POTENTIAL ROLE OF MEMBRANE INTERNALIZATION AND VESICLE FUSION IN ADHESION OF NEUTROPHILS IN RESPONSE TO LIPOPOLYSACCHARIDE AND TNF

Human polymorphonuclear leukocytes (PMN) respond to LPS with strongly increased integrin-mediated adhesion. While the first step of this pro cess has been identified as the interaction of LPS with CD14 on the ce ll surface, subsequent steps remain to be elucidated, The experiments presented here suggest that monomeric LPS is internalized in vesicles, and uptake may be required for signaling. Fluorescently labeled LPS p resented as monomeric complexes with soluble CD14 appeared in the plas ma membrane of PMN by 5 min and was concentrated in cytoplasmic vesicl es by 20 min. Adhesion in response to LPS/soluble CD14 occurred only a fter a 15- to 20-min lag period, consistent with endocytosis occurring before signal generation, In contrast, there was no time lag for adhe sion in response to the formyl peptide formyl-norleucyl-leucyl-phenyla lanine (fNLLP), Adhesion in response to LPS but not fNLLP, war complet ely blocked by lowering the temperature to 19 degrees C, a procedure t hat prevents vesicle fusion, These studies indicated that an event wit h the time and temperature dependence of endocytosis precedes signalin g by LPS. Cytochalasin D, an inhibitor of phagocytosis, and wortmannin , an inhibitor of phosphatidylinositol 3-kinase that blocks vesicle fu sion and phagocytosis, both completely blocked adhesion in response to LPS but not in response to fNLLP. These results support the idea that LPS internalization and early endosomal fusion may be required for si gnal transduction. Parallel studies showed that the adhesion response to TNF had time, temperature, and inhibitor sensitivities nearly ident ical with those of LPS, suggesting that responses to TNF may also incl ude an obligate vesicle fusion step.