Dexamethasone prevents interleukin-1beta-mediated inhibition of rat islet insulin secretion without decreasing nitric oxide production

The deleterious effects of interleukin 1 (IL-1) on insulin-producing beta-cells are partly mediated by the generation of the free radical nitric oxide (NO). We aimed to assess the effect of several steroidal hormones on IL-1beta-induced inhibition of rat islet insulin secretion in vitro, and their possible regulatory effects on NO production. Incubation of newborn rat islets for 24 h in the presence of 150 pg/ml IL-1beta revealed that dexamethasone dose-dependently attenuated the inhibitory effect of IL-1beta on insulin release in response to a 2-h glucose challenge. Physiological and supraphysiological concentrations of testosterone, 17beta-estradiol, progesterone, 1,25-dihydroxyvitamin-D3 and vitamin D analogues (KH1060 and MC1288) were ineffective. Dexamethasone (1 microM) increased the production of NO in IL-1beta-treated rat islets, as measured by the concentration of nitrite in the media. However, 1-5 microM dexamethasone inhibited IL-1beta-induced NO production by RIN cells. Dexamethasone (1 microM) did not affect the inhibitory action of the NO donor S-nitroso penicillamine (500 microM) on rat islet insulin secretion. We conclude that dexamethasone partially protects against IL-1beta-induced inhibition of rat islet insulin secretion, an effect which is not mediated through modulation of the NO pathway.     

Inhibitory effects of streptozotocin, tumor necrosis factor-alpha, and interleukin-1beta on glucokinase activity in pancreatic islets and gene expression of GLUT2 and glucokinase

Treatment of streptozotocin (ST), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) resulted in destroying insulin-secreting beta-cells of pancreatic islets and impairment of islet glucose oxidation and glucose-induced insulin secretion. IL-1beta and TNF-alpha inhibited insulin release and glucose utilization and oxidation. It was shown that the inhibitory effects of ST, IL-1beta, and TNF-alpha were due to impaired glucokinase activity. Glucokinase activity was severely impaired by ST, IL-1beta, and TNF-alpha treatments, as confirmed by assaying enzymes and nucleotides associated with glycolysis and glucose oxidation. On the other hand, nitric oxide was a factor of the deleterious effects of IL-1beta, TNF-alpha, and ST on pancreatic islets. Incubation of mouse pancreatic islets with ST at various concentrations of impairing insulin secretion resulted in generation of nitrite, stimulation of islet guanylyl cyclase and accumulation of cGMP, and inhibition of pancreatic islet mitochondrial aconitase activity to degree similar to those raised by IL-1beta and TNF-alpha. When the effects of IL-1beta and TNF-alpha on the gene expression of pancreatic GLUT2 and glucokinase were examined, the level of GLUT2 and glucokinase mRNA in pancreatic islets was significantly decreased. This suggested that IL-1beta and TNF-alpha downregulate gene expression of GLUT2 and glucokinase in pancreatic beta-cells.     

Ciliary neurotrophic factor potentiates the beta-cell inhibitory effect of IL-1beta in rat pancreatic islets associated with increased nitric oxide synthesis and increased expression of inducible nitric oxide synthase

Proinflammatory cytokines are implicated as effector molecules in the pathogenesis of IDDM. Interleukin-6 (IL-6) alone or in combination with IL-1beta inhibits glucose-stimulated insulin release from isolated rat pancreatic islets by unknown mechanisms. Here we investigated 1) if the effects of IL-6 are mimicked by ciliary neurotrophic factor (CNTF), another member of the IL-6 family of cytokines signaling via gp130, 2) the possible cellular mechanisms for these effects, and 3) if islet endocrine cells are a source of CNTF. CNTF (20 ng/ml) potentiated IL-1beta-mediated (5-150 pg/ml) nitric oxide (NO) synthesis from neonatal Wistar rat islets by 31-116%, inhibition of accumulated insulin release by 34-49%, and inhibition insulin response to a 2-h glucose challenge by 31-36%. CNTF potentiated IL-1beta-mediated NO synthesis from RIN-5AH cells by 83%, and IL-1beta induced islet inducible NO-synthase (iNOS) mRNA expression fourfold. IL-6 (10 ng/ml) also potentiated IL-1beta-mediated NO synthesis and inhibition of insulin release, whereas beta-nerve growth factor (NGF) (5 or 50 ng/ml) had no effect. mRNA for CNTF was expressed in rat islets and in islet cell lines. In conclusion, CNTF is constitutively expressed in pancreatic beta-cells and potentiates the beta-cell inhibitory effect of IL-1beta in association with increased iNOS expression and NO synthesis, an effect shared by IL-6 but not by beta-NGF. These findings indicate that signaling via gp130 influences islet NO synthesis associated with iNOS expression. We hypothesize that CNTF released from destroyed beta-cells during the inflammatory islet lesion leading to IDDM may potentiate IL-1beta action on the beta-cells.     

Role of iron in the potentiation of anthracycline cardiotoxicity: identification of heart cell mitochondria as a major site of iron-anthracycline interaction

The effect of nitric oxide on the lipopolysaccharide (LPS)-induced cytokine production by alveolar macrophages was studied. When alveolar macrophages were cultured, substantial amounts of interleukin-1(IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha(TNF-alpha), and nitric oxide are produced upon stimulation with LPS. Inhibition of the nitric oxide production by the L-arginine analogue N(G)-monomethyl-L-arginine (NMMA), resulted in an increase of IL-1(beta) and IL-6, whereas the TNF-alpha concentrations remained unchanged, suggesting specific inhibitory effects of nitric oxide on the LPS-stimulated cytokine production by alveolar macrophages. The observed cytokine-modulation properties of nitric oxide did not result from cytotoxic actions of the oxidation of L-arginine on macrophages, since nitric oxide synthesis did not affect the viability of the alveolar macrophages. Conversely the nitric oxide donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) induced dose-dependent inhibition of IL-1 production in LPS-stimulated alveolar macrophages in which endogenous nitric oxide production was blocked. The results indicate that nitric oxide can affect the LPS-induced IL-1beta and IL-6 secretion by alveolar macrophages in an autoregulatory way and are discussed in view of the important physiologic consequences this autoregulation by nitric acid oxide may have.     

Studies on hyperthermia combined with arterial blockade for treatment of tumors: (Part I) effectiveness of hyperthermia combined with arterial ligation

The role of nitric oxide (NO) in glucose-induced insulin secretion was studied in pancreatic beta-cells, HIT-T15. A role for NO is suggested since glucose stimulated NO production in a concentration-dependent manner. NG-monomethyl-L-arginine, a potent inhibitor of nitric oxide synthase, significantly inhibited glucose-induced nitric oxide production as well as insulin release in HIT-T15. Furthermore, this inhibitory effect can be reversed by sodium nitroprusside (SNP), a well known NO donor. While SNP alone did not stimulate insulin release, it potentiated the secretory response of HIT-T15 cells to glucose by approximately two-fold. Potentiation by SNP appears to be mediated by NO, since (i) the potentiation was completely abolished by 10 microM hemoglobin, a scavenger of NO; and (ii) was not affected by rhodanese plus sodium thiosulphate. Neither hemoglobin alone nor the combination of rhodanese and sodium thiosulphate had any effect on glucose induced insulin release. These results are consistent with the hypothesis that glucose-induced formation of NO may potentiate the effect of glucose by a positive feedback mechanism.     

Protein serine/threonine kinases (PKA, PKC and CaMKII) involved in ischemic brain pathology

Triptoquinone A (TQA), which is an anti-inflammatory constituent in plants, was studied for its suppressive effect on nitric oxide production by LPS. TQA significantly suppressed smooth muscle relaxation and increase in cyclic GMP levels by nitric oxide (NO) in an L-arginine-induced relaxation experiment. The mechanistic studies showed that TQA did not directly inhibit NO radicals and inducible nitric oxide synthase (iNOS) enzyme but suppressed IL-1 beta and iNOS mRNA expression by LPS. The suppression level of iNOS gene expression by TQA was comparable to that by dexamethasone. TQA may be a useful candidate for the development of a drug as a potent inhibitor of iNOS gene over-expression.     

Induction of a macrophage-like nitric oxide synthase in cultured rat aortic endothelial cells. IL-1 beta-mediated induction regulated by tumor necrosis factor-alpha and IFN-gamma

We investigated the effects of murine rTNF-alpha, human rIL-1 beta, and rat rIFN-gamma in various concentrations and/or combinations on inducible nitric oxide (NO) production in primary cultures of rat aortic endothelial cells. Northern blot analysis of total RNA from induced and control cultures using the cloned mouse macrophage gene of inducible NO synthase as probe as well as polymerase chain reaction using a specific primer sequence gave a positive signal for activated cells only. A RNA approximately 4.4 kb of length similar to the inducible form of NO synthase in macrophages was labeled. The concentration of nitrite as a stable reaction product of NO in culture supernatants was determined 24 h after incubation with the various cytokines. IL-1 beta alone (40 to 1000 U/ml) induced formation of increasing amounts of nitrite with increasing concentrations of IL-1 beta present. Neither TNF-alpha alone (10 to 2000 U/ml) nor IFN-gamma alone 25 to 500 U/ml) showed significant effects on nitrite production. Simultaneous incubation with low concentrations of TNF-alpha (< or = 100 U/ml) and IL-1 beta abrogated the induction effect of IL-1 beta. Conversely, addition of high concentrations of TNF-alpha (> or = 500 U/ml) led to near maximal levels of nitrite formation even at lowest IL-1 beta concentrations (40 U/ml). In addition, simultaneous incubation of endothelial cells with IFN-gamma plus IL-1 beta and/or TNF-alpha led to near maximal NO production of endothelial cells, even at lowest IFN-gamma concentrations (25 U/ml). We hypothesize that the regulating effect of TNF-alpha may in vivo help to prevent local inflammatory responses from spreading to intact sites.     

Interleukin 1 beta, but not tumor necrosis factor, enhances neurogenic vasodilatation in the rat skin: involvement of nitric oxide

In phenobarbitone-anesthetized rats the effects of interleukin 1 beta (IL-1 beta) and tumor necrosis factors (TNFs) were examined on the capsaicin-induced increase of plantar cutaneous blood flow in the rat hind paw as measured by laser Doppler flowmetry. IL-1 beta (0.5-500 pg) or TNF alpha or TNF beta (50-500 pg) was injected subcutaneously into the left paws, while the right paws received vehicle (10 microL) only. IL-1 beta was without effect on blood flow by its own but dose dependently enhanced the hyperemia due to capsaicin (0.3 microgram). TNFs failed to enhance the capsaicin-induced vasodilatation although 5000 pg TNF alpha produced a transient increase of local blood flow. Indomethacin (10 mg/kg, i.p.) did not alter the capsaicin-induced vasodilatation but prevented IL-1 beta (50 pg) from augmenting the hyperemic response to capsaicin. Likewise, blockade of nitric oxide formation by NG-nitro-L-arginine methyl ester (L-NAME) failed to affect the capsaicin-evoked vasodilatation but abolished its amplification by IL-1 beta. Systemic pretreatment with a neurotoxic dose of capsaicin reduced the capsaicin-induced hyperemia and prevented the facilitatory effect of IL-1 beta. The hyperemia evoked by intraplantar calcitonin gene related peptide (0.038-3.8 ng) was not altered by IL-1 beta (50 pg). These data indicate that IL-1 beta but not TNF enhances the cutaneous hyperemic response to capsaicin. This proinflammatory action arises from sensitization of afferent nerve endings and depends on nitric oxide and cyclooxygenase products as essential intermediates.     

Effects of tiludronate and ibandronate on the secretion of proinflammatory cytokines and nitric oxide from macrophages in vitro

Bisphosphonates inhibit osteoclastic bone resorption and are used for the treatment of bone diseases. Some bisphosphonates, such as clodronate and tiludronate, can be incorporated into non-hydrolysable ATP analogues in cells, whereas the more potent anti-resorptive aminoalkylbisphosphonates are not metabolised. Furthermore, clodronate inhibits proinflammatory cytokine and nitric oxide (NO) secretion from activated macrophages in vitro and has anti-inflammatory properties in vivo, especially when delivered into cells by liposomes. By contrast, aminobisphosphonates can induce an acute phase response and fever in vivo, which appears to involve the induction of cytokine secretion. In this study we examined the effect of liposome-mediated intracellular delivery of one aminobisphosphonate, ibandronate, and one metabolizable bisphosphonate, tiludronate, on the secretion of inflammatory mediators. The intracellular uptake of bisphosphonates by macrophages was enhanced by a factor of 20-200 by using liposomes. Tiludronate dose-dependently inhibited both cytokine and NO secretion from activated macrophages, and liposomal tiludronate was more potent than the free drug. By contrast, ibandronate enhanced LPS-induced secretion of IL-1beta and IL-6 but did not affect TNFalpha or NO secretion at non-cytotoxic concentrations. The present results, together with our previous studies, strongly suggest that bisphosphonates can be grouped into those that are metabolised by cells and that are capable of inhibiting cytokine and NO secretion from macrophages, thus having potential anti-inflammatory properties, and those that are not metabolised but can actually enhance the production of cytokines following macrophage activation.     

Tumor-derived recognition factor (TDRF) induces production of TNF-alpha by murine macrophages, but requires synergy with IFN-gamma alone or in combination with IL-2 to induce nitric oxide synthase

A constitutively produced soluble activity, designated tumor-derived recognition factor (TDRF), from L1210, P815 and EL4 tumor targets, was previously shown to synergize with interferon-gamma (IFN-gamma) and subactivating concentrations of interleukin-2 (IL-2) to induce murine macrophage production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) for cytotoxicity of the target of origin. Another study had suggested that TDRF upregulated both TNF-alpha receptor (TNF-alpha R) and IFN-gamma receptor (IFN-gamma R) mRNA synthesis, as well as increased TNF-alpha and IFN-gamma binding to their receptors. In the present study, we have further characterized the concentration-dependent macrophage activating potential of TDRF alone and in synergy with IFN-gamma or IFN-gamma and subactivating concentrations of IL-2. Higher concentrations of TDRF acted independently on inflammatory C3H FeJ mouse macrophage to induce expression of TNF-alpha mRNA and release of TNF-alpha, but failed to induce nitric oxide synthase (NOS) mRNA expression and NO generation. At lower concentrations, TDRF synergized with either IFN-gamma alone or in combination with IL-2 to stimulate a dose-related increase in the expression of TNF-alpha mRNA and secretion of TNF-alpha, as well as increased induction of NOS mRNA and cytotoxic NO generation by macrophage. MCA tumor targets which did not produce TDRF activity were killed by macrophage that had been activated by exogenously added L1210-derived TDRF in synergy with IFN-gamma or in combination with subactivating concentrations of IL-2, but not by TDRF alone. Taken together, our results indicate that TDRF acted independently in a dose-dependent fashion to induce macrophage synthesis and release of TNF-alpha, but in the absence of IFN-gamma or in combination with IL-2 failed to induce the NOS enzyme which was necessary for cytotoxic NO generation. Thus TDRF appears to be a sufficient second signal for IFN-gamma-primed macrophage or alternatively a sufficient third signal for IFN-gamma and IL-2 treated macrophage to culminate the activation process for NOS mRNA synthesis and NO-mediated tumor cytotoxicity.     

HindIII liposomes suppress delayed-type hypersensitivity responses in vivo and induce epidermal IL-10 in vitro

The purpose of this study was to evaluate the effects of resident islet macrophage activation on beta cell function. Treatment of freshly isolated rat islets with TNF-alpha and LPS results in a potent inhibition of glucose-stimulated insulin secretion. The inhibitory actions of TNF + LPS are mediated by the intraislet production and release of IL-1 followed by IL-1-induced inducible nitric oxide synthase (iNOS) expression by beta cells. The IL-1R antagonist protein completely prevents TNF + LPS-induced nitrite production, iNOS expression and the inhibitory effects on glucose-stimulated insulin secretion by rat islets. Resident macrophages appear to be the source of IL-1, as a 7-day culture of rat islets at 24 degrees C (conditions known to deplete islets of lymphoid cells) prevents TNF + LPS-induced iNOS expression, nitrite production, and the inhibitory effects on insulin secretion. In addition, macrophage depletion also inhibits TNF + LPS-induced IL-1alpha and IL-1beta mRNA expression in rat islets. Immunocytochemical colocalization of IL-1beta with the macrophage-specific marker ED1 was used to provide direct support for resident macrophages as the islet cellular source of IL-1. IL-1beta appears to mediate the inhibitory actions of TNF + LPS on beta cell function as TNF + LPS-induced expression of IL-1beta is fourfold higher than IL-1alpha, and Ab neutralization of IL-1beta prevents TNF + LPS-induced nitrite production by rat islets. These findings support a mechanism by which the activation of resident islet macrophages and the intraislet release of IL-1 may mediate the initial dysfunction and destruction of beta cells during the development of autoimmune diabetes.     

Aspirin and salicylate enhances the induction of inducible nitric oxide synthase in cultured rat smooth muscle cells

Aspirin and sodium salicylate enhance to a similar extent the production of nitric oxide (NO) in cultured smooth muscle cells following stimulation by interleukin-1beta (IL-1beta). The similar potencies of aspirin and sodium salicylate indicate that acetylation of cellular macromolecules is not essential for the enhancement of NO production. The failure of added prostaglandin E2 (PGE2) or Thromboxane A2 (TXA2) to overcome the effects of aspirin or sodium salicylate indicates that these effects are not simply the result of inhibition of prostaglandin synthesis. The enhancement of NO production occurs dependent of the effects of these agents on induction of inducible nitric oxide synthase (iNOS) expression by IL-1beta. Aspirin and sodium salicylate enhance the induction of iNOS expression by IL-1beta. We previously reported that pretreatment of vascular smooth muscle cells (VSMCs) with high glucose decreased the response of the cells by IL-1beta, that is, the induction of iNOS expression and NO production. We investigated the effect of aspirin and sodium salicylate on the response by IL-1beta of VSMCs pretreated with high glucose (25 mM). Aspirin and sodium salicylate ameliorate the down-regulation of iNOS expression and the decrease of NO production caused by pretreatment with high glucose (25 mM). These results suggest a possible therapeutic role in atherosclerotic disease and diabetes mellitus for aspirin and sodium salicylate by enhancing the level of iNOS expression and NO production.     

The morphogenic/cytotoxic and prostaglandin-stimulating activities of interleukin-1 beta in the rat ovary are nitric oxide independent

Nitric oxide (NO) has been implicated as a mediator of physiologic and pathologic cellular injury. Since the cytokine interleukin-1 beta (IL-1 beta) induces nitric oxide synthase (NOS) activity as well as effects morphogenic/cytotoxic changes and increased prostaglandin (PGE2) levels in cultured whole ovarian dispersates, we set out to determine whether these actions are interrelated. Treatment with IL-1 beta resulted in a marked increase in media nitrite and nitrate accumulation, morphological alterations, and increased release of lactate dehydrogenase (LDH) into media. Addition of IL-1 receptor antagonist (RA) eliminated these IL-1 beta effects. In contrast, specific inhibitors of NOS failed to reverse IL-1 beta-induced morphogenic changes or LDH release in spite of complete reduction of media nitrite to control levels. Similarly, treatment with transforming growth factor beta 1, inhibited IL-1 beta-induced nitrite accumulation, but had no effect on the morphologic or cytotoxic endpoints. Moreover, the addition of sodium nitroprusside, an NO generator, resulted in progressive increments in media nitrite content without a corresponding increase in the IL-1 beta-associated morphogenic changes or media LDH content. Furthermore, IL-1-induced PGE2 accumulation remained unaffected by specific NOS inhibition. These observations support the view that NO does not mediate the morphogenic/cytotoxic or inflammatory-like (e.g., PGE2 inducing) properties of IL-1 beta in cultured whole ovarian dispersates. Although the precise role of NO in ovarian physiology remains unknown, it is possible that NO participates in the periovulatory modulation of ovarian blood flow by virtue of its potent vasodilatory activity.     

Reconstitution of hyperacetylated, DNase I-sensitive chromatin characterized by high conformational flexibility of nucleosomal DNA

The normal pancreatic beta-cell population exhibits intercellular differences in its responsiveness to glucose. This cellular heterogeneity allows glucose to regulate, in a dose-dependent manner, total rates of insulin synthesis and release. It may also predispose to intercellular differences in susceptibility to dysregulating agents. The present study examines whether this is the case for interleukin 1beta (IL-1beta), which is known to suppress glucose-induced insulin synthesis and release. The effects of the cytokine were compared on beta-cell subpopulations with, respectively, high and low sensitivity to glucose. These subpopulations were separated on the basis of differences in the cellular metabolic responsiveness to an intermediate glucose concentration (7.5 mmol/liter) and then cultured for 20 h at 5 or 20 mmol/liter with or without IL-1beta. The suppressive action of IL-1beta (0.1 ng/ml) occurred predominantly in glucose-activated beta cells, reducing their high rates of insulin synthesis and release by more than 80%. Glucose-unresponsive cells became subject to a similar inhibition after their activation during culture at 20 mmol/liter glucose. On the other hand, IL-1beta induced or enhanced the expression of several noninsulin proteins in both subpopulations. The IL-1beta-stimulated expression of inducible nitric oxide synthase (iNOS) and heat shock protein 70 was more marked in the glucose-responsive subpopulation; that of heme oxygenase and Mn superoxide dismutase was comparable in the two subpopulations. Exposure to IL-1beta resulted in 10-fold higher medium nitrite levels in both subpopulations; this effect was prevented by the iNOS blocker, N(G)-methyl-L-arginine, which also prevented the IL-1beta-induced suppression in the glucose-responsive subpopulation. This study demonstrates that the cellular heterogeneity in glucose responsiveness predisposes to intercellular differences in the IL-1-induced suppression of insulin synthesis and release. While the cytokine induces the expression of noninsulin proteins such as iNOS in both glucose responsive and unresponsive cells, the subsequent nitric oxide production appears to predominantly affect glucose-stimulated functions in the glucose-activated cells.     

In the rat, endogenous nitric oxide modulates the response of the hypothalamic-pituitary-adrenal axis to interleukin-1 beta, vasopressin, and oxytocin

Nitric oxide (NO) synthase (NOS), the enzyme responsible for NO formation, is found in hypothalamic neurons containing oxytocin (OT), vasopressin (VP), and to a lesser extent corticotropin-releasing factor (CRF). Because NO is reported to modulate endocrine activity, we have investigated the hypothesis that endogenous NO participates in ACTH released by various secretagogues in the rat. In the adult male rat, the intravenous injection of interleukin-1 beta (IL-1 beta; 0.2-0.3 micrograms/kg), VP (0.3-0.9 micrograms/kg), and OT (30 micrograms/kg) significantly increased plasma ACTH and corticosterone levels. Pretreatment with the L-form, but not the D-form, of N omega nitro-L-arginine-methylester (L-NAME; a specific inhibitor of NOS) markedly augmented the effects of these secretagogues whether it was injected acutely or over a 4 d period. Blockade of NOS activity also caused significant (P < 0.01) extensions of the duration of action of IL-1 beta, VP, and OT. In contrast, L-NAME did not significantly alter the stimulatory action of peripherally injected CRF, or centrally administered IL-1 beta. Administration of L-arginine, but not D-arginine (100 mg/kg), used as a substrate for basal NO synthesis and which did not by itself alter the activity of the hypothalamic-pituitary-adrenal (HPA) axis, blunted IL-1-induced ACTH secretion, and reversed the interaction between L-NAME and IL-1 beta. The stimulatory action of endotoxin, a lipopolysaccharide that releases endogenous cytokines, was also augmented by inhibition of NO formation.(ABSTRACT TRUNCATED AT 250 WORDS)