Distribution of mast cells in human synovial tissue of patients with osteoarthritis and rheumatoid arthritis

Mast cells are demonstrated in synovial membranes of patients with osteo-arthritis and rheumatoid arthritis using a new staining principle based on interaction of heparin in mast cell granules with peroxidase labeled avidin. It was found that mast cell numbers in the subsynovial layer of patients with rheumatoid arthritis were significantly lower than those in patients suffering from osteo-arthritis. This decrease can be mainly attributed to patients with rheumatoid arthritis whose synovitis was characterized by a distinct histomorphological pattern consisting of lining cell ulcers and granulation tissue. However, when mast cell numbers in rheumatoid arthritis and osteo-arthritis patients were compared without respect to mast cell distribution in the subsynovial layer or the stratum fibrosum, no statistical differences between the diseases could be observed.     

Selective elimination of synovial inflammatory macrophages in rheumatoid arthritis by an Fcγ receptor I–directed immunotoxin

Objective

To determine whether monocyte/macrophages from rheumatoid arthritis (RA) patients can be selectively eliminated by a toxin‐conjugated antibody CD64–ricin A (CD64‐RiA) directed toward the high‐affinity receptor for IgG (FcγRI), exploiting the capacity of FcγRI to efficiently endocytose antibody which it has bound.

Methods

Mononuclear cells from peripheral blood (PB) and synovial fluid (SF) obtained from RA patients were cultured in the presence of CD64‐RiA. Cell death of monocyte/macrophages was measured by phenotypic changes (light‐scatter patterns and CD14 and FcγRI expression) and apoptosis (nuclear DNA fragmentation). We then tested whether CD64‐RiA–induced cell death of macrophages affected their capacity to stimulate antigen‐induced lymphocyte proliferation and to secrete cytokines. Additionally, the capacity of CD64‐RiA to inhibit proinflammatory activity and cartilage degradation by RA synovial tissue explants was evaluated.

Results

Inflammatory macrophages from RA SF expressed elevated levels of FcγRI and were selectively eliminated by CD64‐RiA via apoptotic cell death. Monocyte/macrophages from RA PB, which had lower levels of FcγRI expression, were much less affected. Induction of SF macrophage apoptosis was associated with efficient inhibition of antigen‐induced lymphocyte proliferation and a reduction in tumor necrosis factor α (TNFα) release. Consistent with these effects on SF macrophages, CD64‐RiA also inhibited TNFα production, interleukin‐1β production, and cartilage‐degrading activity of RA synovial tissue explants.

Conclusion

Together, these data underscore the crucial role of synovial macrophages in RA joint inflammation and indicate that selective elimination of these cells through FcγRI‐directed immunotoxins could be a novel approach to the treatment of RA.

     

Fcγ receptor type IIIA genotype and response to tumor necrosis factor α–blocking agents in patients with rheumatoid arthritis

Objective

To determine whether a functional single‐nucleotide polymorphism in the gene encoding Fcγ receptor type IIIA (FcγRIIIA) correlates with the response to treatment with tumor necrosis factor α inhibitors in rheumatoid arthritis (RA).

Methods

The study population comprised 282 Swedish patients with RA in whom the therapeutic efficacy of conventional disease‐modifying antirheumatic drugs had been insufficient. Infliximab or etanercept treatment was initiated, and patients were evaluated after 3 months, using the American College of Rheumatology 20% improvement criteria (ACR20), the ACR50, and the ACR70 or the European League Against Rheumatism (EULAR) criteria. The chi‐square test was used to compare response rates across FcγRIIIA genotypes.

Results

No differences in genotype distribution were observed among nonresponders compared with ACR20 responders (P = 0.80), ACR50 responders (P = 0.56), or ACR70 responders (P = 0.91). Similar results were observed when analyzing infliximab and etanercept separately or when using the EULAR response criteria.

Conclusion

Unlike the findings of a previous study, the results of the current study suggest that the 158V/F polymorphism of FcγRIIIA is very unlikely to influence the clinical efficacy of infliximab or etanercept in patients with RA.

     

Interleukin-1 receptor antagonist production in cultured synovial cells from patients with rheumatoid arthritis and osteoarthritis.

OBJECTIVE--To measure the amounts of interleukin-1 receptor antagonist (IL-1ra) protein produced by cultured synovial cells obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS--Synovial cells obtained from patients with either RA or OA were cultured and the supernatants were measured for IL-1ra by enzyme linked immunosorbent assay. RESULTS--The synovial cells obtained from patients with RA produced significantly smaller amounts of IL-1ra than did those obtained from patients with OA, in a late passage (third to fifth) without stimulation and a first passage both with and without stimulation (p < 0.025, respectively). In addition, when the patients with RA were divided into two groups according to the maximum number of lining cell layers, the amounts of IL-1ra produced by the proliferative type were smaller than those produced by the less proliferative type (p < 0.025). CONCLUSIONS--The above findings suggest that IL-1ra production in RA synovial cells is suppressed, and that reduced IL-1ra protein production is one of the causes which leads to the proliferation of lining cells and persistent joint inflammation.     

C5a receptor enables participation of mast cells in immune complex arthritis independently of Fcγ receptor modulation

Objective

Mast cells are tissue‐resident immune sentinels that are implicated in the pathogenesis of inflammatory joint disease. The aim of this study was to test our hypothesis that complement fragments could be key activators of synovial mast cells in autoimmune arthritis.

Methods

In vivo studies used the murine K/BxN arthritis model, a distal symmetric polyarthritis mediated by IgG immune complexes. Expression of C5aR on synovial mast cells was determined by immunohistochemical and functional studies. C5aR^−/− and control mast cells were engrafted into mast cell–deficient WBB6 F1‐Kit^w/Kit^W‐v (W/Wv) mice to examine the requirement for this receptor in arthritis. C5aR‐dependent activation of mast cells was investigated in C5aR^−/− animals and in murine and human mast cell cultures.

Results

Murine synovial mast cells express functional C5aR. Unlike their wild‐type counterparts, C5aR^−/− mast cells adoptively transferred into W/Wv mice were not competent to restore arthritis, despite equivalent synovial engraftment. Activation of C5aR^−/− mast cells by K/BxN serum in vivo remained intact, indicating that C5aR is dispensable for normal IgG‐mediated triggering. Consistent with this result, cultured mast cells treated with C5a failed to modulate the expression of Fcγ receptors (FcγR) or to otherwise alter the activation threshold. In human mast cells, C5a promoted the production of the neutrophil chemotaxin interleukin‐8, and recruitment of neutrophils at 24 hours after serum administration was impaired in C5aR^−/− mice, suggesting that enhanced neutrophil chemoattractant production underlies the requirement for C5aR on mast cells in arthritis.

Conclusion

Stimulation via C5aR is required to unleash the proinflammatory activity of synovial mast cells in immune complex arthritis, albeit via a mechanism that is distinct from C5a‐modulated expression of FcγR.

     

Spontaneous and aggregated IgG induced rheumatoid factor producing cells in rheumatoid arthritis

Summary Seropositive rheumatoid arthritis (RA) patients were found to have high numbers of spontaneously occurring cells making rheumatoid factor (RF) reactive with human IgG as measured by a RF plaque forming cell (RF-PFC) assay. There was a significant positive correlation between the number of RF-PFC and both disease activity measured by the sedimentation rate and RF titer measured by the RA latex test. Aggregated IgG and pokeweed mitogen were equally effective stimulators of RF-PFC in cultures of RA peripheral blood mononuclear leukocytes. The rheumatoid ratio of helper (T4): suppressor (T8) T lymphocytes was also significantly increased over the ratio of normal controls, but this ratio did not correlate with the number of RF-PFC. Aggregated IgG or immune complexes may be responsible for stimulating RA RF-PFC in vivo.     

Structure of t cell antigen receptor β chain in synovial fluid cells from patients with rheumatoid arthritis

We attempted to identify a clonal proliferation of T cells from synovial fluid samples from patients with rheumatoid arthritis, using techniques of restriction fragment length polymorphism. We used probes for the β chain of the T cell receptor to analyze restriction fragments prepared from the genomic DNA of synovial fluid mononuclear cells from 10 patients and synovial fluid T cell preparations from 5 additional patients. The results demonstrated unarranged (germline) T cell receptor gene fragments of DNA in all cell preparations, indicating the lack of clonality of rheumatoid arthritis synovial fluid T cells.     

Quantitation of human synovial mast cells in rheumatoid arthritis and other rheumatic diseases

We examined sections of synovial membranes from 14 patients with rheumatoid arthritis (RA), 7 with other rheumatic diseases, and 10 with no apparent joint disease. Patients with RA and other rheumatic diseases had significantly more synovial mast cells/vessel than patients with no joint disease (0.49 and 0.20, respectively, versus 0.03). They also had significantly more total mast cells/10 fields than patients with no joint disease (9.9 and 5.0, respectively, versus 0.4). Within the rheumatoid group, patients with active disease had more total mast cells/10 fields than patients clinically considered to have end-stage disease (P < 0.05). Synovial basophils were not identified in any patient. Synovial vascularity was similar for all groups (2.3 vessels/field). The role of the synovial mast cell in RA and other rheumatic diseases remains to be determined.     

Expression of membrane-type matrix metalloproteinases in synovial tissue from patients with rheumatoid arthritis or osteoarthritis

We investigated the expression of membrane-type matrix metalloproteinase (MT-MMP) and matrix metalloproteinase (MMP) mRNAs in synovial tissue from patients with rheumatoid arthritis (RA, n = 5) or osteoarthritis (OA, n = 5) by Northern blot analysis. Northern analysis demonstrated strong expression of MT1-MMP, MT3-MMP, MMP-1, and MMP-3 and weak expression of MT2-MMP and MMP-8 in synovial tissue from patients with RA or OA. MT4-MMP was not detected. No significant difference was shown in the expression of MT-MMP mRNAs between RA and OA. Synovial tissue of RA or OA patients expressed MT-MMPs as well as MMPs. These results indicate that, in addition to MMPs, MT1-MMP, MT3-MMP, and probably MT2-MMP may play a role in the degradation of bone and cartilage matrix in RA and OA. Such information may provide a clue to the development of a novel therapeutic approach targeted on the prevention of joint destruction. Received: April 30, 2000 / Accepted: September 19, 2000     

Phospholipase activity in synovial fluid from patients with rheumatoid arthritis, osteoarthritis and crystal-associated arthritis

Phospholipase activity was assayed in cell-free synovial fluid (SF) from patients with rheumatoid arthritis (RA, n = 28), osteoarthritis (OA, n = 10), and crystal-associated arthritis (C, n = 7) by measuring the release of either [14C]oleic acid or [3H]arachidonic acid from radiolabeled E. coli phospholipids. Activity measured by oleic acid release was not significantly different between the three groups of patients (RA = 571 +/- 43.3, OA = 460 +/- 54.7 and C = 718 +/- 162.6 pmol/min/mg). Arachidonic acid release was significantly (p less than 0.005) less in OA (31 +/- 7.3) than RA (61 +/- 4.7) which was similar to C (58 +/- 17.6 pmol/min/mg). Arachidonic acid release correlated significantly with the SF white blood cell count (r = 0.483, p less than 0.01). This study shows the importance of the type of substrate used to measure phospholipase activity and indicates that differences in the capacity to release arachidonic acid may exist between RA and OA disease states.     

Genetic polymorphism H131R of Fcγ receptor type IIA (FcγRIIA) in a healthy Finnish population and in patients with or without platelet-associated IgG

Abstract: Patients (n = 113) with histories of thrombocytopenia and with different profiles for platelet-associated IgG (PA-IgG) were subdivided according to the genetic polymorphism H131R in the Fcγ receptor type IIA (FcγRIIA). PA-IgG was measured by the direct platelet immunofluorescence test (PIFT), and GP IIbIIIa and/or GP Ib-specific PA-IgG was investigated by a modified version of the direct monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay. As a control, the distribution of FcγRIIA polymorphism H131R was determined among 93 healthy Finnish blood donors. The frequencies for H131 and R131 were 0.56 and 0.44 (CI: 0.37–0.51), respectively, which did not differ significantly from those in other Caucasian populations. The distribution of the genotypes HH131, HR131 and RR131 in the patients and controls did not differ significantly. In the HH131 group, the PA-IgG was higher than in the RR131 group (p = 0.082). Female patients with the genotype RR131 seemed to be younger than those with HH131 (p = 0.065). Among the female patients, a significantly greater number were under 40 yr old in the RR131 group than in the HH131 group (p = 0.0060). Within the RR131 group, the female patients were far younger than the male patients (median 29 vs. 61 yr; p = 0.0021). The results point to the heterogeneity of immune thrombocytopenia, which may partly explain the poor predictive value of PA-IgG studies.     

Secretion of antibodies to types I and II collagen by synovial tissue cells in patients with rheumatoid arthritis

Production of antibodies to IgG and to type I and type II collagen (CI and CII) was analyzed by enzyme-linked immunospot assay in patients with rheumatoid arthritis (RA) and patients with other inflammatory or degenerative joint diseases. Anti-CII-secreting cells, generally in high numbers, were found among mononuclear cells eluted from inflamed synovial tissue in 12 of 13 patients with seropositive RA and 9 of 14 patients with seronegative RA or with undetermined serum rheumatoid factor levels. In contrast, no anti-CII-producing B cells were present among synoviocytes from 4 patients with other joint diseases. In none of 7 RA sera did we find significant levels of anti-CII. Synovial B cells secreting antibodies specific for CI were observed less frequently in patients with RA. These results indicate that measurement of serum antibody levels is not adequate to assess actual autoantibody production in rheumatoid joints and that local autoimmune reactions to CII are common in RA, which implies that collagen-reactive T cells are present within the inflamed joints of RA patients. The possible role of a local collagen autoimmunity in RA is discussed, particularly in relation to its putative role in rheumatoid factor production.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis

OBJECTIVE: Matrix metalloproteinases (MMPs) are expressed in joint tissues of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). The objective of this study was to define the steady state levels of seven different MMPs and two tissue inhibitors of metalloproteinases (TIMPs) as well as the potential metalloproteinase activity in the synovial fluid (SF) to provide more insight into the role of MMPs in cartilage destruction in RA and OA. METHODS: Levels of MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-13, TIMP-1, and TIMP-2 in SF aspirated from knee joints of 97 patients with RA and 103 patients with OA were measured by the corresponding one step sandwich enzyme immunoassays. Proteolytic activity of MMPs in these SFs was examined in an assay using [(3)H]carboxymethylated transferrin substrate in the presence of inhibitors of serine and cysteine proteinases after activation with p-aminophenylmercuric acetate (APMA). Destruction of RA knee joints was radiographically evaluated. RESULTS: Levels of MMP-1, MMP-2, MMP-3, MMP-8, and MMP-9 were significantly higher in RA SF than in OA SF. MMP-7 and MMP-13 were detectable in more than 45% of RA SFs and in less than 20% of OA SFs, respectively. Among the MMPs examined, MMP-3 levels were extremely high compared with those of other MMPs. Direct correlations were seen between the levels of MMP-1 and MMP-3 and between those of MMP-8 and MMP-9 in RA SF. Although the levels of MMP-1 and MMP-3 increased even in the early stage of RA, those of MMP-8 and MMP-9 were low in the early stage and increased with the progression of RA. Molar ratios of the total amounts of the MMPs to those of the TIMPs were 5.2-fold higher in patients with RA than in OA, which was significant. APMA-activated metalloproteinase activity in SF showed a similar result, and a direct correlation was seen between the molar ratios and the activity in RA SF. CONCLUSIONS: Our results show that high levels of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, and TIMP-1 are present in RA SF and suggest that once these MMPs are fully activated, they have an imbalance against TIMPs, which may contribute to the cartilage destruction in RA.     

RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via Toll-like receptor 3

OBJECTIVE: To assess the expression of Toll-like receptor 3 (TLR-3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR-3 ligands. METHODS: TLR-3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescence-activated cell sorting and real-time polymerase chain reaction techniques. TLR-3 signaling was assessed by incubating RASFs with poly(I-C), lipopolysaccharide, palmitoyl-3-cysteine-serine-lysine-4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon-beta (IFNbeta), CXCL10, CCL5, and interleukin-6 (IL-6) protein production in the culture supernatants was performed by enzyme-linked immunosorbent assays. RESULTS: TLR-3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR-3 expression was localized predominantly in the synovial lining, with a majority of the TLR-3-expressing cells coexpressing fibroblast markers. Stimulation of cultured RASFs with the TLR-3 ligand poly(I-C) resulted in the production of high levels of IFNbeta, CXCL10, CCL5, and IL-6 protein. Similarly, coincubation of RASFs with necrotic synovial fluid cells from patients with RA resulted in up-regulation of these cytokines and chemokines in a TLR-3-dependent manner. CONCLUSION: Our findings demonstrate the expression of TLR-3 in RA synovial tissue and the activation of RASFs in vitro by the TLR-3 ligand poly(I-C) as well as by necrotic RA synovial fluid cells, and indicate that RNA released from necrotic cells might act as an endogenous TLR-3 ligand for the stimulation of proinflammatory gene expression in RASFs.     

RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via toll‐like receptor 3

Objective

To assess the expression of Toll‐like receptor 3 (TLR‐3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR‐3 ligands.

Methods

TLR‐3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescence‐activated cell sorting and real‐time polymerase chain reaction techniques. TLR‐3 signaling was assessed by incubating RASFs with poly(I‐C), lipopolysaccharide, palmitoyl‐3‐cysteine‐serine‐lysine‐4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon‐β (IFNβ), CXCL10, CCL5, and interleukin‐6 (IL‐6) protein production in the culture supernatants was performed by enzyme‐linked immunosorbent assays.

Results

TLR‐3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR‐3 expression was localized predominantly in the synovial lining, with a majority of the TLR‐3–expressing cells coexpressing fibroblast markers. Stimulation of cultured RASFs with the TLR‐3 ligand poly(I‐C) resulted in the production of high levels of IFNβ, CXCL10, CCL5, and IL‐6 protein. Similarly, coincubation of RASFs with necrotic synovial fluid cells from patients with RA resulted in up‐regulation of these cytokines and chemokines in a TLR‐3–dependent manner.

Conclusion

Our findings demonstrate the expression of TLR‐3 in RA synovial tissue and the activation of RASFs in vitro by the TLR‐3 ligand poly(I‐C) as well as by necrotic RA synovial fluid cells, and indicate that RNA released from necrotic cells might act as an endogenous TLR‐3 ligand for the stimulation of proinflammatory gene expression in RASFs.