Cytokine-inducing macromolecular glycolipids from Enterococcus hirae: Improved method for separation and analysis of its effects on cellular activation

Previously, we showed that several minor macromolecular glycolipids account ing for less than 5% of the lipoteichoic acid (LTA) fraction from Enterococ cus hirae ATCC 9790 possess cytokine-inducing activity, whereas the purifie d LTA does not. In other words, the immunobiological activity of the LTA fr action reported in the 1980s was not attributable to LTA itself, but to oth er glycolipids coexisting in the fraction. In the present study, we improve d the procedure of separation of the active glycolipids and evaluated their effects on cellular activation. The immunobiologically active glycolipids were separated from the crude glycolipid fraction obtained by hot phenol-wa ter extraction of the cells. The total yield of active glycolipids was abou t fivefold higher than that separated by the previous method. Interleukin-6 -inducing activities of the active glycolipids from 1,25-dihydroxy vitamin D-3-differentiated human monocytic leukemia cells, THP-1, were inhibited by anti-CD14 mAbs in a dose-dependent manner. Macrophages from Toll-like rece ptor (TLR)-2-deficient or -4-deficient mice completely lacked the ability t o produce tumor necrosis factor-alpha on stimulation with active glycolipid s. These observations indicated that the cellular activation by the active glycolipids from E. hirae is mediated by CD14 and by both TLR2 and TLR4. (C ) 2000 Academic Press.