Spontaneous production of PDGF A-chain by rat lung fibroblasts in vitro

homodimer

JAMES P. FABISIAK, MARLENE ABSHER, JOHN N. EVANS, AND JASON KELLEY Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; Departments of Physiology and Biophysics and Medicine, University of Vermont School of Medicine, Burlington, Vermont 05405; Department of Medicine, Loyola University Medical Center, Maywood 60153; and Medicine Service, Department of Veterans Affairs Hospital, Hines, Illinois 60141 Fabisiak, James P., Marlene Absher, John N. Evans, and Jason Kelley. Spontaneous production of PDGF A-chain homodimer by rat lung fibroblasts in vitro. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L185-L193, 1992.-Plateletderived growth factor (PDGF) is considereda decisivemediator of fibroblast growth and phenotype within the lung. The cellular sourcesof PDGF within the lung remain undefined. The ability of lung fibroblasts themselvesto produce PDGF in vitro was therefore investigated. Northern and Western blot analysesrevealed the expression of PDGF-A mRNA and secretion of A-chain containing proteins by fibroblasts derived from adult and fetal rat lung. PDGF-A gene or protein expressionwere below the limits of detection in two human lung fibroblast lines examined in a similar manner. PDGF-B transcripts or proteins were not detected in any lung fibroblast line examined. Conditioned medium (CM) was collected from thesesamelung fibroblast linesand tested for its ability to promote cell growth using human fetal lung fibroblasts astargets. Both adult and fetal rat lung fibroblasts were found to produce a potent and efficacious stimulus for cell growth. Growth-promoting activity in rat fibroblast-derived CM functioned as a “competence” factor and was partially inhibited by anti-PDGF antibody. Thus rat lung fibroblasts in vitro produce potent growth factors of which at leastone appearsto be PDGF-AA. Differences in the expression of PDGF-AA between rat and human lung fibroblasts exist. Growth factor-producing fibroblasts may play a role in lung repair and remodelingthrough production PDGF-AA in vivo. platelet-derived growth factor A-chain; cytokines; lung cell proliferation; pulmonary fibrosis; rat lung fibroblasts; human lung fibroblasts; autocrine growth factors

of a number of cytokine proteins or their respective mRNAs have been documented in the lung where they are thought to modulate critical aspects of tissue growth and remodeling (see Ref. 23 for review). These include platelet-derived growth factor (PDGF) (14), transforming growth factor (TGF)-P (52), and interleukin (IL)-1 (3) among others. They also may play a role in directing homeostasis of the normal lung as several cytokine genes are expressed at low levels in control adult tissue (48). To date much of the effort to identify the source of pulmonary cytokines has been on immune effector cells such as lymphocytes and alveolar macrophages. There is growing recognition, however, that cells of mesenchymal origin can also serve as important producers of growth-regulating cytokines. Several cytokines .have now been shown to be produced by lung fibroblasts. These include among others IL-6 (12), insulin-like growth factor (IGF)-I (45), and granulocyte-macrophage colony stimulating factor (GM-CSF) (48). The abundance of interstitial fibroblasts relative to macrophages THE PRESENCE

1040-0605/92

$2.00

Copyright

in the lung coupled with their ability to produce growthregulating cytokines suggests that these structural lung cells cannot be viewed simply as passive target cells whose phenotypic behavior is solely determined by an independent population of immune effector cells. Originally recognized as the principal mitogen in serum for mesenchymal cells, PDGF is a cationic dimeric protein cytokine (~30 kDa) composed of two peptide chains that are covalently linked by multiple disulfide bonds (see Ref. 40 for review). The two-component peptides have distinct amino acid sequences and are the translational products of separate genes located on different chromosomes (2). The A-chain peptide and the B-chain can combine in any of three dimers to yield a biologically active molecule. Hence, active PDGF can have any of three chain compositions: A-A, B-B, and A-B (18, 40, 46). Human PDGF as found in serum and platelets originally thought to be PDGF-AB is now known to contain a mixture of all three isomers (17)~ Despite its name PDGF is now recognized to be produced by a variety of cells including macrophages (26, 43) and endothelial cells (11). The presence of these cells within the lung and their role in the response to lung injury have suggested that PDGF-like cytokines may play a role in lung repair and remodelling. Our laboratory has recently observed increased expression of PDGF-B mRNA in rat lung that precedes DNA synthesis during chronic hyperoxia (14). This result suggests that the production of PDGF-like cytokines by cells within the lung itself initiates or modulates various aspects of lung injury and repair. The studies presented here were designed to test the potential of lung interstitial fibroblasts to produce PDGF-like cytokines. The expression of PDGF mRNAs in rat and human lung fibroblasts were assessed by Northern blot analysis using cDNA probes specific for the A- and B-chain mRNAs of PDGF. Conditioned media obtained from cultured fibroblasts derived from human and rat lung were tested for their ability to stimulate cell proliferation using lung fibroblast targets. Growth factor activity detected in this assay was further characterized in a ‘standard PDGF bioassay using BALB/c-3T3 cells. Immunologic relationship of lung fibroblast-derived growth factor to PDGF was assessed by neutralization studies as well as Western blot analyses of proteins present in conditioned media using anti-PDGF antibodies. l PDGF-like human platelet

0 1992 the American

cytokines regardless Physiological

refers to PDGF of the dimeric Society

from sources composition.

other

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the L185

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L186

PDGF

PRODUCTION

BY LUNG

FIBROBLASTS

4 x lo3 cells/well and treated essentially according to the protocol describedabove. At the times indicated, cell monolayers Muterials. Human PDGF-AB purified by high-pressureliq- were fixed with 10% formaldehyde solution for at least 30 min uid chromatography from human platelets, IGF-I, and anti- and stained with 1% methylene blue in 0.01 M borate buffer for human PDGF-AB polyclonal immunoglobulin (Ig) G antibody 30 min. Excess stain was removed by repetitive rinses with were obtained from Collaborative Research(Lexington, MA). borate buffer. Cell-associateddye was then eluted in 0.3 ml of Polyclonal antibodies specific for AA and BB homodimeric ethanol-HCl (I:1 vol/vol). Optical density was read at a waveforms of human PDGF were from Genzyme (Boston, MA). Cell length of 658 nm in a microplate reader (Bio-Tek Instruments, culture mediawere obtained from GIBCO (Grand Island, NY). Winooski, VT). Cell number was related to optical absorbance Cell culture plasticware came from Corning. Recrystalized bo- (A6& using a standard curve vine serum albumin (BSA) (Pentex) was purchasedfrom ICN A 650 = 10A6[72.6 X cellno. + 251, r2 = 0.98 Immunobiologicals(Lisle, IL). Cell lines. Adult rat lung fibroblasts (RL-87) were isolatedin our laboratory by enzymatic dissociation of Fischer 344 rat This standardcurve waslinear in the rangeof l,OOO-8,000cells. Competence factor bioassay. The standard PDGF bioassay lungs. Tissue was obtained in a sterile fashion and processed essentially as describedby Waymouth (50) with the exception describedby Pledger and colleagues(37) with minor modificathat a combination of 0.1% (wt/vol) trypsin and 0.1% collage- tions wasusedto demonstratethe ability of lung fibroblast CM nase was employed for tissue disaggregation.After initial cell to act as a competencegrowth factor. This assayrequires denselectionby attachment to tissue culture plastic and prolifera- sity-inhibited quiescent BALB/c-3T3 (A31) cells to undergo tion in 10%fetal bovine serum(FBS), culturesbelowpopulation DNA synthesisand cell division in responseto added PDGF in doubling level 2 were frozen and stored in liquid nitrogen. the presenceof a progressionfactor such as IGF-I. The modiThrough N 20 population doublings these cells demonstrate fication of this assaydescribedby Gratzner (16) which involves a morphology, growth cycle, and aging profile typical of fibro- the replacement of [3H] thymidine with nonradioactive broblasts. They stain positively for actin and vimentin but nega- modeoxyuridine (BrdU) or fluorodeoxyuridine (FrdU) wasemployed. BALB/c-3T3 cellswere seededat 5 x lo4 cells/well on tively for keratin and are able to produce both types I and III Lab-Tek 8-chamber sterile glassculture slides (Nunc, Napercollagen. The BALB/c-3T3 (A31), rat fetal lung fibroblast (RFL-6), ville, IL). The cells were cultured in an incubator (37”C, 95% and human osteosarcoma(U-2 OS) cell lineswere obtained from air-5% CO,) in 0.4 ml Dulbecco’s modified Eagle’s medium American Type Culture Collection (Rockville, MD). Human (DMEM)-Ham’s F-12 (Fl2) (1:l) containing 10% FBS for fetal lung fibroblasts (IMR-90) and human adult lung fibro- 3 days without refeeding, at which time they were confluent. blasts (AG02603) came from the Coriell Institute for Medical Medium was removed, cells were washedtwice with sterile Research(Camden,NJ). All cell lines employedwere shownto PBS, and 0.4 ml of fresh DMEM-F12 containing 15 mM 2v-2be free of mycoplasma contamination using a DNA fluoro- hydroxyethylpiperazine-N’-2-ethanesulfonic acid (pH 7.4), anchrome test kit (Bionique Laboratories, Saranac Lake, NY). tibiotics as above, 0.45% BSA, IGF-I (20 rig/ml), and 100 ~1of For simplicity, the various fibroblast cultures tested are referred test substancewasadded.To allow immunohistochemicalquanto hereafter as HAL (human adult lung, AG02603), HFL (hu- titation of mitotic activity, BrdU and FrdU (IO and 1 PM, man fetal lung, IMR-90), RAL (rat adult lung, RL-87), and RFL respectively) wasaddedto be incorporated into DNA of dividing (rat fetal lung, RFL-6). cells.BALB/c-3T3 fibroblasts were cultured for 24 h, at the end To harvest conditioned medium (CM) from lung fibroblasts, of which the medium wasdecanted, the plastic chamberswere 7.5 x lo5 human lung fibroblasts (population doubling level removedfrom the slide,and the cell layer was rinsed with PBS. 14-25) or 3 x IO5 rat lung fibroblasts (population doubling Slideswere fixed with methanol containing 1.5% hydrogen perlevel 7-22) were seededin each of two T-75 flasks and allowed oxide for 30 min and rinsed three times with PBS. They were then incubated for 1 h with a mousemonoclonal antibody to to grow to confluence in a minimal essentialmedium (MEM) containing 10% FBS, penicillin (100 U/ml), and streptomycin BrdU-FrdU-containing DNA (Amersham, Arlington Heights, (100 pg/ml). After 6 days of culture in an incubator at 37°C IL) and rinsed with PBS three times, followed by a peroxidaseunder an atmosphere of 95% air-5% carbon dioxide the cell conjugated secondantibody raised against mouseIgG. Slides layer was washed with phosphate-buffered saline (PBS) and were reacted with diaminobenzidine with an enhancer for fresh MEM containing either 1% platelet-poor plasma (PPP) IO min and rinsed three times with water, after which color (49) or 0.1% BSA wasadded.After 48 h of further culture, CM allowed to develop. Nuclei staining positively for the BrdUwasharvested,filtered (pore size 0.45 pm), aliquoted, and stored FrdU complex were counted using a Quantex QX-7 imageanaat -20°C until use.Cell numberswere assessed in selectedcul- lyzer system (Sunnyvale, CA) connectedto a ZeissAxiovert 35 microscope at ~10 magnification. Eight hundred cells were tures by nuclei counts as describedbelow. counted in each well. IMR-90 growth assay. The growth of preconfluent IMR-90 target cells was employed to detect growth factors secretedby RNA isolation. RNA was isolated from cells exposedto esthe various lung fibroblast lines. For initial studies, IMR-90 sentially the sameconditions asthose during the production of cellswere seededin 24-wellplates (2 x lo* cells/well) in medium conditioned medium. Lung fibroblasts were seededin IOO-mm depletedof exogenousgrowth factors containing 1% PPP and round dishes(5.5 x lo5 for human lines, 2.2 x lo5 for rat lines) incubated for 2 or 3 days. At this time medium waschangedto in 30 ml MEM with 10% FBS. After 6 days the medium was MEM containing various dilutions of test CM with 1% PPP or decanted, the monolayer was washedwith PBS, and the mefresh MEM alone containing either 10% FBS or 1% PPP for dium was replaced with 18 ml of MEM containing either 1% maximal and minimal growth controls, respectively. At the PPP or 0.1% BSA. Cellswereincubated for 48 h, at which point times indicated cell number wasassessed by counting cell nuclei medium was removed and monolayerswere washedtwice with from washedmonolayersdirectly in a hemacytometer following PBS and then processedfor RNA isolation. RNA was isolated essentially as describedby Chomcyznski cell lysis in an appropriate volume of 0.1% citric acid-0.1% and Sacchi (6). After the last precipitation, the RNA pellet was gentian violet. Alternatively, a larger number of experimental points could redissolvedin IO mM tris (hydroxymethyl) aminomethane(pH be consideredusing a calorimetric assayto quantify increasesin 8.0)-l mM EDTA. RNA wasquantitated by measuringoptical cell number (33). IMR-90 cellswere seededin 96-well plates at density at 260 and 280 nm. RNA was stored at -20°C as a METHODS

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PDGF PRODUCTION

L187

BY LUNG FIBROBLASTS

precipitate after the addition of 0.1 vol of 3 M sodium acetate and 2.5 vol of ethanol. RNA isolated from the U-2 OS cell line served as positive control for PDGF-A and -B gene expression(2). U-2 OS cells (HTB 96, American Type Culture Collection) were maintained and processedfor RNA isolation as previously described(14). cDNA probes. The following cDNA probes were employed for analysisof geneexpressionby Northern blot hybridizations. A 1.3-kb human PDGF-A probe (clone Dl) inserted into the EcoR I site of pUC 13 wasthe generousgift of Dr. C. Betsholtz (2). PDGF-B mRNA was detected using a 1.2-kb v-si.scDNA probe (I-ssv-11 clone 1) (American Type Culture Collection) contained within the Pst I site of pBR 322 as originally describedby Robbinsand co-workers(39). The pA1 P-actin probe, kindly provided by D. Cleveland, representsa 2.0-kb sequence contained in the Pst I site of pBR 322 and is complementaryto nearly the entire /3-actin mRNA and recognizestranscripts for isoforms of actin (8). Probes were propagated, amplified, and isolated using standard techniques (30). PDGF-B and actin probeswere radiolabeledwith [cu-32P]dCTPby nick translation using a kit from Boehringer Mannheim (Indianapolis, IN). PDGF A-chain probe was labeled by random priming method with a kit from International Biotechnologies (New Haven, CT). Northern blot analysis. The electrophoresis, transfer, and hybridization protocols employed by our laboratory have previously been described in detail (14). Ethidium bromide (0.1 pg/ml) wasincluded in the 1% agarosegels(containing 1 M formaldehyde solution) to permit visualization of the 18s and 28s ribosomal subunits to assureequivalency of RNA loading and assessRNA integrity. Only gels containing equivalent amounts of intact RNA were subjected to further analysis. RNA was transferred to a charged nylon membrane (Nytran, Schleicher & Schuell, Keene, NH) electrophoretically with a TransBlot Cell apparatus (Bio-Rad, Richmond, CA). The blot was then prehybridized and hybridized in a plastic bag (Omniblot System, American Bionetics, Hayward, CA) according to the protocol previously described. After hybridization blots were washedunder high-stringency conditions and then autoradiographedfor periods of time indicated. Western blot analyses. For direct analysisof specific proteins exported by lung fibroblasts, CM was collected from confluent cells under serum-freeconditions (MEM with 0.1% BSA) and stored frozen (-70°C) until use. Samplesof CM were dialyzed exhaustively against 1 M acetic acid and lyophilized to dryness by centrifugation under vacuum. Sampleswere resuspendedin Ha0 to yield a 7.5-fold concentration above original material. Twenty microliters were electrophoresedby sodiumdodecyl sulfate-polyacrylamide gel as described by Laemmli (27) under nonreducing conditions with the exclusion of /3-mercaptoethanol. Proteins were transferred to nitrocellulose (0.2 pm, Schleicher & Schuell) using a Profile (Schleicher & Schuell) semidry electrophoretic transfer apparatus. Membranes were blocked by overnight incubation in buffered 1% BSA and 3% gelatin. The membranewas then incubated with specified antibody for another 24 h. Antibody-antigen complexeswere then detected using a preformed biotinylated goat anti-rabbit IgGstreptavidin-alkaline phosphataseImmunoSelect kit (GIBCOBRL, Gaithersburg, MD) according to the manufacturer’s instructions. RESULTS The expression of PDGF genes were measured in both rat and human lung fibroblasts. Figure 1 shows a Northern blot hybridization of PDGF-A (A) and PDGF-B (B) cDNA probes to RNA derived from RAL and HFL. PDGF-A mRNA was detected in RAL cells primarily as

2 a

ml a a

4.0 kb-

Fig. 1. Expression of platelet-derived growth factor (PDGF)-A and -B mRNA by rat adult lung (RAL) and human fetal lung (HFL) fibroblasts. Northern blot hybridization to 50 pg total cellular RNA was performed as described in METHODS. RNA was isolated from RAL and HFL fibroblasts immediately after collection of conditioned media. RNA derived from U-2 OS human osteosarcoma cells (U2-OS) is included as a positive control for PDGF gene expression. Identical blots were hybridized with either PDGF-A (A) or PDGF-B (B) cDNA probes radiolabeled with [32P]dCTP (3 X lo6 cpm/ml), washed under high-stringency conditions, and exposed to X-ray film for 14 days.

a 1.9-kb transcript. This transcript size represents the minor form expressed in human U-2 OS cells, which express 2.8- and 2.3-kb mRNAs predominantly. We have previously noted that the 1.9-kb transcript represents the primary PDGF-A mRNA expressed within rat whole lung tissue (13). Other workers (5,44) using cells or tissue from rat also have observed species differences in sizes of the

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L188

PDGF PRODUCTION

BY LUNG FIBROBLASTS

predominant A-chain transcript. PDGF-A mRNA was below the limits of detection in fibroblasts derived from human fetal lung. Neither fibroblast cell line expressed B-chain transcripts as measurable by Northern blot analysis. The expression of cytoplasmic ,f3-and y-actin genes (2,l kb) were easily detected in both RAL and HFL RNA (Fig. 2A), indicating that failure to detect PDGF gene expression was not due to RNA degradation. Interestingly, the RAL fibroblasts also expressed a significant amount of muscle-specific a-actin mRNA (1.7 kb). Faint expression of cu-actin mRNA can also sometimes be detected in human lung fibroblasts; however, its level is always quantitively less than observed in rat lung fibroblasts. Ethidium bromide stained gels before transfer are shown in Fig. 2B to verify equal sample loading for these experiments. PDGF-A mRNA present in growth factor producing cells appears to be translationally active and ultimately leads to the appearance of mature PDGF A-chaincontaining peptides in their CM. Figure 3 represents a Western blot of proteins contained in CM collected from the four lung fibroblast cell lines examined in this study and probed with an antibody specific for PDGF-AA. Significant immunoreactivity of a peptide (-30 kDa)

A

J’

LL a I a

was observed in both RAL and RFL fibroblasts but not in those derived from human lung (HFL and HAL). The identity of this peptide as PDGF-AA is substantiated by its comigration with a similar protein present in CM of PDGF-AA producing osteosarcoma cells as well as, 10 ng purified recombinant human PDGF-AA. Similar blots using anti-PDGF-BB antibody failed to detect any immunoreactive material in any of the fibroblast lines tested (data not shown). The presence of growth factor bioactivity was then examined in media conditioned by rat and human lung fibroblasts. CM collected from RAL fibroblasts in the presence of 1% PPP for 48 h was capable of stimulating cell proliferation when placed on IMR-90 target fibroblasts (Fig. 4). As a positive control, IMR-90 cells showed a vigorous growth response in the presence of 10% FBS with a >lO-fold increase in cell number over the &day period tested. Minimal cell growth was observed with fresh medium containing 1% PPP but not conditioned by rat lung fibroblasts. Plasma prepared in this way is depleted of serum-derived competence growth factors, most notably, PDGF (49). Undiluted CM from RAL fibroblasts with 1% PPP, however, increased cell number to 80% of that observed with 10% FBS. This represents a greater

B

28s

2.1 kb-

18s

Fig. 2. Expression of actin mRNAs by RAL and HFL fibroblasts. A: actin cDNA hybridization to 5 pg RNA from the same samples in Fig. 1 after a 3-day autoradiographic exposure. RNA integrity in these samples was verified by the expression of P-actin mRNA and visualization of intact ribosomal subunits stained with ethidium bromide (B: H, HFL; R, RFL; 50 and 5 &lane) before transfer. Note expression of cu-actin mRNA (1.7 kb) in ratlung fibroblasts which also exnress PDGF-A transcripts in Fig. 1. -

1.7 kb -

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PDGF PRODUCTION

AA

> /I’

29.L 18.L

L189

BY LUNG FIBROBLASTS

-30-33

kD-

’

Fig. 3. Western blot analysis of PDGF-A peptides present in conditioned media (CM) from various lung fibroblast cell lines. CM was obtained under serum-free conditions, dialyzed, and concentrated as described in METHODS. Reconstituted CM material (20 ~1) from the cell lines indicated was electrophoresed under nonreducing conditions by sodium dodecyl sulfate-polyacrylamide gel (13% acrylamide) and transferred to nitrocellulose. Blot was incubated overnight with 1:lOO dilution of anti-PDGF-A antibody and further developed as described in METHODS. Individual lanes are as follows: lane 1, U-2 OS (OS); he 2, HFL; he 3, human adult lung (HAL); he 4, rat fetal lung (RFL); lane 5, RAL. Lane 6 represents 10 ng of recombinant human PDGF-AA. Position of prestained molecular mass standards is shown for reference. Immunoreactive PDGF-A is present in CM from both rat lung fibroblast cell lines but not in those derived from human lung. These results corroborate results obtained by bioassay (Figs. 4-6).

I RAL

DAYS IN CULTURE

Fig. 4. Effect of adult rat lung fibroblast-conditioned medium on growth of IMR-90 target lung fibroblasts. Confluent RAL cells were cultured for 48 h in medium containing 1% platelet-poor plasma (PPP). CM was harvested and applied to IMR-90 target fibroblasts freshly plated in 24-well plates as described in METHODS. Data represent means of quadruplicate determinations of cell number by nuclei counts determined at each of indicated time points. SEs are omitted for sake of clarity but in most cases represent ~15% of mean value. Maximal growth supported by 10% fetal calf serum (FCS) is shown for reference. All dilutions of CM tested (undiluted, 50% CM, 20% CM, and 10% CM) yielded more rapid growth than 1% PPP.

than fivefold increase in cell number above media with 1% PPP alone at day 3. Dilutions of CM with fresh media containing 1% PPP showed that growth-promoting activity observed in RAL CM was dose dependent; significant stimulation was still observed with as little as 10% CM. Cell number changes in response to CM were observed to be maximal at 3 days; hence, this single time point was adopted for further examination of CM growthpromoting activity. The ability of lung fibroblast cell lines derived from several other sources to produce mitogenic activity is shown in Fig. 5. In this experiment, CM collected from RAL fibroblasts showed a titratable stimulation of

RFL

HAL

“FL

CM SOURCE

Fig. 5. Variable production of growth factor activity by lung fibroblasts cultured from 4 different sources. Sources of conditioned media were as follows: HAL, HFL, RAL, and RFL fibroblasts. Confluent cultures of each cell line were allowed to condition media containing 1% PPP for 48 h. Media was collected and applied in various dilutions indicated to IMR-90 target fibroblasts freshly-plated in 96-well plates. Cell numbers were determined 3 days later using methylene blue binding assay described in METHODS. Increases in cell number above those receiving 1% PPP alone (mean 2.5 x lo3 cells/well) are normalized to the maximal response observed to 10% FCS (8.54 X lo3 cells/well). Data represent means + SE determined from quadruplicate samples for each point. * Statistically significant difference from 1% PPP alone (P < 0.01) by l-way analysis of variance and Dunnett’s multiple comparison to control. Note dose-dependent growth-promoting activity present in CM derived from RAL and RFL.

IMR-90 growth after 3 days of exposure similar to that observed in Fig. 4. In addition, fibroblasts derived from RFL likewise exhibited a growth-promoting activity in CM. In contrast, CM obtained from HFL failed to produce any stimulation of IMR-90 growth above 1% PPP alone at any of the dilutions tested. Fibroblasts derived from HAL also failed to produce growth factor activity observed in those of rat origin. Analysis of growth curves at longer and shorter times also failed to reveal a stimulatory effect of human fibroblast CM. A small dose-unrelated stimulation observed at 50% HAL CM probably

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Ll90

PDGF

PRODUCTION

BY LUNG

represents random variation or enhanced cell attachment at earlier time points. Thus the demonstration of growth factor bioactivity is correlated with both PDGF-A gene expression and the presence of PDGF-A immunoreactive proteins detected in CM. We do not know the stability of the growth factorproducing phenotype over time in culture. We have, however, performed similar experiments on cells of different culture ages and found that RFL cells express growth factor activity at later culture ages (PDL 22) and HAL cells tested as early as PDL 14 do not. Essentially identical results were obtained when CM was collected under serum-free conditions (MEM with 0.1% BSA) and tested after the addition of fresh 1% PPP (data not shown). Both RAL and RFL produced a growth-promoting substance, but neither comparable human lung fibroblast line did under these conditions. These data indicate that growth factor activity present in CM is not due to the ability of RAL or RFL fibroblasts to convert an inactive cytokine precursor molecule present in PPP into an active form. Therefore the most likely source of growth-stimulating activity manifest in rat lung fibroblast CM represents a cytokine produced and exported by these fibroblasts themselves. Several approaches were used to further characterize the nature of the mitogenic activity produced by rat lung fibroblasts and determine its identity as a PDGF-like cytokine. These included assessing the presence of growth factors possessing competence activity and determining the susceptibility of fibroblast-derived growth factors to inhibition by anti-PDGF antibodies. When CM obtained from each of the four lung fibroblast cell lines was tested in the competence factor bioassay described by Pledger et al. (37), similar results were obtained to those of the IMR-90 growth assay. Figure 6 shows that both RFL and RAL secrete a competence factor that replaces the need for PDGF to stimulate DNA synthesis in BALB/c-3T3 cells in the presence of IGF-I (20 rig/ml). Omission of IGF-I blocked the growth-promoting activity of RFL and RAL CM in this study (data not shown). There was a good correlation between the fibroblast CM content of growth-promoting activity for IMR-90 fibroblasts and the ability to provide a competence factor for BALB/c3T3 DNA synthesis. In addition, at least a portion of the growth stimulation of IMR-90 cells by RAL fibroblast CM appears to involve the action of PDGF-like cytokines. The effect of inclusion of polyclonal anti-human PDGF antibody on 3day IMR-90 growth in response to RAL CM was examined. CM (1:3 dilution) was applied to IMR-90 targets in the absence or presence of several concentrations of antiPDGF-AB antibody for 3 days, at which time cell number was determined. Two typical experiments shown in Table 1 demonstrate that inclusion of anti-PDGF antibody (200 pg/ml) significantly reduced RAL CM stimulation of growth by up to 51 t 7% (expt 1) and 43 t 7% (elcpt 2) (higher antibody concentrations not tested). This effect was not observed with inclusion of similar concentrations of nonimmune rabbit IgG.

FIBROBLASTS

-1%

0

PPP

RAL RFL HAL HFL

Fig. 6. Ability of conditioned media from various lung fibroblast cell lines to function as a competence factor for BALBc/3T3 cells. Samples of fibroblast CM were applied to confluent BALBc/3T3 cells in presence of excess IGF-I (20 rig/ml) and DNA synthesis measured as described in METHODS. At least 800 cells/well were scored as either positive or negative based on immunoperoxidase staining for bromodeoxyuridine (BrdU) incorporation into nuclei. Number of positive cells with unconditioned medium without 1% PPP alone is denoted by arrow. Data represent a single representative experiment and are expressed as means * Significant difference compared t SD of triplicate determinations. with fresh MEM with 1% PPP alone by Student’s t test (P < 0.05%). Note that only CM samples from RAL and RFL contain measurable competence factor activity, which parallels growth-promoting effects on IMR-90 target fibroblasts (Fig. 5).

Table 1. Effect of anti-PDGF antibody on growth factor activity in RL-87 CM Anti-PDGF Antibody, dml

% of 3-Day Expt

None 0.3 3 30 200

loot9 109t6 106t5 122&8 49t7’

1

CM

Response Expt

2

lOOt7 132212 113kl2 103&E 5727”

Values are means t SE. PDGF, platelet-derived growth factor; CM, different from CM alone (P < 0.01) conditioned media. * Significantly by l-way analysis of variance and Dunnett’s multiple comparison to control. DISCUSSION

The proliferation and activation of mesenchymal cell populations undoubtedly plays a key role in normal tissue repair after injury as well as the development of various lung pathologies. Increased proliferative potential of interstitial fibroblasts as well as their collagen-synthesizing capacity produce the thickening of the alveolar wall and rearrangement of lung architecture seen in idiopathic pulmonary fibrosis (15). Similar changes have also been observed after a variety of lung injuries and have been extensively described in such experimental models as exposure of animals to bleomycin (25), hyperoxia (9), and hypoxia (32). The regulation of cell growth and phenotype during tissue remodeling is a complex process mediated through a variety of positive and negative controls. The central role of soluble polypeptide growth factors in the initiation of cell division and modulation of cell phenotype has

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PDGF

PRODUCTION

BY LUNG

become increasingly appreciated. PDGF represents one of the most important cytokines for cells of mesenchymal origin. The early cell cycle transitions induced by PDGF are a prerequisite for the mitogenic actions of other cytokines termed progression factors, such as epidermal growth factor and IGF. In addition, PDGF shows chemotactic activity for a variety of cells involved in tissue inflammation and repair including fibroblasts (42) and leukocytes (10). Because of their proliferative activity in response to injury, fibroblasts have historically been thought of primarily as targets of PDGF produced by various immune effector cells such as alveolar macrophages. Rat and human activated alveolar macrophages have been shown to elaborate PDGF-like cytokines in vitro (26, 43). In addition, alveolar macrophages obtained from patients with idiopathic fibrosis appear to release more PDGF-like cytokines than those from normal subjects (31). Several studies, however, have begun to indicate that fibroblasts themselves may participate in these responses as active effector cells capable of directing cell responses via cytokine elaboration. It has been known for some time that human diploid skin fibroblasts in vitro can produce endogenous IGF-I and thus grow in the presence of a competence factor alone (7). IGF-I production has also recently been described for human lung fibroblasts (45). A partial list of other cytokines produced by lung fibroblasts include IL-6 (12), GM-CSF (48), and an epithelialspecific growth factor (41). This is the first study to demonstrate constitutive basal expression of PDGF-like cytokines by normal lung fibroblasts in vitro. A skin fibroblast cell line derived from a patient with Hutchinson-Gilford premature aging syndrome (progeria) was found to constitutively express translationally active PDGF A-chain genes; however, this was not observed in other progeria cell lines or those from normal human subjects (51). Several cytokines appear to be able to induce the transient expression of PDGF-A genes in mesenchymal cells. These include IL-1 (38), tumor necrosis factor (34), and PDGF itself (13,35). It has been postulated that autocrine elaboration of PDGF-like cytokines by these agents may contribute to their mitogenie activity. It is noteworthy that anti-PDGF antibodies failed to completely neutralize the growth factor activity observed in RAL CM; thus it is difficult to precisely assess the contribution of PDGF-AA to the overall bioactivity observed in CM. One possible explanation is that PDGF may be released in complex with carrier molecules such as cxz-macroglobulin (4, 19). Such complexes have been shown to possess biological activity but lack the immunoreactivity of free PDGF (19). Acidification could release this protein-bound PDGF and thus allow its detection on our Western blots. The bioactivity of PDGF in acidified CM, however, would be very difficult to assess due to the activation of latent TGF-P also produced by rat lung fibroblasts (24). Furthermore, the precise neutralization activity of these anti-human PDGF antibodies against their rat homologues is unknown. The situation is further complicated by the fact that in our hands IgGs

FIBROBLASTS

L191

themselves sometimes have been observed to promote growth to a small extent. In addition, the presence of other growth factors in addition to PDGF may also be produced by these fibroblasts. These may include basic fibroblast growth factor, TGF-P, or other, as yet unidentified, growth-promoting substances. The PDGF produced by rat fibroblasts is likely to be of biological importance. PDGF is an exceedingly potent mitogen. If we assume the signal intensity of RFL and RAL CM on our Western blots is lo-fold less than our lo-ng PDGF-AA standard, we approximate a PDGF-AA concentration of 5 rig/ml, which is well within the active range of this cytokine. In addition, small amounts of PDGF-AA that are not directly mitogenic significantly potentiate the response of other mitogens on smooth muscle cells (28). It remains to be shown that rat lung fibroblasts produce PDGF-like cytokines in vivo. Our laboratory has observed that PDGF-B mRNA is expressed in whole rat lung tissue and appears to be upregulated during hyperoxic lung injury (14). We have also recently detected the expression of PDGF-A mRNA in whole rat lung tissue, which appears to be regulated independently during oxygen exposure (13). Jones and co-workers (20) have reported the production of growth factors by lung fibroblasts derived from oxygen-exposed hamsters and baboons. These studies indicate that PDGF production by resident cells of the lung play a role in normal as well as injured lung. Lung fibroblasts represent a sizable fraction of total lung cells and are obviously centrally located to tissue compartments undergoing active remodeling. Therefore, if similar PDGF production by lung fibroblasts occurs in vivo, it is likely to represent an important source of this cytokine within the lung. The data presented here point to striking quantitative differences between fibroblasts derived from rat and human lung with respect to the production of PDGF-like cytokines. It is possible that the differences observed between rat lung fibroblasts and those of human origin may reflect a fundamental species difference in the mechanisms of growth factor production within the lung. In this case, studies of cytokine action during experimental animal models of lung injury may not accurately reflect cellular events in the human. On the other hand, differences in growth factor production between fibroblasts may reflect differential selection in vitro of various fibroblast phenotypes. The existence of several specific fibroblast subpopulations has been postulated for a number of tissues including lung (1, 21, 22, 36). Differences in cell shape, proliferative ability, response to various hormones, and expression of various cell surface proteins all indicate the potential of fibroblasts to adopt a variety of phenotypes. It is not known whether these subpopulations represent stable phenotypes of defined lineage or whether fibroblasts demonstrate plasticity by adopting one form or another depending on various environmental influences. The expression of PDGF-AA appears to play a role in growth control of vascular smooth muscle cells. Arterial smooth muscle cells appear to express PDGF-AA mRNA

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L192

PDGF

PRODUCTION

BY LUNG

and proteins under a variety of culture conditions (29, 44). The growth-promoting effects of TGF-P on confluent smooth muscle cells involves the autocrine elaboration of PDGF-A by the target cells (28). Although the rat lung fibroblasts utilized in these studies do not resemble mature smooth muscle cells in regard to a number of histochemical, morphological, or functional criteria, it is possible that they represent an intermediate phenotype such as myofibroblasts or pericytes (1). The expression of muscle-specific cu-actin mRNA by these cells supports this concept. In conclusion these data indicate that cultured fibroblasts derived from normal rat lung produce growthpromoting cytokines in vitro. A portion of this activity appears to represent PDGF-A homodimer. Growth factor-producing rat fibroblast lines express PDGF-A mRNA as assessed by Northern blot as well as immunoreactive PDGF-A in their conditioned medium. Furthermore, this growth factor appears active on lung fibroblast targets. This activity was present in CM from rat lung fibroblasts of both fetal and adult origin but was not detected in the two comparable human fibroblast lines studied. The nature of these differences remain unknown; however, it is possible that the rat lung fibroblasts employed here represent a phenotype of interstitial cell which can produce growth-regulating cytokines in vivo and thus play a role in lung remodeling. The authors thank Linda Baldor and Karen Hawes for technical assistance in the performance of these experiments and Linda Dunbar for help in preparing the manuscript. The authors are especially grateful to Drs. C. Betsholtz and C.-H. Heldin for their generous gift of the Dl PDGF-A cDNA probe. This study was supported by National Heart, Lung, and Blood Institute Pulmonary Specialized Center of Research Grant HL-14212, Grants HL-38924 and HL-41213, the Vermont Lung Association, and a Department of Veterans Affairs Merit Review Award. J. P. Fabisiak is the recipient of a Parker B. Francis Fellowship in Pulmonary Research. Address for reprint requests: J. P. Fabisiak, Div. of Pulmonary and Critical Care Medicine, Dept. of Medicine, University of Pittsburgh, 440 Scaife Hall, Pittsburgh, PA 15261. Received

29 October

1990; accepted

in final

form

26 February

1992.

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Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature Lond. 227: 680685, 1970. Majack, R. A., M. W. Majesky, and L. V. Goodman. Role of PDGF-A expression in the control of vascular smooth muscle cell growth by transforming growth factor-p. J. CeZZ Biol. 111: 239-247, 1990. Majesky, M. W., E. P. Benditt, and S. M. Schwartz. Expression and developmental control of platelet-derived growth factor A-chain and B-chain/Sk genes in rat aortic smooth muscle cells. Proc. Natl. Acad. Sci. USA 85: 1524-1528, 1988. Maniatis, T., E. F. Fritsch, and J. Sambrook. MoZecular Cloning: A Laboratory 1McznuaZ. Cold Spring Harbor, NY: Cold Spring Harbor Lab., 1983. Martinet, Y., W. N. Rom, G. R. Grotendorst, G. R. Martin, and R. G. Crystal. Exaggerated spontaneous release of plateletderived growth factor by alveolar macrophages from patients with idiopathic pulmonary fibrosis. N. Engl. J. Med. 317: 202-209, 1987. Meyrick, B., and L. Reid. Hypoxia and incorporation of 3Hthymidine by cells of the rat pulmonary arteries and alveolar walls. Am. J. Pathol. 96: 51-70, 1979. Oliver, M. H., N. K. Harrison, J. E. Bishop, P. J. Cole, and G. J. Laurent. A rapid and convenient assay for counting cells cultured in microwell plates: application for assessment of growth factors. J. Cell Sci. 92: 513-518, 1989. Paulsson, Y., R. Austgulen, E. Hofsli, C.-H. Heldin, B. Westermark, and J. Nissen-Meyer. Tumor necrosis factorinduced expression of platelet-derived growth factor A-chain messenger RNA in fibroblasts. Exp. Cell Res. 180: 490-496, 1989. Paulsson, Y., A. Hammacher, C.-H. Heldin, and B. Westermark. Possible autocrine feedback in the prereplicative phase of human fibroblasts. Nature Lond. 328: 715-717, 1987. Phipps, R. P., D. P. Penney, P. Keng, H. Quill, A. Paxhia, S. Derdak, and M. E. Felch. Characterization of two major populations of lung fibroblasts: distinguishing morphology and discordant display of Thy 1 and class II MHC. Am. J. Respir. CeLZ Mol. Biol. 1: 65-74, 1989. Pledger, W. J., C. D. Stiles, H. N. Antoniades, and D. Scher. An ordered sequence of events is required before BALB/c-3T3 cells become committed to DNA synthesis. Proc. Natl. Acad. Sci. USA 75: 2839-2843, 1978. Raines, E. W., S. K. Dower, and R. Ross. Interleukin-1 mitogenic activity for fibroblasts and smooth muscle cells is due to PDGF-AA. Science Wash. DC 243: 393-396, 1989. Robbins, K. C., S. G. Devare, and S. A. Aaronson. Molecular cloning of integrated simian sarcoma virus: genome organization of infectious DNA clones. Proc. Natl. Acad. Sci. USA 78: 29182922, 1981.

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