Isolation and Surface Labeling of Murine PoIy mor pho nuc Iea r Neut rophi Is SUZANNE M. WATT, ANTONY W. BURGESS AND DONALD METCALF Cancer Research Unit, The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, P. 0.3050, Victoria, Australia

ABSTRACT Methods for the induction of an exudate of polymorphonuclear neutrophilic leukocytes (PMN) in the peritoneal cavity of C57BL, BALB/c, SJL and CBA mice were analysed. Peritoneal exudates in male mice were highly enriched for PMN (80-90%)three hours after a single injection of calcium caseinate whereas eosinophils comprised less than 1%of the exudate population. Female mice were a less satisfactory source of PMN because the proportion of eosinophils in the exudate was variable. Purification of PMN from peritoneal exudate cells was performed on the basis of light scattering using a BectonDickinson cell sorter or by density gradient centrifugation with graded polyvinylpyrrolidone-coated silica particles (Percoll). Both techniques yielded approximately 97% pure PMN preparations. Electrophoretic analysis of the PMN proteins revealed an abundance of lactoferrin and actin, but several other proteins were also present in high concentrations. Proteolytic degradation of several high molecular weight proteins (>90,000) was prevented by the addition of phenylmethylsulphonyl fluoride (PMSF) and ethylene diamine tetracetic acid (EDTA). Surface iodination, using diphenyl, tetrachloroglycouril (IODO-DEN), indicated that there were six tyrosine-containing proteins present on the external cell membrane. The apparent molecular weights of these surface proteins ranged from 185,000 to 90,000 and the major lZ5I-labeledprotein had an apparent molecular weight of 90,000. Neither actin nor lactoferrin was labeled with Iz5I unless cell viability was lost during the iodination procedure. Standard conditions for labeling the cell surface only, required low iodide and IODO-GEN concentrations. Biosynthetic labeling of PMN using 35Smethionine increased the sensitivity of detection for most of the proteins, but some of the granule storage proteins (such as lactoferrin) were not effectively labeled within three hours. The cellular aspects of the differentiation of polymorphonuclear leukocytes (PMN) from their hemopoietic progenitor cells (Bradley and Metcalf, '66; Pluznik and Sachs, '66) have been studied in considerable detail (Metcalf, '77). However, there is limited information only on the molecular changes which occur during the formation of PMN. PMN are known to be prominent in the destruction of micro-organisms (Murphy, '76) and also appear to produce regulatory macromolecules which influence the development of other hemopoietic cells (Broxmeyer e t al., '77; Czarnetzki e t al., '78). A comparison of both the internal and surface proteins of PMN with other hemopoietic cells and cells within the granJ. CELL. PHYSIOL.(1979) 100: 1-22.

ulocytic series is needed to understand the changes during the expression of the differentiation program. PMN (purity >95%) purified from human peripheral blood (Beyum, '68; Mahmoud, '76; Segal and Peters, '77; Shapiro, '77) and guinea pig peritoneal exudate PMN (purity 60-97%) (Nelson and Boyden, '63; Oren et al., '63; Simpson and Ross, '71; Stossel and Pollard, '73; DePierre and Karnovsky, '74) have been studied earlier. However, PMN from mice Received Sept. 20, '78. Accepted Feb. 21, '79. ' This work was supported by the Carden Fellowship Fund of the Anti-Cancer Council of Victoria. the National Health and Medical Research Council of Australia and Grant No. I ROI CA 22556-01 from the National Institute of Health. *To whom correspondence should he sent.

1

2

SUZANNE M. WATT, ANTONY W. BURGESS AND DONALD METCALF

have not been purified sufficiently for definitive differentiation marker or biosynthetic labeling studies to be performed. Murine peritoneal exudate cells have been enriched for PMN by the use of inducing reagents such as glycogen, polyvinylpyrrolidone, sodium caseinate and endotoxin (Gallin et al., '74; Yoshinaga et al., '75; Lord, '75; Vassalli et al., '781, but these preparations contained variable proportions of lymphocytes and macrophages (1020%).Yoshinaga et al. ('75) found that a 9093%pure PMN preparation could be obtained if the peritoneal cells were harvested three hours after a single intraperitoneal injection of sodium caseinate. However, these exudates also contained a t least 7-10%contaminating lymphocytes and macrophages and no mention was made of the proportion of eosinophilic polymorphonuclear leukocytes present. The first section of this report describes the preparation of peritoneal exudate PMN which are of suitable purity for electrophoretic analysis of their proteins. The second segment of the paper describes the methods which have been developed for radiolabeling both the internal and external proteins of mouse PMN. Attempts to radioiodinat,e the plasma membrane of human PMN by either the lactoperoxidase method or with the Bolton-Hunter reagent have generally been unsuccessful (Segal and Peters, '77). Recently, a new surface labeling procedure using a sparingly soluble chloramide, 1,3,4,6-tetrachloro-3a, 6 a diphenylglycoluril (IODO-GEN), has been developed (Fraker and Speck, '78). This procedure is extremely gentle, protecting cells from the powerful oxidants and strong reducing agents which are present when using chloramine-T to label cells (Munford and Gotschlich, '77). This new method also avoids the use of extraneous proteins, such as lactoperoxidase, which may become highly labeled during the iodination process (Hubbard and Cohn, '72) and hydrogen peroxide, which is known to damage membrane components. Investigations with erythrocytes (Fraker and Speck, '78; Markwell and Fox, '78) nucleated eukaryotic cells and viruses (Markwell and Fox, '78) have shown that 125J-labeling in the presence of IODO-GEN occurs only a t t h e exterior cell surface (Markwell and Fox, '78). The pattern of labeling of the cells and viruses with IODOGEN closely resembles results obtained with the lactoperoxidase technique (Hubbard and Cohn, '72, '75; Markwell and Fox, '78). Dif-

ficulties, such as t h e ability of endogenous intracellular myeloperoxidase to iodinate inter. nal proteins (Klebanoff and Clark, '771, have been encountered during attempts to radioiodinate PMN with the lactoperoxidase-glu. cose oxidase method. However, the use of IODO-GEN, relatively short iodination times, low iodide concentrations, low temperatures and the ability to use inhibitors of endocytosis have allowed the surface radiolabeling of PMN reported here. This data should be useful as a basis for studying the molecular changes during the formation of PMN when precursor cells are stimulated by granulocyte-macrophage colony stimulating factor (Metcalf, '77; Burgess et al., '78). Recently some studies have defined the sequential appearance of individual proteins (e.g., lyozyme, Krystosek and Sachs, '76; Fc receptor, Rabellino et al., '78; C3 receptor, Lotem and Sachs, '74; Ia antigens, Kaplan e t al., '78) in cultures of developing PMN. The 1251-surfaceand 35S-radiolabeling techniques described will allow a more general approach to the identification of sequential changes during differentiation. MATERIALS AND METHODS

Mice C57BL/6f/J Wehi, BALBlcflAnd Bradley Wehi, CBAfICaH Wehi and SJLflj Wehi male and female mice were bred under specific pathogen-free conditions and conventionalised a t five weeks of age. Mice aged between 8 and 12 weeks old were used throughout the experiments. Induction of peritoneal exudates Peritoneal exudates in male and female C57BL and in male BALBIc, CBA and SJL mice were analysed a t intervals after t h e injection of either 1 ml of polyvinylpyrrolidone (PVP; 15% w/v in 0.02 M sodium phosphate containing 0.149 M NaCl (pH 7.3, PBS) or 2 ml of calcium caseinate (0.2% w/v in a solution of 0.168 M NaC1, pH 7.2) at zero time and after a second injection a t 15 hours. Purification ofpolymorphonuclear neutrophils (PMN) Male mice received a single intraperitoneal injection of 2 ml of 0.2% (w/v) calcium caseinate. Mice were killed by cervical dislocation three hours after injection and 5 ml of isotonic saline (0.168 M) injected into the peritoneal

SURFACE IODINATION OF PURIFIED MOUSE NEUTROPHILS

cavity. The peritoneum was massaged gently and the exudate cells removed with a syringe. Exudates containing a large number of contaminating erythrocytes were discarded. The peritoneal exudate cells (PEC) were washed three times with PBS at 4°C and the contaminating erythrocytes removed by suspending the cells a t room temperature for ten minutes in NH,Cl solution (0.168 M) (Shortman e t al., '72). Where possible all further procedures were carried out at 4°C. Polymorphonuclearneutrophils (PMN) were purified from the peritoneal exudate cells by continuous density gradient centrifugation on Percoll (reviewed by Schmitt e t al., '74) or by the intensity of light scatter using a BectonDickinson I1 cell sorter. a. Density gradient centrifugation Separation of PMN from peritoneal exudate cells was obtained using a self-generated continuous density gradient of colloidal silica sol (Percoll) prepared as described by Pertroft e t al. ('68). Diluted isotonic Percoll was prepared by mixing 9 ml of Percoll with 1ml of sodium phosphate buffer (0.2 M, pH 7.3) containing NaCl(1.49 M) and 1ml of PBS. Peritoneal exudate cells (2 to 5 x lo7) in 1ml of PBS were mixed with the diluted, isotonic Percoll (8ml). A gradient was formed by centrifuging the cells at 60,OOOg,, for 20 minutes in a Beckman 50 Ti angle-head rotor (26" angle) in a Beckman L2 65B ultracentrifuge at 4°C. This program of centrifugation resulted in (96 -+ 8) X l o 4rotor revolutions. Fractions (0.2-0.5 ml) were collected from the gradient with a syringe and the densities measured gravimetrically. For routine preparations PMN were collected from the gradient with a Pasteur pipette. b. Cell separation using light scatter PMN could be separated from other peritoneal exudate cells (PEC) using a light scatteractivated cell sorter. After lysis of the erythrocytes, PEC (5 x 10Vml) in PBS were streamed past the tunable argon ion laser beam (488 nm, 0.2W) of a Becton-Dickinson FACS I1 cell sorter a t 2,000 cells per second. The intensity of scatter perpendicular to and in the direction of the laser beam was measured without filters with the low angle gain set at 4, the high angle gain at 2 and the photomultiplier tube (EM1 9524) a t 220 V. Cells scattering light into the low angle chan-

3

nels 108 - 200 and the high angle channels 71 180 were collected, washed and counted.

Preparation of a cell-freeextract Purified PMN were washed three times in PBS (pH 7.3) and resuspended a t 4 x lo7cells per ml of PBS. Cells were disrupted by three cycles of freezing and thawing. The viscosity was reduced either by syringing the cell-free extract five times through a 26-gauge needle or alternatively, DNAse I (1 pg/ml), together with MgSO, (0.2 mM), was added and the extract incubated a t 4°C for 15 minutes. Protease inhibitors EDTA (20 mM) and PMSF (10 mM in ethanol) were then added. An equal volume of SDS sample buffer was added to the extract to give final concentrations of SDS, 2mercaptoethanol and glycerol of 1.5%, 2.5% and 7.5%respectively. Extracts were stored a t - 20°C. Effect of protease inhibitors The effect of protease inhibitors was tested by preparing a PMN-cell free extract (see previous section) of 3 x lo7 cells per ml in the presence of PMSF, EDTA, iodoacetamide (IAA), chloroquine, tosyl-L-phenylalanyl chloromethane (TPCK) , n-ethylmaleimide (NEM) or pepstatin a t final concentrations ranging from 1-20 mM. The extract was then incubated at 37°C for 30 minutes prior to the addition of the SDS sample buffer and subsequent analysis of the PMN proteins by polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis The method of Laemmli ('70) as modified by Atwell ('74) consisting of an upper stacking gel and a lower running gel was used. A running gel was made so that the acry1amide:bisacrylamide ratio was 40:l. Samples for electrophoresis were boiled a t 100°C for four minutes to allow complete reduction and denaturation of samples. The protein solutions were electrophoresed a t 1 mA/gel to allow stacking of the samples in the upper gel and then at 2 mA/gel for electrophoresis in the lower running gel for cylindrical gels (100 x 5 mm). Slab gels (160 x 160 X 2 mm or 25 X 20 X 0.35 mm) were run at 50 volts per gel for stacking and 100 volts per gel for electrophoresis. Myeloperoxidase was detected on the gels using the method of Thomas et al. ('76) excepting that no sodium dodecyl sulfate was added to the sample before electropho-

4

SUZANNE M. WATT, ANTONY W. BURGESS AND DONALD METCALF

resis. The position of the bromophenol blue dye marker indicated the completion of electrophoresis. Gels were stained with Coomassie blue (0.05% w/v, Coomassie blue R-250, 50% v/v, methanol, 10% v/v, acetic acid) for a t least one hour at room temperature and destained in 7% (v/v) acetic acid a t 4OOC. Protein labeling a. Cell surface iodination Glass tubes (10 x 130 mm) were coated with IODO-GEN (0.1 mg/ml) dissolved in chloroform, which was evaporated under a stream of nitrogen gas, as described by Markwell and Fox ('78). It was important to remove all of the chloroform and to coat the bottom of the tube evenly with IODO-GEN. When the IODO-GEN dried as a spot, it tended to break away from the surface during the labeling. PMN or PEC (2-5 x l o 7 cells in 1ml of PBS) were incubated in the IODO-GEN coated tubes for 15 minutes a t 4°C in the presence of 200 pCi of carrier-free The reaction was terminated by transferring the cells into PBS containing cold NaI or KI (5 mM). The cells were either washed three times in PBS or mixed with diluted isotonic Percoll and separated by density gradient centrifugation. b. 35S-methionineincorporation PMN purified by Percoll density gradient centrifugation were incubated in methioninefree Dulbecco's Modified Eagles medium (DME) containing 5% (v/v) dialysed fetal calf serum, and supplemented with 10 pM I-methionine and 250 pCi/ml of ,'%-methionine (2-5 x lo7cells/ml) (Jones, '77). After washing the cells four times with PBS ( 5 ml) containing methionine (10 mM), the kinetics of 35S-methionine incorporation into TCA-precipitable protein was determined by the method of Burgess and Metcalf ('77a).

Lipid extraction Iodinated PMN (200 p l ; 2.5 X 107/ml)were mixed with washed sheep red blood cells (200 p1; 50%,v/v), the cells pelleted and the supernatant fluid removed. The cell pellet was extracted essentially according to the method of Folch et al. ('57): once with chloroform: methanol (200 pl; 2:1, v/v) at room temperature for 30 minutes; twice with chloroform: methanol (200 p1; 1:2, v/v) under the same conditions. A correction for the reduction in counting efficiency (1.5)caused by the organic solvent was made (Eisenberg e t al.,

'75). lZ5I2was extracted from the organic phase by the method of Mersel et al. ('76) using aqueous KI (2 x lo-' MI.

Autoradiography of electropherograms Slab gels and vertically sliced cylindrical gels were dried under vacuum on to Whatman No. 1 filter peper and autoradiographed using Kodak RP/S X-Omat film or Industrex A film. The autoradiographs were scanned using a Canalco model J micro-densitometer. Measurement of serum colonystimulating factor To test the effect of casein injection on GMCSF levels in serum, C57BL mice were bled three hours after a single intraperitoneal injection of casein. Uninjected mice were used as controls. Sera from both casein-injected and normal mice were diluted 1:5, 1 : l O or 1:20 with normal saline and sterilized by millipore filtration prior to GM-CSF assay. The sera were assayed for GM-CSF by adding 0.1ml of the diluted serum to duplicate 1ml of cultures of 75,000 C57BL bone marrow cells (Metcalf, '77). Granulocyte-macrophage colony formation was scored after seven days of incubation. Chemicals, buffers and solutions Iz5I(5.3 X M) and 35S-methionine(800 Ci/mmole) were purchased from the Radiochemical Centre, Amersham, Bucks., England. IODO-GENTM (1,3,4,6-tetrachloro-3a, 6a-diphenylglycouril) was obtained from Pierce, Rockford, Illinois, U.S.A. Percoll, concanavalin A-Sepharose, wheat germ agglutinin-Sepharose and the molecular weight markers for electrophoresis were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Insolubilized fucose-binding protein was obtained from Miles-Yeda, Israel. Sodium dodecyl sulphate, N, N, N', N1-tetramethyl-ethylene-diamine (TEMED), ethylene diamine tetracetic acid (EDTA, disodium salt) and ammonium persulphate were bought from British Drug House Chemicals Ltd., England. Acrylamide, N, N '-methylene bisacrylamide, Kodak RPSX-Omat and Industrex A films and 2-Napthtol-6, 8-disulfonic acid (dipotassium salt, Acid-G) were purchased from EastmanKodak, Rochester, New York, U S A . Calcium caseinate (casein) was obtained from Glaxo Laboratories, Australia. Phenylmethylsulphony1 fluoride (PMSF), pepstatin, chloroquine, N-ethylmaleimide (NEM), idoacetamide (IAA), rabbit skeletal muscle actin, cy-

5

SURFACE IODINATION OF PURIFIED MOUSE NEUTROPHILS

cloheximide and puromycin were obtained from Sigma Chemical Co., St. Louis, Missouri, U.S.A. Tosyl-L-phenylalanyl chloromethane (TPCK) was purchased from Calbiochem, San Diego, California, U.S.A. Dulbecco’s modified Eagles medium (DME) for the GM-CSF cultures was obtained from Grand Island Biological Company, New York. Methionine-free DME was purchased from the Commonwealth Serum Laboratories, Parkville, Australia. Fetal calf serum was obtained from Flow Laboratories, Australia. The molecular weight marker proteins for gel electrophoresis were prepared as stock solutions of 0.5 mg/ml in SDS sample buffer glycerol (7.5%), SDS (1.5%),2-mercaptoethanol (2.5%) in 0.034 M Tris-HC1 (pH 6.8).

(PVP-BDH) was used, the PEC contained 6580%PMN (table 1).Three hours after a single injection of the PVP-BDH, the percentage of PMN had increased to a maximum of 65%,at which time the nucleated cell number was 4 x lo6 cells per mouse. The number of cells harvested from the peritoneum increased to 1.3 x lo7 cells per mouse three hours after the second PVP injection (at 15 hours). The contaminating cells in the exudate were mainly lymphocytes and macrophages (table 1). On microscopic examination, the PMN appearing in response to PVP did not appear to be vacuolated. When casein was used as the inducing agent, the kinetics of the appearance of PMN in the peritoneal cavity resembled those obtained with PVP-BDH (table 1; figs. 1, 2). Cell morphology and counts However, the injection of casein into the periNucleated cell counts were made in a hemo- toneal cavity routinely induced an exudate cytometer using the eosin dye exclusion test to containing a higher percentage of PMN than estimate viability. Cells were resuspended in was obtained for exudates induced by any of fetal calf serum, smeared, fixed and stained the PVP preparations. The proportion of PMN with May-Grunwald-Giemsastain prior to dif- in the peritoneal cavity increased rapidly to a ferential counting of a t least 200 cells. maximum three hours after the injection of casein. Initially, the percentage of mononuRESULTS clear cells in the exudate was inversely reThe induction of peritoneal PMN lated to the percentage of PMN and had deInitially polyvinylpyrrolidone (PVP) was creased to a minimum within three hours. used as the inducing agent for peritoneal exu- Thereafter, the number of mononuclear cells date cells. A series of PVP reagents were ob- increased gradually. A similar response was tained from various manufacturers (British observed when a second casein injection was Drug Houses, Koch Light, Hopkins-Williams). administered 15 hours after the first injection These preparations of PVP were of varying av- (table 1; figs. 1, 2 ) . Compared to a single injection, two injecerage molecular weights (11,000 to 700,000) and purity. Most of the PVP preparations tions of casein resulted in a slightly lower perinduced cell exudates which contained less centage of PMN in both male (fig. 2A) and than 60% PMN. However, when a laboratory female mice (fig. 2B), although almost three reagent grade PVP, manufactured by BDH times as many cells had migrated into the TABLE 1

Cellular composition ofperitoneal exudates induced bypolyuinylpyrrolidone or casein Material injected

Percent cell8 Time after Injection (hr.)

Sex of

mice

No. of cells ( X 10-61

PMN

Macrophage

Eosinophil

Lymphocyte ~~

Polyvinylpyrrolidone Casein Casein Casein Casein

3 18 3

18 3 18

F F M M

42 5 13-t 7 8-t 4 49-tl8 17210 44t15

65213 772 4 762 8 762 5 got 5 8O-t 2

lot10 92 4 52 3 122 3 4 t 3 14t 1

222 120 8t7 724 121 221

~

23k7 13t7 1121 5-t2 5t3 4-t2

Mean values f standard deviations for five mice. Polyvinylpyrrolidone was a BDH laboratory reagent of unknown molecular weight. PVP was prepared as a 15%(w/v) solution in PBS and C57BL mice were injected with 1 ml intraperitoneally at zero time and 15 hours later. C57BL mice were injected intraperitoneally with 2 ml of casein (0.2%,wlv) in isotonic saline (pH 7.2) at zero time and 15 hours later. Cells were washed from the peritoneal cavity using 5 ml of normal saline at the times indicated, Cell viability was greater than 98%as estimated by eosin dye exclusion.

6

SUZANNE M. WATT, ANTONY W. BURGESS AND DONALD METCALF

peritoneal cavity (table 1). However, the induced PMN harvested a t 18 hours were highly vacuolated and were contaminated with large, vacuolated macrophages. Lymphocytes and macrophages comprised the major contaminating cell type in the exudate. However, the peritoneal exudate often contained significant numbers of eosinophils. The proportion of eosinophils harvested from the peritoneum of age-matched female mice after casein injection fluctuated considerably (fig. 3), but appeared to increase up to three hours. In exudates from male mice, the percentage of eosinophils reached a maximum one and one-half hours after casein injection, but had decreased to 1%of the cell population by three hours (fig. 3). There was considerable variation in the total cell counts of exudates induced by the intraperitoneal injection of casein. Some variation was also noted in the composition of exudate populations. Whilst approximately 90% of the cells induced in male C57BL and CBA mice were PMN, lower percentages of PMN were observed in the peritoneal exudates from SJL and BALB/c male mice (table 2). The kinetic responses for the induction of peritoneal exudates were similar in all strains of mice examined (S. M. Watt, unpublished ob-

TIME AFTER PRIMARY CASEIN INJECTION [HOURS)

Fig. 1 Induction of peritoneal exudates using casein (0.2% w/v, 2 ml) in C57BL male mice. Injections were given at the times indicated by the arrows and the cells harvested by washing the peritoneal cavity with isotonic saline (5 ml). Each point represents the average results macrophage; for at least 5 mice: 0-0, PMN; 0---0, A---A, lymphocyte; and A - - - A , eosinophil.

I

CASEIN

'

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I

7

A

1

80

--

VASE"

ASEIN

B

T

-

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5 2

60-

u Ly

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0

,

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TIME AFTER PRIMARY CASEIN INJECTION [HOURS) Fig. 2 Induction of peritoneal exudates in male (A) and female (B) C57BL mice using casein (0.2%w/v, 2 ml): 0-0, PMN; 0---0, macrophages.

7

SURFACE IODINATION OF PURIFIED MOUSE NEUTROPHILS

Serum colony stimulating factor Three hours after a single injection of casein, the diluted serum from C57BL mice stimulated 680 k 260 colonies per ml. This was approximately a two-fold increase over the number of colonies stimulated by normal mouse sera (290 f 160 colonies per ml), but considerably less than the number of colonies stimulated by post-endotoxin mouse serum (6,000 -t 600 colonies per ml).

Isolation of peritoneal exudate PMN a. Light-scatter Analysis of the size distribution of the peritoneal exudate cells using light scatter a t 0" and 90" (fig. 4) indicated that there was no change in the physical properties of the PMN as a result of the red cell lysis (figs. 4b,c). As expected, the PMN and eosinophils were assoTIME AFTER PRIMARY CASEIN INJECTION IHOURSl ciated with Area I1 (fig. 4b) and the lymphoFig. 3 Induction of peritoneal exudate eosinophils in cytes with Area I (fig. 4b; table 3). PMN were female (A) and male (B)C57BL mice using casein (0.2% separated from the lymphocytes and monow/v, 2 ml). cytes using the cell sorter by selecting for the servations), but fewer cells were produced by cells in Area I1 (fig. 412). For male mice, PNM the second injection of casein in SJL and were enriched to 95%(table 3) or higher when Area I1 was harvested. It was important to use BALB/c mice than in C57BL and CBA mice. Although the peritoneal exudates from a clean pre-filter for the separation of PMN, male C57BL mice harvested three hours after but even with this precaution and the use of casein injection generally yielded cell popula- earthed siliconized tubes, the recovery of tions containing 90% or more PMN, the rela- sorted cells was usually only 60%. When the tive proportions of PMN and macrophages in pre-filter became partially blocked, the lymthe exudates changed rapidly (fig. 2). For this phocytes would pass through preferentially reason, when the collection period was long and the recovery of PMN dropped to 20%.Simk e . , >30 minutes) because peritoneal exu- ilar results were obtained for female C57BL date cells from 20 or more mice were being mice. No separation of eosinophils from the pooled, the frequency of PMN in the exudate PMN was obtained for either male or female was often less than for the analytical experi- mice on the basis of light scattering (table 3). ments reported in tables 1and 2. In such large scale experiments, the peritoneal exudates b. Density centrifugation usually contained between 80%and 90%PMN. PMN could also be purified from caseinTABLE 2

Leukocyte distribution in peritoneal exudatesproduced in different mouse strains Strain

C57BL

Time after initial injection (hr)

3 18

BALB/c SJL CBA Mean values

3 18 3 18 3 18

Percent cells No. of cells (X

1 8 2 10 44?15 52 1 9210 11211 9e 5 8 t 5 202 9

PMN

Macrophage

Eosinophil

Lymphocyte

90k5 80k2 83k6 8057 8453 81k5 88f6 8724

423 13t1 8-t7 14f8 4-t2 1325 3f2 10f4

1 t1 321 ltl 121 1f1 If1 ltl

5-3 4f2 8t3 5t3 11*2 522 823 2* 1

l-tl

* standard deviations for five mice. Mice were injected intrapentoneally at zero and 15 hours with 2 ml of casein

(0.2%,w/v) in isotonic saline (pH 7.2). Cell viability was greater than 98%as estimated by eosin dye exclusion.

8

SUZANNE M. WATT, ANTONY W. BURGESS AND DONALD METCALF

either the viability or degree of purity (97%)of the cells obtained (table 3). Similarly erythrocyte lysis could be carried out either before or after the density gradient centrifugation without interfering with the purification or viability of the PMN. As expected the PMN (density = 1.12 g/cc) were more dense than the monocytes and lymphocytes and less dense than the erythrocytes (fig. 6A). When harvesting the PMN, care was taken to avoid contamination by the lighter density cells by collecting the bands from the highest to the lowest density cells. When more than 5 x l o 7 peritoneal exudate cells were separated using the Percoll gradients (8 ml), the cells tended to aggregate. This caused a broadening of the PMN band, but did not appear to interfere with the preparation of pure PMN. No aggregation was seen when 2 x l o 7cells were separated in this way and the PMN band was always well defined. Acid G used as described by Shortman ('72) did not prevent the aggregation of PMN. The PMN harvested from the Percoll density gradient appeared to be quite normal morphologically (fig. 6B). Analysis of PMN proteins a. The effect of protease inhibitors PMN contain many proteolytic enzymes which might interfere with an analysis of the functional and structural properties of membrane and cytoplasmic proteins. Initial studies were made to determine the effect of various inhibitors on the proteins extracted from PMN. After reduction and denaturation with SDS, the PMN proteins were analyzed by electrophoresis on polyacrylamide gels (5.612% acrylamide). By this method, polypeptides with molecular weights ranging from 11,400 to more than 200,000 were detected by Fig. 4 Separation of peritoneal exudate cells (PEC) Coomassie blue staining. A comparison of the from C57BL male mice using the FACS I1 light scatter electrophoretic patterns revealed several difactivated cell sorter with a laser wavelength of 488 nm ferences in the protein composition of the and output power of 0.2W. a. Untreated PEC; three-dimensional isometric diagram of cell numbers plotted as a PMN extract prepared in the presence of abfunction of the intensity of light scattered in the direction sence of protease inhibitors (fig. 7). The SDS of (0") and perpendicular to (90")the light bea: E, erythpolyacrylamide gel (8%)electrophoretic patrocyte; PMN, polymorphonuclear neutrophils and eoterns of two PMN extracts stained with sinophils; L, lymphocytes and M, macrophages/monoeytes. b. Two-dimensional contour representation of a. c. TwoCoomassie Brilliant Blue are shown in figure dimensional contour representation of a after erythrocyte 7. Under these electrophoretic conditions, the lysis. Areas of high cell density are white. Cells in the molecular weight dispersion ranged from areas labeled I (lymphocytes), I1 (neutrophils) and 111 15,000 to more than 200,000. In the absence of (erythrocytes) were collected for analysis (table 3). protease inhibitors, 40 protein bands were induced peritoneal exudate cells using density detected with Coomassie blue staining (fig. gradient centrifugation (fig. 5 ) . The density 7B). Several proteins appeared to be absent gradient could be preformed or formed a t the from the PMN extracts in the absence of the time of the separation without affecting protease inhibitors. This was especially evi-

9

SURFACE IODINATION OF PURIFIED MOUSE NEUTROPHILS TABLE 3

Separation of casein-induced peritoneal exudate cells using light scatter or buoyant density centrifugation Percent cells Cell distribution

PMN

Macrophage

Eosinophil

Lymphocyte

Males Unfractionated Area I Area I1 Percoll

82% 6 32 952 3 97* 2

422 0,7 If1 121

2" 1 0-0 221 121

12t 4 97,91 2-t 2 12 1

Unfractionated Area I Area I1

71k10 4% 4 87% 5

Females 3-t2 323 1k1

1127 0 1024

15-tlO 932 4 2-t 1

Mean data & standard deviations from 5 experiments with unfractionated cells; 5 experiments with FACS-fractionatedcells and 14 experiments with Percoll-fractionated cells. For male mice, cell counts from Area I were performed twice only. Cells were collected from th e areas indicated on t h e 0"-90" scatter plan (fin. 4) which was determined using the cell sorter. Area 111 contained only red blood cells.

I

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W

I

0

'I w a

n

"I

Y

0

a w m

5 z

3.0

4.0 5.0 CUMUlATlVE FRACTION VOLUME (ml)

Fig. 5 Separation of PEC using density gradient centrifugation on colloidal silica sol (Percoll). Cells ( 5

X

lo7 in 1 ml of PBS) were mixed with 8 ml of isotonic Percoll (81%,v/v) and centrifuged a t 60,000 g,, for 20 minutes. A. The number of PMN per ml 0-0 ; macrophages 0- --0; eosinophils A- --A and lymphocytes A . , . A . B, Total number of erythrocytes per ml 0-0, the number of PMN per ml 0-0, and density X. . . X of each fraction.

10

SUZANNE M. WATT, ANTONY W. BURGESS AND DONALD METCALF

Fig. 6 Preparation of PMN purified by density gradient centrifugation of casein-induced peritoneal exudate cells (PEC). A. Photograph of density gradient separation of untreated PEC: S, silica pellet; E, erythrocytes; PMN, polymorphonuclear neutrophils; L, M, lymphocytes, macrophages. B. Photomicrograph of peritoneal exudate PMN separated by density gradient centrifugation.

dent for some high molecular weight proteins (e.g., band I, figs. 7A,B). While some low molecular weight polypeptides (such as band IV; figs. 7A,B) were noticeably absent after incubation without inhibitors, other lower molecular weight polypeptides appeared to be present in larger amounts. Two major protein bands (band 11, 111) with apparent molecular weights 76,000 and 42,000 respectively dominated the profile. The abundance and characteristic molecular weights of the protein bands I1 and 111 suggested that band I1 corresponded to lactoferrin (Kincade et al., '76; Murphy, '76) and band I11 to actin (Stossel and Pollard, '73; Jones, '77). To confirm these assignments two dimensional electrophoretic analysis of the PMN proteins was performed (A. W. Burgess and S. M. Watt, work in progress). Band I1 was a basic protein migrating rapidly on non-equilibrium pH (7-10) gradient electrophoresis (O'Farrell et al., '77) in a region corresponding to a n isoelectric point near 9. This is the isoelectric point reported for murine lactoferrin (Kincade et al., '76). Band I11 focussed (OFarrell, '75) at pH 5.7 corresponding to the reported isoelectric point for actin (Storti et al., '78; Jones, '77). Band I11 co-migrated with the major component of rabbit skeletal muscle actin. Although several protease inhibitors (TPCK,

pepstatin, EDTA, IAA, NEM and chloroquine) caused some alteration in the protein profile, these inhibitors were relatively ineffective in preventing proteolysis. However, the addition of PMSF a t concentrations of either 5 or 10 mM to the PMN lysate led to the appearance of proteins in the high molecular weight region of the gels. At least 45 Coomassie blue stained bands could be observed in the PMN protein profile when the lysis buffer contained 10 mM PMSF alone or 10 mM PMSF, 20 mM EDTA and 20 mM IAA (fig. 7A). In addition, proteins with apparent molecular weights of 195,000 (band I) and 37,000 (band IV) were prominent, with the low molecular weight polypeptides considerably reduced in amount. Lactoferrin (band 11) and actin (band 111) again appeared to be major constituents of the PMN protein extract representing 6%and 10% respectively of the stained proteins. b. Biosynthetic labeling of PMN Biosynthetic labeling of PMN proteins with 35S-methionine increased the sensitivity of detection of some proteins 10-fold. The incorporation of W-methionine into PMN was found to be linear for at least three hours even a t cell concentrations of 2 x lo7 and 5 x lo7 cells per ml (fig. 8). After three hours, approximately 6 cpm of 35Sradioactivity was associ-

SURFACE IODINATION OF PURIFIED MOUSE NEUTROPHILS

Fig. 7 Effect of protease inhibitors on polyacrylamide gel electrophoresis profile of PMN proteins. PMN cell free lysates were prepared in the presence of PMSF, EDTA and IAA (A) or without protease inhibitors (B). The electrophoresis was performed in the presence of SDS (0.1%)using 8%polyacrylamide gels and the protein stained using Coomassie blue R250. The gels were analysed quantitatively using an Isco gel scanner. Pyronin yellow was used as the tracking dye (TD).

11

12

SUZANNE M. WATT, ANTONY W. BURGESS AND DONALD METCALF

- 1.0 .

I

I

I

r

1

-

2 2 W 1 u

2 0.8 52 W

z

z 0 I0.6 L m CT)

u 0

0.4

z k-

0

1.0 2.0 INCUBATION TIME [HOURS)

3.0

.---.,

Fig. 8 Biosynthetic incorporation of 35S-methionineinto PMN proteins. PMN were incubated with methionine-free DME containing fetal calf serum (5%v/v) and the cells washed before estimating the TCA precipitable 35S by liquid scintillaton counting: 0-0, 2 X lo7 PMN/ml; 5 X lo7 PMN/ml.

ated with each cell (fig. 8 ) and 15 -+ 4%of this was associated with protein as determined by TCA precipitation. In order to estimate the concentration of methionine required for optimal 35S-methionineincorporation into the PMN, the effect of the addition of carrier methonine (0-200pM) to the cell cultures was examined. The acid insoluble 35Sincorporated into these cells increased by approximately 0.6% with the addition of 0.5 pM carrier methionine and plateaued up to a methionine concentration of 10 p M .Thereafter, a gradual decrease in the 35S-methionineuptake was observed. 35S-methioninewas used a t 250 pCi per 5 x l o 7 cells per ml and the culture supplemented with 10 pM cold methionine to maximize the incorporation of 35S-methionine into the PMN. Pre-incubation of the PMN with cycloheximide (50 pg/ml) or puromycin (50 pg/ml) for 20 minutes a t 37°C prior to the addition of methionine decreased the incorporation of radioactivity into acid insoluble protein by 98.5%without loss of cell viability. At higher concentrations of cycloheximide (100 pg/ml) t h e W-methionine incorporation was decreased by 99.4% but the cell viability decreased to 83%. Analysis of the 35S-labeled PMN-proteins by polyacrylamide gel electrophoresis and subsequent autoradiography indicated that most proteins were labeled dur-

ing the 3-hour incubation period (e.g., bands I and 111)but some of the proteins which appear to represent granule proteins such as lactoferrin (band 11) did not appear to be labeled with 35S-methionine(fig. 9). c. Surface labeling of PMN Surface proteins of PMN were labeled using IODO-GEN. Initial studies were directed towards establishing iodination conditions for PMN which were suitable for labeling the surface proteins only. The incorporation of lZ5I into the PMN depended on the amount of IODO-GEN, the concentration of lZ5I, the reaction time and the cell concentration. In the iodination experiments presented in table 4,IODO-GEN kindly supplied by Dr. C. F. Fox (University of California, Los Angeles) could be used at concentrations of 20 p g for 5 x lo6 cells per ml without loss of cell viability ( >98%). All subsequent experiments however were performed using IODO-GEN obtained from Pierce Chemical Co. and cell death became evident when more than 5 p g of IODO-GEN was used to label cells a t a concentration of 3 x lo7 per ml. The Coomassie blue and lZ5I-labeledprofiles of the proteins extracted from PMN iodinated in the presence of varying concentrations of IODO-GEN and subsequently analysed on SDS polyacrylamide gels (12%)are presented

SURFACE IODINATION OF PURIFIED MOUSE NEUTROPHILS

in figure 10 (C-G). With low concentrations of IODO-GEN (2.5pg; fig. lOD), high molecular weight (apparent molecular weights ranging from approximately 90,000 to 185,000) proteins appeared to be labeled with lZ5I.On the 12%polyacrylamide gels, four major l Z 5 1bands could be easily detected (fig. 10D) provided the cell viability remained above 98%as determined by the eosin dye exclusion technique. Further analysis of the high molecular weight proteins on 8%gels (figs. 10A,B) or on 5.6% gels (S.M. Watt, unpublished data) resulted in a greater degree of resolution of the high molecular weight proteins and 6 lZ51bands could be observed clearly (fig. 10B). Under these labeling conditions, t h e Coomassie blue stained proteins with molecular weights equal to or above 195,000 and which were prominent in the gel profiles (e.g., band 1, figs. 8, 10A) were not iodinated. Whilst the I Z 5 1 label appeared in six bands with molecular weights of 185,000 or less, the major polypeptide labeled had a n apparent molecular weight of 90,000. A comparison of t h e Coomassie blue stained profile (fig. 10A) with the lZ5Iprofile (fig. 10B) showed that the 90,000 molecular weight band was not heavily stained with Coomassie blue. Approximately 40% of the lZ51labeled material which entered the SDS gels was detected in the 90,000 molecular weight band. In addition to the lZ5Iappearing in the high molecular weight protein bands, some lZ51ran with and in front of the bromophenol blue dye marker. This represented 20% of the lZ5Iapplied to the gel and presumably contained both free lZ5Iand lipid. Extraction of neutral and polar lipids with chloroform-methanol yielded 23 % 1%of the initial radioactivity. However, most of this 1251was extractable with 2 X M KI, leaving 1.0 -t 0.2%of t h e 1251 which presumably associated with the lipids. This represented 9 f 1%of the TCA insoluble IZ5I.The remainder of the radioac-

Fig. 9 35S-methioninelabeled PMN analysed by p l y acrylamide gel electrophoresis in the presence of SDS. PMN were labeled with 35S-methionine(250 pCi/ml) for three hours, washed and the lysate (50 pl, 5 X lo7 PMN/ ml) electrophoresed in the presence of SDS on polyacrylamide gels (8%).A and D show the Pharmacia molecular weight standards stained with Coomassie blue. A. Protein standards: phosphorylase b (94,000); bovine serum albumin (67,000);ovalbumin (43,000); carbonic anhydrase (30,000). B. PMN protein profile stained with Coomassie blue. C. SsS-methioninelabeled PMN autoradiograph (exposed for 72 hours using Kodak Industrex A film). D. Protein standards: thyroglobulin (330,000); ferritin (220,000); albumin (67,000); catalase (57,000);lactate dehydrogenase (36,000).

13

tivity running in front of the tracking dye presumably represents free lZ5I.The amount of free lZ51associated with the cells was often variable and appeared to depend on the extent of the washing procedure following iodination. The prominent Coomassie blue bands with molecular weights of 76,000 and 42,000 (fig. 10A:

14

SUZANNE M. WATT, ANTONY W. BURGESS AND DONALD METCALF TABLE 4

Incorporation o f

into PMNsurfaceproteins Incorporation (%I

Reaction time (min)

Iz5I pCi/ml 200

5 15 30

1,000

500

'

Con A

TCA

Con A

TCA

Con A

0.16 0.75 0.85

0.02 0.10 0.10

0.28 0.72 0.91

0.04 0.12 0.21

0.39 1.00 1.20

0.06 0.20 0.20

TCA

Tabular entries are the average values for the percentage incorporation of the initial lZsI for three experiments in which 5 X lo6 PMN in 1 ml were labeled with 20 p g of IODO-GEN (supplied by Dr. C. F. Fox) at 4°C. The cells were washed four times with PBS containing NaI (5mM) before counting the lzsIassociated with the cells. In the absence of IODO-GEN,less than 0.1%of the initial was associated with the PMN after 30 minutes. I An aliquot of the lz5Ilabeled cells was mixed with PBS (1ml) containing carrier fetal calf serum (5%, v/v) and an equal volume of a solution of trichloroacetic acid (20%.wiv, TCA). The precipitate was washed three times with TCA (5%,wiv) and once with ethanol-water (7:3). Tabular entries are the average values for the percentage of the initial lP5I added which was acid insoluble. liSIlabeled cells were lysed with PBS containing 0.5%wiv Triton X-100. The proportionof the initial Iz5Iadded which bound specifically to concanavalin A-Sepharose was determined as described in MATERIALS AND METHODS.

bands I1 and 111) which presumably represent the azurophil granule protein, lactoferrin, and the cytoplasmic protein, actin, respectively did not label with l Z 5 1 under these conditions. Similarly after staining enzymically to locate the myeloperoxidase on polyacrylamide slabs, autoradiography showed that there was no '*'I associated with this enzyme. When higher concentrations ( > l o pg) of IODO-GEN were used the cell viability decreased rapidly from 90% for 10 p g of IODOGEN to less than 40% for 100 p g of IODOGEN. Initially, when IODO-GEN concentrations of 20 p g (figs. 10F,G) were used, a series low molecular weight bands were labeled (fig. 10F,G) while the high molecular weight bands were more extensively labeled. However, refractionation of the "'1-labeled cells on Percoll resulted in the removal of the dead cells and the recovered cells were 99-100% viable. At the same time the low molecular weight lZ51bands disappeared from the electrophoretic profile (fig. 10E). Although the level of PMN iodination could be increased using 100 p g of IODO-GEN, cell death invariably followed, together with extensive labeling of low molecular weight polypeptides and the appearance of I Z 5 I in band I11 (actin). PMN were labeled with I Z 5 1 using IODOGEN (5 pg) both before and after density centrifugation on Percoll (fig. 11).Qualitatively neither the Coomassie blue stained proteins nor the proteins labeled with lZ5Iappeared to be affected by the labeling procedure. There was considerably more @fold) Iz5Ilabel incorporated into the surface proteins of the PMN when labeling was performed after the density centrifugation (cf. figs. 11E,D).No lzsIwas as-

sociated with the actin protein (band 111; fig. 10A) even when the PMN were labeled after the density separation. Thus, 1251-labeling of internal proteins was detected when there was a considerable loss in viability during the iodination. "'1-labeling of actin was not detected when the surface proteins were labeled routinely using 5 p g of IODO-GEN together with 200 pCi/ml Iz5I. Some actin labeling was detected when the Iz5Iconcentration exceeded 500 pCi/ml. The standardization of the labeling technique was essential for reproducible iodination patterns to be obtained. It was essential to use low concentrations of IODO-GEN ( < 10 (200 pCi/ml), p g per 3 x lo7cells/ml) and lZ51 as well as to minimize endocytosis by performing all labeling experiments at 4°C. IncorporaM) into the tion of sodium azide (3 X iodination buffer did not alter the level of iodination nor did the electroptoretic pattern of Iz5I-labeledproteins change. In initial experiments using 20 p g of IODOGEN, supplied by Dr. C. F. Fox, to label 5 x lo6cells in 1ml PBS, cell labeling increased linearly for up to 15 minutes at 4°C and then began to plateau by 30 minutes (table 4).With an incubation time of 15 minutes, 0.75%of the initial 200 pCi of carrier-free Iz5Iwas incorporated into TCA precipitable material. Less than 0.1% of the initial IZ5Iwas associated with acid insoluble material in cells when IODO-GEN was absent from the reaction mixture. Using 200 or 500 pCi of lz5I,equivalent to a concentration of 0.13 and 0.26 p M iodide respectively, the efficiency of incorporation was not greatly altered. However, higher rates of incorporation were obtained with high io-

SURFACE IODINATION OF PURIFIED MOUSE NEUTROPHILS

15

Fig. 10 'T-surface labeled PMN proteins analysed using reduced SDS polyacrylamide electrophoresis. A. labeled PMN proteins (5p g IODOPMN protein profile stained with Coomassie blue (8%gel). B. 1*51-surface GEN; 8%gel). C. PMN protein profile stained with Coomassie blue (12% gel). D-G. 1*51-surface labeled PMN proteins analysed on 12% gels. PMN were labeled with varying IODO-GEN concentrations (D2.5 pg; E, F 10 pg; G 20 p g ) . E. PMN were surface labeled with lZ5I using 10 p g IODO-GEN. Non-viable cells were then removed by Percoll density gradient centrifugation after t h e idination.

dide concentrations. With 1 mCi of lZ5I (0.52 p M iodide) the incorporation was increased by approximately 1.4-fold over t h a t found with 200 or 500 FCi of lZ5I.However, under these

conditions, some lZ5I appeared in the actin band (111, fig. 10). Additionally, there was an apparent change in t h e level of incorporation of into glycoproteins which bound to con-

16

SUZANNE M. WATT, ANTONY W. BURGESS AND DONALD METCALF

330K-

220K'-94K 67K 57K

- 6 7K

-

-4 3 K

36K

-30K __ 20 K

18K A

B

C

D

E

F

labeled PMN proteins analysed using reduced SDS polyacrylamide electrophoresis on Fig. I1 1251-surface 12%micro gels, A and F represent the Pharmacia protein standards described in the legend of figure 9. B and using 5 p g E. PMN protein profile stained with Coomassie blue. C. PMN proteins were labeled with IODO-GEN prior t o Percoll centrifugation. D. PMN proteins were labeled with l Z 5 I using 5 pg IODO-GEN after Percoll purification.

canavalin A-Sepharose, both as a function of time of incubation and iodide concentration. However, the proportion of glycoproteins which bound to insolubilized wheat germ agglutinin (5% of TCA precipitable 1251) and fucose binding protein (1%of TCA precipitable Iz5I) decreased when the labeling time was increased beyond 30 minutes. DISCUSSION

Peritoneal exudate cells (PEC) harvested after the injection of glycogen, polyvinylpyrrolidone or sodium caseinate have been reported previously to contain high proportions of polymorphonuclear neutrophils (PMN) (Gallin et al., '74; Yoshinaga et al., '75). Our results agreed qualitatively with these previous studies, but the proportion of PMN in the PEC rarely exceeded 90%- particularly when more than 20 mice were used to prepare the PEC. The percentage of PMN in exudates induced with polyvinylpyrrolidone (PVP) depended on the source of the material. Highly purified preparations of PVP with well char-

acterised molecular weight properties did not appear to induce a high proportion of PMN. The only batch of PVP to induce PEC containing more than 60% PMN was an unfractionated laboratory grade reagent (manufactured by BDH) which is no longer commercially available. More satisfactory results were obtained using casein to induce the peritoneal exudate. Eosinophils were often a major subpopulation of cells in the PEC from female mice. The proportion of eosinophils in the PEC from female mice was variable but appeared to rise after casein injection and remain elevated for at least 24 hours. In male mice, the proportion of eosinophils in the PEC increased during the first two hours after injection of casein, but by Thus, eosinophil three hours was less than 1%. contamination of PMN could be minimized simply by using male mice. The percentage of mast cells in the peritoneal cavity also rewhen mice younger than 12 mained low (