Neutrophils play an essential role via phagocytosis in host defense against microbial infections. However, little is known about molecular mechanisms underlying phagocytosis in neutrophils, because of the difficulty in genetically manipulating these cells. Here, we provide the first comprehensive description of phospholipid metabolism during phagocytosis in human neutrophils, which we have efficiently transfected with cDNAs encoding lipid-probing protein modules. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), an F-actin organizer abundant in the plasma membrane, diminishes progressively from phagosomes during phagosome formation and vanishes after phagosome closure with F-actin disappearance. Diacylglycerol, a metabolite of PtdIns(4,5)P(2), appears at phagocytic cups and remains associated with nascent (closed) phagosomes; it may function with phosphatidylserine, present in both plasma and phagosomal membranes, to recruit phagocytosis-associated proteins. From PtdIns(4,5)P(2), PtdIns(3,4,5)P(3) is also produced at phagocytic cups but becomes undetectable shortly after phagosome sealing, consistent with its proposed roles in pseudopod extension and phagosome closure. PtdIns(3)P, a putative participant in phagosome maturation, emerges at closed phagosomes as does the class III PtdIns 3-kinase Vps34. Although the small GTPases Rab5 and Rab7 are thought to contribute to phagosome maturation in macrophages, Rab5 but not Rab7 fails to accumulate at phagosomes in neutrophils, suggesting a difference in phagocytic mechanism between the two phagocytes.
