BACTERIAL LIPOPOLYSACCHARIDE BINDS TO CB14 IN LOW-DENSITY DOMAINS OF THE MONOCYTE-MACROPHAGE PLASMA-MEMBRANE

We report that gram-negative bacterial lipopolysaccharide (LPS) binds to CD14 on lipid-enriched, low-density domains of the human monocyte-m acrophage (THP-1 cell) plasma membrane. After brief incubation with [H -3]LPS under conditions that prevent its internalization, THP-1 cells were disrupted using a detergent-free method and plasma membrane fragm ents were separated on density gradients. The [H-3]LPS-binding fragmen ts had low bouyant densities and were enriched, when compared to high- density membrane fragments, in CD14 (a receptor for LPS and other micr obial molecules), p53/56(lyn), GTP-binding proteins, ouabain-inhibitab le Na+/K+ ATPase, shingomyelin, and GM(1) ganglioside. Monoclonal anti -CD14 antibody 60bca blocked [H-3]LPS binding to these membrane fragme nts. Immunoelectron microscopic analysis identified clusters of CD14 o n both large (200-1,000 nm) and small (less than or equal to 200 nm) l ow-density membrane fragments. GM(1) and CD14 were usually found on th e same fragments, yet their distributions on those fragments infrequen tly overlapped. These cells seem to lack arrays of caveolae, the order ed membrane structures that harbor glycosylphosphatidyl-anchored prote ins and GM(1) in many other cell types. Finding that LPS binds to CD14 predominantly in low-density plasma membrane domains suggests, howeve r, that discrete regions of the monocyte-macrophage plasma membrane ma y be organized to facilitate rapid responses to, and internalization o f, molecules that bind CD14. (C) 1997 Wiley-Liss, Inc.