Neuropeptides SP and CGRP Diminish the Moraxella catarrhalis Outer Membrane Vesicle- (OMV-) Triggered Inflammatory Response of Human A549 Epithelial Cells and Neutrophils

Figure 5

Modulation of neutrophil degranulation triggered by OMVs from M. catarrhalis strain Mc6 and neuropeptides. Human neutrophils at ~1 × 10⁷ cell/ml were primed with cytochalasin D (5 μg/ml), incubated for 30 min with various stimulants, and assayed for the release of azurophilic (a, c) and specific (b, d, e) granule markers. The release of elastase, NGAL, and CD66b expression was assessed as described in Materials and Methods. (a, b) The dose-dependent increase in granule content release upon stimulation with OMVs. Azurophilic (c) and specific (d, e) granule release triggered by stimulants in concentrations: OMVs (50 μg/ml), SP and CGRP (10⁻⁸ M), and fMLP (1 μM) as positive control. Data in the inset of (c) performed with cells stimulated with whole bacteria Mc6 (MOI 5). (e) CD66b cell surface expression on neutrophils after activation by various stimuli, measured by flow cytometry. The panels show (upper panel) the representative FACs dot plots of analyzed cells (from two donors) after priming followed by 30 min incubation with medium alone (R1 gate refers to neutrophils, and R2 gate refers to lymphocytes and other debris) and (middle and lower panel) representative histograms of gated neutrophils after treatments. Cells were stained for CD66b with FITC-conjugated mAb or isotype-matched control and measured. FACs histograms are representative of two independent experiments performed in duplicates. Data for azurophilic degranulation are expressed as the -fold increase in elastase activity measured in RLU/s relative to unstimulated control. Error bars indicate the SD of the mean values obtained from one representative (a, b) and at least two (c, d) independent experiments performed in duplicates on neutrophils from different donors. Asterisks in (c) and (d) denote values significantly different from the control (). Asterisks in (d) denote values significantly different from OMVs samples treated with CGRP (). Differences were analyzed by one-way ANOVA followed by Tukey’s post hoc test.

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