Functions and Rac2 Gene Dosage Effect on Neutrophil Neutrophils

The Journal of Immunology

Chemoattractant-Stimulated Rac Activation in Wild-Type and Rac2-Deﬁcient Murine Neutrophils: Preferential Activation of Rac2 and Rac2 Gene Dosage Effect on Neutrophil Functions1

Shijun Li,*‡ Akira Yamauchi,*‡ Christophe C. Marchal,*‡ Jason K. Molitoris,*‡ Lawrence A. Quilliam,† and Mary C. Dinauer2‡

The hemopoietic-specific Rho family GTPase Rac2 shares 92% amino acid identity with ubiquitously expressed Rac1. Neutrophils from rac2؊/؊ mice have multiple defects, including chemoattractant-stimulated NADPH oxidase activity and chemotaxis, which may result from an overall reduction in cellular Rac or mechanisms that discriminate Rac1 and Rac2. We show that murine neutrophils have similar amounts of Rac1 and Rac2, unlike human neutrophils, which express predominantly Rac2. An affinity precipitation assay for Rac-GTP showed that although FMLP-induced activation of both isoforms in wild-type neutrophils, Ϸ4-fold more Rac2-GTP was detected than Rac1-GTP. Wild-type and Rac2-deficient neutrophils have similar levels of total Rac1. FMLP-induced Rac1-GTP in rac2؊/؊ neutrophils was Ϸ3-fold greater than in wild-type cells, which have similar levels of total .Rac1, yet FMLP-stimulated F-actin, chemotaxis, and superoxide production are markedly impaired in rac2؊/؊ neutrophils -Heterozygous rac2؉/؊ neutrophils, which had intermediate levels of total and FMLP-induced activated Rac2, exhibited interme diate functional responses to FMLP, suggesting that Rac2 was rate limiting for these functions. Thus, phenotypic defects in FMLP-stimulated Rac2-deficient neutrophils appear to reflect distinct activation and signaling profiles of Rac 1 and Rac2, rather than a reduction in the total cellular level of Rac. The Journal of Immunology, 2002, 169: 5043Ð5051.

mall GTPases function as molecular switches, alternating tions that either Rac1 or Rac2 was essential for high level super- between inactive GDP- and active GTP-bound states in the oxide production in cell-free NADPH oxidase assays (7, 8). The S relay of signals from cell surface receptors. This GDP/ GTP-bound form of Rac binds to p67phox and probably to flavo- GTP cycle is tightly regulated by the interaction of the GTPases cytochrome b in the assembled oxidase complex (9–15). Rho with guanine-nucleotide exchange factors (GEFs)3 that catalyze GTPases have subsequently been implicated in the regulation of the GDP-GTP exchange reaction, by GTPase-activating proteins other phagocyte functions that are activated in response to inflam- that enhance the intrinsic GTPase activity, and, for the Rho matory signals, including actin remodeling, chemotaxis, and phago- GTPase subfamily, by guanine-nucleotide dissociation inhibitors cytosis (16–19). that interfere with nucleotide exchange and also maintain the in- There are three Rac isoforms, Rac1, Rac2, and Rac3 (20–23). active GDP-bound form in the cytoplasm (1–5). The Rho GTPase Rac1 is the most studied isoform and is ubiquitously expressed, subfamily includes Rho, Rac, and Cdc42, which control a wide whereas Rac2 expression is highly restricted to hemopoietic cells spectrum of cellular functions, including cytoskeletal organization, (20, 22, 23). These two isoforms share 92% identity overall and transcription, superoxide production, and cell growth and prolif- differ primarily in the C-terminal 10 residues, where Rac1, but not eration (3, 4, 6). The first cellular function ascribed to Rac was the Rac2, contains a highly basic sequence adjacent to a prenylated activation of the phagocyte NADPH oxidase, based on observa- cysteine that can insert into cellular membranes. Murine Rac1 and Rac2 differ by only one and two amino acids from human Rac1 and Rac2, respectively (20, 23). The more recently discovered *Herman B Wells Center for Pediatric Research and Departments of Pediatrics (He- matology/Oncology), Microbiology/Immunology, and Medical and Molecular Genet- Rac3 shares 72% identity with Rac1 and 83% identity with Rac2 ics, †Biochemistry and Molecular Biology, and †Walther Oncology Center and (20–23); like Rac1, it is expressed in a variety of tissues but its Walther Cancer Institute, ‡James Whitcomb Riley Hospital for Children, Indiana University Medical School, Indianapolis, IN 46202 functions are relatively uncharacterized. Ϫ/Ϫ Received for publication June 13, 2002. Accepted for publication August 19, 2002. rac2 mice generated by gene targeting exhibit multiple func- The costs of publication of this article were defrayed in part by the payment of page tional defects in different hemopoietic lineages (24–27). In vivo, Ϫ/Ϫ charges. This article must therefore be hereby marked advertisement in accordance rac2 mice had decreased exudate formation and increased mor- with 18 U.S.C. Section 1734 solely to indicate this fact. tality in invasive aspergillosis (27). rac2Ϫ/Ϫ neutrophils displayed 1 This work was supported by National Institutes of Health Grants RO1HL45635 and impaired F-actin generation, lamellipodia formation, and directed PO1HL069974 and the Riley Memorial Association (to M.C.D.) and American Can- cer Society Grant 00-125-01-TBE (to L.A.Q.). The Wells Center for Pediatric Re- cell movement in response to FMLP and other agonists signaling search is a Center for Excellence in Molecular Hematology funded by Grant through G-protein-coupled receptors, decreased NADPH oxidase P50DK4921. activity in response to FMLP, phorbol ester, and IgG-opsonized 2 Address correspondence and reprint requests to Dr. Mary C. Dinauer, Wells Center particles and diminished L-selectin-mediated adhesion (24, 27). for Pediatric Research, 1044 West Walnut Street, R4, Room 402A, Indianapolis, IN 46202-5225. E-mail address: [email protected] The requirement for Rac2 in these functions was not absolute and 3 Abbreviations used in this paper: GEFs, guanine-nucleotide exchange factors; BM, was selective for specific signaling pathways. For example, F-actin bone marrow; PBD, p21-binding domain; CRIB motif, Cdc42/Rac interactive binding formation in neutrophils stimulated with tyrosine kinase-coupled motif; PMNs, polymorphonuclear neutrophils; PI3K, phosphatidylinositol 3-kinase; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PP1, 4-amino-5-(4- growth factors was normal, as was opsonized zymosan-stimulated methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. NADPH oxidase activity (24, 27). Rac2 is also likely to play an

important role in human neutrophil function, given that a patient ified water ad libitum. Both male and female mice 8–12 wk of age were with recurrent bacterial infections and a neutrophil phenotype sim- used in these experiments. Ϫ/Ϫ ilar to that of the rac2 mouse was found to have a dominant- Isolation of neutrophils negative Rac2 mutation (28, 29). Taken together, these observations demonstrate that Rac is a critical regulator of specific phagocyte sig- Murine neutrophils were purified from the bone marrow (BM) storage pool using sequential Percoll and Histopaque 1119 gradients as described (24, naling pathways and suggest the hypothesis that Rac2 has nonover- 27), except that PBS buffers were used instead of HBSS in some experi- lapping functions with other Rac isoforms. Alternatively, the func- ments. Freshly isolated murine neutrophil preparations (Ϸ80% neutrophils tional defects in Rac2-deficient cells could reflect an overall reduction as assessed by Diff-Quik staining (Dade, Miami, FL)) were resuspended in 2ϩ 2ϩ in the cellular level of Rac, and the impaired neutrophil function re- PBS (without Ca and Mg ) and kept on ice until further use. Human neutrophils were isolated from heparinized whole blood using Poly- sulting from expression of a dominant-negative Rac2 mutation in- morphprep according to the manufacturer’s protocol. cludes effects on other Rac isoforms. In the current study, to better define the relative role of the Quantification of Rac1 and Rac2 by immunoblot assay different Rac isoforms in regulating neutrophil functions, we used Cell lysates of diisofluorophosphate-treated murine BM neutrophils and an affinity precipitation assay for Rac-GTP to compare Rac1 and human peripheral blood neutrophils were prepared and subjected to 12% Rac2 activation in chemoattractant-stimulated wild-type and Rac2- SDS-PAGE and immunoblotting as previously described (24, 31). For quantification of Rac1 and Rac2 levels in cells, serial dilutions of recom- deficient murine neutrophils. We found that similar amounts of binant isoprenlyated Rac1 and Rac2 were loaded in adjacent lanes. Blots Rac1 and Rac2 were present in wild-type murine neutrophils, un- were probed with either a mouse mAb for Rac1 or a rabbit polyclonal Ab like human neutrophils in which Rac2 is the predominant isoform for Rac2 and with either an anti-mouse or anti-rabbit secondary Ab con- (14). However, although FMLP induced the activation of Rac1 and jugated with HRP and developed using ECL (Amersham Pharmacia Bio- Rac2 in wild-type murine neutrophils, Ϸ4-fold more activated tech, Piscataway, NJ). Integrated densitometry was used to determine the intensity of signals using the Eagle Eye II Still Video System and associ- Rac2 was detected than in Rac1. Both basal and FMLP-stimulated ated software (Stratagene, La Jolla, CA) or, for scanned films, NIH Image Ϫ/Ϫ levels of Rac1-GTP were increased in rac2 neutrophils com- software (Research Services Branch, National Institute of Mental Health, pared with wild-type neutrophils, although FMLP-stimulated che- Bethesda, MD). Multiple exposures were analyzed to ensure that relative motaxis and superoxide production are markedly impaired in signal intensities measured were in the linear range. Ϫ/Ϫ ϩ/Ϫ rac2 neutrophils. Heterozygous rac2 mice had intermedi- Measurement of F-actin and chemotaxis ate levels of FMLP-stimulated F-actin formation, chemotaxis, and Neutrophil F-actin content and chemotaxis were examined as previously NADPH oxidase activity, with decreased levels of activated Rac2 described (27). To measure relative F-actin levels, neutrophils were incu- and similar Rac1 activation compared with wild-type neutrophils. bated in the absence or presence of 10 ␮M FMLP at 37°C for either 10 or These data suggest that the hemopoietic-specific Rac2 plays a 30 s and then fixed with PBS containing 4.6% paraformaldehyde and 0.1% dominant role in FMLP-activated responses in murine neutrophils, BSA on ice, stained with 160 nM FITC-phalloidin for 30 min, and ana- which appears to reflect in part a greater activation of Rac2 and in lyzed by flow cytometry. The results are reported as mean cellular fluorescence. For chemotaxis assays, 27 ␮l of FMLP (at 10, 1, or 0.1 ␮M) or part a preferred role for Rac2 in stimulating downstream functional DMSO vehicle diluted in HBSS (with Ca2ϩ,Mg2ϩ, and glucose) were responses that cannot be compensated for by enhanced Rac1 ac- placed in each lower chamber of a 48-well microchemotaxis device tivation in Rac2null neutrophils. (NeuroProbes, Cabin John, MD), and 1 ϫ 105 neutrophils in 50 ␮lof HBSS (with Ca2ϩ,Mg2ϩ, and glucose) were placed in each upper chamber, which were separated by a 3-␮m pore size polycarbonate filter. The cham- Materials and Methods ber was incubated at 37°C for 45 min; then the filter was fixed and stained Abs, reagents, and buffers with Diff-Quik. The number of migrated cells per high power view field ϫ A polyclonal Rac2 Ab raised in rabbits was a gift from G. Bokoch and U. ( 400) was counted for a minimum of three fields per well, and a mean Knaus (The Scripps Research Institute, La Jolla, CA). A mouse mAb estimate for individual samples was calculated from data of replicate wells. against Rac1 was purchased from Upstate Biotechnology (Lake Placid, Measurement of NADPH oxidase activity NY). Highly purified recombinant prenylated human Rac1 and Rac2 were provided by E. Pick (Tel Aviv University, Tel Aviv, Israel) and R. Erick- Superoxide dismutase-inhibitable FMLP-elicited superoxide production son and J. Curnutte (Genentech, South San Francisco, CA), respectively. was measured by an isoluminol chemiluminescence assay (32) in 96-well An expression vector for a Ϸ6.4-kDa fragment from p21-activated kinase plates using an Lmax microplate luminometer (Molecular Devices, Sunny- 3 containing the Cdc42/Rac binding motif and tagged with glutathione-S- vale, CA). A 2 ϫ 106 cells/ml suspension (50 ␮l) in PBSG was added to transferase was from R. Cerione (Cornell University, Ithaca, NY) (30). each well with 80 ␮l of 125 ␮M isoluminol in PBSG, 40 ␮l of 100 U/ml BL21 competent cells were obtained from Novagen (Madison, WI). PBS HRP (Roche Applied Science, Indianapolis, IN) in 0.9% NaCl, and either ␮ (pH 7.2), ddH2O, glycerol, HBSS, and HEPES (125 mM, pH 7.5) were 5 l of 3 mg/ml superoxide dismutase or PBSG. After cells were incubated from Life Technologies (Gaithersburg, MD). Polymorphprep was pur- at 37°C for 10 min, 25 ␮lof80␮M FMLP (final concentration 10 ␮M) in chased from Accurate Chemical and Scientific (Westbury, NY). Other PBSG or 25 ␮l of PBSG were injected into each well by the automatic chemicals were purchased from Sigma (St. Louis, MO) unless otherwise injector of the luminometer. Chemiluminescence was detected as relative stated. Other buffers used in this article included: PBS with 0.1% BSA and luminescence units by fast kinetic mode, and the relative total amount of

1% glucose (pH 7.2–7.4); PBS with 0.9 mM CaCl2, 0.5 mM MgCl2, and superoxide produced during 1 min was determined using SoftMax PRO 7.5 mM glucose (PBSG); Triton IPB lysis buffer (20 mM Tris-Cl (pH 8.0), software (Molecular Devices). Under these conditions, ϳ97.5% of chemi- 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 20 ␮g/ml chymostain, 2 luminescence was inhibited by superoxide dismutase. Superoxide dis- mM PMSF, 10 ␮M leupeptin, and 1 mM 4-(2-aminoethyl)benzenesulfonyl mutase-inhibitable superoxide production elicited by 200 ng/ml PMA was ﬂuoride); and 5ϫ MLB lysis buffer (125 mM HEPES (pH 7.5), 750 mM measured using a quantitative kinetic assay based on the reduction of cy-

NaCl, 5% Igepal CA-630, 25 mM MgCl2, 25 mM EDTA, 50% glycerol, tochrome c, as previously described (24, 31, 33). 100 ␮g/ml chymostatin, and 10 mM PMSF). Rac activation assays Animals An affinity precipitation or pull-down assay for Rac activation was per- C57BL/6J mice purchased from The Jackson Laboratory (Bar Harbor, ME) formed as described (34) with slight modifications. The p21-binding do- or 129SV mice from an in house colony were used for wild-type controls. main (PBD) of p21-activated kinase 3 was expressed as a fusion protein rac2Ϫ/Ϫ mice had previously been generated by targeted disruption of the with glutathione-S-transferase in the BL21 strain of Escherichia coli (30). rac2 gene (27). Strains of rac2Ϫ/Ϫ and heterozygous rac2ϩ/Ϫ mice used in GST-PBD was purified from glutathione-Sepharose beads with 10 mM this study either had been backcrossed into C57BL/6J mice for Ͼ12 gen- reduced glutathione and stored at Ϫ80°C in 25 mM Tris-HCl (pH 8.0), 0.2

erations or were descendants from a 129SV cross with the original blas- M DTT, 1 mM MgCl2, and 10% glycerol until use. Diisoﬂuorophosphate- tocyst injection founder. Mice were housed in microisolator cages under treated murine neutrophils (6 ϫ 106/tube) were suspended in 0.5 ml of speciﬁc pathogen-free conditions and were fed autoclaved food and acid- PBSG and incubated at 37°C for 5 min before the addition of 5 ␮l of either The Journal of Immunology 5045

DMSO or 200 ␮M FMLP in DMSO (final concentration, 2 ␮M) or 20 with Rac2, and vice versa (Ref. 27 and unpublished observations), ␮g/ml PMA in DMSO (final concentration, 200 ng/ml). In some experi- this should have only a negligible effect on our analyses. Rac1 and ments, cells were pretreated at 37°C for 20 min before stimulation with the ␮ ␮ Rac2 were detected in approximately equal amounts in murine following inhibitors: 100 M genistein; 20 M 2-(4-morpholinyl)-8-phe- Ϸ nyl-4H-1-benzopyran-4-one (LY294002); 100 nM wortmannin; or 10 ␮M, neutrophils (Fig. 1B), whereas human neutrophils contained 3- 25 ␮M, or 50 ␮M 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4- fold more Rac2 than Rac1, consistent with our previous results d]pyrimidine (PP1). Stimulus- or DMSO-treated cells were incubated at (24) and also in general agreement with published data for human 37°C for the indicated times and then lysed with ice cold 125 ␮l5ϫ MLB neutrophil Rac2 (14). Somewhat more Rac1 was detected in hu- lysis buffer containing a total of 10 ␮g GST-PBD. Cell lysates were im- mediately placed on ice, clarified by centrifugation at 10,000 rpm for 5 min man neutrophils than in the study of Heyworth et al. (14) which at 4°C, and transferred to a fresh microfuge tube. For precipitation of could reflect differences in the approaches used to estimate the GST-PBD-bound Rac, 20 ␮l of glutathione-Sepharose 4B beads (50%) was quantity of Rac1 and/or the small degree of Ab cross-reactivity. As added to the 0.5 ml of clarified cell lysate and incubated for1hat4°C with we have previously reported (24, 27), the amount of Rac1 in agitation before centrifugation for 2 min at 9000 rpm. The bead pellet was Ϫ/Ϫ ϩ/Ϫ ϫ rac2 (Fig. 1) and heterozygous rac2 (not shown) neutro- then washed three times with 1 MLB lysis buffer before the final resus- ϩ/Ϫ pension in 35 ␮l of Laemmli sample buffer and stored, if needed, at phils was similar to that in wild-type cells, and rac2 neutro- Ϫ80°C. Before SDS-PAGE, samples were heated to 100°C for 10 min. phils contained Ϸ50% of wild-type levels of Rac2 (not shown). GST-PBD affinity-precipitated proteins were separated by 12% SDS- The similar levels of Rac1 in primary neutrophils isolated from all PAGE in parallel with aliquots of total MLB cell lysate, then transferred to three rac2 genotypes contrast with the increased expression of nitrocellulose membrane, and probed for Rac1 or Rac2, followed by den- Ϫ/Ϫ sitometry, as described above. Rac1 observed in long term rac2 mast cell cultures (26) or in marrow progenitor-derived rac2Ϫ/Ϫ neutrophils generated in vitro Statistical analysis using high concentrations of hemopoietic cytokines (35).

Student’s t test (either paired or unpaired, as indicated) was performed Heterozygous rac2ϩ/Ϫ mice are deficient in FMLP-induced using Microsoft Excel software (Redmond, WA) and correlation analysis performed using Cricket Graph III (Computer Associates International, F-actin formation, chemotaxis, and NADPH oxidase activity New York, NY). To examine whether there was a rac2 gene dosage effect on neutrophil responses induced by the chemoattractant FMLP, we com- Results pared F-actin formation, chemotaxis, and NADPH oxidase activity Rac1 and Rac2 isoforms are expressed at similar levels in in rac2ϩ/ϩ, rac2ϩ/Ϫ, and rac2Ϫ/Ϫ neutrophils. We previously ob- wild-type murine neutrophils served a small but not statistically significant decrease in PMA- Ϫ ϩ/Ϫ We previously reported (24) that murine neutrophils have 3 to 5 elicited O2 production in rac2 neutrophils and failed to detect times more Rac1 on a milligram of protein basis compared with any differences in FMLP-elicited F-actin formation or chemotaxis human neutrophils, in which Rac2 has been estimated at Ϸ60 ng/ between wild-type and rac2ϩ/Ϫ mice (27). However, our original 106 cells and accounts for at least 90% of total cellular Rac (14). studies were done in mixed 129SV ϫ C57BL/6J littermates, in Murine neutrophils do not express Rac3 (U. Knaus, personal com- which variability introduced by strain to strain differences might munication). To directly compare total cellular levels of the Rac1 have obscured any gene dosage effect. In subsequent studies, we Ϫ and Rac2 isoforms in murine neutrophils, we used serial dilutions observed that the defect in PMA-elicited O2 production in of recombinant isoprenylated Rac1 and Rac2 as standards on im- rac2Ϫ/Ϫ exudate neutrophils became more severe as mice were munoblots to estimate the quantity of Rac1 and Rac2 (Fig. 1A), backcrossed into C57BL/6J (24). Therefore, we re-examined re- using Abs that are relatively specific for Rac1 and Rac2. Although sponses elicited by either FMLP or PMA in freshly isolated BM there is a small amount (Ϸ5%) of cross-reactivity of the Rac1 Ab neutrophils containing differing levels of Rac2 in an otherwise homogeneous genetic background, using C57BL/6J rac2ϩ/ϩ, rac2ϩ/Ϫ, and rac2Ϫ/Ϫ mice. Fig. 2A shows the mean basal levels of F-actin and at 10 and 30 s after stimulation with 10 ␮M FMLP, in a representative experiment using BM neutrophils prepared on a single day from each of three mice for each genotype. In resting neutrophils, F-actin levels were decreased in rac2Ϫ/Ϫ compared with wild-type neutrophils, as previously reported (27), and intermediate levels were detected in heterozygous rac2ϩ/Ϫ neutrophils (Fig. 2A). Furthermore, the FMLP- stimulated increase in F-actin followed a similar hierarchy, with very little change from basal levels observed in rac2Ϫ/Ϫ neutrophils compared with the progressive increase seen in rac2ϩ/Ϫ and wild-type neutrophils (Fig. 2A). These data suggest that Rac2 activation may be rate limiting for this early burst of F-actin generation stimulated by FMLP. We next examined FMLP-induced chemotaxis in rac2ϩ/ϩ, rac2ϩ/Ϫ, and rac2Ϫ/Ϫ neutrophils, finding a similar marked dosage effect for the rac2 gene (Fig. 2B). Finally, we found that superoxide production elicited by FMLP (Fig. 2C) or PMA (Fig. 2D)inrac2ϩ/Ϫ neutrophils was also intermediate between wild-type levels and the substantial defects in rac2Ϫ/Ϫ neutrophils. Linear regression analysis FIGURE 1. Expression of Rac1 and Rac2 in human (Hu) and murine showed a highly significant correlation of F-actin content, chemo- (mu) neutrophils. A, Representative immunoblot of neutrophil extracts (10 ␮g/lane) and isoprenylated recombinant Rac1 and Rac2 used as standards taxis, and superoxide production with the number of functional rac2 Ͻ (10, 20, and 40 ng/lane). Blots were probed with either a mAb for Rac1 or genes ( p 0.003). We also observed a similar rac2 gene dosage a polyclonal rabbit Ab for Rac2. B, Rac1 and Rac2 expression levels were effect for FMLP-stimulated chemotaxis (r ϭ 0.9, p ϭ 0.001) and estimated by densitometry of immunoblots of neutrophil extracts and sev- superoxide production (r ϭ 0.76, p ϭ 0.017) in the 129SV strain, eral dilutions of isoprenylated Rac standards. Means Ϯ SD are shown. n Ն 5. although there were slight differences in the absolute magnitude of the 5046 Rac ACTIVATION IN MURINE NEUTROPHILS

FIGURE 2. Heterozygous rac2ϩ/Ϫ mice are deficient in FMLP-induced F-actin formation, chemotaxis, and NADPH oxidase activity. Data are expressed as means Ϯ SD. A, FMLP-elicited F-actin formation. Freshly isolated rac2ϩ/ϩ, rac2ϩ/Ϫ,orrac2Ϫ/Ϫ BM neutrophils were incubated for the indicated times either with 10 ␮M FMLP or without stimulation (time 0) and then fixed and stained with 160 nM FITC-phalloidin, followed by flow cytometry. Data are ,p Ͻ 0.001 ,ءءء ;p Ͻ 0.005 ,ءء ;p Ͻ 0.05 ,ء .shown for the mean cellular fluorescence (MCF) from three independent samples analyzed on a single day as indicated. One of four representative studies. For the combined data from all four experiments (not shown), the F-actin content, as determined by FITC-phalloidin staining intensity, was significantly increased at both 10 and 30 s after FMLP stimulation for both rac2ϩ/ϩ (p Ͻ 0.007) and rac2ϩ/Ϫ (p Ͻ 0.02) cells. B, FMLP-elicited chemotaxis. Neutrophil chemotaxis was assayed in a modified Boyden chamber using different FMLP concentrations (100 p Ͻ 0.001 (vs rac2ϩ/ϩ); #, p Ͻ ,ءء ;(p Ͻ 0.01 (vs rac2ϩ/ϩ ,ء .nM, 1 ␮M, 10 ␮M FMLP) or HBSS, as indicated. Data from five independent experiments 0.001 (vs rac2ϩ/Ϫ). C, FMLP-elicited NADPH oxidase activity. Superoxide dismutase-inhibitable superoxide anion production in neutrophils stimulated p Ͻ ,ء .with 10 ␮M FMLP was measured by isoluminol chemiluminescence. Data are shown for the relative luminescence units integrated over 60 s. n ϭ 6 p Ͻ 0.0005. D, PMA-elicited NADPH oxidase activity. Superoxide dismutase-inhibitable superoxide anion production from ,ءءء ;p Ͻ 0.005 ,ءء ;0.05 .p Ͻ 0.0005 ,ءءء ;p Ͻ 0.005 ,ءء .neutrophils stimulated with 0.2 ␮g/ml PMA was measured by the cytochrome c reduction assay. n ϭ 10 responses compared with the C57BL/6J strain (data not shown). Rac1- and Rac2-GTP within 10 s of stimulation, with maximal levels Taken together, these results suggest that the cellular level of Rac2 detected at 30–60 s of stimulation, which began to decline by 2 min can be rate limiting for F-actin formation, chemotaxis, and NADPH (Fig. 3A) and approached basal levels by 5 min (data not shown), oxidase activation induced by FMLP in murine neutrophils. similar to what has been described for Rac2 activation in FMLP- stimulated human neutrophils (37, 38). At 60 s after FMLP stimula- Activation of Rac1 and Rac2 in wild-type and Rac2-deficient tion, 4.5 Ϯ 1.7% of total cellular Rac2 was recovered as Rac2-GTP murine neutrophils (Fig. 3B), in the same range that has been reported for Rac2-GTP Using the p21-binding domain of PAK kinase that contains the levels in stimulated human neutrophils (37, 38). At all time points Cdc42/Rac interactive binding (CRIB) motif and interacts specifically after stimulation, the amount of Rac2-GTP detected in FMLP-acti- with GTP-bound Rac and Cdc42, several groups have established that vated murine neutrophils was significantly higher than the amount of FMLP and PMA induce formation of Rac2-GTP in human neutro- Rac1-GTP, which peaked at 1.1 Ϯ 0.4% of total Rac1 at 60 s (Fig. phils (36–38). We used this affinity precipitation or pull-down assay 3B). Because similar amounts of the Rac1 and Rac2 isoforms are to examine the relative levels of Rac1-GTP and Rac2-GTP in DMSO- present in wild-type murine neutrophils (Fig. 1), we therefore infer and FMLP-stimulated wild-type murine neutrophils, in which total that the Rac2-GTP levels are Ϸ4-fold higher than Rac1-GTP after cellular levels of Rac1 and Rac2 are similar (Fig. 1). As previously FMLP stimulation. Neither Rac1 nor Rac2 was detected in the deter- observed in freshly isolated human peripheral blood neutrophils (37, gent-insoluble pellet isolated after the initial solubilization of DMSO- 38), a small amount of activated Rac2, and to a lesser extent Rac1, or FMLP-stimulated neutrophils in the affinity precipitation assay was detected in DMSO-treated murine neutrophils (Fig. 3), which (data not shown). varied from experiment to experiment and probably reflects a basal We next compared the activation of Rac1 and Rac2 in FMLP- level of activation in resting neutrophils or induced by the purification stimulated rac2ϩ/ϩ and heterozygous rac2ϩ/Ϫ murine neutrophils. procedure. Stimulation with FMLP induced the formation of both Results from a representative experiment are shown in Fig. 3C. The Journal of Immunology 5047

Although FMLP elicited an increase in Rac2-GTP in rac2ϩ/Ϫ neutrophils, the absolute amount of Rac2-GTP was Ϸ2-fold lower than that detected in FMLP-stimulated wild-type neutrophils (Fig. 3, C and D). Thus, a compensatory increase in Rac2 activation does not occur in the face of the Ϸ2-fold reduction in cellular Rac2 levels in rac2ϩ/Ϫ neutrophils. Rac1 activation in rac2ϩ/Ϫ neutrophils was similar to that in wild-type neutrophils. Finally, we used the PBD-GST precipitation assay to compare the activation of Rac1 and Rac2 in wild-type and rac2Ϫ/Ϫ murine BM neutrophils that were stimulated with either 2 ␮M FMLP or 200 ng/ml PMA. Each agonist induced Rac1 activation in rac2Ϫ/Ϫ neutrophils with kinetics that was similar to that of wild-type neutrophils. Compared with FMLP, the PMA-induced increases in Rac1-GTP were never as high as those detected after FMLP stimulation in either wild-type or rac2Ϫ/Ϫ neutrophils, and very little Rac activation was detected before 1 min (not shown), although elevated Rac1-GTP levels persisted for at least 5 min. In wild-type neutrophils, the kinetics and relative magnitude of Rac2 activation after FMLP or PMA stimulation paralleled that observed for Rac1 (Fig. 4, A and B). Because PBD-GST- precipitated samples were loaded on adjacent lanes and probed on the same immunoblot, Rac1-GTP levels detected in wild-type and Rac2null genotypes can be directly compared. Sub- stantially higher amounts of Rac1-GTP were present in FMLP- or PMA-stimulated rac2Ϫ/Ϫ neutrophils relative to wild-type neutrophils (Fig. 4, A and B). Peak Rac1-GTP levels were Ϸ3-fold higher (range, 2- to 5-fold) in FMLP-stimulated rac2Ϫ/Ϫ cells than in wild- type cells (Fig. 4C). The basal level of Rac1-GTP in DMSO-treated polymorphonuclear neutrophils (PMNs) was also consistently higher in rac2Ϫ/Ϫ than in wild-type PMNs (Fig. 4C).

FMLP-induced activation of Rac1 and Rac2 in murine neutrophils is sensitive to inhibitors of phosphatidylinositol 3-kinase (PI3K) and Src family tyrosine kinases ␣ ␤␥ The neutrophil FMLP receptor is coupled to heterotrimeric G i GTP-binding proteins and transduces signals via multiple pathways, including the src-related kinases, Lyn and Hck, and PI3K (6, 39–41). In chemoattractant-stimulated human neutrophils, Rac2 activation has been reported to be sensitive to either the tyrosine kinase inhibitor genistein or PI3K inhibitors wortmannin or LY294002 (37, 38). Consistent with these observations, we found that the increase in activated Rac above basal levels in FMLP- stimulated wild-type murine neutrophils was substantially inhibited by LY294002 or the src family tyrosine kinase inhibitor PP1 (Fig. 5A). The activation of Rac1 in both wild-type and rac2Ϫ/Ϫ neutrophils appeared to be more sensitive to either LY294002 or PP1 than did Rac2 activation ( p Ͻ 0.03), with Rac1-GTP levels FIGURE 3. FMLP-stimulated activation of Rac1 and Rac2 in wild-type decreasing in inhibitor-treated FMLP-stimulated cells to even be- ϫ 6 ϫ 7 murine neutrophils. Aliquots of 6 10 (A and B)or1.2 10 (C and D) low the basal levels detected in DMSO-only treated cells (Fig. 5A). puriﬁed BM neutrophils were stimulated with 2 ␮M FMLP or with vehicle DMSO only for the indicated times, followed by solubilization and afﬁnity precipitation using PBD-GST. A, Immunoblot from a representative experiment showing activation of Rac1 and Rac2 in rac2ϩ/ϩ murine BM to 25-kDa proteins (including p22phox), for reasons that are uncertain. If neutrophils. SN (supernatant) represents an aliquot of total cell extract doublets were present, both bands were scanned by densitometry as a sin- obtained from 2 ϫ 105 cells. B, Percentage of Rac1-GTP and Rac2-GTP gle unit. D, Relative percentage of Rac1-GTP and Rac2-GTP detected in ϩ ϩ ϩ Ϫ compared with total Rac1 and Rac2 in rac2ϩ/ϩ murine BM-derived neu- DMSO- or FMLP-stimulated rac2 / and rac2 / murine neutrophils, as trophils was calculated by comparing the relative amounts of PBD-GST- indicated, was calculated by comparing the relative amounts of PBD-GST- precipitated Rac1 or Rac2 to the total amount of the corresponding isoform precipitated Rac1 or Rac2 to the total amount of the corresponding isoform ϩ ϩ detected in unstimulated neutrophils. Student’s t test was used for statistical detected in unstimulated rac2 / neutrophils. Means Ϯ SD are shown. ϩ ϩ p Ͻ 0.05, for the relative percentage of Student’s t test was used for statistical analysis. n ϭ 8 for rac2 / and n ϭ ,ءء ;p Ͻ 0.005 ,ء .analysis. n ϭ 6 p Ͻ 0.02, for rac2ϩ/ϩ vs ,ءء ;p Ͻ 0.002 ,ء .Rac1-GTP vs Rac2-GTP at each time point. C, Immunoblot from a rep- 4 for rac2ϩ/Ϫ neutrophils resentative experiment showing activation of Rac1 and Rac2 in rac2ϩ/ϩ rac2ϩ/Ϫ at each time point. The increases in Rac1-GTP and Rac2-GTP in and rac2ϩ/Ϫ murine BM neutrophils. SN (supernatant) represents an ali- rac2ϩ/ϩ neutrophils at 30 and 60 s were statistically different from at time quot of total cell extract obtained from 1 ϫ 105 cells. Although some of the zero (p Ͻ 0.002). The increases in Rac1-GTP and Rac2-GTP in rac2ϩ/Ϫ bands appear to be doublets (see also Fig. 5A), we believe this is likely to neutrophils at 30 s were statistically different from those at time zero (p Ͻ be an artifact of our gel system; we occasionally see doublets for other 20- 0.025) but not at 60 s. 5048 Rac ACTIVATION IN MURINE NEUTROPHILS

dose dependent (Fig. 5B). PP1 (10 ␮M) inhibited Ϸ50% of the FMLP-induced increase in Rac2-GTP over basal levels, which increased to Ϸ90% inhibition of the FMLP response with 50 ␮M PP1. Rac1 activation in wild-type neutrophils was relatively more sensitive to PP1 at all doses tested (Fig. 5B), which was statistically signiﬁcant at 50 ␮M PP1 ( p Ͻ 0.001).

Discussion In contrast to human neutrophils, where Rac2 is the predominant Rac isoform (14), we here show that murine neutrophils contain comparable levels of Rac1 and Rac2 (Fig. 1). We further demonstrate that stimulation of wild-type murine neutrophils with either FMLP or PMA results in activation of both Rac isoforms but found that substantially more Rac2-GTP was detected than Rac1-GTP (Figs. 3 and 4). This is the first evidence to indicate that these highly similar GTPases are differentially activated by agonist-induced signals, which suggests one mechanism that contributes to a nonoverlapping role for Rac1 and Rac2 in coupling chemoattractant receptor signals to downstream functions. Analysis of FMLP-stimulated Rac2-deficient neutrophils indi- cates that more efficient activation of downstream pathways by Rac2 accounts for a second mechanism leading to a preferred role for Rac2 in regulating chemoattractant-induced responses. The se- verity of defects in FMLP-elicited F-actin generation, chemotaxis, and superoxide production was strongly correlated with the level of Rac2 expression and number of functional rac2 genes (Fig. 2), despite the presence of Rac1. In rac2ϩ/Ϫ neutrophils, total activated Rac2 levels after FMLP stimulation were Ϸ2-fold less than in wild-type neutrophils (Fig. 3), consistent with reduced total levels of Rac2 protein in the rac2 heterozygotes and suggesting that Rac2 activation is rate limiting for chemoattractant-induced responses. There was not always a 1:1 relationship between the magnitude of each response and Rac2 levels, but this likely reflects the complexity of the signaling cascades leading to these functional Ͻ ϩ/ϩ Ϫ/Ϫ responses. Because 5% of Rac2 is activated in stimulated FIGURE 4. Activation of Rac1 and Rac2 in rac2 and rac2 mu- ϩ ϩ rac2 / neutrophils, one might also anticipate that the 50% re- rine BM-derived neutrophils. BM neutrophils were purified using sequen- ϩ/Ϫ tial Percoll and Histopaque gradients. Aliquots of 6 ϫ 106 cells were stim- duction in Rac2 protein level in rac2 cells would still be ad- ulated by 2 ␮M FMLP or 200 ng/ml PMA or with vehicle DMSO only for equate to generate a full response. However, subcellular compart- the indicated times, followed by solubilization and Rac-GTP affinity pre- mentalization or the efficient activity of Rac GTPase-activating cipitation using PBD-GST as described in Materials and Methods. A, Rep- proteins may contribute to the observed gene dosage effect. In resentative immunoblot for PBD-GST-precipitated samples after stimula- rac2Ϫ/Ϫ neutrophils, higher levels of activated Rac1 were detected ϩ ϩ Ϫ Ϫ tion of rac2 / or rac2 / neutrophils with either FMLP or PMA and after FMLP stimulation relative to wild-type or rac2ϩ/Ϫ neutro- probed with Abs for either Rac1 or Rac2, as indicated. The PBD-GST- phils (Figs. 3 and 4). Comparing the relative amounts of Rac1- Ϫ/Ϫ precipitated samples from wild-type and rac2 neutrophils were loaded GTP and Rac2-GTP after FMLP stimulation, we estimate that the on adjacent lanes, transferred, and probed on the same immunoblot with Ϫ/Ϫ total cellular level of activated Rac in wild-type neutrophils still Abs for either Rac1 or Rac2. Rac2-GTP was not detected in rac2 neu- Ϫ Ϫ exceeds that in rac2 / neutrophils by up to Ϸ2-fold, despite the trophils, as expected (data not shown). B, Densitometry analysis of immu- Ϫ/Ϫ noblot shown in A. Genotypes and agonists used for neutrophil activation increased amount of Rac1-GTP in rac2 neutrophils. Hence, it are as indicated. C, Comparison of Rac1 activation in rac2ϩ/ϩ and rac2Ϫ/Ϫ is possible that an overall decrease in total cellular levels of acti- Ϫ/Ϫ murine BM-derived neutrophils stimulated by 2 ␮M FMLP. Means Ϯ SD vated Rac in rac2 neutrophils contributes to the observed im- are shown. The values obtained for Rac1-GTP at the indicated times were pairment in FMLP-elicited chemotaxis and superoxide production. averaged after normalization of the 30-s value for rac2Ϫ/Ϫ cells to an However, the functional defects in rac2Ϫ/Ϫ neutrophils are sub- arbitrary value of 1. Student’s t test was used for statistical analysis. n ϭ stantially more severe compared with rac2ϩ/Ϫ cells (Fig. 2), in- Ϫ p Ͻ 2 ϫ 10 5, for the normalized values of Rac1-GTP dicating that activation of even a limited amount of Rac2 is more ,ءء ;p Ͻ 0.007 ,ء .6 ϩ/ϩ Ϫ/Ϫ in rac2 vs rac2 neutrophils. The increases in Rac1-GTP at 30, 60, effective at stimulating downstream functions than Rac1-GTP. By in- ϩ/ϩ Ͻ and 120 s were statistically different from time zero for both rac2 (p ference, this suggests that Rac2-GTP plays a preferred role compared 0.05) and rac2Ϫ/Ϫ (p Ͻ 0.010). with Rac1-GTP in signaling to downstream effectors that regulate actin polymerization, chemotaxis, and superoxide production. The mechanism(s) which underlies the apparent differential activa- In two additional experiments, similar results were obtained for tion of Rac1 and Rac2 in FMLP-stimulated neutrophils is unknown inhibition of FMLP-induced Rac1 and Rac2 activation using either but likely reflects different affinities for proteins that regulate overall wortmannin (100 nM), another PI3K inhibitor, or the tyrosine ki- level of Rac-GTP (GEFs or GTPase-activating proteins), or subtle nase inhibitor, genistein (100 ␮M) (data not shown). Inhibition of differences in membrane binding or subcellular location that influence Rac1 and Rac2 activation in wild-type neutrophils by PP1 was accessibility to these regulatory proteins. Similar mechanisms may The Journal of Immunology 5049

FIGURE 5. Effect of PI3K and src family kinase inhibitors on FMLP-induced activation of Rac1 and Rac2. Wild-type and rac2Ϫ/Ϫ BM neutrophils were pretreated for 20 min with either buffer alone or LY294002 or PP1 at 37°C, as indicated, and stimulated for 60 s with 2 ␮M FMLP, followed by the addition of lysis buffer and affinity precipitation with PBD-GST as described in Materials and Methods. PBD immunoblots were quantified by densitometry, and the values were normalized for each genotype and isoform by assigning the relative amount of Rac-GTP at 60 s after FMLP stimulation an arbitrary value of 1. A, Representative immunoblot showing effect of Ly294002 (20 ␮M) or PP1 (50 ␮M). Bar graphs show relative densitometry values obtained for Rac1-GTP or Rac2-GTP in the indicated genotypes averaged after normalization of values from FMLP-treated (without inhibitor) to an arbitrary value of 1. Mean Ϯ SD are shown (n ϭ 7 for rac2ϩ/ϩ, n ϭ 3 for rac2Ϫ/Ϫ). B, Dose-dependent inhibition of Rac activation by PP1, used at 10, 20, or 50 ␮M. Representative immunoblot and relative densitometry with normalization as performed as in A. n ϭ 7 for rac2ϩ/ϩ, n ϭ 3 for rac2Ϫ/Ϫ. account for a preferential role for Rac2-GTP in activating downstream neutrophil cytosol (42), suggesting that the cytosol contains a fac- responses. Of interest in this latter regard are the findings of Heyworth tor(s) that either suppresses Rac1 activity or promotes Rac2 et al. (42), who compared the ability of purified recombinant pre- function. nylated preparations of Rac1 or Rac2 to stimulate NADPH oxidase Although Rac1 and Rac2 isoforms have 92% sequence identity, activity under cell-free conditions in the presence of GTP-␥S and there are a number of candidate regions that could permit discrim- neutrophil membranes. Both isoforms were equivalent when using ination between the two isoforms. These regions lie outside of the recombinant p47phox and p67phox. However, Rac2 was substan- N-terminal switch I and II regions that change conformation on tially more active in the presence of otherwise limiting amounts of guanine nucleotide exchange, which are identical in Rac1 and 5050 Rac ACTIVATION IN MURINE NEUTROPHILS

Rac2 except for a Gly vs Ser residue at position 49, located be- trophils is P-Rex1 (for PIP3-dependent Rac exchanger), which is di- tween the ␤2 and ␤3 strands linking switch I and II. These do- rectly activated by G␤␥ subunits and by PI3K␥, in both an indepen- mains, particularly switch I, constitute a major binding interface dent and synergistic manner (66). Stimulation of guanine nucleotide with target effector proteins and with Rac GEFs (5, 43, 44). The exchange on Rac by G␤␥-activated P-Rex1 at early time points after greatest divergence between Rac1 and Rac2 is in the polybasic chemoattractant stimulation may explain why PI3K␥-null mice and region located just upstream of the C-terminal CAAX box, where PI3K inhibitor-treated human neutrophils have normal Rac activation Rac1 has six adjacent basic amino acids compared with Rac2, in at 5–10 s after FMLP (36, 67), in contrast to the sensitivity to PI3K which three of these residues are uncharged. The four mammalian inhibitors at later times (60 s) that we and others have observed for Ras proteins also differ primarily in this region, termed the “hy- murine and human neutrophils (37, 38). pervariable” domain, and isoform specificities of Ras and Rac may These studies provide new insights into the basis of the pheno- at least in part be related to differences in localization to subcel- typic defects in Rac2-deficient murine neutrophils and provide the lular membrane compartments or microdomains known to be con- first direct evidence that Rac1 and Rac2 have distinct activation ferred by this domain (45, 46). In the case of Ras, the hypervari- and signaling profiles in agonist-activated cells, despite their high able domain has been linked to differential activation by the Ras degree of sequence homology. Two different mechanisms were exchange factors Ras GRF1 and Ras GRP2 (47, 48), efficiency of identified that may account for the lack of functional redundancy activating PI3K or the Raf serine-threonine kinase (49–51), and between these two Rac isoforms. Although murine neutrophils specific biologic responses (52–56). The Rac polybasic region may have similar amounts of Rac1 and Rac2, Rac2 appears to be pref- also modulate interactions with downstream protein targets inde- erentially activated through the FMLP receptor. The relative level pendent from effects on membrane localization, as has been shown of activated Rac2, in turn, was rate limiting for coupling FMLP- for Pak1 (57). Regions outside of the hypervariable domain in Ras induced signals to F-actin formation, chemotaxis, and superoxide also strongly influence the oncogenic potency of H-Ras (54) and in production, which could not be compensated for by enhanced Rac1 an analogous manner may contribute to isoform-specific Rac func- activation in rac2Ϫ/Ϫ neutrophils. tions. There are three amino acid differences between Rac1 and Rac2 clustered in residues 145–151, a domain shown to be impor- Acknowledgments tant for transducing signals through PAK (58). Rac1 and Rac2 also We thank Gary Bokoch, Ulla Knaus, Rich Erickson, John Curnutte, Edgar have a single amino acid difference in the insert domain (residues Pick, and Rick Cerione for reagents; Wade Clapp for helpful discussions; and 116–136) which has been implicated in regulation of electron Shari Upchurch and Donna Fischer for assistance with manuscript preparation. transport in the NADPH oxidase and in mitogenesis (15, 59–61). 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