S100A9 mediates neutrophil adhesion to fibronectin through activation of beta2 integrins

Abstract

Neutrophil migration from the blood to inflammatory sites follows a cascade of events, in which adhesion to endothelial cells and extracellular matrix proteins is essential. S100A8, S100A9, and S100A12 are small abundant proteins found in human neutrophil cytosol and presumed to be involved in leukocyte migration. Here we investigated the S100 proteins' activities in neutrophil tissue migration by evaluating their effects on neutrophil adhesion to certain extracellular matrix proteins. S100A9 induced adhesion only to fibronectin and was the only S100 protein that stimulated neutrophil adhesion to this extracellular matrix protein. Experiments with blocking antibodies revealed that neither beta1 nor beta3 integrins were strongly involved in neutrophil adhesion to fibronectin, contrary to what the literature predicted. In contrast, neutrophil adhesion to fibronectin was completely inhibited by anti-beta2 integrins, suggesting that S100A9-induced specific activation of beta2 integrin is essential to neutrophil adhesion.

Figure 1

S100A9 induces neutrophil adhesion to fibronectin. Neutrophils (5 × 10⁴ cells/well) were incubated with S100A8, S100A9, S100A8/A9 or S100A12 (20 μg/mL), fMLF 10^-6M (positive control) or M199 (negative control) and let to adhere on fibronectin (A), vitronectin (B), laminin (C), and collagen (D) for 30 min at 37°C. The number of adhered neutrophils was determined as described in materials and methods. Data shown represent mean + SEM of ≥ 3 experiments performed on neutrophils from different donors. ★ p < 0.05, ★★ p < 0.01, One way ANOVA, Dunnett multiple comparison test (compared with control).

Figure 2

S100A9 has no effect on β1 and β3 integrins, but upregulates β2 integrins surface expression. Expression of β1, β2, and β3 integrins was analyzed on S100A9-stimulated neutrophils by flow cytometry analysis. Neutrophils were stimulated with S100A9 (20 μg/mL) for 30 min at 37°C and incubated with specific Abs directed against the different subunits of β1, β2, and β3 integrins. Histograms of S100A9-stimulated neutrophils are illustrated in gray, while unstimulated neutrophils (HBSS) appear in open histograms. Nonspecific stainings are illustrated by a dotted line. Data shown are from an experiment representative of 2 others done on cells from different donors.

Figure 3

S100A9 stimulates neutrophil adhesion to fibronectin via β 2, but not β1 nor β3 integrins. Neutrophils were incubated with blocking Abs directed against A) β1 integrins, B) β3 integrins, or C) β2 integrins and their adhesion to fibronectin was stimulated with S100A9 (20 μg/mL) or M199 (negative control) for 30 min at 37°C. Data shown represent mean + SEM of ≥ 3 experiments performed on neutrophils from different donors. ★ p < 0.05, ★★ p < 0.01, One way ANOVA, Dunnett multiple comparison test (compared with isotype control).