Lectins and Antibodies to Blood Group Antigens as Markersfor the Basal Cells of the Human Respiratory EpitheliumROBERT BALS1* AND ULRICH WELSCH2

1Anatomische Anstalt (Chair II, Dept. Cytology, Histology, and Microscopical Anatomy)2University of Munich, Pettenkoferstr. 11, D-80336 Munich, Germany

KEY WORDS bronchial epithelium; basal cell; lectinhistochemistry; immunohistochemistry

ABSTRACT We used a pattern of 30 lectins and antibodies against antigens of the ABO-bloodgroup system to find specific and sensitive markers for the basal cells of the human respiratorysurface epithelium. Three lectins always stained the basal cells: Aaptos papillata agglutinin I (APAI), peanut agglutinin (PNA), and wheat germ agglutinin (WGA): Other lectins and the antibodiesgave positive results only in tissue of secretors (blood group antigens in secretions) and these weredependent on the ABO-blood group. Griffonia simplicifolia agglutinin (GSA I B4) bound to basalcells of humans with blood group B and AB, Helix pomatia agglutinin (HPA), Soy bean agglutinin(SBA), and Dolichos biflorus agglutinin (DBA) bound to blood group A and AB, Lens tetragonolobusagglutinin (LTA) and Ulex europaeus agglutinin I (UEA) bound to secretors in every case, andstrongly to blood group O. The antibodies bound to basal cells only in the tissue of secretors,dependent on the ABO-blood group.

The results show that lectins and antibodies may be used as markers for the detection of basalcells in the human respiratory epithelium. Furthermore they suggest that the glycosylation of someglycocomponents of the basal cells is under the control of the genes of the secretor- and ABO-bloodgroup system. Microsc. Res. Tech. 38:505–511, 1997. r 1997 Wiley-Liss, Inc.

INTRODUCTIONThe human respiratory surface epithelium is com-

posed of many different cell types (Jeffery, 1990). Besidesecretory and ciliated cells, basal cells are an importantcell type. Nevertheless the functional significance of thebasal cells is not well known. In early studies they havebeen considered to be the progenitors of other epithelialcells (for review see Baldwin, 1994; Evans & Moller,1991). Recent studies and hypotheses indicate andsuggest a participations of the basal cells in the attach-ment of columnar cells (Evans et al., 1989; Evans &Plopper, 1988).

Basal cells are of significant clinical interest, sincethey have important roles in the pathophysiology ofdifferent diseases, e.g. in inflammatory diseases asasthma (Laitinen & Laitinen, 1994) or in neoplasticdiseases (Dail & Hammar, 1987). To investigate thebiology of the basal cells, phenotypic markers arenecessary to select this cell type in situ and in vitro. Inanimal models several markers have been found: e.g.terminal a-galactosyl (Randell et al., 1991) and keratin14 (Randell et al., 1991; Shimizu et al., 1992). The lectinGriffonia simplicifolia agglutinin I B4 (GSA I B4) wasfound to mark the basal cell in the respiratory epithe-lium of the rat (Shimizu et al., 1991). Some antibodiesagainst antigens from small cell carcinomas were foundto react with basal cells (Bernal et al., 1983). In humantissue the cytokeratins 5, 7, 8, 14, 18, and 19 werelocalized in the basal cells (Broers et al., 1989; Ramaek-ers et al., 1987). The use of these marker substances onliving dissected cells is difficult, since the markers arelocalized in the cell. To our knowledge, none of thesemarker substances has been used to identify living

basal cell of the human respiratory surface epitheliumso far.

It is the aim of this study to investigate the potentialof lectins and antibodies against carbohydrate struc-tures to be used as markers specific for the basal cells ofthe human respiratory epithelium on histological sec-tions. If these substances detect surface markers of thebasal cells, it would be likely that basal cells can beisolated with the help of lectins or antibodies. Parts ofthis study have already been presented in abstract form(Bals, 1995).

MATERIAL AND METHODSTissue Collection, Fixation, and Embedding

Twenty three bronchi (main bronchus or lobe bron-chus) were obtained from routine pathology. The mate-rial used for this study was macroscopically and micro-scopically healthy (no tumor infiltration, no signs ofinflammation). The ABO blood group status of thepatients was assessed by routine blood agglutinationmethods, the secretor-status by the detection of thepresence of blood group antigens (results of immunohis-tochemistry) in the secretions or secretory cells: 18secretors, 5 non-secretors; blood group A: 8, B: 3, AB: 1,O: 11. The material was fixed in 5% formalin (inphosphate buffered saline, PBS, 0.1M) for 5–10 hours at4°C. After washing in PBS for 8 hours (hr) at 4°C the

*Correspondence to: Dr. R. Bals, Anatomische Anstalt, Chair II, Dept. Cytology,Histology, and Microscopical Anatomy Pettenkoferstr. 11 D-80336 Munchen,Germany

Contract grant sponsor Friedrich-Baur-Stiftung.Received 2 September 1995; accepted in revised form 28 October 1995

MICROSCOPY RESEARCH AND TECHNIQUE 38:505–511 (1997)

r 1997 WILEY-LISS, INC.

tissue was dehydrated through a graded series ofalcohols and xylenes and embedded in paraffin.

Tissue Processing and Staining ProceduresFor staining or lectin- or immunohistochemical proce-

dures serial sections of 5 µm thickness were dewaxedand rehydrated. Histological and histochemical proce-dures applied were: hematoxylin/eosin (HE), periodicacid Schiff reaction (PAS), and Alcian blue (AB) at pH 1and pH 2.5 and with different concentrations of MgCl2(0.03, 0.3, 0.5, 0.7, and 0.9 M).

For lectin staining rehydrated sections were rinsed inPBS (0.1 M) containing 0.1 M CaCl2 and MgCl2, pH 7.4for 10 min at room temperature (RT). Lectins conju-gated with biotin or peroxidase were used in concentra-tions of 5–20 µg/ml. The lectins used in this study aresummarized in Table 1, they were obtained from ME-DAC (Hamburg, D) or SIGMA (Deisenhofen, D). Abbre-viations for sugars are: Man 5 D-mannose, Fuc 5L-fucose, Gal 5 D-galactose, GalNAc 5 N-acetyl-D-galactosamine, Glc 5 D-glucose, GlcNAc 5 N-acetyl-D-glucosamine. Sections were incubated for 1 hr at RT,visualized by 0.05% 3,38-diaminobenzidine (DAB) and0.003% H2O2 in PBS for 10 min at RT. Sections stainedwith biotin-conjugated lectins were incubated with anavidin-peroxidase-complex (ABC-kit, SIGMA) for 30min at RT, the visualization was performed as de-scribed above. Finally the sections were counterstainedwith hematoxylin, washed in aqua destillata, dehy-drated and mounted in DePX. Control experimentswere carried out by incubation with the lectins togetherwith the corresponding inhibitory sugar (0.1 M) or byomitting the lectin or theABC-complex. For immunohis-tochemistry we used an indirect technique. After rins-ing in PBS the sections were treated with PBS contain-

TABLE 2. Results of the immuno- and lectinhistochemistry of thebasal cell. Quantity of the results is based on a semiquantitative

subjective score of lectin binding: 0 no staining, 1 weak, 2 moderate,3 strong, 4 very strong; percentages indicate the amount

of the marked cells

Lectin-Antibody

Bloodgroup A-secretor

Bloodgroup B-secretor

Bloodgroup AB-secretor

Bloodgroup O-secretor

Non-secretor

Con A 0 0 0 0 0sCon A 0 0 0 0 0LCA 0 0 0 0 0PSA 0 0 0 0 0GSA II 0 0 0 0 0WGA 100% 4 100% 4 100% 4 100% 4 100% 4DSA 0 0 0 0 0APA I 100% 2 100% 2 100% 2 100% 2 100% 2STA 0 0 0 0 0Jac 0 0 0 0 0MPA 0 0 0 0 0EEA 0 0 0 0 0GSA I B4 0 100% 3 100% 3 0 0VAA 0 0 0 0 0RCA I 0 0 0 0 0PNA 70% 2 70% 2 70% 2 70% 2 70% 2ECA 0 0 0 0 0SBA 100% 3 0 100% 3 0 0DBA 100% 3 0 100% 3 0 0SJA 0 0 0 0 0WFA 0 0 0 0 0HPA 100% 3 0 100% 3 0 0VVA 0 0 0 0 0LTA 100% 1 100% 1 100% 1 100% 3 0UEA I 100% 1 100% 1 100% 1 100% 3 0LFA 0 0 0 0 0LPA 0 0 0 0 0SNA 0 0 0 0 0PHA-L 0 0 0 0 0PHA-E 0 0 0 0 0anti A 100% 3 0 100% 3 0 0anti-B 0 100% 3 100% 3 0 0anti-H 100% 1 100% 1 100% 1 100% 3 0

TABLE 1. Lectins used in this study: name, abbreviation, source, specificity, and inhibitory sugar

Lectin Abbreviation Source SpecificityInhibitory

sugar

Canavalia ensiformis agglutinin Con A Canavalia ensiformis a-Man, a-Glc, a-GlcNAc Mansuccinylated Canavalia ensiformis agglutinin sCon A Canavalia ensiformis a-Man, a-Glc, a-GlcNAc ManLens culinaris agglutinin LCA Lens culinaris a-Man, a-Glc, a-GlcNAc, a-Fuc ManPisum sativum agglutinin PSA Pisum sativum a-Man, a-Glc, a-GlcNAc, a-Fuc ManGriffonia simplicifolia agglutinin type II GSA II Griffonia simplicifolia a-GlcNAc, b-GlcNAc GlcNAcWheat germ agglutinin WGA Triticum vulgaris b-GlcNAc, Sialinsaure GlcNAcDatura stramonium agglutinin DSA Datura stramonium b-GlcNAc GlcNAcAaptos papillata agglutinin APA I Aaptos papillata b-GlcNAc GlcNAcSolanum tuberosum agglutinin STA Solanum tuberosum b-GlcNAc GlcNAcJacalin Jac Artocarpus integrifolia b-Gal GalMaclura pomifera agglutinin MPA Maclura pomifera a-Gal, a-GalNAc GalEuonymus europeus agglutinin EEA Euonymus europeus a-Gal GalGriffonia simplicifolia agglutinin type I B4 GSA I B4 Griffonia simplicifolia a-Gal GalViscum album agglutinin VAA Viscum album b-Gal GalRicinus communis agglutinin type I RCA I Ricinus communis b-Gal, b-GalNac GalPeanut agglutinin PNA Arachis hypogaea b-Gal GalErythrina cristagalli agglutinin ECA Erythrina cristagalli a-Gal, b-Gal, a-GalNAc, b-GalNAc GalSoy bean agglutinin SBA Glycine max a-GalNAc, b-GalNAc GalNAcDolichos biflorus agglutinin DBA Dolichos biflorus a-GalNAc GalNAcSophora japonica agglutinin SJA Sophora japonica b-GalNac GalNAcWistaria floribunda agglutinin WFA Wistaria floribunda a-GalNAc, b-GalNAc GalNAcHelix pomatia agglutinin HPA Helix pomatia a-GalNAc GalNAcVicia villosa agglutinin VVA Vicia villosa b-GalNAc GalNAcLotus tetragonolobus agglutinin LTA Lotus tetragonolobus a-Fuc FucUlex europaeus agglutinin type I UEA I Ulex europaeus a-Fuc FucLimax flavus agglutinin LFA Limax flavus sialic-acid sialic-acidLimulus polyphemus agglutinin LPA Limulus polyphemus sialic-acid sialic-acidSambucus nigra agglutinin SNA Sambucus nigra sialic-acid sialic-acidPhaseolus vulgaris agglutinin type L PHA-L Phaseolus vulgaris complex —Phaseolus vulgaris agglutinin type E PHA-E Phaseolus vulgaris complex —

506 R. BALS AND U. WELSCH

ing 1% goat serum. Then they were incubated with theantibodies directed to antigens of the ABO-blood groupsystem (see Table 2) at a dilution of 1:100 for 4 hours atRT. After washing in PBS, the sections were incubatedwith a peroxidase-conjugated goat antibody directed tomouse-IgM diluted 1:50 (for 1 hr, at RT). After washingin PBS the binding sites of the antibodies were visual-ized by DAB/H2O2 as described above. Finally thesections were counterstained with hematoxylin, washedin aqua destillata, dehydrated and mounted in DePX.Control experiments were carried out by omitting thefirst antibody or both antibodies. The specificities of theIgM monoclonal antibodies were: Anti-A (code/clone:81 FR 2.2): trisaccharide a-D-GalNAc-(1 . 3)-(a-L-Fuc-(1 . 2))-b-D-Gal; anti-B (3 E 7): a-D-Gal-(1 . 3)-(a-L-Fuc-(1 . 2))-b-D-Gal; anti-H (92FR A2): a-L-Fuc-(1 . 2)-b-D-Gal-(1 . 4)-b-D-GlcNAc. They werepurchased from DAKO (Hamburg, D). The peroxidase-conjugated second antibody (IgG) from the goat wasdirected to mouse-IgM (SIGMA, Deisenhofen, D).

Cell Type RecognitionBasal cells of the epithelium were detected by their

typical morphology: basal location, elongated, globular,or triangular from, large nucleus, little cytoplasm.

RESULTSBasal cells were detected by their morphology and

their localization in the epithelium. In the materialinvestigated they formed a dense, but non-continuouslayer above the basal lamina. With conventional histo-chemical carbohydrate stains (PAS orAB (pH or MgCl2))only secretory cells in the epithelium were stained(Figure 1). The staining characteristics of the basalcells with lectins and antibodies are summarized inTable 2.

Binding of Lectins to Basal CellsThe lectins WGA, PNA, and APA I always bound to

the basal cells (Figures 2, 3). All basal cells seem to bemarked by WGA and APA I, whereas PNA did not stainall cells. The binding of all three lectins occurredindependent of the secretor status or the ABO blood

group. The cytoplasm of the cells as well as the intercel-lular space between the basal cells were marked (Fig-ure 2, 3). The lectins LTA and UEA I bound to the basalcells in the tissue of secretors, independently of theABO blood group. The lectins GSA I B4, HPA, SBA, andDBA bound only in the tissue of secretors, dependent onthe ABO blood group: in blood group A: HPA, SBA, andDBA; in blood group B: GSA I B4; in blood group AB:HPA, SBA, DBA, and GSA I B (Figure 2, 3). In humanswith the blood group O the lectins UEA I and LTAbound in a particular high degree to basal cells. Thelectins that bind in correlation with the secretor statusor the ABO blood group, seem to stain all basal cells.Control experiments by incubation together with thecorresponding inhibitory sugar or by omitting the lectinor the avidin-peroxidase-complex showed negative re-sults, i.e. the basal and secretory cells were not stained.

Binding of Antibodies to Basal CellsAntibodies against blood group antigens of the ABO-

system bound only to the tissue of secretors (Figure 3,4). The binding was strictly dependent on the ABOblood group of the donor. All of the basal cells weremarked. Again, the cytoplasm and the intercellularspace were marked. Control experiments performed byomitting the first or both antibodies gave negativeresults.

Binding of lectins and antibodiesto other structures

Secretory cells of the big airways (mucous and serouscells of the bronchial glands, goblet cells of the surfaceepithelium) were marked by several lectins and theantibodies against blood group antigens (Figure 5).Mucous cells of the glands and goblet cells exhibited thesame binding patterns, they were marked strongly byWGA, APA I, Jac, MPA, EEA, VAA, RCA I, PNA, ECA,SBA, DBA, GSA I B4, SJA, HPA, VVA, LTA, UEA I,LFA, LPA, SNA, and the antibodies to blood groupantigens. The binding of some of the lectins and theantibodies was dependent on the secretor status andthe ABO-blood group. The serous cells showed clearpositive reactions with ConA, sConA, PSA, LCA, WGA,

Fig. 1. Histological staining of bronchial epithelium with A) Alcian blue at pH 2.5 and B) periodic acidSchiff sequence (PAS); 3325.

507BASAL CELL MARKERS

Fig. 2. Results of the lectinhistochemistry: staining of basal cells with A) DBA and B) APA I in thetissue of a secretor with blood group A; bc 5 basal cell, gc 5 goblet cell; A) 31050, B) 31320.

Fig. 3. Results of the lectin- and immunohistochemistry: staining of basal cells with A) antibodies toblood group A antigen and B) WGA in the tissue of a secretor with blood group A; epithelium is partlydamaged (left side of figure) thus exposing the basal cells. 3800.

Fig. 4. Results of the immunohistochemistry: staining of basal cells with antibodies to A) blood groupsubstance B and B) blood group substance H in the tissue of a secretor with blood group B; bc 5 basal cell,X 5 structures in prismatic cells; 31320.

PNA, and LFA. The cartilage and the connective tissuewere marked by the lectins RCA I, PNA, LTA, ECA,ConA, and WGA. These lectins bound independently ofthe secretor status and the ABO-blood group. Controlexperiments gave positive results at the cartilage andthe connective tissue with ConA and WGA.

DISCUSSIONFormer studies used lectins in human airway tissue

to investigate the products of secretory cells. Theresults of these studies allowed to mark different cellstypes, e.g. mucous and serous cells of the glands(Mazzuca et al., 1982). The basal cell type was investi-gated by lectinhistochemistry in the rat. Here Shimizuet al. (1991) found the lectin GSA I B4 to be a sensitiveand specific marker for the basal cells. In the presentstudy we used 30 lectins and antibodies against theantigens of the ABO-blood group system to mark thebasal cells; the results allow to analyze the potential oflectins and antibodies to be used as phenotypic markerfor this cell type. Some of the lectins bind always tobasal cells (WGA, PNA, and APA I), some bind independency of the ABO-blood group and the secretorstatus (UEA I, LTA, GSA I B 4, DBA, SBA, and HPA).The sensitivity of the lectins WGA, PNA, and APA I ishigh, since in every case all or most of the basal cellswere marked. With other lectins and the antibodies thebasal cells can be detected only if certain constellations

of secretor status and ABO blood group are fulfilled.The specificity of the marker substances depends onother cell types marked in the epithelium. Only gobletcells are marked with the lectins WGA, PNA, and APAI. The goblet cells can be distinguished in situ frombasal cells by their typical localization and morphology.Also conventional mucin staining procedures—as PASor AB—can be used to distinguish both cell types. Thus,lectins and antibodies can be used as sensitive andspecific markers for the basal cells of the humanrespiratory epithelium. Since the binding of the lectinsand antibodies is localized also at the cell surface, itseems likely that the markers can be used for markingor sorting living cells, too.

Furthermore, the results allow to consider the carbo-hydrate components of the basal cells. The bindingspecificities of the lectins that show positive resultsreveal the presence of the terminal sugars b-GlcNAcand b-Gal. Dependent on the secretor status and theABO-blood group the antigens of the ABO-systems canbe detected. In secretors the terminal sugars a-GalNAc,a-Gal, and/or a-Fuc are present additionally, certainlyas part of the blood group structures.

The glycosylation of some glycocomponents of thebasal cells obviously is under the control of the genes ofthe secretor system and the ABO-blood group system. Aglycosylation dependent on blood group systems wasonly analyzed by histochemical methods in secretorycell types so far: in the human submandibular gland(Ito et al., 1989; Nakayima et al., 1988) and in thepancreas (Ito et al., 1986 & 1990). Since the other celltypes of the respiratory surface epithelium—except thesecretory goblet cell—do not exhibit such carbohydratecomponents, it seems likely that the marked sub-stances are a characteristical feature of the status ofthe basal cells. The marked glycocomponents may beimportant for the attachment of other cell types to thebasis of the epithelium. Blood group substances at theterminal region of the glycosubstances of the basal cellsmay indicate the presence of membrane bound mucin-type glycoproteins, since mucins in the respiratorytract are known to carry antigens of the ABO-bloodgroup system (Bals, 1990; Strous & Dekker, 1992).

In conclusion, lectins and antibodies against bloodgroup antigens can be used as specific and sensitivemarkers for the basal cells of the respiratory surfaceepithelium. The results further indicate the presence ofdifferent carbohydrate components. Some of these car-bohydrate components are synthesized by glycosyltrans-ferases coded by genes of the secretor—and ABO—blood group systems.

Lectins and antibodies as markers for the basal cellsof the respiratory epithelium may be useful for therecognition of this cell type in tissue sections as well asin cell suspensions. Experiments with cell sorting proce-dures should be performed to verify the potential oflectins and antibodies to be used as markers.

ACKNOWLEDGMENTSThe authors wish to thank Prof. Dr. W. Woeckel and

Dr. A. Morresi for making the tissue available for ourstudy and Ms. A. Kubacki for her excellent technicalassistance.

Fig. 5. Results of the lectinhistochemistry: staining of bronchialglands with the lectin DBA in the tissue of a secretor with blood groupA; 3800.

510 R. BALS AND U. WELSCH

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