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Originally published online as doi:10.1189/jlb.0705371 on November 7, 2005

Published online before print November 7, 2005
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(Journal of Leukocyte Biology. 2006;79:223-234.)
© 2006 by Society for Leukocyte Biology

Impaired NADPH oxidase activity in Rac2-deficient murine neutrophils does not result from defective translocation of p47phox and p67phox and can be rescued by exogenous arachidonic acid

Chaekyun Kim*,{dagger} and Mary C. Dinauer*,1

* Herman B. Wells Center for Pediatric Research, Department of Pediatrics (Hematology/Oncology), Microbiology/Immunology, and Medical and Molecular Genetics, James Whitcomb Riley Hospital for Children, Indiana University Medical Center, Indianapolis; and
{dagger} Inha University College of Medicine, Incheon, Korea

1 Correspondence: Cancer Research Institute, Indiana University School of Medicine, 1044 West Walnut Street, R4 402C, Indianapolis, IN 46202. E-mail: mdinauer{at}iupui.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rac2 is a hematopoietic-specific Rho-GTPase that plays a stimulus-specific role in regulating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and other functional responses in neutrophils. In this study, rac2-/- neutrophils were shown to have significantly decreased NADPH oxidase activity and actin remodeling in response to exogenous arachidonic acid (AA), as previously observed for phorbol 12-myristate 13-acetate (PMA) or formyl-Met-Leu-Phe (fMLP) as agonists. PMA-, fMLP-, or AA-induced translocation of p47phox and p67phox to the plasma membrane was not impaired in rac2-/- neutrophils. Combined stimulation of rac2-/- neutrophils with exogenous AA and PMA had a synergistic effect on NADPH oxidase activity, and superoxide production increased to a level that was at least as high as wild-type cells and had no effect on fMLP-elicited enzyme activity. Membrane translocation of p47phox and p67phox as well as Rac1 activation was not increased further by combined PMA and AA stimulation. Inhibitor studies were consistent with important roles for phorbol ester-activated protein kinase C (PKC) isoforms and an atypical isoform, PKC{zeta}, in superoxide production by wild-type and rac2-/- neutrophils stimulated with AA and PMA. In addition, PMA-stimulated release of AA and cytoplasmic phospholipase A2 expression in rac2-/- neutrophils were similar to wild-type, suggesting that deficient AA production by PMA-stimulated rac2-/- neutrophils does not explain the effect of exogenous AA on oxidase activity. Although not required for translocation of p47phox and p67phox, Rac2 is necessary for optimal activity of the assembled oxidase complex, an effect that can be replaced by exogenous AA, which may act directly or via an exogenous AA-induced mediator.

Key Words: cPLA2 • PKC • superoxide anion • Rho-GTPase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The membrane-associated phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates superoxide anion during the respiratory burst by catalyzing the transfer of electrons from NADPH to molecular oxygen. The NADPH oxidase includes two membrane-bound components, gp91phox and p22phox, which together, comprise the flavocytochrome b558 and two cytosolic subunits p47phox and p67phox. Upon stimulation, p47phox and p67phox, along with the Rac GTPase, translocate to the plasma membrane and associate with the membrane flavocytochrome b558 [1 2 3 ]. Membrane translocation of the p47phox and p67phox subunits is activated by phosphorylation of multiple serine residues on p47phox [1 , 2 , 4 ]. Concurrent activation and membrane translocation of Rac, which binds to p67phox and flavocytochrome b558, are required to complete NADPH oxidase assembly and activation [3 , 5 6 7 ]. Although the Rac1 and Rac2 GTPases are 92% identical, the Rac2 isoform is critical for normal regulation of cellular NADPH oxidase activity. Under cell-free conditions using purified flavocytochrome b558 and human neutrophil cytosol, Rac2 is more active than Rac1 in stimulating NADPH oxidase activity [8 ]. Furthermore, studies using murine neutrophils with genetic deletion of either isoform indicate that Rac2 but not Rac1 is essential for high-level superoxide production in response to phorbol ester, the chemoattractant formyl-Met-Leu-Phe (fMLP), or immunoglobulin G (IgG)-opsonized particles, whereas opsonized, zymosan-elicited oxidase activity is unaffected by the absence of Rac2 [9 10 11 12 13 ]. The underlying mechanism(s) that contribute to the agonist-selective impairment of NADPH oxidase activity in Rac2-deleted neutrophils remain unclear.

Phorbol esters are among the most potent activators of the neutrophil respiratory burst, acting as analogs of diacylglycerol (DAG) and directly activating many members of the serine-threonine protein kinase C (PKC) family [14 ]. At least 12 different isoforms of PKC have been characterized so far, and these can be grouped into three categories: conventional PKCs ({alpha}, ßI, ßII, and {gamma}), novel PKCs ({delta}, {epsilon}, {theta}, {eta}, and µ), and atypical PKCs ({iota}, {lambda}, and {zeta}), based on the requirement for Ca+2 for activation (conventional) and DAG-binding activity (conventional and novel) [15 , 16 ]. Neutrophil NADPH oxidase activation by most physiologic agonists involves activation of PKCs [14 ]. The ß, {delta}, and {zeta} isoforms appear to be the main PKC isoforms involved, as shown by studies using selective inhibitors, genetic deletion, or antisense technologies [17 18 19 20 21 22 ]. Downstream effects of PKC include direct phosphorylation of p47phox, which leads to membrane translocation of cytosolic components in a cell-free system and intact cells [21 22 23 24 25 26 27 ]. As mentioned above, phorbol 12-myristate 13-acetate (PMA)-elicited superoxide production is decreased substantially in Rac2-deficient neutrophils [9 , 10 , 28 ]. This observation indicates that NADPH oxidase activation by PKCs is also dependent on Rac2.

Many agonists, which stimulate superoxide production in neutrophils, cause the release of arachidonic acid (AA) from membrane phospholipids via the action of phospholipase A2 (PLA2) on glycerolphospholipids or by hydrolysis of DAG [29 , 30 ]. AA levels are also high in ischemic tissue such as myocardium [31 ]. AA can act as a second messenger and is believed to regulate many neutrophil functions, although the underlying mechanisms and its physiologic role are poorly understood. Stimulation of intact neutrophils with exogenous AA leads to activation of PKCs, phosphatidylinositol 3-kinases (PI-3K), PLC, PLD, and mitogen-activated protein kinases (MAPK) [32 33 34 35 36 37 38 39 40 ]. Exogenous AA has long been known to activate neutrophil superoxide production [41 , 42 ], which can be inhibited by antagonists of PI-3K or PLC [33 , 37 ].

In the present study, we further explored the regulation of the neutrophil NADPH oxidase by Rac2. We found that NADPH oxidase activation and actin remodeling stimulated by exogenous AA were decreased significantly in rac2-/- neutrophils, similar to our previous observations for PMA- or fMLP-stimulated responses. To further investigate the basis of reduced NADPH oxidase activity in rac2-/- neutrophils, we examined membrane translocation of the cytosolic p47phox and p67phox subunits, finding that PMA-, fMLP-, or AA-induced translocation is not impaired. It is surprising that NADPH oxidase activity in rac2-/- neutrophils stimulated with AA and PMA was increased by approximately sixfold and was at least as high as wild-type neutrophils. Membrane translocation of p47phox and p67phox subunits to the membrane, along with Rac1 activation, was not augmented by combined stimulation with AA and PMA. These data indicate that Rac2 is not required for translocation of p47phox or p67phox in response to these agonists but is necessary for optimal activity of the membrane-assembled oxidase complex. The marked enhancement of PMA-elicited superoxide production in rac2-/- neutrophils by exogenous AA appears to be exerted on the NADPH oxidase complex by a direct effect or by an AA-induced mediator.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies, reagents, and buffers
Rabbit polyclonal antibodies against PKC{alpha}, PKCßII, PKC{delta}, and PKC{zeta} and a mouse monoclonal antibody (mAb) for cytoplasmic (c)PLA2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and a rabbit polyclonal antibody against Rac2 was a kind gift from Gary Bokoch and Ulla Knaus (The Scripps Research Institute, La Jolla, CA). A rabbit polyclonal antibody for PKC{zeta} and a mouse mAb against Rac1 were purchased from Upstate Biotechnology (Lake Placid, NY); those for p47phox and p67phox were purchased from BD PharMingen (San Diego, CA). Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Distilled water, phosphate-buffered saline (PBS), pH 7.2, Hanks’ balanced salt solution (HBSS; without Ca2+, Mg2+ and phenol red), and HBSS with 1.26 mM Ca2+, 0.4 mM Mg2+ were purchased from Gibco-BRL (Grand Island, NY). HBSS with 0.1% bovine albumin (BSA) and 1% glucose, pH 7.25–7.4, PBS with 0.9 mM CaCl2, 0.5 mM MgCl2, and 7.5 mM glucose (PBSG), and lysis buffer [20 mM Tri-Cl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 50 mM NaF, 2 mM Na3VO4, 0.01 mM phenylarsine oxide, 20 µg/mL chymostatin, 10 µM leupeptin, 2 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF)] were used in this study. AA (Sigma Chemical Co., Cat. #A8798) was freshly diluted from sealed ampules in 1 min before use to avoid alteration from oxidation or contamination.

Animals
Rac2-/- mice were generated by targeted gene disruption and back-crossed into the C57BL/6J mice for ≥10 generations [9 ]. C57BL/6J mice used for wild-type controls were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed in microisolator cages under specific pathogen-free conditions, fed autoclaved food and acidified water ad libitum, and used in experiments at 8–16 weeks of age. The Indiana University Animal Care and Use Committee (Indianapolis) approved all studies.

Isolation of neutrophils
Murine neutrophils were purified from bone marrow (BM) using Percoll gradients as described [10 ]. The final cell preparation was kept on ice in PBS (without Ca2+, Mg2+) until further processing. All reagents used were endotoxin-tested (<0.1 ng endotoxin/ml by the limulus lysate assay) to minimize inadvertent priming during the isolation procedure. Human neutrophils were separated from peripheral venous blood [10 ] obtained from healthy volunteers as approved by the Institutional Review Board of the Indiana University School of Medicine.

NADPH oxidase activity
Superoxide production was measured in a quantitative kinetic assay following stimulation with PMA, AA, based on the superoxide dismutase-inhibitable reduction of cytochrome c [10 ], and an isoluminol chemiluminescence assay [28 ]. To investigate whether cPLA2 and PKC inhibitors affect superoxide production, PKC inhibitors, GF109203x, RO-31-8220, staurosporine, chelerythrine chloride (Alexis Biochemicals, San Diego, CA), and myristoylated PKC{zeta} pseudosubstrate (Calbiochem, San Diego, CA), and PLA2 inhibitors, arachidonyltrifluoromethyl ketone (AACOCF3) and methyl arachidonyl fluorophosphonate (MAFP; Calbiochem) were used. BM neutrophils were incubated with each inhibitor for 0–30 min at 37°C prior to activation. Following incubation, cells were analyzed for superoxide production using the cytochrome c reduction assay in response to various stimuli.

Filamentous actin (F-actin) measurements
The relative amount of F-actin was measured by flow cytometry using fluorescein isothiocyanate-phalloidin as described previously [9 , 43 ]. The results are reported as mean cellular fluorescence (MCF).

Expression of cPLA2 and PKC isoforms
Lysates of mouse BM neutrophils and human peripheral blood neutrophils were prepared, and protein was measured by the bicinchoninic acid (BCA) protein kit (Pierce, Rockford, IL) using BSA as a standard. Lysates were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblots were probed with antibodies for cPLA2 or PKC isoforms, PKC{alpha}, -ßII, -{delta}, and -{zeta} [9 , 10 ].

Translocation of cytosolic NADPH oxidase components
BM neutrophils were treated with 5 mM diisopropylfluorophosphate (DPI), suspended to 3 x 107 cells/ml in PBSG, and stimulated with 200 ng/ml PMA, 10 µM AA, or 200 ng/mL PMA plus 10 µM AA for 3 or 10 min at 37°C. The reaction was stopped by the addition of cold PBS, and then cells were pelleted by centrifugation at 1300 rpm for 10 min at 4°C. Cells were resuspended in relax buffer (100 mM KCl, 10 mM HEPES, 3.5 mM MgCl2, 3 mM NaCl, 1.2 mM EDTA, 25 mM NaF, 5 mM Na3VO4, 1 mM p-nitrophenyl phosphate, 20 µg/mL chymostatin, 2 mM PMSF, 10 µM leupeptin, and 1 mM AEBSF) and were disrupted using 40 strokes at a rate of one stroke/s in a 7-ml Dounce homogenizer. Unbroken cells and nuclei were pelleted by centrifugation at 1300 rpm for 10 min at 4°C, and the cleared supernatant was centrifuged at 40,000 rpm for 30 min (TLA 100.2, Beckman Instruments, Palo Alto, CA). The cytosol fraction was collected in a separated tube, and then the pellet was washed with relax buffer. The pellet was dissolved in solubilization buffer (150 mM NaCl, 5 mM EDTA, 10 mM Tris, pH 7.5, 1% sodium deoxycholate, and 1% Nonidet P-40), and samples were boiled in 5x Laemmli sample buffer and electrophoresed on 12% SDS-PAGE. Immunoblots were probed with antibodies for NADPH oxidase components or those for PKCßII and -{delta} in some experiments. Integrated densitometry was used to determine the intensity of scanned films using National Institutes of Health (NIH) Image software (Research Service Branch, National Institute of Mental Health, Bethesda, MD) or Scion Image (Scion Corp. Frederick, MD).

Immunoprecipitation
DPI-treated BM neutrophils (5x107 cells) were stimulated with the appropriate stimulus for 3 min at 37°C. Membrane and cytosol fractions were prepared as described above and were incubated with mouse mAb against cPLA2 overnight at 4°C with gentle shaking and precipitated with protein A Sepharose beads (Amersham, Little Chalfont, UK). Washed immunoprecipitates were electrophoresed on 10% SDS-PAGE and probed with antibodies for p47phox, p67phox, p22phox, Rac, and cPLA2.

PKCßII and Rac activation
Activation of PKCßII in BM neutrophils was determined by stimulating cells (5x106 cells) with the appropriate stimulus for various amounts of time. Cells were lysed in 100 µl lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10 mM HEPES, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 10% glycerol, 1 mM Na3VO4, 50 mM NaF, 20 µg/ml chymostatin, 2 mM PMSF, 10 µM leupeptin, and 1 mM AEBSF) as described previously [44 ]. The protein lysates were equalized for total protein concentration using a BCA assay. Lysates were incubated with 10 µl anti-PKCßII antibody and 90 µl 1:1 slurry of protein A Sepharose beads in lysis buffer overnight at 4°C. Beads were separated from lysate by centrifugation and washed twice in lysis buffer and twice in kinase buffer (20 mM 3-[N-morpholino]propanesulfonic acid, pH 7.2, 30 mM ß-glycerophosphate, 5 mM EGTA, 20 mM MgCl2, 1 mM Na3VO4, 1 mM dithiothreitol, 20 µg/mL chymostatin, 2 mM PMSF, 10 µM leupeptin, and 1 mM AEBSF). After the final centrifugation, the kinase assay was started by addition of 30 µl kinase buffer containing 5 µCi [32P]adenosine 5'-triphosphate (Amersham) and myelin basic protein (MBP) as a substrate. The reaction was stopped after 30 min at 30°C by addition of 10 µl 5x Laemmli buffer and boiling at 95°C for 5 min. The kinase reaction was resolved on 4–20% SDS-PAGE (Novex, San Diego, CA). Gels were transferred onto polyvinylidene difluoride membrane and subjected to autoradiography.

An affinity precipitation (or pull-down) assay for Rac activation and immunoblotting was performed as described previously [28 , 43 ].

Measurement of AA release
To determine whether cPLA2 activity is different between wild-type and rac2-/- BM neutrophils, the release of AA was measured with a modification of a previously reported method [45 , 46 ]. Cells (1x108/ml) were labeled with [3H]AA (NEN, Boston, MA) for 30 min at 37°C in PBS containing 1 µCi [3H]AA (180–240 mCi/mmol). The labeled cells were washed once with PBS containing 0.1% fatty acid-free BSA and twice with PBS. The pellet was resuspended to 1 x 107 cells/ml in PBSG containing 0.1% fatty acid-free BSA and incubated at 37°C for 10 min with 200 ng/ml PMA or 200 ng/mL PMA plus 1 µM A23187. The reaction was terminated by centrifugation at 4°C, and the supernatants were counted for radioactivity in 10 ml Ultima Gold XL (Packard, Netherlands) by liquid scintillation (Packard). Between 60% and 70% of the total radioactivity added was incorporated by the cells, of which 2–9% was released following stimulation (data not shown).

Statistical analysis
Statistical analysis was performed using Instat (GraphPad, San Diego, CA) or Excel (Microsoft, Redmond, WA) software. Data are expressed as mean ± SD or mean ± SEM, and a value of P < 0.05 was considered significant. The two-tailed Student’s t-test, one-way ANOVA followed by Tukey-Kramer multiple comparison test or an ANOVA and Dunnett’s post-test was used to determine the difference between groups.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NADPH oxidase activation and actin remodeling in response to exogenous AA are decreased in rac2-/- neutrophils
As previously reported [9 , 10 , 28 ], NADPH oxidase activity in rac2-/- neutrophils stimulated with 200 ng/mL PMA was more than threefold lower than wild-type cells (wild-type, 14.6±2.8 nmoles/min/107 cells; rac2-/-, 4.2±1.7 nmoles/min/107 cells; Fig. 1 A ). We found that superoxide production in rac2-/- neutrophils stimulated with exogenous AA was also decreased (wild-type, 6.6±1.7 nmoles/min/107 cells; rac2-/-, 3.5±2.7 nmoles/min/107 cells, P=0.0047, paired Student’s t-test), suggesting that Rac2 is required for AA-induced NADPH oxidase activation. Rac2 is also required for the rapid increase in F-actin content in chemoattractant-stimulated murine neutrophils [9 ]. Rac2-/- neutrophils exhibited poor F-actin formation in response to PMA or AA (Fig. 1B) . The diminished ability of AA to induce actin polymerization in rac2-/- cells further suggests that Rac2 is important for AA-activated signaling events in murine neutrophils.



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  		Figure 1. Rescue of NADPH oxidase activity by PMA and AA costimulation in rac2-/- BM neutrophils. Solid bars, Wild-type (WT); open bars, rac2-/-. Data are expressed as mean ± SD. (A) Superoxide production by freshly isolated BM neutrophils following stimulation with 200 ng/ml PMA (n=17), 10 µM AA (n=8), or 10 µM AA plus 200 ng/ml PMA (n=12) was monitored by reduction of ferricytochrome c. ANOVA followed by Tukey-Kramer multiple comparison test was performed. §, P < 0.001, wild-type PMA versus rac2-/- PMA; *, P< 0.01, compared with wild-type PMA; #, P < 0.001, compared with rac2-/- PMA; {dagger}, P< 0.001, compared with wild-type AA; {ddagger}, P < 0.001, compared with rac2-/- AA. (B) Diminished PMA- or AA-elicited F-actin generation was not rescued by costimulation of PMA (P) with AA (A; n=2). (C) The dose effect of AA in combination with 200 ng/ml PMA (n=3). (D) Superoxide production following stimulation with fatty acids and AA metabolites in combination with 200 ng/ml PMA (n≥2–3). *, P < 0.001, wild-type versus rac2-/- (unpaired t-test). LA, ; PA, ; SA, ; LTB4, leukotriene B4; PGE2, prostaglandin E2. (E) Superoxide production was measured using isoluminol chemiluminescence following stimulation with 10 µM fMLP plus 10 µM AA (n=3). *, P< 0.001, wild-type versus rac2-/- (unpaired t-test). RLU, Relative luminescence unit; LA, linoleic acid; PA, palmitic acid; SA, stearic acid.

 
PMA and AA costimulation rescues NADPH oxidase activity in rac2-/- neutrophils
It is surprising that the combination of exogenous AA and PMA had a synergistic effect and increased NADPH oxidase activity by approximately sixfold in rac2-/- cells, and superoxide production even exceeded wild-type levels (27.1±7.4 nmoles/min/107 cells vs. 19.3±4.6 nmoles/min/107 cells, respectively), although this did not quite reach statistical significance, comparing directly using the paired Student’s t-test (P=0.064) or ANOVA followed by the Tukey-Kramer test (Fig. 1A) . In contrast, production of superoxide by wild-type neutrophils stimulated with PMA and AA appeared only additive (Fig. 1A) . For both genotypes, superoxide production upon combined stimulation with PMA and AA was significantly higher compared with cells activated by PMA or AA alone (wild-type: P<0.05 compared with PMA and P<0.001 compared with AA; rac2-/-: P<0.001 compared with PMA or AA, see Fig. 1A ). The maximal level of superoxide production by rac2-/- neutrophils stimulated with 200 ng/ml PMA in combination with AA occurred at 10 µM AA, although 5 µM or 15 µM AA was almost as effective (Fig. 1C) .

We next investigated the effect of other fatty acids, LTB4 or PGE2, on PMA-elicited superoxide production by rac2-/- neutrophils. The addition of linoleic acid restored NADPH oxidase activity in response to PMA in rac2-/- neutrophils, and there was no effect of palmitic acid or stearic acid (Fig. 1D) . These data are consistent with previous studies, which found that cis-unsaturated but not saturated fatty acids elicit superoxide generation in human neutrophils [41 , 42 ]. AA serves as a precursor for the generation of PGs and LTs, including LTB4 and PGE2. We chose these as representative cyclooxygenase and lipoxygenase products to examine the effect on NADPH oxidase activity in rac2-/- neutrophils. Stimulation with LTB4 or PGE2 did not rescue PMA-elicited superoxide production deficiency in rac2-/- neutrophils. These results suggest that AA plays a specific role in reconstituting neutrophil NADPH oxidase activity in rac2-/- neutrophils.

We also examined whether the effect on rac2-/- neutrophil NADPH oxidase activity by combined stimulation with AA and PMA extended to other neutrophil responses. However, costimulation with 10 µM AA did not improve fMLP-elicited superoxide production in rac2-/- neutrophils (wild-type, 198±70 RLU integrated over 100 s; rac2-/-, 69±43 RLU, n=3; Fig. 1E ). Costimulation with AA and PMA also failed to reconstitute the F-actin response in rac2-/- cells (Fig. 1B) . Taken together, the above results suggest that the mechanisms that underlie the functional defects in rac2-/- neutrophils are heterogeneous, and AA costimulation does not always reconstitute impaired responses.

Translocation of cytosolic components of NADPH oxidase and activation of Rac
To further characterize the basis of deficient superoxide production in the absence of Rac2 and to evaluate possible mechanisms for the marked improvement in NADPH oxidase activity with combined stimulation with PMA and AA, we examined agonist-stimulated translocation of p47phox, p67phox, and Rac. We found that AA or PMA each induced translocation of p47phox, p67phox, and Rac1 to rac2-/- neutrophil membranes (Fig. 2 A ). Although statistical comparisons are difficult as a result of experiment-to-experiment variability in the relative amount of translocation detected, basal and agonist-induced levels of membrane-associated p47phox and p67phox were consistently 1.5- to 2.5-fold greater in rac2-/- neutrophils compared with wild-type neutrophils studied in parallel. Translocation of p47phox (Fig. 2B) and p67phox (data not shown) induced by fMLP was also not impaired in rac2-/- neutrophils. Finally, we found that translocation of p47phox, p67phox, and Rac1 in rac2-/- cells was not further enhanced by combined stimulation with PMA and AA (Fig. 2A) .



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  		Figure 2. Translocation of the cytosolic components of NADPH oxidase. Neutrophils were stimulated and disrupted using the Dounce homogenizer and then separated to cytosol and membrane fraction. The membrane fraction was loaded and separated by 12% SDS-PAGE, and immunoblotting was performed with antibodies against NADPH oxidase components. (A) BM neutrophils were stimulated with 200 ng/ml PMA, 10 µM AA, or a combination of PMA and AA for 10 min at 37°C. Representative immunoblot from 10 independent experiments. Bar graphs represent relative levels of p47phox and p67phox, which were estimated by densitometry of immunoblot signals, normalized to membrane-bound p22phox (used as a control for loading). Mean ± SEM (n=7); *, P< 0.05, compared with the density of wild-type p47phox control (paired t-test). (B) Representative immunoblot showing translocation of p47phox after stimulation with 10 µM fMLP for 5 min at 37°C. Bar graphs represent relative level of p47phox based on densitometry of immunoblot signals, again normalized to p22phox; mean ± SEM (n=3).

 
We also examined the effect of AA on activation of Rac1 in rac2-/- neutrophils using an affinity precipitation assay for Rac-guanosine 5'-triphosphate. We previously demonstrated that although wild-type murine neutrophils express similar amounts of Rac1 and Rac2, the latter isoform is preferentially activated in wild-type cells, whereas Rac1 activation is about threefold higher in rac2-/- neutrophils compared with wild-type [28 ]. Consistent with these previous results, stimulation with PMA or AA induced relatively more Rac1 activation in rac2-/- BM neutrophils compared with wild-type (data not shown). However, the combination of AA and PMA did not further increase activation of Rac1.

These results show that Rac2 is not required for the translocation of p47phox and p67phox or for the activation of Rac1 following neutrophil stimulation and that the marked increase in NADPH oxidase activity with a combination of PMA and AA is not related to an AA-induced increase in membrane levels of p47phox and p67phox or of activated Rac1. Thus, the enhancement of PMA-elicited superoxide production in rac2-/- neutrophils by the addition of exogenous AA appears to involve a direct effect of AA or a downstream mediator on the membrane-assembled oxidase complex.

Signaling pathways involved in activation of NADPH oxidase by PMA, AA, and costimulation with AA and PMA
We next investigated whether specific signaling pathways might be involved in the reconstitution of NADPH oxidase activity in rac2-/- neutrophils costimulated with PMA and exogenous AA. As PMA-induced NADPH oxidase activation and F-actin formation are impaired in the absence of Rac2 (Fig. 1) , we first examined the expression of PKC isoforms by immunoblotting with specific PKC antibodies to verify that reduced responses do not simply reflect decreased PKC expression in rac2-/- neutrophils. PKCs implicated in neutrophil NADPH oxidase activation include PKCßII and -{delta}, both of which are capable of being activated directly by phorbol esters, as well as the atypical PKC{zeta} isoform [18 , 20 , 21 , 24 ]. Human neutrophils express PKC{alpha}, -ßI, -ßII, -{delta}, and -{zeta} [18 , 47 48 49 ]. The relative levels of these PKC isoforms in human and murine neutrophils have not yet been reported.

We found that wild-type and rac2-/- murine neutrophils expressed similar levels of PKC{alpha}, PKCßII, and PKC{delta}, which were also comparable with human neutrophils (Fig. 3 ). PKC{delta} was detected as a doublet in murine neutrophils, as previously observed [26 ]. It is unexpected that a small amount of PKC{zeta} was detected in human neutrophils compared with mouse (Fig. 3) . This finding may reflect a true difference in the relative amount of neutrophil PKC{zeta} between the two species or may simply be related to the relative specificity of the antibody, which was raised against a peptide derived from the carboxy terminus of rat PKC{zeta} and differs by one amino acid in human PKC{zeta} compared with mouse and rat. PKCß activation, measured with MBP as a substrate, was similar between murine wild-type and rac2-/- neutrophils, and translocation of PKCß and -{delta} to the membrane was also not impaired in rac2-/- neutrophils (data not shown).



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  		Figure 3. Expression of PKC isoforms in human and murine neutrophils. The indicated amounts (10, 20 µg) of whole cell lysates from human and murine neutrophils were separated by 10% Tris-glycine gel, transferred to nitrocellulose, and probed with polyclonal antibodies specific for PKC{alpha}, -ßII, -{delta}, and -{zeta}. To confirm equal sample loading, blots were reprobed with antibody against total p38 MAPK. Data shown are representative of three independent experiments.

 
We next examined the effects of inhibitors that act on enzymes activated downstream of PMA or AA, hoping to identify a pathway activated by exogenous AA, which complemented a signaling defect downstream of PMA, and PMA, by acting as a DAG analog, activates conventional and novel PKCs, and ß and {delta} isoforms appear to regulate PMA-activated NADPH oxidase activity [18 19 20 , 22 ]. In human neutrophils, AA stimulates PI-3K and PLC activation, and AA-induced superoxide production is decreased by inhibitors of these enzymes [37 , 50 ]; although AA is a well-known activator of PKCs [32 ], there currently is little direct evidence in the literature that establishes a link between PKCs and AA-induced NADPH oxidase activation in intact neutrophils.

Treatment of murine neutrophils with GF109203x, a PKC inhibitor with selectivity for PKC{alpha}, -ßI, -ßII, -{gamma}, and -{epsilon} (conventional and most novel PKCs), suppressed PMA-induced superoxide production in both genotypes, as expected, as well as PMA plus AA-induced NADPH oxidase activity (Fig. 4 A ). Inhibition was dose-dependent and essentially complete at 2 µM (Fig. 4A) . However, AA-induced superoxide production was only partially inhibited by GF109203x (Fig. 4A) . Similar results were obtained following treatment of cells with RO-31-8220, a second PKC inhibitor acting on conventional and novel PKCs, and staurosporine, a broad spectrum inhibitor of protein kinases including PKC (data not shown).



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  		Figure 4. Effect of PKC inhibitors on PMA- and AA-induced NADPH oxidase activation in murine neutrophils. Solid bars, Wild-type; open bars, rac2-/-. Murine BM neutrophils were pretreated with the indicated concentration of (A) GF109203x (10 min), (B) chelerythrine chloride (10 min), or (C) PKC{zeta} pseudosubstrate (30 min) at 37°C for the indicated time and stimulated with 200 ng/ml PMA, 100 µM AA, or PMA plus AA. Superoxide production was monitored by reduction of cytochrome c. Data are expressed as mean ± SD (n=4 for GF109203x and n=5–6 for chelerythrine chloride and PKC{zeta} pseudosubstrate). ANOVA followed by Dunnett’s post-test was performed. *, P < 0.05; **, P< 0.01, wild-type control versus inhibitor-treated sample; #, P < 0.05; ##, P< 0.01, rac2-/- control versus inhibitor-treated sample.

 
We next examined whether atypical PKCs regulate NADPH oxidase activation by exogenous AA, which is currently unknown. Chelerythrine chloride, a PKC inhibitor whose spectrum includes PKC{zeta} [51 52 53 54 ], produced a dose-dependent inhibition of superoxide production elicited by PMA, AA, or their combination (Fig. 4B) . AA or AA plus PMA-induced oxidase activity was sensitive to moderate concentrations of this inhibitor (Fig. 4B) , suggesting involvement of an atypical PKC isoform. A cell-permeant myristoylated peptide, which acts as a pseudosubstrate to inhibit PKC{zeta}, also substantially inhibited superoxide production elicited by AA, the combination of PMA and AA, or PMA alone, and AA-elicited NADPH oxidase in rac2-/- neutrophils were particularly sensitive (Fig. 4C) .

Taken together, these results indicate that multiple classes of PKCs mediate PMA-induced NADPH oxidase activation in murine neutrophils in the absence or presence of AA, whereas AA-induced superoxide production appears to be regulated by PKC{zeta} but not conventional or novel isoforms. In general, wild-type and rac2-/- cells displayed a similar pattern of sensitivity, with the exception of chelerythrine chloride and the PKC{zeta} pseudosubstrate, where PMA-activated superoxide production was increased modestly in rac2-/- neutrophils at low concentrations of inhibitor.

Inhibition of PMA-stimulated NADPH oxidase activity in murine neutrophils by the PKC{zeta} pseudosubstrate was unexpected. In human neutrophils, similar studies have shown that PMA-induced integrin-dependent adhesion involves PKC{zeta} [52 ], but PMA-activated superoxide production does not (refs. [21 , 52 ] and C. Kim and M. C. Dinauer, unpublished observations). Hence, there may be a species difference for involvement of PKC{zeta} in PMA-activated superoxide production.

Up to one-half of AA-induced NADPH oxidase activity in both genotypes was inhibited by the PI-3K inhibitors, LY924002 or wortmannin, with maximal inhibition at 50 µM or 100 nM, respectively (data not shown). Hence, murine AA-induced activation of NADPH oxidase appears to be partially dependent on PI-3Ks, consistent with results obtained for human neutrophils [50 ]. However, neither PMA- nor PMA + AA-induced superoxide production was inhibited by LY924002 or wortmannin (data not shown).

The PLC inhibitor U73122 diminished AA-stimulated superoxide production in wild-type and rac2-/- murine neutrophils in a dose-dependent manner (Fig. 5 ), similar to reports for human neutrophils [37 ]. PMA-activated oxidase activity in murine neutrophils was also inhibited by U73122, in contrast to human neutrophils, where PMA-stimulated superoxide production is insensitive to this agent [55 ]. This appears to be another species-specific difference, in addition to the above results implicating PKC{zeta} in regulation of PMA-activated superoxide production in murine neutrophils. Superoxide production induced by PMA and AA costimulation of murine neutrophils was only inhibited minimally by U73122, an effect that was seen in both genotypes. This suggests that combined stimulation with PMA and AA eliminates a requirement for PLC, necessary for activation by PMA or AA alone, although the underlying mechanism is unclear.



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  		Figure 5. Effect of PLC inhibitor U73122 on PMA- and AA-induced NADPH oxidase activation in murine neutrophils. Solid bars, wild-type; open bars, rac2-/-. Murine BM neutrophils were pretreated with the indicated concentration of U73122 for 10 min at 37°C and stimulated with 200 ng/ml PMA, 100 µM AA, or PMA plus AA. Superoxide production was monitored by reduction of cytochrome c. Data are expressed as mean ± SD (n=3); ANOVA followed by Dunnett’s post-test was performed. **, P < 0.01, wild-type control versus inhibitor-treated sample; #, P < 0.05; ##, P < 0.01, rac2-/- control versus inhibitor-treated sample (unpaired t-test).

 
cPLA2 activity and AA release in rac2-/- neutrophils
Activation of Rac leads to AA release in fibroblasts, suggesting that cPLA2 can be a downstream target of Rac [56 , 57 ]. Furthermore, studies in neutrophils have implicated intracellular AA as a critical regulator of the assembled NADPH oxidase [58 59 60 ], although this has been disputed [61 ]. We therefore examined whether rac2-/- neutrophils may have defects in activation of cPLA2. Expression of cPLA2 was similar between wild-type and rac2-/- BM neutrophil cell lysates based on immunoblot analysis (data not shown). To investigate the potential role of Rac2 as a regulator of cPLA2, we next measured AA release. As shown Figure 6 A , incubation of BM neutrophils with PMA induced a modest but significant increase of [3H]AA release in wild-type cells, and PMA-elicited [3H]AA release in rac2-/- neutrophils was similar to wild-type. Calcium ionophore A23187 is a strong activator of cPLA2 and further stimulates AA release when used in combination with PMA in neutrophils [62 , 63 ]. Incubation of cells with PMA and A23187 induced a relatively large [3H]AA release compared with PMA alone; however, there was again no significant difference between genotypes. Of note, PMA plus A23187 costimulation did not rescue superoxide production (wild-type, 14.2±9.3 nmoles/min/107 cells; rac2-/-, 5.4±0.6 nmoles/min/107 cells; Fig. 6B ). These results further indicate that deficient superoxide production in PMA-stimulated rac2-/- neutrophils is not related to an underlying defect in release of AA.



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  		Figure 6. Activation of cPLA2 in BM neutrophils. Solid bars, wild-type; open bars, rac2-/-. (A) cPLA2 activity was measured by a [3H]AA-release method. BM neutrophils were preincubated with [3H]AA for 30 min and stimulated with 200 ng/ml PMA or 200 ng/ml PMA plus 1 µM A23187 for 10 min at 37°C. Data are expressed as mean ± SD (n=7–10). *, P< 0.05 (unpaired t-test). (B) Superoxide production by BM neutrophils taken after [3H]AA incorporation. Data are expressed as mean ± SD (n=6–10) *, P < 0.01; **, P < 0.001, wild-type versus rac2-/- (unpaired t-test).

 
We also investigated whether the rescue of AA-mediated superoxide production in PMA-stimulated rac2-/- BM neutrophils was linked to cPLA2 activation. BM neutrophils were treated with cPLA2 inhibitors, AACOCF3 (0.2–200 µM) or MAFP (0.2–200 µM). Neither inhibitor affected superoxide production in PMA, AA, or PMA plus AA-stimulated rac2-/- cells (data not shown). These results indicate that AA release through cPLA2 activation is not required for superoxide production in murine neutrophils, confirming recent studies about murine and human neutrophils [61 ] and murine macrophages [64 ].


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The hematopoietic-specific Rho GTPase Rac2 regulates a variety of neutrophil functions despite the presence of the highly homologous Rac1. Distinct roles of Rac1 and Rac2 have been identified in studies using Rac1- or Rac2-deficient mouse neutrophils. Rac1 and Rac2 play an important role in chemotaxis, and Rac1 is implicated in regulating the directionality of movement in a chemoattractant gradient, whereas Rac2 regulates speed of movement [9 , 54 ]. However, only Rac2 plays an essential role regulating NADPH oxidase activation in response to phorbol esters, fMLP, or IgG-opsonized particles, in that rac2-/- neutrophils have defects in superoxide production, whereas rac1-/- neutrophils do not [9 , 10 , 13 ].

In the current study, we further investigated the role of Rac2 in regulation of the NADPH oxidase. We showed that NADPH oxidase activity in rac2-/- murine neutrophils was deficient in response to exogenous AA, but superoxide production upon combined stimulation with PMA and AA was synergistically enhanced and at least as high as in wild-type neutrophils. Additional studies demonstrated that membrane translocation of p47phox and p67phox, in response to PMA, fMLP, or AA, was consistently increased in rac2-/- neutrophils compared with wild-type cells along with Rac1 activation and was not further increased with combined stimulation of AA and PMA. These results establish that Rac2 is not required for the initial membrane recruitment of p47phox and p67phox, consistent with prior studies by Dorseuil and colleagues [65 ]. Thus, we conclude that Rac2 regulates oxidase activation at the membrane, an effect that can be replaced by exogenous AA.

As noted above, translocation of p47phox and p67phox in response to PMA, fMLP, or AA appeared to be enhanced in the absence of Rac2 compared with wild-type neutrophils. The underlying basis of this observation is unknown. It is possible that there is competition between Rac2 and p47phox/67phox for binding to flavocytochrome b. Another possible mechanism is that Rac2 may regulate events that result in loss of p47phox and p67phox from the membrane as part of a negative feedback loop leading to termination of NADPH oxidase activity. Events associated with termination of superoxide production are poorly understood, but at least during phagocytosis, this is correlated with the disappearance of p47phox and p67phox from the membrane [66 ]. However, superoxide production is not prolonged in rac2-/- neutrophils. Finally, as translocation of p47phox is not enhanced in p67phox-deficient chronic granulomatous disease [67 , 68 ], a decrease in superoxide production per se seems unlikely to account for the observed increase in translocation in rac2-/- neutrophils.

Although stimulated rac2-/- neutrophils generate activated Rac1 [28 ], the preferred role for Rac2 in activating the NADPH oxidase could reflect more efficient incorporation of Rac2 into the enzyme complex or alternately, activation of another mediator that acts at the membrane. We recently showed that the composition of the C-terminal polybasic domain is sufficient for determining Rac isoform specificity in the NADPH oxidase [11 ]. As the polybasic domain can mediate Rac localization to specific subcellular membrane compartments or microdomains [69 , 70 ], this may be important in localizing activated Rac in the vicinity of the assembled NADPH oxidase complex. In addition, the active enzyme Rac enhances electron transfer via interactions with flavocytochrome b and with p67phox [3 , 7 ]. Although Rac1 and Rac2 have similar binding affinities to the Rac-binding domain of p67phox [71 ], Rac2 has a higher affinity for p67phox in yeast two-hybrid studies [72 ], and Rac2 is more active than Rac1 in cell-free NADPH oxidase assays performed in the presence of neutrophil cytosol [8 ].

The strong enhancement of PMA-activated superoxide production by rac2-/- neutrophils upon the addition of exogenous AA may reflect a direct effect of AA on the assembled oxidase complex. Although difficult to prove experimentally, several lines of evidence support this general mechanism, which could be mediated by a direct effect on flavocytochrome b or via enhanced binding of p67phox, p47phox, or Rac1. Studies in cell-free systems have shown that NADPH oxidase activity is sensitive to the composition of lipids surrounding the flavocytochrome b [73 , 74 ]. The addition of exogenous AA to purified flavocytochrome b preparations induces a conformational change, which may promote electron transfer [75 , 76 ]. Other studies have shown that AA can bind to p47phox, potentially synergizing with the effects of p47phox phosphorylation, to promote optimal binding to the flavocytochrome [25 , 73 ]. NADPH oxidase activity can be stimulated by the AA-binding proteins S100A8/A9, which interact with p67phox, and Rac [77 ]. Thus, relatively inefficient superoxide production in the absence of Rac2 could be enhanced by one or a combination of these direct effects of AA on constituents of the NADPH oxidase complex.

The restoration of high-level NADPH oxidase activity in rac2-/- neutrophils by AA may also, at least in part, be mediated indirectly via downstream mediators of AA signaling, which perhaps complement defects in rac2-/- neutrophils to enhance activity of the PMA-assembled oxidase complex. As an approach to identify possible mediators, we examined the effect of specific enzyme inhibitors. These studies implicated PKC{zeta}, PI-3K, and PLC in coupling exogenous AA stimulation to NADPH oxidase activation in wild-type and rac2-/- neutrophils. This confirmed the importance of signaling via PI-3K and particularly, PLC for activation of the NADPH oxidase by exogenous AA [33 , 37 ]. In addition, this is the first study to show that activation of the NADPH oxidase by exogenous AA requires PKC{zeta} activation. The PKC{zeta} isoform also plays an important role in regulating fMLP-induced NADPH oxidase activation [21 ]. However, PKC{zeta}, along with conventional and novel PKC isoforms, was also important in PMA-induced superoxide production by murine neutrophils, and PI-3K and PLC were dispensable when neutrophils were costimulated with PMA and AA. Thus, we were unable to identify a specific target pathway that was regulated in a complementary manner by AA and not by PMA.

Although cPLA2 has been reported to regulate the phagocyte NADPH oxidase [58 , 60 , 78 , 79 ], we determined that reduced NADPH oxidase activity in rac2-/- neutrophils does not reflect defects in cPLA2 activation. The absence of Rac2 did not alter cPLA2 expression or PMA-stimulated AA release in murine neutrophils, and furthermore, cPLA2 inhibitors had no effect on superoxide production in wild-type cells, confirming other recent studies using cPLA2 inhibitors or genetic deletion of murine cPLA2 [61 , 64 ]. Thus, the marked enhancement of NADPH oxidase activity in rac2-/- neutrophils by exogenous AA does not reflect effects of impaired endogenous AA production in the absence of Rac2.

In conclusion, this study provides new insights into the role of Rac2 in regulating neutrophil oxidant production. We establish that Rac2 is not required for membrane translocation of p47phox and p67phox in murine neutrophils but is necessary for optimal activity of the assembled complex. Whether p47phox and p67phox translocation requires Rac1 remains to be determined; at least in heterologous cell models of NADPH oxidase assembly, activation of Rac1 can promote p47phox translocation [80 ]. We also show that exogenous AA allows the PMA-assembled enzyme to function at a high level in the absence of Rac2.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH Grants PO1HL69974 and R01HL45635, the Riley Children’s Foundation, and Korea Research Foundation Grant (KRF-2004-041-E00133). We greatly thank Natalie Stull and Hyung Sim Choi for taking care of mice, Dr. Kerry Bemis (Indiana Center for Applied Protein Science) for helping with statistical interpretations, and Shari Upchurch for managing preparation of this manuscript.

Received July 8, 2005; revised August 22, 2005; accepted August 23, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nauseef, W. M. (2004) Assembly of the phagocyte NADPH oxidase Histochem. Cell Biol. 122,277-291[CrossRef][Medline]
  2. Babior, B. M. (1999) NADPH oxidase: an update Blood 93,1464-1476[Free Full Text]
  3. Dinauer, M. (2003) Regulation of neutrophil function by Rac GTPases Curr. Opin. Hematol. 10,8-15[CrossRef][Medline]
  4. Groemping, Y., Rittinger, K. (2005) Activation and assembly of the NADPH oxidase: a structural perspective Biochem. J. 386,401-416[CrossRef][Medline]
  5. Koga, H., Terasawa, H., Nunoi, H., Takeshige, K., Inagaki, F., Sumimoto, H. (1999) Tetratricopeptide repeat (TPR) motifs of p67(phox) participate in interaction with the small GTPase Rac and activation of the phagocyte NADPH oxidase J. Biol. Chem. 274,25051-25060[Abstract/Free Full Text]
  6. Diekmann, D., Abo, A., Johnston, C., Segal, A. W., Hall, A. (1994) Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity Science 265,531-533[Abstract/Free Full Text]
  7. Diebold, B. A., Bokoch, G. M. (2001) Molecular basis for Rac2 regulation of phagocyte NADPH oxidase Nat. Immunol. 2,211-215[CrossRef][Medline]
  8. Heyworth, P. G., Knaus, U. G., Xu, X., Uhlinger, D. J., Conroy, L., Bokoch, G. M., Curnutte, J. T. (1993) Requirement for posttranslational processing of Rac GTP-binding proteins for activation of human neutrophil NADPH oxidase Mol. Biol. Cell 4,261-269[Abstract]
  9. Roberts, A., Kim, C., Zhen, L., Lowe, J., Kapur, R., Petryniak, B., Spaetti, A., Pollock, J., Borneo, J., Bradford, G., Atkinson, S., Dinauer, M., Williams, D. (1999) Deficiency of the hematopoietic cell-specific Rho-family GTPase, Rac2, is characterized by abnormalities in neutrophil function and host defense Immunity 10,183-196[CrossRef][Medline]
  10. Kim, C., Dinauer, M. C. (2001) Rac2 is an essential regulator of neutrophil nicotinamide adenine dinucleotide phosphate oxidase activation in response to specific signaling pathways J. Immunol. 166,1223-1232[Abstract/Free Full Text]
  11. Yamauchi, A., Marchal, C. C., Molitoris, J., Pech, N., Knaus, U., Towe, J., Atkinson, S. J., Dinauer, M. C. (2005) Rac GTPase isoform-specific regulation of NADPH oxidase and chemotaxis in murine neutrophils in vivo: role of the C-terminal polybasic domain J. Biol. Chem. 280,953-964[Abstract/Free Full Text]
  12. Yamauchi, A., Kim, C., Li, S., Marchal, C. C., Towe, J., Atkinson, S. J., Dinauer, M. C. (2004) Rac2-deficient murine macrophages have selective defects in superoxide production and phagocytosis of opsonized particles J. Immunol. 173,5971-5979[Abstract/Free Full Text]
  13. Glogauer, M., Marchal, C. C., Zhu, F., Worku, A., Clausen, B. E., Foerster, I., Marks, P., Downey, G. P., Dinauer, M., Kwiatkowski, D. J. (2003) Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions J. Immunol. 170,5652-5657[Abstract/Free Full Text]
  14. McPhail, L. C., Waite, K. A., Regier, D. S., Nixon, J. B., Qualliotine-Mann, D., Zhang, W. X., Wallin, R., Sergeant, S. (1999) A novel protein kinase target for the lipid second messenger phosphatidic acid Biochim. Biophys. Acta 1439,277-290[Medline]
  15. Nishizuka, Y. (1995) Protein kinase C and lipid signaling for sustained cellular responses FASEB J 9,484-496[Abstract]
  16. Newton, A. C. (1995) Protein kinase C: structure, function, and regulation J. Biol. Chem. 270,28495-28498[Free Full Text]
  17. Majumdar, S., Kane, L. H., Rossi, M. W., Volpp, B. D., Nauseef, W. M., Korchak, H. M. (1993) Protein kinase C isotypes and signal-transduction in human neutrophils: selective substrate specificity of calcium-dependent ß-PKC and novel calcium-independent nPKC Biochim. Biophys. Acta 1176,276-286[Medline]
  18. Korchak, H. M., Rossi, M. W., Kilpatrick, L. E. (1998) Selective role for ß-protein kinase C in signaling for O-2 generation but not degranulation or adherence in differentiated HL60 cells J. Biol. Chem. 273,27292-27299[Abstract/Free Full Text]
  19. He, R., Nanamori, M., Sang, H., Yin, H., Dinauer, M. C., Ye, R. D. (2004) Reconstitution of chemotactic peptide-induced nicotinamide adenine dinucleotide phosphate (reduced) oxidase activation in transgenic COS-phox cells J. Immunol. 173,7462-7470[Abstract/Free Full Text]
  20. Dekker, L. V., Leitges, M., Altschuler, G., Mistry, N., McDermott, A., Roes, J., Segal, A. W. (2000) Protein kinase C-ß contributes to NADPH oxidase activation in neutrophils Biochem. J. 347,285-289[Medline]
  21. Dang, P. M., Fontayne, A., Hakim, J., El Benna, J., Perianin, A. (2001) Protein kinase C {zeta} phosphorylates a subset of selective sites of the NADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst J. Immunol. 166,1206-1213[Abstract/Free Full Text]
  22. Brown, G. E., Stewart, M. Q., Liu, H., Ha, V. L., Yaffe, M. B. (2003) A novel assay system implicates PtdIns(3,4)P(2), PtdIns(3)P, and PKC {delta} in intracellular production of reactive oxygen species by the NADPH oxidase Mol. Cell 11,35-47[CrossRef][Medline]
  23. Lopes, L. R., Hoyal, C. R., Knaus, U. G., Babior, B. M. (1999) Activation of the leukocyte NADPH oxidase by protein kinase C in a partially recombinant cell-free system J. Biol. Chem. 274,15533-15537[Abstract/Free Full Text]
  24. Fontayne, A., Dang, P. M., Gougerot-Pocidalo, M. A., El-Benna, J. (2002) Phosphorylation of p47phox sites by PKC {alpha}, ß II, {delta}, and {zeta}: effect on binding to p22phox and on NADPH oxidase activation Biochemistry 41,7743-7750[CrossRef][Medline]
  25. Shiose, A., Sumimoto, H. (2000) Arachidonic acid and phosphorylation synergistically induce a conformational change of p47phox to activate the phagocyte NADPH oxidase J. Biol. Chem. 275,13793-13801[Abstract/Free Full Text]
  26. Tsao, L. T., Wang, J. P. (1997) Translocation of protein kinase C isoforms in rat neutrophils Biochem. Biophys. Res. Commun. 234,412-418[CrossRef][Medline]
  27. Lopez-Lago, M., Lee, H., Cruz, C., Movilla, N., Bustelo, X. R. (2000) Tyrosine phosphorylation mediates both activation and downmodulation of the biological activity of Vav Mol. Cell. Biol. 20,1678-1691[Abstract/Free Full Text]
  28. Li, S., Yamauchi, A., Marchal, C., Molitoris, J., Quilliam, L. A., Dinauer, M. (2002) Chemoattractant-stimulated Rac activation in wild-type and Rac2-deficient murine neutrophils: preferential activation of Rac2 and Rac2 gene dosage effect on neutrophil functions J. Immunol. 169,5043-5051[Abstract/Free Full Text]
  29. Henderson, B., Hardingham, T., Blake, S., Lewthwaite, J. (1993) Experimental arthritis models in the study of the mechanisms of articular cartilage loss in rheumatoid arthritis. Joint Destruction in Arthritis and Osteoarthritis, ,15-26
  30. Dana, R., Malech, H. L., Levy, R. (1994) The requirement for phospholipase A2 for activation of the assembled NADPH oxidase in human neutrophils Biochem. J. 297,217-223[Medline]
  31. Chien, K. R., Han, A., Sen, A., Buja, L. M., Willerson, J. T. (1984) Accumulation of unesterified arachidonic acid in ischemic canine myocardium. Relationship to a phosphatidylcholine deacylation-reacylation cycle and the depletion of membrane phospholipids Circ. Res. 54,313-322[Abstract/Free Full Text]
  32. Khan, W. A., Blobe, G. C., Hannun, Y. A. (1995) Arachidonic acid and free fatty acids as second messengers and the role of protein kinase C Cell. Signal. 7,171-184[CrossRef][Medline]
  33. Hii, C. S., Huang, Z. H., Bilney, A., Costabile, M., Murray, A. W., Rathjen, D. A., Der, C. J., Ferrante, A. (1998) Stimulation of p38 phosphorylation and activity by arachidonic acid in HeLa cells, HL60 promyelocytic leukemic cells, and human neutrophils. Evidence for cell type-specific activation of mitogen-activated protein kinases J. Biol. Chem. 273,19277-19282[Abstract/Free Full Text]
  34. Cui, X. L., Douglas, J. G. (1997) Arachidonic acid activates c-jun N-terminal kinase through NADPH oxidase in rabbit proximal tubular epithelial cells Proc. Natl. Acad. Sci. USA 94,3771-3776[Abstract/Free Full Text]
  35. Rao, G. N., Baas, A. S., Glasgow, W. C., Eling, T. E., Runge, M. S., Alexander, R. W. (1994) Activation of mitogen-activated protein kinases by arachidonic acid and its metabolites in vascular smooth muscle cells J. Biol. Chem. 269,32586-32591[Abstract/Free Full Text]
  36. Smith, R. J., Sam, L. M., Justen, J. M., Leach, K. L., Epps, D. E. (1987) Human polymorphonuclear neutrophil activation with arachidonic acid Br. J. Pharmacol. 91,641-649[Medline]
  37. Liu, J., Liu, Z., Chuai, S., Shen, X. (2003) Phospholipase C and phosphatidylinositol 3-kinase signaling are involved in the exogenous arachidonic acid-stimulated respiratory burst in human neutrophils J. Leukoc. Biol. 74,428-437[Abstract/Free Full Text]
  38. McPhail, L. C., Clayton, C. C., Snyderman, R. (1984) A potential second messenger role for unsaturated fatty acids: activation of Ca2+-dependent protein kinase Science 224,622-625[Abstract/Free Full Text]
  39. O’Flaherty, J. T., Chadwell, B. A., Kearns, M. W., Sergeant, S., Daniel, L. W. (2001) Protein kinases C translocation responses to low concentrations of arachidonic acid J. Biol. Chem. 276,24743-24750[Abstract/Free Full Text]
  40. Grenier, S., Flamand, N., Pelletier, J., Naccache, P. H., Borgeat, P., Bourgoin, S. G. (2003) Arachidonic acid activates phospholipase D in human neutrophils; essential role of endogenous leukotriene B4 and inhibition by adenosine A2A receptor engagement J. Leukoc. Biol. 73,530-539[Abstract/Free Full Text]
  41. Abramson, S. B., Leszczynska-Piziak, J., Weissmann, G. (1991) Arachidonic acid as a second messenger. Interactions with a GTP-binding protein of human neutrophils J. Immunol. 147,231-236[Abstract]
  42. Badwey, J. A., Curnutte, J. T., Karnovsky, M. L. (1981) cis-Polyunsaturated fatty acids induce high levels of superoxide production by human neutrophils J. Biol. Chem. 256,12640-12643[Abstract/Free Full Text]
  43. Kim, C., Marchal, C. C., Penninger, J., Dinauer, M. C. (2003) The hemopoietic Rho/Rac guanine nucleotide exchange factor Vav1 regulates N-formyl-methionyl-leucyl-phenylalanine-activated neutrophil functions J. Immunol. 171,4425-4430[Abstract/Free Full Text]
  44. Ingram, D. A., Yang, F. C., Travers, J. B., Wenning, M. J., Hiatt, K., New, S., Hood, A., Shannon, K., Williams, D. A., Clapp, D. W. (2000) Genetic and biochemical evidence that haploinsufficiency of the Nf1 tumor suppressor gene modulates melanocyte and mast cell fates in vivo J. Exp. Med. 191,181-188[Abstract/Free Full Text]
  45. Hazan, I., Dana, R., Granot, Y., Levy, R. (1997) Cytosolic phospholipase A2 and its mode of activation in human neutrophils by opsonized zymosan. Correlation between 42/44 kDa mitogen-activated protein kinase, cytosolic phospholipase A2 and NADPH oxidase Biochem J. 326,867-876[Medline]
  46. Kramer, R. M., Roberts, E. F., Hyslop, P. A., Utterback, B. G., Hui, K. Y., Jakubowski, J. A. (1995) Differential activation of cytosolic phospholipase A2 (cPLA2) by thrombin and thrombin receptor agonist peptide in human platelets. Evidence for activation of cPLA2 independent of the mitogen-activated protein kinases ERK1/2 J. Biol. Chem. 270,14816-14823[Abstract/Free Full Text]
  47. Kent, J. D., Sergeant, S., Burns, D. J., McPhail, L. C. (1996) Identification and regulation of protein kinase C-{delta} in human neutrophils J. Immunol. 157,4641-4647[Abstract]
  48. Smallwood, J. I., Malawista, S. E. (1992) Protein kinase C isoforms in human neutrophil cytoplasts J. Leukoc. Biol. 51,84-92[Abstract]
  49. Dang, P. M., Hakim, J., Perianin, A. (1994) Immunochemical identification and translocation of protein kinase C {zeta} in human neutrophils FEBS Lett 349,338-342[CrossRef][Medline]
  50. Hii, C. S., Moghadammi, N., Dunbar, A., Ferrante, A. (2001) Activation of the phosphatidylinositol 3-kinase-Akt/protein kinase B signaling pathway in arachidonic acid-stimulated human myeloid and endothelial cells: involvement of the ErbB receptor family J. Biol. Chem. 276,27246-27255[Abstract/Free Full Text]
  51. Thompson, L. J., Fields, A. P. (1996) ßII Protein kinase C is required for the G2/M phase transition of cell cycle J. Biol. Chem. 271,15045-15053[Abstract/Free Full Text]
  52. Laudanna, C., Mochly-Rosen, D., Liron, T., Constantin, G., Butcher, E. C. (1998) Evidence of {zeta} protein kinase C involvement in polymorphonuclear neutrophil integrin-dependent adhesion and chemotaxis J. Biol. Chem. 273,30306-30315[Abstract/Free Full Text]
  53. Chodniewicz, D., Zhelev, D. V. (2003) Chemoattractant receptor-stimulated F-actin polymerization in the human neutrophil is signaled by 2 distinct pathways Blood 101,1181-1184[Abstract/Free Full Text]
  54. Sun, C. X., Downey, G. P., Zhu, F., Koh, A. L., Thang, H., Glogauer, M. (2004) Rac1 is the small GTPase responsible for regulating the neutrophil chemotaxis compass Blood 104,3758-3765[Abstract/Free Full Text]
  55. Myhre, O., Vestad, T. A., Sagstuen, E., Aarnes, H., Fonnum, F. (2000) The effects of aliphatic (n-nonane), naphtenic (1,2, 4-trimethylcyclohexane), and aromatic (1,2,4-trimethylbenzene) hydrocarbons on respiratory burst in human neutrophil granulocytes Toxicol. Appl. Pharmacol. 167,222-230[CrossRef][Medline]
  56. Peppelenbosch, M., Qiu, R., de Vries-Smits, A., Tertoolen, L., de Laat, S., McCormick, F., Hall, A., Symons, M., Bos, J. (1995) Rac mediates growth factor-induced arachidonic acid release Cell 81,849-856[CrossRef][Medline]
  57. Peppelenbosch, M. P., Tertoolen, L. G., Hage, W. J., de Laat, S. W. (1993) Epidermal growth factor-induced actin remodeling is regulated by 5-lipoxygenase and cyclooxygenase products Cell 74,565-575[CrossRef][Medline]
  58. Dana, R., Leto, T., Malech, H., Levy, R. (1998) Essential requirement of cytosolic phospholipase A2 for activation of the phagocyte NADPH oxidase J. Biol. Chem. 273,441-445[Abstract/Free Full Text]
  59. Pessach, I., Leto, T. L., Malech, H. L., Levy, R. (2001) Essential requirement of cytosolic phospholipase A(2) for stimulation of NADPH oxidase-associated diaphorase activity in granulocyte-like cells J. Biol. Chem. 276,33495-33503[Abstract/Free Full Text]
  60. Shmelzer, Z., Haddad, N., Admon, E., Pessach, I., Leto, T. L., Eitan-Hazan, Z., Hershfinkel, M., Levy, R. (2003) Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes J. Cell Biol. 162,683-692[Abstract/Free Full Text]
  61. Rubin, B. B., Downey, G. P., Koh, A., Degousee, N., Ghomashchi, F., Nallan, L., Stefanski, E., Harkin, D. W., Sun, C., Smart, B. P., Lindsay, T. F., Cherepanov, V., Vachon, E., Kelvin, D., Sadilek, M., Brown, G. E., Yaffe, M. B., Plumb, J., Grinstein, S., Glogauer, M., Gelb, M. H. (2005) Cytosolic phospholipase A2-{alpha} is necessary for platelet-activating factor biosynthesis, efficient neutrophil-mediated bacterial killing, and the innate immune response to pulmonary infection: cPLA2-{alpha} does not regulate neutrophil NADPH oxidase activity J. Biol. Chem. 280,7519-7529[Abstract/Free Full Text]
  62. Xing, M., Wilkins, P. L., McConnell, B. K., Mattera, R. (1994) Regulation of phospholipase A2 activity in undifferentiated and neutrophil-like HL60 cells. Linkage between impaired responses to agonists and absence of protein kinase C-dependent phosphorylation of cytosolic phospholipase A2 J. Biol. Chem. 269,3117-3124[Abstract/Free Full Text]