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T Kobata

Publications and source records attributed to T Kobata.

At least 55 records · Page 3Linked to original sources
PubMed1998

Induction of apoptosis in the rheumatoid synovium by Fas ligand gene transfer.

We have recently reported that local administration of anti-Fas monoclonal antibody (MAb) in human T cell leukemia virus type 1 (HTLV-1) carrying mice improved arthritis due to the induction of apoptosis. This finding strongly indicated the beneficial therapeutic effect of Fas-mediated apoptosis in rheumatoid arthritis (RA). To establish further the therapeutic effect of Fas-mediated apoptosis on RA taking into consideration safety and practicality, we investigated the effect of cells transfected with human Fas ligand (hFasL) gene on proliferating human rheumatoid synovium engrafted in severe combined immunodeficiency (SCID-RA) mice. The hFasL transfectants exhibited cytotoxic activity against RA synoviocytes via the Fas/FasL system in vitro. Histopathological and immunohistochemical studies showed that local injection of irradiated-hFasL transfectants eliminated synoviocytes and mononuclear cells in engrafted human rheumatoid synovium of SCID-RA mice. Furthermore, in situ nick and labeling analysis confirmed that the cells in engrafted synovium frequently underwent apoptosis by irradiated-hFasL transfectants. Our results clearly demonstrated that hFasL transfectants induced apoptosis by cell-to-cell interaction via the Fas/FasL system. Thus, ex vivo gene transfer of FasL may represent a novel therapeutic strategy for RA.

Animals↗
PubMed1998

Characterization of murine CD70 by molecular cloning and mAb.

CD27, a member of the tumor necrosis factor (TNF) receptor family, has been implicated in T cell activation, T cell development and T-dependent antibody production by B cells. Its ligand CD70 has been identified only in humans, and, thus, physiological and pathological roles of the CD70-CD27 interaction remain to be determined in an experimental animal system. In the present study, we identified murine (m) CD70 by molecular cloning, and characterized its expression and function by generating an anti-mCD70 mAb. The mCD70 cDNA encoded a type II transmembrane glycoprotein of the TNF family, having 56.5% identity to the human CD70 amino acid sequence. The mCd70 gene was assigned in the central region of chromosome 17. To explore the expression and function of mCD70, we generated cDNA transfectants and anti-mCD70 mAb (FR70), which inhibited binding of a murine CD27-Fc fusion protein (mCD27-Ig) to mCD70 transfectants. FR70, as well as mCD27-Ig, immunoprecipitated a 30-33 kDa surface protein from A20 and mCD70-P815 cells but not from P815 cells. The mCD70 transfectants exhibited a potent co-stimulatory activity for anti-CD3-stimulated T cell proliferation, which was blocked by FR70 far more efficiently than mCD27-Ig. FR70 also abrogated the CD28-independent co-stimulatory activity of A20 cells. The expression of mCD70 was detected on splenic T cells after stimulation with anti-CD3 and anti-CD28 mAb, and on splenic B cells after stimulation with anti-CD40 mAb. Cross-linking of surface Ig by anti-IgM mAb did not induce the mCD70 expression but enhanced the anti-CD40-induced mCD70 expression on splenic B cells. These results suggest a contribution of CD70 to murine T-B cognate interaction as proposed in the human system. FR70 will be useful for further investigating the physiological and pathological roles of the CD70-CD27 interaction in T cell development, T-dependent antibody production and various disease models in the murine system.

Amino Acid Sequence↗
PubMed1998

Deletion of signaling molecule genes resembling the cytoplasmic domain of Igbeta in autoimmune-prone mice.

The P8.6 gene is encoded upstream of the mouse TCR Valpha1 gene in the anti-sense strand and its gene product contains the proline-rich region and tyrosine-isoleucine (Y-I) motif, which are consensus sequences for the SH2 and SH3 binding motifs respectively. It was found that this protein is highly homologous to Igbeta. We also found that the P8.6 protein associates with a 170 kDa phosphorylated protein in vivo. To examine the polymorphism of the P8.6 gene, we carried out a single-strand conformation polymorphism analysis and found that the P8.6 gene comprises a family of at least 13 independent genes arising from single or multiple mutations. The mutated P8.6 gene with the Y-L motif was deleted, especially in NZW and BXSB mice, whereas normal BALB/c mice have a P8.6 gene bearing both the Y-I and Y-L motifs, suggesting a dysregulation in signaling through the B cell receptor or TCR in these two autoimmune mice. Functional analysis using transfectant cells carrying P8.6-1 (Y-I motif) and P8.6-3 (Y-L motif) clearly demonstrated that P8.6-3 gene inhibited the signal transduction upon the stimulation of ionomycin and cell growth. The TANYSNI sequence has been proposed as the immunoreceptor tyrosine-based inhibitory motif, this motif is replaced by AANYSNI in the NZW mice. In conclusion, some P8.6 are deleted in particular autoimmune-prone mice.

Amino Acid Sequence↗
PubMed1997

CD154/CD40 and CD70/CD27 interactions have different and sequential functions in T cell-dependent B cell responses: enhancement of plasma cell differentiation by CD27 signaling.

CD40, a TNF receptor family member, plays a central role in T cell-mediated B cell activation. We have recently demonstrated that CD27, another TNF receptor family member, was also involved in B cell regulation and enhanced Ig production. In this report we compare CD27 and CD40 signals in B cell function. We selectively mimicked the effect of T cell help by addition to peripheral blood B cells activated with Staphylococcus aureus Cowan I strain and IL-2 of irradiated 300-19 cells transfected with either the CD70 (CD27 ligand) gene or the CD154 (CD40 ligand) gene, the vector alone, or both CD70 and CD154 genes. CD27 ligation induced only a slight increase in B cell proliferation compared with the dramatic enhancement induced by CD40 ligation; double ligation proved to be less efficient than CD40 ligation alone. In contrast, IgG production was increased only by CD27 ligation alone. Moreover, the CD27 signal was more efficient when it was given on day 2 of the culture rather than on day 0. Phenotypic analysis of the activated cells showed that CD27 ligation increased the percentage of cells showing a plasma cell profile (CD19-, CD38+), whereas upon CD40 ligation most of the cells still had a germinal center-like phenotype (CD19+, CD38+). Our results suggest that the CD27 and CD40 signals are not synergistic but, rather, are complementary and involve distinct steps of T cell-dependent B cell activation. CD27 may be more important in the induction of plasma cell differentiation at a time when the expansion phase has already occurred.

B-Lymphocytes↗
PubMed1997

CD27/CD70 interaction contributes to the activation and the function of human autoreactive CD27+ regulatory T cells.

CD27, a tumor necrosis factor receptor family member, is a constimulatory molecule for T and B cell activation. We demonstrate here that CD27 signaling is critical for T cell activation in the autologous mixed lymphocyte reaction (aMLR) and for aMLR-induced generation of regulatory T cells that suppress pokeweed mitogen-driven immunoglobulin G synthesis by B cells. Moreover, CD27, expressed on CD45RA+ CD4+ T cells, is directly involved in the suppressor function of aMLR-activated CD4+ T cells, probably by interfering with the interaction between CD70, the CD27 ligand, expressed on CD45RO+ CD4+ helper T cells, and CD27, expressed on B cells. CD8+ T cells, which are required in this system to obtain suppression, on the other hand, do not need CD27 engagement to exert their suppressor function.

Antigens, CD↗
PubMed1997

Accumulation of soluble Fas in inflamed joints of patients with rheumatoid arthritis.

OBJECTIVE: To examine the concentration of the soluble form of the Fas molecule (sFas) in the serum and synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). METHODS: The concentration of sFas in the serum of 15 normal subjects and in the synovial fluid and serum of 45 RA patients and 13 OA patients was determined. The erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, and the level of several cytokines in serum and synovial fluid were also determined. RESULTS: The synovial fluid concentration of sFas was higher in RA than in OA patients (P < 0.005). The synovial fluid level of sFas correlated weakly with serum levels of CRP (r = 0.541), the ESR (r = 0.499), and with synovial fluid levels of interleukin-2 (IL-2) receptor (r = 0.544), IL-6 (r = -0.529), and intercellular adhesion molecule 1 (r = 0.514). Reverse transcription-polymerase chain reaction analysis revealed that synovial cells and infiltrating mononuclear cells expressed sFas messenger RNA in RA patients. CONCLUSION: Our data suggest that accumulation of sFas in the joint cavity of RA patients may inhibit apoptosis and exacerbate the inflammatory process.

Aged↗
PubMed1997

Direct evidence of high DNA binding activity of transcription factor AP-1 in rheumatoid arthritis synovium.

OBJECTIVE: To investigate the possible activation of transcription factor AP-1 in rheumatoid arthritis (RA) and its involvement in the pathogenesis of RA. METHODS: Synovial tissues and peripheral blood samples were obtained from 25 patients with RA and 5 patients with osteoarthritis (OA) during arthroplasty and synovectomy. The synovial tissue was digested with collagenase and separated into adherent and nonadherent cells by plastic-adhesion methods. Nuclear extracts obtained from each sample were examined by electrophoretic mobility shift assay to determine the DNA binding activity of AP-1. The expression of c-fos and c-jun messenger RNA (mRNA) was examined by in situ reverse transcription assay. RESULTS: A markedly high DNA binding activity of AP-1 was detected in the synovial tissues of RA patients, while virtually no activity or only a little activity was observed in OA patients. Following separation of adherent and nonadherent cells, the AP-1 activity was mainly detected in adherent cells, which consisted of synovial cells and macrophages. However, the activity was significantly higher in the mononuclear cells infiltrating into RA synovium than in RA peripheral blood mononuclear cells. The high DNA binding activity of AP-1 in RA correlated with the expression of c-fos and c-jun mRNA in situ. Furthermore, AP-1 binding activity also correlated with disease activity. CONCLUSION: In RA synovium, AP-1 DNA binding activity was constitutively up-regulated. These findings suggest that AP-1 may play an important role in the pathogenesis of RA, including synovial hyperplasia and abnormal immune responses.

Aged↗
PubMed1997

Selective activation of the JNK/AP-1 pathway in Fas-mediated apoptosis of rheumatoid arthritis synoviocytes.

OBJECTIVE: To evaluate the Fas-dependent signaling pathway, we examined the involvement of protein tyrosine phosphorylation and the DNA binding activity of AP-1 in rheumatoid arthritis (RA) cultured synovial cells. METHODS: The number of dead cells was counted after treatment with anti-Fas antibody in the presence of protein tyrosine kinase or phosphatase inhibitor. Protein tyrosine phosphorylation in synoviocytes after Fas ligation was examined by immunoblot and immunoprecipitation analyses. The DNA binding activity of AP-1 was examined by electrophoretic mobility shift assay. RESULTS: Treatment with the protein tyrosine phosphatase inhibitor, orthovanadate, significantly enhanced the apoptosis of RA synoviocytes after Fas ligation. Ligation of the Fas molecule on RA synoviocytes induced a rapid tyrosine phosphorylation of JNK (c-Jun amino-terminal kinase) and formation of the AP-1 transcription factor. CONCLUSION: Our results strongly suggest that the JNK/AP-1 signaling pathway is activated during the process of Fas-mediated apoptosis of RA synovial cells.

Apoptosis↗
PubMed1997

B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B cells and helper T cells in immunoglobulin production.

B cell immunoglobulin production is regulated by helper T cells through direct interaction and secreted cytokines. In the present study, we functionally analyzed CD27 in cord and peripheral blood B cells. Adult peripheral blood B cells were separated into CD27+ and CD27- cells, which differed in their morphology. Cord blood B cells did not express CD27, and CD27 expression on peripheral blood B cells increased with age. Only CD27+ B cells had the ability to produce immunoglobulin, which was increased by contact with a tumor necrosis factor-related transmembrane ligand, CD70. Adult peripheral blood CD27+ B cells can be further subdivided into two discrete subtypes: IgD- CD27+ and IgD+ CD27+ B cells. IgD- CD27+ B cells produce IgG, IgM and IgA, whereas IgD+ CD27+ B cells predominantly produce IgM. The addition of activated CD4+ CD45RO T cells expressing CD70 caused down-regulation of CD27 expression on activated B cells, and this down-modulation was completely blocked by anti-CD70 monoclonal antibody, indicating direct T-B cell contact via CD27/CD70. The triggering via CD27 and CD40 additively increased the immunoglobulin production under Staphylococcus aureus Cowan strain plus interleukin-2 stimulation. Taken together, our findings demonstrate that peripheral blood B cells are separated into subpopulations by CD27 and IgD expression and that CD27+ B cells produce large amounts of immunoglobulin by interaction with the CD70 molecule.

Adult↗
PubMed1997

Apoptosis with FasL+ cell infiltration in the periphery and thymus of corrected autoimmune mice.

Fas (CD95) ligand (L) is a death factor that binds to its receptor, Fas, and induces apoptotic cell death, a crucial process in immunological tolerance. gld (generalized lymphoproliferative disorder) mice, which have a point mutation in the FasL gene, develop spontaneous systemic autoimmune syndromes characterized by hypergammaglobulinaemia and lymphoid hyperplasia owing to accumulation of abnormal B220+ CD3+ cells. Transplantation of wild-type (wt) bone marrow cells into old gld mice on the same strain background results in normalization of autoimmune syndromes. We characterized the cellular mechanisms (functionally and histologically) of the above phenomena in gld mice after bone marrow transplantation (BMT) to determine the role of apoptosis via Fas/FasL interactions in inducing and maintaining self-tolerance in vivo. Activated splenocytes from wt and BMT (wt to gld) mice showed significant cytotoxic activity against Fas transfectant cells while those from BMT (gld to gld) mice did not. Cells in the thymus, spleen and lymph nodes of gld mice uniformly upregulated Fas expression and were sensitive to Fas-mediated apoptosis compared with those in wt mice. Cells sensitive to Fas-mediated apoptosis in gld mice resided not only among abnormal B220+ CD3+ cells but also among conventional lymphocytes. More importantly, histological analysis revealed that cells in the spleen, lymph nodes and thymus frequently underwent apoptosis with infiltration of FasL+ cells in BMT (wt to gld) mice compared with BMT (gld to gld) mice. Our results indicated that apoptosis via Fas/FasL interactions can directly eliminate pathogenic cells responsible for autoimmunity in the periphery and possibly in the thymus in vivo.

Animals↗
PubMed1997

Long-term persistent accumulation of CD8+ T cells in synovial fluid of rheumatoid arthritis.

OBJECTIVE: To characterise the type and kinetics of T cell clones in synovial lesions of patients with rheumatoid arthritis (RA). METHODS: Mononuclear cells from serial samples of synovial fluid (SF) and peripheral blood from nine RA patients were separated phenotypically using antibody coated magnetic beads. After mRNA preparation, reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify V-D(N)-J (that is, the third complementarity determining, CDR3) regions of their T cell receptor beta chain genes. This was followed by single strand conformation polymorphism (SSCP) analysis to detect the clonotypes of accumulating T cells. Amino acid sequences of the dominant clones were also determined. RESULTS: Although peripheral T cells were heterogeneous, accumulation of oligoclonal T cells was detected in SF. The predominant accumulating clone was the CD8 subset, which was persistently present in serial samples obtained over almost one year of follow up. A proportion of these cells expressed CD25 or CD45RO, or both, suggesting they are 'memory' clones. CONCLUSION: The persistent presence of CD8+ T cell clones in RA joints indicates that they may be involved in the perpetuation of the chronic inflammatory process in RA joints.

Adult↗
PubMed1997

In situ expression of protooncogenes and Fas/Fas ligand in rheumatoid arthritis synovium.

OBJECTIVE: To examine the relationship among the expression of protooncogenes such as c-fos and c-myc, Fas antigen, Fas ligand, and apoptosis in the synovial tissue of patients with rheumatoid arthritis (RA). METHODS: The expression of c-fos, c-myc, Fas antigen, and Fas ligand was examined in synovial tissues of 6 patients with RA and 4 with osteoarthritis (OA) using in situ reverse transcriptase (RT) assay and immunohistochemical staining. Apoptosis was detected by TUNEL method in situ. RESULTS: Expression of protooncogenes, c-fos, and c-myc was detected in all samples from patients with RA, but in only a few cells of OA synovium. 30 to 90% of cells in RA synovium positive for these protooncogenes also coexpressed Fas antigen. Fas positive cells in RA synovium underwent apoptosis to a significant degree. Fas ligand mRNA was detected only in mononuclear cells in RA synovium. CONCLUSION: The expression of protooncogenes is closely related to Fas mediated apoptosis in RA synoviocytes.

Aged↗
PubMed1996

Fas-mediated stimulation induces IL-8 secretion by rheumatoid arthritis synoviocytes independently of CPP32-mediated apoptosis.

In this study, we investigated the IL-1 beta converting enzyme (ICE) family cysteine proteases responsible for the Fas-mediated apoptosis of rheumatoid arthritis (RA) synoviocytes and their involvement in proinflammatory cytokine production. CPP32 inhibitor, but not ICE inhibitor, was capable of inhibiting the Fas-mediated apoptosis of RA synovial cells. CPP32, but not ICE, was activated in response to anti-Fas stimulation. IL-8, but not IL-1 beta, was secreted from the anti-Fas-stimulated RA synoviocytes even in the presence of CPP32 inhibitor. These results demonstrated that CPP32, but not ICE, is the predominant cysteine protease that mediates the Fas-mediated apoptosis of RA synovial cells. We also demonstrated that anti-Fas stimulation of RA synoviocytes leads to IL-8 secretion independently of the CPP32-mediated apoptosis, which would accelerate inflammation.

Apoptosis↗
PubMed1996

Therapeutic effect of the anti-Fas antibody on arthritis in HTLV-1 tax transgenic mice.

We have recently demonstrated Fas-mediated apoptosis in the synovium, of patients with rheumatoid arthritis (RA) and suggested that it may be one factor responsible for the regression of RA. To examine whether the induction of apoptosis caused by anti-Fas mAb may play a potential role as a new therapeutic strategy for RA, we investigated the effect of anti-Fas mAb (RK-8) on synovitis in an animal model of RA, the human T cell leukemia virus type I (HTLV-1) tax transgenic mice. We report here that administration of anti-Fas mAb into mice intra-articularly improved the paw swelling and arthritis within 48 h. Immunohistochemical study and in vitro culture studies showed that 35% of synovial fibroblasts, 75% of mononuclear cells, and some of polymorphonuclear leukocytes infiltrating in synovium underwent apoptosis by anti-Fas mAb. In situ nick end labeling analysis and electron microscope analysis clearly showed that many cells in synovium were induced apoptosis by anti-Fas mAb administration. However, local administration of anti-Fas mAb did not produce systemic side effects. Results demonstrated that administration of anti-Fas mAb in arthritic joints of the HTLV-1 tax transgenic mice produced improvement of arthritis. These findings suggest that local administration of anti-Fas mAb may represent a useful therapeutic strategy for proliferative synovitis such as RA.

Alanine Transaminase↗
PubMed1996

Time course analysis of alpha+ beta+ T cell clones during normal pregnancy.

During normal pregnancy, the fetus continues to mature inside the uterus without rejection. Inherited paternal antigens could be targeted by the maternal immune system. These reactions are believed to play a role in a number of habitual abortions. However, the precise maternal mechanisms preventing fetal tissue rejection are not well understood. Maternal T cells should recognize fetal antigens, so it is conceivable that antigen-specific T cell response to fetal antigens would occur by proliferation and accumulation of certain T cell clones in the pregnant mother. To elucidate the maternal immune response to the fetus we investigated the clonality of expanded T cells in peripheral blood lymphocytes in ten normal pregnant women. We employed reverse transcriptase-polymerase chain reaction for T cell receptor beta chain gene and subsequently analyzed the PCR product by single-strand conformation polymorphism analysis. A large number of distinctly expanded T cell clones were detected during pregnancy. These accumulations were observed as early as the ninth to tenth week post-conception and reached a maximum during the second trimester, suggesting the existence of dynamic antigen-specific T cell responses in the pregnant mother. However, after the 30th week of gestation, nearly all expanded T cell clones disappeared before parturition and the degree of clonality reached almost normal levels. Our results clearly indicate the existence of dynamic maternal T cell responses during pregnancy.

Adult↗
PubMed1996

Expression of Fas antigen and Fas ligand in the rheumatoid synovial tissue.

To understand the role of apoptosis through Fas/Fas ligand (Fas-L) interaction in the pathogenesis of rheumatoid arthritis (RA), we examined the expression of Fas antigen, Fas-L, and apoptosis in synovial tissue obtained from eight patients with RA and five patients with osteoarthritis (OA). Immunohistochemical staining demonstrated the significant expression of Fas antigen and Fas-L in RA synovial tissue compared with that in OA synovial tissue. Immunohistochemical staining and the DNA nick end labeling (TUNEL) method were combined and revealed that approximately 10 to 30% of Fas antigen-expressing cells in the RA synovium showed DNA fragmentation characteristic for apoptosis. In double-staining analysis, Fas-L was expressed on up to 10% of CD45RO-, CD4-, CD8-, or CD56-positive mononuclear cells in RA synovial tissue. Our results suggest that activated T cells and natural killer cells infiltrating into the RA synovium may contribute to the induction of apoptosis of RA synovial and mononuclear cells through Fas/Fas-L interaction.

Aged↗
PubMed1996

Clonal dominance of human autologous cytotoxic T lymphocytes against gastric carcinoma: molecular stability of the CDR3 structure of the TCR alphabeta gene.

In our previous study, RT-PCR suggested that cytotoxic T lymphocyte (CTL) clones may specifically recognize human autologous gastric signet ring cell tumor (HST2) by using TCR products of Valpha7 and Vbeta20 subfamilies. In this report, we first determined the TCR nucleotide sequences of one such CTL from patient's peripheral blood lymphocytes (PBL), the PBL were newly stimulated with a mixed lymphocyte-autologus tumor cell (HST2) culture (MLTC) and cytotoxic T cell lines, such as HPBL3x, were obtained. RT-PCR and the nucleotide sequence data indicated that HPBL3x also showed TCR Valpha7 and Vbeta transcripts, and that HPBL3x TCR was composed of the exact same CDR3 gene structures as those of the TcHLT2 clone. T cells with same TCR structures were also detected in patient's non-treated peripheral blood, although they were infrequent. These data indicated that functional cytotoxic T cells with these distinct CDR3 equivalent structures were the dominant effector cells against HST2 autologous tumor cells. Moreover, the highly dominant and reproducible clonal expansion of T cells bearing heterodimeric TCR with identical variable, N diversity and constant region structures suggest that the molecular nature of governing antigenic peptide to TcHDT2 may be stable and perhaps immunologically dominant in the interaction between CTL and HST2 autologous tumor cells.

Amino Acid Sequence↗