Proc. Nati Acad. Sci. USAVol. 79, pp. 6033-6037, October 1982Medical Sciences

Transepithelial transport by pulmonary alveolar type II cells inprimary culture

(sodium transport/pulmonary edema)

ROBERT J. MASON*t, MARY C. WILLIAMS*, JONATHAN H. WIDDICOMBE*, MARTIN J. SANDERSt,DAYTON S. MISFELDTt, AND LEONARD C. BERRY, JR.**Cardiovascular Research Institute, Department of Medicine, Physiology, and Anatomy, University of California, San Francisco, California 94143; and*Department of Medicine, Palo Alto Veterans Administration Hospital, Stanford University, Palo Alto, California 94305

Communicated by John A. Clements, June 25, 1982

ABSTRACT Fluid and electrolyte transport by epithelial cellsin vitro can be recognized by the ability of cultured cells to formdomes and by the electrical properties ofmonolayer cultures. Pul-monary alveolar epithelial cells are thought to be partially re-sponsible for fluid movement in the fetal lung, but their role inelectrolyte transport in the adult lung is not known. We isolatedalveolar type II cells from adult rat lung and maintained them onplastic culture dishes alone, on plastic culture dishes coated withan extracellular matrix, and on collagen-coated Millipore filters.Numerous large domes were formed on culture dishes coated withthe extracellular matrix; smaller domes were formed on uncoatedplastic culture dishes. Sodium butyrate (3 mM) stimulated domeformation. Transmission electron microscopy showed that the ep-ithelial cells had flattened but still retained lamellar inclusions andthat the cells were polarized with microvilli on the apical surfacefacing the culture medium. The electrical properties of the mono-layers maintained on collagen-coated Millipore filters were testedin two laboratories. The transepithelial potential differences were0.7 ± 0.1 mV (24 filters, seven experiments) and 1.3 ± 0.1 mV(13 filters, two experiments) apical side negative, and the corre-sponding resistances were 217 + 11 ohm.cm2 and 233 ± 12ohm.cm2. Terbutaline (10 FM) produced a biphasic response witha transient decrease and then a sustained increase in potentialdifference. Amiloride (0.1 mM) completely abolished the potentialdifference when it was added to the apical side but not when it wasadded to the basal side, whereas 1 mM ouabain inhibited the po-tential difference more effectively from the basal side. Thus, typeII cells form a polarized epithelium in culture, and these cells ac-tively transport electrolytes in vitro.

The alveolar space of adult mammalian lungs is lined by onlya thin film of fluid. Although the amount of fluid is not knownprecisely, one estimate is that 20 ml of alveolar fluid is distrib-uted over a surface area ofabout 70 m2 (1). When lungs are fixedfor electron microscopy by vascular perfusion, the only areasthat appear to contain fluid are the corners ofthe alveoli, wherethe radius ofcurvature is short (2). At the corners, the net forceof surface tension, which is a function of the radius ofcurvatureand the actual surface tension, is directed to draw fluid into thealveolus (3-7). How this force is counterbalanced is not known.Although most investigators have tended to discount the pos-sibility of active transport across alveolar epithelium (7, 8), re-cent observations in vivo suggest that there may be active trans-port of fluid from the airspace into the interstitium (9, 10).

Cell culture techniques have been used to study transepi-thelial fluid movement. Epithelial cells that transport fluid formdomes or hemicysts in culture (11, 12). Dome formation isthought to require active transport to create the force to lift the

monolayer from the growth surface, tight junctions betweenepithelial cells, and attachment points to the culture surface toprevent the fluid from flowing underneath the monolayer.Dome formation is a sensitive indicator of active transport butis difficult to study quantitatively (12). For more quantitativestudies, monolayers ofepithelial cells can be mounted in Ussingtype chambers and the electrical properties of the monolayerscan be determined by the methods used for excised epitheliain vitro.

In this paper, we describe dome formation in primary cul-tures of alveolar type II cells from adult rat lungs and the elec-trical properties of type II cells maintained on collagen-coatedMillipore filters. Our results indicate that alveolar type II cellshave the ability to actively transport sodium and suggest thatactive as well as passive forces are important in keeping thealveoli relatively free of fluid. These observations have beenreported in abstract form (13, 14).

MATERIALS AND METHODSCell Isolation and Culture. Alveolar type II cells were iso-

lated from adult Sprague-Dawley male rats by tissue dissocia-tion with elastase and partial purification on a metrizamide den-sity gradient (15). The cells were suspended at 106/ml inDulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum, 2 mM glutamine, gentamicin at 10 pg/ml,penicillin at 100 units/ml, and streptomycin at 50 ug/ml withand without amphotericin at 2.5 kug/ml. All observations weremade in the presence and absence of amphotericin; there wasno apparent effect of this concentration of amphotericin ondome formation or on the electrical properties of the monolayers.

Extracellular matrices were prepared in 35-mm plastic cul-ture dishes from confluent cultures of bovine corneal endothe-lial cells by the method of Gospodarowicz (16). Extracellularmatrices prepared from L-2 cells, A549 cells, and lung fibro-blasts (American Type Culture Collection) were made by grow-ing the cells in Dulbecco's modified Eagle's medium supple-mented with 10% fetal bovine serum, glutamine, and antibioticsand by extracting the monolayers with 20 mM ammonium hy-droxide (17).Dome Formation and Analysis. The partially purified type

II cells were plated at a density of 2 x 105/cm' in 35-mm cul-ture dishes that did or did not contain an extracellular matrix.After the type II cells had adhered overnight, the monolayerswere rinsed with medium and the nonadherent cells were dis-carded. The resultant adherent cells cultured directly on plasticdishes were >90% type II cells and those cultured on extra-cellular matrices made from bovine corneal endothelial cells

t Present address: Dept. of Medicine, National Jewish Hospital, Den-ver, CO 80206.

6033

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment' in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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were >95% type II cells as judged by the modified Papanico-laou stain or phosphine 3R (18). After domes had formed, themonolayers were fixed with 1.25% glutaraldehyde in phos-phate-buffered saline, stained with a polychrome stain of azureII and methylene blue (19), and counted under a dissectingmicroscope. Monolayers were fixed and embedded for electronmicroscopy as described (20). In some experiments, the cellswere stained with tannic acid prior to dehydration (21).

Electrical Characterization of the Monolayers. Type II cellswere maintained on Millipore filters that were coated with rattail collagen and then fixed with glutaraldehyde (22). Becauseadherence to floating collagen gels is enhanced with porcineserum and dexamethasone (23), the type II cells maintained onthe collagen-coated filters were cultured with 10% porcineserum and 0.1 AM dexamethasone. The spontaneous potentialdifference across the monolayers was low and therefore theelectrophysiologic studies were verified independently in twolaboratories (at the University of California and at StanfordUniversity). The filters were mounted in Ussing type chambersfor measurements of resistance and transepithelial potentialdifference (22, 24, 25).

In the University of California laboratory, potential differ-ences were measured with 3 M KCI/agar bridges positioned2 mm from either side of the tissue. Tissue resistance was cal-culated from the change in potential difference produced bypassing 90-_uA.cm-2 current pulses of 1-sec duration. Currentwas passed via 150 mM NaCI/agar bridges positioned 1.5 cmfrom either side of the tissue. Collagen coated filters alone hadan insignificant resistance and no potential difference. With notissue present, the resistance of the solution between the 3 MKCI/agar bridges was determined at the start and end of eachexperiment and was 12-18 ohm-cm2. With tissues in place, thechange in potential difference accounted for by the solutionresistance was only about 7% of the total potential differencechange and was routinely corrected for in determining tissueresistance. Similar techniques were used in the Stanford lab-oratory (22, 25).

Materials. Reagents were obtained as reported (15, 23).Denis Gospodarowicz kindly supplied fibroblast growth factorfor culturing corneal endothelial cells. Terbutaline was pro-vided by Astra Pharmaceutical Products (Framingham, MA).

RESULTSAfter 2 to 3 days in culture, type II cells maintained directly onplastic culture dishes formed a few small domes. However, typeII cells maintained on an extracellular matrix formed by cornealendothelial cells adhered better, flattened more rapidly, andformed numerous large domes, typically 1 mm in diameter. Alow-power light micrograph ofdomes formed on an extracellularmatrix is shown in Fig. 1. When the medium was removed, thedomes were visible to the unaided eye and appeared like grainsofsand sprinkled on the culture dish. The domes formed by typeII cells on the corneal extracellular matrix are larger than thoseformed by MDCK cells grown on a plastic culture surface (ourobservations and refs. 26 and 27). Type II cells formed fewerand smaller domes when they were cultured on extracellularmatrices prepared from lung epithelial cells (L-2 cells or A549cells), lung fibroblasts, or primary cultures of type II cells.When L-2 cells, A549 cells, and lung fibroblasts were culturedon extracellular matrices prepared from corneal endothelialcells, these cell types did not form domes.

Although dome formation is generally thought to be a resultof active fluid transport (11, 12) we wanted to show that domeformation was reversibly blocked by inhibitors oftransport, that

dome formation could be stimulated by pharmacologic agents,and that the fluid under a dome was different from the culture

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FIG. 1. Light micrograph of domes in primary culture. Type I cellswere grown for 3 days on culture dishes coated with corneal extracel-lular matrix. The domes were fixed with glutaraldehyde and stainedwith a polychrome stain (19). (x40.)

medium. Dome formation was inhibited by 1 mM ouabain andby 0.1 mM amiloride. However, overnight incubation was nec-essary to cause these effects. The cells appeared damaged, andthe effects were irreversible. In a series of experiments to eval-uate drugs for their ability to stimulate dome formation, wefound that 3 mM sodium butyrate greatly enhanced dome for-mation (Table 1). Butyrate has been used to stimulate differ-entiated functions in a variety of cell types, but the mechanismof its action is not known. David Warnock (University of Cali-fornia, San Francisco) sampled dome fluid by micropunctureand showed that the bicarbonate concentration under thedomes was the same as that of the medium. He verified thathe had sampled the contents of a dome by adding radioactiveinulin to the culture medium; no inulin was found in his sam-ples. Concentrations ofother electrolytes were not determined.

Because these observations were made with primary cul-tures, we needed to establish that the cells in the domes hadthe morphologic characteristics of type II alveolar epithelialcells. Phosphine 3R is a fluorescent compound that causes thelamellar bodies of type II cells to fluoresce intensely (18). Thecells in the domes appeared like type II cells in culture and werenot distinguishable from the other type II cells in the monolayer(Fig. 2). Electron micrographs showed that the cells formed apolarized epithelium with microvilli on the apical surface andtightjunctions between the epithelial cells (data not shown) andthat cells contained membrane-bound lamellar inclusions thatwere structurally similar to those of type II cells of intact lung(Figs. 3 and 4).

Table 1. Effects of drugs on dome formationDomes per dish, no.

Addition Exp. 1 Exp. 2 Exp. 3None 3 ± 1 13 ± 2 74 ± 10Phorbol 12-myristate

13-acetate(1OnM) 2 ± 1 14 ± 5 66 ± 15Cholera toxin (0.1 Ag/ml) 9 ± 3 8 ± 2 79 ± 8Terbutaline (10 uM) 1 ± 1 10 ± 2 122 ± 29Sodium butyrate (3mM) 120 ± 8* 47 ± 10* 194 ± 23*

Cultures were maintained in Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum for overnight adherenceand then rinsed and incubated with fresh medium containing variousdrugs. Fresh medium and drugs were again added 24 hr later and themonolayers were fixed with glutaraldehyde after incubation for 48 hrwith drugs. Results represent mean ± SEM of four separate dishes foreach of three independent experiments.*P < 0.01 by Friedman's two-way analysis of variance of ranks andthe Newman-Keuls multiple comparison procedure (28).

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FIG. 2. Dome stained with phosphine 3R. Type II cells were cul-tured on a glass coverslip coated with corneal extracellular matrix. Thelamellar bodies of type II cells fluoresce intensely after incubationwith phosphine 3R (18). These two micrographs show a small dome andthe monolayer focused at different levels. (x380.)

To show that dome formation was due to electrolyte trans-port, type II cells were maintained on Millipore filters coatedwith rat tail collagen and studied in Ussing type chambers. Thespontaneous potential difference showed that the apical surfacewas consistently negative with respect to the basolateral surface.The transepithelial potential differences, measured in two lab-oratories, were 0.7 ± 0.1 mV (24 filters, seven experiments) and1.3 ± 0.1 mV (13 filters, two experiments), and the correspond-ing resistances were 217 ± 11 and 233 ± 12 ohm.cm2. As shownin Fig. 5, terbutaline, a 3-adrenergic agonist, produced a tran-sient decrease and then a prolonged increase in the potentialdifference. In the experiment shown, the transepithelial resis-tance was not changing at the time when terbutaline was added.In a number of other experiments, however, terbutaline wasadded at a time when the resistance was decreasing. Becauseof this complicating factor, we determined the effect of terbu-taline, not on the potential difference, but on the apparentshort-circuit current, where short-circuit current is given by theratio of open-circuit potential difference to transepithelial re-sistance. Terbutaline was found to increase short-circuit currentby 28%, from 3.58 ± 0.30 to 4.58 ± 0.30 uAAcm-2 (n = 21;

FIG. 3. Light micrographs of type II cells attached to substratumand in a dome. Type II cells were grown for 3 days on a coverslip coveredwith corneal extracellular matrix. (A) Type II cells on the flat portionof the monolayer. (B) Type II cells in a dome. Type II cells appear asflattened epithelial cells with apical microvilli and lamellar inclusionsnear the nucleus. The extracellular matrix can be seen beneath thecells. (x850.)

P < 0.05 by paired t test). Amiloride (0.1 mM), which blocksmembrane sodium channels (29), rapidly brought the potentialdifference to zero when it was added to the apical side but not

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FIG. 4. Transmission electron micrographs of cultured type IIcells. A large lamellar inclusion is present in the cytoplasm and theextracellular matrix is visible beneath the cell. The apical surface iscovered by microvilli that contain an array of microfilaments (Inset).(Main, x11,400; Inset, x39,900.)

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FIG. 5. Potential differences across monolayers of type II cells. Thetype II cells were cultured for 7 days on collagen-coated Millipore fil-ters and then placed in Ussing type chambers. At time zero, 10 I-Mterbutaline was added. At the end of the experiment, 1 mM ouabain(0) or 0.1 mM amiloride (A) was added to the apical/luminal side (L)or to the basal/serosal side (S).

when it was added to the basal side. On the other hand, 1 mMouabain, which inhibits Na+, K+-ATPase, inhibited the poten-tial difference more effectively when it was added to the basalside. The potential difference was not affected by adding phlor-izin or furosemide to either side or by adding glucose to theapical side. Thus, both morphologic and electrophysiologic datashow that type II cells formed a polarized epithelium in culture.The negative spontaneous apical surface potential differenceand the effect ofamiloride are consistent with sodium resorptionas the dominant transport process.

DISCUSSIONBoth dome formation and the electrical properties of the mono-layers indicate that type II alveolar epithelial cells activelytransport fluid and electrolytes. Although most cells in mono-layer cultures do not form domes, epithelial cells that transportfluid-e.g., epithelial cells from the kidney, mammary gland,colon, trachea, and choroid plexus-form domes in monolayerculture (11, 12). Definitive proofofactive transport is, however,more difficult to establish. Ouabain and amiloride inhibiteddome formation, but in our experiments these drugs appearedto be cytotoxic. We measured the bicarbonate ion concentrationunder the domes to see whether the pH was different orwhether there was a gross change in electrolyte concentration.These experiments were designed to see whether type II cellsreabsorb bicarbonate, which might account for the acid pH ofalveolar fluid (30, 31). The bicarbonate concentration was thesame as that of the culture medium, which indicates that thetransport process is not selective or restrictive for bicarbonate

or that the domes are relatively permeable to bicarbonate overthe time span required for dome formation. We tested severaldrugs for their ability to stimulate or to inhibit dome formation.From other studies, butyrate (27), terbutaline (32), and choleratoxin might be expected to increase dome formation and phor-bol 12-myristate 13-acetate (33) to decrease dome formation.Because dome formation is a complex process of active trans-port, interaction with the growth surface, maintenance of tightjunctions, and other aspects of cellular metabolism, stimulationof dome formation cannot be attributed simply to increased ac-tive transport. The butyrate effect, however, indicates thatdome formation can be stimulated by drugs. An unlikely alter-native explanation for dome formation is hydrolysis of the ex-tracellular matrix and the creation of an osmotic pressure gra-dient. The electrical properties of the monolayer and theobservation of dome formation by cells maintained on plasticalone strongly weigh against this alternative explanation.The electrical properties of monolayer cultures of alveolar

type II cells are similar to some other transporting epithelialcells in culture. The apical side is electrically negative. Thespontaneous potential difference and transepithelial resistanceare similar to those of MDCK cells (25, 34, 35) and LLC-PK1renal cells (22) but less than those of mammary cells on floatingcollagen gels (36) and toad bladder cells (37). As expected fromstudies of epithelial cells in vitro, amiloride was most activewhen it was added to the apical side and ouabain was most activewhen it was added to the basal side of the monolayer (38, 39).In almost all epithelial cells, the Na',K+-ATPase is found pre-dominately on the basolateral membrane.Dome formation had not been described in cultures of type

II cells before these experiments were completed. This is pre-sumably because dome formation requires a complete mono-layer of nearly pure type II cells, the domes take several daysto form, and the domes formed on tissue culture plastic aresmall. The use of the extracellular matrix increases the size ofthe domes dramatically. The matrix improves the plating effi-ciency and cellular spreading and thereby the formation of themonolayer. Corneal extracellular matrices have been used pri-marily to improve the proliferative response of endothelial cellsand smooth muscle cells (16, 17). Independently, Goodman etaL (40) recently reported dome formation and inhibition in pri-mary cultures of alveolar type II cells.

Because of the heterogeneity of lung cells, the complete ar-ray of physiological functions of individual pulmonary cell typesis difficult to define from studies with intact lung. We proposethat transepithelial transport and dome formation in vitro aredistinctive functions of alveolar type II cells in addition to theirmajor function of synthesis, storage, and secretion of surface-active material. In this regard, two epithelial cell lines, L-2 cellsand A549 cells, that have some similarities to type II cells, donot form domes in culture (41). The type II cells in our culturesare, however, large flat cells and, in this regard, are similar totype I epithelial cells. Nevertheless, the cells in our cultureshave microvilli on their surface and do not have the array ofsmall pinocytotic vesicles under their plasma membrane thatare typically seen in type I cells in intact adult rat lungs. Hence,although type II cells have been shown to differentiate into typeI cells in vivo and in the process develop into "transitional" cells,which still contain lamellar inclusions but are beginning to flat-ten (42), we will refer to the cells in our cultures simply as typeII cells, until definitive biochemical or antigenic markers fortype I cells are discovered and can be tested with these cells.

Recent studies in vivo and theoretical considerations supportthe concept that the alveolar epithelium actively transportsfluid and electrolytes from the alveolar side (apical side) to theinterstitium (basal side). In fetal lung at the beginning of labor,

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there is not only a cessation of secretion but net resorption ofthe alveolar fluid, and this resorption is stimulated by a-ad-renergic agonists (32). In the fetal lung, the alveolar side iselectrically negative and the potential difference across the ep-ithelium is increased with 3-adrenergic agonists (9). Matthayet aL (10) found that protein-rich fluid instilled into the distallung of anesthetized sheep was absorbed against a large oncoticpressure gradient. They concluded that either the interstitialpressure was very negative or there was active fluid resorption.In the initial physiologic considerations of pulmonary surface-active material, Pattle (4) and Clements (5) noted that surfacetension would tend to draw fluid into the alveolus and that thiseffect was minimized by a low surface tension at the air/liquidinterphase. The initial calculations of the force due to surfacetension assumed the radius ofan entire alveolus (5). Guyton andMoffatt (6) showed that its force becomes much greater as theradius decreases and, if one assumes that the radius is as smallas 0.5 pum (2) and the surface tension is 10 mN/m, the force isenormous [greater than 300mm Hg (1 mm Hg = 133 Pa)]. Thus,although the magnitude of this force is not known and will varywith alveolar geometry and surface tension during the respi-ratory cycle, the force at the corners ofthe alveoli due to surfacetension is probably significantly larger than 4 to 5 mm Hg (5,43). The increased surface tension in disease states has beenproposed as a mechanism for the formation ofpulmonary edema(5, 44). We propose that active sodium transport by the alveolarepithelium is one of the forces that counterbalance the effectof surface tension. Thus, theoretical considerations, recentphysiologic studies in vivo, and our studies with type II cellsin primary culture, all suggest that active as well as passiveforces are involved in the regulation of alveolar fluid in mam-malian lungs.

We thank Dr. Denis Gospodarowicz for providing fibroblast growthfactor and showing us how to prepare extracellular matrices, Dr. DavidWarnock for carrying out the micropuncture and for determining thebicarbonate concentration in the dome fluid, Lynne D. Calonico forphotographing the domes, and Yuki Kubo-Hendricks and Marcia Han-sen for technical assistance. This work was supported by National In-stitutes of Health Grants HL-24075 and HL-24136 and National CancerInstitute Grant 5 T32 CA 90287.

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