Paul P. Tak

Detection of bacterial DNA in joint samples from patients with undifferentiated arthritis and reactive arthritis, using polymerase chain reaction with universal 16S ribosomal RNA primers

OBJECTIVEnBacteria are considered to be important in the pathogenesis of several forms of arthritis. The goal of this study was to apply the 16S ribosomal RNA (rRNA)-polymerase chain reaction method for the detection of bacterial DNA in synovial fluid (SF) and synovial tissue (ST) from inflamed joints.nnnMETHODSnSamples from 5 patients with septic arthritis and from 7 with osteoarthritis or arthritis secondary to joint trauma were used as controls. Samples from 6 patients with spondylarthropathy (SpA) and from 20 with undifferentiated arthritis (UA) were analyzed for the presence of bacterial DNA using universal 16S rRNA primers. Automated sequencing and comparative data analysis were performed to identify the species.nnnRESULTSnIn the positive control group, the bacterial species cultured from the synovium could be identified in all cases. No bacterial DNA was detected in the SF and ST from patients in the negative control group. In 4 of 6 patients with SpA and 7 of 20 with UA, the analysis of joint samples revealed the presence of bacterial DNA. Sequence analysis indicated the presence of multiple species, which was confirmed by sequencing of cloned products.nnnCONCLUSIONnWhen the the above techniques were used with a stringent regimen, we were able to demonstrate that it is possible to collect and analyze joint samples without contaminating bacterial DNA. The accumulation of phagocytic cells that contain bacterial DNA of various species could play a role in the pathogenesis of both SpA and UA.

INCREASED EXPRESSION OF IL-15 IN THE SYNOVIUM OF PATIENTS WITH RHEUMATOID ARTHRITIS COMPARED WITH PATIENTS WITH YERSINIA-INDUCED ARTHRITIS AND OSTEOARTHRITIS

Recently, a new player in the cytokine network has been described that is produced by monocytes and can be detected in the rheumatoid synovium: interleukin‐15 (IL‐15). Since this cytokine may play a role in the accumulation and activation of T‐cells, B‐cells, and natural killer (NK) cells characteristic of synovial tissue (ST) from patients with rheumatoid arthritis (RA), the expression of IL‐15 was studied in ST from RA patients in comparison with ST from patients with reactive arthritis (ReA) and osteoarthritis (OA) and the phenotype of IL‐15‐positive cells was determined. IL‐15 expression was investigated by immunohistochemical analysis of ST from ten patients with RA, ten patients with Yersinia enterocolitica‐induced ReA, and nine patients with OA. The immunohistological findings were quantified and the results obtained in the different patient groups were compared. To determine the phenotype of IL‐15‐expressing cells, double‐labelling immunofluorescence was performed. The expression of IL‐15 was significantly higher in ST from patients with RA than in ST from patients with ReA or OA. In double‐label experiments, co‐expression was observed with markers for macrophages, T‐cells, and NK cells. The composition of the cellular infiltrate in the synovium of patients with RA might be partly explained by the specific increase in expression of IL‐15 in rheumatoid ST. It can be speculated that IL‐15 production by inflammatory cells other than macrophages may occur in the rheumatoid synovium.

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

OBJECTIVEnTo 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.nnnMETHODSnSamples 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.nnnRESULTSnSynovial 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.nnnCONCLUSIONnMost 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.

Expression of the thioredoxin-thioredoxin reductase system in the inflamed joints of patients with rheumatoid arthritis

OBJECTIVEnTo examine the expression of the thioredoxin (TRX)-thioredoxin reductase (TR) system in patients with rheumatoid arthritis (RA) and patients with other rheumatic diseases.nnnMETHODSnLevels of TRX in plasma and synovial fluid (SF) were measured using enzyme-linked immunosorbent assay. Cellular distribution of TRX was determined by flow cytometry and histochemistry. Cellular expression of TR was studied by in situ messenger RNA (mRNA) hybridization. The effect of oxidative stress and tumor necrosis factor alpha (TNF alpha) on TRX expression by cultured rheumatoid fibroblast-like synoviocytes was studied.nnnRESULTSnSignificantly increased TRX levels were found in the SF from 22 patients with RA, when compared with plasma levels in the same patients (P < 0.001) and compared with SF TRX levels in 15 patients with osteoarthritis (P < 0.001), 13 patients with gout (P < 0.05), and 9 patients with reactive arthritis (P < 0.0001). The presence of TRX could be demonstrated within the SF-derived mononuclear cells and synovial tissue (ST) of RA patients. Concordantly, expression of TR mRNA was observed in the ST of these patients. Stimulation of synovial fibroblast-like synoviocytes with either H2O2 or TNF alpha induced an increase in the production of TRX.nnnCONCLUSIONnThe data demonstrate significantly increased concentrations of TRX in the SF and ST of RA patients when compared with the levels in patients with other joint diseases. Evidence is presented that the local environment in the rheumatic joint contributes to increased TRX production. Based on its growth-promoting and cytokine-like properties, it is proposed that increased expression of TRX contributes to the disease activity in RA.

Defective TCR-mediated signaling in synovial T cells in rheumatoid arthritis

In rheumatoid arthritis (RA), the functional status of T cells is incompletely understood. Synovial T cells display phenotypic evidence of former activation, but there is poor production of T cell-derived cytokines in the synovium. In addition, synovial T cell proliferation upon mitogenic and antigenic stimulation was decreased compared with that in peripheral blood T cells. Moreover, previous reports revealed that early Ca2+ rises induced by TCR/CD3 stimulation were decreased in RA T cells compared with those in healthy controls. To investigate the molecular mechanisms of RA synovial T cell hyporesponsiveness, we analyzed the TCR/CD3-mediated protein tyrosine phosphorylation in RA peripheral blood and synovial fluid (SF) T cells. SF T cells exhibited a decreased overall tyrosine phosphorylation pattern upon stimulation. Most notably, the induction of phosphorylation of p38 was virtually absent. Moreover, we found that tyrosine phosphorylation of the TCR zeta-chain, one of the most proximal events in TCR signaling, is clearly diminished in RA SF T cells. The decrease in tyrosine phosphorylation was accompanied by a decrease in detectable levels of zeta-protein within synovial T cells. These results suggest that a defective TCR signaling underlies the hyporesponsiveness of synovial T cells in RA.