A novel epithelial cell from neonatal rat lung: isolation and differentiated phenotype PENELOPE E. ROBERTS, DAVID M. PHILLIPS, AND JENNIE P. MATHER Department of Cell Biology, Genentech, South San Francisco, California 94080; and The Population Council, Center for Biomedical Research, New York, New York 10021

ROBERTS, PENELOPE E., DAVID M. PHILLIPS, AND JENNIE P. MATHER. A novel epithelial cell from neonatal rat lung: isolation and differentiated phenotype. Am. J. Physiol. 259 (Lung Cell. Mol. Physiol. 3): L415-L425, 1990.-A novel epithelial cell from normal neonatal rat lung has been isolated, established, and maintained for multiple passages in the absence of serum, without undergoing crisis or senescence. By careful manipulation of the nutritional/hormonal microenvironment, we have been able to select, from a heterogeneous population, a single epithelial cell type that can maintain highly differentiated features in vitro. This cell type has characteristics of bronchiolar epithelial cells. A clonal line, RL-65, has been selected and observed for >2 yr in continuous culture. It has been characterized by ultrastructural, morphological, and biochemical criteria. The basal medium for this cell line is Ham’s FlZ/Dulbecco’s modified Eagle’s (DME) medium plus insulin (1 pg/ml), human transferrin (10 pg/ml), ethanolamine (10m4 M), phosphoethanolamine (10s4 M), selenium (2.5 X 10B8 M), hydrocortisone (2.5 X 10N7 M), and forskolin (5 PM). The addition of 150 pg/ml of bovine pituitary extract to the defined basal medium stimulates a >lO-fold increase in cell number and a 50- to loo-fold increase in thymidine incorporation. The addition of retinoic acid results in further enhancement of cell growth and complete inhibition of keratinization. We have demonstrated a strategy that may be applicable to isolating other cell types from the lung and maintaining their differentiated characteristics for long-term culture in vitro. Such a culture system promises to be a useful model in which to study cellular events associated with differentiation and proliferation in the lung and to better understand the molecular mechanisms involved in these events. epithelial cultures; defined mones; growth factors

medium;

airway

epithelium;

THE LUNG is a complex organ composed of >40

hor-

different cell types. Work on small cell carcinoma and recent advances in endocrinology have led to the recognition that the lung is the site of production of and target tissue for a number of endocrine, paracrine, and autocrine factors. Although several tissue culture systems have been reported for primary culture of cells from the lung, specifically tracheobronchial epithelium (5, 14, 15, 27, 29, 34), epithelial cell lines that can maintain their differentiated function in vitro have been difficult to establish without viral transformation or immortalization by transfection with various oncogenes. It was felt that epithelial cell lines from normal tissue would be of great use in furthering our understanding of lung endocrinology and physiology. Serum is known to support the growth of many cell 1040-0605/90 $1.50 Copyright

types, however, it is complex and not well defined. In vivo, cells would be exposed to the equivalent of serum only under special circumstances involving tissue injury and blood coagulation. In vitro, serum may not support the growth of some cell types, due to specific inhibition or a failure to provide an adequate concentration of stimulatory factors. For this reason, we sought to utilize hormone-supplemented, serum-free medium as a method of selecting for specific cell types from the lung. Mather and Sato (22) demonstrated that a serum-free hormonally defined medium for melanoma cells could be used to select for that same cell type when used as the culture media for a mixed cell population. Piltch et al. (25) were able to select for differentiated epithelial cells from rat thymus and maintain these cells continuously in a defined serum-free medium supplemented with hormones. Loo and co-workers (15) have described a serum-free, hormonally defined culture system for the establishment of a mouse embryo cell, selected from whole embryos. These cultures, when carried in the presence of serum, undergo a well-defined crisis or senescence (16). This senescencedoes not occur when these cultures are carried continuously in serum-free, hormonally defined culture. The lungs function as an endocrine organ, but one that has been difficult to study in vitro, largely because of the many diverse cell types. It was important to ascertain whether or not novel epithelial cell types of the lung could be isolated and established in vitro by careful manipulation of the nutritional/hormonal culture environment. We report here a serum-free, hormone-supplemented cultured system that will initially select for a single epithelial cell type from normal, neonatal rat lung. These cells will undergo multiple passageswithout crisis or senescence. A clonal cell line established in this fashion has been designated RL-65 and its properties are described below. MATERIALS

AND METHODS

Animals. Five-day-old male Sprague-Dawley rats used for these experiments were obtained from Simonsen Labs, Gilroy, CA. Materials. Dulbecco’s modified Eagle’s (DMEM) medium, high glucose, and Ham’s F12 medium (F12) were obtained in powder form from GIBCO, Grand Island, NY; porcine insulin (pins), human transferrin (hTF), hydrocortisone (HC), progesterone, ethanolamine (Eth), phosphoethanolamine (PEth), triiodothyronine (T3), and soybean trypsin inhibitor (STI) were obtained from

0 1990 the American

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L415

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Sigma Chemical, St. Louis, MO; trypsin (0.05% + 0.053 mM EDTA) was obtained from GIBCO; epidermal growth factor (EGF) was from Collaborative Research, Waltham, MA; forskolin (FK) was obtained from Calbiothem, La Jolla, CA; bovine lipoprotein [predominantly high-density lipoprotein (HDL) (Excyte)] was obtained from Miles Laboratories, Napierville, IL; sodium selenite (Sel) was from Johnson Matthey (Aesar, Seabrook, NH); whole mixed sex bovine pituitaries were obtained form Pel Freeze, Rogers, AR; human plasma fibronectin was obtained from Bethesda Research Labs (GIBCO), Bethesda, MD; tubulin, desmin, and vimentin monoclonal antibodies (MABs) were obtained from Chemicon, Los Angeles, CA; rabbit antihuman keratin and rabbit antichicken actin were obtained from Polysciences, Warrington, PA; fluorescein isothiocyanate conjugated, F(abl)2 fragments of goat antimouse and antirabbit immunoglobin G (H- and L-chains specific) were obtained from Jackson ImmunoResearch Laboratories, West Grove, PA; Dil-Ac-LDL was obtained from Biomedical Technologies, Stoughton, MA. The following pituitary factors were obtained from Sigma Chemical: growth hormone, follicle-stimulating hormone (FSH), thyroid-stimulating hormone, human-luteinizing hormone, human prolactin, adrenocorticotropic hormone, oxytocin, vasopressin, cyand ,&melanocyte-stimulating hormone, ,&lipotropin fragments, cu,P,y-endorphin. Fibroblast growth factor (FGF) was obtained from Collaborative Research. Radiochemicals were purchased from New England Nuclear. Recombinant human TGFPl was supplied by Genentech. Culture media and conditions. DME and Ham’s F12 (1:l wt/wt) were dissolved in Milli-Q water, and supplemented with 1.2 g/l NaHC03, 0.4 g/l glutamine, and 15 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES). Hormones, trace elements, vitamins, and phospholipids used to supplement the serum-free medium are shown in Table 1. All of these factors are prepared as sterile stock solutions at lOO- to l,OOO-fold final concentration and added to the medium just before use. Bovine pituitary extract (BPE) was prepared by the method of Tsao et al. (30). Cells were incubated in a 5% COZ-95% air, H20-saturated atmosphere at 37OC. Primary culture. Five-day-old male Sprague-Dawley rats were killed by COe asphyxiation, the lungs were removed, the trachea was excised, and the entire lung TABLE 1. Factors required for proliferation of RL-65 cells Factor

7F

Insulin (porcine) Transferrin (human) Epidermal growth factor Ethanolamine Phosphoethanolamine Selenium Hydrocortisone Forskolin Progesterone Triiodothyronine Bovine lipoprotein

1 Pdml 10 cLg/ml

Bovine ml protein for growth

pituitary extract concentration. in 7F.

(BPE) Retinoic

1 x 1O-4 M 1 x 1O-4 M 2.5 x lo-’ M 2.5 x 1O-7 M 5PM

11F

10 /-%/ml 10 Pdml 5 rig/ml 1 x lO+ 1 x lo+ 2.5 x lo-’ 1 x lo-’ 1PM 1 x lo-’ 5 x lo-l2 0.5%

used at a concentration acid (0.05 PM) + BPE

M M M M M M

of 150 pg/ is optimal

EPITHELIAL

CELLS

was washed briefly in serum-free medium containing 20 pg/ml gentamicin. Peripheral lung tissue was minced into fragments that were then resuspended in serum-free medium containing 0.05% (wt/vol) collagenase-dispase (Boehringer Mannheim, Indianapolis, IN) and incubated for 30-45 min at 37OC.The tissue fragments were washed twice with serum-free medium and allowed to settle for 15 min after which time the supernatant was removed. The fragments were dispersed by repetative pipetting in serum-free, hormone-supplemented medium (llF, see Table 1) and aliquoted into fibronectin-coated 60-mm tissue culture dishes. After 24 h, tissue fragments still in suspension were transferred to new fibronectin-coated dishes. This was repeated daily, transferring the remaining tissue fragment four to five times. Medium was changed every 3 days, and the cultures were maintained in 11F medium for 1 mo. At this point, BPE, at 150 pg/ ml protein concentration, was added. Colonies became densely packed monolayers within 7-10 days from the day of addition of BPE and were passaged at this stage. By this protocol, we have successfully isolated the cell type seven times. Serial passage. Highly cornified colonies from 60-mm dishes were initially passaged by several trypsinizations (0.05% trypsin-0.53 mM EDTA) for 10 min each at 37OC. After neutralization with ST1 (1 mg/ml), cells were washed twice by centrifugation in serum-free F12/DME to remove residual ST1 and plated in fibronectin-coated 12-well trays (usually one 60-mm dish per well) containing 1lF medium supplemented with BPE. Cells were subsequently passaged at near confluency and seeded into sequentially larger dishes at each passage. Continuous culture was then maintained by passaging at near confluency and at a high-seeding density (1:l or 1:2 split ratio). At no time after the addition of the BPE did the cells undergo a reduction in growth rate or “crisis.” Establishment of a cloned lung epithelial cell line. Lung epithelial cells grown continuously in this way for several months do not require that the dishes be fibronectin coated for attachment. Cloned populations were selected by plating 100-500 cells in a loo-mm dish containing 15% (vol/vol) conditioned per fresh 11F medium and changing the medium every 3 days. By day 10, colonies were of sufficient size to be cloned by trypsinizing in 6mm stainless steel cloning rings (penicylinders, Bellco). Each colony was then seeded into 1 well of a 24-well tray, grown to near confluency, and the entire well was passaged into sequentially larger surface areas at each trypsinization until stock cultures could be maintained in loo-mm dishes. All of the colonies picked had a similar morphology. One of these clones, designated RL-65, was chosen for further characterization, and the optimal nutrient and hormonal requirements were determined. This ceil line has been carried in continuous culture for X50 passages and can thus be considered an established cell line. The cell line is aneuploid, with chromosome counts in the hypotriploid range. The RL-65 stocks are currently maintained by passaging every 4-6 days at a 1:50 or 1:200 split ratio. Indirect immunofluorescence. Cells were grown to near confluency on 12-mm glass cover slips. The cover slips were moved to fresh serum-free F12/DME, and paraformaldehyde was added to a final concentration of 2%.

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After 20 min, the cover slips were washed with phosphate-buffered saline (PBS) and placed in 0.1 M glycine for an additional 20 min. After washing twice in PBS, 1% Triton-X 100 was added and left on for 6 min. The cover slips were then washed twice with PBS and exposed to the first antibody (diluted 1:25) for 30 min at 37°C. After rinsing four times in PBS (5 min/rinse), the second antibody (1:lO) was added, and the above procedure was repeated. The cover slips were then drained, air-dried, mounted in Aquamount, and examined with a Nikon Microphot FX epifluorescence microscope. Electron microscopy. Near-confluent cultures of primary lung cells or the established RL-65 line were prepared for electron microscopy by washing the cells with serum-free FlB/DME, diluting the medium 1:l with 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.2), then fixing overnight in 2.5% glutaraldehyde. Alternatively, cells were grown on collagen-coated Transwell mem-

EPITHELIAL

L417

CELLS

brane inserts (Costar) in six-well trays and fixed as above. Cultures were postfixed in 1% buffered Os04, dehydrated in alcohol, and embedded in Polybed (Polysciences). Sections cut on a Reichart OmU3 ultratome were stained in 3% aqueous uranyl acetate at 50°C for 1 h and examined with a Phillips 300 microscope. Quantitative assay of growth. Cells were seeded into 60-mm dishes, with the indicated growth supplements, and counted on day 5. Cells were dispersed by trypsinization and counted with a Coulter counter (model ZF). Viability was determined by the ethidium bromide-acridine orange method (24). Thymidine incorporation studies were done at 24, 48, and 72 h after plating. Cultures that were seeded at 5 x lo4 tells/60-mm dish (4 ml/dish) were pulsed for 3 h with 1 pCi/ml [3H]thymidine. Dishes were rinsed once in serum-free FlS/DME and then rinsed twice in 5% trichloroacetic acid. One milliliter of 0.1 N NaOH was added to each dish, and after 15 min

FIG. 1. Morphology of cells from primary lung explant of &day-old rat. Initial outgrowth of selected cell 12 days in culture in 11F (A, ~65); heterogeneous cell population after 12 days in culture in 11F + BPE selected cell type, 1 wk after addition of BPE on day 21 (C, X65); heterogeneous population after 12 days in 7F + BPE (D, ~65); fibroblast overgrowth when plated in FlP/DME + 10% fetal bovine serum, day 12 RL-65 clonal line in 7F + BPE (F, X 130). See text for definitions to abbreviations.

type after (B, X65); in culture (E, x65);

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FIG. 2. Growth of RL-65 in 7F + bovine pituitary extract (BPE) vs. 10% fetal bovine serum (FBS). Cells were plated at 5 x lo4 cells/21 cm2 and counted on day 5. Inset: thymidine incorporation 72 h after plating.

.

7F

IO4

0

FBS

7F + BPE

I

I

I

I

2

4

6

8

Days in Culture

7F

FIG. 3. Effect of FBS, BPE, and individual growth factors in 7F on growth of RL-65. Concentrations are given in Table 1. Each factor and BPE were omitted individually, and growth in remaining components was compared with growth in optimal medium (7F + BPE). Also compared: 10% FBS in presence and absence of 7F + BPE (&SE). Definitions as in Fig. 2 legend.

-TF +BPE

’ -Eth

’ -oEth ’ -Sel

’ -HC

‘-For&

-BPE

1

I

-

I

FBS

I

FBS +?F +BPE

+BPE +RA

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PROPERTIES

7F

7F +BPE

FGFa

FGFb

ECE3

Fbn

BSA

OF LUNG EPITHELIAL

Fbn +BSA

FIG. 4. Effect of fibroblast growth factor (FGF) acidic (FGFa, 5 ng/ ml), FGF basic (FGFb, 5 r&ml). endothelial cell growth sunnlement (ECGS, 100 pg/ml), fibronectin (Fbn 30 rg/21 cm*; and bovine serum albumin (BSA, 15 mg/ml).

Bpe 106

M

Fyi

TSJi

htH

hPRL

KTH

oxy

AMI

ahw

-r

T

f 2 = = IO5 c 104 RMSH

1 10

39 45

OLipotropm

88 91

Fragments

REndor

aEntir

aE!xbr

FG

B

BPe

0=7F

FIG. 5. Effect of pituitary growth factors on growth of RL-65. Each factor was added in presence of 7F and compared with 7F supplemented with bovine pituitary extract (BPE) or 7F alone (*SE). Growth hormone (GH), 1 rg/ml; follicle- and thyroid-stimulatory hormones (FSH and TSH), 10 rg/ml; human luteinizing hormone (hLH), human prolactin (hPRL), 5 rig/ml; adrenocorticotropic hormone (ACTH), o&otin (Oxv), vasonressin (ADH). (Y- and /3-melanocvte hormone (orMSH and PMSH), lb rig/ml, Plipotropin fragments, ,&cu,y-endorphin, 100 rig/ml, fibroblast growth factor (FGF), 5 rig/ml.

0.5 ml from each dish was transferred to a 7.5-ml minivial. To each vial, 3.5 ml of scintillation fluid and 200 ~1 of 40% acetic acid were added. Vials were counted for 1 min each in a Beckman 3800 counter. RESULTS

Growth and morphology of primary cultures. The initial cell population, which attaches and spreads after plating the dispersed lung tissue, is heterogeneous. By choosing very specific initial culture conditions, followed by a growth medium, we have been able to select against a large number of cell types in the lung as well as allow the survival of the particular cell type we designate as

CELLS

L419

RL-65 (Fig. lA). If serum is present initially, a heterogeneous culture is obtained and, with time, the culture is eventually overgrown by fibroblastic cell types (Fig. 1E). If BPE is present initially, even in the absence of serum, a fairly heterogeneous population of nonfibroblastic ceils is evident, and the population remains heterogeneous with time (Fig. 1B). The protocol described first uses a serum-free, defined medium (llF, see Table 1) to allow the survival, but not growth of the desired cell type, while not supporting the survival of the majority of cells in the original culture (Fig. 1A). The selection seems to occur both by providing an environment inadequate for the growth of some cells while actively inhibiting the survival/growth of others. This is followed by the addition of a critical mitogenic component (BPE) and the subsequent optimization of a medium for enhanced growth of the surviving cells (Fig. 1, C and F). If the optimized medium (7F and BPE) is used in the initial protocol, a diverse population of cells is supported, which may obscure or outstrip the growth of the desired cell type (Fig. 1D). Thus, in selecting for, and in the establishment of the RL-65 cell, the chronology of events and the timely addition/deletion of the proper supplements is critical. Continuous culture. Long-term cultures were established by serially passaging the cells at high density. This was done in 15% (vol:vol) conditioned medium, supplemented with fresh 11F and BPE. These cultures became progressively more homogeneous even before cloning and grew without fibronectin precoating of the dish. Optimal growth for the RL-65 clonal line was found to require only pins, hTF, Eth, PEth, Sel, HC, FK (7F), and BPE at the concentrations shown in Table 1. The remaining factors, progesterone, T3, bovine lipoprotein, and EGF, showed no further growth stimulation in the presence of the optimal 7F and BPE supplements and were omitted. RL-65 exhibited a doubling time of 17 h and had a 50to loo-fold increase in [3H]thymidine incorporation in this medium (Fig. 2). Neither 7F, in the absence of bovine pituitary extract, nor pituitary extract alone could stimulate cell division to the same extent as the combination. Each of the eight components used in the optimized medium have proved to be essential to achieving an optimal doubling time, BPE being the most crucial. Fetal bovine serum (10%) alone did not stimulate to optimal growth levels. This may be less due to inhibitory substances in serum than to a lack of essential growth factors, either absent in serum, or not provided at the optimal concentration. In the presence of those factors required for growth, serum was not growth limiting (Fig. 3). Bovine serum albumin, fibronectin, endothelial cell growth supplement, and FGF, all present in pituitary extract, could not alone, or in combination, account for the response to BPE (Fig. 4). Initial screening of all known and commercially available pituitary factors, at varying concentrations, alone and in combination, demonstrated no stimulation over control (Fig. 5). Although a crude preparation of FSH appeared to stimulate growth (Fig. 5), two different more highly purified preparations had no growth stimulatory activity. Nutrient and hormone interactions. Serum-free media was prepared by supplementing FlB/DME with a mixture of hormones, growth factors, and trace elements.

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FIG. 6. A: primary culture grown on a filter in llF-supplemented with bovine pituitary extract in presence of retinoic acid. Section has been cut perpendicular to filter. Cytological features of cells proximal to filter (bottom) and distal to filter are similar. Cells have prominent microvilli and occasional desmosomes. ~5,000. B: primary culture grown as in A but in absence of retinoic acid. Epidermal fibers are observed in epithelium. Apical cells are more flattened than in cultures with retinoic acid, desmosomes, and tonofilaments are more frequent and highly developed. ~5,000. C, D: phase-contrast micrograph in presence (C) and absence (D) of retinoic acid. x150.

FlB/DME, mixed l:l, was initially selected as the basal medium because of its widespread use in supporting the growth of a large variety of mammalian cells. Basal media formulations previously shown to be optimal for hamster and human tracheal epithelial cells and human bronchial epithelial cells were either inhibitory or not optimal for

the growth of RL-65. These included some of the MCDB media (MCDB 151, 152, 153, 301, and 302) originally developed in Ham’s lab for the growth of human keratinocytes (30) and modified by other labs for the growth of airway epithelial cells. Other cell types of the lung, such as the epithelial mink lung cell line, were unable to grow

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FIG. 7. A: with increasing time in retinoic acid-deficient medium epithelium becomes higher. Epidermal filaments increase in concentration, especially in apical cells. Granules similar to keratohyalin granules are observed, and cells most distal to filter (top) die just as in a keratinized squamous epithelium. ~3,000. H: section cut parallel to membrane reveals elaborate highly organized keratin filaments in absence of retinoic acid. Circular profiles are often observed when sections are cut precisely parallel to the membrane. ~3,500.

FI(:. 8. A: desmosomes and tonofilaments in cultures grown in absence of retinoic acid appear identical to desmosomes in normal skin. x62,000. B: granules in a primary culture are similar to keratohyalin granules of normal skin. ~23,000. C: swirls of epidermal filaments in a primary culture in 11F supplemented with bovine pituitary extract. There are epidermal filaments in absence of retinoic acid. ~43,000.

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PROPERTIES

FIG. 9. RL-65 cell line. Indirect immunofluorescent (C), tubulin (D), laminin (E), and fibronectin (F). x380.

OF

staining

LUNG

EPITHELIAL

of antibodies

or survive in the optimized RL-65 medium. The addition of 0.05 PM retinoic acid further increased cell number in the presence but not in the absence of BPE (Fig. 3). Ultrastructure. The most prominent features of the primary cultures grown in 11F supplemented with BPE are expanded endoplasmic reticulum (ER) and numerous microvilli. Additionally, there are many microfilaments, connected together by desmosomes on cellular processes. Cells grown in the presence of retinoic acid had a markedly different morphology exhibiting more densely packed colonies and a complete absence of cornification or keratinization (Fig. 6).

to keratin

CELLS

(A),

desmin

(B),

vimentin

In the presence of retinoic acid (0.05 PM), the epithelium resembles a low nonkeratinized transitional or squamous epithelium. As in a typical epithelium of this type, basal cells are cuboidal in shape and apical cells more squamous (Fig. 6A). The absence of retinoic acid in the culture medium totally alters the morphological appearance of the cultures. Cells grown on membrane inserts exhibit an epithelium that is very obviously keratinized. In cultures that have been grown long enough to be a few cells deep, the apical cells (distal to the membrane) become flattened, whereas the basal cells are cuboidal (Fig. 6B). Desmosomes are prominent as are numerous epidermal filaments. Filaments are observed throughout

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demonstrated in the RL-65 cells. Although this protease cleaved the substrate [leu-enkephalin (D-ala2)], the proteolytic activity was not inhibited by the enkephalinase inhibitor, thiorphan, and is therefore probably not enkephalinase. When compared with other cell types, such as mouse sertoli and leydig cells, rat capillary endothelial cells, and mouse kidney cells, dil-AC-low-density lipoprotein (LDL)-labeled RL-65 cells demonstrated that they can internalize and metabolize acetylated LDL at an accelerated rate (Fig. 10). DISCUSSION

PIG. 10. RL-65 cell line. Abundance rescence as determined by labeling with

X380.

of punctate fluorescent

perinuclear fluoprobe dil-ac-LDL.

the epithelium, even in the basal cells (Fig. 6B). When cultures in the absence of retinoic acid are allowed to grow longer, the epithelium becomes more stratified and so highly keratinized that the apical cells die (Fig. 7A). Sections cut parallel to the filter membrane reveal elaborate networks of epidermal filaments (Fig. 7B). Viewed at high magnification, the organelles of this highly differentiated, keratinized, stratified epithelium are seen to be highly developed. Desmosomes, indistinguishable from normal skin, are associated with typical tonofilaments (Fig. 8A). Large granules, similar to keratinophylan granules, are also observed in these cells, especially in cells near the apical surface of the epithelium (Figs. 7B and 8B). Epidermal filaments are particularly well developed in these cells. In sections cut parallel to the filter membrane, they appear in semiregular patterns of swirls and circles (Figs. 7B and 8C). Phenotype of RL-65. The primary and secondary lung cultures, as well as the established cell line RL-65, grown in the absence of retinoic acid, exhibit characteristics typical of epithelial cells. The cells were found to contain the cytoskeletal proteins, desmin, vimentin, and tubulin. Cells grown in the absence of retinoic acid stained more or less brightly for a-keratin, depending on their level of differentiation (Fig. 9). In addition, the cells reacted positively with antisera against the rat cell attachment proteins fibronectin and laminin (Fig. 9). Through the use of specific radioimmunoassays (RIAs) and bioassay, it was determined that the RL-65 cells also secrete transforming growth factor (TGFP) and insulinlike growth factor (IGFl). Preliminary experiments to ascertain which cyclooxygenase products are secreted by the RL-65 cells have shown that they produce prostaglandin E, as determined by RIA. Bombesin, the gastrinreleasing peptide found in small cell carcinoma of the lung (6), was not found in these cells. A membrane bound metalloprotease-like activity was

The distribution and frequency of 10 morphologically distinct cell types have been described in the surface epithelium of the rat intrapulmonary airways (8). Eight of these cell types are epithelial, differentiating into secretory cells, ciliated cells, and cells in which the main recognized function is to provide a large surface area for gas exchange. The maturation of the alveolar type 2 cell and the surfactant system is achieved prenatally, whereas maturation of the epithelium of the respiratory bronchioles and the small conducting airways occurs just after birth before restructuring of the lung parenchyma and alveolarization (days 4-13) (3, 21). The RL-65 cell line, derived from the lungs of 5-dayold rats, has phenotypic characteristics typical for cells of the airway epithelium. Studies are currently underway to further define the specific origin of this cell type. It may be a type of progenitor cell with the capacity to differentiate along several pathways, depending on alterations in the cell culture microenvironment. This cell type is not readily observed during the first week in culture but can easily be identified after 12-14 days. This may be due to a change in morphology after time in culture or its appearance may be facilitated by the death of most of the other cell types in the heterogeneous population. Alternatively, the late appearance of this cell type may be due to continued differentiation in vitro. Growth control and differentiation may be regulated by changes in culture conditions. The careful and timely addition/deletion of such components as BPE or retinoic acid, for example, may result in a culture condition in which cells either become committed to squamous differentiation and cornification or to further cell division. Although RL-65 cells have a phenotype distinct from that reported for other types of long-term cultures of lung cells, and other established lung cell lines, they do share reported characteristics of respiratory epithelial cells in culture. They exhibit numerous tonofilaments and desmosomes, and they are highly keratinized in the absence of retinoic acid. The RL-65 cell line grows optimally in the basal medium, FlB/DME plus the addition of insulin, transferrin, Eth, PEth, selenium, hydrocortisone, FK, and BPE. All of these components have been used in various combinations or concentrations to grow primary cultures of tracheal and bronchial epithelial cells from rabbit, hamster, and human tissue. Although it is not surprising that these various cell types along the airway require similar components for growth, the subtle differences in requirements for optimal growth and differentiation cannot be accounted for by species specific-

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L424

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ity alone. Differences in basal media formulations (i.e., Ca ‘+, amino acid concentration) in combination with differences in concentration and type of growth factor used have been shown to be responsible for the optimal growth or inhibition of a particular cell type. Moreover, in this report, we have shown that such differences can select for a particular cell type by differential regulation of growth. Bombesin, the gastrin-releasing peptide found in neuroendocrine cells of the lung, was not produced in the RL-65. This suggests that they are probably not of neuroendocrine origin. In addition, bombesin, found to be stimulatory for human bronchial epithelial cells (32), had no effect on growth of the RL-65. Retinoic acid, on the other hand, weakly stimulatory for the growth of tracheal epithelial cells, and necessary for mucociliary differentiation, is markedly stimulatory for the RL-65 cell line, as it is for human bronchial epithelial cells. EGF, a factor required for growth by both tracheal epithelial and bronchial epithelial cell cultures, and a powerful mitogen for a number of cell types, showed no growth stimulation in the RL-65. Lamellar inclusions, typical of the alveolar type II cell, were not observed in the RL-65, even at the early passages. Serum has been shown to be inhibitory for some primary cultures of normal human bronchial epithelial cells (14), normal rabbit and hamster tracheal epithelial cells (33,34), and human bronchogenic epidermoid carcinoma cells (23). Emura et al. (7) found serum to be stimulatory for cloning efficiency and colony size of a fetal respiratory epithelial cell line from hamster. Schumann et al. (27) have reported long-term cultivation of bovine tracheal epithelial cells in media containing 10% fetal bovine serum, and Thomassen and co-workers (29) demonstrated a similarity in population doubling time and colony-forming efficiency in serum-free and serum-containing primary cultures of rat tracheal epithelial cells. The RL-65 cell type is neither inhibited nor stimulated to optimal growth levels by serum. The proteolytic activity of the RL-65, as well as aldehyde dehydrogenase production, points toward a potentially important role in the detoxification of reactive compounds in the lung. This is further supported by the fact that this cell type has a large number of acetylated LDL receptors, perhaps for the purpose of scavenging and degrading extracellular molecules. It, therefore, may possess a “scavenger cell” pathway of LDL metabolism similar to that found in macrophages, endothelial cells, and microglia (10-12, 31). Preliminary data have indicated that the RL-65 are active secretory cells, as indicated by two-dimensional gel analysis of medium conditioned by the RL-65 (data not shown). Although little is known about secretory products of the airway epithelium, future identification and characterization of such components may further elucidate the neurohumoral regulation of secretion in these cells and the function of these products in lung cell metabolism. The role of vitamin A in the control of cell proliferation and differentiation has been well documented, both for cells of the tracheobronchial epithelium (4, 17-19, 28, 33, 35), as well as cells from other tissues (1, 4, 17). both at the Exneriments to date have demonstrated,

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CELLS

ultrastructural and light microscopy level, the striking inhibitory effect of retinoic acid on keratinization in the RL-65. Moreover, at concentrations varying from 0.01 to 0.5 PM, retinoic acid had a marked stimulatory effect on cell growth. Experiments are underway to further define the role of retinoids on the expression of the differentiated phenotype of the RL-65. BPE has been demonstrated to be stimulatory for growth in a number of culture systems (2, 8, 12, 24, 27), including the airway epithelium (29). Studies on the effect of pituitary extract on RL-65 have shown X0-fold increase in growth. We have tested the known and commercially available pituitary growth factors, none of which, by themselves or in combination, have demonstrated a growth effect equal to that of BPE. Moreover, because the extract is a homogenate of whole pituitaries, factors found in clotted blood serum, such as plateletderived growth factor, and the hematopoietic factors interleukin-1 and IL 2 were also tested and found not to have stimulatory activity. The growth-stimulatory activity by pituitary extract may thus be due to a novel mitogenic factor. The above data, taken together, suggest that the RL65 cells have the properties of airway epithelial cells. The lack of lamellar inclusions, bombesin production, and cilia (in the initial explant) suggest that it is not derived from a neuroendocrine, ciliated, or alveolar epithelial cell. The removal of the trachea before culturing the lung tissue precludes the cell being derived from the tracheal epithelium. The cell may be derived from the bronchial or bronchiolar epithelium or the epithelial cells present in &day rat lung that are the developmental precursors to these cell types. The properties reported here are distinct from, as well as similar to, properties previously described for primary and long-term cultures of airway epithelial cells. Further work on the properties of the RL-65 and other lung cell cultures should clarify this point. In a complex tissue such as the lung, each cell type, even within the same region, may require a different combination or concentration of nutrients, hormones, and/or growth factors. We have demonstrated that a carefully tailored serum-free environment can lead to the isolation and identification of a new type of cell from the lung that has not previously been established in vitro. This should provide the opportunity to isolate and characterize the unique gene products of these specific lung cells, to investigate cell-type specificity of physiology and gene control, and to study normal differentiated function of these cells in vitro. The authors are indebted to Dr. Cecilia M. Smith for insightful discussions and to Mary C. Tsao for comments and suggestions on pituitary extract in mammalian cell systems. Address for reprint requests: P. E. Roberts, Dept. of Cell Biology, Genentech, Inc., 460 Pt. San Bruno Blvd., South San Francisco, CA 94080. Received

5 April

1990; accepted

in final

form

5 June

1990.

REFERENCES T. R., S.E. SELONICK, AND S.J. of differentiation of the human promyelocytic

1. BREITMAN,

COLLINS.

leukemia

Induction cell line

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