MFG-E8 inhibits neutrophil migration through αvβ₃-integrin-dependent MAP kinase activation.
We have previously demonstrated the involvement of milk fat globule-epidermal growth factor-factor 8 (MFG‑E8) in reducing neutrophil infiltration in a murine model of acute lung injury (ALI). In the present study, we aimed to delineate the mechanisms through which MFG‑E8 attenuates neutrophil migration. Recombinant human MFG‑E8 (rhMFG‑E8) was expressed and purified in our facility. The human differentiated neutrophil cell line, dHL‑60, was treated with rhMFG‑E8 and cell migration assay was performed in a Boyden chamber using recombinant interleukin‑8 (IL‑8) as the chemoattractant. Surface CXCR2 and intracellular G protein‑coupled receptor kinase 2 (GRK2) levels were evaluated by flow cytometry or western blot analysis. The levels of mitogen‑activated protein (MAP) kinases were determined by western blot analysis. Treatment with rhMFG‑E8 resulted in a significant inhibition of dHL‑60 cell migration in a dose‑dependent manner. There was a 46% decrease in CXCR2 expression in the rhMFG‑E8‑treated dHL‑60 cells, which was associated with a 32% increase in GRK2 expression. In the dHL‑60 cells, treatment with rhMFG‑E8 promoted the phosphorylation of p38 and extracellular signal-regulated kinase (ERK) within 10‑30 min. The use of SB203580, a p38 inhibitor, and PD98059, an ERK inhibitor, resulted in the restoration of dHL‑60 cell migration which was significantly inhibited treatment with rhMFG‑E8. Furthermore, blocking the MFG‑E8 receptors, αvβ3/αvβ5‑integrins, by anti‑αv‑integrin neutralizing antibody (Ab) inhibited the activation of p38 and ERK, and reversed the rhMFG‑E8‑induced inhibition of dHL‑60 cell migration. Finally, treatment of the dHL‑60 cells with SB203580 and PD98059 neutralized the rhMFG‑E8‑induced downregulation of CXCR2 expression and upregulation of GRK2 expression, as well as the inhibitory effects on cell migration. Our findings reveal a novel mechanism of action of MFG‑E8 through which it inhibits neutrophil migration through αvβ3-integrin-dependent MAP kinase activation.
Center for Translational Research, The Feinstein Institute for Medical Research and Department of Surgery, Hofstra North Shore‑LIJ School of Medicine, Manhasset, NY, USA.
Monowar
M
Aziz
Center for Translational Research, The Feinstein Institute for Medical Research and Department of Surgery, Hofstra North Shore‑LIJ School of Medicine, Manhasset, NY, USA.
Weng-Lang
WL
Yang
Center for Translational Research, The Feinstein Institute for Medical Research and Department of Surgery, Hofstra North Shore‑LIJ School of Medicine, Manhasset, NY, USA.
Lana M
LM
Corbo
Center for Translational Research, The Feinstein Institute for Medical Research and Department of Surgery, Hofstra North Shore‑LIJ School of Medicine, Manhasset, NY, USA.
Wayne W
WW
Chaung
Center for Translational Research, The Feinstein Institute for Medical Research and Department of Surgery, Hofstra North Shore‑LIJ School of Medicine, Manhasset, NY, USA.
Shingo
S
Matsuo
Center for Translational Research, The Feinstein Institute for Medical Research and Department of Surgery, Hofstra North Shore‑LIJ School of Medicine, Manhasset, NY, USA.
Ping
P
Wang
D057134:Antibodies, Neutralizing / Q000276:immunology / Q000494:pharmacology; D000954:Antigens, Surface / Q000494:pharmacology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005419:Flavonoids; D005434:Flow Cytometry; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000096:biosynthesis / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D039302:Integrin alphaVbeta3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016209:Interleukin-8 / Q000276:immunology; D008894:Milk Proteins / Q000494:pharmacology*; D020556:Neutrophil Infiltration / Q000187:drug effects / Q000276:immunology; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D023063:Receptors, Interleukin-8B / Q000096:biosynthesis / Q000378:metabolism; D019030:Receptors, Vitronectin / Q000037:antagonists & inhibitors; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D057134:Antibodies, Neutralizing; D000954:Antigens, Surface; C508606:CXCL8 protein, human; D005419:Flavonoids; D007093:Imidazoles; D039302:Integrin alphaVbeta3; D016209:Interleukin-8; C098942:MFGE8 protein, human; D008894:Milk Proteins; D011725:Pyridines; D023063:Receptors, Interleukin-8B; D019030:Receptors, Vitronectin; C064660:integrin alphaVbeta5; C496567:GRK2 protein, human; D054769:G-Protein-Coupled Receptor Kinase 2; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one