Lyn‐coupled LacCer‐enriched lipid rafts are required for CD11b/CD18‐mediated neutrophil phagocytosis of nonopsonized microorganisms

The integrin CD11b/CD18 plays a central role in neutrophil phagocytosis. Although CD11b/CD18 binds a wide range of ligands, including C3bi and β‐glucan, and transmits outside‐in signaling, the mechanism of this signaling responsible for phagocytosis remains obscure. Here, we report that lactosylceramide (LacCer)‐enriched lipid rafts are required for CD11b/CD18‐mediated phagocytosis of nonopsonized zymosans (NOZs) by human neutrophils. Anti‐CD11b and anti‐LacCer antibodies inhibited the binding of NOZs to neutrophils and the phagocytosis of NOZs. During phagocytosis of NOZ, CD11b and LacCer were accumulated and colocalized in the actin‐enriched phagocytic cup regions. Immunoprecipitation experiments suggested that CD11b/CD18 was mobilized into the LacCer‐enriched lipid rafts during phagocytosis of NOZs. DMSO‐treated, neutrophil‐like HL‐60 cells (D‐HL‐60 cells) lacking Lyn‐coupled, LacCer‐mediated signaling showed little phagocytosis of NOZs. However, loading of D‐HL‐60 cells with C24 fatty acid chain‐containing LacCer (C24‐LacCer) reconstructed functional Lyn‐associated, LacCer‐enriched lipid rafts, and restored D‐HL‐60 cell NOZ phagocytic activity, which was inhibited by anti‐LacCer and anti‐CD11b antibodies. Lyn knockdown by small interfering RNA blocked the effect of C24:1‐LacCer loading on D‐HL‐60 cell phagocytosis of NOZs. CD11b/CD18 activation experiments indicated phosphorylation of LacCer‐associated Lyn by activation of CD11b. Taken together, these observations suggest that CD11b activation causes translocation of CD11b/CD18 into Lyn‐coupled, LacCer‐enriched lipid rafts, allowing neutrophils to phagocytose NOZs via CD11b/CD18.