- Methodology article
- Open Access
Gene transfer and expression in human neutrophils. The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes
BMC Immunology volume 7, Article number: 28 (2006)
Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection.
Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX) domain of p47phoxlocalizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47phox, is translocated to the membrane of mature phagosomes.
We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils.
Study of a gene product by expressing its constitutively active or dominant negative mutant in a cell is a powerful tool of investigation. However, neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. Researchers have partially overcome these difficulties by performing studies in well-developed cell-free systems , with permeabilized neutrophils  and with cell lines that undergo some neutrophil functions, such as HL-60 cells or B-lymphoblasts [3, 4]. Others have used virus-based expression systems, but these systems require laborious cloning into specific vectors and the procedures for viral infection are time consuming and potentially hazardous . Here we report the transfection and expression of genes into neutrophils by nucleofection. Using this technology, which delivers the vector directly into the nucleus , exogenous genes can be expressed in neutrophils in as short as 2 h after transfection, overcoming the difficulties associated with the short lifetime of these cells.
In innate immunity, neutrophils are crucial for the destruction of bacteria and fungi [7, 8]. To combat bacterial infection, neutrophils must perform functions that include migration to the inflammatory site, phagocytosis of invading microorganisms, and generation of reactive oxygen species (ROS) that contribute to killing. The NADPH oxidase of neutrophils is a multisubunit enzymatic complex responsible for the monoelectronic reduction of oxygen to produce superoxide anion (O2- . Free radical production is directly related to the bactericidal capacity of these cells since patients with chronic granulomatous disease (CGD), whose NADPH oxidase is inactive , suffer recurrent bacterial and fungal infections. The NADPH oxidase comprises the cytosolic factors p47phox, p67phoxand p40phox, the membrane-associated cytochrome b558 and the accessory proteins Rac2 and Rap1a. The cytochrome b558, consisting of the glycoprotein gp91phoxand the protein p22phox, localizes in the plasma membrane as well as in the membrane of secretory vesicles, specific and tertiary granules. In resting neutrophils, the oxidase remains unassembled and, therefore, inactive. In response to adequate stimuli, the cytosolic factor p47phoxis phosphorylated at serine residues located at its carboxy terminus, then the phox homology (PX) domain located in its amino terminus is unmasked  and p47phox, together with p67phox, translocates to the membrane-associated cytochrome b558. This switches the NADPH oxidase to its active form . The activation of the NADPH oxidase by a soluble stimulus like the formylated peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) involves both early trafficking of the cytochrome b558 to the plasma membrane through degranulation and the subsequent translocation of the cytosolic factors to assemble the oxidase. The exposure of neutrophils to phorbol 12-myristate 13-acetate (PMA) stimulates a larger degree of degranulation than that observed in response to fMLP and induces the assembly of the NADPH oxidase not only at the plasma membrane but also at the membrane of the intracellular vesicles/granules . If neutrophils are exposed to particulate stimulus like opsonized microorganisms, phagocytosis takes place, and specific and azurophilic granules fuse with the phagosome to integrate the cytochrome b558 with the phagosomal membrane  and to release their contents into the phagolysosome . In all cases, the activation of the NADPH oxidase requires the interaction of p47phoxand p22phox, which is mediated by the SH3 domains located in the carboxy terminus of p47phox. Recent studies suggested that the PX domain of p47phox, a phosphoinositide-binding module , plays an important role in the translocation of p47phoxtowards biological membranes . We show that the PX domain of p47phoxdoes not translocate to the membranes of mature phagosomes during phagocytosis of opsonized-zymosan particles.
Results and discussion
Transfection efficiency and cellular viability
To optimize the transfection of human neutrophils, we used the β-galactosidase expression vector pCMV-βgal, previously described . After transfection, cells were incubated for 2, 4, 6 or 8 hours at 37°C and at the appropriate times were centrifuged, washed twice with PBS and lysed by sonication in 2% Nonidet P-40. β-Galactosidase activity was then measured using the β-gal Reporter Gene assay kit (Roche). Maximum galactosidase activity was observed at 2 h after gene transfer when using the proprietary transfection solution ''T'' (amaxa biosystems, Germany) and an electrical setting corresponding to program T27 (Fig. 1A). The transfection efficiency under these experimental conditions was 0.4 to 1%. The viability of neutrophils after the electrical pulse and after the 2-hour recovery period was 78.8 ± 2.5% and 78.5 ± 2.3%, respectively, evaluated using Trypan Blue exclusion. Next, we compared the efficiency of transfection of human neutrophils under various electrical settings corresponding to a series of second generation programs for amaxa nucleofectors. In figure 1B, we show that the electrical settings corresponding to programs U14 and Y01 resulted in higher transfection efficiency for similar experimental conditions based on flow cytometric analysis of neutrophils transfected with the vector pmaxGFP. Also in figure 1B, we show that the second generation programs (U14 and Y01) better preserve cell viability when evaluated by propidium iodide. For consistency and since the initial experiments were performed using program T27, this electrical setting was employed in subsequent experiments unless stated otherwise. Importantly, no differences in the subcellular distribution of the fluorescent chimeras were observed when using program Y01 instead of T27. Next, we used confocal microscopy to examine the transfection and expression of enhanced green fluorescent proteins encoded by the pEGFP (EGFP, enhanced green fluorescent protein) expression vector (Clontech®) in neutrophils. To demonstrate further that the population of transfected neutrophils was fully functional, we transfected neutrophils with the NADPH oxidase cytosolic factor p47phoxas an EGFP chimera, and we stimulated cells with phorbol ester or with the formylated peptide fMLP which mimics bacteria-derived peptides and stimulates neutrophils through binding to the membrane receptor formyl peptide receptor 1 (FPR1) . In figure 2, we show that transfected neutrophils stimulated with PMA or fMLP can undergo morphological changes represented by the appearance of protrusions of the plasma membrane, presumably membrane ruffles. These membrane structures were enriched in EGFP-p47phox(Fig. 2), which is in agreement with previous reports describing the localization of NADPH oxidase factors at membrane ruffles after activation in non-primary cell lines . The distribution pattern for EGFP was different from that of EGFP-p47phox. EGFP was detected in the nucleus and cytosol, and was also detected in membrane protrusions in stimulated cells (Fig. 2).
To evaluate the effect of nucleofection on NADPH oxidase activation, we first analyzed the ability of nucleoporated neutrophils to produce ROS using the luminol-dependent chemiluminescence detection system. In figure 3A, we show that electroporated cells have relatively low basal luminol-dependent chemiluminescence activity in the absence of stimuli. Importantly, they maintain their capacity to respond to stimuli. Nucleoporated neutrophils show ROS production kinetics similar to that of non-electroporated cells in response to PMA (Fig. 3A). Furthermore, electroporated neutrophils expressing an exogenous protein respond to stimulus (Fig. 3B). The response was detected using the nitroblue tetrazolium reaction. Formazan, the blue-black precipitate generated by superoxide anion-dependent NBT reduction, was evident in stimulated cells but undetectable in unstimulated, transfected cells (Fig. 3B). These results suggest that electroporated neutrophils conserve an intact NADPH oxidase. To further support this idea, we transfected neutrophils with a vector expressing wild type p47phoxdownstream of EGFP and then stimulated the cells with PMA. The translocation of p47phoxto the plasma membrane was followed in real time by confocal microscopy analysis (Fig. 4). The results indicate that EGFP-p47phox, but not the EGFP control, undergoes translocation during activation further supporting the idea that electroporated neutrophils are functional and responsive.
To evaluate whether nucleofection induces more subtle degrees of activation in neutrophils, we examined the surface expression of CD11b, a human leukocyte integrin subunit that is mainly present in the membrane of secretory vesicles and tertiary granules in resting neutrophils. In figure 5, we show that nucleoporated neutrophils have, in fact, a marked increase in CD11b molecules on their cellular surface. The level of CD11b at the plasma membrane after nucleoporation was even larger than that triggered by fMLP stimulation. The increase in the surface expression of CD11b was evident using either program T27 or the second generation program Y01 (Fig. 5A and 5B). In some experiments, two populations of cells were identified according to their level of surface expression of CD11b after nucleoporation (Fig. 5B). Cells expressing GFP were mainly distributed with the subpopulation of cells showing higher surface expression of CD11b (Fig. 5C). These results suggest that nucleofection induces the mobilization of secretory vesicles and, probably, tertiary granules, which are the subpopulation of neutrophil secretory organelles with the highest tendency to undergo exocytosis.
The PX domain of p47phoxdoes not translocate to the phagosomal membrane in human neutrophils
The cytosolic factors p47phoxand p40phoxeach contain a PX domain at their amino terminus . PX domains are also present in proteins involved in vesicular trafficking and cellular signaling including Vam7p  and PI3K-C2 [22, 23], respectively. PX domains are phosphoinositide binding modules . The affinity of the various PX domains for several phosphoinositides differs from protein to protein. In particular, the PX domain of p47phoxhas been shown to bind preferentially to PI(4)P, PI(3,4)P2 , and PI(3,4,5)P3 . The mechanism mediated by the PX domain of p47phoxin the activation of the oxidase is controversial. One group has suggested that the phosphoinositide-binding activity of the p47phoxPX domain is essential for the membrane translocation of this protein and activation of the phagocyte NADPH oxidase . Other groups have indicated that the translocation of the p47phoxPX domain to the plasma membrane is not due to interactions with phospholipids but to association with the actin cytoskeleton  and have shown in a variety of cell lines that the PX domain of p47phoxtargets to cell membranes via interaction with moesin [24, 25]. Furthermore, while some researchers propose that a single residue substitution in the PX domain of p47phoxdecreases its affinity to phosphoinositides and its binding to cell membranes in resting or PMA-stimulated cells , others show that the PX domain of p47phoxtranslocates to the plasma membrane after activation in a phosphoinositide-independent manner in COS cells . The subcellular localization of the PX domain of p47phoxin human neutrophils has not yet been shown. To analyze this, we transfected human neutrophils with a vector for the expression of a chimera composed of EGFP upstream of the p47phoxPX-domain (EGFP-p47-PX, Fig. 6). The visualization of the subcellular distribution of the PX domain is somewhat difficult due to the relatively small size of these cells. However, the fluorescence intensity plots helped to identify an increase in the distribution of fluorescence towards the edge of the cells. We concluded that the p47phoxPX domain partially localizes at the plasma membrane in nucleoporated neutrophils. Localization of EGFP-p47-PX in intracellular structures was also evident, a phenomenon previously described in COS cells . Differently from the fusion protein EGFP-p47phox(full-length) which required phorbol ester-mediated neutrophil activation for plasma membrane localization (Fig. 4), the subcellular distribution of EGFP-p47-PX was not markedly affected by cellular stimulation (Fig. 6). Although the concentrations of PI(3,4)P2 , and PI(3,4,5)P3 in resting neutrophils are considered to be relatively low , it could be possible that PI3-kinases undergo some level of activation during nucleoporation thus increasing the membrane distribution of the PX domain in unstimulated cells. However, pretreatment of neutrophils with the PI3-kinase inhibitor LY294002 did not alter the pattern of protein distribution in EGFP-p47-PX transfected neutrophils or prevent the increase in the surface expression of CD11b after nucleoporation (not shown). Another possibility is that once exposed (the PX domain is masked in unphosphorylated full-length p47phox), the PX domain could recognize pre-existent structures at the plasma membrane, possibly moesin, PI(4)P , phosphatidic acid  or even basal levels of PI 3-kinase products. From our experiments, it seems that the plasma membrane of nucleoporated neutrophils has the necessary molecular machinery to sequester, at least in part, the p47phoxPX-domain. The molecular basis of this mechanism remains to be clarified.
During particle engulfment, the NADPH oxidase complex is assembled at the membrane of the phagosome so that superoxide anion is pumped into the core of the phagosome where the engulfed microorganism is killed. In particular, p47phoxhas been shown to localize towards nascent and mature phagosomes in neutrophils after phagocytosis of opsonized zymosan particles . This poses the question of whether the PX domain of p47phox, which has been proposed to mediate the translocation of p47phoxtowards the cytochrome b558-containing plasma membrane and to participate in the activation of the NADPH oxidase in cells stimulated with soluble stimuli , could also be implicated in the assembly of the oxidase at the phagosome membrane. In an attempt to clarify this, we analyzed the distribution of the PX domain of p47phoxduring phagocytosis. We transfected neutrophils with the expression vector pEGFP-p47-PX or with cDNA encoding a chimera of EGFP and RhoB, a small GTPase thought to function in the regulation of endocytosis  and phagocytosis . Then, we exposed the transfected cells to Texas Red-conjugated opsonized zymosan A particles, and protein distribution during phagocytosis was evaluated by confocal microscopy. In Figure 7, we show that neutrophils expressing EGFP-RhoB or EGFP-p47-PX can undergo phagocytosis. From those experiments, it becomes clear that nucleoporated neutrophils conserve their ability to phagocytose opsonized particles. The accumulation of green fluorescence around the phagosome is evident in EGFP-RhoB-expressing neutrophils but was not observed in cells expressing EGFP-p47-PX (Fig. 7). Similar results were observed in HL-60 promyelocytic cells when differentiated to granulocytes (Fig. 8). These data suggest that the mechanisms of translocation of p47phoxto the plasma membrane and to the phagosome membrane are different. It is likely that the translocation of p47phoxto the phagosome does not involve the PX domain. One possibility is that the SH3 domains of p47phox, which have been largely shown to bind to the membrane-localized cytochrome b558 through interaction with the cytosolic domain of p22phox, are sufficient to translocate p47phoxto the phagosome. However, p47phoxwas clearly detected in forming phagosomes in X-linked CGD neutrophils which lack cytochrome b558, indicating that the recruitment of this protein to the nascent phagosome is independent of cytochrome b558 . In the same work, p47phoxwas undetectable in phagosome membranes of mature phagosomes in neutrophils from X-linked CGD patients , suggesting that p47phoxcan not be retained at the phagosome in the absence of cytochrome b558. Likewise, the PX domain of p47phoxlocalizes at the plasma membrane in the proximity of the opsonized particle in the nascent phagosome (Fig. 7), but is not retained in the membrane of mature phagosomes (Fig. 7 and 8) despite the fact that moesin, the actin-binding protein shown to bind to the PX domain of p47phox, is present in phagosome membranes . Therefore, the reason why the PX domain of p47phoxis not retained in the mature phagosome may reside in the differential role that the products of class I and class III phosphatidylinositol 3-kinase play during phagosome formation and maturation . The product of class I PI 3-kinase, PI(3,4,5)P3, has been shown to be rapidly synthesized during phagosomal formation [32, 33], but the accumulation was sharply restricted to the phagosomal cup . Conversely, PI(3)P, a product of class III PI 3-kinase, was only detected in sealed phagosomes . These data correlate with our observation that the PI(3,4,5)P3-binding domain of p47phox(PX domain) is present in membranes in the forming phagosome but is absent from the membrane of the mature phagosome. Although not explored here, it is also possible that the PI(3)P-binding domain of p40phox(p40phox-PX domain) is implicated in maintaining the cytosolic complex of the NADPH oxidase at the mature phagosome.
We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation on the study of neutrophil function; however, the relatively low efficiency of transfection/expression observed after 2 h restricts the subsequent analysis to experiments that use single cells. Since phagocytosis of opsonized particles by neutrophils as well as their response to stimuli requires intact downstream signaling machinery, the results presented here suggest that transfected neutrophils are viable and fully functional. Using this methodology, we showed that the PX domain of p47phoxis translocated to the plasma membrane but is not retained at the membrane of mature phagosomes suggesting that distinct mechanisms may operate during the activation of the NADPH oxidase at different subcellular sites in human neutrophils.
Transfection of human neutrophils and confocal microscopy
For our studies, we isolated neutrophils from healthy human donors as previously described  and resuspended them in phosphate-buffered saline (PBS). Neutrophils were then stored on ice for 30 min or less before use. Immediately before transfection, cells were centrifuged at 1,800 rpm (800 × g) and 4°C for 5 min then resuspended in transfection buffer as indicated below. Ninety μL of neutrophil suspension (2 × 106 cells) were transferred to a nucleoporation cuvette (amaxa Biosystems, Germany), and 5 μg of the indicated cDNA were added to complete a final volume of 100 μl. We found that this concentration of cells is essential to achieve maximum transfection efficiency. Cells were transfected in an amaxa nucleofector apparatus then immediately transferred to 8-well poly-L-lysine–coated chambered glass slides (Lab-Tek) containing RPMI medium supplemented with 10% fetal calf serum (50,000 to 100,000 cells per well in 400 μl of medium). Neutrophils were maintained for 2 h at 37°C in 5% CO2/air then fixed with 3.7% paraformaldehyde for 10 min. Fixed cells were washed three times with PBS and stored in Fluoromount-G (Southern Biotechnology, CA) until analysis by laser-scanning confocal microscopy on either a Bio-Rad MRC1024 attached to a Zeiβ Axiovert S100TV microscope or a Zeiss (BioRad) Radiance 2100 Rainbow laser scanning confocal microscope (LSCM) attached to a Nikon TE2000-U microscope with infinity corrected optics. Images were collected using the Bio-Rad LaserSharp (v3.2) software. Images were taken at constant exposure times pre-determined to be sub-saturating for the brightest sample. The images were processed and analyzed for the distribution of the fluorescence intensity using the NIH image processing and analysis program IMAGE/J software, IMARIS software (Bitplane AG) and Image-pro plus (MediaCybrnetics®). In some experiments, neutrophils were stimulated 2 h after transfection using the formylated peptide fMLP (1 μM) or PMA (0.1 μg/ml) for 5 or 20 min, respectively, at 37°C. Where indicated, the subcellular localization of the EGFP-p47phoxchimera was followed in real time using a Zeiss Radiance 2100 Rainbow LSCM attached to a Nikon TE2000-U microscope equipped for viewing live specimens with a Neue temperature and CO2 controlled live chamber (LiveCell Inc., PA) and a Bioptechs Objective heater (Biotechs, Inc., PA).
Neutrophils were transfected as described above with the expression vectors EGFP-RhoB or EGFP-p47phox-PX. After transfections, the cells were maintained for 2 h at 37°C in RPMI medium at a cellular concentration of 100,000 neutrophils per 400μl of RPMI in 8-well poly-L-lysine – coated chambered glass slides (Lab-Tek). Then, the cells were incubated in the presence of Texas Red-conjugated zymosan A (S. cerevisiae) BioParticles (Invitrogen, CA), previously opsonized using the Fluorescent Particles Opsonizing Reagent (Invitrogen, CA) as described by the manufacturer. The fluorescent particles were added in a ratio of particles to phagocytes 15:1 or 100:1, the slides were immediately spun down at 1,500 rpm for 5 min at 4°C then incubated for 10 or 15 min at 37°C. Cells were fixed, washed with PBS and stored in Fluoromount-G (Southern Biotechnology) at 4°C until analyzed by laser-scanning confocal microscopy as described above. The quantification of the fluorescent intensity (FI) at the phagosomal membrane versus the cytosolic distribution of the fluorescent chimera for the green channel was assessed using the NIH image processing and analysis program IMAGE/J software. Briefly, two lines were drawn on each phagosome using the straight line tool. These generated four points where the lines intersected the phagosomal membrane. The intensity of fluorescence at the end of the lines (~ 1 μm outside the phagosome into the cytosol) was subtracted from the fluorescence intensity at the point where the lines intersected the phagosome (ΔFI). The four independent values were averaged and used as representative of the ΔFI for that particular phagosome. At least three phagosomes from different cells were analyzed for each chimera.
Nitroblue tetrazolium test
Human neutrophils were nucleoporated as described above and maintained in RPMI medium for 2 h at 37°C in 8-well chambered coverglass slides at 50.000 cells/well). Then, cells were incubated with 1 mg/ml Nitroblue tetrazolium (NBT) (Bio-Rad Laboratories, CA) in the presence or absence of 0.1 μg/ml PMA, for 30 min at 37°C. After stimulation, cells were washed with PBS, fixed and analyzed by confocal microscopy.
Luminol-dependent chemiluminescence assay
Neutrophils were nucleoporated in solution "T" using an EGFP expression vector and an electrical setting corresponding to program T27. The cells were recovered in serum-free RPMI containing 0.1% gelatin (Sigma, MO) for 2 h at 37°C. Neutrophils (2 × 106) were resuspended in PBS containing 5 mM glucose and 0.1% gelatin. Luminol was added to a final concentration of 1 μM. Cells were left untreated or stimulated with 0.1 μg/ml PMA. Luminol-dependent chemiluminescence was continuously recorded for 65 minutes.
Flow cytometry analysis
Neutrophils were nucleoporated in solution "T" using an electrical setting corresponding to programs T27, U14 or Y01 in the presence or absence of the expression vector pmaxGFP (amaxa biosystems). The cells were recovered in serum-free RPMI containing 0.1% gelatin (Sigma, MO) for 2 h at 37°C. Untransfected cells were also incubated in RPMI and used as a control. Where indicated, control cells were stimulated with fMLP (1 μM). The cells were spun down, washed and resuspended in flow cytometer diluent buffer (PBS containing 0.5% BSA and 3 mM NaN3). Cells were incubated with a specific antibody anti-CD11b or with an isotype-matched control (BD Pharmingen, CA). Next, they were incubated with a fluorescein isothiocyanate-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, PA) then fixed in 1% paraformaldehyde. Expression of CD11b antigen on the surface of treated and untreated neutrophils was analyzed by flow cytometry (FACSCalibur BD Biosciences, CA). The data was analyzed using CellQuest™ software (Becton Dickinson, CA). For flow cytometry based viability analysis, cells were labeled for 15 min with propidium iodide (final conc. 10 μg/ml) and analyzed by flow cytometry. GFP fluorescence was detected in the FL-1 channel and propidium iodide using the FL-3 channel.
The various steps in the cloning of the constructs used in this work were performed by standard techniques, and all constructs were verified by sequencing using an automated fluorescent dye-terminator sequencer. The full-length p47phoxand the PX domain of p47phox(residues 1–130) cDNA were amplified from the full-length cDNA using pfu polymerase (Stratagene, La Jolla, CA) and the following primers: 5' primer GAATTC ATGGGGGACACCTTCATCCGT; p47phox 3' primer GGTACC GACGGCAGACGCCAGCTTCCG and PX domain 3' primer, GGTACC GTCTGTGGGGAGCTTGAGGT. The Eco RI I and Kpn I sites are underlined. The fragments were purified and ligated into the pEGFP-C2 vector (Clontech).
Transfection of HL-60 promyelocytic cells
The promyelocytic leukemia human HL-60 cell line (American Type Culture Collection (ATCC), VA) was cultured in Dulbecco's Modified Eagle Medium (D-MEM) (Gibco) supplemented with 20% fetal bovine serum (Hyclone), 0.292 mg/ml glutamine, 50 units/ml penicillin and 50 μg/ml streptomycin at 37°C in 5% CO2/air. HL-60 cells were differentiated to granulocytes by incubation in the presence of 1.3% DMSO for 48 h. For transfections, 5 × 106 cells were resuspended in 100 μl of Solution "V" (amaxa biosystems) in the presence of 5 μg of the vectors expressing EGFP-p47-PX domain or EGFP-RhoB and nucleofected in the nucleoporator apparatus (amaxa Biosystems, Germany) using the T01 electrical setting. The cells were then re-plated in complete medium in the presence of 1.3% DMSO, incubated at 37°C in 5% CO2/air and used for analysis 24 h post-transfection. For phagocytosis assays, transfected HL-60 cells were seeded at 70% confluence in an eight-well chambered coverglass (pre-treated with 0.01% poly-L-lysine in PBS) in D-MEM medium for 30 min at 37°C and incubated in the presence of Texas Red®-conjugated zymosan A (S. cerevisiae) BioParticles® (Invitrogen), that had been opsonized using the Fluorescent Particles Opsonizing Reagent (Invitrogen) as described by the manufacturer. The fluorescent particles were added in a ratio of particles to phagocytes approximately 15:1 and the slides were immediately spun down at 1,500 rpm for 5 min at 4°C then incubated for 15 min at 37°C. Next, cells were fixed with 3.7% PAF, washed with PBS, and stored in Fluoromount-G (Southern Biotechnology) at 4°C until analysis by laser-scanning confocal microscopy
Control of protein expression
HL-60 granulocytes were transfected with the expression vectors pEGFP, pEGFP-p47phoxor pEGFP-p47phox-PX as described above. The cells were lysed using M-PER mammalian protein extraction reagent (Pierce) in the presence of anti-proteases and 40 μg of total protein was resolved by SDS-PAGE, transferred to nitrocellulose and detected using a monoclonal antibody raised against the GFP tag (B-2, Santa Cruz Biotechnology).
All procedures regarding human subjects have been reviewed and approved by the Human Subjects Research Committee at The Scripps Research Institute and were conducted in accordance with the requirements set forth by the mentioned Human Subjects Research Committee.
El Benna J, Park JW, Ruedi JM, Babior BM: Cell-free activation of the respiratory burst oxidase by protein kinase C. Blood Cells Mol Dis. 1995, 21: 201-206. 10.1006/bcmd.1995.0023.
Brown GE, Stewart MQ, Liu H, Ha VL, Yaffe MB: 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. 2003, 11: 35-47. 10.1016/S1097-2765(03)00005-4.
Chanock SJ, Faust LR, Barrett D, Christensen B, Newburger PE, Babior BM: Partial reconstitution of the respiratory burst oxidase in lymphoblastoid B cell lines lacking p67-phox after transfection with an expression vector containing wild-type and mutant p67 -phox cDNAs: Deletions of the carboxy and amino terminal residues of p67-phox are not required for activity. Exp Hematol. 1996, 24: 531-536.
Leto TL, Adams AG, de M: Assembly of the phagocyte NADPH oxidase: binding of Src homology 3 domains to proline-rich targets. Proc Natl Acad Sci U S A. 1994, 91: 10650-10654. 10.1073/pnas.91.22.10650.
Zhong B, Jiang K, Gilvary DL, Epling-Burnette PK, Ritchey C, Liu J, Jackson RJ, Hong-Geller E, Wei S: Human neutrophils utilize a Rac/Cdc42-dependent MAPK pathway to direct intracellular granule mobilization toward ingested microbial pathogens. Blood. 2003, 101: 3240-3248. 10.1182/blood-2001-12-0180.
Gershan JA, Johnson BD, Weber J, Schauer DW, Natalia N, Behnke S, Burns K, Maloney KW, Warwick AB, Orentas RJ: Immediate transfection of patient-derived leukemia: a novel source for generating cell-based vaccines. Genet Vaccines Ther. 2005, 3: 4-10.1186/1479-0556-3-4.
Babior BM: NADPH oxidase. Curr Opin Immunol. 2004, 16: 42-47. 10.1016/j.coi.2003.12.001.
Beutler B: Innate immunity: an overview. Mol Immunol. 2004, 40: 845-859. 10.1016/j.molimm.2003.10.005.
Babior BM: Activation of the respiratory burst oxidase. Environ Health Perspect. 1994, 102 Suppl 10: 53-56.
Allen LA, DeLeo FR, Gallois A, Toyoshima S, Suzuki K, Nauseef WM: Transient association of the nicotinamide adenine dinucleotide phosphate oxidase subunits p47phox and p67phox with phagosomes in neutrophils from patients with X-linked chronic granulomatous disease. Blood. 1999, 93: 3521-3530.
Ago T, Kuribayashi F, Hiroaki H, Takeya R, Ito T, Kohda D, Sumimoto H: Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation. Proc Natl Acad Sci U S A. 2003, 100: 4474-4479. 10.1073/pnas.0735712100.
Cross AR, Segal AW: The NADPH oxidase of professional phagocytes--prototype of the NOX electron transport chain systems. Biochim Biophys Acta. 2004, 1657: 1-22.
Karlsson A, Dahlgren C: Assembly and activation of the neutrophil NADPH oxidase in granule membranes. Antioxid Redox Signal. 2002, 4: 49-60. 10.1089/152308602753625852.
Reeves EP, Lu H, Jacobs HL, Messina CG, Bolsover S, Gabella G, Potma EO, Warley A, Roes J, Segal AW: Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature. 2002, 416: 291-297. 10.1038/416291a.
Ago T, Nunoi H, Ito T, Sumimoto H: Mechanism for phosphorylation-induced activation of the phagocyte NADPH oxidase protein p47(phox). Triple replacement of serines 303, 304, and 328 with aspartates disrupts the SH3 domain-mediated intramolecular interaction in p47(phox), thereby activating the oxidase. J Biol Chem. 1999, 274: 33644-33653. 10.1074/jbc.274.47.33644.
Ago T, Takeya R, Hiroaki H, Kuribayashi F, Ito T, Kohda D, Sumimoto H: The PX domain as a novel phosphoinositide- binding module. Biochem Biophys Res Commun. 2001, 287: 733-738. 10.1006/bbrc.2001.5629.
Kanai F, Liu H, Field SJ, Akbary H, Matsuo T, Brown GE, Cantley LC, Yaffe MB: The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nat Cell Biol. 2001, 3: 675-678. 10.1038/35083070.
Johnson JL, Raney AK, McLachlan A: Characterization of a functional hepatocyte nuclear factor 3 binding site in the hepatitis B virus nucleocapsid promoter. Virology. 1995, 208: 147-158. 10.1006/viro.1995.1138.
Niedel JE, Cuatrecasas P: Formyl peptide chemotactic receptors of leukocytes and macrophages. Curr Top Cell Regul. 1980, 17: 137-170.
Zhan Y, Virbasius JV, Song X, Pomerleau DP, Zhou GW: The p40phox and p47phox PX domains of NADPH oxidase target cell membranes via direct and indirect recruitment by phosphoinositides. J Biol Chem. 2002, 277: 4512-4518. 10.1074/jbc.M109520200.
Lu J, Garcia J, Dulubova I, Sudhof TC, Rizo J: Solution structure of the Vam7p PX domain. Biochemistry. 2002, 41: 5956-5962. 10.1021/bi020050b.
Sato TK, Overduin M, Emr SD: Location, location, location: membrane targeting directed by PX domains. Science. 2001, 294: 1881-1885. 10.1126/science.1065763.
Ponting CP: Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3-kinases: binding partners of SH3 domains?. Protein Sci. 1996, 5: 2353-2357.
Zhan Y, He D, Newburger PE, Zhou GW: p47(phox) PX domain of NADPH oxidase targets cell membrane via moesin-mediated association with the actin cytoskeleton. J Cell Biochem. 2004, 92: 795-809. 10.1002/jcb.20084.
Wientjes FB, Reeves EP, Soskic V, Furthmayr H, Segal AW: The NADPH oxidase components p47(phox) and p40(phox) bind to moesin through their PX domain. Biochem Biophys Res Commun. 2001, 289: 382-388. 10.1006/bbrc.2001.5982.
Stephens LR, Jackson TR, Hawkins PT: Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)- trisphosphate: a new intracellular signalling system?. Biochim Biophys Acta. 1993, 1179: 27-75. 10.1016/0167-4889(93)90072-W.
Karathanassis D, Stahelin RV, Bravo J, Perisic O, Pacold CM, Cho W, Williams RL: Binding of the PX domain of p47(phox) to phosphatidylinositol 3,4-bisphosphate and phosphatidic acid is masked by an intramolecular interaction. EMBO J. 2002, 21: 5057-5068. 10.1093/emboj/cdf519.
Ellis S, Mellor H: Regulation of endocytic traffic by rho family GTPases. Trends Cell Biol. 2000, 10: 85-88. 10.1016/S0962-8924(99)01710-9.
Zhang J, Zhu J, Bu X, Cushion M, Kinane TB, Avraham H, Koziel H: Cdc42 and RhoB activation are required for mannose receptor-mediated phagocytosis by human alveolar macrophages. Mol Biol Cell. 2005, 16: 824-834. 10.1091/mbc.E04-06-0463.
Defacque H, Egeberg M, Habermann A, Diakonova M, Roy C, Mangeat P, Voelter W, Marriott G, Pfannstiel J, Faulstich H, Griffiths G: Involvement of ezrin/moesin in de novo actin assembly on phagosomal membranes. EMBO J. 2000, 19: 199-212. 10.1093/emboj/19.2.199.
Vieira OV, Botelho RJ, Rameh L, Brachmann SM, Matsuo T, Davidson HW, Schreiber A, Backer JM, Cantley LC, Grinstein S: Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation. J Cell Biol. 2001, 155: 19-25. 10.1083/jcb.200107069.
Marshall JG, Booth JW, Stambolic V, Mak T, Balla T, Schreiber AD, Meyer T, Grinstein S: Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc gamma receptor-mediated phagocytosis. J Cell Biol. 2001, 153: 1369-1380. 10.1083/jcb.153.7.1369.
Czech MP: Dynamics of phosphoinositides in membrane retrieval and insertion. Annu Rev Physiol. 2003, 65: 791-815. 10.1146/annurev.physiol.65.092101.142522.
Johnson JL, Park JW, Benna JE, Faust LP, Inanami O, Babior BM: Activation of p47(PHOX), a cytosolic subunit of the leukocyte NADPH oxidase. Phosphorylation of ser-359 or ser-370 precedes phosphorylation at other sites and is required for activity. J Biol Chem. 1998, 273: 35147-35152. 10.1074/jbc.273.52.35147.
Supported by U.S. Public Health Service Grant AI-024227 and by the Sam and Rose Stein Endowment Fund. We would like to thank Dr. William Kiosses from the Core Microscopy Facility at TSRI for technical assistance with the processing of confocal images and Deborah Noack for technical assistance. This work is dedicated to the memory of Dr. Bernard. M. Babior.
There is not any competing financial or other interest in relationship to this work.
JLJ contributed to the experimental design, set up conditions for the transfection experiments, optimized the transfection and expression of the β-galactosidase gene in human neutrophils, performed some of the phagocytosis experiments and contributed to the writing of the manuscript. BAE isolated human neutrophils, contributed to the optimization of the phagocytosis assays and performed flow cytometry assays. DBM contributed to the analysis of the confocal microscope images, performed Western blots, phagocytosis assays and NBT tests. AAB designed and performed flow cytometry assays and analyzed results. SDC designed nucleofection experiments for transfection of human neutrophils, performed research, collected and processed confocal microscopy images and wrote the manuscript. All of the authors read and approved the final manuscript.
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
About this article
Cite this article
Johnson, J.L., Ellis, B.A., Munafo, D.B. et al. Gene transfer and expression in human neutrophils. The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes. BMC Immunol 7, 28 (2006) doi:10.1186/1471-2172-7-28
- NADPH Oxidase
- Human Neutrophil
- Cytochrome B558
- Chronic Granulomatous Disease Patient
- Electrical Setting