Background and objectives Several functions of neutrophilic granulocytes such as migration, phagocytosis and production of reactive oxygen compounds depend on the state of the small GTPase Rac. The GTP-bound active state is the result of the balance of three types of regulatory proteins: guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs). Recently we identified a novel GAP protein, ARHGAP25 which is expressed mostly in leukocytes and regulates Rac. Our previous results show that ARHGAP25 plays a regulatory role in phagocytosis and superoxide production. We also find increased filamentary actin level in ARHGAP25 knock down cells which suggest that ARHGAP25 participates in signalization of actin rearrangement, and probably in regulation of neutrophil migration.
Materials and methods The ARHGAP25-/- mouse strain used for this research project was created from ES cell clone (EPD0085_1_C10) obtained from the NCRR-NIH supported KOMP Repository (www.komp.org) and generated by the CSD consortium for the NIH funded Knockout Mouse Project (KOMP). In vivo migration upon TNFα-stimulation was investigated by intravital microscopy of postcapillar venules from the cremaster muscle. Ex vivo flow chamber assay was carried out with rectangular glass capillaries coated with rmE-selectin, rmCXCL1, and rmICAM-1. No. of extravasated cells was calculated by the analysis of Giemsa-stained whole-mount cremaster muscles. ICAM-1 binding assay and cell surface receptor expression as well as filamentary actin level were measured with flow cytometry after labelling with specific antibodies.
Results We found significant decrease of rolling velocity in the ARHGAP25-/- (KO) mice in vivo compared to ARHGAP25+/+ (WT), however, the No. of rolling and adherent cells showed increased tendency. The velocity and euclidean distance of crawling was significantly higher in KO animals than in WT. These results were also confirmed by flow chamber experiments. We observed marked increase of extravasation in KO animals compared to WT. Neither cell surface expression of the receptors involved in migration nor binding of ICAM-1 differed between KO and WT cells. However, filamentary actin level showed significant increase in KO neutrophils.
Conclusions The major steps of neutrophil migration from rolling to extravasation require completely different signalization processes and our data suggest that ARHGAP25 is involved in all of them. Our results suggest that ARHGAP25 provides this varied role through the regulation of actin rearrangement.
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