Kazuhiko Amano

Evidence for Initial Involvement of Macrophage in Development of Insulitis in NOD Mice

Macrophages have been shown to be the major population of infiltrated immunocytes at the early stage of insulitis in diabetes-prone BB rats. This study was undertaken to investigate the role of macrophages in the development of insulitis in nonobese diabetic (NOD) mice. Administration of cyclophosphamide to NOD mice resulted in a significant increase in the incidence of overt diabetes and severity of insulitis compared with that in untreated NOD mice. Intraperitoneal injections of silica completely prevented the development of diabetes and insulitis in both cyclophosphamide-treated and untreated animals. Because silica is selectively toxic to macrophages, the results suggest that macrophages play an important role in the initiation of insulitis in NOD mice.

Preferential Infiltration of Macrophages During Early Stages of Insulitis in Diabetes-Prone BB Rats

The subpopulation of lymphoid cells at the different stages of insulitis in BB rats was determined by immunohistochemical techniques with various monoclonal antibodies, including the recently developed OX41, which distinguishes macrophages from T-lymphocytes, OX19 for pan–T-lymphocytes, W3/25 for both helper T-lymphocytes and macrophages, OX8 for cytotoxic T-lymphocytes and natural killer cells, and OX12 for B-lymphocytes. The major population of infiltrated cells found during the early stages of insulitis appeared to be macrophages. This preceded invasion by a mixed population of cells, including both T- and B-lymphocytes and/or natural killer cells. The preferential infiltration of macrophages during the early stages of insulitis strongly suggested that there might be an initial change in the target β-cells that precedes their immune destruction, although the amplification of immune response by activated T-lymphocytes and natural killer cells at a later stage seemed to be required for the clinical expression of the disease.

Expression of intercellular adhesion molecule 1 on pancreatic beta-cells accelerates beta-cell destruction by cytotoxic T-cells in murine autoimmune diabetes

Intercellular adhesion molecule 1 (ICAM-1) plays an important role in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) by being involved in the extravasation of lymphocytes from the circulation into the inflamed pancreas. However, the mechanism of β-cell destruction by which expression of ICAM-1 on β-cells may facilitate adhesion of effector cells still remains to be elucidated. Several lines of evidence suggest that this adhesion molecule is involved in the destruction of pancreatic β-cells by killer lymphocytes in the NOD mouse, which shows an autoimmune diabetic syndrome similar to that of human IDDM. Immunohistochemical study under light microscopy demonstrated that all of the mononuclear cells infiltrating the islets strongly expressed ICAM-1 and leukocyte function-associated antigen 1 (LFA-1), a counterreceptor of ICAM-1, whereas ICAM-1 expression on islet cells was not apparent. However, immunohistochemical staining under electron microscopy revealed that islet β-cells adjacent to infiltrating lymphocytes were clearly stained by an anti-ICAM-1 monoclonal antibody (mAb). Flow cytometric analysis showed that the ICAM-1 expression on NOD islet cells and NOD-derived insulinoma cells (MIN6N8a) was inducible by interferon (IFN)-γ or tumor necrosis factor-α. These cytokines had an additive effect on the ICAM-1 induction. Susceptibility of MIN6N8a cells to lysis by a NOD islet-derived CD8+ cytotoxic T-cell clone was greatly enhanced by IFN-γ pretreatment, and this enhancement was abolished by anti-ICAM-1 and anti-LFA-1 mAbs. When both mAbs were administered into NOD mice with spontaneous or adoptively transferred diabetes, the development of diabetes was significantly prevented. These results suggest that cytokines secreted by isletinfiltrating mononuclear cells could induce the ICAM-1 expression on γ-cells, which accelerates the γ-cell destruction by cytotoxic T-cells. Therefore, immunointervention of the ICAM-l/LFA-1 pathway would be an excellent strategy to prevent human IDDM.

Studies on Autoimmunity For Initiation of β-Cell Destruction: V. Decrease of Macrophage-Dependent T Lymphocytes and Natural Killer Cytotoxicity in Silica-Treated BB Rats

Administration of silica, which is selectively toxic to macrophages, to young BB rats resulted in the prevention of insulitis and diabetes. However, the mechanism leading to the prevention of an autoimmune process in silica-treated BB rats is not known. This study was undertaken to investigate the mechanism involved in prevention of insulitis and diabetes. Neonates of diabetes-prone BB (DPBB) rats injected with concanavalin A (ConA)-activated spleen cells from silica-treated DPBB (STDPBB) rats did not develop insulitis or diabetes, whereas DPBB neonates injected with ConA-activated spleen cells from the untreated DPBB rats developed both insulitis and diabetes. Not only was there a decrease of natural killer (NK) cell activity in splenocytes from STDPBB rats, but there was also a significant reduction in the number of immunocytes such as T lymphocytes (helper/inducer and cytotoxic/suppressor) and NK cells. The number of macrophages in both spleen and peripheral blood was significantly decreased in STDP rats compared with untreated DP rats. In contrast to the changes in T lymphocytes and NK cell activity, there was no change in target β-cells in STDPBB rats with regard to the susceptibility to adoptive transfer of insulitis. It is concluded that the prevention of insulitis and diabetes in STDPBB rats is due to a decrease in macrophage-dependent T lymphocytes and NK cell cytotoxicity.

CD8 cytotoxic T-cell clone rapidly transfers autoimmune diabetes in very young NOD and MHC class I-compatible scid mice.

Summary A CD8 T-cell clone (YNK1.3) generated from acutely diabetic NOD mouse islets, showed proliferation and cytotoxicity when challenged with NOD and BALB/c islet cells and NOD-derived insulinoma cells. When 1–2 × 107 YNK1.3 cells were administered to 7–10-day-old NOD mice, the cells transferred overt diabetes very rapidly in each of the 16 recipients within 4 days of cell transfer. However, of 14 recipients receiving YNK 1.3 cells above 14 days of age none became diabetic. Fluorescent dye-labelled YNK1.3 cells extensively accumulated in the islets by 36 h after transfer in 7-day-old NOD recipients, while no significant insulitis was seen in 21-day-old recipients. Over half of NOD-scid recipients (5/9) rapidly became diabetic within 5 days after transfer of 1–2 × 107 YNK1.3 cells at 7 days of age, whereas only one of 12 recipients over 14 days of age became diabetic. Furthermore, YNK1.3 cells also transferred diabetes to H-2Kd-matched very young BALB/c-scid and CB17-scid mice, but not to C57BL/6-scid mice. Thus, optimally activated islet-specific CD8 T-cell clones are able to rapidly transfer diabetes to NOD and MHC class I compatible scid mice when a large enough number is administered at 7 days of age. Administration of monoclonal antibodies against adhesion molecules involved in the trafficking of lymphocytes from the circulation into the inflammatory tissues, could not prevent the cellular infiltration of YNK1.3 cells into the islets in 7-day-old NOD recipients. The results indicate that islet cells in the mouse around 7 days of age are generally susceptible to cytotoxic CD8 T cells, suggesting, therefore, that CD8 T cells may play an important role in the initiation of autoimmune diabetes in NOD mice. [Diabetologia (1997) 40: 1044–1052]

Insulin-degrading enzyme is capable of degrading receptor-bound insulin

In the investigation of the intracellular sites of insulin degradation, it might be important whether receptor-bound insulin could be a substrate for insulin-degrading enzyme (IDE). Insulin receptor and IDE were purified from rat liver using a wheat germ agglutinin column and monoclonal anti-IDE antibody affinity column, respectively. [125I]insulin-receptor complex was incubated with various amounts of IDE at 0 degree C in the presence of disuccinimidyl suberate and analyzed by reduced 7.5% SDS-PAGE and autoradiography. With increasing amounts of IDE, the radioactivity of 135 kd band (insulin receptor alpha-subunit) decreased, whereas that of 110 kd band (IDE) appeared then gradually increased, suggesting that IDE could bind to receptor-bound insulin. During incubation of insulin-receptor complex with IDE at 37 degrees C, about half of the [125I]insulin was dissociated from the complex. However, the time course of [125I]insulin degradation in this incubation was essentially identical to that of free [125I]insulin degradation. Cross-linked, non-dissociable receptor-bound [125I]insulin was also degraded by IDE. Rebinding studies to IM-9 cells showed that the receptor binding activity of dissociated [125I]insulin from insulin-receptor complex incubated with IDE was significantly (p less than 0.001) decreased as compared with that without the enzyme. These results, therefore, show that IDE could recognize and degrade receptor-bound insulin, and suggest that IDE may be involved in insulin metabolism during receptor-mediated endocytosis through the degradation of receptor-bound insulin in early neutral vesicles before their internal pH is acidified.

Identification of Antigenic Differences Between the Diabetogenic and Non-diabetogenic Variants of Encephalomyocarditis Virus Using Monoclonal Antibodies

The M variant of encephalomyocarditis (EMC) virus consists of two biologically distinct variants: one, diabetogenic D variant (EMC-D) and the other, non-diabetogenic B variant (EMC-B). These two variants cannot be distinguished by hyperimmune sera. Monoclonal antibodies were generated against EMC-D or EMC-B to identify antigenic differences between these two variants. Fourteen independent hybrid cell lines, selected from seven separate fusions of mouse myeloma cells to spleen cells isolated from mice immunized with EMC-D, consisted of 12 hybrids which produced monoclonal antibodies that neutralized both EMC-D and EMC-B, and two hybrids (ED-HJ-23 and ED-HJ-31) which produced monoclonal antibodies that neutralized EMC-D but not EMC-B. Similarly, 16 independent hybrid cell lines, selected from eight separate fusions using spleen cells prepared from mice immunized with EMC-B, consisted of 15 hybrids which produced monoclonal antibodies neutralizing both EMC-D and EMC-B, and one hybrid (EB-48A-F1) which produced antibody that neutralized EMC-B, but not EMC-D. The specificities of these monoclonal antibodies (ED-HJ-23, ED-HJ-31, EB-48A-F1) were further confirmed using an immunofluorescent technique. The D variant-specific monoclonal antibodies reacted with cells infected with EMC-D but not EMC-B. In contrast, the B variant-specific monoclonal antibody reacted with the cells infected with EMC-B but not EMC-D. It is concluded that the EMC-D- and EMC-B-specific monoclonal antibodies are able to identify antigenic differences between diabetogenic and non-diabetogenic variants of EMC virus which cannot be distinguished by hyperimmune sera.

A novel function of islet-derived CD8+T cells in initiating and developing autoimmune insulin-dependent diabetes mellitus in non-obese diabetic (NOD) mice

Accumulated studies revealed that CD4+T cells were initially required for diabetes in NOD mice, whereas interaction of CD4+T/CD8+T cells is not fully understood. To address this question, we established islet-derived CD4+T cells and CD8+T cells from NOD mice. One NOD neonate that received CD4+T cells developed diabetes and insulitis with CD8+T cells. Administration of cyclophosphamide to non-diabetic recipients accelerated the development of diabetes, while none of the mice with anti-CD8 antibody did so. Similarly, it was observed that neonates that received islet-derived CD8+T cells developed diabetes and obvious insulitis mainly with CD4+T cells. Administration of anti-CD4 antibody with transfer of CD8+T cells inhibited insulitis. These results imply that CD8+T cells function as an initial element to recruit CD4+T cells to islets as well as a final effector.

Effect of T-cell receptor VΒ-specific monoclonal antibodies on cyclophosphamide-induced diabetes mellitus in non-obese diabetic mice

SummaryThe expression of specific T-cell receptor gene segments by T lymphocytes appears to be critically important for the induction of several experimental autoimmune diseases mediated by these cells. We examined whether this situation also applied to non-obese diabetic mice by using various T-cell receptor VΒ-specific monoclonal antibodies. No significant age- or sex-related differences were observed in VΒ usage by peripheral and splenic T lymphocytes. CD8+ T lymphocytes among the islet-derived mononuclear cells isolated from 20-week-old female non-obese diabetic mice showed heterogeneity of their VΒ gene usage. In order to examine the role of T lymphocyte subsets expressing specific T-cell receptor VΒ gene segments in the development of diabetes mellitus, T-cell receptor VΒ-specific monoclonal antibodies were administered to 10-week-old male non-obese diabetic mice treated with cyclophosphamide. None of the antibodies used could significantly diminish the incidence of cyclophosphamide-induced diabetes and the severity of insulitis [anti-VΒ3 (11 of 22 mice became diabetic, 50%), anti-VΒ5 (9 of 14, 64%), anti-VΒ8 (9 of 21, 43%), anti-VΒ11 (12 of 23, 52%), anti-VΒ14 (7 of 12, 58%), and anti-VΒ5 + anti-VΒ11 (6 of 12, 50%)] when compared with control mice (12 of 21, 57%). In addition, there were no significant differences in T-cell receptor VΒ usage between diabetic and non-diabetic cyclophosphamide-treated mice. These results suggest that five T-lymphocyte subsets expressing different T-cell receptor VΒ gene segments, considered to be candidates involved in the pathogenesis of autoimmune diabetes, do not individually contribute to the development of cyclophosphamide-induced diabetes in non-obese diabetic mice.

Effect of antiserum to monoclonal anti-islet cell surface antibody on pancreatic insulitis in non-obese diabetic mice

Immunotherapeutic intervention has been studied in non-obese diabetic (NOD) mice. Twenty-five male NOD mice aged 6 weeks were treated with anti-mouse T lymphocyte serum (ATS), N-(2-carboxyphenyl)-4-chloroanthranilic acid disodium salt (CCA); a non-specific immunostimulant which seems to potentiate the suppressor T cell activity, and antiserum raised against previously reported monoclonal antibody 3A4 to the surface of islet cells. Pancreatic islets from NOD mice sacrificed at 12 weeks of age were scored morphologically for the severity and the frequency of insulitis. Both the severity of insulitis in each islet and the frequency of insulitis-positive islets in each pancreas were reduced in the groups treated with ATS (group B), antiserum to 3A4 (group D) and a combination of antiserum plus CCA (group E) in comparison with other groups (control: group A and CCA: group C). At 8 weeks of age, the binding capacities of sera to insulinoma cells measured by protein A radioligand assay were significantly decreased in the groups treated with antiserum to 3A4 (groups D and E) as compared with those in the other groups. These results suggest that both ATS and antiserum to 3A4 prevent the occurrence and the progress of insulitis in NOD mice, but the immunosuppressive mechanisms differ from each other; therefore, combination therapy with these suppressants may be more effective in the prevention of Type 1 diabetes mellitus.