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(Journal of Leukocyte Biology. 2001;69:57-62.)
© 2001 by Society for Leukocyte Biology

Neutrophil secretory vesicles are the intracellular reservoir for GPI-80, a protein with adhesion-regulating potential

Claes Dahlgren*, Anna Karlsson* and Fujiro Sendo{dagger}

* The Phagocyte Research Laboratory, Department of Medical Microbiology and Immunology, University of Göteborg, S-413 46 Göteborg, Sweden
{dagger} Department of Immunology and Parasitology, Yamagata University School of Medicine, Yamagata, Japan

Correspondence: Claes Dahlgren, Department of Medical Microbiology and Immunology, University of Göteborg, Box 435, 405 30 Göteborg, Sweden. E-mail: Claes.Dahlgren{at}microbio.gu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The subcellular localization of GPI-80, a novel, adhesion-regulating protein, was investigated in human neutrophils. Surface expression of GPI-80 was determined by FACS analysis as well as by the ability for phospholipase C to cleave the protein from the cell surface. Increasing amounts of GPI-80 were exposed on the cell surface after weak stimulation with the chemoattractant fMLF, suggesting that the protein can be translocated to the plasma membrane from intracellular stores. By subcellular fractionation of the neutrophils, GPI-80 was defined as a component of a light membrane fraction, containing secretory vesicles and plasma membranes, and it was absent from the neutrophil granule fractions. Separation of the plasma membranes from the secretory vesicles by flotation gradient fractionation confirmed that the GPI-80 was localized in the mobilizable secretory vesicles by approximately 50%, and the rest was plasma membrane-bound. Thus, we identify secretory vesicles as the reservoir of GPI-80 from which it may translocate to the plasma membrane after weak stimulation of the cells.

Key Words: granulocytes • subcellular localization • granule • cell adhesion


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The neutrophil granulocytes are inflammatory cells of great importance for eliminating invading microorganisms [1 ]. Adhesion to endothelial cells (during diapedesis) and recognition of chemoattractants and foreign material are dependent on a dynamic expression/shedding of different cell-surface molecules and receptors. A new cell-surface glycosylphosphatidyl-inositol-(GPI)-anchored glycoprotein, GPI-80, was identified recently in human neutrophils [2 ]. The precise function of GPI-80 is not known, but the high degree of homology with Vanin-1 (a GPI-linked protein involved in thymus homing) suggests involvement in the regulation of cell adhesion and consequently regulatory effects on leukocyte trafficking. This suggestion gains support from the fact that treatment of human neutrophils with an antibody to GPI-80 modulates ß2-integrin-dependent neutrophil adhesion and also modulates transendothelial migration of neutrophils in vitro [2 ]. The mechanisms involved in GPI-80-induced modulation of ß2-integrin-mediated functions are not known, but intermolecular reactions between the ectodomain of the ß2-integrin and other GPI-anchored proteins have been recognized recently as important in the regulation of ß2-integrin function [3 4 5 6 ]. Many of the neutrophil surface-effector proteins (adhesion molecules as well as different chemoreceptors) required for adhesion to an activated endothelium and transmigration into an inflamed tissue are present in internal stores and mobilized to the neutrophil plasma membrane during neutrophil-endothelial interaction and migration [7 ]. Neutrophil function thus relies on different subcellular organelles to take part in fusion processes with the plasma membrane.

The neutrophil contains several types of subcellular organelles: peroxidase-positive granules (termed primary or azurophil granules) that in many respects (but not all) resemble classical lysosomes [8 , 9 ] and at least two types of peroxidase-negative granules/vesicles (secondary or specific granules and secretory vesicles, respectively) [7 , 10 , 11 ]. The membrane of the specific granules contains a variety of different receptors such as CD11b/CD18 (Mac1), CEACAMs (CD66a and b), the formylpeptide receptor (FPR), the fibronectin receptor, and the laminin receptor. These are all up-regulated on the cell surface as a consequence of granule fusion with the plasma membrane. However, several neutrophil receptors, including Mac-1 and FPR, can be mobilized to the cell surface without corresponding exocytosis of specific granule contents. This is explained by the fact that these receptor molecules are stored not only in the membrane of the specific granules but also in the membrane of the most easily mobilizable neutrophil organelle, the secretory vesicle [7 , 10 , 11 ]. The aim of the present study was to determine the subcellular localization of GPI-80 in human neutrophils. We found that the secretory vesicles are the intracellular reservoir for this novel, adhesion-regulating protein.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and antibodies
Percoll and Ficoll-Paque were from Pharmacia (Uppsala, Sweden). Adenosine 5'-triphosphate (ATP), ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA), nitroblue tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), p-nitrophenyl phosphate, and diisopropylfluorophosphate (DFP) were products from Sigma Chemical Co. (St. Louis, MO). The polylvinylidene difluoride (PVDF) membrane was from Millipore (Bedford, MA). Cyanocobalamin ([57Co]-vitamin B12) was from Amersham Laboratories (Buckingham, England), and phosphatidylinositol-specific phospholipase C (PLC) was from Boehringer Mannheim (Mannheim, Germany). The monoclonal antibody (mAb) 3H9 immunoglobulin G1 (IgG1), recognizing GPI-80 [12 ], was obtained by immunizing BALB/c mice with phorbol myristate acetate-treated human peripheral blood neutrophils. The basic properties of the antibody have been described earlier [12 ]. Mouse monoclonals to human CD11b as well as the fluorescein isothiocyanate (FITC)-labeled rabbit anti-mouse IgG antibodies were obtained from DAKO (Glostrup, Denmark). Antibodies against human gelatinase (MMP9) were obtained from Chemicon International Inc. (Temecula, CA).

Isolation of cells
Human polymorphonuclear leukocytes (neutrophil granulocytes) were isolated from buffy coats as described by Bøyum et al. [13 ]. The purified neutrophils were washed twice and resuspended in Krebs-Ringer medium (120 mM NaCl, 4.5 mM KCl, 1.2 mM MgSO4, 1.0 mM CaCl2, 1.7 mM KH2PO4, 8.3 mM Na2HPO4, 10 mM glucose, pH 7.3). Cells used for subcellular fractionation were resuspended in physiological saline and treated with the serine protease inhibitor DFP (5 mM, 10 min on ice). The cells were then washed and resuspended in relaxation buffer [10 ml, 100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, 10 mM Pipes, 1 mM ATP(Na)2, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.4].

Subcellular fractionation
Neutrophils (5x108 cells) in relaxation buffer were disrupted in a nitrogen bomb (375 psi; Parr Instruments, Moline, IL). To separate azurophil granules, specific granules, and plasma membrane/secretory vesicles, the postnuclear supernatant was layered on top of two-step (1.05 and 1.12 g/ml), Percoll-density gradients and fractionated according to the technique described by Borregaard et al. [14 ] and Sjölin et al. [15 ]. A modification of the flotation technique described earlier [8 ] was used to separate secretory vesicles from plasma membranes. In short, the postnuclear supernatant was mixed with twice the vol of a heavy Percoll solution (1.12 g/ml), and this mixture (15 ml) was underlaid 15 ml of a light (1.04 g/ml) Percoll solution. Relaxation buffer (5 ml) was applied on top of the gradient, which was then centrifuged at 37,000 g for 30 min (4°C) using a fixed-angle Beckman JA-20 rotor. Gradient fractions (1.5 ml) were collected from the bottom of the gradients.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting
SDS-PAGE was performed essentially according to Laemmli [16 ] using homogenous 10% polyacrylamide gels. The gradient fractions were applied to the gels in vol corresponding to the fractionated content of 2.5 x 105 cells. The gels were blotted electrophoretically onto PVDF membranes, and the membranes were blocked and incubated with antibodies as described [15 ]. The blots were then developed with substrate buffer containing NBT (0.3 mg/ml) and BCIP (0.15 mg/ml). The blots were replicated using a UMAX C12 scanner, and analysis of the images was performed using the public domain NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/).

Quantification of granule markers
The Percoll fractions were assayed for myeloperoxidase (MPO) [17 ], vitamin B12 binding protein (B12BP) [18 ], and alkaline phosphatase (ALP) in the presence and absence of Triton X-100 as described [19 ]. The molecules analyzed constitute markers for azurophil granules (MPO), specific granules (B12BP), secretory vesicles (latent ALP), and plasma membranes (nonlatent ALP activity), respectively. ALP latency was determined by subtracting the activity measured in the absence of Triton X-100 (nonlatent pool) from the activity obtained in the presence (total pool) of detergent.

Surface up-regulation of complement receptor 3 (CR3) and GPI-80
Cells were kept at 4°C (resting cells) or stimulated with formylmethionyl-leucyl-phenylalanine (fMLF; 10-7 M final concentration) at 15°C for 10 min followed by incubation at 37°C for another 10 min. In one set of experiments, the expression of GPI-80 on the cell surface was analyzed by adding 10 µl mouse mAbs (3H9 diluted 1/100) to cell pellets (around 106 cells). The cells were incubated at 4°C for 30 min, washed twice, and incubated with a FITC-labeled secondary antibody, washed again, and examined in a flow cytometer (FACS Scan; Becton Dickinson, Mountain View, CA). Phycoerythrin-conjugated mAbs against CD11b (Becton Dickinson) were used to determine mobilization of CR3 [20 ]. In another set of experiments, PLC (50 ng/ml, final concentration) was added to the cells that were then incubated for 10 min at 37°C. The proteins released from the cell surface by PLC were isolated by brief centrifugation of the cells in a microfuge. The resulting supernatants were analyzed with respect to ALP activity and content of GPI-80.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mobilization of GPI-80 to the cell surface
Mobilization of neutrophil storage organelles (granules and secretory vesicles) can be determined indirectly by the exposure of membrane receptors present in the membrane of these organelles. In neutrophils, the adhesion protein CR3 (Mac-1) is localized by a small fraction in the plasma membrane, and the major part of these molecules is found in the secretory vesicles and the specific granules [10 , 11 ]. As illustrated in Figure 1 , neutrophil interaction with the chemotactic peptide fMLF results in an increased surface exposure of Mac-1 (Fig. 1a) concomitant with a low level of granule-marker release (Fig. 1c) , suggesting that the newly exposed receptors originate mainly from the secretory vesicles. Mobilization of the secretory vesicles was accompanied also by an increased surface exposure of GPI-80, as illustrated by an increased binding to the cells of anti-GPI antibodies (Fig. 1b) .



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  		Figure 1. Mobilization of secretory vesicles. Surface up-regulation of Mac-1 (a) and GPI-80 (b) on human neutrophils, expressed as mean fluorescence intensity of cells labeled with antibodies against CD11b (a) and GPI-80 (b). Cells were kept at 4°C (control; solid lines) or preactivated with fMLF (see Materials and Methods; 10-7 M final concentration; dotted lines). A representative experiment out of six is shown, and the increased expression of Mac-1 and GPI-80, respectively, calculated as the ratio, and absolute difference between the mean fluorescence value, the fMLF, and control cell populations from the six experiments is given. Panel (c) shows the release into the medium of markers for specific granules (vitamin B12-binding protein; B12bp) and azurophil granules (MPO) after fMLF-induced activation of the cells. The release is expressed as percent of the total amount in control cells.

 
Inositolphosphate-linked proteins exposed on the cell surface can be cleaved from their lipid anchor by the action of an externally added phosphatidyl inositol (PI)-specific PLC. Mobilization of the secretory vesicles (by fMLF pretreatment) was accompanied by an increased exposure of GPI-80 as well as of the secretory-vesicle marker ALP, illustrated by increased amounts of these proteins in the cell-free supernatant following cleavage with PI-specific PLC (Fig. 2 ). We could not detect any PLC-induced rise in intracellular Ca2+ measured by Fura2 fluorescence (not shown by figure), suggesting that cleavage is restricted to PI-linked molecules exposed on the cell surface, and that the plasma membrane was not permeabilized by the lipase.



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  		Figure 2. Cleavage of GPI-80 and alkaline phosphatase by a PI-specific PLC. PI-PLC was added to neutrophils that were preactivated with fMLF (10-7 M final concentration) as well as to nontreated control cells. The cells were removed by centrifugation, and the amounts of ALP (determined by the enzyme activity in the samples) and GPI-80 (determined by Western blotting and quantification by densitometric measurements of the blots), respectively, were determined. The figure shows an immunoblot for GPI-80 from one representative experiment out of four. The figures represent the mean ratios obtained for ALP and GPI-80, respectively, between amounts in the supernatants of the fMLF and control cell populations in the four experiments.

 
Localization of GPI-80 in neutrophil-subcellular granules
By use of the mouse mAb raised against GPI-80, we could detect a major 80 kD band in Western blots of human neutrophils (Figs. 2 and 3 ). To prove that a cell component is localized to a particular organelle, a biochemical approach has to be added to the observation that the protein is mobilized to the cell surface during cell activation. To determine the subcellular localization of GPI-80, neutrophils were disintegrated and fractionated on discontinuous Percoll gradients. In an ordinary two-step discontinuous Percoll gradient, four subcellular fractions are easily identified: the cytosol (S2), plasma membrane/secretory vesicles (the {gamma}-band), specific granules (the ß-band), and azurophil granules (the {alpha}-band; Fig. 3a ). No GPI-80 was detected in the S2 fraction, which is what was to be expected because GPI-80 is a membrane protein. GPI-80 was not found in the azurophil-granule fraction either (the {alpha}-band) or the specific-granule fraction (the ß-band). A modified, two-step gradient, allowing an even better separation of the plasma membrane/secretory vesicles from the gelatinase granules, revealed that no GPI-80 was present in any of the granule subtypes. Instead it was found exclusively in the light membrane fraction (the {gamma}-band), enriched for secretory vesicles and plasma membranes (Figs. 3 and 4 ).



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  		Figure 3. Identification of GPI-80 in neutrophil subcellular fractionations. A postnuclear supernatant of disrupted neutrophils was fractionated on a discontinuous, two-layer, Percoll-density gradient (14 ml 1.12 g/ml and 14 ml of 1.05 g/ml). Three bands were visible, which were denoted {alpha}, ß, and {gamma} in order of decreasing density. Cytosol components were located in the upper portion of the gradient (S2). a) Fractions were analyzed for myeloperoxidase (a marker for azurophil granules; {blacksquare}), B12BP (marker for peroxidase negative-specific granules; •), nonlatent ALP (marker for the plasma membranes; {blacktriangleup}), and latent ALP (marker for plasma the secretory vesicles; {triangleup}). Each fraction was also analyzed by SDS-PAGE under nonreducing conditions in 10% (w/v) polyacrylamide gels and immunoblotting with anti-GPI-80 antibodies (b). The results of one representative experiment of six are shown in (b). The arrowheads designate the peak fractions of the {alpha}, ß, and {gamma} bands, respectively.

 


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  		Figure 4. Localization of gelatinase and GPI-80 in neutrophil-subcellular fractionations. A postnuclear supernatant of disrupted neutrophils was fractionated on a discontinuous, two-layer, Percoll-density gradient (3 ml 1.12 g/ml and 25 ml 1.05 g/ml Percoll, respectively). The peak fractions for MPO and vitamin B12BP are marked {alpha} and ß, respectively. The fractions (1–25) were analyzed by SDS-PAGE under nonreducing conditions in 10% (w/v) polyacrylamide gels and immunoblotting with anti-gelatinase antibodies (upper panel) and anti-GPI-80 antibodies (lower panel), respectively.

 
Localization of GPI-80 in neutrophil-secretory vesicles
To clarify further the identity of the GPI-80-containing membranes in the {gamma}-fraction, the postnuclear material was fractionated on a flotation gradient. Two light membrane bands were seen in the gradient after centrifugation, and based on the distribution profiles for MPO (azurophil-granule marker) and vitamin B12BP (specific-granule marker), we conclude that the modified gradient permits a clear separation of the light membranes from the granules. The nonlatent ALP activity and latent ALP activity were also clearly separated. Most of the alkaline phosphatase present in the {gamma}1-fraction (Fig. 5 ) was latent (80.6±4.7%; mean±SD, n=5) and thus derived from the secretory vesicles, whereas most of the activity present in the {gamma}2-fraction (Fig. 5) was nonlatent (84±10%; mean±SD, n=5) and thus derived from the plasma membrane. We conclude that our gradient system permits a clear separation of the secretory vesicles ({gamma}1) from the plasma membranes ({gamma}2). GPI-80 was found in the secretory-vesicle fraction and the plasma-membrane fraction, respectively, in similar amounts (Fig. 6 ).



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  		Figure 5. Separation of secretory vesicles and plasma membranes. A postnuclear supernatant of disrupted neutrophils was fractionated on a modified, Percoll-density gradient. Fractions were analyzed for nonlatent ALP activity (plasma membrane marker; solid symbols) and latent ALP activity (marker for secretory vesicles; open symbols). The positions in the gradient of the azurophil granules ({alpha}) and the specific granules (ß) are indicated by arrows. Fractions enriched in secretory vesicle ({gamma}1) and plasma membrane ({gamma}2), respectively, were pooled, concentrated, and analyzed further by SDS-PAGE and immunoblotting (Fig. 6) .

 


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  		Figure 6. Identification of GPI-80 in plasma membrane and secretory vesicles. Fractions enriched in secretory vesicles ({gamma}1) and plasma membrane ({gamma}2), respectively, were prepared as described (see the legend to Fig. 5 ) and analyzed by SDS-PAGE under nonreducing conditions in 10% (w/v) polyacrylamide gels. Immunoblotting was performed with anti-GPI-80 antibodies. The results of one representative experiment out of five are shown, together with the ratio (mean±SD) between the amount GPI-80 present in the secretory vesicles and the plasma membranes calculated from five experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study designates secretory vesicles as the main intracellular reservoir of GPI-80. The protein was not found in the azurophil granule fraction or the specific-granule fraction, which in our fractionation system contains the lactoferrin-containing specific granules (secondary granules) and the gelatinase (tertiary) granules [21 ]. Separation of plasma membranes from the secretory vesicles revealed that GPI-80 is fairly equally distributed between these two membrane fractions, and mobilization of the secretory vesicles results also in a doubling of GPI-80 exposed on the cell surface. The interpretation that about 50% of the cellular GPI-80 is stored in the secretory vesicles should possibly be taken with some caution. The figure may be somewhat under-estimated, because the secretory vesicles may be partly mobilized during isolation of the cells even when extreme care is taken during the isolation procedure. Nevertheless, the fact that CR3 was up-regulated 3.5-fold by fMLF implies that no more than 20% of the vesicles could have been secreted before.

The secretory vesicles are of endocytic origin. Accordingly, the vesicle matrix contains plasma proteins [22 ], and the membrane is derived from the plasma membrane. During vesicle formation, the plasma membrane proteins are sorted to become included (and sometimes even enriched) or excluded from the invagination that forms the vesicle. The membrane of the secretory vesicles has subsequently been shown to contain a number of different proteins including CR3/Mac-1, the b cytochrome of the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, CR1, FPR, Fc receptor for IgG (Fc{gamma}R)III, and the UPA receptor, and we can now add GPI-80 to this group of membrane proteins. The presence in the secretory vesicles of this array of functionally important proteins implies that these organelles, when incorporated into the plasma membrane in response to inflammatory mediators, furnish the cell surface with new proteins. This alters the functional repertoire of the neutrophil markedly and enables it to interact with endothelial cells, extracellular matrix, and microorganisms. Secretory vesicles are therefore believed to be of prime importance in the early neutrophil activation, leading to endothelial adhesion and transmigration. The subcellular localization of GPI-80 is in line with its proposed function as a regulator of neutrophil adhesion and migration events. Although the secretory vesicles resemble the so-called caveolae, in that they contain several GPI-anchored molecules, the two are different in nature. This is illustrated clearly because the secretory vesicles are not primary-located to the subplasmalemmal region, and they lack caveoline [23 ].

GPI-80 is probably involved in the regulation of neutrophil adhesion, but very little is known about the precise mechanisms involved in the GPI-80-mediated sequential up- and down-regulation of ß2-integrin-dependent neutrophil functions. A GPI-induced modulation of the avidity of the ß2-integrin for its ligands has been suggested [24 ], and this gains support from the fact that another GPI-anchored protein (UPAR) has been shown to be of importance for leukocyte recruitment via the ß2-integrin [25 ]. It is worth noting that UPAR is also present in the secretory vesicles [26 ]. Taken together, the presented data show that GPI-80 is present in the membrane of the secretory vesicles and in the plasma membranes of resting neutrophils. GPI-80 may, when present on the surface of activated/extravasating phagocytes, have a potentially important role as modulator of neutrophil ß2-integrin-mediated adhesion, but further investigations are needed to clarify the precise role of GPI-80 in neutrophil adhesion, the functional significance of a 50% increase in surface GPI-80, and the mechanism(s) by which it affects the properties of other adhesion molecules.


   ACKNOWLEDGEMENTS
 
Grants from the Swedish Medical Research Council, King Gustaf the V 80-Year Foundation, Vrdalstiftelsen, the Fredrik and Ingrid Thuring Foundation, the Swedish Society for Rheumatological Research, the Swedish Society for Medical Research, and the Swedish Society for Medicine supported the work. The technical assistance of Lisbeth Björck is greatly appreciated.

Received May 30, 2000; revised September 15, 2000; accepted September 20, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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K. Kaldi, J. Szeberenyi, B. K. Rada, P. Kovacs, M. Geiszt, A. Mocsai, and E. Ligeti
Contribution of phopholipase D and a brefeldin A-sensitive ARF to chemoattractant-induced superoxide production and secretion of human neutrophils
J. Leukoc. Biol., April 1, 2002; 71(4): 695 - 700.
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