S. Stosic-Grujicic

Anti-glioma action of aloe emodin: the role of ERK inhibition.

Abstract.The effect of aloe emodin (AE), a herbal anthraquinone derivative, on the rat C6 glioma cell line was investigated. In addition to cell cycle block and caspasedependent apoptosis, AE led to the formation of intracytoplasmic acidic vesicles indicative for autophagic cell death. Moreover, differentiation of surviving cells toward the astrocytic lineage was confirmed by typical morphological changes and increased expression of glial fibrillary acidic protein (GFAP). AE did not affect the activation of mitogen-activated protein kinase p38, Jun-N-terminal kinase, or transcription factor NF-κB, but markedly inhibited the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in C6 cells. A selective inhibitor of ERK activation, PD98059, mimicked the effects of AE on glioma cell morphology and GFAP expression, but failed to induce either apoptosis or autophagy. Taken together, these results indicate that the anti-glioma action of AE involves ERK-independent induction of both apoptosis and autophagy, as well as ERK inhibition-mediated differentiation of glioma cells.

Interleukin-17 stimulates inducible nitric oxide synthase-dependent toxicity in mouse beta cells.

Abstract.The influence of the proinflammatory cytokine interleukin (IL)-17 on inducible nitric oxide (NO) synthase (iNOS)-mediated NO release was investigated in the mouse insulinoma cell line MIN6 and mouse pancreatic islets. IL-17 markedly augmented iNOS mRNA/protein expression and subsequent NO production induced in MIN6 cells or pancreatic islets by different combinations of interferon-γ, tumor necrosis factor-α, and IL-1β. The induction of iNOS by IL-17 was preceded by phosphorylation of p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK activation completely abolished IL-17-stimulated NO release. IL-17 enhanced the NO-dependent toxicity of proinflammatory cytokines toward MIN6 cells, while IL-17-specific neutralizing antibody partially reduced the NO production and rescued insulinoma cells and pancreatic islets from NO-dependent damage induced by activated T cells. Finally, a significant increase in blood IL-17 levels was observed in a multiple low-dose streptozotocin model of diabetes, suggesting that T cell-derived IL-17 might be involved in NO-dependent damage of beta cells in this disease.

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, leflunomides 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.

Induction of experimental autoimmune encephalomyelitis in Dark Agouti rats without adjuvant

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.

Down-regulation of multiple low dose streptozotocin-induced diabetes by mycophenolate mofetil

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.

Iron down-regulates macrophage anti-tumour activity by blocking nitric oxide production

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, FeSO4 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 FeSO4 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.

Mycophenolic acid inhibits activation of inducible nitric oxide synthase in rodent fibroblasts

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.

The Production of TNF, IL-1 And IL-6 in Cutaneous Tissues During Maturation and Aging

Epithelial cells (EC) resident to cutaneous tissues are important producers of various immunostimulatory, proinflammatory and inhibitory cytokines1,2 Locally produced cytokines in epithelial tissues mediate events relevant to growth, differentiation and function of surrounding cells. Based on their capacity to induce various cell adhesion molecules, as well as “secondary” cytokines,3 they play an important role in intercellular communications. Since immunocompetence of epithelial tissues may be influenced by maturation and aging,4-6 the aim of the present study was to obtain more information on the age-related changes of production of relevant cytokines. To examine this, we investigated age-related expression of TNF, IL-1 and IL-6 by comparing secretory potential of EC normally resident to skin of fetal, neonatal and adult rats.

Strain-dependent Induction and Modulation of Autoimmunity by Mercuric Chloride in Two Strains of Rats

Publisher Summary This chapter presents a study to examine the strain differences in susceptibility to mercuric chloride-induced autoimmune syndrome in Albino Oxford (AO) and Dark August (DA) rats and the possible mechanisms responsible for the development of and/or resistance to mercury disease. For this study, mercuric chloride was administered to inbred AO and DA male rats, of 3–4 month old. Multiple subtoxic doses of mercuric chloride have a potent effect on the immune system of rats, and the outcome of this treatment is strain dependent. In the AO strain, which is resistant to Th1 type organ-specific autoimmune diseases, administration of mercuric chloride results in the production of various autoantibodies, followed by systemic autoimmune disorders and damages to renal tissue. In contrast, the DA strain, which is susceptible to Th1-mediated autoimmune diseases, did not develop autoantibodies and related manifestations in response to mercuric chloride. Although this treatment in DA rats probably promoted Th2 cytokine synthesis, indirectly proved by eosinophilia and inhibition of autoimmune diabetes, as in Th1-mediated disease, the net effect could be down-modulation of Th2 response due to the overproduction of Th1 cytokines.

Antidiabetogenic Effect of Mycophenolate Mofetil Is Associated with Down-Regulation of Adhesive Interactions and Autoreactive Cell Activation

Abstract: We have investigated the immunomodulatory potential of mycophenolate mofetil (MMF) on the development of autoimmune diabetes induced by multiple low doses of streptozotocin (MLD‐SZ) in genetically susceptible DA rats. Administration of 25 mg/kg MMF continuously during the 7 weeks of MLD‐SZ treatment, or as a 10‐day treatment starting together with MLD‐SZ (early treatment), or 5 days after the last dose of SZ (late treatment) significantly suppressed the development of hyperglycemia. Ex vivo analyses performed on day 10 after diabetes induction showed that MMF treatment down‐regulates adhesive interactions and proliferation of autoreactive spleen cells. Similar effects were observed in vitro when MLD‐SZ‐derived splenocytes were cultured with an active metabolite of MMF, mycophenolic acid (MPA). These results suggest that the antidiabetogenic effect of MMF involves inhibition of the expansion and migration of autoreactive cells.