Experimental and Molecular Pathology 71, 13–33 (2001)

doi:10.1006/exmp.2001.2376, available online at http://www.idealibrary.com on

TNF-a, PDGF, and TGF-b1 Expression by Primary Mouse Bronchiolar–AlveolarEpithelial and Mesenchymal Cells: TNF-a Induces TGF-b1

G. Sakuntala Warshamana, Miriam Corti, and Arnold R. Brody1

Lung Biology Program, Department of Pathology, Tulane University Health Sciences Center,New Orleans, Louisiana 70112

among the multitude of mediators that are released during lung cell

Received January 8, 2001

The bronchiolar–alveolar epithelium (BAE) is a primary target sitefor inhaled agents that cause lung injury. These cells, consequently,release a broad range of mediators that influence other cell populations,including interstitial lung fibroblasts that are central to the developmentof interstitial pulmonary fibrosis (IPF). A number of peptide growthfactors (GF) have been postulated to be essential in the pathogenesisof IPF. We demonstrate here that primary populations of mouse BAEand mesenchymal cells, maintained in culture, synthesize four potentGF. These are platelet-derived growth factor isoforms (PDGF) A andB, transforming growth factor beta-1 (TGF-b1), and tumor necrosisfactor alpha (TNF-a). A mouse lung epithelial cell isolation techniquepioneered in this laboratory has been used to purify the BAE cells togreater than 85% (80 6 5.6% alveolar type II and 9 6 2.3% Claracells) in culture. Northern analysis, RNase protection assay, and immu-nocytochemistry (ICC) were used to establish mRNA and proteinexpression of the GF over time in the cultured BAE and mesenchymalcells. We show for the first time in these primary mouse lung cellsthat treatment of both cell types with TNF-a upregulates expressionof TGF-b1. The four GF are produced by both epithelial and mesenchy-

mal cells but with different temporal patterns. TGF-b1 is expressedconstitutively by BAE and mesenchymal cells, whereas TNF-a expres-sion wanes over time. The findings by ICC were consistent with levelsof mRNA expression in both cell types. As genetically defined andaltered mouse strains are becoming increasingly valuable for modelinglung disease, studying the gene expression patterns of target cells fromthese animals in vitro would be useful in sorting out the complexresponses by individual cell types of the lung and the interactions

1To whom correspondence should be addressed at Department ofPathology, Tulane University Health Sciences Center, 1430 TulaneAvenue, SL-79, New Orleans, LA 70112-2699. Fax: (504) 588-5707E-mail: abrody@tulane.edu.

0014-4800/01 $35.00 13Copyright q 2001 by Academic PressAll rights of reproduction in any form reserved.

injury. q 2001 Academic Press

Key Words: alveolar type II cells; growth factors; RNase protectionassay; Northern analysis; immunocytochemistry.

INTRODUCTION

It has become clear over the past 2 decades that the lungepithelium lining conducting airways and alveolar surfacesactively produces numerous bioactive molecules (Zhang andPhan, 1996; Simon and Paine, 1995; Adler et al., 1994).Some examples of these are surfactant-associated proteins(Hermans and Bernard, 1999), reactive oxygen species (Bar-rett et al., 1999), arachidonic acid metabolites (Hunter etal., 1985), cytokines (Finkelstein et al., 1997), and peptidegrowth factors (Zhang et al., 1999; Morris and Brody, 1998;Brody et al., 1997). Similarly, pulmonary mesenchymal cellsrelease a complex array of chemical mediators (Morris and

Brody, 1998). There is little doubt that these agents, amongothers, are responsible for normal lung development andhomeostasis, as well as varied responses to lung injury.Investigators have a daunting task when attempting to sortout which of these potential mediators are essential for anypathobiological response. One approach to this problem has

animal lungs, thus providing an opportunity to correlateevents in vitro with pathogenic events associated with GF

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been to isolate the various lung cell populations and maintainthem in vitro, induce these cells to release a class of media-tors, and then draw inferences from these observations andfrom correlations in vivo. Mesenchymal and circulating cellpopulations are particularly amenable to this approach(Zhang and Phan, 1996; Lasky et al., 1995; Kumar et al.,1991; Bonner et al., 1991). Primary lung fibroblasts fromhumans and a variety of animal lungs have been maintainedover many passages in vitro and offer few technical chal-lenges for long-term study. In addition, airway epitheliumfrom animals and humans (Krunkosky et al., 2000) hasbeen maintained in long-term culture. Less conducive to thisapproach have been the primary epithelial cells lining thesmallest bronchioles and alveolar surfaces. Several investi-gators have isolated pure populations of bronchiolar epithe-lium from rabbit, which consists of Clara and cuboidal cili-ated cells. These cells remain active in culture and readilydifferentiate under the proper conditions (Brody et al., 1987).In vitro, Type I and II alveolar epithelial cells exhibit culturecharacteristics similar to those of the bronchiolar Clara cells.A number of investigators have purified and cultured TypeII cells from humans (Bingle and Tetley, 1990) and rats(Dobbs et al., 1997; Simon et al., 1993), and there are reportsof a modest degree of proliferation (Leslie et al., 1993). RatType I cells have been derived from Type II cells in culture(Vanderbilt and Dobbs, 1998). We had shown previouslythat viable murine alveolar type II cells could be isolatedto ,90% purity and cultured (Corti et al., 1996).

The goal of most of the studies referenced above is todetermine the roles played by the various cell types in lungdevelopment or response to injury through elaboration ofvarious mediators. We and others have postulated (Liu etal., 1996, 1997, 1998; Zhang and Phan, 1996) that peptidegrowth factors (GF) are the primary mediators of interstitialpulmonary fibrosis (IPF) (Daly et al., 1998). Again, thereare numerous such GF known to be synthesized and secretedby lung cells in vivo (Mills et al., 1999; Dai et al., 1998;Liu et al., 1997; Stadnyk, 1994; Blau et al., 1994). In vitro,production of growth factor protein in alveolar macrophagesor epithelial cells from rats or humans is shown in severalstudies (Shimizu et al., 2000; Cheng et al., 1999; McRitchie

et al., 1999). Thus, in order to establish a role for any givenpeptide, we must develop approaches that allow for themanipulation of GF expression with various substances thatinitiate lung cell inflammation and injury in controlled cellpopulations. This task has become more approachable sincegenetically altered and defined mouse strains are being em-ployed increasingly in experimental systems. Primary cul-tures of bronchiolar–alveolar epithelial and mesenchymal

WARSHAMANA ET AL.

cells from these animals would facilitate the study and ma-nipulation of GF expression by eliminating many complexi-ties.

In the work presented here, we show that a primary mouselung epithelial cell population, consisting of Type II alveolarcells (80%) and a few Clara cells (9%), expresses the mRNAand protein of tumor necrosis factor alpha (TNF-a), trans-forming growth factor beta 1 (TGF-b1), and the platelet-derived growth factor A and B isoforms (PDGF A and PDGFB). Primary lung fibroblasts from these animals produce thesame array of these factors, but with a different temporalpattern. This shows that in primary culture, the cells are ableto express the GF that have been identified in human and

bioactivity in vivo. Finally, we show for the first time thattreatment of primary mouse lung alveolar epithelial cellswith TNF-a upregulates expression of TGF-b1 and PDGFB, suggesting that TNF-a acts as a trigger or “master switch”(see Discussion) which activates other, more downstreamgrowth factors in IPF.

MATERIALS AND METHODS

Cell Isolation, Purification, and Culture

Bronchiolar–alveolar epithelial cells. Lung bronchio-lar–alveolar epithelial (BAE) cells from pathogen-free, 20-to 25-g male C57BL/6 mice (Charles River Laboratories,Wilmington, MA) were isolated according to Corti et al.(Corti et al., 1996; Corti and Brody, 1996). Briefly, the lungswere perfused with 15–20 ml of 0.9% NaCl injected throughthe pulmonary artery until the lungs were cleared of blood.Three milliliters of dispase (Collaborative Research, Inc.,Bedford, MA) was instilled into the lungs with a 3-ml syringeattached to a 20-gauge luer stub adapter (Becton–Dickinson,Sparks, MD) through the trachea. The syringe was thenremoved to dispel any excess dispase from the airways and0.45 ml of a solution of 1% low melting agarose (Sigma,

St. Louis, MO) in water was instilled into the lungs usinga 1-ml syringe through the luer stub adapter. This was donevery gently to avoid the agarose mixing with the dispase inthe distal lung. The agarose was then allowed to polymerizeby placing crushed ice over the lungs for 2 min. The lungswere removed to a sterile tube with 1 ml of dispase andincubated at room temperature for 45 min for enzymaticdigestion to occur.

GROWTH FACTOR EXPRESSION BY LUNG EPITHELIUM

Lung tissue was separated from the main airways andteased apart in 7 ml of Dulbecco’s modified Eagle’s medium(DMEM, Sigma) with 0.01% type II DNase I (Sigma) in a60-mm petri dish. The cell isolate was filtered successivelythrough nylon mesh filters (Falcon, Becton–Dickinson,Franklin Lakes, NJ, and Tetko, Inc., Lancaster, NY) of sizes100, 40, and finally 25 mM. The cell suspension thus obtainedwas centrifuged at 130g for 12 min at 48C to collect the cellpellet, which was resuspended in DMEM supplemented with10% fetal bovine serum (FBS, Sigma).

The cells were purified and cultured by a slight modifica-tion (Corti and Brody, 1996) of the method described inCorti et al. (Corti et al., 1996). Approximately 30 millioncells of the crude cell suspension in 10% FBS were platedon 100-mm tissue-culture-treated dishes coated with rat anti-mouse anti-CD 45 (42.5 mg) and rat anti-mouse anti-CD 32(16.5 mg) (both from Pharmingen, San Diego, CA) andincubated for 2 h in a humidified, 10% CO2 incubator at378C. The unattached cells were collected by panning thedish and the suspension was centrifuged as before. The cellpellet was resuspended in Ham’s F12 medium (Cellgro,Fisher Scientific, Pittsburgh, PA) supplemented with 15 mMHepes (Sigma), 0.8 mM CaCl2 (Fisher Scientific), 0.25%BSA, pH 7.4 (Sigma), 5 mg/ml insulin, 5 mg/ml transferrin,5 ng/ml sodium selinite (ITS, Boehringer Mannheim Bio-chemicals, Indianapolis, IN), and 2% mouse serum (Sigma).

The cells were plated and cultured on Type IV collagen(Sigma) coated 75-mm (18–20 3 106 cells) or 6.4-mm (2.53 105 cells) diameter, 0.4-mm-pore-size polyethylene orpolycarbonate transwell filters (Corning Costar, Cambridge,MA) or 8-well Permanox chamber slides (5 3 105 cells perwell) (Nunc, Inc., Naperville, IL) in the above medium ina humidified, 10% CO2 incubator. Four days after the cellswere plated, the medium was changed to serum-free definedmedium (SFDM, which was composed of Ham’s F12 me-dium supplemented with 15 mM Hepes, 0.8 mM CaCl2,0.25% BSA, pH 7.4, 5 mg/ml insulin, 5 mg/ml transferrin,and 5 ng/ml sodium selinite) and maintained in culture inthe same medium.

Mesenchymal cells. Mouse lung fibroblasts (MLF) wereisolated in a method similar to that described for the epithe-lial cells with the following modifications. After the lungs

were removed consequent to instillation of dispase and agar-ose, they were incubated at 378C for 45 min in 1 ml ofdispase in a sterile tube. The cells were teased apart asdescribed above, filtered only through a 100-mm filter toremove tissue debris, and plated on a petri dish in DMEMsupplemented with 10% FBS (Gibco BRL, Gaithersburg,MD) and incubated in a humidified, 5% CO2 incubator at378C for 30 min to remove as many of the macrophages

15

which attach to the plastic as possible. The supernatant wasthen removed to a tissue culture dish and incubated as beforefor 2 days. The unattached cells and debris were removedand the attached cells were rinsed twice in phosphate-buf-fered saline (PBS) and refed with DMEM supplementedwith 10% FBS. The cells were trypsinized and replated afterabout 5 days in culture. After passage one, the survivingcells in culture were composed mainly of fibroblasts andmyofibroblasts, characterized as such by immunocytochem-istry (ICC) (see Results). The cells were passaged as neededand maintained in DMEM supplemented with 10% FBS.All treatments were done with cells of passages two to four.Cells for treatment were seeded in 100-mm tissue culturedishes (for RNA analysis) and in 8-well chamber slides(for ICC).

Determination of Cell Viability, Cell Type, andAssessment of Cell Purity in Primary Epithelial CellIsolates and Cultures

Cell viability in the freshly isolated cells was determinedby trypan blue dye exclusion. Cells were counted using ahemocytometer. Purity (% alveolar type II (ATII) or Claracells) was evaluated by Pap stain (Dobbs, 1990) and nitroblue tetrazolium (NBT) staining (Walker et al., 1989) of air-dried cytospin cell smear preparations of freshly isolatedcells. Cell smears were prepared in 1 M cacodylate bufferon glass slides in a Shandon cytospin centrifuge by centrifug-ing at 600 rpm for 3 min. The percentage of granulated cells(AT II, in Pap-stained cell smears) or purple-colored cells(Clara cells, in NBT-stained cell smears) of a total 500 cellswas counted when assessing the cell type. Cultures wereprepared for scanning electron microscopy (SEM), osmiumtetroxide (OsO4)/tannic acid staining, and immunocyto-chemistry as described.

Scanning electron microscopy. Polyethylene transwellinsert filters (6.4 mm) containing epithelial cell cultures ofday 7 or 14 were prepared for SEM. Transwells were cooledat 48C for 20 min and rinsed in cold 1 M phosphate bufferand fixed in 2% glutaraldehyde (Electron Microscopy Sci-ences, Ft. Washington, PA) and 2% formalin (Sigma) inphosphate buffer for 1 h at 48C. The filters were then rinsed

in phosphate buffer twice to rinse off excess fixative anddehydrated through 25, 50, 70, 80, 95, and 100% cold etha-nol. The filters were removed from the plastic support usinga sharp blade and dried at the critical point in a Samdri-790 semi-automatic critical point drying apparatus (TousimisResearch Corp. Rockville, MD), according to the manufac-turer’s instructions. The filters were then mounted on carbonplanchets with carbon adhesive and sputter coated with gold

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in a SEM coating unit (Polaron Instruments) according tothe manufacturer’s instructions and observed under a JOELJSM35 scanning electron microscope.

Osmium tetroxide/tannic acid staining. Cell monolayersof culture day 3 epithelial cells in 8-well chamber slideswere stained with OsO4/tannic acid according to Mason etal. (Mason et al., 1985) to characterize the ATII nature ofthe cells in the initial cell monolayer.

Immunocytochemistry. Cytokeratin and vimentin immu-nocytochemistry (Franke et al., 1978) was performed tocharacterize the nature of the cell cultures and to monitorfibroblasts as the major contaminants in epithelial cell cul-tures after 6 or 7 days. Cell monolayers in 8-well chamberslides were rinsed in PBS and fixed with 95% ethanol for30 min. Epithelial cell cultures were immunostained forcytokeratin with anti-cytokeratin (cam 5.2, Becton–Dickinson Immunocytochemistry Systems, San Jose, CA)and vimentin with anti-vimentin (13.2, Sigma) using a linkedstreptavidin–biotin immunoperoxidase system, Dako ARK(Dako Corporation, Carpinteria, CA), according to the manu-facturer’s instructions. The cells were then counterstainedwith Lerner-3 hematoxylin (Lerner Laboratories, Pittsburgh,PA), dehydrated through an alcohol gradient, and mountedwith Permount (Fisher Scientific, Fair Lawn, NJ).

Determination of Cell Type in Primary LungFibroblast Cultures

Primary MLFs were stained for cytokeratin (with anti-cytokeratin, cam 5.2, Becton–Dickinson Immunocytochem-istry Systems), vimentin (with anti-vimentin, 13.2, Sigma),alpha-smooth muscle actin (Skalli et al., 1986) (with antialpha-smooth muscle actin, IA4, Sigma), and desmin (Lazar-ides, 1982) (with anti-desmin, D33, Dako Corp.) using thesame methods as above.

Treatment of Cell Monolayers

Day-4 epithelial cell cultures in 75-mm polycarbonateor 6.4-mm polyethylene transwell filters were quiesced inSFDM for 48 h and then exposed to either chrysotile asbestos

(2 mg/cm2) or latex beads (Sigma) in SFDM or left untreated(only SFDM). In another set of experiments, the quiescedcell monolayers were treated with TNF-a (0, 20, or 50 ng/ml) or with TGF-b1(5 ng/ml) (both from R&D Systems Inc.,Minneapolis, MN) in SFDM. The treatment duration was48 h.

MLF cultures in 100-mm tissue culture dishes at 80–90%confluence were quiesced in SFDM for 48 h and then treated

WARSHAMANA ET AL.

with TNF-a (0, 5, or 50 ng/ml) or TGF-b1 (5 ng/ml). Treatedand untreated cells were incubated at 378C for 4, 12, 24,and 48 h.

Analysis of Total Cell RNA

Total cell RNA was isolated from freshly isolated epithe-lial cells (day 0) and from cells in culture on days 7 and 14or from cells treated with asbestos, TNF-a or TGF-b1. TheRNA was quantitated with a spectrophotometric measure-ment and an equal amount of each sample was run on a 1%agarose gel to check the integrity of the RNA and to confirmthe spectrophotometric quantitation. The RNA was analyzedby either ribonuclease protection assay (RPA) or Northernanalysis (see below). Ribonucleoside 58-triphosphates anddeoxyribonucleoside 58triphosphates were purchased fromPharmacia Biotech, Inc. (Piscataway, NJ). All enzymes werepurchased from Promega (Madison, WI) or New EnglandBiolabs (Beverly, MA). [a32P] UTP was from ICN Pharma-ceuticals, Inc. (Costa Mesa, CA). All other chemicals usedin RNA analysis work were molecular biology grade andpurchased from Sigma Chemical Co. or Fisher Scientific(Pittsburgh, PA) unless otherwise noted.

Total cell RNA from 7- or 14-day cultures of MLFs orfrom MLFs treated with TNF-a or TGF-b1 was isolated,purified, quantitated, and analyzed as above.

Ribonuclease protection assay. RPAs were carried outas commonly described (Ausubel et al., 1994). A total of10–15 mg of total cell RNA was used to hybridize to a[a32P] UTP (3000 Ci/mmol) -labeled anti-sense RNA probeof PDGF A, TGF-b1, TNF-a, or GAPDH.

A 339-bp EcoRI/PstI fragment containing a mouse PDGFA cDNA fragment from plasmid pF9A5 (Mercola et al.,1990) was resubcloned into pGEM 1 vector (Promega). Invitro transcription of the linearized plasmid using SP6 RNApolymerase was used as the PDGF A riboprobe. A 233-bpSmaI/AlwNI fragment containing a murine TGF b1 cDNAfragment from plasmid pmTGFb1-1a (Derynck et al., 1986;Pelton et al., 1991) was subcloned into pBluescript KS1vector (Stratagene, La Jolla, CA) and in vitro transcriptionof the linearized plasmid using T3 RNA polymerase pro-vided the TGF-b1 riboprobe. The TNF-a riboprobe was

made by in vitro transcription of a custom template set(MCK-3, Pharmingen) containing a mouse TNF-a 287-bpcDNA fragment. For GAPDH riboprobe, linearized pTR1-GAPDH from Ambion, Inc. (Austin, TX), which containsa 154-bp cDNA fragment of mouse GAPDH, was used asthe template. All riboprobes were purified by separating thein vitro transcription reaction products on a 5% polyacryl-amide gel and eluting the correct sized transcripts from the

Lerner Laboratories).

GROWTH FACTOR EXPRESSION BY LUNG EPITHELIUM

polyacrylamide in a solution of 0.5% SDS in Tris–EDTA,pH 7.4. tRNA was used as a negative control in RPAs andtotal lung RNA from mice treated with LPS was used asthe positive control for all growth factors. The hybridizedfragments were digested with ribonuclease T1 (Calbiochem–Novabiochem Corp. La Jolla, CA) and separated on a 7 M,8% urea polyacrylamide gel. The bands were visualized byautoradiography and/or by exposure to a phosphorimagerplate. Levels of mRNA were quantitated using the instrumentsoftware of a phosphorimager (Fuji Medical Systems, Stan-ford, CT).

Northern analysis. Northern analysis was carried out aspreviously described (Sambrook et al., 1989). A total of15–30 mg of total cell RNA was electrophoresed on a 1%agarose, 2.2 M formaldehyde gel, transferred to a PVDFmembrane (Immobilon-N, Millipore Corp., Bedford, MA),and hybridized to [a32P] dCTP (3000 Ci/mmol) -labeledDNA probes of TGF-b1, PDGF B, pro-a 1 (I) collagen,GAPDH, or 36B4. A 974-bp TGF-b1 probe was made fromthe plasmid pmTGFb1-1a (Derynck et al., 1986; Pelton etal., 1991) containing the murine TGF-b1 cDNA. The 550-bp probe for PDGF B was made from a NotI/SalI digest ofa plasmid containing the rat PDGF B cDNA in pBluescript(Katayose et al., 1992). A 180-bp probe for GAPDH wasmade by a SacI/KpnI fragment of a plasmid, which containsthe rat GAPDH in pBluescript. A 740-bp probe for 36B4(human acidic ribosomal phosphoprotein, PO (Laborda,1991)) was made from a PstI digest of plasmid p36B4 (Masi-akowski et al., 1982). A 320-bp pro-a1(I) collagen probe wasmade from a BamHI/HindIII digest of plasmid pMCOL1a1-1(Metsaranta et al., 1991). The bands were visualized andquantitated as described under Ribonuclease ProtectionAssay.

Immunocytochemistry

On days 7 and 14, epithelial and mesenchymal cell mono-layers in 8-well chamber slides were rinsed in PBS and fixedwith 95% ethanol for 30 min. Cells were stained for PDGFA (with anti-PDGF A, H77), PDGF B (with anti-PDGF B,

H55), TNF-a (with anti/TNF-a H156), and TGF-b1 (withanti-TGF-b1, V) protein using rabbit anti-mouse polyclonalantibodies (all antibodies were from Santa Cruz Biotechnol-ogy, Santa Cruz, CA) with a labeled streptavidin–biotinimmunoperoxidase system, Dako LSAB (Dako Corp.) ac-cording to the manufacturers’ instructions. The color reac-tion was developed using 3-amino-9-ethylcarbazole as achromagen, which results in a red-colored precipitate at the

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antigen site. The cells were then counterstained with Lerner-3 hematoxylin and mounted with Aquamount (both from

Statistical Analysis

All values are expressed as means 6 SEM. A nonparamet-ric Student’s t test was used to compare band intensity valuesin RNA analysis experiments where n 5 3. Differences wereconsidered statistically significant when P , 0.05.

RESULTS

Cell Preparation and Characterization

Yields for the crude preparation of epithelial cells wereconsistent at (23 6 3) 3 106 (n 5 10) cells per mousewith a viability of 95 6 3% (n 5 5). The fibroblasts andmacrophages in the crude cell isolate attached rapidly toplastic surfaces, leaving an essentially pure population ofBAE cells. After purification, there were (6.3 6 1.3) 3 106

(n . 50) bronchiolar–alveolar epithelial cells per mousewith a viability of 93 6 4% (n 5 5). These cell prepscontained 80 6 5.6% (n 5 30) Type II cells (Fig. 1A),9 6 2.3% (n 5 12) Clara cells (Fig. 1B), and a few macro-phages or unidentified cells. The cells spread readily onType IV collagen-coated dishes (Figs. 1C–1F). The Type IIcells were identified for enumeration by Papanicolaou stain-ing of lamellar bodies (Fig. 1A). Nitro blue tetrazoliumshowed the numbers of Clara cells (Fig. 2B), and osmiumtetroxide staining revealed the lipid-containing granules ofthe great majority of the Type II cells soon after they hadadhered (Figs. 1C and 1D). Scanning electron microscopywas used to observe the epithelial cell surfaces over the 2-week period studied. Microvilli typical of Type II cells wereobserved (Fig. 1E), but this was not a predominant featureof the epithelial surfaces in 2-week-old cultures. At this 2-

week time, approximately 30% of the epithelium appeareddifferentiated into clusters composed of domed Clara cellsand cuboidal ciliated cells (Ci, Figs. 1E and 1F). After 2weeks, the majority of the epithelial surfaces were relativelyfeatureless, reminiscent of the Type I epithelium in vivo.

Anti-cytokeratin staining was used to assess the distribu-tion of epithelial cells throughout the cultures (Figs. 2A–2D). Since lamellar bodies are retained only in early cultures

18 WARSHAMANA ET AL.

FIG. 1. (A) Freshly isolated BAE cell smear subjected to Pap staining shows ATII cells with darkly stained cytoplasmic granules clearly visiblein 80% of the cells. Magnification bar 5 10 mM. (B) Freshly isolated BAE cell smear stained with nitro blue tetrazolium (NBT) to visualize Claracells which are identified by the presence of the diffuse purple stain. The isolates typically contained about 9% NBT-positive cells. Magnificationbar 5 10 mM. (C) OsO4/tannic acid staining of day-3 cultures of BAE cells show cytoplasmic inclusions characteristic of ATII cells. With thismethod granules were clearly visible in about 80% of the cell population. Magnification bar 5 50 mM. (D) OsO4/Tannic acid staining of day-3cultures of BAE cells at higher magnification. Magnification bar 5 10 mM. (E) Scanning electron micrograph of BAE cultures at day 7 showsthe surface microvilli typical of Type II cells (II). Newly differentiating ciliated cells (Ci) are also seen, as other cells that are flat and featurelessother than a few microvilli as seen on Type I epithelial cells (I). Magnification bar 5 10 mM. (F) Scanning electron micrograph of BAE culturesat day 14 shows ciliated and non-ciliated domed epithelial cells (*) much like that of the terminal bronchiolar epithelium.

GROWTH FACTOR EXPRESSION BY LUNG EPITHELIUM 19

FIG. 2. (A, B) BAE cell cultures at day 7 immunostained with anti-cytokeratin show the characteristic monolayer and staining pattern ofepithelial cells. Magnification bar 5 100 mM (A); 20 mM (B). (C, D) BAE cell cultures at day 14 immunostained with anti-cytokeratin continueto exhibit positive staining in a majority of the cells. Magnification bar 5 20 mM. (E, F). Anti-vimentin immunostaining was clearly positive forprimary MLFs on day 7. Magnification bar 5 50 mM (E); 20 mM (F). (G) BAE cells immunostained with IgG2a, negative control for anticytokeratin. Magnification bar 5 50 mM. (H) MLFs immunostained with IgM, negative control for anti vimentin. Magnification bar 5 50 mM.

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(Corti et al., 1996), expression of the cytoskeletal protein,cytokeratin, specific for epithelial cells (Franke et al., 1978)was assayed. The intense positive staining for cytokeratin(Figs. 2A–2D) and minimal anti-vimentin and anti-desminstaining (results not shown) are evidence that the culturescontinued to be predominantly epithelial through the 2-weektime period. A normal IgG blocked all background staining(Fig. 2G).

Anti-vimentin staining demonstrated the mesenchymalcell nature of the fibroblast cultures (Figs. 2E and 2F), andan appropriate IgM blocked this reaction (Fig. 2H). About30% of the cells were positive for a-smooth muscle actinand 5% were positive for desmin (results not shown), whichshowed the nature of the mesenchymal cell cultures to bea mixture of fibroblast and myofibroblast cells.

Time-Related Growth Factor mRNA Expression

Figure 3 demonstrates the results of an RNase protectionassay carried out on total cell RNA from the epithelial andfibroblast cultures at 1 and 2 weeks after initial plating. Forcomparison, total cell RNA from freshly isolated cells wastreated in an identical manner. This experiment was repeatedthree separate times, and the results shown here are typicalfor all lanes and at all times. The zero time point was forcells just isolated from lung and purified, but still in suspen-sion. These cells apparently were agitated by the treatmentnecessary to remove them from the lung, as demonstratedby high levels of gene expression for all peptides (Fig. 3).We have shown previously that expression of these genesin vivo in normal control rats and mice is very low to unde-tectable by analysis of whole lung mRNA and by in situhybridization (Liu et al., 1996, 1997, 1998). The levels ofgene expression were assessed by a semi-quantitative analy-sis in which the intensity of the various bands was normal-ized to the appropriate loading control.

In the experiments reported here, it is apparent that thegrowth factor mRNA for PDGF A, TNF-a, and TGF-b1 areexpressed in BAE cells as soon as the cells are removedand continues through the first 2 weeks studied. (Figs. 3Aand 3B). These data show that gene expression in BAE cellsis highest when the cells are freshly isolated, and the levels

of PDGF A and TNF-a tend to go down over time. TGF-b1 expression in BAE cells remained at a steady level (Fig.3B) through the 2-week time period studied. In MLFs, PDGFA, TNF-a, and TGF-b1 expression was reduced at 14 days(Figs. 3A and 3C). MLFs were maintained in medium thatwas serum free or that contained 10% FBS (results notshown). These two conditions produce no apparent differ-ences on expression levels of the three growth factors.

WARSHAMANA ET AL.

As we reported previously (Lasky et al., 1995), PDGF BmRNA and protein are difficult to detect as a product of ratlung fibroblasts. We show here that primary mouse lungfibroblasts follow this pattern. However, very low but clearlyvisible expression of PDGF B mRNA was observed afterlonger exposure of the hybridized membrane to film. Incontrast, BAE cells exhibit strong hybridization of mRNAfor PDGF B, which is most prominent at 1 week of culture(Figs. 4A and 4B).

Particle Treatment

Our previous studies showed that treating lung fibroblastswith asbestos induced increased production of PDGF B(Lasky et al., 1995). Here we show a similar but slightresponse by the primary BAE cells at the 7-day time period(Figs. 5A and 5B). Northern analysis of mRNA from un-treated, latex-treated, and asbestos-exposed cells revealedan increased level (,70%) of hybridization in the latter.

Treatment of MLFs and BAEs with TNF-a and TGF-b1

A series of experiments was carried out to determinewhether TNF-a and TGF-b1 treatment induced changes inexpression of the growth factors as well as pro-a1(I) col-lagen.

Mouse lung fibroblasts (Northern analysis of pro-a1(I)collagen and TGF-b1). Levels of expression by untreatedMLFs in SFDM served as a baseline control for comparisonof time and treatment effects. A total of 5 ng/ml of TNF-acaused a rapid (by 4 h) and sustained increase in TGF-b1

expression through the 48-h period studied (Figs. 6A and6B). A similar effect of the 5 ng/ml dose was seen for pro-a1(I) collagen expression (Figs. 6A and 6B). The 12-h timepoint exhibited the highest expression of both mRNAs. The50 ng/ml dose appeared to induce inconsistent increases inTGF-b1 over time, and there was a small effect on collagenexpression only at the 4-h time period. TGF-b1 treatmenthad little effect on its own expression at both 4 and 48 h(Figs. 6A and 6B). TGF-b1 induced collagen gene expressionat the early 4-h time period, but surprisingly was not apparent

at any later point in the experiment.

Bronchiolar–alveolar epithelial cells (Northern analysisof PDGF B and TGF-b1). As in the MLFs, TNF-a treat-ment at both 20 and 50 ng/ml induced a clear expression ofTGF-b1 (Figs. 7A and 7B). Earlier experiments using 5 ng/ml showed no effect over background. TNF-a also had aclear effect on PDGF B expression at the 50 ng/ml dose.TGF-b1 had a marked effect on increasing PDGF B (Figs.

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FIG. 3. (A) Comparison by RNase protection assay (RPA) of the eLane 0 represents RNA from epithelial cells immediately after isolatioalveolar epithelial cells; MLF, mouse lung fibroblasts. (B) Quantitationof growth factor mRNA level to that of GAPDH. The data representfrom day 0 value. (C) Quantitation of mRNA levels in MLF cells. Ththat of GAPDH. The data represent mean values 6 SEM (n 5 3). * D

7A and 7B) at the same 5 ng/ml dose that caused increasesin collagen at 4 h after treatment in MLFs (Figs. 6A and 6B).

Mouse lung fibroblasts and bronchiolar–alveolar epithe-lial cells (RNase protection assay for PDGF A and TNF-a). TNF-a and PDGF A are best detected by the moresensitive RNase protection assay since the levels of TNF-ain untreated cultures of cells are very low and Northern

ssion profile of PDGF A, TNF-a and TGF-b1 in BAE and MLF cells.nd purification. M, marker (positive control RNA). BAE, bronchiolar–mRNA levels in BAE cells. The bars are ratios of band intensity valuesn values 6 SEM (n 5 3) * Denotes statistically significant differencers are ratios of band intensity values of growth factor mRNA level totes statistically significant difference from day 7 value.

GROWTH FACTOR EXPRESSION BY LUNG EPITHELIUM 21

analysis probes for PDGF A give rise to unclear results dueto multiple hybridization bands of different intensities.

In MLFs the 5 ng/ml dose of TNF-a induced a sharpincrease in expression of PDGF A at the 12-h time period(Figs. 8A and 8B), similar to the pattern seen for collagenand TGF-b1 expression (Figs. 6A and 6B). The 50 ng/mldose caused little effect on PDGF A expression throughout

long exposures of autoradiogram). Day 0 represents RNA from cells

immediately after isolation and purification of BAE cells before plating.Presence of serum in the culture medium for MLFs did not make adifference in the expression of PDGF B. BAE, bronchiolar–alveolarepithelial cells; MLF, mouse lung fibroblasts. SFDM, serum-free de-fined medium. FBS, fetal bovine serum. (B) Quantitation of mRNAlevels in (A), in SFDM. The bars are ratios of band intensity valuesof growth factor mRNA level to that of 36B4. The data represent mean values 6 SEM (n 5 3). BAE, bronchiolar–alveolar epithelial cells;MLF, mouse lung fibroblasts.

the 48 h of treatment. TGF-b1 caused a rapid increase inthe level of PDGF A expression at 4 h but the effect hadwaned by 48 h after treatment (Figs. 8A and 8B). Interest-ingly, both the 5 and the 50 ng/ml doses of TNF-a causedclear upregulation of TNF-a itself, but the high concentration

FIG. 5. (A) Northern analysis of total cell RNA from 6-day BAEcultures in SFDM exposed to 2 mg/cm2 chrysotile asbestos or latexbeads for 24 h. SF, SFDM; lat., latex; asb., asbestos. (B) Quantitationof PDGF B mRNA levels in (A). The PDGF B mRNA levels areratioed to the 36B4 mRNA levels in the same lane and expressed asa percentage of the same value from the “untreated” (SF) lane. Thedata represent mean values 6 SEM (n 5 3).

22

FIG. 4. (A) Comparison by Northern analysis of the expressionprofile of PDGF B in BAE and MLF cells over time. MLFs expressedvery low levels of PDGF B at 7 and 14 days (observed only after

WARSHAMANA ET AL.

induced the increase by 4 h and the lower 5-ng dose upregu-lated a clear increase at 12 h after treatment (Figs. 8Aand 8B).

There was obvious expression of both PDGF A and TNF-a by the BAE cells after 8 days of culture (Figs. 9A and9B). However, treatment with TNF-a and TGF-b1 caused

GROWTH FACTOR EXPRESSION BY LUNG EPITHELIUM 23

FIG. 6. (A) Northern analysis of total cell RNA from MLFs after treatment with TNF-a or TGF-b1. Numbers underneath the lanes show theconcentration of treatment used in nanograms per milliliter. At all time points tested, both TGF-b1 and pro a-1(I) collagen gene expression wereupregulated by 5 ng/ml TNF-a (lanes 3, 6, 9, and 13). At 50 ng/ml (lanes 4, 7, 10, and 14), both transcripts were up only at 4 h after treatment,whereas at other time points only TGF-b1 is upregulated. TGF-b1 upregulates itself and pro a-1(I) collagen only at 4 h (lane 2). (B) Quantitationof mRNA levels in (A) after treatment with 5 ng/ml of TNF-a or 5 ng/ml of TGF-b1. The mRNA levels are ratioed to the 36B4 mRNA levelsin the same lane and expressed as a percentage of the same value from the corresponding “untreated” lane.

b1 mRNA expression remained high through the 2-weektime period, and this is reflected in the staining pattern which

FIG. 7. (A) Northern analysis of total cell RNA from BAE culturesafter 6 days in SFDM. The cells were treated with TNF-a (20 or 50ng/ml) or TGF-b1 (5 ng/ml) for 48 h. TNF-a treatment increased the

TGF-b1 levels (lanes 3 and 4) over that of the untreated cells (lane 1).TGF-b1 clearly increased the levels of PDGF B (lane 2) over theuntreated cells (lane 1). A dose of 50 ng/ml of TNF-a increased thelevel of PDGF B by about 80% (lane 4) over the untreated cells (lane1). (B) Quantitation of mRNA levels in (A). The mRNA levels areratioed to the 36B4 mRNA levels in the same lane and expressed asa percentage of the same value from the corresponding “untreated” lane.

little change in gene expression after 48 h of treatment. Adose of 5 ng/ml of TNF-a appeared to downregulate PDGFA expression while 50 ng/ml showed a slight upregulationof itself. TGF-b1 treatment caused a small increase in PDGFA expression.

WARSHAMANA ET AL.

Immunohistochemistry of Growth Factor Proteins

The BAE cells stained brilliantly for all of the growthfactor proteins at 7 days of culture, however, at 14 days,there was no visible TNF-a expression (Figs. 10A–10H).Even though no quantitation is possible, the intensity ofstaining generally followed the pattern observed for the cor-relative mRNA expression. For example, staining for PDGFA and B was more intense at day 7 than day 14 (Figs. 10Aand 10B), consistent with the mRNA expression in BAEcells at these time points (Figs. 3 and 4). In contrast, TGF-

24

remained intense (Figs. 10E and 10F). TNF-a staining wasmore intense at the earlier time point (Figs. 10G and 10H),although this would have been difficult to predict from thelevels of mRNA expression (Figs. 3A and 3B).

The MLFs stained intensely for all four growth factors at7 days and all but TNF-a at 14 days (Figs. 11A–11H).

DISCUSSION

There has been a recent focus on the respiratory epithe-lium, both airway (Mills et al., 1999; Dai et al., 1998;Stadnyk, 1994) and alveolar (Blau et al., 1994; Liu et al.,1996, 1997, 1998), as targets of lung injury and consequentproducers of potent mediators that contribute to disease de-velopment. This is an important concept because the epithe-lial barriers also provide potential targets for therapeuticintervention (Korfhagen et al., 1994; Sime et al., 1998;Huang et al., 1998; Bellocq et al., 1999). We and othershave postulated for over a decade that a broad array ofinteracting cytokines and growth factors are central to thepathogenesis of interstitial pulmonary fibrosis. Because thereare so many potential mediators, their individual functions,sources, and patterns of expression must be sorted out. Thus,in the studies presented here, we have begun a characteriza-tion of the expression of four peptide growth factors invitro, which we had shown previously are synthesized bythe bronchiolar–alveolar epithelium in vivo in rat and mouse

models of asbestos-induced lung injury and fibrogenesis (Liuet al., 1996, 1997, 1998; Perdue and Brody, 1994; Brass etal., 1999). We had shown that these factors are also producedby mesenchymal cells and macrophages in this model sys-tem. There is very little information available that wouldallow us to predict whether the primary bronchiolar–epithelial cells isolated from mice would continue to synthe-size the growth factors in vitro. It is clear that macrophages

-d i, 7PDne

FIG. 8. (A) RNase protection assay of total cell RNA from TNF-aNumbers underneath the lanes show the concentration of treatment useafter TNF-a treatment (5 ng/ml, lanes 6, 9, and 13; or 50 ng/ml, lanes 4a (lanes 4, 6, 9, 13, and 14). A dose of 5 ng/ml of TGF-b1 increasedThe mRNA levels are ratioed to the GAPDH mRNA levels in the same la

“untreated” lane. Maximum induction of both PDGF A and TNF-a were

from rats and mice readily synthesize and secrete growthfactors and cytokines in vitro. TNF-a production in primaryRat alveolar epithelial cells (McRitchie et al., 1999) andPDGF expression in human epithelial and fibroblast cell

and TGF-b1-treated MLF cells to analyze PDGF A and TNF-a levels.n nanograms per milliliter. TNF-a expression was noticeably increased, 10, and 14). PDGF A levels also increased in MLFs treated with TNF-GF A levels at 4 h (lane 2). (B) Quantitation of mRNA levels in (A).and expressed as a percentage of the same value from the corresponding

GROWTH FACTOR EXPRESSION BY LUNG EPITHELIUM 25

seen at 12 h after treatment with 5 ng/ml of TNF-a.

lines (Shimizu et al., 2000) using different approaches forgrowth factor stimulation have been reported. But at a timewhen a host of genetically altered and defined mouse strainsare being used in experimental systems, there is far less

a levels. Concentrations (in ng/ml) appear beneath the respective lanes.Both concentrations of TNF-a increased the TGF-b1 expression levels(lanes 2 and 3; also see Figs. 7A and 7B)). TNF-a (50 ng/ml) upregu-lated itself slightly after 48 h of treatment (lane 3). TGF-b1 (5 ng/ml)increased PDGF A levels in BAE cells after 48 h of treatment (lane4). Lane 1 shows positive control signals for the three growth factors.(B) Quantitation of mRNA levels for PDGF A and TNF-a in (A). ThemRNA levels are ratioed to the GAPDH mRNA levels in the same laneand expressed as a percentage of the same value from the corresponding“untreated” lane.

WARSHAMANA ET AL.

information on primary epithelial and mesenchymal cellsseparated from the lungs of mice. This is an important reasonwhy the data presented here include our experiments withboth primary epithelial and mesenchymal cells, and interest-ing comparisons can be made between growth factor expres-sion profiles of the two cell types, both in vitro and in vivoafter lung injury.

The four growth factors we have selected for study, PDGFA and B, TGF-b1, and TNF-a, all exhibit potent biologicalactivities and have been demonstrated in human and animallung tissue following injury (Lasky et al., 1995; Liu et al.,1996, 1998; Korfhagen et al., 1994; Sime et al., 1998; Huanget al., 1998; Bellocq et al., 1999). To our knowledge, thedata presented here are the first to show the expression ofthese factors by primary mouse lung epithelial and mesen-chymal cells in vitro. As genetically altered mice becomemore useful as experimental models (Sime et al., 1998;Bellocq et al., 1999; Korfhagen et al., 1994; Whitsett andKorfhagen, 1996; Hoyle et al., 1999), it will become increas-ingly important to isolate the individual cell types from thoseanimals to understand the genes and cognate proteins thatare expressed under various conditions. For example, weknow that the four factors remain at levels so low in normalrat and mouse lung that mRNA expression or protein produc-tion cannot be detected by Northern analysis of total cellRNA, in situ hybridization, or immunohistochemistry (Liuet al., 1996, 1997, 1998; Brass et al., 1999; Perdue andBrody, 1994).

Rat primary alveolar epithelial type II cell cultures havebeen shown to differentiate to a type I phenotype within48–72 h after plating (Dobbs et al., 1985). Such studieshave not been fully carried out with these primary mousetype II cells, but when they are cultured on a collagen sub-strate in 2% mouse serum, epithelial cell characteristics aremaintained (Corti and Brody, 1996) and the cells exhibithigh transepithelial resistances, a measure of the integrityof tight junctions, with an average maximum resistance of4.0 KV?cm2 on day 14 and greater than 2 KV?cm2 throughculture week 5 (Brody et al., 1997). These values are approx-imately 50% higher than those previously reported for mono-layers of rat alveolar type II cells (Cheek et al., 1989). Thisfinding and characterization of the cultures shown above are

26

A

FIG. 9. (A) Total cell RNA from TNF-a- and TGF-b1-treated BAEcells were subjected to RPA to analyze TGF-b1, PDGF A, and TNF-

consistent with the viability and epithelial nature of the cellswe are studying here.

Simply isolating the epithelial and mesenchymal cellsfrom the lung stimulates gene expression (Figs. 3 and 4).The four factors clearly were activated upon initial isolation,probably as a result of removal from the normal substrateby treatment with the various enzymes and media used incell preparation, although cells immediately after isolation

GROWTH FACTOR EXPRESSION BY LUNG EPITHELIUM 27

FIG. 10. Immunostaining of BAE cultures to visualize the expression of growth factor protein. Magnification bar 5 20 mM. (A, B) PDGF A,days 7 and 14; (C, D) PDGF B, days 7 and 14; (E, F) TGF-b1, days 7 and 14; (G, H) TNF-a, days 7 and 14. Staining was consistent with mRNAexpression except for TNF-a, which was clearly apparent by ICC at day 7. (See Figs. 11I and 11J for IgG background control).

28 WARSHAMANA ET AL.

FIG. 11. Immunostaining of MLF cultures to visualize the expression of growth factor protein. Magnification bar for A–H 5 50 mM. (A, B)PDGF A, days 7 and 14; (C, D) PDGF B, days 7 and 14; (E, F) TGF-b1, days 7 and 14; (G, H) TNF-a, days 7 and 14. Staining was consistentwith mRNA expression except for PDGF B, which was hardly detectable by Northern analysis with normal exposure times, but apparent by ICC.(I, J) Days 7 and 14 BAE cultures immunostained with IgG. Magnification bar 5 20 mM. (K, L) Days 7 and 14 MLF cultures immunostainedwith IgG. Magnification bar 5 50 mM.

GROW 9

—C

FIG. 11

and purification from the lung could most represent in vivocharacteristics. However, the different genes exhibited vari-ous responses over the 2-week period in culture compared

to the freshly isolated cells. PDGF A and TNF-a decreasedat the 2-week time, but TGF-b1 remained in an essentiallysteady state (Figs. 3 and 4). This is consistent with otherstudies showing that TGF-b typically is constitutively ex-pressed by a number of cell types (Zhang et al., 1997; Khalilet al., 1996; Zhang and Phan, 1996). In addition, proteinproduction as indicated by ICC generally followed the levelsof mRNA expression. A good example of this can be seen

ontinued

in the high levels of both mRNA and protein staining forTGF-b, but a lack of staining for TNF-a in both the fibro-blasts and the BAE cells at 2 weeks (Figs. 10 and 11),

TH FACTOR EXPRESSION BY LUNG EPITHELIUM 2

consistent with an apparent lack of mRNA expression (Figs.3 and 4). Information on release of growth factor proteininto the medium is currently incomplete (Brody et al., 1997)and will require additional data before this complex compo-nent of the story is known.

Thus, we now know that the four growth factors are ex-pressed in vitro, both mRNA and protein, apparently acti-vated by the original isolation procedure and continuously

2 weeks in primary culture, and study variations in geneexpression and correlated protein production. These investi-

This work was supported by NIH Grants RO1ES60766, RO1

30

stimulated by culture conditions through the 2-week period.Therefore, we then asked whether additional treatment withparticulates or the growth factors themselves would furtherstimulate mRNA expression. As indicated above, we hadshown previously that asbestos inhalation in rats and miceupregulated growth factor expression at sites of initial fiberdeposition and lung injury (Liu et al., 1996, 1997, 1998;Brass et al., 1999). Studies from our laboratory and fromBonner’s group also showed that primary lung fibroblastsexpress substantial amounts of PDGF A and its receptor,but little PDGF B, after treatment with asbestos (Lasky etal., 1995; Bonner et al., 1993, 1995) or with dexamethasone(Warshamana et al., 1998). In contrast, we show here thattreatment of the epithelial cells with asbestos (but not latex)upregulates PDGF B expression. This is consistent with ourearlier studies showing clear expression of PDGF B by thebronchiolar–alveolar epithelium in asbestos-exposed ratsand mice (Liu et al., 1997, 1998).

There are conflicting data as to whether TNF-a influencesexpression of TGF-b1. Several publications have indicatedno effect, whereas others show strong influences of TNF-aon TGF-b1 in a variety of cell types (Phillips et al., 1996;Pekary et al., 1995; Pang et al., 1994; Chao et al., 1995;Phan et al., 1992). Here we show that TNF-a clearly upregu-lates TGF-b1 expression in both primary mesenchymal andepithelial cells. This is an important concept because it isnecessary to explain how the various cytokine cascades areperpetuated and controlled during the development of dis-ease. If it can be shown that TNF-a is necessary to activatethe expression of TGF-b or other growth factors, potentialtargets for therapy might be at hand. Indeed, mice withboth receptors for TNF-a knocked out are resistant to thefibrogenic effects of asbestos (Liu et al., 1998), silica, andbleomycin (Ortiz et al., 1999). Most interesting is that thesemice exhibit reduced expression of TGF-b1 (Liu and Brody,2000) PDGF A, and TGF-a (Liu et al., 1998). This is consis-tent with the data shown here (Figs. 6–8), where TNF-aupregulates TGF-b1, PDGF A, and PDGF B. TGF-b1, inturn, can increase PDGF B levels in BAE cells (Fig. 7),which has been reported previously in a mouse fibroblast cellline (Leof, 1986). The intracellular signaling mechanismsthrough which inhaled particles such as asbestos (Robledo

et al., 2000; Mossman et al., 1997) and silica (Ortiz et al.,1999) influence the expression of other genes provide animportant research focus for a number of laboratories, in-cluding studies on cytokine-induced genes in lung epithe-lium (Schwiebert et al., 1997). Finally on this topic, it isabundantly clear that TGF-b1 is a potent inducer of extracel-lular matrix gene expression (Zhang et al., 1997; Khalil etal., 1996). However, there is little information on whether

WARSHAMANA ET AL.

TNF-a also upregulates these genes directly. While thisquestion is not answered here, TNF-a treatment of MLFsdid cause rapid (at 4 h) increases in pro-a1(I) collagenmRNA expression that increased over time and persistedthrough the 48-h period studied (Fig. 6). The question couldnot be answered with these experiments because TNF-aincreases TGF-b1 expression (Fig. 6), therefore not allowingthe source of collagen gene upregulation to be established,i.e., TNF-a upregulates collagen, or TNF-a upregulatesTGF-b1 which, in turn, is known to increase collagen levels.We are carrying out similar experiments on fibroblasts iso-lated from TGF-b1 knockout mouse embryos, and interest-ingly enough, preliminary findings show a direct effect ofTNF-a on collagen production by these primary cells.

In conclusion, we have demonstrated that it is feasible toisolate bronchiolar–alveolar epithelial and interstitial mesen-chymal cells from the lungs of mice, maintain them for

gations have allowed us to demonstrate that TNF-a upregu-lates expression of PDGF A, PDGF B, TGF-b1, and TNF-a. This could be a mechanism through which TNF-a influ-ences the expression of other growth factors in vivo andplays a potential role as a “master switch” in mediating thepathogenesis of interstitial fibrogenesis.

ACKNOWLEDGMENTS

HL60532, and NRSA HL09691-01. The authors thank the followinginvestigators for their generous gifts of the plasmids used to generateprobes for Northern analysis or RNase Protection assays: Dr. M. Mer-cola of Harvard Medical School (Boston, MA) for pF9A5, Dr. H. L.Moses of Vanderbilt University (Nashville, TN) for pmTGFb1-1a, andDr. E. Vuorio of The University of Turku, Finland, for pMCOL1a1-1. We also thank Ms. Odette Marquez for preparation of the manuscriptand Mr. Ellis Diaz for photographic services.

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