Heat shock protein 70 gene polymorphisms in rheumatoid arthritis

A role for heat shock proteins (hsp) in rheumatoid arthritis has been suggested. In addition, the specific binding of human HSP70 protein to QKRAA and RRRAA motifs within the HV3 region of disease-associated DRB1*0401 and DRB1*1001 molecules, respectively, has been proposed as being relevant to rheumatoid arthritis. The purpose of this work was to analyze the influence of HSP70 gene polymorphism on the susceptibility to or severity of rheumatoid arthritis and to investigate the possible contribution of these HSP70 polymorphisms in determining HLA-DRB1*0401/*1001 disease association. The frequencies of the HSP70-1, HSP70-2 and HSP70-hom genotypes were analyzed by PCR-RFLP using BsrBI , Pst I and Nco I enzymes, respectively, in patients with heumatoid arthritis and in healthy controls. No significant differences were observed when HSP70 alleled istribution between the groups under study were compared. Moreover, we did not observe any significant difference in HSP70 allele frequencies between patients positive for HLA-DRB1*0401/*1001 alleles and matched controls. Our data indicate that HSP70 gene polymorphisms do not appear to be relevant in the susceptibility to or severity of rheumatoid arthritis.     

Cytokine profile in systemic lupus erythematosus,rheumatoid arthritis,and other rheumatic diseases

We investigated serum levels of interleukin-6 (IL-6), interferon-gamma (IFN-), and tumor necrosis factor alpha (TNF) from patients with systemic lupus erythematosus (SLE) and its various clinical manifestations of disease and from patients with rheumatoid arthritis (RA) and other rheumatic diseases. The serum levels of IL-6 and IFN- were highly elevated from patients with SLE associated with lymphadenopathy (LN) or nephrotic syndrome (NS). On the contrary, the serum levels of TNF were elevated from most patients with SLE associated with thrombocytopenia (TP). However, serum levels of TNF were in the normal range from patients with SLE associated with NS, LN, or central nervous system disease. Of interest, patients with SLE associated with humoral immunodeficiency disorder, hypogammaglobulinemia, had highly elevated levels of serum IL-6. The concanavalin A-stimulated mononuclear cells (MNC) of patients with SLE associated with TP secreted highly elevated levels of TNF compared to other patient groups. We suggest that abnormal production of various cytokines in SLE is an intrinsic defect of MNC and the immune system that may be the key element for a variety of clinical manifestations of this disease.     

Analysis of depressed cell-mediated immunity in asbestos workers

To explore the mechanisms of asbestos-related perturbations of the immune system, we evaluated the in vitro cell-mediated immunity of five asymptomatic asbestos workers with hypergammaglobulinemia and decreased T-cell numbers. These results were compared with those in 10 matched controls. Analysis of T-lymphocyte populations revealed decreased absolute numbers of OKT4+ (helper/inducer) T cells in the peripheral blood and phytohemagglutinin (PHA)-stimulated mononuclear cell cultures of the workers. When chrysotile asbestos was added to PHA cultures, expansion of OKT4+ cell populations was disproportionately inhibited in workers' cultures. Furthermore, control proliferative responses to PHA became indistinguishable from initial worker responses. These effects were incompletely explained by the cytotoxic effects of asbestos on cultured lymphocytes. We conclude that both in vivo and in vitro exposure of mononuclear cell populations to asbestos may lead to a diminution of helper-inducer T-cell numbers. In asbestos-exposed individuals, this latter lymphocyte subpopulation appears to be especially sensitive to in vitro asbestos exposure. Although the clinical implications of these findings are unclear, we hypothesize that many of the immunologic abnormalities that occur in asbestos workers could be explained by direct asbestos effects on the OKT4+ immunoregulatory population.     

风湿性关节炎患者T细胞表达CCR5和CXCR3的三色流式细胞分析

目的 在单细胞水平上，研究类风湿性关节炎（RA）患者滑液及外周血中T淋巴细胞上CCR5及CXCR3的表达，并结合临床资料分析其在RA发病中的可能作用机制及临床应用。方法 分离15例RA患者的滑液单个核细胞（SFMC）、外周血单个核细胞（PBMC），及15正常人PBMC（对照），以三色荧光素标记物进行流式细胞术分析T细胞上CCR5及CXCR3的表达。结果 与PBMC相比较，RA患者SFMC中T细胞上CCR5及CXCR3的表达显著增高（特别是CCR5）；而CXCR3的表达个体差异较大；与正常人相比较，RA患者初发或活动期未治时，PBMC中CCR5及CXCR3的表达明显增多。CCR5及CXCR3的表达率与患者的ESR及CRP相关。结论 CCR5^ T细胞积聚在RA患者的病变关节内，且与RA的病情密切相关。     

类风湿性关节炎患者关节滑膜液浸润的T细胞表达特性

目的 :为研究类风湿性关节炎 (RA)患者关节滑膜液浸润的淋巴细胞介导自身免疫病的特性 ,分析了 2 2例RA患者滑膜液中淋巴细胞的免疫表型、对II型胶原的反应频率及IL 10、IL 12的分泌格局。方法 :用流式细胞术分别测定滑膜液和外周血淋巴细胞表型 ,并采用国际标准半有限稀释法分析了关节滑膜液中浸润的淋巴细胞对II型胶原 (CII)的反应频率 ,同时用ELISA方法检测了滑膜液与外周血中IL 10与IL 12含量。结果 :滑膜液中的T淋巴细胞的表型分别为CD4 (39 6 %±10 5 % )和CD8细胞 (36 4 %± 16 4 % ) ,CD4 CD8细胞比值显著低于外周血 ,且同时表达CD16和CD5 6的活化NK细胞占15 5 %± 11 1%。T细胞受体谱取用表明仍以αβTCR为主 (6 9 6 %± 15 7% )。有意义的是 :滑膜液中的T细胞对CII的反应频率为 15 2× 10 - 6 ,远远高于外周血 (4 0× 10 - 6 )。IL 12含量为 (4 19 9± 89 2 )pg ml,IL 10含量为 (187 7± 34 5 )pg ml,与外周血中这 2种细胞因子的含量〔分别为IL 12 :(6 5 32± 34 2 )pg ml和IL 10 :(85± 12 7)pg ml〕比较 ,具有显著的统计学差异。结论 :上述实验结果表明这种具有表达特性的浸润T细胞介导了RA患者关节滑膜组织的免疫损伤。     

Collagen-induced arthritis in TNF receptor-1-deficient mice: TNF receptor-2 can modulate arthritis in the absence of TNF receptor-1

TNF is a potent proinflammatory cytokine important for the development of arthritis in human and animals. We have investigated the roles of TNF receptor-1 (TNFR1) and TNF receptor-2 (TNFR2) in collagen-induced arthritis (CIA) by inducing CIA in mice genetically deficient in TNFR1. TNFR1-/- mice developed arthritis with similar incidence and severity as TNFR1+/- littermates, indicating that TNFR1 is redundant for the development of CIA. Anti-type II collagen (CII) antibody levels and T cell responses to CII did not differ between TNFR1-/- mice and controls. Neutralization of TNF with soluble TNF binding protein suppressed the development of arthritis in TNFR1+/- mice but not in TNFR1-/- mice, indicating that TNFR2 cannot substitute for TNFR1 for the proinflammatory function. To further investigate the functions of TNFR2, TNFR1-/- mice were injected with murine TNF-alpha at different stages during the course of CIA. Repeated TNF-alpha injection during the early induction phase enhanced the development of arthritis, but inhibited arthritis when administered during the late progression phase. These results show that the engagement of TNFR2 by TNF is involved in the development of CIA in the absence of TNFR1 and that opposing signals can be transduced by TNFR2.     

Genetic dissection of the complex pathological manifestations of collagen disease in MRL/lpr mice

An MRL strain of mice bearing a Fas-deletion mutant gene, lpr, MRL/MpJ-lpr/lpr (MRL/lpr) develops collagen disease involving vasculitis, glomerulonephritis, arthritis and sialoadenitis, each of which has been studied as a model for polyarteritis, lupus nephritis, rheumatoid arthritis and Sjögren’s syndrome, respectively. Development of such lesions seems dependent on host genetic background since the congenic C3H/HeJ-lpr/lpr (C3H/lpr) mice rarely develop them. To identify the gene loci affecting each lesion, a genetic dissection of these complex pathological manifestations was carried out. First, histopathological features in MRL/lpr, C3H/lpr, (MRL/lpr × C3H/lpr) F1 intercross, and MRL/lpr × (MRL/lpr × C3H/lpr) F1 backcross mice were analyzed. Genomic DNA of the backcross mice were subjected to association studies by Chi-squared analysis for determining which polymorphic microsatellite locus occurs at higher frequency among affected compared to unaffected individuals for each lesion. As a result, gene loci recessively associated with each lesion were mapped on different chromosomal positions. We concluded that each of these lesions in MRL/lpr mice is under the control of a different set of genes, suggesting that the complex pathological manifestations of collagen disease result from polygenic inheritance.     

Inflammatory cytokine levels in paw tissues during development of rat collagen-induced arthritis: Effect of FK506, an inhibitor of T cell activation

Objective and design: To characterize rat collagen-induced arthritis (CIA) on the basis of levels of inflammatory cytokines, tumor necrosis factor (TNF)-, interleukin (IL)-1 and IL-6 in paw tissues, and further investigate the effect of FK506 (tacrolimus), a potent inhibitor of T cell activation, on cytokine levels.Methods: CIA was induced in female Lewis rats. The volume of hindpaws was measured before and after collagen immunization. TNF-, IL-1 and IL-6 levels in paw tissue extracts were determined by ELISA. Proteoglycan contents of cartilage in femoral heads was measured as an indication of cartilage destruction. To assess the effect of FK506 on inflammatory cytokine levels, rats were orally treated with 5 mg/kg of FK506 from days 14–21.Results: TNF- a level in paw tissues did not significantly change compared to levels found before collagen immunization, throughout development of CIA. In contrast, IL-1 and IL-6 levels in paw tissues significantly increased between day 14 and day 28 after collagen imuninization, when the arthritis was at a developed stage. Therapeutic treatment with FK506 reduced the elevated level of IL-6, but not IL-1, in paw tissue. FK506 treatment was effective in suppressing paw swelling and also recovering the loss of proteoglycan contents in the cartilage.Conclusions: Levels of IL-1 and IL-6, but not TNF- , in paw tissue were upregulated in association with the development of arthritis in rat CIA. These results suggest that IL-1 and IL-6, rather than TNF- , may play important roles at local inflammatory sites in producing joint destruction in rat CIA. FK506 may improve arthritis in established stages of CIA, by reducing the elevated level of IL-6.Received 4 March 2004; returned for revision 2 April 2004; accepted by M. J. Parnham 9 April 2004     

Antigen presentation of Type II collagen in rats

Collagen-induced arthritis (CIA) is a T-cell dependent disease of rats which follows immunization with bovine type II collagen (bCII). Susceptibility to CIA is linked to the genes encoding the major histocompatibility complex (MHC), suggesting that antigen presentation is important in disease pathogenesis. Antigen-presenting cells (APC) (macrophages, dendritic cells (DC) and B cells) were prepared from WA/KIR/KCL rats and presentation of antigen, in the form of native protein (bCII) or synthetic peptide (bCII:184-198), was assessed in T-cell proliferation assays. Whilst macrophages inhibited proliferative responses to bCII, splenic or thymic low density cells, enriched for DC, presented both bCII and bCII(184-198) peptide. However, bone marrow-derived DC, which stimulated T-cell responses to OVA, failed to present bCII, suggesting differences in processing of these two antigens. B-cell depletion from lymph node cells abrogated the proliferative response to bCII and reconstitution of a T-cell population with B cells restored the proliferative response, indicating that B cells are important for stimulating T-cell responses to bCII. B cells play a critical role in CIA by producing pathogenic anti-bCII antibodies, and we propose that B cells are also important APC which present bCII to CD4+ T cells.     

The role of COX-2 in angiogenesis and rheumatoid arthritis

Recent evidence suggests that cyclooxygenase (COX)-2 is a mediator of angiogenesis, and COX-2 activity is known to be upregulated in the rheumatoid arthritis (RA) synovium. We examined whether mediation of angiogenesis by COX-2 was occuring in cells of the RA synovium and in microvascular endothelial cells (ECs) that are similar to those found in the RA synovium. We demonstrate that rofecoxib, a selective COX-2 inhibitor, acts directly on human dermal microvascular ECs (HMVECs) to inhibit their chemotactic and tube forming ability. Likewise, pretreatment of HMVECs with rofecoxib significantly inhibited their ability to form tubes induced by conditioned media (CM) of activated RA synovial fibroblasts. When RA synovial fibroblasts were pretreated with rofecoxib for 16 h and then stimulated with interleukin (IL)-1beta, their CM induced significantly less HMVEC tube formation when compared with CM from vehicle-treated RA synovial fibroblasts. ELISAs performed on activated RA fibroblast CM for known proangiogenic factors demonstrated a significant reduction in bFGF, in addition to the expected decrease in PGE(2). Our studies suggest that COX-2-induced angiogenic activity is an active mechanism within diseased synovium and may provide an additional rationale for the use of COX-2 inhibitors in RA.