Hiroko Nagafuchi

Characterization of T cells specific for an epitope of human 60-kD heat shock protein (hsp) in patients with Behcet's disease (BD) in Japan

BD is prevalent in the area of the Silk Route. It has been shown that hsp are involved in the T cell activation in patients with BD in the UK, where this disease has developed sporadically. We have thus examined whether the T cell response to the hsp‐derived peptides may be induced in patients with BD in Japan, an east pole of the Silk Route. As with patients in the UK, the human 60‐kD hsp peptide 336–351 also yielded vigorous proliferation of T cells in Japanese patients with BD, but neither in normal subjects nor in patients with rheumatoid arthritis (RA); there was significant association between proliferation by this peptide and the presence of ocular lesion, but not any other symptoms of BD. To clarify whether the peptide stimulates T cells as a polyclonal activator, a specific antigen or a superantigen‐like substance, we analysed T cell receptor (TCR) usage of responding T cells by means of MoAbs specific for TCR Vβ subfamily and polymerase chain reaction (PCR)‐single‐strand conformation polymorphism (SSCP)‐based technique. We found that T cells with certain TCR Vβ subfamilies (including Vβ5.2–3, 8, 13.6, 18, 21.3) were increased in circulation and responded to the hsp peptide in an antigen‐specific fashion. In addition, TCR Vβ gene‐amplified products of freshly isolated T cells of patients with BD formed several bands in the PCR‐SSCP analysis; some of them became prominent after stimulation with the peptide. This suggests that T cells in patients with this disease have already been expanded oligoclonally in vivo, which may be a result of stimulation by triggering antigens, including the hsp peptide. In addition, hsp peptide stimulation induced proinflammatory cytokine mRNA expression in peripheral blood mononuclear cells, including IL‐8, tumour necrosis factor‐alpha (TNF‐α) and TNF‐β in eight out of eight patients studied. Taken together, the results suggest that hsp antigen may play a role in the pathogenesis of BD, not only in the area of the Silk Route, but also outside the Silk Route area.

Modulation by proinflammatory cytokines of Fas/Fas ligand-mediated apoptotic cell death of synovial cells in patients with rheumatoid arthritis (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.

Txk, a Member of Nonreceptor Tyrosine Kinase of Tec Family, Acts as a Th1 Cell-Specific Transcription Factor and Regulates IFN-γ Gene Transcription

Precise mechanisms responsible for Th1 cell activation and differentiation are not fully elucidated. We have recently reported that Txk, a member of Tec family nonreceptor tyrosine kinase, is expressed on Th1/Th0 cells, and Txk regulates specifically IFN-γ gene expression. In this study, we found that Txk bound to IFN-γ promoter region. Txk transfection increased transcriptional activity of IFN-γ promoter plus luciferase constructs severalfold, including IFN-γ promoter −538, −208, and −53. IFN-γ promoter −39 was refractory to the Txk transfection. The actual site to which Txk bound was the element consisting of −53 and −39 bp from the transcription start site of human IFN-γ gene, a site distinct from several previously characterized binding sites. We found that the entire −53/−39 region was necessary for the binding to and function of Txk, because mutant promoter oligoDNA that contained contiguous five base substitutions dispersed throughout the −53/−39 inhibited the binding, and the mutant promoters did not respond to the Txk transfection. Similar sequences of this element are found within the 5′ flanking regions of several Th1 cell-associated protein genes. Thus, Txk is expressed on Th1/Th0 cells with the IFN-γ production and acts as a Th1 cell-specific transcription factor.

Anatomical and functional recovery by embryonic stem cell-derived neural tissue of a mouse model of brain damage

We have treated undifferentiated mouse embryonic stem (ES) cells with all-trans retinoic acid (RA) to induce differentiation in vitro into neuron-like cells with good cell viability for use as a graft. Furthermore, we asked whether the RA-induced neuron-like cells restored neurological dysfunction. To this end, the cells were transplanted into right hemiplegia model of mice, developed by a cryogenic injury of motor cortex. Motor function of the recipients was gradually improved, whereas little improvement was observed in control mice. The lesion showed clustering of mature and almost mature neuron-like cells in mice transplanted with the RA-treated cells. The grafted cells had synaptic vesicles. This finding may suggest their maturation and synaptic connection in the recipient brain. Even though further study is necessary to elucidate molecular and cellular mechanisms responsible for the functional recovery, we consider that the ES cells may have advantage for use as a donor source in various neurological disorders including motor dysfunction.

Excessive expression of Txk, a member of the Tec family of tyrosine kinases, contributes to excessive Th1 cytokine production by T lymphocytes in patients with Behcet's disease

Excessive Th1 cell function is importantly involved in the pathogenesis of Behcets disease (BD). We previously found that Txk, a member of the Tec family of tyrosine kinases, acts as a Th1 cell specific transcription factor. To investigate immune aberration in the pathogenesis of BD, we studied the expression of Txk and Th1 cytokines in peripheral blood lymphocytes (PBL) and skin lesions in patients with BD. Cytokine production by the lymphocytes was assessed using ELISA. PBL produced excessive Th1 associated cytokines including IFN‐γ and IL‐12 spontaneously and in response to exogenous HSP60‐derived peptide stimulation, which was shown to induce proliferation of PBL, in patients with BD. Circulating CD4+ T cells expressed excessive Txk protein. A majority of cells infiltrating into skin lesions expressed IFN‐γ in the BD specimens. IL‐12 and IL‐18 were also expressed in the mononuclear cell aggregates. Lymphocytes accumulating in the skin lesion expressed higher levels of Txk as compared with atopic dermatitis lesions, a typical Th2 disease. IFN‐γ, IL‐18 and Il‐12 were detected in the BD skin lesions, which may induce preferential development of Th1 cells in patients with BD. The mononuclear cell aggregates contained Txk expressing cells in such skin lesions. Collectively, Txk expressing Th1 cells and the Th1 associated cytokines may play a critical role in the development of skin lesions in BD.

Flk1 cells derived from mouse embryonic stem cells reconstitute hematopoiesis in vivo in SCID mice

OBJECTIVE Embryonic stem (ES) cells are pluripotent and can differentiate into any cell type, including the hematopoietic lineage. We examined whether hematopoietic progenitor cells derived from ES cells reconstitute hematopoiesis in irradiated SCID mice. MATERIALS AND METHODS ES cells (E14.1, H2K(b)) were cultured for 4 days in semisolid medium containing methylcellulose. Irradiated SCID mice were used as recipients of hematopoietic progenitor cells. Cell surface antigen expression was analyzed by flow cytometry. The spleens of the recipient mice were studied by hematoxylin and eosin staining and immunohistochemical staining. RESULTS After cell culture of ES cells in methylcellulose for 4 days, the cells expressing Flk1 (VEGF receptor 2), a tentative marker of hemangioblasts, were increased, whereas cells expressing CD31 (PECAM-1) and E-cadherin (nonmesodermal adhesion molecule) were dramatically reduced. Flk1+ cells expressed c-kit predominantly. Circulating leukocytes and thrombocytes were increased in irradiated SCID (H2K(d)) mice transplanted with ES cell-derived Flk1+ cells compared with vehicle-injected control mice. H2K(b+) and VE-cadherin(+) vascular endothelial cells were prominent in spleens of the recipient mice. Flow cytometric analysis demonstrated that H2K(b+) cells were increased in the bone marrow of recipient mice. In addition, Flk1+ cells accompanying enhanced c-kit expression preferentially repopulated in the bone marrow, and leukopoiesis and thrombopoiesis of the recipient mice were evident. CONCLUSION The Flk1+ hematopoietic cells derived from ES cells reconstitute hematopoiesis in vivo and may become an alternative donor source for bone marrow transplantation.

Abnormal killer inhibitory receptor expression on natural killer cells in patients with Behçet's disease

Viral infection has been assigned some role in the pathogenesis of Behçets disease (BD). Defects in natural killer (NK) cell repertoire may be involved in impaired antiviral immunity, leading to the development of BD. We studied killer inhibitory receptor (KIR) expression in 40 patients with BD. CD94 and CD158b expression of NK cells was normal in a great majority of BD patients. NKB1 expression was reduced in eight and increased in six. Twelve of these 14 patients (86%) had severe eye disease. Some had reduced NKB1 and enhanced CD158a expression simultaneously, or enhanced NKB1 and reduced CD158a simultaneously, suggesting a skewed NK cell repertoire in BD. Collectively, KIR expression was abnormal in the BD patients with severe eye disease. This may result from genetic predisposition, or certain viruses may affect the KIR repertoire formation in BD patients. Abnormal KIR expression of NK cells may be associated with the development of BD.

Lupus antibodies to the HMGB1 chromosomal protein: epitope mapping and association with disease activity

The high mobility group box 1 (HMGB1) protein is a non-histone chromosomal protein that acts as a potent proinflammatory cytokine when actively secreted from LPS- or TNF-activated macrophages, monocytes, and other cells. Anti-HMGB1/2 antibodies have been previously identified in sera from a high proportion of patients with autoimmune diseases. In this study, we examined anti-HMGB1 antibody titers in sera of patients with systemic rheumatic diseases and the correlations between the presence of anti-HMGB1 antibodies and disease activity in systemic lupus erythematosus (SLE) patients by enzyme-linked immunosorbent assay and western blotting. We detected increases in both the levels and the frequency of anti-HMGB1 antibodies in sera from SLE and polymyositis/dermatomyositis (PM/DM) patients, and observed that the presence of anti-HMGB1 antibodies positively correlates with SLE disease activity index. Through epitope mapping, we found that multiple HMGB1 epitopes were recognised in SLE sera, with the major epitope mapping to box A. Another epitope, the joiner region of HMGB1, was preferentially recognized by SLE sera, but not by PM/DM sera. Collectively, these observations suggest that the presence of anti-HMGB1 antibodies correlates with disease activity in SLE patients.

Comparison of phenotype and outcome in microscopic polyangiitis between europe and Japan.

Objective. There are differences between Europe and Japan in the incidence and antineutrophil cytoplasmic antibody (ANCA) serotype of patients with microscopic polyangiitis (MPA). However, differences in phenotype or outcome have not been explored. We aimed to identify differences in phenotype and outcome of MPA between Europe and Japan. Methods. Sequential cohorts of patients with MPA and renal limited vasculitis were collected from European and Japanese centers (n = 147 and n = 312, respectively). Trial databases from the European Vasculitis Society and the Japanese patients with Myeloperoxidase (MPO)-ANCA-Associated Vasculitis (JMAAV) trial were studied (n = 254 and n = 48, respectively). We evaluated baseline characteristics including ANCA status and organ involvement, treatment, survival, and renal survival. Differences in survival and renal survival were studied using multivariate analysis. Results. The non-trial cohorts showed patients with MPA in Japan had a higher age at onset, more frequent MPO-ANCA positivity, lower serum creatinine, and more frequent interstitial pneumonitis than those in Europe (all p < 0.01). Comparisons between the trial databases demonstrated similar results. Cumulative patient survival and renal survival rates were not different between Europe and Japan (p = 0.71 and p = 0.38, respectively). Multivariate analysis identified age at onset, serum creatinine, gastrointestinal, and respiratory involvement as factors with higher risk of death. For endstage renal failure, serum creatinine and use of plasma exchange were identified as factors with higher risk, and immunosuppressant use as lower risk factors. Conclusion. Phenotypes in patients with MPA were different between Europe and Japan. However, the outcomes of patient survival and renal survival were similar.

Involvement of simultaneous multiple transcription factor expression, including cAMP responsive element binding protein and OCT-1, for synovial cell outgrowth in patients with rheumatoid arthritis

OBJECTIVE To elucidate possible roles of several transcription factors in the pathogenesis of rheumatoid arthritis (RA), the transcription factor expression in RA synovial tissue and their contribution to RA synovial cell functions were studied. METHODS Single cell suspension of dissociated synovial tissue was cultured to induce in vitro tissue outgrowth of RA synovial cells. Transcription factors were immunohistochemically identified in RA synovial tissue obtained by joint surgery and in the in vitro tissue outgrowth, and confirmed by western blotting and gel shift assays. RESULTS Immunohistochemical examination of RA synovial tissue revealed simultaneous expression of various transcription factors (NF-κB, c-Jun (a component of AP-1), cAMP responsive element binding protein (CREB), and OCT-1). The same set of transcription factors was expressed in the in vitro tissue outgrowth of RA patients. The early passage RA synovial cells were treated with interleukin 1β (IL1β) and confirmed translocation of transcription factors into the nucleus by western blotting, and their DNA binding activity by gel shift assays. CONCLUSION This study emphasises the importance of the simultaneous expression of several transcription factors for the hyperactivity of RA synovial cells that leads to tissue outgrowth.