Shedding of L-selectin from the neutrophil surface is responsible for its role in outside-in signaling. Adhesion-dependent oxidative burst of WT neutrophils and WT neutrophils pretreated with IgG F(ab)2 or Mel14 F(ab)2 (A), WT neutrophils pretreated with IgG or 4RA10 antibody (B), or WT and Sell−/− neutrophils pretreated with scrambled or L-selectin Tat peptides (C) prior to plating on fibrinogen alone or in the presence of TNF-α. WT and Sell−/− neutrophils preincubated for 2 hours in 1× or 0.65× PBS were plated on pRGD for 10 minutes and lysates were immunoblotted with anti–phosphorylated (p)-PLCγ2 and total PLCγ2 (D-E), p-Syk and total Syk (D-F), or anti–p-Akt or total Akt (D,G). (H) Levels of soluble L-selectin in the supernatant of unstimulated or pRGD-stimulated (10 minutes) WT and Sell−/− neutrophils and WT neutrophils that were preincubated for 2 hours with 0.65× PBS or for 30 minutes with dimethyl sulfoxide (DMSO) or the metalloproteinase inhibitor GM6001. (I-L) WT neutrophils pretreated with DMSO or GM6001 were plated on pRGD for 10 minutes, and lysates were immunoblotted with anti–p-PLCγ2 or total PLCγ2 (I-J), anti–p-Syk or total Syk (tSyk) (I,K), or anti–p-Akt or total Akt (I-L). (M) Phagocytosis of pHrodo-labeled K pneumoniae particles by isolated WT neutrophils and neutrophils of L(E) mutant mice. (N) Adhesion-dependent oxidative burst of isolated WT and L(E) mutant neutrophils plated on fibrinogen in the presence of TNF-α. n = 3. Data are mean ± standard error of the mean. *P = .05, **P = .01, ***P = .001, 1-way or 2-way ANOVA.