Neither CD14 nor serum is absolutely necessary for activation of mononuclear phagocytes by bacterial lipopolysaccharide

Abstract

The stimulation of mononuclear phagocytes by lipopolysaccharide (LPS) is facilitated by the binding of complexes of LPS and LPS-binding protein to CD14. Although it is clear that CD14 is involved in LPS-induced signaling, other investigators have hypothesized the existence of additional signaling pathways in macrophages. We sought to determine whether CD14-independent pathways of monocyte activation might exist. Washed human mononuclear cells responded with reduced sensitivity to LPS in the absence of serum. Anti-CD14 monoclonal antibody (MAb) inhibited the response to LPS in serum-free conditions, but this was easily reversed 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 of abundant cell surface CD14. Differentiated SFM cells responded to LPS despite the complete absence of serum proteins for > 20 generations of 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 that these compounds compete with LPS at a site distinct from CD14. Undifferentiated SFM cells (expressing minimal CD14) still responded to LPS in serum-free conditions, and anti-CD14 MAb had little inhibitory effect. The addition of purified LPS-binding protein or human serum promoted a CD14-dependent pathway of monocyte activation by LPS in these cells. We conclude that monocytes do not absolutely require serum proteins to be stimulated by LPS and that CD14-independent LPS signaling pathways exist which are inhibitable by lipid IVa and R. sphaeroides lipid A.