage in light of the heterogeneity of synovial inflammation among different RA patients, since it allows potentially meaningful analysis in a relatively small group of patients. The demonstration of statistically significant correlations for all markers in the synovial sublining supports the view that it was possible to avoid a falsenegative result due to interindividual variability. Synovial tissue analysis is increasingly being used in pathogenetic studies and in studies evaluating the effects of treatment of inflammatory arthropathies. The interpretation of these studies depends in part on the assumption that synovial tissue samples from one joint may represent systemic disease. This notion is supported by the correlation between synovial inflammation and disease activity in cross-sectional studies (10,11), the association between changes in the features of serial synovial biopsy samples and the clinical response to treatment (1,12), and the relationship between synovial tissue characteristics and disease outcome (13). Further support for the view that the features of one joint may represent systemic disease comes from the observation

5 2038 KRAAN ET AL that there is a relationship between different joints with regard to joint damage, as has been shown radiographically (14). The data presented here provide direct evidence that cell infiltration of the synovial sublining is part of a systemic process. It is important to note that the majority of the inflammatory cells in rheumatoid synovial tissue are found in the synovial sublining rather than in the intimal lining layer, as confirmed in this study. Interestingly, intimal lining layer hyperplasia appears to depend on local processes. There was no correlation of the numbers of intimal macrophages or fibroblastlike synoviocytes between the different joints. One possible explanation for the increase in fibroblast-like synoviocytes in individual joints could be the induction of genotoxic changes in response to the toxic environment, which could enhance focal hyperplasia of fibroblast-like synoviocytes in individual joints due to impaired apoptosis and increased proliferation (15). Successful treatment with diseasemodifying antirheumatic therapies is associated with decreased mononuclear cell infiltration and reduced expression of proinflammatory cytokines, especially in the synovial sublining (1,7). Previous studies have not shown a clear-cut relationship between changes in fibroblast-like synoviocyte hyperplasia and the clinical response to treatment. These cells may be more important in the process of joint destruction than they are in producing clinical signs of inflammation. We have recently used sublining cellularity and cytokine expression in serial synovial samples from knee joints as substitute end points to screen for potential antirheumatic effects of novel treatments in RA patients. The results presented here suggest that serial biopsy samples from inflamed small joints can be used in a similar way. REFERENCES 1. Tak PP. Lessons learnt from the synovial tissue response to antirheumatic treatment. Rheumatology (Oxford) 2000;39: Reece R, Emery P. Needle arthroscopy. Br J Rheumatol 1995;34: Hukkanen M, Konttinen YT, Rees RG, Santavirta S, Terenghi G, Polak JM. Distribution of nerve endings and sensory neuropeptides in rat synovium, meniscus and bone. Int J Tissue React 1992;14: Ceponis A, Konttinen YT, Mackevicius Z, Solovieva SA, Hukkanen M, Tamulaitiene M, et al. Aberrant vascularity and von Willebrand factor distribution in inflamed synovial membrane. J Rheumatol 1996;23: Simkin PA. Why this joint and why not that joint? Scand J Rheumatol Suppl 1995;101: Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31: Kraan MC, Reece RJ, Barg EC, Smeets TJM, Farnell J, Rosenburg R, et al. Modulation of inflammation and metalloproteinase expression in synovial tissue by leflunomide and methotrexate in patients with active rheumatoid arthritis: findings in a prospective, randomized, double-blind, parallel-design clinical trial in thirtynine patients at two centers. Arthritis Rheum 2000;43: Tak PP, van der Lubbe PA, Cauli A, Daha MR, Smeets TJM, Kluin PM, et al. Reduction of synovial inflammation after anti- CD4 monoclonal antibody treatment in early rheumatoid arthritis. Arthritis Rheum 1995;38: Kraan MC, Haringman JJ, Ahern MJ, Breedveld FC, Smith MD, Tak PP. Quantification of the cell infiltrate in synovial tissue by digital image analysis. Rheumatology (Oxford) 2000;39: Tak PP, Smeets TJM, Daha MR, Kluin PM, Meijers KAE, Brand R, et al. Analysis of the synovial cell infiltrate in early rheumatoid synovial tissue in relation to local disease activity. Arthritis Rheum 1997;40: Kraan MC, Versendaal H, Jonker M, Bresnihan B, Post WJ, t Hart BA, et al. Asymptomatic synovitis precedes clinically manifest arthritis. Arthritis Rheum 1998;41: Smith MD, Kraan MC, Slavotinek J, Au V, Weedon H, Parker A, et al. Treatment-induced remission in rheumatoid arthritis patients is characterized by a reduction in macrophage content of synovial biopsies. Rheumatology (Oxford) 2001;40: Cunnane G, FitzGerald O, Hummel KM, Youssef PP, Gay RE, Gay S, et al. Synovial tissue protease gene expression and joint erosions in early rheumatoid arthritis. Arthritis Rheum 2001;44: Hulsmans HMJ, Jacobs JWG, van der Heijde DMFM, van Albada-Kuipers GA, Schenk Y, Bijlsma JWJ. The course of radiologic damage during the first six years of rheumatoid arthritis. Arthritis Rheum 2000;43: Tak PP, Zvaifler NJ, Green DR, Firestein GS. Rheumatoid arthritis and p53: how oxidative stress might alter the course of inflammatory diseases. Immunol Today 2000;21:78 82.