Biochemical characterization of the interaction of PR3 with the plasma membrane. (A) Flow cytometry analysis of membrane expression of PR3, HNE, and MPO in isolated resting neutrophils. Neutrophils were stained with the MoAb anti-PR3 CLB 12.8, 2 HNE MoAbs, or a polyclonal anti-MPO shown by FITC-conjugated antibodies to visualize PR3, HNE, or MPO surface labeling, respectively. In this representative experiment performed in a given individual, flow cytometry analysis shows that 75% of the neutrophils are labeled with the MoAb anti-PR3 (mPR3⁺), whereas no surface labeling was observed for HNE and MPO under the same conditions. (B) Effect of modification of membrane charge on membrane PR3 expression: isolated neutrophils having a mPR3⁺ subset of 70% and 30% were treated either with acid pH (50 mmol/L glycine, 150 mmol/L Tris, pH 3) or with basic pH (100 μmol/L protamine, pH 10.7), respectively. The treatment resulted in an increase in PR3 surface labeling (bold line) as compared with untreated neutrophils (plain line) with reference to control IgG1 (dotted line). On the right, isolated neutrophils from an individual having a 25% mPR3⁺ subset (control IgG1 in dotted line) were treated with neuraminidase, resulting in an increase in PR3 surface labeling (bold line) as compared with untreated neutrophils (plain line); the insert shows the positive control for neuraminidase activity on CD43 expression shown by an MoAb whose epitope is sialic acid dependent.