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Phagocytosis of modified erythrocytes by macrophages and L2 cells
Experimental Cell Research 56 (1969) 326-332
PHAGOCYTOSIS
OF MODIFIED
BY MACROPHAGES
ERYTHROCYTES
AND L2 CELLS
M. RABLNOVITCH The Rockefeller University, New York, N. Y. 10021, USA
SUMMARY Glutaraldehyde-treated horse red cells (GRC) are taken up by mouse peritoneal macrophages and by L2 mouse “fibroblasts” in the absence of added antibody or complement. The surface of GRC was modified in order to examine their interaction with the two cell types. Coating of the erytbrocytes with ovalbumin, bovine albumin, lysozyme or cytochrome c, inhibited both attachment to and ingestion by, macrophages; and ingestion by L2 cells. Digestion of GRC with chymotrypsin, pronase, subtilisin or papain, stimulated their uptake by macrophages and L2 cells. Chymotrypsinogen was ineffective. The stimulation by chymotrypsin was dependent on enzyme concentration, temperature, and was inhibited by the addition of phenylmethyl sulfonyl fluoride (PSF). Chymotrypsin was stimulatory when GRC were treated with PSF either before or after digestion by the enzyme. Treatment of the GRC with 80 % methanol prior to or following exposure to chymotrypsin, reduced the stimulation of phagocytosis. The parallel behavior of macrophages and L2 cells supports a common mechanism of non-immunological “recognition” by the two cell types.
Phagocytosis of certain particles by macrophages or leucocytes can take place in the absenceof serum antibody and complement. No generalization can be made at present of the characteristics of the particles that permit this form of “recognition”, nor is it known whether separate phagocytic receptors are available for different kinds of particles. When serum factors participate in the recognition, it may be assumed that specific sites of immunoglobulin molecules or on complement interact with appropriate phagocytic receptors [l 11.It appears that different receptors mediate the attachment to phagocytes of particles that have or have not been coated with antibody
WI. Little information is available on particle uptake by cells other than macrophages and leucocytes. We have found that mouse L2 fibroblasts ingested the sameparticles that were taken up by macrophages in the absence of serum, namely, aldehyde-treated or heat denatured erythrocytes, iron, alumina, glass or polystyrene latex beads ([9] and unpublished). However, Exptl Cell Res 56
immunoglobulin G antibody inhibited the uptake of the aldehyde treated erythrocytes by L2 cells. This was taken to indicate that L2 cells, in contrast to macrophages, do not possess receptors for the antibody [12]. Particle uptake that bypassesthe requirement for antibody or complement may represent a more primitive form of recognition which is shared by a variety of cell types. Macrophages and leucocytes would in addition possess receptors for particle-bound antibody and complement [9]. One way of testing this hypothesis is to compare the uptake of different particulates, in the presenceor absenceof serum factors, by a variety of phagocytic cells. The present report makes use of glutaraldehyde-treated horse red cells (GRC) to examine particle uptake by macrophages and L2 cells in the absence of added antibody or complement. It will’be shown that the two cell types responded similarly to two modifications of the GRC surface. The uptake of particles coated with protein was reduced, whereas treatment of GRC
Phagocytosis by macrophages and L2 cells
with proteolytic enzymes increased their uptake. The stimulation of phagocytosis of proteolytic enzyme treated GRC was related to the digestion of a surface component.
MATERIAL
AND METHODS
Media Buffered saline (PBS) was prepared by adding 1 vol of 0.07 M phosphate buffer pH 7.3 to 10 vol of 0.85 % sodium chloride. Fetal bovine serum (FBS), Gey, Puck saline A, tissue culture medium 199, Eagles minimum essential medium for stationary (MEM) or for suspension (SMEM) cultures were obtained from Grand Island Biological Co.
Chemicals Phenylmethyl sulfonyl fluoride (PSF), obtained from Sigma, was dissolved in “Tris” buffer pH 7.5, with the aid of methanol. The 1 x 1O-s M solution contained less than 5 % methanol.
Proteins and enzymes The following crystallized preparations were used (unless otherwise stated the data are those supplied by the manufacturers). Bovine plasma albumin (Armour Pharmaceutical Co.); ovalbumin (Worthington Biochem. Corp.); lysozyme (General Biochemicals); cytochrome c (horse heart, type III). o-Chymotrypsinogen (type II, 26.5 benzoyl-L tyrosine ethyl ester-BTEE-units/mg protein after activation; 0.03 units/mg protein prior to activation), papain (1 mg split 4.0 PM NH3/min from benzoylt-arginine amide at pH 6.0 at 37”C), and subtilisin (subtilopeptidase A, type VII; 1 mg liberated 1.6 mg tyrosine/min from casein at pH 7.5, 37”C), from Sigma. One sample of a-chymotrypsin was obtained from Mann Research Laboratories and assayed 7000 units/mg towards N-acetyl+tyrosine ethyl ester. Three other samples of chymotrypsin were obtained from Sigma on different occasions, with a stated activity between 40 and 49 BTEE units/mg. One sample of chymotrypsin assayed in this laboratory by the method of Hummel [4], hydrolyzed 64 PM of BTEE per min/mg protein at 25°C. The activity was entirely inhibited when the chymotrypsin (1 mg) was pretreated for 20 min with 1 x 1O-4M phenylmethyl sulfonyl fluoride in PBS. Pronase (B grade, Calbiochem) and V. cholerae neuraminidase (Hoechst Pharmaceutical Co.) were semi-purified preparations.
327
Treatment of GRC with proteins Bovine albumin or ovalbumin were dissolved in 0.1 M acetate buffer pH 5.0. Lysozyme or cytochrome c were dissolved in 0.07 M phosphate buffer pH 7.3. Five x 10’ GRC were incubated with 2 ml of the protein solutions for 40 min at 37°C and washed three times in PBS. Control GRC were treated with buffers alone.
Treatment of GRC with enzymes or with chymotrypsinogen Five times 10’ GRC in 50 ~1 PBS were added to solutions (2 ml) of trypsin, chymotrypsin, subtilisin, pronase or chymotrypsinogen in “Tris” 0.05 M, pH 7.5. The medium for papain was acetate 0.1 M with 0.001 M EDTA and 0.005 M dithiotbreitol (Calbiochem), and that for neuraminidase was acetate 0.1 M pH 6.0, with 0.01 M CaCl,. GRC were incubated for 2 to 4 h at 36°C and washed three times in PBS. Controls were treated with the enzyme diluents alone. In some experiments, treatment with chymotrypsin was done in the presence of PSF. The inhibitor was incubated with the enzyme for 30 min in PBS at room temperature prior to the addition of GRC. In other experiments, GRC were treated with solutions of PSF in PBS for 2 to 4 h either prior to or following exposure to chymotrypsin.
Treatment of GRC with methanol GRC were suspended in 25 % cold methanol and the concentration of methanol increased to 80 % by dropwise addition. After 1 h at 5”C, the GRC were washed three times with PBS.
Collection of peritoneal macrophages [8] The macrophages were used without cultivation.
Attachment of GRC to macrophages Macrophages attached to coverglasses were rinsed in PBS and overlaid for 20 min at 31°C with 0.1 ml of a suspension of 5000 to 20,000 GRC/,ul in PBS [lo]. The macrophages were washed free of unattached GRC, fixed, stained, and the % of cells with attached GRC was obtained -by scoring 200 or more successive phagocytes. Alternatively, the number of GRC attached to 200 consecutive macrophages (i.e. macrophages with zero, 1,2 or more GRC) was scored and the average attachment expressed as GRC/macrophage. In some experiments GRC were suspended in Gey, Puck saline A, or MEM-10% FBS.
Animals Mice of the NCS strain, Rockefeller University colony, of both sexes, 20-25 g body weight, were used as a source of macrophages.
Red cells Horse erythrocytes (Animal Blood Centre, Syracuse, N. Y.) were washed three times with PBS and treated with glutaraldehyde as previously described [7].
Ingestion assay for macrophages [8] After attachment of GRC (8000 per yl) as above, macrophages were incubated in medium 199 for 20 min at 35°C in a 10% CO,-air atmosnhere. The monolavers were rinsed, fixed, stained, and 200 or more GRC were scored as either attached or ingested [7j. Percent ingestion = ingested GRC x lOO/attached + ingested GRC. a ingestion % = ingestion % of enzyme-treated GRC, minus ingestion % of control GRC. Exptl Cell Res 56
328 M. Rabinovitch
Table 2. Attachment of ovalbumin-treated GRC
Single step phagocytosis assay for macrophages
to macrophages in different media
Macrophages on coverglasses were incubated for 30 min at 35°C in one ml of MEM or MEM-10 % FBS, with the addition of 1000 CRC/p1 in a 10 % CO,-air atmosphere. The macrophages were rinsed, fixed, stained, and the % ingestion of GRC determined as above.
Cultivation of L2 cells Stocks were obtained from Dr S. Dales (Public Health Research Institute, New York) and Dr L. S. Sturman (Rockefeller University). The cells were grown in suspension in S-MEM with 10 % FBS, containing 20,000 units penicillin, 10 mg streptomycin and 1 ml tylosin tartrate (Grand Island Biological Co.) per 100 ml. For the phagocytosis experiments, the L2 cells were suspended in MEM10 % FBS and 1 x 104 cells in 3 ml were dispensed in 20 x 110 mm Leighton type tissue culture tubes containing one round 18 mm coverglass [12]. Cells were grown overnight prior to incubation with GRC. All media were gassed with 10% COz in air. Phagocytosis was found to be less active when the tubes were seeded with cells exposed to trypsin.
Phagocytosis assay for L2 cells [12] CRC were added to the Leighton tubes to a final concentration of 1 x 1Oaper ,UI. & each experiment, control and treated GRC were adjusted to the same concentration. After 4 h incubation at 37°C with gentle shaking every hour, coverglasses were rinsed in medium 199 containing 1 % FBS, fixed and stained with Giemsa. Two hundred or more successive L2 cells were scored and the % of L2 containing ingested GRC was determined. In some experiments, the L2 cells were also scored as to their content of 0, l-2, 3-4 and 5 or more GRC and the results expressed as per cent of L2 with stated numbers of ingested GRC. Only the data for the first and the last classes are shown in the tables. It should be noted that no agglutinating antibody against fresh or aldehydetreated erythrocytes was detected in the FBS used. Furthermore the FBS contained no detectable hemolytic complement.
Table 1. Attachment of protein treated GRC to macrophages Treatment of GRC
Attachment=
PBS, pH 7.2 Acetate, pH 5.0 Bovine albumin, 25 mg/ml, pH 5.0 Ovalbumin, 10 mg/ml, pH 5.0 Ovalbumin, 20 mg/ml Lysozyme, 2 mg/ml, pH 7.2 Lysozyme, 10 mg/ml Cytochrome c, 2 mg/ml, pH 7.2
lOOk5.4 (5)b 100+ 8.4 (5) 5.6kO.13 (4) 17.7k3.1 (4) 7.OF0.7 (4) 22.Ok3.3 (4) 12.9 k4.6 (4) 28.2kll.l (4)
a In % of attachment of GRC treated with the buffers alone. Actual values for the % of macrophages with GRC attachment were 78.0 k4.2 and 76.Ok6.4 for the PBS and acetate groups respectively. b Mean + S.E. (no. of samples). Exptl Cell Res 56
Medium
Attachmenta
PBS Gey Puck A MEM MEM-10 % FBS
1.8 i0.7b 2.120.8 2.6* 1.5 19.2k5.3 38.5 + 10.7
a Attachment of ovalbumin-GRC in % of the attachment of control GRC. Ovalbmnin: 50/mg/ml, 1 h, pH 5.0. b Mean I S.E. Averages of 4 replicates.
RESULTS Attachment to macrophages of GRC with purified proteins
Table 1 shows that the attachment in PBS was markedly reduced when GRC were treated with bovine albumin, ovalbumin, lysozyme or with cytochrome c. GRC coated with ovalbumin were used to examine attachment in different media (table 2). Attachment was minimal in PBS, Gey or Puck’s saline A (a Ca2+,Mg”+-free medium), but significantly more extensive in MEM or in MEM-10 % FBS. Uptake by macrophages of GRC treated with ovalbumin
The extent of the interaction of coated GRC with macrophages in MEM or in MEM-10% FBS, permitted the study of ingestion in a “single step” assay. In one experiment, ingestion per cent of control GRC was 15.4+ 3.4 in MEM and 29.5k3.5 in MEM-10% FBS. The figures for GRC treated with 50 mg/ml ovalbumin were, respectively, 1.5k 0.6, and 4.7 + 0.9 (averages of 4 samples + standard error). Therefore, the ingestion of ovalbumin coated GRC by macrophages was also markedly inhibited. Uptake by L2 cells of GRC treated with purified proteins
Table 3 presents the results of an experiment in which GRC were incubated with several proteins prior to uptake by L2 cells. The table shows that treatment with the proteins decreased
Phagocytosis by macrophages and L2 cells
329
Table 3. Uptake by L2 cells of GRC treated with protein
Treatment of GRC
% of L2 without ingested GRC
PBS= 14.6 k 1.6 (7)b Ovalbumin, 30 mg/ml 52.6 + 3.0 (5) Bovine albumin, 30 mg/ml 52.2 f 6.0 (4) Lysozyme, 15 mg/ml 52.7 k4.5 (4)
% of L2 with 5 or more ingested GRC 41.713.8 (7) 2.2kl.l (5) 6.7 + 2.4 (4) 9.0 + 2.9 (4)
a Ingestion of GRC treated with acetate was not significantly different from the values given. b Mean +_ S.E. (no. samples).
the proportion of L2 cells that ingested GRC and reduced the percentage of cells that ingested 5 or more GRC.
Fig. 1.
Fig. 2.
Abscissa: pg/ml; ordinate: A ingestion %. Fig. 1. Relationship between chymotrypsin concentration used to treat GRC (4 h, 379, and the stimulation of the ingested of GRC attached to macrophages. A ingestion % = ingestion % of chymotrypsin-treated GRC, minus ingestion % of control GRC. Abscissa: Hours; ordinate: A ingestion %. Fig. 2. Time course of the chymotrypsin effect on GRC ingestion. Treatment of the GRC with chymotrypsin (1.1 mg/ml) at different temperatures. Ordinates as in fig. 1.
Ingestion by macrophages of GRC treated with chymotrypsinogen or with enzymes
this experiment. Results are corrected for the ingestion of untreated GRC. It can be seen that Table 4 shows that several proteolytic enzymes stimulated the ingestion of GRC attached to there is a clear log dose- effect relationship. In macrophages. The GRC were treated for 3 h view of the possibility that chymotrypsin mowith the enzymes and washed prior to their dified the attachment of GRC and thus could attachment to the phagocytes. Ingestion then have influenced the ingestion assays,attachment proceeded in medium 199. All of the proteolytic to macrophages of the same GRC preparations enzymes tested were stimulator-y. Chymotryp- used in the experiments shown in fig. 1 was studied. CRC were used at 15,000per ,~l in PBS, sinogen was inactive. attached for 12 min at 31°C and the number of In fig. 1, the ingestion of GRC attached to GRC bound to 200 macrophages was determacrophages is plotted against the dose of chymotrypsin used to pretreat the erythrocytes. mined. Attachment in GRC/macrophage -t standCRC were incubated with the enzyme for 4 h in ard error (no. samples) was 1.18? 0.10 [7]; 1.24kO.03 [5]; 1.14kO.14 [5]; 1.4510.09 [5] Table 4. Ingestion by macrophages of GRC and 1.20$0.04 [5], respectively, for control treated with chymotrypsinogen or with proteolytic GRC and for GRC treated with 100,250,500 pg and 1 mg/ml chymotrypsin. Thus, treatment of enzymes GRC with chymotrypsin did not significantly Treatment of GRC’ Dose (ug/ml) Ingestion %b influence their attachment to macrophages. Fig. 2 presents the time course of the effect “Tris” pH 7.5 8.5Ll.l (5)’ of chymotrypsin on GRC at three different Go 40.5d Chymotrypsin Subtilisin 100 39.0 temperatures. Stimulation of the GRC ingestion Papain (pH 6, EDTA was clearly dependent on the temperature. The DW 250 40.8 Pronase 100 35.2 time course at 37°C departs markedly from Chymotrypsinogen 500 6.8 linearity after 1 h treatment with the enzyme. Phenyl-methyl sulfonylfluoride (PSF) was a 3 h, 37°C. b 100 x ingested GRC/attached + ingested GRC. used in order to inhibit the activity of chymoc Mean f S.E. (no. samples). trypsin during or after exposure of the GRC to d Each figure is an average of 3 determinations. Exptl Cell Res 56
330 M. Rabinovitch Table 5. Effect of PSF on the stimulation by chymotrypsin of the ingestion of GRC by macrophages Treatment of GRC’ Experiment I Tris, pH 7.5 Chymotrypsin idem with PSF 2 x 10m4M idem with PSF 1 x 10e6 M PSF, 2 x 1O-4 PSF, 1 x 1O-5
Table 6. Effect of treatment of GRC with methanol on the ingestion-stimulatory activity of proteolytic enzymes (macrophage assay)
Ingestion Ykb
1st treatment of GRCa
9.OkO.6 (4)’ 3;f.i: 2.9 (4)
PBS Methanol 80 % Chymotrypsin, 1 mg/ml PBS
17:s 9.3 10.5
Tris, pH 7.5 PSF. 5 x 1O-4M Chymotrypsin PSF 5 x lO-4 followed by chymotrypsin
14.Ok1.4 (4) 13.5 59.2 57.5
Experiment 3 Tris, pH 7.5 Chymotrypsin idem, followed by PSF 5 x 10e4
11.8 45.7 43.7
a Chymotrypsin, 1 mg/ml, 2 h 37°C. PSF, 2 h, 25°C. In expt 1 enzyme was pretreated with PSF for 30 min prior to the addition of GRC. b Ingested GRC x lOO/attached + ingested GRC. c Mean i S.E. (no. samples). d Each figure is an average of 3 determinations.
2nd treatment of GRC=
PBS PBS PBS chymotrypsin, 1 mg/ml Chymotrypsin, 1 mg/ml methanol, 80 % Methanol 80 % Chymotrypsin Subtilisin 50 pg/ml PBS Subtilisin 50 pg/ml 80 % methanol
Ingestion on per centb 15.1 kO.6’ 15.1 + 1.4 55.5 +2.5 59.1 k2.4 21.5k1.8 17.9 + 1.2 49.8 +2.0 26.1 k2.4
a Treatment with PBS or enzymes, 2 h at 37°C; treatment with methanol, 1 h 5°C. b 100 x ingested GRC/ingested + attached GRC. ’ Mean + S.E. Each mean based on 4 determinations.
No stimulation of GRC ingestion by macrophages was obtained when the red cells were treated for 3 h with 3 to 250 units/ml of neuraminidase.
Ingestion by L2 cells of GRC treated with proteolytic enzymes the enzyme. Table 5 (expt 1) presents the results Table 7 shows that the uptake of GRC was inof an experiment in which chymotrypsin was creased by treatment of the red cells with subtreated with PSF for 30 min prior to the addition tilisin (expt I) or with chymotrypsin (expt II). of GRC. It can be seen that 2 X 1O-4 M PSF Table 7 also shows (expt II) that only minor abolished the stimulatory effect of chymotrypsin. At 1 X 1O-5 some stimulation of ingestion Table 7. Uptake by L2 cells of GRC treated with was obtained. As in this experiment 4 x 1O-5M subtilisin or with chymotrypsin (effect of temchymotrypsin was used, it is assumed that only perature and of PSF) less than 50 % of the enzyme was active. Treat% L2 cells ment of GRC with PSF either prior to (expt 2) % L2 cells with 35 with no ingested or following (expt 3) incubation with chymoTreatment of GRCa ingestion GRC trypsin (table 5) did not inhibit the stimulation by the enzyme of the ingestion of GRC attached Experiment I to macrophages. Tris, pH 7.5 27.7k3.1 (5) 17.2k3.3 (5) 10.8 k 1.6 (5) 35.2k3.1 (5) Table 6 shows that treatment of GRC with Subtilisin 50 ,ug/ml 80 % aqueous methanol, either prior to or fol- Experiment 2 44.6 + 3.2 (6)b 3.2kO.9 (6) lowing incubation with chymotrypsin or with Tris, pH 7.5 CT, 1 mgjml, 36°C 18.2k2.5 (4) 19.7k3.1 (4) subtilisin, inhibited the stimulatory effect of the CT, 1 mg/ml, 2°C 34.1 k4.4 (4) 8.8 + 2.4 (4) enzymes. In another experiment, pretreatment CT, 1 mg/ml, 36” plus PSF 2 x lo-” MC 37.6 _+5.5 (4) 7.Ok3.3 (4) with 25 % methanol did not influence the effect of chymotrypsin, whereas 50 % methanol in- a 2 h, 36°C. hibited by 30 % the stimulatory action of the ’ Mean k S.E. (no. samples). ’ CT incubated with PSF for 30 min prior to addition of enzyme. GRC. Exptl Cell Res 56
Phagocytosis by macrophages
stimulation of GRC ingestion by L2 cells was obtained when the red cells were incubated at either 2°C with the enzyme alone or at 37°C with the enzyme in the presenceof 2 x lo-* PSF. DISCUSSION Interactions of GRC with macrophages or with L2 cells in the absence of added antibody or complement, were both similarly influenced by modifications of the particle surface. Phagocytosis of GRC treated with purified proteins was reduced. Whereas uptake was increased when erythrocytes were exposed to proteolytic enzymes. Aldehyde-treated erythrocytes have been shown to bind a variety of proteins [l]. We can thus assume that the addition of a protein coat led to diminished interactions of the GRC with macrophages (tables 1, 2) and with L2 cells (table 3). The attachment to macrophages of GRC coated with ovalbumin was found to depend on the medium employed. More attachment was obtained in MEM or in MEM-FBS than in simpler salines, whether or not the latter contained divalent cations (table 2). The reason for this difference is not clear at present. Data from table 3 confirm and extend the results of prior experiments with L2 cells and formaldehyde-chicken erythrocytes, treated with ovalbumin, bovine albumin or hemoglobin [12]. It is of interest that proteins of diverse isolectric points (below 5.0 for bovine albumin or ovalbumin; above 10.0 for cytochrome c or lysozyme), were able to similarly influence the interaction of GRC with the phagocytes. We assume that, in the absenceof antibody and complement, macrophages and L2 cells “recognize” the cross-linked or denatured [5] surface protein of GRC. The surface of GRC treated with the native proteins would not be “recognized” by the phagocytes. In the absence of information on the binding sites for the proteins on the GRC surface, on the GRC sites that interact with the phagocytes or on the kinds of adhesion involved in the interaction [2] it is not possible to choose between the following mechanisms [12]. The proteins
and L2 cells
331
may cover the GRC sites that interact with the phagocytes, or (2) the protein coat may change some overall property of the GRC, e.g. charge, in such a way as to inhibit particle attachment [2]. Thus, the protein coat added to the GRC may be analogous to the anti-phagocytic capsules of several bacteria [l I]. This analogy is strengthened by the finding that ovalbumin coated GRC do attach to macrophages in the presence of antiserum to ovalbumin (unpublished). Phagocytosis of GRC by both macrophages and L2 cells was stimulated by the treatment of the particles with proteolytic enzymes (table 4 to 7). In view of the high doses and duration of treatment with the enzymes, experiments were performed with macrophages and chymotrypsin treated GRC in order to examine the following possibilities: (1) The particle-bound enzymes acted as opsonins; (2) The surface of the phagocytes was acted upon by enzyme bound to the particles, leading to the labilization of the membrane, or to the liberation of some “kinin”like material that would trigger red cell ingestion; or (3) The GRC surface was digested, with exposure of a component that would better interact with the phagocytes than with the surface of untreated GRC. The first alternative implies that in analogy to some basic polypeptides [l 11, chymotrypsin as a protein could have opsonizing activity. This alternative does not gain support from the ineffectiveness of chymotrypsinogen, a protein that shares several common features with its derivative, chymotrypsin [14]. The second hypothesis does not fit with the unhindered stimulation of phagocytosis when the chymotrypsin inhibitor, PSF [3] was applied after exposure of the erythrocytes to chymotrypsin (table 5). The third possibility is consistent with the results of the temperature study (fig. 2), and with the outcome of experiments in which GRC were treated with chymotrypsin in the presence of PSF (table 5). Results of phagocytosis by L2 cells of GRC treated with chymotrypsin at 2°C or with the enzyme at 37°C in the presence of PSF, (table 7) are in agreement with more extensive data obtained Exptl Cell Res 56
332 M. Rabinovitch with macrophages. Proteolytic enzymes may thus remove some as yet unidentified “antiphagocytic” material from the surface of the particles. The analogy with bacterial systems again seems pertinent. Enzymatic removal of protein or polysaccharide capsular components of certain bacteria leads to increased phagocytosis in the absence of antibody [I I]. Proteolytic enzymes liberate neuraminic acid containing material from fresh or from aldehydetreated erythrocytes [13]. In the present experiments, exposure of GRC to neuraminidase had no effect on the uptake of GRC. This finding indicates that the stimulation of GRC ingestion by proteolytic enzymes is not mediated by the removal of neuraminic acid. Proteolytic enzymes did not stimulate GRC ingestion by macrophages when the particles were treated with methanol either prior to or following incubation with the enzymes (table 6). The membrane component uncovered by the proteolytic enzymes is therefore susceptible to methanol. Whether the solvent extracted some membrane component, conceivably a phospholipid [6], or whether it denatured or otherwise
Exptl Cell Res 56
modified a protein, requires further investigation. The author is grateful to Dr Z. A. Cohn for helpful discussion and to Miss Mary Jo de Stefano for dedicated technical assistance. This work was supported by grants Al 07012 and Al 01831 from National Institute of Allergy and Infectious Diseases, National Institutes of Health.
REFERENCES 1. Bing, D H, Weygand, J G M & Stavitsky, A B, Proc sot exptl biol med 124 (1967) 1166. 2. Curtis, A S G, The cell surface, p. 113. Academic Press, New York (1967). 3. Fahmey, D E & Gold, A M, J Am them sot 85 (1963) 997. 4. Hummel, B C W, Can j biochem physiol 37 (1959) 1393. 5. Lenard, J & Singer, S J, J cell biol 37 (1968) 117. 6. Napolitano, L, Lebaron, F & Scaletti, J, J cell bio134 (1967) 817. 7. Rabinovitch, M, Exptl cell res 46 (1967) 19. 8. -J immuno199 (1967) 1115. 9. - J cell biol 35 (1967) 108a. Abstract. 10. - Proc. sot exptl biol med 127 (1968) 351. 11. - Seminars in hematol 5 (1968) 134. 12. - Exptl cell res 54 (1969) 210. 13. Seaman, G V F & Cook, G M W, Cell electrophoresis (ed E J Ambrose) p. 48. Little, Brown & Co, Boston (1965). 14. Stryer, L, Ann rev biochem 37 (1968) 25. Received November 25, 1968
PHAGOCYTOSIS
OF MODIFIED
BY MACROPHAGES
ERYTHROCYTES
AND L2 CELLS
M. RABLNOVITCH The Rockefeller University, New York, N. Y. 10021, USA
SUMMARY Glutaraldehyde-treated horse red cells (GRC) are taken up by mouse peritoneal macrophages and by L2 mouse “fibroblasts” in the absence of added antibody or complement. The surface of GRC was modified in order to examine their interaction with the two cell types. Coating of the erytbrocytes with ovalbumin, bovine albumin, lysozyme or cytochrome c, inhibited both attachment to and ingestion by, macrophages; and ingestion by L2 cells. Digestion of GRC with chymotrypsin, pronase, subtilisin or papain, stimulated their uptake by macrophages and L2 cells. Chymotrypsinogen was ineffective. The stimulation by chymotrypsin was dependent on enzyme concentration, temperature, and was inhibited by the addition of phenylmethyl sulfonyl fluoride (PSF). Chymotrypsin was stimulatory when GRC were treated with PSF either before or after digestion by the enzyme. Treatment of the GRC with 80 % methanol prior to or following exposure to chymotrypsin, reduced the stimulation of phagocytosis. The parallel behavior of macrophages and L2 cells supports a common mechanism of non-immunological “recognition” by the two cell types.
Phagocytosis of certain particles by macrophages or leucocytes can take place in the absenceof serum antibody and complement. No generalization can be made at present of the characteristics of the particles that permit this form of “recognition”, nor is it known whether separate phagocytic receptors are available for different kinds of particles. When serum factors participate in the recognition, it may be assumed that specific sites of immunoglobulin molecules or on complement interact with appropriate phagocytic receptors [l 11.It appears that different receptors mediate the attachment to phagocytes of particles that have or have not been coated with antibody
WI. Little information is available on particle uptake by cells other than macrophages and leucocytes. We have found that mouse L2 fibroblasts ingested the sameparticles that were taken up by macrophages in the absence of serum, namely, aldehyde-treated or heat denatured erythrocytes, iron, alumina, glass or polystyrene latex beads ([9] and unpublished). However, Exptl Cell Res 56
immunoglobulin G antibody inhibited the uptake of the aldehyde treated erythrocytes by L2 cells. This was taken to indicate that L2 cells, in contrast to macrophages, do not possess receptors for the antibody [12]. Particle uptake that bypassesthe requirement for antibody or complement may represent a more primitive form of recognition which is shared by a variety of cell types. Macrophages and leucocytes would in addition possess receptors for particle-bound antibody and complement [9]. One way of testing this hypothesis is to compare the uptake of different particulates, in the presenceor absenceof serum factors, by a variety of phagocytic cells. The present report makes use of glutaraldehyde-treated horse red cells (GRC) to examine particle uptake by macrophages and L2 cells in the absence of added antibody or complement. It will’be shown that the two cell types responded similarly to two modifications of the GRC surface. The uptake of particles coated with protein was reduced, whereas treatment of GRC
Phagocytosis by macrophages and L2 cells
with proteolytic enzymes increased their uptake. The stimulation of phagocytosis of proteolytic enzyme treated GRC was related to the digestion of a surface component.
MATERIAL
AND METHODS
Media Buffered saline (PBS) was prepared by adding 1 vol of 0.07 M phosphate buffer pH 7.3 to 10 vol of 0.85 % sodium chloride. Fetal bovine serum (FBS), Gey, Puck saline A, tissue culture medium 199, Eagles minimum essential medium for stationary (MEM) or for suspension (SMEM) cultures were obtained from Grand Island Biological Co.
Chemicals Phenylmethyl sulfonyl fluoride (PSF), obtained from Sigma, was dissolved in “Tris” buffer pH 7.5, with the aid of methanol. The 1 x 1O-s M solution contained less than 5 % methanol.
Proteins and enzymes The following crystallized preparations were used (unless otherwise stated the data are those supplied by the manufacturers). Bovine plasma albumin (Armour Pharmaceutical Co.); ovalbumin (Worthington Biochem. Corp.); lysozyme (General Biochemicals); cytochrome c (horse heart, type III). o-Chymotrypsinogen (type II, 26.5 benzoyl-L tyrosine ethyl ester-BTEE-units/mg protein after activation; 0.03 units/mg protein prior to activation), papain (1 mg split 4.0 PM NH3/min from benzoylt-arginine amide at pH 6.0 at 37”C), and subtilisin (subtilopeptidase A, type VII; 1 mg liberated 1.6 mg tyrosine/min from casein at pH 7.5, 37”C), from Sigma. One sample of a-chymotrypsin was obtained from Mann Research Laboratories and assayed 7000 units/mg towards N-acetyl+tyrosine ethyl ester. Three other samples of chymotrypsin were obtained from Sigma on different occasions, with a stated activity between 40 and 49 BTEE units/mg. One sample of chymotrypsin assayed in this laboratory by the method of Hummel [4], hydrolyzed 64 PM of BTEE per min/mg protein at 25°C. The activity was entirely inhibited when the chymotrypsin (1 mg) was pretreated for 20 min with 1 x 1O-4M phenylmethyl sulfonyl fluoride in PBS. Pronase (B grade, Calbiochem) and V. cholerae neuraminidase (Hoechst Pharmaceutical Co.) were semi-purified preparations.
327
Treatment of GRC with proteins Bovine albumin or ovalbumin were dissolved in 0.1 M acetate buffer pH 5.0. Lysozyme or cytochrome c were dissolved in 0.07 M phosphate buffer pH 7.3. Five x 10’ GRC were incubated with 2 ml of the protein solutions for 40 min at 37°C and washed three times in PBS. Control GRC were treated with buffers alone.
Treatment of GRC with enzymes or with chymotrypsinogen Five times 10’ GRC in 50 ~1 PBS were added to solutions (2 ml) of trypsin, chymotrypsin, subtilisin, pronase or chymotrypsinogen in “Tris” 0.05 M, pH 7.5. The medium for papain was acetate 0.1 M with 0.001 M EDTA and 0.005 M dithiotbreitol (Calbiochem), and that for neuraminidase was acetate 0.1 M pH 6.0, with 0.01 M CaCl,. GRC were incubated for 2 to 4 h at 36°C and washed three times in PBS. Controls were treated with the enzyme diluents alone. In some experiments, treatment with chymotrypsin was done in the presence of PSF. The inhibitor was incubated with the enzyme for 30 min in PBS at room temperature prior to the addition of GRC. In other experiments, GRC were treated with solutions of PSF in PBS for 2 to 4 h either prior to or following exposure to chymotrypsin.
Treatment of GRC with methanol GRC were suspended in 25 % cold methanol and the concentration of methanol increased to 80 % by dropwise addition. After 1 h at 5”C, the GRC were washed three times with PBS.
Collection of peritoneal macrophages [8] The macrophages were used without cultivation.
Attachment of GRC to macrophages Macrophages attached to coverglasses were rinsed in PBS and overlaid for 20 min at 31°C with 0.1 ml of a suspension of 5000 to 20,000 GRC/,ul in PBS [lo]. The macrophages were washed free of unattached GRC, fixed, stained, and the % of cells with attached GRC was obtained -by scoring 200 or more successive phagocytes. Alternatively, the number of GRC attached to 200 consecutive macrophages (i.e. macrophages with zero, 1,2 or more GRC) was scored and the average attachment expressed as GRC/macrophage. In some experiments GRC were suspended in Gey, Puck saline A, or MEM-10% FBS.
Animals Mice of the NCS strain, Rockefeller University colony, of both sexes, 20-25 g body weight, were used as a source of macrophages.
Red cells Horse erythrocytes (Animal Blood Centre, Syracuse, N. Y.) were washed three times with PBS and treated with glutaraldehyde as previously described [7].
Ingestion assay for macrophages [8] After attachment of GRC (8000 per yl) as above, macrophages were incubated in medium 199 for 20 min at 35°C in a 10% CO,-air atmosnhere. The monolavers were rinsed, fixed, stained, and 200 or more GRC were scored as either attached or ingested [7j. Percent ingestion = ingested GRC x lOO/attached + ingested GRC. a ingestion % = ingestion % of enzyme-treated GRC, minus ingestion % of control GRC. Exptl Cell Res 56
328 M. Rabinovitch
Table 2. Attachment of ovalbumin-treated GRC
Single step phagocytosis assay for macrophages
to macrophages in different media
Macrophages on coverglasses were incubated for 30 min at 35°C in one ml of MEM or MEM-10 % FBS, with the addition of 1000 CRC/p1 in a 10 % CO,-air atmosphere. The macrophages were rinsed, fixed, stained, and the % ingestion of GRC determined as above.
Cultivation of L2 cells Stocks were obtained from Dr S. Dales (Public Health Research Institute, New York) and Dr L. S. Sturman (Rockefeller University). The cells were grown in suspension in S-MEM with 10 % FBS, containing 20,000 units penicillin, 10 mg streptomycin and 1 ml tylosin tartrate (Grand Island Biological Co.) per 100 ml. For the phagocytosis experiments, the L2 cells were suspended in MEM10 % FBS and 1 x 104 cells in 3 ml were dispensed in 20 x 110 mm Leighton type tissue culture tubes containing one round 18 mm coverglass [12]. Cells were grown overnight prior to incubation with GRC. All media were gassed with 10% COz in air. Phagocytosis was found to be less active when the tubes were seeded with cells exposed to trypsin.
Phagocytosis assay for L2 cells [12] CRC were added to the Leighton tubes to a final concentration of 1 x 1Oaper ,UI. & each experiment, control and treated GRC were adjusted to the same concentration. After 4 h incubation at 37°C with gentle shaking every hour, coverglasses were rinsed in medium 199 containing 1 % FBS, fixed and stained with Giemsa. Two hundred or more successive L2 cells were scored and the % of L2 containing ingested GRC was determined. In some experiments, the L2 cells were also scored as to their content of 0, l-2, 3-4 and 5 or more GRC and the results expressed as per cent of L2 with stated numbers of ingested GRC. Only the data for the first and the last classes are shown in the tables. It should be noted that no agglutinating antibody against fresh or aldehydetreated erythrocytes was detected in the FBS used. Furthermore the FBS contained no detectable hemolytic complement.
Table 1. Attachment of protein treated GRC to macrophages Treatment of GRC
Attachment=
PBS, pH 7.2 Acetate, pH 5.0 Bovine albumin, 25 mg/ml, pH 5.0 Ovalbumin, 10 mg/ml, pH 5.0 Ovalbumin, 20 mg/ml Lysozyme, 2 mg/ml, pH 7.2 Lysozyme, 10 mg/ml Cytochrome c, 2 mg/ml, pH 7.2
lOOk5.4 (5)b 100+ 8.4 (5) 5.6kO.13 (4) 17.7k3.1 (4) 7.OF0.7 (4) 22.Ok3.3 (4) 12.9 k4.6 (4) 28.2kll.l (4)
a In % of attachment of GRC treated with the buffers alone. Actual values for the % of macrophages with GRC attachment were 78.0 k4.2 and 76.Ok6.4 for the PBS and acetate groups respectively. b Mean + S.E. (no. of samples). Exptl Cell Res 56
Medium
Attachmenta
PBS Gey Puck A MEM MEM-10 % FBS
1.8 i0.7b 2.120.8 2.6* 1.5 19.2k5.3 38.5 + 10.7
a Attachment of ovalbumin-GRC in % of the attachment of control GRC. Ovalbmnin: 50/mg/ml, 1 h, pH 5.0. b Mean I S.E. Averages of 4 replicates.
RESULTS Attachment to macrophages of GRC with purified proteins
Table 1 shows that the attachment in PBS was markedly reduced when GRC were treated with bovine albumin, ovalbumin, lysozyme or with cytochrome c. GRC coated with ovalbumin were used to examine attachment in different media (table 2). Attachment was minimal in PBS, Gey or Puck’s saline A (a Ca2+,Mg”+-free medium), but significantly more extensive in MEM or in MEM-10 % FBS. Uptake by macrophages of GRC treated with ovalbumin
The extent of the interaction of coated GRC with macrophages in MEM or in MEM-10% FBS, permitted the study of ingestion in a “single step” assay. In one experiment, ingestion per cent of control GRC was 15.4+ 3.4 in MEM and 29.5k3.5 in MEM-10% FBS. The figures for GRC treated with 50 mg/ml ovalbumin were, respectively, 1.5k 0.6, and 4.7 + 0.9 (averages of 4 samples + standard error). Therefore, the ingestion of ovalbumin coated GRC by macrophages was also markedly inhibited. Uptake by L2 cells of GRC treated with purified proteins
Table 3 presents the results of an experiment in which GRC were incubated with several proteins prior to uptake by L2 cells. The table shows that treatment with the proteins decreased
Phagocytosis by macrophages and L2 cells
329
Table 3. Uptake by L2 cells of GRC treated with protein
Treatment of GRC
% of L2 without ingested GRC
PBS= 14.6 k 1.6 (7)b Ovalbumin, 30 mg/ml 52.6 + 3.0 (5) Bovine albumin, 30 mg/ml 52.2 f 6.0 (4) Lysozyme, 15 mg/ml 52.7 k4.5 (4)
% of L2 with 5 or more ingested GRC 41.713.8 (7) 2.2kl.l (5) 6.7 + 2.4 (4) 9.0 + 2.9 (4)
a Ingestion of GRC treated with acetate was not significantly different from the values given. b Mean +_ S.E. (no. samples).
the proportion of L2 cells that ingested GRC and reduced the percentage of cells that ingested 5 or more GRC.
Fig. 1.
Fig. 2.
Abscissa: pg/ml; ordinate: A ingestion %. Fig. 1. Relationship between chymotrypsin concentration used to treat GRC (4 h, 379, and the stimulation of the ingested of GRC attached to macrophages. A ingestion % = ingestion % of chymotrypsin-treated GRC, minus ingestion % of control GRC. Abscissa: Hours; ordinate: A ingestion %. Fig. 2. Time course of the chymotrypsin effect on GRC ingestion. Treatment of the GRC with chymotrypsin (1.1 mg/ml) at different temperatures. Ordinates as in fig. 1.
Ingestion by macrophages of GRC treated with chymotrypsinogen or with enzymes
this experiment. Results are corrected for the ingestion of untreated GRC. It can be seen that Table 4 shows that several proteolytic enzymes stimulated the ingestion of GRC attached to there is a clear log dose- effect relationship. In macrophages. The GRC were treated for 3 h view of the possibility that chymotrypsin mowith the enzymes and washed prior to their dified the attachment of GRC and thus could attachment to the phagocytes. Ingestion then have influenced the ingestion assays,attachment proceeded in medium 199. All of the proteolytic to macrophages of the same GRC preparations enzymes tested were stimulator-y. Chymotryp- used in the experiments shown in fig. 1 was studied. CRC were used at 15,000per ,~l in PBS, sinogen was inactive. attached for 12 min at 31°C and the number of In fig. 1, the ingestion of GRC attached to GRC bound to 200 macrophages was determacrophages is plotted against the dose of chymotrypsin used to pretreat the erythrocytes. mined. Attachment in GRC/macrophage -t standCRC were incubated with the enzyme for 4 h in ard error (no. samples) was 1.18? 0.10 [7]; 1.24kO.03 [5]; 1.14kO.14 [5]; 1.4510.09 [5] Table 4. Ingestion by macrophages of GRC and 1.20$0.04 [5], respectively, for control treated with chymotrypsinogen or with proteolytic GRC and for GRC treated with 100,250,500 pg and 1 mg/ml chymotrypsin. Thus, treatment of enzymes GRC with chymotrypsin did not significantly Treatment of GRC’ Dose (ug/ml) Ingestion %b influence their attachment to macrophages. Fig. 2 presents the time course of the effect “Tris” pH 7.5 8.5Ll.l (5)’ of chymotrypsin on GRC at three different Go 40.5d Chymotrypsin Subtilisin 100 39.0 temperatures. Stimulation of the GRC ingestion Papain (pH 6, EDTA was clearly dependent on the temperature. The DW 250 40.8 Pronase 100 35.2 time course at 37°C departs markedly from Chymotrypsinogen 500 6.8 linearity after 1 h treatment with the enzyme. Phenyl-methyl sulfonylfluoride (PSF) was a 3 h, 37°C. b 100 x ingested GRC/attached + ingested GRC. used in order to inhibit the activity of chymoc Mean f S.E. (no. samples). trypsin during or after exposure of the GRC to d Each figure is an average of 3 determinations. Exptl Cell Res 56
330 M. Rabinovitch Table 5. Effect of PSF on the stimulation by chymotrypsin of the ingestion of GRC by macrophages Treatment of GRC’ Experiment I Tris, pH 7.5 Chymotrypsin idem with PSF 2 x 10m4M idem with PSF 1 x 10e6 M PSF, 2 x 1O-4 PSF, 1 x 1O-5
Table 6. Effect of treatment of GRC with methanol on the ingestion-stimulatory activity of proteolytic enzymes (macrophage assay)
Ingestion Ykb
1st treatment of GRCa
9.OkO.6 (4)’ 3;f.i: 2.9 (4)
PBS Methanol 80 % Chymotrypsin, 1 mg/ml PBS
17:s 9.3 10.5
Tris, pH 7.5 PSF. 5 x 1O-4M Chymotrypsin PSF 5 x lO-4 followed by chymotrypsin
14.Ok1.4 (4) 13.5 59.2 57.5
Experiment 3 Tris, pH 7.5 Chymotrypsin idem, followed by PSF 5 x 10e4
11.8 45.7 43.7
a Chymotrypsin, 1 mg/ml, 2 h 37°C. PSF, 2 h, 25°C. In expt 1 enzyme was pretreated with PSF for 30 min prior to the addition of GRC. b Ingested GRC x lOO/attached + ingested GRC. c Mean i S.E. (no. samples). d Each figure is an average of 3 determinations.
2nd treatment of GRC=
PBS PBS PBS chymotrypsin, 1 mg/ml Chymotrypsin, 1 mg/ml methanol, 80 % Methanol 80 % Chymotrypsin Subtilisin 50 pg/ml PBS Subtilisin 50 pg/ml 80 % methanol
Ingestion on per centb 15.1 kO.6’ 15.1 + 1.4 55.5 +2.5 59.1 k2.4 21.5k1.8 17.9 + 1.2 49.8 +2.0 26.1 k2.4
a Treatment with PBS or enzymes, 2 h at 37°C; treatment with methanol, 1 h 5°C. b 100 x ingested GRC/ingested + attached GRC. ’ Mean + S.E. Each mean based on 4 determinations.
No stimulation of GRC ingestion by macrophages was obtained when the red cells were treated for 3 h with 3 to 250 units/ml of neuraminidase.
Ingestion by L2 cells of GRC treated with proteolytic enzymes the enzyme. Table 5 (expt 1) presents the results Table 7 shows that the uptake of GRC was inof an experiment in which chymotrypsin was creased by treatment of the red cells with subtreated with PSF for 30 min prior to the addition tilisin (expt I) or with chymotrypsin (expt II). of GRC. It can be seen that 2 X 1O-4 M PSF Table 7 also shows (expt II) that only minor abolished the stimulatory effect of chymotrypsin. At 1 X 1O-5 some stimulation of ingestion Table 7. Uptake by L2 cells of GRC treated with was obtained. As in this experiment 4 x 1O-5M subtilisin or with chymotrypsin (effect of temchymotrypsin was used, it is assumed that only perature and of PSF) less than 50 % of the enzyme was active. Treat% L2 cells ment of GRC with PSF either prior to (expt 2) % L2 cells with 35 with no ingested or following (expt 3) incubation with chymoTreatment of GRCa ingestion GRC trypsin (table 5) did not inhibit the stimulation by the enzyme of the ingestion of GRC attached Experiment I to macrophages. Tris, pH 7.5 27.7k3.1 (5) 17.2k3.3 (5) 10.8 k 1.6 (5) 35.2k3.1 (5) Table 6 shows that treatment of GRC with Subtilisin 50 ,ug/ml 80 % aqueous methanol, either prior to or fol- Experiment 2 44.6 + 3.2 (6)b 3.2kO.9 (6) lowing incubation with chymotrypsin or with Tris, pH 7.5 CT, 1 mgjml, 36°C 18.2k2.5 (4) 19.7k3.1 (4) subtilisin, inhibited the stimulatory effect of the CT, 1 mg/ml, 2°C 34.1 k4.4 (4) 8.8 + 2.4 (4) enzymes. In another experiment, pretreatment CT, 1 mg/ml, 36” plus PSF 2 x lo-” MC 37.6 _+5.5 (4) 7.Ok3.3 (4) with 25 % methanol did not influence the effect of chymotrypsin, whereas 50 % methanol in- a 2 h, 36°C. hibited by 30 % the stimulatory action of the ’ Mean k S.E. (no. samples). ’ CT incubated with PSF for 30 min prior to addition of enzyme. GRC. Exptl Cell Res 56
Phagocytosis by macrophages
stimulation of GRC ingestion by L2 cells was obtained when the red cells were incubated at either 2°C with the enzyme alone or at 37°C with the enzyme in the presenceof 2 x lo-* PSF. DISCUSSION Interactions of GRC with macrophages or with L2 cells in the absence of added antibody or complement, were both similarly influenced by modifications of the particle surface. Phagocytosis of GRC treated with purified proteins was reduced. Whereas uptake was increased when erythrocytes were exposed to proteolytic enzymes. Aldehyde-treated erythrocytes have been shown to bind a variety of proteins [l]. We can thus assume that the addition of a protein coat led to diminished interactions of the GRC with macrophages (tables 1, 2) and with L2 cells (table 3). The attachment to macrophages of GRC coated with ovalbumin was found to depend on the medium employed. More attachment was obtained in MEM or in MEM-FBS than in simpler salines, whether or not the latter contained divalent cations (table 2). The reason for this difference is not clear at present. Data from table 3 confirm and extend the results of prior experiments with L2 cells and formaldehyde-chicken erythrocytes, treated with ovalbumin, bovine albumin or hemoglobin [12]. It is of interest that proteins of diverse isolectric points (below 5.0 for bovine albumin or ovalbumin; above 10.0 for cytochrome c or lysozyme), were able to similarly influence the interaction of GRC with the phagocytes. We assume that, in the absenceof antibody and complement, macrophages and L2 cells “recognize” the cross-linked or denatured [5] surface protein of GRC. The surface of GRC treated with the native proteins would not be “recognized” by the phagocytes. In the absence of information on the binding sites for the proteins on the GRC surface, on the GRC sites that interact with the phagocytes or on the kinds of adhesion involved in the interaction [2] it is not possible to choose between the following mechanisms [12]. The proteins
and L2 cells
331
may cover the GRC sites that interact with the phagocytes, or (2) the protein coat may change some overall property of the GRC, e.g. charge, in such a way as to inhibit particle attachment [2]. Thus, the protein coat added to the GRC may be analogous to the anti-phagocytic capsules of several bacteria [l I]. This analogy is strengthened by the finding that ovalbumin coated GRC do attach to macrophages in the presence of antiserum to ovalbumin (unpublished). Phagocytosis of GRC by both macrophages and L2 cells was stimulated by the treatment of the particles with proteolytic enzymes (table 4 to 7). In view of the high doses and duration of treatment with the enzymes, experiments were performed with macrophages and chymotrypsin treated GRC in order to examine the following possibilities: (1) The particle-bound enzymes acted as opsonins; (2) The surface of the phagocytes was acted upon by enzyme bound to the particles, leading to the labilization of the membrane, or to the liberation of some “kinin”like material that would trigger red cell ingestion; or (3) The GRC surface was digested, with exposure of a component that would better interact with the phagocytes than with the surface of untreated GRC. The first alternative implies that in analogy to some basic polypeptides [l 11, chymotrypsin as a protein could have opsonizing activity. This alternative does not gain support from the ineffectiveness of chymotrypsinogen, a protein that shares several common features with its derivative, chymotrypsin [14]. The second hypothesis does not fit with the unhindered stimulation of phagocytosis when the chymotrypsin inhibitor, PSF [3] was applied after exposure of the erythrocytes to chymotrypsin (table 5). The third possibility is consistent with the results of the temperature study (fig. 2), and with the outcome of experiments in which GRC were treated with chymotrypsin in the presence of PSF (table 5). Results of phagocytosis by L2 cells of GRC treated with chymotrypsin at 2°C or with the enzyme at 37°C in the presence of PSF, (table 7) are in agreement with more extensive data obtained Exptl Cell Res 56
332 M. Rabinovitch with macrophages. Proteolytic enzymes may thus remove some as yet unidentified “antiphagocytic” material from the surface of the particles. The analogy with bacterial systems again seems pertinent. Enzymatic removal of protein or polysaccharide capsular components of certain bacteria leads to increased phagocytosis in the absence of antibody [I I]. Proteolytic enzymes liberate neuraminic acid containing material from fresh or from aldehydetreated erythrocytes [13]. In the present experiments, exposure of GRC to neuraminidase had no effect on the uptake of GRC. This finding indicates that the stimulation of GRC ingestion by proteolytic enzymes is not mediated by the removal of neuraminic acid. Proteolytic enzymes did not stimulate GRC ingestion by macrophages when the particles were treated with methanol either prior to or following incubation with the enzymes (table 6). The membrane component uncovered by the proteolytic enzymes is therefore susceptible to methanol. Whether the solvent extracted some membrane component, conceivably a phospholipid [6], or whether it denatured or otherwise
Exptl Cell Res 56
modified a protein, requires further investigation. The author is grateful to Dr Z. A. Cohn for helpful discussion and to Miss Mary Jo de Stefano for dedicated technical assistance. This work was supported by grants Al 07012 and Al 01831 from National Institute of Allergy and Infectious Diseases, National Institutes of Health.
REFERENCES 1. Bing, D H, Weygand, J G M & Stavitsky, A B, Proc sot exptl biol med 124 (1967) 1166. 2. Curtis, A S G, The cell surface, p. 113. Academic Press, New York (1967). 3. Fahmey, D E & Gold, A M, J Am them sot 85 (1963) 997. 4. Hummel, B C W, Can j biochem physiol 37 (1959) 1393. 5. Lenard, J & Singer, S J, J cell biol 37 (1968) 117. 6. Napolitano, L, Lebaron, F & Scaletti, J, J cell bio134 (1967) 817. 7. Rabinovitch, M, Exptl cell res 46 (1967) 19. 8. -J immuno199 (1967) 1115. 9. - J cell biol 35 (1967) 108a. Abstract. 10. - Proc. sot exptl biol med 127 (1968) 351. 11. - Seminars in hematol 5 (1968) 134. 12. - Exptl cell res 54 (1969) 210. 13. Seaman, G V F & Cook, G M W, Cell electrophoresis (ed E J Ambrose) p. 48. Little, Brown & Co, Boston (1965). 14. Stryer, L, Ann rev biochem 37 (1968) 25. Received November 25, 1968