The Role And Related Mechanism Of TNF-α Production Of Schwann Cell In The Course Of Inflammation

【Background and Objective】Schwann cells (SCs) are glia of the peripheral nervous system (PNS). Besides their roles in myelination, trophic support and regeneration of axons, SCs exhibit potential for some immune functions, similarly to non-myelinating glia of the central nervous system (CNS), SCs can be induced to produce cytokines and chemokines, express MHC class II molecules, adhesion molecules and serve as antigen presenting cells. Tumor necrosis factor-alpha (TNF-α), a pleiotropic cytokine with both secreted and transmembrane forms, predominately mediates immune and inflammatory responses. In vivo, SCs at the site of injury may also produce TNF-α, as SCs are activated and/or damaged following nerve injury.However, the precise mechanisms of TNF-αsynthesis are still not clear. The biological properties of TNF-αare signaled through two distinct cell surface receptors: TNF receptor typeⅠ(TNFRⅠ, p55) and TNF receptor typeⅡ(TNFRⅡ, p75).To certificate the fact that SCs, as immunocompetent cells, may participate the inflammatory and immune reaction following injury or infection of peripheral nerve, we firstly begin with the important cytokine-TNF-α, further analysis the molecular mechanism of synthesis TNF-αby SC, then investigate the biological properties of autocrine TNF-αon SC. These results may establish a basis for SC, as immunocompetent cells, meanwhile, may provide a theoretical evidence for the involvement of SC in pathogenesis of neurodegenerative disorders.【Methods】(1) Schwann cells were treated with LPS at different concentration and variant time. TNF-αin cytoplasm and supernatant were quantified by sandwich enzyme-linked immunosorbent assay (ELISA). TNF-αmRNA were detected by RT-PCR. Intracellular location of TNF-αwere detected under confocal microscope. Effects of LPS on cell viability were detected by TUNEL.(2) The protein level of phosphorylated-MAPK,NF-κB P65,IκB-α,P-IκB-αand IκB-βwere determined by Western blot analysis. SCs were pretreated with the inhibitor of ERK (PD98059),P38(SB202190) and SAPK/JNK (SP600125) before treated with LPS, Intracellular concentrations of TNF-αwere detected by sandwich enzyme-linked immunosorbent assay (ELISA), TNF-αmRNA,LITAF mRNA,CD14 mRNA,TLR4 mRNA and MyD88 mRNA were detected by RT-PCR. Intracellular location of phosphorylated-MAPK and NF-κB P65 were observed by indirect-immunofluorescence staining.(3) The expression and cellular localization of TNFRⅠand TNFRⅡin SC are detected by RT-PCR and flow cytometry, Cytokines secreted from SC were detected by RT-PCR and ELISA in order to examine the function of TNF-αas an autocrine mediator in SC activation. The effects of TNF-αon cell viability were detected by Cytometry, cell counting kit and terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay.【Results】(1) LPS could predominantly induce TNF-αexpression in cytoplasm of rat Schwann cells, after 10μg/ml for 2h, also, TNF-αwas detected in supernatant. Analysis through indirect-immunofluorescent staining also showed a cytoplasmic staining of TNF-αin the SCs after stimulation with LPS. We treated the SCs at 100μg/ml LPS for 24 h. There was a significant increase in the number of apoptotic cells in LPS-treated cells(2) The activities of ERK, P38 and SAPK/JNK were significantly increased in the LPS treated SCs. The activated peak of phosphorylated-ERK was detected after stimulation with LPS for 30~45 min. The level of phosphorylated-ERK began to fall-off after 60 min. Specifically, significant phosphorylation of P38 and SAPK/JNK were observed at 30 min, followed by a gradual decline reaching basal levels after 45 min, and the maximum response occurred again following persistent stimulation of LPS for 120 min. Inhibition of MAPK activation by using its inhibitor PD98059,SB202190 and SP600125 predominantly suppressed not only the phosphorylation of ERK, P38 and SAPK/JNK, but also expression of TNF-αor TNF-αmRNA. Analysis with immunofluorescent staining also showed a cytoplasmic staining for pERK, pP38 and pSAPK/JNK in the SCs after stimulation with LPS. We also used specific inhibitors of MAPKs, and pretreatment cell with these inhibitors weakened the intensity of fluorescence. NFκB P65 only in the cytoplasma in resting. SC was subjected to LPS for 20 min, IκB-αand IκB-βwere phosphorylated by IκB kinase (IKK) and degraded in the cytoplasm. NFκB P65 subunit was moving to peripheral region of nucleus after stimulated by LPS for 10 min. Finally, it located in nucleus at about 40 min after the treatment. Our results also showed that SCs exhibited a relatively a high level of expression of TLR4 mRNA and MyD88 mRNA after the treatment of LPS. However, SCs expressed CD14 mRNA no matter they were treated with LPS or not.(3) RT-PCR and flow cytometry displayed the high expression of TNFRⅠ, the expression of TNFRⅡwas weak on SC. TNF-α, as an autocrine mediator in SCs activation, can induced TNF-αproduction by SCs through ligation of TNFR, TNF-αalso increased the production of other inflammatory mediators, including iNOS,IL-6 and IL-10. Cytometry and TUNEL assay displayed TNF-αcan induced the apoptosis of SCs.【Conclusions】(1) Bacterial causative agent-LPS could induce the expression and secretion of TNF-αin rat SCs, which imply that SCs, as immunocompetent cells, may play an important role in the immune regulation in the peripheral never system.(2) LPS activated the MAPK and translocation of NF-κB P65 following ligation of CD14 and Toll-like Receptor4 (TLR4), then initiate the transcription of TNF-α. Activation of MAPK-pathway and translocation of NF-κB P65 might be a precondition for the inducement of TNF-αexpression. Reducing biosynthesis of TNF-αand other cytokines by blocking the cell signal pathway, which may give us a new strategy against inflammatory and immune reaction after injury or inflammation of peripheral nerve.(3) In the peripheral nervous system, TNF-αderived from activated SCs plays a critical role as an inflammatory mediator. TNF-α, as an autocrine mediator, induced TNF-αor other inflammatory mediators production by SCs, meanwhile, induced the apoptosis of SCs, through ligation of TNFR. The neuroprotective or cytotoxic effects of TNF-αare mediated through two distinct cell surface receptors.