Zorica Ramić

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.

Astrocyte-induced regulatory T cells mitigate CNS autoimmunity.

Although astrocytes presumably participate in maintaining the immune privilege of the central nervous system (CNS), the mechanisms behind their immunoregulatory properties are still largely undefined. In this study, we describe the development of regulatory T cells upon contact with astrocytes. Rat T cells pre‐incubated with astrocytes completely lost the ability to proliferate in response to mitogenic stimuli. The cells were blocked in G0/G1 phase of the cell cycle, expressed less IL‐2R, and produced significantly lower amounts of interferon‐γ (IFN‐γ), but not interleukin‐2 (IL‐2), IL‐10, or tumor necrosis factor (TNF). These anergic cells completely prevented mitogen‐induced growth of normal T lymphocytes, as well as CNS antigen‐driven proliferation of autoreactive T cells. The suppressive activity resided in both CD4+ and CD8+ T‐cell compartments. Heat‐sensitive soluble T‐cell factors, not including transforming growth factor‐β (TGF‐β) or IL‐10, were solely responsible for the observed suppression, as well as for the transfer of suppressive activity to normal T cells. The administration of astrocyte‐induced regulatory T cells markedly alleviated CNS inflammation and clinical symptoms of CNS autoimmunity in rats with experimental allergic encephalomyelitis. Finally, the cells with suppressive properties were readily generated from human lymphocytes after contact with astrocytes. Taken together, these data indicate that astrocyte‐induced regulatory T cells might represent an important mechanism for self‐limitation of excessive inflammation in the brain.

Strain difference in susceptibility to experimental autoimmune encephalomyelitis between Albino Oxford and Dark Agouti rats correlates with disparity in production of IL-17, but not nitric oxide

Albino Oxford (AO) rats, unlike Dark Agouti (DA) rats are resistant to the induction of experimental autoimmune encephalomyelitis (EAE). The reason for the resistance could be some restraining mechanism preventing auto‐aggressive cell activation at the level of draining lymph nodes (DLN) during the induction phase of the disease. Such a mechanism could be anti‐proliferative action of nitric oxide (NO), which has already been shown of importance for the resistance of several rat strains to the induction of the disease. Importantly, number of AO DLN cells (DLNC) is markedly lower and with lower proliferative response to myelin basic protein (MBP) ex vivo in comparison to DA DLNC in the inductive phase of EAE, thus implying that in AO rats DLNC do not proliferate as extensively as in DA rats. We show that AO rats do not produce larger quantities of NO than DA rats after immunization. Further, DLNC of immunized AO rats have significantly lower mRNA expression and synthesis of interferon (IFN)‐γ and interleukin (IL)‐17 compared to DLNC of DA rats. Collectively, these results suggest that there is a substantial difference between EAE‐resistant AO rats and EAE‐prone DA rats in the initiation of autoimmune response. This difference seems to be independent of anti‐proliferative actions of NO, but correlates with impaired IL‐17 production in AO rats.

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.

Analysis of T cell subsets after induction of experimental autoimmune encephalomyelitis in susceptible and resistant strains of rats

T cell subsets in the peripheral blood, draining lymph node (DLN) and spinal cord lesions were analysed after the induction of experimental autoimmune encephalomyelitis (EAE) in susceptible (DA) and relatively resistant (AO) rats. In DA rats, a significantly higher number of CD4+ cells were generated in the DLN, in response to both nervous tissue antigens and complete Freunds adjuvant (CFA), compared to AO rats. In the peripheral blood of DA rats, the percentage as well as absolute number of CD4+ cells increased in the preclinical phase of EAE, but declined as the disease developed. The percentage of CD8+ cells decreased in both these phases of EAE. In resistant AO rats, however, there were no significant changes in the T lymphocyte subset percentages after EAE induction, although the absolute number of peripheral blood CD4+ cells again increased in the preclinical stage of EAE. In the CFA-treated control DA rats, the absolute number of CD4+ cells was increased in the preclinical phase. However, no decline comparable to that seen in diseased animals followed. It is concluded that the generation of CD4+ cells in response to this antigen is strain specific and, since the cells are released into the circulation, will affect the balance between the T cell subsets in the peripheral blood during the development of EAE.

Methylprednisolone inhibits interleukin-17 and interferon-gamma expression by both naive and primed T cells

BackgroundInterleukin-17 (IL-17)-producing cells are increasingly considered to be the major pathogenic population in various autoimmune disorders. The effects of glucocorticoids, widely used as therapeutics for inflammatory and autoimmune disorders, on IL-17 generation have not been thoroughly investigated so far. Therefore, we have explored the influence of methylprednisolone (MP) on IL-17 expression in rat lymphocytes, and compared it to the effect of the drug on interferon (IFN)-γ.ResultsProduction of IL-17 in mitogen-stimulated lymph node cells (LNC) from non-treated rats, as well as in myelin basic protein (MBP)-stimulated draining LNC from rats immunized with spinal cord homogenate and complete Freunds adjuvant was significantly reduced by MP. The reduction was dose-dependent, sustained through the follow-up period of 48 hours, and was not achieved through anti-proliferative effect. Additionally, MP inhibited IL-17 production in purified T cells as well, but to less extent than in LNC. In its influence on IL-17 production MP inhibited Ror-γT transcription factor expression, as well as Jun phosphorylation, but not ERK or p38 activation in mitogen-stimulated LNC. Importantly, MP collaborated with IFN-γ in inhibiting IL-17 generation in LNC.ConclusionThe observed difference in the effect of MP on IL-17 and IFN-γ could be important for the understanding of the variability in the efficiency of glucocorticoids in the treatment of autoimmune diseases.

Antiproliferative Effect of Vitamin A and D Analogues on Adult Human Keratinocytes in vitro

Background: Vitamin A and D analogues play an important role in epidermal homeostasis and are used in the treatment of various skin diseases. The failure of retinoid and vitamin D treatments is sometimes difficult to explain. Methods: We analyzed the effect of all-trans retinoic acid (all-trans RA), 13-cis retinoic acid (13-cis RA), ergocalciferol and cholecalciferol in keratinocyte cultures established from adult donors, on the cell proliferation by means of [3H]thymidine incorporation and apoptosis after fluorescein diacetate/trypan blue staining. Results: All tested agents exerted a dose-dependent inhibition of keratinocyte proliferation in the concentration range of 1.25–5 µM. Based on IC50 values, the antiproliferative efficiency was as follows: cholecalciferol > ergocalciferol = all-trans RA > 13-cis RA. The observed effect of cholecalciferol and ergocalciferol, but not retinoids, involved the induction of apoptotic cell death. Combining vitamins A and D did not further increase the proliferation block and even displayed an antagonistic effect. Conclusion: The susceptibility of keratinocytes to the antiproliferative action of vitamins A and D was markedly different in cell cultures derived from different donors, indicating a possible predictive value of the in vitro testing for the efficiency of the clinical response to these agents.

Strain difference in susceptibility to experimental autoimmune encephalomyelitis in rats correlates with TH1 and TH17-inducing cytokine profiles

Albino Oxford (AO) rats are resistant to induction of experimental autoimmune encephalomyelitis (EAE), in contrast to susceptible Dark Agouti (DA) rats. We have previously shown that draining lymph node cells (DLNC) obtained from immunized DA rats before the onset of the clinical disease produced more interferon (IFN)-gamma and interleukin (IL)-17 (signature cytokines of T(H)1 and T(H)17 responses, respectively) compared to DLNC from AO rats. In this study, we extend our analysis to entire induction phase of EAE with the emphasis on the T(H)1 and T(H)17-inducing cytokines. As a result, we show that throughout the inductive phase of the disease DLNC of DA rats, not only expressed higher levels of IFN-gamma and IL-17, but also of T(H)1-inducing cytokine-IL-12. As for T(H)17-inducing cytokines, DLNC of DA rats expressed more mRNA for p19, specific subunit of IL-23, but the expression of transforming growth factor (TGF)-beta in both strains was similar. Interestingly, the analysis of IL-6 expression revealed striking difference: while all DA DLNC were positive for IL-6 mRNA, cells from none of AO rats expressed detectable levels of mRNA for this cytokine. Taken together, our data suggest that the differential regulation of production of T(H)1 and T(H)17 cytokines, and IL-6 in particular, during the induction phase of disease could be responsible for the discrepancy in susceptibility to EAE between these two rat strains.

Down-regulation of experimental allergic encephalomyelitis in DA rats by tiazofurin

The immunomodulatory potential of tiazofurin (TR) on experimental autoimmune encephalomyelitis (EAE) was investigated. Given continuously, TR dose-dependently suppressed the development of EAE in Dark Agouti (DA) rats immunized with either rat spinal cord homogenate (SCH) or myelin oligodendrocyte glycoprotein (MOG). Amelioration of clinical signs was also obtained when the drug was administered during the inductive phase only (day 0 to 8), or during the effector phase (day 10 to 20) of the disease. Efficacy of TR was further evaluated by adoptive transfer of the disease with myelin basic protein (MBP)-sensitized draining lymph node cells (DLNC). Cells from TR-protected rats failed to transfer the disease into naive syngeneic recipients; in addition, TR treatment of recipient rats that had received MBP-sensitized lymphoid cells diminished the adoptively transferred EAE. A reduction of clinical EAE in TR-treated rats was accompanied with the absence of mononuclear infiltration in the spinal cord and defective adhesive cell-cell interactions. The anti-MOG autoAb production was also decreased. Importantly, no evidence for a generalized impairment of the T cell activity, nor decreased in vitro proliferative antigen specific response of LNC from TR-treated animals was found. These results suggest that TR exerts its EAE protective and suppressive effects by limiting adhesive interactions involved in the autoimmune pathogenic process, and due to the lack of general immunosuppressive activity, it should be considered as a candidate drug for the treatment of neuroinflammatory diseases like multiple sclerosis (MS).

Cell-type dependent response of melanoma cells to aloe emodin.

Intrinsic characteristics of melanoma cells such as expression of inducible nitric oxide synthase (iNOS), redox status, and activity of signaling pathways involved in proliferation, differentiation and cell death define the response of the cells to the diverse treatments. In this context we compared the effectiveness of herbal antaquinone aloe emodin (AE) against mouse B16 melanoma and human A375, different in initial activity of ERK1/2, constitutive iNOS expression and basal level of reactive oxygen species (ROS). Both cell lines are sensitive to AE treatment. However, while the agent induces differentiation of B16 cells toward melanocytes, in A375 cells promoted massive apoptosis. Differentiation of B16 cells, characterized by enhanced melanin production and tyrosinase activity, was mediated by H(2)O(2) production synchronized with rapid p53 accumulation and enhanced expression of cyclins D1 and D3. Caspase mediated apoptosis triggered in A375 cells was accompanied with Bcl-2 but not iNOS down-regulation. In addition, opposite regulation of Akt-ERK1/2 axis in AE treated B16 and A375 cells correlated with different outcome of the treatment. However, AE in a dose-dependent manner rescued both B16 and A375 cells from doxorubicin- or paclitaxel-induced killing. These data indicate that caution is warranted when AE is administrated to the patients with conventional chemotherapy.