Experssion Of Toll-Like Receptor 4 On Microglia In The CNS And The Secretion Of Cytokines Induced By IgG

Central nervous system (CNS) is an immune privileged area which is lack of lymphocytic system and is protected by blood-brain barrier (BBB). Microglia is residential phagocyte of the CNS. Many physiologic and pathologic factors such as blood pressure challenge, immune stimulation and traumatic injury could lead to BBB compromise and thereafter the extravasation of circulating proteins. How these proteins would affect the CNS immune reaction was not very clear. Pattern recognition receptor (PRR) has been intensively studied and shed light in innate immune reaction mechanisms. Toll-like receptor (TLR) 4, an important member of PRR, is expressed mainly by microglia, and plays important role in innate immune reaction in the CNS. We investigated TLR4 expression and IgG extravasation in several non-infectious CNS injury models; and stimulated primary cultured microglia and mixed glia from neonatal rats with rat IgG to see the TLR4 expression and cytokine secretion.1. TLR4 expression in the microglia with BBB opening-induced IgG extravasation. In this part, we observed TLR4 expression in the rat model of transient hypertension, immune stimulation, ischemia/reperfusion and sterile spinal cord crush injury. Using immunofluorescent staining and RT-PCR, we found TLR4 was expressed in the area that IgG extravasated compared with normal control. Peripheral LPS stimulation induced TLR4 mRNA upregulation as well as the protein expression in the endothelia-like cells within the IgG extravasated area. By transient hypertension induced by adrenalin injection, this effect was enhanced and the TLR4 immunoreactive product was found in the microglia besides blood vessel cells. These results indicate TLR4 expressed in microglia was closely related to IgG extravasation in the CNS, and suggest that TLR4 might be involved in IgG-induced innate immune reaction in the CNS.2. TLR4 expression in cultured microglia and cytockine secretion incduced by IgG. With primary cultured purified microglia isolated from neonatal rats, we examined TLR4 and CD64 (FcγR I) expression and pro-inflammatory cytokines production as responses to rat IgG stimulation, using immunofluorescent staining and ELISA methods. Both CD64 and TLR4 were upregulate by IgG stimulation, and these two receptors were co-locoalized. As compared to LPS stimulation and blank control, IgG upregulated TLR4 expression, meanwhile, the stimulated cells became larger and with many obvious pseudopods, indicating activation and phagocytosis of the microglia. Compared with control group(normal medium without IgG), tumor necrosis factor alpha (TNF-α) level in the culture medium was elevated 24 hours after IgG exposure and appeared to be dose-depended, although it was lower than LPS stimulated group. But interferon gamma (IFN-γ) was below the blank control both in LPS and IgG stimulated groups. These results indicated that IgG induced TLR4 expression and TNF-αincrease in cultured microglia, suggesting TLR4 may be activated by IgG from the same species so as to trigger the MyD88-dependent signal pathway.3. TLR4 expression in cultured mixed glial and cytockine secretion incduced by IgG. Primary glial cells were obtained from neonatal rats as the above. Mixed glial celles were exposed to IgG or LPS for 24 hours. Then the medium was collected for ELISA test and the glial cells were fixed to see TLR4 and CD64 expression by immunofluorescent staining. As in the purified microglia, both CD64 and TLR4 were upregulate by IgG stimulation, and these two receptors were co-locoalized in microlia of mixed CNS glial culture. Mixed glial culture showed undetectable TNF-αand IFN-γin the medium of IgG stimulated group, while LPS elevated TNF-αlevel. Interleukin-1 beta (IL-1β) was significantly elevated by higher dose of IgG stimulation and moderate dose of LPS in the medium of mixed culture. It was also expressed in cytoplasma of microlia and astrocytes of the mixed glial cultured, indicated by immunofluorescent staining. These results show three points for mixed glial cultures: (1) Microglia is the most important immunoreactive cells in the CNS glial cells and could respond to IgG stimulation by up-regulated TLR4 and CD64 expression. (2) There has interaction among different glial cell types.The results suggestion that endogenous IgG extravasation could induce microgial TLR4 and CD64 up-regulation in CNS, and lead to pro-inflammatory cytokine produce though activtion the TLR4 downstream singnal pathway. At the same time it prompted that molecule protein could permeate into CNS induced microglia TLR4 and CD64 up-regulation initiation the inherent immunity reaction when BBB opened.,which might elucidate the pathogenic mechanisms of some CNS autoimmune disorders.