Cross Talk between Rho GTPases Is Critical for Hematopoietic-Derived Inflammatory Processes.

Neutrophils are a critical cell in inflammatory processes by moving rapidly to tissue sites of inflammation to perform phagocytosis, cytokine and reactive oxygen species release. Members of the small Rho GTPase family, Rac1, Rac2, CDC42 and RhoA, are central regulators of cell movement via cytoskeleton rearrangement. We have previously demonstrated that the Rho family GTPase Rac2 is a critical regulator of neutrophil functions in vitro and in vivo (Roberts et al, Immunity 1999). We have also demonstrated that in response to formyl-methionyl-leucyl-phenylalanine (fMLP), the related GTPase Rac1 plays a distinct, but as yet ill-defined role in tail retraction during cell movement and cell spreading in vitro (Gu and Filippi et al, Science 2003). Here, we further demonstrate that Rac1 appears to be critical for β2-integrin mediated adhesion and migration likely via cross talk with another Rho GTPase, RhoA. Although, Rac1^−/− PMNs show normal in vitro migration in response to fMLP using the Boyden chamber assay, Rac1^−/− PMNs demonstrate a dramatic defect compared with WT cells in haptotaxis using transwell precoated with fibrinogen (1.3±0.3x10³ vs 9.8±0.5x10³). In addition, Rac1^−/− PMNs displayed increased frequency in pseudopodia formation associated with lack of cell body contraction upon integrin ligation compared with WT (80% vs 40%). We noted that this phenotype closely mimics deregulation of the related Rho GTPase, RhoA. Remarkably, Rac1-deficiency leads to mislocalization of RhoA in neutrophils after integrin ligation and reintroduction of Rac1 into Rac1^−/− cells completely restores the correct localization of RhoA. These data are consistent with the hypothesis that Rho GTPases interact in a time- and space-dependent manner. Because fMLP-induced PMN migration into the lung has previously been shown to be beta2-integrin dependent (Mackarel, Am. J. Respir. Cell. Mol. Biol 2000), we used a model of neutrophil associated lung inflammation induced by intratracheal (IT) injection of fMLP to address the physiological role of Rac1 in neutrophil-derived inflammatory processes in vivo,. To study the role of Rac1 specifically in bone marrow-derived cells, we reconstituted C57BL/6 mice with either wild type or Rac1^Flox/Flox bone marrow cells. After Cre-mediated deletion of Rac1, reconstituted mice were treated with one dose of fMLP (20mg) IT. One day after fMLP exposure, bronchoalveolar lavage (BAL) from reconstituted animals showed complete loss of Rac1 expression and demonstrated significantly reduced numbers of migrated neutrophils in BAL compared with mice reconstituted with WT cells (3.1±0.65 vs 9.56±2, p<0.05). Importantly, 5 weeks after fMLP exposure IT, Rac1^−/− recipients displayed a significant reduction in emphysematous lesions as compared with WT as assessed by morphometric measurement of alveolar spaces (57.6±7.8 vs 73.3±3.04, p<0.05), demonstrating the physiological relevance of Rac1 in neutrophil-related inflammatory responses in vivo.

Taken together, these results suggest that Rac1 activity regulates b2 integrin-induced cell shape change and RhoA subcellular localization in PMNs and demonstrate the existence of physiological cross talk between Rac1 and RhoA where RhoA activity depends at least in part on Rac1. Thus, Rac1 and RhoA appear to coordinately regulate PMN migration into the lung during inflammation.