Proc. Nati. Acad. Sci. USAVol. 77, No. 8, p. 4831-4835, August 1980Developmenta Biology

A cell surface molecule involved in aggregation of embryonicliver cells

(cell adhesion molecules/hepatocytes/tissue formation/antibodies)

ROGER BERTOLOTTI*, URS RUTISHAUSER, AND GERALD M. EDELMANtThe Rockefeller University, 1230 York Avenue, New York, New York 10021

Contributed by Gerald M. Edelman, May 16,1980

ABSTRACT Agegation of chicken embryo hepatocytescan be inhibited by Fab' fragments of antibodies preparedagainst the cells. An aqueous extract of liver cell membranescontained antigens that neutralized the adhesion-blockingproperties of the Fab' fragments. This neutralization activitywas. associated with a polypeptide of Mr 68,000 in NaDodSO4;the polypeptide was distinct from serum albumin. Specific an-tibodies prepared against the 80-fold purified active fractioninhibitedliver cell adesion and immunoprecipitated the 68,000Mr polypeptide from active fractions as well as from a detergentextract of liver cell membranes. In hepatocyte cultures, Fab'fragments of antibodies against the liver molecule preventedbot colony formation and appearance of histotypic patterns.Liver cell adhesion was compared at the cellular and molecularlevels to that of embryonic neural retina cells. Antibodiesagainst the cell adhesion molecule from neural tissue inhibitedretinal but not liver cell aggregation; conversely, antibodiesagainst the liver polypeptide inhibited liver but not retinal cellaggregation. By means of antibody absorption and immu-noprecipitation, it was confirmed that the two cell adhesionmolecules are antigenically unrelated.

The developmental significance and chemical basis of cell-celladhesion are poorly understood. In previous studies on adhesionamong embryonic cells (1-4), we initially focused on the re-aggregation of cells obtained from nerve tissue, although pre-liminary comparisons with liver cell aggregation were also re-ported (3). Several important features were revealed by theimmunologically based approach used in these studies. Theaggregation of nerve cells appears to require the function of anerve-specific surface protein now called neural cell adhesionmolecule or N-CAM.t In particular, specific antibodies againstN-CAM prevent binding among nerve cells from either retinaor brain (3), inhibit the fasciculation of nerve fibers (4), and alterthe formation of histotypic patterns in both cultured aggregatesand tissues (4, 5).

This report describes parallel studies carried out with em-bryonic liver, whose cells also will reaggregate spontaneouslyin vitro (6, 7). The same paradigm developed to define andpurify N-CAM was used for liver: antibodies that inhibit celladhesion were used to identify antigens involved in the bindingprocess (1). This approach has previously been used to char-acterize the contact sites A of slime molds (8) and was subse-quently used to detect surface components involved in adultrat hepatocyte adhesion (9). In the present studies, a liver celladhesion molecule (L-CAM) has been identified, purified, andcharacterized. Specific antibodies against L-CAM and againstN-CAM were used to show that the liver adhesion mechanismis distinct from that of nerve cells. With these antibodies wehave also examined the function of the liver molecule in theformation of colonies by cultured hepatocytes.

MATERIALS AND METHODSPreparation of Cells. Hepatocytes from livers of 10-day

chicken embryos were prepared by treatment with a mixtureof 0.1% collagenase (CLS, Worthington), 0.1% trypsin (1-250,Nutritional Biochemicals), and 10% (vol/vol) fetal bovine serum(10). The livers were incubated for 5 min at 370C on a rotaryshaker (150 rpm): twice in 20 ml of calcium- and magnesium-free balanced salt solution and then twice in 10 ml of enzyme.The tissue was dispersed by gentle trituration and washed threetimes in Eagle's minimal essential medium with spinner salts(GIBCO) by centrifugation in a clinical centrifuge for 3 minat 900 rpm.

Antibodies and Fab' Fragments. Rabbit antibodies againsthepatocytes (anti-Hep) were prepared by two weekly injectionsof 4 X 107 cells in complete Freund's adjuvant at multiple sitesand two to six injections in incomplete adjuvant. Antibodiesagainst the liver adhesion molecule were produced by four tosix injections of 15-50,ug of protein at 10-day intervals with thesame adjuvants described above. Fab' fragments were preparedas described (1).

Preparation and Extraction of Plasma Membranes. Liverplasma membranes were prepared from 14-day embryos bya modification of the procedure described by Ray (11). Twohundred livers were homogenized in a Dounce homogenizerin 300 ml of 1 mM NaH2CO3/0.5 mM CaC12, pH 7.5, and di-luted to 3.6 liters in the same buffer. The crude membraneswere washed three times by centrifugation (twice for 20 minat 4000 rpm and once for 30 min at 2500 rpm) in a Sorvalll G53rotor and then pelleted by centrifugation (10 min at 19,000rpm) in a Sorvall SS34 rotor. The membranes were purified bysucrose density centrifugation; plasma membranes were ob-tained at the interface between 37% and 41% (wt/wt) su-crose.

For extraction, membranes were incubated for 1 hr at 370Cin 20mM Tris-HCI, pH 7.4/30mM NaCI/50mM EDTA. Themembranes were removed by centrifugation at 40C for 1 hr at105,000 X g, and the soluble extract was dialyzed at 40C against10 mM Hepes, pH 7.4/137 mM NaCl/4.7 mM KCI/0.8 mMMgSO4/0.8 mM CaCI2.

Assays for Cell Aggregation and Cell Adhesion Molecules.Cell aggregation was measured by the rate of decrease in par-ticle number (1, 12). The assay was carried out at 25"C with 2X 106 cells in 2 ml of Eagle's minimal essential medium

Abbreviations: N-CAM, cell adhesion molecule from neural tissue;L-CAM, cell adhesion molecule from liver; anti-Hep, rabbit antibodiesagainst chicken hepatocytes.* Present address: Centre de Genetique Moleculaire, CNRS 91190 Gifsur Yvette, France.

t To whom reprint requests should be addressed.t This molecule was previously called CAM (2); the new designationallows discrimination between cell adhesion molecules from dif-ferent tissues.

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The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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4832 Developmental Biology: Bertolotti et al.

(GIBCO) containing 20 jig of deoxyribonuclease II per ml(Worthington) by rotation (90 rpm) on a New Brunswick in-cubator-shaker. Aliquots were diluted 1:6 with 1% glutaral-dehyde before particles were counted. The rate of aggregationwas expressed as the percent decrease in particle number after20 min. Inhibition of aggregation by Fab' was tested bypreincubation of the cells with the antibody for 15 min at40C.The assay for liver cell adhesion molecules paralleled the

Fab'-neutralization procedure used in our previous studies (1).Anti-Hep Fab' fragments were incubated with membraneantigens for 20 min at 250C and then added to the cells. After15 min at 40C, the rate of aggregation was determined.

Fractionation of Membrane Extract. The membrane ex-

tract was concentrated by using Amicon PM-10 membranes andfractionated on a column (1.2 X 75 cm) of Sephacryl S-300(Pharmacia) equilibrated in the pH 7.4 Hepes/salt buffer. Thecolumn eluate was tested for neutralizing antigens; activefractions were pooled and dialyzed against 20 mM Tris-HC1(pH 7.4). The active material was adsorbed to a column (1.2 X7 cm) of w-aminohexylagarose (Miles) equilibrated with theTris buffer. After the column was washed with 25 ml of buffer,material was eluted with a 60-ml linear gradient of NaCl from0 to 0.5 M. Fractions that contained neutralizing antigens werepooled and dialyzed against the pH 7.4 Tris buffer. This poolwas applied to a 0.5 X 6 cm column of DEAE-Sephadex(Pharmacia). After the column was washed with 15 ml of Trisbuffer, the activity was eluted by the same NaCl gradient usedfor hydrophobic chromatography.To evaluate the purity of active fractions, we labeled samples

with 125I (13) and fractionated them by electrophoresis on 7.5%polyacrylamide slab gels containing NaDodSO4 and 2-mer-captoethanol or by two-dimensional gel isoelectric focusing andelectrophoresis (14).Removal of Albumin and Antibodies Against Albumin. To

remove albumin, we dialyzed the purified activity against 0.1M sodium phosphate buffer (pH 7.0) and passed it through a

Blue Sepharose (Pharmacia) column (2.2 X 16 cm) equilibratedwith the same buffer. Albumin-coated Blue Sepharose was usedto remove anti-albumin antibodies. Removal of albumin or

anti-albumin by these procedures was confirmed by immu-nodiffusional analysis and radioimmunoprecipitation.

Immunoprecipitation. Proteins that bound to antibodieswere identified by labeling of the proteins with 1251 (13) fol-lowed by specific immunoprecipitation and gel electrophoresis(15). With intact cells iodination was carried out enzymatically(16) in 0.25% sucrose/10mM Hepes, pH 7.4, and the proteins

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Anti-Hep Plasma Soluble extract, mlFab', mg membrane, Ml

FIG. 1. Inhibition of liver cell aggregation by anti-Hep Fab' (A)and neutralization of the Fab' by membrane vesicles (B) and solublemembrane extract (C). Data refer to a 20-min aggregation periodduring which particle number decreased by 50%. Neutralizing activitywas titrated against a concentration ofanti-Hep Fab' that produceda 50% inhibition of aggregation.

Table 1. Fractionation of neutralizing activityActivity, Protein, Specific Activity

Fraction units mg activity yield, %

Membranes from 60 gof tissue 2600 32 81 100

Soluble extract 780 5.1 150 30Gel filtration 700 2.0 350 27Hydrophobicchromatography 570 0.27 2100 22

Ion-exchangechromatography 320 0.05 6400 12

were solubilized with a detergent mixture containing 1% eachof sodium deoxycholate, Triton X-100, and NaDodSO4.

RESULTSAssay for Molecules Involved in Liver Cell Aggregation.

Hepatocytes obtained from 8- to 14-day embryos reaggregatedrapidly, and this aggregation was inhibited by anti-Hep Fab'fragments (Fig. 1A). Neutralization of the adhesion-blockingactivity of anti-Hep Fab' was used to assay for cell adhesionmolecules (Fig. 1), with 1 unit of activity defined as the amountof antigen required to give 25% neutralization. Absorption ofthe Fab' with plasma membrane vesicles (Fig. 1B) suggestedthat such antigens exist on the surface of liver cells. Whereas0.6 mg of anti-Hep Fab' decreased the rate of aggregation of2 X 106 hepatocytes in 2 ml by 50% (Fig. 1A), absorption of theFab' by membranes obtained from 1/5th of a 14-day chickenliver decreased the inhibition (Fig. 1B) to about 25% (a 50%neutralization).

Solubilization, Characterization, and Fractionation ofFab' Neutralizing Activity. The best yield of soluble neutral-izing activity (Fig. 1C) was obtained by extraction of plasmamembranes with a dilute salt buffer (pH 7.4) containing 50mMEDTA. Activity yields fluctuated from 20% to 37%; longer or

serial extractions did not improve the yield. Neutralizing ac-

tivity was lost after treatment with papain or pepsin or byheating to 90'C for 4 min, suggesting that the antigen is a

protein.The neutralizing activity was fractionated by gel filtration,

hydrophobic chromatography, and ion-exchange chromatog-raphy (Table 1). On Sephacryl S-300, the activity was elutedin the apparent Mr range of 70,000-500,000 (Fig. 2). This

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FIG. 2. Gel filtration of soluble membrane extract on SephacrylS-300. Fractions (1 ml) were assayed for neutralizing activity (@-*),absorbance at 280 nm (-), and the presence of 12I-labeled rabbitimmunoglobulin G (- - -) as a molecular weight standard. Molecularweight estimates were based on the positions of elution of ferritin,immunoglobulin G, serum albumin, and cytochrome c. The bar in-dicates fractions that were pooled for further purification.

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FractionFIG. 3. Fractionation of the activity obtained by gel filtration.

The pooled activity was fractionated first by hydrophobic chroma-tography on w-aminohexylagarose (A) and then by ion-exchangechromatography on DEAE-Sephadex A-50 (B). Fractions (1 ml) were

assayed for neutralizing activity (0-0) and the presence of the1251-labeled immunoglobulin G standard (--- ). Fractions 1-13 and51-54 did not contain activity. -, NaCl gradient.

material was pooled for subsequent fractionation. At low ionicstrength, the activity bound to w-aminohexylagarose and was

eluted by a linear gradient of NaCI (Fig. 3A). Additionalfractionation was obtained by salt gradient elution from a

DEAE-Sephadex A-50 column (Fig. SB).Characterization of Liver Cell Activity. Electrophoretic

analysis of the active fractions (Fig. 4) and, in particular, theeffluent of the ion-exchange column (Fig. 5) showed that the

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FIG. 4. Gel electrophoresis in NaDodSO4 of radioiodinated

samples from fractions (Table 1) containing the Fab'-neutralizing

activity from liver. Lane a, soluble membrane extract; lane b, gel fil-

tration (see Fig. 2); lane c, hydrophobic chromatography (see Fig. 3A);

lane d, ion-exchange chromatography (see Fig. 3B).

FIG. 5. Comparison of gel electrophoresis patterns and Fab'-neutralizing activity of fractions obtained by ion-exchange chroma-tography (see Fig. 3B).

activity was correlated with two components with similarelectrophoretic mobilities corresponding to Mr in NaDodSO4of about 70,000. The more slowly migrating of the two bandswas serum albumin. Material in this band was selectively pre-cipitated by antibodies against chicken serum but not by theanti-Hep antibodies, and antibodies against the purified activitygave a strong precipitin line after immunodiffusion againstserum albumin. Although it comigrated with activity, serum

albumin did not itself neutralize, the anti-Hep Fab' nor didanti-albumin Fab' affect liver cell aggregation.

Albumin was completely removed by passing active fractionsthrough a Blue Sepharose column; over 80% of the neutralizingactivity remained in the eluate. The two molecules were moreclearly separated by two-dimensional gel isoelectric focusingand electrophoresis (Fig. 6), the albumin again being com-

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FIG. 6. Analysis of purified activity by two-dimensional iso-electric focusing and gel electrophoresis. Radioiodinated samples weresubjected to isoelectric focusing in the first (horizontal) dimensionand NaDodSO4polyacrylamide gel electrophoresis in the second(vertical) dimension. (A) Two components were present in the verticalposition corresponding to a Mr in NaDodSO4 of about 70,000. Thesewere serum albumin and a slightly more acidic molecule (arrow). (B)Same sample from which the albumin, but not the activity, had beenremoved by treatment with Blue Sepharose. The two other compo-nents are internal standards.

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4834 Developmental Biology: Bertolotti et al.

Table 2. Inhibition of liver and retinal cell aggregation byanti-liver activity and anti-(N-CAM) Fab'

Inhibition, %Fab' Hepatocytes Retinal cells

Anti-liver activity 47-80 0-2Anti-(N-CAM) 0-8 56-97Ranges of inhibition shown are for 0.25-2 mg of Fab' per assay.

pletely removed by treatment with Blue Sepharose. The activityhad an isoelectric point of pH 5.2.

Antibodies to Purified Activity. Active material from ion-exchange chromatography was used to immunize rabbits; Fab'fragments of the elicited antibodies strongly inhibited liver cellbut not retinal cell aggregation (Table 2). Both these antibodiesand anti-Hep precipitated the 68,000 Mr band from the activefraction (Fig. 7). Anti-albumin antibodies were easily removedby passing the antibody preparation through a Blue Sepharosecolumn that had been exposed to albumin.The cell surface components recognized by antibodies against

the purified activty are shown in Fig. 8. Whereas anti-Hepprecipitated a large number of molecules, the more specificantibodies reacted with relatively few components, the mainone of which had an apparent Mr in NaDodSO4 of 68,000.Among the other components were three with apparent Mr inNaDodSO4 of about 90,000, 135,000, and 160,000. The im-munogen was free of material with these electrophoretic mo-bilities (Fig. 4, lane d).Comparison of L-CAM and N-CAM. N-CAM purified from

neural retina is present primarily, if not entirely, in nerve tissues(3). Antibodies against N-CAM did not immunoprecipitate theactivity isolated from liver (Fig. 7), and antibodies against theliver activity did not precipitate N-CAM. These results suggestthat N-CAM is antigenically unrelated to the liver molecule.Cell aggregation assays further supported this conclusion. As

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FIG. 8. Radioiodinated liver cell surface proteins precipitated

by anti-Hep immunoglobulin (lane a) and by anti-liver neutralizing

activity before (lane b) and after (lane c) removal of antibodies againstserum albumin.

shown in Table 2, Fab' from antibodies against the liver activity

inhibited liver cell but not retinal cell aggregation, and anti-

(N-CAM) Fab' inhibited aggregation of retinal cells but not

liver cells. Similarly, the inhibition of cell adhesion by anti-liver

activity was reduced 44-81% by absorption of the antibody with107 hepatocytes, but was unaffected by treatment with 3 x i10retinal cells; conversely, the inhibition by anti-(N-CAM) was

reduced 84-100% by absorption with retinal cells but not byabsorption with hepatocytes.

Effects of Antibodies Against Liver Activity on Hepato-cyte Cultures. When cultured on plastic dishes, embryonic

'12

FIG. 7. Immunoprecipitates of radioiodinated active fractions.Active fractions from ion-exchange chromatography were immu-noprecipitated with: lane a, immunoglobulin from nonimmune rab-bits; lane b, anti-Hep; lanes c and d, anti-liver activity; lane e, anti-liver activity adsorbed by albumin bound to a Blue Sepharose column;and lane f, anti-(N-CAM). The band at Mr 35,000 represents labeledmaterial that migrated with the front of the unlabeled immunoglob-ulin.

FIG. 9. Phase-contrast photomicrographs, at the same magnifi-cation, of 24-hr hepatocyte cultures in medium supplemented with10%D (vol/vol) fetal calf serum: (Left) control with 0.5 mg of normalrabbit Fab' per ml; (Right) with 0.5 mg of anti-liver activity Fab' perml. Compare the compact architecture of the control colony, withpossible intercellular channels (arrows), with the flattened andunorganized monolayer obtained in the presence of the anti-liveractivity. (X150.)

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hepatocytes formed colonies with a distinct histotypicpatterzx.(Fig. 9), including extracellular channels that have structuralsimilarities to bile canaliculi (17). When anti-liver activity Fab'was included in the medium, however, the cultures assumeda different morphology (Fig. 9). Most conspicuous was the in-ability of the cells to associate into compact, three-dimensionalcolonies. Instead, they flattened out onto the substrate to forma monolayer; in the absence of colonies the size and shape of thecells were altered and specialized channels did not appear.Nonetheless, the cells remained viable in the cultures and theircytoplasmic and nuclear characteristics did not seem to begrossly changed.

DISCUSSIONThe main purpose of these studies was to detect, purify, andcharacterize a cell surface component involved in the re-aggregation of embryonic liver cells. Antibodies preparedagainst this component, which we have called liver cell adhesionmolecule or L-CAM, were used to probe the mechanism of livercell adhesion, compare it to adhesion among embryonic neuralcells, and investigate the role of L-CAM in the formation ofhistotypic liver cell colonies. Starting from liver plasma mem-branes, an 80-fold purification of L-CAM was achieved via afour-step fractionation. This purification, along with affinitychromatography to remove contaminating albumin, allowedthe tentative identification of L-CAM as a protein migratingas a single band in NaDodSO4 gel electrophoresis and enabledthe production of antibodies of restricted specificity that inhibitliver cell aggregation.

This work represents the second purification of a cellaggregation molecule (CAM) by means of an assay involvingneutralization of a complex antibody by fractionated cell an-tigens. The first CAM, which was obtained from neural tissues,is now called N-CAM to distinguish it from the liver mole-cule.Whereas the anti-Hep Fab' neutralizing activity was con-

sistently correlated with a polypeptide having a Mr in Na-DodSO4 of 68,000, the apparent native Mr of the activity, asestimated by gel filtration, ranged from about 70,000 to severalhundred thousand. This observation raises the possibility thatthe L-CAM polypeptide can exist in multimeric or oligomericstructures. Similar interactions might account for the presenceof the high molecular weight components obtained in immu-noprecipitates of radiolabeled cell surface molecules withanti-(L-CAM). The observed cofractionation of serum albuminand L-CAM might also be consistent with the formation of acomplex, although the two molecules were ultimately separableby absorption of the albumin to Blue Sepharose. The relevanceof such interactions,- however, remains undetermined.L-CAM is clearly distinct from N-CAM: they differ in ap-

parent Mr in NaDodSO4, in antigenic determinants, and intissue distribution. Whereas neural cells synthesize N-CAM andexpress it on their surfaces, the present data do not define thesite of synthesis of L-CAM and thus cannot exclude the possi-bility that L-CAM might have been passively absorbed by theliver cells. It is also possible that L-CAM is related genetically(but not antigenically) to serum albumin or structurally toa-fetoprotein, which has a similar molecular weight and is

expressed during liver development and regeneration (18). Atthe phenomenological level, the adhesive mechaniss inhibitedby anti-(L-CAM) and anti-(N-CAM) are distinguishable by thefact that the former requires the presence of Ca2+ (7) whereasthe latter does not (unpublished data).

Essentially all hepatocytes in embryonic liver can aggregateby the mechanism associated with L-CAM; similarly, all neuralcells appear to share an adhesive mechanism involving N-CAM.It is therefore unlikely that the difference in adhesive specificityindicated by these two molecules contributes to formation ofcell patterns within either tissue alone. Such a specificity couldbe important during early stages of embryo development,however, and it is likely that each adhesive mechanism is byitself an important parameter in tissue assembly.

As shown previously with anti-(N-CAM), antibodies againsta cell adhesion molecule can be a useful probe in investigatingthe role of cell adhesion in tissue formation. In the presentstudies, we observed that the appearance of histotypic liver cellpatterns in culture was altered by anti-(L-CAM) withoutcausing cell death or drastic changes in cell structure. Althoughconsiderably more information will be required to interpretsuch experiments in detail, the results suggest that these anti-bodies may prove to be of substantial value in analyzing mo-lecular events in liver development

This work was supported by U.S. Public Health Service GrantsHD-09635 and AI-11378, a Scholars Award to U.R. from the McKnightFoundation, and a French-U.S. Exchange Award to R.B. from theNational Science Foundation.

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