Figure 2.

Surface exposition of PR3 on activated healthy and patient neutrophils before and after treatment with α1PI.A, PR3^mb on purified quiescent blood neutrophils from a healthy volunteer was stained using murine anti-PR3 WGM-2 (25 μg/ml). Flow cytometry analysis showed two populations of neutrophils with low and high amounts of constitutive PR3^mb. Quiescent cells were then stimulated using PMA (100 ng/μl) or calcium ionophore A23187 (1 μM) for 15 min at 37 °C and the load of PR3^mb before and after treatment with α1PI (50 μM) was quantified by flow cytometry using anti-PR3 WGM-2 (25 μg/ml). Flow cytometry studies showed that after neutrophil stimulation, PR3^mb load was increased and as expected, the proportion of low and high PR3-exposing cells remained stable (14, 27). The displacement of fluorescence of the histogram plots to the left shows that induced but not constitutive PR3^mb was cleared from the surface of triggered neutrophils by α1PI. Two populations of neutrophils with low and high amounts of constitutive PR3^mb remained unchanged. Similar results were obtained in n ≥ 10 independents experiments using 10 or 50 μM of α1PI. B, purified blood neutrophils from two patients were stimulated using PMA or calcium ionophore A23187. The load of PR3^mb before and after treatment with α1PI (50 μM) or plasma (dilution 1/3) was quantified by flow cytometry using murine anti-PR3 WGM-2 (25 μg/ml, epitope 3) or MCPR3-2 (40 μg/ml, epitope 4). A monomodal exposition of PR3^mb on GPA patient neutrophils was observed as documented in (13, 14, 33). The gray lines represent nonspecific binding of mouse isotype-specific IgG control antibody used at the same concentration. C, PR3^mb amount on PMA- or A23187-activated neutrophils after α1PI treatment by flow cytometry (PMA-activated cells, n = 5 independent experiments; A23187-activated cells, n = 5 independent experiments; See Fig. S1). Individuals results and the means ± SD are given. The results showed that the induced PR3^mb was removed from the cell surface after α1PI treatment of activated healthy and patient neutrophils. α1PI, alpha-1 protease inhibitor; GPA, granulomatosis with polyangiitis; PMA, phorbol myristate acetate; PR3, proteinase 3.