Figure 6: Intracellular nonoxidised SLPI exerts immunomodulatory activity. (a) Neutrophils (1 × 107/mL) were exposed to unlabeled fMLP (10 μM) or SLPI (480 nM) for 1 min, followed by FITC-labeled fMLP (fMLP′′) (1 μM), and the level of bound fMLP′′, quantified by FACS. The negative control (unlabeled cells) is illustrated in dark grey and a total of 10,000 events were collected. (b) Results in mean fluorescence intensity units (MFI) demonstrate that pre-incubation with unlabeled fMLP, but not rhSLPI, prevented binding of fMLP′′ to the cell membrane (* compared to fMLP/fMLP′′ cells). (c) and (d); Neutrophils (1 × 107/mL) remained untreated or treated with 480 nM SLPI or 480 nM oxidised SLPI (ox-SLPI) for 5 min, prior to stimulation with fMLP ((c), 1 μM) or IL-8 ((d), 1.2 nM). Ox-SLPI did not inhibit the fMLP or IL-8 induced Ca2+ flux (* between SLPI and ox-SLPI). Each experiment was performed in triplicate, each point is the mean ± S.E. and statistical significance was calculated by 2-way ANOVA followed by Bonferroni test or Student’s -test.