Induction of caspase- and reactive oxygen species-independent phosphatidylserine externalization in primary human neutrophils: role in macrophage recognition and engulfment.

Macrophage recognition and disposal of neutrophils are important steps in the resolution of inflammation. Externalization of phosphatidylserine (PS) on the cell surface serves as a common recognition signal for macrophages and is associated with the apoptosis program in neutrophils. Here, we report that macrophage-differentiated PLB-985 cells induce rapid, caspase-independent PS externalization in human neutrophils. A similar degree of PS externalization was seen when neutrophils were cocultured with gp91(phox)-deficient PLB-985 macrophages, thus demonstrating that macrophage-induced PS externalization was NADPH oxidase-independent. Macrophage-induced PS externalization required cell-to-cell contact and kinase activation and was shown to correlate with neutrophil degranulation. Of note, the degree of engulfment of such PS-positive neutrophils by activated human monocyte-derived macrophages was considerably lower than for neutrophils undergoing constitutive apoptosis, indicating that PS externalization alone is not sufficient for macrophage disposal of neutrophils. However, addition of recombinant milk fat globule epidermal growth factor 8, a PS-binding protein, restored engulfment of the macrophage-cocultured target cells. Finally, neutrophils undergoing spontaneous apoptosis but not macrophage-cocultured neutrophils displayed surface expression and release of annexin I, and the addition of N-t-Boc-Phe-D-Leu-Phe-D-Leu-Phe (Boc1), a formyl peptide receptor/lipoxin receptor antagonist, suppressed clearance of apoptotic neutrophils. Conditioned medium from apoptotic neutrophils also promoted the engulfment of macrophage-cocultured neutrophils, and Boc1 blocked this process. Taken together, these studies highlight a novel pathway of PS externalization in primary human neutrophils and also provide evidence for an auxiliary function of annexin I in macrophage clearance of neutrophils.