Rho-GTPase Cdc42 Regulates Neutrophil Polarity Via Crosstalk Between Wiskott Aldrich Syndrome Protein (WASp), Lipid Raft Reorganization, CD11b and Microtubules

Abstract 16

Neutrophils (PMNs) are the first line of immune defense by moving toward the infection site. A key event of cell migration is the maintenance of a polarized morphology characterized by a single protrusive leading edge (i.e. lamellipodia) and a contractile uropod. Using mice with a conditional Cdc42 (flox) allele, we have previously reported that the small Rho GTPase Cdc42 controls neutrophil polarity via CD11b integrin signaling at the uropod (Szczur; Blood 2009). In the present study, we seek to dissect the mechanism of Cdc42-CD11b axis in neutrophil polarity. We first examined the role of the Cdc42 effector Wiskott Aldrich Syndrome protein (WASp), as WASp^−/− neutrophils showed defective neutrophil migration and integrin clustering (Yang; Immunity 2006). A Cdc42 mutant for WASp Binding (Cdc42S71P) was expressed in Cdc42^−/− cells using retroviral transduction. Rescue of Cdc42 functions was analyzed compared to WT cells and Cdc42^−/− cells transduced with an empty vector, and Cdc42^−/− cells transduced with the wild type form of Cdc42 (wtCdc42). While expression of wtCdc42 rescued abnormal polarity and CD11b clustering in Cdc42^−/− neutrophils to WT levels, expression of Cdc42S71P did not, suggesting that Cdc42 controls neutrophil polarity via WASp. We further confirmed the involvement of WASp in neutrophil polarity using WASp^−/− neutrophils. WASp^−/− neutrophils exhibited all characteristics of loss of polarity (ie frequent changes in direction during migration as assessed by video microscopy, multiple F-actin protrusions at the uropod). We next examined how Cdc42/WASp regulates neutrophil polarity and explored the roles of microtubules (MTs), since MTs are oriented toward the uropod in migrating WT neutrophils and Cdc42 is known to regulate MT polarity. Cdc42^−/− as well as WASp^−/− neutrophils showed loss of MT polarity with cells extending MT towards both the front and the uropod. They also exhibited less stabilized MTs (ie, defect in detyrosinated MT). Interestingly, MTs made contact with CD11b clusters at the uropod in WT, but not in Cdc42^−/− and WASp^−/− neutrophils. Enforcing CD11b clustering by CD11b antibody crosslinking in Cdc42^−/− and WASp^−/− neutrophils rescued polarity and MT capture at the plasma membrane to WT levels. Thus, Cdc42 regulates CD11b clustering via WASp, which, in turn, may capture and stabilize MTs at the uropod. To further examine this possibility, we examined in detail the plasma membrane of these cells. Indeed, on activation, the uropod of neutrophils reorganizes into detergent resistant membrane (DRM) domains – something, which is essential for polarity. Immunostaining with the DRM marker cholera toxin indicated loss of DRM assembly at the uropod of Cdc42^−/− and WASp^−/− neutrophils. Biochemistry analysis showed that DRM fraction of WT cells contained CD11b, WASp, tubulin and the microtubule end-binding protein EB1. In contrast, Cdc42^−/− and WASp^−/− DRMs had significantly lower amount of CD11b, WASp, tubulin, EB1 and CD11b, tubulin, EB1, respectively. Remarkably, CD11b crosslinking in WASp^−/− cells rescued CD11b, tubulin and EB1 whereas CD11b crosslinking in Cdc42^−/− cells rescued CD11b, tubulin and EB1 but not WASp, suggesting that Cdc42 and WASp controls DRM formation containing CD11b, and that CD11b recruits EB1 to capture and stabilize microtubules downstream of WASp.

Together, our study uncovers a novel mechanism of neutrophil migration in which Cdc42 recruits WASp to the uropod to reorganize the plasma membrane into discrete DRM domains and induce CD11b clustering. CD11b clusters, in turn, capture and stabilize microtubules via EB1 – something that is critical for maintaining neutrophil polarity during directed migration. Our study thus reveals a new function for WASp in the control of neutrophil polarity via crosstalk of DRM/CD11b and microtubules.

We used a 3-D migration model that mimics the extravasation cascade ex vivo to show that Cdc42/WASp/CD11b-dependent neutrophil polarity regulates the crawling step of neutrophils toward endothelial cell junction for efficient transmigration on HUVECs. Using a model of LPS-induced acute lung injury, we then show that Cdc42 loss dramatically reduced neutrophil recruitment to lung alveolar cavities as well as lung tissue inflammation – an indication for the patho-physiological importance of this pathway during the neutrophil extravasation cascade and subsequent inflammation in vivo.