Clin Exp Immunol 1994; 96:170-176

Characterization of EN4 monoclonal antibody:a reagent with CD31 specificity

V. L. BURGIO, S. ZUPO*, S. RONCELLA*, M. ZOCCHIt, L. P. RUCO & C. D. BARONI Dipartimento diMedicina Sperimentale, Sezione di Immunopatologia, II Cattedra di Anatomia Patologica, Universitai "La Sapienza", Rome,

*Istituto Nazionale per la Ricerca sul Cancro, Genova, and tlstituto Scientifico "S. Raffaele", Milan, Italy

(Acceptedfor publication 27 October 1993)

SUMMARY

EN4 MoAb was originally described as a MoAb that reacts specifically with human endothelial cells,and the reagent was not assigned to any of the presently known CD. Here, we provide evidenceindicating that EN4 reacts with the CD31 antigen. Thus, EN4 stains strongly murine fibroblaststransfected with the human CD3 1 gene. Furthermore, SDS-PAGE analysis of immunoprecipitatesof cell lysates from surface-iodinated Jurkart T cells demonstrated that EN4 and reference CD31MoAb recognized the same antigen, of 130 kD mol. wt. Finally, both EN4 and CD3 1 gave the samepattern of reactivity when tested on tonsillar or peripheral blood lymphoid cells by FACS analysis orby immunohistochemistry on sections of a variety of human tissues. EN4, however, provedconsistently more efficient than the reference anti-CD31 MoAb as judged by both the intensity offluorescence or of tissue staining. This property has thus allowed a better characterization of thetissue and cellular distribution of CD3 1.

Keywords EN4 CD31 adhesion moleculesperipheral lymphoid tissues

INTRODUCTIONEN4 (MAS336) has been described as a MoAb specificallystaining endothelial cells lining blood and lymphatic vessels [1-4]. This MoAb has been prepared by immunizing CPA/BALB/cmice with isolated human umbilical cord endothelial cells [1].Recently, we observed [5] also that several non-vascular,mononucleated haematopoietic and lymphoid cells present inthe bone marrow were strongly EN4-positive. This preliminaryobservation prompted us to characterize better the expressionand distribution at cellular level of the antigen recognized byEN4. The collected evidence using surface iodination methodsfollowed by immunoprecipitation and SDS-PAGE analysis andFACS studies demonstrates that EN4 is a CD3 1 MoAb. Becauseof the stronger reactivity of this reagent both in the FACSanalysis and in the staining of tissue section, it has been possibleto characterize better the reactivity of anti-CD31 MoAb.

MATERIALS AND METHODS

Tissue analysisTissue samples, surgically removed for diagnostic purposes,were collected from central and peripheral lymphoid tissues and

Correspondence: Professor Carlo D. Baroni, II Cattedra di Anato-mia ed Istologia Patologica, Sezione di Immunopatologia, Diparti-mento di Medicina Sperimentale, Policlinico Umberto I, Viale ReginaElena 324, Roma, Italy.

bone marrow thymus

from mucosa-associated lymphoid tissue (MALT), and used inthe present study. They included two thymuses, 10 bonemarrows, 20 lymph nodes involved by several types of chroniclymphadenitis ofdifferent nature and etiology, including follicu-lar hyperplastic lymphadenitis, diffuse lymphadenitis, tubercu-losis, sarcoidosis and toxoplasmosis, two spleens, five tonsilsand two appendices. None of the tissue samples was involved byprimary or secondary neoplasia. Representative fragments of allthe tissues included in the present investigation were embeddedin paraplast for morphological analysis.

Additional fragments ofthe same tissues were snap-frozen inoct compound in liquid nitrogen for immunophenotyping oncryostat sections, using the alkaline phosphatase anti-alkalinephosphatase (APAAP) method [6]. New Fuxin (Merk, Darm-stadt, Germany) was used as chromogen in the single primaryMoAb immunohistochemical staining method, as well as for thedevelopment of positivity of the second primary MoAb used inthe double-staining procedure. In this procedure the secondreagent (rabbit anti-mouse; Dako, Glostrup, Denmark) and theAPAAP complex (Dako) were repeated twice after the firstMoAb used as primary antibody, whose positivity was devel-oped using fast blue salt (Sigma-Aldrich, Milan, Italy) aschromogen, followed by a second 60-min incubation in thedevelopment solution without chromogen. Bone marrow sec-tions prepared from glycol-methacrylate embedded biopsieswere immunostained using a method recently described in our

170

EN4 MoAb reacts with CD31

laboratory [5]. Sections treated with the second antibody andwith the APAAP complex alone, and sections treated withirrelevant isotype-matched antibodies were used as controls.

Cell preparationsMononuclear cells (MNC) from surgically removed tonsils wereisolated by gentle mincing followed by centrifugation on Ficoll-Hypaque density gradient [7]. The MNC suspensions wererosetted with neuraminidase-treated sheep erythrocytes, and therosetting cells (T cells) were separated from the non-rosettingcells by Ficoll-Hypaque density gradient centrifugation [8].Non-rosetting cells (non-T cells) were subsequently depleted ofCD15 (Leu-M1) and CD16 (Leu-1lb)-positive cells usingMoAbs (Becton Dickinson, San Jose, CA) and complement(Cederlane, Ontario, Canada) [8]. The resulting cell population(B cells) always contained more than 95% B cells as determinedby indirect immunofluorescence using CD20 (Bi) MoAb.Peripheral blood (PB) T cells were obtained from MNC byrosetting with sheep erythrocytes (that had not been treated withneuraminidase) at 29'C (low-affinity rosettes) [8]. Monocyte-enriched suspensions were prepared from normal PB MNC by asingle step centrifugation on a 50-6% Percoll (Pharmacia LKB-Biotechnology, Inc., Uppsala, Sweden) density gradient [9].These cell preparations consistently contained more than 90%CD3+ T cells and 85-90% CD14+ cells respectively, as deter-mined by immunofluorescence.

Radiolabelling, immunoprecipitation and SDS-PAGE analysisFor cell surface iodination, 50 x 106 cells of Jurkart line werewashed in PBS (0-02 M sodium phosphate pH 7 2) and labelledwith I mCi 1251 (Amersham International, Aylesbury, UK) in250 ml ofPBS using 50 pl of lactoperoxidase at 2 mg/ml (Sigma)as a catalyst. After labelling, cells were solubilized and immuno-precipitated as described previously [10]. Briefly, the cells werewashed three times in cold PBS and lysed with 1% Triton X-100for 1 h at 40C. The lysate was pretreated with normal mouseimmunoglobulin coupled to protein A-Sepharose (Pharmacia)and then incubated for 4 h at 40C with 10 pg of the appropriateantibody. The precipitation was carried out using ProteinA-Sepharose (Pharmacia) for 1 h. After extensive washing inPBS containing 1% Triton, immunoprecipitates were eluted byboiling for 5 min in SDS sample buffer. The samples were loadedand run on a 8% polyacrilamide gel, under reducing conditions.Autoradiography took place at -70°C, using Kodak XAR-5film in combination with intensifier screens for 20 days.

PECAM-J (CD31) transfectantsCell lines were made with PECAM-1 and neomycin resistanceon the same plasmid. PECAM was subcloned into pcDNAI/Neo (Invitrogen, San Diego, CA) at the Xhol (5') Nsil (3') sitesfrom the original pGem7 vector (PECAM-l/pGEM7 waskindly provided by Dr Peter Newman, Blood Centre ofSoutheastern Wisconsin, Milwaukee, WI). NIH/3T3 transfec-tion was performed by calcium phosphate-DNA co-precipi-tation. Confluent T75 flasks ofNIH/3T3 cells were split 1: 5 andplated on 100-mm Petri dishes in D-MEM with 10% fetal calfserum (FCS). After 12 h, the cells were transfected with 10 pg ofPECAM/pcDNAI/Neo plasmid and incubated at 37°C for 12 h.Cells were then washed with PBS, shocked with 10% glycerol inD-MEM 10% FCS. The plates were maintained for an addi-tional 36 h before immunofluorescence test. Stable transfectants

were selected by addition of neomycin analogue G418 to a finalconcentration of 0-8 mg/ml. Neomycin-resistant colonies werepicked up 10 days later, expanded and tested for PECAMexpression by immunofluorescence using MoAb 5.6E (Immuno-tech, Luminy-Marseille, France) and JC70A (Dako).

Mock transfected line and wild type NIH/3T3 cells wereused as negative controls.

Monoclonal antibodies and immunofluorescenceThe MoAbs used were: anti-CD3 (OKT3) (Ortho Diagnostics,Raritan, NJ); anti-CD20 (Bi), anti-CDllb (Mol) (CoulterCorp., Hialeah, FL); anti-CD31 (JC70A) (Dako); (9G1 1) (BBL,Cowley, UK) and EN4 (Seralab, Crawley Down, UK).

Indirect immunofluorescence was performed with FITC-conjugated goat anti-mouse immunoglobulin isotype anti-bodies (Southern Biotechnologies, Birmingham, AL) as secondreagent. The samples were analysed using a FACStar cytofluori-meter (Becton Dickinson). Cells treated with irrelevant isotype-matched MoAbs were used as negative control.

RESULTS

EN4 reactivity on CD31 transfectant cellsThe eukariote expression vector pGEM7 containing the CD3 1cDNA and neomycin-resistant gene (PECAM-l/pGEM7) wastransfected in the murine NIH/3T3 fibroblast line by calcium-phosphate DNA coprecipitation. The NIH/3T3 fibroblastswere similarly transfected with empty pGEM7 vector contain-ing only neomycin gene, as negative control. The cytometricanalysis of the transfected clones with JC70A (anti-CD31)indicated that CD31 was expressed on the PECAM-1 trans-fected cells (Fig. 1, top), while the cells transfected with theempty vector were negative (Fig. 1, bottom). We tested the EN4reactivity on the transfected lines. EN4 stained strongly thePECAM- I transfected line (Fig. 1, top), and was negative on the

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Log fluorescence intensityFig. 1. Flow cytometric analysis with EN4 and JC70A (anti-CD31)MoAbs ofNIH/3T3 transfected cells. Indirect immunofluorescence wasperformed with EN4 or JC70A followed by FITC-conjugated goat anti-mouse immunoglobulin (solid line) on the PECAM-l/NIH/3T3-trans-fected cells (top) or empty vector NIH/3T3-transfected cells (bottom).Broken lines represent control stainings performed with irrelevantisotype-matched MoAb followed by FITC-conjugated goat anti-mouseimmunoglobulin.

171

control transfected line (Fig. 1, bottom). Of note is the findingthat EN4 staining was I log brighter than the staining obtainedby JC70A.

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Molecular characterization of the surface structure reactive withEN4The above results indicate that the antigen recognized by EN4was CD331. To confirm the nature of antigen defined by EN4,immunoprecipitation studies were carried out. '251I-labelledJurkart cells were immunoprecipitated with JC70A and EN4under reducing conditions. Both the MoAbs precipitated a

single molecule from the surface of Jurkart cells with an

estimated mol. wt of 130 kD (Fig. 2). In comparison with JC70A,EN4 proved inferior in its capacity to precipitate the labelledantigen. Unfortunately, sequential immunoprecipitation studies(not shown) were inconclusive, possibly owing to the relativelylow avidity of both the JC70A and EN4 MoAbs, that were notcapable of removing all of the antigen from the cell lysates.

Immunofluorescence analysis of EN4 reactivity on lymphoidpopulationsB cells were purified from tonsils and comprised 95 + 3%CD20+ B lymphocytes (mean + s.d., four experiments) (Fig. 3a).43 + 7% of the CD20+ B cells (mean+ s.d. of four experiments)were stained by EN4 mAb. The intensity of staining with JC70A(anti-CD3 1) MoAb was much weaker (Fig. 3a); a precise

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Fig. 3. Flow cytometric analysis of purified lymphoid populations stained with EN4 and JC70A (anti-CD3 1). Indirect immunofluores-

cence was performed on purified B lymphocytes (a), monocytes (b), T lymphocytes (c) using the indicated MoAb (solid lines) or control

irrelevant immunoglobulin (broken lines) followed by FITC-conjugated goat anti-mouse immunoglobulin isotypes as second reagent.

172 V. L. Burgio et al.

EN4 MoAb reacts with CD31

Table 1. Staining of lymphoid tissues by EN4 and JC70A MoAbs

Thymuscortical thymocytesmedullary thymocytesinterstitial macrophagesHassall's body macrophagesepithelial cells

Bone marrowmegakaryocytes and plateletsimmature myeloid cellsmonocytesscavenger macrophagestissue PMNcirculating PMNerythroblastslymphocytesplasma cellsfibroblastoid cells

Spleen andperipheral lymphoid tissues (lymph nodes, tonsils, appendix)primary follicles and mantle zone lymphocytesgerminal centre B cellsT cells wandering in germinal centremacrophages in germinal centrefollicular dendritic cellslymphocytes of interfollicular areasplasma cellsinterfollicular macrophages and interdigitating cellsspleen periartheriolar lymphocyteslymph node sinus and spleen red pulp macrophagesepithelioid and giant cells in granulomasintraepithelial tonsil and appendix lymphocytesLangerhans cellsepithelial tonsil and appendix cells

PMN, Polymorphonuclear cells.

percentage of JC70A+ cells could not be determined, owing toboth the low fluorescence observed and the large overlapping(shift) between the curves of the control and of the JC70AMoAb.

Monocytes and T cells were purified from PB MNC. Thesesuspensions comprised 90+3% CDllb+ monocytes and95+2% CD3+ T cells, respectively (mean+s.d. of threeexperiments) (Fig. 3b, c). As shown in Fig. 3b, both EN4 andJC70A stained virtually all of the monocytes. Again, theintensity of staining of JC70A was weaker than that of EN4.

Of the cells in the T cell suspensions, 35 + 13% were EN4+,while only a lower proportion of the cells (13+15%) were

JC70A+ (mean+s.d., three experiments) (Fig. 3c). When thecells were simultaneously stained with EN4 and JC70A MoAbs,the same proportion of positive cells, as detected with EN4, was

recorded, thus indicating that the two MoAbs stained the samecell population (data not shown). Unfortunately, two colourstainings could not be performed since EN4 and JC70A were ofthe same isotype.

Lymphoid tissue stainingsDifferent lymphoid tissues were stained with EN4 and in parallelwith the reference anti-CD31 MoAb JC70A and 9G1 1. EN4

immunostains blood and lymphatic vessel endothelial cells in allthe tissues under investigation. This pattern of positivity issuperimposable on that obtained with anti-CD31, JC70A and9G1 1, and it is in line with previous observations about CD31distribution in the tissue.

As shown in Table 1 that summarizes the data obtained bythe EN4 and anti-CD31 stainings, all the MoAbs gave almostidentical staining patterns, except that EN4 displayed a strongerreactivity. Moreover, EN4 recognized medullary thymocytesand CD3+ T cells wandering through tonsil epithelium andgerminal centres, as shown in Fig. 4.

DISCUSSION

EN4 MoAb was previously described as anti-endothelial cellreagent and widely used in immunopathology to detect vascularstructures [1-4]. The reagent was not assigned previously to anyof the known CD. The present experiments, intended tocharacterize EN4 further, clearly demonstrated that this MoAbbelongs to the CD3 1 group. These conclusions are based uponthe following observations: (i) EN4 stained mouse fibroblaststransfected with human CD31 gene; (ii) EN4 precipitated thesame '25I-labelled molecular structures from lymphoid cell

173

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V. L. Burgio et al.

Fig. 4. (a) Lymph node. Blood and lymphatic vessels are lined by EN4+ cells. Dilated sinuses (A) are characterized by EN4+ liningmacrophages; the lumens of the sinuses are filled with several EN4+ macrophages. Arrows indicate EN4+ paracortical lymphocytes.(APAAP, x 250.) (b) Secondary follicle ofa lymph node immunostained with EN4 (left) and CD31/JC70A (right) MoAbs. Mantle zonelymphocytes are immunostained by both antibodies, although with a lower intensity for CD31/JC70A. (APAAP, x 1000.) (c) Lymphnode. There is an EN4- germinal centre surrounded by mantle lymphocytes EN4+ (blue stain). Few CD3+ T lymphocytes (red stain) arepresent in mantle zone and the germinal centre, some of which are double stained (arrows). (APAAP, double staining, x 1000.) (d)Normal thymus showing EN4+ cells in the cortex (A) and in the medulla (B). The arrow indicates an EN4- Hassall's body. (APAAP,x 250.)

lysates as the reference reagent; (iii) the pattern of staining oflymphoid cell suspensions as well as of lymphoid tissue sectionswas very similar for EN4 and anti-CD31 MoAbs. In compari-son with the two reference reagents used throughout, EN4proved consistently more powerful and was able to stain a largernumber of a given cell type in suspension or to detect additional

cells in tissue sections. In this respect, EN4 has proved as strongand reliable as LAK-I antibody, another reagent of the CD3 1MoAb family.

The availability of the reagent that seems particularlysuitable for tissue section staining has allowed a better definitionof tissue distribution of CD31 antigen. This antigen is an

174

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adhesion molecule of the immunoglobulin superfamily, closelyrelated to carcinoembriogenic antigen [11], and similar in sizeand function to the molecules of the chaderin family [12,13].Usually, these molecules subserve the role of maintainingcontact between cohesive cells of the same lineage [14-16]. Byusing EN4, besides the already defined CD3 1 + cell populations,we also showed that lymphocytes wandering through the tonsilepithelial layers and CD3+ T cells within germinal centresexpress CD31.

Further studies on normal and pathological tissues usingparticularly suitable reagents such as EN4 or LAK-1 maydemonstrate new localizations of CD31 antigenic structures.

ACKNOWLEDGMENTS

This study was supported by AIRC and Fondazione Istituto Pasteur-Cenci Bolognetti. The authors are particularly grateful to ProfessorManlio Ferrarini for critically reading the manuscript. The authors wishalso to thank Marina Fabbi for help in the immunoprecipitationexperiments, and Maria Grazia Saladino and Teresa Tavilla formanuscript preparation.

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