Proc. Natl. Acad. Sci. USAVol. 75, No. 8, pp. 3831-3835, August 1978Cell Biology

Membrane sialic acid on target particles modulates theirphagocytosis by a trypsin-sensitive mechanism on human monocytes

(alternative complement pathway/natural and modified activators/opsonin-independent ingestion)

JOYCE K. Czop, DOUGLAS T. FEARON, AND K. FRANK AUSTEN*Department of Medicine, Harvard Medical School, and the Robert B. Brigham Division of the Affiliated Hospitals Center, Inc., Boston, Massachusetts 02120

Contributed by K. Frank Austen, May 22, 1978

ABSTRACT Monolayers of human peripheral blood mo-nocytes in the absence of exogenous proteins ingest a varietyof natural particulate activators of the human alternativecomplement pathway. Shee erythrocytes, which do not ordi-narily activate the human alternative complement pathway orinitiate a direct monocyte phagocytic response, can be modifiedto exhibit both functions by the deletion or alteration of mem-brane sialic acid residues. Enzymatic removal of the sialic acidresidues with sialidase or their conversion to heptulosonic acidderivatives by limited oxidation with NaIO4 and reduction withBH4- have equivalent dose-response effects on the capacity ofthe altered sheep erythrocytes to initiate the phagocytic re-sponse by human monocytes or to activate the alternativepathway in human serum. The deposition of C3b on nativesheep erythrocytes had little effect on their ingestion by humanmonocytes, whereas the fixation of C3b on desialated sheeperythrocytes had a synergistic effect on the percentage of mo-nocytes ingesting such a particle. The monocyte receptor es-sential for ingestion of desialated sheep erythrocytes or desia-lated sheep erythrocytes bearing C3b was inactivated by con-centrations of trypsin that also prevented the monocytes fromingesting natural activators of the human alternative comple-ment pathway, but did not alter the receptors for C3b or the Fcportion of IgG. The capacity of the nonimmune host to respondto desialated particles by initiating the monocyte ingestiveprocess and by activating the alternative complement pathwayto provide the synergy afforded by C3b deposition on that par-ticle represents a primitive biochemical basis for differentiationof nonself from self.

Human peripheral blood monocytes in a protein-free mediumdirectly ingest a variety of particulate activators of the humanalternative complement pathway, such as zymosan and rabbitand mouse erythrocytes, and do not ingest the nonactivatingparticles, sheep and guinea pig erythrocytes (1). Ingestion ismediated by monocyte receptors, which are diminished bytreatment of the cell with concentrations of trypsin that do notaffect the functional integrity of Fc and C3b receptors. Thesetrypsin-sensitive receptors are regenerated during culture oftrypsin-treated monocytes for 48 hr under serum-free condi-tions. The rank order of the relative capacities of the activatingparticles to stimulate their phagocytosis by human monocytesis the same as their capacities to activate the human alternativecomplement pathway, suggesting that the same surface char-acteristics of those particles regulate both reactions. Membranesialic acid was identified as having a critical role in alternativepathway activation when sheep erythrocytes, normally a non-activating particle, acquired this capacity after membrane sialicacid moieties were removed by sialidase or chemically con-verted to their heptulosonic acid derivative (2). This finding,coupled with the realization that the natural activators, zymosan(3) and rabbit erythrocytes (4), have absent (5) and diminished(6) amounts of sialic acid, respectively, led to an examinationof the effects of this carbohydrate on the capacity of a particleto initiate the monocyte phagocytic response.The present study demonstrates that sheep erythrocytes (ES)

are directly ingested by human monocytes through a trypsin-

3831

sensitive receptor after quantitative removal of sialic acidresidues from Es or conversion to their heptulosonic acid de-rivative. Addition of the major cleavage product of the thirdcomplement component, C3b, to the surface of these modifiedEs increases the number of human monocytes capable of in-gesting these particles, thereby revealing a synergistic inter-action between monocyte C3b receptors and monocyte tryp-sin-sensitive receptors for activators of the alternative pathway.Since particles that activate the alternative complement path-way would naturally become coated with C3b in vivo, thesynergistic interaction of these two monocyte receptors wouldnormally be operative for phagocytosis of such particles.

MATERIALS AND METHODSAssay of Phagocytic Function of Monolayers of Human

Monocytes. Monolayers of human monocytes were preparedand their phagocytic function was quantitated as described (1).Normal human peripheral blood monocytes were isolated fromcitrated and dextran-treated blood, purified by gradient cen-trifugation on Ficoll-Hypaque cushions (7), resuspended inRPMI 1640 Medium (Grand Island Biological Co., GrandIsland, NY) that had been supplemented with 0.5% bovineserum albumin (crystallized, Miles Laboratories, Inc., Elkhart,IN) (RPMI-BSA), and counted on a model ZF Coulter counter(Coulter Electronics, Hialeah, FL). Samples of S X 105 mono-nuclear cells in 0.25 ml of RPMI-BSA were layered onto raised,siliconized, circular, glass coverslips (15 mm diameter, SGAScientific Inc., Bloomfield, NJ), and incubated at 370 in ahumidified atmosphere of 95% air/5% CO2 for 50 min. The twocoverslips were rinsed in 50-ml volumes of RPMI to removenonadherent cells, were again layered (8) with 5 X 105 mono-nuclear cells in 0.25 ml, and were incubated as before. About50% of the layered cells remained attached to the coverslips,and more than 90% of the total cells were identified as mono-cytes by their morphology and by their capacity to ingestIgG-coated Es.The assay for phagocytosis by monolayers of monocytes used

particles that had been washed with RPMI, resuspended inRPMI supplemented with 5 mM MgCI2 (RPMI-Mg), andcounted on the Coulter counter. Monolayers were incubatedwith 0.25 ml of various concentrations of each particle for 45-60min at 370 in the CO2 chamber, rinsed twice in RPMI, treated

Abbreviations: RPMI-BSA, RPMI 1640 medium supplemented with0.5% bovine serum albumin; RPMI-Mg, RPMI supplemented with 5mM MgCI2; Es, sheep erythrocytes; Er, New Zealand White rabbiterythrocytes; GVB, veronal-buffered saline, pH 7.5, containing 0.1%gelatin; GVB-EDTA, GVB containing 40 mM EDTA; EA, erythro-cyte-IgG complex; GVB2+, GVB containing 0.5 mM Mg2+ and 0.15mM Ca2+; DGVB2+, half-isotonic GVB2+ containing 2.5% dextrose;EPMBb, Es bearing .C3b; EsC3b,Bb, Es bearing the amplification con-vertase C3b,Bb.* To whom reprint requests should be addressed.

Proc. Natl. Acad. Sci. USA 75 (1978)

with 0.84% NH4Cl for 4 min to lyse adherent but not ingestederythrocytes, and rinsed again. The monolayers were then fixedto the coverslips by sequential passage in a series of ethanol/formaldehyde solutions (1) and stained for 4 min in 4% Giemsasolution. The number of particles ingested by at least 500 mo-nocytes on each coverslip was determined by visual enumera-tion at X1000 with a light microscope. The data are expressedas the percentage of monocytes ingesting either one or moreor three or more particles, as specified in each experiment. Toassess binding of C3b-bearing particles, the NH4Cl step waseliminated, the numbers of bound erythrocytes were counted,and the percentage of monocytes binding three or more wasdetermined.

Monolayers of monocytes were treated with trypsin that hadbeen purified by affinity chromatography as described (1).Monolayers were treated with 0.25 ml of 3-31 ,g of trypsin perml for 30 min at 370 in the CO2 chamber, rinsed, incubatedwith 10 ;ig of trasylol per ml for 15 min, rinsed, and layeredwith particles for assay of phagocytic capacity.

Preparation of Erythocytes for Phagocytosis. Erythrocytes(E) from sheep (Es) and New Zealand White rabbits (Er) werecollected into, washed in, and stored in veronal-buffered saline(pH 7.5) containing 0.1% gelatin (9) (GVB) and 40mM EDTA(GVB-EDTA). Zymosan (Schwarz/Mann, Orangeburg, NY)was boiled, washed, and stored in 0.15 M NaCl at 40. Eryth-rocyte-antibody (EA) complexes were prepared by incubatingEs (1 X 109) with rabbit 7S anti-sheep hemolysin (Cordis Lab-oratories, Inc., Miami, FL) diluted 1:200 in GVB-EDTA for 30min at 370 and then for 30 min at 40.The Es were treated with Clostridium perfringens sialidase

(acylneuraminyl hydrolase, EC 3.2.1.18) purified (2) to a spe-cific activity- of 16 units/mg (1 unit of enzyme is defined as thatwhich releases 1 Mmol of N-acetylneuraminic acid from N-acetylneuraminlactose per min at 37°). Four samples of 2 X1010 Es were washed with 50 mM Na acetate/120 mM NaCl,pH 6.5, and sedimented by centrifugation for 10 min at 1300X g. Three samples were resuspended in 1-ml portions of thesame buffer containing 0.370, 0.009, and 0.002 unit of sialidase,respectively. The fourth sample was resuspended in 1 ml ofbuffer containing 0.2 ,mol of synthetic N-acetylneuraminicacid (Calbiochem, Lajolla, CA). The Es were incubated for 30min at 370 and sedimented by centrifugation at 1300 X g for10 min at 20. The supernatant fluids were removed for assayof sialic acid by the thiobarbituric acid method (10) and theerythrocytes were washed four times with GVB and stored astarget particles for phagocytosis. Stroma were prepared from3 X 101° Es and used for the determination of total sialic acidassociated with Es membranes. These stroma were hydrolyzedin 0.1 M HC1 at 800 for 60 min, and free sialic acid was assayedby the thiobarbituric acid method (10). The sialic acid enzy-matically released from each of the three samples of erythro-cytes was calculated on the basis of the recovery of the N-acetylneuraminic acid that was incubated with the fourthsample of Es and expressed as nmol of sialic acid released per1010 Es. This value was converted to percent sialic acid releasedby dividing by the total number of sialic acid residues per 1010Es in the acid-hydrolyzed Es stroma.The Es were treated with NaTO4 (2, 11) and the number of

sialic acid residues oxidized was assayed by the amount offormaldehyde released (12). Four samples of 2 X 1010 Es werewashed with 50 mM Na acetate/120 mM NaCl, pH 6.5, andsedimented by centrifugation at 1300 X g for 10 min. Threesampleswere resuspended in 1-ml portions of the same bufferat 00 containing 3.0,1.5, and 0.75,umol of NalO4, respectively;the fourth sample was resuspended in 1 ml containing 0.12,umolof formaldehyde. After incubation at 00 for 10 min in the dark,

the Es were sedimented by centrifugation at 1300 X g for 10min at 20 and the supernatants were removed for subsequentassay of formaldehyde. Each sample of Es was immediatelyresuspended in 10 ml of acetate buffer to which was added 0.2ml of 10 mM NaOH containing 10 Mmol of NaBH4. After in-cubation for 45 min at 220, the Es were washed three times withGVB and stored as target particles for phagocytosis. The proteinwas removed by precipitation with trichloroacetic acid and theformaldehyde in the supernatants was assayed as described (12),except that 1 ml of the chromotropic acid solution (325 mg ofchromotropic acid in 100 ml of 16.4 M H2SO4) was added to0.8 ml of the supernatants, a modification that increased de-tection of formaldehyde more than 4-fold (2). A standard ref-erence curve was established with commercial formaldehyde(Merck and Co., Inc., Rahway, NJ). Reagent blanks were pre-pared with the amounts of NaIO4 used in the experiments. Aftersubtraction of the reagent blank, the nmol of formaldehyde inthe experimental supernatant were determined from the ref-erence curve and corrected for the recovery of formaldehydein the control sample. This result was then expressed as nmolof sialic acid oxidized per 1010 ES.

Es bearing variable numbers of C3b were prepared by atwo-step procedure. B (13), CS (14), and D (15) were preparedfree of detectable contaminating proteins, as assessed bypolyacrylamide gel electrophoresis of their reduced forms inthe presence of sodium dodecyl sulfate (16), and were quanti-tated as described. C3 was trace-labeled with 125I (New En-gland Nuclear, Boston, MA) by insoluble lactoperoxidase(Worthington Biochem. Corp., Freehold, NJ) (17) to a specificactivity of 83,000 cpm/Mg without loss of specific functionalactivity. Es (1 X 1010) and 1 X 1010 Es from which 85% of themembrane sialic acid residues had been enzymatically removedwere each incubated with 1 mg of B, 2Mg of D, 3mg of CS, and1 Mug of 125I-labeled C3 in 1 ml of GVB containing 0.5mM Mg2+and 0.15 mM Ca2+ (GVB2+) (9) for 45 min at 300. The cellswere washed four times at 250 with GVB2+ and assessed forcell-bound radioactivity. These EsCtb intermediates were in-cubated with 100Mug of B and 1 ,ug of 5 in 2 ml of half-isotonicGVB2+ containing 2.5% dextrose (DGVB2+) (9) for 30 min at300, sedimented by centrifugation at 20, and resuspended in3.3 ml of ice-cold DGVB2+. One-milliliter portions that con-tained 3 X 109 EsC3b,Bb or desialated ECSb,Bb were addedto 1 ml of DGVB2+ containing 30 gg of C3/1 Ag of 125I-labeledCS, 150 ug of C3/i gg of 125I-labeled C3, or no CS, and incu-bated for 45 min at 300. The cells were washed and assayed forcell-bound radioactivity. Nonspecific uptake of 125I-labeled CSwas determined at each step with Es, ECS~b, desialated Es, anddesialated EsC3b by incubation with the appropriate concen-trations of 125I-labeled CS and C3 in the absence of B and D.This amount was subtracted to define the net C3b uptake ineach step. The total C3b per cell was calculated from the125I-labeled CS specifically taken up in the first and secondsteps.

RESULTSEffect of quantitative removal or modification of sialicacid residues from Es on ingestion by humanmonocytesEs, treated with concentrations of sialidase that removed 48nmol/1010 Es (37%), 70nmol/1010 Es (54%), and 120 nmol/1010Es (92%) of the sialic acid residues, respectively, and withconcentrations of NaIO4 followed by BH4- reduction thatconverted 53 nmol/1010 Es (42%), 73 nmol/1010 Es (57%), and100 nmol/1010 Es (77%), respectively, of the sialic acid moietiesto a heptulosonic acid, were each suspended to 3.3 X 108/ml

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Proc. Natl. Acad. Sci. USA 75 (1978) 3833

in RPMI-Mg. These modified Es and buffer-treated Es werelayered in duplicate onto monolayers of monocytes frotdonors previously shown to differ in the proportion of mono-cytes ingesting Er (1). None of the monocytes from any of thedonors ingested Es until more than 60-65 nmol/1010 Es of sialicacid residues had been removed or modified (Fig. 1). Once thisthreshold, which represented an alteration of 50% of the sialicacid residues, had been reached, the proportion of monocytesingesting the modified Es increased in a fashion that wasdose-related to further reduction of intact membrane sialic acidresidues on ES. The chemical cleavage of two carbon atoms fromthe exocyclic polyhydroxylated side chain of sialic acid on Eswas equivalent to enzymatic removal of the entire sialic acidmoiety in terms of the capacity of modified Es to initiate themonocyte phagocytic process. The different capacities of themonocytes from different donors to ingest Er, which had ledto the original designation of the donors as low, intermediate,or high (1), were maintained in the same rank order when thetarget particles were Es with modified cell-surface sialic acidresidues.The effect of trypsin treatment of monolayers of monocytes

on their capacity to ingest desialated Es was examined with Esthat had 120 nmol/1010 Es (92%) of their sialic acid residuesremoved by treatment with sialidase. Ingestion of ET was againused for comparison. Monolayers of monocytes were treatedwith increasing concentrations of trypsin, washed, exposed to10 Atg of trasylol per ml, and washed again. These monolayerswere incubated with 1.5 X 108 Er, 1.5 X 108 desialated Es, and0.8 X 108 EA in 0.25 ml of RPMI-Mg for 60 min under the usualassay conditions. Treatment of the monolayers with 31 ,g oftrypsin per ml did not affect their capacity to ingest EA (Fig.2). The capacity of monocytes to ingest Er and desialated Es was

50 -

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cm 40C

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020

O 10_

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I Er0 40 80 120Sialic acid removed ormodified, nmol/1010 Es

FIG. 1. Percentage of human monocytes from three donors (A,B, and C) ingesting one or more Es whose sialic acid residues were

modified in a dose-response fashion by treatment with NaIO4 (0---0)or sialidase (0-0). Numbers of sialic acid residues removed or al-tered on the target particles are indicated on the abscissa. Ingestionofone or more Er is indicated by the bars. Data are plotted as the meanof duplicate determinations.

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o 40E

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0110 20 30Trypsin, pg/ml

FIG. 2. Effect of pretreatment with various concentrations oftrypsin on the capacity of monolayers of monocytes to ingest one ormore EA (A), one or more (0) and three or more (0) desialated ES,and one or more (v) and three or more (v) Er.

decreased in a dose-related fashion by trypsin treatment. Theproportion of monocytes ingesting one or more of both typesof erythrocytes was decreased to one-half of control levels with8 ,g of trypsin per ml and was further reduced to one-third with31 Mg of trypsin per ml. Thus, there was comparable andmarked trypsin sensitivity of the monocyte recognition unitinvolved in the ingestion of Er and desialated Es.Effect of C3b on ingestion of sialidase-treated anduntreated EsThe C3b fragment enhances ingestion of IgG-bearing particlesby human monocytes (18, 19) and, thus, the effect of C3b oningestion of sialidase-treated Es was assessed. Proteins of thealternative pathway were used in a two-step procedure to de-posit various known amounts of C3b on untreated Es and on Esfrom which 85% of the membrane sialic acid had been re-moved. Untreated and trypsin-treated monolayers of monocyteswere incubated for 60 min with 0.8 X 108 Es, 0.8 X 108 EsC3bbearing 560, 3460, or 15,710 C3b per cell, 0.8 X 108 desialatedEs, 0.8 X 108 desialated EsC3b bearing 450, 1280, or 4550 C3bper cell, 0.8 X 108 Er, or 0.8 X 108 EA under the usual assayconditions. Trypsin treatment of monocytes did not alter theircapacity to ingest EA, whereas treatment with only 8 ,g oftrypsin per ml decreased the percentage of monocytes ingestingEr from 42% to 12% (Fig. 3A).Less than 1% of the monocytesingested Es that had no C3b or 560 C3b per cell, and fixationof 3460 and 15,710 molecules of C3b per Es increased theproportion of ingesting monocytes to only 5% and 8%, respec-tively. This minimal proportion of monocytes ingesting C3b-bearing Es was reduced by treatment of the monocytes with 8Mg of trypsin per ml. Since 80-5% of the untreated monocytes,as well as those treated with 8 ,ug of trypsin per ml, formed ro-settes with Es bearing 15,710 C3b per cell, C3b adherence tomonocytes did not lead to substantial phagocytosis and the re-ceptor was not sensitive to these concentrations of trypsin.

Twenty-five percent of the monocytes ingested desialated

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Proc. Nati. Acad. Sci. USA 75 (1978)

100

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(A0)CYa,co0

0

E

I I

0 10 20 30 0 10 20 30

Trypsin, mg/mlFIG. 3. Dose-response effect of trypsin on the capacity of human

monocytes to ingest one or more of the following particles: (A) EA (4),Er (v), Es (),EIC3b bearing 560 (0), 3460 (v), and 15,710 (o) C3bper cell. (B) ES, 85% desialated (@) and bearing 450 (0), 1280 (v), and4550 (D) C3b per cell.

Es (Fig. SB); this result was not appreciably altered by therandom distribution of 450 C3b on the Es membrane. Thefixation of C3b through the focal action of cell-bound C3b,Bbconvertase so as to deposit 830 and 4100 additional C3b per

desialated Es resulted in doubling the percentage of the mo-

nocytes ingesting these particles. Trypsin treatment of mono-cytes reduced their capacity to ingest desialated Es irrespectiveof whether or not C3b had been fixed to the surface. The trypsineffect was dose related, and a concentration of 8 ,ug/ml reducedto one-half the proportion of monocytes ingesting each categoryof particle. Thus, the presence of C3b on the desialated Essubstantially- increased the proportion of monocytes ingestingthese cells by a trypsin-sensitive mechanism.

DISCUSSIONThe capacity of human monocytes to ingest directly particulateactivators of the alternative complement pathway in the ab-sence of any exogenous factors by a trypsin-sensitive process

reflects a monocyte recognition mechanism available to thenonimmune host. The evidence that the monocyte recognizedsome cell-surface characteristic of the alternative pathwayactivators was circumstantial and based entirely on the findingthat the rank order of ingestion paralleled the alternativepathway-activating capacity of these particles and that non-

activating particles were not ingested (1). More direct evidencethat the monocyte phagocytic response is initiated by the samesurface characteristics that enable a particle to activate the al-ternative complement pathway is derived from the finding thatchemical modification or enzymatic deletion of membranesialic acid residues confers an ability to both activate the al-ternative complement pathway and to be directly ingested byhuman monocytes.

Es do not ordinarily activate the alternative complementpathway in whole serum or initiate a monocyte phagocyticresponse; however, this cell can be modified to exhibit both

functions by the deletion or alteration of more than 40-50% ofthe membrane sialic acid residues. Further, once the extent ofmodification exceeds this threshold, there is a quantitative re-lationship between the further reduction of intact sialic acidmoieties and the capacity of the particles to elicit a phagocyticresponse and to activate the alternative complement pathway.Both enzymatic removal of the sialic acid residues with sialidaseand their conversion to heptulosonic acid derivatives by mildoxidation with periodate and reduction with borohydride gaveequivalent dose-response effects on both functions (ref. 2 andFig. 1). Because removal of the C8 and C9 carbon atoms of thepolyhydroxylated side chain of sialic acid by limited oxidationwith periodate and reduction with borohydride was function-ally equivalent to deletion of the entire sialic acid moiety, sec-ondary effects of the latter reaction, such as diminution of netnegative charge or exposure of penultimate galactose residues,are not considered to be responsible for initiation of thephagocytic response of the monocyte. In a molecular modelsystem using the preformed amplification convertase, C3bBb,there is a linear relationship between the deletion or chemicalmodification of Es sialic acid residues and the decrease in thecapacity of the regulatory protein, ,31H, to cause extrinsic decaydissociation of Bb from the membrane-bound C3b,Bb (2). Itseems likely that plasma proteins of the alternative pathwayresponding to and regulating the effects of the alternativepathway activators are the prototypes of the monocyte mem-brane proteins involved in the ingestion of activators.The monocyte recognition mechanism for the ingestion of

natural activators of the human alternative complementpathway is inactivated by concentrations of trypsin that do notaffect the function of its C3b or Fc receptors (1). The dose-related effects of trypsin on the capacity of the monocyte toingest native Er and desialated Es were essentially parallel (Fig.2). Thus, the monocyte receptor for particles that activate thealternative pathway by virtue of in vitro deletion of their sialicacid residues appears to be the same as that involved in therecognition of natural activators of the pathway.The literature contains several examples in which the in-

gestion of a particle by monocytes appears to be facilitated bytwo distinct receptor mechanisms on the monocyte. Normalmouse peritoneal macrophages adhere to C3b-bearing particles(20) without ingesting them, unless they are also coated withIgG so as to interact with the macrophage Fc receptors as well(21, 22). The ingestion is greater than that obtained with theIgG opsonin alone (21) and thus reflects the synergy affordedby the interaction of the particle with two monocyte receptors.Es are not ingested in the presence of mouse serum unless theyhave been first treated with sialidase (23), suggesting, on thebasis of current evidence, that ingestion is dependent on thecombined effect of reduced membrane sialic acid and adher-ence by C3b deposited by activation of the alternative pathway.In another study, sialidase treatment of Es alone did not leadto their ingestion by human monocytes (19), but did increasethe ingestion observed with a fixed dose of IgG opsonin. Thedeposition of C3b on Es, which in their natural state are notingested by human monocytes, increased the percentage ofmonocytes ingesting the particles to only 8% (Fig. 3A), eventhough 80% of the monocytes exhibited adherence to the EsC3b.In contrast, whenC3b was fixed to desialated Es, the percentof monocytes ingesting such particles increased from 25% to50% (Fig. 3B). The C3b effect was not seen when the fragmentwas randomly distributed at 450 molecules of C3b per cell andwas nearmaximal when an additional 830 C3b molecules weredeposited in a more focal fashion by a cell-bound amplificationconvertase. The augmentation in the percentage of humanmonocytes ingesting desialated Es bearing C3b was reversed

3834 Cell Biology: Czop et al.

Proc. Natl. Acad. Sci. USA 75 (1978) 3835

with trypsin treatment of the monocytes at concentrations thatdid not affect the C3b receptor as defined by adherence$S'Wenatural activators of the alternative pathway are relativelydeficient in membrane sialic acid, it could be assumed that theiringestion in vivo by monocytes would use not only the directrecognition of the activating particle, but also the synergy af-forded by the C3b deposition on that particle. The superim-

position of an immune response with immunoglobulin mole-cules of the IgG class would add immunochemical specificityto this biochemical differentiation of self from nonself basedupon the number of particle-bound sialic acid residues.

This work was supported by Grants AI-07722, AM-05577, andRR-05669 from the National Institutes of Health-and a grant from theNew England Peabody Home Foundation. J.K.C. is a PostdoctoralFellow of The Arthritis Foundation. D.T.F. is a Research Career De-velopment Awardee (K04 Al 00245-01) from the National Institutesof Health.

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J. Henatol. 1, 419-432.

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110, 128-138.14. Tack, B. F. & Prahl, J. W. (1976) Biochemistry 15, 4513-

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Muiller-Eberhard, H. J. (1968) Science 162, 1281-1283.19. Ehlenberger, A. G. & Nussenzweig, V. (1977) J. Exp. Med. 145,

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