A study of the attachment phase of phagocytosis by murine macrophages

Experimental Cell Research 59 (1970) 105-I 16

A STUDY

OF THE ATTACHMENT BY MURINE JENNIFER

PHASE OF PHAGOCYTOSIS

MACROPHAGES

M. ALLEN

and G. M. W. COOK

Strangeways Research Laboratory,

Cambridge, UK

SUMMARY A macroglobulin component of calf serum was found to interact opsonically with motile bacteria, mediating their adherence to mouse peritoneal macrophages in culture. The nature of the receptor site for the opsonised bacteria on the macrophage surface was studied by treating the cells with enzymes and chemicals known to modify certain components of cell surface membranes. After treatment of the macrophages with phospholipases, bacterial adherence was destroyed only when the cells had been extensively damaged by the enzyme. Neuraminidase treatment increased bacterial adherence, possibly by bringing about a reorientation of membrane material, whilst the proteolytic enzymes pronase, trypsin and chymotrypsin destroyed receptor sites for the opsonised bacteria on the macrophage surface. It is deduced that these sites are probably associated with a surface protein though the involvement of surface glycoproteins cannot be excluded. Iodoacetamide was found to be ineffective in blocking the receptor site, indicating that SH groups are not involved.

The plasma membrane of the macrophage possesses special characteristics which enable the cell to select certain components from the surrounding tissue fluids for phagocytosis. This has been demonstrated and certain aspects of the phenomenon have been analysed in vivo by experiments on blood-stream clearance of particles and bacteria by the reticula-endothelial system [3, 15, 18, 201. The process of phagocytosis by macrophages can be divided into two main stages: first, attachment of the particle to the surface of the phagocyte, and second, invagination of the plasma membrane with subsequent ingestion of the adhering particle. The initial stage of adherence has been studied in detail by Rabinovitch [29, 301 and by Lee & Cooper [23] using mouse peritoneal macrophages and chemically modified red cells of various species.

In the present work the attachment phase of phagocytosis has been studied in a system in which mouse peritoneal macrophages, cultured on coverslips, were allowed to interact with motile Gram-positive bacteria. The nature of a necessary serum factor has been elucidated, and some information on the chemical nature of the attachment site on the surface of the macrophage has been obtained by enzymatic pretreatment of the cells before application of opsonised bacteria; evidence is presented which suggests that the attachment site on the surface of the macrophage is peculiar to this cell.

MATERIALS

AND METHODS

Cell culture system Mouse peritoneal macrophages were obtained from a subline of the AK strain of mice maintained in this Exptl Cell Res 59

106 Jennifer M. Allen & G. M. W. Cook laboratory and kindly made available by Dr W. Jacobson. For some experiments 1.2 % sodium caseinate in physiological saline was used to stimulate the production of macrophages [27]. Mice were killed by cervical dislocation and pinned out in a supine position. The peritoneal cavity was washed out under aseotic conditions with medium 1415ATM [16] or in phosphate-buffered saline pH 7.2 1131. both containing heoarin (5 units/ml). The cell suspension was diluteh tdapprox. 2 x lt$ cells/ml and 0.2 ml aliquots were pipetted onto glass coverslips whose edges had been treated with silicone grease to prevent the drop from spreading 1261. The coverslips were enclosed in Petri dishes 7 cm in diameter. The cells were allowed to settle out at 37°C in a moist atmosphere for 30 min, the coverslips were washed once with shaking to remove cells, principally lymphocytes, that were not adhering to the glass, and the medium was replaced by medium 199 [25] (supplied by Glaxo Ltd. without added antibiotics) supplemented with 20% v/v new-born calf serum (NBCS) obtained from the Ministry of Agriculture Animal Research Station, Compton, Berkshire, through the kindness of Dr A. McDermid, or 2.5 % w/v crystalline bovine serum albumin [14] (BA), from Armour Pharmaceutical Co. The cultures were maintained at 37°C in a moist atmosphere containing 2 % COz. The medium was changed again with further shaking after 24 h, and then every second day. Before the cultures, which now consisted almost entirely of macrophages, were submitted to experimental procedures, the culture medium was removed, and the monolayers were washed three times with excess phosphate-buffered saline, applied with a Pasteur pipette. To obtain mouse polymorphonuclear leucocytes, four doses of 2 ml of a 0.1 % w/v solution of rabbit liver glycogen (British Drug Houses) in pyrogen-free saline was inoculated intraperitoneally on alternate days. Two hours after the 4th inoculation, a final injection of 2 ml was given and the cells harvested 2 h after this. They were allowed to settle out on coverslips in the same manner as were macrophages and were used without further cultivation. Two strains of mouse fibroblasts, C57S/l and C3HSIl. kindlv provided bv Dr M. R. Daniel of this laboratory, were cultured- for short periods in a manner similar to that employed for macrophages.

Bacterial culture system Two strains of B. subtilis and one strain of B. cereus were used for the experiments. B. subtilis “A” was isolated at this laboratory and kindly identified by Dr T. Gibson of the Edinburgh School of Agriculture. B. subtilis “B” and B. cereus were obtained from the National Collection of Type Cultures (NCTC nos. 6276 and 8035). To obtain cultures of rapidly swimming rods the bacteria were grown in nutrient broth for 18 h at 18-20°C. B. subtilis “A” was employed for the quantitative experiments.

Opsonisation procedure Bacteria were suspended in medium 1415ATM or phosphate-buffered saline at pH 7.s7.2 at a conExptl Cell Res 59

centration of 2.5 x lo8 cells/ml. New-born calf serum (NBCS) was added to make a 20 % v/v solution, and the preparation was immediately centrifuged for 5 min in an MSE (Measuring and Scientific Equipment Ltd., London) bench centrifuge at approx. 1200 g.av., taken up in the required volume of phosphatebuffered saline and immediately applied to the cells. Ten samples of serum from new-born and foetal calves were made available to us through the kindness of Dr D. Franks of the Patholoav Deoartment. Cambridge University, and were t&ted ‘for their ability to promote adherence of B. subtilis to macrophages. Results were expressed as a percentage of the adherence produced by NBCS.

Chemicals Iodoacetamide was supplied by Sigma Chemical Co. and was used at 5.0 and 0.5 mM/ml for times varying from 2 min to 30 min at 37°C. Mercaptoethanol was obtained from Koch-Light Laboratories Ltd. Whole calf serum was incubated with 0.1 M mercaptoethanol [12] for 1 h at 37°C. The serum was then dialysed against 0.145 M NaCl+ PO, [S] buffer for 24 h before use.

Enzyme treatments Neuraminidase (EC 3.2.1.18, lot No. 1165A, 966B) from Vibrio cholerae was obtained from Behringwerke (Marburg/Lahn) as an aqueous solution containing 500 units/ml, where the number of units of neuraminidase activity is defined as being equivalent to the number of pg of N-acetyl neuraminic acid released from c+ acid glycoprotein in 15 min at 37°C and pH 5.5 in an appropriate medium. This preparation &stated by the manufacturer to be free of lecithinase C and neither protease nor aldolase activity could be demonstrated. Using Azocoll (Calbiochem.) as a general substrate for proteolytic activity, under the incubation conditions described here we were unable to detect proteolytic activity in the neuraminidase preparations. Neuraminidase was used at a concentration of 50 units/ml of medium (enzyme/cell ratio in excess of 10 units/lo6 cells) at 37°C for 30 min or 1 h. Phospholipase A (EC 3.1.1.4) from Nuju naja venom was suoolied by Koch-Light Laboratories Ltd, Batch No.-19999. The enzyme-was dissolved in Sorenson’s phosuhate buffer uH 6 at 10 ma/ml, and was then heated-to 100°C for 15 min to destroy any proteolytic activity. Phospholipase A (EC 3.1.1.4) from wheat germ (Type 1, lot 114B.8610), and phospholipase C (EC 3.1.4.3) from Clostridium welchii were supplied from Sigma Chemical Co. (lot 115B-1090). Trypsin (EC 3.4.4.4) (crystallised) was from Armour Pharmaceutical Co. Diisopropyl fluorophosphate (DFP)-treated trypsin, soya bean trypsin inhibitor, chymotrypsin (EC 3.4.4.5) and DFP-treated chymotrypsin were all from Worthington Biochemical Corporation. Pronase (B grade) was from Calbiochem (Batch No. 53702, lot 44579). All reagents were dissolved in medium 1415ATM or phosphate-buffered saline [13], and according to

Macrophage cell surface the reagent the pH was adjusted to 6.8-7.2 with 0.3 M NaOH. Before and after treatment the cultures were washed three times with a large excess of 1415ATM or phosphate-buffered saline.

Procedure for evaluating attachment of bacteria to macrophages An aliquot (0.1 ml) of bacterial suspension containing 2.5 x 108 bacteria per ml of buffered saline was placed between wax strips painted on 3 x 1 in. microscope slides. Monolayers of macrophages on coverslips were washed three times in medium 1415ATM or buffered saline, and inverted over the bacterial suspension. Cells and bacteria were allowed to interact for 5 min at 1820°C; under these conditions the ingestion rate was negligible. The coverslips were fixed in place by the application of wax dots at each corner, and given one brief wash in saline to remove bacteria that were not adhering to the cells. The washing was done by introducing the saline carefully under the coverslip and simultaneously allowing the bacterial suspension to be drawn out by gentle suction applied to the opposite side of the coverslip. Fixation with 4% form01 saline was performed in a similar manner, and the preparations remained in the fixative, sealed round with liquid paraffin [26]. Counts were made under a x 45 oil immersion objective of numbers of bacteria adhering to 200 or 500 cells, in random fields, and the results were expressed as a percentage of control counts. With standardised procedures the number of bacteria adhering per cell does not vary greatly over the whole area of the monolayer, so that satisfactory results have been obtained by counting 200 macrophages in random fields. Rabinovitch [29, 301 assayed the attachment of glutaraldehyde-treated red cells on 200 macrophages and Davey & Asherson [l l] have presented consistent results based on counts of only 100 macrophages.

Characterisation

of opsonin

Antibodies to the opsonin were prepared in New Zealand white rabbits. A suspension (1 ml) of heatkilled (60°C 30 min) bacteria, opsonised by the standard procedure after heat-killing, was inoculated into the rabbit via the ear vein every second day for 6 days. One week after the last inoculation the animals were bled and the serum collected. Parallel controls were made in which unopsonised bacterial suspensions were used for inoculation. Antiserum against whole calf serum was prepared by adding potassium aluminium sulphate to calf serum according to the method of Proom [28] and injecting this suspension into rabbits according to the timetable of Hirschfeld [17]. Rabbit antisera to bovine yG (R2472) and whole bovine serum (R-3191) were obtained from Mann Research Laboratories, New York. Immunoelectrophoresis of the opsonising calf serum against the various rabbit antisera was carried out in 2 % w/v agar gel, with an applied voltage between the ends of the slide of 60 volts (approx. 8 V/cm). The precipitin patterns were fixed in 2 % acetic acid solution followed by staining with amido black 10B (G.T. Gurr, London).

107

For further investigation, the opsonising calf serum was fractionated on linear sucrose gradients (10-35 %) in 0.145 M phosphate-buffered saline (pH 7.3) formed in 23 ml centrifuge tubes (MSE, rotor 2418 3 x 23 ml swing out tvoe). The serum was diluted with an equal volume of buffer and 1 ml was pipetted onto the top of each gradient. The preparations were centrifuged in a Super Speed 50 ultracentrifuge (MSE) at 4°C at 120,000 g max., for 20 h, and were then fractionated by drop collection from the base of the tube which was punctured by a commercial tube piercer (MSE). The fractions were suitably diluted with buffer for optical density measurement in 1 cm path length cells in a Beckman DB spectrophotometer. The fractions under each peak were collected and the sucrose removed by exhaustive dialysis against phosphate-buffered saline before addition to cultures.

RESULTS Adherence of bacteria to macrophages

Application of rapidly swimming B. subtilis (strain A or B) or B. cereus to mouse macrophages in the absence of serum resulted in a low incidence of phagocytosis, and when the living cultures were observed by phase contrast microscopy, continual movement of the bacteria among the cells was seen. When the bacteria were applied in the presence of NBCS, any bacterium that came in contact with the surface of a macrophage immediately adhered to it, and the general appearance of the living culture was comparatively static; the bacteria, stuck fast to the macrophages, were either rendered quite motionless or continued to rotate on one spot. Adherence appeared to involve the flagella in the first instance, since bacterial rods were often only loosely attached to the cell surface and continued to wriggle and gyrate for some minutes after contact. At 37°C attached bacteria rapidly induced active folding of the surface in the immediate vicinity of the organisms which by this time, were usually immobilised by multiple contact. Ingestion and digestion then took place (figs l-8). Ingestion was arrested at temperatures of 18-20°C. Interaction of opsonised bacteria with Exptl

Cell Res 59

108 Jennifer M. Allen & G. M. W. Cook

Figs I-4. Adherence and ingestion of opsonised 8. subtilis “A” (arrow) by mouse peritoneal macrophage in culture at 37°C. Times are indicated in the top right hand corner of each figure. Adherence initiates violent movements of the adjacent plasma membrane (2, 3) leading to intake (4) after 5 min. Phase contrast.

mouse fibroblasts C57S/l and C3HS/l or mouse polymorphonuclear leucocytes resulted in no adherence of the organisms to the cells. Due to size of rod and speed of swimming, B. subtilis “A” was found to be more suitable for quantitative studies than B. subtilis “B” and was therefore used in all experiments. Exptl Cell Res 59

B. cereus was toxic to the cells. When B. subtilis “A” were applied to the cells in serial dilutions of NBCS, increasing numbers of bacteria adhered to the cells above & dilution of serum. To determine whether at 18-20°C the serum factor involved was acting as a cytophilic antibody on the macrophage surface,

Macrophage cell surface

IO9

Figs 5-8. Continuation of the series seen in figs 14. The ingested bacterium remains within the cytoplasm for almost 20 min before digestion produces a sudden alteration of phase density of the bacterial rod (figs 7, 8). Phase contrast.

or whether it was combining opsonically with the bacteria, cells in monolayer which had been cultivated in 199 +20 % v/v NBCS for 8 days, were washed three times with 199 +2.5 Y0 BA, and the coverslip was inverted over a bacterial suspension in the same medium. As observed by phase contrast microscopy of the living culture, bacterial

adherence was negligible, but could be restored by the addition of calf serum to the bacterial suspension. If, however, the bacteria were first opsonised (as described in Methods) and then applied to (a) cells that had been maintained in serum-free medium (199+2.5% BA) for 8 days, or (b) cells that had been maintained in 199 + 20 % NBCS Exptl Cell Res 59

110 Jennifer M. Allen & G. M. W. Cook Table 1. Effects of various treatments on the opsonising properties of NBCS Serum treatment of bacteria

Average no. of bacteria/cell (200 cells counted)

Applied to the cells in medium 1415 ATM (No serum) Medium 1415 ATM containing 10 % NBCS Opsonised with 10 % NBCS and applied in serum-free medium 1415 ATM Opsonised with 10 % NBCS and applied in medium 1415 ATM

0.17 5.32 5.07 5.25 Bacterial adherence index Y;,

Treatment of serum used in opsonisation None Heated to 56°C 30 min Incubated with 0.1 M mercaptoethanol for 1 h at 37°C

for 8 days and then washed three times in serum-free medium, the organisms adhere. This implied that a component of the calf serum had combined with the bacteria and, without appreciably affecting their mobility, had rendered them compatible with some component on the surface membrane of the macrophage, thus leading to adherence on contact. It was found that bacterial adherence was of the same order whether the organisms were suspended in a serum-containing medium or whether they were opsonised by centrifugation through the medium and resuspended in the same volume of serum-free medium before being applied to the cell monolayers (table 1). In the experiments reported in this paper, bacteria were routinely opsonised and then applied to the cells in the absence of serum. Of ten other samples of calf sera tested for opsonic activity, two proved to possess a level comparable to that in the NBCS used in the experiments described here. Identqication serum

of the opsonising factor in calf

Heating of the calf serum to 56°C for 30 min had no effect on its opsonising properties Exptl Cell Res 59

Expt 1 loo 86.4 3.9

Expt 2 100 102.9 3.5

(table l), indicating that complement was not involved, but treatment with mercaptoethanol destroyed its ability to promote adherence (table l), suggesting that the opsonising agent was a macroglobulin. This possibility was investigated immunoelectrophoretically, and a single precipitin line was produced that was identifiable as an IgM globulin (fig. 9). In some experiments an additional very faint line corresponding to IgG was visible. Fractionation of the calf serum on linear lo-35 % sucrose density gradients to yield two protein fractions (fig. 10) was achieved. Identical peaks were obtained in three separate experiments. From the position of the smaller peak in the gradient, this fraction was identified as 19s macroglobulin [22] and was found to partially restore bacterial adherence in serum-free media while the fraction making up the larger peak was conpletely lacking in activity. Investigation of the chemical nature of the receptor on the surface of the macrophages

Different components of the macrophage’s surface were modified by means of enzymes, in an attempt to determine the nature of the receptor sites responsible for adherence of opsonised bacteria.

Macrophage cell surface

111

Fig. 9. Immunoelectro-

phoresisof NBCS. Slides stained with amido black. Slide 1: NBCS (in the well) against rabbit anti-opsonin serum and rabbit antiwhole calf serum. Slide 2: NBCS against commercial rabbit anti-bovine yG and rabbit antiopsonin serum. Slide 3: Culture medium (199 f 2.5 % bovine albumin) against rabbit anti-opsonin and rabbit antiwhole calf serum.

Treatment of the monolayers with neuraminidase at a concentration of 50 units/ml for 30 min at 37°C increased bacterial adherence (table 2). Analysis by students t-test suggests that this increase is probably significant (~~0.05 for 8 degrees of freedom) when compared to the inactivated (boiled) neuraminidase preparation. Treated cells showed no gross morphological changes even after an hour’s exposure to enzyme at this concentration. The application of phospholipases had a profound effect on cell morphology and viability. After treatment with phospholipase A (from Naja naja venom) at 100 pug/ml for 10 min most of the cells were killed, their nuclei were pycnotic and the cytoplasm disintegrated. Similar treatment with phospholipase C (from Clostridium welchii) also killed

many of the cells in the monolayers; the dead cells became unevenly detached from the glass and assumed cylindrical or spindle shapes. After treatment of the monolayer with these enzymes it was found that bacteria adhered only to those cells that remained morphologically intact; this was reflected in an increasing adherence after treatment with lower concentrations of enzyme (table 2). A second source of phospholipase A (wheat germ lipase) had little or no effect on either cellular morphology or bacterial adherence. Pronase when applied to 1000 ,ug/ml for 30 min removed all the cells from the surface of the coverslips and was lethal at concentrations down to 250 ,ug/ml. At 100 ,ug/ml the cells were considerably altered morphologically, assuming a stellate form with angular extensions; after 50 pg/ml the nuclei were still Exptl Cell Res 59

112

Jennifer M. Allen dz G. M. W. Cook

Abscissa: tube number; ordinate: optical density at 280 m,u. Fig. 10. A typical fractionation of NBCS on a l&35 % linear sucrose density gradient. Tube 1 contains the fraction from the bottom of the centrifuge tube. Each tube contained 20 drops of gradient- medium to which 1.2 ml of phosphate-buffered saline (PBS) was added before making the optical density measurement. Fractions 1623 (macroglobulin) and 26-37 were each pooled, dialysed exhaustively against PBS and then concentrated with lyphogel (Gelman Instrument Co.). The macroglobulin fraction produced in three exoeriments. 50.2 k 11.5( %) adherence index when compared to an equivalent concentration of whole serum. The other fraction proved to be negative.

intact, the cytoplasmic extensions were fewer, but bacterial adherence was much reduced (table 2). Trypsin treatment caused little morphological damage to’ the cells which assumed a flattened, discoidal shape and remained viable, but the enzyme almost entirely eliminated bacterial adherence when applied at concentrations of 500-1000 pg/ml for 30 min at 37°C (table 2). At lower concentrations the effect on adherence was less, approaching a minimum at 10 kg/ml. To determine whether this was due to the specific action on trypsin, experiments were made with trypsin that had been specifically inactivated by d&isopropyl fluorophosphate (DFP), or by applying trypsin together with soya bean inhibitor. The sample of DFPtreated trypsin used was not completely without activity, as confirmed @ titration against Azocoll, but the manufacturers found Exptl CeN Res 59

the sample to be at least 98 % inactivated and to contain no chymotryptic activity. The residual proteolytic activity, however, which may be clearly demonstrated with Azocoll, was sufficient to cause some degradation of the membrane (table 2). Soya bean inhibitor, however, entirely prevented tryptic removal of the receptor sites (table 2). The application of trypsin for varying periods showed a progressive reduction of adherence with time indicating that the observed effect is unlikely to be due to adsorption of enzyme to the cells. After 15 min the adherence index was 27.8k6.6 (4), after 20 min the index had fallen to 7.6k3.5 (4). By 30 min the adherence properties of the membrane were almost completely destroyed, the index being 0.8+ 0.5 (7), and in two experiments no adherence was observed; at 40 min the result was 0.4+ 0.2 (4). Chymotrypsin also destroyed the adherence properties of the cells, and after 30 min treatment with 500 pg/ml at 37°C adherence was completely abolished. DFP only partially prevented the enzyme’s activity. After exposure to chymotrypsin the cells had assumed a similar morphology to trypsin-treated cells, and likewise remained viable. After 30 min incubation at 37°C with 5.0 mM iodoacetamide, macrophages were found to be grossly damaged. The effect was lessened by reduction of the concentration of the chemical to 0.5 mM and opsonised bacteria were found to adhere to the treated cells to the same extent as controls. However, if the bacteria were applied at 37°C ingestion was prevented if the cells had been treated for more than 5 min with 0.5 mM iodoacetamide. DISCUSSION The experiments described here were designed to study those properties of the surface membrane that are peculiar to the macro-

Macrophage cell surface

113

Table 2. The adherence of opsonised bacteria to macrophages following treatment of the cells with various enzymes Concentration (,&-nl)

Enzyme Control Neuraminidase Neuraminidase Phospholipase Phospholipase Phospholipase Phospholipase Phospholipase Phospholipase Pronase Pronase Trypsin

Incubation time (min)

Number of experiments

Bacterial adherence index %

30 30

6 4

100 146.3 k 36.3 106.4f 11.0 59.8 100.4 105.3 42.9 103.7 109.9 2.5 5.3

Nil

A A A C C C

Trypsin Trypsin Trypsin Trypsin Trypsin (return to culture for 24 h after treatment) Trypsin + Soya bean inhibitor Trypsin DFP treated Chymotrypsin

5OU 5OU (Boiled preparation) 100 50 1: 50 10 100 50 1000

10 10 10 10 10 10 30 30 30

500 250 100 50 500

30 30 30 30 30

5 5 I

54.7; 90.7; 93.2; 28.9; 92.5; 105.3; 2.4; 2.9; Nil 0.61 Nil o.s+ 21.7+ 35.7F 80.1 + 54.0+

30

A

119.0*

32.1

30 30

2 2

53.3; Nil;

54.4 0.1

500 (of each) 500 500

0.3 0.5 3.6 7.0 22.4 23.0

Bacterial adherence index was determined at 18-20X as described in the text. Results are presented as the mean + S.D. where appropriate. a Concentration quoted in units of neuraminidase activity at the enzyme pH optima of 5.5. At pH 7.2kO.2 60 “b of the original activity is retained.

phage and which enable this cell type to select and retain particles from the surrounding medium for subsequent phagocytosis; opsonised bacteria were used as a test particle. A convenient source of opsonin was present in a sample of new born calf serum which was routinely used in culture media. Immunoelectrophoretic studies, fractionation on sucrose gradients and mercaptoethanol reduction indicated that the principal opsonising factor from the calf serum was an IgM. Cohn & Parkes [5] found a macroglobulin in bovine serum which, while causing cytolysis of mouse macrophages in the presence of complement, in its absence S-691809

induced pinocytosis by a stimulation of the plasma membrane. In our experiments, at a concentration of serum that did not noticeably increase pinocytosis and which was beneficial for the maintenance of macrophages in culture, the macroglobulin component was present in sufficient amounts to act as an effective opsonin for B. subtilis and B. cereus when these organisms were either added to culture medium containing serum or opsonised by being centrifuged through diluted serum and resuspended in serum-free medium. Whilst in general IgM is not opsonic unless complement is present [lo], heating at 56°C for 30 min did not affect the opExptl

Cell Res 59

114 Jennifer M. Allen & G. M. W. Cook sonising properties of the serum in the system described here. It must be emphasised that phagocytosis is essentially a two-stage process, namely an initial adsorption of the particle to the phagocyte followed by uptake and ingestion. The present experiments were designed to examine the adsorption phase of phagocytosis as a distinct but essential step for the subsequent process of invagination and digestion. Since his first stage can take place at room temperature whereas the second requires a temperature of 37”C, it was possible to study adherence uncomplicated by the onset of ingestion, merely by performing the experiment at 1820°C. On raising the temperature to 37”C, the bacteria. adhering to the cell surface were ingested by normal phagocytosis, with subsequent killing and digestion of the organisms (figs l-8). In the present experiments inversion of the cell monolayers over a suspension of activelyswimming opsonised bacteria ensured that most of the bacteria that adhered to the cells did so because of the opsonic properties of the serum component. It also minimised the number of moribund or slowly-swimming bacteria which fall onto cells lying underneath a bacterial suspension or become trapped by pseudopodia; under these conditions bacteria and other particles can be ingested by phagocytic cells in the absence of serum [l]. This phenomenon resembles the “surface phagocytosis” described by Wood et al. [34] and Sawyer et al. [31] by which phagocytes could ingest capsulated pneumococci in the absence of serum, provided that the cells were attached to certain types of surface. Employing the calf serum component as a tool to promote adherence of bacteria to the macrophage surface, it was possible to examine the effect of removal or chemical modification of some of the known constiExptl Cell Res 59

tuents of cell membranes on the ability of the cells to combine with the opsonised bacteria. Recent studies on animal-cell surfaces, especially by cell electrophoresis, have stressed the important role played by carbohydratecontaining macromolecules in cell surfaces [6]. In particular, proteins, especially glycoproteins, have been shown to occur quite generally at the cell surface, possibly providing the cell with an efficient means for cellular recognition [7]. The action of neuraminidase, trypsin, chymotrypsin and pronase, enzymes that are known to degrade specifically membrane proteins and glycoproteins [8, 91 were investigated together with various phospholipase preparations which were considered by Davey & Asherson [l I] to degrade a receptor site for cytophilic antibody. Weiss et al. [33] have demonstrated that the phagocytosis of plastic particles by human blood monocytes is enhanced after neuraminidase treatment as also is the contact phase between cells, and Bona et al. [4] have implicated glycoproteins as receptors for sheep erythrocytes on the guinea pig polymorphonuclear leucocyte membrane. In the present experiments, at a concentration at which it will degrade surface antigens and will release sialic acid from blood group glycopeptides from red cells [24], neuraminidase had no inhibitory effect on bacterial adherence, and indeed caused an increase. The removal of charged sialic acid residues may well be accompanied by a change in the orientation of membrane constituents allowing for a sterically more favourable configuration of the bacterial receptor sites. Both phospholipases and proteolytic enzymes on the other hand altered the cell membrane in a manner that reduced bacterial adherence. In the case of the phospholipases, the reduction in adherence occurred

Macrophage cell surface

only at a level of enzymic activity that caused gross damage to the cells, making it difficult to prove that any specific removal of receptor component had taken place. Pronase likewise severely injured the cells, and at concentrations that produced minimum damage, bacterial adherence was reduced. Trypsin and chymotrypsin almost entirely prevented bacterial adherence at concentrations that caused no gross injury to the cells, and the removed receptors were resynthesised after 24 h in culture. Vaughan [32] observed that trypsin treatment of guinea pig macrophages destroyed their ability to attach effete autochthonous red blood cells in the absence of “free” antibody. Rabinovitch [29, 301 found that the attachment of erythrocytes whose surface had been altered chemically to mouse macrophages was abolished or reduced by trypsin treatment of the macrophages. On the other hand, if the chemically altered erythrocytes had first been exposed to antiserum, trypsin treatment of the macrophages did not reduce adherence. Rabinovitch, therefore, postulates two different receptor sites on the macrophage surface for chemically altered red cells that have or have not been treated with antibody. The relationship between opsonising and cytophilic antibody appears to be fairly close [2] and it is possible that the receptors in the macrophage surface for the two types of antibody might be similar. Attachment sites for guinea pig cytophilic antibody, however, have been shown to be resistant to treatment with proteolytic enzymes by Howard & Benacerraf [ 181 and Davey & Asherson [l 11. Kossard & Nelson [21] have found different sensitivities to proteolytic enzymes, in the attachment to mouse macrophages of cytophilic antibodies against sheep red cells from homologous mouse sera obtained at different times after immunisation. Thus attach-

115

ment of antibody from serum obtained seven days after primary immunisation was susceptible to trypsin or papain treatment of the macrophages, while that from hyperimmune serum (obtained 28 days after primary immunisation) was not; this result indicated that there were different receptor sites on the macrophages for globulins from the two sera. Howard & Benacerraf [ 181 reported that treatment of guinea pig macrophages with phospholipase C at a concentration of 2 mg/ml for 1 h at 37°C inhibited the uptake of cytophilic antibody by the cells which appeared “superficially normal”. Their interpretation that phospholipase C had destroyed the integrity of the cell membrane agrees with our observations that treatment of mouse macrophages with 0.1 mg/ml of this enzyme for 10 min at 37°C at pH 7.2 (kO.2) in buffered saline, has a drastic effect on the cell’s morphology, and that opsonised bacteria adhere only to those cells that have not yet succumbed to the action of the enzyme. A similar interpretation may be applied to the effect of the phospholipase A from Naja naja venom which, at a concentration of 0.1 mg/ml for 1 h, was found by Davey & Asherson [l l] to destroy the receptor for cytophilic antibody on guinea pig macrophages, and which in our hands killed most of the cells within 10 min. Phospholipase A from wheat germ, on the other hand, had no apparent effect on either cellular morphology or bacterial adherence when applied to the cells for 15 min at 1.0 mg/ml. Howard & Benacerraf [18] made similar observations on guinea pig macrophages. There appears to be a considerable species difference in the activity of both phospholipase A and C on mouse and guinea-pig macrophages respectively, but the present experiments on mouse macrophages indicate that loss of the property of bacterial adherence by a cell after treatment with either of these Exptl

Cell Res 59

116 Jennifer M. Allen Q G. M. W. Cook enzymes coincides with the onset of the cell’s general disruption, making it impossible to attribute lack of bacterial adherence to removal of any specific receptor site. Lack of any concomitant changes in bacterial adherence following removal by neuraminidase of free sialic acid would suggest that this material does not form an integral part of a receptor site for bacteria opsonised with bovine macroglobulin. However, the results obtained with the various proteolytic enzymes would suggest that the receptor site is either a surface protein or that a carbohydrate moiety, other than sialic acid, linked to a protein, might be important. The effective loss of receptor sites due to conformational changes at the cell surface as a result of the action of the proteolytic enzymes cannot be excluded. The implication of sulphydryl groups in the receptor site for cytophilic antibody [18] does not appear to hold for the receptor site investigated here. The authors wish to acknowledge the generous encouragement and continuous hip of Dame Honor B. Fell, D.B.E., F.R.S., and the kind advice of Professor R. R. A. Coombs, F.R.S. They would also like to thank Mrs J. Emmines for her able technical assistance, Mrs J. Wright and Miss E. Stewart for secretarial help and Mr M. Applin for help in the preparation of the figures. J. M. A. and G. M. W. C. are members of the External Scientific Staff, Medical Research Council.

REFERENCES 1. Allen, J, M. Unpublished observations. 2. Berken, (1966) 119. A & Benacerraf, B, J exptl med 123 3. Biozzi, G, Stiffel, C, Halpern, B W, Le Minor, L & Mouton, D, J immunol 87 (1961) 296. 4. Bona, C, Ghyka, G & Vranialici, D, Exptl cell res 53 (1968) 519.

Exptl

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