Increased cell surface expression of CD63 and reduced elastase levels in HPS2 patients. (A) Circulating neutrophils were separated as described in “Patients, materials, and methods,” washed twice, and analyzed by a flow cytometer for expression of CD63. CD63 staining (thick line) in comparison with mouse IgG antibody (thin line) is presented on a histogram plot. Red fluorescence intensity is shown on the x-axis expressed in a log scale; the number of cells per channel is shown on the y-axis. Experiment shown is representative of 3 independent experiments performed. (B) Immunofluorescence staining for NE in circulating neutrophils from HPS2 patients. PMN cells were obtained from peripheral blood, cytocentrifuged, and stained with anti-NE. In cells from a control subject, a strong expression of NE is observed in the cytoplasm with the presence of cytoplasmic dots (top panel). In patients (Pt 1 and Pt 2), only scattered NE-positive dots are evident (middle and bottom panels) in rare PMN cells. NE was revealed using FITC-conjugated secondary antibody. Experiment shown is representative of 2 performed. (C) Western blot analysis for NE in circulating neutrophil content for HPS2 patients. Cell lysates from separated neutrophils of Pt 1, Pt 2, their father, and a healthy control were separated by SDS-PAGE and probed with NE antibody (see “Patients, materials, and methods”). β-actin was used to compare protein levels. (D) Confocal microscopy analysis of double immunofluorescence staining of PMN cells from HPS Pt 1 with antielastase (green) and anti-CD43 (red) was performed as described in “Patients, materials, and methods.” Experiment shown is representative of 2 performed. Sections were examined using an Olympus BX60 fluorescence microscope and objectives with numeric apertures of 0.40 (10 ×), 0.70 (20 ×), 0.85 (40 ×), and 0.90 (60 ×), equipped with a DP-70 Olympus digital camera (Olympus, Melville, NY). Images were acquired using analySIS Image Processing software (Soft Imaging System, Münster, Germany).