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The non-obese diabetic (NOD) mouse is an animal model of insulin-dependent diabetes mellitus (IDDM), in which 80% of the females become diabetic after the age of 12 weeks. Using an in vitro assay we investigated the capacity of spleen lymphocytes from NOD mice to inhibit the insulin secretion of normal islet cells after stimulation by theophylline plus arginine. Spleen cells from diabetic NOD mice inhibited the insulin release of DBA/2 islet cells. Depletion experiments using monoclonal antibodies demonstrated that inhibitory ceils belonged to the Lyt2 positive T lymphocyte subset. The phenomenon was not restricted by the MHC class I K region, shared by NOD and DBA/2 mice, since lymphocytes from diabetic NOD mice also inhibited the insulin secretion of normal Wistar rat islet cells. Inhibitory T cells were detected in overtly diabetic mice but also in non-diabetic females aged 5-11 weeks indicating that they are not secondary to metabolic disturbances and might contribute to their onset. Conversely they were not found in male NOD mice although some of these mice show insulitis. The presence of these inhibitory T ceils might thus represent an early and sensitive marker of anti-islet cell-mediated autoimmunity. [ABSTRACT FROM AUTHOR]

The insulin response of isolated islet cells to glucose and theophylline in vitro was studied after incubation with lymphocytes. The test was employed to explore cell-mediated immunity in diabetics. A significant inhibition of insulin response to glucose and theophylline as compared to insulin release in a 'basal' medium was found after incubation with blood lymphocytes from 21 out of 23 insulin-dependent diabetics (mean secretion index 18±8 versus 118±8 (SEM) % in control subjects). Most of the patients studied had associated autoimmune diseases: all of these displayed inhibition of insulin release. In six cases, the diabetes had a recent onset with no associated autoimmune disease: four of them displayed the same inhibition. No inhibition was found in the 26 control subjects and in seven non-insulin-dependent diabetics (mean secretion index 134±17 versus 145±23 (SEM) % in four control subjects). Lymphocytes inhibiting islet cell response were not cytotoxic against mouse fibroblasts. Twenty-two insulin-dependent diabetics showed islet cell antibodies to human and/or mouse pancreatic islets.. However, an inhibition of insulin release was found with no detectable islet cell antibodies in one case, and the converse in two cases. Lymphocyte cytotoxicity to islet cells could play a role in the natural history of insulin-dependent diabetes. [ABSTRACT FROM AUTHOR]

Mixed lymphocyte cultures were performed before renal transplantation in 36 patients, using the cells from the recipient and the prospective living donor. A highly significant correlation was found between the results of mixed leucocyte cultures and (a) the renal function at one year or two years; (b)the occurrence of early or late rejection crises. A good correlation was also found between the results of mixed cultures and leucocyte typing based on HL-A haplotypes (but not with conventional typing based on the number of antigenic differences). As applied to the selection of the donors for kidney transplantation, the mixed culture has the advantage of recognizing all the 'good' cases, including the favourable cases of the HL-A semi-identical group; it will also select however a small number of poor cases. In contrast, the determination of HL-A haplotypes will select almost only 'good' cases but will discard a significant number of cases which will eventually prove to be favourable. [ABSTRACT FROM AUTHOR]

The NOD mouse is studied as an animal model of human insulin-dependent diabetes mellitus (IDDM). To evaluate the role of IFN gamma in the pathogenesis of the disease, we have studied the effect of anti-IFN gamma mAb on the expression of insulitis and clinical diabetes. Treatment of mice with anti-IFN gamma mAb prevented the induction of early IDDM by cyclophosphamide as well as the adoptive transfer of diabetes by spleen cells from diabetic NOD mice. The protection against induction of diabetes by cyclophosphamide was observed in animals treated with the anti-IFN gamma mAb within 24 h following the first cyclophosphamide injection but not in animals in which mAb treatment was started 7 days later. Transfer of disease was prevented both in adult irradiated and in newborn recipients. The absence of clinical signs in these mice was corroborated by a significant reduction of both the extent and severity of insulitis. Over-expression of Ia antigen on endothelial cells lining the islets was also considerably reduced in mice treated with mAb. These data strongly suggest a role for IFN gamma during the autoimmune process leading to beta cell destruction in diabetes and prompt further investigation of the use of such antibodies in the immunoprevention of IDDM.

A significant inhibition of insulin response was found after incubation of islet cells with blood lymphocytes from 18 out 20 insulin-dependent diabetics. No inhibition was found in 22 control subjects.

CBA Mice were immunized by two intraperitoneal injections of 30 X 10(6) DBA/2 or C57BL/6 spleen cells at days--12 and--2. Peritoneal cell population was obtained at day zero by washing the peritoneal cavity of Mice. Adherent cells were then separated using a 2 hrs. incubation in "Falcon" plates followed by washing. This macrophage-rich peritoneal cell population was found nonspecifically cytotoxic against 51Cr labeled tumoral target cells: P815 X DBA/2 mastocytoma cells, EL4 X C57BL/L lymphoma cells and spontaneous lymphoma AKR cells (same H--2k as CBA). This adherent peritoneal cell cytoxicity was demonstrated after 24 hrs. incubation with the target cells. It was found in nonspecific combination as well as when using target cells syngeneic to the donor. These findings suggest that adherent peritoneal cell cytotoxicity could be at least partly due to macrophages and result from factor (s) released by sensitized lymphocytes in vivo in the same way as has been previously demonstrated in vitro.

Antilymphocyte sera, either allogeneic or xenogeneic, may retain the antigen used for the immunization of the animal producing the serum in the form of antigen-antibody complexes, at least when the animal is bled within the first two weeks following the last immunizing injection. Evidence for this has been obtained in vivo and in vitro (3). Thus, antilymphocyte injections may contain not only antibodies but the antigens used to prepare the serum as well, and active immunization to these antigens can result in the human or animal recipient. In the heart-allografted rat, pre-transplantation injection of immune complexes prepared from alloantisera prolongs graft survival. The filtrate from these complexes, presumably containing antigens, also prolongs graft survival when administered in conjunction with nonspecific immunosuppresive drugs after transplantation. These results, however, do not allow determination of whether the effect is active or passive, or perhaps both, nor what is the action of the complexes themselves.

It is usually accepted that macrophages "activated" by lymphokines may be found cytotoxic against tumoral target cells but show no detectable cytotoxicity in in vitro tests using normal non tumoral cells as target cells. These data have been obtained mainly with the chromium-release test. The present paper describes a new test using normal isolated pancreatic cells as target cells and evaluating the effect of activated or non-activated macrophages on the insulin secretion response to glucose stimulation. The results show a striking decrease in this response following an 18-hr incubation of pancreatic islet cells with activated macrophages, as compared to that of the same cells incubated with control macrophages. This is clear evidence that activated macrophages may alter normal cells and suggests that their cytotoxic properties are not restricted to tumoral target cells.

The development of IDDM correlates with the presence of biologic markers pointing to the involvement of the immune system in the disease process. In addition to clinical observations of association of IDDM with other autoimmune disease and morphologic evidence of a mononuclear cell infiltration of the islets of Langerhans at the onset of the disease, anti-islet cell antibodies are detected in the serum of IDDM patients. Moreover, a strong genetic association with HL-A DR3 and DR4 identifies a genetic background compatible with autoimmune phenomena. Whether autoimmune phenomena are primary or secondary to an initial damage of the islets by infectious agents or other environmental factors is unknown. Whether or not the autoimmune response participates in the selective destruction of insulin-secreting cells has been a major issue in the past five years. The presence of T lymphocytes and anti-islet cell antibodies, which selectively inhibit or lyse insulin-secreting cells in vitro, strongly suggests that it may be the case. A definitive demonstration is difficult to provide in human IDDM. The development of animal models for IDDM has allowed useful insight into the pathogenetic mechanisms responsible for IDDM. In both the BB rat and the low-dose streptozotocin mouse model, the role of the immune system in the destruction of the islets of Langerhans is supported by the prevention of the disease by treatments interfering with the immune system. The BB rat develops a spontaneous autoimmune disease on a genetic background defined by the association with a major histocompatibility complex allele without any evidence for a role in initial damage of islets of a triggering infectious or chemical process. The low-dose streptozotocin model is an autoimmune IDDM secondary to the selective damage of islet cells by a toxin. The present scheme of an islet cell target and specific autoreactive T and B lymphocyte clones raises two major issues: what is the target antigen on islet cells and what is the role at the molecular level of class II major histocompatibility complex genes in susceptibility for IDDM? The first issue is presently being addressed in several laboratories using the hybridoma technology. The second issue is addressed at the biochemical level by studying restriction site polymorphism of major histocompatibility genes in susceptible individuals and IDDM patients, and at the functional level by studying the action of monoclonal antibodies to class II antigen on the development of IDDM in animal models. These steps are likely to be a prerequisite to antigen-specific immunotherapy in IDDM.

In order to explore complement-dependent humoral immunity in insulin-dependent diabetic patients, their serum was incubated with Mouse isolated pancreatic islets in the presence of complement, after which the response of these islets to glucose was estimated by measuring insulin secretion. Out of 13 sera from insulin-dependent diabetic patients, 5 caused a significant inhibition of the insulin response. Morphological observation of islets suggested that this inhibition was due to a cytotoxic effect of the sera. In the absence of complement, the same sera were not able to suppress insulin release. No beta-cell inhibition or morphological damage was detectable on islets incubated with sera from 13 healthy subjects and 4 non-insulin dependent diabetic patients, in the presence or in the absence of complement. Complement-dependent humoral immunity may play a role in the beta-cell injury of insulin-dependent diabetes.

Increasingly, experimental results are underlining the role played by autoimmune mechanisms in the pathogenesis of type I insulin-dependent diabetes mellitus (IDDM). It has appeared logical to attempt to preclude the onset of IDDM by suppressing immune responses, but trials using steroids or azathioprine were unequivocal. Subsequently, a Canadian and a French group performed pilot studies to assay a new immunosuppressive drug, cyclosporine A (CyA) in human diabetes (Stiller et al., 1984; Assan et al., 1985). However, further testing is required to evaluate and confirm its potential benefits and possible risks.

Mice peritoneal macrophages obtained after i.p. injection of a medium containing thioglycollate (Institut Pasteur, Paris) have increased levels of beta glucuronidase and acid phosphatase, when compared with macrophages obtained without previous thioglycollate injection. The maximum increase is observed between 7 and 10 days after injection and reaches up to 3 to 4 times the normal activity. When using separately the various components of the medium, the same phenomenon may be observed but never to the same degree. The injection of thioglycollate also produces: a) an increase in total number of peritoneal cells, and b) an increase in the percentage of macrophages among these cells. Kinetic studies suggest that thioglycollate injection first induces an increase in the number of peritoneal macrophages and subsequently macrophage modifications resulting in lysosomal enzyme increase.

Macrophage-rich adherent peritoneal cells from mice sensitized by two i.p. injections of allogeneic cells at days -- 12 and --2, were found non-specifically cytotoxic against (a) tumoral target cells, as evaluated by the chromium release test, and (b) normal target cells, as evaluated by a functional test based on the insulin secretion response to glucose stimulation of isolated pancreatic islet cells. This non-specific cytotoxicity was identical to that of macrophages "activated" by an in vitro incubation with supernatants of MLC between allograft donor and recipient as reported elsewhere. Moreover this cytotoxicity was not abolished by anti-theta serum treatment of adherent peritoneal cells before contact with target cells. It is concluded that an in vivo macrophage activation was obtained in our experiment. Using this same model, we found that the survival time of skin grafts from male C57BL/6 to female C57BL/6 mice was shortened if the graft was performed at the time when the recipient macrophages were found non-specifically cytotoxic (two days after the second allogeneic cell injection). The role of macrophage cytotoxicity in this accelerated allograft rejection is suggested by (a) the lack of any detectable cross-histocompatibility antigens between the immunizing cells and the skin graft, and (b) the absence of any alteration of the graft survival if the second immunizing injection two days prior to grafting is omitted (in that case only lymphocytes are found specifically cytotoxic against the strain of injected cells, while the non-specific cytotoxicity of macrophages is lacking).

It has been suggested that the immune system may be responsible for the destruction of insulin secreting cells in some types of diabetes. In order to test this hypothesis, we studied the consequences of immune-mediated reactions on the function of pancreatic islet cells in vitro. A model was set up in vitro where mouse pancreatic islet cells are exposed to human lymphocytes or sera + complement then stimulated for the release of insulin or glucagon. A selective inhibition of insulin secretion, but not of glucagon secretion, was observed in the presence of lymphocytes from 37 out of 40 insulin-dependent diabetic (IDD) patients and in the presence of sera (+ complement) from 22 out of 40. Lymphocytes were found inhibitory in almost all patients in both groups, with and without associated autoimmune diseases. In contrast, inhibitory sera were observed almost only in patients with associated autoimmune diseases or recent onset diabetes. The selective inhibition of insulin secretion, but not of glucagon secretion, suggests that lymphocytes or sera may be involved in a destructive process of insulin secreting cells in vivo. This cell-mediated effect depends on direct T lymphocyte cytotoxicity, rather than antibody-dependent cell cytotoxicity, as suggested by the lack of any effect of aggregated immunoglobulins on the reaction. In contrast, when C57BL/6 mice were immunized by mastocytoma cells from a DBA2 strain, their lymphocytes and sera blocked both secretions of insulin and glucagon when incubated in vitro with DBA2 islet cells. This non-selective inhibition may be due to anti-H2 immunity, rather than immunity directed against insulin secreting cells.

Monoclonal autoantibodies cytotoxic for murine islet cells have been obtained by cell fusion using spleen cells from non-immunized diabetic C57BL/KsJ (db/db) Mice. These monoclonals, of IgG2a subclass, blocked in vitro insulin secretion induced by arginine in Mouse pancreatic cells.

We previously showed that circulating lymphocytes from more than 90% of insulin-dependent diabetics, block extra insulin secretion induced by stimulatory media in mouse pancreatic cells in vitro, without altering the secretion of glucagon. The present work demonstrates that this phenomenon depends on lymphocytes having the OKT3 marker, i.e., thymodependent lymphocytes. However, OKT4+ T helper cells are not required for the above phenomenon as proved by experiments using monoclonal sera against the OKT4 marker. When diabetes is associated with other autoimmune diseases, the pancreatic lymphocyte cytotoxicity observed in vitro is inhibited by the addition of a normal lymphocyte population; this could indicate that a "suppressor" factor is lacking in these patients. Conversely, addition of normal lymphocytes does not prevent lymphocyte cytotoxicity in diabetics without associated autoimmune diseases. Such a difference confirms the present trend to make a distinction between these two categories of diabetes.