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Originally published In Press as doi:10.1074/mcp.M500336-MCP200 on January 14, 2006.
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Molecular & Cellular Proteomics 5:620-634, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Maturation of Human Neutrophil Phagosomes Includes Incorporation of Molecular Chaperones and Endoplasmic Reticulum Quality Control Machinery *,S

Christopher Burlak{ddagger}, Adeline R. Whitney{ddagger}, David J. Mead§, Ted Hackstadt§ and Frank R. DeLeo{ddagger},

From the {ddagger} Laboratory of Human Bacterial Pathogenesis and § Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 59840


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A Human neutrophils are an essential component of the innate immune response. Although significant progress has been made toward understanding mechanisms of phagocytosis and microbicidal activity, a comprehensive analysis of proteins comprising neutrophil phagosomes has not been conducted. To that end, we used subcellular proteomics to identify proteins associated with human neutrophil phagosomes following receptor-mediated phagocytosis. Proteins (n = 411 spots) resolved from neutrophil phagosome fractions were identified by MALDI-TOF MS and/or LC-MS/MS analysis. Those associated with phagocytic vacuoles originated from multiple subcellular compartments, including the cytosol, plasma membrane, specific and azurophilic granules, and cytoskeleton. Unexpectedly several enzymes typically associated with mitochondria were identified in phagosome fractions. Furthermore proteins characteristic of the endoplasmic reticulum, including 11 molecular chaperones, were resolved from phagosome preparations. Confocal microscopy confirmed that proteins representing these major subcellular compartments were enriched on phagosomes of intact neutrophils. Notably calnexin and glucose-regulated protein 78 co-localized with gp91phox in human neutrophils and were thus likely delivered to phagosomes by fusion of specific granules. We conclude that neutrophil phagosomes have heretofore unrecognized complexity and function, which includes potential for antigen processing events.

Polymorphonuclear leukocytes (PMNs1 or neutrophils) are a first line of defense against bacterial and fungal infections. PMNs are recruited to sites of infection and ingest microbes by a process known as phagocytosis. Uptake of microorganisms is facilitated efficiently by complement and antibody receptors present on the cell surface (1). Receptor-mediated phagocytosis triggers production of reactive oxygen species and granule-phagosome fusion, thereby enriching the phagocytic vacuole with antimicrobial molecules (2–4). The ability of neutrophils to ingest and kill microbes is essential for human innate host defense (5).

Phagocytosis and subsequent maturation of phagosomes involves coordinated molecular processes, including multiple vesicle fusion events (for a review by Desjardins, see Ref. 66; also see Vieira et al. (7)) and reorganization of the actin cytoskeleton (8–11). The plasma membrane is a key component of newly forming phagocytic vacuoles (12), and membrane proteins are continually recycled in and out of the vacuoles as they mature (13, 14). Studies using macrophages and macrophage-like cell lines have revealed that phagosomes mature with sequential endosome-lysosome fusion events culminating in the formation of a mature phagocytic vacuole competent to kill microbes (7, 13, 15–19). Notably, several recent studies indicate that the endoplasmic reticulum (ER) fuses with forming phagosomes, thereby providing an additional source of membrane and machinery required for antigen processing (6, 20, 21).

Much of our current understanding of phagosome maturation is derived from model systems using macrophages or macrophage-like cell lines. As such, there is a paucity of information for this process in human neutrophils. To gain an enhanced understanding of mechanisms that promote phagocytosis and microbicidal activity in phagocytes, we performed a comprehensive analysis of proteins associated with human neutrophil phagosomes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Human Neutrophils—
Human neutrophils were isolated from fresh venous blood of healthy individuals as described previously (22). Studies were performed in accordance with a protocol approved by the Institutional Review Board for Human Subjects, NIAID, National Institutes of Health. Heparinized blood was mixed 1:1 with 0.9% sodium chloride (injection, United States Pharmacopoeia, Baxter Healthcare, Deerfield, IL) containing 3.0% dextran T-500 (Amersham Biosciences) and then incubated for 20 min at room temperature to sediment erythrocytes. The resulting leukocyte-rich supernatant was centrifuged at 670 x g for 10 min, and cells were resuspended in 35 ml of 0.9% sodium chloride (Baxter Healthcare). The leukocyte suspension was underlayed with 10 ml of Ficoll-PaquePLUS (1.077 g/liter, Amersham Biosciences) and centrifuged for 25 min at 350 x g to separate neutrophils from peripheral blood mononuclear cells. Peripheral blood mononuclear cells were aspirated from the Ficoll-PaquePLUS-saline interface, and sides of the gradient tubes were wiped with sterile cotton swabs to remove any residual cells. After standard hypotonic lysis of erythrocytes, purified PMNs were suspended in RPMI 1640 medium (Invitrogen) buffered with 10 mM Hepes, pH 7.2 and placed on ice until used. PMNs were enumerated with a hemocytometer, and purity of cell preparations and viability were assessed by flow cytometry (FACSCalibur, BD Biosciences). PMN preparations typically contained ~94% neutrophils with the remaining cells being predominantly eosinophils (22, 23). For simplicity, the terms neutrophil and PMN are used interchangeably. All reagents used contained <25.0 pg/ml endotoxin (Limulus amebocyte lysate assay, Fisher).

Neutrophil Phagocytosis and Phagosome Isolation—
Antibody- and complement-coated latex beads (IgG/C3bi-LBs) were prepared as described previously (22). Synchronized phagocytosis was performed as reported by Kobayashi et al. (22) using a ratio of ~8 IgG/C3bi-LBs/neutrophil. At the indicated times, phagocytosis was terminated either by chilling PMNs on ice or adding cold paraformaldehyde (4%) to each well of the assay. Cells destined for subcellular fractionation were immediately placed on ice and treated with diisopropylfluorophosphate (4 mM, Sigma). Neutrophils were centrifuged at 350 x g for 6 min at 4 °C and suspended in relaxation buffer (1 mM PIPES, 100 mM KCl, 3 mM NaCl, and 3.5 mM MgCl2) containing ATP (1 mM) to a final concentration of 108 cells/ml. PMNs were disrupted by standard N2 bomb cavitation at 400 p.s.i. for 20 min as reported previously (24). Subcellular fractions were separated with a Percoll step gradient as described originally by Borregaard et al. (24). IgG/C3bi-LB phagosomes sedimented to a region of the gradient between the plasma membrane- and specific granule-enriched fractions. To remove Percoll, phagosome-enriched fractions were centrifuged at 288,490 x g for 10 min (4 °C), and the phagosome pellet was aspirated and dispersed in a new tube containing 1 ml of relaxation buffer. This procedure was performed three times. Purified phagosomes were precipitated with ice-cold acetone and lyophilized as described below and then resuspended in SDS-PAGE sample buffer (10% SDS, 30 mM Tris-HCl, pH 6.8, 50% glycerol, 0.05% bromphenol blue, and 144 mM ß-mercaptoethanol) or isoelectric focusing solubilization buffer (7 M urea, 2 M thiourea, CHAPS, and ultrapure water). Samples were stored at –80 °C until used.

SDS-PAGE and Immunoblotting—
Proteins were separated by SDS-PAGE and transferred to PVDF or nitrocellulose. Membranes were blocked with 5% nonfat milk in Dulbecco’s PBS (DPBS) (Sigma) overnight at 4 °C. Blots were probed with antibodies specific for human gp91phox (monoclonal antibody 54.1) (25), p47phox (26), myeloperoxidase (MPO) (monoclonal antibody MPO-7, DakoCytomation California Inc., Carpinteria, CA), and rabbit IgG (MP Biomedicals, Irvine, CA) for 1–2 h at ambient temperature or overnight at 4 °C. Blots were washed in DPBS containing Tween 20 and incubated with secondary antibodies conjugated to horseradish peroxidase for 1–2 h at ambient temperature. Proteins were visualized with enhanced chemiluminescence (SuperSignal West Pico, Pierce) using Eastman Kodak Co. X-Omat film.

IEF and Second Dimension SDS-PAGE—
Phagosome proteins were precipitated overnight in polypropylene tubes with 8 volumes of cold acetone at –20 °C. Precipitated proteins were centrifuged at 3273 x g for 30 min at 4 °C and resuspended in ice-cold ethanol. Samples were chilled for 30 min at –20 °C and centrifuged as described above. Proteins were air-dried and then solubilized with IEF buffer (7% urea, 2% thiourea, 4% CHAPS, and 200 mM tributylphosphine). Protein concentration was measured with the 2-D Quant kit (Amersham Biosciences), and purified phagosome proteins were stored at –80 °C.

For IEF, samples were treated with Destreak rehydration solution (25% of total sample volume) (Amersham Biosciences), 200 mM tributylphosphine, and 1% ampholytes. The appropriate rehydration volume for each IPG Ready Strip (Bio-Rad) size was attained by adding solubilization buffer. IEF was performed with 11-, 17-, or 24-cm IPG Ready Strips for 40, 60, or 80 kV-h, respectively. IPG Ready Strips were actively rehydrated at 50 V overnight prior to first dimension separation. Moistened filter paper wicks (Whatman No. 1 paper) were added between each electrode and strip prior to focusing (after rehydration). Wicks were changed four times in the first 4 h of IEF after which the voltage was maintained at 8000 V (11-cm IPG Ready Strips) or 10,000 V (17- and 24-cm IPG Ready Strips). Following IEF, IPG Ready Strips were stored at –80 °C until used for SDS-PAGE. For second dimension SDS-PAGE, IPG Ready Strips were thawed in SDS equilibration buffer (5% SDS, 30 mM Tris, pH 8.8, 4 M urea, and 0.01% bromphenol blue) containing dithiothreitol (65 mM DTT) for 20 min followed by incubation in SDS equilibration buffer containing 135 mM iodoacetamide for an additional 20 min. IPG Ready Strips were placed immediately in IPG wells of 11 or 18 cm (precast 12.5% acrylamide or 10–20% gradient gels, Bio-Rad) or 24 cm (precast 12.5% acrylamide gels, Bio-Rad) and sealed with 1% agarose prepared with running buffer (25 mM Tris, pH 8.3, 192 mM glycine, and 0.1% SDS) containing 0.01% bromphenol blue. Electrophoresis was performed at 200 V until the dye front reached the bottom of each gel (~1 h for 11-cm gels, 6.5 h for 18-cm gels, and 10.5 h for 24-cm gels). Electrophoresis with 18- and 24-cm gels was performed at 20 °C. Gels were incubated with Bio-Safe Coomassie Blue stain (Bio-Rad), and protein spots were excised in water containing 3% acetic acid and placed directly into 96-well Zip plates (Millipore Corp.). Protein plugs were subjected to sequential dehydration in 5, 50, and 100% acetonitrile and rehydrated in buffer containing 200 units/ml porcine trypsin (Promega Corp., Madison, WI). Peptides were eluted from Zip plates with 50% acetonitrile and 0.2% trifluoroacetic acid (for MALDI-TOF analysis) or 100% acetonitrile and 1% formic acid (for LC-MS/MS analysis) and stored at –80 °C until analysis.

Immunofluorescence and Confocal Laser-scanning Microscopy—
Acid-washed coverslips (No. 1, 13 mm, round) were flamed and then coated with 20% normal human serum in 24-well tissue culture plates for 1 h. Coverslips were washed twice with DPBS, and synchronized phagocytosis was performed in 24-well plates as described above. In some assays, culture medium was exchanged with that containing 250 nM Mitotracker Red CMXRos (Molecular Probes, Eugene, OR) 15 min after cell activation. At 30 min, plates were chilled on ice, and cells were fixed with cold RPMI 1640 medium containing 4% paraformaldehyde for 30 min. Fixed PMNs were washed three times in DPBS and then permeabilized with 0.2% Triton X-100 for 5 min. After three more washes in DPBS, cells were incubated with blocking buffer (DPBS containing 5% BSA and 0.02% sodium azide) for 1 h. Samples were labeled with goat anti-rabbit polyclonal antibody conjugated to FITC (Zymed Laboratories Inc.) singly or in combination with antibodies specific for glucose-regulated protein 78 (GRP78) (5 µg/ml) (Santa Cruz Biotechnologies, Santa Cruz, CA), peroxiredoxin 3 (5 µg/ml) (Santa Cruz Biotechnologies), prohibitin (5 µg/ml) (Santa Cruz Biotechnologies), F1 ATPase (5 µg/ml) (Santa Cruz Biotechnologies), karyopherin ß1 (5 µg/ml) (Santa Cruz Biotechnologies), protein-disulfide isomerase (PDI) (5 µg/ml) (Abcam, Cambridge, MA), actin-related protein C2 (2 µg/ml) (Abcam), resistin (5 µg/ml) (Chemicon International Inc., Temecula, CA), ERp61 (5 µg/ml) (BD Biosciences), CD16 (5 µg/ml) (BD Biosciences), CD11b (5 µg/ml) (DakoCytomation California Inc.), and/or MPO (2 µg/ml) (DakoCytomation California Inc.). Samples were subsequently labeled with donkey anti-goat or goat anti-mouse antibody conjugated with AlexFluor594 (2 µg/ml) (Molecular Probes). In some experiments, PMNs were stained with 4',6-diamidino-2-phenylindole (300 nM in DPBS, Molecular Probes) or DRAQ5 (1.25 µM in DPBS, Biostatus Ltd., Leicestershire, UK) prior to mounting coverslips onto slides. Slides were analyzed with a Zeiss LSM510 confocal laser-scanning microscope coupled to an Axiovert 200M inverted microscope (Carl Zeiss, Inc., Thornwood, NY). Images were acquired using a 100x Plan-Apochromat oil immersion objective (1.4 numerical aperture) at 512 x 512 pixel resolution with averaging (2x). Images were adjusted equally for brightness and contrast in Adobe Photoshop CS (Adobe Systems Inc., San Jose, CA).

Transmission Electron Microscopy—
Electron microscopy was performed as described previously (27) except that the sections were examined on a Hitachi H7500 transmission electron microscope operated at 80 kV. Images were recorded with a charge-coupled device camera (Advanced Microscopy Technologies, Danvers, MA) and adjusted with Adobe PhotoShop CS as described above.

Mass Spectrometry—
Peptides from trypsin digests were diluted 1:10 in a matrix solution containing saturated sinapinic acid in acetonitrile/water/trifluoroacetic acid (30:70:0.1, v/v), and a 1-µl aliquot was spotted and air-dried on a sample plate for MALDI-TOF MS analysis. Mass spectra were obtained by using a Voyager-DE STR MALDI-TOF instrument (Applied Biosystems, Foster City, CA). The ion spectra were collected with the following instrument settings: mode of operation, linear; extraction mode, delayed; extraction delay time, 240 ns; polarity, positive; accelerating voltage, 25,000 V; grid voltage, 69%; acquisition mass range, 600–3000 Da; and laser repetition rate, 20 Hz. The spectra were calibrated by using internal standards of autolytic trypsin peptides (molecular masses used were 842.5, 1045.5, 1940.8, 2211, and 2807.3 Da).

Capillary LC-MS/MS was performed with a Micromass CapLC and a quadrupole- time-of-flight mass spectrometer (QTof-2, Micromass, Manchester, UK) at the NIAID, National Institutes of Health, Mass Spectrometry Laboratory (Rockville, MD). Peptides from trypsin digests were separated with a Zorbax C18 SBW reverse phase column (100 mm x 0.15 mm inner diameter) at a flow rate of 1 µl/min. The mobile phase consisted of a gradient prepared from solvent A (0.2% formic acid) and solvent B (99.8% acetonitrile and 0.2% formic acid). Peptide separations were performed at ambient temperature. Computer-controlled data-dependent automated switching to MS/MS provided peptide sequence information. MassLynx and Global Server software (Waters Corp., Milford, MA) was used for data acquisition and processing, and the search analysis was performed using Mascot software version 2.1 (Matrix Science, London, UK) using the National Center for Biotechnology Information non-redundant database (NCBInr, updated February 14, 2005 at 15:54:28 Greenwich mean time).

Proteins analyzed by MALDI-TOF MS were identified using the MS-Fit module of Protein Prospector version 4.0.5 (Mass Spectrometry Facility, University of California, San Francisco, CA) by searching against the Homo sapiens subset of the NCBI non-redundant protein database (database number 030311). Search parameters included analysis of peptides for carbamidomethylation, modification of peptide N-terminal glutamine to pyroglutamine, oxidation of methionine, acrylamide-modified cysteine, a mass tolerance of ±25 ppm, a maximum of one missed tryptic cleavage, and a minimum of four peptides identified.

Proteins analyzed by LC-MS/MS were identified with the MS/MS Ion Search module of Mascot Search (Matrix Science, London, UK) using the NCBI non-redundant database. Search parameters included monoisotopic masses, analysis of peptides for carbamidomethylation and/or propionamidylation of cysteine, oxidation of methionine, peptide and fragment mass tolerance of ±0.5 Da, and a maximum of one missed tryptic cleavage. The significance threshold for positive identification was determined by the Mascot Search program. A workflow diagram summarizes the approach used to identify proteins associated with neutrophil phagosomes (Fig. 1).


Figure 1
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  		FIG. 1. A workflow for the identification of neutrophil phagosome proteins. Following receptor-mediated phagocytosis, IgG/C3bi-LB phagosomes were isolated by Percoll density gradient centrifugation. Phagosome proteins were separated by IEF coupled with second dimension SDS-PAGE (A) or SDS-PAGE alone (B). Proteins were removed from polyacrylamide gels and identified by mass spectrometry as indicated. TBP, tributyl phosphine.

 

   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation of Neutrophil Phagosomes following Receptor-mediated Phagocytosis—
To determine an optimal time point at which to isolate neutrophil phagosomes, we measured phagocytosis of IgG/C3bi-LBs. Consistent with previous studies (22), there was rapid ingestion of IgG/C3bi-LBs by human neutrophils (phagocytosis was 92.84 ± 0.7% by 30 min, n = 5) (Fig. 2A). Therefore, phagosome isolation was conducted at the 30-min time point. We next determined that IgG/C3bi-LB phagosomes could be isolated with a Percoll step gradient developed originally by Borregaard et al. (24) (Fig. 2B). Analysis of phagosome fractions by transmission electron microscopy indicated that vacuole membranes were at least partially intact (Fig. 2C, red arrows). In accordance with this observation, IgG/C3bi-LBs obtained from phagosome fractions were enriched for p47phox and gp91phox, components of the NADPH-dependent oxidase that localize typically to phagosome membranes (28) (Fig. 2D). To examine further the purity of phagosome fractions, subcellular fractions from unstimulated or phagocytosing neutrophils were probed for gp91phox (plasma membranes and membranes of secretory vesicles and specific granules), MPO (azurophilic granule matrix), p47phox (cytosol), and IgG (IgG/C3bi-LB phagosomes) (Fig. 2E). By immunoblot analysis, there was clear redistribution of p47phox, gp91phox, and MPO to phagosome fractions. Inasmuch as MPO was redistributed from azurophilic granules to phagosomes only and p47phox was redistributed from cytosol to phagosomes, isolation per se of IgG/C3bi-LB phagosomes did not compromise integrity of the vacuole or perturb key protein-protein interactions (Fig. 2E). We note that equivalent fractions from unstimulated PMNs had little or no positive staining for these proteins, confirming that phagosome-containing fractions were of high purity.


Figure 2
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  		FIG. 2. Isolation and characterization of neutrophil phagosomes. A, phagocytosis of IgG/C3bi-LBs by human neutrophils. Results are the mean ± S.E. of five separate experiments. B, isolation of neutrophil phagosomes. IgG/C3bi-LB-containing phagosomes (phg) sediment to a region of the Percoll gradient between plasma membrane- ({gamma}) and specific granule-enriched (ß) fractions. Mock gradient (without IgG/C3bi-LBs) is shown on the right (Unstim.). cyt, cytosol fraction; {alpha}, azurophilic granule-enriched fraction. C, transmission electron micrographs of IgG/C3bi-LB phagosomes isolated from Percoll density gradients (left panel). Red arrows indicate phagosomal membrane. Black arrows indicate the boundary of latex beads at the point of embedding in resin. Right panel, IgG/C3bi-LBs (LB) (not incubated with neutrophils). D, isolated phagosomes contain NADPH oxidase components. Purified phagosomes were labeled with monoclonal antibodies specific for gp91phox and p47phox and then visualized by confocal laser-scanning microscopy (left panel). Right panel, IgG/C3bi-LBs not incubated with neutrophils. E, redistribution of gp91phox, p47phox, and MPO to phagosomes. Unstimulated neutrophils or those activated by phagocytosis of IgG/C3bi-LBs were fractionated by Percoll density gradient sedimentation. 106 cell equivalents of each particulate fraction were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with the indicated antibodies. {gamma}, plasma membrane-enriched fraction; phg, phagosome-enriched fraction; ß, specific granule-enriched fraction; {alpha}, azurophil granule-enriched fraction. Results are representative of three experiments. DIC, differential interference contrast; IgG Hch, rabbit immunoglobulin G heavy chain.

 
Identification of Neutrophil Phagosome Proteins Using Subcellular Proteomics—
The formation and maturation of neutrophil phagosomes involve proteins from multiple subcellular compartments and the cytosol, which in concert mediate uptake of microbes and killing of pathogens. As a step toward gaining an enhanced understanding of mechanisms of phagocytosis and bactericidal activity, we used high resolution subcellular proteomics to identify proteins associated with neutrophil phagosomes. Greater than 500 protein spots were resolved using IEF coupled with second dimension SDS-PAGE of which 411 were positively identified by MALDI-TOF and/or LC-MS/MS. In addition, we used SDS-PAGE in combination with LC-MS/MS to identify proteins that might not be easily resolved by IEF, such as those with high net charge. Excluding protein isoforms and subunits translated from the same gene, we identified 198 unique proteins associated with neutrophil phagosomes (Figs. 3, Table I, and Supplemental Table 1, which contains details for identification of proteins by MS). To facilitate subsequent analyses, phagosome proteins were separated into categories based on functional annotation (Fig. 3 and Table I).


Figure 3
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  		FIG. 3. Function of proteins associated with neutrophil phagosomes. Proteins identified by proteomics were categorized based upon functional annotation. The number in each category precedes the percentage (of 198 unique proteins).

 

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  		TABLE I Neutrophil phagosome proteins identified by MALDI-TOF MS and/or LC-MS/MS

One-hundred ninety-eight unique proteins were identified from one-dimensional (see Footnote f) or two-dimensional gels coupled with MS analysis as described under "Experimental Procedures." Protein isoforms and/or subunits and proteins comprising IgG/C3bi-LBs were not included in the final protein count. Aliases or common protein names are presented as appropriate (after semicolons). SC, sequence coverage; MP, matched peptides.

 
Newly Formed Phagosomes Contain Dozens of Proteins Involved in Signal Transduction, Cell Mobility, and Innate Host Defense—
We identified 28 phagosome-associated proteins known to promote signal transduction (Table I). For example, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAB), a member of the 14-3-3 family of proteins, dimerizes in response to mitogen-activated protein kinase-dependent inflammatory signaling (29) and binds CD11/CD18 in human neutrophils, possibly regulating cell adhesion (30). Localization of signaling molecules, such as 14-3-3 proteins, to the phagosome is novel but not unexpected as they reflect the complex molecular rearrangements taking place.

At least 27 unique proteins involved in cell motility and/or those that comprise the cytoskeleton were identified in phagosome fractions (Fig. 3 and Table I). Consistent with these results, actin, ezrin, gelsolin, moesin, L-plastin, profilin, tropomyosin, tubulin, and radixin have been shown to participate directly or indirectly in phagocytosis (9, 31–37). Collectively the data indicate that three classes of well characterized cytoskeletal components, i.e. microfilaments (actins), intermediate filaments (desmin and vimentin), and microtubules (tubulins), are enriched on neutrophil phagosomes (38). These elements work together to facilitate phagocytosis and likely promote fusion of granules and other organelles with maturing phagosomes.

Proteins directly involved in host defense comprise 14.6% (29 of 198) of those identified on or within PMN phagosomes (Table I). We identified at least 17 phagosome-associated proteins that were originally derived from neutrophil granules, including {alpha}1-antitrypsin, arginase 1, azurocidin 1, bactericidal/permeability-increasing protein, cathepsin S, cathepsin G, CD11b, collagenase, {alpha}-defensin, elastase, FALL-39, gelatinase, lactoferrin, lipocalin, lysozyme, MPO, proteinase-3, and V-type H+-ATPase (2). These findings indicate that there was fusion of azurophilic, specific, and gelatinase granules with IgG/C3bi-LB phagosomes. In addition, phagosomes were enriched with calgranulins A–C (S100A8, S100A9, and S100A12), chitotriosidase, ficolin, granulocyte peptide A, haptoglobin, lactotransferrin, S100P, and peptidoglycan recognition protein 1 (PGLYRP1). Taken together, the data provide direct evidence for the notion that neutrophil phagosomes are enriched with numerous antimicrobial molecules.

Proteins That Facilitate Metabolism and Vesicle Trafficking Are Associated with Neutrophil Phagosomes—
Forty-nine (24.7%) of the proteins associated with phagosomes are known to participate in cell metabolism and detoxification/redox processes (Fig. 3 and Table I). Enzymes that regulate glycolysis and/or the pentose phosphate pathway, including enolase 1, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase, pyruvate kinase 3, transketolase, and triose-phosphate isomerase 1 were found in PMN phagosome fractions (Table I). Glyceraldehyde-3-phosphate dehydrogenase is known to associate with actin filaments (39) and has been identified previously in macrophage phagosomes (40). The finding that other key enzymes of glucose metabolism were associated with neutrophil phagosomes suggests glycolysis occurs in or around the phagocytic vacuole.

The identification of at least 20 proteins typically associated with mitochondria was unexpected (Table I). Several subunits of the mitochondrial respiratory chain, including cytochrome c oxidase Va (COX5A), cytochrome c oxidase VIIa (COX7A2), NADH coenzyme Q reductase (NDUSF3), Rieske iron-sulfur polypeptide (UQCRFS1), ubiquinol-cytochrome c reductase complex core protein I (UQCRC1), and electron transfer flavoprotein, {alpha} polypeptide (ETFA), were identified in phagosome fractions (Table I). Because we failed to identify these proteins in comparable fractions from unstimulated cells (data not shown) and mitochondrial proteins co-localize with phagosomes in intact cells (see below), these proteins are not likely derived from mitochondria simply co-sedimenting with phagosomes during the purification process. Garin et al. (40) found mitochondrial proteins associated with phagosomes from macrophage-like cells, but a function for those molecules was not proposed. It is possible that incorporation of selected components of the mitochondrial electron transport chain promotes the generation of oxidative potential across the phagosome membrane.

Inasmuch as phagosomes are dynamic organelles that undergo high levels of vesicle fusion, it might be predicted that molecules linked to vesicle trafficking and/or fusion would be associated with phagosomes. However, we identified only a limited number of proteins directly involved in vesicle fusion events (n = 7). This may be due to the relatively mature state of the phagosomes analyzed (30 min). Our observation that several annexin proteins were associated with phagosomes is consistent with the known role of these proteins in regulation of the actin-based cytoskeleton (32, 35, 41–45).

Neutrophil Phagosomes Are Enriched with Proteins Characteristic of the ER and Protein Quality Control Machinery—
It has been proposed that the ER fuses with the developing phagocytic vacuole to contribute membrane and possibly provide machinery for antigen processing (6, 21). Consistent with that idea, molecular chaperones normally found in the ER, such as ER protein 29 precursor (ERp29), glucose-regulated protein 58 (GRP58, ERp61, or ERp57), GRP78 (or BiP), PDI, and protein-disulfide isomerase-associated 6 (PDIA6), were identified in neutrophil phagosome fractions (Table I). Recent studies indicate that macrophage and dendritic cell phagosomes process antigens for MHC class I cross-presentation, a phenomenon facilitated presumably by proteasomes and molecular chaperones (21, 46–48). In addition, previous work has shown that phagosomes are competent for MHC class II presentation, (19) although a direct role for the proteasome in that process has not been demonstrated. Thus it is possible that the phagosome-associated molecular chaperones are required for antigen processing by neutrophils. Consistent with that notion, we identified an MHC class II molecule (HLA-DPB1), five proteasome subunits, including a subunit of the 11 S immunoproteasome (PA28{alpha}/PSME1), ubiquitin, and ubiquitin C, in phagosome-enriched fractions from human neutrophils (Table I). These observations provide strong support to the idea that ER protein processing and associated quality control machinery are involved in the generation of peptide antigens within phagosomes. Further studies are needed to investigate antigen processing in neutrophil phagosomes.

Phagosomes of Intact Human Neutrophils Are Enriched with Molecular Chaperones and Proteins Typically Found in Mitochondria—
We next used confocal laser-scanning microscopy to confirm that proteins representative of selected subcellular compartments, i.e. granules, ER, and mitochondria, were enriched on phagosomes in intact human neutrophils (Fig. 4). MPO, CD11b, and CD16, proteins derived from granules, secretory vesicles, and the plasma membrane, were enriched in or around most PMN phagosomes as expected (Fig. 4). CD11b and CD16 were positive on most but not all phagosomes (91 ± 5% positive) and appeared present in relatively low abundance (Fig. 4). The reduced levels (compared with MPO) of these key receptors on mature phagosomes may be due to receptor recycling. Importantly we confirmed that GRP58, GRP78, and PDI were enriched on or within phagosomes from intact human neutrophils (Fig. 4). In addition, F1 ATPase subunits, peroxiredoxin 3 (PRDX3), and prohibitin, markers for mitochondria, were clearly localized to neutrophil phagosomes (Fig. 4). We note that subcellular compartments other than phagosomes stained positively with antibodies specific for these proteins. To determine whether the organelles in question were mitochondria, we labeled phagosome-containing PMNs with Mitotracker Red CMXRos, a dye whose accumulation is dependent on the oxidative potential of the mitochondrial membrane. PMNs stained with Mitotracker Red CMXRos had patterns of fluorescence similar to those of cells labeled with antibodies specific for F1 ATPase, peroxiredoxin 3, and prohibitin (Fig. 5). Although many of these subcellular compartments are likely neutrophil mitochondria, it is possible that the proteins originate in part from other organelles such as granules (49).


Figure 4
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  		FIG. 4. Confirmation of proteomic data by confocal laser-scanning microscopy. Following synchronized phagocytosis of IgG/C3bi-LBs (LB) (30 min), neutrophils were labeled with the indicated antibodies, and proteins were visualized by confocal laser-scanning microscopy. The number of percent positive phagosomes is indicated within each panel. Karyopherin ß1, a protein typically present in the nucleus, was used as a negative control. Yellow arrows indicate representative phagosomes. Dotted lines indicate the outline of each PMN. Results are the mean ± S.D. of 50 cells from three separate experiments. Ab, antibody.

 

Figure 5
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  		FIG. 5. Subcellular distribution of F1 ATPase, prohibitin, PRDX3, and mitochondria in human neutrophils. Following synchronized phagocytosis of IgG/C3bi-LBs (30 min), neutrophils were labeled with the indicated antibodies and Mitotracker Red CMXRos (CMXRos). Proteins, mitochondria, and phagosomes were visualized by confocal laser-scanning microscopy. White arrows indicate organelles co-labeled with antibody and Mitotracker Red CMXRos. Yellow arrows indicate organelles labeled with antibody only. Dotted lines indicate the outline of each neutrophil. Results are the mean ± S.D. of 50 cells from three separate experiments. Ab, antibody.

 
Proteins Characteristic of Mitochondria and ER Originate in Part from Neutrophil Granules—
As a step toward determining whether molecular chaperones and selected proteins of mitochondria reside in specific granules, we stained phagocytosing human PMNs with antibodies specific for calnexin, GRP78, prohibitin, F1 ATPase, and gp91phox (Fig. 6). Neutrophil phagosomes were highly enriched for each of the proteins 15 min after initiation of phagocytosis (Fig. 6B). There was partial co-localization of calnexin and GRP78 with gp91phox, indicating that a pool of these proteins is located in specific or gelatinase-containing granules (Fig. 6A). The finding that calnexin co-localized with neutrophil granules is consistent with studies by Lominadze et al. (49). Although prohibitin and F1 ATPase co-localized in part with gp91phox, there were other subcellular compartments, including phagosomes, that stained singly with the mitochondrial proteins (Fig. 6A). On the other hand, the kinetics of phagosome enrichment indicates that each of the proteins was delivered to phagosomes in a relatively synchronous manner (Fig. 6B).


Figure 6
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  		FIG. 6. Co-localization of specific granules with GRP78, calnexin, F1 ATPase, and prohibitin in human neutrophils. A, following synchronized phagocytosis of IgG/C3bi-LBs (30-min time point is shown), neutrophils were labeled with the indicated antibodies, and proteins were visualized by confocal laser-scanning microscopy. White arrows indicate co-localization of proteins. Yellow arrows indicate organelles that labeled with one antibody only. B, number of enriched phagosomes at each time point. Results are the mean ± S.D. of 50 cells from three separate experiments. Ab, antibody; DIC, differential interference contrast; phago., phagosomes.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Using high resolution subcellular proteomics we generated a comprehensive view of proteins associated with human neutrophil phagosomes. Although previous studies have identified individual proteins or small groups of proteins associated with neutrophil phagosomes or reported proteomic analyses of macrophage-like cells, the work herein represents the first proteomic analysis of these organelles from human PMNs.

Comparison of Phagosomes Derived from Human PMNs and Macrophage-like Cells—
Inasmuch as PMNs and macrophages have overlapping roles in the destruction of invading microorganisms, the finding that a number of proteins associated with phagosomes of macrophage-like cells (J774 macrophages) were also present on or in those of neutrophils is not unexpected (compare Table I in the current studies with Table I I in Ref. 40). For example, proteins comprising the actin-based cytoskeleton or those involved in cell motility, such as ß- and {gamma}-actin, {alpha}-actinin, actin-related protein 2/3 complex subunits, casein {alpha}S1, syntenin, tubulin {alpha}6, tropomyosin, myosin, vimentin, and 14-3-3 proteins (YWHAB and YWHAZ), were associated with phagosomes of human PMNs and J774 macrophages (40). One of the notable findings of our studies was that proteins typically found in the ER, including calnexin, ERp29, GRP58/ERp57, GRP78/BiP, and PDI, were associated with neutrophil phagosomes (Figs. 4–6 and Table I). Previous studies have shown that these molecular chaperones are associated with phagosomes of macrophage-like cells (20, 40), albeit a role for those proteins was not elucidated. There is ongoing debate regarding the role of ER in macrophage phagocytosis (50, 51). Recent studies by Gagnon et al. (20), Houde et al. (21), Ackerman et al. (46), and Guermonprez et al. (47) revealed how antigens processed within phagosomes undergo "cross-presentation" by MHC class I molecules. In those studies, proteasome subunits, ubiquitin, and polyubiquitin proteins were found associated with macrophage or dendritic cell phagosomes (21). The authors proposed that protein processing and quality control machinery (i.e. proteasomes, molecular chaperones, etc.) function within phagosomes to modify proteins for antigen presentation (21). This hypothesis is supported by our analysis of human neutrophil phagosomes (Figs. 3–6 and Table I) in which we identified ubiquitin, 11 molecular chaperones, and five proteasome subunits associated with these organelles. Thus, it will be important in future studies to investigate the role of neutrophils in this process.

There are also significant differences in phagosome composition between neutrophils and macrophage-like cells (40). For instance, Garin et al. (40) identified at least nine Rab proteins associated with phagosomes from macrophage-like cells, whereas we failed to identify these proteins on phagosomes derived from human PMNs. This disparity may be related to the differences in times analyzed (30 min in neutrophils versus 60 min plus in J774 macrophages) or cell types, although granules were clearly delivered to neutrophil phagosomes by the time point analyzed in our studies (see Fig. 1 and Table I). Compared with macrophage like-cells, a greater number of proteins known to participate in microbicidal activity were found in neutrophil phagosomes, most likely reflecting the known enhanced capacity of PMNs to kill bacteria and fungi compared with macrophages. Consistent with that notion, many proteins that moderate effects of reactive oxygen species, including catalase, glutathione S-transferase, and several peroxiredoxins, were associated with neutrophil phagosomes but not identified in or around those of macrophage-like cells (40).

Are Mitochondrial Proteins Bona Fide Components of Neutrophil Phagosomes?—
Each of the mitochondrial proteins we identified on or within phagosomes is encoded by the cell nucleus, and these proteins are normally imported into mitochondria by translocases of inner and outer mitochondrial membranes (for a review by Wiedemann et al., see Ref. 52). As visualized by confocal microscopy, F1 ATPase, prohibitin, and peroxiredoxin 3 co-localized in part with organelles labeled with Mitotracker Red CMXRos, confirming their presence within mitochondria (Fig. 5, white arrows). However, there were instances when staining for these proteins failed to correspond with that of mitochondria (Fig. 5, yellow arrows). These observations indicate that distribution of F1 ATPase, prohibitin, and peroxiredoxin 3 is not restricted to mitochondria, and therefore the proteins might have been delivered to phagosomes by fusion with other organelles or by direct import. The recent identification of mitochondrial proteins in gelatinase granules, including F1 ATPase, by Lominadze (49) et al. supports this notion (see below). Of note, recent studies by Fossati et al. (53) revealed that mitochondria of human neutrophils are involved either directly or indirectly in functions such as chemotaxis, respiratory burst activity, and maintenance of cell shape. Given that mitochondria have these unexpected roles in neutrophil function it is also possible that proteins are enriched on phagocytic vacuoles by direct interaction with mitochondria. This hypothesis remains to be tested.

Delivery of Proteins to Phagosomes by Fusion with Granules—
Lominadze et al. (49) recently performed a comprehensive analysis of proteins comprising human neutrophil granules. Many of the neutrophil phagosomes proteins we report herein were also identified as granule proteins by Lominadze et al. (49), including host defense molecules, components of the actin-based cytoskeleton, and histones, a finding most compatible with the idea that the proteins were delivered to forming phagosomes by fusion with granules. Furthermore the observation that molecular chaperones, such as calnexin, GRP78, and PDI, reside in granules correlates with our microscopy data and provides an explanation in part for enrichment in or on neutrophil phagosomes (Fig. 6).

On the Issue of Contaminating Organelles or Proteins—
As with studies performed by Garin et al. (40) in macrophage-like cells, we considered carefully the possibility that proteins typical of ER and mitochondria were contaminants of our PMN phagosome preparations. Although we cannot exclude the possibility that ER or mitochondria bound nonspecifically to phagosomes during the subcellular fractionation process, we failed to identify ER or mitochondrial proteins in comparable fractions from unstimulated cells (data not shown) nor did we find evidence for contaminating organelles by transmission electron microscopy analysis of purified neutrophil phagosomes (Fig. 1C and data not shown). Furthermore, several proteins representative of ER and mitochondria co-localized with phagosomes in intact cells and have been described as neutrophil granule proteins (Figs. 4–6) (49). Therefore, these proteins were not likely derived from ER or mitochondria simply co-sedimenting with phagosomes during the purification process.

Some of the proteins identified by our proteomic analysis were components of IgG/C3bi-LBs (Table I). For example, rabbit IgG and human serum albumin were used to generate IgG/C3bi-LBs and were thus identified by our phagosome analysis. In a recent proteomic analysis of human neutrophil granules, Lominadze et al. (49) proposed that hemoglobin from lysed erythrocytes was a contaminant in their granule preparations because it has affinity for phospholipid membranes. Thus, it is possible that we identified hemoglobin in our studies because that from lysed erythrocytes was bound nonspecifically to either the plasma membrane of purified PMNs or IgG/C3bi-coated latex beads during opsonization in fresh human serum, which may have residual red cell debris.

Proteins of Uncharacterized Function—
Another key discovery was that at least 11 proteins with no reported function were associated with neutrophil phagosomes (Table I). In addition, molecules with known function but no characterized role in neutrophils were found in or around phagosomes (Table I). For example, resistin, an adipocyte and mononuclear cell-derived protein associated with adipocyte differentiation, is linked to insulin resistance in a mouse model (54). Human resistin is expressed in circulating monocytes and appears to have significant proinflammatory properties (55, 56). It is possible that resistin and other phagosome proteins identified herein play prominent roles in phagocytosis and/or microbicidal activity in neutrophils.

Taken together, our results suggest that previously unappreciated processes facilitate phagosome maturation and function in human neutrophils. The finding that neutrophil phagosomes contain components of the proteasome, endoplasmic reticulum, and mitochondria evokes a complex picture of the phagosome beyond the generation of reactive oxygen species and microbial killing.


   ACKNOWLEDGMENTS
 
We thank Dr. Carl H. Hammer and Dr. Mary Ann Robinson for analysis of protein samples by LC-MS/MS at the NIAID, National Institutes of Health, Mass Spectrometry Laboratory (Rockville, MD), Mr. Stanley F. Hayes for analysis of phagosomes by electron microscopy at the NIAID Rocky Mountain Microscopy Branch (Hamilton, MT), and Dr. Mark T. Quinn (Montana State University, Bozeman, MT) for critical review of the manuscript.


  FOOTNOTES
 
Received, October 11, 2005, and in revised form, November 30, 2005.

Published, MCP Papers in Press, January 14, 2006, DOI 10.1074/mcp.M500336-MCP200

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 The abbreviations used are: PMN, polymorphonuclear leukocyte; DPBS, Dulbecco’s PBS; ER, endoplasmic reticulum; ERp29, ER protein 29 precursor; GRP, glucose-regulated protein; IgG/C3bi-LB, antibody- and complement-coated latex bead; MPO, myeloperoxidase; NCBI, National Center for Biotechnology Information; PDI, protein-disulfide isomerase; PRDX, peroxiredoxin; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); MHC, major histocompatibility complex. Back

* This work was supported by the Intramural Research Program of the NIAID, National Institutes of Health. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

To whom correspondence should be addressed: Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, NIAID, National Institutes of Health, Hamilton, MT 59840. Tel.: 406-363-9448; Fax: 406-363-9394; E-mail: fdeleo{at}niaid.nih.gov


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