MFG-E8 inhibits neutrophil migration through α_vβ₃-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, α_vβ₃/α_vβ₅-integrins, by anti-α_v-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 α_vβ₃-integrin-dependent MAP kinase activation.