FIGURE 6.
Functional analyses identify Nexinhibs as efficient inhibitors of neutrophil exocytosis and extracellular superoxide anion production. A–D, up-regulation of the neutrophil adhesion molecule CD11b, the receptors CD66b and CD35, and the NADPH oxidase membrane-associated subunit gp91phox at the plasma membrane was evaluated by flow cytometry using specific antibodies that detect extracellular epitopes of the indicated markers. Human neutrophils were incubated with the indicated Nexinhibs (NEI) at 10 μm or DMSO and subsequently treated with GM-CSF and fMLP or vehicle (Unstimulated). Symbols correspond to individual donors, and error bars indicate mean ± S.E. of nine independent donors. *, p < 0.02; **, p < 0.003; ***, p < 0.0003, versus DMSO, stimulated. Paired Student's t test. E, production of extracellular superoxide anion by phorbol ester-stimulated human neutrophils treated either with the indicated compounds at 10 μm or vehicle for 1 h was analyzed using the cytochrome c reduction assay. Mean ± S.E. from 7 to 10 independent donors. *, p < 0.04; **, p < 0.001, versus DMSO. Unpaired Student's t test. F, superoxide anion production was measured as in E except that, where indicated, the Nexinhib or DMSO-treated cells were washed twice in PBS before the addition of stimuli. Mean ± S.E. (n = 4). *, p < 0.02; **, p < 0.002, versus same compound, no wash condition.