Murine GM-CSF (mGM-CSF), FMLP, luminol, DPI, and HRP were from Sigma-Aldrich. Murine TNF-α (mTNF-α) was from R&D Systems. All buffer components were from Sigma-Aldrich and were endotoxin free or low endotoxin, as available. The following antibodies were used: anti–phospho- Ser473 PKB (Ab4802; Abcam), anti-PKB (9272; Cell Signaling), anti–phospho-T180/Y182 p38 (9211S; Cell Signaling), anti-p38 (9212; Cell Signaling), anti–phospho-T202/Y204 p42/44 (9106S; Cell Signaling), and anti-p42/22 (606-259-1550; TransLabs).
Generation of p40phox−/− mice.
Several clones encoding the p40phox genomic sequence were isolated from the RPCI mouse PAC library 21 (Pieter de Jong, UK HGMP Resource Centre) by Southern screens using an NT-cDNA probe. An 11.9-kb SpeI–SpeI fragment encompassing exons 1–4 was isolated from clone RP21-641C7 and inserted into the low copy number plasmid pSC-3Z to form the basis of a p40phox gene-targeting vector (Fig. S1 A).
A smaller fragment containing exon 3 was subcloned into pBS, and site-directed mutagenesis was conducted to alter codon 73 to a translational stop and to introduce silent mutations creating additional XhoI and ApaI sites (Fig. S1 B), which were used to track the presence of the mutated sequence. This modified segment was sequenced and reintroduced into the targeting vector. This strategy was adopted because the translational stop or p40phox−/− mouse described here was one of several knock-in mutations planned for exon 3.
A loxP-flanked cassette containing a tACE-Cre expression module and a NeoR
expression module (pACN; A. Plagge, Babraham Institute, Cambridge, UK) (74
) was inserted into the SnaBI site in the intron between exons 3 and 4 (Fig. S1 A). The tACE promoter is expected to operate in the testis and drive Cre/Lox-mediated deletion of this cassette on the breeding of targeted chimeras. Deletion is predicted to leave 59 bp of foreign DNA remaining in the intron.
The final targeting vector was digested with DraI and SalI (in the 3′ polylinker) to remove excess pSC-3Z vector sequence and used to transfect E14 129s/v embryonic stem cells by the Gene-Targeting Facility at the Babraham Institute. 500 clones were initially screened for homologous recombination using a 3′ Southern screen (probe 3′ to targeted sequence, EcoRI digest, 4.6–8.3 kb transition), and positive clones were rescreened by a 5′ Southern screen (probe 5′ to targeted sequence, SpeI digest, 11.9–10.0 kb transition) and for single insertion of the NeoR cassette (Neo probe, EcoRI/8.3 kb, and SpeI/5.7 kb; Fig. S1, A and C). Three clones were taken forward for blastocyst injection, and male chimeras from these mice were bred with female C57BL/6 animals to generate p40phox+/− heterozygotes on a mixed 129s/v/C57BL/6 background. Deletion of the NeoR cassette was confirmed by appropriate Southern and PCR analyses, and separate p40phox−/− colonies were created from each of the original targeted embryonic stem cell lines and housed under specific pathogen-free conditions in the SABU facility at the Babraham Institute. Genotyping of the mice was routinely performed by PCR amplification of an approximately 850-bp region flanking exon 3 (to include the inserted translational stop and additional XhoI and ApaI sites) and subsequent diagnosis by susceptibility of the product to cleavage by XhoI or ApaI (to yield ~525- and 325-bp fragments; Fig. S1 D). All mice used were 2–8-mo old and showed no age-dependent variation. This work was covered by UK Home Office Project License PPL 80/1875.
Preparation of BMNs.
BMNs were prepared as described previously with minor modifications (75
). Bone marrow, from at least three mice per preparation, was dispersed in HBSS (without Ca2+
), 0.25% fatty acid–free BSA, 15 mM Hepes, pH7.4, at room temperature and purified over discontinuous Percoll (GE Healthcare) gradients. After washes, mature neutrophils were resuspended in Dulbecco's PBS with Ca2+
, 1 g/liter glucose, 4 mM sodium bicarbonate (DPBS+
). Purity was typically 70–80% as assessed by cytospin and REASTAIN Quick-Diff (Reagena) staining (non-neutrophils were ~50% immature white cells, 25% monocytes, and 25% lymphocytes). All assays were performed in DPBS+
. BMNs were primed at 37°C with 500 U/ml mTNF-α, 100 ng/ml mGM-CSF, and 10% mouse serum for 60 min. In some experiments, as indicated, BMNs were primed individually with 500 U/ml mTNF-α for 30 min, 100 ng/ml mGM-CSF for 60 min, or 500 U/ml mTNF-α and 100 ng/ml mGM-CSF for 60 min.
Neutrophil and multiple tissue Western blots.
5 × 106 BMNs were sonicated into 1× SDS loading buffer, and 5 × 105 cell equivalents were subjected to SDS-PAGE, transferred, and blotted for p40phox (05-539 monoclonal; Upstate Biotechnology; sc-18252 and sc-18253 polyclonals; Santa Cruz Biotechnology, Inc.), p47phox (07-500 polyclonal; Upstate Biotechnology), and p67phox (07-502 polyclonal; Upstate Biotechnology). Signal was detected (Image Reader LAS-1000; Fugifilm) and quantified using Aida Image Analyser 2.2. For tissue Westerns, tissues were collected and immediately machinated into ice-cold 20 mM Hepes, pH7.1, at 4°C, 0.1% SDS, 0.4% cholate, 0.1% NP-40, 0.1M NaCl, 0.2 mM PMSF, and 10 μg/ml each of pepstatin A, leupeptin, antipain, and aprotinin and clarified by centrifugation (14,000 g for 30 min at 4°C). Protein concentrations were determined (BCA; Pierce Chemical Co.), and 30 μg of each protein was subjected to SDS-PAGE, transferred, and immunoblotted for p40phox.
PKB, p38 MAPK, and p42/44 Erk activation assays.
5 × 106 unprimed BMNs from p40phox−/− and p40phox+/+ mice were prewarmed for 3 min at 37°C at 5 × 107/ml in DPBS+ in duplicate. After 1 min of stimulation with prewarmed FMLP (10 μM final) or salts, reactions were stopped by the addition of excess ice-cold PBS, followed by immediate centrifugation (12,000 g for 10 s). Cell pellets were lysed in 20 mM Tris, pH 7.5 at 4°C, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 0.2 mM PMSF, and 10 μg/ml each of pepstatin A, leupeptin, antipain, and aprotinin and incubated on ice for 10 min. Cytoskeletal debris was removed by centrifugation (12,000 g for 20 min at 4°C). Lysates were split between two SDS-PAGE gels and blotted for phospho- and total protein, respectively. Blots were imaged and quantified as described above.
Preparation of mouse serum.
Mouse blood was collected and left to clot at room temperature for 45 min in a glass container before transferal to a 15-ml tube followed by centrifugation (1,500 g for 10 min at room temperature). Serum was removed to a fresh tube, recentrifuged, recovered, placed on ice, aliquoted, and stored at −80°C.
Chemiluminescent detection of ROS.
ROS production was measured by luminol-dependent chemiluminescence in polystyrene 96-well plates (no. 23300; Berthold Technologies Ltd.) as described previously (75
) in DPBS+
, except final concentrations of luminol and HRP were 150 μM and 18.75 U/ml, respectively. Prewarmed stimuli were added manually and measurement started immediately. Assays using soluble stimuli (PMA, FMLP, and mTNF-α) were conducted in the presence of exogenous added HRP; the signal was >95% HRP dependent, indicating predominantly extracellular ROS production (unpublished data). Assays using particulate stimuli (zymosan, IgG latex beads, and live S. aureus
) were conducted without HRP and thus represent intracellular ROS production. The addition of HRP revealed little extracellular ROS production (unpublished data). Final particle/BMN ratios were as follows: zymosan, 5:1; S. aureus
, 20:1; and IgG latex beads, 50:1. In some S. aureus
experiments, BMNs were preincubated with varying concentrations of DPI or vehicle (DMSO) alone before stimulation. Data output is in relative light units per second (rlu/s).
Surfaces (96-well plates or 12-well tissue culture plates) were coated overnight at 4°C with sheep fibrinogen (F9754; Sigma-Aldrich) at 150 μg/ml in PBS (100% FCS was used as a nonspecific control). Before use, surfaces were washed three times with PBS. For adhesion-dependent ROS assays, neutrophils at 5 × 106/ml were incubated at 37°C for 1 h before the addition of prewarmed 2× HRP/luminol followed by mTNF-α (final concentration of 20 ng/ml), anti-CD18 (anti–β2 integrin, M18/2; Chemicon), or IgG2a isotype control antibodies (final concentration of 18 μg/ml). Cells were immediately aliquoted into the plate (100 μl/well) and counted. For spreading experiments, preincubated neutrophils were incubated at 37°C in coated 12-well tissue culture plates (6.25 × 105/well) in the presence of 20 ng/ml mTNF-α. After 40 min, wells were aspirated and cells were fixed in 3.8% formaldehyde. Non-adhered cells were washed away and remaining cells were visualized by light microscopy. In both ROS and spreading assays, FCS-coated wells produced minimal responses (unpublished data). For analysis of β2 integrin expression, neutrophils were stimulated with mTNF-α for 30 min at 37°C, placed on ice, and processed for FACS analysis using the anti-CD18 antibody, its isotype control, and an FITC anti–rat secondary.
Preparation of particulate stimuli.
IgG-opsonized zymosan particles (IgG-Zym) were prepared as per the manufacturer's instructions (unlabeled zymosan A, Z-2849, and rabbit anti-zymosan A, Z-2850; Invitrogen). Zymosan and S. aureus
were serum opsonized or mock opsonized by incubation in DPBS+
with or without 50% mouse serum at 37°C with end-over-end mixing for 1 h (zymosan) or 15 min (S. aureus
) followed by washing. Carboxylate-modified latex beads (0.9-μm diameter; Sigma-Aldrich) were cross-linked to sulfhydryl-modified BSA and coated with an anti-BSA monoclonal antibody (Sigma-Aldrich) or not, as described previously (IgG latex beads) (76
). Where appropriate, S. aureus
was washed and resuspended in DPBS+
(4 × 108
/ml), heat killed at 60°C for 30 min, and opsonized in mouse serum as described above.
S. aureus phagocytosis assay.
primed BMNs were allowed to adhere to glass coverslips for 20 min at 37°C. They were then aspirated, 107
FITC-labeled, serum-opsonized S. aureus
was added, and they were returned to 37°C (FITC labeling of bacteria as described previously [77
] and opsonization as detailed above). After 40 min, coverslips were washed, fixed in 4% paraformaldehyde, and mounted. Postfixation probing with a rabbit anti–S. aureus
antibody (S-2860; Invitrogen) and goat anti–rabbit Alexa Fluor 568 secondary antibody (Invitrogen) revealed that >95% of bacteria present were internalized. Phagocytosed bacteria were visualized by fluorescence microscopy and enumerated.
Oxygen consumption was measured in a Clark-type oxygen electrode (Rank Brothers Ltd.) at 37°C with rapid stirring. Primed BMNs were added to the rapidly stirred chamber at 5 × 106/ml and equilibrated for 5 min before the addition of prewarmed stimuli. Final concentrations were 1 μM PMA; IgG-Zym, 20:1; and heat-killed S. aureus, 20:1 (particles/BMNs).
Primed BMNs were adhered to a coverslip in 0.5 mg/ml NBT at 37°C. IgG-Zym particles were added, and dark purple formazan deposition was followed during phagocytosis by bright field microscopy.
In vitro bacterial killing assays.
Bacteria (S. aureus Wood 46 and E. coli E2348169) were subcultured at 37°C to logarithmic growth from an overnight culture. 4 × 107 bacteria were washed in DPBS+ and opsonized as described above. Opsonized bacteria (1.5 × 106 S. aureus and 6 × 106 E. coli) were added to 6.2 × 106 primed BMNs (2.5 × 107/ml) at 37°C with rapid orbital mixing. After the indicated times, 50-μl aliquots were removed to 950-μl ice-cold Luria broth (LB) containing 0.05% saponin. Samples were sonicated (output 1.5 for 10 s; Sonicator 3000; Misonix) to liberate intracellular bacteria and returned to ice. Suspensions were serially diluted and plated on LB-agar to enumerate surviving bacteria. A parallel bacterial incubation was also run in the absence of neutrophils. In some experiments, neutrophils were incubated for 5 min with DPI at varying concentrations or vehicle (DMSO) alone before the addition of bacteria (oxidant-dependent killing control).
For S. aureus killing assays in whole blood, 2.5 × 106 bacteria in 1 ml DPBS+ were added to 1 ml of fresh whole blood (mixed from at least three animals) and incubated for 20 min at 37°C with end-over-end mixing. 0.8 ml of blood/bacteria mix was added to tubes containing lysostaphin (a final concentration of 2.5 U/ml to kill extracellular bacteria) in duplicate and returned to mixing. Samples were taken after 1 h, added to ice-cold PBS, and pelleted by centrifugation, and the pellets were resuspended in 0.5 ml nutrient broth, 0.05% saponin. Samples were then processed and quantified as described above.
In vivo S. aureus survival assays.
S. aureus (LS-1) was subcultured at 37°C to logarithmic growth from an overnight culture. Bacteria were washed and resuspended in injection-grade PBS at 2.5 × 108/ml. Three animals of each genotype per time point were injected intraperitoneally with 0.2 ml of bacterial suspension (5 × 107 bacteria). After 4 or 24 h, mice were killed and the peritoneal cavity was thoroughly flushed with 10 ml ice-cold PBS, 5 mM EDTA, and 5 U/ml heparin. Aliquots were diluted, sonicated, and plated, and bacteria were enumerated as for the in vitro killing assays.
Online supplemental material.
Fig. S1 illustrates the targeting strategy used to generate p40phox−/− mice and the subsequent screening strategies used. Fig. S2 denotes normal organ weights of p40phox−/− animals. Fig. S3 describes the S. aureus killing deficiency of whole blood from p40phox−/− animals.