Inflammatory immune cell responses and Toll-like receptor expression in synovial tissues in rheumatoid arthritis patients treated with biologics or DMARDs

Biologic antirheumatic drugs (BIO) have been reported to be potent therapeutic agents in the prevention of inflammatory joint destruction in rheumatoid arthritis (RA). The aim of this study was to investigate the immune-inflammatory cells, including Toll-like receptor (TLR)-equipped cells, in synovial tissue samples from RA patients on BIO compared to patients, who are only on conventional disease-modifying antirheumatic drug (DMARD). We analyzed immune-inflammatory cells in RA synovitis in patients of BIO group (n?=?20) or DMARD group (n?=?20). The grading scores of synovitis was 1.7 and 1.8 in each BIO and DMARD group and correlated best with the CD3⁺ T (r?=?0.71/0.70, p?<?0.05) and CD20⁺ B (r?=?0.80/0.84, p?<?0.05) cells in the both groups, but less well with the CD68⁺ macrophages and S-100⁺ dendritic cells (DCs). Interestingly, both T (116 vs. 242, p?<?0.05) and B (80 vs. 142, p?<?0.05) cell counts were lower in the BIO than in the DMARD group, whereas macrophage and DC counts did not differ. In contrast, the C-reactive protein (CRP) and disease activity score DAS28-CRP did not show clear-cut correlations with the inflammatory grade of the synovitis (r range, 0–0.35). Similar numbers of cells immunoreactive for TLR-1 to TLR-6 and TLR-9 were found in synovitis in both groups. Patients clinically responding to biologics might still have the potential of moderate/severe local joint inflammation, composed in particular of and possibly driven by the autoinflammatory TLR⁺ cells.     

Ia+ T cells in synovial fluid and tissues of patients with rheumatoid arthritis

Markedly elevated levels of T cells expressing Ia antigens were found in the synovial membranes and synovial fluids of patients with rheumatoid arthritis. The primary increase in expression of the Ia antigens was on the OKT 8+ (suppressor/cytotoxic) T cell subset. In addition, the total percentage of OKT 8+ T cells in intraarticular sites was usually greater than levels in peripheral blood. Small numbers of OKT 4+ (helper/inducer) cells bearing Ia antigens were also identified. The characteristic increase in the Ia+ T cells in peripheral blood was not encountered in most patients treated with D-penicillamine.     

Analysis of cell populations in rheumatoid arthritis synovial tissues.

Knee synovium, taken from patients with rheumatoid arthritis at the time of arthroplasty, was studied immunohistologically. Focal perivascular lymphoid infiltrates of different sizes were examined in detail to evaluate changes in cell populations as the infiltrate size increased. T cells formed the largest component of mononuclear cells of all aggregates. The large grade 3 aggregates contained substantial numbers of B cells arranged around a central venule and cells bearing the CD45RA+ phenotype. In contrast, the small grade 1 aggregates contained few B cells and the T-cell population contained relatively greater numbers of CD8+ cells. Cells bearing the CD45RO+ phenotype exceeded CD45RA+ cells in grade 1 aggregates. Detailed analysis of mononuclear cell aggregates of different sizes in the rheumatoid synovium suggests that the composition of each aggregate depends on the total number of mononuclear cells it contains.     

Epstein-Barr virus-specific cytotoxic T cell responses in rheumatoid arthritis patients

Summary The level of Epstein-Barr (EB) virus-specific cytotoxic T cell responsiveness was measured in 21 patients with active, progressive rheumatoid arthritis (RA). A significant number (8 out of 20) of EB virus sero-positive patients showed a markedly impaired responsiveness when compared with a control group of healthy individuals. Serological responses of the RA patients to EB virus antigens were not significantly different from the control group. The defect in EB virus-specific cellular immunity shown by these results is of interest in the light of previous evidence of an alteration in the virus-host balance in RA patients.     

T cell responses to citrullinated self-peptides in patients with rheumatoid arthritis

Antibodies to citrullinated peptides(ACPA) have high specificity for diagnosis and prognosis in rheumatoid arthritis (RA). ACPA are of IgG isotype and have an association with shared epitope-bearing HLA DR allele, suggesting that T cell help is needed for their generation. In mice models, T cell reactive to citrullinated self-peptide have been reported however, the human data is limited. Patients with RA satisfying ACR criteria were included and peripheral blood obtained for lymphoproliferative assay, antibody level and HLA typing. Citrullinated (Cit) and native peptides of Vimentin and Aggrecan were used for stimulating peripheral blood mononuclear cells in 5-day cultures. A SI value above >2.0 was taken as significant. HLA typing was done by SSCP and ACPA were tested by ELISA. A total of 50 patients (45 females; mean age 42 years; mean duration of disease 7 years) with RA were included in the study. A total of 90 % were RF positive and 78 % were ACPA positive. A total of 28 patients showed response to Agg peptide with 21 of them showing higher response to CitAgg as compared to native Agg peptide as well as the median SI was higher with CitAgg (6.07 Vs. 5.09; p = 0.009). A total of 31 patients showed response to Vim peptide with response to native peptide being higher than CitVim peptide in 22 of the patients. There was no association of T cell response with presence of shared epitope. Nearly half the patients with RA show T cell response to aggrecan and vimentin peptides; however, citrullination is not crucial for T cell response.     

Polyamine levels in synovial tissues and synovial fluids of patients with rheumatoid arthritis.

We determined the polyamine contents of the synovial tissues from 11 patients with rheumatoid arthritis (RA), and the free putrescine levels in the synovial fluids (SF) from 10 patients with RA, 7 with osteoarthritis (OA), 5 with posttraumatic arthritis, and 3 with infectious arthritis. Putrescine levels in the synovial tissues correlated with serum C reactive protein concentration in patients with RA. Free putrescine levels in SF were significantly elevated in patients with infectious arthritis, compared with those found in RA, OA, and posttraumatic arthritis. Free putrescine levels in SF from patients with RA were significantly higher than in those with OA. Our findings suggest that polyamines may play an important role in RA.     

IgE-containing immune complexes in synovial fluid of patients with rheumatoid arthritis

Summary The prevalence and composition of IgE-containing immune complexes in paired synovial fluid and serum of 42 patients with classical or definite rheumatoid arthritis were studied. IgE-containing immune complexes were found in 15/42 synovial fluids; 15 sera were also positive. The correlation between serum and synovial fluid complexed IgE levels was high (r=0.77). The mean ratio of synovial fluid/serum levels was 1.96, i.e. significantly higher than 0.33, the synovial fluid/serum ratio for alpha-2-macroglobulin (molecular weight 820 kD), which was taken as high molecular weight control protein (p<0.0001). Apart from IgE in immune complex form, monomeric IgE was also significantly higher in synovial fluid compared to serum (ratio = 2.94). Other constituents which could be found in the immune complexes, i.e. anti-IgE antibodies, rheumatoid factors and anticollagen antibodies, were also higher in synovial fluid than predicted. Our results suggest intra-articular production of IgE-containing complexes in the synovial fluid, in addition to possible exudation of the complexes from the serum. These findings provide further evidence for the role of IgE-containing immune complexes in rheumatoid synovitis.     

Inflammatory synovial T cells in different activity subgroups of patients with rheumatoid arthritis and juvenile rheumatoid arthritis

Mononuclear cells were eluted from synovial membranes of 39 patients with rheumatoid arthritis and 12 patients with juvenile rheumatoid arthritis. A considerable cell loss, about 50% or more, was seen during the various isolation steps. The CD4/CD8 ratio just after enzyme treatment (stage I) was significantly higher than at later stages, i.e. after removal of adherent cells (stage II, p less than 0.05) and after Isopaque Ficoll gradient centrifugation (stage III, p less than 0.01). This indicates a selective loss of CD4+ cells during isolation. In addition, stages I and II had higher CD4/CD8 ratios than peripheral blood of normal controls (p less than 0.01 and p less than 0.03), but not significantly higher than in peripheral blood of patients (p greater than 0.05). The CD4/CD8 ratio in eluted synovial membrane cells did not differ between patients with high and patients with low disease activity (p greater than 0.05). No correlation was found between any of the CD4/CD8 ratios and individual disease activity variables. Furthermore, a laboratory activity index and a disease outcome index were determined for each patient and no correlation was found between these indices and the CD4/CD8 ratios.     

T cell homeostasis in patients with rheumatoid arthritis

The immune system is equipped with an extremely large spectrum of structurally diverse receptors to recognize all potential antigens. This fundamental principle of receptor diversity is no longer upheld in patients with rheumatoid arthritis (RA), who have a marked contraction of the T cell receptor repertoire. In this study, the ability of RA patients to produce T cells and to maintain T cell homeostasis was examined. CD4 T cells containing T cell receptor rearrangement excision circles (TREC) were substantially reduced in RA patients; TREC levels in young adult patients matched those of controls 20 years older. Increased self-replication of T cells in RA was indicated by age-inappropriate erosion of telomeres in circulating T cells with almost complete attrition of telomeric reserves in patients 20-30 yr of age. The degree of telomere loss was not related to disease duration or the use of disease-modifying medication and was most pronounced in CD4(+)CD45RO(null) (naive) T cells. The loss of TREC-positive T cells could be a consequence of a primary defect in peripheral T cell homeostasis. Alternatively, RA patients may have impaired thymic function with the increased turnover of peripheral T cells being a secondary compensatory event.     

Distribution of protein nitrotyrosine in synovial tissues of patients with rheumatoid arthritis and osteoarthritis

OBJECTIVE: Because nitric oxide related species have been found in the inflamed joints of patients with arthritis, we investigated whether protein nitrotyrosine (a marker of tissue exposure to peroxynitrite) is present in their synovial tissues. METHODS: Protein nitrotyrosine was detected immunohistochemically and by Western blot analysis. Synovial tissues removed surgically from 12 patients with rheumatoid arthritis (RA) (mean age 63.7 yrs) and 20 with osteoarthritis (OA) (mean age 66.6 yrs) were studied. RESULTS: Nitrated proteins were detected immunohistochemically in all of 18 tissues examined. Diffuse staining of the stroma was seen in all patients, with more extensive staining in RA than OA (p = 0.008). Intense staining was detected in some lymphocytes, but not in others, even within a single lymphoid aggregate. Neutrophils did not stain for nitrotyrosine. Vascular endothelial cells stained for nitrotyrosine but adjoining smooth muscle cells did not. Both cytoplasmic and nuclear staining was seen in macrophages, endothelial cells, and lymphocytes. Numerous bands of nitrated proteins were detected by Western blot analysis of 15 synovial tissue extracts. Inducible nitric oxide synthase (iNOS) was detected immunohistochemically in endothelial cells, macrophages, vascular smooth muscle cells, and synoviocytes. CONCLUSION: Nitrotyrosine-containing proteins were found in essentially all synovia from RA and OA patients. The most prominent site of nitration in all cases was the stroma. iNOS, the likely source of the nitrating species, was found in a variety of cell types.     

T cell receptor expression and activation of synovial lymphocyte subsets in patients with rheumatoid arthritis. Phenotyping of multiple synovial sites

Two-color flow cytometry analysis of peripheral blood and synovial lymphocytes from rheumatoid arthritis patients was performed using monoclonal antibodies directed against T cell subsets, T cell activation markers, and T cell receptors. The results showed an abnormally high percentage (greater than 15%) of CD3+, CD4-, and CD8- T cells expressing a specific receptor containing a gamma chain. Phenotypic analysis of lymphocytes infiltrating both knee joints of individual rheumatoid arthritis patients revealed very similar subset distribution and activation levels, despite strong differences in the clinical status between the 2 sites.     

Persistent synovial lymphocyte responses to cytomegalovirus antigen in some patients with rheumatoid arthritis

Synovial lymphocytes from 6 of 40 patients with rheumatoid arthritis responded to cytomegalovirus antigen stimulation. 3H-thymidine uptakes were more than 3 times greater than were those of the responses to 13 other microbial antigens. Similar results were obtained in 1 patient on 7 occasions over 17 months, and in the 5 other patients on each of 2 occasions. In 3 of the 6 patients, synovial lymphocyte responses to cytomegalovirus antigen were markedly different from simultaneous peripheral blood lymphocyte responses.     

Evaluation of T cell subsets with monoclonal antibodies in synovial fluid in rheumatoid arthritis

Monoclonal antibodies that react with all T cells (OKT3), the inducer-helper T cell subset (OKT4), and the suppressor-cytotoxic T cell subset (OKT8) have been used to evaluate T cell and T cell subpopulations in synovial fluid (SF) from patients with rheumatoid arthritis (RA). We found that the immunoregulatory ratio of SF lymphocytes ranged from normal values to extremely low values indicating normal to excess numbers of OKT8+ cells. The rheumatoid synovium in patients with RA is characterized by the presence of lymphoid aggregates in which HLA-DR+ interdigitating cells form close contacts with a large number of OKT4+ T cells, while the OKT8+ T cells are sparse. It may be that the localizing mechanisms for OKT8+ suppressor cells in lymphoid aggregates are deficient in RA and the cells are thus permitted to pass into synovial exudates, resulting in an increase of OKT8+ cells in the SF.     

Structure of T cell antigen receptor beta 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 beta 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.     

类风湿关节炎滑膜细胞的增殖及细胞周期的研究

目的　研究类风湿关节炎 (RA )患者滑膜细胞增殖变化及其机制。方法　应用MTS/PMS比色法和流式细胞分析技术分别测定RA患者滑膜细胞的细胞增殖水平和细胞周期 ,以骨关节炎 (OA)病人及因外伤截肢的正常人作对照。结果　RA患者滑膜细胞接种后第 4天增殖水平 (0 .5 3 63± 0 .0 0 7)显著高于对照组 (0 .4187± 0 .0 0 8,P <0 .0 5 ) ,细胞周期分析显示RA患者滑膜细胞G1期的比例 (61.92 % )比对照组明显降低 (74.2 8% ) ,P <0 .0 5 ,而S、G2期的细胞比例比对照组则明显增加 ;加入RA患者滑液能以剂量依赖性明显提高滑膜细胞的增殖水平 ,并使G1期细胞进一步降低 ,S、G2期细胞进一步增加。结论　含有多种免疫调节因子的滑液使RA患者滑膜细胞在G2、S期的滞留 ,是滑膜细胞过度增殖 ,从而导致RA发病的重要因素。