EXPERIMENTAL

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MOLECULAR

A Rat Monoclonal

PATHOLOGY

57,235-246 (1992)

Antibody Specific for Murine Type 1 Pneumocytes

JON A. HOTCHKISS,'STEPHEN J. KENNEL,'ANDJACK

R. HARKEMA'

‘Inhalation Toxicology Research Institute, P.O. Box 5890, Albuquerque, New Mexico 87185; *Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831

and

Received October 3, 1991, and in revised form October 21, 1992 A rat monoclonal antibody (MAb), 411-52, that binds specifically to murine pulmonary alveolar type 1 cells was developed. The cell-binding specificity of MAb 41 l-52 was assessed by light microscopy on immunoperoxidase-labeled tissue sections, electron microscopy on immunogold-labeled tissue blocks, and by flow cytometric analysis and fluorescenceactivated cell sorting of immunofluorescently labeled cells enzymatically dissociated from murine lungs. The epitope recognized by MAb 411-52 was first detected in immunoperoxidase-stained sections of neonatal lungs of mice approximately 3 weeks after birth. In adult mice, the MAb 41 I-52-directed, immunoperoxidase-staining pattern was uniform throughout the lung parenchyma, was restricted to the luminal surfaces of alveoli, and was absent from type 2, endothelial, and interstitial cells, as well as from the epithelial cells of conducting airways. Electron microscopic analysis of immunogold-labeled lung tissue confirmed the type 1 cell binding specificity of MAb 411-52. Analysis by multiparameter, laser flow cytometry indicated that MAb 411-52 binds to 4.6 * 0.5% (mean ? SD) of enzymatically dissociated cells from the lungs of normal adult mice. The absence of immunogold-labeling of type 2 cells suggested that the epitope recognized by MAb 41 l-52 might be a differentiation marker for the type 1 cell phenotype. With this MAb and standard immunohistochemical techniques, it is possible to visualize directly type 1 cells in paraffin sections. 0 1992 Academic

Press. Inc.

INTRODUCTION The epithelial lining of the adult pulmonary alveolus is composed primarily of large squamous type 1 cells and smaller cuboidal type 2 cells. Type 2 cells synthesize and secrete surfactant (Mason and Dobbs, 1980; Williams and Benson, 1981; Dobbs et al., 1982), are involved in the transepithelial transport of fluid and electrolytes (Cott er al., 1978), and are the progenitor cells for type 1 pneumocytes (Adamson and Bowden, 1974; Evans et al., 1973, 1975). Type 1 cells represent only 5-8% of lung parenchymal cells, but cover greater than 90% of the alveolar surface and have the largest average volume of any cell in the lung (Kauffman et al., 1974; Kikkawa and Yoneda, 1974; Crapo et al., 1978). Little is known of type 1 cell function or physiology. These cells from junctional complexes with neighboring type 1 and type 2 cells (Hirai et al., 1977; Weller and Kamovsky, 1986), contain numerous smooth and clathrin-coated endocytic vesicles (Gordon and Puszkin, 1989), and presumably are the primary barrier through which oxygen must pass before entering the pulmonary capillaries. Type 1 cells are injured in vivo by systemically administered (Hirai et al., 1977; Adamson et al., 1977; Dinsdale and Verschoyl, 1989) or inhaled (Strauss et al., 1976) chemicals, by inhalation of pollutant gases (Evans et al., 1973, 1975; Plopper et al., 1973; Stephens et al., 1973), and by the high levels of oxygen used in clinical respiratory therapy (Adamson and Bowden, 1974; Kapanci et al., 1969; Bowden and Adamson, 1971). Thus, the susceptibility of type 1 cells to injury would seem to provide a sensitive indicator of acute pulmonary toxicity. However, these 235 00144800192 $5.00 Copyright 8 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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extremely thin, attenuated cells are difficult to quantitate accurately in routine paraffin or semithin-plastic sections. To study the mechanisms of acute toxicant-induced pulmonary injury and repair, it is important to identify and isolate specific cell types for detailed biochemical and molecular biological analyses. Methods have been developed to isolate type 1 cells from rabbits (Picciano and Rosenbaum, 1978; Rosenbaum and Picciano, 1978) and rats (Weller and Kamovsky, 1986), but similar methods have not been developed for mice. Ideally, a cell-specific marker would be identified that could be used to monitor or aid in the isolation of a cell population. Brandt (1982) reported that the alveolar surfaces of type 1 and type 2 cells have different lectinbinding properties. Much-u pomiferu binds to type 2, but not to type 1 cells, while Ricinus communis Z (RCA I) binds to type 1, but not type 2 cells in situ. However, the binding of the latter is not specific for type 1 epithelial cells, as other cell types also bind RCA I (Dobbs et al., 1985). Dobbs et al. (1988) have developed monoclonal antibodies that bind to the apical surfaces of rat type 1 cells. However, to our knowledge, there are no similar immunochemical reagents available for specifically labeling murine type 1 cells. We have developed a rat monoclonal antibody (MAb) that binds specifically to a membrane-associated epitope found on the apical surfaces of murine type 1 cells. This MAb is useful in identifying type 1 cells in both routine paraffinembedded lung tissue and enzymatically dissociated lung cells. METHODS Immunization Specific-pathogen-free F344 rats (Oak Ridge National Laboratory colony) were immunized with normal (Balb/c) mouse lung (NML) homogenate on a schedule based on the method of Cianfriglia et al. (1983). The homogenate was prepared from adult Balb/c lungs perfused in situ via the right ventricle with 10 ml of phosphate-buffered saline (PBS), pH 7.4. Lung lobes were removed below the tracheal bifurcation and were homogenized in ice-cold PBS with 0.02% phenylmethylsulfonyl fluoride (PBSF), using several bursts of a Polytron homogenizer (Brinkman Instruments, Westbury, NY) until no whole cells remained. The homogenate was then centrifuged at 400g for 10 min. Protein concentration of the supernatant was determined by the method of Lowry et al. (1951). The NML homogenate (400 ~1) emulsified with complete Freund’s adjuvant in a 1 ml volume was administered to the specific-pathogen-free rats intraperitoneally (ip) on Days 15 and 8 prior to cell fusion. Three days prior to fusion, rats were injected (ip) with 400 pg of NML homogenate in 1 ml of PBS. On Day 2 and again on Day 1 before cell fusion, rats received 400 kg of NML homogenate protein in PBS, but in a split dose (200 pg ip/200 kg iv). Cell Fusion Spleen cells from an NML homogenate-immunized rat were fused with mouse myeloma SP2/0 cells as described previously (de St. Groth and Scheidegger, 1980; Kennel et al., 1983). Parent myeloma cells and rat-mouse hybridomas were cultured in Dulbecco’s modified Eagle’s medium (GIBCO, Grand Island, NY), supplemented with 2 mM glutamine, 100 l&ml streptomycin, 100 U/ml penicillin, and 20% fetal bovine serum (DME). Fusion products were selected from parent pop-

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ulations in DME supplemented with hypoxanthene, aminopterin, and thymidine (Littlefield, 1964). Hybridomas were screened for specific antibody production by an indirect binding assay (see below) against enzyme-dissociated Balb/c lung cells (DLC) (Urade ef al., 1982). Selected hybridomas were cloned by limiting dilution before being used to produce ascites fluid in n&u mice (Life Science, FL) previously primed with pristane (Kennel et al., 1981). MAb Binding

Assay

An indirect, solid-phase radioimmunoassay was used to identify hybridomas producing antibody that preferentially reacted with enzyme-dissociated lung cells. DLC antigen was bound to Immulon II microtiter wells (Dynateck Labs, Alexandria, VA) by a poly-L-lysine/glutaraldehyde bridge (Kennet, 1980; Kennel, 1982). NML homogenate and normal mouse serum were bound to microtiter wells in a similar manner. Fifty microliters of hybridoma culture supematant or ascites fluid diluted in DME were added to test wells, and the plates were incubated on a rocker platform at 37°C for 2 hr. After washing the wells with PBS, bound MAb was detected with radioiodinated, goat anti-rat Ig secondary antibody in 50 t.~l of DME. Two hours later, the wells were washed with PBS and analyzed for bound “‘1 secondary antibody in a Searle gamma counter. Values for the amount of secondary antibody bound were calculated based on the specific activity of the radiolabeled antibody (approximately 10,000 cpmlng secondary antibody). Tissue Sample Preparation

Fourteen Balb/c mice (Oak Ridge National Laboratory colony) 3, 7, 10, 14, 21, 28, or 35 days old (2 per age group) were deeply anesthetized with Nembutal (Abbott Laboratories, North Chicago, IL) and exsanguinated by severing the abdominal artery. The lungs were inflated in situ by tracheal perfusion with 10% neutral buffered formalin. The trachea was ligated, and the lungs were exposed, removed, and placed in an excess of formalin for 48 hr. Tissue samples from each lung lobe were embedded in paraffin, sectioned at 4-5 km, and processed for immunoperoxidase staining and light microscopic observation. Two B6C3F, mice, 30 weeks old, from the Inhalation Toxicology Research Institute’s colony were deeply anesthetized with 4% halothane in O2 and exsanguinated by severing the abdominal artery. The lungs were fixed by tracheal perfusion with modified Karnovsky’s fixative (Dungworth et al., 1976) at a constant 25 cm of fixative pressure for 2 hr. Samples from each lung lobe were embedded in paraffin, sectioned at 4-5 pm, and processed for immunoperoxidase staining and light microscopic observation. Two or three transverse slices from each lung lobe (l-2 mm thick) were cut perpendicularly to the main axial airway and processed for immunogold labeling as described below. Zmmunohistochemistry Light microscopy. Lung sections were deparaffinized in xylene and rehydrated through a series of graded ethanol solutions. Endogenous peroxidases were inhibited by incubating the sections for 30 min in 3% H20z in methanol. Tissue sections were then incubated for 20 min with 2% normal serum in DME prior to applying the primary antibody; the normal serum corresponded to the species in which the secondary antibody was raised. Balb/c lung sections were incubated

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with a 1:50 dilution of MAb 411-52 in DME or with DME alone (negative control) for 2 hr at 37°C. Bound MAb 411-52 was detected with a 1: 1 mixture of horseradish peroxidase (HRP)-conjugated, rabbit anti-rat Ig diluted 1:30 in DME and HRP-conjugated, swine anti-rabbit Ig diluted 1:50 in DME (Accurate Chemical and Scientific Corp., Westbury, NY) (Km-tin and Pinkus, 1985). Bound secondary antibody was visualized by incubating the slides for 5 min with HRP substrate (0.5 mg/ml3,3’-diaminobenzedene, 0.01% H202 in 0.05 M Tris-HCl, pH 7.2). B6C3F1 lung sections were treated as described above, except that the sections were incubated overnight at 4°C with a I:250 dilution of MAb 411-52. Bound MAb was visualized using Vectastain ABC immunoperoxidase reagents (rabbit anti-rat Ig) and procedures (Vector Laboratories Inc., Burlingame, CA). Electron microscopy. Tissue slices from lungs fixed with modified Karnovsky’s fixative were washed 4 x 15 min in PBS and 2 x 15 min in PBS with 0.1 M glycine to block any remaining reactive aldehyde groups. The washed tissue slices were blocked with 5% normal rabbit serum in PBS (bPBS) for 2 hr and were then incubated sequentially in (1) MAb 411-52 or 411-201 (an unrelated monoclonal antibody that reacts specifically with murine pulmonary endothelial cells (Rorvik et al., 1988) ascites (1:250 in bPBS), or in bPBS alone (negative control) overnight at 4°C; (2) biotinylated rabbit anti-rat Ig (mouse Ig adsorbed, Vector Laboratories Inc., Burlingame CA; 1:200 in PBS) for 4 hr at 37°C; and (3) succinyl avidin conjugated to lo-nm colloidal gold particles (Sigma Chemical Co., St. Louis, MO; I:100 in PBS with 0.5% bovine serum albumin and 0.05% Tween 20) for 4 hr at 37°C with 4 x 15 min PBS washes between steps. Lung slices were then postfixed, first in 1% glutaraldehyde and then in 1% OsO,, dehydrated through ethanol and propylene oxide, and then embedded in Epon/Araldite. Sections with gold/ silver interference colors were floated onto 90-mesh hex grids, stained with lead citrate and uranyl acetate, and observed with an Hitachi H-7000 scanning transmission electron microscope. Flow Cytometric

Analysis

Flow cytometric analysis of MAb binding to monodispersed DLC was performed with an Ortho Model 50 H cytofluorograph (Ortho Instrumentation, Westwood, MA). The cells were labeled as follows: 1 x 10’ DLC were incubated in 200 pl of MAb 411-52 ascites fluid diluted 1:50 in DME, for 30 min at 37°C. Following this incubation, the cells were washed in PBS containing 2% fetal bovine serum in 0.001% NaN, (PBSF) and then incubated in FITC-conjugated, goat anti-rat IgM (Cooper Biomedical, West Chester, PA), diluted 1:25 in DME, for an additional 20 min at 37°C. The cells were again washed with PBSF and then immediately fixed in 70% ethanol for a minimum of 1 hr. The fixed cells were pelleted by centrifugation and resuspended in 2 ml PBS, to which a final concentration of 50 &ml of ribonuclease II (Cal-Biochem, LaJolla, CA) were added. This mixture was incubated for 30 min at room temperature, after which the cells were stained with propidium iodide (15 pg/ml; to determine DNA content) for an additional 30 min. Immediately following this incubation, the DNA-content and MAb-binding profdes of the cell population were determined. Fluorescence-Activated

Cell Sorting (FACS)

Immunofluorescence staining and FACS were performed on viable DLC. The primary and secondary antibody incubations were performed as described above,

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except that NaN, formed for 1 hr at for DNA content. FITC-fluorescence Electron

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was omitted from the wash medium, all incubations were per4”C, and the FITC-stained DLC were neither fixed nor stained Cells were sorted on the basis of forward light scatter and intensity.

Microscopy

of Sorted Ceils

Sorted cells were pelleted at 400g and fixed overnight at 4°C in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. The following day, the cells were rinsed two times by centrifugation in buffer and then postfixed for 30 min in 1% 0~0, on ice. The fixed cells were embedded in 2% aqueous agar, dehydrated in ethanol, and embedded in Epon according to the method of Luft (1961). Silvergold sections were mounted on bare, 200-mesh grids, stained with uranyl acetate and lead citrate, and examined with a Seimens Model 101 transmission electron microscope. RESULTS Binding of MAb 411-52 to Normal Adult Murine Lung MAb 411-52 is a rat, IgM-class antibody, as determined by its reactivity with class- and subclass-specific anti-rat Ig antisera. Figure 1 shows the characteristic immunohistochemical (IHC) staining pattern of MAb 411-52 on normal, adult lung sections, as observable by light microscopy. MAb 411-52 produces a thread-like, cell-associated immunoperoxidase stain on both luminal sides of alveolar septae viewed in cross section. These IHC-stained cells appear as lightly stained, anuclear plates when seen in plane view. Although the overall MAb 411-52-staining pattern was uniform throughout the lung, not every type 1 cell was stained. Occasionally, individual type 1 cells within an alveolus were unstained. There was no apparent IHC staining of epithelial cells lining conducting airways, type 2 cells, endothelial or interstitial cells, or alveolar macrophages. Omission of the primary antibody eliminated all IHC staining. Electron microscopy was used to localize ultrastructurally MAb 41 l-52 binding within the tissue sections. Tissue blocks incubated with MAb 411-52 had lo-nm gold particles associated with the luminal surfaces of type 1 cell apical membranes (Fig. 2), but no gold particles associated with airway epithelial cells, endothelial cells, or pulmonary alveolar macrophages. In particular, there was no immunogold labeling of type 2 cells. Omission of MAb 411-52 eliminated immunogold labeling of all cells, while substitution of an unrelated primary antibody (MAb 41 l-201) resulted in specific labeling of endothelial cell surfaces, but no labeling of type 1 cell membranes. Binding of MAb 411-52 to Developing Lung DeparafIinized lung sections of mice between 3 and 35 days old were also incubated with MAb 41 l-52. There was no detectable immunoperoxidase staining of alveolar epithelial cells in lung sections from mice 3,7, 10, or 14 days old. The epitope recognized by MAb 41 l-52 was detectable by IHC beginning 21 days after birth (Fig. 3); however, not all alveolar epithelial (type 1) cells simultaneously expressed the MAb 411-52 epitope. Rather, cells that appeared morphologically similar by light microscopy to neighboring type 1 cells that were immunoperoxidase stained often were unstained. With increasing lung maturity, more type 1 cells stained positive for the MAb 411-52 epitope. Lungs of 35-day-old mice were I

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FIG. 1. Light photomicrographs of immunoperoxidase-stained, normal Balbk lung sections. (A, C, and E) The characteristic MAb 411-52 immunoperoxidase staining pattern. (B, D, and F) Similar regions stained without the primary antibody (41 l-52). AV, alveolar lumen; BR, bronchiolar lumen; T,, type 1 cell; T,, type 2 cell; CE, capillary endothelial cell; CL, capillary lumen.

indistinguishable IHC staining.

from adult murine lungs, in terms of their MAb 41 I-52-directed

Flow Cytometric Analysis and FACS Isolation of DLC

Fixed DLC were analyzed for bound MAb 411-52 (FITC fluorescence) and DNA content (propidium iodide fluorescence). Compared to negative controls

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AL

FIG. 2. Electron photomicrographs of murine lung immunogold labeled with MAb 41 l-52. (A and B) Alveolar type l/type 2 junction. (C) Bronchiolar epithelium. Arrows indicate location of lo-nm gold particles. T,, type 1 cell; T,, type 2 cell; AV, alveolar lumen; AL, airway lumen; CL, Clara cell; CI, ciliated cell.

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FIG. 3. Light photomicrographs of neonatal Balbk mouse lung sections immunoperoxidase-stained with MAb 41 l-52. (A) 3 days old. (B) 14 days old. (C) 21 days old. (D) 35 days old. Immunoperoxidase staining of alveoli with MAb 41 l-52 was first detected in lung sections from 21-day-old mice (C). By 35 days of age the majority of type 1 cells were stained (D). AV, alveolar lumen; BR, bronchiolar lumen; CL, capillary lumen; T,, type 1 cell.

(primary antibody omitted), 4.6 * 0.5% of DLC bound MAb 41 l-52, and greater than 95% of these cells had a diploid DNA content. Cells were sorted on the basis of forward light scatter and FITC-fluorescence and examined by transmission electron microscopy (Fig. 4). The cells had scant amounts of perinuclear cytoplasm and often had extensive membrane-bound cytoplasmic leaflets with ribosome-studded endoplasmic reticulum and small cytoplasmic vesicles. The primary cellular contaminants in the sorted samples were lymphocytes. DISCUSSION We have developed a rat monoclonal antibody that binds specifically to the apical surface of murine type 1 epithelial cells. Immunoperoxidase staining of formalin- or glutaraldehyde-fixed, deparaffinized lung sections revealed that MAb 411-52 binds exclusively to the luminal surfaces of alveoli throughout the lung. There was no detectable immunoperoxidase staining associated with conducting airway epithelia, type 2 cells, endothelial cells, or interstitial cells. Macrophages are the primary resident phagocytic cell in the alveolar compartment. As such, they may be involved in clearing cellular debris, resulting from normal cell tum-

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FIG. 4. Electron photomicrograph of murine pneumocyte isolated by fluoresence-activated sorting. Cells stored on the basis of cell size and MAb 411-52 binding.

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over or acute injury, from the lung. In agreement with the observations reported by Dobbs et al. (1988), we could not detect MAb 41 l-52 immunoreactivity within resident alveolar macrophages from normal mice. This may have been due to destruction of the MAb 41 l-52 epitope within alveolar macrophage phagolysosomes, a very slow rate of type 1 cell turnover, or perhaps an inability of macrophages to phagocytize type 1 cell fragments. Additional studies involving sequential sacrifice of mice treated with a type 1 cell toxicant such as butylated hydroxytoluene (Hirai ef al., 1977; Adamson et al., 1977), are needed to resolve this issue. Immunogold labeling of tissue slices was performed prior to plastic embedding. Repeated attempts to label previously embedded and sectioned lung tissue were unsuccessful. The epitope recognized by MAb 411-52 may have been destroyed by the embedding procedures or, if the surface density of the antigen on type 1

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cells is low, there may have been insufficient antigen exposed at the surface of the thin sections to provide a detectable antigen-antibody interaction. Because en bloc staining may result in limited penetration of the immunoreagents into intracellular and interstitial compartments, we cannot rule out the possibility that the antigen recognized by MAb 41 I-52 is uniformly distributed on both the apical and basolateral surfaces of type 1 cells. The epitope recognized by MAb 411-52 is not detectable by means of IHC in the lungs of neonatal mice. The appearance of detectable quantities of 411-52 antigen is delayed until approximately 21 days after birth, well after the time that cells with characteristic type 1 cell morphology are present (Ten-Have Opbroek, 1975, 1979). This observation suggests that the antigen recognized by MAb 411-52 may be a phenotypic marker of fully mature, terminally differentiated type 1 cells. However, the late appearance of this particular marker may also limit its usefulness in studies of lung development or the early stages of alveolar repair following injury. The fact that not every type 1 cell in an adult animal can be immunoperoxidase stained with MAb 41 l-52 is to be expected if the expression of the antigen is in fact related to the maturity or state of differentiation of the cell. The alveolar epithelium, like any other epithelium, is constantly being renewed, and because this is not a synchronous event, there must be a continuum of type 1 cell developmental ages present in this epithelium. We have yet to characterize fully the antigen that bears the 411-52 epitope. Type 1 cell immunoreactivity with MAb 411-42 is lost after extensive treatment of lung tissue with pronase or protease V8, but not after the limited digestion with the trypsin and collagenase solutions used for lung cell dissociation in the present study. The monoclonal antibodies specific for rat type 1 cells, reported by Dobbs et al. (1988), recognize proteins of 40 and 42 kDa. We have attempted to identify the antigen recognized by MAb 411-52 by means of immunoprecipitating radiolabeled, NP-40-solubilized lung cells and then analyzing the precipitates by SDSPAGE gel electrophoresis and autoradiography, as well as by performing western blot analysis of SDS-PAGE gel electrophoresis of lung homogenates, with no success. This may indicate that the antigen is not a protein, but perhaps a glycolipid, or that the MAb is sensitive to changes in the conformation of the epitope resulting from the nonionic or ionic detergents used in these procedures. In summary, we have developed a rat monoclonal antibody that binds specititally to murine type 1 cells and have confirmed the binding specificity of the antibody by immunoelectron microscopy. This MAb allows accurate identilication of murine type 1 cells in routine histological sections and thus eliminates the need for electron microscopy to identify regions of type 1 cell necrosis and exfoliation following toxic insult. ACKNOWLEDGMENTS This research was supported by the Offke of Health and Environmental Research, U.S. Department of Energy, under Contracts DE-ACO4-76EV01013 and DE-ACOS840R21400 and by a National Institutes of Health Grant GM7438, in facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care.

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A rat monoclonal antibody specific for murine type 1 pneumocytes.

A rat monoclonal antibody (MAb), 411-52, that binds specifically to murine pulmonary alveolar type 1 cells was developed. The cell-binding specificity...
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