Phenotypic analysis of chemokine-driven actin reorganization in primary human neutrophils

The chemokine-driven activation of CXC-type chemokine receptors 1/2 (CXCR1/2) and the subsequent reorganization of the neutrophilic actin are early key events in the induction of neutrophil migration toward centers of inflammation. In this study, an image analysis algorithm was developed to detect subtle chemokine-induced changes in the actin cytoskeleton of primary human neutrophils. By this means, a discrete early step of neutrophil activation was dissected that could be initiated by concentrations of growth-related oncogen α (Gro-α) or interleukin-8 (IL-8) just above their resting-state plasma levels. The associated half-maximal effective concentration (EC50) values for Gro-α and IL-8 of 8 and 22 pM, respectively, are between two and three orders of magnitude below the so-far reported EC50 values of these chemokines for the induction of neutrophilic calcium release, integrin expression, degranulation, and receptor internalization. Sch527123, a known inhibitor of CXCR2 (KD=49 pM) and with a lower potency/affinity also of CXCR1 (KD=3.9 nM), antagonized actin remodeling with half-maximal inhibitory concentration (IC50) values of 400 pM for the CXCR2-specific agonist Gro-α and of 36 nM for the CXCR1/2-promiscuous agonist IL-8. This observation indicates that the here-described early step of chemokine-driven actin reorganization is modulated by both CXCR1 and CXCR2. Thus, the imaging-based assay format, as developed in this work, may be employed in a phenotypic screening campaign to identify inhibitors of an early step in CXCR1/2-induced neutrophilic chemotaxis.