What can we learn from the synovium in early rheumatoid arthritis?

Rheumatoid arthritis (RA) affects approximately 1% of the population and is a chronic inflammatory joint disease resulting in joint destruction, increased morbidity and mortality. Although the aetiology of this disease is unknown, the pivotal role played by cytokines and degradative enzymes in mediating inflammation and joint destruction, particularly early in the disease process, has been the focus of recent literature and will be the focus of this review. Up until recently, studies on early RA were limited as there was an inherent delay in patients reaching the rheumatologist's care and initial diagnostic confusion may have compounded these problems. In particular, the observation that early intervention improves outcome, has driven the study of early RA. It is difficult to define early RA but most studies have defined this as disease duration of less than 12 months from symptom onset. Clearly, it is important to study the synovial membrane in early disease, in particular to try and answer the important questions: (1) What are the earliest changes to occur in the RA synovium? (2) Can we distinguish RA on the basis of synovial membrane pathology? (3) Can synovial immunopathology predict outcome? (4) What is the role of arthroscopic biopsy in early RA?

Expression of chemokines and matrix metalloproteinases in early rheumatoid arthritis.

OBJECTIVE: To compare macrophage infiltration and expression of chemokines and matrix metalloproteinases (MMPs) in synovial tissue between patients with early and long-standing rheumatoid arthritis (RA). METHODS: Knee synovial biopsies were taken from 22 patients with early (<1 yr) and 22 patients with long-standing (>5 yr) RA and immunostained with antibodies specific for CD68; macrophage inflammatory protein (MIP)-1alpha and monocyte chemoattractant protein (MCP)-1; MMP-1 and -3 and the tissue inhibitors of metalloproteinases (TIMP)-l and -2. Immunostaining was quantified using a colour video image analysis system. RESULTS: CD68+ macrophage infiltration and the expression of MIP-1alpha, MCP-1, MMP-1, MMP-3, TIMP-1, and TIMP-2 were observed in synovial tissue of patients with early RA. In long-standing RA, there was a further increase in CD68+ macrophage infiltration and MIP-1alpha expression in the synovial lining layer. CD68 expression correlated with MIP-1alpha (R=0.39, P=0.01), but not with MCP-1 expression. CONCLUSION: Macrophage accumulation, and the expression of chemokines and MMPs in synovial tissue occur in early RA. Targeting chemokines which play a role in the migration of macrophages into the joints may be of therapeutic benefit in RA patients.

Synovial tissue protease gene expression and joint erosions in early rheumatoid arthritis.

OBJECTIVE: To relate the expression of proteases in the lining and sublining layers of the synovial membrane to the rate of joint damage during 1 year in patients with early inflammatory arthritis. METHODS: Samples of synovial membrane were obtained by closed-needle biopsy or needle arthroscopy from inflamed knees of 20 patients with early inflammatory polyarthritis (mean disease duration 9.6 months, range 2 weeks to 18 months). Expression of matrix metalloproteinase 1 (MMP-1), cathepsin B (CB), and cathepsin L (CL) was examined using in situ hybridization. Immunohistochemistry was used to identify infiltrating mononuclear cell populations. Radiographs of the hands and feet, performed at presentation and after 1 year, were evaluated for the development of new erosions. RESULTS: Twelve patients had rheumatoid arthritis (RA), 6 had psoriatic arthritis (PsA), 1 had gout, and 1 had an undifferentiated arthritis. Six patients had erosions at presentation. Eleven patients (10 with RA, 1 with PsA) demonstrated at least 1 new erosion after 1 year of followup. MMP-1, CB, and CL messenger RNA (mRNA) were expressed in the synovial membrane of all patients and were present throughout the lining layer, as well as in perivascular cellular infiltrates and endothelial cells in the sublining layer. In the lining layer, the mean percentages of protease mRNA-positive cells per high-power field were higher in those patients who developed new joint erosions than in those without evidence of joint damage. A similar pattern was observed in the sublining layer, where mean numbers of protease mRNA-positive cells were also greater in patients with new joint erosions. There were significant differences between the two groups in MMP-1 mRNA expression in both the lining and sublining layers (P = 0.0007 and P = 0.0027, respectively), as well as in sublining layer CL mRNA expression (P = 0.017), but not in CB mRNA expression. Numbers of lining layer CD68+ cells correlated positively with lining layer MMP-1 mRNA expression (P = 0.043) and with the development of new joint erosions (P = 0.002). CONCLUSION: The detection of MMP-1, CB, and CL in the synovium soon after the onset of symptoms highlights the potential for early joint destruction in patients with RA. High levels of MMP-1 mRNA expression in the lining layer distinguished patients with more rapidly progressive erosive disease. This is the first study to demonstrate features of early synovial pathophysiology that may identify patients at increased risk of developing new joint erosions.

Analysis of the cell infiltrate and expression of matrix metalloproteinases and granzyme B in paired synovial biopsy specimens from the cartilage-pannus junction in patients with RA.

OBJECTIVES: Examination of synovial tissue (ST) obtained at surgery because of end stage destructive rheumatoid arthritis (RA) showed that macrophages and fibroblasts are the major cell types at the cartilage-pannus junction (CPJ). This study aimed at defining the cell infiltrate and mediators of joint destruction in ST selected at arthroscopy from the CPJ in patients with RA who did not require joint surgery. METHODS: Paired synovial biopsy specimens were obtained at arthroscopy from ST adjacent to the CPJ and the suprapatellar pouch from the knee joints of 17 patients with RA. Immunohistological analysis was performed using monoclonal antibodies to detect T cells, B cells, plasma cells, macrophages, fibroblast-like synoviocytes, mast cells, and granzyme B+ cytotoxic cells as well as the expression of metalloproteinase (MMP)-1, MMP-3, and MMP-13. The sections were evaluated by computer assisted image analysis and semiquantitative analysis. RESULTS: The cell infiltrate comprised mainly T cells, macrophages, and plasma cells. The ST was also infiltrated by the other cell types, but at lower numbers. Expression of MMPs was abundant, especially MMP-3. The features of ST at the CPJ were generally similar to those at the suprapatellar pouch. CONCLUSIONS: The synovium at the CPJ in patients with RA who did not require joint surgery exhibits, in general, the same type of cell infiltrate and expression of MMPs and granzymes as ST from the suprapatellar pouch. The pathological changes that have been described at the CPJ in patients with RA with end stage, destructive disease may well reflect the transition to a process in which macrophages, fibroblast-like synoviocytes, and other cell types become increasingly important.

Effects of intraarticular glucocorticoids on macrophage infiltration and mediators of joint damage in osteoarthritis synovial membranes: findings in a double-blind, placebo-controlled study.

OBJECTIVE: To investigate the effects of intraarticular glucocorticoid treatment on macrophage infiltration, the expression of the chemokines monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1alpha (MIP-1alpha), and the expression of matrix metalloproteinases 1 and 3 (MMPs 1 and 3) and their inhibitors, the tissue inhibitors of metalloproteinases 1 and 2 (TIMPs 1 and 2), in osteoarthritis (OA) synovial membranes. METHODS: Forty patients underwent arthroscopic biopsy before and 1 month after intraarticular injection of glucocorticoids. Twenty-one patients received 120 mg of methylprednisolone acetate (Depo-Medrol; Upjohn, Kalamazoo, MI), and 20 patients received placebo (1 patient received placebo in 1 knee and methylprednisolone acetate in the other). Immunoperoxidase staining for the expression of CD68, MCP-1, MIP-1alpha, MMP-1, MMP-3, TIMP-1, and TIMP-2 was performed, and the immunostaining was quantified by color video image analysis. RESULTS: CD68, MCP-1, MIP-1alpha, MMP-1, MMP-3, TIMP-1, and TIMP-2 immunostaining was observed in all synovial membranes. Intraarticular glucocorticoid treatment was associated with a small (30%) but statistically significant (P = 0.048) reduction in CD68+ macrophage staining in the synovial lining layer, but there was no change in the CD68 expression in the synovial sublining layer. No significant differences were observed for MCP-1, MIP-1alpha, MMP-1, MMP-3, TIMP-1, and TIMP-2 immunostaining in the synovial lining or sublining layers. CONCLUSION: Intraarticular glucocorticoids may reduce CD68+ macrophage infiltration into the synovial lining layer, but not the expression of MCP-1, MIP-1alpha, MMP-1, MMP-3, TIMP-1, and TIMP-2 in the synovial membrane, in patients with OA.

Effects of pulse methylprednisolone on macrophage chemotactic protein-1 and macrophage inflammatory protein-1alpha in rheumatoid synovium.

OBJECTIVE: To determine the effect of pulse methyprednisolone (PMP; 1000 mg) on the expression of monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha in rheumatoid synovial membrane. METHODS: Seven patients with rheumatoid arthritis (RA) were studied. Arthroscopically-directed synovial biopsies were taken before and 24 hours after treatment with intravenous PMP. Synovial membranes were stained by immunohistochemical techniques with monoclonal antibodies against MCP-1, MIP-1alpha and CD68 (a macrophage marker). Quantitation of staining was performed by computer-assisted color video image analysis. RESULTS: PMP therapy was associated with a rapid (within 24 hours) and substantial decrease in the expression of MCP-1 and MIP-1alpha expression by a mean of 55% (p = 0.05) and 45% (p = 0.03), respectively, with no effect on CD68 expression in the synovial lining layer. There was no significant change in MCP-1, MIP-1alpha or CD68 expression in the synovial sublining. CONCLUSION: PMP therapy rapidly reduces MCP-1 and MIP-1alpha levels in the synovial lining layer without a fall in macrophage numbers. It thus appears that the initial effect of PMP is that of reducing macrophage activation.

Microscopic measurement of cellular infiltration in the rheumatoid arthritis synovial membrane: a comparison of semiquantitative and quantitative analysis.

Microscopic measurement of inflammation in synovial tissue may be important in studies of clinical status, prognosis and response to treatment. The aim of this study was to compare quantitative microscopic analysis of inflammation with a semiquantitative grading system in rheumatoid arthritis (RA) synovial membrane. Knee synovial membrane samples from 16 patients with RA, including paired samples taken before and after treatment in nine patients, were immunostained with anti-CD68 and anti-CD3 monoclonal antibodies using standard techniques. The intensity of macrophage and T-lymphocyte infiltration was measured both by quantitative and semiquantitative techniques, and the results were compared. In a cross-sectional comparison, both methods correlated significantly for lining layer macrophage infiltration, as well as sublining layer macrophage and T-cell infiltration. However, in some patients demonstrating a clinical response to treatment, semiquantitative analysis lacked sensitivity to biologically relevant changes in mononuclear cell infiltration. These observations have important implications for future studies of therapeutic modalities.

Quantitative microscopic analysis of inflammation in rheumatoid arthritis synovial membrane samples selected at arthroscopy compared with samples obtained blindly by needle biopsy.

OBJECTIVE: To evaluate microscopic measures of inflammation in rheumatoid arthritis synovial tissue samples selected at arthroscopy compared with those obtained blindly by needle biopsy from the suprapatellar pouch (SPP) of the same joint. METHODS: Samples were selected at knee arthroscopy from the SPP and the lateral and medial gutters. Immediately following arthroscopy, a biopsy needle was inserted through the same portal into the SPP by a second investigator, and 3 further samples were obtained blindly. Using standard immunohistologic methods, all samples were analyzed by a single investigator without knowledge of the original tissue location and biopsy technique. Following staining with anti-CD3 and anti-CD68 monoclonal antibodies, T lymphocyte and macrophage infiltration were measured by quantitative analysis. RESULTS: Synovial tissues from 14 patients were analyzed. In comparing microscopic measures of inflammation using the 2 procedures, mean scores of lining cell depth and the percentage of CD68+ cells in the lining layer correlated positively (tau = 0.59, P = 0.003 and tau = 0.73, P = 0.0003, respectively). In the sublining layer, CD3+ cell counts also correlated significantly (tau = 0.71, P = 0.0004). Sublining CD68+ cell counts did not correlate. This was explained by the observation that CD68+ cell infiltration in areas adjacent to articular cartilage was significantly greater than in the SPP (P = 0.01), suggesting preferential trafficking to this site by macrophages, but not by T lymphocytes. Macroscopic appearance at arthroscopy did not predict microscopic features. CONCLUSION: Most microscopic measures of inflammation in synovial tissue samples obtained blindly from the SPP were similar to those determined in samples selected at arthroscopy. However, measurements in samples from the SPP may underestimate the intensity of macrophage infiltration in areas more adjacent to cartilage. These observations have important implications for future study of macrophage function in synovial tissue.

Effects of pulse methylprednisolone on inflammatory mediators in peripheral blood, synovial fluid, and synovial membrane in rheumatoid arthritis.

OBJECTIVE: To establish whether the clinical efficacy of pulse methylprednisolone (MP; 1,000 mg intravenously) is related to the modulation of proinflammatory cytokines within the peripheral blood, synovial membrane, or synovial fluid compartments. METHODS: Eighteen patients with active rheumatoid arthritis (RA) were studied. Peripheral blood (11 patients) and knee synovial fluid (9 patients, 10 knees) were obtained before and at 4 and 24 hours after MP therapy. Interleukin-1beta (IL-1beta), IL-8, and tumor necrosis factor alpha (TNFalpha) were measured by enzyme-linked immunosorbent assay and biologic assays; prostaglandin E2 (PGE2) was measured by competitive radioimmunoassay. In 10 patients, arthroscopically directed synovial biopsies were obtained before and at 24 hours after treatment, at disease relapse (4 patients), and after retreatment (1 patient). Membranes were stained by immunohistochemical techniques with monoclonal antibodies against TNFalpha, IL-8, IL-1beta, and the IL-1 receptor antagonist protein (IL-1Ra). RESULTS: MP therapy was associated with a rapid (within 24 hours) and substantial decrease in the expression of TNFalpha in the lining and sublining regions of the synovial membrane, as well as substantial decreases in the levels of TNFalpha in serum and synovial fluid. There was also reduced IL-8 expression in the synovial lining, as well as reduced synovial fluid IL-8 levels. No effect on synovial membrane IL-1beta and IL-1Ra or synovial fluid IL-1beta and PGE2 was found. CONCLUSION: MP therapy rapidly reduces IL-8 and TNFalpha levels in the synovial compartment, with cytokine changes in the serum and synovial fluid reflecting the changes in the synovial membrane. Alterations in TNFalpha expression in the synovial membrane correlated with clinical response to, and subsequent relapse after, MP therapy.

Synovial membrane cytokine profiles in reactive arthritis secondary to intravesical bacillus Calmette-Guérin therapy.

We describe the cellular infiltrate and cytokine profile in sequential synovial membrane biopsies from a patient with acute followed by chronic synovitis after intravesical bacillus Calmette-Guérin (BCG) therapy for an in situ transitional cell carcinoma of the bladder. Histological and immunohistochemical analysis of 3 synovial biopsies were done sequentially over a 9 month period. The patient was HLA-B27 positive, but HLA-DR4 negative, and did not have the "shared epitope." Unlike other cases, this patient's arthritis did not respond initially to nonsteroidal antiinflammatory drugs and was exacerbated by corticosteroid therapy. The synovitis took a neutrophilic form, with marked synovial membrane content of interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha). It subsequently developed into chronic lymphoplasmacytoid synovitis, similar to rheumatoid arthritis (RA), with decreased IL-8 but continuing IL-1 and TNF-alpha production in the synovial membrane. The synovitis resolved to a fibrotic synovium with residual thickening of the synovial lining layer and continued production of TNF-alpha. Thus, during the evolution of this arthritis, the synovial layer and continued production of TNF-alpha. Thus, during the evolution of this arthritis, the synovial membrane yielded a cellular infiltrate and cytokine content that had marked similarities with that seen in RA; however, the arthritis eventually remitted spontaneously.

Synovial membrane inflammation and cytokine production in patients with early osteoarthritis.

OBJECTIVE: To establish the presence of inflammation in and cytokine production by synovial membranes from patients with various stages of early osteoarthritis (OA), with knee pain, normal knee radiographs, and arthroscopic evidence of chondral damage. METHODS: Synovial membrane samples were obtained from the knees of 63 patients at the time of arthroscopy for unexplained knee pain or at the time of joint replacement surgery. Evaluations of synovial membrane variables including thickness of lining layer, vascularity, and inflammatory cell infiltrate were by a blinded observer. In a subset of 20 patients, production of interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), and IL-1 receptor antagonist (IL-1ra) at the mRNA and protein levels was determined using in situ hybridization with biotin labeled ribo-probes and immunohistochemistry. RESULTS: There was evidence of thickening of the lining layer, increased vascularity, and inflammatory cell infiltration in synovial membranes from patients with all grades of OA, with the most marked changes seen in synovial tissue from patients with advanced grades of OA. Similarly, production of IL-1 alpha, IL-1 beta, and TNF-alpha was present in synovial membranes from all patients with OA, irrespective of the degree of articular cartilage damage. There was a trend to decreased levels of IL-1ra in synovial membranes from patients with OA that did not attain statistical significance. Similarly, there was a decrease in the ratio of IL-1ra to IL-1 alpha and beta with increasing grades of OA. CONCLUSION: Chronic inflammatory changes with production of proinflammatory cytokines are a feature of synovial membranes from patients with early OA, with the most severe changes seen in patients at the time of joint replacement surgery resembling those seen in rheumatoid arthritis. This low grade synovitis results in the production of cytokines that may contribute to the pathogenesis of OA.

Variability in cytokine and cell adhesion molecule staining in arthroscopic synovial biopsies: quantification using color video image analysis.

OBJECTIVE: To investigate the variability in immunostaining for cytokines and cell adhesion molecules using multiple arthroscopically directed synovial biopsies from within a rheumatoid knee joint, quantitated by color video image analysis. METHODS: Needle arthroscopic biopsies were taken from multiple sites (4-7 sites) around a knee joint in 8 patients with rheumatoid arthritis (RA). In 5 patients, immunoperoxidase staining for the cytokines tumor necrosis factor alpha (TNF-alpha), interleukin 8 (IL-8), and IL-1beta as well as the IL-1 receptor antagonist protein (IL-1ra) was performed. In 3 patients, immunoperoxidase staining for the cell adhesion molecules E-selectin (CD62E), P-selectin (CD62P), intercellular adhesion molecule 1 (ICAM-1, CD54), and platelet endothelial cell adhesion molecule (PECAM, CD31) was performed. Immunostaining was quantified using color video image analysis. RESULTS: The overall probability of paired biopsies from the same RA knee joint being significantly different from each other due to sampling variation was at most 22% for cytokine staining (usually less than 10%). There were no significant differences between intrabiopsy and interbiopsy variability for cell adhesion molecule staining of the sublining and vessels. CONCLUSION: The variability in cytokine and cell adhesion molecule staining within any single biopsy usually reflects the variability between biopsies taken from different sites in the same rheumatoid joint when the immunostaining is quantified using color video image analysis. Therefore, only a small number of synovial biopsies are required to accurately determine the cytokine and cell adhesion molecule expression in a single joint.

Effects of pulse methylprednisolone on cell adhesion molecules in the synovial membrane in rheumatoid arthritis. Reduced E-selectin and intercellular adhesion molecule 1 expression.

OBJECTIVE: To investigate the effects of a 1,000-mg intravenous pulse of methylprednisolone succinate (MP) on cell adhesion molecule expression on the synovial vascular endothelium in patients with rheumatoid arthritis (RA). METHODS: Sequential arthroscopic biopsy samples were taken before and 24 hours after MP administration (10 patients) and at the time of RA flare (2 patients) and after retreatment with MP (1 patient). Immunoperoxidase staining for E-selectin (CD62E), P-selectin (CD62P), intercellular adhesion molecule 1 (ICAM-1; CD54) and platelet-endothelial cell adhesion molecule (PECAM; CD31) was performed, and the staining was quantified by color video image analysis. RESULTS: MP caused a rapid (within 24 hours) and substantial decrease in the expression of E-selectin on the synovial vascular endothelium, with a smaller reduction in ICAM-1 expression on synovial vascular endothelium and the synovial lining. There were no similar effects on synovial membrane P-selectin or PECAM expression. CONCLUSION: A potential mechanism by which MP impairs neutrophil trafficking into inflamed RA joints might be by reducing E-selectin, and possibly, ICAM-1, expression in the synovial membrane.

Tumor necrosis factor priming of peripheral blood neutrophils from rheumatoid arthritis patients.

Recently it was shown that tumor necrosis factor-alpha (TNF) receptors on neutrophils may be down-regulated after stimulation with proinflammatory mediators. Since in rheumatoid arthritis neutrophils are likely to encounter these mediators in the circulation, we tested the hypothesis that rheumatoid arthritis neutrophil TNF receptors are down-regulated. Peripheral blood neutrophils from patients with rheumatoid arthritis and healthy subjects were compared with respect to their TNF binding activity and ability to be primed by TNF. There were no differences between rheumatoid arthritis and control neutrophils in receptor-mediated TNF binding, superoxide release in response to agonist, and TNF priming of this respiratory burst or in the ability to degrade cartilage in vitro and TNF priming for increased cartilage damage. It is evident that rheumatoid arthritis blood neutrophils retain the ability to bind TNF and can be primed by TNF for increased oxygen radical production and augmented cartilage damage. These findings further implicate the role of neutrophils in the pathogenesis of arthritis.