We investigated the capability of an L-form derived from Staphylococcus aureus to induce tumor necrosis factor alpha (TNF-ax) production in murine peritoneal macrophages. The activity for TNF-a induction was found in the membrane fraction of the L-form but not in the cytoplasmal fraction purified by the sucrose step gradient centrifugation. TNF-at mRNA was also detected in macrophages stimulated with L-form membranes. L-form induced TNF-a production in macrophages from both lipopolysaccharide-responsive and-unresponsive mouse strains. Regardless of the presence of polymyxin B, the activity of TNF-ai induction of L-form was mostly found in the phenol layer, but not in the aqueous layer, both of which were prepared by phenol extraction method. Fractions of L-form membranes representing molecular masses of approximately between 29 and 36 kDa were primarily responsible for inducing the production of TNF-aI consistently. Moreover, this stimulatory effect was abolished by digestion with Streptomyces griseus protease. In Western blot (immunoblot) analysis with anti-lipoteichoic acid antibody, two bands (65 and 45 kDa) were observed in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phenol layer, whereas one band (14 kDa) was observed in either the aqueous layer or lipoteichoic acid of S. aureus. These results suggest that the component in the membrane of the L-form, distinct from cell wall components such as teichoic acid or lipopolysaccharide, possesses the capability to stimulate TNF-a production by macrophages.

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