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 beta-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.
Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Tomioka, Urayasu, Chiba 279-0021, Japan.
Hitoshi
H
Nakayama
Fumiko
F
Yoshizaki
Alessandro
A
Prinetti
Sandro
S
Sonnino
Laura
L
Mauri
Kenji
K
Takamori
Hideoki
H
Ogawa
Kazuhisa
K
Iwabuchi
D000818:Animals; D015703:Antigens, CD / Q000502:physiology*; D039481:CD11b Antigen / Q000502:physiology*; D018821:CD18 Antigens / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D018922:HL-60 Cells; D006801:Humans; D007790:Lactosylceramides / Q000494:pharmacology*; D021962:Membrane Microdomains / Q000502:physiology*; D051379:Mice; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D010587:Phagocytosis / Q000187:drug effects / Q000502:physiology*; D010588:Phagosomes / Q000502:physiology / Q000648:ultrastructure; D034741:RNA, Small Interfering / Q000235:genetics; D015054:Zymosan / Q000502:physiology; D019061:src-Family Kinases / Q000502:physiology*
D015703:Antigens, CD; D039481:CD11b Antigen; D018821:CD18 Antigens; D007790:Lactosylceramides; D034741:RNA, Small Interfering; D015054:Zymosan; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases