Alex Rabinovitch

An update on cytokines in the pathogenesis of insulin-dependent diabetes mellitus.

Correlation studies between cytokines expressed in islets and autoimmune diabetes development in NOD mice and BB rats have demonstrated that beta-cell destructive insulitis is associated with increased expression of proinflammatory cytokines (IL-1, TNF alpha, and IFN alpha) and type 1 cytokines (IFN gamma, TNF beta, IL-2 and IL-12), whereas non-destructive (benign) insulitis is associated with increased expression of type 2 cytokines (IL-4 and IL-10) and the type 3 cytokine (TGF beta). Cytokines (IL-1, TNF alpha, TNF beta and IFN gamma) may be directly cytotoxic to beta-cells by inducing nitric oxide and oxygen free radicals in the beta-cells. In addition, cytokines may sensitize beta-cells to T-cell-mediated cytotoxicity in vivo by upregulating MHC class I expression on the beta-cells (an action of IFN gamma), and inducing Fas (CD95) expression on beta-cells (actions of IL-1, and possibly TNF alpha and IFN gamma). Transgenic expression of cytokines in beta-cells of non-diabetes-prone mice and NOD mice has suggested pathogenic roles for IFN alpha, IFN gamma, IL-2 and IL-10 in insulin-dependent diabetes mellitus (IDDM) development, and protective roles for IL-4, IL-6 and TNF alpha. Systemic administrations of a wide variety of cytokines can prevent IDDM development in NOD mice and/or BB rats; however, a given cytokine may retard or accelerate IDDM development, depending on the dose and frequency of administration, and the age and the diabetes-prone animal model studied (NOD mouse or BB rat). Islet-reactive CD4+ T-cell lines and clones that adoptively transfer IDDM into young NOD mice have a Th1 phenotype (IFN gamma-producing), but other islet-specific Th1 clones that produce TGF beta can adoptively transfer protection against IDDM in NOD mice. NOD mice with targeted deletions of IL-12 and IFN gamma genes still develop IDDM, albeit delayed and slightly less often. In contrast, post-natal deletions of IL-12 and IFN gamma, also IL-1, TNF alpha, IL-2, and IL-6--by systemic administrations of neutralizing antibodies, soluble receptors and receptor antagonists, and receptor-targeted cytotoxic drugs--significantly decrease IDDM incidence in NOD mice and/or BB rats. These cytokine deletion studies have provided the best evidence for pathologic roles for proinflammatory cytokines (IL-1, TNF alpha, and IL-6) and type 1 cytokines (IFN gamma, IL-2 and IL-12) in IDDM development.

Cytokines and Their Roles in Pancreatic Islet β-Cell Destruction and Insulin-Dependent Diabetes Mellitus

Insulin-dependent diabetes mellitus (IDDM) is a disease that results from autoimmune destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. The autoimmune response against islet beta-cells is believed to result from a disorder of immunoregulation. According to this concept, a T helper 1 (Th1) subset of T cells and their cytokine products, i.e. Type 1 cytokines--interleukin 2 (IL-2), interferon gamma (IFNgamma), and tumor necrosis factor beta (TNFbeta), dominate over an immunoregulatory (suppressor) Th2 subset of T cells and their cytokine products, i.e. Type 2 cytokines--IL-4 and IL-10. This allows Type 1 cytokines to initiate a cascade of immune/inflammatory processes in the islet (insulitis), culminating in beta-cell destruction. Type 1 cytokines activate (1) cytotoxic T cells that interact specifically with beta-cells and destroy them, and (2) macrophages to produce proinflammatory cytokines (IL-1 and TNFalpha), and oxygen and nitrogen free radicals that are highly toxic to islet beta-cells. Furthermore, the cytokines IL-1, TNFalpha, and IFNgamma are cytotoxic to beta-cells, in large part by inducing the formation of oxygen free radicals, nitric oxide, and peroxynitrite in the beta-cells themselves. Therefore, it would appear that prevention of islet beta-cell destruction and IDDM should be aimed at stimulating the production and/or action of Type 2 cytokines, inhibiting the production and/or action of Type 1 cytokines, and inhibiting the production and/or action of oxygen and nitrogen free radicals in the pancreatic islets.

Immunoregulatory and Cytokine Imbalances in the Pathogenesis of IDDM: Therapeutic Intervention by Immunostimulation?

The autoimmune response that leads to destruction of pancreatic islet β-cells and insulin-dependent diabetes mellitus (IDDM) has a genetic basis; however, environmental factors can exert profound modulating effects on the genetic predisposition to this autoimmune response. Recent studies in animal models for human IDDM, the genetically diabetes-prone NOD mouse and BB rat, have revealed that microbial agents—including certain viruses and extracts of bacteria, fungi, and mycobacteria—often have a protective action against diabetes development. Many of these microbial preparations are immune adjuvants, which are agents that stimulate the immune system. The protective effects of these agents against diabetes appear to involve perturbations in the production of cytokines, which are polypeptides produced by and acting on cells of the immune system. Thus, recent studies in NOD mice suggest that the islet β-cell-directed autoimmune response may be mediated by a T-helper 1 (Th1) subset of T-cells producing the cytokines interleukin-2 (IL-2) and interferon-γ. These studies also suggest that the diabetes-protective effects of administering microbial agents, adjuvants, and a β-cell autoantigen (GAD65 [glutamic acid decarboxylase]) may result from activation of a Th2 subset of T-cells that produce the cytokines IL-4 and IL-10 and consequently downregulate the Th1-cell-mediated autoimmune response. The clinical implication of these findings is that the autoimmune response leading to islet β-cell destruction and IDDM may be amenable to prevention or suppression by therapeutic interventions aimed at stimulating the hosts own immunoregulatory mechanisms.

Interferon Expression in the Pancreases of Patients With Type I Diabetes

We have used a reverse transcriptase–polymerase chain reaction (RT-PCR) protocol to examine the expression of cytokines in the pancreases and islets of patients with type I diabetes. We detect a significant increase in the level of expression of interferon (IFN)-α in the pancreases of the diabetic patients as compared with the control pancreases. In contrast, IFN-β was detected at comparable levels in both groups, while IFN-γ was detected in three of four control pancreases and one of four pancreases from the diabetic individuals. The IFN-α cDNAs generated by the RT-PCR were cloned and sequenced to determine which α-subtypes were being expressed. We found that the repertoire of subtypes was quite limited in any one individual (diabetic or not), although each individual was different with respect to the pattern of subtypes expressed. We also examined these pancreases for the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-2, IL-4, and IL-6. We found no detectable expression of TNF-α or IL-2 in any pancreases, and the expression of the other cytokines was variable, with no pattern emerging from the comparison of the diabetic and nondiabetic individuals. We conclude that, of the cytokines examined, only IFN-α was significantly increased in the diabetic patients, a result that is consistent with the possibility that this cytokine is directly involved in the development of type I diabetes.

Th1 to Th2 Cytokine Shifts in Nonobese Diabetic Mice: Sometimes an Outcome, Rather Than the Cause, of Diabetes Resistance Elicited by Immunostimulation

Numerous immunostimulatory protocols inhibit the development of T cell-mediated autoimmune insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse model. Many of these protocols, including treatment with the nonspecific immunostimulatory agents CFA or bacillus Calmette-Guérin (BCG) vaccine, have been reported to mediate protection by skewing the pattern of cytokines produced by pancreatic β-cell autoreactive T cells from a Th1 (IFN-γ) to a Th2 (IL-4 and IL-10) profile. However, most of these studies have documented associations between such cytokine shifts and disease protection rather than a cause/effect relationship. To partially address this issue we produced NOD mice genetically deficient in IFN-γ, IL-4, or IL-10. Elimination of any of these cytokines did not significantly alter the rate of spontaneous IDDM development. Additional experiments using these mice confirmed that CFA- or BCG-elicited diabetes protection is associated with a decreased IFN-γ to IL-4 mRNA ratio within T cell-infiltrated pancreatic islets, but this is a secondary consequence rather than the cause of disease resistance. Unexpectedly, we also found that the ability of BCG and, to a lesser extent, CFA to inhibit IDDM development in standard NOD mice is actually dependent upon the presence of the Th1 cytokine, IFN-γ. Collectively, our studies demonstrate that while Th1 and Th2 cytokine shifts may occur among β-cell autoreactive T cells of NOD mice protected from overt IDDM by various immunomodulatory therapies, it cannot automatically be assumed that this is the cause of their disease resistance.

Normoglycaemia after transplantation of freshly isolated and cryopreserved pancreatic islets in Type 1 (insulin-dependent) diabetes mellitus

SummaryPurified islets of Langerhans and a kidney were transplanted into a 36-year-old patient who suffeded from renal failure secondary to a 25 year history of Type 1 (insulin-dependent) diabetes mellitus. The islet graft contained 243 000 fresh islets (mean islet diameter 150 μm) that were syngeneic with the kidney fraft and 368 000 cryopreserved islets that had been collected from four other donors. The total of 10 000 islets/kg body weight was infused into the liver via the umbilical vein. Immunosupperession was induced with antilymphocyte globulin and maintained with prednisone, cyclosporine and azathioprine. Serum C-peptide levels (ng/ml) during fasting and after standard mixed metal feeding (Sustacal) were <0.12 preoperatively. Postoperatively, insulin secretion was restored: fasting C-peptide rose during the first 4 weeks to levels of 4 to 5 and Sustacal elicited a further rise to 6 to 7. Transplant renal function was stable. Dialy fasting glucose (mmol/l, mean±SD) was 5.6±1 and 5.3±0.6 during the first and second months respectively and post-Sustacal glucose was 5.7+-0.8. Exogenous insulin therapy was progressively withdrawn and stopped duting the ninth week. Thereafter, fasting glucose was 4.7+-0.5, 24 h mean glucose was 6.6+-0.5, and normoglycaemia was maintained after Sustacal. These data show that this mass of freshly isolated and cryopreserved islets from multiple donors provided sustained function (3 months) that reversed insulin-dependence in an immunosuppressed Type 1 diabetic patient treated with simultaneous islet-kedney transplantation.

Development of autoimmune diabetes in NOD mice is associated with the formation of peroxynitrite in pancreatic islet β-cells

Peroxynitrite (ONOO−) is a highly reactive oxidant species produced by the reaction of the free radicals superoxide (O2·–) and nitric oxide (NO·). Here we report a marked increase in nitrotyrosine (NT), a marker of peroxynitrite, in islet cells from NOD mice developing spontaneous autoimmune diabetes. By using specific antibodies and immunohistochemical methods, we found that NT-positive cells were significantly more frequent in islets from acutely diabetic NOD mice (22 ± 6%) than in islets from normoglycemic NOD mice (7 ± 1%) and control BALB/c mice (2 ± 1%). The NT+ cells in islets were identified to be macrophages and also β-cells. Most of the β-cells in islets from acutely diabetic NOD mice were NT+ (73 ± 8%), whereas significantly fewer β-cells were NT+ in islets from normoglycemic NOD mice (18 ± 4%) and BALB/c mice (5 ± 1%). Also, the percentage of β-cells in islets from NOD mice (normoglycemic and diabetic) correlated inversely with the frequency of NT+ β-cells. This study demonstrates for the first time that peroxynitrite, a reaction product of superoxide and nitric oxide, is formed in pancreatic islet β-cells of NOD mice developing autoimmune diabetes. This suggests that both oxygen and nitrogen free radicals contribute to β-cell destruction in IDDM via peroxynitrite formation in the islet β-cells.

Oxygen free radical scavengers protect rat islet cells from damage by cytokines.

SummaryA possible role for oxygen free radicals in mediating the cytotoxic effects of cytokines in islets was sought by the use of agents that scavenge free radicals. Rat islet cell monolayer cultures were incubated for 6 days with t-butylhy-droperoxide, alloxan, streptozotocin, or the cytokines, interleukin 1, tumour necrosis factor, and interferon gamma, without and together with the oxygen free radical scavenger combination of dimethylthiourea and citiolone, and islet cell lysis was measured in a 15chromium cytotoxicity assay. The free radical scavengers significantly inhibited the islet cell cytotoxic effects of t-butylhydroperoxide and alloxan, but not streptozotocin. Similarly, the cytotoxic effects of the cytokine combinations of interleukin 1 plus tumour necrosis factor, interferon gamma plus tumour necrosis factor, and interferon gamma plus interleukin 1 were significantly inhibited by the free radical scavenger combination of dimethylthiourea and citiolone. These results suggest that the cytokine products of macrophages and lymphocytes infiltrating islets in Type 1 (insulin-dependent) diabetes may contribute to B-cell damage by inducing the production of oxygen free radicals in the islet cells.

Combination Therapy With Glucagon-Like Peptide-1 and Gastrin Restores Normoglycemia in Diabetic NOD Mice

OBJECTIVE—Glucagon-like peptide-1 (GLP-1) and gastrin promote pancreatic β-cell function, survival, and growth. Here, we investigated whether GLP-1 and gastrin can restore the β-cell mass and reverse hyperglycemia in NOD mice with autoimmune diabetes. RESEARCH DESIGN AND METHODS—Acutely diabetic NOD mice were treated with GLP-1 and gastrin, separately or together, twice daily for 3 weeks. Blood glucose was measured weekly and for a further 5 weeks after treatments, after which pancreatic insulin content and β-cell mass, proliferation, neogenesis, and apoptosis were measured. Insulin autoantibodies were measured, and adoptive transfer of diabetes and syngeneic islet transplant studies were done to evaluate the effects of GLP-1 and gastrin treatment on autoimmunity. RESULTS—Combination therapy with GLP-1 and gastrin, but not with GLP-1 or gastrin alone, restored normoglycemia in diabetic NOD mice. The GLP-1 and gastrin combination increased pancreatic insulin content, β-cell mass, and insulin-positive cells in pancreatic ducts, and β-cell apoptosis was decreased. Insulin autoantibodies were reduced in GLP-1–and gastrin-treated NOD mice, and splenocytes from these mice delayed adoptive transfer of diabetes in NOD-scid mice. Syngeneic islet grafts in GLP-1–and gastrin-treated NOD mice were infiltrated by leukocytes with a shift in cytokine expression from interferon-γ to transforming growth factor-β1, and β-cells were protected from apoptosis. CONCLUSIONS—Combination therapy with GLP-1 and gastrin restores normoglycemia in diabetic NOD mice by increasing the pancreatic β-cell mass and downregulating the autoimmune response.

Combination Therapy With Sirolimus and Interleukin-2 Prevents Spontaneous and Recurrent Autoimmune Diabetes in NOD Mice

Sirolimus is an immunosuppressant that inhibits interleukin (IL)-2 signaling of T-cell proliferation but not IL-2-induced T-cell apoptosis. Therefore, we hypothesized that administration of IL-2, together with sirolimus, might shift T-cell proliferation to apoptosis and prevent autoimmune destruction of islet beta-cells. We found that sirolimus and IL-2 therapy of female NOD mice, beginning at age 10 weeks, was synergistic in preventing diabetes development, and disease prevention continued for 13 weeks after stopping sirolimus and IL-2 therapy. Similarly, sirolimus and IL-2 were synergistic in protecting syngeneic islet grafts from recurrent autoimmune destruction after transplantation in diabetic NOD mice, and diabetes did not recur after stopping sirolimus and IL-2 combination therapy. Immunocytochemical examination of islet grafts revealed significantly decreased numbers of leukocytes together with increased apoptosis of these cells in mice treated with sirolimus and IL-2, whereas beta-cells were more numerous, and significantly fewer were apoptotic. In addition, Th1-type cells (gamma-interferon-positive and IL-2(+)) were decreased the most, and Th2-type cells (IL-4(+) and IL-10(+)) and Th3-type cells (transforming growth factor-beta1(+)) were increased the most in islet grafts of sirolimus and IL-2-treated mice. We conclude that 1) combination therapy with sirolimus and IL-2 is synergistic in protecting islet beta-cells from autoimmune destruction; 2) diabetes prevention continues after withdrawal of therapy; and 3) the mechanism of protection involves a shift from Th1- to Th2- and Th3-type cytokine-producing cells, possibly due to deletion of autoreactive Th1 cells.