IgG and IgM antibody to native type ii collagen in rheumatoid arthritis serum and synovial fluid evidence for the presence of collagen-anticollagen immune complexes in synovial fluid

Levels of IgG and IgM antibody to native type II collagen, a component of articular cartilage, were measured by a specific solid-phase radioimmunoassay, in 48 rheumatoid arthritis patients. Good correlations were found between the levels in sera and those in synovial fluids. Collagenase digestion caused a significant rise in specific antibody levels in synovial fluids, indicating the presence of intraarticular collagen-anticollagen complexes. These immune complexes may be one mechanism for perpetuation of inflammation in some rheumatoid arthritis patients.     

Immunohistologic demonstration of type II collagen in synovial fluid phagocytes of osteoarthritis and rheumatoid arthritis patients

We were able to demonstrate type II collagen in synovial phagocytes of osteoarthritis (OA) and rheumatoid arthritis (RA) patients, using a monoclonal antibody to human type II collagen and immunoperoxidase staining. In addition, using immunoelectron microscopy, we demonstrated labeled fragments in synovial phagocytes of both RA and OA patients. This immunohistochemical assay may prove to be a sensitive indicator of cartilage erosion in patients with OA and RA.     

IgG and IgM antibody to native type II collagen in rheumatoid arthritis serum and synovial fluid. Evidence for the presence of collagen-anticollagen immune complexes in synovial fluid

Levels of IgG and IgM antibody to native type II collagen, a component of articular cartilage, were measured by a specific solid-phase radioimmunoassay, in 48 rheumatoid arthritis patients. Good correlations were found between the levels in sera and those in synovial fluids. Collagenase digestion caused a significant rise in specific antibody levels in synovial fluids, indicating the presence of intraarticular collagen-anticollagen complexes. These immune complexes may be one mechanism for perpetuation of inflammation in some rheumatoid arthritis patients.     

Accumulation of T cells reactive to type II collagen in synovial fluid of patients with rheumatoid arthritis

OBJECTIVE: To determine if type II collagen (CII) reactive T lymphocytes selectively accumulate in the inflamed joint of patients with rheumatoid arthritis (RA) and to study the specificity of the CII reactive cells. METHODS: Synovial fluid (SF) cells or peripheral blood lymphocytes were cultured with interleukin 2 (IL-2) for 24 h and then cultured at limiting dilution concentrations in the presence of filler cells and IL-2. The outgrowing cell lines were screened for their responses to CII. The percentages of the CII reactive T cells from SF were compared with those from peripheral blood of patients with RA. The CII reactive T cell lines were tested for their responses to different types of collagen. RESULTS: CII reactive T cell lines were identified in the SF of 3 RA patients; the frequencies were 5.0% (11/219), 3.7% (5/134), and 3.5% (3/86), respectively. In contrast, none of CII-specific T cell lines were identified in peripheral blood of the patients. The T cell lines recognized both human and bovine CII and, to a lesser extent, type I collagen. CONCLUSION: CII reactive T cells are present in high frequency in the inflamed joint of patients with RA, where they may play an important role in the pathogenesis of RA.     

Homologous type ii collagen—induced arthritis in rats

Arthritis was induced in Lewis and DA rat strains after immunization with native homologous type II collagen (CII). Differences were noted in the clinical signs of arthritis in the 2 rat strains, which were immunized with the same arthritogenic preparation of CII. DA rats showed disease onset characterized by symmetric involvement of the interphalangeal joints of the hind feet. Lewis rats showed disease onset characterized by involvement of only the ankle or knee joints. Moreover, the arthritis tended to be chronic (i.e., persistent and variable redness and swelling seen in interphalangeal joints) in DA rats, but not in Lewis rats. Analysis of the delayed-type hypersensitivity reaction to CII and anti-CII autoantibody production demonstrated the presence of T cell, as well as B cell, reactivity to rat CII in both strains of rat. A spontaneous T cell reactivity to CII, as measured by rat CII-induced ear swelling, was observed in nonimmunized DA rats, but not in nonimmunized Lewis rats. The demonstration that clinical features of arthritis induced with identical arthritogenic stimuli are different depending on the genetic background of the affected animal may be relevant in understanding the heterogeneity of the clinical features of human inflammatory joint disease.     

Diagnostic value of synovial fluid anti-cyclic citrullinated peptide antibody for rheumatoid arthritis

Early and accurate diagnosis and treatment of rheumatoid arthritis (RA) improves disease outcome. Anti-cyclic citrullinated peptide antibody (anti-CCP) which is highly specific for RA is produced locally from inflamed synovium. The present study was designed to assess the diagnostic performance of synovial fluid anti-CCP (sf-CCP) for RA. A total of 128 patients consisted of 37 RA confirmed by the American College of Rheumatology revised criteria, 91 non-RA (50 non-RA inflammatory arthritis and 41 osteoarthritis) entered the study. Serum anti-CCP (sm-CCP) and Sf-CCP were measured by the ELISA method. Receiver operating characteristics curves were constructed to determine the optimal cutoff point levels for sf-CCP and sm-CCP to discriminate RA from non-RA. Diagnostic characteristics of both variables were determined by comparison of RA patients with non-RA controls. Mean levels of sf-CCP and sm-CCP were significantly higher in RA than in non-RA (P < 0.001). Sf-CCP discriminated RA from non-RA at the optimal cutoff value of 10 U/mL with high accuracy at AUC value of 0.897 ± 0.039, P < 0.001) sensitivity of 83.7% and specificity of 95.6%. Sm-CCP diagnosed RA at optimal cutoff level of 14.6 U/mL with respective sensitivity, specificity and AUC values of 84.8, 94.3% and 0.895 ± 0.049, P < 0.001). Sm-CCP was strongly correlated with sf-CCP (r = 0.75, r ² = 0.57, P < 0.0001). Two of 5 sm-CCP negative RA and 25.7% of serum rheumatoid factors negative RA were sf-CCP positive. These findings indicate that sf-CCP yields diagnostic ability as comparable as sm-CCP for RA. Respecting to local production of sf-CCP prior to disease onset, therefore sf-CCP determination may offer earlier as well as additional diagnostic information which may be more helpful in recognizing RA particularly among recent onset arthritis.     

High seroprevalence of anti–HTLV-I antibody in rheumatoid arthritis

Objective. To investigate the association between human T lymphotropic virus type I (HTLV-I) infection and rheumatoid arthritis (RA) in Nagasaki, an area highly endemic for HTLV-I infection. Methods. Sera from 113 female patients with RA and 19,796 female blood donors were screened for anti–HTLV-I antibodies with a gelatin particle agglutination kit and confirmed using an immunoblotting kits. Results. The age-adjusted summary odds ratio of HTLV-I infection among RA patients, as compared with blood donors, was 2.8 (95% confidence interval [95% CI] 1.8–4.6). The etiologic fraction, i.e., the proportion of RA in the study population that is attributable to HTLV-I infection, was estimated to be 13.2% (95% CI 5.1–21.2). There was no significant difference in the clinical and laboratory findings between HTLV-I–infected and HTLV-I–uninfected RA patients. Conclusion. These epidemiologic findings support the idea that HTLV-I infection is a risk factor for RA, and suggest that ∼13% of the cases of RA in females living in Nagasaki are associated with HTLV-I infection.     

Resistin in rheumatoid arthritis synovial tissue, synovial fluid and serum

BACKGROUND: Resistin is a newly identified adipocytokine which has demonstrated links between obesity and insulin resistance in rodents. In humans, proinflammatory properties of resistin are superior to its insulin resistance-inducing effects. OBJECTIVES: To assess resistin expression in synovial tissues, serum and synovial fluid from patients with rheumatoid arthritis, osteoarthritis and spondylarthropathies (SpA), and to study its relationship with inflammatory status and rheumatoid arthritis disease activity. METHODS: Resistin expression and localisation in synovial tissue was determined by immunohistochemistry and confocal microscopy. Serum and synovial fluid resistin, leptin, interleukin (IL)1beta, IL6, IL8, tumour necrosis factor alpha, and monocyte chemoattractant protein-1 levels were measured. The clinical activity of patients with rheumatoid arthritis was assessed according to the 28 joint count Disease Activity Score (DAS28). RESULTS: Resistin was detected in the synovium in both rheumatoid arthritis and osteoarthritis. Staining in the sublining layer was more intensive in patients with rheumatoid arthritis compared with those with osteoarthritis. In rheumatoid arthritis, macrophages (CD68), B lymphocytes (CD20) and plasma cells (CD138) but not T lymphocytes (CD3) showed colocalisation with resistin. Synovial fluid resistin was higher in patients with rheumatoid arthritis than in those with SpA or osteoarthritis (both p<0.001). In patients with rheumatoid arthritis and SpA, serum resistin levels were higher than those with osteoarthritis (p<0.01). Increased serum resistin in patients with rheumatoid arthritis correlated with both CRP (r=0.53, p<0.02), and DAS28 (r=0.44, p<0.05), but not with selected (adipo) cytokines. CONCLUSION: The upregulated resistin at local sites of inflammation and the link between serum resistin, inflammation and disease activity suggest a role for resistin in the pathogenesis of rheumatoid arthritis.     

Differences in synovial tissue infiltrates between anti–cyclic citrullinated peptide–positive rheumatoid arthritis and anti–cyclic citrullinated peptide–negative rheumatoid arthritis

Objective

To compare synovial tissue infiltrates from patients with anti–cyclic citrullinated peptide (anti‐CCP)–positive rheumatoid arthritis (RA) with those from patients with anti‐CCP–negative RA.

Methods

Synovial tissue samples were obtained arthroscopically from the inflamed knee joints of 57 patients with RA (34 of whom were anti‐CCP positive) and examined for several histologic features along with immunohistologic expression of cell markers. Joint damage was assessed using the Kellgren/Lawrence (K/L) scale (range 0–4) on standard anteroposterior radiographs. In 31 patients (18 of whom were anti‐CCP positive), synovial tissue was available from an earlier time point, allowing analysis of temporal changes.

Results

Synovial tissue from anti‐CCP–positive patients was characterized by a higher mean number of infiltrating lymphocytes (61.6 versus 31.4/high‐power field [hpf] [400×]; P = 0.01), less extensive fibrosis (mean score of 1.2 versus 2.0; P = 0.04), and a thinner synovial lining layer (mean score of 2.1 versus 3.3; P = 0.002) compared with synovial tissue from anti‐CCP–negative patients. Anti‐CCP–positive patients expressed more CD3, CD8, CD45RO, and CXCL12. More anti‐CCP–positive patients had a K/L score >1 compared with anti‐CCP–negative patients. The difference in the mean lymphocyte counts was already present a mean of 3.8 years before the index biopsy (76.7 lymphocytes/hpf and 26.7 lymphocytes/hpf in anti‐CCP–positive patients and anti‐CCP–negative patients, respectively; P = 0.008) and was independent of disease duration and K/L score.

Conclusion

Synovitis in patients with anti‐CCP–positive RA differs from that in patients with anti‐CCP– negative RA, notably with respect to infiltrating lymphocytes, and is associated with a higher rate of local joint destruction.

     

Beta-2-microglobulin in synovial fluid of rheumatoid arthritis

The concentration of beta-2-microglobulin was determined by radioimmunoassay in the synovial fluid of 12 patients affected with rheumatoid arthritis and in 10 with other arthropathies. The white cell count, total protein concentration, total hemolytic activity of complement and acid phosphatase activity were also determined. Only beta-2-microglobulin was found to be significantly higher in rheumatoids compared to non-rheumatoid cases. In contrast, the other parameters showed no significant differences between the 2 groups of patients. These preliminary observations suggest that the measurement of beta-2-microglobulin concentrations in synovial fluid may be of diagnostic value.     

Plasminogen activator in synovial fluid from patients with rheumatoid arthritis

The plasminogen activator in synovial fluid from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) was analyzed on a molecular basis. The level of plasminogen activator in RA was found to be higher than in OA. The plaminogen activators of both RA and OA revealed 3 different molecular weights: 90,000, 55,000 and 33,000. RA demonstrated the 3 plasminogen activators in broadly comparable ratios, but OA had the 55,000 form dominantly. The 90,000 plasminogen activator was a tissue-type plasminogen activator, while the 55,000 and 33,000 plasminogen activators were of the urokinase-type. beta-Methasone suppressed the tissue-type plasminogen activator, and urinary trypsin inhibitor suppressed the urokinase-type plasminogen activators. When urinary trypsin inhibitor was injected clinically into the joint space of a patient with RA, the urokinase-type plasminogen inhibitor was suppressed as in the in vitro study, and the clinical signs and symptoms were markedly improved. Open trials of intraarticular injections of urinary trypsin inhibitor demonstrated improvement of the clinical signs and symptoms.     

Hydroxypyridinium collagen crosslinks in serum,urine, synovial fluid and synovial tissue in patients with rheumatoid arthritis compared with osteoarthritis

OBJECTIVE: To investigate the relationship between inflammation markers and content of pyridinium crosslinks in hydrolysates of synovial tissue and to specify the significance of urinary excreted pyridinoline, released primarily from collagen I and II of bone and cartilage, and deoxypyridinoline released especially from collagen I of bone and dentin, dependent on disease activity in rheumatoid arthritis (RA). METHODS: Synovial tissue and fluid from knee endoprosthesis surgery, as well as simultaneously obtained serum and urine, were collected from 12 patients with inactive RA or RA with low disease activity [iRA: C-reactive protein (CRP) <28 mg/l], 10 with active RA (aRA: CRP > or =28 mg/l) and 21 with OA. After preparation of the synovial tissue, including hydrolysis, completely released synovial pyridinoline and deoxypyridinoline crosslinks as well as those from synovial fluid, serum and urine were investigated using a gradient ion-paired reversed-phase HPLC method. Crosslink levels in synovial tissue are expressed as mol/mol collagen, assuming 300 residues of hydroxyproline per collagen molecule, also measured by HPLC. RESULTS: In the synovial tissue of aRA patients we found significantly elevated total pyridinoline concentrations and pyridinoline/deoxypyridinoline (Pyr/Dpyr) quotients compared with the iRA and OA controls, indicating an elevated crosslinking density of mature synovial tissue collagen with increased activity of RA. Pyridinoline levels and the Pyr/Dpyr ratio were correlated with those of urine and with acute-phase reactants in RA patients. Compared with serum crosslink levels, which were unrelated to disease activity, the urinary concentration of pyridinoline was increased by a factor of 2 and showed a simultaneous increase with increasing synovitis. CONCLUSION: Both crosslinking density and degradation of mature collagen from synovial tissue depend on the disease activity in RA. Urinary excretion of associated crosslinks, expressed as the Pyr/Dpyr ratio, correlates with those in synovial tissue and may be confirmed as a marker of synovial tissue collagen degradation. We suggest that increased crosslinking of mature collagen in the synovial tissue of RA is related to an inflammation-dependent regulation of collagen synthesis in activated synovial fibroblasts, in which lysyl oxidase represents the final enzymatic step for crosslinking.     

Isoelectric focusing studies of synovial fluid in rheumatoid arthritis]

Synovial fluids obtained at operation in several different diseases were studied by means of isoelectrical focussing. All the punctates of patients with rheumatoid arthritis showed bands which migrated towards the cathode and which were not found in synovial fluid from other diseases, except in 2 meniscus lesions. In the range of pH 6.81-7.30, typical synovial proteins were demonostrated.     

Interleukin-1 lymphocyte chemotactic activity in rheumatoid arthritis synovial fluid

We examined the role of interleukin-1 (IL-1) in the chemotactic activity of rheumatoid arthritis (RA) synovial fluid (SF). Crude RA SF was found to be chemotactic for B cells and T cells. After AcA 54 gel filtration, the principal peaks of chemotactic activity were found in the 5-kd, 16-kd, and 60-kd fractions. The majority of the chemotactic activity for both the B cells (74-85%) and the T cells (69-78%) was removed from these fractions by treatment with anti-IL-1 antibody. However, in crude SF, approximately 60% of the chemotactic activity for B cells and 40% of that for T cells was removed, indicating the presence of additional chemotactic factors in RA SF. IL-1 activity, measured by the thymocyte proliferation assay, was demonstrated in RA SF AcA 54 Ultrogel fractions after separation from inhibitors of thymocyte proliferation that are present in crude SF. On chromatofocusing of the 16-kd fraction, the principal peaks of both thymocyte proliferation activity and chemotactic activity were present in the same fractions with pI values of 6.8, 5.7, and 5.2, which are characteristic of IL-1. The demonstration of IL-1-associated chemotactic activity in RA SF may reflect the presence in the RA synovial membrane (including both the lining layer and the subsynovial layer) of activated macrophages, interstitial histiocytic cells, and other IL-1-producing cells, such as endothelial cells. These findings suggest that such cells may attract lymphocytes to their environment by secretion of IL-1.