Differential effects of proinflammatory cytokines on cell death and ER stress in insulin-secreting INS1E cells and the involvement of nitric oxide

Proinflammatory cytokines produced by immune cells destroy pancreatic beta cells in type 1 diabetes. The aim of this study was to investigate the cytokine network and its effects in insulin-secreting cells. INS1E cells were exposed to different combinations of proinflammatory cytokines. Cytokine toxicity was estimated by MTT assay and caspase activation measurements. The NFκB-iNOS pathway was analyzed by a SEAP reporter gene assay, Western-blotting and nitrite measurements. Gene expression analyses of ER stress markers, Chop and Bip, were performed by real-time RT-PCR. Cytokines tested in this study, namely IL-1β, TNFα and IFNγ, had deleterious effects on beta cell viability. The most potent toxicity exhibited IL-1β and its combinations with other cytokines. The toxic effects of IL-1β towards cell viability, caspase activation and iNOS activity were dependent on nitric oxide and abolished by an iNOS blocker. IL-1β was the strongest inducer of the NFκB activation. An iNOS blocker inhibited IL-1β-mediated NFκB activation in the first, initial phase of cytokine action, but did not affect significantly NFκB activation after prolonged incubation. Interestingly iNOS protein expression was induced predominantly by IL-1β and decreased in the presence of an iNOS blocker in the case of a short time exposure. The changes in the expression of ER stress markers were also almost exclusively dependent on the IL-1β presence and counteracted by iNOS blockade. Thus cytokine-induced beta cell death is primarily IL-1β mediated with a NO-independent enhancement by TNFα and IFNγ. The deleterious effects on cell viability and function are crucially dependent on IL-1β-induced nitric oxide formation.     

Cytokine-induced inhibition of insulin release from mouse pancreatic beta-cells deficient in inducible nitric oxide synthase

Cytokines may participate in islet destruction during the development of type 1 diabetes. Expression of inducible nitric oxide synthase (iNOS) and subsequent NO formation induced by IL-1 beta or (IL-1 beta + IFN-gamma) may impair islet function in rodent islets. Inhibition of iNOS or a deletion of the iNOS gene (iNOS -/- mice) protects against cytokine-induced beta-cell suppression, although cytokines might also induce NO-independent impairment. Presently, we exposed wild-type (wt, C57BL/6 x 129SvEv) and iNOS -/- islets to IL-1 beta (25 U/ml) and (IL-1 beta (25 U/ml) + IFN-gamma (1000 U/ml)) for 48 h. IL-1 beta and (IL-1 beta + IFN-gamma) induced a significant increase in NO formation in wt but not in iNOS -/- islets. Both IL-1 beta and (IL-1 beta + IFN-gamma) impaired glucose-stimulated insulin release and reduced the insulin content of wt islets, while (IL-1 beta + IFN-gamma) reduced glucose oxidation rates and cell viability. IL-1 beta exposure to iNOS -/- islets impaired glucose-stimulated insulin release, increased insulin accumulation and reduced the insulin content, without any increase in cell death. Exposure to (IL-1 beta + IFN-gamma) had no effect on iNOS -/- islets except reducing the insulin content. Our data suggest that IL-1 beta may inhibit glucose-stimulated insulin release by pathways that are not NO-dependent and not related to glucose metabolism or cell death.     

Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.     

Interleukin-1beta-induced nitric oxide production in rat aortic endothelial cells: inhibition by estradiol in normal and high glucose cultures.

Expression of inducible nitric oxide synthase (iNOS) and the resultant increased nitric oxide (NO) production are associated with septic shock, atherosclerosis, and cytokine-induced vascular injury. Estrogen is known to impact vascular injury and vascular tone, in part through regulation of NO production. In the current study, we examined the effect of physiological concentrations of estradiol on interleukin-1beta (IL-1beta)-induced NO production in rat aortic endothelial cells (RAECs). 17Beta-estradiol significantly decreased IL-1beta-induced iNOS protein levels and reduced NO production in RAECs. High glucose (25 mM) elevated the increase in IL-1beta-induced iNOS protein and NO production. Nevertheless, estradiol still inhibited IL-1beta-induced iNOS and NO production even in the presence of high glucose. These data suggest that estradiol may exert its beneficial effects in part by inhibiting induction of endothelial iNOS, a possible mechanism for the protective effect of estradiol against diabetes-associated cardiovascular complications.     

Amomum xanthoides extract prevents cytokine-induced cell death of RINm5F cells through the inhibition of nitric oxide formation

We previously showed that Amomum xanthoides extract prevented alloxan-induced diabetes through the suppression of NF-kappaB activation. In this study, the preventive effects of A. xanthoides extract on cytokine-induced beta-cell destruction were examined. Cytokines produced by immune cells infiltrating pancreatic islets are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. A. xanthoides extract completely protected interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma)-mediated cytotoxicity in rat insulinoma cell line (RINm5F). Incubation with A. xanthoides extract resulted in a significant reduction in IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding that correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism by which A. xanthoides extract inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. Our results revealed the possible therapeutic value of A. xanthoides extract for the prevention of diabetes mellitus progression.     

Mechanisms of cytokine induced NO-mediated cardiac fibroblast apoptosis

This study examined the role of nitric oxide (NO) in cytokine-induced apoptosis in adult cardiac fibroblasts (CFbs). In cultured adult rat CFbs, IL-1beta (5 ng/ml), but not interferon-gamma (10 ng/ml) or tumor necrosis factor-alpha (10 ng/ml), induced inducible NO synthase (iNOS) expression and NO production that was associated with an increase in caspase-3 activity and apoptotic cell death. Apoptotic frequency was reduced by the iNOS inhibitor S-methylisothiourea (3 x 10(-5) M). Apoptosis in response to IL-1beta was attenuated by the caspase-3 inhibitor [Z-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DVED-FMK)] but not by inhibition of guanylyl cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). IL-1beta-induced CFb apoptosis was associated with an increase in p53 and Bax protein expression with no changes in Bcl-2 or Bcl-x(L). Nuclear condensation and fragmentation occurred when isolated nuclei were exposed to an NO donor [Z-1[N-(2-aminoethyl)-N-(2-ammonoethyl)amino]diazen-1-ium-1,2-dioate (DETA-NONOate) 10(-5) M], an effect that was not blocked by the peroxynitrite scavenger Mn(III)tetrakis(4-benzoic acid) porphyrin chloride. Moreover, Mn(III)tetrakis(4-benzoic acid) porphyrin chloride attenuated but did not eliminate IL-1beta-induced CFb apoptosis, indicating that the proapoptotic effect of NO can occur independently of its conversion to peroxynitrite. Our results demonstrate that IL-1beta-induced iNOS expression can trigger NO-dependent apoptosis in adult CFbs, which appears to result from DNA damage and may be mediated by a p53-dependent apoptotic pathway.     

Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide

Mesenchymal stem cells (MSCs) can become potently immunosuppressive through unknown mechanisms. We found that the immunosuppressive function of MSCs is elicited by IFNgamma and the concomitant presence of any of three other proinflammatory cytokines, TNFalpha, IL-1alpha, or IL-1beta. These cytokine combinations provoke the expression of high levels of several chemokines and inducible nitric oxide synthase (iNOS) by MSCs. Chemokines drive T cell migration into proximity with MSCs, where T cell responsiveness is suppressed by nitric oxide (NO). This cytokine-induced immunosuppression was absent in MSCs derived from iNOS(-/-) or IFNgammaR1(-/-) mice. Blockade of chemokine receptors also abolished the immunosuppression. Administration of wild-type MSCs, but not IFNgammaR1(-/-) or iNOS(-/-) MSCs, prevented graft-versus-host disease in mice, an effect reversed by anti-IFNgamma or iNOS inhibitors. Wild-type MSCs also inhibited delayed-type hypersensitivity, while iNOS(-/-) MSCs aggravated it. Therefore, proinflammatory cytokines are required to induce immunosuppression by MSCs through the concerted action of chemokines and NO.     

LPS/IFN-γ-Induced RAW 264.7 Apoptosis is Regulated by Both Nitric Oxide–Dependent and –Independent Pathways Involving JNK and the Bcl-2 Family

Lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) stimulate macrophages to produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS) and activate stress signaling cascades including the c-jun-N-terminal kinase (JNK) pathway. These events trigger an apoptotic cascade that ultimately results in death. Since JNK regulates pro-apoptotic and anti-apoptotic Bcl-2 family members, the role of NO in LPS/IFN-γ-induced activation of JNK and its effects on the Bcl-2 family was examined in RAW 264.7 macrophage-like cells. Inhibition of JNK by siRNA verified a role for JNK in LPS/IFN-γ-induced apoptosis. Suppression of NO production by a pharmacologic agent, i.e. iNOS inhibitor L-NIL, altered the kinetics of JNK activation by LPS/IFN-γ. Examination of mitochondrial and nuclear compartments of RAW 264.7 cells demonstrated NO-dependent activation of mitochondrial JNK by LPS/IFN-γ, but NO-independent, cytokine-induced phosphorylation of Bim. NO did not affect phosphorylation, but did inhibit Bax phosphorylation. These results suggest a novel mechanism of LPS/IFN-γ-induced apoptosis in macrophages involving NO-independent phosphorylation of Bim and NO-dependent dephosphorylation of Bax.     

Activation of peroxisome proliferator-activated receptor-gamma protects pancreatic beta-cells from cytokine-induced cytotoxicity via NF kappaB pathway

Diabetes mellitus is characterized by cytokine-induced insulitis and a deficit in beta-cell mass. Ligands for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) have been shown to have anti-inflammatory effects in various experimental models. We questioned whether activation of endogenous PPAR-gamma by either PPAR-gamma ligands or adenoviral-directed overexpression of PPAR-gamma (Ad-PPAR-gamma) could inhibit cytokine-induced beta-cell death in RINm5F (RIN) cells, a rat insulinoma cell line. Treatment of RIN cells with interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) induced beta-cell damage through NF kappaB-dependent signaling pathways. Activation of PPAR-gamma by PPAR-gamma ligands or Ad-PPAR-gamma inhibited IL-1 beta and IFN-gamma-stimulated nuclear translocation of the p65 subunit and DNA binding activity. NF kappaB target gene expression and their product formation, namely inducible nitric oxide synthase and cyclooxygenase-2 were decreased by PPAR-gamma activation, as established by real-time PCR, Western blots and measurements of NO and PGE(2). The mechanism by which PPAR-gamma activation inhibited NF kappaB-dependent cell death signals appeared to involve the inhibition of I kappa B alpha degradation, evidenced by inhibition of cytokine-induced NF kappaB-dependent signaling events by Ad-I kappaB alpha (S32A, S36A), non-degradable I kappaB alpha mutant. I kappaB beta mutant, Ad-I kappaB beta (S19A, S23A) was not effective in preventing cytokine toxicity. Furthermore, a protective effect of PPAR-gamma ligands was proved by assaying for normal insulin secreting capacity in response to glucose in isolated rat pancreatic islets. The beta-cell protective function of PPAR-gamma ligands might serve to counteract cytokine-induced beta-cell destruction.     

Activation of extracellular signal-regulated kinase (ERK)1/2 contributes to cytokine-induced apoptosis in purified rat pancreatic beta-cells

Cytokines may contribute to beta-cell apoptosis in the early stages of type 1 diabetes mellitus. It has been reported recently that interleukin-1 beta (IL-1 beta) induces activation of the mitogen-activated protein kinases (MAPK) p38 and ERK1/2 in neonatal rat islets. Since these kinases may participate in cytokine-induced apoptosis, we evaluated whether cytokines induce activation of MAPKs in FACS-purified primary rat beta-cells, and whether blockers of p38 and/or ERK1/2 prevent beta-cell death. IL-1 beta, but not interferon-gamma (IFN-gamma), caused phosphorylation of the substrates Elk-1, ATF-2 and hsp25, and the phosphorylation of both Elk-1 and hsp25 were decreased by the p38 blocker SB203580 (p38i) and the MAPK/ERK blocker PD 098059 (MEKi). When added together, p38i and MEKi decreased IL-1 beta-induced nitrite production over 24 hours by 60%, but did not affect IL-1 beta-induced manganese superoxide dismutase (MnSOD) mRNA expression. To test the effects of MAPK inhibitors on beta-cell death by necrosis or apoptosis, these cells were exposed for 6 or 9 days to IL-1 beta + IFN-gamma. This treatment induced cell death, mostly by apoptosis. The MEKi, but not the p38i, significantly decreased cytokine-induced apoptosis, thus decreasing the total number of dead cells. This protection was only partial, suggesting that ERK1/2 activation is not the only mechanism by which cytokines induce beta-cell apoptosis. We conclude that IL-1 beta induces activation of both p38 and ERK1/2, and that ERK1/2 contributes to the pro-apoptotic effects of the cytokine in primary beta-cells.     

Defined inflammatory states in astrocyte cultures: correlation with susceptibility towards CD95-driven apoptosis

A complete cytokine mix (CCM) or its individual components tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) were used to switch resting murine astrocytes to reactive states. The transformation process was characterized by differential up-regulation of interleukin-6 (IL-6), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) mRNA and protein and a subsequent release of prostaglandin E2, nitric oxide (NO) and IL-6. Both CD95L and anti-CD95 antibodies triggered caspase activation followed by apoptotic death in fully pro-inflammatory astrocytes, whereas resting cells were totally resistant. Two other death-inducing ligands, TNF and TNF-related apoptosis-inducing ligand (TRAIL) did not induce apoptosis in reactive astrocytes. The switch in astrocyte sensitivity was accompanied by up-regulation of caspase-8 and CD95 as well as the capacity to recruit Fas-associated death domain (FADD) to the activated death receptor complex. Neither CD95-mediated death, nor other inflammatory parameters were affected by inhibition of iNOS or COX, respectively. Accordingly, IFN-gamma was absolutely essential for up-regulation of iNOS, but not for the switch in apoptosis sensitivity. In contrast, p38 kinase activity was identified as an important controller of both the inflammatory reaction and apoptosis both in astrocytes stimulated with CCM and in glia exposed to TNF and IL-1 only.