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(Journal of Leukocyte Biology. 2002;72:970-977.)
© 2002 by Society for Leukocyte Biology

The presence of stomatin in detergent-insoluble domains of neutrophil granule membranes

Elisabeth Feuk-Lagerstedt*, Marie Samuelsson*, Wilhelm Mosgoeller{dagger}, Charlotta Movitz*, Åsa Rosqvist*, Jörgen Bergström{ddagger}, Thomas Larsson{ddagger}, Marianne Steiner§, Rainer Prohaska|| and Anna Karlsson*

* The Phagocyte Research Laboratory, Department of Rheumatology and Inflammation Research, and
{ddagger} Institute of Medical Biochemistry, Göteborg University, Sweden; and Institutes of
{dagger} Cancer Resarch,
§ Histology and Embryology, and
|| Medical Biochemistry, University of Vienna, Austria

Correspondence: Anna Karlsson, The Phagocyte Research Laboratory, Department of Rheumatology and Inflammation Research, Göteborg University, Guldhedsgatan 10, S-413 46 Göteborg, Sweden. E-mail: anna.karlsson{at}microbio.gu.se


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ABSTRACT
 
Neutrophil azurophil granules, traditionally regarded as the neutrophil counterpart to lysosomes, lack the lysosomal marker lysosome-associated membrane glycoprotein and have recently been suggested to be nonlysosomal secretory organelles. The membrane of the azurophil granules is poorly characterized—CD63 and CD68 are the only membrane proteins identified so far. Here, azurophil granule membranes were isolated by Percoll gradient subcellular fractionation. Using matrix-assisted laser desorption ionization time of flight mass spectrometry of tryptic peptides from an isolated protein, stomatin was identified in these membranes. Using immunoelectron microscopy and immunoblot analysis of isolated organelles, stomatin was found to be subcellularly localized, not only to the azurophil granules but also by a major part to the specific granules and by a minor part to the secretory vesicles/plasma membrane. We also show the presence of detergent-insoluble, low-density membrane domains in the plasma membrane and the granule membranes and found stomatin to be localized to these domains.

Key Words: membrane proteins • subcellular organelles • membrane rafts


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INTRODUCTION
 
The human polymorphonuclear leukocyte (neutrophil) plays an important role in eliminating invading microorganisms [1 ]. To fulfil its task, the cell contains different bactericidal proteins and mobilizable cell-surface receptors, which are stored in at least four types of intracellular granules and vesicles. By mobilizing these organelles to various extents, the neutrophil can communicate with its environment and combat microbial invaders. The mobilization is sequential, starting with the secretory vesicles that are the most easily mobilized, followed by the gelatinase granules, and subsequently the specific granules [2 , 3 ]. Mobilization of the secretory vesicles and gelatinase granules is crucial for neutrophil interaction with endothelium and migration into the tissue, and specific and azurophil granules are more of importance for phagocytosis and killing of microbes by fusing with the phagosome [4 ]. Differences in functional repertoire among the granules are reflected in their contents of matrix and membrane proteins.

The azurophil granules are traditionally regarded as the neutrophil counterpart to lysosomes. They contain, in addition to myeloperoxidase (MPO), antibacterial agents such as defensins, bactericidal/permeability-increasing protein, and lysosomal enzymes such as proteases and hydrolases [4 ]. These enzymes are primarily delivered to the phagosome, as azurophil granules are not mobilized to the cell surface to any higher extent. Not much is known about the membrane composition of the azurophil granules. So far, the only known membrane proteins are CD63 and CD68 [5 6 7 ]. The lysosome-associated membrane glycoproteins-1 and -2 (Lamp-1 and Lamp-2), which are universal markers for lysosomes, are not found in the azurophil granules [8 ] but have instead been identified in multilaminar compartments and multivesicular bodies, suggested to correspond to prelysosomes [9 ]. Together with the fact that the azurophil granules contain proteins still carrying phosphorylated mannose epitopes, the absence of Lamps indicates that the azurophil granules are not true lysosomes. Instead, they are beginning to be regarded as regulated secretory granules [9 ].

In an attempt to increase our understanding of azurophil granule function by studying the composition of its membrane, we aimed to identify new membrane proteins. During this work, we found the integral membrane protein stomatin to be a component of the azurophil granules as well as of the specific granules and the light membranes comprising the plasma membrane and secretory vesicles. We also report that detergent-insoluble membrane domains exist not only in the plasma membrane but also in the granule membranes and that stomatin is localized to these domains.


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MATERIALS AND METHODS
 
Isolation and subcellular fractionation of human neutrophils
Neutrophils were isolated from buffy coats obtained from healthy blood donors. The buffy coats were subjected to dextran sedimentation at 1 g, followed by hypotonic lysis of the remaining erythrocytes, and centrifugation in a Ficoll-Paque gradient [10 ]. The purified neutrophils (1x109 cells) were fractionated according to Borregaard et al. [11 ]. In short, the cells were treated with diisopropylfluorophosphate (DFP; final concentration 8 mM), resuspended in 10 mL relaxation buffer [100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 10 mM piperazine-N,N'-bis [2-ethanesulfonic acid], 1 mM adenosine 5'-triphosphate (ATP), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.4], and disrupted in a nitrogen bomb (Parr Instruments, Moline, IL) at 400 pounds per square inch (2500 kPa). The post-nuclear supernatant was layered on top of a two-step Percoll gradient (1.05 and 1.12 g/L) and centrifuged at 15,000 g for 45 min in a fixed-angle JA-20 Beckman rotor. The material was collected in fractions of 1.5 mL from the bottom of the centrifuge tube or in one portion for each organelle using a syringe.

Quantification of granule markers
To identify the localization in the gradient of the granules and plasma membrane, specific markers were measured in the gradient fractions. Vitamin B12-binding protein [12 ] was used as marker for the specific granules, and alkaline phosphatase [13 ] was used as marker for the secretory vesicles/plasma membrane. MPO, marker for the azurophil granules, was determined by enzymatic activity. The peroxidase substrate 1,2-phenylenediamine dihydrochloride (DAKO, Glostrup, Denmark) was dissolved according to instructions from the manufacturer and mixed with H2O2 prior to use. Peroxidase substrate (100 µL) was mixed with 25 µL from each fraction of the gradient in a 96-well plate and incubated for 30 min at room temperature. The reaction was stopped by adding 100 µL 0.1 M H2SO4 to each well, and the absorbance was measured at 492 nm.

Isolation of proteins from purified azurophil granule membranes
After performed subcellular fractionation, the fractions containing the azurophil granules were pooled and centrifuged at 100,000 g for 90 min. To separate the granule membranes from the matrix, the organelles were resuspended in 2 mL relaxation buffer and submitted to freeze-thawing 10 times. The membrane fraction was collected by centrifugation at 100,000 g for 90 min. To ensure complete separation of the membrane from the matrix, the remaining MPO activity in the membrane fraction was determined. No MPO could be detected in the pelleted membrane fraction after freeze-thawing and centrifugation.

The pelleted membranes were suspended in relaxation buffer containing 1 M NaCl to remove peripheral membrane proteins and were kept on ice for 15 min, centrifuged at 100,000 g for 90 min, and washed once in relaxation buffer. The membranes were resuspended in solubilization buffer (0.5% Triton X-100, 80 mM 3-[N-morpholino]propanesulfonic acid, pH 7.2) and kept on ice for 1 h. The mixture was centrifuged at 100,000 g for 15 min, and the pellet was resuspended in nonreducing sodium dodecyl sulfate (SDS)-containing sample buffer and boiled for 5 min. Samples corresponding to 5 x 107 cell equivalents (CE) were applied to polyacrylamide gels, and SDS-polyacrylamide gel electrophoresis (PAGE) was performed according to Laemmli [14 ]. After electrophoresis, the gels were stained with Coomassie blue according to a modified method by Jagow and Schägger [15 ]. Although the membrane preparation was devoid of the soluble matrix protein MPO (see above), the Coomassie blue staining showed a large, low molecular weight component that probably corresponds to contaminating defensins (see Fig. 1 ).



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  		Figure 1. Identification of a 31-kDa protein in the azurophil granule membrane. Azurophil granule membranes prepared by subcellular fractionation, freeze-thawing, and centrifugation were resuspended in solubilization buffer containing Triton X-100. After centrifugation, the pellet was subjected to SDS-PAGE followed by Coomassie blue staining. The protein band with a molecular mass of approximately 31 kDa (arrow) was excised for further analysis by MS. All peptide masses identified as belonging to stomatin were within 100 ppm.

Enzymatic digestion of isolated proteins for mass spectrometry (MS)
A distinct band with a molecular weight of approximately 31 kDa was excised from the gel, placed in a siliconized tube, and destained by washing the pieces three times on a shaker with 100 µL 25 mM NH4HCO3 in 50% CH3CN for 30–60 min. The destained gel pieces were dried for 40 min in a vacuum concentrator (SpeedVac, Thermo Savant, Holbrook, NY) and were rehydrated with trypsin solution (10 ng/µL in 25 mM NH4HCO3, pH 8) before incubation for 16 h at 37°C. The peptides were extracted from the gel by shaking for 30 min with 15 µL 15% CF3COOH in 75% CH3CN followed by centrifugation for 2 min at 5000 g [16 ].

MS
Samples were analyzed using a Micromass TofSpecE matrix-assisted laser desorption ionization time of flight (MALDI-TOF) MS (Micromass, Manchester, UK) equipped with a pulsed 337-nm nitrogen laser, a delayed extraction ion source, and a reflector. A 0.5-µL sample was mixed with a 0.5-µL matrix solution ({alpha}-cyano-4-hydroxy-cinnamic acid, 10 mg/mL, in CH3CN/H2O, 1:1) directly on the MALDI target and was allowed to dry at ambient conditions. Peptide spectra were acquired in reflectron mode at an accelerating voltage of 20 kV and a sum of 200 laser shots. External calibration using angiotensin II and ACTH-clip (18–39) was used together with an internal lock mass from autodigested trypsin. Resulting values for monoisotopic peaks were used for identification against the NCBI nr database using ProFound, available on the Internet at <http://prowl.rockefeller.edu/cgi-bin/ProFound>. The searches were made on homosapiens with no limitation in size or isoelectric point and an error tolerance of 200 parts per million (ppm).

Light microscopic immunocytochemistry
For light microscopic immunostaining, we used blood smears from healthy donors stained according to Pappenheim (described in Romeis [17 ]). We recorded the positions on the slide of positively identified neutrophils and then destained the smears in acidic ethanol. For immunostaining, the slides were rinsed three times with phosphate-buffered saline (PBS), after which the cells were extracted in 0.5% Triton X-100 in PBS (pH 7.3) for 30 min at 4°C. This extraction prior to antibody incubation recognizably improved signal intensity. The cells were rinsed in PBS, and unspecific binding was blocked with PBS containing 1% bovine serum albumin (BSA) and 0.05% Tween-20 (PBS-ST, pH 7.3). This was followed by incubation with mouse monoclonal anti-human stomatin antibody (ascites 1:750 in PBS-ST) for 1 h. The primary antibody was detected using the Chem-Mate horseradish peroxidase (HRP)-3'-diaminobenzidine tetrahydrochloride system designed for automated immunostaining (DAKO) with one modification. To reliably block endogeneous peroxidase activity, the slides were preincubated with 3% H2O2 in 50% methanol for 15 min at room temperature. After the immunostaining procedure, the slides were dehydrated in ethanol and xylene and mounted with Eukitt (Merck, Darmstadt, Germany). Previously identified and position-recorded neutrophils were photographed with a Nikon Mikrophot FX4 using black and white film.

Ultrastructural immunostaining
Neutrophils were washed in PBS and fixed in 2% freshly depolymerized paraformaldehyde containing 0.2% glutaraldehyde in 0.15 M phosphate buffer (pH 7.4). The cells were dehydrated, embedded into hydrophilic LR-White resin, sectioned, and mounted on gold grids as published earlier [18 ]. Immunostaining for stomatin was performed as previously described [19 ]. Pale gold (about 90 nm), thin sections were rinsed in PBS and PBS-ST and incubated with mouse monoclonal anti-human stomatin antibody (GARP-50; supernatant diluted 1:2 in PBS-ST) or antistomatin immunoglobulin (Ig) from mouse ascites (1:750 in PBS-ST) for 1 h. The primary antibody was detected by secondary goat anti-mouse Ig with 10-nm colloidal gold grains (Biocell, Cardiff, UK), diluted 1:40 in PBS-ST (pH adjusted to 8.0). As negative controls, the primary antibody was omitted or preabsorbed by incubation with 100- to 1000-fold excess of the stomatin N-terminal peptide sequence (amino acids 1–24). Both procedures abolished the signal, indicating the specificity of the assays.

SDS-PAGE and Western blotting
SDS-PAGE was performed essentially according to Laemmli [14 ]. Samples corresponding to 2 x 106 CE were separated on the gels. The gels were developed using Silver stain [20 ] or electrotransferred to polyvinylidene difluoride (PVDF) membranes (Immobilion P, Millipore, Bedford, MA) using a Tris-glycine transfer buffer [21 ]. For immunoblotting, the following antibodies were used: monoclonal mouse anti-human stomatin antibody (GARP-50; supernatant diluted 1/200 [22 ]), mouse-anti human CD63, mouse anti-human CD11b, and mouse anti-human CD35 (all diluted 1/1000; DAKO). The PVDF membranes were blocked (1% BSA in PBS with 0.05% Tween) for 1 h and then incubated with the primary antibody for 90 min in blocking solution. After washing, the membranes were incubated with HRP-labeled rabbit anti-mouse IgG (1/2000; DAKO) for 1 h. The blots were washed and developed using a peroxidase substrate (VIP; Vector Laboratories, Burlingame, CA).

Neutrophil stimulation by lipopolysaccharide (LPS)
To investigate the up-regulation of stomatin localized in mobilizable granules and vesicles to the cell surface, neutrophils (107/ml) were incubated in the presence or absence of LPS from Escherichia coli serotype O111:B4 (10 µg/ml) for 30 min at 37°C [23 ]. The cells were then subcellularly fractionated as stated above. The fractions containing the plasma membrane were pooled, and the material was analyzed for the amount of stomatin present by SDS-PAGE and Western blot.

Isolation of Triton X-100 insoluble membrane domains
Cell membranes have been shown to contain membrane domains or "rafts" that are enriched in (glyco)sphingolipids, cholesterol, specific membrane proteins, and glycosylphosphatidylinositol (GPI)-anchored proteins [24 ]. To isolate these rafts, sucrose gradients, in which detergent-insoluble membrane domains are allowed to float, have originally been used. Here, we have instead used a Percoll gradient, allowing for the flotation to occur faster and under isotonic conditions.

Membranes from granules or plasma membrane/secretory vesicles corresponding to 109 cell equivalents were washed in relaxation buffer and resuspended in 2 mL relaxation buffer containing Percoll (1.10 g/L) and Triton X-100 (1%). The mixture was kept on ice for 20 min, overlayered with 24 mL Percoll (1.07 g/L) and Triton X-100 (1%), and centrifuged at 3000 g for 90 min at 4°C using a fixed-angle Beckman JA20 rotor. Fractions of 1.5 mL were collected from the bottom of the tube. To prove that the Percoll-based separation technique allowed for separation of membrane rafts, we investigated the distribution in the gradient of the GPI-linked protein alkaline phosphatase (ALP) that is known to localize to detergent-insoluble membrane domains [24 , 25 ]. As expected, ALP was recovered in the low-density, upper region of the gradient, and several membrane-spanning proteins such as CD63, CD11b, and CD35 (CR1) remained in a high-density part of the gradient (see Results). Hence, ALP was present in detergent-insoluble membrane domains, and the other proteins tested were solublized in the Triton X-100 and thus remained in the bottom of the tube. This shows that the Percoll gradient can separate membrane rafts containing GPI-linked proteins on the basis of density.

To further evaluate the Percoll technique for isolation of detergent-insoluble membrane domains, we attempted to disrupt the domains in isolated plasma membranes by depleting the cholesterol with methyl-ß-cyclodextrin (1 mM, 30 min, 37°C). However, only 20% of the cholesterol was solubilized by this procedure, and subsequently, no shift could be seen in the membrane domain-localized proteins (ALP, stomatin) to the soluble fraction.

Reagents
The ATP, EGTA, paranitrophenyl phosphate, Triton X-100, PMSF, MOPS, and PIPES were obtained from Sigma Chemical Co. (St. Louis, MO). The SDS and DFP were from Fluka Chemie AG (Buchs, Switzerland). BSA was supplied by Boehringer Mannheim (Mannheim, Germany). Dextran, Ficoll-Paque, and Percoll were from Pharmacia (Uppsala, Sweden). The polyacrylamide and molecular weight standard proteins were from Bio-Rad Laboratories (Richmond, CA). The [57Co]vitamin B12 was supplied by Amersham Laboratories (Buckinghamshire, UK).


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RESULTS
 
Identification of stomatin by MS
To investigate the presence of other membrane components in the azurophil granules, besides CD63 and CD68, we first isolated the azurophil granule membrane. Neutrophils were subjected to disruption by nitrogen cavitation and subcellular fractionation on a two-step, discontinuous Percoll gradient. The visible band containing the azurophil granules was collected, freeze-thawed, and centrifugated. After removing peripheral membrane proteins in 1 M NaCl, the pellet was solublized in Triton X-100-containing buffer. After centrifugation, the nonsolubilized pellet was submitted to SDS-PAGE and stained by Commassie blue. In the Coomassie-stained gel, we identified a band with a molecular mass of approximately 31 kDa (Fig. 1 ), which most probably was not CD63 (53 kDa) or CD68 (110 kDa). The band was excised, submitted to trypsin digestion, and analyzed by MALDI-TOF MS. By peptide mass-mapping and database searches, the protein was identified as stomatin (accession numbers gi|4758276, gi|181184, gi|1103842, gi|14715077) with a molecular weight of 32 kDa (Fig. 2 ). The mass mapping had a z score of 2.26, corresponding to a percentile well above the 95% level and covered 41% of the protein. Six of the peptides were analyzed by MS/MS on a Q-TOF (Micromass), and the resulting spectra were identified by MASCOT (available at <www.matrixscience.com>) with a combined score of 434, where 43 corresponds to the 95% probability level.



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  		Figure 2. MALDI-TOF MS spectrum of the 31-kDa protein. The 31-kDa protein was subjected to trypsin digestion, and the peptides were analyzed by MALDI-TOF MS. T, Peptide from trypsin autolysis. Annotated masses are monoisotopic. All masses were within 100 ppm. Sequences validated by electrospray ionization-MS/MS are noted in the figure. Intensity is given in arbitrary units.

Microscopic detection of stomatin in neutrophils
The distribution of stomatin in neutrophils was first examined using light and electron microscopy. Light microscopic immunocytochemistry revealed an intense cytoplasmic staining of the neutrophils, and the cell nucleus was devoid of labeling (Fig. 3a ). Basophils and eosinophils were negative for stomatin staining (data not shown). At the ultrastructural level, two preferential subcellular locations of the immunogold labeling were observed. Most of the staining was observed along the membranes of intracytoplasmic granules, and some was associated with the cytoplasmic membrane (Fig. 3b) .



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  		Figure 3. Light microscopical and ultrastructural immunodetection of stomatin. Stomatin immunostaining in a neutrophil granulocyte from a blood smear (a) and isolated from a buffy coat (b). At the light microscopic level (a), the signal reveals a patchy distribution within the cytoplasm. Although the nuclear segments appear light and unstained, there is a tendency for enhanced signal intensity in the perinuclear areas. (b) The subcellular distribution of stomatin is shown at the ultrastructural level. Stomatin, as revealed by colloidal gold grains, is mainly detected over the membranes of the granules (arrowheads). Some signal is associated with the plasma membrane (arrows). n, Nucleus. Original bars: (a) 5 µm; (b) 0.5 µm.

Localization of stomain in neutrophil subcellular fractions
To determine if stomatin is present in neutrophil membranes other than the azurophil granule membranes, we again used the subcellular fractionation technique. As described above, neutrophils were fractionated on a Percoll gradient. Here, the gradient was collected in 24 1.5-mL fractions from the bottom of the tube. To determine the localization of neutrophil organelles in the gradient, the fractions were analyzed for the presence of marker molecules (Fig. 4 ).



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  		Figure 4. Subcellular localization of stomatin in neutrophils. Human neutrophils were fractionated on a Percoll gradient as described in Materials and Methods. The localization of neutrophil organelles in the gradient is shown by marker analysis of the fractions. The top panel shows the distribution of myeloperoxidase (marker for the azurophil granules; {blacklozenge}), vitamin B12-binding protein (marker for the specific granules; {blacksquare}), and alkaline phosphatase (marker for the plasma membrane and secretory vesicles; {blacktriangleup}). The bottom panel shows a Western blot of the fractions probed with antistomatin antibody.

The gradient fractions were then submitted to SDS-PAGE and immunoblotting with antibodies to stomatin (Fig. 4) . The antistomatin antibody detected a 31-kDa protein that was present in the azurophil granules (fractions 2 and 3), the specific granules (fractions 7–10), and the plasma membrane/secretory vesicles (fractions 14–17). No stomatin was found in the cytosol (fractions 19–24), as expected. The relative amount of stomatin varied between the organelles, being most prominent in the specific granules, somewhat less in the azurophil granules, and quite minute in the light membranes corresponding to plasma membrane/secretory vesicles (Fig. 4) .

Up-regulation of stomatin to the neutrophil cell surface after LPS treatment
The presence of the membrane protein stomatin in granules suggests that this protein should be up-regulated on the cell surface after fusion of the granules with the plasma membrane (exocytosis). To investigate this, neutrophils were treated with LPS for 30 min at 37°C, a protocol that has earlier been shown to induce exocytosis of gelatinase granules and secretory vesicles [23 ]. These cells were then subcellularly fractionated on Percoll density gradients as described above. The fractions corresponding to the plasma membrane/secretory vesicles (as determined by marker analysis; data not shown) were pooled and immunoblotted with antistomatin antibody. The stomatin content in the LPS-treated plasma membrane was increased as compared with the nontreated cells (Fig. 5 ). Thus, we conclude that the stomatin found to colocalize with the specific/gelatinase granules by subcellular fractionation indeed is localized to the membrane of these organelles and can be mobilized to the cell surface upon exocytosis.



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  		Figure 5. Exposure of stomatin on the plasma membrane after LPS treatment. Neutrophils were treated in the presence or absence of E. coli LPS (10 µg/ml) for 30 min at 37°C. After subcellular fractionation in Percoll gradients, the plasma membrane/secretory vesicle fractions (as determined by marker analysis; not shown) were pooled and concentrated by centrifugation, and 1.2 x 107 cell equivalents were analyzed by SDS-PAGE and immunoblotting with antistomatin antibody. Densitometric analysis was performed using a UMAX C12 scanner and the public domain NIH Image program (developed at the U.S. National Institute of Health and available on the Internet at <http://rsb.info.nih.gov/nih-image/>). The relative intensity of the stomatin band of the LPS-treated cells was 165% of that of nontreated neutrophils.

Presence of detergent-insoluble domains in neutrophil membranes and localization of stomatin in these domains
Stomatin has recently been shown to colocalize with Lamp-2 and CD63 in pericentrosomal vesicles, corresponding to late endosomes of the epithelial cell line UAC [25 ]. These endosomes were shown to have detergent-insoluble domains in which stomatin is present. Such domains are enriched in laterally close-packed sphingolipids and cholesterol and can be isolated from the membrane by nonionic detergents, such as Triton X-100 [26 27 28 ].

To investigate if detergent-insoluble membrane domains are present in the neutrophil granules and whether stomatin is localized to these, granules and plasma membrane/secretory vesicles were isolated by subcellular fractionation as described. The organelles were freeze-thawed and centrifuged to obtain membrane fractions. The membranes were resuspended in relaxation buffer containing Percoll and Triton X-100, transferred to the bottom of a centrifuge tube containing Percoll, and centrifuged (as described in Materials and Methods). To determine the localization in the gradients of detergent-insoluble membrane domains, fractions from the plasma membrane/secretory vesicle gradient were analyzed for the presence of the GPI-linked protein ALP. The ALP activity was recovered in the top fractions of the gradient, thus containing the floating detergent-insoluble membrane domains (Fig. 6 ). Next, all gradients were investigated for the distribution of stomatin and marker proteins. Fraction samples from each gradient were separated by SDS-PAGE, after which the proteins were transferred to PVDF membranes and probed with antibodies. The azurophil granule marker CD63 was localized in the lower partof the Percoll gradient containing the detergent-soluble material, in line with its distribution in UAC cells [25 ]. Conversely, stomatin was localized in the upper part of the gradient, in fractions corresponding to those containing the plasma membrane ALP, indicating that the protein is localized in detergent-insoluble membrane domains (Fig. 6) . Also in the specific granules and light membranes, stomatin was localized to the detergent-insoluble domains; however, part of the protein was found in the lower part of the gradient, indicating that a fraction of the protein was localized to detergent-soluble domains. The specific granule marker molecule CD11b was found in the detergent-soluble fraction as was CD35 (CR1), marker for the secretory vesicles.



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  		Figure 6. Distribution of stomatin and marker proteins in detergent-soluble domains of neutrophil membranes. Isolated membranes from azurophil granules, specific granules, and plasma membrane/secretory vesicles, respectively, were resuspended in relaxation buffer containing Triton X-100, layered under Percoll, and centrifuged as described in Materials and Methods. The collected fractions were analyzed for alkaline phosphatase activity or submitted to SDS-PAGE and immunoblotting using antibodies toward markers for the different organelles and stomatin. Triton-insoluble membrane domains have floated to the upper part of the gradient (fractions 15–17), and Triton-soluble membrane constituents remain in the lower fractions.

In conclusion, the data implicate that detergent-insoluble domains are present not only in the plasma membrane of neutrophils but exist also in the azurophil and specific granule membranes and that stomatin is localized in these detergent-insoluble domains.


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DISCUSSION
 
The neutrophil azurophil granules have been considered classical examples of lysosomes that play an important role in killing of phagocytosed microbes. The matrix accordingly contains many well-defined bactericidal agents. However, not much is known about the membrane content of these organelles, and so far, CD63 and CD68 are the only known membrane components [5 6 7 ]. The background to the present investigation was our results published some years ago, showing that the azurophil granule membrane lacks the lysosome markers Lamp-1 and Lamp-2 [8 ]. This was later supported by the study of Cieutat et al. [9 ] in which it was reported that the Lamps in fact reside in organelles other than the granules, corresponding to multivesicular bodies and multilaminar compartments. These data suggest that the azurophil granules are not the neutrophil counterpart to classical lysosomes but may instead be an entirely different organelle (or a nonclassical lysososome) with specific characteristics and functional repertoire. Based on these facts, we found reason to attempt to identify novel membrane components of the azurophil granules.

Here, we show that the neutrophil azurophil granules, specific granules, and plasma membrane/secretory vesicles, contain the integral membrane protein stomatin. Stomatin, also known as band 7.2b, is a 31-kDa integral membrane protein that exists in abundance in different mammalian tissues and cell types, among them human erythrocytes. The function of the protein is so far not entirely understood. Absence of stomatin from the red cell membrane is observed in the rare hereditary hemolytic anemia called overhydrated hereditary stomatocytosis [22 , 29 30 31 ]. In such anemia, erythrocytes have a major defect in membrane permeability to the univalent cations Na+ and K+, which makes the cells leaky to these ions [32 ]. Thus, stomatin appears to function as an ion channel regulator [33 ]. In the human epithelial cell line UAC, stomatin colocalizes with cortical actin microfilaments, indicating a connection between stomatin and the membrane-associated cytoskeleton and a possible function as a cytoskeletal anchor [30 ]. It has been suggested that these two effects (ion channel regulator and cytoskeletal anchor) may work in concert if the protein acts as an information relay between sensors of stretch in the membrane and the ion channels, which are embedded in the lipid bilayer, perhaps to influence channel stability and organization in the plasma membrane [33 , 34 ]. Stomatin localized in neutrophil granule membranes may, through its cytoskeletal interactions, take part in transport and fusion of the granules with the plasma membrane. Whether the protein also has channel-regulating features in the granule membranes remains to be investigated.

In UAC cells, stomatin is also present in the late endosomes, where it colocalizes with CD63 [25 ]. However, the two proteins are expressed in different types of domains in the membrane. CD63 is localized to detergent-soluble domains, and stomatin is found in detergent-insoluble domains [25 ]. Also in red blood cells, stomatin is a major component of the lipid rafts [35 ]. Here, we investigated whether the neutrophil granules contain such membrane domains. For the azurophil granules, we used stomatin and CD63 as marker proteins for this purpose. After solubilization of the granules and centrifugation in a Percoll gradient, CD63 remained in the lower part of the gradient, corresponding to detergent-soluble membrane-domain constituents. In contrast, stomatin floated upward in the gradient and localized to the same fractions as the GPI-linked protein ALP, indicating that it is localized to detergent-insoluble domains (Fig. 4) . We found a similar pattern in the light membranes (plasma membrane and secretory vesicles) and the specific granule membranes. In the latter, stomatin was divided between the detergent-soluble material and the detergent-insoluble material. This could depend on the larger content of stomatin in these membranes as compared with the other membranes, resulting in an excess pool of stomatin that is excluded from the domains. It could also be an effect of differential palmitoylation of stomatin [36 ], which may regulate the lipid raft association.

It is generally believed that membrane rafts are microdomains present in the plasma membrane. However, detergent-insoluble membrane domains have been isolated from Golgi membranes [37 ] and are implicated in recycling endosomal compartments [38 ]. Here, we show that neutrophil granules contain detergent-insoluble membrane domains, and the implications of this finding for the function of the neutrophil granules can at this time only be speculated on. In general, membrane rafts have been suggested to be involved in signal transduction, mostly through GPI-anchored proteins but also through transmembrane receptors [39 40 41 ]. They are also thought to be involved in lipid and protein sorting and in secretory and endocytic pathways [27 ]. Thus, the presence of detergent-insoluble domains in neutrophil granules strongly suggests that these organelles are more sophisticated than only being storage organelles for lysosomal and bactericidal proteins (azurophil granules) or matrix-degrading enzymes and receptor proteins (specific granules). As discussed above, the presence of stomatin in a membrane implicates the existence of a control function of ion channels, mechanoreception, and lipid domain organization [34 ]. Thus, receptor aggregation and lipid rafts may be functional properties of importance also in the azurophil granule of neutrophils. What this implicates with regards to phagolysosome formation and bacterial killing has yet to be investigated.

In conclusion, this study provides original data proving the presence of stomatin in human neutrophils, it defines a new azurophil granule membrane component, and it presents evidence for the existence of detergent-insoluble domains in neutrophil granules.


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ACKNOWLEDGEMENTS
 
Financial support was obtained from the Swedish Medical Research Council, King Gustav the V 80-Year Foundation, Swedish Rheumatism Association, Inga Britt and Arne Lundbergs Foundation, Knut and Alice Wallenberg Foundation, Magnus Bergvall Foundation, and Swedish Society for Medicine.

Received September 13, 2001; revised July 4, 2002; accepted July 18, 2002.


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