Figure 3.

Inhibition and surface exposition of PR3 on fMLP-activated neutrophils treated with α1PI.A, residual activity of soluble PR3 (gray bars) and PR3^mb (black bars) after incubation with increasing amounts of α1PI (0.01–10 μM). Neutrophils were primed with CB (10 μg/ml) for 10 min and stimulated with chemotactic N-formyl-methionyl-leucyl-phenylalanine (fMLP, 1 μM) for 20 min at 37 °C and the activity of PR3^mb was measured using FRET substrate ABZ-VAD(nor)VADYQ-EDDnp (20 μM) (43, 57) after incubation with increasing amounts of α1PI in comparison with that of soluble purified PR3. The starting rates of hydrolysis were adjusted on that of a 1 nM PR3 solution. Individuals results and the means ± SD are given (n = 4 independents experiments). B, representative flow cytometry analysis of fMLP-activated neutrophils as analyzed using anti-PR3 WGM-2 (25 μg/ml) showing a partial decrease of the load in PR3^mb after incubation with α1PI (50 μM). The proportion of PR3^mb over the total neutrophil population remained unchanged. The gray line represents nonspecific binding of mouse isotype-specific IgG control antibody used at the same concentration. C, PR3^mb amount on fMLP-activated neutrophils after α1PI treatment by flow cytometry (n = 5 independents experiments, See Fig. S2). Individuals results and the means ± SD are given. α1PI, alpha-1 protease inhibitor; fMLP, formyl-methionyl-leucyl-phenylalanine; PR3, proteinase 3.