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

A novel antigen that induces nitric oxide (NO) synthesis by murine peritoneal exudate cells (PEC) was prepared from a culture supernate of Streptococcus anginosus NCTC 10713 in dialysed medium by column chromatography with DEAE-Sephacel followed by size-exclusion high performance liquid chromatography (HPLC). A chemical analysis of the S. anginosus antigen (SAA) revealed that it mainly consisted of carbohydrates (rhamnose, N-acetylglucosamine, glucose and galactose), smaller quantities of protein and a trace amount of phosphorus. The SAA stimulated PEC from C57BL/6N mice to produce NO and accumulate induced NO synthetase (iNOS) mRNA in a dose-dependent manner, reaching a plateau with 10-30,mug/ml. Furthermore, a reverse transcription-PCR assay revealed that SAA 10 mug/ml could induce mRNA accumulation of tumour necrosis factor-alpha, interleukin (IL)-1 beta and IL-6 as well as iNOS. In contrast, Rantz-Randall antigen (RRA), a carbohydrate antigen prepared from the organisms, could not induce NO synthesis or cause the accumulation of iNOS 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 immunodiffusion, SAA reacted with both anti-SAA and anti-RRA antibodies, and the precipitin bands formed crossing lines, suggesting that SAA could possess two different antigenic components - one that reacts specificially with anti-SAA antibodies and another that has an identity similar to that of RRA. Taken together, SAA, a novel antigen of S. anginosus, was found to induce NO synthesis as well as produce inflammatory cytokines in murine PEC. It is suggested that the protein molecule of SAA may exclusively induce NO synthesis, and its carbohydrate component(s) could have a relationship to cytokine production.