Browsing by Author "Stosic-Grujicic, S. (7004253020)"

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 IFNγ- 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-γ- 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. (C) 2000 Elsevier Science B.V.

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 IFNγ- 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-γ- 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. (C) 2000 Elsevier Science B.V.

The new immunosuppressive agent mycophenolate mofetil (MMF) has been shown recently to exert a protective effects in certain animal models of autoimmunity, including diabetes in diabetes-prone bio-breeding (BB) rats. In the present study, the immunomodulatory potential of MMF was investigated in autoimmune diabetes induced by multiple low doses of streptozotocin (MLD-STZ) in genetically susceptible DA rats [20 mg STZ/kg body weight (b.w.) for 5 days] and CBA/H mice (40 mg STZ/kg b.w. for 5 days). In both species, short time treatment of animals with MMF (25 mg/kg) during the early development of the disease, as well as continuous MMF treatment, prevented the appearance of hyperglycaemia and inflammatory infiltrates in the pancreatic tissue. Moreover, clinical manifestations of diabetes were suppressed by application of the drug after the onset of clinical symptoms. Treatment with guanosine (1 mg/kg) in parallel with MMF completely reversed MMF activity in vivo, indicating that inhibition of inosine monophosphate dehydrogenase (IMPDH) was responsible for the observed suppressive effects. MMF-mediated protection from diabetes correlated with reduced ex vivo spontaneous spleen mononuclear cell (MNC) proliferation and defective adhesive cell interactions. MMF-treated animals also had lower local production of IFN-γ, as well as IL-12 and nitric oxide (NO) production by peripheral tissues (spleen and peritoneal cells), compared to that in control diabetic groups, while IL-10 level was elevated. Together, these data demonstrate that MMF interferes with autoimmune process in streptozotocin-induced diabetes at multiple levels, including lymphocyte proliferation and adhesion, as well as pro/anti-inflammatory cytokine balance.

Pretreatment of epidermal cells (EC) with hydrocortisone or dexamethasone abolishes their capacity to produce interleukin-1 (IL-1) and therefore reduces their capacity to support proliferative response of lectin-stimulated T cells. Additionally, glucocorticoid-pretreated keratinocytes produce an inhibitor of IL-1 activity. This factor is a non-dialysable product of radioresistant epidermal cells, does not represent a non-specific inhibitor of DNA synthesis and appears to be specific for IL-1 since it did not interfere with IL-2-dependent T-cell proliferation. It affects both IL-2 production and the induction of IL-2-receptor expression. Finally, it blocks binding of IL-1 to its receptors on D10S subclone as evaluated by competitive binding assay. Thus, we have provided evidence which indicates that immunosuppressive effects of glucocorticoids in the skin may also be mediated by an IL-1 receptor antagonist(s) produced by keratinocytes.

Pretreatment of epidermal cells (EC) with hydrocortisone or dexamethasone abolishes their capacity to produce interleukin-1 (IL-1) and therefore reduces their capacity to support proliferative response of lectin-stimulated T cells. Additionally, glucocorticoid-pretreated keratinocytes produce an inhibitor of IL-1 activity. This factor is a non-dialysable product of radioresistant epidermal cells, does not represent a non-specific inhibitor of DNA synthesis and appears to be specific for IL-1 since it did not interfere with IL-2-dependent T-cell proliferation. It affects both IL-2 production and the induction of IL-2-receptor expression. Finally, it blocks binding of IL-1 to its receptors on D10S subclone as evaluated by competitive binding assay. Thus, we have provided evidence which indicates that immunosuppressive effects of glucocorticoids in the skin may also be mediated by an IL-1 receptor antagonist(s) produced by keratinocytes.

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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)-γ/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-γ/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. © 2002 Published by Elsevier Science Ltd.

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)-γ/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-γ/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. © 2002 Published by Elsevier Science Ltd.

[No abstract available]

Experimental autoimmune encephalomyelitis (EAE) is a well-recognized model for multiple sclerosis (MS) in humans. However, adjuvants used with encephalitogens to induce EAE produce non-specific effects interfering with the mechanisms involved in the autoimmune response to the central nervous system (CNS) tissue. It is therefore important to establish a more suitable model of EAE for analysis of autoimmune phenomena resembling those operative in MS. Here we report that EAE can be induced regularly in Dark Agouti (DA) strain of rats with spinal cord tissue without any adjuvant, as judged by both clinical and histological parameters. The incidence and severity of EAE depended on the origin of the encephalitogen, the rat versus guinea pig spinal cord homogenate being more efficient. Furthermore, EAE could be reinduced in animals which had recovered from disease that had been induced actively with encephalitogen alone, suggesting the role of adjuvant-generated non-specific mechanisms in resistance to reinduction of EAE. Thus, EAE induced in DA rats with encephalitogen alone provides a reproducible model for defining pathogenically relevant events in CNS autoimmunity devoid of the potentially misleading effects of adjuvants.

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[No abstract available]

[No abstract available]

Although the inhibitory effect of iron on macrophage production of tumoricidal free radical nitric oxide (NO) has been reported, its possible influence on macrophage anti-tumour activity has not been established. In the present study, FeSO 4 markedly reduced IFN-γ+ LPS-induced NO synthesis in mouse and rat macrophages. The effect of iron coincided with the loss of macrophage cytotoxic activity against NO-sensitive C6 rat astrocytoma and L929 mouse fibrosarcoma cell lines, as measured by MTT assay for cellular respiration and the crystal violet test for cell viability. Tumour cell survival did not improve further in the presence of FeSO 4 if macrophage NO release and cytotoxicity were already blocked by aminoguanidine. In accordance with the results obtained with exogenous iron, cell membrane permeable iron chelator o-phenanthroline enhanced both macrophage NO release and anti-tumour activity. Iron also down-regulated NO production and increased the viability of L929 fibrosarcoma cells stimulated with IFN-γ+ LPS in the absence of macrophages. However, neither NO release nor cell viability was affected by iron addition to cultures of the C6 astrocytoma cell line. Iron was unable to prevent L929 and C6 cell death induced by the NO releasing chemicals SNP and SIN-1, indicating that iron-mediated inhibition of NO synthesis, rather than interference with its cytotoxic action, was responsible for the protection of tumour cells. Collectively, these results indicate that iron might protect tumour cells by reducing both macrophage and tumour cell-derived NO release.

Although the inhibitory effect of iron on macrophage production of tumoricidal free radical nitric oxide (NO) has been reported, its possible influence on macrophage anti-tumour activity has not been established. In the present study, FeSO 4 markedly reduced IFN-γ+ LPS-induced NO synthesis in mouse and rat macrophages. The effect of iron coincided with the loss of macrophage cytotoxic activity against NO-sensitive C6 rat astrocytoma and L929 mouse fibrosarcoma cell lines, as measured by MTT assay for cellular respiration and the crystal violet test for cell viability. Tumour cell survival did not improve further in the presence of FeSO 4 if macrophage NO release and cytotoxicity were already blocked by aminoguanidine. In accordance with the results obtained with exogenous iron, cell membrane permeable iron chelator o-phenanthroline enhanced both macrophage NO release and anti-tumour activity. Iron also down-regulated NO production and increased the viability of L929 fibrosarcoma cells stimulated with IFN-γ+ LPS in the absence of macrophages. However, neither NO release nor cell viability was affected by iron addition to cultures of the C6 astrocytoma cell line. Iron was unable to prevent L929 and C6 cell death induced by the NO releasing chemicals SNP and SIN-1, indicating that iron-mediated inhibition of NO synthesis, rather than interference with its cytotoxic action, was responsible for the protection of tumour cells. Collectively, these results indicate that iron might protect tumour cells by reducing both macrophage and tumour cell-derived NO release.

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-γ+LPS-stimulated primary astrocytes, but not in macrophages. While A77 1726 did not alter iNOS enzymatic activity, it markedly suppressed IFN-γ+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-γ+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-γ+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. © 2001 Elsevier Science B.V.

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-γ+LPS-stimulated primary astrocytes, but not in macrophages. While A77 1726 did not alter iNOS enzymatic activity, it markedly suppressed IFN-γ+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-γ+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-γ+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. © 2001 Elsevier Science B.V.

To investigate the role of IL-1 in nitric oxide (NO)-mediated destructive processes in the multiple low dose streptozotocin (MLD-STZ) induced model of autoimmune diabetes, CBA/H mice received 10 consecutive i.p. injections of either rat IL-1 inhibitor (IL-1 INH), or human recombinant IL-1 receptor antagonist (IL-1ra). In contrast to the control MLD-STZ group (40-45 mg/kg for 5 days) which developed persistent hyperglycemia associated with insulitis and islet cell destruction, both IL-1 INH- and rIL-1ra-treated mice were protected from the induction of the disease. Preliminary immunohistochemical studies indicated that the high local concentration of NO-producing enzyme NO synthase, present in control MLD-STZ islets, is significantly reduced in both experimental groups of mice where IL-1 activity was blocked either by IL-1 INH or rIL-1ra.

Mycophenolate mofetil (MMF) is an immunosuppressive drug that acts as a selective inhibitor of inosine monophosphate dehydrogenase (IMPDH). MMF has recently been shown to inhibit the enzymatic activity of inducible NO synthase (iNOS) and subsequent production of the cytotoxic free radical nitric oxide (NO) in endothelial cells. We here investigated the effect of bioactive MMF compound mycophenolic acid (MPA) on iNOS-mediated NO synthesis in fibroblasts, which are important source of NO in rheumatoid arthritis and during rejection of solid organ transplants. MPA exerted dose-dependent inhibition of NO synthesis, measured as nitrite accumulation, in IFN-γ+ LPS-stimulated L929 mouse fibroblast cell line and rat primary fibroblasts. The effect of MPA was not mediated through interference with IMPDH-dependent synthesis of iNOS co-factor BH4 and subsequent suppression of iNOS enzymatic activity, as direct BH4 precursor sepiapterin failed to block the action of the drug. MPA suppressed the IFN-γ+ LPS-induced expression of fibroblast iNOS protein, as well as mRNA for iNOS and its transcription factor IRF-1, as assessed by cell-based ELISA and semiquantitative RT-PCR, respectively. MPA suppression of fibroblast NO release, iNOS, and IRF-1 activation, was efficiently prevented by exogenous guanosine, indicating that the drug acted through reduction of IMPDH-dependent synthesis of guanosine nucleotides. These results suggest that MPA inhibits NO production in fibroblasts by blocking guanosine nucleotide-dependent expression of iNOS gene, through mechanisms that might involve the interference with the induction of iNOS transcription factor IRF-1.

Mycophenolate mofetil (MMF) is an immunosuppressive drug that acts as a selective inhibitor of inosine monophosphate dehydrogenase (IMPDH). MMF has recently been shown to inhibit the enzymatic activity of inducible NO synthase (iNOS) and subsequent production of the cytotoxic free radical nitric oxide (NO) in endothelial cells. We here investigated the effect of bioactive MMF compound mycophenolic acid (MPA) on iNOS-mediated NO synthesis in fibroblasts, which are important source of NO in rheumatoid arthritis and during rejection of solid organ transplants. MPA exerted dose-dependent inhibition of NO synthesis, measured as nitrite accumulation, in IFN-γ+ LPS-stimulated L929 mouse fibroblast cell line and rat primary fibroblasts. The effect of MPA was not mediated through interference with IMPDH-dependent synthesis of iNOS co-factor BH4 and subsequent suppression of iNOS enzymatic activity, as direct BH4 precursor sepiapterin failed to block the action of the drug. MPA suppressed the IFN-γ+ LPS-induced expression of fibroblast iNOS protein, as well as mRNA for iNOS and its transcription factor IRF-1, as assessed by cell-based ELISA and semiquantitative RT-PCR, respectively. MPA suppression of fibroblast NO release, iNOS, and IRF-1 activation, was efficiently prevented by exogenous guanosine, indicating that the drug acted through reduction of IMPDH-dependent synthesis of guanosine nucleotides. These results suggest that MPA inhibits NO production in fibroblasts by blocking guanosine nucleotide-dependent expression of iNOS gene, through mechanisms that might involve the interference with the induction of iNOS transcription factor IRF-1.