Antigenic characterisation of a novel Streptococcus anginosus antigen thatinduces nitric oxide synthesis by murine peritoneal exudate cells

A novel antigen that induces nitric oxide (NO) synthesis by murine peritone al exudate cells (PEC) was prepared from a culture supernate of Streptococc us anginosus NCTC 10713 in dialysed medium by column chromatography with DE AE-Sephacel followed by size-exclusion high performance liquid chromatograp hy (HPLC). A chemical analysis of the S. anginosus antigen (SAA) revealed t hat it mainly consisted of carbohydrates (rhamnose, N-acetylglucosamine, gl ucose and galactose), smaller quantities of protein and a trace amount of p hosphorus. The SAA stimulated PEC from C57BL/6N mice to produce NO and accu mulate induced NO synthetase (iNOS) mRNA in a dose-dependent manner, reachi ng a plateau with 10-30,mug/ml. Furthermore, a reverse transcription-PCR as say revealed that SAA 10 mug/ml could induce mRNA accumulation of tumour ne crosis factor-alpha, interleukin (IL)-1 beta and IL-6 as well as iNOS. In c ontrast, Rantz-Randall antigen (RRA), a carbohydrate antigen prepared from the organisms, could not induce NO synthesis or cause the accumulation of i NOS mRNA, although cytokine production was observed after stimulation. The SAA-induced NO synthesis, but not the cytokine production, was sensitive to heat. Furthermore, an immunoblot analysis of SAA indicated that the 43-kDa protein band reacted with anti-SAA but not anti-RRA antibodies. In immunod iffusion, SAA reacted with both anti-SAA and anti-RRA antibodies, and the p recipitin bands formed crossing lines, suggesting that SAA could possess tw o different antigenic components - one that reacts specificially with anti- SAA antibodies and another that has an identity similar to that of RRA. Tak en together, SAA, a novel antigen of S. anginosus, was found to induce NO s ynthesis as well as produce inflammatory cytokines in murine PEC. It is sug gested that the protein molecule of SAA may exclusively induce NO synthesis , and its carbohydrate component(s) could have a relationship to cytokine p roduction.