JOURNAL OF CELLULAR PHYSIOLOGY 14M36-ti44 (1989)

Preparation and Characterization of Plasma Membrane Vesicles From Human Polymorphonuclear Leukocytes

BRIAN J. DEL B U O N O , FRANCIS W. LUSCINSKAS, AND ELIZABETH R. SIMONS* Department of Biochemistry, Borton University School of Medicine,

Boston, Massachusetts 02 I 1 8

It would be advantageous to prepare models of the neutrophil plasma membrane in order to examine the role of the plasma membrane in transmembrane signal transduction in the human neutrophil and to dissect ligand-receptor interactions and structural changes in the cell surface upon stimulation. A number of inves- tigators have prepared neutrophil membrane vesicles by homogenization, soni- cation, or centrifugation-techniques that can result in the loss of substantial amounts of surface membrane material, disruption of lysosomes causing prote- olysis of membrane proteins, and contamination of the plasma membrane frac- tion by internal membranes. These limitations have been overcome in the present studies by employing a modification of the method previously developed in this laboratory. Human neutrophils were suspended in a buffer simulating cytoplas- mic ionic and osmotic conditions and disrupted by nitrogen cavitation. The re- sultant cavitate was freed of undisrupted cells and nuclei and then centrifuged through discontinuous isotoniciisoosmotic Percoll gradients, which resolved four fractions: a (intact azurophilic granules), p (intact specific granules), y (mem- brane vesicles), and 6 (cytosol). The y fraction was highly enriched in alkaline phosphatase, a marker of the plasma membrane. In addition, this fraction con- tained <5% of the amounts of lysosomes (indicated by lysozyme activity) and nuclei (indicated by DNA content) found in intact cells or in unfractionated cavitate. Furthermore, the y fraction contained < I 0% of the levels of endoplas- mic reticulum, Colgi, mitochondrial, and lysosomal membranes in cells or cav- itates, as determined by assays for glucose 6-phosphatase, galactosyl transferase, monoamine oxidase, and M o l (CD11 b/CD18; Mac-I), respectively. Finally, 75% of the membrane vesicles were sealed, as indicated by assay of ouabain-sensitive (Na',K+) ATPase activity, and 55% were oriented right-side-out, as determined by exposure of concanavalin A (ConA) receptors and sialic acid residues on the surfaces of the vesicles. These heterogeneous preparations could be enriched for right-side-out vesicles by their selective adherence to ConA-coated plates and subsequent detachment by rinsing the surfaces of the plates with a-mcthylman- noside. This enrichment protocol did not affect the integrity of the vesicles and resulted in populations in which >85% of the vesicles were oriented right-side- out. This procedure thus permits the preparation of sealed, right-side-out mem- brane vesicles that may be used as valid experimental models of the neutrophil plasma membrane in a variety of functional studies.

The binding of agonists to specific receptors on the surfaces of human polymorphonuclear leukocytes (PMN) elicits a variety of cellular responses, including turnover of membrane lipids, alterations in transmem- brane potential, cation fluxes, initiation of the oxida- tive burst, cytoskeletal restructuring, chemotaxis, phagocytosis, and degranulation (reviewed by Gold- stein, 1979; Tauber and Simons, 1983; Lazzari et al., 1986; Dillon et al., 1987; Omann et al., 1987a,b; Kor- chak et al., 1988a,b; Luscinskas et al., 1988). Although the mechanisms by which these responses are elicited remain undefined, the plasma membrane and its in- trinsic components are undoubtedly involved. Such a role for the plasma membrane in stimulus-response

coupling could be more precisely investigated using membrane vesicles as models of the PMN surface.

Membrane vesicles have been prepared from PMN by several methods, including techniques that employ mechanical shear forces to disrupt cells, e.g., homoge- nization and sonication (Volpi et al., 1982; Painter et

Received April 21, 1989; accepted August 7, 1989. *To whom reprint requestsicorrespondence should be addressed. Francis W. Luscinskas is now at the Department of Pathology, Vascular Research Division, Harvard Medical School and Brigham and Women's Hospital, Boston, M A 02115.

Q 1989 ALAN R. LISS, INC.

CHARACTERIZATION OF NEUTROPHIL MEMBRANE VESICLES 637

al., 1987; Rotrosen et al., 1988). These methods, how- ever, generally effect an incomplete rupture of cells and cause a significant disruption of subcellular or- ganelles (Wallach and Lin, 1973; Wallach and Schmidt-Ullrich, 1973; Klempner e t al., 19801, causing contamination of the plasma membrane fraction with organellar membranes and exposure of membrane components to intracellular hydrolases. These findings imply that membrane vesicles prepared by these protocols are less than ideal models of the plasma membranes of unstimulated PMN.

As an alternative to these physically disruptive tech- niques, Roos et al. (1983) prepared cytoplasts from enu- cleated PMN. These cytoplasts contained lower quan- tities of granular enzymes than did intact PMN, and responded to stimuli with a respiratory burst, Ca + +

flux, cytoskeletal activation, phagocytic index, and ad- hesion to endothelium, all analogous to the responses of intact PMN (Roos et al., 1983; Lutter et al., 1984; Omann et al., 1987b; Vedder and Harlan, 1988). Dur- ing preparation, however, a large proportion of the sur- face membrane was shed with the extruded nucleus. Cytoplast membranes have also been shown to contain relatively high amounts of cytochrome b and Mol (Petrequin et al., 1986), which are markers of the mem- branes of intracellular granules (Borregaard et al., 1983; Todd et al., 1984; Petrequin et al., 1986; Vedder and Harlan, 1988). These results suggest that, as was the case for the physical methods described above, dis- ruption of subcellular organelles occurred during cyto- plast preparation, thus limiting their usefulness as models of the PMN surface.

To circumvent the technical limitations of these methods, an alternative technique for the preparation of membrane vesicles was developed. This method, modified from that previously developed in this labo- ratory (Borregaard et al., 1983), provides quantitative disruption of PMN at a controlled temperature in an inert atmosphere by nitrogen cavitation, and subse- quent separation of plasma membrane vesicles from intact intracellular organelles by differential centrifu- gation on isotonic, isoosmotic Percoll gradients.

The present studies assess the purity, integrity, and orientation of PMN plasma membrane vesicles. These vesicles are enriched in markers of the cell surface and contain low amounts of organellar contents and mem- branes. Furthermore, most of the membrane vesicles are sealed, and the preparations can be enriched for vesicles having the same (right-side-out) orientation as the plasma membranes of intact PMN. These mem- brane vesicles therefore are ideal models for the study of the structure and function of the cell surface of hu- man PMN.

MATERIALS AND METHODS Reagents and media

All reagents were purchased from Sigma (St. Louis, MO), except Percoll, which was from Pharmacia (Pis- cataway, NJ), nitrocellulose from Bio-Rad (Rockville Centre, NY), 3,5-diaminobenzoate dihydrochloride from Aldrich (Milwaukee, WI), and 14C-UDP-galactose (specific activity 272.8 mciimmol) from New England Nuclear (Boston, MA). Anti-human Mol monoclonal antibody (MAb 904) was the generous gift of Dr. James D. Griffin (Dana-Farber Cancer Institute, Boston, MA).

Relaxation buffer contained 125 mM KC1, 5 mM NaC1,l mM ATP, 3.5 mM MgCl,, 10 mM PIPES, 5 mM EGTA, 1 mM PMSF, 10 pM leupeptin, and 5 pM pep- statin A, pH 7.4. Phosphate-buffered saline (PBS) con- sisted of 140 mM NaC1, 8 mM Na,HPO,, and 2 mM NaH,PO,, pH 7.4. Tris-buffered saline (TBS) was 150 mM NaCl and 100 mM Tris, pH 7.4.

Preparation of plasma membrane vesicles PMN were isolated as described previously (Luscin-

skas et al., 1988). Plasma membrane vesicles were pre- pared from PMN by nitrogen cavitation and density gradient centrifugation on discontinuous Percoll gra- dients by the method of Borregaard et al. (1983), with the following modifications. PMN were cavitated at 425 psi for 30 min at 4°C. To remove nondisrupted cells, nuclei, and debris, the cavitate was centrifuged at 500g for 10 min a t P C , and pellets saved at 4°C for enzyme assays. Subcellular fractions were prepared on discon- tinuous Percoll gradients centrifuged at 50,OOOg for 30 min at 4"C, and purified fractions were resuspended in -2 ml of relaxation buffer or PBS and stored at 4°C until use.

Assays for vesicle and granule constituents Total protein concentration was determined using

the 37"C/30 min BCA method (Pierce Chemical Co., Rockford, IL), with BSA in relaxation buffer as the standard. Alkaline phosphatase activity was assayed according to DeChatelet and Cooper (1970), except that the assay buffer was 1 mM MgCl,, 50 mM sodium bar- bital, pH 10.5, and the reaction was allowed to proceed for 45 min and was terminated with 1.0 ml of 0.9% NaC1. Activities of lysozyme, DNA, glucose 6-phos- phatase, and galactosyl transferase were determined as described by Metcalf et al. (1986), Puzas and Good- man (1978), Swanson (1955), and Fleischer (19711, re- spectively. Monoamine oxidase activity was deter- mined as described by Lovenberg et al. (19621, except that samples contained 1 mg of purified aldehyde de- hydrogenase, and enzyme activities were determined by linear regression from a standard fluorescence curve of indoleacetic acid.

The content of granular membranes in subcellular fractions was determined by a dot blot assay for Mol (CDllblCD18; Mac-1). Samples of the subcellular frac- tions, each containing 500 p,g of protein, were bound to nitrocellulose at room temperature for 60 min. Nitro- cellulose was blocked for 6-24 hr a t 4°C in 3% (w/v) BSA in TBS (TBS/BSA) and washed three times with TBS for 30 min each. Blots were then incubated with mouse anti-human Mol (1:250 in TBS/BSA) at room temperature for 60 min with constant rocking, washed three times with TBSiBSA for a total of 30 min, incu- bated in peroxidase-conjugated anti-mouse IgG (1500) for 60 min at room temperature, and washed as above with TBSIBSA. For visualization of Mol antigen, blots were incubated for 5 min at room temperature in 20 ml of TBS containing 600 pgiml of 4-chloro-l-napthol and 0.01% (vh, final concentration) H,02. Blots were then removed into distilled water for 5 min to terminate the reaction and dried overnight in the dark. Mol antigen was quantitated by densitometric scans of the spots on the nitrocellulose, and the quantity in each fraction was expressed as a relative percentage of the amount

638 DEL BUONO ET AL.

contained in whole cavitates. Linearity of densitometer response over the range of concentrations was ensured by scanning a blot of serial dilutions of mouse IgG probed as for Mol blots (not shown).

Characterization of vesicle orientation The orientation (right-side-out or inside-out) of mem-

brane vesicles was determined by two independent techniques: 1) assessment of the percentage of total vesicle sialic acid that was externally cleavable by neuraminidase and 2) measurement of the percentage of total vesicles that adhered to concanavalin A (ConA) affinity columns. In the former technique, neuramini- dase was used to cleave externally exposed sialic acid residues on membrane vesicles by a method modified from that of Steck and Kant (1974). Aliquots of vesicles (250-500 pg of protein) were suspended in 100 mM Tris-acetateil50 mM NaC1, pH 5.2 (TABS), containing 100 pg of neuraminidase either with (sample A) or without (sample B) saponin (100 pgiml final concentra- tion). A separate aliquot (sample C) was suspended in TABS, and H,SO, added to a final concentration of 200 mM. Samples A and B were incubated at 37°C for 40 min, and sample C at 88°C for 60 min. After incuba- tion, samples were vortexed and centrifuged at 13,800g for 15 min a t 4°C on a Fisher 235A microcentrifuge, and supernatants were stored a t -20°C until being as- sayed for sialic acid by the method of Warren (1959).

ConA affinity chromatography was performed at 4°C as described by Ochs and Reed (19831, with minor mod- ifications. ConA-Sepharose 4B was added to polypropy- lene or siliconized glass columns to give a packed bed volume of 3 ml, and columns were exhaustively washed with degassed relaxation buffer. To reduce nonspecific binding of vesicles, the columns were treated with 8 ml of 0.1% (wiv) BSA in relaxation buffer for 15 min and extensively washed with relaxation buffer until the el- uate contained no BSA (OD,,,, <.002). Samples of membrane vesicles that were untreated, or permeabi- lized by treatment with 100 pg/ml saponin or by pas- sage through four freeze-thaw cycles, were added to the columns in a volume of 1-3 ml and incubated for 10 min. Nonadherent vesicles were eluted with ice-cold relaxation buffer; the first 6 ml of the eluate contained >95% of total eluted protein. The percentage of adher- ent vesicles in each sample was calculated after deter- mination of the amount of protein eluted vs. protein added to each column, using the 37”Ci30 min BCA as- say. Protein in samples containing saponin was first extracted with 0.015% sodium deoxycholate/7.2% tri- chloroacetic acid and then assayed for protein by the method of Peterson (1977).

Characterization of vesicle integrity Vesicle integrity (degree of sealing) was determined

by assay of ouabain-sensitive (Na+ ,KC) ATPase activ- ity in vesicles in the presence or absence of 1 mM oua- bain and/or 100 pg/ml of saponin (Seiler and Fleischer, 1982). These assays were based on the assumption that in sealed membrane vesicles, ouabain-sensitive (Na + , K f ) ATPase activity cannot be determined, since the binding sites for ouabain and ATP on the enzyme are located on opposite sides of the plasma membrane; in nonsealed vesicles, however, ATP and ouabain can freely diffuse across the membrane (Caldwell and

Keynes, 1959; Perrone and Blostein, 1973). Release of inorganic phosphate from untreated or saponin-perme- abilized vesicles was determined according to Otto- lenghi (19751, and the percentage of sealed vesicles in a given preparation calculated as the percentage of to- tal ATPase activity (from permeabilized vesicles) that was present in untreated vesicles (Ochs and Reed, 1983).

Enrichment of vesicle preparations Preparations of membrane vesicles were enriched for

those oriented right-side-out using a modified “affinity panning” technique based on those described for lym- phocyte separation (Mage et al., 1977; Wysocki and Sato, 1978). Briefly, 5 ml of a ConA solution (5 mg ConAiml of TBS, pH 8.5) were added to 60 mm poly- styrene petri plates (Falcon; Boston, MA), and the plates were incubated for 18-24 h r at 4°C to allow bonding of lectin to plate surfaces. After incubation, the ConA solution was removed and plates rinsed three times with PBS or relaxation buffer; samples of mem- brane vesicles (or intact PMN for controls) were then added to plates in a volume of 3-5 ml. Plates were incubated at 4°C for 30 min, gently agitated, and re- placed for an additional 30 min. Nonadherent (inside- out) vesicles were aspirated with the supernatant; plate surfaces were gently rinsed once with 3 ml of PBS or relaxation buffer, and eluate saved with the nonad- herent vesicles. Adherent (right-side-out) vesicles were detached from plate surfaces by adding 3 ml of 100 mM a-methyl-D-mannoside in PBS or relaxation buffer, pH 7.4 (AMDM), incubating for 15 min a t 4”C, collecting the vesicles with the media, and gently rinsing plates twice with 1 ml each of AMDM (saving the washes). The percentage of vesicles with each orientation was determined by protein assay (or by hemacytometer counting for cells) of the two subpopulations using AMDM as the blank, and vesicles in both subpopula- tions were characterized with respect to orientation and integrity as described above.

RESULTS Disruption and fractionation of polymorphonuclear leukocytes

Plasma membrane vesicles were obtained from hu- man PMN by a modification of the method of Borre- gaard et al. (1983). Disruption of PMN was effected by N, cavitation, and nuclei and undisrupted cells were removed from the cavitates by low-speed centrifuga- tion prior to fractionation on Percoll gradients. The resultant pellet contained <15% of the total cavitate protein (Table l ) , indicating that >85% was loaded onto the Percoll gradients.

Density gradient centrifugation of these postnuclear cavitates resolved four fractions, denoted as (in order of decreasing density) a (azurophilic granules), p (specific granules), y (membrane vesicles), and 6 (cytosol), in confirmation of results previously obtained in this lab- oratory (Borregaard et al., 1983). As shown in Table 1, the membrane vesicle fraction (7) contained the least protein (5-lo%), and nearly 90% of the total cavitate protein was recovered from the gradients.

As an initial step in the characterization of the mem- brane vesicles, the subcellular fractions were assayed for alkaline phosphatase activity, a marker of the

CHARACTERIZATION OF NEUTROPHIL MEMBRANE VESICLES 639

TABLE 1. Content of protein and organellar markers in subcellular fractions from N,-cavitated human PMN

Fraction total protein' phosphatase' Lysozyme3 DNA4 phosphatase5 oxidase6 Percentage of Alkaline Glucose 6- Monoaniine Galactosyl

transferase7

Whole cavitate 100.0 1.00 1.00 1.00 1.00 1.00 1.00 Low-speed pellet 11.3 0.07 0.52 2.73 0.31 1.61 1.18 a 15.1 0.07 1.11 0.57 0.04 0.84 0.37

6.4 8.93 0.02 0.04 0.07 0.08 0.00 B 11.6 0.67 4.53 0.17 0.11 0.17 0.64

S 45.2 0.13 0.00 0.06 1.70 0.05 1.36 Y

'Values represent means of six separate experiments, normalized to amount of protein (determined by BCA assay) in whole cavitates prior to fractionation on Percoll gradients.

3Relative specific activities (pgimg of protein), obtained by linear regression from a standard curve (n = 5 ) . *Relative specific activities (nmolimg of protein), obtained by linear regression from a standard curve (n = 4). 'Relative specific activities (pmol P, released from glucose 6-phosphatelminimg of protein) (n = 4). 'Relative specific activities (pmol of indoleacetic acid formedimidmg of protein) in = 41. 7Relative specific activities (nmol of galactose transferred/hr/mg of protein) (n = 3).

Relative specific activities (unitdmg of protein); one unit of alkaline phosphatase liberates 1 pmol of nitrophenol/min (n = 51

plasma membrane (DeChatelet and Cooper, 1970). As shown in Table 1, the y fraction contained much greater levels of alkaline phosphatase than the whole cavitate and other subcellular fractions, while the low- speed nuclei/debris pellet contained <lo% of this amount. These results indicate that most of the plasma membrane material was located in the vesicles in the y fraction and very little was discarded with the nuclear pellet.

Content of organelles in membrane vesicles To determine the content of intracellular organelles

within the vesicles, subcellular fractions were exam- ined for lysozyme activity, a marker of azurophilic and specific granules (Metcalf et al., 1986). As shown in Table 1, the a and p layers contained the highest amounts of lysozyme, while the y layer contained <5% of the levels of lysozyme found in whole cavitates. The 6 layer also contained very little lysozyme activity, in- dicating that disruption and/or fusion of granules with the plasma membrane did not occur during cavitation or centrifugation. Together, these results indicate that lysosomal granules remained intact and were effi- ciently separated from the membrane vesicles and that few granules remained enclosed within the vesicles fol- lowing their formation.

As a second method to determine the organellar con- tent of membrane vesicles, the presence of nuclei in the fractions was determined by fluorimetric quantitation of DNA (Puzas and Goodman, 1978). As shown in Table 1, the low-speed centrifugation prior to Percoll frac- tionation of cavitates removed most of the nuclear ma- terial. Importantly, the y and 6 fractions contained only -5% of the levels of DNA in whole cavitates and <2% of that contained in the nuclear pellets. Approx- imately 57% of the amount of DNA contained in cavi- tates was found in the a fraction, suggesting that nu- clei remaining in the cavitate cosedimented with the most dense azurophilic granules. Together with those above, these results indicate that the membrane vesi- cles contained very low levels of the intracellular or- ganelles found in intact PMN.

Content of organellar membranes in membrane vesicles

To determine whether membrane vesicles were con- taminated with intracellular membranes, the subcel- lular fractions were assayed for the presence of mark-

ers of endoplasmic reticulum (ER) and mitochondrial and granular membranes. The presence of ER was de- termined by assaying for glucose 6-phosphatase activ- ity (Swanson, 1955). The y fraction was found to con- tain 7%, and the granular fractions 4-11%, of the levels of glucose 6-phosphatase found in whole cavi- tates (Table l); most of the enzyme activity was con- tained in the nuclear pellet and the 6 fraction, probably arising from uncavitated cells in the former and from microsomes in the latter. These results indicate that the membrane comprising the vesicles was not largely derived from ER membrane and, further, that only small amounts of ER were enclosed within the mem- brane vesicles.

Mitochondria1 membranes were determined by as- saying for monoamine oxidase (Lovenberg et al., 1962). As was the case for ER, a very low level of monoamine oxidase was contained in the y fraction (Table 1); most of the activity was recovered in the nuclear pellet and the a fraction, with lesser amounts in the p and 6 fractions. These results indicate that the membrane vesicles contained only low levels of mitochondrial membranes and intact mitochondria.

To determine the levels of Golgi membrane, the frac- tions were tested for galactosyl transferase activity (Fleischer, 1971). As shown in Table 1, the y fraction was devoid of galactosyl transferase, most of the activ- ity being recovered in the nuclear pellet and the 6 frac- tion, and somewhat less in the granular fractions. These results indicate that the vesicles contained very little Golgi membrane.

Finally, to measure the levels of granular mem- branes in the membrane vesicles, monoclonal antibod- ies were used to assay the fractions for Mol (CDllb/ CD18; Mac-1). Mol is an antigen expressed on specific (Todd et al., 1984) and/or tertiary (Lacal et al., 1988) granule membranes, but in only small amounts on the surfaces of unstimulated PMN until its translocation to the cell surface upon stimulation and degranulation (Todd et al., 1984; Petrequin et al., 1986). In two sep- arate preparations from unstimulated cells (Fig. la), the y fraction contained levels of Mol that were only slightly above background. (The activity seen in back- ground (buffer) lanes probably resulted from nonspe- cific reactivity of the primary and/or secondary anti- bodies with the peptide protease inhibitors contained in the relaxation buffer.) When quantitated by densi- tometry, the y fraction was found to have <5% of

640 DEL BUONO ET AL.

m 3

0 0 Lo

C 3

6

Cavilate

Pellet

a

P

Y

C a v i t ate

a

m I

0 0 v)

d

b

2.00

1 .m

0.00 Cavltate Pellet a p y 6

Fraction

Fig. 1. Content of Mol (CDllb/CD18; Mac-l), a marker of granular membranes, in subcellular fractions from human neutrophils. a: Dot blot of Mol content in subcellular fractions. Cells were unstimulated, or stimulated with A23187 plus cytochalasin B, prior to nitrogen cav- itation. Purified subcellular fractions (or relaxation buffer) were spot- ted onto nitrocellulose, incubated with anti-human Mol monoclonal antibodies, and probed with peroxidase-conjugated anti-mouse IgG

and color substrate. Since cavitates often contained large particulates that increased the uncertainty of the amount of protein blotted, two lanes of cavitate were applied to blots and amounts of Mol in cavi- tates calculated as the average of these lanes. b Quantities of Mol in subcellular fractions, relative to cavitates from unstimulated cells, as determined by densitometric analysis of dot blot lanes containing 500 yg of protein. Data are representative of five separate experiments.

the amount of Mol contained in whole cavitates from unstimulated cells (Fig. lb). The 01 and p layers in unstimulated cells contained substantially higher amounts of Mol than did the y fraction, while very low amounts of antigen were found in the 6 layer. Low-speed pellets from unstimulated cells contained nearly the same amounts of Mol as whole cavitates, suggesting that these pellets contained substantial quantities of intracellular granules, in concurrence with the relatively high lysozyme levels observed in this fraction (Table 1). As a positive control, mem- brane vesicles were prepared from PMN stimulated with cytochalasin B and the C a t + ionophore A23187, a treatment that increases the cell surface expression of Mol (Petrequin et al., 1986). As expected, mem- brane vesicles prepared from these stimulated cells contained over ten times the levels of Mol found in those prepared from unstimulated cells (Fig. 1). The a and 6 fractions from these cells contained approxi- mately the same amounts of Mol as their counterparts from unstimulated cells. Interestingly, the p fractions from stimulated cells contained about twice as much Mol as those from unstimulated cells; this result may indicate that stimulation of PMN induced a preferen- tial colocalization of unfused tertiary granules con- taining Mol (Lacal et al., 1988) with the specific granules in the p fraction.

In conjunction with those for alkaline phosphatase activity and organellar content, these results provide

strong evidence that these membrane vesicles origi- nate from the plasma membrane of cavitated PMN and contain only low levels of intracellular organelles and organellar membranes.

Determination of vesicle orientation The membrane vesicles were also characterized with

respect to orientation, i.e., whether they were right- side-out or inside-out. Based on the assumptions that sialic acid residues and ConA receptors are found ex- clusively on the extracellular surface of the plasma membrane (Ochs and Reed, 1983), the fraction of right- side-out vesicles was determined by assessing the per- centage of total vesicle sialic acid that was externally cleavable by neuraminidase and by measuring the percentage of vesicles that adhered to ConA aEnity columns.

As shown in Table 2, -55% of the total acid-hydro- lyzable sialic acid residues were found to be accessible to neuraminidase. If vesicles were incubated with neuraminidase in the presence of detergent, 95% of the total sialic acid residues were cleaved. Thus, 50-55% of the total sialic acid residues in membrane vesicles were externally cleavable by neuraminidase. Similarly, 50- 55% of the applied vesicle protein was retained on ConA affinity columns (Table 2). Disruption of the ves- icles by treatment with saponin or by passage through four rapid freeze-thaw cycles before addition to col- umns resulted in retention of 91% and 86% of the ves-

CHARACTERIZATION OF NEUTROPHIL MEMBRANE VESICLES 641

TABLE 2. Determination of orientation of plasma membrane vesicles by surface expression of ConA receptors and sialic acid residues

Vesicle protein retained by Total sialic

Treatment of ConA Sialic acid acid external% vesicles columns (%a)’ released (nmol)’ accessible (30)

None 53.6 2 3.0 1.8 f 0.2 54.5 Saponin3 91.3 -t_ 2.1 3.1 t 0.4 93.9 Acid4 ND6 3.3 -+ 0.4 100.0 Freeze-thaw5 86.9 ? 6.7 ND ND

‘Values represent means I standard deviation of five separate experiments. ?otal sialic acid determined in each experiment from acid-hydrolyzed samples. 3Samplee treated with 50 Fg/ml of saponin prior to addition to ConA columns and extracted with deoxycholate prior to protoin ass;iy. ‘Samples treated with H,SO, at a final concentration of 200 mM. ’Samples passed through four rapid freeze-thaw cycles prior to addition to Cow4 columns. 6ND. not determined.

icles, respectively. Together, these results indicate that 5 0 4 5 % of the isolated plasma membrane vesicles are oriented right-side-out.

Enrichment for right-side-out vesicles In order to be useful as models of the plasma mem-

branes of PMN, vesicle preparations must be enriched in right-side-out vesicles, above the 55% observed above. Such an enrichment was accomplished using an “affinity panning” technique modified from that used for the separation of lymphocytes (Wysocki and Sato, 1978) and thymocyte membrane vesicles (Resch et al., 198l), using ConA-coated plates.

When samples of vesicles were applied to these plates within 6 hr after preparation, and adherent ves- icles separated from nonadherent, approximately the same percentages of vesicles were found in both sub- populations (Table 3); this result concurred with those obtained with ConA columns. As a control, intact cells were incubated on ConA plates; nearly 90% of these PMN were adherent to the plates. Storage of cells and vesicles overnight at 4°C prior to plating did not sig- nificantly affect their adherence, indicating that both cells and vesicles retained their orientation with stor- age.

The purified adherent and nonadherent subpopula- tions of vesicles were then assayed with respect to ori- entation. As shown in Table 4, nearly 90% of the vesicles in the plate-adherent subpopulations were re- tained by ConA columns and displayed externally ac- cessible sialic acid residues, while only 5-10% of the vesicles in the nonadherent subpopulations displayed these characteristics. These results indicate that prep- arations of membrane vesicles from PMN can be en- riched in right-side-out vesicles by ConA panning, thus providing vesicles that resemble intact PMN with re- spect to orientation.

Determination of vesicle integrity To assess the integrity (i.e., degree of sealing) of

membrane vesicles, ouabain-sensitive (Na+ ,K +) ATPase activity was determined by a procedure modi- fied from that of Ochs and Reed (1983). By the ratio- nale outlined above in Materials and Methods, the per- centage of unsealed vesicles in the preparation was calculated by determining the fraction of the total oua-

TABLE 3. Enrichment of vesicle preparations for right-side-out vesicles by ConA panning.’

Total protein in each subpopulation (30)’

Sample, hr Adherent Nonadherent after DreDaration Vesicles, 6 52.8 iz 10.2 52.1 i 11.8 Vesicles, 20 40.3 ? 12.3 62.9 2 14.8 Cells, 6 88.9 t 4.7 5.1 ? 3.2 Cells. 20 91.8 2 6.9 4.4 k 4.0

‘Samples of membrane vesicles (500 Fg of vesicle proteidsample) or intact PMN (10’ cellsisample) were applied to ConA-coated petri plates either 6 or 20 h r after preparation, Plates were incubated for 1 hr a t 4°C and rinsed three times with PBS or relaxation buffer to remove nonadherent vesicles and cells, pooling the rinses. Adherent vesicles were removed from plates by rinsing with u-methyl- D-mannoside as described in Materials and Methods. The percentage of applied vesicle protein that was in adherent and nonadherent suhpopulations was de- termined by BCA protein assay; percentage of cells in each supopulation was determined by mimoscopic counting. ‘Values represent means 2 standard deviation of five separate experiments.

bain-sensitive (Na + ,K+ ) ATPase activity (obtained from saponin-treated vesicles) that was present in ves- icles not treated with detergent.

As shown in Table 5, 80% of the vesicles in unen- riched samples examined within 6 hr of preparation were sealed, compared to 83% in preparations enriched for right-side-out vesicles. However, if vesicles from unenriched or enriched preparations were stored over- night at 4°C and then examined, less than 50% of the vesicles in each sample remained sealed. Vesicle integ- rity was not significantly improved if 5 FM ATP or 10 mM glucose were included as sources of energy in the storage buffer (data not shown). Together with those above, these data indicate that plasma membrane ves- icles may be prepared such that most are sealed and oriented right-side-out, thereby resembling the plasma membranes of intact PMN, but that vesicle integrity (but not orientation) decays rapidly upon storage.

DISCUSSION The current studies describe a novel method for the

preparation of sealed, right-side-out vesicles from the plasma membranes of unstimulated PMN, based on that developed previously in this laboratory (Borre- gaard et al., 1983). Since these vesicles contain fewer intracellular organelles and membranes than those prepared by sonication (Gay and Stitt, 1988a,b; Wright et al., 1988), homogenization (Volpi et al., 1982; Painter et al., 19871, or enucleation (Roos et al., 1983; Korchak et al., 1983; Lutter et al., 19841, they may be an ideal model system for the study of the role of the cell surface in stimulus-response coupling in human PMN.

Others have used similar techniques to isolate PMN membranes either with (Klempner et al., 1980; Record et al., 1985; Krause and Lew, 1987; Clark et al., 1987) or without (Painter et al., 1987; Babior et al., 1988; Bokoch and Parkos, 1988; Matsumoto et al., 1988) den- sity gradient centrifugation. The cavitation buffers used in most of these previous studies, however, simu- lated extracellular rather than cytoplasmic ionic and osmotic conditions, resulting in osmotic disruption of intracellular granules (Borregaard et al., 1983) and thereby exposing the membrane vesicles to lysosomal enzymes and to contamination by nonsurface mem- branes. In addition, neither the orientation nor the in-

642 DEL BUONO ET AL.

TABLE 4. Determination of orientation of plasma membrane vesicles in enriched populations by surface expression of ConA receptors and sialic acid residues’

Vesicle protein retained Sialic Total sialic acid by ConA columns (%I2 acid released (nmol)2 externally accessible (’%)

Treatment of v e 8 i c 1 e s A N A N A N None 88.9 i: 5.7 6.4 i 2.2 3.3 t 0.5 0.3 f 0.1 86.8 7.3 Saponin 93.3 k 6.0 7.0 ? 3.1 3.6 2 0.4 3.7 2 0.6 94.7 90.2 Acid ND3 ND 3.8 2 1.0 4.1 2 1.1 100.0 100.0 Freeze-thaw 90.2 i: 4.8 6.9 i 2.7 ND ND ND ND

‘Populations enriched for adherent (A) right-side-out and nonadherent (N) inside-out vesicles by C o d panning as described in Materials and Methods ‘Values represent means ? standard deviation of five separate experiments. 3ND. not determined.

TABLE 5. Determination of integrity of plasma membrane vesicles by activity of ouabain- resistant (Na+,K+) ATPase

ATPase activitv’

Sample, hr Saponin’: after ureuaration2 Ouabain4:

A - B - +

C + -

n + + Percents e sealed c Unenriched, 6 2.31 i: 0.27 2.04 i: 0.13 2.76 t 0.21 1.42 2 0.13 79.9 Enriched, 6 2.18 2 0.16 1.95 f 0.11 2.58 * 0.20 1.20 i 0.13 83.3 Unenriched, 20 2.76 2 0.25 1.44 -t 0.18 3.53 50.29 1.39 k 0.17 38.3 Enriched, 20 2.62 -+ 0.29 1.35 t 0.19 3.40 i- 0.33 1.21 2 0.12 42.0

‘Specific activity (pmol P, released from ATF’/mg of protein) of (Na ‘ ,K+) ATPase. Values represent means ? standard deviation of five separate experiments. ‘Samples were unenriched or enriched for right-side-out vesicles by “ConA panning” and assayed 6 or 20 hr after cavitation. 3Samples untreated (-1 or treated ( + ) with 50 pg/ml of saponin during reaction. 4Samples untreated (-1 or treated ( + 1 with 100 p M ouabain during reaction. 5Percentage of vesicles that were sealed was calculated from the mean values for each sample using the formula % sealed = 100- descrihed in text.

1100 x ((A - BMC - D))1, as

tegrity of the membrane vesicles was characterized in these previous studies.

The vesicles prepared in the present studies, however, have been so characterized. While they are derived nearly exclusively from the cell surface and contain only insignificant amounts of intracellular membranes and organelles, only 50-60% of the mem- brane vesicles are oriented right-side-out. Vesicle prep- arations of such heterogeneous orientation are obvi- ously of limited usefulness as models of the plasma membranes of intact PMN, which are, by definition, oriented right-side-out. As shown here, however, these preparations may be enriched for right-side-out vesi- cles by selective adherence to ConA-coated plates, thus providing a source of membrane vesicles with the same orientation and structural features as the plasma membranes of intact PMN.

The present results indicated that Mol is found in very low amounts on the surfaces of unstimulated PMN, but is increased approximately tenfold upon stimula- tion, in agreement with other reports (Todd et al., 1984; Petrequin et al., 1986). However, the finding that Mol is expressed in relatively large amounts in layers con- taining azurophilic granules is of some concern, since others have recently shown that intracellular pools of Mol are mainly contained on specific and tertiary granules (Jones et al., 1988; Lacal et al., 1988). Our findings do not necessarily conflict with those of the earlier studies, but may instead suggest that our alter- ation of cavitation and centrifugation conditions to in- crease the yield and purity of membrane vesicles may have decreased the resolution of the azurophilic, spe- cific, and tertiary granules into distinct layers. Be- cause the goal of the present studies-the preparation of vesicles devoid of intracellular organelles and mem-

branes-was achieved, the apparent nonresolution of the different granule layers is of less consequence.

Another concern that often arises when preparing membrane vesicles is their degree of leakiness (Steck et al., 1970; Wallach and Schmidt-Ullrich, 1973; Ochs and Reed, 1983). Ideally, to be useful as models of the surface of intact PMN, the vesicles should be predom- inantly sealed. In the present studies, 8 0 4 5 % of the membrane vesicles were found to be sealed when ex- amined within 6 hr of preparation. Overnight storage at 4“C, however, even in buffers containing ATP or glucose as energy sources, caused most of the vesicles to become leaky. These findings indicate that vesicle structural integrity decays rapidly over time, perhaps precluding long-term storage under conventional con- ditions, and may necessitate the performance of func- tional studies within 6-12 hr of vesicle preparation to ensure that they remain structurally similar to native PMN membranes. In preliminary studies, however, we have found that the membrane-impermeant form of the fluorescent Ca+ * indicator indo-1 is retained within vesicles for up to 72 hr after preparation, indicating that the vesicles may be sealed t o molecules at least as large as indo-1 (M, = 635 daltons). Other storage methods are currently being examined in the attempt to overcome these time limitations.

In summary, the current studies have provided a method for the preparation of plasma membrane vesi- cles from human PMN that overcomes the technical limitations of other protocols. Since they are predomi- nantly right-side-out and sealed, the vesicles resemble the plasma membranes of intact PMN. In preliminary experiments, the vesicles have been observed to main- tain a transmembrane potential, to bind fluorescent ligands (immune complexes and chemotactic peptides),

CHARACTERIZATION OF NEUTROPHIL MEMBRANE VESICLES 643

and to exhibit C a t + fluxes and turnover of membrane lipids, but no change in membrane potential, in re- sponse to these stimuli. These studies are currently being extended to more fully examine the efficacy of the vesicles as models for use in studies of the role of the plasma membrane in stimulus-response coupling in human PMN.

ACKNOWLEDGMENTS We thank Dr. James D. Griffin for his generous gift

of anti-human Mol antibody and Dr. Burton F. Dickey for his help in performing galactosyl transferase as- says. This research was supported by NIH grants HL07501, HL19717, and AM31056.

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