ABSTRACT
Between one-third and one-half of all cases of sepsis are known to be caused by gram-positive microorganisms through the cell wall component, e.g. lipoteichoic acid (LTA). Gram-positive bacteria are also known to induce encephalomyelitis and meningeal inflammation, and enhance the production of nitric oxide (NO) via expression of inducible nitric oxide synthase (iNOS) in murine tissue macrophages. It remains to be explored if LTA could activate microglia considered to be resident brain macrophages. We report here that LTA derived from gram-positive bacteria (Staphylococcus aureus) significantly induces NO release and iNOS expression in primary microglia. LTA-induced NO accumulation was detected at 2 h in microglial culture and was significantly attenuated by pretreatment with anti-CD14, complement receptor type 3 (CR3) or scavenger receptor (SR) antibodies. LTA activated mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase, p38 MAPK or c-Jun N-terminal kinase in cultured microglia. LTA-elicited microglial NO production was also drastically suppressed by SB203580 (p38 MAPK inhibitor) or pyrrolidine dithiocarbamate (an inhibitor of nuclear factor kappaB), indicating that p38 MAPK and nuclear factor kappaB were involved in microglial NO release after LTA challenge. These results suggest that gram-positive bacterial product such as LTA can activate microglia to release NO via the signal transduction pathway involving multiple LTA receptors (e.g. CD14, CR3 or SR), p38 MAPK and nuclear factor kappaB. The in vivo study further confirmed that administered intracerebrally LTA induced considerable noticeable iNOS, phospho-IkappaB and phospho-p38 MAPK expression in microglia/macrophages.
Subject(s)
Lipopolysaccharides/pharmacology , Microglia/drug effects , Nitric Oxide/metabolism , Signal Transduction/drug effects , Teichoic Acids/pharmacology , Animals , Antibodies/pharmacology , Blotting, Western/methods , Carbidopa/immunology , Cells, Cultured , Dose-Response Relationship, Drug , Drug Combinations , Eye Proteins/immunology , Fluorescent Antibody Technique/methods , Gene Expression Regulation/drug effects , I-kappa B Proteins/metabolism , Indoles , Lectins/metabolism , Levodopa/immunology , Lipopolysaccharide Receptors/immunology , Microglia/enzymology , Nerve Tissue Proteins/immunology , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Nitrites/metabolism , Rats , Rats, Wistar , Time Factors , gamma-Synuclein , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
HIV stimulates strong immune CD8(+) cytotoxic T lymphocytes (CTL) response in infected people, despite causing an immunodeficiency. It has been demonstrated that this response could be very important for the control of the virus. We have shown previously that a recombinant fowlpox virus (rFWPV), expressing the multi-epitope polypeptide (MEP) from HIV-1 TAB9, induces strong and protective Th1 and CTL responses in Balb/c mice. Here, we have studied the CTL response against MEPs TAB9 and CR3 after immunizing with rFWPVs, where these genes are under the control of a strong synthetic early/late promoter or the 7.5 kDa promoter from vaccinia virus. TAB9 expression was increased by more than 9-fold using the strong promoter, which was translated into a two times increase in CTL response. The overall expression of CR3 was already ten times higher when compared with TAB9 with the 7.5 kDa promoter, but the use of a stronger promoter showed no effect either on the expression or CTL response. Moreover, rFWPV expressing TAB9 induced a stronger CTL response than those expressing CR3, measured as the number of interferon- gamma -secreting splenocytes, in spite of its lower antigen expression levels. These results suggest that the capacity of a stronger promoter to increase the MEP expression and/or CTL response against their epitopes is highly dependent on the nature of the polypeptide used.
