Rac1 Deletion in Mouse Neutrophils Has Selective Effects on Neutrophil Functions 1 Michael Glogauer, 2 * † Christophe C. Marchal, ‡ Fei Zhu, † Aelaf Worku,* Bjo ¨ rn E. Clausen, § Irmgard Foerster, ¶ Peter Marks,* Gregory P. Downey,  Mary Dinauer, ‡ and David J. Kwiatkowski* Defects in myeloid cell function in Rac2 knockout mice underline the importance of this isoform in activation of NADPH oxidase and cell motility. However, the specific role of Rac1 in neutrophil function has been difficult to assess since deletion of Rac1 results in embryonic lethality in mice. To elucidate the specific role of Rac1 in neutrophils, we generated mice with a conditional Rac1 deficiency restricted to cells of the granulocyte/monocyte lineage. As observed in Rac2-deficient neutrophils, Rac1-deficient neu- trophils demonstrated profound defects in inflammatory recruitment in vivo, migration to chemotactic stimuli, and chemoattrac- tant-mediated actin assembly. In contrast, superoxide production is normal in Rac1-deficient neutrophils but markedly diminished in Rac2 null cells. These data demonstrate that although Rac1 and Rac2 are both required for actin-mediated functions, Rac2 is specifically required for activation of the neutrophil NADPH oxidase. The Journal of Immunology, 2003, 170: 5652–5657. T he Rho family of small GTPases consists of three major members: Cdc42, Rac, and Rho (1). Within the Rac sub- class there are three members; Rac1, Rac2, and Rac3 (2). Rac1 is ubiquitously expressed while Rac2 expression is restricted to cells of the hemapoietic lineage. Rac3, the most recently de- scribed Rac isoform, is expressed in brain, lung, liver, and pan- creas (2) but not in neutrophils (U. Knauss, unpublished observa- tions). Although Rac2 is the predominant isoform in human neutrophils (3), murine neutrophils express similar amounts of Rac1 and Rac2 (4). Rac1 and Rac2 share 92% amino acid identity with the major divergence occurring in the C terminus. Impor- tantly, this region determines the subcellular localization and in- teractions of Rac with some downstream effector proteins (5, 6). Racs are known to be key regulators of the actin cytoskeleton and of the NADPH oxidase system in neutrophils (7–10). Recent studies using Rac2-deficient mice and neutrophils from a patient with a naturally occurring mutation in Rac2 have demonstrated that this isoform is a key regulator of several antimicrobial func- tions including dynamic alterations of the actin cytoskeleton re- quired for cell migration, cell shape, and generation of reactive oxygen species by the NADPH oxidase complex (11–14). How- ever, the specific role of Rac1 in neutrophils remains obscure. The high degree of homology in the effector regions of Rac1 and 2 has led to the hypothesis that these two proteins function interchange- ably. Furthermore, in in vitro cell-free assays of the NADPH ox- idase, prenylated Rac1 and Rac2 are indistinguishable. Using pu- rified neutrophil membranes and recombinant Rac1 and Rac2, Heyworth et al. (10) demonstrated that both isoforms have equal activity in reconstitution of superoxide production, although Rac2 was more efficient in the presence of neutrophil cytosol. In per- meabilized human neutrophils, Rac1 and Rac2 activated actin as- sembly similarly and catalytically inactive forms of Rac1 and Rac2 were equally effective at inhibiting fMLP-mediated actin assembly (15). However, the conditions used for all these in vitro systems may not replicate conditions present in intact cells. Recent studies using Rac2-deficient neutrophils suggest that Rac1 and Rac2 have discrete functions inasmuch as activation and signaling profiles for each isoform in intact neutrophils are unique (4). In fMLP-activated murine neutrophils, 4-fold more Rac2 is activated compared with Rac1. In addition, using Rac2 null and Rac2 heterozygous mice, Li et al. (4) demonstrated that the level of activated Rac2 is rate limiting for fMLP-induced F-actin poly- merization, chemotaxis, and superoxide generation (4). However the role of Rac1 in regulating neutrophil functions has not been evaluated. Investigation of the specific roles of these two Rac iso- forms and specifically Rac1 in primary neutrophils requires an alternative model to the in vitro models used previously. Targeted gene disruption in the mouse is an alternate approach to elucidate the distinct functions of Rac1 and 2, because in theory each gene can be disrupted individually. However, Rac1 defi- ciency results in embryonic lethality (16). To examine the specific role of Rac1 in neutrophil function, we generated mice in which the rac1 gene is selectively disrupted in cells of granulocyte/mono- cyte lineage. As described herein, this was accomplished using a conditional (“floxed”) allele of rac1 and a mouse expressing the Cre recombinase under the control of a neutrophil/monocyte-spe- cific promoter (Lysozyme M). We demonstrate here for the first time that Rac1 null neutrophils have significant defects in inflam- matory recruitment in vivo, migration to chemotactic stimuli, and chemoattractant-mediated actin assembly. However, superoxide production is normal in Rac1-deficient neutrophils in contrast to *Brigham and Women’s Hospital, Boston, MA 02115; † University of Toronto, To- ronto, Canada; ‡ Department of Pediatrics (Hematology/Oncology), Indiana Univer- sity School of Medicine, Indianapolis, IN 46202; § Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; ¶ Institute for Medical Microbiology, Immunology and Hygiene, Tech- nical University of Munich, Munich, Germany; and  Department of Medicine, Divi- sion of Respirology, and The Toronto General Hospital Research Institute of the University Health Network, Toronto, Canada Received for publication December 12, 2002. Accepted for publication March 20, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Canadian Institutes of Health Research grants (to M.G. and G.P.D.) and National Institutes of Health Grants HL54188 (to D.J.K.), ROI HL45635 (to M.D.), and POI HL069974 (to M.D.). M.G. is a Canadian Institutes of Health Research Clinician Scientist. 2 Address correspondence and reprint requests to Dr. Michael Glogauer, University of Toronto, Room 241, 150 College Street, Toronto, Ontario, Canada M5S 1A8. E-mail address: michael.glogauer@utoronto.ca The Journal of Immunology Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00