Monoclonal antibody (Mab) markers for subpopulations of rat tracheal epithelial (RTE) cells.

Experimental Lung Research

ISSN: 0190-2148 (Print) 1521-0499 (Online) Journal homepage: http://www.tandfonline.com/loi/ielu20

Monoclonal Antibody (Mab) Markers for Subpopulations of Rat Tracheal Epithelial (RTE) Cells T. Shimizu, P. Nettesheim, E. M. Eddy & S. H. Randell To cite this article: T. Shimizu, P. Nettesheim, E. M. Eddy & S. H. Randell (1992) Monoclonal Antibody (Mab) Markers for Subpopulations of Rat Tracheal Epithelial (RTE) Cells, Experimental Lung Research, 18:3, 323-342, DOI: 10.3109/01902149209031688 To link to this article: http://dx.doi.org/10.3109/01902149209031688

Published online: 02 Jul 2009.

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Monoclonal Antibody (Mab) Markers for Subpopulations of Rat Tracheal Epithelial (RTE) Cells

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T. Shimizu, I?. Nettesheim, E. M. Eddy, and S. H. Randell

ABSTRACT: We sought monoclonal antibodies (Mabs) that would recognize distinct subsets of rat tracheal epithelial (R TE) cells. Mice were immunized with pronase-dissociated R TE cells and hybridomas whose supernatants immunocytochemically stained subpopulations of tracheal cells were selected. We report the immunohistochemical staining properties of the antibodies and give the results of preliminary biochemical characterization of the antigens. Four dflerent types of antibodies were produced. Antibody RTE 1 stained most RTE cells. Three antibodies (RTE 2, 7, and 13) recognized a subpopulation of nonciliated cells, both columnar and basal cells. Antibody RTE 3 intensely labeled the surface of ciliated cells. Three antibodies reacted with granule components of secretory cells; antibodies RTE 9 and I1 reacted with mucous-type secretory cells and antibody R TE 12 stained all tracheal surface secret0ry cells. As described in detail, some antibodies were RTE cell specific while others also reacted with cells and secretions in other organs; the antibodies did not cross react with guinea pig or rabbit tissues. Periodate sensitivity of the antigens suggested that some antibodies recognized carbohydrate moieties while others detected peptide epitopes. In some cases, Western blotting revealed the molecular weights of the antigens, but some antigens were denatured by sodium dodecyl sulfate (SDS) and heat treatment. These antibody probes provide a usefNl means to immunochemically study changes in cell type distribution and/or epitope expression during development, injury, and regeneration.

From the Laboratory of Pulmonary Pathobiology and Laboratory of Reproductive and Developmental Toxicolom National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina. Address all correspondence to Dr. Scott H. Randell, NIEHS, PO. Box 12233, LPl? MD 02-01, Research Triangle Park, N C 27709. Received 23 September 1991; accepted 28 September 1991.

Experimental Lung Research 18:323-342 (1992) Copyright 0 1992 by Hemisphere Publishing Corporation

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INTRODUCTION The epithelium of the large conducting airways consists of three major cell types: basal, secretory, and ciliated. Both the basal and secretory cell compartments are heterogeneous. There are small and large basal cells; the latter have more cytoplasm and endoplasmic reticulum and less tonofilaments than the former [I, 21. Among the secretory cells, serous and mucous types (and subtypes of both) can be identified using ultrastructural and histochemical criteria [3]. The biochemical diversity of the secretory cell population has also been demonstrated with lectins [4-61 and monoclonal antibodies (Mabs) [7-lo]. There are also small numbers of other cell types such as brush cells and neuroepithelial cells, and, as a result of injury or vitamin A deficiency, epidermoid cells may appear [ll, 121. There are many unanswered questions regarding the pathways of cellular differentiation in this epithelium during fetal and neonatal development and during normal and pathological cell renewal. For example, the relationship between the serous and mucous secretory cell phenotypes is not known. Are they distinct cell types that belong to different cell lineages, are they different generations derived from the same progenitor, or are they merely different functional stages of the same secretory cell? Another unresolved issue is the relationship between basal and secretory cells. Both are capable of replication [l, 2, 13-15] and are endowed with specialized functions [16], but the differentiation potential of both cell types is not clear; the ultimate tracheobronchial epithelial stem cell has not been identified. One reason that these and related questions have not been answered is the lack of a sufficient number of markers useful for defining the stages of differentiation. However, there has been some recent progress in this regard. Certain lectins have been shown to be relatively cell-type specific [17, 181, and keratin expression was shown to be linked closely to cell type and differentiation state in several epithelia [1, 19-22]; monoclonal antibodies to mucous components are useful for identifying secretory cell types [7-lo], and for quantifying mucin glycoprotein content of various fluids [23, 241. Mabs against ferret tracheal cells that detect subpopulations of live dissociated cells in a fluorescence activated cell sorter have been produced, but these do not react with rat antigens [25]. Because differentiation involves the activation/inactivation of many different genes and altered expression of gene products, several different markers are needed to define specific cell phenotypes. The main goal of the studies described here was to obtain antibodies that could be used to identify specific cell types in RTE cell suspensions, cell cultures, and on tissue sections of tracheas. Whole RTE cells were used as the immunogen and splenocytes of immunized mice were fused to mouse myeloma cells to create hybridomas. Hybridomas whose supernatants immunocytochemically stained subsets of RTE cells were selected. We report the immunohisto-

Monoclonal Antibody Markers

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chemical staining properties of antibodies produced by eight stable hybridoma cell lines and give the results of preliminary biochemical characterization of the antigens involved.

MATERIALS AND METHODS


Animals Adult (10-14 weeks old) male Fischer 344 rats and female BALB/c mice from the National Institute of Environmental Health Sciences (NIEHS) breeding colony were used and handled under an Institutional Animal Care and Use Committee approved protocol. Mice were killed with an overdose of sodium pentobarbital given intraperitoneally and rats were killed by CO, asphyxiation.

Isolation of Rat Tracheal Epithelial (RTE) Cells Rat tracheal epithelial (RTE) cells were obtained by pronase dissociation as described previously [26]. Briefly, the trachea was cannulated with polyethylene tubing and excised. It was infused with a 1% solution of pronase (Sigma Chemical Co., St. Louis, MO) in Ham’s F-12 media and was incubated overnight at 4OC. The epithelial cells were then flushed out with Ham’s F-12 media and filtered through a 20-pm nitex mesh. Erythrocytes were eliminated by hypotonic lysis (2 min in 0.83% NH,Cl, 0.1% KHCO,, and 0.0037% EDTA solution). RTE cells were washed and resuspended in 0.01 M phosphate buffered saline (PBS).

Preparation of Monoclonal Antibodies Freshly isolated RTE cells were used as the immunogen. For the initial injection, four mice received 2.8 x lo6 RTE cells each in Ribi’s adjuvant (200 pl s.c., 200 pl i.p., Ribi Immunochem Research, Inc., Hamilton, MT). O n days 7, 14, 28, 42, 56, and 77, they received booster immunizations of 1 x lo6 RTE cells each in Ribi’s adjuvant (200 p1 i.p.). O n day 87, immunized mice were bled from the tail vein and their serum was titered for antiRTE cell antibodies immunocytochemically as described below. The two best responders showed a positive staining reaction with a 1 : 500000 dilution of serum (a 1 : 500 dilution of pre-immune serum was negative), and received an additional booster of 2.5 x lo6 RTE cells in PBS (200 p1 i.v.) on day 105; they were killed for spleen harvest 5 days later. Splenocytes from the two mice described above were combined with mouse myeloma cells (NS-1, American Type Culture Collection, Rockville, MD) at a ratio of 18 : 1 in hybridization medium described previously [27], and fused by the addition of 50% polyethylene glycol (MW 1300-1600,


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American Type Culture Collection) as described by Galfre and Milstein [28]. Fusion products were transferred to 96-well tissue-culture plates, and splenocyte-myeloma cell hybridomas were selected by using hypoxanthineaminopterin-thymidine (HAT) media. Screening of the hybridoma supernatants was performed using RTE cell coated slides on which 16 separate immunocytochemical staining reactions could be performed. To prepare these slides, 16 circles (5-mm diameter) were made on poly-L-lysine coated glass microscope slides using a hydrophobic pen (PAP pen, Kiyota International Inc., Arlington Heights, IL). Freshly prepared, dissociated RTE cells (8000 cells in 5 pl PBS) or rat thymocytes (negative control) were spotted on the center of each circle. The slides were air dried and stored in acetone at - 20°C. The hydrophobic circles prevented cross-contamination of different hybridoma supernatants during the immunostaining procedure (described below). The slides were used within 3 weeks. Positive hybridomas were cloned by limiting dilution. Antibody subclass was determined with an enzyme linked immunoassay (ELISA) using rabbit antimouse typing sera (Mouse monoclonal sub-typing kit, Hyclone Inc., Logan, UT).

Immunostaining Cell coated slides or tissue sections were incubated in 0.3% H,O, in methanol for 20 min to inhibit endogenous peroxidases. Following washing with PBS, nonspecific antibody binding was blocked by a 10-min incubation with NS-1 myeloma cell conditioned medium. The slides were then incubated with hybridoma supernatant for 30 min at room temperature and washed with 1% bovine serum albumin (BSA)-PBS. The slides were incubated with peroxidase conjugated goat antimouse IgM and IgG (Jackson Immunoresearch, West Grove, PA) diluted 1 : 100 in 1% BSA-PBS for 30 min. Peroxidase activity was visualized by a 6-min incubation in diaminobenzidine (DAB)-H,O, solution (20 mg DAB and 100 p1 3% H,O, in 100 ml of 0.05 M Tris buffer, pH 7.6). The slides were counterstained with 1% methyl green. As described in Results, several antibodies that reacted with granule components of secretory cells were obtained. To compare immunoreactivity with histochemical properties of the granules, dual staining with antibodies and alcian blue (pH 2S)-periodic acid-Schiff (AB-PAS) staining was performed. Following immunostaining as described above, and prior to counterstaining, slides were stained with AB-PAS as described previously [18].

Cell Counts Estimations of the percent of the total dissociated RTE cell population that reacted with a given antibody were performed by counting a minimum of 300 cells in random fields on RTE cell coated slides at high-power magnification ( x 1000). Cells prepared on four separate days were stained and


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counted. Cells that stained much stronger than background were considered positive. Data are shown as the mean kSD.

Immunocytochemical and Immunohistochemical Properties of the Antibodies


In previous studies, we separated RTE cells into subpopulations based on Griffonia simplicifoliu I (GSI) lectin binding using flow cytometry (1). The GSI positive cells were >90% basal cells and the GSI negative cells contained secretory (!%%), unidentified (30%), and ciliated (16%) cells. To explore the cell-type specificity of the antibodies, cell coated slides using these cell fractions were made, stained with hybridoma supernatants, and scored as described above. Since antigenicity is influenced markedly by tissue preparation methods, the effects of different fixatives [including acetone; 4% formaldehyde; freshly prepared from paraformaldehyde (PFA); 10% neutral buffered formalin (NBF); and Bouin's solution] were tested with tracheal sections. Frozen, paraffin, and methacrylate sections (JB-4 embedding kit, Polysciences) were obtained for immunostaining. To study the reactivity of each antibody with different organs and species, rat bronchus (intrapulmonary left lobar bronchus), lung, nose, buccal mucosa, esophagus, intestine (duodenum and jejunum), colon, submandibular salivary gland, endocrine and exocrine pancreas, liver, and kidney were immunostained with all antibodies. Tracheal sections of guinea pig and rabbit were also immunostained.

Periodate Sensitivity of Antigens Sodium periodate in 50 mM sodium acetate buffer was applied to RTE cell coated slides in the dark. Three conditions of periodate oxidation were used as described by Basbaum et al. [29]: mild (10 mM, 10 min, 4OC), moderate (50 mM, 1 hr, 4OC), and harsh (100 mM, 12 h, room temperature). Slides were rinsed in 10 mM sodium borohydride in PBS for 30 min. Following five washes with PBS, immunostaining was performed as described above. Controls were incubated in buffer without periodate.

Western Blots Isolated RTE cells were sonicated and boiled for 3 min in 0.0625 M Tris buffer (pH 6.8) containing 2% sodium dodecyl sulfate (SDS) and 5% 2mercaptoethanol. Aliquots of RTE cells were also prepared in distilled water and protein content was measured (BioRad protein assay, BioRad, Richmond, CA) using bovine serum albumin as a standard. Cell extracts (50 pg proteidlane) and prestained molecular weight markers were separated on

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4-20% polyacrylamide gradient gels according to Laemmli [30], and transferred to Immobilon P paper (Millipore, Bedford, MA) using a semi-dry electroblotter. The paper was cut into strips, and antigens were visualized by sequential incubation in: (1) 5% normal donkey serum in PBS containing 0.05% Tween 20 (PBS-Tween 20) for 1 h at room temperature; (2) hybridoma supernatants diluted 1 : 10 in 1% nonfat dry milk, PBS-Tween 20 overnight at 4OC; (3) peroxidase conjugated donkey anti-mouse IgG (Jackson Immunoresearch) diluted 1 : 3000 in PBS-Tween 20 for 1 hr at room temperature; and (4) 10 mg DAB, 100 p1 3% H,O, in 100 ml of 0.05 M Tris buffer, p H 7.6.

Slot Blots

RTE cell extracts were prepared as described above except that in some cases SDS or heat denaturation were avoided. Tracheal grafts inoculated with RTE cells were prepared as described previously [2] and were implanted subcutaneously in isogenic rats. After 14 days, the luminal contents were harvested and used as a source of RTE cell secretions. Intestinal mucin was obtained by lavage of the intestines of fasted rats. Solutions containing 5 pg protein from these preparations were applied to nitrocellulose paper using a slot-blot apparatus (Schleicher & Schuell, Keene, NH). The nitrocellulose membrane was stained using each antibody as described for Western blots.

RESULTS Screening of Hybridoma Supernatants Initial screening of hybridoma supernatants using immunocytochemical staining of RTE cell coated slides revealed 649 positive wells out of 1,344 wells. The staining patterns were mainly of four kinds: (1) most RTE cells were stained, diffuse, or membranous pattern; (2) a subpopulation of nonciliated cells was stained, diffuse pattern; (3) ciliated cells were stained, membranous pattern; and (4) secretory cells were stained, secretory granule pattern. We selected hybridomas of each type for further study. After cloning by limiting dilution, eight stable antibody-producing hybridomas were obtained. Seven of the antibodies were classified as IgG, subtype and one as IgG,. Because the objective of our studies was to obtain antibodies reacting with specific cell types of rat tracheal epithelium, we tested the antibodies on slides coated with three different cell preparations: (1) dissociated RTE cells containing all subtypes, (2) a GSI-positive cell fraction containing > 90% basal cells, and (3) a GSI-negative cell fraction containing > 50% secretory cells (see Materials and Methods).


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Effects of Different Fixatives and Embedding Methods on Staining with Monoclonal Antibodies To study the tissue distribution of the antigens reacting with anti-RTE cell antibodies, we first determined the best immunohistochemical conditions; the results are given in Table 1. All eight antibodies reacted with frozen tracheal sections and with frozen sections of PFA prefixed rat trachea; however, the PFA fixation weakened the staining intensity for some of the antibodies. Antibodies RTE 1, 13, 3, and 11 stained paraffin-embedded sections of tracheas treated with four different fixatives, although some fixativedependent loss of staining intensity was observed. Antibodies RTE 2 and 7 stained paraffin-embedded sections from tissue fixed either in acetone or Bouin’s solution. Antibody RTE 9 stained paraffin-embedded sections from tissue fixed in acetone, and antibody RTE 12 faintly stained paraffinembedded sections from tissues fixed in PFA. Methacrylate sections of rat trachea, regardless of the method of fixation, lost almost all reactivity with any of the antibodies, and trypsin pretreatment of the sections (0.25% trypsin, 10 min, 37°C) did not restore it. In summary, the best results for antibodies RTE 1 , 2 , 7 , 13,9, and 11 were obtained with acetone-fixed paraffin sections, and 4% PFA or 10% NBFfixed paraffin sections were the best for antibody RTE 3. Antibody RTE 12 did not work well in any type of paraffin section, and 4% PFA-fixed frozen sections were used.

Antibody Reacting with Most RTE Cells; RTE 1 Antibody RTE 1 stained 92 k 3% of the cells on slides coated with RTE cells (unfractionated tracheal cells), although there were marked differences in the staining intensity between the cells (Fig. Id). Small cells tended t o stain weakly, and larger cells, particularly ciliated cells, stained heavily. The antibody strongly stained all cells in the GSI-negative cell fraction (loo%), but only 31 k 7% of the cells in the GSI-positive cell fraction (Table 2). One possibility for the reduced staining in the GSI-positive fraction is that the lectin used to separate the cells blocked antibody binding to the antigen. We tested this by applying the lectin to unsorted RTE cells on cell coated slides and then performing immunostaining. Lectin applied in this manner did not reduce RTE 1 immunostaining intensity (not shown). Therefore, the loss of reactivity of the GSI-positive cells with antibody RTE 1 remains unexplained. In acetone-fixed paraffin sections, antibody RTE 1 stained all epithelial cells in the nose, trachea (Fig. lb), bronchi, and bronchioles with varying intensity, and it also stained alveolar type I1 cells very faintly. Of the nonrespiratory tract tissues tested, the antibody reacted with most epithelial cells in the buccal mucosa, esophagus, intestine, and colon. It also stained the

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Table 1 Effects of Fixation and Embedding on Immunostaining of Rat Tracheal Sections with Monoclonal Antibodies Frozen sections

Paraffin sections

Met hacrylate sect ions


Unfixed 4% PFA RTE 1 RTE 2 RTE 7 RTE 13 RTE 3 RTE 9 RTE 11 RTE 12

+ + + + + + + +

Staining intensity: formalin.

+ f f

+ + + + +

+ , positive; f , weakly positive; - , negative; PFA, paraformaldehyde; NBF, neutral buffered

epithelial lining of the pancreatic duct, bile duct, and submandibular salivary gland duct (but not the parenchymal epithelium of these glands). All tubular epithelial cells in the outer stripe of the renal medulla were stained (these are mostly proximal tubule zone three and distal tubules). Collecting duct epithelial cells and the transitional epithelial cells lining the renal pelvis were also stained.

Nonciliated Cell Antibodies; RTE 2, 7, and 13 Antibodies RTE 2, 7, and 13 diffusely stained approximately half of the nonciliated RTE cells on slides coated with unfractionated RTE cells (Fig. lc) and also stained about half of the cells both in the GSI-positive and negative cell fractions (Table 2). In acetone-fixed paraffin sections of the upper trachea (rings 1-5 below the larynx, Fig. ld), these antibodies stained most nonciliated epithelial cells; both secretory and basal cells were reactive. However, staining was uniformly lost in all but a few cells in the lower trachea (rings 16-20 below the larynx). The antibodies did not stain other parts of the airways or any other organs tested.

Ciliated Cell Antibody; RTE 3 Using a 1 : 200 dilution of hybridoma supernatant, antibody RTE 3 stained only the cilia and plasma membrane of ciliated cells on RTE cell coated slides (Fig. le and Table 2). With a 1 : 10 dilution on cell coated slides, the plasma membrane of most RTE cells stained weakly, although staining of


Figure 1 Mab staining of dissociated RTE cells and rat tracheal epithelium. Acetone-fixed RTE cell coated slides (u,c, e, g, i, k), acetone-fixedparaffin sections (b, d, h, I), 4% paraformaldehyde-fixed paraffin 20 pm. Antibody RTE 1 reacted section 0, and 4% paraformaldehyde-fixed frozen section (J), bar with most epithelial cells both on RTE cell coated slides and tracheal sections (a b). Antibody RTE 2 stained approximately half of the dissociated nonciliated cells, and both basal cells and nonciliated columnar cells on tracheal sections. Ciliated cells did not stain (arrows) (c, d). Antibody RTE 3 stained cilia and the plasma membrane of ciliated cells on RTE cell coated slides. On tracheal sections, cilia and the apical plasma membrane stained (e, A. Antibody RTE 11 reacted with granules of all mucous-type secretory cells and a subpopulation of serous-type cells (g, h). Antibody RTE 12 stained secretory granules of almost all tracheal surface secretory cells (i,I).Control slides stained with NS-1 myeloma cell conditioned medium displayed negligible staining (k,I).

-

33 1

RTE 1 RTE 2 RTE 7 RTE 13 RTE 3 RTE 9 RTE 11 RTE 12

Antibody reacting with most RTE cells Nonciliated cell antibodies

Antibody subclass 1:l 1:l 1:l 1:l 1 : 200 1 : 10 1 : 10 1 : 10

Dilution"

+

f

& &

+++ + + +

RTE cells

GSI-positive fractionb

+ ++

f

&

+++ + + +

GSI-negative fractionb

Percent cells staining: - , 0-1%; ?, 1-20%; + , 20-50%; + + , 50-90%; + + + , 90-100%. "Dilution of hybridoma supernatants used for immunostaining. k T E cell subpopulations were separated with GSI lectin (1). The GSI-positive fraction was >90% basal cells. The GSI-negative fraction was depleted of basal cells and contained 54% secretory, 30% unidentified, and 16% ciliated cells.

Ciliated cell antibody Secretory cell antibodies

Hybridoma

Antibody

Table 2 Reactivity of Monoclonal Antibodies on Slides Coated with RTE Cells




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cilia and the plasma membrane of ciliated cells was much more intense. In 4% PFA-fixed paraffin sections with a 1 : 10 dilution of hybridoma supernatant, the antibody stained only cilia and the apical plasma membrane of ciliated cells throughout the respiratory tract from nose to terminal bronchioles (Fig. If). Clara cells and pulmonary alveolar cells did not stain. The suprabasal layer of the buccal mucosa and esophagus also stained, but the epithelial cells of the intestine and colon did not stain. The antibody weakly stained the apical surface of pancreatic and submandibular salivary gland ducts and mucous cells of the submandibular salivary gland. In the kidney, scattered tubule epithelial cells in the cortex and most tubular epithelial cells in the inner stripe of the medulla (a pattern most consistent with distal tubules) were stained; collecting duct epithelial cells and the transitional epithelium lining the renal pelvis were also reactive.

Secretory Cell Antibodies; RTE 9, 11, and 12 Three antibodies that stained secretory granules were obtained. Antibodies RTE 9 and 11 detected a subpopulation of secretory cells, especially those that had many and large granules including all typical goblet cells on RTE cell coated slides (Fig. lg). Antibody RTE 9 stained 10 f 1% of all RTE cells, 19 f 3% of the cells in the GSI-negative cell fraction, and < 1% of the cells in the GSI-positive cell fraction. Antibody RTE 11 stained 18 f 3% of all RTE cells, 29 k 5% of the cells in the GSI-negative cell fraction, and < 1% of the cells in the GSI-positive cell fraction. Antibody RTE 12 stained the granules of most secretory cells on cell coated slides (Fig. 12); 41 f 3% of all RTE cells, 79 k 6% of the cells in the GSI-negative fraction, and < 1% of the cells in the GSI-positive cell fraction. Dual staining with alcian blue (AB; p H 2.5)-PAS and antibodies RTE 9, 11, and 12 was performed. Antibodies RTE 9 and 11 stained all AB positive granules and a small subpopulation of PAS-positive but AB-negative granules. Antibody RTE 12 stained almost all AB- or PAS-positive granules. According to the tracheobronchial secretory cell classification by Spicer et al. [ 3 ] , it appears that antibodies RTE 9 and 11 stained all mucous-type tracheal secretory cells but only a subpopulation of serous-type cells, while antibody RTE 12 stained almost all tracheal mucous- and serous-type cells. Immunologic reactivities of antibodies RTE 9, 11, and 12 with acetonefixed paraffin sections (RTE 9 and 11) and 4% PFA-fixed frozen sections (RTE 12) in various tissues are given in Table 3 . Antibodies RTE 9 and 11 stained all AB-positive cells and a subpopulation of AB-negative and PASpositive cells in trachea (Fig. lh), bronchi, bronchioles (Fig. 2), and tracheal gland. Antibody RTE 12 stained almost all surface secretory cells in the trachea (Fig. I]), but did not stain tracheal glands. In the bronchi and bronchioles, staining with antibody RTE 12 was limited to only a small subpopulation of goblet cells (weakly). Clara cells and pulmonary alveolar cells did

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not stain with any of these three antibodies. Antibodies RTE 9 and 11 stained a subpopulation of nasal secretory cells including a few goblet cells and Bowman's gland in the olfactory epithelium. Antibody RTE 12 did not stain secretory cells in the nose. Gland cells below the respiratory epithelium of the nasal septum did not stain with any of these three antibodies. Goblet cells in intestine and colon did not stain with antibody RTE 9, but RTE 11 detected a small subpopulation of intestinal goblet cells and RTE 12 stained all goblet cells in intestine and colon. Buccal salivary glands stained with only antibodies RTE 9 and 11, but submandibular salivary gland and pancreas did not stain with any of these three antibodies.

Table 3 Reactivity of Antibodies RTE 9, 11, and 12 with Secretory Cells in Various Tissues Location

Trachea Tracheal gland Broncus and bronchiole

Alveolus Nose Gland of nasal septum Bowman's gland Intestine Colon Submandibular salivary gland Buccal salivary gland Pancreas

Cell type"

RTE 9 and l l b

RTE 12'

Serous Mucous

+d

+ +

Serous Mucous Clara Type I1 Secretory

+d

-

-

kd -

+d

-

Goblet Goblet

+ + +

-

-

+

N.D.'

+f

+ +

-

-

+

N.D.e

-

-

"Tracheobronchial surface secretory ceIl types were classified as serous or mucous according to Spicer et al. [3]. bStaining intensity: +, positive; +, weakly positive; -, negative. Antibodies RTE 9 and 11 are combined because they demonstrated nearly identical staining patterns. Acetone-fixed paraffin sections were used. '4% paraformaldehyde-fixed frozen sections were used. dOnly a small subpopulation of this cell type was stained with this antibody. 'Not done because bony structures prevented frozen sections from being obtained. k T E 11, but not RTE 9, stained a small subpopulation of intestinal goblet cells.

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-

Figure 2 Staining of the rat lobar bronchus and segmental bronchiole with antibody RTE 11. Secretory granules of all goblet-type cells and other secretory cells with many and large granules were stained. Bar 200 pm, and 20 pm (inset).

Cross Reactivity of the Monoclonal Antibodies with Tracheal Epithelium of Other Species None of the antibodies reacted with frozen sections of guinea pig or rabbit trachea.

Periodate Sensitivity Periodate oxidation of vicinal diols of carbohydrates to aldehydes can be used t o determine if these groups are critical antigenic determinants. Which type of carbohydrate structure is affected depends on the concentration, time, and temperature of periodate treatment [29]. Slides coated with RTE cells were subjected to periodate treatment of increasing severity and immunostained (see Materials and Methods). As presented in Table 4, the antigen reacting with antibody RTE 1 was resistant to even harsh periodate treatment; the antigens reacting with antibodies RTE 2, 7, 13, 3, 11, and 12 were destroyed by harsh periodate treatment, whereas the antigen reacting with antibody RTE 9 was destroyed by moderate periodate treatment.

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Table 4 Characterization of Antigens Reactive with Monoclonal Antibodies ~


Periodate sensitivity Hybridoma

of antigen‘

RTE 1 RTE 2 RTE 7 RTE 13 RTE 3 RTE 9 RTE 11 RTE 12

-

~~

42-37 69, 36, 33.5, 26, 21.5, 19 69, 36, 33.5, 26, 24, 21.5, 19 69, 42, 34.5 SDS and boiling treatment sensitive

+ + + + ++ + + ~

Molecular weights (kD)of antigenb

SDS treatment sensitive SDS treatment sensitive SDS and boiling treatment sensitive ~

“Slides coated with RTE cells were submitted to periodate treatment of different severity as described in Materials and Methods; - , not sensitive; , sensitive to harsh periodate treatment; + +, sensitive to moderate periodate treatment. bApparent molecular weight of bands stained on Western blots. In cases where no bands were observed, antigens were applied directly to nitrocellulose paper using a slot blot apparatus with or without SDS or heat pretreatment. Western and slot blots were stained with the antibodies as described in Materials and Methods.

+

Immunoblot Analysis of RTE Cell Extracts Western blot analysis of RTE cell extracts separated by SDS-PAGE showed positive reactions with four of the eight antibodies (RTE 1, 2, 7, and 13). Representative Western blot results are shown in Fig. 3, and approximate molecular weights of the antigens are given in Table 4. Antibody RTE 1 reacted with several diffuse bands from 42- to 37-kD. Antibodies RTE 2, 7 , and 13 reacted strongly with the same 69-kD band and several lower molecular weight bands were also observed. Antibodies RTE 2 and 7 stained the same bands except for a 24-kD band that stained with antibody RTE 7 only. Antibody RTE 13 reacted with two other completely different bands. When rat serum was run on the gel, no bands were observed (data not shown). Antibodies RTE 3, 9, 11, and 12 produced no bands on Western blots. Antigens from RTE cell extracts, luminal contents of tracheal grafts, and rat intestinal lavage were applied to nitrocellulose paper using a slot blot apparatus. Pretreatment of the antigens showed that the epitopes for these four antibodies were sensitive to SDS and the epitopes for antibodies RTE 3 and 12 were sensitive to boiling. The antigens for antibodies RTE 9 and 11 were secreted into the lumen of 14-day old tracheal grafts, and intestinal lavage from rats was an abundant source of the antigen for antibody RTE 12.


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DISCUSSION We sought to develop immunologic markers capable of identifying specific subsets of RTE cells. Our approach was to immunize mice with RTE cells and produce hybridomas from their splenocytes. Hybridoma supernatants were screened for immunocytochemical reactivity with subpopulations of dissociated RTE cells. The choice of immunogen and screening method were critical variables affecting the outcome of this study. Pronasedissociated RTE cells, a mixture of several cell types, were chosen as the immunogen with the hope of obtaining markers for several different cell types. We recognized that pronase treatment likely destroyed many external membrane markers. However, a major intended use for the antibodies was to identify cells in dissociated cell preparations, possibly for cell sorting. Thus selection for pronase-resistant antigens was, in some ways, desirable.

Figure 3 Western blot analysis of RTE cell extracts using antibodies RTE 1,2, 7, and 13. NS-1 myeloma cell conditioned medium was used as a negative control. Reduced and SDS-denatured proteins were separated on a 4-20°/o polyacrylamide gel, transferred to Immobilon P paper, and stained with indicated antibodies as described in Materials and Methods. Arrows indicate the positions of prestained molecular weight standards (kD); myosin 200.0, bovine serum albumin 69.0, ovalbumin 46.0, carbonic anhydrase 30.0, and trypsin inhibitor 21.5.


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Immunocytochemical screening of hybridoma supernatants, although tedious to perform, was chosen for several reasons: (1) only 8,000 RTE cells were required per assay as opposed to several hundred thousand needed for an ELISA, (2) the staining reaction permitted detection of antibodies against small and specific subpopulations of RTE cells, and (3) important information regarding subcellular antigen localization (e.g., staining of cell surface, cytoplasm, or granules) could be used as a criterion for hybridoma selection. The purpose of this report is to describe the immunohistochemical staining properties of the antibodies obtained and the results of biochemical characterization of the antigens. Eight antibodies representing four distinct staining patterns were obtained; they did not cross react with rabbit or guinea pig tracheal epithelial cells, but other species have not been tested. Antibody RTE 1 reacted with almost all RTE cells and with many other surface epithelial cells throughout the respiratory and digestive tracts. The antigen was resistant even to harsh periodate treatment, suggesting that the epitope was not a carbohydrate; the antibody labeled several diffuse 37-42 kD bands on Western blots. The staining intensity was variable among different RTE cells. Because the antigen was expressed in most epithelial cell types in the airways, antibody RTE 1 will not be particularly useful for studies of airway cell subpopulations. However, it may be of interest as a marker of a “common epithelial antigen” because it reacted with epithelial cells in many organs but did not react with any mesenchymal cells. Antibodies RTE 2, 7, and 13 reacted with an antigen expressed by both columnar and basal cells, but mainly in the upper trachea. In the lower trachea, fewer cells exhibited this antigen, and in the bronchi and bronchioles it was either absent or masked. We could not detect the antigen in any other tissues studied. This highly restricted expression pattern is intriguing because the antigen is apparently both abundant and highly immunogenic, as suggested by the very strong 69-kD band on Western blots, and because it was recognized by three of the antibodies obtained in this study. The lower molecular weight bands seen on Western blots may be proteolytic products of the 69-kD protein, but this has not been tested. The fact that the antigen is expressed in both basal and columnar cells, but not in ciliated cells, suggests that expression of the epitope is suppressed or masked during terminal differentiation. The striking and uniform loss of staining from upper to lower trachea was not due to changes in cell populations because there are only small differences in cell population densities between these two locations [ 181. The topographic distribution of the antigen suggests functional differences between cells of the upper and lower trachea. One of the most useful antibodies produced was RTE 3, which intensely labeled the cilia and apical plasma membrane of ciliated cells throughout the respiratory tract. Immunostaining of dissociated RTE cells and paraffin sections of rat trachea with 1 : 200 and 1 : 10 dilutions of hybridoma supernatant, respectively, demonstrated exclusive staining of ciliated cells. Higher



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antibody concentration produced very faint staining of other airway cell types. It is possible that the intense staining of ciliated cells is partially a function of their much higher density of plasma membrane at their apical, ciliated pole. However, on cell coated slides with a 1 : 200 dilution of RTE 3, the entire plasma membrane of ciliated cells, including the nonciliated areas, was stained while the other cell types were negative. The presence of faint staining on nonciliated cells with much higher primary antibody concentrations indicates that the antigen may also be expressed, but probably at a much lower level on other airway cells. The sensitivity of the antigen to denaturation by SDS and its presence in only a small percentage of dissociated RTE cells present obstacles in the biochemical characterization of the epitope. Imrnunoaffinity purification or immunoprecipitation techniques will be needed. We obtained three antibodies reacting with granule components of secretory cells. Antibodies RTE 9 and 11 stained all morphologically defined goblet-type secretory cells. Dual staining with antibodies RTE 9 or 11 and AB-PAS demonstrated that, for the most part, these antibodies react with components of secretory granules that also contain sulfated and carboxylated PAS-positive glycoconjugates, properties usually attributed to mucin glycoproteins [3]. Preliminary evidence indicates that the antigens for RTE 9 and 11 are secreted together with mucin glycoproteins. Antibody RTE 9 was sensitive to moderate periodate treatment, suggesting a carbohydrate determinant. Denaturation of RTE cell extracts or secretions with SDS abolished antibody binding, which precluded molecular weight determination of the antigens with the methods employed in this study. The fact that antibodies RTE 9 and 11 immunohistochemically stained the subpopulation of secretory cells traditionally thought of as mucous cells [3] suggests that they react with mucin glycoproteins. However, t o confirm this, further biochemical characterization of the antigens is necessary. Antibody RTE 12 reacted with most secretory cells of the tracheal surface and with an abundant antigen in the digestive tract, but not with tracheal gland cells. Interestingly, with few exceptions, secretory cells in the intrapulmonary bronchi and bronchioles did not stain. This suggests that expression of certain antigens is not only specific to cell type but is also topographically restricted. The fact that tracheal surface, but not gland epithelial cells, reacted with antibody RTE 12 is another example of the marked biochemical heterogeneity among the secretory cell compartment. In summary, we produced Mab markers for RTE cells including one whose reactivity is limited to ciliated cells and three that stain different subpopulations of secretory cells. These will be useful probes for immunochemical studies of changes in cell type distribution and/or epitope expression during development, injury, and regeneration. The authors thank W. D. Willis for excellent training and technical support.

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