We detected a novel nuclear protein, MRF, that binds to multiple sites on the modulator which is located upstream of the human cytomegalovirus major immediate early gene enhancer. The expression of MRF is differentiation specific; the DNA binding activity is present in nuclear extracts from undifferentiated Tera-2 and THP-1 cells, but significantly reduced after these cells are induced to differentiate. In undifferentiated cells the enhancer activity is repressed by the modulator and upon differentiation the enhancer becomes active. Competitive binding assays demonstrate that MRF requires the presence of multiple A+T stretches for binding to DNA, rather than binding to a specific DNA sequence. Mutations of these stretches in the modulator reduce the binding activity of MRF, as well as the repressing activity on the enhancer. These results suggest that MRF may act as a repressor of enhancer function. We propose that MRF binds over the entire modulator and exerts repressor activity.

Objective. To elucidate the role of neurologic, endocrine, and immune system interactions in the development of pathologic responses in patients with rheumatoid arthritis (RA), we studied somatostatin (SOM) production and somatostatin receptor (SOMR) expression in RA synovium and its function in patients with RA.Methods. The effects of SOM on proinflammatory cytokine (interleukin-6 [IL-6] and IL-8) and collagenase production by RA synovial cells were estimated by enzyme-linked immunosorbent assay, and their messenger RNA expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR) using limiting dilutions of the complementary DNA. The expression of SOMR by RA synovial cells was also studied by RT-PCR. Local production of SOM was estimated by RT-PCR and immunohistochemical staining. Results. Physiologic concentrations (-lO-''W ofSOM inhibited proliferation of RA synovial cells. The production of proinflammatory cytokines and matrix metalloproteinases by RA synovial cells was also modulated by SOM. SOMR subtypes 1 and 2 were expressed on fibroblast-like synovial cells, and the expression of SOMR-2 was up-regulated by proinflammatory cytokine treatment of the synovial cells from patients with RA. RA fibroblast-like cells synthesized SOM by themselves, suggesting that SOM acts as an autocrine regulator of synovial cell function in patients with RA.

Synovial cell hyperplasia is a characteristic of patients with RA. Excessive proliferation of RA synovial cells is, in part, responsible for the synovial cell hyperplasia. In addition, synovial cell death that would reduce synovial cell number may be defective, leading to the hyperplasia. Thus, the defective control of cell death as well as cell proliferation may be of central importance in the pathogenesis of RA. In this study we analysed effects of proinflammatory cytokines on Fas/Fas ligand (FasL)‐induced synovial cell apoptosis, and evaluated apoptosis‐associated protein expression in the synovial cells in patients with RA. RA synovial cells expressed Fas antigen and lymphocytes infiltrating into RA synovium expressed FasL. Apoptotic synovial cells were detected within the sublining layer of RA synovium. Anti‐Fas MoAb induced apoptosis of RA synovial cells in vitro, and proinflammatory cytokines tumour necrosis factor‐alpha (TNF‐α) and IL‐1β, but not IL‐6 or IL‐8, inhibited the anti‐Fas‐induced apoptosis accompanying up‐regulation of Bcl‐2 protein expression and reduced expression of CPP32 and ICH‐1L. Immunohistochemical study revealed that CPP32 and ICH‐1L were expressed weakly in the RA synovial lining cells compared with osteoarthritis (OA) synovial lining cells. Thus, we found that although RA synovial cells could die via apoptosis through Fas/FasL pathway, apoptosis of synovial cells was inhibited by proinflammatory cytokines present within the synovium. Inhibition of apoptosis by the proinflammatory cytokines may contribute outgrowth of synovial cells that leads to pannus formation and the destruction of joints in patients with RA.

Objective. To elucidate the possible involvement of the nervous system in the regulation of pathophysiologic responses in patients with rheumatoid arthritis (RA), we examined the expression of peripheral nerves containing the neuropeptides calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) in RA synovium and their effects on RA synovial cell functions. Methods. The effects of CGRP and VIP on proin-flammatory cytokine and matrix metalloproteinase (MMP) production by RA synovial cells were estimated by enzyme-linked immunosorbent assay, and their messenger RNA (mRNA) expression by reverse transcription-polymerase chain reaction (RT-PCR) using limiting dilutions of the complementary DNA. Expression of CGRP receptors (CGRPRs) and VIP receptors (VIPRs) on RA synovial cells was assessed by RT-PCR and radioreceptor assays. The functions of CGRPRs and VIPRs of the synovial cells were studied by using a CGRPR antagonist and a VIPR antagonist, respectively. Results. CGRP and VIP inhibited the proliferation of, and the proinflammatory cytokine and MMP production by, RA synovial cells at the level of mRNA expression. Expression of CGRPR and VIPR on RA fibroblast-like synovial cells was confirmed by RT-PCR and radioreceptor assays. Functions of the neuropeptide receptors were inhibited by their receptor antagonists. CGRP and VIP inhibited nuclear translocation and phosphorylation of the transcription factor cAMP response element binding protein in RA synovial cells. Conclusion. CGRP and VIP inhibited excessive synovial cell functions, which suggests neural regulation of inflammatory responses in patients with RA and possible clinical application of the neuropeptides.

Disease-modifying antirheumatic drug (DMARD) combination therapies are used widely, but there have been few reports clearly demonstrating that combination therapy is more effective than DMARD monotherapy. We conducted a multicenter, double-blind controlled trial in order to clarify that the combination of methotrexate and bucillamine is more effective than either alone. The subjects of this study were 71 patients with active rheumatoid arthritis within 2 years of onset. Dosages were 8 mg methotrexate with 5 mg folic acid per week (MTX group), 200 mg bucillamine per day (BUC group), or both MTX and BUC (combination group). Clinical effects and adverse reactions were observed for 96 weeks. The ACR 20 response rate was 79.2% in the combination group, significantly higher than the rates of 43.5% for the MTX group (P = 0.008) and 45.8% for the BUC group (P = 0.0178). The cumulative survival curve of maintaining the ACR 20 response was significantly higher in the combination group than in the MTX and BUC groups (P = 0.0123 and P = 0.0088, respectively). The mean increase in the total Sharp score over 96 weeks was 12.6 +/- 9.0 in the combination group, significantly lower (P = 0.0468) than the value of 28.0 +/- 28.3 for the single DMARD (combined MTX and BUC) group. The incidence of adverse reactions did not differ significantly between the three groups. It was concluded that the combination therapy with MTX and BUC showed significantly higher clinical efficacy than either of the single DMARD therapies.