Immunosuppressants leflunomide and mycophenolic acid inhibit fibroblast IL-6 production by distinct mechanisms.

Mycophenolic acid (MPA) and A77 1726, the active components of the immunosuppressants mycophenolate mophetil and leflunomide, respectively, in a dose-dependent manner inhibited interferon (IFN)-gamma/LPS-induced interleukin (IL)-6 release in confluent cultures of mouse L929 fibrosarcoma cells. In addition, both drugs markedly reduced the production of the free radical gas nitric oxide (NO), without affecting the viability of L929 cells. The inhibitors of NO synthase, aminoguanidine and L-NMMA, but not L-NMMA inactive counterpart D-NMMA, mimicked the effects of A77 1726 and MPA on IL-6 generation in L929 fibroblasts. Furthermore, NO-releasing substance SNP completely reverted IL-6 accumulation in L929 cultures treated with A77 1726, while only partial recovery of IL-6 production was observed in the presence of MPA. MPA, but not A77 1726, significantly suppressed NO-independent IL-6 release triggered by cAMP-elevating agent rolipram. Thus, while A77 1726 effect on IL-6 production was mediated through concomitant reduction of NO synthesis, MPA action was mainly independent of the interference with NO generation. Finally, both agents inhibited IFN-gamma/LPS-triggered IL-6 production in mouse primary fibroblasts, but not in mouse peritoneal macrophages, indicating cell-specificity of this novel anti-inflammatory action of A77 1726 and MPA.

Nitric oxide metabolites and interleukin-6 in cerebrospinal fluid from multiple sclerosis patients.

Interleukin-6 (IL-6) and nitric oxide (NO) are implicated in the pathology of multiple sclerosis (MS). We have investigated the levels of these mediators in the cerebrospinal fluid (CSF) from 50 patients with MS and 23 control subjects. Mean CSF IL-6 level was higher in the total MS group in comparison with controls, but not significantly, whilst the difference between patients with stable MS and controls reached the level of statistical significance. Mean CSF nitrite/nitrate level was significantly higher in the total MS group compared with the control group, as well as in active MS patients versus controls. There was significant difference neither in the mean CSF IL-6 nor in nitrite/nitrate levels between active and stable MS patients. Interestingly, we observed a significant negative correlation between IL-6 and nitrite/nitrate levels in the CSF in the total MS group. Such a trend existed in both subgroups with active and stable MS, but without reaching the level of statistical significance. Our data further support the involvement of IL-6 and NO in ongoing pathological processes in MS, suggesting their potential interplay within the central nervous system in this disease.

Interleukin-17 stimulates inducible nitric oxide synthase activation in rodent astrocytes.

The effect of interleukin-17 (IL-17) on production of nitric oxide (NO) in rodent astrocytes was investigated. While IL-17 by itself did not induce NO production, it caused a dose-dependent enhancement of IFN-gamma-triggered NO synthesis in both mouse and rat primary astrocytes. In contrast, IL-17 was unable to stimulate NO synthesis in either murine or rat macrophages. IFN-gamma-triggered expression of mRNA for iNOS, but not for its transcription factor interferon regulatory factor-1 (IRF-1), was markedly elevated in IL-17-treated astrocytes. The induction of iNOS mRNA by IL-17 in IFN-gamma-pretreated astrocytes was abolished by antagonists of nuclear factor-kappaB (NF-kappaB) activation--a proteasome inhibitor MG132 and an antioxidant agent PDTC, as well as with specific p38 MAP kinase inhibitor SB203580. While IL-17 stimulated both IL-1beta and IL-6 production in astrocytes, only IL-1 was partly responsible for IL-17-induced NO release. Finally, IL-17 synergized with exogenous IL-1beta and TNF-alpha for astrocyte NO production. Having in mind a well-known neurotoxic action of NO, these results suggest a possible role for IL-17 in the inflammatory diseases of the CNS.

Amphotericin B potentiates the activation of inducible nitric oxide synthase and causes nitric oxide-dependent mitochondrial dysfunction in cytokine-treated rodent astrocytes.

Because the neurotoxic effects of the antifungal drug amphotericin B (AMB) closely resemble those ascribed to the highly reactive gaseous free radical nitric oxide (NO), we investigated the effect of AMB on NO production in rodent astrocytes. AMB caused a dose-dependent increase of NO generation in interferon-gamma (IFN-gamma)-stimulated rat and mouse astrocytes, as well as in IFN-gamma + tumor necrosis factor-alpha (TNF-alpha)-activated rat astrocytoma cell line C6. Treatment of rat astrocytes with AMB markedly potentiated IFN-gamma-triggered expression of mRNA for iNOS, but not for its transcription factor IRF-1. The activation of transcription factor NF-kappaB was apparently required for AMB-induced iNOS mRNA expression, as the latter was abolished by NF-kappaB inhibitors: pyrrolidine dithiocarbamate and MG132. AMB-mediated enhancement of astrocyte NO production was partly dependent on endogenous IL-1, as shown by partial inhibition of AMB effect with IL-1 receptor antagonist. IFN-gamma + AMB treatment led to reduction of astrocyte mitochondrial respiration (measured by MTT assay) that has been completely reverted by selective iNOS inhibitor aminoguanidine. AMB toxicity toward IFN-gamma-stimulated astrocytes was dependent on both AMB and NO action, since AMB and NO-releasing substance SNP synergized in inducing astrocyte mitochondrial dysfunction. These results suggest that the enhancement of cytokine-induced iNOS activation in astrocytes and the subsequent release of high amounts of NO might be at least partly responsible for AMB neurotoxicity.

Pentoxifylline inhibits the synthesis and IFN-gamma-inducing activity of IL-18.

The effect of phosphodiesterase-inhibiting anti-inflammatory drug pentoxifylline (PTX) on LPS-induced IL-18 synthesis and IL-18-mediated IFN-gamma-induction were investigated. In a dose-dependent manner PTX inhibited production of IL-18 in LPS-treated cultures of murine spleen cells and bone marrow-derived macrophages. Similarly, PTX treatment significantly reduced blood IL-18 levels and expression of spleen IL-18 mRNA in LPS-challenged mice. The inhibitory effect of PTX was specific for IL-18, since LPS-induced IL-12 p40 release was not suppressed either in splenocyte cultures or blood of LPS-injected animals. Synergistic induction of IFN-gamma by combined IL-12/IL-18 treatment was also inhibited by PTX in vitro and in vivo. Experiments with IL-12 pretreatment of splenocytes, followed by IL-18 stimulation, revealed that PTX suppressed both IL-12 and IL-18 signals responsible for IFN-gamma induction. These results suggest that interference with IL-18 synthesis and IFN-gamma-inducing activity might contribute to anti-inflammatory actions of PTX.

Tumor cell-specific inhibition of inducible nitric oxide synthase activation by tiazofurin.

The effects of tiazofurin (TR) on proliferation and cytokine-induced nitric oxide (NO) production in the L929 fibrosarcoma cell line and murine embryonic fibroblasts were investigated. Treatment with TR inhibited the growth of nonconfluent L929 cells in a dose-dependent manner. TR, at concentrations not affecting cell viability or proliferation, markedly decreased IFN-gamma + LPS-induced expression of inducible NO synthase (iNOS) mRNA and, subsequently, NO production in confluent L929 cultures. However, TR did not interfere with the IFN-gamma-triggered expression of mRNA for IRF-1, an important iNOS transcription factor, implying that TR interferes with some other intracellular pathway involved in iNOS induction triggered by IFN-gamma + LPS. In contrast to the results obtained in L929 cells, iNOS mRNA expression induced by IFN-gamma + LPS in murine embryonic fibroblasts was resistant to TR, indicating a tumor-selective action of this agent.

Possible virulence factors of Staphylococcus sciuri.

Staphylococcus sciuri is an opportunistic pathogen of controversial clinical significance. The factors that contribute to colonization and/or infection caused by this bacterium have not been studied intensively so far. The present research was carried out in order to study the presence of potential virulence factors in 121 human and animal isolates of this bacterium. Isolates were examined for biofilm formation, hemagglutination, presence of clumping factor, production of spreading factors and exotoxins, cytotoxicity and capacity to stimulate nitric oxide production. The results showed that S. sciuri is highly capable of biofilm production, that it displays strong proteolytic and DNase activities, produces hemolysins and stimulates nitric oxide production by rat macrophages. Although the present study showed existence of a wide spectrum of possible virulence determinants of S. sciuri, their exact contribution to virulence of this bacterium in vivo remains to be determined.

Raised cerebrospinal fluid nitrite and nitrate levels in patients with multiple sclerosis: no correlation with disease activity.

A growing body of evidence implicates excessive generation of nitric oxide (NO) within the central nervous system (CNS) in multiple sclerosis (MS). The aim of our study is to analyse nitrite and nitrate as end products of NO in the cerebrospinal fluid (CSF) from MS patients and correlate the concentrations with clinicol characteristics of the disease. CSF nitrite and nitrate concentrations were measured after reduction of nitrate, by Griess reaction, in 105 MS potients, 27 patients with non-inflammatory neurological disorders (NIND) and 13 individuals without neurological disorder (Co). Mean CSF nitrite and nitrate concentrations were significantly higher in patients with MS and NIND compared with the Co patients (9.44 and 8.68, respectively, versus 6.85 microM; P=0.0001 and P=0.031, respectively). There was no significant correlation between CSF nitrite and nitrate concentrations and activity, phase, severity and duration of MS. Our data are in agreement with the results of previous studies which have demonstrated raised concentrations of CSF NO metabolites in MS patients, providing further evidence for NO involvement in MS. The lack of correlation between NO metabolites and disease activity speaks in favour of the possible dual role of NO, as both immunoregulatory and pro-inflammatory molecule, in the pathogenesis of MS.

STAT1 is required for iNOS activation, but not IL-6 production in murine fibroblasts.

The role of transcription factor STAT1 in production of pro-inflammatory mediators nitric oxide (NO) and IL-6 was examined in murine embryonic fibroblasts. While cells from wild-type animals released large amounts of NO after stimulation with IFN-gamma in combination with LPS, TNF-alpha or IL-1, their STAT1-deficient counterparts failed to synthesise detectable levels of this free radical gas. Inability of STAT1-/- fibroblasts to produce NO was accompanied by complete absence of mRNA for iNOS and its transcription factor IRF-1, both readily upregulated in wild-type cells. However, treatment with cytokines (IFN-gamma, TNF-alpha, IL-1, IL-17) significantly increased IL-6 generation in STAT1-deficient fibroblasts. These results indicate that STAT1 activation and subsequent IRF-1 transcription are required for induction of iNOS, but not IL-6 in murine fibroblasts.

Antidiabetogenic effect of pentoxifylline is associated with systemic and target tissue modulation of cytokines and nitric oxide production.

We have shown recently that xanthine derivative pentoxifylline (PTX) downregulates an inflammatory autoimmune process triggered in genetically susceptible Dark Agouti rats by multiple low doses of streptozotocin (MLD-SZ, 20 mg/kg/day ip for 5 days). We studied the cellular and molecular consequences of PTX treatment during MLD-SZ-induced diabetes with special emphasis on local vs. systemic production of inflammatory mediators. Administration of PTX (200 mg/kg/day for 10 days) during induction of the disease reduced clinical signs of diabetes and protected rats from development of destructive intrainsulitis. Pentoxifylline did not affect diabetogenic effect of single high dose of SZ (100 mg/kg SZ). Ex vivo analysis of the islets of Langerhans performed in early disease development revealed that PTX downregulates production of proinflammatory cytokines IFN-gamma and TNF, as well as inducible nitric oxide synthase (iNOS) expression and NO production. In addition, PTX treatment suppressed splenocyte autoreactivity, as well as the frequency of cells expressing IL-2R and MHC class II antigens. There was no evidence of any changes in proportion of ICAM-1 and LFA-1 expressing splenocytes in comparison to control MLD-SZ-treated animals. In contrast to suppressed intraislet production, high peripheral expression of both iNOS mRNA and NO was found in MLD-SZ rats treated with PTX. Taken together, the data indicate that the effect on both systemic and intra-islet production of NO, suppression of autoreactive cell activation and of local type 1 cytokine release may contribute to the therapeutic benefit achieved by PTX in the rat.

Leflunomide inhibits activation of inducible nitric oxide synthase in rat astrocytes.

Highly reactive gaseous free radical nitric oxide (NO), generated by astrocytes and infiltrating macrophages is implicated in inflammatory destruction of brain tissue, including that occurring in multiple sclerosis. Therefore, the influence of immunosuppressive drug leflunomide on inducible nitric oxide synthase (iNOS)-dependent NO production in rat astrocytes and macrophages was investigated. Under the same cultivating conditions, leflunomide's active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-gamma+LPS-stimulated primary astrocytes, but not in macrophages. While A77 1726 did not alter iNOS enzymatic activity, it markedly suppressed IFN-gamma+LPS-triggered expression of iNOS mRNA in astrocytes. In the presence of transcription inhibitor actinomycin D, A77 1726 failed to inhibit astrocyte NO production, suggesting transcriptional regulation of iNOS by leflunomide. This assumption was further supported by the ability of A77 1726 to inhibit IFN-gamma+LPS-induced expression of mRNA for an important iNOS transcription factor IRF-1. PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MAPKK/MEK), but not genistein, an unselective protein tyrosine kinase inhibitor, completely mimicked cell type-specific inhibition of NO synthesis by A77 1726. Therefore, previously described inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for A77 1726-mediated suppression of iNOS activation in astrocytes. Accordingly to results obtained with primary astrocytes, both A77 1726 and PD98059 significantly reduced IFN-gamma+LPS-induced NO synthesis in the cultures of rat astrocytoma cell line C6. The ability to suppress iNOS induction in astrocytes supports potential use of leflunomide in the treatment of multiple sclerosis and other NO-dependent inflammatory brain disorders.

Muramyl dipeptide potentiates cytokine-induced activation of inducible nitric oxide synthase in rat astrocytes.

We investigated the influence of muramyl dipeptide (MDP), a cell wall component of Gram-positive bacteria, on cytokine-induced nitric oxide (NO) production in rat primary astrocytes. MDP alone did not induce NO release in astrocyte cultures. However, MDP increased astrocyte NO production and subsequent nitrite accumulation triggered by IFN-gamma. IFN-gamma-activated expression of mRNA for inducible NO synthase (iNOS) and iNOS transcription factor interferon regulatory factor-1 (IRF-1) was markedly enhanced in astrocytes treated with MDP. The potentiating effect of MDP on IFN-gamma-induced NO production in astrocytes was completely blocked with protein tyrosine kinase (PTK) inhibitor genistein or mitogen activated protein kinase (MAPK) inhibitor PD98059. In contrast, protein kinase C (PKC) inhibitor calphostin C did not affect ability of MDP to augment IFN-gamma-triggered astrocyte NO synthesis. These results suggest that MDP synergizes with IFN-gamma in the induction of iNOS gene in astrocytes through mechanisms involving PTK and MAPK, but not PKC activation. Finally, MDP also augmented NO production and iNOS mRNA expression in astrocytes treated with IL-1beta.

Cryptococcus neoformans neutralizes macrophage and astrocyte derived nitric oxide without interfering with inducible nitric oxide synthase induction or catalytic activity - possible involvement of nitric oxide consumption.

The effect of Cryptococcus neoformans on the accumulation of nitrite, an indicator of nitric oxide (NO) synthesis, was investigated in cytokine (interferon-gamma [IFN-gamma] and interleukin [IL]-1)-stimulated cultures of rat peritoneal macrophages and C6 astrocytoma cells. Cytokine-induced nitrite generation in cultures of both cell types was inhibited in a dose-dependent manner by live C. neoformans, but not by heat-killed cryptococcal cells or conditioned medium from yeast cultures. C. neoformans-mediated reduction of nitrite formation coincided with impairment of NO-dependent macrophage tumoricidal activity. Cytokine-triggered induction of inducible NO synthase (iNOS) was unaffected in C6 cells, and only marginally reduced in macrophages. When cells were pretreated with cytokines for 24 h to induce iNOS, and any further induction was prevented by inhibition of protein synthesis, C. neoformans was still able to reduce nitrite accumulation in cultures of both cell types. Finally, live C. neoformans, but not heat-killed yeast cells or yeast culture supernatant, significantly reduced nitrite production in a culture solution of NO-releasing compound S-nitrosoglutathione (GSNO). Thus, it appears that cryptococcal reduction of nitrite formation in macrophage and C6 cultures was caused by the consumption of NO by some yeast molecule, rather than by the inhibition of cellular NO synthesis.

Cell-specific inhibition of inducible nitric oxide synthase activation by leflunomide.

The influence of a novel immunomodulating drug, leflunomide, on iNOS-dependent nitric oxide (NO) production in rodent macrophages and fibroblasts was investigated. Leflunomide's active metabolite A77 1726 caused a dose-dependent decrease of NO production in IFN-gamma-treated L929 fibroblasts. The observed effect was cell-specific, as well as stimulus-specific, since A77 1726 did not affect NO production in IFN-gamma-stimulated murine peritoneal macrophages or db-cAMP-treated L929 cells. A77 1726 reduced expression of IFN-gamma-induced iNOS and IRF-1 mRNA in L929 cells, while iNOS enzymatic activity remained unchanged. Specific inhibitor of MAP kinase kinase (MEK), PD98059, but not unselective protein kinase inhibitor genistein, completely mimicked cell-type-specific and stimulus-specific NO-inhibitory action of leflunomide. Therefore, the recently described inhibition of MEK/MAP pathway by leflunomide could present a possible mechanism for its suppression of iNOS activation in L929 fibroblasts. Finally, a similar inhibitory effect of A77 1726 on both NO production and iNOS mRNA expression was observed also in IFN-gamma + LPS-activated murine and rat primary fibroblasts.

Differential regulation of nitric oxide production by increase of intracellular cAMP in murine primary fibroblasts and L929 fibrosarcoma cell line.

The effect of intracellular cAMP rise on nitric oxide (NO) production was compared in murine primary fibroblasts isolated from the spleens of CBA mice, and L929 fibrosarcoma cell line. Treatment of confluent L929 cells with cAMP analogues -dibutyryl-cAMP (db-cAMP) or 8-Cl-cAMP caused dose-dependent augmentation of inducible NO synthase (iNOS)-mediated NO production, which has been abrogated by inhibition of protein synthesis with cycloheximide or addition of selective iNOS inhibitor aminoguanidine. In contrast, under the same cultivating conditions, cAMP analogues were not able to upregulate NO synthesis in primary fibroblasts. Treatment with cAMP analogues or non-selective phosphodiesterase (PDE) inhibitor pentoxifylline affected IFNgamma-induced NO synthesis in both cell types, but in the opposite manner-enhancing in L929 cells and suppressive in primary fibroblasts. The induction of iNOS, but not its catalytic activity, was impaired in cAMP-treated primary fibroblasts. Finally, PDE type IV inhibitor rolipram enhanced IFN-gamma-triggered NO synthesis in L929 cells, but was unable to mimic cAMP analogue or PTX-mediated suppression of NO synthesis in spleen fibroblasts. These results suggest that, in contrast to L929 fibrosarcoma cell line, intracellular cAMP rise might have a role in downregulation of NO production in murine primary fibroblasts.

Cyclosporin A suppresses the induction of nitric oxide synthesis in interferon-gamma-treated L929 fibroblasts.

The effects of immunosuppressant cyclosporin A (CsA) on nitric oxide (NO) production and inducible NO synthase (iNOS) activity in murine L929 fibroblasts were investigated. IFN-gamma-induced NO production in L929 cells was mediated through an iNOS-dependent L-arginine-NO pathway, since it was abrogated by a selective inhibitor of iNOS, aminoguanidine. CsA applied simultaneously with IFN-gamma caused a dose-dependent reduction of NO synthesis in L929 cells. However, CsA did not influence the enzymatic activity of iNOS, since it failed to affect NO production in cells in which iNOS had already been induced with IFN-gamma and any further induction was blocked by the protein-synthesis inhibitor cycloheximide. IFN-gamma-triggered expression of mRNA for interferon regulatory factor-1 was not reduced by CsA-treatment, suggesting that this iNOS transcription factor is not a target in CsA-mediated inhibition of NO synthesis. Finally, FK506 was not able to mimic the inhibitory effect of CsA on NO production in L929 cells, indicating the calcineurin-independent mechanism of CsA action. These results indicate that CsA suppresses NO synthesis in L929 cells independent of calcineurin inhibition, and interfering with intracellular pathways involved in the iNOS induction, rather than inhibiting its enzymatic activity.