| Part I Erythropoietin ameliorate the clinical symptoms and pathological signs of experimental autoimmune encephalomyelitis miceObjective:To observe the role of erythropoietin (EPO) on mice of experimental autoimmune encephalomyelitis (EAE).Methods:EAE mice were grouped randomly as EAE control group(EAEcontrol), preventively premorbid therapy group(EPOpremorbid) and postmorbid therapy group(EPOpostmorbid), which were injected intraperitoneally with EPO and PBS of the same amount respectively; The weight and clinical symptoms scores of all EAE mice were recorded during 30 days after EAE establishment. Spinal cords of EAE mice in different groups were sliced and stained by HE staining to observe the numbers of focus infiltrating cells, which were also extracted for analysis of flow cytometry to detect the T cells subgroups and cytokines secreted by CD4+T cells.Results:Mice in EPOpremorbid and EPOpostmorbid groups presented with later onset of diseases, improved symptoms of EAE and fewer infiltrating cells numbers in spinal cords compared against EAEcontrol mice(P<0.05). The percentages of CD4+T cells, IL-17+CD4+T and IFN-γ+CD4+T cells of infiltrating cells extraction were lower in EPOpremorbid mice than those in EAEcontrol mice(P<0.05).Conclusion:EPO can improve EAE symptoms by reducing the numbers of infiltrating cells while inhibiting inflammatory cytokines of those cells.Objective:To observe EPO receptor expression on mononuclear cells(MNC) and glial cells.Methods:MNC and pure CD4+T cells were prepared, by differential centrifugation and purified CD4+T cells kit, from spleens and peripheral lymph nodes of EAE mice. Astrocytes and microglial cells were cultured and identified according to classical methods. EPO receptors on MNC and glial cells were detected by RT-PCR.Results:EPO receptors were found on MNC but not pure CD4+T cells; Astrocytes and microglial cells, in resting or activated condition, express EPO receptors.Conclusion:Both mononuclear cells and glial cells express EPO receptors.Objective:To observe the effects of EPO on immunity of EAE mononuclear cells.Methods:MNC prepared from EAEcontrol and EPOpremorbid group(MNC and MNCe) were applied to flow cytometry to analyze T cells subgroups, MHC-Ⅱexpression and cytokines secreted by CD4+T cells. After being stimulated by Phytohemagglutinin(PHA) or MOG35-55 peptide, cells proliferation and cytokines production of MNC and MNCe were examined by the methods of MTT, ELISA or RT-PCR.Results:The percentages of CD4+T cells, IL-17+CD4+T, IFN-y+CD4+T cells and MHC-Ⅱexpression of MNCe were lower than those in MNC(P<0.05). Being stimulated with PHA, IL-17, L-23, IL-6 and IFN-γproduced by MNCe were lower than those produced by MNC(P<0.05) while the proliferation rates of MNC and MNCe were about the same(P>0.05); Being stimulated with MOG35-55 peptide, IL-17, L-23 and IFN-y produced by MNC treated with EPO were lower than those produced by MNC(P<0.05); CD4+T cells treated with EPO and MOG35-55 peptide produced less IFN-y than those treated with MOG35-55 peptide only(P<0.01).Conclusion:EPO can inhibit pro-inflammatory cytokines, such as IL-17, IFN-γ, through down-regulating IL-23 and MHC-Ⅱ.Objective:To observe the effects of EPO on interaction between astrocytes and immunocytes from EAE mice.Methods:Cytokines of resting and activated astrocytes, treated with or without EPO, were detected by ELISA or RT-PCR, while proliferation rates were assessed with MTT methods and MHC-Ⅱexpressions were detected by immunocytochemical technique and flow cytometry. Resting and activated astrocytes, treated with or without EPO, were mixed and cultured with MNC or CD4+T cells at the rate of 1:1 and 1:5, cytokines in medium of co-culture were detected with ELISA kits and MHC-Ⅱexpressions of astrocytes were examined by flow cytometry.Results:1) Resting astrocytes treated with EPO produced less IL-6 than those untreated with astrocytes(P<0.05); 2) EPO could inhibit the expression of IFN-γ, IL-6, IL-17, IL-23 and MHC-Ⅱof LPS-activated astrocytes(P<0.05); 3) EPO promoted the proliferation and nitric oxide(NO) release of astrocytes; 4) Co-culture of resting astrocytes with MNC at the rate of 1:1 produced more IFN-γ, IL-10 and IL-6 than mixed cells of EPO-treated resting astrocytes with MNC (P<0.05); Co-culture of resting astrocytes with MNC at the rate of 1:5 produced more IFN-γ, IL-6, IL-17, IL-23 than mixed cells of EPO-treated resting astrocytes with MNC (P<0.05); 5) Co-culture of activated astrocytes with CD4+T cells at the rate of 1:1 produced more more IFN-γ, IL-6, IL-17, IL-23 than mixed cells of EPO-treated activated astrocytes with CD4+T cells(P<0.05).Conclusion:EPO promote the proliferation and NO release of astrocytes, inhibit production of IL-17, IL-23, IL-27 and the expression of MHC-Ⅱ; EPO-treated astrocytes, through down-regulating IL-23 and (or) MHC-Ⅱof mixed cells, make the co-culture with immunocytes produce less IL-17 and (or) IFN-γ.Objective:To observe the effects of EPO on interaction between microglia and immunocytes from EAE mice.Methods:Cytokines of resting and activated microglia, treated with or without EPO, were detected by ELISA or RT-PCR, while death rates were assessed with LDH methods and MHC-Ⅱexpressions were detected by immunocytochemical technique and flow cytometry. Resting and activated microglia, treated with or without EPO, were mixed and cultured with MNC or CD4+T cells at the rate of 1:1 and 1:5, cytokines in medium of co-culture were detected with ELISA kits and MHC-Ⅱexpressions of microglia were examined by flow cytometry.Results:1) EPO could inhibit the expression of IFN-γ, IL-6, IL-17, IL-23 and MHC-Ⅱof LPS-activated microglia (P<0.05); 2) EPO inhibited the death of microglia; 3) Co-culture of resting microglia with CD4+T cells at the rate of 1:1 produced more IL-6, IL-17, IL-23 than mixed cells of EPO-treated resting microglia with CD4+T cells (P<0.05); Co-culture of resting microglia with CD4+T cells at the rate of 1:5 produced more IFN-γ, IL-6, IL-23 than mixed cells of EPO-treated resting microglia with CD4+T cells (P<0.05); Co-culture of resting microglia with MNC at the rate of 1:1 produced more IFN-γ, less IL-10 than mixed cells of EPO-treated resting microglia with MNC (P<0.05); 4) Co-culture of activated microglia with CD4+T cells at the rate of 1:1 produced more IFN-γ, IL-6, IL-23 than mixed cells of EPO-treated activated microglia with CD4+T cells(P<0.05); Co-culture of activated microglia with MNC at the rate of 1:1 produced more IFN-γ, IL-6 than mixed cells of EPO-treated activated microglia with MNC(P<0.05); 5) In co-culture, EPO could inhibit MHC-Ⅱexpression of microglia induced by MNC.Conclusion:EPO inhibit the death of microglia, supress production of IL-17, IL-23, IL-27 and the expression of MHC-Ⅱ; EPO-treated microglia, through down-regulating IL-23 and MHC-Ⅱof mixed cells, make the co-culture with immunocytes produce less IL-17 and (or) IFN-γ.1. EPO reduce the focus infiltrating cells and pro-inflammatory cytokines produced by them and ameliorate symptoms of EAE.2. Both mononuclear cells and glial cells express EPO receptors, so EPO can act on them.3. Activated astrocytes and microglia produce IL-17, IL-23, IL-27 and express MHC-Ⅱ, EPO suppress all of these molecules.4. EPO promote the proliferation and NO release of astrocytes, inhibit the death of microglia.5. Mononuclear cells in spleen and peripheral lymph nodes were the major immunological response cells in EAE. EPO inhibit pro-inflammatory cytokines produced by them, such as IL-17, IFN-γ.6. Compared against co-culture of immunocytes with resting/activated astrocytes or microglia, mixture of immunocytes with EPO-treated resting/activated astrocytes or microglia produce less IL-17 or (and) IFN-γ, through down-regulating IL-23 and (or) MHC-Ⅱof mixed cells. |
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