Fig. 7

LCN-2 regulates neutrophil adhesion and transmigration by modulating the Dab2/integrin β1 axis. a Pull down assay from neutrophils exposed to conditioned media from RPE cells overexpressing IFNλ (1:1, for 6 h) or to recombinant IFNλ (200 U/mL, for 2 h) showed increased association between LCN-2 and Dab2 upon IFNλ treatment, relative to control. n = 3. *P < 0.05 and **P < 0.01 (one-way ANOVA and Tukey’s post hoc test). b–d Flow cytometry assay showed increased extracellular and decreased intracellular expression of integrin β1 (FITC-A Median) respectively, in WT neutrophils treated with either recombinant IFNλ (200 U/mL, 2 h) or conditioned media from RPE cells overexpressing IFNλ (1:1 diluted, 6 h), compared to controls. Absence of LCN-2 in neutrophils (LCN-2^−/−) led to a reversal in the expression of both extracellular and intracellular levels on integrin β1, even after IFNλ treatment, relative to WT neutrophils. n = 3. *P < 0.05, **P < 0.01, and ^#P < 0.05 (one-way ANOVA and Tukey’s post hoc test). e, f WT and LCN-2^−/− neutrophils exposed to IFNλ (recombinant 200 U/mL for 2 h or 1:1 diluted conditioned media from RPE cells overexpressing IFNλ for 6 h), showed rapid adhesion to fibrinogen (20 mg/mL) coated plates (top panel, arrows: graph denotes adherent cells, counted in 0.2 mm²) and transmigration across fibrinogen (150 mg/mL) coated plates (bottom panel, arrows: graph denotes relative migration (%) of cells, representative of cell count at the bottom of the insert using a computer assisted cell counter system). Integrin β1 shRNA transfected and LCN-2^−/− neutrophils do not show changes in adhesion and transmigration even after IFNλ exposure (asterisk) n = 3. *P < 0.05 and **P < 0.01 (one-way ANOVA and post hoc test). Scale bar, 50 μm