Studies on phagocytosis of unopsonized rabbit erythrocytes by human monocytes

113,25 l-260 (1988)

CELLULARIMMUNOLOGY

Studies on Phagocytosis of Unopsonized Rabbit Erythrocytes by Human Monocytes’

Division ofMolecular Immunology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, California 92037 Received June I I, 1987;accepted November 16, 1987 Monolayers of freshly isolated human monocytes are known to ingest particulate activators of the human alternative complement pathway. The ingestion of rabbit erythrocytes, Ex, by human monocytes in serum-free medium was studied. The processis Mg’+-dependent and optimum phagocytic activity was obtained at -20 mMMgC1,. Preincubation of mononuclear leukocytes increased the number of monocytes ingesting Ea by at least twofold and this involved de novo protein synthesis, as evidenced by inhibition with cycloheximide. However, preincubation of the mononuclear leukocytes for longer periods (14 hr) caused a decreasein the percentage of mgesting monocytes. No inhibition of ingestion of ER was observed by cobra venom factor (CVF) or F(ab’), rabbit anti-human C3 or F(ab’)z murine monoclonal anti-human Bb, known 1.0 inhibit C3 convertase activity. The ingestion was also not inhibited by (a) rabbit antihuman CR 1, (b) OKM 1 or anti-MOl, two monoclonal anti-CR3 antibodies, (c) goat anti-human IgG F, receptor, or(d) mannan, a competitive inhibitor of ligand uptake by the mannosylfucosyl Ireceptor(MFR). In contrast. ingestion was inhibited by glucan particles of yeast. o 1988 Academc

Press. Inc.

INTRODUCTION Particles opsonized with IgG or fragments of C3 are known to adhere to or to be ingested by human monocytes (l-3). In 1978, Czop and co-workers reported that particulate activators of the human alternative complement pathway, e.g., zymsoan particles, rabbit erythrocytes (ER),2mouse erythrocytes, and desiaylated sheeperythrocytes, can be ingested by human monocytes without prior opsonization (4, 5). Ezekowitz et al. (6) have observed that binding and ingestion of unopsonized zymo’ This is Publication 45 lo-IMM from the Department of Immunology, Research Institute of Scripps Clinic. This investigation was supported by U.S. Public Health Service Grants Al 17354, CA 27489, and HL 16411. * Abbreviations used: En, rabbit erythrocytes; CR3, complement receptor type 3; MFR, mannosylfucosyl receptor: CVF, cobra venom factor: IgG, immunoglobulin G; EDTA, ethylenediaminetetraacetate; HBSS, Hanks’ balanced salt solution; supplemented RPM1 1640, RPM1 1640 supplemented with 2 mA4 L-glutamine, 1 &sodium pyruvate, penicillin, streptomycin mixture (25 units of penicillin G and 25 pg of streptomycin sulfate), and 450 mM NaHCO,; BSA, bovine serum albumin; CNBr, cyanogen bromide; PBS, sodium phosphate-buffered saline, pH 7.2; EnC3b. C3b-coated rabbit erythrocytes; EsC3b, C3bcoated sheep erythrocytes; EsC3bi, C3bi-coated sheep erythrocytes; EA, sheep erythrocyte-IgG antibody complex. 251 0008-8749/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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san particles proceed via the macrophage receptor for C3bi (CR3) and the mannosylfucosyl receptor. In these studies binding to CR3 was afforded by macrophage-derived C3 and local opsonization of the zymosan particles. Johnson et al. (7) also suggestedthat monocyte-derived C3 and C3 convertase play a role in the uptake of unopsonized zymosan particles. More recently Ross ef al. (8) have reported that ingestion of unopsonized zymosan particles and rabbit erythrocytes by neutrophils proceeds via binding to the 01chain of CR3, and that this binding site is different from that involved in the binding of C3bi. They also reported that the ingestion of these particles did not require opsonization by cell-derived C3. But Ezekowitz et al. failed to observe any ingestion of unopsonized zymosan by neutrophils as those particles could not be locally opsonized by C3 and C3 convertase which are not known to be produced by neutrophils (9). The purpose of the present study was to investigate the mechanism of ingestion of ERby human monocytes. The results suggestthat neither monocyte-derived complement proteins nor CRl, CR3 (at least the epitopes recognized by OKM 1 and anti-MO I), IgG F, receptor, and the mannosylfucosyl receptors are involved in ERingestion by human monocytes. Part of the results described here have been presented in the form of abstracts (10, 11). MATERIALS AND METHODS Isolation of human monocytes. Freshly drawn blood in EDTA from adult human donors was diluted twofold in sterile 0.9% NaCl, layered on Lymphopaque (Nyegaard and Co., Oslo, Norway), and centrifuged at 800g for 18 min. The mononuclear leukocytes layered at the interphase were collected and washed four times with HBSS without calcium and magnesium (M. A. Bioproducts, Walkersville, MD) at 200g for 10 min. The mononuclear leukocytes were suspendedin supplemented RPM1 1640 (M. A. Bioproducts) + 0.5% BSA (Sigma Chemical Co., St. Louis, MO). Usually 2.5 X lo6 mononuclear leukocytes were adhered to each plastic well ( 16 mm diameter) of a 24well tissue culture tray (Costar, Cambridge, MA) for 1 hr at 37°C in a humidified atmosphere of 95% air and 5% COz. Then, nonadhered cells were washed away with supplemented RPM1 1640 and counted in a hemacytometer. Usually 1O-20% of the mononuclear leukocytes became adherent to the plastic well. Assay of phagocytosis. Phagocytosis was carried out by overlaying monolayers of monocytes with 0.5 ml of ER (5 X 10s/ml) in supplemented RPM1 1640 in the presence of 20 mM MgClz. Incubation was carried out at 37°C for 1 hr in a humidified atmosphere of 95% air and 5% CO*. Initially each well was washed with supplemented RPM1 1640, then with 0.84% NH&l for 4 min to lyse noningested ER, and finally with supplemented RPM1 1640 followed by supplemented RPM1 1640 + 0.5% BSA. To quantify phagocytosis, the monocytes were directly analyzed visually under an inverted phase-contrast microscope (Olympus, Tokyo, Japan). Usually 300-500 monocytes from at least three different microscopic fields were analyzed. The percentage of monocytes ingesting ERand the average number of ERingested by monocytes were determined. The total number of ERingested by monocytes in a particular well was calculated from the total number of monocytes per well, the percentage of ingesting monocytes, and average number of ERper monocyte. In initial experiments, the total number of ERingested in a particular well was also quantitated using “Cr-labeled ER. The two methods gave comparable results.

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Preparation oftarget particles,fi,r ingestion. For ER, rabbit blood was collected in EDTA from New Zealand white rabbits and used fresh or stored at 4°C for 2-3 days. The erythrocytes were washed with supplemented RPM1 1640 before use. For E,C3b, human complement components C3 ( 12) factors B ( 13) and D ( 14) were isolated as(describedpreviously; E&3b were prepared by C3b deposition with nickelstabilized C3 convertase formed with factor B and factor D ( 15, 16). For quantitation of the number of C3 molecules per ER, C3 was radiolabeled with “‘1 (Amersham, Arlington Heights, IL) by the iodogen technique (Pierce Chemical Co., Rockford, IL) ( 17) and a small amount of ‘251-C3was added to the reaction mixture. E&3b contained approximately 2500 molecules ofC3b per ER. EA were prepared by incubating sheep erythrocytes with an equal volume of l/400 dilution of rabbit 7 S anti-sheep hemolysin for 30 min at 37°C and then for another 30 min at 4°C. The cells were washed and stored in gelatin Verona]-buffered saline at 4°C until used. Anti-C3, anti-CR], and anti-IgG F, receptorpolyclonal antibodies, rabbit IgG, and murine Zg(;. Antiserum to purified human C3 ( 12) was raised in rabbits. Antiserum to human CR1 was provided by Dr. R. D. Schreiber (Department of Pathology, Washington University Medical School, St. Louis, MO). Antiserum to IgG F, receptor of human mononuclear phagocytes (18) was provided by Professor C. L. Anderson (Department of Medicine, College of Medicine, Ohio State University, Coiumbus, OH). The immunoglobulin fraction was purified by ammonium sulfate precipitation ant1 subsequent affinity chromatography on protein A-agarose (E. Y. Laboratories Inc., San Mateo, CA) for anti-C3 and anti-CR 1, and by ion-exchange chromatog,raphy on diethylaminoethyl cellulose 52 (Whatman Ltd., Kent, U.K.) for anti-IgG F, receptor. The purified anti-C3, tested by Ouchterlony double diffusion using 1% agarose (w/v) against normal human serum or purified C3, produced a single immunoprecipitation line. Rabbit IgG and murine IgG were obtained from Sigma Chemical Company and Calbiochem (La Jolla. CA), respectively. Anti-Bb monoclonal antibody. Murine monoclonal antibody to human Bb (10D 11-F12) was produced as described previously ( 19). The immunoglobulin fraction was purified by ammonium sulfate precipitation and affinity chromatography on protein A-agarose. Anti-CR3 monoclonal antibodies. Murine monoclonal antibodies OKM 1 and antiMO 1, which bind to the cychain of CR3 (20, 2 1), were purchased from Ortho Diagnostics (Raritan, NJ) and Coulter Immunology (Hialeah, FL), respectively. Preparation and pkjication sf F(ab’)2fragments of anti-C3 and anti-Bb. Purified IgG was digested with pepsin (22) in 100 mM sodium acetate, pH 4.5, for 24 hr at 37°C. The F(ab’)z fragments were purified by gel filtration on Sephacryl S-200 (Pharmacia Fine Chemicals, Piscataway, NJ) and affinity chromatography on protein Aagaroseto remove F, fragments. Purification of’CVF. CVF was purified as described previously (23). Preparation of C3-Sepharose 4B. Eleven milligrams of purified C3 ( 12) was coupled to 1 g of CNBr-activated Sepharose 4B (Pharmacia Fine Chemicals) according to manufacturer’s instructions. The coupling efficiency was >90%. FurtherpuriJication ofF(ah’)2fragment of anti-C3. The F(ab’), fragment of anti-C3 (40 mg) was further purified by affinity chromatography on C3-Sepharose 4B. The antibody was loaded onto a C3-Sepharose column (0.7 X 7.5 cm), preequilibrated with PBS, pH 7.2. The column was washed with PBS, pH 7.2, and the adsorbed

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Time (hr) FIG. I. Kinetics of ingestion of ER by human monocytes. Two different donors (donor A, 0; donor B, 0) were studied. The data shown are the averagesof duplicate determinations.

was eluted with 200 mMglycine-HCl fragment of anti-C3 was 2.5 mg.

protein

buffer, pH 2.5. The yield of the F(ab’)*

RESULTS Kinetic Analysis of Ingestion of ER by Human Monocytes The rate of ingestion of unopsonized ER by human monocytes was studied. Ingestion was found to proceed linearly with time up to 1 hr and then reached a plateau after 1.5 hr (Fig. 1). Visual analysis under the inverted phase microscope revealed that the increase in total number of ingested ER with time was due to simultaneous increases in the percentage of ingesting monocytes and in the average number of ER per monocyte (Table 1). Also, only a fraction of monocytes ingested ERwhile the rest of the monocytes did not ingest any ER. In all further experiments, incubation of human monocytes with ERwas carried out for 1 hr. Eflect qf MgClz Addition of MgC& to the medium greatly increased ingestion (Fig. 2). The increase in the total number of ER ingested in the presence of increasing concentrations of MgC& was also due to the increase in the percentage of ingesting monocytes as well as the average number of ER ingested per monocyte (data not shown). However, at high concentrations of MgCl* (-40 mM), there was a decreasein the total number of ER ingested. In all further experiments, 20 mM MgCl* was added during incuba-

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1

The Percentage of Monocytes Ingesting ER and the Number of ER per Ingesting Monocyte with Progress of Ingestion Time (W

Average percentage of monocytes ingesting ER

A

0.25 0.5 1.0 1.5 2.0 2.5 3.0

12 28 46 38 31 41 32

1.5 2.0 3.5 3.5 3.5 3.5 3.5

B

0.25 0.5 1.0 1.5 2.0 2.5 3.0

6 8 II IO II 10 12

1.0 2.0 2.5 3.0 3.0 3.0 3.5

Donor

Average No. of ER ingested per monocyte

tion of human monocytes with ER. When Es, EsC3b, or EsC3bi were used as target particles, MgCl* did not induce ingestion of these particles. Preincubation qfMononuclear Leukocytes Preincubation of mononuclear leukocytes for 2 hr increased the number of ER ingested two to three times (Fig. 3). This observed increase was due to the increase in the percentage of ingesting monocytes. However, preincubation for longer periods (>4 hr) led to abrogation of the ability of the cells to ingest Ea. When Es, EsC3b, or EsC3bi, we:reused as target particles, preincubation did not induce ingestion of these particles. Preincubation of Peripheral Mononuclear Leukocytes in the Presence of Cycloheximide Mononuclear leukocytes were incubated for 2 hr in the presence or absence of cycloheximide. As is evident from Fig. 4, there was inhibition of ingestion of Ea by monocytes which were obtained from mononuclear leukocytes preincubated in presence of cycloheximide. This indicates that during preincubation, some de novo protein synthesis took place in the mononuclear leukocytes that was necessaryfor ingestion to occur. Using EA as the target particle, there was no inhibition of ingestion of these particles by monocytes obtained from mononuclear leukocytes incubated in the presenceof cycloheximide compared to that incubated in the absenceof cycloheximide. Eflect ofCVF, F(ab’)2Fragments qfAnti-C3 andAnti-Bb, Anti-CR], OKMl anddntiMOl, Anti-IgG F, receptor, Mannan, and Glucan To examine the possibility that monocyte-derived C3 and C3 convertase play any part in the ingestion of unopsonized ER, the effect of CVF, F(ab’), rabbit anti-human

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FIG. 2. Effect of magnesium ion concentration on ingestion of ERby human monocytes. Two different donors (donor C, 0; donor D, 0) were studied. The data shown are the averages of duplicate determinations.

C3, and F(ab’)* murine anti-human Bb (a murine monoclonal antibody known to inhibit C3 convertase activity of C3b, Bb) on ingestion was studied. The effect of antiCR 1, OKM 1, and anti-MO 1, which bind to the (Ychain of CR3, and that of mannan, a competitive inhibitor of ligand uptake by MFR, was also studied. As seen in Table 2, none of the above compounds showed any significant inhibition, suggesting that the ingestion of unopsonized En (a) does not involve opsonization with monocytederived C3; (b) does not proceed via CR 1 or the LYchain of CR3; (c) does not proceed via MFR. However, addition of glucan particles from yeast almost completely inhibited ingestion. Although F(ab’)Zrabbit anti-human C3 did not produce any inhibition of ingestion of unopsonized ER, it inhibited the ingestion of EnC3b. In presence of F(ab’)z rabbit anti-human C3, the percentage of human monocytes ingesting E&3b was reduced and became equal to that of unopsonized En. Although the F(ab’), fragments of the antibodies employed did not produce any inhibition, high concentrations ( 100-400 pg/ml) of the intact antibodies, or nonimmune rabbit or mouse IgG, produced significant but variable inhibition (50-90%) of ingestion. However, a significant difference in the dose response of inhibition in IgG-dependent, opsonin-independent ingestion was obtained using nonimmune rabbit IgG and mouse IgG. In the case of ingestion of EA, maximum inhibition (-85%) was obtained at 50 pg/ml with mouse IgG and maximum inhibition (-90%) was obtained at 25 pg/ml with rabbit IgG. However, in the case of ingestion of En, both mouse and rabbit IgG produced only 60 to 70% inhibition at a much higher dose (200 yg/ml). Apart from that, 5 pg of a polyclonal goat antibody to the IgG F, receptor (18) inhibited (>90%) ingestion of EA but did not inhibit the ingestion of En even in the presence of a lo-fold excessof the antibody (Table 2). These results clearly rule out the possibility that ingestion of En takes place via the F, receptor.

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2

4

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6

MONOCYTES

257

6

Tima of Preincubationfhrl

FIG. 3. Efiect of preincubation of peripheral mononuclear leukocytes on ingestion of ER by human monocytes. F’reincubation was performed in serum-free medium at 37°C. Two different donors (donor E, 0: donor F, Cl) were studied. The values shown are the averagesof duplicate determinations.

DISCUSSION Previouls studies by Czop and co-workers have established that freshly isolated human monocytes contain a trypsin-sensitive recognition mechanism for the phagocytosis of the unopsonized particulate activators of the human alternative complement pathway (4, 5) which involves a /!I-glucan-inhibitable receptor on the monocyte cell surface (24). Other investigators have observed (6,7) that for unopsonized zymosan particles, the ingestion takes place after local opsonization of zymosan by monocytederived C3 and C3 convertase. In the caseof freshly isolated human monocytes, the receptor involved was reported to be CR3, as the processwas inhibited by MO 1 antibody. For monocytes cultured for 7 days, both CR3 and MFR were shown to participate since inhibition was effected by MO 1 antibody or mannan. Although zymosan was ingested by human monocytes, Johnson et al. (7) failed to observe any ingestion of unopsonized ERby human monocytes. In the caseof neutrophils, the ingestion of unopsonized zymosan and ER was reported to be mediated by the 01chain of CR3, but the sitlewas reported to be distinct from that involved in the binding of C3bi, and cell-derived C3 did not play any role in the ingestion (8). However, Ezekowitz et al. failed to observe ingestion of zymosan particles by neutrophils provided they are not exogenously opsonized (9). We observed that in the case of ingestion of unopsonized ER by freshly isolated human peripheral blood monocytes, cell-derived C3 and C3 convertase did not play a role becausethere was no inhibition of ingestion in the presenceof F(ab’)z fragments of anti-Cl, anti-Bb, or CVF (Table 2). However, high concentrations of the corresponding purified intact IgG molecules as well as equivalent amounts of purified nonimmune rabbit and mouse pooled IgG produced variable inhibition of ingestion by 50-90%. It has been clearly demonstrated that the ingestion of ER does not take

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Concenfra~mnof Cyclohexlmide Ipglmll

FIG.4. Inhibition of ingestion ofE, by human monocytes obtained from mononuclear leukocytes preincubated in the presence of cycloheximide. The peripheral mononuclear leukocytes (1 X lO’/ml) were incubated for 2 hr in presence of different concentrations of cycloheximide (2 to 10 pg/ml). More than 95% ofthe cells were viable in the presenceofcycloheximide as determined by trypan blue exclusion. After 2 hr, 25 ~1of mononuclear leukocytes was layered on plastic wells and allowed to adhere for 1 hr in the presenceof cycloheximide (concentration of cycloheximide added was diluted 20-fold during adherence), and then the drug was washed away together with the nonadherent cells before addition of En. Two different donors (donor G, 0; donor H, Cl) were studied. The results shown are the average of duplicate determinations.

place via the F, receptor on monocytes. Antibody to the IgG F, receptor of human mononuclear phagocytes ( 18) does not inhibit the ingestion of En by human monocytes (Table 2). It can only be speculated that high occupancy of F, receptors on monocytes by IgG may hinder approach of ER to its monocyte surface receptor by steric or any other spatial interactions. Neither CR I nor the cychain of CR3, at least the epitopes recognized by OKM 1 and anti-MO 1 antibodies, or MFR was involved in the ingestion of unopsonized En by human monocytes asthe processwas not inhibited by (a) OKM 1 and anti-MO 1, two monoclonal antibodies which react with the a: chain of CR3 and produced inhibition of ingestion of unopsonized zymosan by neutrophils (8); and (b) mannan, which is a competitive inhibitor of ligand uptake by MFR (Table 2). However, the processwas inhibited by glucan particles from yeast. The lack ofinhibition by mannan was in accordance with the fact that freshly isolated human monocytes had very little MFR activity (6). In fact, in the case of ingestion of unopsonized zymosan particles by freshly isolated human monocytes, MFR did not play any part (6). We have also observed that the process required Mg2+ ions, approximately 20 mM Mg*+ was required for optimal phagocytic activity. However, at higher concentrations of MgC12, the ingestion of ER by human monocytes was inhibited (Fig. 2). If no exogenous MgC12was added, very few monocytes (~5%) ingestedEn. This fact might be the reason some other investigators (7) did not observe significant phagocytosis of ERby human monocytes.

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TABLE 2 Effecit of CVF, Antibodies,

ICompound added

Mannan, and Glucan on Ingestion of ER by Human Monocytes”

-

Amount w

Effect (%)

25 50 10 100 20 200 20 5 10 20 5 10 20 5 50 100 1000 IO 100 500

-7 +7 +8 +5 +5 -9 0 -12 +6 +6 0 +8 +8 0 0 +10 +15 ~25 -85 ~98

CVF F(ab’)z anti-C3 F(ab’)z anti-Bb Anti-CR I * OKMl

Anti-MO

1’

Anti-F, recepto@ Mannan C&can

a Compounds and adherent monocytes were incubated for 30 min at 37°C before addition of ER which remained present during ingestion. b Twenty micrograms of rabbit anti-CR1 was present in the incubation mixture: 5 pg of anti-CR1 was able to completely inhibit the rosetting of EsC3b (2500 molecules of C3b per Es) by monocytes. ’ Five micrograms of anti-MO 1 in presence of 5 pg of anti-CR 1 was able to completely inhibit the rosetting of EsC3bi (2500 molecules of C3bi per Es) by monocytes. d Five micrograms of anti-F, receptor was able to inhibit (>90%) the ingestion of EA by monocytes.

The ingestion of unopsonized ER reached a plateau value within 1.5 hr (Fig. 1). which is much longer than that required (20 min) for zymosan particles (4). Preincubation of mononuclear leukocytes for 2 hr increased the percentage of monocytes ingesting ER(Fig. 3) and protein synthesis was necessaryfor ingestion to occur (Fig. 4). Cycloheximide at the concentrations used was not toxic to the mononuclear leukocytes asjudged by exclusion of trypan blue. Further study is in progress to clarify whether protein synthesis is needed in monocytes or lymphocytes or in both cell populations. ACKNOWLEDGMENT The author wishes to thank Dr. Hans J. Muller-Eberhard

for encouragement and support.

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H. J., Science 162,

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3. Fearon, D. T., and Wong, W. W., -innu. Rev. Immunol. 1,243, 1983. 4. Czop, J. K., Fearon, D. T., and A&en, K. F., J. Immunol. 120, 1132, 1978. 5. Czop, J. K., Fearon, D. T., and Austen, K. F., Proc. Natl. Acad. Sci. USA 75,383 1, 1978. 6. Ezekowitz, R. A. B., Sim, R. B., Hill, M., and Gordon, S., J. Exp. Med. 159,244, 1983. 7. Johnson, E., Eskeland, T., and Bertheussen, K., &and. J. Immunol. 19,3 1, 1984. 8. Ross, G. D., Cain, J. A., and Lachmann, P. J., J. Immunol. 134,3307, 1985. 9. Ezekowitz, R. A. B., Sim, R. B., MacPherson, G. G., and Gordon, S., J. Clin. Invest. 76,2368, 1985. 10. Chakravarti, B., Schreiber, R. D., and Muller-Eberhard, H. J., Fed. Proc. 44,533, 1985. Il. Chakravarti, B., Schreiber, R. D., and Muller-Eberhard, H. J., Fed. Proc. 45, 1106, 1986. 12. Tack, B. F., and Prahl, J. W., Biochemistry 15,45 13, 1976. 13. Gotze, O., and Muller-Eberhard, H. J., J. Exp. Med. 134,9Os, 1971. 14. Lesavre, P. H., Hugli, T. E., Esser,A. F., and Muller-Eberhard, H. J., J. Immunol. 123,529, 1979. 15. Fishelson, Z., and Muller-Eberhard. H. J., J. Immunol. 129,2603, 1982. 16. Fishelson, Z., and Muller-Eberhard, H. J., Mol. Immunol. 20,309, 1983. 17. Fraker, P. J., and Speck, J. C., Biochem. Biophys. Rex Commun. 80,849, 1978. 18. Anderson, C. L., Spence,J., Edwards, T. S., and Nusbacher, J., J. Immunol. 134,465, 1985. 19. LaMotte, G. B., Tamer&, J. D., and Muller-Eberhard, H. J., Fed. Proc. 40,963, 1981. 20. Wright, S. D., Rao, P. E., vanvoorhis, W. C., Craigmyle, L. S., Iida, K., Talle, M. A., Westberg, E. F., Goldstein, G., and Silverstein, S. C., Proc. Natl. Acad. Sci. USA 80,5699, 1983. 2 1. Dana, N., Todd, R. F., III, Pitt, J., Springer, T. A., and Amaout, M. A., J. Clin. Invest. 73, 153, 1984. 22. Hudson, L., and Hay, F. C., “Practical Immunology,” 2nd ed., p. 197. Blackwell Scientific Publications, Oxford, 1980. 23. Vogel, C. W., and Muller-Eberhard, H. J., J. Immunol. Methods 73,203, 1984. 24. Czop, J. K., and Austen, K. F., J. Immunol. 134,2588, 1985.