Rac2 Is an Essential Regulator of Neutrophil Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activation in Response to Specific Signaling Pathways1

Chaekyun Kim and Mary C. Dinauer2

Rac2 is a hematopoietic-specific Rho family GTPase implicated as an important constituent of the NADPH oxidase complex and shares 92% amino acid identity with the ubiquitously expressed Rac1. In bone marrow (BM) neutrophils isolated from rac2؊/؊ mice generated by targeting, we previously reported that PMA-induced superoxide production was reduced by about 4-fold, which was partially corrected in TNF-␣-primed BM neutrophils and in peritoneal exudate neutrophils. We investigated receptor- mediated activation of the NADPH oxidase in the current study, finding that superoxide production in rac2؊/؊ BM and peritoneal exudate neutrophils was normal in response to opsonized zymosan, reduced to 22% of wild type in response to IgG-coated SRBC, and almost absent in response to fMLP. In wild-type murine BM neutrophils, phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and Akt was induced by PMA or fMLP, which was decreased in rac2؊/؊ neutrophils for ERK1/2 and p38. Activation of p38 by either opsonized zymosan or IgG-coated ,SRBC was similar in wild-type and rac2؊/؊ cells. Inhibition of ERK1/2 or p38 activation using either PD98059 or SB203580 respectively, had only a modest effect on fMLP-elicited superoxide production and no effect on the PMA-induced response. These data provide genetic evidence supporting an important role for Rac2 in regulating neutrophil NADPH oxidase activation down- stream of chemoattractant and Fc␥ receptors. The effect of Rac2 deficiency on superoxide production is probably exerted through multiple pathways, including those independent of mitogen-activated protein kinase activation. The Journal of Immunology, 2001, 166: 1223–1232.

he phagocyte NADPH oxidase (respiratory burst oxidase) vitro (4, 5), has also been found to be essential for high level is a membrane-associated enzyme complex that generates superoxide production in cell-free NADPH oxidase assays (6, 7), T superoxide during the respiratory burst by catalyzing the although its role in the NADPH oxidase complex is not fully transfer of electrons from NADPH to molecular oxygen. The ac- defined. tive NADPH oxidase is made up of two membrane-bound com- The Rho family regulate a wide spectrum of cellular ponents, gp91phox and p22phox, that together form the oxidase fla- functions, including cytoskeletal organization, transcription, cell vocytochrome b, and three cytoplasmic subunits, p47phox, p67phox, growth, development, and superoxide production (8–13). Alter- and p40phox, that translocate to the membrane upon cellular acti- nating between GDP- and GTP-bound states, Rho GTPases func- vation (1–3). In addition to these five phox components, two small tion as intermediary switches, mediating the transfer of signals GTPases, Rap1A and Rac, have been implicated in regulation of from receptors and their associated kinases to downstream effec- the NADPH oxidase complex. Flavocytochrome b and the p47phox tors (12). Three Rho GTPase subclasses based on homology to the and p67phox subunits are required for superoxide production, since three prototypic members, RhoA, Rac1, and Cdc42Hs, are cur- a deficiency in any one of these components results in chronic rently recognized. These encompass seven distinct proteins, in- granulomatous disease, an inherited disorder characterized by ab- cluding Rho (A, B, and C isoforms), Rac (1, 2, and 3 isoforms), sent phagocyte NADPH oxidase activity and current bacterial and Cdc42 (Cdc42Hs and G25K isoforms), RhoD, RhoG, RhoE, and fungal infections (3). Flavocytochrome b is the redox center of the Tc10. Rac1 and Rac2 share 92% identity and differ primarily in the enzyme and appears to be activated upon binding of the p47phox C-terminal 10 residues, where Rac1, but not Rac2, contains a po- and p67phox subunits. Rac GTPase, which can bind to p67phox in lybasic sequence. Rac1 and share 77% identity, and Rac2 and Rac3 share 83% identity (14–18). Murine Rac1 has 100% amino acid identity and 92% nucleotide homology with human The Herman B Wells Center for Pediatric Research, Department of Pediatrics (He- Rac1, and murine Rac2 differs by only two amino acids from hu- matology/Oncology) and Medical and Molecular Genetics, The James Whitcomb Riley Hospital for Children, Indiana University Medical Center, Indianapolis, IN man Rac2 (14, 15). Rac1 and Rac3 are expressed in a wide variety 46202 of tissues, whereas Rac2 expression is highly restricted to hema- Received for publication August 17, 2000. Accepted for publication October topoietic cells (14–18). 25, 2000. One of the first cellular functions ascribed to Rac was the acti- The costs of publication of this article were defrayed in part by the payment of page vation of the phagocyte NADPH oxidase (6, 7). The GTP-bound charges. This article must therefore be hereby marked advertisement in accordance phox with 18 U.S.C. Section 1734 solely to indicate this fact. form of Rac binds to both p67 and probably also to flavocy- 1 This work was supported by National Heart, Lung, and Blood Institute Grant tochrome b in the assembled oxidase complex (4, 5, 19–22). Su- RO1HL45635. The Wells Center for Pediatric Research is a Center for Excellence in peroxide generation in cell-free assays is substantially increased by Molecular Hematology funded by National Institute of Diabetes and Digestive and the addition of Rac (6, 7), and studies using either Rac2 transgenes Kidney Diseases Grant P50DK49218. or antisense oligonucleotides to manipulate Rac levels suggest that 2 Address correspondence and reprint requests to Dr. Mary C. Dinauer, Cancer Re- search Institute R4, Indiana University School of Medicine, 1044 West Walnut Street, Rac regulates NADPH oxidase activity in intact cells (23, 24). Room 402A, Indianapolis, IN 46202. E-mail address: [email protected] Neutrophil activation with either fMLP or phorbol ester stimulates

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 1224 SELECTIVE REGULATION OF NEUTROPHIL NADPH OXIDASE BY Rac2 the formation of Rac-GTP (25, 26). Rac2 is the predominant form Animals in human neutrophils (22, 27). In the yeast two-hybrid system, Ϫ/Ϫ phox Rac2 knockout (rac2 ) mice had previously been generated by targeted p67 has higher affinity for Rac2 than for Rac1 (19). Recom- disruption of the rac2 gene (11). Rac2 knockout mice used in this study had binant isoprenylated Rac1 and Rac2 were equipotent in a cell-free been backcrossed into C57BL/6J mice for Ն11 generations. C57BL/6J NADPH oxidase assay using purified flavocytochrome b and re- mice were used for wild-type controls. Mice were housed in microisolator combinant cytosolic phox proteins, but Rac2 was more active than cages under specific pathogen-free conditions and were fed autoclaved food and acidified water ad libitum. Mice used in these experiments were Rac1 when neutrophil cytosol was added (28). 8–16 wk of age. To examine whether Rac2 has a specialized function in the NADPH oxidase and other neutrophil functions, mice with a tar- Isolation of neutrophils geted disruption of the hematopoietic-specific rac2 gene were gen- Murine neutrophils were obtained from the BM and peritoneal cavity as erated (11). Rac2-null mice exhibited decreased exudate formation previously described (11). BM neutrophils were isolated from femurs and in vivo and an increased susceptibility in invasive Aspergillosis. tibias flushed with 3 ml of HBSS-BG using a 22-gauge needle (Becton Dickinson, Franklin Lakes, NJ). The cells were pelleted by centrifugation Phorbol ester-activated NADPH oxidase activity in bone marrow ϫ 3 Ϫ/Ϫ at 600 g for 10 min at 4°C and suspended in 3 ml of 45% Percoll (BM) rac2 neutrophils was reduced by about 4-fold. How- (Pharmacia, Uppsala, Sweden). Two milliliters each of the 62, 55, and 50% ever, the requirement for Rac2 was not absolute, in that deficient Percoll solutions was layered successively onto 3 ml of the 81% solution. superoxide production by rac2Ϫ/Ϫ BM neutrophils could be par- One hundred percent Percoll stock solution was made by mixing 9 vol of ϫ tially overcome by first priming with TNF, and phorbol ester-elic- Percoll with 1 vol of 10 HBSS. Finally, cells in 45% Percoll were layered Ϫ/Ϫ on top of the gradient. After centrifugation at 1600 ϫ g for 30 min at 10°C, ited superoxide production in rac2 exudate neutrophils was the cell band between the 81 and 62% layer was collected and washed Ϫ/Ϫ almost normal. Other functional defects in rac2 neutrophils twice in 10 ml of HBSS-BG and suspended in 3 ml of HBSS-BG. These include diminished L-selection-mediated adhesion to glycam-1, cells were layered over 3 ml of Histopaque 1119 and centrifuged at 1600 ϫ impaired actin polymerization, and chemotaxis in response to g for 30 min at 10°C to remove contaminating RBC. The cells between the fMLP and other chemoattractants signaling through -cou- Histopaque and HBSS-BG layers were harvested and washed twice in 10 ml of HBSS-BG. The resulting cell population was 73–78% neutrophils by pled receptors, and decreased fMLP-induced activation of p42/p44 assessment of nuclear morphology of Diff-Quick (Dade, Miami, FL)- and p38 mitogen-activated protein (MAP) kinase (11). Taken to- stained cells, with the remainder being mononuclear cells. The final cell gether, these findings suggested that Rac2 regulates multiple cel- preparation was kept on ice in PBS (without Ca2ϩ and Mg2ϩ) until further lular functions in neutrophils. Additional studies in mast cells (29) processing. For some studies BM neutrophils were cultured for 24 h at 37°C in DMEM containing 20% FCS and 1 ng/ml IL-3 before measuring and T cells (30) have demonstrated a variety of functional abnor- NADPH oxidase activity. To elicit peritoneal exudate neutrophils, mice malities associated with intracellular signaling defects. These in- were injected with 1 ml of 3% thioglycolate. Peritoneal cells were har- clude reduced chemotaxis and growth factor-induced survival in vested after 18 h as previously described (31). All reagents used were mast cells, with impaired activation of Akt (29). endotoxin tested (Ͻ0.1 ng endotoxin/ml by the Limulus lysate assay) to In the present study, we used rac2Ϫ/Ϫ mice as a genetic ap- minimize inadvertent priming during the isolation procedure. Human neutrophils were separated from peripheral venous blood ob- proach to investigate the role of Rac2 in receptor-mediated acti- tained from healthy volunteers as approved by the institutional review vation of the neutrophil NADPH oxidase. The results support the board of the Indiana University School of Medicine. Heparinized whole concept that Rac2 plays an important, but stimulus-specific, role in blood was sedimented using 6% dextran 70 (McGaw, Irvine, CA), and the regulating NADPH oxidase activation and other functional re- granulocytes were purified from the leukocyte-rich serum by Ficoll- Hypaque. Remaining erythrocytes were removed by hypotonic lysis. sponses of neutrophils, particularly those downstream of chemoat- tractant receptors and Fc␥ receptors. Fc␥RII/III expression Flow cytometry was used to quantitate the relative amount of cell surface Materials and Methods Fc␥RII/III expression per neutrophils. BM neutrophils (2 ϫ 105/ml) in PBS Abs, reagents, and buffers containing 0.1% BSA were incubated with 2 ␮g/ml FITC-conjugated anti- mouse CD16/CD32 or FITC-conjugated anti-mouse IgG2b isotype Rac2 and p47phox Abs were gifts from G. Bokoch and U. Knaus (The (PharMingen) for 30 min on ice. After washing cells twice with PBS con- Scripps Research Institute, La Jolla, CA) and D. Lambeth and D. Uhlinger taining 0.1% BSA, flow cytometric analysis was performed using a FAC- (Emory University, Atlanta, GA), respectively. Rabbit polyclonal Abs Scan (Becton Dickinson). A minimum of 10,000 neutrophil events was against p42/44, Thr202/Tyr204-phosphorylated p42/44, Thr183/Tyr185-phos- recorded and analyzed using CellQuest (Becton Dickinson). phorylated c-Jun N-terminal kinase (JNK)/SARK, p38 MAP kinase, Thr180/Tyr182-phosphorylated p38 MAP kinase, Akt, and Ser473-phosphor- Opsonization of zymosan and preparation of IgG-sensitized ylated Akt were purchased from New England Biolabs (Beverly, MA). SRBC Mouse mAbs against Rac1 and phosphotyrosine (4G10) were purchased from Upstate Biotechnology (Lake Placid, NY), and those for JNK1/2 and Zymosan particles were prepared as described previously (32), opsonized CD16/CD32 were purchased from PharMingen (San Diego, CA). Highly with normal human serum for1hat37°C, washed with PBS, and then purified recombinant human Rac1 and Rac2 were provided by D. Lambeth. diluted in PBS. Ab-coated SRBC (IgG-SRBC) were freshly prepared in Chemicals were purchased from Sigma (St. Louis, MO) unless otherwise magnesium- and calcium-free PBS by incubating 109/ml SRBC (Cappel; stated. PBS (pH 7.2), HBSS (without Ca2ϩ,Mg2ϩ, and phenol red), and ICN Biomedicals, Costa Mesa, CA) with an equal volume of 2 ␮g/ml IgG HBSS with 1.26 mM Ca2ϩ and 0.4 mM Mg2ϩ were purchased from Life rabbit-anti-SRBC Ab (Cappel) as previously described (33). Technologies (Grand Island, NY). Other buffers used in this study include HBSS with 0.1% BSA and 1% glucose (pH 7.25–7.4) (HBSS-BG); PBS Immunoblot analysis of Rac1 and Rac2 expression with 0.9 mM CaCl2, 0.5 mM MgCl2, and 7.5 mM glucose (PBSG); and Lysates of mouse BM neutrophils and human peripheral blood neutrophils lysis buffer (20 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% were prepared, and protein was measured with the bicinchoninic acid pro- Triton X-100, 50 mM NaF, 2 mM Na3VO4, 0.01 mM phenylarsine oxide, ␮ ␮ tein kit from Pierce (Rockford, IL) using BSA as a standard. Lysates were 20 g/ml chymostatin, 2 mM PMSF, 10 M leupeptin, and 1 mM 4-(2- subjected to SDS-PAGE and immunoblotting as previously described (11, aminoethyl)benzenesulfonyl fluoride). 34). Blots were probed with either mouse mAb for Rac1 or a rabbit poly- clonal Ab for Rac2 and developed with the ECL method (Amersham, Ar-

3 lington Heights, IL) as previously described (35). The Ab specificity of the Abbreviations used in this paper: BM, bone marrow; MAP, mitogen-activated pro- Rac1 and Rac2 Abs was confirmed by immunoblotting Escherichia coli- tein; JNK, c-Jun N-terminal kinase; HBSS-BG, HBSS with 0.1% BSA and 1% glu- produced human recombinant Rac1 and Rac2, supplied by D. Lambeth cose (pH 7.25–7.4); PBSG, PBS with 0.9 mM CaCl2, 0.5 mM MgCl2, and 7.5 mM glucose; OpZ, opsonized zymosan; IgG-SRBC, Ab-coated SRBC; NBT, nitroblue (Emory University). Integrated densitometry was employed to measure the tetrazolium; MEK-1, MAP kinase kinase-1; ERK, extracellular signal-regulated ki- relative intensity of signals using an Eagle Eye II Still Video System and nase; PI3K, phosphoinositide-3 kinase; DAG, diacylglycerol. associated software (Stratagene, La Jolla, CA). The Journal of Immunology 1225

Measurement of NADPH oxidase activity Superoxide production was measured in a quantitative kinetic assay based on the reduction of cytochrome c. The assay was performed at 37°C using a Thermomax microplate reader and associated SOFTMAX version 2.02 software (Molecular Devices, Menlo Park, CA) as reported previously (34, 35). Briefly, for the soluble stimuli, cells were suspended (2.5 ϫ 105 cells/ well) in 250 ␮l of PBSG containing 75 ␮M ferricytochrome c and activated by the addition of 200 ng/ml PMA or 10 ␮M fMLP. For opsonized particle stimulation, cells were preincubated for 30 min at 37°C with 100 ␮g/ml opsonized zymosan (OpZ) or a 10-fold excess of IgG-SRBC (2.5 ϫ 106 IgG-SRBC in 0.25 ml for 2.5 ϫ 105 BM neutrophils) before adding PBSG containing ferricytochrome c.Fc␥R-activated oxidant production in BM neutrophils was also measured using the Fc OxyBurst reagent (Molecular Probes, Eugene, OR), according to the manufacturer’s instructions. Briefly, FIGURE 1. Expression of Rac1 and Rac2 in human and murine neu- 6 BM neutrophils were suspended at 2 ϫ 10 /ml in PBSG and then stimu- trophils. The indicated amounts (5, 10, and 20 ␮g) of whole-cell lysates ␮ lated with Fc OxybBurst at a final concentration of 140 g/ml. Flow cy- from human and murine neutrophils were separated by 12% SDS-PAGE, tometric analysis was performed using a FACScan (Becton Dickinson), transferred to nitrocellulose, and probed with either a mAb specific for and data were analyzed using CellQuest. For measurement of nitroblue Rac1 or a polyclonal rabbit Ab specific for Rac2. To confirm equal sample tetrazolium (NBT) reduction, neutrophils suspended in IMDM were placed phox a glass chamber slide (Nunc, Naperville, IL) for 1 h before activation with loading, blots were reprobed with Ab against p47 . Band intensities different stimuli. After an additional incubation for 20 min at 37°C, slides were determined by densitometry (Eagle Eye II). The data shown are rep- were fixed and counterstained with safranin, and the percentage of NBT- resentative of four independent experiments. positive neutrophils and the intensity of staining were determined by eval- uating 100 randomly selected cells (34).

MAP kinase activation peroxide production in response to fMLP was ϳ10-fold higher in Ϯ To examine the phosphorylation of MAP kinases, paired samples of wild- freshly isolated wild-type BM neutrophils (2.5 2.3 nmol/min/ Ϫ Ϫ type and rac2Ϫ/Ϫ BM neutrophils were suspended in HBSS (1 ϫ 107 107 cells) than in those isolated from rac2 / mice (0.2 Ϯ 0.7 cells/ml) and stimulated for varying periods of time with 200 ng/ml PMA, nmol/min/107 cells; Fig. 2A). BM neutrophils were also cultured ␮ ␮ 10 M fMLP, 100 g/ml OpZ, or a 10-fold excess of IgG-SRBC at 37°C. with 1 ng/ml IL-3 for 24 h at 37°C to enhance responsiveness to The activation was terminated by addition of cold PBS and placement on ice. Thereafter, cells (1 ϫ 107 cells/ml) were pelleted and lysed in 200 ␮l fMLP stimulation (36, 37) (Fig. 2B). fMLP-elicited superoxide of lysis buffer at 4°C for 30 min. Cell lysates were clarified by centrifuging production by IL-3-treated wild-type BM neutrophils increased for 2 min at 18,000 ϫ g at 4°C. Samples then were heated at 95°C for 5 ϳ2-fold compared with that by freshly isolated BM neutrophils min with 0.2 vol of 5ϫ Laemmli sample buffer and 10 ␮g of cell lysate (10 ( p Ͻ 0.05), but was still virtually undetectable in rac2Ϫ/Ϫ cells. ␮ l for IgG-SRBC samples) resolved on 10% SDS-PAGE. Immunoblot Consistent with previous observations (11), PMA-elicited NADPH analysis was conducted as described above. Expression of total p42/44, p38, JNK, and Akt was detected using rabbit polyclonal Abs against p42/ oxidase activity was reduced by about 4-fold in freshly isolated Ϫ Ϫ 44, p38, and Akt and a mouse mAb against JNK1/2. Activation of p42/44, rac2 / BM neutrophils (3.6 Ϯ 1.2 nmol/min/107 cells) compared p38, JNK1/2 MAP kinases, and Akt and tyrosine phosphorylation were with wild-type neutrophils (17.3 Ϯ 3.8 nmol/min/107 cells) (Fig. determined using phospho-specific p42/p44, p38, JNK, Akt, and tyrosine 2A). Culture in IL-3 resulted in a modest increase in PMA-elicited Abs. Integrated densitometry was employed to measure the relative inten- Ϯ sity of the protein signal using an Eagle Eye II Still Video System. superoxide production by BM neutrophils (22.5 6.5 nmol/min/ 107 cells), although this was still significantly reduced in cells MAP kinase inhibitors isolated from rac2Ϫ/Ϫ mice (9.6 Ϯ 3.2 nmol/min/107 cells). To investigate whether MAP kinase inhibitors affect superoxide production Activation of NADPH oxidase in response to opsonized parti- Ϫ Ϫ in murine BM neutrophils, the p38 MAP kinase inhibitor, SB203580 (Cal- cles was also evaluated in wild-type and rac2 / BM neutrophils biochem, La Jolla, CA) and the MAP kinase/extracellular signal-regulated (Fig. 3). Superoxide production elicited by IgG-SRBC was re- kinase (ERK) kinase (MEK) inhibitor, PD98059 (Calbiochem), were used. duced by ϳ5-fold in rac2Ϫ/Ϫ BM neutrophils compared with wild- BM neutrophils were incubated with SB203580 (1, 5, and 10 ␮M) or Ϯ 7 PD98059 (5, 25, and 50 ␮M) for 30 min at 37°C. Following incubation, type neutrophils (wild-type, 6.9 2.5 nmol/min/10 cells; Ϫ/Ϫ 7 cells were analyzed for cytochrome c superoxide production using the re- rac2 , 1.5 Ϯ 2.4 nmol/min/10 cells). The expression of Fc␥R, duction assay and for MAP kinase activation in response to various stimuli as measured by flow cytometry using an Ab against Fc␥ III/II as described above. receptor (CD16/CD32), was similar for both wild-type and rac2Ϫ/Ϫ genotypes (data not shown). This suggests that the sig- Results nificant impairment in activation of the NADPH oxidase by IgG- Expression of Rac1 and Rac2 in mouse and human neutrophils Ϫ Ϫ SRBC in rac2 / BM neutrophils does not reflect reduced expres- It has previously been shown that Rac2 is the predominant Rac sion of Fc␥ receptors. In contrast, activation of the respiratory ␤ isoform in human neutrophils (22). We compared the expression of burst by serum OpZ, which can also interact with the 2 integrin Rac1 and Rac2 in mouse BM neutrophils to those in human neu- CR3 receptor for activated complement protein C3bi, was pre- trophils by immunoblotting with specific Abs for Rac1 and Rac2. served in rac2Ϫ/Ϫ BM neutrophils. There was no difference in At least three to five times more Rac1 was expressed in mouse OpZ-induced superoxide release between genotypes (wild-type, neutrophils on a milligram protein basis compared with human 7.3 Ϯ 3.0 nmol/min/107 cells; rac2Ϫ/Ϫ, 6.0 Ϯ 2.2 nmol/min/107 neutrophils, whereas similar levels of Rac2 were detected in mouse cells; Fig. 3). and human neutrophils (Fig. 1). As previously reported (11), the We next examined receptor-mediated activation of superoxide expression of Rac1 in murine rac2Ϫ/Ϫ neutrophils was similar to production in peritoneal exudate neutrophils from wild-type and that in wild-type neutrophils. rac2Ϫ/Ϫ mice. NADPH oxidase activity in response to IgG-SRBC was substantially reduced in rac2Ϫ/Ϫ peritoneal exudate neutro- Receptor-mediated activation of neutrophil NADPH oxidase in Ϯ 7 Ϫ/Ϫ Ϯ Ϫ/Ϫ phils (wild type, 5.04 1.52 nmol/min/10 cells; rac2 , 0.08 rac2 mice 0.08 nmol/min/107 cells), while it was normal in response to OpZ To examine the activation of the NADPH oxidase by physiological (wild type, 5.0 Ϯ 1.8 nmol/min/107 cells; rac2Ϫ/Ϫ, 5.0 Ϯ 2.1 agonists, three different receptor-mediated stimuli were tested. Su- nmol/min/107 cells; Fig. 4). Stimulation with fMLP did not induce 1226 SELECTIVE REGULATION OF NEUTROPHIL NADPH OXIDASE BY Rac2

FIGURE 2. Activation of NADPH oxidase by fMLP receptor in BM neutrophils. f, Wild type; Ⅺ, rac2Ϫ/Ϫ. Data are expressed as the mean Ϯ p Ͻ 0.001, wild type vs rac2Ϫ/Ϫ (unpaired t test). A, Superoxide ,ء .SD production by freshly isolated BM neutrophils following stimulation with 200 ng/ml PMA or 10 ␮M fMLP was monitored by reduction of ferricy- tochrome c (n ϭ 8). B, Superoxide production by BM neutrophils cultured with 1 ng/ml IL-3 for 24 h at 37°C before stimulation with either PMA or fMLP (n ϭ 11). sufficient superoxide production that could be detected in the cy- tochrome c assay in either wild-type or rac2Ϫ/Ϫ peritoneal exudate cells (data not shown). In contrast to our initial observation that PMA-activated superoxide production was not significantly re- duced in rac2Ϫ/Ϫ peritoneal exudate neutrophils (11), we found an ϳ2-fold reduction in rac2Ϫ/Ϫ peritoneal exudate neutrophils com- pared with wild-type (wild type, 14.9 Ϯ 2.4 nmol/min/107 cells; rac2Ϫ/Ϫ, 7.7 Ϯ 2.8 nmol/min/107 cells; p Ͻ 0.01). This may re- flect differences in the mouse genetic background. In the original ϫ study, we used C57BL/6J 129Sv F2,F3, and F4 littermate mice, FIGURE 3. Activation of BM neutrophil NADPH oxidase by opsonized whereas in the current study, mice were in the C57BL/6J particles. A, BM neutrophils were preincubated with 100 ␮g/ml OpZ (n ϭ background. 9) or 10ϫ IgG-SRBC (n ϭ 7) for 30 min at 37°C before adding ferricy- As another approach to evaluating NADPH oxidase activity in tochrome c to monitor superoxide production. f, Wild type; Ⅺ, rac2Ϫ/Ϫ. p Ͻ 0.001, wild type vs rac2Ϫ/Ϫ ,ء .BM and peritoneal exudate neutrophils, we used the NBT test, a Data are expressed as the mean Ϯ SD sensitive semiquantitative assay for monitoring superoxide pro- (unpaired t test). B, Representative FACScan of BM neutrophils incubated duction in individual cells. In this assay NADPH oxidase is acti- with Fc OxyBurst at 37°C. The shaded area shows fluorescence obtained Ϫ/Ϫ vated in the presence of NBT, which forms an insoluble purple immediately after mixing, which was the same for wild-type and rac2 mice. The open curves show fluorescence after 30 min of incubation, with precipitate when reduced by superoxide. The relative numbers of the dark line indicating fluorescence of wild-type neutrophils, and the thin- NBT-positive cells in response to PMA and OpZ were similar Ϫ/Ϫ Ϫ/Ϫ ner line indicating fluorescence for rac2 neutrophils. C, The mean cel- between wild-type and rac2 BM and peritoneal exudate neu- lular fluorescence for the highly fluorescent subpopulation of Fc OxyBurst- trophils (Table I). However, the relative numbers of NBT-positive activated neutrophils (see B) is shown after either 20 (n ϭ 4–5) or 30 (n ϭ p ϭ ,ء .cells detected upon activation with either fMLP or IgG-SRBC 3–5) min of incubation, as indicated. f, Wild type; Ⅺ, rac2Ϫ/Ϫ Ϫ Ϫ .p ϭ 0.011 ,ءء ;were significantly decreased in rac2 / cells (Table I). Activation 0.022 The Journal of Immunology 1227

Table I. NADPH oxidase activity in BM and exudate neutrophilsa

Peritoneal Exudate BM Neutrophils Neutrophils

Wild type rac2Ϫ/Ϫ Wild type rac2Ϫ/Ϫ

No stimulus 10 Ϯ 52Ϯ 213Ϯ 86Ϯ 5 PMA 81 Ϯ 587Ϯ 378Ϯ 673Ϯ 4 FMLP 59 Ϯ 10 7 Ϯ 7* 42 Ϯ 10 10 Ϯ 6* OpZ 61 Ϯ 655Ϯ 14 63 Ϯ 11 54 Ϯ 14 IgG-SRBC 38 Ϯ 314Ϯ 1** 24 Ϯ 98Ϯ 4**

a NADPH oxidase activity was evaluated in individual neutrophils using the NBT test. Shown are the percentages of NBT-positive cell (mean Ϯ SD, n ϭ 3–4). .(p Ͻ 0.05 wild type vs rac2Ϫ/Ϫ (unpaired t test ,ءء ;p Ͻ 0.001 ,ء

that decreased superoxide production by Fc␥R-activated rac2Ϫ/Ϫ FIGURE 4. Activation of peritoneal exudate neutrophil NADPH oxi- neutrophils is due to both a decrease in cellular oxidant production dase. f, Wild type; Ⅺ, rac2Ϫ/Ϫ. Superoxide production was measured in as well as a smaller proportion of responding cells. peritoneal exudate neutrophils harvested 18 h following i.p. instillation of ϭ 3% thioglycolate. Cells were stimulated with 200 ng/ml PMA (n 8), 100 Activation of intracellular kinases in activated murine ␮g/ml OpZ (n ϭ 8–9), or 10ϫ IgG-SRBC (n ϭ 4) as described in the neutrophils ;p Ͻ 0.01 ,ء .legends to Figs. 2 and 3. Data are expressed as the mean Ϯ SD p Ͻ 0.05 (wild type vs rac2Ϫ/Ϫ, unpaired t test). We previously found that phosphorylation of ERK1/2 and p38 ,ءء MAP kinases induced by fMLP was diminished in rac2Ϫ/Ϫ BM neutrophils (11). To expand on these initial observations, we com- with either PMA or OpZ produced intense cell-associated pared chemoattractant-activated MAP kinase activation in mouse formazan deposits in both wild-type and rac2Ϫ/Ϫ neutrophils, BM neutrophils to stimulation by PMA, OpZ, or IgG-SRBC, using which were concentrated in the phagosome in the case of OpZ- Abs to detect the corresponding phosphorylated form of the protein activated neutrophils, whereas fMLP or IgG-SRBC stimulation (Fig. 5). Preliminary studies (see also Fig. 6A) determined that PMA- produced less intense formazan staining of cells (data not shown). induced phosphorylation of ERK1/2 and p38 was initiated within 2 Phagocytosis of serum-opsonized zymosan particles was equiva- min, which increased further by 10 min. fMLP-induced phosphory- lent in wild-type and rac2Ϫ/Ϫ neutrophils. The number of particles lation of ERK1/2 and p38 peaked at 2 min, with phosphorylated ingested per 100 neutrophils was 131 Ϯ 19 for wild-type and ERK1/2 declining by 5 min (data not shown). Phosphorylated p38 137 Ϯ 26 for rac2Ϫ/Ϫ (n ϭ 5 for both genotypes). Ingestion of was detected 5 min after the addition of IgG-SRBC and persisted IgG-SRBC was rarely seen for either wild-type or rac2Ϫ/Ϫ neu- for at least 60 min; we did not detect ERK1/2 phosphorylation in trophils, perhaps because the rabbit IgG used for opsonization is a response to IgG-SRBC over this same period (data not shown). In poor stimulus inducing phagocytosis in murine neutrophils under subsequent studies, we examined agonist-induced MAP kinase our conditions. protein phosphorylation at 2 min for fMLP, 10 min for PMA, and The differences seen between wild-type and rac2Ϫ/Ϫ neutrophils 30 min for opsonized particles. As shown in the representative in the NBT assay for superoxide production are overall consistent blots in Fig. 5, phosphorylation of ERK1/2 induced by either Ϫ Ϫ with the results from the cytochrome c assay, except for the re- fMLP or PMA was markedly reduced in rac2 / BM neutrophils sponse to PMA. However, we have previously observed that in- compared with wild-type neutrophils. Only a small decrease in p38 tense NBT reduction can be observed when NADPH oxidase ac- phosphorylation was typically seen for fMLP or PMA-activated Ϫ Ϫ tivity is only 20–30% of wild-type levels (34, 38). This could rac2 / BM neutrophils compared with wild-type cells, although account for the similar percentage of NBT-positive cells for this difference was more prominent in some experiments (compare rac2Ϫ/Ϫ and wild-type PMA-activated neutrophils despite a re- Figs. 5 and 6A). Activation of p38 by either OpZ or IgG-SRBC Ϫ Ϫ duced amount of superoxide release by rac2Ϫ/Ϫ cells. was similar in wild-type and rac2 / cells. Phosphorylation of The decreased fraction of NBT-positive neutrophils in rac2Ϫ/Ϫ JNK was also detectable after stimulation with either PMA or mice in response to fMLP or IgG-SRBC also indicates that re- fMLP, which was consistently decreased in fMLP-activated Ϫ Ϫ duced superoxide release measured in the cytochrome c assay rac2 / cells. Previous studies have suggested that Rac is down- could reflect either fewer numbers of activated rac2Ϫ/Ϫ neutro- stream of phosphoinositide-3 kinase (PI3K) activation induced by Ϫ Ϫ phils and/or decreased neutrophil NADPH oxidase activity. To ex- chemoattractants and other signals (39, 40). In rac2 / mast cells, amine this question directly for Fc␥R-elicited NADPH oxidase PI3K activation by stem cell factor is normal, but phosphorylation activity, we used the Fc OxyBurst reagent to monitor oxidant pro- of PI3-dependent kinase Akt (41) is decreased (29). Phosphoryla- Ϫ Ϫ duction by flow cytometry. As shown in Fig. 3B, incubation with tion of Akt was not decreased in rac2 / BM neutrophils stimu- Fc Oxyburst produced a shift in fluorescence, corresponding to lated with either PMA or fMLP, and actually appeared to be in- Ϫ Ϫ oxidant production, in a subpopulation of BM neutrophils. The creased in fMLP-treated rac2 / cells compared with wild-type shift in fluorescence was significantly decreased in rac2Ϫ/Ϫ neu- cells (Fig. 5), confirming our initial observations of fMLP-acti- trophils compared with wild-type cells (666 Ϯ 76 vs 1753 Ϯ 501 vated neutrophils (11). Under the conditions used, Akt phosphor- U after 30 min; n ϭ 3–5; p ϭ 0.011; Fig. 3C). The relative number ylation was not observed in response to OpZ or IgG-SRBC. of responding neutrophils was also decreased in rac2Ϫ/Ϫ mice, with 27 Ϯ 9% of rac2Ϫ/Ϫ cells exhibiting a shift in fluorescence by Effect of MAP kinase inhibitors on superoxide production 30 min compared with 48 Ϯ 25% for wild-type cells, although this We next investigated whether the decreased phosphorylation of difference was not statistically significant. These results suggest ERK1/2 or p38 observed in rac2Ϫ/Ϫ BM neutrophils activated by 1228 SELECTIVE REGULATION OF NEUTROPHIL NADPH OXIDASE BY Rac2

FIGURE 5. Intracellular kinase activation in wild-type and rac2Ϫ/Ϫ BM neutrophils. Ten micrograms of cell lysate (or for the IgG-SRBC sample, 15 ␮l; 7.5 ϫ 105 cell equivalents) was loaded and separated by 10% SDS-PAGE. A representative blot and arbitrary density from one of three independent experiments are shown. A, Activation of MAP kinases and Akt. Blot was probed for phosphorylated and total ERK1/2, p38, JNK MAP kinase, and Akt in cell lysates of BM neutrophils stimulated with 200 ng/ml PMA (10 min), 10 ␮M fMLP (2 min), 100 ␮g/ml OpZ (30 min), and 10ϫ IgG-SRBC (30 min) at 37°C. The positions of the phosphorylated kinases are indicated by the solid line. B, Blots quantified by densitometry, as represented by arbitrary density units. Note that arbitrary density units for phospho-ERK 1/2 are shown on a logarithmic scale. fMLP or PMA was functionally linked to NADPH oxidase acti- vivo by baseline neutrophilia, reduced inflammatory peritoneal ex- vation. Studies in human neutrophils have found variable results as udate formation, and increased mortality infected with Asperigillus to whether NADPH oxidase activity can be suppressed by inhib- fumigatus (11). The importance of Rac2 in regulating neutrophil iting phosphorylation of either ERK1/2 using the MEK-1 inhibitor function has also been highlighted by the recent discovery of a new PD098059 or of p38 using SB203580 (42–47). In initial studies genetic immunodeficiency syndrome in humans due to a dominant we established that phosphorylation of ERK1/2 or p38 could be negative mutation of Rac2 (48, 49). Neutrophils isolated from the effectively inhibited by the corresponding inhibitor (Fig. 6A). affected patient exhibited functional abnormalities similar to those However, PD098059 or SB203580 did not significantly inhibit seen in rac2Ϫ/Ϫ neutrophils, with defects in chemotaxis and NADPH PMA-elicited superoxide production in either wild-type or oxidase activation in response to some, but not all, agonists along with Ϫ/Ϫ rac2 BM neutrophils, either when given alone (Fig. 6B)orin diminished L-selectin-mediated adhesion. Chemoattractant-in- combination (data not shown). fMLP-induced superoxide produc- duced superoxide production, chemotaxis, and degranulation were tion was diminished by about 30% in wild-type BM neutrophils markedly deficient (48, 49) as was fMLP-primed phagocytosis of treated with either inhibitor, although this reached statistical sig- IgG-SRBC (48). However, only a small decrease was observed in nificance only for PD098059 (Fig. 6C). The residual superoxide- Ϫ Ϫ the phorbol ester-activated respiratory burst (48, 49). generating activity in fMLP-activated rac2 / BM neutrophils In the current study, we examined receptor-mediated activation was also partially sensitive to inhibition by PD098059 or Ϫ/Ϫ SB203580. These results suggest that the effect of Rac2 deficiency of the NADPH oxidase in rac2 neutrophils to further charac- on activation of the NADPH oxidase in murine neutrophils may be terize the role of Rac2 in the regulation of neutrophil superoxide mediated largely through non-MAP kinase-dependent pathways. production. NADPH oxidase activity was normal in opsonized zy- mosan-activated rac2Ϫ/Ϫ neutrophils, but was deficient in re- Discussion sponse to fMLP or to IgG-opsonized particles despite the expres- Genetic deficiency of Rac2, a hematopoietic-specific Rho GTPase, sion of Rac1 at substantially higher levels compared with human is associated with a variety of functional defects in murine BM- neutrophils. The agonist-specific defects observed in activation of Ϫ/Ϫ derived cells due to abnormalities in intracellular signaling path- the respiratory burst in murine rac2 neutrophils are consistent ways (11, 29, 30) despite the presence of the highly homologous with those reported for human neutrophils expressing a dominant Rac1 (Fig. 1) and, probably, Rac3 (18). In neutrophils, the absence negative Rac2 mutant. We had previously observed that the im- of Rac2 was associated with significant defects in chemotaxis, L- paired phorbol ester-induced activation of superoxide production selectin-mediated rolling, F-actin generation, and phorbol ester- in rac2Ϫ/Ϫ neutrophils could be partly surmounted by in vivo or in induced superoxide production. These defects were reflected in vitro priming with inflammatory cytokines (11). Taken together, The Journal of Immunology 1229

FIGURE 6. Effect of p38 inhibitor SB203580 and MEK inhibitor PD98059 on MAP kinase activation and NADPH oxidase activity. BM neutrophils were incubated for 30 min at 37°C and in some cases treated with 10 ␮M SB203580 or 50 ␮M PD098059 before stimulation with either 200 ng/ml PMA or 10 ␮M fMLP. Inhibitors were present throughout the assay. A, Blots were probed with either phosphorylated p42/p44 Ab or phosphorylated p38 Ab and were reprobed with an Ab for p38 MAP kinase to monitor protein loading. The results are one of two to four experiments. B, Effects of p38 and MEK p Ͻ 0.001, significant difference wild type vs rac2Ϫ/Ϫ ,ء .(inhibitors on PMA-elicited superoxide production. Data expressed as the mean Ϯ SD (n ϭ 4 p Ͻ ,ء .(paired t test). C, Effects of p38 and MEK inhibitors on fMLP-elicited superoxide production. Data are expressed as the mean Ϯ SD (n ϭ 4–5) .(p Ͻ 0.05, no inhibitors vs SB203580 or PD098059 (paired t test ,ءء ;significant difference wild type vs rac2Ϫ/Ϫ ,0.001 these data show that the requirement for Rac2 in superoxide pro- Why Rac1 is unable to substitute for Rac2 in superoxide pro- duction is not absolute and suggest that specific signaling path- duction, chemotaxis, and other functional responses that are im- ways leading to activation of the neutrophil respiratory burst are paired in Rac2-deficient blood cells also remains to be defined. selectively regulated by Rac2. These two isoforms may be selectively activated by specific ex- There are at least two mechanisms by which the absence of change factors or interact differentially with downstream effectors. Rac2 could lead to defects in neutrophil superoxide production. For example, the Rac-GEF Tiam-1 (51) is a strong inducer of Rac is a component of the enzymatically active NADPH oxidase p21-activated kinase 1 activation, but not of JNK (52), two kinases complex, and either Rac1 or Rac2 is required for enzyme activity that are both strongly activated by Rac-GTP. This selectivity could in recombinant cell-free oxidase assays. The GTP-bound form of arise either through differences in subcellular localization or in Rac can bind directly to p67phox and to at least one other site in the relative affinities for different Rac isoforms with interacting pro- NADPH oxidase (5, 19, 50). Decreased respiratory burst activity in teins. For example, Rac1 has been shown to bind to and stimulate rac2Ϫ/Ϫ neutrophils might reflect a preferred role for Rac2 due p21-activated kinase 1 activity more efficiently that Rac2 (53). In either to increased affinity to one or more other subunits of the addition, studies using the yeast two-hybrid system have suggested NADPH oxidase and subsequent enhanced enzymatic activity or to that the affinity of Rac2 for p67phox is higher than that for Rac1 differences in activation or subcellular localization of Rac2 com- (19), and Rac2 is more active than Rac1 in stimulating NADPH pared with Rac1. Alternatively, Rac2 deficiency may lead to sig- oxidase activity in the presence of neutrophil cytosol (28). Another naling abnormalities that affect phosphorylation of p47phox and potential mechanism for functional defects in rac2Ϫ/Ϫ cells is that subsequent translocation of p47phox/p67phox/p40phox and/or recruit- the overall level of Rac-GTP might be a rate-limiting step in the ment of Rac-GTP to the enzyme complex. At the current time it is activation of downstream functions, and signaling cascades trig- unknown whether the predominant effect of Rac2 deficiency on the gered by different neutrophil agonists may vary in the amount of respiratory burst reflects its function in the NADPH oxidase com- activated Rac generated. plex itself or in the preceding steps leading to enzyme assembly. Rac has been implicated as a participant in many of the multiple However, since multiple functional abnormalities in other BM- signaling pathways that are triggered upon neutrophil activation by derived cells have been identified in rac2Ϫ/Ϫ mice (11, 29, 30), we soluble or particulate agonists (1, 39, 54, 55). How these complex speculate that defects in intracellular signaling pathways account and often redundant signals are ultimately linked to specific func- for at least some of the observed decrease in superoxide production tional responses, such as activation of the otherwise dormant in response to specific agonists. NADPH oxidase, remains incompletely understood. However, 1230 SELECTIVE REGULATION OF NEUTROPHIL NADPH OXIDASE BY Rac2 from studies using biochemical, pharmacologic, or genetic ap- PKC-␤ (68). In either case, an ϳ2-fold reduction in neutrophil proaches, it is clear that different agonists activate both overlap- superoxide production elicited by IgG-coated particles or PMA ping and distinct signaling cascades (1, 39, 54, 56–59) that could was observed. Inhibition of PKC-␤ expression in HL-60 cells us- potentially lead to selective activation of Rac2 and the respiratory ing antisense oligonucleotides also resulted in decreased NADPH burst. oxidase activity (69). The deficient respiratory burst in rac2Ϫ/Ϫ Signaling defects downstream of -cou- neutrophils in response to PMA, fMLP, or IgG-opsonized particles pled receptors for chemoattractants are a prominent feature of may reflect a common defect in Rac2-dependent events down- Rac2 deficiency. We previously reported that that chemotaxis and stream of PKC that promote oxidase assembly. F-actin polymerization in response to fMLP or leukotriene B4 was We hypothesized that the differential activation of MAP kinase substantially diminished in rac2Ϫ/Ϫ neutrophils (11) and now cascades might explain the agonist-dependent effects on neutrophil show that fMLP-induced activation of superoxide production also NADPH oxidase activation in murine rac2Ϫ/Ϫ neutrophils. MAP significantly impaired. These features of the rac2Ϫ/Ϫ phenotype kinase activation is a prominent response in human neutrophils have some overlap with mice deficient in the p110 catalytic sub- activated by either soluble agonists or opsonized particles (54, 70, unit of PI3K␥ (57–59). PI3K␥-deficient leukocytes are unable to 71). ERK2/1 (p42/44) MAP kinase is activated via the Ras/Raf generate phosphatidyl-inositol 3,4,5-trisphosphate upon G protein- pathway, although cross-regulation with Rac-dependent pathways coupled receptor stimulation, in association with deficient che- can also occur (72, 73). Many groups have reported that JNK and moattractant-induced chemotaxis and NADPH oxidase activity. p38 MAP kinase pathways are downstream of Rac and Cdc42 in fMLP-induced Rac activation was reported to be unaffected in various cell types, suggesting an analogous role to that played by PI3K␥-deficient mice (58). This suggests that PI3K␥-dependent Ras in the ERK MAP kinase cascade (12, 13, 17, 74, 75). signaling and a parallel pathway(s) linked to Rac2 activation are We found that both fMLP and PMA induced phosphorylation of both required for inducing a respiratory burst by fMLP and related ERK1/2 and p38 in murine BM neutrophils, which was decreased agonists. in rac2Ϫ/Ϫ cells compared with wild-type cells, particularly for The rac2Ϫ/Ϫ neutrophils also exhibited a defect in NADPH ox- ERK1/2. However, activation of these MAP kinases appears to be idase activation upon activation through Fc␥R using Ig-opsonized only in part functionally linked to downstream activation of su- particles, but not with zymosan opsonized with serum opsonins peroxide production. Pharmacologic inhibition of either ERK1/2 ␤ (C3bi, IgG), which can bind to both the 2 integrin receptor CR3 or p38 phosphorylation had no significant effect on PMA-induced and Fc␥R (60–64). This is the first direct evidence that Rac2 plays superoxide production in wild-type BM neutrophils. Hence, al- an important role in regulating the Fc␥R-dependent respiratory though PMA activated both ERK1/2 and p38 in murine BM neu- burst in neutrophils, which is likely to occur downstream of the trophils in a Rac2-dependent manner, these events do not play a activation of receptor-associated tyrosine kinases, including Syk role in NADPH oxidase activation and are unlikely to account for and Hck, that are prominent proximal events upon Fc␥R ligation the defect in superoxide generation in PMA-activated rac2Ϫ/Ϫ (65). Our data also indicate that signals induced either downstream neutrophils. These results are generally consistent with observa- of the CR3 receptor or through CR3 costimulation of Fc␥Rs (66) tions in human neutrophils, where inhibitors of either ERK1/2 or are sufficient to activate NADPH oxidase independently of Rac2 in p38 have had variable results on PMA-induced superoxide pro- murine neutrophils. Signaling through Rho GTPases has previ- duction (42, 43, 45–47). With regard to the fMLP-activated respi- ously been shown to play an important role in the phagocytosis of ratory burst, inhibition of ERK1/2 has also had inconsistent effects opsonized particles by macrophages, and different Rho family in human neutrophils (43–45, 76), whereas at least a partial de- members appear to play distinctive roles in this process (10, 65, crease in NADPH oxidase activity has been reported in the pres- 67). In macrophages, Cdc42 and Rac regulate uptake of Ig-opso- ence of p38 inhibitors (42, 43, 47). In fMLP-activated wild-type nized particles via Fc␥R, whereas Rho plays a prominent role in murine BM neutrophils, we found that inhibition of either ERK1/2 CR3-mediated phagocytosis (67). Our findings that superoxide or p38 produced only an ϳ30% decrease in NADPH oxidase ac- production in rac2Ϫ/Ϫ neutrophils is impaired in response to Fc␥R tivity. The residual respiratory burst detected in fMLP-stimulated ligation but not when CR3 binding is also involved are consistent rac2Ϫ/Ϫ BM neutrophils exhibited a similar partial sensitivity to with these observations. p38 and ERK1/2 inhibitors. These data suggest that events down- Although PMA is not a physiologic stimulus for neutrophil ac- stream of ERK1/2 and p38 regulate the fMLP-activated respiratory tivation, it is noteworthy that PMA-elicited superoxide production burst in murine BM neutrophils, but that the absence of Rac2 is was substantially decreased in rac2Ϫ/Ϫ neutrophils. PMA is a di- likely to affect additional or redundant fMLP-induced signaling rect activator of protein kinase C (PKC) and is one the most potent pathways important for NADPH oxidase activation. In contrast to activators of the neutrophil respiratory burst. Neutrophil activation studies on human neutrophils (76–78) or murine macrophages (79, by most physiologic agonists is accompanied by activation of 80), we were unable to detect phosphorylated ERK1/2 in murine PKC, and specific inhibitors of PKC decrease NADPH oxidase BM neutrophils stimulated with opsonized particles. However, p38 activity (reviewed in Refs. 54 and 55). An early event downstream MAP kinase was activated with either OpZ or IgG-SRBC, but to of agonist binding to chemoattractant or Fc␥ receptors is the ac- a similar extent in wild-type and rac2Ϫ/Ϫ cells. Therefore, al- tivation of phospholipase C, which catalyzes the formation of di- though IgG-SRBC-induced superoxide release was markedly de- acylglycerol (DAG) and inositol 1,4,5-trisphosphate by hydrolysis creased in rac2Ϫ/Ϫ neutrophils, this effect appears to mediated by of membrane phospholipids (54). In turn, DAG directly activates downstream pathways unrelated to p38. PKC, and inositol 1,4,5-trisphosphate promotes the release of in- We also observed activation of JNK in murine BM neutrophils tracellular calcium, which is important for activation of many PKC in response to either fMLP or PMA, which was decreased in isoforms, including the PKC-␤ and -␦ species present in neutro- fMLP-activated rac2Ϫ/Ϫ neutrophils. Our data agree with previous phils (54, 55). DAG can also be generated through a parallel path- reports that formyl receptors in neutrophils can couple to the JNK way involving activation of phospholipase D. Direct evidence for pathway (44, 58) and are also consistent with the important role for PKC-␤ regulation of NADPH oxidase activity was found in a re- Rac/Cdc42 in pathways leading to JNK activation (12, 13). The cent study in which PKC-␤ activity was ablated either using a failure to detect neutrophil JNK activation in response to fMLP in specific inhibitor or genetically using a knockout mouse lacking our earlier study (11) may reflect the use of a less sensitive Ab for The Journal of Immunology 1231 detecting the phosphorylated form of JNK MAP kinase. The func- 22. Heyworth, P., B. Bohl, G. Bokoch, and J. Curnutte. 1994. Rac translocates in- phox phox tional consequences of defective JNK activation in rac2Ϫ/Ϫ neu- dependently of the neutrophil NADPH oxidase components p47 and p67 . J. Biol. Chem. 269:30749. trophils are currently unknown. However, JNK is believed to play 23. Dorseuil, O., A. Vazquez, P. Lang, J. Bertoglio, G. Gacon, and G. Leca. 1992. a role in the activation of stress-induced gene expression and ap- Inhibition of superoxide production in B lymphocytes by antisense oligonu- cleotides. J. Biol. Chem. 267:20540. optosis (12, 13). 24. Gabig, T., C. Crean, P. Mantel, and R. Rosli. 1995. Function of wild-type or In conclusion, the present study provides direct genetic evidence mutant Rac2 and Rapla GTpases in differentiated HL60 Cell NADPH oxidase that Rac2 is a critical regulator of specific signaling pathways that activation. Blood 85:804. 25. Benard, V., B. P. Bohl, and G. M. Bokoch. 1999. Characterization of rac and activate the neutrophil NADPH oxidase, particularly those down- activation in chemoattractant-stimulated human neutrophils using a novel stream of chemoattractant receptors and Fc␥ receptors, despite the assay for active GTPases. J. Biol. Chem. 274:13198. concomitant presence of the highly homologous Rac1 isoform. 26. Akasaki, T., H. Koga, and H. Sumimoto. 1999. Phosphoinositide 3-kinase-de- pendent and -independent activation of the small GTPase Rac2 in human neu- The effect of Rac2 deficiency on NADPH oxidase activity is prob- trophils. J. Biol. Chem. 274:18055. ably exerted through multiple pathways, including those indepen- 27. Knaus, U., P. Heyworth, B. Kinsella, J. Curnutte, and G. Bokoch. 1992. Purifi- dent of MAP kinase activation. Ongoing studies are directed at cation and characterization of rac2. J. Biol. Chem. 267:23575. 28. Heyworth, P., U. Knaus, X. Xu, D. Uhlinger, L. Conroy, G. Bokoch, and further identifying these pathways and the specific Rac2 sequences J. Curnutte. 1993. Requirement for posttranslational processing of rac GTP-bind- important for regulation of NADPH oxidase activation. ing proteins for activation of human neutrophil NADPH oxidase. Mol. Biol. Cell 4:261. 29. Yang, F. C., R. Kapur, A. J. King, W. Tao, C. Kim, J. Borneo, R. Breese, Acknowledgments M. Marshall, M. C. Dinauer, and D. A. Williams. 2000. 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