Proc. Natl. Acad. Sci. USAVol. 74, No. 3, pp. 1077-1081, March 1977Cell Biology

Adhesive specificity of juvenile rat and chicken liver cells andmembranes

(plasma membranes/intercellular adhesion/cell-cell recognition)

BJORN OBRINK*, MARK S. KUHLENSCHMIDT, AND SAUL ROSEMANDepartment of Biology and the McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland 21218

Contributed by Saul Roseman, December 10, 1976

ABSTRACT Liver cells, isolated from either juvenile ratsor chickens by a collagenase perfusion technique, reaggregatedwhen maintained in suspension. The cells exhibited markedadhesive specificity; when suspensions contained both celltypes, the aggregates consisted primarily of either rat or chickencells. Adhesive specificity was also observed with plasmamembrane fractions isolated from rat liver homogenates, andwith comparable fractions from chicken liver. These mem-branes stimulated aggregation of the homologous but not theheterologous cell type. Other membrane fractions had little or

no effect on the aggregation of the homologous cell type. Theseand other prope ies of the liver cell and membrane preparationssuggest that biochemical studies on cell-cell recognition andadhesion can most effectively be conducted with cells fromjuvenile and adult animals.

Several mechanisms have been proposed to explain the adhesivespecificities of vertebrate cells (1-5), but the relevant surfacemolecules remain to be identified. A major limitation in our

work and that of others (6-9) has been that the biological sys-

tems thus far used, embryonic and tissue culture cells, provideonly limited quantities of material for biochemical analysis.The present report shows that liver cells from juvenile rats

and chickens appear to be. ideal for studying intercellular ad-hesion. They are easily obtained in large quantity, and thepopulations consist primarily of one cell type (in contrast to cellsfrom embryonic tissues). In addition, the cells show adhesivespecificity in that they adhere rapidly to the homologous butnot the heterologous cell type, and readily yield membranefractions that also show adhesive specificity.

EXPERIMENTAL PROCEDUREMaterials. Male Sprague-Dawley rats (Flow Laboratories,

Dublin, Va., 24084) 6-8 weeks old (175-250 g) and WhiteLeghorn chickens (Truslow Farms, Inc., Chestertown, Md.,21620) 3-4 weeks after hatching were used for preparing livercells and liver subcellular fractions. Collagenase type I, bovineserum albumin fraction V, ethylene glycol bis(,B-aminoethylether)-N,N'-tetraacetic acid (EGTA), and N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (Hepes) were obtainedfrom Sigma Chemical Co.; collagenase CLS III, from Worth-ington Biochemical Co. Dry powder tissue culture media, chickserum, and postnatal calf serum were obtained from Gibco,Inc.Medium A (modified Eagle's medium) used for the adhesion

assays consisted of Gibco G-14, adjusted to 1 liter and pH 7.4after addition of the following components: 2.4 g of Hepes, 220mg of NaHCO3, 50 mg of alanine, 150 mg of arginine, 50 mg

Abbreviations: BSA, bovine serum albumin; EGTA, ethylene glycolbis(fl-aminoethyl ether)-N,N'-tetraacetic acid; Hepes, N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid.* Present address: Department of Medical and Physiological Chem-istry, The Biomedical Center, University of Uppsala, Sweden.

1077

of asparagine, 120 mg of aspartic acid, 90 mg of cysteine, 15mg of cystine, 150 mg of glutamic acid, 100mg of glycine, 150mg of histidine, 100 mg of isoleucine, 100 mg of leucine, 240mg of lysine, 100 mg of methionine, 100 mg of phenylalanine,100 mg of proline, 100 mg of serine, 150 mg of threonine, 50mg of tryptophan, 100 mg of tyrosine, and 150 mg of valine.In some experiments postnatal calf serum (final concentration10%) or bovine serum albumin (BSA) (final concentration 2%)was added as indicated. F-10 was Ham's F-10 medium madeup as described by Gibco, Inc.

Preparation of Liver Cells. Hepatocytes were prepared bya collagenase perfusion method (10) and modified as described(11). Collagenase type I and CLS III were usually used for therat and chicken, respectively, and were removed from the cellsby a final 3 min perfusion with Buffer 1 (11). Buffer 1 contains8.3 g of NaCI, 0.5 g of KCI, 2.4 g of Hepes, and water to 1 liter,adjusted to pH 7.4 with 1 M NaOH.The cells (suspended in Buffer 1 supplemented with 1.5%

BSA) were filtered through a 100 mesh silk screen, diluted to75 ml with the same medium, and filtered sequentially throughnylon (Nitex) filters of pore sizes 35 and 25 ,um. The chickenliver cells were also filtered through a 15 ,um filter. The cellswere further purified at room temperature by four centrifu-gations in polycarbonate tubes at 70 X g for 2 min each(swinging bucket rotor). The cell pellets obtained in each cen-trifugation (except the last) were gently resuspended in 75 mlof Buffer 3 (11). Buffer 3 contains 8.0 g of NaCl, 0.35 g of KCI,0.16 g of MgSO4-7H2O, 0.18 g of CaCl2.2H20, 2.4 g of Hepes,15 g of BSA, and water to 1 liter, adjusted to pH 7.4 with 1 MNaOH. The final cell preparations were suspended in MediumA.The average cell diameters were 23 and 13 ,um for the rat and

chicken cells, respectively. Viability by trypan blue exclusionand by lactate dehydrogenase activity (12) was 80-95%; 70-90%of the cells were single cells, and the remainder were in smallaggregates of two to three cells each. Both chicken and rat cellscould be stored in Medium A for up to 5 hr in an ice bath withno apparent decrease in either viability or adhesive proper-ties.

Preparation of Subcellular Fractions. Homogenization,fractionation, and dialysis steps were conducted at 0-40. Thefollowing media were used. Ca-medium; 145 mM NaCI, 4 mMKCI, 2 mM CaCl2, 2 mM MgCl2, 5 mM Tris-HCI at pH 7.4, oradjusted to pH 9.6 (Ca-medium, pH 9.6). EGTA-medium: 145mM NaCl, 4 mM KCl, 2 mM MgC12, 1 mM EGTA, 5 mMTris-HCl at pH 7.4. Phosphate-buffered saline: 8.0 g of NaCl,0.2 g of KCI, 1.15 g of Na2HPO4, 0.2 g of KH2PO4, water to 1liter, pH 7.4.

Rat livers were homogenized and fractionated in 0.25 Msucrose containing 5 mM Tris-HCI, pH 7.4, or in 1 mMNaHCO3, 0.5 mM CaCI2 at pH 7.4, essentially as described (13,

1078 Cell Biology: Obrink et al.

A. RAT CELLS B. CHICKEN CELLS C CHICKEN CELLS

T IME ( MIN UT E S )FIG. 1. Aggregation of rat and chicken liver cells. Assays were conducted with the Coulter counter; the ordinate represents the number of

single cells remaining in the population (as percentage of the initial single cell number) and the abscissa represents time of incubation. In eachcase, 4 X 105 cells were rotated at 70 rpm in flat-bottomed Coulter counter vials. Each vial contained 0.3 ml of Medium A with or without BSAor calf serum (as indicated). (A) Rat hepatocytes: 0, 370; 0, 10% calf serum, 370; &, 270; *, 10% calf serum, 270. (B) Chicken liver cells: 0,

2% BSA, 370; 0, 2% BSA, 270; a, 2% BSA, 210. (C) Chicken liver cells: *, 2% BSA, 210; 0, 10% calf serum, 210; ,, 10% chicken serum, 210. Theranges shown in A and B indicate the variation in duplicate incubations. Similar ranges were observed in all experiments given in the other fig-ures.

14). Thus, a nuclear pellet Pi, a mitochondrial pellet P2, a heavymicrosomal pellet P3, a light microsomal pellet P4, and a su-pernatant S were obtained. Each pellet was suspended in thesolution used for homogenization, and along with the super-natant fraction was dialyzed sequentially against the followingmedia: EGTA-medium, phosphate-buffered saline, and Me-dium A. Chicken livers were homogenized in 0.25 M sucrosecontaining 0.01 M Hepes, pH 7.5, fractionated in the samemanner, dialyzed against phosphate-buffered saline, andwashed and suspended in Medium A.

Rat liver plasma membranes were isolated by the procedureof Ray (15). The last step, isopycnic centrifugation, gave thefollowing fractions at the indicated interfaces: plasma mem-branes (Band 1), 37-41% sucrose (wt/wt); Band 2,41-45% su-crose; Band 3, 45-48% sucrose. Bands 2 and 3 consisted pri-marily of mitochondria and mitochondrial membranes (16),although Band 2 contained some plasma membrane. Markerenzyme analyses showed that the plasma membranes (Band 1)were of the same degree of purity as reported by Ray andothers. The Ray procedure was also followed for isolation ofmembrane fractions from chicken liver homogenates, althoughwe do not know if the resulting membrane fractions are en-

riched in plasma membranes, because we have not yet founda good enzyme marker for such membranes in chicken liver.

Bands 1 to 3 from rat liver were routinely dialyzed againstCa-mediumn, centrifuged and washed twice with the same so-

lution to remove adsorbed proteins (17, 18). Each of the prep-arations was then divided into two portions, one dialyzed againstCa- and the other against EGTA-medium. The membranefractions obtained from chicken liver were treated in the sameway or were dialyzed against Ca-medium, pH 9.6. For some

experiments, rat liver membranes were also dialyzed againstthis alkaline solution. Prior to use, all membrane fractions werewashed and suspended in Medium A.Measurement of Cell Adhesion. The rate of formation of

stable intercellular adhesive bonds was measured by describedprocedures (19, 20), using the Coulter counter assay to deter-mine the rate of disappearance of single cells from a shakensuspension.Measurement of Intercellular Adhesive Specificity. Rat

and chicken liver cells are readily distinguishable by theirdifference in size. Adhesive specificity could therefore bemeasured when the two cell types were incubated together in

suspensions.Selected ratios of chicken and rat cells were incubated as

follows: (a) 8 X 105 cells in 0.3 ml of Medium A containing 10%calf serum were rotated at 70 rpm and 370 in Coulter vials asdescribed in Fig. 1; (b) 4 X 107 cells in 5 ml of Medium A con-taining 10% calf serum were incubated at 370 in 60mm Falcontissue culture plastic petri plates, and shaken on a reciprocalshaker at 80 strokes per min; (c) 4 X 107 cells in 5 ml of F-10containing 10% calf serum were incubated in similar dishes butwithout shaking and in humidified air, 5% CO2 at 37°.

After incubation, aliquots of the suspensions were carefullytransferred to microscope slides and covered with cover slips,and the numbers of rat and chicken cells in aggregates were

determined (see Figs. 2 and 3).Other Methods. Protein was determined according to the

method of Lowry et al. (21) with BSA as the standard, andmarker enzymes for subcellular fractions were assayed as de-scribed (18).

RESULTSCell-Cell Adhesion and Specificity. The juvenile liver cells

rapidly adhered to each other (Fig. 1). The following resultswere obtained: (a) Chicken cells adhered to the Coulter vialsduring the assay. This was prevented by adding either BSA orserum to the incubation medium (22). Rat cells occasionallyadhered to the vials when incubated in the presence of calfserum; if this occurred, the samples were discarded. (b) Rathepatocytes were more fragile than chicken liver cells. Viabilityof the rat cells in suspension was improved by adding calf serumto the medium. (c) Interestingly, calf serum showed oppositeeffects on the two cells types (Fig. 1). It stimulated the adhesiverate of rat but inhibited that of the chicken cells. Chicken serum,however, stimulated chicken cell adhesion. (d) Intercellularadhesion is sensitive to changes in temperature (20). To facilitatestudies on the effects of subcellular fractions and other potentialactivators, the rapid rates at 370 were reduced by lowering thetemperature to 270 and 210 for the rat and chicken cells, re-spectively (Fig. 1).

Adhesive specificity was studied by mixing different pro-portions of the two cell types and analyzing the resulting ag-gregates. Three sets of conditions gave the same results (Figs.2 and 3). The method for analyzing the data has an inherent biasin favor of nonspecific adhesion. For example, no correctionis applied to show that cells in aggregates containing both celltypes were not distributed randomly, but were usually presentas clusters of homologous cell types (Fig. 2). Nevertheless, evenby the most conservative estimate (Fig. 3), the two cell types

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Proc. Natl. Acad. Sci. USA 74 (1977)

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FIG. 2. Specific aggregation of rat and chicken liver cells. Cells (4 X 107) were incubated in 5 ml ofF-10 medium containing 10% calfserumin 60mm petri dishes at 370 without agitation as described in the text. Thin arrows indicate chicken cells or aggregates, and thick arrows indicaterat cells or aggregates. The bar represents 100 ,m. The mixed suspension contained chicken and rat cells in a ratio of 1.1:1. (A) Mixture of chickenand rat cells before incubation. (B) Rat cells incubated for 60 min. (C) Chicken cells incubated for 60 min. (D-F) Chicken and rat cells incubatedtogether for 60 min. The asterisks show aggregates consisting of a cluster of chicken cells adhering to a cluster of rat cells. Such aggregates werescored as mixed aggregates in the quantitative analysis shown in Fig. 3.

showed a high degree of adhesive specificity. As observed with Fractions. Membrane fractions were tested for their effect onother vertebrate cells, a low level of heterologous adhesion was rat hepatocyte adhesion at 370 and 27° and chicken liver cellalso found. adhesion at 370, 270, and 21° (Figs. 4 and 5).

Effects of Plasma Membranes and Other Subcellular Except for purified rat plasma membranes and fractions

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AGGREGATE CLASS (% HOMOGENEITY)FIG. 3. Adhesive specificity of rat and chicken liver cells. Cells were mixed and incubated under the conditions described below, and aggregates

were analyzed by microscopic examination. Only aggregates containing four or more cells were analyzed. For a given sample, at least 100 aggregateswere picked at random and assayed by counting the number of rat and chicken cells in each aggregate. The abscissa represents aggregate classes,presented as degree of homogeneity with respect to cell type: 0-10, 11-20,... 91-10096. Left to right on the abscissa represents increasing ho-mogeneity of aggregates with respect to rat cells, while right to left represents increasing homogeneity with respect to chicken cells. The ordinaterepresents the number of rat (or chicken) cells in an aggregate class normalized to percentages of the total rat (or chicken) cells counted in allaggregate classes. Each bar therefore represents the relative number of rat (or chicken) cells within a given aggregate class. For example, anaggregate containing eight rat and three chicken cells would be scored as 8 in the 71-80% homogeneous rat cell aggregate class, and 3 in the 21-30%homogeneous chicken cell aggregate class. The values 8 and 3 are converted to percentage, with 100% representing the total number of rat orchicken cells counted, respectively. A total of 4 X 107 cells were incubated in 5 ml of Ham's F-10 medium containing 10% calf serum for 60-120min, without shaking, as described in the text. The arrows correspond to the ratios used in the mixtures of cells, and where the major aggregateclass would be expected, if the cells formed aggregates in a random manner.

77

1080 Cell Biology: Obrink et al.

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FIG. 4. Effect of rat liver subcellular fractions on aggregation of rat hepatocytes. For each experiment 4 X 105 cells were incubated in 0.3ml of Medium A containing the indicated quantities of subcellular fractions (as amount ofmembrane protein added per vial) and assayed withthe Coulter counter. (A) 370 or (B) 270: *, no additions; 0, plus 0.05 mg of Ca2+-dialyzed plasma membrane; A, plus 0.05 mg of EGTA-dialyzedplasma membrane. (C) all EGTA-dialyzed fractions, 270; 0, no additions; A, plus 0.08 mg of plasma membrane, Band 1; a, plus 0.06 mg ofBand2; 0, plus 0.08 mg of Band 3 (EGTA-dialyzed fractions PI through P4 and supernatant fraction S gave the same results as Band 3, i.e., they hadno significant effect on the aggregation).

enriched in such membranes (i.e., Band 2 and the nuclear pelletfrom liver homogenized in bicarbonate/CaCl2), none of the ratliver subcellular or membrane fractions showed stimulatory or

inhibitory effects on rat hepatocyte adhesion. Rat liver plasmamembranes were routinely active when dialyzed against EGTA(Fig. 4), and occasionally active when prepared in the presenceof Ca2+. Dialysis against alkali did not activate rat liver plasmamembranes when tested with rat hepatocytes. The stimulatoryeffects (Figs. 4 and 5) were obtained with 0.05-0.10 mg (asmembrane protein) of EGTA-dialyzed membranes, but activitywas detected at 0.01 mg, a level calculated to be close to thatof the membrane protein in the intact cells (4 X 105) used in theassay.

Similar results were obtained with chicken liver cells. Noneof the subcellular organelle or membrane fractions stimulatedadhesion of chicken cells even when dialyzed at pH 9.6. Frac-tions obtained by the Ray procedure (Bands 1-3) and presumedto be rich in plasma membranes were active (Fig. 5) when firstdialyzed in Ca-medium at pH 9.6. They were inactive whendialyzed against EGTA or Ca2+ at pH 7.4.The heterologous systems were studied to determine whether

the membranes retained adhesive specificity (Fig. 5). Rathepatocyte adhesion was not stimulated or inhibited by chickenliver membrane Bands 1-3 dialyzed against EGTA or Ca2+ or

alkali. Chicken liver cell adhesion was not affected by ratplasma membranes dialyzed against EGTA or Ca2+. However,some preparations of chicken liver cells and alkali-extractedrat plasma membranes did not show this specificity. In abouthalf of the experiments, these membranes stimulated adhesion(data not shown), although much less than did comparablelevels of chicken liver membranes. When the rat membranesshowed no stimulatory effect, they were inactive even whentested at levels 10 times higher than the chicken membranes.The nonspecific interactions between membranes and cells mayreflect the low level of heterologous cell-cell adhesion discussedabove.

DISCUSSION

Few reports describe the adhesive properties of juvenile or adultvertebrate cells. Porcine thyroid cells have been reported toaggregate in vitro (23), and liver cells from adult rats re-

aggregate (24, 25). The adult rat liver cells form intercellularjunctions when plated on solid substrata (26, 27), and stablelateral contacts on collagel gels (K. Rubin, L Kjellen, and B.Obrink, unpublished data).

In the present studies, chicken liver cells and rat hepatocyteswere obtained from juvenile animals by perfusing the intactlivers with collagenase. The cells rapidly adhered to each otherat 370. In fact, the adhesive rate obtained with the chicken livercells was greater than that of any other cell type thus far studiedin this laboratory, including cells from embryonic chick liverand other embryonic chick tissues, and of tissue culture cells(19, 20). As in the case of the embryonic chick liver and neuralretina cells (20), preliminary experiments indicate that twotypes of intercellular bonds, "reversible" and "stable," are

formed between the juvenile liver cells; only the kinetics offormation of the stable bonds are presented here. The rat livercells consist almost exclusively of hepatocytes (27-29). Thechicken liver cells are uniform in diameter, but require furthercharacterization. Preliminary experiments suggest that theyare primarily hepatocytes. Recent studies (30) with similarpreparations (i.e., collagenase digestion of minced chicken liver)show that they synthesize and secrete very low density lipo-proteins at rates comparable to the intact liver in vivo. (In fact,such cell preparations, kindly provided by Dr. M. D. Lane,Department of Physiological Chemistry, The Johns HopkinsUniversity, were the first chicken liver cells tested here.)The hepatocytes show a high degree of viability and stability,

consist primarily of single cells, and are readily prepared inlarge quantity. Even more important, they meet an essentialcriterion for work on intercellular adhesion in that they exhibitmarked adhesive specificity.t It is only this parameter that canbe used to assay for cell-cell recognition. The extent of adhesivespecificity observed with the rat and chicken liver cells is at leastequal to and probably better than that previously detected herewith embryonic and tissue culture cells.A variety of liver membrane fractions were tested for their

ability to stimulate (or inhibit) hepatocyte aggregation. Rat livermembranes were inactive with the exception of purified plasmamembranes or fractions enriched in such membranes. Theactive preparations stimulated rat hepatocyte but not chickenliver cell aggregation. Chicken liver membranes were likewiseinactive with chicken (or rat) cells with the exception of thoseprepared by the method of Ray (presumably rich in plasmamembranes) and only after dialysis at pH 9.6. These mem-branes stimulated chicken but not rat cell aggregation. Thus,

t Conceivably, the adhesive specificities represent differences betweenthe two species. To determine whether the preparations describedhere exhibit tissue specificity, we are currently investigating othercell types from the same animals.

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Proc. Natt. Acad. Sci. USA 74 (1977)

Proc. Natl. Acad. Sci. USA 74 (1977) 1081

A. RAT CELLS B. CHICKEN CELLS

TIME (MINUTES )

FIG. 5. Adhesive specificity of cell membrane fractions from rat and chicken liver. Assays were performed as described in Fig. 4. (A) Ratliver cells, Medium A, 27°: 0, no additions; 0, plus 0.07 mg of chicken liver membranes dialyzed against Ca-medium, pH 9.6 (similar resultswere obtained with chicken membranes dialyzed against Ca- or EGTA-medium at pH 7.4); &, plus 0.08 mg of rat liver plasma membranes dialyzedagainst EGTA. (B) Chicken liver cells, Medium A containing 10% calf serum, 210 (similar results were obtained with Medium A containing2% BSA and also at 270 and 370): 0, no additions; 0, plus 0.05 mg of chicken liver membrane (Band 1) dialyzed against EGTA; *, plus 0.05mg of chicken liver membrane dialyzed in Ca-medium at pH 9.6 (similar results were obtained with membranes dialyzed against Ca2+_ andMg2+-free medium, pH 9.6); &, plus 0.1 mg of rat plasma membrane dialyzed against EGTA. Bands 2 and 3 (chicken liver membrane fractions)gave results qualitatively similar to those obtained with Band 1.

the active membranes stimulated aggregation of the homolo-gous but not the heterologous cell type, and by this criterion themembranes show adhesive specificity.

Earlier biochemical studies on intercellular adhesion havebeen severely hampered by the availability of only limitedquantities of material showing adhesive specificity (such as

membrane fractions). The present results indicate that thismajor technical problem has been resolved. The use of cellsfrom juvenile and possibly adult animals should now substan-tially aid studies on the mechanisms underlying adhesion.

Note Added in Proof. A method similar to the one described in thisreport has recently been used to study adhesive specificity (31). Ra-dioactive 32P- and 3H-labeled suspensions of single cells were mixedand allowed to aggregate, and the individual aggregates were analyzedfor both isotopes. Mixtures of embryonic chick and mouse mesen-

cephalon cells showed no adhesive specificity, whereas specificity wasobserved with mixtures of embryonic liver cells from the twospecies.

This work was supported by National Institutes of Health Grants CA15161 and AM 09851, Contract NO1-CB43985, and a Public HealthService International Fellowship (FO5 TW02143) to B.O., who was

also supported by the Swedish Medical Research Council. M.S.K. issupported by an Olive V. Levin Fellowship from the Leukemia Societyof America. This is contribution No. 889, McCollum-Pratt Institute.We thank W. Loh, M. Kogan, and B. Smith for expert assistance, andDr. E. N. Moudrianakis for help in preparing the photomicro-graphs.

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