NEITHER CD14 NOR SERUM IS ABSOLUTELY NECESSARY FOR ACTIVATION OF MONONUCLEAR PHAGOCYTES BY BACTERIAL LIPOPOLYSACCHARIDE

The stimulation of mononuclear phagocytes by lipopolysaccharide (LPS) is facilitated by the binding of complexes of LPS and LPS-binding prot ein to CD14. Although it is clear that CD14 is involved in LPS-induced signaling, other investigators have hypothesized the existence of add itional signaling pathways in macrophages. We sought to determine whet her CD14-independent pathways of monocyte activation might exist. Wash ed human mononuclear cells responded with reduced sensitivity to LPS i n the absence of serum. Anti-CD14 monoclonal antibody (MAb) inhibited the response to LPS in serum-free conditions, but this was easily reve rsed at higher concentrations of LPS. We established a human monocytic cell line, designated SFM (derived from THP-1), in serum-free medium to examine LPS responses under defined conditions. Differentiation of SFM cells with 1,25-dihydroxycholecalciferol promoted the expression o f abundant cell surface CD14. Differentiated SFM cells responded to LP S despite the complete absence of serum proteins for >20 generations o f growth. LPS stimulation of differentiated SFM cells was inhibited by anti-CD14 MAbs only when serum was present. In contrast to anti-CD14 MAb, the LPS antagonists lipid IVa and Rhodobacter sphaeroides lipid A inhibited monocyte activation under serum-free conditions, implying t hat these compounds compete with LPS at a site distinct from CD14. Und ifferentiated SFM cells (expressing minimal CD14) still responded to L PS in serum-free conditions, and anti-CD14 MAb had little inhibitory e ffect. The addition of purified LPS-binding protein or human serum pro moted a CD14-dependent pathway of monocyte activation by LPS in these cells. We conclude that monocytes do not absolutely require serum prot eins to be stimulated by LPS and that CD14-independent LPS signaling p athways exist which are inhibitable by lipid IVa and R. sphaeroides li pid A.