| Backgrounds: It has been evidenced for the past decade, that apolipoprotein E (apoE) plays an immune regulatory role in the autoimmune disorders of central nervous sytem. Schwann Cells (SCs) as the myelin-forming cells in the peripheral nervous system (PNS) are involved in the pathogenesis of autoimmue disorders and also are main target cells in the autoimmune response of the PNS. So far no study has focussed on the isoform-specific effect of ApoE on the immunomodulatory properties of SCs in the PNS.Objectives and methods: In the present study, we cultured SCs from human ApoE2, E3 and E4 transgenic (Tg) mice, stimulated them with pro- inflammatory agents and compared the expression of cytokines and production of NO among different strains of mice to see whether there is an isoform- dependent effect of ApoE upon SCs in response to inflammatory stimuli. Intracellular signaling pathways were further detected to find specific molecules that might be involved in the process.Results:Significant morphological changes were observed on cultured SCs after stimulation, which were characterized by enlarged cell body, reduced cell processes and blurred cell margins. Cultured SCs manifested a homogenous population when detected by flow cytometry and more than 95% of cultured SCs expressed ApoE protein. The purity of SCs was more than 96% as measured by flow cytometry with anti-S100 antibodies.ApoE3 increased IL-6, IL-10 and NO production from SCs. To explore the role of ApoE isoforms in inflammatory responses, we detected levels of cytokines (TNF-α, IFN-γ, IL-4, IL-6 and IL-10) within SCs and in the culture supernatants (IL-6, IL-10 and NO only) in the three strains of Tg mice. Intracellular TNF-α, IFN-γ, IL-4, IL-6 and IL-10 were almost undetectable by flow cytometry before stimulation. After 72 hr stimulation with LPS plus IFN-γ, the expressions of above five cytokines were up-regulated in all group mice. SCs from ApoE3 mice expressed higher levels of IL-6 and IL-10 when compared with SCs from ApoE2 and ApoE4 Tg mice. Similar patterns of IL-6, IL-10 and NO production were found in the culture supernatants after stimulation. Concentrations of IL-6, IL-10 and nitrite/nitrate were clearly elevated in all groups. As compared with ApoE3 group, lower levels of IL-6, IL-10 and NO production were detected in ApoE4 group. The expression of iNOS by SCs was undetectable before stimulation. After stimulation, the iNOS positive cell percentage was more than 99% when detected by flow cytometry. The levels of iNOS expression (as shown either by postive cell percentage or geometric mean fluorescence intensity) were insignificantly different among groups.ApoE isoforms did not affect the expression of co-stimulatory molecules and ApoE. Expression of co-stimulatory molecules, i.e. CD80 and CD86, MHC-II, CD1d and TLR-4 on/in SCs is a first critical step in the immune-inflammatory response. Before stimulation, the expression of these molecules was as low as undetectable. After stimulation, the expression of CD80, CD86, MHC-II, CD1d and TLR-4 was up-regulated. However, none of their expressions was significantly different among groups. The intracellular expression of ApoE, as assessed with mean fluorescence intensity from flow cytometry, or concentration of ApoE in the SC culture supernatants as detected by ELISA, was not significantly different among groups.ApoE 3 inhibited the expression of signaling pathways NFκB and Akt. To obtain a further insight into the mechanism of ApoE isoform modulation of inflammation, we studied the role of intracellular signaling molecules, i.e. NFκB and Akt in inflammatory responses. After stimulation, NFκB was differentially expressed by SCs in the three groups, with the lowest level in ApoE3 group, followed by ApoE4 and ApoE2 groups; SCs from ApoE3 and ApoE4 Tg mice expressed lower levels of Akt as compared with ApoE2 Tg mice.Conclusions: SCs respond to inflammatory insults in vitro in an ApoE-isoform-dependent fashion. SCs from ApoE2 and ApoE4 Tg mice bear some insufficiency in producing cytokines and NO whereby modulating local inflammatory responses as compared with their ApoE3 counterparts. Our findings may help to clarify the effects of ApoE genotypes on inflammatory disorders of the PNS.
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