Surface and intracellular Fas expression associated with cytokine-induced apoptosis in rodent islet and insulinoma cells.

During the process of insulitis in the pathogenesis of type I (insulin-dependent) diabetes mellitus, proinflammatory cytokines induce expression of the death receptor Fas on the surface of pancreatic beta-cells and thereby contribute to the enhanced susceptibility of beta-cells for apoptosis. The aim of this study was to compare cell-surface and intracellular Fas expression associated with cytokine-induced apoptosis in commonly used beta-cell models such as isolated islets and insulinoma lines derived from mouse and rat. The cell line NIT-1 responded to the interleukin (IL)-1beta+interferon (IFN)-gamma stimulus with translocation of Fas to the cell surface. Likewise, islet cells from non-obese diabetic (NOD) mice and BB/OK rats expressed increasing amounts of the Fas receptor on their surfaces after exposure to IL-1beta in combination with IFN-gamma and tumour necrosis factor-alpha. Moreover, islets obtained from BB/OK rats at an age near the onset of diabetes had an increased surface expression of Fas compared with young rats. In contrast, western blot analysis of cell lysates from cytokine-exposed islets and insulinoma cells revealed total Fas expression levels comparable to those of untreated controls. In conclusion, islets from BB/OK rats and NOD mice, in addition to NIT-1 insulinoma cells, responded to cytokine exposure with surface expression of the Fas receptor, whereas in cell lysates the levels of expression of Fas were found to be independent of cytokine exposure. Taken together, the findings indicate that cytokine-treated beta-cells might possess two pools of Fas protein, one of which is inducible by cytokines and accounts for surface Fas expression, whereas the other is constitutively expressed in cytoplasmic compartments. The underlying mechanisms, including possible interactions between these two sources of cellular Fas expression, need to be investigated in future studies.

Insulin treatment improves islet function in type 2 diabetic Chinese hamsters.

To study whether normalization of hyperglycemia improves islet function in long-standing type 2 diabetes, hyperglycemic CHIG/Han subline of the genetic type 2 diabetic Chinese hamster (>15 mmol/l: n=23) were either treated with insulin implants (liberating 1 U/day) or vehicle for two weeks. Islets were isolated and incubated for 3 h in the presence of 10 mmol/l glucose with or without 0.1 mmol/l 3-isobutyl-1-methylxanthine (IBMX). Specimens were also taken for immunocytochemical analysis of insulin cells. Glucose-stimulated insulin secretion was reduced by 83% in the vehicle-treated diabetic hamsters compared to non-diabetic controls (p<0.001). This impairment was not improved by the two-week insulin treatment. IBMX potentiated glucose-stimulated insulin secretion; this effect was markedly reduced in vehicle-treated diabetics compared to controls (p<0.001). In fact, the linear relation between IBMX-potentiated and glucose-stimulated insulin secretion in controls was absent in islets from diabetic animals. The two week insulin treatment normalized this relation, although still the total insulin secretory response to IBMX and glucose was lower than in controls. Furthermore, the islet insulin content was significantly increased by the 2 week normalization of glucose and, finally, the severe degranulation and lowering of insulin staining in islet beta cells in diabetic animals were markedly improved by insulin treatment. The results suggest that two-weeks of normalization of glycemia in long-standing type 2 diabetes in non-obese Chinese hamster improves beta cell signaling induced by the cyclic AMP pathway in conjunction with improved islet insulin content and beta cell morphology.

Immunoadsorption of immunoglobulins alters intracytoplasmic type 1 and type 2 T cell cytokine production in patients with refractory autoimmune diseases.

Intracellular cytokine staining and flow cytometry were used to investigate whether immunoadsorption (IA) of immunoglobulins alters intracytoplasmic cytokine production in CD4+ and CD8+ T cells from the blood of patients with refractory rheumatoid arthritis (n = 7), membrane proliferative glomerulonephritis (n = 1), and Goodpasture's syndrome (n = 1). Four patients (Group 1) showed severely depressed production of TNF-alpha, IL-2, IFN-gamma, and IL-4 by CD4+ and CD8+ T cells and responded to 3 IA sessions with significant increases in CD4+TNF-alpha+, CD4+IL-2+, and CD8+IL-2+ T cells. Also, a tendency toward increased percentage levels of CD4+ T cells producing IFN-gamma or IL-4 and of CD8+ T cells producing either TNF-alpha or IFN-gamma was seen, but due to the small number of patients investigated, these differences did not attain statistic significance. Group 2 (n = 5) showed unimpaired intracellular cytokine levels and responded to IA with a heterogeneous pattern of changes in TNF-alpha, IL-2, IFN-gamma, and IL-4 production, but these alterations were smaller than those in Group 1. The present findings indicate that the extracorporeal removal of immunoglobulins by anti-IgG or protein A adsorber columns has an impact on T cell immunity and suggest that modulating effects on cellular immune system function are involved in the mode of action of IA.

Islet neuronal abnormalities associated with impaired insulin secretion in type 2 diabetes in the Chinese hamster.

This study examined the relationship between islet neurohormonal characteristics and the defective glucose-stimulated insulin secretion in genetic type 2 diabetic Chinese hamsters. Two different sublines were studied: diabetes-prone CHIG hamsters and control CHIA hamsters. The CHIG hamsters were divided into three subgroups, depending on severity of hyperglycemia. Compared to normoglycemic CHIG hamsters and control CHIA hamsters, severely hyperglycemic CHIG hamsters (glucose > 15 mmol/l) showed marked glucose intolerance during i.p. glucose tolerance test and 75% impairment of glucose-stimulated insulin secretion from isolated islets. Mildly hyperglycemic CHIG animals (glucose 7.2-15 mmol/l) showed only moderate glucose intolerance and a 60% impairment of glucose-stimulated insulin secretion from the islets. Immunostaining for neuropeptide Y and tyrosine hydroxylase (markers for adrenergic nerves) and for vasoactive intestinal peptide (marker for cholinergic nerves) revealed significant reduction in immunostaining of islets in the severely but not in the mildly hyperglycemic animals, compared to control CHIA hamsters. The study therefore provides evidence that in this model of type 2 diabetes in Chinese hamsters, severe hyperglycemia is accompanied not only by marked glucose intolerance and islet dysfunction but also by reduced islet innervation. This suggests that islet neuronal alterations may contribute to islet dysfunction in severe but not in mild diabetes.

Relationship between the histopathology of the endocrine-exocrine pancreas parenchyma and beta-cell function in the Chinese hamster CHIG/Han subline.

To investigate pancreatic histopathology in relation to islet function in type 2 diabetes, pancreatic tissue was examined at disease onset and after death in the genetically diabetic Chinese hamster CHIG/Han subline. The pancreatic islets displayed vacuolation, intraislet fibrosis, variable stages of degranulation, and beta-cell necrosis, but no insulitis. These lesions were associated with changes in immunostaining of major islet peptides, impaired glucose-stimulated (10 mM) insulin secretion, reduced islet insulin content, and the severity of hyperglycemia. The exocrine pancreas was characterized by peri- and intrapancreatic fat and mononuclear cell infiltration. Biopsy of the pancreas had a marked effect on plasma glucose such that at 2 weeks after excision, in 9 and 18% of the severely hyperglycemic hamsters, plasma glucose levels decreased to <7.2 and 15 mM, respectively, and 45% of the mildly hyperglycemic hamsters became transiently normoglycemic (<7.2 mM). The results showed that insulitis is not involved in beta-cell failure of diabetic CHIG/Han hamsters. Vacuolation of islets was the most prominent lesion associated with a functional islet abnormality and development of hyperglycemia. Attenuation of hyperglycemia after biopsy suggests that the pancreas, in type 2 diabetes, maintains the ability to respond to impairment with amelioration of the diabetic state.

Differential expression of fructosyllysine-specific receptors on monocytes and macrophages and possible pathophysiological significance.

A differing individual expression of fructosyllysine-specific receptors has been found on the monocytes of 90 insulin-dependent diabetic patients and 101 healthy control subjects. The degree of receptor expression is neither age- nor sex-dependent; however, in the diabetic group it correlates significantly with the severity and age of onset of diabetic microangiopathy. To interpret the results of the human study, spontaneously diabetic and non-diabetic BB/OK rats were used to estimate tissue content of glucose-modified proteins and capillary basement membrane thickness in relation to the receptor expression on macrophages. In non-diabetic and diabetic rats no correlation was found between receptor expression and tissue content (i.e. artery, nerve) of fructosyllsine and fluorescent advanced glycation end products. However, animals which express the fructosyllysine receptor showed a greater increase in muscle capillary basement membrane thickness. There are indications that fructosyllysine receptor expression is positively associated with indices of diabetic complications such as microangiopathy and/or capillary basement membrane thickening.

Antibody response to islet antigens in anti-CD4/prednisolone immune intervention of type 1 diabetes.

Cytoplasmic islet cell antibodies, glutamic acid decarboxylase autoantibodies, spontaneous insulin autoantibodies, and insulin-induced antibodies were analyzed in a 1-year follow-up study of 12 newly diagnosed patients with insulin-dependent diabetes mellitus aged 14 +/- 2 years (range 7-20 years) who had been initially treated with either multiple injections of insulin alone (control group) or, in addition, anti-CD4 monoclonal antibody/prednisolone (treatment group). Despite individual variations in islet cell antibody titers, there were no significant differences in the prevalence or changes in the mean titers between the two groups. Glutamic acid decarboxylase autoantibodies remained almost unchanged, but correlated with levels of islet cell antibodies. While at initiation of treatment only 50% of the patients from both groups had spontaneous insulin autoantibodies, all patients developed insulin-induced antibodies upon conventional insulin therapy during the course of follow-up. This was not related to islet cell antibody or glutamic acid decarboxylase antibody levels. The insulin requirement was markedly reduced through the period of follow-up, but did not significantly differ between the two groups. A correlation between islet cell antibody levels and insulin requirement was observed in the control group but not in the treatment group. Plasma levels of the antibodies were not associated with changes in stimulated C-peptide or hemoglobin A1 concentrations. Activated T-lymphocytes persisted in both groups of patients, but their mean levels were not significantly different. The reason for the absence of statistically significant differences between treatment and control groups could be due to the small number of patients in the study. In conclusion, short-term immune intervention with anti-CD4 monoclonal antibody in addition to insulin therapy did not suppress autoimmune reactions towards the beta cells.

Aberrant activation of CD8+ T-cell and CD8+ T-cell subsets in patients with newly diagnosed IDDM.

Two- and three-color cytofluorimetric techniques were used to study the expression patterns of the activation antigen HLA-DR on peripheral blood immunoregulatory T-cells from 25 patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) and 14 age- and sex-matched control subjects. The mean percentage of total activated (CD3+HLA-DR+) T-cells was significantly elevated in the IDDM group compared with the control group (P < 0.001). In control subjects, basal activation of CD4+ and CD8+ lymphocytes accounted for the low percentage levels of activated T-cells. In contrast, the majority of IDDM patients showed an unbalanced activation of CD4+ and CD8+ lymphocytes with predominant activation of the CD8+ lymphocyte subset. The composition of the activated T-cell fraction was dependent on the composition of the total (activated + nonactivated) T-cell population, as indicated by the positive correlation between the CD4+/CD8+ T-cell ratios in these two cell populations (r = 0.714; P < 0.001). Excessive activation of CD8+ T-cells was attributable to similar increases in the proportions of CD8+ CD45RA+HLA-DR+ (naive) and CD8+CD45RA-HLA-DR+ (memory) cells. Analysis of the CD11b-defined subsets revealed predominant activation of CD8+ CD11b- (cytotoxic) T-cells; CD8+ CD16+ HLA-DR+ natural killer cells were unchanged. The distribution of HLA-DR+ cells among subsets of CD4+ T-cells differed from the pattern in the CD8+ population in that selective activation of CD4+ CD45RA- (memory, helper-inducer) cells accounted for the small increase in activated CD4+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired beta-cell function in the Chinese hamster CHIG/Han subline.

Plasma glucose and insulin levels were measured in the genetically diabetic CHIG/Han and the diabetes-resistant CHIA/Han subline of the Chinese hamster. At 31 +/- 8 wk of age, the CHIG hamsters were grouped into nondiabetic, mildly and severely diabetic, according to their levels of glycemia. Hyperinsulinemia, occurring in nondiabetic and mildly diabetic CHIG hamsters, was attenuated in severely diabetic animals. Light microscopy and immunohistochemistry revealed initial beta-cell hyperplasia, followed by extensive degranulation and loss of immunoreactive insulin in islets of severely diabetic animals. Staining intensity of glucagon-immunoreactive cells was unchanged in nondiabetic and mildly diabetic animals, but was increased in islets from the severely diabetic hamsters. A static incubation system was used to examine the insulin response of pancreatic islets isolated from the diabetic and nondiabetic CHIG hamsters, and the diabetes-resistant CHIA subline. Compared with the nondiabetic CHIG hamsters, islets from mildly and severely diabetic animals displayed increased basal insulin release at 1.5 mmol/l and a deficient response at 10 mmol/l glucose which was associated with 61 and 77% decreases (p < 0.01 and p < 0.001) in the islet insulin content. The addition of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) enhanced glucose-stimulated insulin release from islets of nondiabetic and mildly diabetic CHIG hamsters, although the response elicited was lower than from CHIA islets. However, IBMX failed to significantly increase the glucose-stimulated insulin response of islets from severely diabetic hamsters. A negative correlation (r = -0.73, p < 0.001, n = 48) was found between islet insulin content and plasma glucose levels. The data suggest that the reduced secretory capacity represents an early islet beta-cell dysfunction, and the decrease in the insulin content contributes to the islet abnormalities in the diabetes-susceptible CHIG hamsters.

The detection of autoantibodies to pancreatic islet cells by immunoenzyme histochemistry.

Using horseradish peroxidase- or alkaline phosphatase-conjugated secondary antibodies, an immunohistochemical assay was established for the detection of islet cell cytoplasmic antibodies (ICA). Determination of end-point titers showed a significant correlation between the conventional immunofluorescence and either immunocytochemistry assay. The assays with the enzyme-conjugated antibodies were more sensitive than the indirect immunofluorescence assay. Because of its simplicity, specificity, and easy microscopic evaluation of the chromogenic reaction product at the site of ICA binding, the indirect immunoperoxidase technique proved to be most suitable. This technique detected frequencies of ICA positives among newly diagnosed insulin-dependent (IDDM), noninsulin-dependent, and at-risk subjects that were comparable with previous studies. Preabsorption of ICA-positive sera with either rat or porcine brain extracts, containing the glutamate decarboxylase antigen, differently blocked, reduced or did not affect ICA reactivity with human or porcine pancreas sections. Testing of sera on human, bovine, and porcine pancreas sections demonstrated heterogeneity in ICA-binding with a high proportion of ICA false-positives on bovine pancreas. The results demonstrated that immunohistochemical techniques for detecting ICA are, in several aspects, preferable to indirect immunofluorescence and that individual serum ICA identify various antigens on pancreas from different species. However, bovine or porcine pancreas could not substitute for human pancreas in the ICA assay.

Modulation of the effect of humoral-mediated cytotoxicity on isolated rat pancreatic islets by glucose.

Rat islets of Langerhans exposed for 20 h at high glucose (20 mmol/l) to 50% or 20% experimentally raised rabbit anti-rat islet cell surface antiserum (ICSA-positive serum) plus complement exhibited an irreversible loss of glucose-stimulated insulin secretion. In contrast, islets treated with 50% ICSA-positive serum at low glucose (5.5 mmol/l) could overcome this alteration within a subsequent 48 h recovery period at 10 mmol/l glucose in the absence of ICSA, and islets affected at 5.5 mmol/l glucose by 20% ICSA-positive serum even retained the insulin secretory potential and responded on glucose challenge already immediately after the removal of ICSA. The islet insulin content was reduced by the effect of 50% as well as of 20% ICSA-positive serum and complement irrespective of whether the glucose level amounted to 5.5 or 20 mmol/l during serum influence. However, islets altered in a normoglycaemic environment at 5.5 mmol/l glucose by 20% ICSA-positive serum restored their insulin content up to the level of control islets, whereas those islets affected under hyperglycaemic conditions at 20 mmol/l glucose only partially recovered. Thus, beta-cell loss and/or impairment of the insulin secretory mechanisms result from the simultaneous action of humoral-mediated anti-islet cytotoxicity and elevated glucose level, and cause the diminished insulin secretory potential of the islets. These results support the hypothesis that decreasing the insulin secretory activity of beta cells may protect them from cytotoxic immunological attacks.

Effect of syngeneic islet antigen administration on complement-dependent antibody-mediated cytotoxicity to islet cells and diabetes onset in diabetes-prone BB/OK rats.

Diabetes-prone BB/OK rats aged 30 to 35 days were subjected to three sequential intrasplenic injections of unfractionated homogenate prepared from Langerhans' islets of newborn syngeneic BB/OK rats. Syngeneic islet antigen administration resulted in increased complement-dependent antibody-mediated cytotoxicity (C'AMC) to rat pancreatic islet cells in serum, compared to buffer-treated control animals as detected by 51Cr-release assay. However, the increase of anti-islet-cell cytotoxicity neither impaired glucose tolerance nor affected the incidence of diabetes and the age at manifestation. In contrast to BB/OK rats developing diabetes, animals remaining long-term normoglycaemic did not show an enhancement of cytotoxicity to islet cells within twelve days after the first islet antigen injection as revealed retrospectively. In conclusion, humoral mediated anti-islet-cell cytotoxicity is not decisively involved in pancreatic beta-cell destruction and promotion of diabetes development in BB/OK rats, but animals becoming diabetic seem to be characterized by a stronger immunological reactivity upon syngeneic islet antigen challenge as indicated by an increase of anti-islet C'AMC compared to long-term normoglycaemic rats.

Humoral-mediated cytotoxicity to rat pancreatic exocrine cells in serum of diabetes-prone BB/OK rats--predictive value for diabetes onset.

Diabetes-prone BB/OK rats were checked in a follow-up study between 40 and 200 days of age for the appearance of humoral-mediated cytotoxicity to rat pancreatic exocrine cells in serum by 51Cr-release assay. BB/OK rats maintaining normoglycaemia were characterized by a pronounced decrease of anti-exocrine cell cytotoxicity in serum at 75 and 105 days of age compared to the initial value at day 40. In contrast, in animals developing diabetes cytotoxicity was found more frequently and the level of cytotoxicity did not likewise decrease during the period up to diabetes onset. With respect to the prediction of diabetes in individual BB/OK rats the retrospective analysis revealed that 81.0% of those animals displaying cytotoxicity at 75 days of age subsequently developed diabetes. If sera from BB/OK rats were identified as non-cytotoxic at day 75 and also at 105 days of age normoglycaemia persisted in 83.3% of these animals. Thus, screening of young diabetes-prone BB/OK rats for the presence of humoral-mediated cytotoxicity to pancreatic exocrine cells in serum seems to facilitate the identification of potential diabetic or long-term normoglycaemic animals in the BB/OK rat population.

Monoclonal antibody-mediated cytotoxicity against rat beta cells detected in vitro does not cause beta-cell destruction in vivo.

Two monoclonal Beta-cell surface antibodies M10H6 und K14D10 were obtained by fusion of spleen cells of Balb/c mice with the myeloma cell line P(3)0. The monoclonal antibody M10H6 was induced by immunization with rat insulinoma cells finally boostered with disintegrated rat islets, whereas the K14D10 was generated after immunization with porcine proinsulin. Both monoclonals belong to the IgG2A isotype and were screened with insulin-producing rat insulinoma cells by an indirect immunofluorescence test as well as by a cellular enzyme linked immunosorbent assay. In addition to the cell surface binding on living Beta cells the monoclonals react with islets on cryostat sections of rat pancreas. The anti-islet cytotoxic potential of these monoclonals was measured by 51Chromium-release in the presence of complement or Fc-receptor bearing leucocytes using 51Chromium-labelled rat islet cells as target. Both antibody secreting hybridomas were propagated in syngeneic mice resulting in high levels of islet cell surface antibodies in ascites and sera from the recipient. High anti-islet cytotoxicity was mediated by ascites fluid, but no mouse developed hyperglycaemia. Furthermore, the repeated injections of the monoclonals into rats did not exert a diabetogenic action and failed to reduce the pancreatic insulin content although the attraction of the K14D10 to the pancreatic islets in vivo could be demonstrated. We conclude that islet cell surface antibody-mediated Beta-cell lysis in vitro may not be relevant to Beta-cell destruction in vivo.

Immunological and beta-cell-specific characteristics of diabetes-prone BB/OK rats and their congenic derivatives--a comparative study.

In a comparative study congenic rat strains bearing either the genetic background of diabetic BB/OK rats and the MHC (RTla) of diabetes-resistant LEW.1A rats (BB.1A/OK) or vice versa carrying the genetic background of LEW.1A rats in combination with the MHC (RTlu) of diabetic BB/OK rats (LEW.1BB/OK) and their parental rat strains BB/OK and LEW.1A were checked for insulin secretion of pancreatic islets, for the number of splenic and peripheral blood mononuclear cells (MNC) as well as for the mitogenic response of splenic MNC. Glucose stimulated insulin secretion of isolated islets of Langerhans was not different in 50, 70 and 100 day-old congenic rats and the progenitor rat strains excluding an impact of the genotype on this beta-cell-specific function. The number of splenic and peripheral blood MNC was reduced in BB/OK and BB.1A/OK rats compared to LEW.1A and LEW.1BB/OK rats. Splenic MNC from BB/OK and BB.1A/OK rats displayed a strongly decreased total [3H]thymidine incorporation under basal conditions as well as upon mitogenic stimulation by ConA in comparison with MNC from LEW.1A and LEW.1BB/OK rats. Thus, the occurrence of lymphopenia and the impairment of mononuclear cell proliferation in BB/OK rats is not related to the RTlu haplotype of the MHC but is linked to non-MHC genes as indicated by the phenotypic expression of these traits in congenic BB.1A/OK rats.

Complement-dependent antibody-mediated cytotoxicity (C'AMC) to pancreatic islet cells in the spontaneously diabetic BB/OK rat: interference from cell-bound and soluble inhibitors.

Sera from 30 newly diagnosed diabetic BB/OK rats were analyzed cross-sectionally for complement-dependent antibody-mediated cytotoxicity (C'AMC) to pancreatic islet cells using different 51Cr release test systems. The sera contained enough active complement to lyse either sheep erythrocytes or 51Cr-labelled rat islet cells that had been sensitized with specific rabbit antibodies, but, in the presence of BB/OK rat islet cell antibody they released significant amounts of islet cell-bound 51Cr only after addition of rabbit complement. In a one-step assay, 19 out of the 30 sera produced lysis significantly above that by sera from the non-diabetes-prone WOK strain. This was increased 2.5-fold (p less than 0.01) by briefly washing the serum-treated cells before adding complement (two-step assay), indicating that C'AMC inhibitory activity was present in the diabetic sera. Some inhibitory activity could still be detected in heated sera but only when they were added to the cells immediately before the rabbit complement. Islet cell lysis was still substantial after preferential inactivation of factor B of the alternative complement activation pathway (by heating at 50 degrees C), and thus mainly depended on the classical pathway. From these findings it is concluded that (a) islet cell surface antibodies in diabetic rats activate heterologous complement via the classical pathway, (b) anti-islet C'AMC is sensitive both to species-restricted interference in interactions between homologous antibody and homologous complement in the target cell membrane, and to a distinct serum inhibitor that impairs the ability of membrane-fixed BB/OK rat antibody to interact with rabbit complement.

Detection and time course of humoral-mediated cytotoxicity to different cell types in diabetes-prone BB/OK rats.

Sera from diabetes-prone BB/OK rats were tested for humoral-mediated cytotoxicity to rat pancreatic islet cells, spleen lymphocytes and exocrine pancreatic cells using 51Cr-release assay systems. At the age of 20, 30 and 40 days all cross-sectionally studied BB/OK rats showed cytotoxicity to islet cells but only 37.5%, 25.0% and 63.3% of them exhibited anti-lymphocyte cytotoxicity, respectively. Neither the time course of cytotoxicity to islet cells nor to lymphocytes differed in BB/OK rats developing diabetes compared to animals maintaining normoglycaemia as evidenced in a follow-up study. The decrease of cytotoxicity to islet cells in vitro as observed in the time course study seems to be due to the appearance of an inhibitor of anti-islet cell cytotoxicity in serum from BB/OK rats older than 70 days. However, under conditions avoiding the influence of inhibitory components the observed time course of anti-islet cell cytotoxicity also did not permit to distinguish potential diabetic BB/OK rats from animals maintaining normoglycaemia. In contrast, long-term normoglycaemic BB/OK rats showed a peak value of cytotoxicity to rat exocrine pancreatic cells between 40 and 50 days of age only whereas animals developing diabetes more frequently displayed cytotoxicity in the prediabetic phase. Inhibitory activity against cytotoxicity to exocrine cells was not likewise detectable in BB/OK rat serum. In conclusion, except of more frequently appearing cytotoxicity to rat exocrine pancreatic cells among the investigated BB/OK rats becoming diabetic the cytotoxicity patterns to islet cells and spleen lymphocytes were not predictive for diabetes onset. Thus, humoral-mediated cytotoxicity seems to appear in BB/OK rats as a sign of immune dysregulation characteristic for this animal model at high risk for diabetes rather than in a manner related to disease manifestation.

Beta cell destruction of pancreatic islets transplanted into diabetic BB/OK rats may be reflected by increased antibody-mediated anti-islet cytotoxicity.

Neonatal rat pancreatic islets can be affected in vitro by ADCC mediated by serum and mononuclear cells from diabetic BB/OK rats or complement-dependent antibody-mediated cytotoxicity (C'AMC) of BB rat serum as revealed by enhanced 51Cr-release. Using a syngeneic islet transplantation system in BB/OK rats this study addressed the question whether the destruction of islets of Langerhans in vivo is reflected by the appearance of ADCC or C'AMC in vitro. The frequency of the appearance of enhanced anti-islet ADCC in newly diagnosed diabetic BB/OK rats amounted to 33% whereas ADCC was not detectable in long-term diabetic rats with a diabetes duration in a range between 50 and 90 days. After transplantation of syngeneic islets beneath the kidney capsule of long-term diabetic BB/OK rats held without immunosuppression the destruction of the islet graft and the persistence of hyperglycaemia was accompanied by an increase of anti-islet ADCC in 66.6% of the animals. While only 18.7% of the long-term diabetic BB/OK rats showed C'AMC against islet cells before transplantation this frequency raised after transplantation and 62.8% of the animals exhibited increased C'AMC within a period of 21 days but with a pronounced individual variability of the time-course. Increased anti-islet cytotoxicity (ADCC or C'AMC) parallels newly initiated or reactivated beta-cell destruction after transplantation and thus seems to reflect this process.

Complement-dependent antibody-mediated cytotoxicity in the spontaneously diabetic BB/OK rat: association with beta cell volume density.

The present study examined in the spontaneously diabetic BB/OK rat whether a relationship exists between the appearance of complement-dependent antibody-mediated cytotoxicity (C'AMC) in serum and the relative beta cell volume density determined in pancreatic biopsies. C'AMC was estimated by 51Cr release from prelabeled major histocompatibility complex-compatible neonatal rat islet cells after exposure to rat serum and rabbit complement. Fifty-one percent (72/141) of sera from BB/OK rats with newly diagnosed diabetes were positive for C'AMC. At onset of hyperglycemia, insulin-immunoreactive beta cells were only detectable in pancreas biopsies of 25% (10/40) of the BB/OK rats who displayed mild hyperglycemia (plasma glucose 8.3-13.0 mmol/l) and low serum C'AMC. A twofold increase (p less than 0.01) of C'AMC and loss of the remaining beta cells was evident in untreated animals upon their reexamination within 1 week after diagnosis of hyperglycemia. Initiation of insulin therapy prevented neither the increase in C'AMC activity nor the decrease in the beta cell volume density. In contrast, three out of four mildly hyperglycemic BB/OK rats treated with cyclosporin A maintained both their initial C'AMC levels and relative beta cell volume density not only throughout the treatment period (4 weeks) but also for at least 4 weeks thereafter. In one additional animal receiving cyclosporin A no protection of the remaining beta cells could be achieved and C'AMC levels were markedly increased. It is concluded that the appearance of increased C'AMC in serum may reflect autoimmune reactions against the islet beta cells of spontaneously diabetic BB/OK rats. The increase of C'AMC seen in untreated as well as insulin-treated BB/OK rats, which were even devoid of beta cells, suggests that C'AMC activity appears secondary to the loss of beta cells. These results do not support the hypothesis of a direct beta cell destruction via intrainsular complement activation.