Rho GTPase Cdc42 Maintains Neutrophil Polarity during Directed Migration by Regulating CD11b Integrin Signals.

Neutrophil (PMN) migration to sites of infection is the first line of cellular defense. Among others, a key event of cell migration is the maintenance of a polarized morphology characterized by a single protrusive leading edge of F-actin and a contractile uropod devoid of F-actin protrusions. Using mice genetically deficient in the Cdc42 negative regulator Cdc42 GTPase Activating Protein, we previously demonstrated that Cdc42 activity suppresses membrane protrusions at the uropod of the cells to maintain stable polarity during directed migration (Szczur et al, Blood 2006). However, the underlying molecular mechanism of Cdc42-mediated neutrophil polarity remains to be understood. Here, using mice with a conditional Cdc42 (flox) allele, we showed by video microscopy that Cdc42^−/− PMNs exhibited multiple membrane extensions in various directions and failed to maintain cell polarity and directionality towards formyl-methionyl-leucyl-phenylalanin (fMLP) gradient compared to wild type (WT) cells. Consistent with this observation, Cdc42^−/− PMNs exhibited increased lamellipodia protrusions of F-actin all around the cells compared to WT PMNs, in response to fMLP stimulation and fibrinogen ligation, confirming that Cdc42 maintains stable polarity by preventing abnormal membrane protrusions outside the leading edge. To understand how Cdc42 orchestrates neutrophil polarity at a mechanistic level, we explored the possibility of a role for integrins in this process since Cdc42 appears to regulate neutrophil polarity in a manner, at least in part, dependent on integrin ligation (Szczur et al, Blood 2006). Expression of the neutrophil integrin, CD11b/CD18, on resting or fMLP-stimulated PMNs was similar between the genotypes. Stimulation of WT PMNs with fMLP and ligation to fibrinogen induced a polarized distribution of CD11b into clusters mostly concentrated at the uropod of the cells. Remarkably, the numbers of CD11b clusters of Cdc42^−/− PMNs were significantly decreased compared to WT cells. Furthermore, inhibition of CD11b clustering in WT PMNs, using anti-CD11b blocking antibody, significantly increased membrane protrusions associated with loss of stable polarity during directed migration, similarly to Cdc42-deficiency. Enforcing CD11b clustering by CD11b cross-linking in Cdc42^−/− PMNs partially rescued cell polarity and F-actin distribution concentrated only at the leading edge of the cells to WT levels. These results strongly suggest that CD11b clustering is regulated by Cdc42 activity and contributes to suppress F-actin protrusions at the uropod of neutrophils during migration. The uropod distribution of CD11b suggests that CD11b may recruit contractile proteins, such as the myosin regulator myosin light chain (MLC), to antagonize membrane protrusions. To test this hypothesis, we analyzed the distribution of phosphorylated MLC (p-MLC). Upon stimulation, p-MLC strongly translocated to the uropod of WT cells. In contrast, p-MLC remained diffuse and non polarized in the cytoplasm of Cdc42^−/− cells. Blocking CD11b function in WT cells abrogated the polarized distribution of p-MLC mimicking Cdc42^−/− PMNs. Enforcing CD11b clustering in Cdc42^−/− PMNs rescued p-MLC signals concentrated at the uropod of the cells. Altogether, this study suggests that Cdc42 activity maintains neutrophil polarity during directed migration by regulating CD11b clustering/distribution and subsequent outside/in signals to suppress lateral membrane protrusions. This study uncovers a critical role for CD11b in maintaining neutrophil polarity during migration.