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The Journal of Immunology, 2004, 173: 7462-7470.
Copyright © 2004 by The American Association of Immunologists

Reconstitution of Chemotactic Peptide-Induced Nicotinamide Adenine Dinucleotide Phosphate (Reduced) Oxidase Activation in Transgenic COS-phox Cells1

Rong He*, Masakatsu Nanamori*, Hairong Sang*, Hong Yin*, Mary C. Dinauer{dagger} and Richard D. Ye2,*

* Department of Pharmacology, College of Medicine, University of Illinois, Chicago, IL 60612; and {dagger} Herman B. Wells Center for Pediatric Research, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, IN 46202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A whole-cell-based reconstitution system was developed to study the signaling mechanisms underlying chemoattractant-induced activation of NADPH oxidase. This system takes advantage of the lack of formyl peptide receptor-mediated response in COS-phox cells expressing gp91phox, p22phox, p67phox, and p47phox, which respond to phorbol ester and arachidonic acid with O

{cjs1138}

2 production. By exogenous expression of signaling molecules enriched in neutrophils, we have identified several critical components for fMLP-induced NADPH oxidase activation. Expression of PKC{delta}, but not PKC{alpha}, -{beta}II, and -{zeta}, is necessary for the COS-phox cells to respond to fMLP. A role of PKC{delta} in neutrophil NADPH oxidase was confirmed based on the ability of fMLP to induce PKC{delta} translocation and the sensitivity of fMLP-induced O

{cjs1138}

2 production to rottlerin, a PKC{delta}-selective inhibitor. Optimal reconstitution also requires phospholipase C-{beta}2 and PI3K-{gamma}. We found that formyl peptide receptor could use the endogenous Rac1 as well as exogenous Rac1 and Rac2 for NADPH oxidase activation. Exogenous expression of p40phox potentiated fMLP-induced O

{cjs1138}

2 production and raised the level of O

{cjs1138}

2 in unstimulated cells. Collectively, these results provide first direct evidence for reconstituting fMLP-induced O

{cjs1138}

2 production in a nonhemopoietic cell line, and demonstrate the requirement of multiple signaling components for optimal activation of NADPH oxidase by a chemoattractant.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The phagocyte NADPH oxidase activity is vitally important for host defense against invading microorganisms. In neutrophils, O_.2 production requires the phagocyte oxidase (phox)3 components gp91phox, p22phox, p67phox, and p47phox (reviewed in Ref.1). Defect in any one of these components compromises the ability of neutrophils to produce O

{cjs1138}

2 and eliminate bacteria, as seen in patients suffering from chronic granulomatous diseases (2, 3). Extensive studies have been conducted to characterize the NADPH oxidase and its activation mechanism. It has been established that the active NADPH oxidase is a complex consisting of the membrane-associated proteins gp91phox and p22phox, and the cytosolic proteins p67phox and p47phox, as well as the small GTPase Rac1 and/or Rac2 (reviewed in Ref.1). Phosphorylation of p47phox and p67phox, and activation of Rac, are critical steps preceding membrane translocation of the cytosolic factors and formation of a functional NADPH oxidase complex at the plasma membrane (1). The exact function of another cytosolic protein, p40phox, remains incompletely understood.

Biological processes that trigger the activation of phagocyte NADPH oxidase include phagocytosis of opsonized bacteria and binding of chemoattractants such as fMLP and C5a to their cell surface receptors. These events induce O

{cjs1138}

2 production through activation of Fc{gamma}Rs and G protein-coupled chemoattractant receptors, respectively. In addition, pharmacological agents such as phorbol esters (e.g., PMA) and amphiphilic molecules (e.g., arachidonic acid and SDS) are potent stimuli of the phagocyte NADPH oxidase. These agents are extensively used in characterization of the NADPH oxidase activation mechanisms in both intact cells and cell-free reconstitution assays. The development of the cell-free assay for in vitro reconstitution of NADPH oxidase activity (4, 5, 6, 7) has greatly accelerated the characterization of individual phox proteins and their interactions during assembly of a functional NADPH oxidase complex. However, reconstitution of receptor-mediated NADPH oxidase activation remains difficult in the cell-free system, and there are discrepancies in data collected from the cell-free assays and whole-cell-based experiments. For example, the Src homology 3 (SH3) domain located near the C terminus of p67phox is essential for NADPH oxidase activation in intact cells, whereas it is not required in the cell-free assay (8). Furthermore, prenylated Rac1 is sufficient to initiate NADPH oxidase assembly in cell-free assays (9) but unable to mediate fMLP-elicited superoxide generation in neutrophils derived from Rac2 knockout mice (10, 11). These findings imply that NADPH oxidase activation in intact cells requires additional signaling components that are not essential in the cell-free assays.

The chemoattractant fMLP is a potent activator of phagocyte NADPH oxidase. fMLP induces a rapid and transient O

{cjs1138}

2 generation in phagocytes through binding to the G{alpha}i-coupled formyl peptide receptor (FPR) (12, 13). It is presumed that the same NADPH oxidase components are used for both fMLP-elicited and PMA-stimulated response, but additional signaling events must exist for receptor-mediated activation of protein kinase C (PKC) and the Rac small GTPase, and possibly for phosphorylation and final assembly of the phox proteins. An understanding of the related signaling mechanisms is important for the control of undesirable activation of phagocytes, which contributes to the release of oxidants and tissue damage. Genetic studies involving targeted deletion of phospholipase C-{beta} (PLC{beta})2/3 and PI3K{gamma} have shown that these enzymes are important for chemoattractant-induced NADPH oxidase activation (reviewed in Ref.14). However it is not clear whether activation of these signaling molecules is sufficient for O

{cjs1138}

2 production, and how they interact with kinases and GTPases that are directly responsible for the phosphorylation and translocation of phox proteins. An area of significant interest is the involvement of individual PKC isoforms in fMLP-induced NADPH oxidase activation. Unlike PMA that can activate multiple PKCs, signaling through FPR may only stimulate selected PKC isoforms. Another unresolved issue is the vast disparity among chemoattractant and chemokine receptors in their ability to activate NADPH oxidase. Although all these receptors are capable of mediating leukocyte chemotaxis, only a small fraction of them can stimulate O

{cjs1138}

2 production. These unanswered questions necessitate the development of reconstitution systems in which receptor-mediated NADPH oxidase can be studied in detail.

To accomplish this goal, we explored several possibilities for reconstituting FPR-mediated NADPH oxidase activation in intact cells. A previous study demonstrates that expression of gp91phox, p67phox, and p47phox in the erythroleukemia cell line K562 renders the cells responsive to PMA in O

{cjs1138}

2 production assay (15). Reconstitution of NADPH oxidase activity was also achieved by expression of p47phox in EBV-transformed B cells that lack this cytosolic factor (16). In both cases, the reconstituted cells produced a relatively small amount of O

{cjs1138}

2 compared with neutrophils. More recently, one of our laboratories generated a stable COS-7 line expressing gp91phox, p22phox, p67phox, and p47phox (the COS-phox cell line) (17). Stimulation of these cells with PMA and arachidonic acid led to potent production of O

{cjs1138}

2, suggesting the possibility of using these genetically amenable cells to identify signaling molecules downstream of the activated FPR. Whereas K562 is a cell line of hemopoietic lineage and may already have the necessary signaling components for FPR signaling, COS-7 is an epithelial cell line that lacks the hemopoietic specific proteins required for FPR signaling (18). We exploited this property of the COS-phox cells to examine the roles of selected signaling molecules in FPR-mediated NADPH oxidase activation by taking a gain-of-function approach. In this study, we report reconstitution of fMLP-induced NADPH oxidase activity in the transgenic COS-phox cells. Our results indicate that the novel PKC isoform, PKC{delta}, plays an important role in fMLP-induced O

{cjs1138}

2 production in both the transfected COS-phox cells and human neutrophils. Our results also suggest a role of p40phox in positive regulation of fMLP-induced NADPH oxidase activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

The N-formyl peptide fMLP, PMA, and isoluminol were purchased from Sigma-Aldrich (St. Louis, MO). HRP and superoxide dismutase (SOD) were purchased from Roche (Indianapolis, IN). Pertussis toxin was obtained from List Laboratories (Campbell, CA). LY294002, GF109203X, and rottlerin were obtained from Calbiochem (San Diego, CA). Polyclonal rabbit serum against G{alpha}i2 was prepared against a synthetic peptide with the sequence CAKNNLKDCGLF. The following Abs were purchased from the indicated sources (in parentheses): mouse mAbs against Myc-epitope tag and HA-epitope tag (Covance, Richmond, CA), rabbit polyclonal Abs against PLC{beta}2 and PKC{beta}II (Santa Cruz Biotechnology, Santa Cruz, CA), a mouse mAb against p40phox (Upstate, Lake Placid, NY), a mouse mAb to {beta}-actin (Santa Cruz), a mouse mAb against Rac1 (BD Pharmingen, San Diego, CA), and rabbit polyclonal Abs against nonphosphorylated and phospho-PKC{delta} (Th505) (Cell Signaling Technologies, Beverly, MA).

Expression vectors

A full-length cDNA for human FPR was subcloned into the pRK5 vector (BD Pharmingen). Plasmids containing cDNA inserts for wild-type G{alpha}i proteins were gifts from Drs. C. Knall and G. Johnson (National Jewish Center, Denver, CO). The PLC{beta}2 expression vector was a gift from Dr. D. Wu (University of Connecticut Health Center, Farmington, CT). Preparation and characterization of the HA-tagged PKC{alpha}, PKC{delta}, and PKC{zeta} expression constructs were described in a previous publication (19). The GFP expression vector EGFP-N1 was from Clontech (Palo Alto, CA). A full-length cDNA for human Rac1 (Guthrie Research Institute, Sayre, PA) was subcloned into the pRK5 expression vector (BD Pharmingen). A Myc-tagged Rac1 expression construct was provided by Dr. U. Knaus (Scripps Research Institute, La Jolla, CA). Myc-tagged p110{gamma} and p101 constructs were provided by Dr. A. Smrcka (University of Rochester, Rochester, NY). The p40phox expression vector was a gift from Dr. S. Chanock (National Cancer Institute, National Institutes of Health, Bethesda, MD).

Preparation of human neutrophils

Peripheral blood was drawn from healthy donors, using a protocol approved by the Institutional Review Board at the University of Illinois (Chicago, IL). Neutrophils were prepared using Percoll gradient centrifugation based on the method of Ulmer and Flad (20), as detailed in a previous publication (21). The prepared cells contained ~97% neutrophils with viability ≥98%. Neutrophils were resuspended in serum-free RPMI 1640 medium at a density of 2 x 106 cells/ml before use. Blood cells from different donors (n ≥ 3) were used in experiments.

Cell culture and transient transfection

The transgenic COS-phox cells were generated as described previously (17). The stable cell line was maintained at 37°C with 5% CO2 in DMEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 IU/ml penicillin, and 50 µg/ml streptomycin. Cells were grown by limiting dilution in the presence of 0.2 mg/ml hygromycin (Sigma-Aldrich), 0.8 mg/ml neomycin (Invitrogen Life Technologies, Carlsbad, CA), and 1 µg/ml puromycin (Calbiochem). LipofectAMINE 2000 reagent (Invitrogen Life Technologies) was used for transient transfection of 6–7 µg of DNA into COS-phox cells grown in a 100-mm culture dish (0.5–1 x 106 cells per dish). Cells were analyzed 21–24 h after transfection. Transient transfection efficiency, determined by flow cytometry based on fluorescence of a cotransfected GFP, was 45–55%.

Measurement of NADPH oxidase activity

Superoxide production by COS-phox cells and neutrophils was determined by an isoluminol-ECL assay (22), in 6-mm diameter wells of 96-well, flat-bottom, white tissue culture plates (E&K Scientific, Campbell, CA). COS-phox cells were harvested with enzyme-free cell-dissociation buffer (Invitrogen Life Technologies), and washed once with 0.5% BSA/HBSS. Cells were then resuspended in 0.5% BSA/RPMI 1640 buffer at the density of 3–5 x 106 cells/ml, and preincubated in the dark with 100 µM isoluminol and 40 U/ml HRP at room temperature for 5 min. Aliquot (200 µl) of the cells was added into the well and assayed for chemiluminescence (CL) at 37°C in a Wallac 1420 Multilabel Counter (PerkinElmer Life Sciences, Boston, MA). The CL counts per second (cps) was continually recorded, at 1-min intervals, for 5–15 min before and 20–30 min after stimulation with PMA or fMLP. Samples containing 250 U of SOD, in addition to the stimulators, were run in parallel. The relative level of superoxide produced was calculated based on the integrated CL during the first 20 min (COS-phox cells) or first 10 min (neutrophils) after agonist stimulation.

Analysis of protein expression

Whole-cell extracts were generated as described previously (21). In brief, the transfected COS-phox cells were lysed with 200–500 µl of PAGE buffer containing protease inhibitors (Protease Inhibitor Mixture Set I; Calbiochem). Each sample was sonicated for 15 s on ice (60 Sonc Dismembrator; Fisher, Hampton, NH) and heated at 95°C for 5 min. Whole-cell extracts were analyzed by 10% denaturing SDS gels, and protein profiles were then transferred to nitrocellulose membranes (Hybond ECL; Amersham Biosciences, Piscataway, NJ) for Western blotting using ECL detection (Pierce, Rockford, IL).

Flow cytometry measurements were used to determine the cell surface expression of FPR. Briefly, the transfected COS-phox cells were incubated with an anti-FPR mAb 5F1 (BD Pharmingen), washed in PBS containing 0.2% BSA, and then incubated with FITC-conjugated anti-mouse IgG (1:200). The green fluorescence of each single cell was detected using a FACScan flow cytometer (BD Biosciences, Mountain View, CA). All incubations were done on ice for 60 min.

Rac activation assay

Activation of Rac was determined as previously described (17), based on the affinity of Rac-GTP for the p21-binding domain (PBD) of PAK1 (23). The PBD-GST fusion protein was expressed in Escherichia coli strain HB101 and purified. Twenty-one hours posttransfection, COS-phox cells were detached with dissociation buffer, washed, and resuspended in RPMI 1640 containing 0.5% BSA to 5 x 106 cells/0.5 ml. The cells were then stimulated with fMLP or left untreated as indicated in the figures. Twenty micrograms of PAK1 PBD-GST recombinant protein was added, and cells were lysed by the addition of lysis/wash buffer (6 mM Na2HPO4, 4 mM NaH2PO4, 1% Nonidet P-40, 150 mM NaCl, 2.5 mM MgCl2) supplemented with 2 mM PMSF, Protease Inhibitor Cocktail III (Calbiochem), 0.1 mM Na3VO4, and 50 mM NaF. The lysate was cleared, 30 µl of glutathione-Sepharose 4B beads (Amersham Biosciences) was added, and the binding reaction was conducted for 1 h at 4°C. Beads were pelleted and washed three times with wash buffer, and then finally resuspended in 30 µl of Laemmli sample buffer. Aliquots of supernatant (Rac-GDP) and pull-down samples (Rac-GTP) were electrophoresed, and the proteins were analyzed as described above.

PKC phosphorylation and translocation

Isolated neutrophils (1–5 x 107 cells) were stimulated with either PMA or fMLP over a time course of 0–30 min, and stopped by addition of a 10-fold excess volume of cold HBSS. Cells were treated with 1 mM diisopropylfluorophosphate, suspended in hypotonic lysis buffer (50 mM Tris-HCl (pH 7.5), 5 mM MgSO4, 0.5 mM EGTA, 0.1% 2-ME) supplemented with 1 mM PMSF and protease inhibitors at a concentration of 1 x 108 cells/ml, and sonicated (5 s, three times, at level 2) on ice. The sonicated samples were centrifuged at 800 x g for 10 min at 4°C, and the supernatants were subjected to differential centrifugation at 150,000 x g for 90 min at 4°C to yield cytosolic fractions (supernatants) and membrane/particulate fractions (pellets). Membrane/particulate fractions were subject to Western blotting to detect PKC{delta} translocation by using a specific anti-PKC{delta} Ab (Cell Signaling Technologies). For PKC phosphorylation assay, the stimulated neutrophils were directly lysed with 200–500 µl of SDS-PAGE buffer containing protease inhibitors, sonicated for 15 s on ice, and heated at 95°C for 5 min. Whole-cell extracts were analyzed by 10% denaturing SDS gels and phospho-PKC{delta} was detected by Western blotting using a specific anti-phospho-PKC{delta} Ab (Cell Signaling Technologies).

Statistics

Paired Student’s t test was performed to determine statistical significance. A p value of <0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of FPR in COS-phox is insufficient for reconstitution of fMLP-induced O

{cjs1138}

2 production

The COS-phox cell line was generated by stable expression of gp91phox, p22phox, p67phox, and p47phox in the monkey epithelial cell line COS-7 (17). Expression of these components enables the epithelial cells to respond to arachidonic acid and PMA with potent O

{cjs1138}

2 production (17) (Fig. 1). The PMA-induced O

{cjs1138}

2 generation was dose dependent, could be inhibited by SOD (Fig. 1, A and B), and displayed kinetics similar to that of neutrophils (Fig. 1A and data not shown). To determine whether COS-phox cells respond to fMLP, we expressed the human FPR by means of liposome-mediated transfection. Twenty-four hours after transfection, 45–55% of the cells expressed FPR on cell surface as detected by an anti-FPR mAb (Fig. 1C). However, fMLP was unable to induce O

{cjs1138}

2 production in the transfected cells (Fig. 1, A and B), suggesting that COS-phox cells lack the necessary components for FPR-mediated signaling leading to NADPH oxidase activation.



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  		FIGURE 1. Transgenic COS-phox cells respond to PMA but not fMLP in O{cjs1138}2 production assay. COS-phox cells were harvested and preincubated with isoluminol as described in Materials and Methods, and stimulated with PMA (50 and 100 ng/ml) or fMLP (1 µM). A, CL (cps) recorded at 37°C for 65 min, in COS-phox without or with the exogenous FPR (R). SOD (250 U) was included in one sample. The histogram is representative of three independent experiments with similar results. B, Bar chart showing CL integrated during the first 20 min after agonist stimulation. Data shown are mean ± SEM from three experiments. C, A representative histogram showing expression of FPR as detected by flow cytometry using an anti-FPR mAb (5F1) and a FITC-conjugated secondary Ab (solid line).

 
Multiple signaling molecules are required for optimal O

{cjs1138}

2 production in fMLP-stimulated COS-phox cells

Previous studies have shown that PLC{beta}2 and PI3K{gamma} are important for fMLP-induced neutrophil functions including O

{cjs1138}

2 production (14). Because COS-phox cells do not contain these hemopoietic cell-specific enzymes, we speculated that exogenous expression of PLC{beta}2 and/or PI3K{gamma} might render the cells responsive to fMLP. To our surprise, COS-phox cells cotransfected to express PLC{beta}2 and FPR, PI3K{gamma} and FPR, or PLC{beta}2 and PI3K{gamma} with FPR still could not respond to fMLP with detectable O

{cjs1138}

2 production (data not shown), suggesting that these two enzymes were insufficient for reconstitution of FPR-mediated NADPH oxidase activation.

Additional signaling molecules including the G{alpha}i proteins, the small GTPases Rac1, and selected PKC isoforms (shown in Fig. 7 below), were expressed in COS-phox either alone or in combinations. Although none of these molecules could rescue FPR-mediated O

{cjs1138}

2 production when expressed alone, the combined expression of FPR, G{alpha}i2, PI3K{gamma}, PLC{beta}2, Rac1, and PKC{delta} (FPR plus six plasmids (6PL)) resulted in the production of substantial amount of O

{cjs1138}

2 when the cells were stimulated with fMLP. After normalization with transfection efficiency (45–55%), the O

{cjs1138}

2 produced in response to 1 µM fMLP (Fig. 2B) was similar to the O

{cjs1138}

2 generated in cells stimulated with 50 ng/ml PMA (Fig. 1B). Inclusion of SOD in the assay buffer abolished fMLP-induced O

{cjs1138}

2 production as well as spontaneous production of O

{cjs1138}

2 in the 6PL-transfected COS-phox (Fig. 2A). The response to fMLP was rapid for both COS-phox cells and neutrophils, whereas the response to PMA was delayed in both cells.



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  		FIGURE 7. Effects of selected PKC isoforms in fMLP-induced O{cjs1138}2 production. COS-phox cells were transfected with FPR plus 5PL (minus PKC), and with expression construct for one of the four PKC isoforms as indicated. A, The ability of these PKC isoforms to potentiate fMLP-induced O{cjs1138}2 production is shown as percentage change relative to the minimal response induced by fMLP (set as 100%, with FPR plus 5PL plus PKC{delta}). Data were based on the integrated CL collected during the first 20 min, from three independent experiments. B, Expression of the exogenous PKC isoforms as determined by Western blotting, using an anti-HA Ab detecting the tagged PKC{alpha}, PKC{delta}, and PKC{zeta}, and a specific Ab against PKC{beta}II. Representative blots from three to four experiments are shown. C, fMLP-induced PKC{delta} translocation in neutrophils. Membrane translocation of PKC{delta} was determined in PMA (100 ng/ml)- and fMLP (100 nM)-stimulated neutrophils, as described in Materials and Methods. A representative blot, taken from three independent experiments, is shown.

 


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  		FIGURE 2. Reconstitution of fMLP-induced O{cjs1138}2 production in transfected COS-phox cells. COS-phox cells were transiently transfected with expression constructs coding for FPR, G{alpha}i2, PLC{beta}2, PI3K{gamma} (p110{gamma} and p101), PKC{delta}, and Rac1 (FPR plus 6PL). Twenty-four hours after transfection, cells were assayed for O{cjs1138}2 generation. A, A representative histogram showing CL (cps) recorded after addition of 1 µM fMLP for the first 30 min. SOD (250 U) was added to the control. B, CL integrated during the first 20 min after agonist stimulation was quantified and expressed as mean ± SEM, from three independent experiments. C, Expression of the transfected components were detected by Western blotting using either specific Abs (for G{alpha}i2 and PLC{beta}2) or Abs against epitope tags (HA-tagged PKC{delta}, myc-tagged Rac1, and myc-tagged p110{gamma} and p101). {beta}-Actin in the same cell lysate was used as a control for sample loading and efficiency of transfer.

 
Requirement of G{alpha}i coupling to FPR for the reconstitution of NADPH oxidase activity

Because FPR-mediated O

{cjs1138}

2 production requires functional coupling of the receptor to the G{alpha}i class of G proteins in neutrophils (12, 13), we next examined a role of the G{alpha}i proteins in the transfected COS-phox cells. Treatment with pertussis toxin that ADP-ribosylates the G{alpha}i proteins and disrupts their interaction with FPR, abolished fMLP-induced O

{cjs1138}

2 production in the transfected COS-phox cells (Fig. 3B) as well as in human neutrophils (A). Likewise, removal of the exogenous G{alpha}i2 expression construct from the transfection mixture reduced the fMLP-stimulated O

{cjs1138}

2 production by ~68% (Fig. 3B), confirming an important role of G{alpha}i2 in coupling FPR to the downstream signaling pathways. We have also observed that a substitution with the exogenous G{alpha}i3 for G{alpha}i2 resulted in similar level of O

{cjs1138}

2 production, suggesting that FPR can couple to either protein for NADPH oxidase activation.



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  		FIGURE 3. FPR-mediated O{cjs1138}2 generation requires G{alpha}i proteins. A, Neutrophils were either treated with pertussis toxin (PTX; 100 ng/ml; 16 h) or left untreated, and stimulated with fMLP. O{cjs1138}2 production was monitored based on isoluminol-ECL. Similar results were obtained with a shorter PTX treatment (400 ng/ml for 4 h). B, COS-phox cells were transiently transfected with FPR plus 6PL as described above, without G{alpha}i2, or with G{alpha}i3 in place of G{alpha}i2 as indicated. Some samples were incubated for 16 h in RPMI 1640 with 0.5% BSA, in the presence or absence of PTX (100 ng/ml). Cells were stimulated with fMLP (1 µM), and O{cjs1138}2 generation was determined based on isoluminol-ECL (CL). The integrated CL during the first 20 min after stimulation was quantified and expressed as mean ± SEM based on three independent experiments. Maximal response (100%) was determined in fMLP-stimulated cells transfected with FPR and all six plasmids.

 
The relative contributions of exogenous vs endogenous PLC{beta} and PI3K to FPR-mediated NADPH oxidase activation

We next investigated whether PLC{beta}2 and PI3K{gamma}, insufficient by themselves for reconstitution of the FPR-mediated response, are necessary for the fMLP-induced O

{cjs1138}

2 production. COS-phox cells were transfected without either the PLC{beta}2 expression vector, or the two PI3K{gamma} expression plasmids coding for the respective catalytic and regulatory subunits. Omission of the PLC{beta}2 expression vector led to a small (~23%) but statistically significant (p < 0.05) reduction in O

{cjs1138}

2 production. The absence of PI3K{gamma} resulted in a ~75% decrease in O

{cjs1138}

2 production (Fig. 4). These findings suggest possible involvement of the two hemopoietic-specific enzymes in O

{cjs1138}

2 production by fMLP in the transfected cells. The results also indicate that the endogenous PLC{beta} and PI3K isoforms can partially fulfill the functions of PLC{beta}2 and PI3K{gamma}.



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  		FIGURE 4. Requirement of PLC{beta}2 and PI3K{gamma} for optimal O{cjs1138}2 generation. The cells were transiently transfected with FPR plus the six plasmids, or without PLC{beta}2 or PI3K{gamma}. The cells were then assayed for fMLP-stimulated O{cjs1138}2 generation. Isoluminol-ECL (CL) recorded during the first 20 min were integrated and presented as percentage of the maximal response, which was determined in the presence of FPR plus the six plasmids. The reduction in response is statistically significant (p < 0.05 for the –PLC{beta}2 sample, and p < 0.01 for the –PI3K{gamma} sample). The data presented are mean ± SEM from three independent experiments.

 
Roles of Rac1 and Rac2 in the FPR reconstitution assay

The small GTPase Rac is essential for NADPH oxidase activation in neutrophils and in cell-free reconstitution assays (24, 25). We investigated whether Rac also plays a critical role in fMLP-induced O

{cjs1138}

2 generation in the transfected COS-phox cells. In the experiments described below, COS-phox cells were transfected with expression constructs coding for FPR, G{alpha}i2, PI3K{gamma}, PLC{beta}2, and PKC{delta}, with or without the expression vectors for human Rac1 and Rac2. Although the initial successful reconstitution included an expression construct of Rac1, substantial amount of O

{cjs1138}

2 could be produced in its absence (Fig. 5A, first group). This result suggests that FPR is able to use the endogenous Rac1 for activation of NADPH oxidase. In recent years, a critical role of Rac2 in neutrophil NADPH oxidase activation has been reported based on studies of Rac2–/– mice (10, 26). We investigated whether activation of Rac2 is a unique property of hemopoietic cells by exogenous expression of Rac2 in the transfected COS-phox cells. As shown in Fig. 5, Rac2 is absent from COS-phox cells and its expression resulted in a statistically significant (p < 0.05) enhancement of the fMLP-induced O

{cjs1138}

2 production. Activation of Rac under these conditions was determined based on the ability of the N-terminal domain of p21-activated kinase 1 to bind and pull down the activated Rac (23). Results shown in Fig. 5C demonstrate that fMLP induced rapid but transient increases in this binding as detected by Western blotting with either an anti-Rac1 Ab for the endogenous Rac1 or an anti-myc Ab for the exogenous Rac1 and Rac2 recovered from the pull-down assay. The kinetics of Rac activation remained unchanged when Rac1 is overexpressed, with peak activation detected at around 1 min after fMLP stimulation. This is consistent with the rapid generation of O

{cjs1138}

2 in fMLP-stimulated neutrophils and reconstituted COS-phox cells. Activation of Rac2 also peaked at ~1 min (Fig. 5C), and O

{cjs1138}

2 production in Rac2-transfected cells was similarly rapid as was observed in Rac1-transfected cells (data not shown).



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  		FIGURE 5. Effect of endogenous and exogenous Rac on fMLP-induced O{cjs1138}2 generation. A, COS-phox cells were transiently transfected with FPR and the five expression constructs without exogenous Rac, or with exogenous Rac1 or Rac2. The integrated CL (first 20 min) showing percent differences in O{cjs1138}2 production without the exogenous Rac (No ExRac), and with the exogenous Rac1 (ExRac1) or Rac2 (ExRac2). Data shown are mean ± SEM from three experiments. B, A representative gel picture derived from RT-PCR detection of the Rac1 and Rac2 transcripts in COS-phox cells. Control, No reverse transcription products added. cDNA std, Rac1 or Rac2 cDNA (10 ng) was used as standards and positive controls. C, Activation of Rac based on binding to PBD-GST, with total Rac proteins shown as reference for the amounts of proteins used in the assays. An anti-Rac1 Ab was used for detecting the endogenous and exogenous Rac1, and the anti-myc Ab 9E10 was used for detecting the expression of Rac2, which was myc-tagged. fMLP stimulation (1 µM) was conducted for 1, 2, and 5 min as indicated. Three experiments were performed, and a set of representative blots is shown.

 
Expression of p40phox enhances fMLP-induced O

{cjs1138}

2 production in reconstituted COS-phox cells

p40phox is a cytosolic, SH3 domain-containing protein found in myeloid cells (27), but not in COS-7 cells (Fig. 6A). Although p40phox is known to form an active complex with p47phox and p67phox for their membrane translocation (27), its role in NADPH oxidase activation has not been fully established. COS-phox cells respond to PMA and arachidonic acid with potent O

{cjs1138}

2 production (17), and our initial reconstitution of fMLP-induced NADPH oxidase activity was achieved without p40phox (Fig. 2). These observations indicate that p40phox is not essential for NADPH oxidase activation in either PMA- or fMLP-stimulated COS-phox cells.



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  		FIGURE 6. Exogenous expression of p40phox augment fMLP-induced O{cjs1138}2 production. COS-phox cells were transiently transfected with expression constructs for FPR and five plasmids (minus Rac), with or without the p40phox expression vector. A, A representative Western blot showing expression of the transfected p40phox construct (p40). Vec, Vector-transfected cells. {beta}-Actin was shown as a control for sample loading and transfer. B, The fMLP-induced change in O{cjs1138}2 production was shown as a function of time, in the reconstituted COS-phox cells with or without p40phox and stimulated with fMLP or buffer. C, Integrated CL (area under curve; shown as mean ± SEM) from experimental data shown in B and two repeating experiments.

 
Because the current literature suggests that p40phox can be either stimulatory or inhibitory in NADPH oxidase activation, we examined its potential involvement in FPR-reconstituted COS-phox cells. Exogenous expression of p40phox led to a marked (2.8-fold) increase in fMLP-induced O

{cjs1138}

2 production when compared with cells without p40phox (Fig. 6, B and C). An elevation of the basal O

{cjs1138}

2 level in control (unstimulated) cells was also apparent (Fig. 6, B and C). Exogenous expression does not seem to affect the kinetics of fMLP-induced O

{cjs1138}

2 generation, because the rise and fall of O

{cjs1138}

2 level follow a similar pattern in the cells without and with p40phox. Taken together, the above data suggest that p40phox enhances fMLP-induced O

{cjs1138}

2 production in the reconstituted COS-phox cells and can also increase the basal O

{cjs1138}

2 level.

PKC{delta} is critical to fMLP-induced O

{cjs1138}

2 production in COS-phox cells and in human neutrophils

Phosphorylation of p47phox by PKC is essential for translocation of the cytosolic factors and assembly of a functional NADPH oxidase complex (28). In neutrophils, the conventional PKC isoforms PKC{alpha} and PKC{beta}II are believed to mediate PMA-induced O

{cjs1138}

2 production (29). The PKC expression profile in COS-7 cells is different from that of neutrophils. COS-7 cells contain a high level of PKC{alpha}, a moderate level of PKC{zeta}, but only small amounts of PKC{delta} and PKC{beta}II. In contrast, neutrophils express high levels of PKC{beta}II and PKC{delta}, a moderate level of PKC{alpha}, and very little PKC{zeta} (17). To determine which PKC is important for the fMLP-induced O

{cjs1138}

2 production, we expressed the four individual PKC isoforms together with other necessary components in COS-phox cells. As shown in Fig. 7, cells transfected to express FPR, G{alpha}i2, PI3K{gamma}, PLC{beta}2, and Rac1 responded to fMLP stimulation with a small increase in O

{cjs1138}

2 generation over basal level. Cotransfection with a PKC{delta} expression vector markedly enhanced fMLP-stimulated O

{cjs1138}

2 production (by 4-fold), whereas cotransfection with vectors encoding the other three PKC isoforms resulted in statistically insignificant (p > 0.05) changes in O

{cjs1138}

2 production.

Because COS-phox is a nonhemopoietic cell line and can have quite different properties than neutrophils, we next examined whether PKC{delta} is important for fMLP-induced NADPH oxidase activation in human neutrophils. Freshly prepared blood neutrophils were stimulated with either PMA or fMLP, and membrane translocation of PKC{delta} was determined at various time points after stimulation (Fig. 7C). Translocation of PKC{delta} was evident 10 min after PMA stimulation. In fMLP-stimulated neutrophils, PKC{delta} translocation appeared much earlier and was detectable after 1 min (Fig. 7C). This profile is consistent with the kinetics of fMLP-induced O

{cjs1138}

2 production.

We next determined the effects of PKC inhibitors on the fMLP-induced O

{cjs1138}

2 production. Treatment of human neutrophils with GF109203X, a broad PKC inhibitor with selectivity for PKC{alpha} and other conventional PKCs, effectively suppressed PMA-induced O

{cjs1138}

2 production at 0.1–1 µM concentrations (Fig. 8, A and B). In comparison, GF109203X was less effective on fMLP-induced O

{cjs1138}

2 production. At 1 µM, GF109203X inhibited the PMA-induced O

{cjs1138}

2 production by ~85%, but only ~15% for the fMLP-induced O

{cjs1138}

2 production. These results contrast sharply with data derived from a parallel study in which rottlerin, a PKC{delta}-selective inhibitor (IC50 = 3–6 µM), was used. Rottlerin inhibited fMLP-induced O

{cjs1138}

2 production by 65–100% at concentrations of 3–12 µM. Under the same experimental conditions, rottlerin had minimal effect on the PMA-induced O

{cjs1138}

2 production (Fig. 8, C and D).



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  		FIGURE 8. Effect of GF109203X and rottlerin on O{cjs1138}2 production induced by fMLP and PMA. Human neutrophils were pretreated with or without the PKC inhibitors at different concentrations for 15 min, and then stimulated with fMLP (100 nM) or PMA (200 ng/ml). A and C, Histogram showing O{cjs1138}2 generation as a function of time, based on isoluminol-ECL, after treatment with GF109203X (GF) (A) or rottlerin (C). B and D, Integrated CL (mean ± SEM based on three independent experiments) showing different inhibitory effects on PMA vs fMLP-induced O{cjs1138}2 production in cells treated with GF109203X (B) or rottlerin (D).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Investigation of chemoattractant-induced O

{cjs1138}

2 generation has been hampered by the lack of a whole-cell model for reconstitution of receptor-mediated signaling pathways. As a result, our current understanding of how chemoattractants activate NADPH oxidase relies heavily on information obtained with pharmacological activators such as PMA and arachidonic acids. The mechanistic information derived from these studies does not accurately reflect signaling events initiated by chemoattractant receptors. In the current study, we attempted to characterize the proximal signaling events immediately downstream of FPR activation and associate these events with kinase and GTPase activities directly responsible for NADPH oxidase activation. Our approach is based on the assumption that key signaling components for fMLP-induced O

{cjs1138}

2 production may be identified by expression of the relevant proteins in cells that lack them. The transgenic COS-phox cell line is excellent for this study, because it is not of hemopoietic origin and thus does not have the special signaling components found in neutrophils. The experimental data presented above confirmed our initial prediction and demonstrated the utility of the COS-phox cells in reconstituting NADPH oxidase activation through a cell surface receptor.

PKC-mediated phosphorylation of NADPH oxidase components is a critical step in the initiation of a series of events that led to membrane translocation of the cytosolic factors (28, 30, 31). Several PKC isoforms are found to interact with and phosphorylate p47phox in its C-terminal region containing two SH3 domains. In PMA-stimulated neutrophils, translocation of PKC{alpha} and PKC{beta}II to the cytoskeletal fraction correlates with p47phox translocation, suggesting that these conventional PKC isoforms are involved in PMA-induced O

{cjs1138}

2 generation (29). PKC-regulated phosphorylation of p47phox is also observed in fMLP-induced NADPH oxidase activation (32, 33, 34, 35), but the responsible PKC isoforms have not been identified. A major observation of the current study is that PKC{delta} plays a key role in FPR-mediated NADPH oxidase activation in both transgenic COS-phox cells and human neutrophils. In the COS-phox cells, exogenous expression of PKC{delta} led to a potent induction of fMLP-elicited O

{cjs1138}

2 production. In comparison, exogenous expression of PKC{alpha}, PKC{beta}II, and PKC{zeta} did not significantly enhance the fMLP-induced response. In human neutrophils, a role of PKC{delta} in fMLP-elicited O

{cjs1138}

2 generation was suggested by two findings. First, fMLP stimulates membrane translocation of this kinase with temporal correlation to the induced O

{cjs1138}

2 production. Second, the PKC{delta}-selective inhibitor rottlerin exerts a much stronger effect on fMLP-induced O

{cjs1138}

2 production than the PMA-stimulated response. PKC{delta} is a novel PKC isoform and is activated in a Ca2+-insensitive and diacyl glycerol-dependent manner. PKC{delta} and PKC{beta}II are the two most abundant PKC isoforms in neutrophils, whereas COS-7 cells express only a low level of PKC{delta} (17). Like several other PKC isoforms, PKC{delta} responds to PMA stimulation with binding to p47phox (36) and translocation to the cytoskeletal fraction (29). However, the lack of temporal correlation with p47phox binding (36) and translocation (29) casts doubt on the importance of PKC{delta} translocation in PMA-induced neutrophil NADPH oxidase activation. More recently, Yaffe and colleagues (37) studied NADPH oxidase activation in cytosol-depleted neutrophil cores and identified PKC{delta} as a key component for reconstitution of PMA-stimulated NADPH oxidase activity. Their conclusion was based on the observations that rottlerin (10 µM) inhibits PMA-induced O

{cjs1138}

2 production in neutrophil cores, and that selective depletion of PKC{delta} from cytosol impairs its ability to restore the PMA-stimulated response in neutrophil cores. These findings provide additional evidence for a role of PKC{delta} in NADPH oxidase activation. A function of PKC{delta} in leukotriene B4-stimulated eosinophil O

{cjs1138}

2 production was also reported (38). However, eosinophils differ from neutrophils, and leukotriene B4 primes but does not directly activate neutrophil NADPH oxidase. Therefore, further investigation is necessary to determine whether PKC{delta} is required for neutrophil NADPH oxidase activation induced by a potent chemoattractant such as fMLP.

Although the above experimental results are important in establishing a correlation between PKC{delta} activation and O

{cjs1138}

2 production, PKC{delta} may not be the only PKC isoform involved in fMLP-elicited NADPH oxidase activation. Extensive cross talk exists between different kinases and may complicate the interpretation of our experimental data. The lack of highly specific inhibitors for the individual PKC isoforms also hinders studies using neutrophils that are not genetically amenable. Published reports suggest that fMLP can activate PKC{beta}II in differentiated HL-60 cells (39) and PKC{zeta} in human neutrophils (40). Based on these observations, it is likely that the fMLP-induced response requires more than one PKC isoform for optimal O

{cjs1138}

2 production. This possibility, as well as the relative position of PKC{delta} in the FPR-mediated signaling cascade, will need to be investigated in future studies.

The development of COS-phox-based reconstitution assay has enabled us to assess the roles of individual signaling molecules in NADPH oxidase activation. These signaling molecules can be divided into two groups. The first group consists of signaling molecules immediately downstream of the activated FPR. Our results demonstrated that G{alpha}i2 and G{alpha}i3 are equally capable of mediating fMLP-induced O

{cjs1138}

2 generation in intact cells. Exogenous expression of G{alpha}i2 or G{alpha}i3 potentiates O

{cjs1138}

2 production in response to fMLP, suggesting that the endogenous G{alpha}i proteins became a limiting factor when FPR is overexpressed. With the development of the COS-phox-based reconstitution assay, it is now possible to examine other G proteins for their potential involvement in G protein coupled receptor-mediated O

{cjs1138}

2 production. PLC{beta}2 is a downstream effector of G proteins. Our experimental data indicate that exogenous expression of PLC{beta}2 is necessary for optimal reconstitution of fMLP-elicited O

{cjs1138}

2 production, but significant amount of O

{cjs1138}

2 can still be generated in the absence of PLC{beta}2 (Fig. 4). This result suggests that FPR is able to use the endogenous PLC{beta}3 in COS-7 cells for downstream signaling. PLC{beta} activation is triggered by the G protein {beta}{gamma} subunits that become available after the activation of G{alpha}i, which serves to link the agonist-occupied receptor with second messenger production and PKC activation. In this regard, it will be interesting to examine whether the difference in the ability to activate NADPH oxidase by FPR and chemokine receptors lies in the activation of PLC{beta} isoforms. Another signaling component downstream of activated G proteins is PI3K{gamma}. This PI3K isoform is expressed primarily in hemopoietic cells and mediates important functions of neutrophils, macrophages, and mast cells based on loss-of-function studies involving PI3K{gamma}–/– leukocytes (14). Our results suggest that, in the absence of PI3K{gamma}, the fMLP-induced O

{cjs1138}

2 production is greatly diminished. However, significant amounts of O

{cjs1138}

2 were produced without the exogenous expression of PI3K{gamma}, indicating the ability of FPR to use endogenous PI3K for signaling.

Another group of proteins studied in the COS-phox cells includes PKC, Rac, and p40phox, factors that directly participate in the modification and assembly of the phox proteins. In addition to demonstrating a role of PKC{delta} in the fMLP-elicited response, we have shown that the FPR-mediated signaling pathway is able to trigger activation of endogenous Rac1. Exogenous expression of Rac1 further increased the level of Rac activation as well as O

{cjs1138}

2 production, suggesting that Rac1 is one of the limiting factors in this pathway. This finding does not conflict with previous observations that Rac2 plays an important role in NADPH oxidase activation in human neutrophils, but suggest that FPR-mediated signaling pathway can lead to Rac1 activation in an epithelial cell line. fMLP may stimulate Rac1 dissociation from Rho GDP-dissociation inhibitor, as seen in primary human monocytes (41), thereby promoting NADPH oxidase activation. We have further demonstrated the ability of fMLP to induce Rac2 activation and to potentiate O

{cjs1138}

2 generation in the reconstituted COS-phox cells expressing human Rac2. This finding suggests that Rac2 activation is not an exclusive property of hemopoietic cells where it is naturally expressed; instead, it can be achieved by the guanine nucleotide factor(s) present in epithelial cells such as COS-7. Further studies will be necessary to compare the different Rac guanine nucleotide exchange factors for their ability to mediate fMLP-induced O

{cjs1138}

2 production.

In addition to the four essential phox proteins and Rac, recent studies suggest that p40phox may be involved in regulating NADPH oxidase activation. Published results indicate that p40phox can enhance O

{cjs1138}

2 generation in cell-free assays (42, 43, 44) and in K562 cells (45). The PX domain of p40phox is considered important in the interaction with the lipid products of PI3Ks (44, 46, 47), which may be one of the mechanisms for the potentiation of O

{cjs1138}

2 production. However, there are also published reports indicating that p40phox is an inhibitory molecule for NADPH oxidase activation, in both K562 cells (48) and cell-free assays (49, 50). In light of the controversy surrounding the exact role of p40phox in NADPH oxidase activation, we sought to examine this cytosolic protein, which is absent from the epithelial COS-phox cells. By exogenous expression, we observed that p40phox enhances the level of fMLP-induced O

{cjs1138}

2 production in the reconstituted COS-phox cell, suggesting that p40phox can be stimulatory for chemoattractant-induced NADPH oxidase activation in intact cells. Expression of p40phox does not alter the kinetics of fMLP-induced O

{cjs1138}

2 production, because the initial rise to peak and subsequent fall of O

{cjs1138}

2 levels follow a similar pattern in the presence or absence of p40phox (Fig. 6B). Overexpression of p40phox affects the basal level of NADPH oxidase activity as well. Because COS-phox cells can be readily transfected with DNA expression constructs, the reconstitution system is suitable for further delineation of the mechanism of p40phox action through analysis of mutants and chimeric proteins.

In summary, the FPR-reconstituted COS-phox cells share many functional features with neutrophils and provide a genetically amenable system for characterization of the individual signaling components and phox proteins. Using the COS-phox reconstitution system, we have identified PKC{delta} as an important kinase for fMLP-induced O

{cjs1138}

2 production, demonstrated the ability of FPR to activate both Rac1 and Rac2 in a nonhemopoietic cell line, and confirmed the requirement of PLC{beta}2 and PI3K{gamma} for optimal NADPH oxidase activity. We further demonstrated that p40phox is a positive regulator of fMLP-induced O

{cjs1138}

2 production. This whole-cell-based reconstitution system is expected to complement the existing cell-free system for more detailed analysis of receptor-mediated signaling pathways leading to O

{cjs1138}

2 generation.


   Acknowledgments
 
We thank Emily Welch for technical assistance, and our colleagues for kindly providing the expression constructs used in this study.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by National Institutes of Health Grants AI33503 (to R.D.Y.), HL45635 and HL69974 (to M.C.D.), and T32 DK07739 (predoctoral fellowship to M.N.). Back

2 Address correspondence and reprint requests to Dr. Richard D. Ye, Department of Pharmacology, M/C 868, University of Illinois, Chicago, IL 60612. E-mail address: yer{at}uic.edu Back

3 Abbreviations used in this paper: phox, phagocyte oxidase; SH3, Src homology 3; FPR, formyl peptide receptor; PKC, protein kinase C; PLC{beta}, phospholipase C-{beta}; SOD, superoxide dismutase; CL, chemiluminescence; cps, counts per second; PBD, p21-activated kinase binding domain; PL, plasmid. Back

Received for publication August 2, 2004. Accepted for publication September 30, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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