INDUCTION OF MACROPHAGE-MEDIATED PRODUCTION OF TUMOR-NECROSIS-FACTOR-ALPHA BY AN L-FORM DERIVED FROM STAPHYLOCOCCUS-AUREUS

We investigated the capability of an L-form derived from Staphylococcu s aureus to induce tumor necrosis factor alpha (TNF-alpha) production in murine peritoneal macrophages. The activity for TNF-alpha induction was found in the membrane fraction of the L-form but not in the cytop lasmal fraction purified by the sucrose step gradient centrifugation. TNF-alpha mRNA was also detected in macrophages stimulated with L-form membranes. L-form induced TNF-alpha production in macrophages from bo th lipopolysaccharide-responsive and -unresponsive mouse strains. Rega rdless of the presence of polymyxin B, the activity of TNF-alpha induc tion of L-form was mostly found in the phenol layer, but not in the aq ueous layer, both of which were prepared by phenol extraction method. Fractions of L-form membranes representing molecular masses of approxi mately between 29 and 36 kDa were primarily responsible for inducing t he production of TNF-alpha. consistently. Moreover, this stimulatory e ffect was abolished by digestion with Streptomyces griseus protease. I n Western blot (immunoblot) analysis with anti-lipoteichoic acid antib ody, two bands (65 and 45 kDa) were observed in the sodium dodecyl sul fate-polyacrylamide gel electrophoresis of the phenol layer, whereas o ne band (14 kDa) was observed in either the aqueous layer or lipoteich oic acid of S. aureus. These results suggest that the component in the membrane of the L-form, distinct from cell wall components such as te ichoic acid or lipopolysaccharide, possesses the capability to stimula te TNF-alpha production by macrophages.