Proc. NatL Acad. Sci. USAVol. 79, pp. 7891-7895, December 1982Medical Sciences

Monoclonal antibody against angiotensin-converting enzyme: Itsuse as a marker for murine, bovine, and human endothelial cells

(cell sorting/cell culture/blood vessels)

R. AUERBACH, L. ALBY, J. GRIEVES, J. JOSEPH, C. LINDGREN, L. W. MORRISSEY, Y. A. SIDKY, M. Tu, ANDS. L. WATTDepartment of Zoology, University of Wisconsin, Madison, Wisconsin 53706

Communicated by M. R. Irwin, September 2, 1982

ABSTRACT A monoclonal antibody has been prepared againstrat angiotensin-converting enzyme (ACE). By selection for anti-body binding to endothelial cells of bovine rather than rat originwe have obtained a reagent that has broad cross-species bindingproperties and that can at the same time serve as a useful markerfor the surface of endothelial cells. The IgM-producing clone thatwe have established, a-ACE 3.1.1, has been grown in ascites formto yield ascites fluid that binds selectively to immobilized ACE ata >1:10,000 dilution. By use of enzyme-linked immunosorbentassays, immunofluorescence histology, and flow cytometry, wehave demonstrated the presence of ACE on endothelial cells ofmurine, bovine, and human origin. By means of a fluorescence-activated cell sorter (FACS-IV) we have been able to selectivelyisolate viable endothelial cells from a mixture of endothelial cellsand fibroblasts. We believe the antibody will be useful not onlyfor the selection and in vitro cultivation ofendothelial cells but alsoas a tool for the identification and pharmacological study of ACE.

Angiotensin-converting enzyme (ACE), or kininase II, whichcleaves the terminal dipeptide from angiotensin I to form vaso-active angiotensin II and which is active as a dipeptidyl hydro-lase in its action on bradykinin and other small peptides, servesas a useful marker for the identification ofendothelial cells fromcapillaries, veins, and arteries (1-4). Because the enzyme isassociated with the cell surface (5), antibodies directed againstACE can serve not only to identify endothelial cells but also canmark them for analysis and isolation without loss of viability.Our interest in the growth and development of murine en-

dothelial cells prompted us to generate monoclonal reagentsdirected at strongly cross-species reactive ACE. Tests carriedout with conventionally generated rabbit anti-rat ACE (6)pointed to rat lung ACE as a suitable immunogen; therefore,we used a rat lung ACE preparation to induce an immune re-sponse in mice. Spleens from these mice then were fused byhybridization to nonsecreting mouse myeloma cells to permitisolation and characterization of hybrids (hybridomas) that pro-duced monoclonal antibodies to ACE.We now report on the properties of one such hybridoma-

its cell-specific and enzyme-directed binding properties and itsuse as a reagent for identifying and isolating murine, bovine,and human endothelial cells.

MATERIALS AND METHODSACE Preparation. ACE was obtained by following the meth-

ods of Lanzillo and Fanberg (6, 7). In brief, rat lungs were dis-sected after lavage, lightly homogenized in 0.02 M potassiumphosphate buffer (pH 8.3), and centrifuged (250 x g; 10 min)to remove cells and debris. The supernatant was recentrifuged

(54,000 X g; 60 min) and the pellets then were rehomogenizedto yield the "crude enzyme preparation." For further purifi-cation, sodium deoxycholate was added; this was followed bycentrifugation and dialysis, filtration through Whatman no. 1paper, and fractionation on DEAE-cellulose columns (7). Frac-tions from the column were assayed for ACE activity and themost positive fractions were pooled for use as the "partially pu-rified enzyme preparation." More highly purified ACE wasobtained from bovine lung, by using ion exchange chromatog-raphy and gel filtration protocols (8, *). The bovine "purifiedACE preparation" was used in enzyme-linked immunosorbentassays (ELISAs).ACE Assays. ACE activity was measured by using radiola-

beled diglycyl hippurate ([3H]Hip-Gly-Gly, Ventrex Labora-tories, Portland, ME) as substrate. Enzyme activity was as-sessed by separating the radioactive peptide on the basis of itsdifferential solubility in ethyl acetate.

Immunization. BALB/cAu mice were immunized with 0.1ml of a 1:1 mixture of crude enzyme preparation and Freund'sincomplete adjuvant, injected intraperitoneally, restimulatedwith 0.1 ml of the partially purified enzyme preparation, andused 4 days later.

Hybridization and Hybridoma Culture Methods. Spleencells were fused with NS-1 (P3 NS1 Ag 4/1) cells (9) that wereoriginally obtained from the Salk Institute and maintained inDulbecco's modified Eagle's minimal essential medium (DMEmedium) with 10% fetal bovine serum. Spleen cell/myelomaratio was 10:1; fusion was effected with 40% polyethylene glycol(Mr 1,050). Cells were selected in hypoxanthine/amethopterin/thymidine (HAT) medium and then were cultured further inhypoxanthine/thymidine (HT) medium. Soft agar cloning tech-niques were used (10). Ascites were produced by injection ofcells into pristane-treated BALB/cAu mice.

Endothelial Cell Cultures. Endothelial cells from adult bo-vine adrenal gland, aorta, pulmonary vein, and retina, from fetalbovine aorta and ovary, and from murine brain and epididymalfat pad were obtained by following published protocols (11-17).In principle, cells were obtained by collagenase treatment, fil-tered (210-pum Nytex) to remove clumps, and cultured in DMEmedium with 20% fetal bovine serum mixed with 20% S180-conditioned medium. DeBault's medium was used for murineendothelial cell cultures (16). Other cell lines (suppliers in pa-

Abbreviations: ACE, angiotensin-converting enzyme; Hip-Gly-Gly,diglycyl hippurate; HAT, hypoxanthine/amethopterin/thymidine; HT,hypoxanthine/thymidine; DME medium, Dulbecco's modified Eagle'sminimal essential medium; TNP, trinitrophenvl; Pi/NaCl, phosphate-buffered saline; ABAE, adult bovine aortic endothelium; BT, bovineturbinate; ELISA, enzyme-linked immunosorbent assay; FITC, fluo-rescein isothiocyanate; FACS, fluorescence-activated cell sorter.* Lindgren, C. (1982) Undergraduate thesis (Univ. of Wisconsin, Mad-ison, WI).

7891

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7892 Medical Sciences: Auerbach et al.

rentheses) included human Ruba endothelial cells (K. Rezni-koff; ref. 18), adult bovine turbinate (BT) cells and bovine fetallung fibroblasts (C. Kanitz), fetal bovine heart endothelial cells(American Type Culture Collection), human and mouse embryofibroblast lines (B. Kahan), and L929 murine fibroblasts (E.Borden).

Flow Cytometry and Cell Sorting. Cells were obtained bycollagenase treatment, washed in DME medium, passedthrough 15- to 40-gm Nytex filters, and centrifuged throughFicoll-Hypaque. Cells were incubated at room temperature for30 min with serum diluted in Ca2+- and Mg2 -free phosphate-buffered saline (Pi/NaCl) containing 1% bovine serum albuminand 0.1% sodium azide. Cells were washed again in 1% bovineserum albumin, incubated with fluorescein isothiocyanate-(FITC) labeled rabbit anti-mouse immunoglobulin (CappelLaboratories, Cochranville,- PA), washed again, and diluted to2 X 105-1 x 106 cells per ml with Pi/NaCl. For viable, sterilesorting the final cell suspension was diluted with Pi/NaCl con-taining 2% fetal bovine serum.

Cell were analyzed or sorted on a Becton Dickinson fluo-rescence-activated cell sorter (FACS-IV) equipped with a 5-Wargon ion laser tuned to 488 nm and operating at 100 mW out-put. Cells were passed through a 70-,im orifice and were as-sessed for relative intensity of fluorescence emission at >515nm as well as for forward-angle.light scatter. Cells were sortedinto DME medium with 1% bovine serum albumin for ACEassays or into tumor-conditioned medium for endothelial cellculture.

Immunohistology. Cryostat sections were cut at 6 Am andplaced on coverslips for processing. Cells from culture weregrown directly on coverslips. Dried sections were used un-stained or stained with hematoxylin in water for 5 min; cells fromculture always were examined unstained.

ELISAs. Test endothelial or control fibroblast cells were per-mitted to grow to confluency in 96-well Linbro plates. To avoidbinding of ACE that was present in fetal bovine serum, cellswere grown in serum-free DME medium for 24-48 hr beforeuse in ELISAs. Cells were fixed.in 0.02% glutaraldehyde, and0.3% gelatin was added during washing procedures to decreasenonspecific adsorption. The response was measured by usinga dual wavelength Microelisa Reader (Dynatech, Alexandria,VA).

Affinity Gels. Affinity columns were made by binding 40 mgof ascites protein (Lowry) to 2 ml of Affi-Gel 10 (Bio-Rad) ac-cording to standard protocols;. 80% of the protein remainedbound to the beads.

Ultrafiltration. Enrichment for IgM was achieved by con-centration and washing of ascites fluid on XM-300 Amicon fil-ters under nitrogen pressure. After filtration the volume wasreadjusted and protein concentration was calculated fromLowry determinations.

RESULTSFrom the spleen ofone ofthe mice immunized against rat ACE,960 microwell cultures (10 Linbro plates) were prepared. Vir-tually all culture wells showed growth ofhybrid cells at the endof a 2-wk culture period. As a primary screen, hybridoma su-pernatants were tested for binding (ELISAs) to bovine pul-monary vein endothelial cells and, on the basis of these tests,cells from nine of the microtiter wells were selected for expan-sion. On retesting, one hybridoma supernatant (isolate no. 3)appeared particularly active; therefore, cells from this isolatewere chosen for expansion, cloning, and generation of ascites.The remaining selected hybrids were frozen for future analysis.Clone 3.1.1, maintained-both in vitro and in ascites form, hasprovided the material for all of the results that we now report.

By Ouchterlony tests a-ACE 3.1.1 was found to be an IgM-producing clone, and therefore, we used another IgM produc-ing hybridoma, prepared against an unrelated antigen (a-trini-trophenyl; a-TNP F1.2.1), as specificity control. Cell bindingstudies were carried out by using ELISAs on cultured endothe-lial cell lines and nonendothelial control cultures and by flowcytometry with a fluorescein-labeled anti-mouse immunoglob-ulin antibody. ELISAs were positive on endothelial cells whenperformed with either ascites fluid or cell culture medium su-pernatants. Mouse L929 fibroblasts were negative as were hu-man foreskin fibroblasts and bovine fetal lung fibroblasts. Theonly inconsistent results were obtained with the bovine BT cellline, which was positive in ELISAs but was negative both forenzyme activity and flow cytometry.

Flow cytometric analysis confirmed the anti-endothelial cellspecificity of clone 3.1.1 (Table 1). The antibody bound with

Table 1. Binding of anti-ACE 3.1.1 ascites fluid to-cultured celllines, fresh isolates, or cryostat sections and its correlation withACE activity

Flow Immuno- EnzymeSource of cells ELISA cytometry histology activity

EndotheliumABAEFresh isolateEarly passage (<10)Late passage (>16)

Fetal bovine aorticFresh isolateEarly passage

Adult bovine adrenalcapillaryFresh isolateEarly passageLate passage

Adult bovine retinalMouse epididymal fat padMouse brainEarly passageLate passage

Human Ruba (endothelialcells)

Human umbilical vein

FibroblastsBTBovine fetal lungMouse embryoMouse L929Human foreskinHuman lung

+ + NT ++ + + +- - NT -

NT + NT ++ + + +

NT

+ NT ++ NT +- NT -

NT + ++ + +

+ +- NT

+

+ NT NT +NT + + NT

+ - NT- NT NTNT - NT

- NT NT- NT NT

Other*Mouse-adrenal gland

Endothelial cellsNonendothelial cells

Mouse kidneyEndothelial cellsProximal tubulesOther cell types

Human umbilical veinEndothelial cellsSubendothelial regionOther nonendothelial

cells

NT, not tested.* Cryostat sections.

NTNTNT

+

++

++

Proc. Natl. Acad. Sci. USA 79 (1982)

Proc. NatL Acad. Sci. USA 79 (1982) 7893

highest fluorescence intensity to mouse brain endothelial cells(Fig. IA), showed distinct binding profiles with bovine aorticand adrenal capillary endothelial cells, and bound effectivelyto a significant fraction of freshly isolated cells from bovine aortaor adrenal cortex. Binding was negative against both BT andbovine fetal lung fibroblasts as well as against murine L929 andembryonic fibroblasts.A direct test for specificity of the antibody was carried out

by adsorbing purified ACE onto microtiter wells for ELISAs.Activity ofascites fluid was seen at dilutions ranging from 1:100to 1:10,000, depending on the preparation, and was always ac-tive at dilutions two or more logarithms higher than that ofneg-ative control ascites fluid (Table 2).

Further tests for specificity were carried out by preparingaffinity columns. Crude ascites fluids obtained from the anti-ACE and anti-TNP hybridomas were used to provide two com-parable small Affi-Gel columns, to each ofwhich 100 1.d of fetalbovine serum (as a source ofACE) was applied. Fractions weretested forACE activity by using the Hip-Gly-Gly cleavage assay(Fig. 2). The a-ACE column selectively retained much of theACE activity at low salt concentration, and the enzyme could

z

VI

Table 2. Binding of anti-ACE 3.1.1 ascites fluid to microtiterplates coated with purified ACE isolated frombovine fetal lung

Optical transmission ratioExperiment Dilution a-ACE 3.1.1 a-TNP F1.2.1

I* 1:1001:1,0001:10,0001:100,000

HAt Direct1:101:1001:1,000

ILBt Direct1:101:1001:1,000

HIM 1:1001:4001:1,6001:6,400

IHB§ 1:6001:2,5001:10,0001:40,000

0.1340.2320.2100

0.4460.3030.136

0

0.5350.4240.1360.012

0.7790.5080.3010.207

0.230000

0.0180.0170.024

0

0.0680.0300.0240.065

0.0830.0530.0720.038

0.2680.2370.1100.047

Dishes were incubated overnight withACE at 100,ug/ml, rinsed, andcovered with 5% calf serum for 30 min prior to addition of ascitesfluid. ELISAs utilized alkaline phosphatase-coupled anti-IgG (H andL chains).

tBlocking with either calf serum (IA) or 1% bovine serum albumin(IIB) for 2 hr at room temperature.Similar to experiment HIB; a different lot of ascites fluid was usedfor preparing the Amicon filter XM300 retentate.

§ Ascites fluid was passed over the Amicon filter XM300. Retentatewas reconstituted to a final protein concentration of 3 mg of totalprotein per milliliter; 4 ml of the original ascites fluid was used toyield 3 ml of the final reagent.

FL UORE SCE NCE

FIG. 1. Flow cytometric analysis of antibody binding to endothe-

lial cells. (A) Comparison of binding of a-ACE 3.1.1 antibody to mouse

embryonic fibroblasts and mouse brain endothelial cells. Fluorescence

intensity (x axis) was obtained by using logarithmic amplification to

permit visualization of the unlabeled fibroblasts and heavily labeled

endothelial cells on the same display. (B) Freshly isolated ABAE la-

beled either with a-ACE 3.1.1 or a-TNP F1.2.1 ascites fluid. Fluores-

cence intensity (x axis), measured on linear scale, distinguishes clearlybetween the two reagents, cells labeling only withthe a-ACE antibody.

be eluted subsequently, indicating reversible binding to theaffinity column.

Immunohistological studies carried out on frozen sectionsfrom murine thyroid, kidney, adrenal, and brain (region of cho-roid plexus) showed that both capillaries and larger blood ves-sels stained dramatically with the anti-ACE reagent (1: 100 forstained sections; 1:1,000 dilution for unstained sections), pro-viding at low magnification a lattice-like pattern of the micro-vasculature. At higher magnifications the antibody was seen tobe clearly associated with endothelial cells lining the blood ves-sels. Fluorescence was associated with the renal epithelium ofthe proximal convoluted tubules. Tissues stained with anti-TNPwere routinely negative. Endothelial cells grown on coverslipsshowed positive fluorescence with a-ACE 3.1.1, except for latepassage adult bovine aortic and late passage murine brain en-dothelium (Table 1). Negative staining with the antibody cor-related with loss ofenzymatic activity as measured by tripeptidecleavage assays.The ability of a-ACE 3.1.1 to bind to endothelial cells was

used next to select endothelial cells from a mixed population.For demonstration purposes adult bovine aortic endothelialcells (ABAE) and murine L929 fibroblasts, similar in their for-ward angle light scatter properties, were used. Individual flu-orescence profiles were obtained for the two cell types, andthese are shown superimposed in Fig. 3. Marker settings were

Medical Sciences: Auerbach et aL

7894 Medical Sciences: Auerbach et al.

6010

l

to

z

00

0

m

w40.

-a TNP F1-2-1501-

401a ACE 3-1-1

30k20H

1o0

CI

2 4 6 8 10 12 14FRACTION NUMBER

FIG. 2. Specificity of a-ACE monoclonal antibody examined byaffinity chromatography. Affinity columns were made either with a-ACE 3.1.1 or a-TNP F1.2.1, fetal bovine serum was applied as a sourceof ACE, and the fractions were assayed for ACE activity (expressedas dpm above background). Arrow indicates change from 0.015 M NaClto 1.5 M NaCl.

determined from the raw counts so that "dim" cells included50% of fibroblasts and 20% of endothelial cells, whereas"bright" cells included 20% of fibroblasts and 50% ofendotheli-al cells. The two cell suspensions then were mixed in ratios of1:1 and 3:1, and for each mixture 106 dim and bright cells wereobtained by sorting. The enzymatic activity of each cell popu-lation was measured by tripeptide cleavage assays, and the ac-tivity was compared to the activity predicted from the fluores-cence profiles (Fig. 3; Table 3). The results established that cellslabeled with the hybridoma reagent had ACE activity, whereasthe unlabeled cells had low or absent activity for this enzyme.We next prepared cell suspensions from freshly obtained

bovine adrenal cortex. After labeling with anti-ACE antibody,cell sorting was carried out by selecting the brightest 25% anddimmest 25% ofcells in the suspension. Bright cells were foundto be enriched for ACE activity, whereas activity among the dim

0:EW L929

z

wj ABAE0

Table 3. Effectiveness of sorting for ACE-positive cellsL929 to ABAE ratio

Cells sorted Results* 1:1 3:1Bright Expected 25,588 16,305

Observed 21,827 15,579Dim Expected 10,249 4,216

Observed 9,656 726

Values are presented as dpm for 106 sorted cells.* Expected dpm were based on an observed value of 35,836 dpm per106 ABAE cells released from [3H]Hip-Gly-Gly (see Fig. 2). The es-timated expected dpm were only approximations because tripeptidecleavage is not a simple linear function of cell numbers.

cells was decreased. Cells from the sorted groups as well asunsorted cells were placed in cell culture. Both dim and brightcells suspensions gave rise to cultures that grew to confluency.The bright cell cultures appeared to be enriched for endothelialcells, as judged by gross microscopic inspection, by continuedACE activity (tripeptide cleavage assay), and by successful invitro passage (Fig. 4).

Finally, we wished to determine whether the antibody alsocould detect ACE on human endothelial cells. Freshly obtainedhuman umbilical vein endothelial cells were cultured for 4 daysand were assayed for antigen by flow cytometry. Specific ad-sorption ofthe a-ACE antibody was seen at a dilution of 1:500;higher dilutions were not tested. The human Ruba endothelialcell line also showed positive labeling at 1:500 dilution.

DISCUSSIONWe have described a monoclonal antibody that is directed spe-cifically against ACE. This antibody has a broad species range,as shown by our extensive documentation of reactivity withmurine, bovine, and human endothelial cells. The reagent hasbeen shown to bind selectively to endothelium and to bind ina quantitative manner to bovine and rat ACE in ELISAs.The ability of this monoclonal reagent to bind to the cell sur-

face has permitted us to use the antibody as a marker both forthe identification of endothelial cells and for the isolation ofthese cells for in vitro cultivation. Enrichment for viable en-dothelial cells using a cell sorter has been achieved. Selectiveadhesion to antibody-coated dishes (panning; ref. 19) or alter-native solid-phase selection methods (20) should be equally fea-sible.

DIM.<

FLUORESCE NCE

FIG. 3. Fluorescence profiles obtained for L929 fibroblasts andABAE used in cell mixing and sorting experiments (see text). Markers(vertical bars) were set so that dim cells included 50% of fibroblastsand -20% of endothelial cells, whereas bright cells included 50% ofendothelial cells and -20% of fibroblasts.

FIG. 4. Bovine adrenal gland capillary endothelial cells grown invitro 1 wk after sorting (FACS-IV) on the basis of bright fluorescenceafter labeling with a-ACE 3.1.1 and FITC-labeled rabbit anti-mouseimmunoglobulin.

Proc. Nad Acad. Sci. USA 79 (1982)

3.01

Ia 5....

............ .................

....... ...

9.9

Proc. Natl. Acad. Sci. USA 79 (1982) 7895

Our own studies of endothelial cells have been directed pri-marily at determining differences that characterize these cellsfrom different organ sources. Our central hypothesis, based ondevelopmental considerations, states that endothelial cells arenot all alike, their dissimilarities being due in part to their di-verse embryonic origins (21, 22, t). That hypothesis predictsthat organ specificity of endothelial cells can be detected by theselective expression of surface-associated organ-limited anti-gens on endothelial cells from different organ sources. Indeed,we have already shown that, for example, mouse brain capillaryendothelial cells differ from capillary endothelial cells obtainedfrom mouse epididymal fat pad by their differential expressionof I region and Thy-i antigens (23).

Interest in the developmental biology of blood vessels andof the endothelial cells of the microvascular system has alreadyprompted us to look at the expression of ACE during the on-togeny ofthe mouse embryo (unpublished data). The high levelsofenzyme present in fetal sera suggest a prominent role for thisenzyme during embryogenesis, and it appears that high enzy-matic activity is associated with the vascular extraembryonicyolk sac of the early mouse embryo. Since the turn of the cen-tury there has been extensive debate concerning the origin ofblood vessels (cf. ref. 23), and the expression of ACE duringearly development and its detection with the anti-ACE antibodymay now permit us to trace at the cellular level the ontogenichistory ofvascular endothelium. We do not need to assume thatall blood vessels, or even all capillaries, necessarily follow a sin-gle pattern of development: intrinsic endothelial cell differen-tiation may be characteristic of specific organ rudiments,whereas in other instances (e. g., the kidney glomerular bed; ref.24) an ingrowth of endothelial cells from previously establishedlarger vessels may be responsible for the establishment of thelocal vascular cells.The isolation of specific endothelial cells from different

sources and with different phenotypes will furnish us with theopportunity to examine several corollaries ofour hypothesis thatorgan-specific, cell-surface-associated antigenic differences areexpressed on endothelial cells. We propose that (i) selectiveadhesion of tumor cells to different endothelial cells may becorrelated with selective tumor metastasis; (ii) organ-specificantigens are responsible for the induction of endothelium-di-rected angiogenesis factors as these are released from sensitizedlymphocytes; and (iii) a variety of specific manifestations ofvas-cular pathology associated with a wide range of disease statesmay result from organ-limited differences in the biology of thevascular endothelial cells.

We appreciate the assistance of Dr. Rodney Pratt in isolation ofACE,Louis Kubai, Sheryl Strong, and Gareth Thomas for technical assistancein many of the procedures used in these experiments, Barbara Houserand Wanda Auerbach for assistance in bibliographical and editorialwork, and J. Scott Cairns and Carol Dahl for helpful suggestions anddiscussions. We are indebted to Dr. B. Fanburg for rabbit a-rat ACEand to J. S. Cairns for a-TNP F1.2.1 monoclonal antibody. We thankDrs. K. Reznikoff, C. Kanitz, B. Kahan, and E. Borden for providingus with some of the cell lines used in our work. These studies weresupported by Grants CA 28626 from the National Cancer Institute, AI14607 from the National Institute of Allergy and Infectious Diseases,and EY 3243 from the National Eye Institute.

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