T cell-extrinsic IL-1 signaling controls long-term gammaherpesvirus infection by suppressing viral reactivation.

Gammaherpesviruses establish life-long infection in over 95% of adults and are associated with several cancers, including B cell lymphomas. Using the murine gammaherpesvirus 68 (MHV68) animal model, we previously showed a pro-viral role of Interleukin-1 (IL-1) signaling that supported viral reactivation during the establishment of chronic infection. Unexpectedly, in this study we found that the proviral effects of IL-1 signaling originally observed during the establishment of chronic gammaherpesvirus infection convert to antiviral effects during the long-term stage of infection. Specifically, IL-1 signaling promoted expansion of antiviral CD8+ T cells and control of viral reactivation in the peritoneal cavity of a long-term infected host. Using a novel mouse model of T cell-specific IL-1 signaling deficiency, we found that the antiviral effects of IL-1 signaling were T cell extrinsic. Our study highlights a dynamic nature of host factors that shape the parameters of chronic gammaherpesvirus infection.

Intraislet release of interleukin 1 inhibits beta cell function by inducing beta cell expression of inducible nitric oxide synthase.

Cytokines, released in and around pancreatic islets during insulitis, have been proposed to participate in beta-cell destruction associated with autoimmune diabetes. In this study we have evaluated the hypothesis that local release of the cytokine interleukin 1 (IL-1) by nonendocrine cells of the islet induce the expression of inducible nitric oxide synthase (iNOS) by beta cells which results in the inhibition of beta cell function. Treatment of rat islets with a combination of tumor necrosis factor (TNF) and lipopolysaccharide (LPS), conditions known to activate macrophages, stimulate the expression of iNOS and the formation of nitrite. Although TNF+LPS induce iNOS expression and inhibit insulin secretion by intact islets, this combination does not induce the expression of iNOS by beta or alpha cells purified by fluorescence activated cell sorting (Facs). In contrast, IL-1 beta induces the expression of iNOS and also inhibits insulin secretion by both intact islets and Facs-purified beta cells, whereas TNF+LPS have no inhibitory effects on insulin secretion by purified beta cells. Evidence suggests that TNF+LPS inhibit insulin secretion from islets by stimulating the release of IL-1 which subsequently induces the expression of iNOS by beta cells. The IL-1 receptor antagonist protein completely prevents TNF+LPS-induced inhibition of insulin secretion and attenuates nitrite formation from islets, and neutralization of IL-1 with antisera specific for IL-1 alpha and IL-1 beta attenuates TNF+LPS-induced nitrite formation by islets. Immunohistochemical localization of iNOS and insulin confirm that TNF+LPS induce the expression of iNOS by islet beta cells, and that a small percentage of noninsulin-containing cells also express iNOS. Local release of IL-1 within islets appears to be required for TNF+LPS-induced inhibition of insulin secretion because TNF+LPS do not stimulate nitrite formation from islets physically separated into individual cells. These findings provide the first evidence that a limited number of nonendocrine cells can release sufficient quantities of IL-1 in islets to induce iNOS expression and inhibit the function of the beta cell, which is selectively destroyed during the development of autoimmune diabetes.

Recognition of allo-peptide is governed by novel anchor imposition and limited variations in TCR contact residues.

Immune specificity of a T cell is determined by the TCR contact residues exposed on the antigenic peptide/MHC complex. Naturally processed, biallelic epitopes from H7 minor histocompatibility (mH) antigen vary in position 7 (p7) from aspartic acid (D) to a glutamic acid (E), which differ by an additional methylene (-CH(2)) in the side chain. Here, we show that this variation generates a strong anti-H7a or anti-H7b cytotoxic T cell responses. Further, the H7 allelic peptides use p6 asparagine as their central anchor residue and amino acid variations in either the canonical p5 or the predicted p6 anchor positions in the antigenic epitope were detrimental for TCR recognition. In addition, introduction of any other amino acids, except asparagine, in the polymorphic p7 significantly abolished the ability of anti-H7b TCR recognition. This demonstrates that only an asparagine with an amine group as a side chain instead of a charged oxygen radical could effectively stimulate the anti-H7b specific T cells. Our findings provide evidence that mH antigen-specific TCRs are highly stringent in recognizing their cognate epitopes.

Nitric oxide mediates interleukin-1-induced cellular cytotoxicity in the rat ovary. A potential role for nitric oxide in the ovulatory process.

Treatment of primary cultures of rat ovarian dispersates with IL-1 beta results in morphologic and cytotoxic changes, thought to reflect tissue remodeling events associated with ovulation. We examined the role that the free radical nitric oxide plays in this process and report that IL-1 beta induces expression of the inducible isoform of nitric oxide synthase in ovarian cells as demonstrated by immunoprecipitation. We show that IL-1 beta treatment results in the formation of nitric oxide (as measured by accumulation of nitrite and cGMP) in both a time- and concentration-dependent manner that is prevented by aminoguanidine, a selective inhibitor of the inducible isoform of nitric oxide synthase. Aminoguanidine also inhibits IL-1-induced ovarian cellular cytotoxicity. These results suggest that nitric oxide is an important mediator of cell death and may act as a physiologically significant mediator of tissue remodeling events that occur in vivo during the ovulatory process.

Interleukin 1 beta induces the formation of nitric oxide by beta-cells purified from rodent islets of Langerhans. Evidence for the beta-cell as a source and site of action of nitric oxide.

Nitric oxide has recently been implicated as the effector molecule that mediates IL-1 beta-induced inhibition of glucose-stimulated insulin secretion and beta-cell specific destruction. The pancreatic islet represents a heterogeneous cell population containing both endocrine cells (beta-[insulin], alpha-]glucagon], gamma[somatostatin], and PP-[polypeptide] secreting cells) and non-endocrine cells (fibroblast, macrophage, endothelial, and dendritic cells). The purpose of this investigation was to determine if the beta-cell, which is selectively destroyed during insulin-dependent diabetes mellitus, is both a source of IL-1 beta-induced nitric oxide production and also a site of action of this free radical. Pretreatment of beta-cells, purified by FACS with IL-1 beta results in a 40% inhibition of glucose-stimulated insulin secretion that is prevented by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine (NMMA). IL-1 beta induces the formation of nitric oxide by purified beta-cells as evidenced by the accumulation of cGMP, which is blocked by NMMA. IL-1 beta also induces the accumulation of cGMP by the insulinoma cell line Rin-m5F, and both NMMA as well as the protein synthesis inhibitor cycloheximide prevent this cGMP accumulation. Iron-sulfur proteins appear to be intracellular targets of nitric oxide. IL-1 beta induces the formation of an iron-dinitrosyl complex by Rin-m5F cells indicating that nitric oxide mediates the destruction of iron-sulfur clusters of iron containing enzymes. This is further demonstrated by IL-1 beta-induced inhibition of glucose oxidation by purified beta-cells, mitochondrial aconitase activity of dispersed islet cells, and mitochondrial aconitase activity of Rin-m5F cells, all of which are prevented by NMMA. IL-1 beta does not appear to affect FACS-purified alpha-cell metabolic activity or intracellular cGMP levels, suggesting that IL-1 beta does not exert any effect on alpha-cells. These results demonstrate that the islet beta-cell is a source of IL-1 beta-induced nitric oxide production, and that beta-cell mitochondrial iron-sulfur containing enzymes are one site of action of nitric oxide.

IL-1 produced and released endogenously within human islets inhibits beta cell function.

Resident macrophages have been suggested to participate in the initiation of beta cell damage during the development of autoimmune diabetes. The purpose of this study was to determine if the endogenous production and release of interleukin 1 (IL-1) in human islets of Langerhans by resident macrophages results in the inhibition of beta cell function. Treatment of human islets with a combination of tumor necrosis factor (TNF) + lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) stimulates inducible nitric oxide synthase (iNOS) expression, nitric oxide production, and inhibits glucose-stimulated insulin secretion. The IL-1 receptor antagonist protein (IRAP) prevents TNF + LPS + IFN-gamma-induced iNOS expression and nitrite production, and attenuates the inhibitory effects on glucose-stimulated insulin secretion by human islets. Inhibition of iNOS activity by aminoguanidine also attenuates TNF + LPS + IFN-gamma-induced inhibition of insulin secretion by human islets. These results indicate that the inhibitory effects of TNF + LPS + IFN-gamma are mediated by nitric oxide, produced by the actions of IL-1 released endogenously within human islets. Reverse transcriptase polymerase chain reaction was used to confirm that TNF + LPS + IFN-gamma stimulates the expression of both IL-1alpha and IL-1beta in human islets. Two forms of evidence indicate that resident macrophages are the human islet cellular source of IL-1: culture conditions that deplete islet lymphoid cells prevent TNF + LPS + IFN-gamma-induced iNOS expression, nitric oxide production, and IL-1 mRNA expression by human islets; and IL-1 and the macrophage surface marker CD69 colocalize in human islets treated with TNF + LPS + IFN-gamma as determined by immunohistochemical analysis. Lastly, nitric oxide production is not required for TNF + LPS + IFN-gamma-induced IL-1 release in human islets. However, cellular damage stimulates IL-1 release by islet macrophages. These findings support the hypothesis that activated islet macrophages may mediate beta cell damage during the development of insulin-dependent diabetes by releasing IL-1 in human islets followed by cytokine-induced iNOS expression by beta cells.

Evidence for the presence of type I IL-1 receptors on beta-cells of islets of Langerhans.

The cytokine interleukin-1beta (IL-1beta) has been shown to inhibit insulin secretion and destroy pancreatic islets by a mechanism that involves the expression of inducible nitric oxide synthase (iNOS), and the production of nitric oxide (NO). Insulin containing beta-cells, selectively destroyed during the development of autoimmune diabetes, appear to be the islet cellular source of iNOS following treatment with IL-1beta. In this study we have evaluated the presence of type I IL-1 signaling receptors on purified pancreatic beta-cells. We show that the interleukin-1 receptor antagonist protein (IRAP) prevents IL-1beta-induced nitrite formation and IL-1beta-induced inhibition of insulin secretion by isolated islets and primary beta-cells purified by fluorescence-activated cell sorting (FACS). The protective effects of IRAP correlate with an inhibition of IL-1beta-induced iNOS expression by islets and FACS purified beta-cells. To provide direct evidence to support beta-cell expression of IL-1 type I signaling receptors, we show that antiserum specific for the type I IL-1 receptor neutralizes IL-1beta-induced nitrite formation by RINm5F cells, and that RINm5F cells express the type I IL-1 receptor at the protein level. Using reverse transcriptase-polymerase chain reaction (RT-PCR), the expression of type I IL-1 signaling receptors by FACS purified beta-cells and not alpha-cells is demonstrated. These results provide direct support for the expression of type I IL-1 receptors by primary pancreatic beta-cells, the cell type selectively destroyed during the development of autoimmune diabetes.

Characterization of the sphingomyelin content of isolated pancreatic islets. Evaluation of the role of sphingomyelin hydrolysis in the action of interleukin-1 to induce islet overproduction of nitric oxide.

Inflammatory cytokines may participate in the destruction of pancreatic islets during the pathogenesis of insulin-dependent diabetes mellitus, and the cytokine interleukin-1 (IL-1) strongly inhibits insulin secretion from rat pancreatic islets by a process which involves induction of expression of the inducible isoform of nitric oxide synthase and the overproduction of nitric oxide. The signaling events between IL-1 receptor occupancy and induction of nitric oxide synthase in rat islets involve activation of the transcriptional activator NFkappa B. Because sphingomyelin hydrolysis has been implicated as a signaling process both in NFkappa B activation and in IL-1 action in some cells, we have examined the potential involvement of sphingomyelin hydrolysis in the induction of islet nitric oxide overproduction by IL-1. Rat islet sphingomyelin pools were radiolabeled with [3H]choline, and sphingomyelin was then isolated by normal phase HPLC. Electrospray ionization-mass spectrometric analysis revealed islet sphingomyelin consists of at least 4 distinct molecular species, and the most abundant of them contained sphingosine as the long chain base and a residue of palmitic acid as the fatty acid substituent. Molecular species containing residues of stearic acid and arachidic acid were also observed. Neither interleukin-1 nor tumor necrosis factor-alpha was found to induce hydrolysis of islet sphingomyelin species, and neither an exogenous, cell-permeant ceramide species (N-acetyl-D-sphingosine) nor exogenous sphingomyelinase mimicked or potentiated the effect of IL-1 to increase rat islet nitric oxide generation, as reflected by nitrite production. Similar findings were obtained with RINm5F insulinoma cells and with mouse pancreatic islets. These findings provide the first information on the molecular species of sphingomyelin in pancreatic islets and suggest that sphingomyelin hydrolysis is not involved in the signaling pathway whereby IL-1 induces the overproduction of nitric oxide by pancreatic islets.

Does nitric oxide mediate autoimmune destruction of beta-cells? Possible therapeutic interventions in IDDM.

Cytokines have been implicated as immunological effector molecules that induce dysfunction and destruction of the pancreatic beta-cell. The mechanisms of cytokine action on the beta-cell are unknown; however, nitric oxide, resulting from cytokine-induced expression of nitric oxide synthase, has been implicated as the cellular effector molecule mediating beta-cell dysfunction. Nitric oxide is a free radical that targets intracellular iron-containing enzymes, which results in the loss of their function. The cytokine IL-1 beta induces the formation of nitric oxide in isolated rat islets and the insulinoma cell line, Rin-m5F. NMMA and NAME, both inhibitors of nitric oxide synthase, completely protect islets from the deleterious effects of IL-1 beta. These inhibitors are competitive in nature and inhibit both the cytokine-inducible and constitutive isoforms of nitric oxide synthase with nearly identical kinetics. This may preclude their use as therapeutic agents because of increases in blood pressure which result from the inhibition of constitutive nitric oxide synthase activity. Aminoguanidine, an inhibitor of nonenzymatic glycosylation of cellular and extracellular constituents associated with diabetic complications, recently has been reported to inhibit nitric oxide synthase. Aminoguanidine is approximately 40-fold more effective in inhibiting the inducible isoform of nitric oxide synthase, suggesting that aminoguanidine or analogues may serve as potential therapeutic agents to block diseases associated with nitric oxide production by the inducible isoform of nitric oxide synthase. In vivo administration of TNF IL-1 has been shown to induce anti-diabetogenic effects in the NOD mouse. This anti-diabetogenic effect of cytokines appears to conflict with evidence suggesting that cytokines mediate beta-cell dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual functional effects of interleukin-1beta on purine nucleotides and insulin secretion in rat islets and INS-1 cells.

Interleukin-1beta (IL-1beta) has been shown to inhibit glucose-induced insulin secretion from rat islets and purified beta-cells, primarily through the generation of nitric oxide (NO). However, the mechanisms by which NO exerts its effects remain unclear. To examine the role of purine nucleotides, we cultured intact rat islets or INS-1 (glucose-responsive transformed rat) beta-cells for 18 h in the presence or absence of IL-1beta. In islets, the exposure to IL-1beta (100 pmol/l) inhibited subsequent glucose-induced insulin secretion by 91% with no significant effect on insulin content or basal insulin release. IL-1beta also diminished insulin secretion induced by pure mitochondrial fuels, 40 mmol/l K+, or a phorbol ester. Concomitantly, IL-1beta significantly decreased islet ATP (-45%), GTP (-33%), ATP/ADP (-54%), and GTP/GDP (-46%). These effects were totally reversed by provision of N(omega)-nitro-L-arginine methyl ester (NAME) in arginine-free media that inhibited NO production. In contrast, in INS-1 cells, IL-1beta (10 or 100 pmol/l) reduced both basal and glucose-induced insulin secretion by 50%, but insulin content was also reduced by 35%. Therefore, the INS-1 cells were still able to respond to glucose stimulation with a 1.8-2.0-fold increase in insulin release in either the presence or absence of IL-1beta. Concomitantly, in INS-1 cells, IL-1beta had no effect on ATP/ADP or GTP/GDP ratios, although it modestly decreased ATP (-25%) and GTP (-22%). As in islets, all effects of IL-1beta in INS-1 cells were prevented by NAME. Thus, in rat islets, IL-1beta (via the generation of NO) abolishes insulin exocytosis in association with large decreases in the ATP/ADP (and GTP/GDP) ratio, implying the impairment of mitochondrial function. Furthermore, IL-1beta inhibits cytosolic synthesis of new purine nucleotides (via the salvage pathway), as assessed by a decrease in their specific activity after labeling with [3H]hypoxanthine. In contrast, in INS-1 cells, IL-1beta appears to impair cytosolic synthesis of purine nucleotides and insulin biosynthesis selectively (both possibly reflecting decreased glycolysis) with little direct effect on insulin exocytosis itself.

Double-stranded RNA-dependent protein kinase is not required for double-stranded RNA-induced nitric oxide synthase expression or nuclear factor-kappaB activation by islets.

Environmental factors, such as viral infection, have been implicated in the destruction of beta-cells during the development of autoimmune diabetes. Double-stranded RNA (dsRNA), produced during viral replication, is an active component of a viral infection that stimulates antiviral responses in infected cells. Previous studies have shown that treatment of rat islets with dsRNA in combination with gamma-interferon (IFN-gamma) results in a nitric oxide-dependent inhibition of glucose-stimulated insulin secretion. This study examines the role of nuclear factor-kappaB (NF-kappaB) and the dsRNA-dependent protein kinase (PKR) in dsRNA + IFN-gamma-induced nitric oxide synthase (iNOS) expression and nitric oxide production by rat, mouse, and human islets. Treatment of rat and human islets with dsRNA in the form of polyinosinic-polycytidylic acid (poly IC) and IFN-gamma resulted in iNOS expression and nitric oxide production. Inhibitors of NF-kappaB activation-the proteasome inhibitor MG-132 and the antioxidant pyrrolidine-dithiocarbamate (PDTC)-prevented poly IC + IFN-gamma-induced iNOS expression and nitric oxide production. Incubation of rat islets for 3 h or human islets for 2 h with poly IC alone or poly IC + IFN-gamma resulted in NF-kappaB nuclear translocation and degradation of the NF-kappaB inhibitor protein, IkappaB, events that are prevented by MG-132. PKR has been shown to participate in dsRNA-induced NF-kappaB activation in a number of cell types, including mouse embryonic fibroblasts. However, poly IC stimulated NF-kappaB nuclear translocation and IkappaB degradation to similar levels in islets isolated from mice devoid of PKR (PKR-/-) and wild-type mice (PKR+/+). Furthermore, the genetic absence of PKR did not affect dsRNA + IFN-gamma-induced iNOS expression, nitric oxide production, or the inhibitory actions of these agents on glucose-stimulated insulin secretion. These results suggest that 1) NF-KB activation is required for dsRNA + IFN-gamma-induced iNOS expression, 2) PKR is not required for either dsRNA-induced NF-kappaB activation or dsRNA + IFN-y-induced iNOS expression by islets, and 3) PKR is not required for dsRNA + IFN-gamma-induced inhibition of glucose-stimulated insulin secretion by islets.

Anti-inflammatory actions of 15-deoxy-delta 12,14-prostaglandin J2 and troglitazone: evidence for heat shock-dependent and -independent inhibition of cytokine-induced inducible nitric oxide synthase expression.

In this study, the anti-inflammatory actions of the peroxisome proliferator-activated receptor (PPAR)-gamma agonists 15-deoxy-delta 12,14-prostaglandin J2 (15-d-delta 12,14-PGJ2) and troglitazone have been examined. Treatment of RAW 264.7 cells and CD-1 mouse peritoneal macrophages with lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) results in inducible nitric oxide synthase (iNOS), inducible cyclooxygenase (COX-2) and interleukin-1 (IL-1) expression, increased production of nitric oxide, and the release of IL-1. In a concentration-dependent manner, 15-d-delta 12,14-PGJ2 inhibits each of these proinflammatory actions of LPS + IFN-gamma, with half-maximal inhibition at approximately 0.5 microg/ml and complete inhibition at 1-5 microg/ml. The inhibitory actions of 15-d-delta 12,14-PGJ2 on LPS + IFN-gamma-induced inflammatory events are not associated with the inhibition of iNOS enzymatic activity or macrophage cell death, but appear to result from an inhibition of iNOS and IL-1 transcription. In addition, the anti-inflammatory actions of 15-d-delta 12,14-PGJ2 are not limited to peritoneal macrophages, as 15-d-delta 12,14-PGJ2 prevents TNF-alpha + LPS-induced resident islet macrophage expression of IL-1beta and beta-cell expression of iNOS stimulated by the local release of IL-1 in rat islets. 15-d-delta 12,14-PGJ2 appears to be approximately 10-fold more effective at inhibiting resident islet macrophage activation (in response to TNF + LPS) than IL-1-induced nitrite production by beta-cells. Two mechanisms appear to be associated with the antiinflammatory actions of both 15-d-delta 12,14-PGJ2 and troglitazone: 1) the direct inhibition of cytokine- and endotoxin-stimulated iNOS and IL-1 transcription; and 2) the inhibition of IL-1 signaling, an event associated with PPAR-gamma agonist-induced activation of the heat shock response (as assayed by heat shock protein 70 expression). These findings indicate that the PPAR-gamma agonists, troglitazone and the J series of prostaglandins, are potent anti-inflammatory agents that prevent cytokine- and endotoxin-stimulated activation of peripheral and resident tissue macrophages and cytokine-induced iNOS expression by beta-cells by the inhibition of transcriptional activation and induction of the heat shock response.

Evidence for involvement of the proteasome complex (26S) and NFkappaB in IL-1beta-induced nitric oxide and prostaglandin production by rat islets and RINm5F cells.

Interleukin-1beta (IL-1beta) has been implicated as an effector molecule of beta-cell destruction in autoimmune diabetes. IL-1beta inhibits insulin secretion from pancreatic beta-cells by stimulating the expression of inducible nitric oxide synthase (iNOS) that generates the free radical nitric oxide. IL-1beta also induces the coexpression of the inducible isoform of cyclooxygenase (COX-2) that results in the overproduction of proinflammatory prostaglandins. The current studies were designed to characterize the involvement of protease(s) in the signaling pathway of IL-1beta-induced iNOS and COX-2 expression by rat islets and transformed rat pancreatic beta-cells. Because of the limitations of cell numbers of purified primary beta-cells obtained from rat islets, biochemical and molecular studies were performed using the rat insulinoma beta-cell line RINm5F. A serine protease inhibitor, Nalpha-P-tosyl-L-lysine chloromethyl ketone (TLCK), and a proteasome complex (26S) inhibitor, MG 132, inhibited IL-1beta-induced nitrite formation, an oxidation product of nitric oxide produced by iNOS, in a concentration-dependent manner, with complete inhibition observed at 100 micromol/l and 10 micromol/l, respectively. Both TLCK and MG 132 also inhibited iNOS gene expression at the level of mRNA and protein. In an analogous manner, TLCK (100 micromol/l) and MG 132 (10 micromol/l) inhibited IL-1beta-induced COX-2 enzyme activity (PGE2 formation) and COX-2 gene expression at the level of mRNA and protein. In human islets, the proteasome inhibitor MG 132 also inhibited the formation of the products of iNOS and COX-2 enzyme activity, nitrite, and PGE2, respectively. These findings suggest that the inhibitory action of TLCK and MG 132 on iNOS and COX-2 expression precedes transcription. The transcription factor NFkappaB is essential for activation of a number of cytokine-inducible enzymes and was evaluated as a possible site of protease action necessary for IL-1beta-induced coexpression of iNOS and COX-2. TLCK and MG 132 inhibited both IL-1beta-induced activation of NFkappaB and degradation of IkappaBalpha by islets and RINm5F cells. These results implicate protease activation as an early signaling event in IL-1beta-induced inhibition of beta-cell function. This study also suggests that IL-1beta-induced iNOS and COX-2 coexpression by pancreatic beta-cells share a common signaling pathway in utilizing the proteasome complex (26S) and the transcription factor NFkappaB, and it identifies sites of intervention to prevent the overproduction of their inflammatory products.

Potential autoantigens in IDDM. Expression of carboxypeptidase-H and insulin but not glutamate decarboxylase on the beta-cell surface.

Insulin, carboxypeptidase-H (CP-H), and glutamate decarboxylase (GAD) have been identified as potential autoantigens in insulin-dependent diabetes mellitus (IDDM). Previous studies have described immunoreactive insulin as a surface molecule on the plasma membrane of rat islet cells and suggested that cell-surface insulin was derived during exocytosis by the fusion of insulin secretory granules with the beta-cell plasma membrane. These findings predict that insulin and other secretory granule-derived proteins such as the putative autoantigen CP-H may be colocalized with insulin at specific sites of exocytosis on the beta-cell surface. In studies to test this hypothesis, cell-surface staining of dispersed rat islet cells occurred in a granule-like pattern with antibodies for CP-H and insulin. The specificity of the CP-H antiserum was confirmed by immunoblotting and indicated that the antiserum was essentially monospecific for CP-H. Confocal laser microscopy confirmed that immunoreactive staining for CP-H and insulin was confined to the beta-cell surface. Colocalization of CP-H and insulin on the cell surface of beta-cells was demonstrated by double staining with antibodies to CP-H and insulin, and the percentage of beta-cells positive for both of these autoantigens increased twofold with increases in insulin secretion. In contrast, islet cells failed to reveal cell-surface staining for GAD65, another putative autoantigen in IDDM, under either basal or insulin stimulatory conditions or following exposure of islet cells to the cytokines interleukin-1 beta, tumor necrosis factor-alpha, and recombinant human interferon-gamma.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of diabetic vascular dysfunction by guanidines. Inhibition of nitric oxide synthase versus advanced glycation end-product formation.

This study was undertaken to compare the ability of two guanidine compounds (aminoguanidine and methylguanidine), with different in vitro effects on NO synthase activity and AGE formation, to inhibit diabetic vascular dysfunction developing early after the onset of diabetes. In rats with STZ-induced diabetes of 5-wk duration, regional vascular [125I]albumin permeation was increased about two- to threefold in ocular tissues, sciatic nerve, and aorta; in general, both guanidine compounds normalized albumin permeation in diabetic rats without affecting it in controls. Methylguanidine was only approximately 7% as effective as aminoguanidine as an inhibitor of AGE formation from L-lysine and G6P; both compounds were poor inhibitors of AR. Methylguanidine was approximately 1-5% as potent as aminoguanidine and L-NMMA as an inhibitor of the cytokine- and endotoxin-inducible isoform of NO synthase. In contrast, the potency of methylguanidine as an inhibitor of the constitutive isoform of NO synthase was comparable to that of aminoguanidine, and both guanidine compounds were much less effective than L-NMMA. These observations suggest a role for a relative or absolute increase in NO production in the pathogenesis of early diabetic vascular dysfunction and raise the possibility that inhibition of diabetic vascular functional changes by aminoguanidine may reflect inhibition of NO synthase activity rather than, or in addition to, prevention of AGE formation.

Aminoguanidine, a novel inhibitor of nitric oxide formation, prevents diabetic vascular dysfunction.

Increased blood flow and vascular leakage of proteins preferentially affect tissues that are sites of diabetic complications in humans and animals. These vascular changes in diabetic rats are largely prevented by aminoguanidine. Glucose-induced vascular changes in nondiabetic rats are also prevented by aminoguanidine and by NG-monomethyl-L-arginine (NMMA), an established inhibitor of nitric oxide (NO.) formation from L-arginine. Aminoguanidine and NMMA are equipotent inhibitors of interleukin-1 beta-induced 1) nitrite formation (an oxidation product of NO.) and cGMP accumulation by the rat beta-cell insulinoma cell line RINm5F, and 2) inhibition of glucose-stimulated insulin secretion and formation of iron-nitrosyl complexes by islets of Langerhans. In contrast, NMMA is approximately 40 times more potent than aminoquanidine in elevating blood pressure in nondiabetic rats. These results demonstrate that aminoguanidine inhibits NO. production and suggest a role for NO. in the pathogenesis of diabetic vascular complications.

K cells: a novel target for insulin gene therapy for the prevention of diabetes.

Recently, gut K cells have been shown to express glucokinase, the glucose sensor of pancreatic beta cells, and transgenic mice expressing human insulin under the control of a K cell-specific promoter are resistant to diabetes development induced by the beta-cell toxin streptozotocin. These novel findings suggest that gut K cells might be a suitable target for gene therapeutic treatment of type 1 diabetes mellitus.

Interleukin 1-induced Fos and Jun do not regulate inducible nitric oxide synthase in rat islets of Langerhans and RINm5F cells.

Recent evidence indicates that nitric oxide (NO) produced after expression of inducible NO synthase (iNOS) mediates cytokine-induced inhibition of insulin secretion by pancreatic islets. The current studies were designed to characterize the involvement of immediate-early response genes, c-fos and c-jun, in interleukin 1 (IL-1)-induced expression of iNOS. iNOS messenger RNA (mRNA) expression by both rat islets and RINm5F cells was time dependent, with maximal expression observed after an approximately 3- to 6-h exposure to IL-1. IL-1 also stimulated rapid and transient expression of c-fos and c-jun by both rat islets and RINm5F cells, with maximal mRNA accumulation detected 30-60 min after IL-1 treatment. IL-1-induced protein synthesis of Fos and Jun was observed as early as 30 min, peaked between 3-5 h, and decreased by 8 h after IL-1 treatment. Temporal correlation of Fos and Jun expression and iNOS gene induction suggested that Fos and Jun might regulate iNOS gene transcription by rodent pancreatic beta-cells. The present study, however, indicates that IL-1 induced expression of Fos and Jun does not seem to participate in the regulation of iNOS and mRNA expression, because: 1) cycloheximide (1 microM) completely inhibited Fos expression but had no inhibitory effect on iNOS mRNA levels; and 2) tyrosine kinase inhibitors genistein and herbimycin A completely inhibited IL-1 induced iNOS expression but did not block c-fos and c-jun expression. These results indicate that two separate signaling pathways may exist for induction of c-fos and c- jun and iNOS genes and that de novo synthesis of Fos and Jun does not participate in the regulation of iNOS gene expression.

Irreversible inhibition of metabolic function and islet destruction after a 36-hour exposure to interleukin-1beta.

The purpose of this study was to identify the duration of exposure of islets to interleukin 1beta (IL-1beta) that results in irreversible damage. Treatment of rat islets for 18 h with IL-1beta results in an inhibition of glucose-stimulated insulin secretion, mitochondrial aconitase activity, and total protein synthesis. The addition of N(G)-monomethyl-L-arginine (NMMA) or aminoguanidine to islets preincubated for 18 h with IL-1beta, followed by continued culture for 8 h (with both NMMA and IL-1beta), results in the recovery of islet secretory function, aconitase activity, and protein synthesis. However, islet metabolic function is irreversibly inhibited after a 36-h incubation with IL-1beta, as an additional 8-h incubation with NMMA or aminoguanidine does not stimulate the recovery of insulin secretion, aconitase activity, or protein synthesis. The irreversible inhibition of metabolic function correlates with the commitment of islets to destruction. Treatment of islets for 96 h with IL-1beta results in islet degeneration. NMMA, added to islets 24 h after the addition of IL-1beta, followed by continued culture for 72 h (with NMMA and IL-1beta), prevents islet degeneration. However, NMMA added to islets 36 h or 48 h after the addition of IL-1beta, followed by continued culture for a total of 96 h, does not prevent islet degeneration. New messenger RNA expression appears to be required for islet recovery from IL-1beta-induced damage as actinomycin D prevents the recovery of islet aconitase activity. Lastly, treatment of human islets with a combination of IL-1beta and interferon-gamma (IFNgamma) results in a potent inhibition of mitochondrial aconitase activity. NMMA, when cocultured with IL-1beta + IFNgamma, completely prevents cytokine-induced inhibition of human islet aconitase activity. NMMA, when added to human islets pretreated for 18 h with IL-1beta + IFNgamma, stimulates the recovery of mitochondrial aconitase activity after an additional 8 h incubation. These findings indicate that nitric oxide-induced islet damage is reversible; however, prolonged production of nitric oxide (after a 36-h exposure to IL-1beta) results in the irreversible inhibition of islet metabolic and secretory function.

Heat shock inhibits cytokine-induced nitric oxide synthase expression by rat and human islets.

In this study the effects of heat shock on interleukin-1beta (IL-1)-induced inhibition of islet metabolic function were examined. Treatment of rat islets for 18 h with IL-1 results in a potent inhibition of glucose-stimulated insulin secretion. The inhibitory effects of IL-1 on insulin secretion are completely prevented if islets are pretreated for 60 min at 42 C before cytokine stimulation. Heat shock also prevents IL-1-induced inhibition of insulinoma RINm5F cell mitochondrial aconitase activity. The protective effects of heat shock on islet metabolic function are associated with the inhibition of IL-1-stimulated inducible nitric oxide synthase (iNOS or NOS II) expression. Islets heat shocked for 60 min at 42 C fail to express iNOS (messenger RNA or protein) or produce nitrite in response to IL-1. IL-1-induced iNOS expression by rat islets requires activation of the transcriptional regulator nuclear factor kappaB (NF-kappaB). Heat shock prevents IL-1-induced NF-kappaB nuclear localization by inhibiting inhibitory protein kappaB (IkappaB) degradation in rat islets. Similar to rat islets, heat shock (stimulated by 90 min incubation at 42 C) prevents IL-1 + interferon gamma-induced iNOS expression and NF-kappaB nuclear localization in human islets. IL-1 also stimulates heat-shock protein 70 (hsp 70) expression by rat islets, and hsp 70 expression is dependent on islet production of nitric oxide. Last, evidence is presented that implicates nitric oxide as a stimulus for the expression of proteins that participate in islet recovery from nitric oxide-mediated damage. These studies indicate that heat shock prevents cytokine-induced islet damage by inhibiting iNOS expression, and suggest that nitric oxide is one effector molecule that stimulates the expression of factors involved in beta-cell recovery from nitric oxide-mediated damage.