α1-Antitrypsin regulates CXCR2 surface expression in human neutrophils via IL-8 release and expression

Neutrophil activation and migration are regulated by IL8 dependent internalization and subsequent re-expression of chemokine receptor CXCR2. A rapid modulation of CXCR2 by IL8 is associated with IL8-driven neutrophil migration. α1-Antitrypsin (AAT) binds IL8 and is a regulator of IL8-induced neutrophil migration, but the effect of AAT on CXCR2 expression is unknown. Isolated human blood neutrophils were treated with AAT (Prolastin, 1 mg/ml) and/or IL8 (50 ng/ml) and/or lipopolysaccharide (LPS, 10 ng/ml) for 1, 4 and 24h. After, neutrophils were stained with CXCR2/IL8 and CXCR1 fluorescein-conjugated antibody and flow cytometry was performed. The levels of IL8 in cell supernatants were quantified by ELISA and <italic>CXCR2</italic> and <italic>IL8</italic> gene expression levels were checked using qRT-PCR. As expected, exposure of neutrophils to IL8 resulted in a transient internalization and a subsequent re-expression (at 4h) of surface CXCR2 without affecting CXCR2 mRNA. In contrast, LPS did not affect surface CXCR2 levels within 1h, but reduced CXCR2 expression at 4 and 24h. Addition of AAT greatly increased the internalization of CXCR2 in IL8 or LPS treated neutrophils at 1h, showed no effect at 4h, and, independent of the presence of stimuli, increased surface levels of CXCR2 at 24h. These changes in CXCR2 levels paralleled the effect of AAT on IL8 release and expression. While AAT in the short term significantly enhanced IL8 and LPS-induced IL8 release, at 4 and 24h AAT inhibited LPS-induced IL8 release. Finally at 24h, AAT by itself reduced IL8 mRNA expression. Our data show that exogenously added AAT regulates CXCR2 expression in neutrophils by inducing time-dependent changes in IL8 release and expression.