Gene transfer and expression in human neutrophils. The phox homology domain of p47 phox translocates to the plasma membrane but not to the membrane of mature phagosomes
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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 p47 phox localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47 phox , 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.
KeywordsNADPH Oxidase Human Neutrophil Cytochrome B558 Chronic Granulomatous Disease Patient Electrical Setting
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 p47 phox , p67 phox and p40 phox , the membrane-associated cytochrome b558 and the accessory proteins Rac2 and Rap1a. The cytochrome b558, consisting of the glycoprotein gp91 phox and the protein p22 phox , 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 p47 phox is phosphorylated at serine residues located at its carboxy terminus, then the phox homology (PX) domain located in its amino terminus is unmasked  and p47 phox , together with p67 phox , 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 p47 phox and p22 phox , which is mediated by the SH3 domains located in the carboxy terminus of p47 phox . Recent studies suggested that the PX domain of p47 phox , a phosphoinositide-binding module , plays an important role in the translocation of p47 phox towards biological membranes . We show that the PX domain of p47 phox does not translocate to the membranes of mature phagosomes during phagocytosis of opsonized-zymosan particles.
Results and discussion
Transfection efficiency and cellular viability
The PX domain of p47phoxdoes not translocate to the phagosomal membrane in human neutrophils
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 p47 phox is 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-p47 phox chimera 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-p47 phox -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 p47 phox and the PX domain of p47 phox (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; p47 phox 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-p47 phox or pEGFP-p47 phox -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.
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.
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