Reactivity of Alveolar Epithelial Cells in Primary Culture with Type I Cell Monoclonal Antibodies Spencer I. Danto, Stephanie M. Zabski, and Edward D. Crandall Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, University of Southern California, Los Angeles, California

An understanding of the process of alveolar epithelial cell growth and differentiation requires the ability to trace and analyze the phenotypic transitions that the cells undergo. This analysis demands specific phenotypic probes to type II and, especially, type I pneumocytes. To this end, monoclonal antibodies have been generated to type I alveolar epithelial cells using an approach designed to enhance production of lung-specific clones from a crude lung membrane preparation. The monoclonal antibodies were screened by a combination of enzyme-linked immunosorbent assay and immunohistochemical techniques, with the determination of type I cell specificity resting primarily on immunoelectron microscopic localization. Two of these new markers of the type I pneumocyte phenotype (II FI and VIII B2) were used to analyze primary cultures of type II cells growing on standard tissue culture plastic and on a variety of substrata reported to affect the morphology of these cells in culture. On tissue culture plastic, the antibodies fail to react with early (days I to 3) type II cell cultures. The cells become progressively more reactive with time in culture to a plateau of approximately 6 times background by day 8, with a maximum rate of increase between days 3 and 5. This finding is consistent with the hypothesis that type II cells in primary culture undergo at least partial differentiation into type I cells. Type II cells grown on laminin, which reportedly delays the loss of type II cell appearance, and on fibronectin, which has been reported to facilitate cell spreading and loss of type II cell features, develop the type I cell markers during cultivation in vitro with kinetics similar to those on uncoated tissue culture plastic. Cells on type I collagen and on tissue culture-treated Nuclepore filters, which have been reported to support monolayers with type I cell-like morphology, also increase their expression of the II FI and VIII B2 epitopes around days 3 to 5. Taken together with available morphologic information, these data suggest that expression of different alveolar epithelial cell phenotypic markers by type II cells in primary culture may be independently regulated. The monoclonal antibody probes described in this report should prove useful in the continued investigation of the mechanisms and regulation of alveolar epithelial cell differentiation.

The epithelium lining the alveoli of the lung, in addition to providing the necessary surface across which gas exchange occurs, subserves the critical role of separating the external environment from the internal milieu and represents the primary barrier to fluid fluxes from the microcirculation, thereby keeping the airspace relatively dry (1, 2). The importance of this barrier to the normal functioning of the lung is

(Received in original form March 5, 1991 and in final form September 18, 1991) Address correspondence to: Spencer I. Danto, M.D., Ph.D., Division of Pulmonary and Critical Care Medicine, University of Southern California, GNH 11-900, 2025 Zonal Avenue, Los Angeles, CA 90033. Abbreviations: Dulbecco's modified Eagle's medium, DMEM; extracellular matrix, ECM; enzyme-linked immunosorbent assay, ELISA; immunoelectron microscopy, immunoEM; immunofluorescence, IF; monoclonal antibody, mAb; phosphate-buffered saline, PBS; short circuit current, SCC; spontaneous potential difference, SPD. Am. J. Respir. Cell Mol. BioI. Vol. 6. pp. 296-306, 1992

demonstrated by the physiologic derangements accompanying lung injury, in which the integrity of the epithelium is violated. After lung injury, a stereotypic sequence of events is initiated in which there is an influx of edema fluid and inflammatory cells, followed by a proliferation of interstitial cells and hypertrophy and proliferation of alveolar epithelial cells. Resolution of injury is accompanied by restitution of the normal interstitium and differentiation of the epithelial cells with possible reconstitution of the normal alveolar wall (3). The mechanisms by which these processes occur and are regulated remain largely obscure. Morphologic analysis of the alveolar epithelium (4,5) has revealed two distinct populations of cells: type II cells, which are cuboidal with apical microvilli and distinctive intracellular inclusions (lamellar bodies), which are the site of surfactant storage; and type I cells, which are expansive flat cells with bulging, protruberant nuclei and highly attenuated cytoplasmic processes. Type II cells synthesize and secrete surfactant (6-8), actively transport solutes (9, 10), and, under

Danto, Zabski, and Crandall: Alveolar Epithelial Cells in Culture

appropriate circumstances (for example, during the response to lung injury), proliferate and differentiate into type I cells (11-13). Type I cells are presumed to have a role in gas exchange, but little more is known about them. Type II cells have been cultivated in vitro (7, 14) which, as noted above, has allowed considerable insight into their function. They gradually undergo a phenotypic transformation in culture in which they lose their distinguishing features (15, 16). It has been suggested that the phenotypic changes observed in these cells in vitro may represent differentiation of type II into type I alveolar epithelial cells (15). This morphologic transition in primary culture appears to be somewhat sensitive to hormonal and other growth factors in the medium (17, 18), as well as to the type of substratum upon which the cells are grown (17, 19-25). Further evaluation of these interesting observations requires differentiation markers of type I and type II cells, few of which are currently available. Our laboratory has long been interested in the cell physiologic properties of the alveolar epithelium, leading to the recent development of a preparation of type II cells that exhibit remarkable bioelectric properties (26). These monolayers may more accurately reflect the barrier properties of the in vivo alveolar epithelium. Morphologically, the cells assume an appearance very reminiscent of type I cells in parallel with the establishment of their remarkable bioelectric parameters (19). In order to investigate the cell biologic events that are occurring in this system, as well as to address the larger issue of alveolar epithelial cell development, we undertook the task of developing monoclonal antibodies (mAbs) to alveolar epithelial cells. Vsing an immunization strategy that maximized production of lung-specific antibodies, we now report the successful generation of several type I cell antibodies. Two of these have been analyzed in detail and used to dissect the growth of type II cells in tissue culture, both on tissue culture-treated plastic as well as on substrata reported to affect the morphologic properties of type II cells in culture. Our results indicate that type II cells grown in primary culture gradually develop the type I cell epitopes defined by these two mAbs, irrespective of the substratum upon which the cells are cultivated, suggesting the cells are undergoing at least partial differentiation from type II to type I cells in this model preparation.

Materials and Methods Materials Rats used in all experiments were Sprague-Dawley, male, specific pathogen-free, and obtained from Hilltop Laboratories (Scottdale, PA) or Taconic Farms (Germantown, NY). Balb/C mice were purchased from Charles River (Wilmington, MA). Elastase was obtained from Worthington Biochemical (Freehold, NJ). Microtiter plates were purchased from Falcon (Lincoln Park, NJ). Microtiter plates precoated with extracellular matrix (ECM) materials (laminin, fibrenectin, and type I collagen) were supplied by Collaborative Research (Bedford, MA). Tissue culture-treated Nuclepore filters (Transwell) were from Costar (Cambridge, MA). Sera were obtained from Flow Laboratories (McLean, VA), and antibiotics and adjuvants from GIBCO (Grand Island, NY). All other tissue culture supplies were purchased from Sigma

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Chemical Co. (St. Louis, MO). Alkaline phosphate-based enzyme-linked immunosorbent assay (ELISA) kits (VECTASTAIN ABC-AP) were purchased from Vector Laboratories (Burlingame, CA). Electron microscope reagents and supplies were obtained from Electron Microscope Sciences (Fort Washington, PA) or Polysciences (Warrington, PA). Gold-labeled second antibodies were purchased from Janssen (Piscataway, NJ). Type II Cell Isolation We have previously described our procedures for the isolation and cultivation of alveolar epithelial cells (26, 27). Briefly, enriched populations of type II cells were prepared from rats weighing 125 to 150 g by elastase (2 to 3 Vlml) disaggregation of the epithelial cells, followed by centrifugation through a discontinuous gradient of metrizamide (p = 1.0401p = 1.090) to separate type II cells (which band at the interface) from macrophages that had been previously laden with fluorocarbon-albumin emulsion and thus pellet through the gradient. At this stage, the cell yield per rat was 10 to 15 X 106 , approximately 65% type II cells, and> 95% viable by trypan blue dye exclusion. These enriched type II cell mixtures were resuspended in MEM (Eagle's modified minimum essential medium containing 2.0 mM t-glutamine, 23.8 mM NaHC03 , 10 mM Hepes, 250 Vlml potassium penicillin G, and 50 mg/ml gentamicin sulfate) supplemented with 10% newborn bovine serum and 0.1 ILM dexamethasone, and seeded either into wells of microtiter plates at a density of 5 x lQ4 cells/well (for ELISA) or into Transwell filter cups at a density of 1.5 x 106 cells/em' (for preparation of high-resistance monolayers). All nutrient media were changed on the second day after plating, removing unattached blood cells (mostly leukocytes) in the process. At this point, on tissue culture-treated plastic, the monolayers were> 90% type II cells. These cultures were maintained in a humidified 5 % COl (in air) incubator at 37° C. Screening of Monolayer Bioelectric Properties Beginning on day 3 in culture, transepithelial resistance (R" kO-cml ) and spontaneous potential difference (SPD, mV) were determined for monolayers grown on Transwell filters using a rapid screening device (Millicell-ERS; Millipore, Bedford, MA). Correction was made for blank filter and solution resistance (about 50 O-cm l ) . Equivalent short circuit current (SCC, ILA/cmZ) can be calculated from the relationship SSC = SPD/R Typically, the monolayers develop R, > 1.5 kO-cm l , SPD > 10 mV, and SCC rv 4 t •

ILA/cm z•

Preparation of P, Membrane Fraction Adult male Sprague-Dawley rats (200 to 250 g) were anesthetized with sodium pentobarbital (50 to 60 mg/kg), the trachea and main pulmonary artery were cannulated, and the lungs perfused with cold phosphate-buffered saline (PBS) (10 mM Na-P04 buffer [pH 7.2], 0.15 M NaCl) while being ventilated with air. The lungs were lavaged with PBS to remove macrophages, quickly excised, and chilled on ice. The parenchyma was dissected free of large airways, minced finely, and homogenized into 5 ml of lysis buffer (25 mM Tris-HCl [pH 7.2], 0.1 M KCl, 1.0 mM csci, 2.5 mM MgClz)/g wet weight tissue in a Dounce homoge-

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nizer (10 strokes each with "1\' and "B" pestles) at 0 to 4 0 C Phenylmethylsulphonyl fluoride was added directly (to 0.1 mM) before homogenization to minimize proteolysis. The homogenate was centrifuged at 500 x g for 20 min. The resulting supernatant was centrifuged at 2,400 x g for 20 min. The supernatant from this spin was centrifuged for I h at 47,000 x g. The pellet from this spin (P2 fraction) was resuspended in lysis buffer and stored at -20 0 C until needed. Protein concentration was estimated by the method of Lowry and associates (28); about I to 2 mg/g wet weight whole lung could be obtained. Membrane fractions were similarly prepared from adult rat livers. Preparation of mAbs Four 6-wk-old Balb/C mice were injected with 107 formalinfixed rat splenocytes in complete Freund's adjuvant. Four days later, at the peak of the primary immune response, they were injected with 40 mg/kg cyclophosphamide. This twostage procedure induces partial tolerance to generic rat xenogeneic antigens, permitting enhanced production of tissue-specific mAbs. The mice were then allowed to recover for 2 wk before being hyperimmunized with 1.0 mg of a lung P2 membrane fraction injected intraperitoneally in incomplete Freund's adjuvant. The mice were reinjected every 2 to 3 wk for at least four cycles before being used for the fusion. Four days after the final immunization, the spleen was removed and the splenocytes were isolated and fused with P3U\ myeloma cells (a nonsecreting, x-chain-producing HAT-sensitive myeloma line; kindly provided by Dr. Donald A. Fischman, Cornell University Medical College, New York, NY) using polyethylene glycol (29). The hybridomas were selected by growth initially in HAT medium (Iscove's supplemented Dulbecco's modified Eagle's medium [DMEM] containing 4.5 g/liter glucose, supplemental 1 mM pyruvate, 2 mM glutamine and 2.5 JLM l3-mercaptoethanol, 20% fetal bovine serum, 100 Ulml penicillin, 100 JLg/ml streptomycin, 0.01 mM aminopterin, 0.Q3 mM thymidine, and 0.1 mM hypoxanthine). They were fed every 2 to 3 days with HAT medium for 2 wk and then weaned into HT medium (HAT medium without aminopterin). After an additional2 wk, the cells were transferred into regular growth medium (medium without HAT containing 10% fetal bovine serum). Positive wells were expanded and cloned at least twice by limiting dilution to ensure clonality. Clones were isotyped with respect to the class of immunoglobulin with specific antisera to the heavy chain (IgG I , IgG 2" IgG zb , 19G 3 IgM) and light chain (K, A) using class-specific second antibodies. For long-term preservation, stocks of cells were established by freezing in 10% dimethyl sulfoxide-90% fetal bovine serum and storage in liquid nitrogen. For elaboration of mAbs, hybridomas were maintained in large-volume tissue culture flasks. Antibodies thus obtained were appropriately diluted and used without further purification. Solid-phase Assays Screening for reactivity to lung and liver P, membrane fractions commenced as soon as visible colonies appeared (about 2 wk). Test plates were prepared by drying 15 JLg (in 25 JLl) of antigen (lung or liver P2)/weIL Nonspecific protein binding sites were blocked by incubation with 3 % horse serum in PBS for 30 min at room temperature. Wells were

incubated with 50 JLI of mAb supernatant or control, and binding was assessed using an alkaline phosphatase.avidinbiotin-complex-based ELISA (VECTASTAIN ABC-AP kit). Absorbances were measured at 490 nm using an automatic plate reader (Titertek Multiskan MCC-340; Flow Laboratories). Controls included substitution of supernatant from the parent myeloma or from a hybridoma known not to react with these antigens (MF-20, a chicken myosin-specific IgG I monoclonal, kindly provided by Dr. Donald A. Fischman) for the hybridoma supernatant, or use of chromatographically purified mouse IgG (Cappel, Malvern, PA) at 10 JLg/mL Positive colonies were operationally defined as producing a minimum of 1.5 times background absorbance with lung membranes and no reaction with liver membranes. Immunohistochemistry To define the cellular specificity of the positive mAbs, histologic localization was accomplished by immunofluorescence (IF) and immunoelectron microscopy (immunoEM). For IF, fresh frozen sections were prepared from unfixed, inflated rat lung. The tissue was embedded in O.CT. compound and snap-frozen in isopentane at -150 0 C For IF, sections 1 to 4 JLm in thickness were cut onto chrome alum-gelatin-coated slides and air-dried. Sections were fixed briefly (5 min) with methanol at 4 0 C, rehydrated in PBS, quenched of endogenous fluorescence by incubation in 1% sodium borohydride, blocked with PBS containing 3 % crystalline bovine serum albumin or 5 % normal goat serum, and incubated with mAb or control for 30 to 60 min at room temperature. The sections were then washed extensively in PBS, blocked again with PBS-albumin, incubated with fluorescein- or tetramethylrhodamine-conjugated second antibody for 60 min, and again washed extensively. The specimens were postfixed with 3.7% formalin in PBS, mounted in 90 % glycerol-IOmM Tris-HCI (pH 8.7) containing 1 mg/ml DABCO (1,4-diazobicyclo[2.2.2]octane; Aldrich, Milwaukee, WI), and the stained specimens viewed with an Olympus BHK-lOmicroscope (Olympus, Lake Success, NY) equipped with epifluorescence optics. Controls included use of MF-20 or 10 JLg/ml chromatographically purified mouse IgG as first antibody. Other tissue specimens (trachea, aorta, pulmonary vein, skeletal and cardiac muscle, kidney, liver, and small intestine) were similarly prepared and processed for IF. For immunoEM, the lungs were prefixed with McLean's fixative (paraforrnaldehyde-Iysine-periodate) (30) by perfusion and tracheal instillation. The tissue was frozen as above, and 6- to 10-JLm-thick sections were cut and processed as for IF, except for the omission of the sodium borohydride treatment and the use of colloidal gold-conjugated second antibodies. Following the immunolabeling steps, the specimens were fixed with 2.5% glutaraldehyde, postfixed with 1% OS04' dehydrated in graded acetones, and embedded in EMbed812. Ultrathin sections were cut and viewed with a lEOL 1200xc electron microscope. In some experiments, ultrathin frozen sections were prepared from tissue and fixed with 1% paraformaldehyde-0.05 % glutaraldehyde, essentially as detailed by Tokuyasu (31). These specimens were stained by.floating the grids successively on droplets of blocking agent (PBS containing 5 % bovine serum albumin [BSA]), mAb, wash solution (PBS), and second antibody as described (31).

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Monolayers on Nuclepore filters were processed for immunoEM in a similar fashion, except that the filters were washed with cold PBS, fixed lightly with 2 % paraformaldehyde, treated with 0.1 M lysine-HCI, and incubated with mAb or control antibody on ice. They were then washed with PBS and incubated with gold-labeled second antibody. After washing, the monolayers were postfixed with glutaraldehyde and osmium, dehydrated, and infiltrated with resin as above. The filters were then removed from the cup and embedded in a flat embedding mold. The controls for nonspecific binding included substitution of 10 Itg/ml chromatographically purified mouse IgG or the myosin heavy chain mAb (MF-20) for the type I cell mAb. Analysis of Alveolar Epithelial Cell Monolayers with mAbs To assess the phenotypic changes occurring during type II alveolar epithelial cell growth in tissue culture with respect to differentiation towards type I cells, type II cell cultures were probed with two of the type I cell mAbs (II FI and VIII B2) as a function of time in vitro. In these experiments, type II cells prepared as described above were seeded into wells of tissue culture-treated polystyrene microtiter plates (50,000 cells/well, plating efficiency, 25 to 40%) and grown under standard conditions. At various intervals in culture, the plates were removed from the incubator, washed rapidly with cold PBS, and air-dried. The wells were then analyzed by ELISA for binding of type I cell mAbs (as described above for P2 membrane fractions) as a function of time in culture. For those experiments in which the influence of ECM was being assessed, microtiter plates precoated with type I collagen, human fibronectin, or laminin were substituted for the standard polystyrene microtiter plates. These precoated plates, purchased from Collaborative Research as noted above, were prepared by standard techniques using protein concentrations similar to those used in other investigations (17, 20, 23, 24). Similar experiments, evaluating the phenotype of cells grown as monolayers on Nuclepore filters, were perfomed using a quantitative immunoEM assay. In these studies, the cell monolayers were immunolabeled with the type I cell mAbs or control (MF-20) antibody and the filters processed for electron microscopy as described above. Sections were prepared from each experimental (type I cell mAb) and control (MF-20) block, and quantitation of labeling of the epithelial cell surface was accomplished by a simple morphometric analysis. Photomicrographs were made of nonoverlapping fields spanning the length of the specimen. Using these photomicrographs, the density of label appearing along the cell surface was measured and expressed as gold particles/lO Itm linear distance. At least 10 randomly selected fields were examined per experimental condition. All monolayer preparations used in these studies were tested for the development of high resistance beginning on day 3 (when confluency is first achieved), with> 90% of those filters attaining tissue resistances > 1 kO-cm2 • Analysis of Other Cells with mAbs LLC-PKI cells (a renal epithelial cell line, kindly provided by Dr. Alicia McDonough, University of Southern California) were maintained in DMEM supplemented with 4 mM glutamine, 7.5% horse serum (Sigma), 2.5% fetal calf se-

Figure 1. Immunofluorescence (IF) on fresh-frozen sectioned adult

rat lung with monoclonal antibody (mAb) II FI. Phase-contrast (panel A) and corresponding fluorescein isothiocyanate (FITC)-IF (panel B) images obtained with mAb II FI are shown. Numerous alveoli are seen, all of which are uniformly labeled with mAb II FI. Note that the bronchus (br) at the upper left is unlabeled. Also unstained are interstitial regions (arrows), some of which contain capillaries and nonepithelial cells. Bar = 10.0 J1.m.

rum (Irvine ScientificIrvine, CA), and penicillin/streptomycin. Cultures were fed every other day and passaged 7 days after reaching confluency. For ELISA studies, 50,000 cells/ well were seeded into 96-well tissue culture plates and assayed for binding of mAbs II Fl and VIII B2 at 1 and 8 days after replating. Primary rat hepatocyte cultures were established from liver cells (kindly provided by Dr. Neil Kaplowitz, University of Southern California) isolated from adult rat livers by collagenase perfusion (32), and maintained in Ham's F12/ DMEM (1:1) supplemented with 4 mM glutamine, 10% fetal calf serum (Irvine Scientific), and penicillin/streptomycin. Cells were plated into 96-well tissue culture plates at 50,000 cells/well and fed every other day. Cultures were assayed for binding of mAbs II Fl and VIII B2 by ELISA at 1 and 8 days after isolation. Statistics Results are expressed as mean ± SEM. Differences in mAb binding among various time points on a given substratum

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Figure 2. IF on fresh-frozen sectioned adult rat long with mAb VIII B2. Phase-contrast (panel A) and corresponding FITC-IF (panel B) images obtained with mAb VIII B2 are shown. Note the

uniform staining of the alveolar septae and the complete nonreactivity of the interstitium, small bronchiole (br), and blood vessel (v), Arrowpoints to a putative type II cell, whichis unlabeled. Bar = 10.0 p.m.

were assessed by one-way ANOVA. Contrasts between points within a time course experiment were assessed by Scheffe's method (33).

Results Preparation and Characterization of Type I Cell Membrane mAbs Following the immunization protocol described above, which was designed to optimize the production of antibodies to lung cell surface epitopes, the immune splenocytes were fused with the myeloma cell line, resulting in > 900 hybridoma colonies. These were screened for reactivity to lung and liver membrane fractions by ELISA. From among these colonies, 67 clones producing lung-specific antibodies (defined by reactivity to lung and not to liver membrane antigens) were eventually isolated. IF on frozen-sectioned adult rat lung revealed 23 clones that selectively label the alveolar wall without staining conducting airways or vasculature structures. To date, six antibodies that preferentially label type I cell membranes by immunoEM have been identified.

Figure 3. Immunoelectron microscopic localizationof mAb II FI epitopes in adult rat lung. Panel A.- Low-power electronmicrograph showing a sectionthrough an alveolar wall including a type II cell (ATII), which contains lamellar bodies (LB), between two airspaces lined by type I cells. Close examination reveals uniform label along the alveolar wall except over the type II cell. Bar = 1 p.m. Panels Band C.- Higher magnifications of the cell in panel A highlighting the junction (bracket) betweenthe type II cell and the type I cell. The gold particles that uniformly decorate the type I cell membrane stop abruptly at the junction (arrowheads). Bar = 1 p.m and 0.3 p.m in panels Band C, respectively.

Although their epitopes have not yet been identified, these antibodies have proved valuable as markers of type I cell phenotypic differentiation. Of the several type I cell antibodies with interesting patterns of binding to alveolar epithelial cells in primary culture, two (II PI and VIII B2) were selected for more intensive investigation. Both of these antibodies are IgGt-K isotypes. IF studies on adult rat lung are depicted in Figures 1 (II PI) and 2 (VIII B2). At this magnification, numerous alveoli are seen in cross section, as well as a small bronchus (Figure 1) and a small bronchiolar-vascular pair (Figure 2). The alveolar septae throughout the sections are diffusely labeled by

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MF-20 or chromatographically purified mouse IgG as first antibody demonstrated no alveolar septal labeling. Pulmonary vasculature, aorta, and trachea were examined independently, and no staining of these structures was seen with either antibody (data not shown). IF analysis of frozen sections of rat kidney, liver, skeletal and cardiac muscle, and small intestine was also conducted to evaluate the tissue distribution of the type I cell epitopes recognized by these mAbs. Neither mAb reacted with these other tissues (data not shown), suggesting that the epitopes are restricted to lung parenchyma. Electron microscopy was employed to define the specificity of the mAbs more precisely. Figures 3 and 4 show a series of low- to high-magnification electron micrographs of sections of alveolar walls labeled by the immunogold technique (see MATERIALS AND METHODS) with II Fl and VIII B2, respectively. In the uppermost (low power) panel of each figure, an alveolar septum containing a type II pneumocyte (recognized by its cuboidal shape, apical microvilli, and remnants of lamellar bodies) is depicted. Close inspection reveals uniform labeling of the epithelial surface adjacent to the airspace, except over the type II cell. At the progressively higher magnifications (shown in the lower panels) of a junction between type I and type II cell surfaces, the uniform labeling of the type I cell surface is clearly demonstrated. This labeling stops abruptly at the junction with the type II cell. Both antibodies appear to label only the apical surfaces of the type I cells. In neither case are interstitial cells, interstitial matrix material, or capillary endothelium stained. Control experiments (with MF-20 or mouse IgG) produced exceedingly rare, randomly distributed gold labeling. Ultrathin frozen sections yielded essentially identical results (data not shown), eliminating the potential problem of inadequate permeation through the thickness of the sections (leading to false negative results). Overall, the patterns of immunolabeling obtained with both II Fl and VIII B2 indicate that both of these mAbs are type I cell-specific. Figure 4. Immunoelectron microscopic localization of mAb VIII B2 epitopes in adult rat lung. Panel A: Low-power electron micrograph showing a section through an alveolar wall including a type II cell, recognized by its lamellar bodies and apical microvilli. Close inspection reveals uniform label along the alveolar surfaces except over the type II cell. Bar = I !-tm. Panels Band C: Higher magnifications of the cell in panel A highlighting the junction (bracket) between the type II cell and the type I cell. The abrupt discontinuation of labeling at the junction (arrowheads) is clearly seen. Bar = I !-tm and 0.3 !-tm in panels Band C, respectively.

both antibodies (although perhaps more intensely by VIII B2), consistent with the fact that> 95% of the alveolar surface is covered by type I cell membrane. Note, however, that there is no reactivity above background to larger vessels or to conducting airways, nor to the connective tissue surrounding these structures, suggesting that the reactivity of these mAbs is limited to the alveolar epithelium. Although precise cellular identification is impossible at the level of resolution afforded by IF, a patch of unlabeled alveolar surface is noted in Figure 2 (arrow) which may correspond to a type II cell location (recognized by its raised silhouette and position in the corner of the alveolus). Control experiments utilizing

Analysis of Alveolar Epithelial Cells Cultivated In Vitro These two type I cell-specific probes were used to reexamine the issue of type II cell development with time in primary culture. Type II alveolar epithelial cells were cultured in tissue culture-treated polystyrene microtiter plates according to our standard protocol (see MATERIALS AND METHODS). After various intervals after plating, the cells were assayed for the presence of type I cell epitopes by ELISA. Figure 5 shows the results obtained from this analysis. As can be seen, neither II Fl nor VIII B2 reacted significantly with freshly isolated (day 0) or day 1 cells in culture. However, there was a temporal and significant increase in binding to the cultured cells, to a maximum of greater than 6 times background by both antibodies. The greatest rate of increase occurred between days 3 and 5, which corresponds to the period of most dramatic morphologic change (19) and the establishment of high transepithelial electrical resistance for monolayers on Nuclepore filters (26). To explore the possibility that the mAbs were recognizing generic epithelial cell antigens induced by maintenance in tissue culture, similar ELISA analyses were performed on LLC-PKI renal epithelial cells at days 1 and 8 after passage, and on primary cultures of rat hepatocytes after 1 and 8 days in culture. No binding was de-

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Figure 5. Timecourseof type I cell mAblabeling of alveolarepithelial cells grown on uncoated tissuecultureplates. Isolatedtype II cells wereseeded in wellsof uncoated tissueculture-treatedmicrotiter plates at 50,000cells/well. Culture was terminated on the indicated day by washing and air-drying the cells to affix them to the plate. Binding of mAbs II FI and VIII B2 was assayed by enzyme-linked immunosorbent assay. Allabsorbances havebeennormalized to background values (range, 0.050 to 0.150 OD) obtained by using a myosin mAb(MF-20) thatdoesnot reactwiththesecells.. Each point represents the mean ± SEM of replicate data from at least threeseparate type II cell preparations. The increases in mAb binding are statistically significant by ANOVA. Curves have been interpolated freehand.

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differences in the effects of these ECM materials upon the appearance of the type I cell epitopes defined by mAbs II Fl and VIII B2 can be detected (although there is a suggestion that the increase in binding on laminin is the most protracted). However, it is possible that the cells cultivated on these substrata (especially collagen and laminin) develop an increased density of II Fl and VIII B2 epitopes compared with cells plated on uncoated polystyrene tissue culture dishes. Evaluation of Monolayers of Alveolar Epithelial Cells on Tissue Culture-treated Nuclepore Filters with Type I Cell mAbs To gain further insight into the biology of alveolar epithelial cell monolayers on Nuclepore filters, we analyzed these monolayers for the development of type I cell epitopes using mAbs II FI and VIII B2, employing a quantitative immunoEM assay. Figure 9 depicts the pattern of reactivity obtained with VIII B2 at days 1, 3, 5, and 8 in culture. Examination of this figure reveals two important points. First, it is clear that the cells become progressively more attenuated and lose lamellar bodies with time in culture. Second, concomitant with the development of the highly flattened morphology, there is a dramatic increase in labeling along the cell surface. Morphometric analysis of these data is presented in Figure 10, which reveals significant temporal increases in immunolabeling with II Fl and VIII B2, with the maximum rate of increase occurring around day 3. High electrical resistance also appears at this time. These highly functional monolayers clearly express markers of type I cell differentiation with time in culture.

Discussion Utilizing a strategy to optimize the production of lung membrane-specific mAbs from a heterogenous antigen preparation, we have succeeded in producing a panel of mAbs to type I alveolar epithelial cells. In this report, the immunohistochemical characterization and binding properties of two

Danto, Zabski, and Crandall: Alveolar Epithelial Cells in Culture

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Figure 7. Timecourseof type I cell mAb labeling of alveolar epithelial cells grown on fibronectin. TypeII cells werecultured and assayed as in Figure5 except that microtiterplates precoated with humanfibronectin wereused insteadof the standard microtiter tissue culture plate. Data are normalized and graphed as for Figure 5. The increases in mAb binding are statistically significant by ANOVA-

DAY IN CULTURE

Figure 8. Timecourseof type I cell mAblabelingof alveolar epithelial cells grownon laminin. TypeII cells were cultured and assayed as in Figure 5 except that microtiter plates precoated with laminin (isolated from Engelbreth-Holm-Swarm cell extracellular matrix) wereused insteadof the standard microtiter tissueculture plate. Data are normalized and graphed as for Figure 5. The increases in mAb binding are statistically significant by ANOVA.

of these mAbs have been described in more detail. By IF, knowledge about them. The inability to assay for type II and, both mAbs II PI and VIII B2 stain the walls of alveoli in a particularly, type I cell properties has impeded investigation relatively uniform manner, but do not react with bronchial of the phenotypic transformation undergone by type II cells in vitro. More importantly, the lack of suitable cellular probes or vascular structures. ImmunoEM localization demonhas limited attempts to address the larger problem of alveolar strates preferential reactivity of both of these mAbs with type I cell apical membranes, with absence of labeling of , epithelial proliferation and differentiation, which the alveolar epithelial cell phenotypic changes in vitro may mimic. type II cells, capillaries, interstitial cells, and ECM. Because alveolar epithelial cell types can be defined reliably by their A number of studies have had the objective of identifying specific markers for type I cells and nonsurfactant markers morphology in situ, we conclude from these histochemical data that both mAbs II Fl and VIII B2 are type I cellof type II cell differentiation that could be used in analyses specific. Using these markers of the type I cell phenotype to of alveolar epithelial cell differentiation. The relative speanalyze type II cells in primary culture, we found that both cificity of the Madura pomifera lectin for type II cells (16) of these mAb epitopes (which are not present on isolated has been confirmed (38), and a 200- to 230-kD glycoprotein identified as the putative receptor (38). However, lectins type II cells) are expressed to a progressively greater extent with time in culture, suggesting at least partial differentiation have proved less useful for type I cell identification (39). Patof type II into type I cells. Furthermore, type II cells cultiterns of cytoskeletal protein expression have been used to distinguish possible stages of alveolar epithelial cell differenvated on various substrata reported to affect the phenotypic tiation (40, 41), but no type'1 cell-specific pattern has yet transformation of type II cells in culture also develop the type been recognized. Although many mAbs reactive with type I cell markers defined by mAbs II PI and VIII B2 with someII cells have been produced using hybridoma technology what similar time courses. (42-49), most of these have not been well characterized One of the major problems confronting the pulmonary and/or are not type II cell-specific. Partially purified type I cell biologist interested in the properties of the alveolar epicells, although perhaps nonviable, have recently been used thelium has been the difficulty in identifying pertinent cell as immunogen in the preparation of mAbs (50). This populations (type II and, especially, type I pneumocytes) ex vivo by nonmorphologic means. Type II cells can be isolated resulted in one antibody that reacts with the semipurified in relatively pure form (7, 34), which has led to an initial unpreparation of type I cells as well as with type I cells in situ. Interestingly, this antibody also appears to recognize a fetal derstanding of their biochemical and functional properties. lung cell that concurrently expresses a type II cell marker For example, type II cells can be recognized by the presence (51). The significance of this crossreaction requires further of lamellar bodies and surfactant phospholipids and apoproclarification, for which additional probes of alveolar epitheteins (8,35,36). However, with time in primary culture, they lial cell phenotypes are likely to be useful. lose these morphologic and biochemical hallmarks (8, 37). On the basis of the appearance of the cultures, it has been Because antigens used in producing mAbs should be as reflective as possible of the native state of type I and type II speculated that they may be differentiating into type I cells cells, the immunogen should preferably not contain elements (15, 16). Evaluation of this hypothesis requires additional knowledge about type II and, especially, type I cells, as well from cells that have been enzymatically disaggregated (which as intermediary cell phenotypes. Unfortunately, type I cells would remove and/or alter cell surface determinants) or culhave not yet been successfully harvested and cultivated in tivated in vitro (as the phenotype is likely to be different). vitro; hence, there is little specific functional or biochemical Consequently, the immunogen used in our studies was de-

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Figure 9. Morphology of monolayers on tissueculture-treated Nuclepore filters labeled with mAb VIII B2. Type II cells (1.5 x lQ6/cm2) were seeded into Transwell filters and cultivated according to our standard technique. On successive days, filters were immunolabeled with mAb VIII B2 and processed for electron microscopy. Panels A through D represent fields at approximately the samemagnification obtained from filters on days 1, 3, 5, and 8, respectively. On days 1 and 3, the cells are easily recognizable as type II cells and contain multiple lamellar bodies. On day 1, there is no labeling detected, while on day 3 occasional grains may be seen. These early cells can be contrasted withthoseon days 5 and 8. Notethe remarkable attenuation ofthe cytoplasmic processes achieved byday 5 in thisexperiment, with their far morenumerous immunogold grains. The day 8 monolayer is similarin appearance and extentof labeling, though the cellular process may be even thinner than on day 5. These filters typically develop electrical resistances greater than 1.5 kO-cm2 by day 4. Bar = 1 p.m.

rived from whole lung, with immunocytochemistry used to define the specificity of the mAbs. The strategy we devised employed an immune tolerance-inducing step designed to enhance production of lung-specific clones, and a panel of lung-specific mAbs was produced. A subset of these proved to be type I cell-specific by the following criteria: reactivity with lung and no reactivity to liver membrane antigens by ELISA; staining of alveolar walls by IF without staining bronchi, vasculature, loose connective tissue, trachea, aorta, or pulmonary artery; and, electron microscopic identification of immunogold labeling on type I cell membranes but no labeling of adjacent type II cells, basement membrane, ECM, interstitial fibroblasts, or capillaries. Macrophages and surfactant (obtained by lung lavage) were evaluated independently and were nonreactive (data not shown). Two approaches were taken to ensure that inadequate permeation of antibodies into the sections was not responsible for the lack of labeling of structures inaccessible to the alveolar airspace. First, the immunolabeled specimens were serially sectioned completely through their 6- to lO-p,m thickness so that all levels of the specimen could be examined. Second, ultrathin frozen sections were prepared and stained after sectioning. In all sections studied by both techniques, only type I cell plasma membrane, and specifically

the apical surface, was labeled, with all other structures remaining negative. Thus, these antibodies appear to be type I cell-specific. Interestingly, no type II cell antibody has yet been identified. In this regard, we hypothesize that by using a membrane preparation, we biased the immunogen to type I cell epitopes that contribute the majority of the alveolar epithelial membrane surface. The studies described in this report focus on two representative mAbs, II FI and VIII B2. Both of these antibodies are type I cell-specific by the criteria detailed above. Furthermore, they appear to be tissue-specific as demonstrated by the absence of IF labeling of rat kidney, liver, muscle, and intestine. Neither antibody reacts with freshly isolated type II cells nor with type II cells in culture for up to 24 to 48 h. However, on tissue culture plastic, both mAbs demonstrate a significant time-dependent increase in binding, with the most dramatic rise occurring between days 3 and 5 and a maximum binding of 6 to 10 times background levels. This interesting observation suggests that the alveolar epithelial cells in primary culture are developing the ability to express type I cell epitopes, consistent with results reported previously with lectins (16) and another type I cell antibody (50). At this time, it is unclear if the antigens recognized by the mAbs II F1 and VIII B2 are homologous with that recognized

Danto, Zabski, and Crandall: Alveolar Epithelial Cells in Culture

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Reactivity of alveolar epithelial cells in primary culture with type I cell monoclonal antibodies.

An understanding of the process of alveolar epithelial cell growth and differentiation requires the ability to trace and analyze the phenotypic transi...
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