PHAGOCYTOSIS OF GRAM-NEGATIVE BACTERIA BY A UNIQUE CD14-DEPENDENT MECHANISM

THP-1-derived cell lines were stably transfected with constructs encod ing glycophosphatidyl-inositol (GPI) -anchored or transmembrane forms of human CD14. CD14 expression was associated with enhanced phagocytos is of serum (heat-inactivated) -opsonized Escherichia coli (opEc). Bot h the GPI-anchored and transmembrane forms of CD14 supported phagocyto sis of opEc equally well. Lipopolysaccharide-binding protein (LBP) pla yed a role in CD14-dependent phagocytosis as evidenced by inhibition o f CD14-dependent phagocytosis of opEc with anti-LBP monoclonal antibod y (mAb) and by enhanced phagocytosis of E. coli opsonized with purifie d LBP. CD14-dependent phagocytosis was inhibited by a phosphatidylinos itol (PI) 3-kinase inhibitor (wortmannin) and a protein tyrosine kinas e inhibitor (wortmannin) and a protein tyrosine kinase inhibitor (tyrp hostin 23) but not a protein tyrosine kinase C inhibitor (bisindolyl-m aleimide) or a divalent cation chelator (ethylenediaminetetraacetate). Anti-LBP mAb 18G4 and anti-CD14 mAb 18E12 were used to differentiate between the pathways involved in CD14-dependent phagocytosis and CD14- dependent cell activation. F(ab')(2) fragments of 18G4, a mAb to LBP t hat does not block cell activation, inhibited ingestion of opEc by THP 1-wtCD14 cells. 18E12 (an anti-CD14 mab that does not block LPS bindin g to CD14 but does inhibit CD14-dependent cell activation) did not inh ibit phagocytosis of LBP-opEc by THP1-wtCD14 cells. Furthermore, CD14- dependent phagocytosis was not inhibited by anti-CD18 (CR3 and CR4 bet a-chain) or anti-Fc gamma receptor mAb.