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Originally published online as doi:10.1189/jlb.0207112 on June 18, 2007

Published online before print June 18, 2007
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(Journal of Leukocyte Biology. 2007;82:551-558.)
© 2007 by Society for Leukocyte Biology

Intracellular pools of Fc{alpha}R (CD89) in human neutrophils are localized in tertiary granules and secretory vesicles, and two Fc{alpha}R isoforms are found in tertiary granules

Na Yin, Min Peng, Yukun Xing and Wei Zhang1

Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing, China

1 Correspondence: Department of Immunology, Institute of Basic Medical Sciences, 5 Dong Dan San Tiao, Beijing 100005, China. E-mail: wzhang{at}pumc.edu.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human Fc{alpha}RI (CD89) is expressed on cells of myeloid lineage and plays an important role in host defense. Neutrophils make up the majority of Fc{alpha}RI-positive cells. Previous reports suggested that Fc{alpha}R was stored in neutrophil intracellular pools, and it could be mobilized quickly once neutrophils were activated. However, the subcellular localization of Fc{alpha}R in neutrophils has not been defined yet. In this study, we identified that Fc{alpha}R was stored in secretory vesicles and tertiary granules of neutrophils by flow cytometry analysis, ELISA, confocal microscopy, and Western blotting. The molecular mass of Fc{alpha}R in secretory vesicles was different from that in tertiary granules. Fc{alpha}R stored in tertiary granules had a molecular mass of 50–70 kDa, whereas Fc{alpha}R in secretory vesicles and membranes had a molecular mass of 55–75 kDa. After treatment by peptide-N-glycosidase F, an enzyme that removes N-glycosylation, Fc{alpha}R from secretory vesicles and tertiary granules revealed a core protein of 32 kDa, which was the same as the backbone of full length of Fc{alpha}R. A smaller Fc{alpha}R variant with a core protein of 29–30 kDa was found in tertiary granules but not in secretory vesicles. The nature of the small variant is not clear at present and remains to be investigated further.

Key Words: IgA receptor • PMN • gelatinase • alternative splicing


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human IgA FcR (Fc{alpha}RI, CD89) is expressed mainly on cells of the myeloid lineage including polymorphonuclear neutrophils (PMNs), monocytes, some macrophages, eosinophils, and subpopulations of dendritic cells [1 2 3 4 5 ]. Binding of antigen-complexed IgA to Fc{alpha}RI elicits a wide range of immunological responses including antibody-dependent, cell-mediated cytotoxicity, phagocytosis, release of cytokines, respiratory bursts, and degranulation, which play important roles in host defense against invading microorganisms and may be involved in inflammation under pathological conditions [5 6 7 ].

Neutrophils make up the majority of Fc{alpha}RI-positive cells in blood. It has been reported that Fc{alpha}R expression can be up-regulated in vitro when neutrophils are exposed to stimuli such as fMLP, C5a, IL-8, TNF-{alpha}, and antigen-complexed IgA [8 9 10 11 12 ]. The receptor up-regulation is a fast process. A three- to fourfold increase of surface Fc{alpha}R was observed within minutes of exposure to fMLP [8 ]. Also, when neutrophils were incubated with IgA immune complexes, Fc{alpha}R was up-regulated within 30 min and reached a plateau after 60 min [12 ]. No de novo protein synthesis could happen in such short time, and it has been proven that the up-regulation did not depend on protein synthesis, as it was not inhibited by cycloheximide or puromycin [8 ]. Therefore, Fc{alpha}R must have been presynthesized and stored in neutrophils. However, the subcellular localization of Fc{alpha}R in neutrophils has not been defined yet.

It is well known that four subsets of membrane-bound compartments exist in neutrophils, i.e., primary or azurophilic granules, secondary or specific granules, tertiary or gelatinase granules, and secretory vesicles. Each type of compartments, containing its specific group of proteins with relative functions, serves different functions [13 ]. Targeting proteins to an individual granule compartment is determined by the timing of protein synthesis during myeloid progenitor cell differentiation [13 14 15 ]. Neutrophils perform immunological tasks efficiently at different stages of the inflammatory process, depending on a sophisticated mechanism to mobilize its four types of granules. Their mobilization depends on the intensity of stimuli, but the order of mobilization is fixed. Secretory vesicles are exocytosed more readily than the other three granules. Tertiary granules are mobilized more easily than secondary granules, which again are mobilized more readily than primary granules [13 14 15 16 17 18 ]. Thus, localization of a protein in granules may provide clues such as to the function of that protein and its time of synthesis.

In this study, we identified the subcellular localization of Fc{alpha}R in neutrophils. Our results showed that Fc{alpha}R was localized in secretory vesicles and tertiary granules. Also, we found that tertiary granules contained two Fc{alpha}R variants, whereas secretory vesicles only had one.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibody
The murine anti-Fc{alpha}R mAb MIP8a, MIP7c, MIP10c, MIP38c, MIP68b, and MIP71a were generated previously [19 ]. F(ab')2 fragments of MIP8a were made by digestion with pepsin (Sigma-Aldrich, St. Louis, MO, USA), and undigested MIP8a was removed by a protein A column. The MIP8a F(ab')2 fragments were labeled with FITC or tetramethylrhodamine isothiocyanate (TRITC; Sigma-Aldrich).

The cDNA encoding the Fc{alpha}RI extracellular domain (EC) was inserted in a pET-28a vector (Novagen, Nottingham, UK) and expressed in BL21 (DE3) Escherichia coli. Then, the protein was purified by SDS-PAGE, and the purity was more than 95%. Polyclonal antibody against the Fc{alpha}RI EC was generated in a rabbit immunized with purified Fc{alpha}RI. The specificity of polyclonal antibody to Fc{alpha}R was determined by comparing its binding property to Chinese hamster ovary (CHO) and CHO stably transfected with Fc{alpha}RI.

Murine anti-human serum albumin (anti-HSA) mAb (KT11, KT23), FITC-conjugated F(ab')2 fragments of goat anti-mouse IgG, and HRP-conjugated goat anti-rabbit IgG were from Absea Biotechnology Ltd. (Beijing, China). HRP-conjugated goat anti-mouse IgG polyclonal antibody and MOPC21 were from Sigma-Aldrich. Goat antigelatinase [matrix metalloproteinase 9 (MMP9)], rabbit antimyeloperoxidase (anti-MPO), FITC-labeled rabbit anti-goat IgG, and TRITC-labeled goat anti-rabbit IgG polyclonal antibody were from Santa Cruz Biotechnology (Heidelberg, Germany). HRP-conjugated rabbit anti-goat IgG polyclonal antibody was from Zhongshan Biotechnology Co. (Beijing, China). Rabbit antilactoferrin polyclonal antibody was made previously [19 ].

Isolation of neutrophils
Blood samples were taken from healthy donors in this laboratory. Neutrophils were isolated from heparinized blood in sterile condition by Dextran T500 containing 5 mM EDTA sedimentation. Neutrophils were then separated from mononuclear leukocytes by density gradient centrifugation through discontinuous Percoll (GE Healthcare, Uppsala, Sweden) gradients. Isolated neutrophils were resuspended in HBSS for further study.

Stimulation of neutrophils
Isolated neutrophils were resuspended at the concentration of 2 x 106 cells/ml in HBSS containing 1 mM CaCl2 and 1.5 mM MgCl2 and stimulated with indicated concentrations of fMLP (Sigma-Aldrich) or PMA (Sigma-Aldrich) for various times at 37°C. The reaction was stopped by adding ice-cold PBS containing 0.1% NaN3 and subsequently centrifuged at 400 g for 2 min at 4°C.

Flow cytometry analysis of Fc{alpha}R on stimulated neutrophils
Stimulated or unstimulated neutrophils were washed with ice-cold 1% BSA/PBS/0.1% NaN3 for three times and incubated with 1 µg MIP8a in 100 µl 1% BSA/PBS/0.1% NaN3 for 1 h on ice and then with 1 µg FITC-conjugated F(ab')2 fragments of goat anti-mouse IgG for 30 min on ice. Nonspecific binding was determined by incubation of cells with MOPC21, an isotype control antibody. The samples were analyzed by a flow cytometer (Coulter Epics XL, Beckman Coulter, Fullerton, CA, USA).

Flow cytometry analysis for intracellular Fc{alpha}R
For cell-surface staining, isolated neutrophils were fixed and incubated with 2 µg FITC-labeled MIP8a in 100 µl 5% horse serum/PBS/0.1% NaN3 for 1 h on ice. For intracellular staining, cells were first incubated with 20 µg MIP8a for 30 min on ice. After five washes with PBS/0.1% NaN3, cells were fixed with 1% paraformaldehyde for 30 min on ice and permeabilized by exposure to 0.2% saponin/5% horse serum/PBS/0.1% NaN3 for 30 min on ice and then incubated with 2 µg FITC-labeled MIP8a for 1 h on ice. Saponin (0.2%) was also included during all washes and incubation steps. Nonspecific binding was determined by incubation of cells with FITC-labeled MOPC21. The samples were analyzed by a flow cytometer (Coulter Epics XL).

Subcellular fractionation
Neutrophil subcellular fractionation was performed as described [20 , 21 ] with a few modifications. Briefly, purified neutrophils (108/ml) were resuspended in ice-cold relaxation buffer (100 mM KCl, 3 mM NaCl, 1 mM ATPNa2, 3.5 mM MgCl2, 10 mM PIPES, 1 mM PMSF, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 0.7 µg/ml pepstatin, pH 7.2) and disrupted by gentle sonication. The homogenate was centrifuged (400 g for 15 min at 4°C) to remove nuclei and large debris. The supernatant added with 1.5 mM EGTA was applied to a 9-ml, three-layer, discontinuous, precooled Percoll gradient (1.05/1.09/1.12 g/ml) in relaxation buffer and then centrifuged at 37,000 g for 30 min at 4°C. The three-layer Percoll separation resulted in well-separated visual four bands, designated {gamma}, ß2, ß1, and {alpha} from the top down as reported [20 ]. The different fractions were then collected. For evaluation of proteins in subcellular fractions by Western blotting, the samples were ultracentrifugated at 100,000 g for 90 min to remove Percoll.

Detection of neutrophil granules by ELISA and enzyme activity assay
The following markers were used for identifying neutrophil granules. MPO was for primary granules, lactoferrin for secondary granules, gelatinase B (MMP9) for tertiary granules, and HSA for secretory vesicles.

MPO activity was determined using substrate 2,2'-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt (Sigma-Aldrich) in 0.1 M citrate buffer (pH 5.0) containing H2O2 as described previously [22 ].

Lactoferrin, HSA, and Fc{alpha}R were determined by sandwich ELISA. For lactoferrin detection, polyclonal antilactoferrin was used as capture antibody, and HRP-labeled rabbit antilactoferrin was used as detection antibody. For HSA detection, KT11 was used as capture antibody, and KT32-HRP was used as detection antibody. For Fc{alpha}R detection, a mixture of anti-Fc{alpha}R EC1 domain mAb (MIP8a, MIP7c, and MIP38c) was used as capture antibodies and a mixture of anti-Fc{alpha}R EC2 domain mAb (MIP10c-HRP, MIP68b-HRP, and MIP71a-HRP) was used as detection antibodies. The capture antibodies (0.5 µg/well) were coated on 96-well plates. Granule fractions were lysed with an equal volume of 1% Nonidet-P40 (NP-40)/PBS. To remove intrinsic, peroxidase activities in granules, 3% H2O2 was added in each well and washed off before adding detection antibodies. The standard curves were determined using lactoferrin (Calbiochem, Nottingham, UK), HSA (Sigma-Aldrich), and soluble Fc{alpha}R [19 ], respectively.

Western blotting
An equal volume of granule fractions was first separated by 10% SDS-PAGE and then transferred onto a nitrocellulose membrane (Millipore Corp., Billerica, MA, USA). After blocking with 5% defatted milk for 2 h at room temperature, the membranes were incubated with rabbit anti-Fc{alpha}R polyclonal antibody (1:500) overnight at 4°C. Then, the membranes were washed with 1% defatted milk and incubated with HRP-conjugated goat anti-rabbit IgG (1:3000) at room temperature for 1 h. After washes, Fc{alpha}R on the membranes were visualized by SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA).

To detect HSA, KT11 was used as primary antibody (1:1000), and HRP-conjugated goat anti-mouse IgG (1:5000) was used as secondary antibody. To detect MMP9, anti-MMP9 was used as primary antibody (1:1000), and HRP-conjugated rabbit anti-goat IgG (1:5000) was used as secondary antibody.

Immunoprecipitation analysis
A mixture of anti-Fc{alpha}R antibodies, MIP8a, MIP7c, MIP10c, MIP38c, MIP68b, and MIP71a, was coupled onto Sepharose beads (GE Healthcare). Granule fractions were lysed with 0.5% NP-40/PBS containing 1 mM PMSF, 2 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.7 µg/ml pepstatin at 4°C for 30 min. The insoluble contents were removed by centrifugation, and the supernatants were incubated with anti-Fc{alpha}R, mAb-coupled beads at 4°C for 2 h. After washing with 0.5% NP-40/PBS, the beads were eluted with 0.05 M glycine at pH 2.8. The eluted proteins were neutralized with Tris·Cl to pH 7.5 and analyzed by Western blotting as described above. To remove N-glycosylation on Fc{alpha}R, the eluted proteins were treated with peptide-N-glycosidase (PNGase F; New England Biolabs, Beverley, MA, USA) for 5 h at 37°C before Western blotting.

Confocal microscopy analysis
After incubation for 15 min at 37°C in the presence or absence of fMLP, neutrophils were resuspended in HBSS at the concentration of 2 x 106 cells/ml. Cells were added to poly-L-lysine-treated coverslips and then fixed with 1% paraformaldehyde for 30 min at room temperature. Fixed cells were permeabilized with 0.15% Triton X-100/3% BSA/PBS for 10 min, followed by three washes with 3% BSA/PBS. Then, cells were blocked with 5% horse serum for 1 h. Subsequently, cells were incubated with FITC- or TRITC-labeled F(ab')2 fragments of MIP8a. Granule structure was detected with corresponding antigranule marker antibody and then FITC- or TRITC-labeled secondary antibodies. After three washes, cells were mounted. Imaging was performed on a confocal laser-scanning microscope system (Leica TCS SP2 SE, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Intracellular Fc{alpha}R expression in neutrophils
Neutrophil activation with stimuli has been reported to result in rapid up-regulation of surface Fc{alpha}R expression [8 9 10 11 12 ]. To confirm this, neutrophils were stimulated with fMLP and PMA. As shown in Figure 1A , surface expression of Fc{alpha}R increased within 15 min after neutrophils were exposed to 10 nM fMLP. No more Fc{alpha}R expression could be detected after 30 min. Neutrophils treated with a higher concentration of fMLP for 15 min resulted in slightly more expression of Fc{alpha}R (Fig. 1B) . In comparison, up-regulation of surface Fc{alpha}R expression was much more significant when cells were treated with PMA (20 ng/ml), a more potent stimulus compared with fMLP. As no de novo protein synthesis could happen in such a short period of time, the up-regulated Fc{alpha}R on neutrophil surface must be presynthesized and stored intracellularly.


Figure 1
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  		Figure 1. Up-regulation of surface Fc{alpha}R on neutrophils by stimuli. Neutrophils were stimulated with fMLP or PMA at an indicated concentration and time at 37ºC. Then, the cells (4x105) were washed and incubated with 1 µg MIP8a, followed by 1 µg FITC-conjugated F(ab')2 fragments of goat anti-mouse IgG. Mean fluorescence intensity was gained by subtracting background fluorescence staining with irrelevant antibody MOPC21. (A) Neutrophils were stimulated with 10 nM fMLP for the indicated time. (B) Neutrophils were stimulated with different concentrations of fMLP and PMA for 15 min. Data shown are representative of three separate experiments.

 
To confirm the intracellular storage of Fc{alpha}R, we examined intracellular Fc{alpha}R directly in permeabilized neutrophils. As shown in Figure 2A , when neutrophils were not permeabilized, surface expression of Fc{alpha}R on neutrophils could be detected by FITC-labeled MIP8a using flow cytometry analysis, and the staining could be abolished completely when neutrophils were preincubated with 20 µg unlabeled MIP8a. Then, neutrophils were preincubated with 20 µg unlabeled MIP8a to block surface staining first, then washed, permeabilized, and stained with FITC-labeled MIP8a. As shown in Figure 2B , neutrophils were still stained, indicating that there were intracellular storages of Fc{alpha}R. This intracellular staining of Fc{alpha}R was specific, as FITC-labeled isotype control antibody caused a much weaker signal. Moreover, preincubation of permeabilized neutrophils with 20 µg unlabeled MIP8a could abolish the intracellular staining completely, further demonstrating that the intracellular staining of Fc{alpha}R was specific. When neutrophils were permeabilized but not preincubated with unlabeled MIP8a, FITC-labeled MIP8a gave the strongest staining, as this detected total Fc{alpha}R, including surface expression and intracellular storages.


Figure 2
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  		Figure 2. Flow cytometry analysis for intracellular expression of Fc{alpha}R. (A) Neutrophils (106) were fixed and incubated with 2 µg MIP8a-FITC. In inhibition of a surface-staining experiment, neutrophils were preincubated with 20 µg unlabeled MIP8a for 30 min on ice and then washed, fixed, and incubated with 2 µg MIP8a-FITC. (B) Neutrophils (106) were fixed and permeabilized: For total Fc{alpha}R staining, neutrophils were incubated with 2 µg MIP8a-FITC; for intracellular Fc{alpha}R staining, before fixed, permeabilized, and incubated with 2 µg MIP8a-FITC, neutrophils were preincubated with 20 µg unlabeled MIP8a for 30 min on ice to inhibit the surface staining; for inhibition of Fc{alpha}R staining, an additional 20-µg unlabeled MIP8a was added to permeabilized neutrophils before incubation with 2 µg MIP8a-FITC. Irrelevant MOPC21-FITC was used as control antibody for nonspecific antibody binding.

 
In addition, we noted that PMA caused more marked surface Fc{alpha}R up-regulation compared with fMLP (Fig. 1B) . As PMA is a more potent stimulus than fMLP, it can completely mobilize tertiary granules, which can only be mobilized partly by fMLP. This observation suggested that tertiary granules may be one of the intracellular pools of Fc{alpha}R. Further experiments were undertaken to determine the subcellular localization of Fc{alpha}R in neutrophils.

Localization of Fc{alpha}R in neutrophil granules by ELISA analysis
Subcellular localization of Fc{alpha}R stored in neutrophils was investigated next. For this purpose, neutrophil granules were separated by a three-layer Percoll gradient. After high-speed centrifugation, four distinct bands were observed and from the top, designated {gamma}-band (plasma membranes and secretory vesicles), ß2-band (tertiary granules), ß1-band (secondary granules), and {alpha}-band (primary granules), as reported previously [20 , 21 ]. The Percoll gradient containing granules was then fractionated, and 30 fractions were collected. The {gamma}-band, ß2-band, ß1-band, and {alpha}-band were bracketed in fractions five to 11, 12–16, 17–24, and 25–30, respectively. Then, the contents of secretory vesicles and secondary and primary granules were analyzed as described in Materials and Methods. As a result of lack of paired antibodies, we were unable to identify the tertiary granule marker by sandwich ELISA. Instead, we identified the tertiary granule marker by Western blotting (see Go Go Fig. 5D below). Figure 3 showed that secretory vesicles and primary granules were separated well, whereas there were some contaminations of secondary granules in ß2-band, possibly as a result of inefficient separating volume of the gradient [20 ]. Detection of Fc{alpha}R by sandwich ELISA showed that it existed in {gamma}-band and ß2-band fractions, indicating Fc{alpha}R was located in plasma membranes and/or secretory vesicles and tertiary granules. There is no doubt that Fc{alpha}R is located in plasma membranes, as it is expressed on neutrophil surface constitutively. Localization of Fc{alpha}R in secretory vesicles was investigated further by confocal microscopy analysis (see below). Fc{alpha}R could hardly be detected in secondary granule fractions, and no Fc{alpha}R could be detected in primary granule fractions.


Figure 3
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  		Figure 3. Subcellular localization of Fc{alpha}R in neutrophils by ELISA and enzyme activity analysis. Neutrophils (108) were disrupted and separated by a three-layer Percoll gradient to 30 fractions. The following proteins were measured in each fraction as described in Materials and Methods: MPO, •; lactoferrin, {blacksquare}; HSA, {blacktriangledown}; and Fc{alpha}R, {blacktriangleup}. Experiments were repeated three times, and mean values were calculated with three parallel wells from one representative experiment.

 

Figure 4
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  		Figure 4. Confocal microscope analysis for subcellular localization of Fc{alpha}R in neutrophils, which were incubated in the presence or absence of fMLP at 37ºC for 15 min. Then, cells were adhered onto coverslips, permeabilized, stained, and subjected to confocal microscope analysis. Data shown are representative of three separate experiments. (A) Mobilization of secretory vesicles, granules, and Fc{alpha}R in neutrophils: For secretory vesicles, HSA was detected by FITC-conjugated KT11; for tertiary granules, MMP9 was detected by goat anti-MMP9 as primary antibody and FITC-conjugated rabbit anti-goat IgG as secondary antibody; for secondary granules, lactoferrin was detected by FITC-conjugated rabbit antilactoferrin; for primary granules, MPO was detected with rabbit anti-MPO as primary antibody and TRITC-conjugated goat anti-rabbit IgG as secondary antibody. Fc{alpha}R was detected using TRITC-conjugated F(ab')2 fragments of MIP8a. (B) Colocalization analysis of Fc{alpha}R with neutrophil granules by double-staining: For secretory vesicles, Fc{alpha}R was detected with TRITC-conjugated F(ab')2 fragments of MIP8a, and HSA was detected by FITC-conjugated KT11; for tertiary granules, Fc{alpha}R was detected with TRITC-conjugated F(ab')2 fragments of MIP8a, and MMP9 was detected by goat anti-MMP9 as primary antibody and then FITC-conjugated rabbit anti-goat IgG as secondary antibody; for secondary granules, Fc{alpha}R was detected with TRITC-conjugated F(ab')2 fragments of MIP8a, and lactoferrin was detected by FITC-conjugated rabbit antilactoferrin; for primary granules, Fc{alpha}R was detected with FITC-conjugated F(ab')2 fragments of MIP8a, and MPO was detected by rabbit anti-MPO as primary antibody and then TRITC-conjugated goat anti-rabbit IgG as secondary antibody.

 

Figure 5
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  		Figure 5. Western blotting analysis of intracellular Fc{alpha}R. Neutrophils (108) were disrupted and separated by a three-layer Percoll gradient. Samples bracketing the peak of the individual fractions were pooled together, and Percoll was removed by ultracentrifugation. Each type of granule was suspended in 100 µl PBS/1% NP-40. (A) Each granule fraction (25 µl) was run on 10% SDS-PAGE, and the molecular weight was analyzed by Western blotting using rabbit anti-Fc{alpha}R polyclonal antibodies followed by HRP-conjugated goat anti-rabbit IgG. (B) The ß2 or {gamma} fraction (50 µl ) was precipitated with anti-Fc{alpha}R, mAb-coupled beads. The eluted Fc{alpha}R was N-deglycosylated by PNGase F and analyzed by Western blotting as above. (C) ß1, ß2, or {gamma} fraction (25 µl) was run on 10% SDS-PAGE and analyzed by Western blotting. HSA was detected by KT11 as primary antibody and HRP-conjugated goat anti-mouse polyclonal antibody as secondary antibody. (D) Each granule fraction (10 µl) was run on 10% SDS-PAGE and analyzed by Western blotting. MMP9 was detected by anti-MMP9 as primary antibody and HRP-conjugated rabbit anti-goat polyclonal antibody as secondary antibody.

 
Localization of Fc{alpha}R by confocal microscopy analysis
Localization of Fc{alpha}R in secretory vesicles and tertiary granules was also proven by confocal microscopy analysis. Neutrophils were treated with 10 nM and 1000 nM fMLP for 15 min. Mobilization of HSA, MMP9, lactoferrin, MPO, and Fc{alpha}R in neutrophils was detected by immunofluorescence staining. As shown in Figure 4A (columns 1 and 2 from the left), HSA disappeared mostly, and MMP9 decreased after stimulation, indicating that almost all secretary vesicles and partial tertiary granules exocytosed. In comparison, there was nearly no change of lactoferrin and MPO, demonstrating that secondary and primary granules were not mobilized following the same stimulation (Fig. 4A , columns 3 and 4 from the left). Concurrently under fMLP stimulation, Fc{alpha}R dispersed in nonstimulated neutrophils, redistributed to the cell edge (surface), suggesting that granules containing Fc{alpha}R were mobilized (Fig. 4A , column 5 from the left). These results indicated that Fc{alpha}R was mobilized along with albumin and MMP9 but not with lactoferrin and MPO following the same stimulation, which demonstrated that Fc{alpha}R shared the same granules with albumin and MMP9 but not with lactoferrin and MPO.

Localization of Fc{alpha}R in secretory vesicles and tertiary granules was proven further by double-staining with FITC- or TRITC-conjugated anti-Fc{alpha}R and antigranule marker antibodies. For resting neutrophils, a greater degree of coincidence was found in cells double-stained with anti-Fc{alpha}R and anti-HSA (for secretory vesicles) or anti-MMP9 (for tertiary granules). There was no overlap between anti-Fc{alpha}R with antilactoferrin (for secondary granules) or anti-MPO (for primary granules; Fig. 4B , row 1). When neutrophils were stimulated with 10 nM or 1000 nM fMLP for 15 min, colocalization of Fc{alpha}R with MMP9 was still detectable, although nearly all secretary vesicles had been exocytosed (Fig. 4B , rows 2 and 3, columns 1 and 2 from the left). Again, there was no colocalization between Fc{alpha}R with lactoferrin or MPO (Fig. 4B , rows 2 and 3, columns 3 and 4 from the left).

Molecular mass of Fc{alpha}R in secretory vesicles and tertiary granules
We also examined molecular mass of Fc{alpha}R stored in secretory vesicles and tertiary granules by Western blotting. As shown in Figure 5A , a 55- to 75-kDa band of Fc{alpha}R was detected in {gamma}-band pools (secretory vesicles). A smaller band, 50–70 kDa, was detected in ß2-band pools (tertiary granules). No Fc{alpha}R was found in ß1-band (secondary granules) and {alpha}-band (primary granules) pools. No protein band was detected with control rabbit IgG, purified from serum taken before immunization (data not shown).

The different-sized Fc{alpha}R in secretory vesicles and tertiary granules might be a result of a different degree of glycosylation or different length of protein backbones. To find out the causes, Fc{alpha}R was immunoprecipitated by anti-Fc{alpha}R, mAb-coupled beads and then treated with excessive PNGase F, followed by Western blotting as above. As shown in Figure 5B , the core protein of Fc{alpha}R from secretory vesicles had a single molecular mass of 32 kDa. However, Fc{alpha}R from the tertiary granules showed two distinct bands after N-deglycosylation. One was 32 kDa, the same as that in secretory vesicles. The other one was smaller, with a molecular mass of 29–30 kDa, and the 32-kDa Fc{alpha}R in tertiary granules was not possibly a contamination by secretory vesicles, as tertiary granules and secretory vesicles were separated well (Figs. 3 and 5C) .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Neutrophils are short-lived cells, which are generated continuously from hematopoietic stem cells in bone marrow by a process called myelopoiesis. Neutrophil granule and vesicle proteins appear to be segregated into different granules and vesicle populations on the basis of their in vivo roles. The granules formed at any given stage of neutrophil development will be composed of a concurrent synthesis of groups of proteins of related function. Accordingly, any localization of a protein of interest with granule or vesicle may provide indications such as to the function of that protein and its time of synthesis [13 14 15 16 17 18 ].

Results from previous studies suggested that there were intracellular storage pools for Fc{alpha}R in neutrophils. However, its localization was unknown. In the present study, we separated plasma membranes/secretory vesicles ({gamma}), tertiary granules (ß2), secondary granules (ß1), and primary granules ({alpha}) from gently disrupted neutrophils by a three-layer Percoll gradient. Fc{alpha}R was found in {gamma}-band and ß2-band, and this indicated Fc{alpha}R was stored in plasma membranes and/or secretory vesicles and tertiary granules. That Fc{alpha}R was stored in plasma membranes was proven by its constitutive expression on neutrophil surface. Then, the mobilization of Fc{alpha}R and four granules in neutrophils following fMLP stimulation was examined. We found that Fc{alpha}R was mobilized along with secretory vesicles and tertiary granules, which suggested that Fc{alpha}R was stored in these two vesicles/granules. Further, the colocalization between Fc{alpha}R with HSA and MMP9 was confirmed by confocal microscopy analysis; this gave the direct proof that Fc{alpha}R was stored in secretory vesicles and tertiary granules. Finally, Western blotting analysis also showed that Fc{alpha}R was stored in secretory vesicles and tertiary granules but not in secondary or primary granules. Taken together, these results demonstrated that Fc{alpha}R was stored in secretory vesicles and tertiary granules.

Theilgaard-Monch et al. [23 ] applied microarray technology to systematically profile the gene expression patterns in myeloblast promyelocytes (PMs), which form primary granules, myelocyte/metamyelocytes (MYs), which form secondary and tertiary granules, and bone marrow PMNs (bm-PMNs), which form secretory vesicles. This study showed that the gene expression level of Fc{alpha}R was low in PM populations, high in MY populations, and medium in the bm-PMN populations [23 ]. Our results showed that Fc{alpha}R was stored in tertiary granules (formed during MYs) and secretory vesicles (formed during bm-PMNs). This is in agreement with the gene expression profile of Fc{alpha}R during neutrophil development identified by Theilgaard-Monch et al. [23 ].

The fact that Fc{alpha}R was expressed at a relative later stage during neutrophil development could be proven by examining its expression on HL-60 cells (human promyelocytic leukemic cell line), which could be induced to differentiate into a mature, neutrophilic-like phenotype by DMSO [24 ]. We examined the expression level of Fc{alpha}R on HL-60 at different stages by flow cytometry and RT-PCR analysis. No Fc{alpha}R expression on HL-60 was detected without DMSO induction, but its expression was detectable after DMSO treatment (data not shown). This result is in agreement with a previous report [25 ].

Fc{gamma}RIII (CD16) is a predominant FcR for IgG in neutrophils, and it was only stored in secretory vesicles [26 , 27 ]. As Fc{alpha}R also existed in tertiary granules, Fc{alpha}R should be synthesized earlier than Fc{gamma}RIII during neutrophil differentiation. The functions of the Fc{alpha}R expressed earlier are not clear. It is possible that immature neutrophils exert their immunological function predominantly via IgA, not IgG. In agreement with this speculation, Otten et al. [28 ] have reported that immature neutrophils mediated tumor cell killing via IgA, but not IgG FcR.

It is more interesting that we found that there were two Fc{alpha}R variants expressed in neutrophils. One had a core protein of 32 kDa and existed in tertiary granules and secretory vesicles. The other one was 2–3 kDa smaller, with a core protein of 29–30 kDa. This small variant only existed in tertiary granules. Expression of the small variant was not an individual variation, as similar results were obtained with neutrophils from other donors (data not shown). To our knowledge, previous studies had not found this variant at the protein level in neutrophils; this may be a result of different detection methods. In those studies, researchers usually detected Fc{alpha}R via surface labeling, which cannot detect any protein stored in neutrophil granules.

It has been reported that there are more than 10 Fc{alpha}R splice variants in human myeloid cells [29 30 31 32 33 34 35 ], and at least six of them (Fig. 6 ) have been detected at the mRNA levels in neutrophils [29 , 31 , 32 , 34 , 35 ]. However, the function of these splice variants is not clear. The Fc{alpha}R variant identified in tertiary granules in this study had a molecular mass close to the {triangleup}66EC2 and {triangleup}S266EC2 variants. {triangleup}66EC2 differs from the full-length Fc{alpha}R by a 22-amino acid deletion in the EC2 domain, and {triangleup}S266EC2 is the same as {triangleup}66EC2 but has an additional deletion at the S2 signal sequence. Patry et al. [33 ] have identified {triangleup}66EC2 in alveolar macrophages, and it is the only variant that has been identified as a natural protein.


Figure 6
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  		Figure 6. Schematic representation of the organization of the Fc{alpha}R gene and transcripts of the full-length form Fc{alpha}R and spliced isoforms. Exons are represented by rectangles and are drawn in scale; S, Signal sequence; TM/C, transmembrane/cytoplasmic domain.

 
The nature of the smaller-sized Fc{alpha}R variant is not clear at present. As neutrophil tertiary granules are not mobilized as quickly as secretory vesicles after stimulation, the smaller variant would neither be constitutively expressed on the cell surface nor mobilized to the cell surface immediately after cells were activated. The small Fc{alpha}R variant may be expressed on the cell surface when tertiary granules were mobilized at a relative later stage of neutrophil activation. Nevertheless, this small variant may have its special functions. Toyabe et al. [35 ] reported that Fc{alpha}R transcripts identified in granulocytes and monocytes from IgAN patients differed significantly with that from healthy volunteers. This finding implied that Fc{alpha}R variants may have different biological functions, and abnormal expression of these variants may lead to pathological conditions. So, the selective expression of the Fc{alpha}R variant in neutrophil suggests that this variant may have immunomodulatory function during the inflammatory process, but this speculation needs to be studied further.

In conclusion, Fc{alpha}Rs are stored in tertiary granules and secretory vesicles. Fc{alpha}R in secretory vesicles is a full-length receptor. There are two Fc{alpha}R isoforms in neutrophil tertiary granules. One of them is a full-length receptor, and the other one is 2–3 kDa smaller. The biological functions of the small variant needs further investigation.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the National Natural Science Foundation of China (30170878 and 30571693). The authors thank Dr. Gang Xu for his help in this study, Xueyu Dong, Qing Zhao, Leiying Dai, and Lin Zhang for technical help, and Qing Zhao, Xu Liu, Laishun Chi, and Fangtao Zhao from the Equipment Centre of this institute for data collection. We also thank Yumei Li from China Academy of Traditional Chinese Medicine for data collection.

Received February 15, 2007; revised May 17, 2007; accepted May 20, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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