A Novel Immunomodulator Different from Endotoxin was Extracted from Prevotella intermedia ATCC 25611 with Hot Phenol-Water

Lipopolysaccharides (LPS) extracted from oral black-pigmented anaerobic rods with hot phenolwater have been reported to exert bioactivities different from those of classical endotoxin. This study was carried out to isolate a novel immunomodulator different from endotoxin from the phenol-water extract of Prevotella intermedia ATCC 25611. The bacterial cells were extracted twice with phenol-water at 67.. for 20 min. The pooled extract in the water phase was dialyzed against distilled water, and ultracentrifuged at 100, 000 x g for 3 hr to remove the lipopolysaccharide (LPS). The resultant supernatant was lyophilized and after being resolved in 0.2% sodium deoxycholate, it was subjected to Sephadex G-100 column chromatography with monitoring of mitogenicity on the splenocytes from C3H/HeN and C3H/HeJ mice, Limulus activity, and then deoxycholate polyacrylamide gel electrophoresis (DOCPAGE), to separate the target material and residual LPS. The first fraction showed mitogenicity on the splenocytes from both strains of mice, only slight Limulus activity, and high-molecular-weight bands, 10-12 K, on DOC-PAGE. The latter fraction showed mitogenicity on the splenocytes from the C3H/HeN but not the C3H/HeJ mice, strong Limulus activity, and a single low-molecularweight band, approximately 3 K, on DOC-PAGE. These properties of the latter fraction were in accordance with those of the LPS (LPS-PCP) extracted from the P. intermedia with the phenol -chloroform-petroleum ether mixture . The former fraction was treated with NP 1 nuclease, and rechromatographed on Sephadex G-100. The resultant Prevotella mitogen (PM) fraction exhibited activities similar to those of the parent fraction, and also stimulated peritoneal macrophages from both the C3H/HeN and C3H/HeJ mice to produce interleukin (IL) -6 and tumor necrosis factor (TNF) -a. The mitogenicity of the PM fraction was resistant to heat (100.. for 1 hr) and protease-treatments. Furthermore, the PM fraction stimulated human peripheral blood cells and gingival fibroblasts to produce IL-6. The activities of the PM on the splenocytes and macrophages from the C3H/HeN mice were scarcely inhibited by polymyxin B. By contrast, the LPS-PCP lacked activity on the cells from C3H/HeJ mice or human gingival fibroblasts and its activities on the cells of C3H/HeN mice were completely inhibited by polymyxin B. The LPS (LPS-PW) extracted from the bacteria with hot phenol-water exhibited both the properties of the PM fraction and LPS-PCP