Toshihiro Utsugi

Perforin-independent beta-cell destruction by diabetogenic CD8(+) T lymphocytes in transgenic nonobese diabetic mice.

Autoimmune diabetes in nonobese diabetic (NOD) mice results from destruction of pancreatic beta cells by T lymphocytes. It is believed that CD8(+) cytotoxic T lymphocytes (CTLs) effect the initial beta-cell insult in diabetes, but the mechanisms remain unclear. Studies of NOD.lpr mice have suggested that disease initiation is a Fas-dependent process, yet perforin-deficient NOD mice rarely develop diabetes despite expressing Fas. Here, we have investigated the role of perforin and Fas in the ability of beta cell-reactive CD8(+) T cells bearing a T-cell receptor (8.3-TCR) that is representative of TCRs used by CD8(+) CTLs propagated from the earliest insulitic lesions of NOD mice, and that targets an immunodominant peptide/H-2Kd complex on beta cells, to effect beta-cell damage in vitro and in vivo. In vitro, 8.3-CTLs killed antigenic peptide-pulsed non-beta-cell targets via both perforin and Fas, but they killed NOD beta cells via Fas exclusively. Perforin-deficient 8.3-TCR-transgenic NOD mice expressing an oligoclonal or monoclonal T-cell repertoire developed diabetes even more frequently than their perforin-competent littermates. These results demonstrate that diabetogenic CD8(+) CTLs representative of CTLs putatively involved in the initiation of autoimmune diabetes kill beta cells in a Fas-dependent and perforin-independent manner.

Hypoxia Induces Transcription of the Plasminogen Activator Inhibitor-1 Gene Through Genistein-Sensitive Tyrosine Kinase Pathways in Vascular Endothelial Cells

A decline in oxygen concentration perturbs endothelial function, which promotes local thrombosis. In this study, we determined whether hypoxia in the range of that observed in pathophysiological hypoxic states stimulates plasminogen activator inhibitor-1 (PAI-1) production in bovine aortic endothelial cells. PAI-1 production, measured by ELISA, was increased by 4.7-fold (P<0.05 versus normoxic control, n=4) at 12 hours after hypoxic stimulation. Northern blot analysis showed the progressive time-dependent increase in the steady-state level of PAI-1 mRNA expression by hypoxia, which reached a 7.5-fold increase (P<0.05 versus control, n=4) at 12 hours. Deferoxamine, which has been known to bind heme protein and to reproduce the hypoxic response, induced PAI-1 production at both the mRNA and protein levels. The half-life of PAI-1 mRNA, as determined by a standard decay assay, was not affected by hypoxia, suggesting that induction of PAI-1 mRNA was regulated mainly at the transcriptional level. Transient transfection assays of the human PAI-1 promoter-luciferase construct indicates that a hypoxia-responsive region lies between -414 and -107 relative to the transcription start site, where no putative hypoxia response element is found. The hypoxia-mediated increase in PAI-1 mRNA levels was attenuated by the tyrosine kinase inhibitors genistein (50 micromol/L) and herbimycin A (1 micromol/L), whereas PD98059 (50 micromol/L, MEK1 inhibitor), SB203580 (10 micromol/L, p38 mitogen-activated protein kinase inhibitor), and calphostin C (1 micromol/L, protein kinase C inhibitor) had no effect on the induction of PAI-1 expression by hypoxia and deferoxamine. Genistein but not daidzein blocked the production of hypoxia- and deferoxamine-induced PAI-1 protein. Thus, we conclude that hypoxia stimulates PAI-1 gene transcription and protein production through a signaling pathway involving genistein-sensitive tyrosine kinases in vascular endothelial cells.

Major Histocompatibility Complex Class I–Restricted Infiltration and Destruction of Pancreatic Islets by NOD Mouse-Derived β-Cell Cytotoxic CD8+ T-Cell Clones In Vivo

NOD mouse-derived β-cell-specific cytotoxic T-cell (β-CTL) clones are diabetogenic in adult NOD mice, but only if co-injected with splenic CD4+ T-cells from diabetic animals. This investigation was initiated to determine whether infiltration of pancreatic islets by β-CTL is a major histocompatibility complex (MHC) class I-restricted response, and whether β-CTL has a direct cytopathic effect on β-cells in vivo. Pancreatic islets from BALB/c (H-2d) or B6 (H-2b) mice were transplanted under the renal capsule of streptozotocin (STZ)-induced diabetic (NOD × BALB/c) Fl (H-2Kd, H-2Dd,b) or (NOD × B6) Fl (H-2Kd,b, H-2Db) mice, respectively. H-2Kd-restricted β-CTL clones from NOD mice were transfused into euglycemic mice within 3 days after transplantation. In all of the H-2d islet-grafted (NOD × BALB/c) Fl mice that received the β-CTL clones, the β-CTLs homed into the grafts, recruited host Mac-1+ cells and CD4+ and CD8+ T-cells, and caused diabetes within 7 days. In contrast, none of the H-2b islet-grafted (NOD × B6) Fl mice who received the β-CTL clones and none of the H-2d islet-grafted (NOD × BALB/c) Fl mice who received a non-β-cell cytotoxic CTL clone (Nβ-CTL) developed graft inflammation or diabetes. Depletion of CD4+ T-cells in H-2d islet-grafted (NOD × BALB/c) Fl mice did not prevent β-CTL clone-induced diabetes but reduced its severity. In contrast, when the β-CTL clones were injected >8 days after transplantation, none of the H-2d islet-grafted (NOD × BALB/c) Fl mice became diabetic or developed graft inflammation. We conclude that (1) isletderived β-CTLs can destroy β-cells in vivo; (2) infiltration of grafted islets by β-CTLs is an MHC class I-restricted response; (3) β-CTLs can recruit naive CD4+ T-cells to the site, leading to further β-cell damage; and (4) revascularized islet grafts are, like pancreatic islets of irradiated adult NOD mice, “sequestered” from circulating β-CTLs.

Clinical significance of neutrophil apoptosis in peripheral blood of patients with type 2 diabetes mellitus.

UNLABELLED Although neutrophils are essential components of the natural immune system, they have also been implicated in the pathogenesis of tissue injuries. We assessed the clinical significance of neutrophil apoptosis in the peripheral blood of patients with type 2 diabetes mellitus (T2DM). PATIENTS AND METHODS The study included 52 patients with T2DM (30 men, 22 women). Control subjects were 16 healthy volunteers without diabetes (7 men, 9 women). Neutrophil apoptosis levels were measured active caspase-3 positive rate by flow cytometry. RESULTS The mean rate of neutrophil apoptosis in patients with T2DM was 15.0% (95% confidence interval [CI]: 9.5% approximately 20.5%), while that in the control group was 5.8% (95% CI: 1.6% approximately 10.0%). There were significant negative correlations between neutrophil apoptosis rate and hemoglobin (Hb) A1c levels (r = -0.352, P < .01). The mean rate of neutrophil apoptosis in the patient group with the 3 major complications (diabetic retinopathy, nephropathy, and neuropathy) was 11.1% (95% CI: 5.5%-16.7%, n = 36) and that of another group without complications was 23.8% (95% CI: 11.4%-36.2%, n = 16). There was a significant difference between these 2 groups (P < .05). CONCLUSIONS The neutrophil apoptosis rate in patients with T2DM was significantly correlated with HbA1C levels. The mean rate of neutrophil apoptosis in the patient group with 3 major diabetic complications remained lower than that in another patient group without complications. The inhibition of neutrophil apoptosis by chronic hyperglycemia is thought to promote tissue injury and to enhance the risk of microangiopathy.

Prevention of recurrent diabetes in syngenic islet-transplanted NOD mice by transfusion of autoreactive T lymphocytes.

Islet transplantation has been considered to be one of the best methods for the cure of type I diabetes, but transplanted islets are eventually destroyed by the hosts cell-mediated autoimmune responses unless immunosuppressive agents are given. This investigation was initiated to develop a method for the prevention of beta cell destruction in transplanted islets without the use of immunosuppressive drugs. We have recently cloned CD4+ autoreactive T cells (NY1.1 and NY4.2) from lymphocytes infiltrating the pancreatic islets of nonobese diabetic (NOD*) mice and have shown that these cells respond to self MHC class II determinants. When the T cell clones (107 cells of either NY1.1 or NY4.2) were transfused into acutely diabetic NOD mice 2 to 3 days before transplantation of syngenic islets (400) into the subrenal space, the transplanted islets were not destroyed, and the animals maintained normoglycemia over 100 days without insulin treatment. In contrast, NOD mice that received syngenic islets (400) without the transfusion of an autoreactive T cell clone showed a recurrence of diabetes and massive mononuclear cell infiltration of the grafted islets within 17 days. On the basis of these observations, it is concluded that CD4+ autoreactive T lymphocytes can prevent the recurrence of insulitis and development of diabetes in pancreatic islet-transplanted NOD mice, without treatment with immunosuppressive drugs.