Gene deletion of Rac2 leads to defective primary granule translocation in neutrophils

Abstract

Rationale Neutrophil degranulation is a crucial event in numerous pulmonary disorders, including severe asthma. Gene deletion of Rac2, a member of the Rho GTPase subfamily, has been shown to result in loss of primary granule exocytosis in murine neutrophils. We hypothesized that Rac2 regulates primary granule exocytosis by controlling granule mobilization from the cytoplasm to the plasma membrane prior to fusion. Methods Bone marrow-derived neutrophils were prepared from rac2 −/− and wild type C57Bl/6 (WT) mice. Mediator release in response to cytochalasin B and f-Met-Leu-Phe (CB/fMLP) was determined by measurement of myeloperoxidase (MPO), a primary granule marker. This was compared with release of secondary granule lactoferrin (LTF) and tertiary granule matrix metalloprotease-9 (MMP-9). Granule translocation was determined by confocal analysis using CD63 as a membrane marker for primary granules. Results Neutrophils from rac2 −/− mice released LTF and MMP-9 in response to CB/fMLP stimulation, but failed to release primary granule MPO. The defect in MPO release was not rescued by priming cells with TNF, although p38 MAP kinase phosphorylation in response to TNF and CB/fMLP was similar in Rac2 −/− and WT neutrophils. CB/fMLP induced mobilization of the primary granule marker CD63 to the cell membrane in WT neutrophils. However, Rac2 −/− neutrophils lacked CD63 + granule translocation during CB/fMLP stimulation. Conclusions These findings suggest a pivotal role for Rac2 in regulation of primary granule translocation required for exocytotic release, and provide important insights into intracellular signalling pathways that regulate neutrophil degranulation.