Neutrophils are the most abundant white blood cell in humans, and form the body's first line of cellular defense against infecting microorganisms. While normally found in the blood stream, neutrophils migrate rapidly to sites of infection in tissue. This migration is tightly regulated;indeed, aberrant accumulation of neutrophils causes tissue injury. The understanding of the molecular mechanism(s) controlling neutrophil migration is thus of considerable clinical importance. This application focuses on understanding the role of the small Rho GTPase Cdc42 in neutrophil polarity during migration. Our analysis of Cdc42-null neutrophils shows that Cdc42 is a critical regulator of polarity in neutrophils. At a mechanistic level, we have identified a new pathway mediating Cdc42 functions and involving the leukocyte integrin CD11b/CD18. CD11b/CD18 is an adhesion molecule associated with a lethal human immunodeficiency disorder Leukocyte Adhesion Deficiency syndrome. CD11b is an important mediator of neutrophil migration into tissue, of ill-defined mechanism. We show CD11b clustering is defective in Cdc42-null cells, and restoring CD11b clustering rescues polarity - thereby suggesting the involvement of CD11b in neutrophil polarity. Interestingly, we show that CD11b-null neutrophils, similarly to Cdc42-null cells, exhibited defective polarity during migration. These data suggest the hypothesis that Cdc42 regulates neutrophil polarity through CD11b signaling. We propose to determine the molecular mechanism and biological consequences of the Cdc42-CD11b axis in modulating neutrophil migration both in vitro and in vivo. We plan to use video microcopy, immunofluorescence imaging and biochemistry experiments to examine (Aim1) the molecular mechanism by which Cdc42 regulates CD11b clustering in polarized neutrophils;
(Aim 2) to determine the signaling pathway that is activated downstream of the Cdc42-CD11b axis in neutrophil polarity.
(Aim 3) Since CD11b is known to play significant roles in neutrophil migration into lungs, if the Cdc42-CD11b axis is critical in neutrophil polarity, then deficiency of Cdc42 should result in equivalent defects in lung inflammatory processes in vivo. We will test this using a model of CD11b-dependent lung injury. We believe that analysis of primary Cdc42-null neutrophils offers a rare opportunity to study the specific function of Cdc42 in neutrophil polarity. If our hypothesis that Cdc42 regulates neutrophil polarity though CD11b signaling is validated, this will significantly change the view of Cdc42 and CD11b functions in neutrophil migration and thus in neutrophil-dependent inflammation. The proposed studies are expected to lead to strategies to understand and ultimately treat neutrophil-related disorders.

Public Health Relevance

Rapid and directed migration of neutrophils to sites of infection is the first line of defense employed by the body. Failure to regulate neutrophil migration leads to an inability to control infection. However, aberrant accumulation of neutrophils in tissues is also a known cause of tissue injury. The lungs are particularly vulnerable to neutrophil mediated inflammation and resulting disorders are amongst the leading causes of morbidity and mortality in the United States of America. These patients respond poorly to current therapeutics modalities. Despite its clinical importance, the molecular mechanisms regulating neutrophil migration remains incompletely understood both in physiological and pathological settings. The studies proposed in this application will provide important information of the physiologic role of Cdc42 in the regulation of neutrophil migration. The inhibition of Cdc42 activity in vivo has just become possible with the development of small pharmacologic inhibitor. Therefore, our study will guide designing strategies to modulate neutrophil functions via inhibition of Cdc42 that could be used in the future for novel therapeutics of inflammatory diseases.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Erythrocyte and Leukocyte Biology Study Section (ELB)
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Reynolds, Herbert Y
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Cincinnati Children's Hospital Medical Center
United States
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