Glycophorin a on normal and leukemia cells detected by monoclonal antibodies, including a new monoclonal antibody reactive with glycophorins A and B

Molecular Immunology, Vol. 22, No. 4, pp. 369-318. 1985 Printed

in Great

0161-5890/85 53.00 + 0.00 Pergamon Press Ltd

Britain

GLYCOPHORIN A ON NORMAL AND LEUKEMIA CELLS DETECTED BY MONOCLONAL ANTIBODIES, INCLUDING A NEW MONOCLONAL ANTIBODY REACTIVE WITH GLYCOPHORINS A AND B ANN REARDEN,*

RAYMOND TAETLE, DONALD

A. ELMAJIAN, JOHN A. MAJDA and

STEPHEN M. BAIRD Departments of Pathology and Medicine, University of California, San Diego and Veterans Administration Medical Center, La Jolla, CA 92103, U.S.A.

(First received 29 May 1984; accepted in revised form 16 August 1984) Abstract-A new hemagglutinating monoclonal antibody, MoAb31, detected glycophorins A and B in Western blots. Results with enzyme-modified erythrocytes indicated the MoAb31 determinants were sialic acid dependent, and resided on glycophorin A on the trypsin-resistant, ficin-sensitive segment, and on glycophorin B on the ficin-sensitive segment. Another new monoclonal antibody, MoAb36, detected the Wrb antigen, located on the non-glycosylated segment of glycophorin A near its insertion into the lipid bilayer. Immunofluorescent staining of normal hematopoietic and leukemia cells with these and other monoclonal antibodies to glycophorin A demonstrated glycophorin A on erythroid cells only. Cytofluorograph analysis showed the majority of cells of the erythroleukemia cell lines K562 and HEL expressed glycophorin A, as indicated by reactivity with the monoclonal glycophorin A antibodies RlO, R18, 6A7 and lOF7. However, reactivity with monoclonal antibodies to glycosylated determinants (MoAb31 and R1.3) and to the non-glycosylated segment near the membrane insertion (MoAb36, and R7.1) was reduced or absent. Expression of “missing” glycophorin A antigens on KS62 and HEL could not be induced using a variety of chemical and biologically active modifiers. We conclude that glycophorin A of erythroleukemia cell lines K562 and HEL differs from glycophorin A at the surface of normal, mature erythrocytes with respect ro reactivity with monoclonal glycophorin A antibodies.

INTRODUCTION

Because glycophorin erythroid

lineage

A expression (Gahmberg,

is restricted

et al.,

1978),

Smith et al. (1983) reported detection of lineage in blast cells from nine out of 20 patients with acute leukemia, including myeloid and erythroid markers on the same leukemia cells. In contrast, Greaves et al. (1983b) used two monoclonal antibodies to different determinants on glycophorin A and found 21 of 27 cases of erythroleukemia to be glycophorin A positive, whereas only 20 of 724 other leukemias (3%) expressed glycophorin A. They interpreted these latter cases as representing occult erythroleukemia. Greaves et al. (1983a) also reported on 21 cases of acute undifferentiated leukemia and found none to express glycophorin A as determined by one of the monoclonal antibodies. Similar results were reported by Liszka et al. (1983) using a different monoclonal antibody to glycophorin A; only two of 229 patients with hematopoietic malignancies reacted with the antibody, and both were diagnosed as erythroleukemia. One possible explanation for the discrepancies noted in these studies of glycophorin A expression by leukemia cells is that the monoclonal antibodies may detect completely glycosylated, mature glycophorin A, whereas the rabbit anti-glycophorin A serum may detect not only mature glycophorin A but also immature, precursor glycophorin A, which is known to be incompletely glycosylated (Jokinen et al., 1979). In this study we have applied a panel of monoclonal infidelity

to the

a number

of studies have used glycophorin A antibodies to detect erythroid differentiation by leukemia cells. Anderson et al. (1979~) detected glycophorin A on leukemia blasts from three out of 15 patients diagnosed as acute myelogenous leukemia. In addition, K562, a line originally established from a chronic myelogenous leukemia patient in blast crisis (Lozzio and Lozzio, 1975), was shown to express glycophorin A (Andersson et al., 19796). Blast cells from nine out of 22 patients with chronic myelogenous leukemia in blast crisis were also shown to express glycophorin A (Ekblom et al., 1983). These studies used rabbit anti-glycophorin A serum obtained by immunization a glycophorin preparation isolated by with chloroform-methanol extraction. The antiserum was absorbed with En(a-) erythrocytes, which lack glycophorin A (Dahr et al., 1976; Gahmberg et al., 1976; Anstee and Tanner, 1976). The authors postulated that undifferentiated leukemias may show features of early erythroid differentiation.

*Correspondence

should be addressed to: Ann Rearden, Assistant Professor of Pathology, Department of Pathology H-720, UCSD Medical Center, 225 Dickinson Street, San Diego, CA 92103, U.S.A. 369

370

ANN REARDEN et

antibodies to glycophorin A and related antigens, including antibodies to determinants which are dependent on the presence of sialic acid, to study glycophorin A expression by normal and leukemia cell lines induced to cells, and by leukemia differentiate along the erythroid line. We also report two new monoclonal antibodies, MoAb31 and 36, and provide additional information regarding MoAb145, described previously (Rearden et al., 1983). MATERIALS

AND METHODS

Monoclonal antibody production The methods of mouse immunization, hybridization, cloning and ascites production have been described (Rearden et al., 1983). Briefly, spleen cells from BALB/c mice (Jackson Laboratory, Bar Harbor, ME) which had been immunized with salinewashed, packed human red blood cells, were fused with the mouse myeloma cell line NS-1 (Kohler and 1975). Hybridoma supernatants were Milstein, screened for hemagglutinating activity both in saline and in the antiglobulin reaction, and positive hybridoma cell lines were twice cloned by limiting dilution. Erythrocytes Human erythrocytes were obtained through the courtesy of the San Diego Blood Bank. Panels of erythrocytes extensively phenotyped for common blood group antigen systems including MNSs, were obtained from commercial sources [Biological Corporation of America (West Chester, PA) and Gamma Biologicals (Houston, TX)]. Erythrocytes from a newly detected En(a-) individual (E.P.) collected in citrate-phosphate-dextrose-adenine were a gift from Dr H. Perkins (Irwin Memorial Blood Bank, San Francisco, CA). Reaction of the monoclonal antibodies with a panel of rare human erythrocytes negative for high incidence blood group antigens was performed at the Blood Services Laboratories of the American National Red Cross (Bethesda, MD), through the courtesy of Dr Moses Shanfield and MS Delores Mallory; the composition of this panel has been published previously (Rearden et al., 1983). In addition, Rh null, -D, and . D. erythrocytes were examined at the Blood Bank of Riverside-San Bernardino Counties (San Bernardino, CA), through the courtesy of Dr Arthur J. Silvergleid. Reaction of the monoclonal antibodies with unmodified and enzymemodified S-s-U- erythrocytes that lack glycophorin B (Dahr et al., 1975) was performed at both the American National Red Cross and the Irwin Memorial Blood Bank. African green monkey, rhesus monkey, bovine, sheep, rabbit, guinea pig, rat, adult chicken and 24-hr old chicken erythrocytes were obtained from Flow Laboratories (Inglewood, CA). Red blood cell ghost membranes were prepared by hypotonic lysis (Dodge et al., 1963). Hemagglutination was performed in saline at room temp., in

al

bovine albumin (Ortho Diagnostic System, Raritan, NJ) at 3°C and in the antiglobulin reaction using goat antimouse immunoglobulin (Miles Laboratories, Elkhart, IN) as previously described (Rearden et al., 1983). Enzymatic modification qf‘ erythrocytes Neuraminidase from Vibrio cholerae (CalbiochemBehring, La Jolla, CA), 200mIU/ml in phosphatebuffered saline, pH 7.4 (PBS), was reacted with an equal vol of packed erythrocytes for 60 min at 37°C; erythrocyte modification was verified by reactivity with anti-T lectin [Arachis hypogaea (Vector Laboratories, Burlingame, CA)]. Three times crystallized chymotrypsin (Sigma Chemical Co., St. Louis, MO) lOmg/ml in PBS, TPCK-trypsin (Millipore Corp., Freehold, NJ), 1% (w/v) in PBS, pronase (Sigma), 58 units/ml in PBS, and proteinase K (Sigma). 160units/ml in PBS, were reacted with equal vols of packed erythrocytes for 60min at 37°C. Ficin solution (Accugenics, Costa Mesa, CA) was reacted with an equal vol of packed erythrocytes for 30 min at room temp. After modification, erthrocytes were washed in PBS. Antibodies Monoclonal antibodies RIO, R18, R1.3 and R7.1 (Edwards, 1980; Anstee and Edwards, 1982) were a gift from Dr P. A. W. Edwards. Antibodies RlO and R18 react with the trypsin-sensitive and trypsininsensitive regions respectively of glycophorin A; the lack of an effect of neuraminidase modification suggests that these determinants are not glycosylated. Antibody R1.3 likely reacts with a sialic acid dependent determinant shared by the trypsin-sensitive region of glycorporin A and the ficin-sensitive region of glycophorin B. Antibody R7.1 detects the Wrh antigen, believed to be located on glycophorin A near its insertion into the membrane (Ridgwell PI ul., 1983). Clones 6A7 and lOF7 reported by Bigbee et ul. (1983) and clone NN5 from Dr M. Vanderlan were obtained from the American Type Culture Collection (Rockville, MD). Antibody 6A7 recognizes blood group M. Antibody lOF7 recognizes both M- and N-types of glycophorin A; like RlO, it detects a determinant 011 the neuraminidase-insensitive trypsin-sensitive region. Antibody NN5 detects blood group N. Monoclonal antibodies to the ABH blood group (anti-A, anti-B and anti-H, type 2) were a gift from Chembiomed Ltd (Edmonton, Alberta, Canada). A monoclonal antibody to monocytes (Mo.1) was obtained from Bethesda Research Laboratories (Gaithersburg, MD). Monoclonal antibodies to Ia were obtained from several manufacturers and pooled prior to use. A monoclonal antibody to a common determinant on the 45,000-dalton polypeptide of HLA-A, B, C was obtained from Cappel Laboratories (Cochraneville, PA). Two nonreactive mouse myeloma proteins were used as controls [MOPC 21

Glycophorin A and MOPC 104E (Litton Bionetics Laboratory Products, Charlottesville, SC)]. Fluorescein-conjugated goat antimouse immunoglobulin was obtained from Antibodies Inc. (Davis, CA), and was absorbed with human spleen cells prior to use. Fluoresceinconjugated goat antimouse IgG, F(ab), fragment, was obtained from Tago Inc. (Burlingame, CA) and was used for experiments with cell lines. Mouse immunoglobulin heavy- and light-chain isotypes were determined as previously described (Rearden et al., 1983). Erythrocyte sialogiycoprotein Purified glycophorin A (GP-A) and a mixture of erythrocyte sialoglycoproteins (GP mix) prepared by the lithium diiodosalicylate-phenol procedure (Marchesi and Andrews, 1971) were a gift from Dr H. Furthmayr. Hemagglutination inhibition Hemagglutination inhibition was demonstrated by addition of serially diluted inhibitors to a dilution of antibody 4 times the minimal hemagglutinating concn, 1 hr prior to addition of erythrocytes. Absorption studies Reaction conditions that produced complete absorption of MoAb31 from supernatant fluid by normal erythrocytes were determined in preliminary experiments. MoAb31 supernatant fluids were absorbed under these conditions (two adsorptions with equal vols of washed, pooled erythrocytes for 1 hr at room temp.), using either normal or En(a-) erythrocytes. Unabsorbed and absorbed MoAb31 supernatant fluids were reacted with normal and En(a-) erythrocytes and their titers in the antiglobulin reaction compared.

371

(Collins et al., 1977) was exposed to 4 units/ml sheep plasma erythropoietin (Connaught Laboratories, Toronto, Ontario, Canada), 10% medium conditioned by phytohemagglutinin-stimulated mononuclear cells (PHA-LCM) as a source of burstpromoting activity, 7.5% placental conditioned mecolony-stimulating dium as factor, 1.25% dimethylsulfoxide (Sigma) and combinations of these reagents. The erythroleukemia cell lines KS62 and HEL (Martin and Papayannopoulou, 1982) were exposed to 20mM hemin (Sigma) and 4 units/ml sheep plasma erythropoietin, or both reagents. Cells were harvested after 4 days of culture and stained by immunofluorescence. HL60 cells were also stained for chloroacetate esterase; K562 and HEL were stained for hemoglobin using diaminobenzidine (Sigma). The myeloid cell line KG-1 and the lymphoid cell lines 8392, 8402 and Jurkat were also stained with MoAb31, 36 and 145. AJinity columns Three monocional antibody affinity columns were prepared by coupling MoAb31, 36 and 145 to cyanogen bromide activated Sepharose 4B (Pharmacia, Piscataway, NJ) according to the manufacturer’s instructions. Briefly, ascites protein was twice precipitated with 40% ammonium sulfate, equilibrated by dialysis against coupling buffer (0.2 M NaHCO,, 0.5 M NaCl) and reacted with affinity matrix at 4C overnight on a rotator. Unbound material was washed away with coupling buffer, and the remaining active groups rendered inactive by incubation with 1 M ethanolamine, pH 8, for 2 hr at room temp. Three wash cycles, alternating 0.1 M acetate, 1.M NaCl, pH 4.0, with 0.1 M borate, 1 M NaCl, pH 8.0, followed. Ten milligrams of ascites protein were reacted per gram of Sepharose 4B; incorporation was greater than 98%.

Immunqfluorescence Binding of monoclonal antibodies to erythrocytes, red blood cell ghost membranes, peripheral blood leukocytes, bone marrow nucleated cells, and cell lines was demonstrated by indirect immunofluorescence, either by fluorescence microscopy or by analysis with a fluorescence-activated cell sorter (Ortho Cytofluorograf 60H) as previously described (Rearden et al., 1983). Normal donors and patients Peripheral blood and bone marrow were obtained from normal human donors according to procedures approved by the Committee on Human Subjects, University of California, San Diego, CA. Excess peripheral blood and bone marrow from patients with hematologic malignancies was obtained from the Department of Pathology, University of California Medical Center, San Diego, California. Cell lines The HL60 acute promyelocytic

leukemia cell line

Erythrocyte iudination Intact erythrocytes were surface-labeled with “‘1 using lactoperoxidase (LPO) (Calbiochem) (Hubbard and Cohn, 1972). Erythrocytes suspended at 6 x IO’jml in PBS were reacted with 1 mCi of 12? (Amersham, Arlington Heights, IL) and 0.1 mg lactoperoxidase/ml erythrocytes. Freshly prepared 0.01% H,O, in PBS was added in a ratio of 10 $/ml erythrocytes every 4 min for 16 min. The reaction was terminated at 20min by addition of 10% sodium azide (Sigma) in PBS in a ratio of 20 $/ml erythrocytes. After a 5-min incubation with sodium azide, the cells were washed twice in cold PBS. Solubilized erythrocyte membranes were prepared from the iodinated erythrocytes by reaction with 6 M urea, 1% Triton X-100 (Sigma) containing 5% fetal calf serum, 77miW aprotinin (Calbiochem) and IOmM phenylmethylsulfonylfluoride (PMSF) (Sigma) for 1 hr on ice. After overnight dialysis in the cold against 0.1% Triton X-100 and 5 mM PMSF in PBS, the sample was layered over 2.5 ml of 25% sucrose in PBS

312

ANN REARDENet

and centrifuged for 90 min at 100,000 g in an ultracentrifuge (Beckman Model L5-65). The sample was recovered from the uppermost layer. Affinity

column elution

An aliquot of ‘2SI-labelled solubihzed erythrocyte membrane containing 2.5 x 1O”cpm was added to 200~1 of monoclonal antibody conjugated affinity matrix. After reaction for 1 hr at room temp., unbound material was washed away with 0.1% Triton X-1OOjPBS. Bound material was eluted by contact with 250 ~1 of 6 M guanidine hydrochloride (Eastman Kodak, Rochester, NY) for 5 min. The eluate was dialyzed against several changes of PBS, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970) on a 10% (w/v) polyacrylamide slab. The gels were exposed to X-ray film [XAR-5 (Eastman Kodak)] for 10 days. Development was in an RP X-Omat Film Processor (Eastman Kodak). Immunoblotting Proteins were transferred from SDS-polyacrylamide gels to nitrocellulose membrane according to the “Western blot” method of Towbin ef al. (1979). Transfer occurred in 25 mM Tris, 192 mM glycine, 20% methanol, pH 8.3, using a constant current of 220 mA for 3 hr. The nitrocellulose membrane was cut into appropriate strips, and remaining active sites were blocked by reaction for 2 hr with 3% gelatin in 20 mM Tris, 500 mM NaCI, pH 7.5 [Tris-buffered saline (TBS)]. The strips were reacted with mouse antihuman erythrocyte serum (positive control), MOPC 21 mouse myeloma protein (negative control), or monoclonal antibody supernatant fluid in 1% gelatin-TBS for 2 hr. Following two washes in TBS, the strips were incubated for 1 hr with a 1: 2000 dilution of affinity-purified peroxidase-conjugated goat antimouse immunoglobulin (BioRad) in 1% gelatin-TBS. After two further washes in TBS, the protein bands recognized by the antibodies were visualized using Peroxidase Color Development Solution containing 4-chloro-1-naphthol (BioRad) according to the manufacturer’s instructions. ELBA Polyvinyl chloride U-bottomed microtiter plates (Titertek, McLean, VA) were coated with 50 ~1 well, 20pgg/ml glycophorin A or glycophorin mix in 15 mM Na,CO,, 35 mM NaHCO,, pH 9.6, overnight at 4’C in a humidified chamber. After three washes with 0.05% Tween-20 in PBS 7.4 (Tween-20/PBS), remaining binding sites were blocked by reaction with 50 pi 3% bovine serum albumin (BSA) (Sigma) in PBS for 30 min at room temp. After three washes in Tween-20/PBS, 50 p I of serial IO-fold dilutions of hybridoma supernatant fluids in 0.1% BSA, Tween20/PBS were added to the wells and incubated for 2 hr at room temp. After three washes in Tween20/PBS, 50 ~1 of alkaline phosphatase conjugated,

al.

affinity-purified goat antimouse IgG, heavy- and light-chain-specific (Cappel) diluted 1: 100 in 0. I’% BSA, Tween-20/PBS was added to each well and incubated for 2 hr. Following three washes in 0.15 mM NaCI, color was developed by the addition of 50 ~1 of 1.6 mM p-nitrophenyl phosphate (Sigma) in 10.0% diethanolamine (Mallinckrodt, Paris, KY), pH 9.8, to each well. The reaction was stopped after 30 min-3 hr by addition of 50 ~1 of 2.0 mM NaOH to each well. Absorbance at 405 nm was determined using a microtiter plate scanner (Dynatech Microelisa AutoReader, MR580). Significant negative controls included substitution of BSA for glycophorin as substrate, substitution of nonreactive mouse myeloma protein MOPC 21 for hybridoma supernatant fluid, and omission of the second antibody, alkaline phosphatase conjugated goat antimouse IgG. RESULTS

MoAb31 MoAb31 reacted by direct hemagglutination and in the antiglobulin reaction with all human erythrocytes tested, including the null-type cells. It also bound to red blood cell ghost membranes as demonstrated by indirect immunofluorescence. MoAb3 1 failed to react with peripheral blood lymphocytes, monocytes or granulocytes from more than 75 normal human blood donors, as determined by indirect immunofluorescence, but did react with a population of small and medium-sized nucleated human bone marrow cells. Fluorescence intensity was reduced on bone marrow cells relative to mature red blood cells. Fluorescence-activated cell sorting analysis using MoAb31-stained normal human bone marrow showed enrichment for nucleated erythroid precursors from 2”/, of the total cells in the unsorted population to 39% in the fluorescein-positive population; sorting conditions were such that unlabelled as well as labelled cells were included in the positive fraction. The fluorescein-negative population contained only unlabelled cells and was devoid of erythroid precursors. MoAb31 did not agglutinate either in direct hemagglutination or in the antiglobulin reaction any of the non-human erythrocytes tested, with the exception of very weak direct and indirect agglutination of adult and 24-hr-old chicken erythrocytes. MoAb31 isotype is IgG,, kappa. Hemagglutination titers were reduced by neuraminidase or ficin modification of human erythrocytes, but were unaffected by trypsin or chymotrypsin modification (Table 1). The effect of enzymatic erythrocyte modification on hemagglutination titer was variable with both neuraminidase and ficin, ranging from insignificant to complete loss of MoAb31 reactivity. Enzymatic modification of a lo-member erythrocyte panel using donors of known MNSs phenotype failed to demonstrate any correlation of loss of reactivity and MNSs type. MoAb31 hemagglutination was also reduced by pronase and proteinase K modification.

Giycophorin Table MOnOClOnal antibody MoAb3

I. Hemagglutination

Agglutination

Unmodified

Direct Antiglobulin Direct Antiglobulin Direct

16.000 64,000 128 128,000 512

1

MoAb36 MoAb145 “No agglutination

A

373

titers with enzyme modified erythrocytes Neuraminidase

16.000 64,oM, 1024 5 12,000 2048

with antibody as ascites fluid used undiluted and at

Table 2. Hemag~ntination

Chymotrypsin

Trypsin

2048 4096 128 128,000 2048

MoAb31 MoAb36 MoAb145 6A7 IOF “Minimum concn glutination.

GP mix

inhibition

0.02” >2.5 > 2.5 0.02 0.02 (mgimlf

GP-A

Table 3. ELISA assay Substrate Neuraminic acid

0.02 >a.25 0.25 0.03 0.06 that

completely

0” 0 1024 512,000 2048

I :128.

Inhibitor Monoclonal antibody

Ficin

8192 ~,OOO 512 128,000 512

>a.5 >o.s >o.s >0.5 20.5 inhibits

Monoclonal antibody

GP mix

GP-A

BSA

MoAb31 MoAb36 MoAbl45 6A7 10F7

0.65” 0 0 0.26 0.59

0.05 0 0 0.19 0.60

0 0 0 0 0

“Absorbance at 405 nm.

hemag-

The MoAb31 immunoglobulin fraction used to prepare the affinity column was titrated against normal and En(a -) erythro~ytes; both erythrocytes gave a titer of 2 million in the antiglobulin reaction. En(a-) erythrocyte reactivity was completely abolished by both neuraminidase and ficin modification as indicated by lack of hemagglutination with the MoAb31 immunoglobulin fraction used at 1: 10 and 1: 100, but was unaltered by trypsin modification. En(a-) erythrocytes were able to absorb a MoAb31 supernatant fluid with antiglobulin titer of 2048 in a comparable fashion to normal erythrocytes; under conditions which produced complete absorption with normal erythrocytes, the supernatant fluid absorbed with En(a-_) erythrocytes produced weak (l-l-)

agglutination in the antiglobulin reaction with a titer of 4 against normal erythrocytes and 2 against En(a-) erythrocytes. An MoAb31 supernatant fluid with a titer of 8192 in the antiglobulin reaction when reacted with normal erythrocytes had an antiglobulin titer of 2048 when reacted with S-s-U- erythrocytes. MoAb31 reactivity was further reduced by papain and ficin, but not by trypsin mo~~cation, of S-s-U- erythrocytes. MoAb31 hemagglutination was inhibited by both GP-A and GP mix (Table 2). In the ELISA assay, MoAb31 reactivity was markedly greater with GP mix than with GP-A (Table 3). Reaction of MoAb31 in the Western blot method (Fig. 1) showed staining of the dimer of glycophorin

W’Ah GPA,B

(GPW, GPA

GPB

4

5

6

7

8

9

10

Fig. 1. SDS-polyacrylamide gels (l-6) and Western blot (7-10). Lanes 1-3 are stained with Coomassie blue; lanes 4-6 are stained with PAS. (1) and (4) Normal human erythrocyte membrane. (2) and (5) GP-A fraction. (3) and (6) GP mix fraction. Western blot of normal human erythrocyte membrane (7) En(a-) erythrocyte membrane (8), GP-A fraction (9), and GP mix fraction (10) stained by immunoperoxidase with MoAb31. Erythrocyte membrane proteins named according to Fairbanks et al. (1971).

ANN REARDENet al

314

0,3 GPA, B

GPA -

GPB -

Fig. 2. Autoradiograph prepared from SDS-polyacrylamide gel. (1) LPO-iodinated normal human erythrocyte membrane. (2) LPO-iodinated normal human erythrocyte membrane plus unlabelled normal human erythocyte membrane. (3) Eluate from MoAb31 affinity column that had been reacted with LPO-iodinated normal human erythrocyte membrane. (4) Eluate in 3 plus unlabelled normal human erythrocyte membrane. (5) Eluate from MoAb31 affinity column that had been reacted with LPOiodinated En(a-) erythrocyte membrane.

A [(GPA),], the heterodimer of glycophorins A and B (GPA, B), the dimer of glycophorin B [(GPB),], the monomer of glycophorin A (GPA) and the monomer of glycophorin B (GPB) in the normal erythrocyte membrane and the GP mix fraction: of (GPB), and GPB only in the En(a-) erythrocyte membrane, and of (GPA), with a trace of GPB in the GP-A fraction. Analysis of the GP-A and GP mix fractions in SDS-polyacrylamide gels (Fig. 1) showed that the GPA fraction contained principally glycophorin A with a trace of glycophorin B, and the GP mix fraction contained glycophorins A, B and C. Bands noted above (GPA), probably represent higher polymers of glycophorin A, and are more prominent in the GP mix than in the GP-A fraction. MoAb31 affinity column eluate prepared from LPO-iodinated normal erythrocyte membrane contained a single iodinated protein that co-migrated with monomeric glycophorin A (Fig. 2). Addition of unlabelled normal erythrocyte membrane to this eluate resulted in three bands in the regions of (GPA),, GPA, B and GPA. The MoAb31 affinity column eluate prepared from iodinated En(a-) erythrocyte membrane contained no detectable iodinated protein. lmmunoprecipitation by MoAb31 of iodinated normal membrane as previously described (Baird, 1979)

showed shown).

precipitation

of glycophorin

A (data

not

MoAb36 MoAb36 reacted weakly in direct hemagglutination and strongly in the antiglobulin reaction with all human erythrocytes tested, except En(a-) erythrocytes and the erythrocytes of M. Fr., who has been designated Wr(b-) (Adams et al., 1971; Issitt et al., 1976). Its isotype is IgG,, kappa. Like MoAb31, MoAb36 bound to red blood cell ghost membranes and a population of human bone marrow cells, and did not react with human peripheral blood lymphocytes, monocytes or granulocytes, as demonstrated by indirect immunofluorescence. MoAb36 also failed to agglutinate the non-human erythrocytes tested, with the exception of weak direct and indirect agglutination of chicken erythrocytes. Unlike MoAb31, however, MoAb36 hemagglutination was not reduced by enzymatic modification (Table l), nor was it inhibitable by either GP-A or GP mix fractions (Table 2). MoAb36 did not react with either GP-A or GP mix in the ELISA (Table 3). Immunoprecipitation of iodinated normal membrane by MoAb36 showed no detectable precipitate, and the MoAb36 affinity column eluate prepared from

Glycophorin A Table 4.

Cytofluorograph analysis

of human

315 erythroleukemia

K562 MO~OClO~d

antibody

o/i positive

MoAb31 MoAb36 MoAb145 RIO RI8 RI.3 R7.1 6A7 (arm-M) NN5 (anti-N) lOF7 Blood group H Blood group A Blood group B HLA-A, B, C la “No reaction

iodinated iodinated

2&40 0 0 60-~70 6&70 20-50 0 60-70 20-30 50-60 0 0 0 0 0

with antibody

normal membrane contained protein (data not shown).

Fluorescence intensity Moderate

Bright Bright Moderate Bright Moderate Moderate

‘A positive 0” 0 0 80-90 S&90 20-30 0 90-100 9c-100 90-100 70-80 0 0 8&90 0

Fluorescence intensity

Bright Bright Weak Bright Moderate Bright Moderate

Moderate

as ascites fluid used at 1:40.

no detectable

MoAb 145 MoAb145, reported previously (Rearden et al., 1983), agglutinated in saline all human erythrocytes tested except En(a-) erythrocytes and the Wr(b-) erythrocytes of M. Fr. Its isotype is IgM, kappa. The present study showed that MoAb145 hemagglutination could be inhibited by purified glycophorin A (Table 2). MoAb145 did not react with either GP-A or GP mix in the ELISA assay (Table 3). Immunoprecipitation of iodinated normal erythrocyte membrane by MoAb145 showed no detectable precipitate, and the MoAb145 affinity column eluate prepared from iodinated normal membrane contained no detectable iodinated protein (data not shown). Leukemia

cell lines

HEL

cells

MoAb31, 36 and 145 failed to react with the myeloid leukemia cell lines HL60 and KG-I, or the lymphoid cell lines 8392, 8402 and Jurkat. Results of immunofluorescent staining of K562 and HEL are shown in Table 4. Attempts to induce expression of antigens recognized by MoAb3 1, 36 and 145, anti-H, anti-Ia and anti-HLA-A, B, C on HL60, K562 and HEL cells using chemical inducers or conditioned media were unsuccessful. Treatment of HL60 cells with PHA-LCM or dimethylsulfoxide resulted in expression of monocyte antigen (MO. 1) and increased chloroacetate esterase reactivity respectively and morphological granulocyte differentiation. Similarly, exposure of HEL and K562 cells to hemin did not increase expression of the antigens defined by these monoclonal antibodies, but did result in hemoglobin production as determined by diaminobenzidine staining. MoAb31, 36 and 145. failed to react with nonerythroid hematopoietic cells from 45 patients including patients with acute non-lymphocytic leukemia (20), chronic lymphocytic leukemia (five), acute lym-

phoblastic leukemia (seven), non-Hodgkin’s lymphoma (six), Hodgkin’s disease (one), reactive lymphadenopathy (one), multiple myeloma (one) and Sezary cell leukemia (one). Peripheral blood cells from three patients with myeloproliferative disorders, without circulating nucleated erythrocytes, also failed to react. DISCUSSION

The major human erythrocyte sialoglycoproteins, glycophorins A and B, have been extensively characterized [for review see Anstee (1981)]. Glycophorin A has been isolated in pure form and its complete sequence of 131 amino acids determined (Tomita et al., 1978). Polymorphism at positions 1 and 5 of the amino terminal end determines the MN blood group; serine and glycine respectively occupy these positions when glycophorin A has M-activity, and leucine and glutamic acid when it has N-activity (Dahr and Uhlenbruck, 1978; Furthmayr, 1978a). Trypsin cleaves the polypeptide at two sites, between amino acids 30 and 3 1, and between amino acids 39 and 40 from the amino terminal end (Furthmayr, 19786). The sequence of the 35 amino-terminal amino acids of glycophorin B is known; the first 26 amino acids are largely identical in sequence to glycophorin A and express N-antigen (Dahr et al., 1980). The Ss blood group is determined by an amino acid polymorphism at residue 29 of glycophorin B, methionine occupying this position in S-type and threonine in s-type (Dahr et al., 1980). Glycophorin B on the intact erythrocyte resists trypsin (Furthmayr, 19786); both glycophorins A and B are cleaved by ficin and other proteases. MoAb31 is a new hemagglutinating mouse monoclonal antibody that detected both glycophorins A and B in Western blots. Results with enzymemodified normal, En(a-) and S-s-Uerythrocytes indicated that the MoAb31 determinant or determinants were sialic acid dependent, and resided on glycophorin A on the segment which resists the action

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of trypsin but is sensitive to the action of ficin, and on glycophorin B on the ficin-sensitive segment. There are three glycosylation sites on glycophorin A in the trypsin-insensitive region, at amino acids 44,47 and 50. MoAb31 most likely recognizes one or more of these oligosaccharides, or a determinant which is the result of protein-carbohydrate interaction. Theoretically a determinant could lack sialic acid at the combining site but be sialic acid dependent because its conformation depends on the presence of sialic acid at some distant site. We feel this is unlikely in the case of the MoAb31 determinant because MoAb31 reactivity with glycophorins A and B in the Western blot method suggests that the antigenic site recognized is not conformation-dependent. Therefore, the MoAb31 determinant most likely contains sialic acid at the combining site; lack of hemagglutination inhibition by sialic acid suggests that the determinant contains other structures in addition to sialic acid. The MoAb31 affinity column eluate prepared from normal erythrocyte membrane contained glycophorin A. However, neither the eluate prepared from normal erythrocyte membrane nor the eluate prepared from En(a-) erythrocyte membrane contained glycophorin B. Failure to elute glycophorin B may result from the low density of glycophorin B in the erythrocyte membrane (estimated 70,000 glycophorin B sites compared to 500,000 glycophorin A sites per erythrocyte) or from different MoAb31 affinities for glycophorins A and B. Binding of IgG antibodies to carbohydrate antigen is frequently of low affinity if only a single antibody combining site binds antigen; however, if antigen is bound by both combining sites (“monogamous bivalency”), the rate of dissociation decreases substantially resulting in functionally higher antibody affinity (Crothers and Metzger, 1972; Hornick and Karush, 1972; Romans et al., 1980). The tendency of glycophorin A to aggregate in solution may provide greater opportunity for bivalent antibody binding. This phenomenon may also explain the observed greater ELISA reactivity of MoAb31 with the GP mix fraction (that contained higher polymers of glycophorin A) than with the GP-A fraction. Surprisingly, MoAb31 titers, both direct and in the antiglobulin reaction, were the same with normal and En(a-) erythrocytes in spite of absence of glycophorin A in En(a-) erythrocytes. One possible explanation is enhanced agglutinability due to the reduced sialic acid content of En(a-) erythrocytes. The strong reactivity of MoAb31 with En(a-) erythrocytes contrasts with the weak reactivity of RI.3 with En(a-) erythrocytes. Anstee and Edwards (1982) proposed that R1.3 recognized a sialic acid dependent determinant shared by glycophorins A and B, because R1.3 reacted with En(a-) and S-Uerythrocytes but did not react with trypsin-modified S-Uerythrocytes. or neuraminidaseor proteasemodified normal erythrocytes. This suggested the shared determinant was on the trypsin-sensitive

region of glycophorin A and the protease-sensitive region of glycophorin B. No immunoprecipitation or electroblotting studies were reported, nor inhibition by tryptic peptides prepared from glycophorin A. In addition to its strong reactivity with En(a-) erythrocytes, MoAb31 differs from R1.3 in that it reacts with trypsin-modified S-s-Uerythrocytes. MoAb36 failed to agglutinate En(a -) erythrocytes and the erythrocytes of M. Fr. designated Wr(b-). The Wrb antigen is believed to be on glycophorin A near its insertion into the membrane (Ridgwell et al., 1983). MoAb36 was indistinguishable from R7.1, which has been designed anti-Wrb by Anstee and Edwards (1982). MoAb36 and R7.1 agglutinated in the antiglobulin reaction unmodified and trypsinmodified erythrocytes; could not be inhibited by glycophorin A; did not bind to K562 or HEL: and did not precipitate glycophorin A from human erythrocyte membranes. Three other monoclonal antibodies having anti-Wrb specificity (NBTSBRIC 13, 14 and 15) precipitate glycophorin A from normal but not Wr(b-) erythrocyte membranes (Ridgwell et al., 1983); presumably they are able to do this while MoAb145, 36 and R7.1 do not because they have higher affinity for glycophorin A. We previously reported (Rearden et (I/., 1983) that MoAb145 hemagglutination could not be inhibited by a sialoglycoprotein fraction prepared by butanol extraction. This was confirmed in the present study using a glycophorin mixture prepared by the lithium diiodosalicylate-phenol procedure. However, MoAb145 hemagglutination was completely inhibited by a purified glycophorin A preparation, indicating that MoAb145 recognizes a determinant on glycophorin A. Failure to achieve inhibition with the sialoglycoprotein mixtures may be related to masking of the MoAbl45 determinant on glycophorin A because of aggregation. The MoAb145 determinant is resistant to ficin modification of the intact erythrocyte, suggesting that it resides on that segment of glycophorin A near the insertion of the molecule into the membrane. MoAb145 failed to react with the erythrocytes of M. Fr., designated Wr(b-). Although MoAb36 and 145 probably recognize determinants on the ficin-insensitive region of glycophorin A near its insertion to the membrane, they do not recognize the same determinant. as evidenced by differences in reactivity with primate erythrocytes. Therefore, there is heterogeneity in the specificity of antibodies that fail to agglutinate Wr(b -) erythrocytes. Cytofluorograph analysis demonstrated the majority of cells of the erythroleukemia cell lines K562 and HEL expressed glycophorin A, as indicated by binding RlO, R18, 6A7 and lOF7. Fluorescence intensity was moderate to bright, suggesting high glycophorin A site density on both K562 and HEL; previously Gahmberg et al. (1979) demonstrated that the number of molecules of glycophorin A on K562 cells was comparable to that on mature erythrocytes.

Glycophorin

However, binding of monoclonal antibodies to glycosylated determinants on glycophorin A (MoAb3 1 and R 1.3) and to the non-glycosylated segment near the membrane insertion (MoAb36, 145 and R7.1) was reduced or absent. These results are not explained by failure of MoAb31. 36 and 145 to react by immunofluorescence, since all three antibodies produced bright fluorescence when reacted with mature erythrocytes. Failure of some monoclonal antibodies to glycophorin A to react with K562 and HEL suggests that glycophorin A conformation is altered or that part of the molecule is absent. Altered conformation could result from differences in glycosylation. Another explanation is different associations of glycophorin A with other molecules in the membrane of erythroleukemia and mature red blood cells. Differences in glycosylation and resultant conformational changes in glycophorin A may account for some of the apparently discrepant results obtained using polyclonal and monoclonal glycophorin A antibodies to detect glycophorin A expression by leukemia cells (Elkblom ez al., 1983; Liszka et al., 1982). Polyclonal antisera may detect multiple determinants on glycophorin A, and therefore be more likely to detect immature or incomplete glycophorin A. This interpretation is consistent with the observed higher incidence of glycophorin A expression in leukemia reported by those using polyclonal antiglycophorin A sera. In this study we examined blast cells from a limited number of patients with leukemia and from patients with other hematopoietic malignancies. We were unable to detect the expression of MoAb31, 36 and 145 antigens on any of these cells. We were also unable to induce expression of any of these antigens by the HL60, K562 or HEL cell lines, using either chemical or biologically active mediators. We conclude from these results that inappropriate expression of these erythroid antigens does not occur or is uncommon. Whether leukemia cells other than erythroleukemia cells can express glycophorin A has theoretical importance. McCulloch (1983) showed reduced remission induction in acute myelogenous leukemia when blast cells expressed markers of more than one lineage; he proposed that transformed cells which express markers of multiple cell lineages are biologically more aggressive. Glycophorin A is a specific molecular marker of one myeloid linage, the erythroid, and as such could be used to better define the genetic programs controlling erythroid differentiation. A growing number of monoclonal antibodies to determinants on gfycophorin A have been produced (Edwards, 1980; Anstee and Edwards, 1982; Fraser et al., 1982a, b; Barsoum et al., 1982; Ridgwell et al., 1983; Ochai et al., 1983; Bigbee et al., 1983). These antibodies may be useful in studies of the molecular development of glycophorin A. The present study shows that glycophorin A produced by the erythroleukemia cell lines

A

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KS62 and HEL differs from glycophorin A at the surface of normal, mature erythrocytes with respect to reactivity with monoclonal glycophorin A antibodies. Whether erythroleukemia cell glycophorin A corresponds to a normal developmental stage in the molecular development of glycophorin A during erythropoiesis remains to be determined. Acknowledgements-This work was supported in part by grants from the National Institutes of Health (HL-249292, AM33463 and CA32094); The Cancer Research Coordinating Committee of the University of California; The Academic Senate, San Diego Division, University of California, and by the Research Service of the Veterans Administration. We wish to thank Dr Paul A. W. Edwards for monoclonal antibodies, Dr Paul Martin for the cell line HEL, Dr Heinz Furthmayr for sialoglycoprotein fractions. and Drs Faith Kung and Alice Yu for bone marrow samples, We are grateful to Dr Herbert A. Perkins and MS Phyllis Walker of the Irwin Memorial Blood Bank (San Franciso, CA) and to Dr Moses Shanfield, MS Delores Mallory and MS Rosia Nesbitt of the American National Red Cross (Bethesda, MD) for studies with rare erythrocytes. We gratefully acknowledge the technical assistance of MS Susan Wormsley who performed the cytofluorograph analysis, and of MS Eileen Bessent, Catherine Stanish and Mary Ann Salapow. We also thank MS Mary Oberg for typing the manuscript.

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