Figure 2.
NADPH oxidase activity in PMNs and monocytes. (a) O2− generation in PMNs and monocytes from healthy controls (C+; n = 5 (PMNs) and n = 6 (monocytes)), a patient with CGD (CGD; n = 1) and patients with the CYBB mutations (P-Q231P (n = 4) and P-T178P (n = 1)), assessed after the addition of PMA (40 ng/ml) with or without catalase (ten conditions: six for PMNs and four for monocytes), measured by an assay for reduction of cytochrome c that can be inhibited by superoxide dismutase. Each symbol represents an individual subject. P = 0.01 (not significant), healthy controls versus patients with the Q231P substitution (nonparametric Wilcoxon exact test, accounting for multiple testing). (b) Fluorometric quantification of H2O2 release from PMNs and monocytes from healthy controls (n = 11 (PMNs) and n = 12 (monocytes)) and in patients with CGD or the CYBB mutations, assessed with the fluorogenic substrate ADHP (N-acetyl-3,7–dihydroxyphenoxazine) after the addition of PMA. (c) Flow cytometry of intracellular H2O2 production (dihydrorhodamine 123 (DHR) assay) in PMNs and monocytes from healthy controls, a patient with X-linked CGD and a patient with the Q231P substitution, assessed before (dotted lines) and after (solid lines) stimulation with PMA (400 ng/ml). (d) Flow cytometry (dihydrorhodamine 123 assay) of PMNs from healthy controls, patients with CGD, a patient with the Q231P substitution and a female heterozygous for the allele encoding Q231P (H), left untreated (NS) or treated with various combinations (above plots) of tumor necrosis factor (TNF), IL-1β, cytochalasin b (Cyt b) and formyl-Met-Leu-Phe (fMLF). (e) Killing of S. aureus by granulocytes from healthy controls (n = 4; 1–4 in parentheses), patients with CGD (n = 3; 1–3 in parentheses) and a patient from kindred A (Q231P; n = 1) and one from kindred B (T178P; n = 1). CFU, colony-forming units. Data are representative of three experiments (a,b; mean of duplicates in b), two experiments (c,e) or two independent experiments (d).