Expression of Transforming Growth Factor-{jt, -{j2' and -{33 mRNA and Protein in the Murine Lung Ron

w. Pelton, Mahlon D. Johnson, Elizabeth A. Perkett, Leslie I. Gold, and Harold L. Moses

Departments of Cell Biology, Pathology, and Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee, and the Department of Pathology, New York University Medical Center, New York

Evidence has accumulated suggesting that the various isoforms of l3-type transforming growth factors (TGF-l3s) regulate important functions in the lung; however, the cellular source of these proteins is not well defined. Northern blot analysis of murine lung tissue demonstrates that mRNA transcripts for all three TGF-13 isoforms are found from birth through adulthood. Although the level of expression for each TGF-13 is variable during the first 2 wk post partum, all three isoforms are equal in the adult lung. Using in situ hybridization and immunohistochemical analysis, we have localized both mRNA and protein expression for all three isoforms of TGF-13 in the adult murine lung. At low magnification, immunohistochemical localization of TGF-13 proteins appears coincident in their pattern of expression with TGF-13 mRNAs in the large proximal conducting airways of the lung. However, on closer analysis, protein expression of all three TGF-13 isoforms is confined to the bronchiolar epithelium, while TGF-13 mRNA transcripts for each of the TGF-13 genes are found in smooth muscle cells and connective tissue fibroblasts lying subjacent to the epithelium. Although the levels of both lGF-13 mRNA and protein expression are high in the proximal bronchiolar tree, their signal intensities completely disappear as the terminal bronchioles progress to respiratory bronchioles. Additionally, in the lung vasculature, there is very high expression of all three lGF-13 mRNA transcripts in the smooth muscle cells of the large vessels. lGF-132 and lGF-133 but not lGF-131 proteins are expressed in these same smooth muscle cells. The temporal and differential cellular localization of mRNA synthesis and protein expression in lung tissue provides evidence for a regulatory role for each of these proteins in cellular processes of the lung. Comparison of lGF-13 mRNA and protein localization suggests that both paracrine and autocrine mechanisms of lGF-13 activity are present in the lung.

The adult lung is impressive both for its high level of functional stability and for its high rate of biosynthetic activity (1, 2). The preservation of a steady equilibrium in the presence of the intense metabolic rate of the lung requires a finetuned interplay of many different cell types. Many of the cellcell interactions in the lung are thought to be mediated by peptide growth factors (3), which control a wide range of cellular activities including proliferation, differentiation, chemotaxis, and extracellular matrix (ECM) production. The transforming growth factor-S (TGf-l3) family of proteins represent one set of peptide growth factors that regulate cellular activities in the lung (reviewed in reference 4). The effects of the lGF-l3s in the lung are many and di(Received in originalform September 14, 1990 and in revisedformMay 14, 1991)

Address correspondence to: Ron W. Pelton, Ph.D., Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232.

Abbreviations: bovine serum albumin, BSA; dithiothreitol, OTT; extracellular matrix, ECM; phosphate-buffered saline, PBS; sodium dodecyl sulfate, SOS; transforming growth factor-d. TGF-I3. Am. J. Respir. Cell Mol. BioI. Vol. 5. pp. 522-530, 1991

verse. When added to in vitro cultures of human lung fibroblasts, lGF-131 alone has little or no effect on proliferation. However, if added in conjunction with epidermal growth factor, TGF-131 augments the stimulatory effect of EGF on cell growth (5). In contrast, lGF-131 is a powerful inhibitor of proliferation in epithelial cell types in the lung; picomolar concentrations of lGF-131 irreversibly inhibit the proliferation of normal human bronchial epithelial cells (6). lGF-131 also causes these cells to adopt a differentiated phenotype. At concentrations as low as 0.4 pM, lGF-131 stimulates the squamous differentiation of human bronchial epithelial cells and rabbit tracheal epithelial cells (6-8). lGF-131 can also effectively inhibit the production of a surfactant-associated protein of M, = 35,000 (SAP-35) by lung epithelial cells (9). The ECM of the lung is a very dynamic structure and is altered in many disease states such as pulmonary fibrosis or emphysema (reviewed in reference 10). The principal components of the ECM in the lung are collagen, fibronectin, elastin, and glycosaminoglycans. lGF-131 regulates the synthesis of each of these molecules in lung-derived cell types; hence control of ECM production may be a major activity of

Pelton, Johnson, Perkett et al.: Expression of l1-type Transforming Growth Factors in the Lung

lGF-111 in the lung. For example, lGF-111 selectively stimulates the production of types I and III collagen, as well as fibronectin and its receptor in human embryonic lung fibroblasts (11, 12). In adult human lung fibroblasts derived from both normal and fibrotic lung specimens, lGF-111 increases the synthesis of types I, Ill, and V collagen (13) and stimulates elastin synthesis by cultured vascular smooth muscle cells and neonatal rat lung fibroblasts (14, 15). lGF-111 also increases the synthesis of various glycosaminoglycans by human lung fibroblasts and alters their intracellular and extracellular distributions (16). In addition to activating the synthesis of ECM components, lGF-111 also prevents their degradation by inhibiting the production of ECM proteases as well as by inducing the synthesis of protease inhibitors. Treatment of human embryonic lung fibroblasts or normal human bronchiolar epithelial cells with lGF-111 stimulates a large increase in plasminogen activator inhibitor-1 mRNA (17, 18) and an overall decrease in both urokinase-type plasminogen activator and tissue-type plasminogen activator (19-21). Recent evidence suggests that lGF-111 may also be involved in the pathogenesis of some forms of lung cancer. Analysis of various human lung cancer cell lines and primary tumors has demonstrated that several of these cell lines and tissues express lGF-111 mRNA (22, 23). Interestingly, only tumors and cell types derived from non-small cell lung carcinomas have been shown to express high levels of lGF-111 mRNA and only the non-small cell lung tumors appear to mount a significant stromal response when injected into nude mice (consistent with the ability of lGF-111 to increase ECM production) (24). Despite a large number of studies that have demonstrated lGF-11 effects on lung-derived cell types, relatively few data concerning the in vivo sites of TGF-11 gene expression have been reported. Previous studies have demonstrated lGF111, -112' and -113 mRNAs in the adult mouse lung using Northern blot analysis; however, the specific cell type(s) producing these mRNAs was not investigated (25). The epithelial lining fluid of the human lung as well as sheep lung lymph both possess TGF-11 activity (26, 27). Furthermore, following bleomycin-induced pulmonary inflammation and fibrosis, TGF-111 protein levels in the adult rat lung increase up to 30-fold over controls (28). Also, there is a concomitant large increase in fibronectin, perhaps a reflection of the increased level of TGF-111. Immunohistochemical staining of this lung tissue reveals increased TGF-111 in the bronchiolar epithelium, ECM, and alveolar macrophages. Using in situ hybridization analysis, we and others have shown that transcripts for TGF-l1h -112' and -113 are localized in the embryonic mouse lung and appear to exhibit different patterns of expression (29, 31). In addition, elegant work by Heine and associates (32) has correlated TGF-111 protein expression in specific cell types in the embryonic lung with the spatial and temporal expression of several ECM molecules. In light of the data outlined above suggesting a role(s) for lGF-111 and the closely related proteins lGF-112 and lGF-113 in lung physiology and pathophysiology, we have used in situ hybridization and immunohistochemical analysis to examine lGF-l1h -112 and -113 gene expression in the adult mouse lung. In contrast to previous reports, these studies are the first to eo-localize TGF-11 mRNAs and proteins for TGF-111,

523

-112, and -113 to specific cell types in the lung. Our results suggest a regulatory role for lGF-111, -112, and -113 in the mouse lung executed through both paracrine and autocrine mechanisms.

Materials and Methods Tissues Adult lung tissue (whole lung) was obtained from outbred ICR female and Swiss-Webster male mice (Taconic Farms). Neonatallung tissues (whole lung) were obtained from the offspring of matings between ICR female and Swiss-Webster male mice. Adult mice or neonatallittermates (at birth, and 3, 8, or 14 days post partem) were killed by cervical dislocation and immediately dissected. Lungs were excised and immediately placed in ice-cold 4 % paraformaldehyde/phosphate-buffered saline (PBS) (for in situ hybridization or immunohistochemical analysis) or were snap-frozen in liquid nitrogen and stored at -70 0 C (for Northern blot analysis) until used. Probe Construction Transcript-specific probes were obtained by deleting the highly conserved regions of the three murine cDNAs in whole (lGF-112 and TGF-113) or in part (TGF-111) (see Figure 1). The resulting fragments were then subcloned into pGEM7Z(f+) (TGF-111 and TGF-113) or pSP73 (TGF-112) so that antisense RNA could be synthesized from the T7 promoter. The TGF-111 construct consists of nucleotides 421 through 1,395 in the murine cDNA (33). This region contains 764 bp of the N-terminal glycopeptide (precursor) region and 210 bp of the mature region. The TGF-112 construct consists of nucleotides 1,511 through 1,953 in the murine cDNA (34) and contains 442 bp of the N-terminal glycopeptide region. The TGF-113 construct contains nucleotides 831 through 1,440 of the murine cDNA (35) and covers 609 bp of the N-terminal glycopeptide region. Single-stranded antisense riboprobes were radiolabeled with [cx- 35S]uridine triphosphate (UTP) (1,400 Cif mM; New England Nuclear, Boston, MA) for in situ hybridization experiments or with [cx-32P]uridine triphosphate (800 Ci/mM; New England Nuclear) for Northern blot analysis to a specific activity of rv2 x 1Q6 dpm/ng. For in situ hybridization analysis, the radiolabeled probes were reduced by limited alkaline hydrolysis to an average size of 100 to 150 bp and used at a final concentration of rv4 X 1(}4 cpm/ ~l. Previous studies havedemonstrated that each probe recognizes a specific TGF-11 isoform and does not cross-hybridize with other lGF-11 transcripts (25, 31). Northern Blot Analysis Poly(Ay RNA was isolated essentially as described (34) from whole mouse lungs and diluted to 1 ~g/ml in 50 mM 3-(N-morpholino)-propanesulfonic acid (MOPS) (pH 7.0), 20 mM sodium acetate, 2.5 mM EDTA (pH 8.0), 6.5% formaldehyde, 50% formamide and was heated to 65 0 C for 5 min. The RNA was then mixed with loading buffer (50% glycerol, 1 mM EDTA, 0.25 % bromophenol blue, 0.25 % xylene cyanol FF, 0.5 ~g/ml ethidium bromide), loaded on a 1% agarose/2.2 M formaldehyde gel at 3 ~g/lane, and run at 40 V for 8 to 10 h. The gel was then photographed on a UV illuminator, and equal loading was judged based on the

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991

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strating the cDNA constructs used in the Northern blot and in situ hybridization analyses. A. DiaA. 5' ffii· gram of a generic transforming PRECURSOR growth factor-S (TGF-tJ) cDNA. The "precursor" or N-terminal b.p. probe lengtb glycopeptide region codes for the 974 B. nonactive portion of the latent TGF-tJ, whereas the "mature" region codes for the biologically ac442 c. tive 112 to 114 amino acid TGF-tJ monomer. The greatest nucleotide sequence identity is found in the 609 D. mature region and the area of least sequence identity is found in the precursor region. B. The TGF-tJ, cDNA contains nucleotides 421 through 1,395 and has a portion of the mature region. C. The TGF-tJ2 cDNA covers nucleotides 1,511 through 1,953. D. The TGF-tJ3 cDNA consists of nucleotides 831 through 1,440.

ethidium bromide-stained gels. The RNA was transferred to nitrocellulose (Nitroplus 2000; Micron Separations Inc.) in 10x SSC by capillary transfer. The RNA was UV-crosslinked to the nitrocellulose (1,200 total joules) and baked at 80° C for 2 h. Blots were prehybridized at 65° C in 50% formamide, 250 j.tg/ml sheared salmon sperm DNA, 1 x Denhardt's, 50 j.tg/ml poly(A), 0.1% sodium dodecyl sulfate (SDS), and sx SSC (lx SSC = 150 mM NaCl, 15 mM sodium citrate) for 4 to 6 h and then hybridized in the same solution with 1 x 1()6 cpm of labeled riboprobe/ml of hybridization buffer for 18 h at 65° C. After hybridization, the blots were washed at room temperature in ix SSC, 0.1% SDS for 20 min and then 3 times at 65° C in o.ix SSC, 0.1% SDS. Blots were then exposed to X-ray film (Kodak XAR 2; Eastman Kodak, Rochester, NY) with intensifying screen at -70° C for 7 days. After exposure, the blots were stripped of hybridizing riboprobe in 70% formamide, 0.1% SDS at 70° C for 30 min and were then rehybridized with another riboprobe to allow a direct comparison of RNA expression for each TGF-~ isoform.

In Situ Hybridization Adult mouse lungs were fixed overnight in 4 % paraformaldehyde/PBS, dehydrated in increasing concentrations of ethanol, and embedded in paraffin wax. Frontal sections of 5 j.tm were cut and floated onto slides coated with 3-triethoxysilylpropylamine. The slides were then incubated at 42 ° C overnight, and the sections were then dewaxed through xylene, rehydrated through decreasing concentrations of ethanol, and refixed in 4 % paraformaldehyde/PBS. They were then treated with proteinase K (20 j.tg/ml in 50 mM Tris, 5 mM EDTA) at room temperature for 5 min, refixed in 4% paraformaldehyde/PBS, acetylated (100 mM triethanolamine, 25 mM acetic anhydride), and dehydrated through ethanol. The sections were then hybridized at 55° C for rv18 h under siliconized coverslips in a solution containing 50% formamide, 10% dextran sulfate, 8 mM dithiothreitol (DTT) , 300 mM NaCl, 10 mM Tris-HCl (pH 7.4), 5 mM EDTA, 10 mM Na 2P04 , ix Denhardt's, and radiolabeled probe. After incubation in a humid chamber for 18 h, the coverslips were removed in sx SSC, 10 mM DTT at 50° C. The slides were then washed at 65° C in ax SSC, 100 mM DTT, 50%

formamide and then treated with RNase A (20 j.tg/ml; Sigma Chemical Co., St. Louis, MO) at 37° C for 20 min followed by washes in 2x SSC and O.1x SSC at 65° C. Slides were then dehydrated through ethanol containing 0.3 M ammonium acetate, dipped in photographic emulsion (Ilford K.5) diluted 1:1 with 2% glycerol/water, and exposed at 4° C for 2 wk in the presence of desiccant. The slides were then developed (Kodak DI9), counterstained with toluidine blue, and analyzed on a Zeiss Axioplan microscope. Photographs were taken with an Olympus OMT (Olympus Corp., Lake Success, NY) on Panatomic X or Ektachrome (Kodak) film using brightfield and darkground optics. Antibody Preparation Peptides of each TGF-~ isoform were synthesized using a 430A Peptide Synthesizer (Applied Biosystems). The following amino acid residues were used: TGF-~, and TGF~z, residues 4 through 19, and TGF-~3' residues 9 through 20. A detailed description of the preparation and characterization of the antisera is described elsewhere (45). Briefly, the peptides were purified by high-performance liquid chromatography and 5.0 mg of each peptide coupled to KLH at a 1:1 ratio (wt/wt) , using 1.25 % glutaraldehyde. Rabbits were immunized with 500 j.tg of each peptide initially, subsequently boosted with 250 j.tg, and antibody titer determined by enzyme-linked immunosorbent assay using appropriate corresponding uncoupled peptide. The antisera did not indicate cross-immunoreactivity with the peptides not used as immunogen, and each antiserum was purified by ammonium sulfate precipitation (31.3 %) followed by peptide affinity chromatography using the respective peptide as imunogen. Each anti-peptide antiserum was tested for both immunoreactivity with the corresponding intact TGF-~ molecule and for cross-reactivity with each other TGF-~ isoform by Western blot analysis (45). Each anti-peptide antiserum to the three isoforms of TGF-~ only reacted with the appropriate and corresponding intact molecule; no cross-reactivity was observed. The specificity of the individual antisera was further demonstrated following complete absorption of the immunoreactivity with 10 M in excess of the corresponding peptide.

Pelton, Johnson, Perkett et al.: Expression of /3-type Transforming Growth Factors in the Lung

Immunohistochemistry Adult lungs were fixed overnight in 4 % paraformaldehyde/PBS, dehydrated in increasing concentrations of ethanol, and embedded in paraffin wax (Fisher Scientific, Fairlawn, NJ). Sections of 5 to 7 ~m were cut and floated onto slides coated with 3-triethoxysilylpropylamine (Sigma). The sections were then incubated at room temperature in PBS/ 0.1% (vol/vol) Triton X-lOO for 15 min, PBS for 5 min, methanol for 2 min, and methanol/0.6 % hydrogen peroxide (vol/ vol) for 30 min. The slides were then washed at room temperature in methanol for 2 min, PBS for 5 min, and 3 times in PBS/O.1 % (wt/vol) bovine serum albumin (BSA) for 3 min each. After treatment with hyaluronidase (l mg/ml in 100 mM sodium acetate, 0.85% [wt/vol] NaCI) and three washes in PBS/O.1 % BSA, nonspecific binding sites were blocked with 5 % normal swine serum in PBS/0.5 % BSA for 15 min at room temperature. The primary antibodies to the synthetic peptides to TGF-/31' TGF-/32' and TGF-/33 were then applied at 5 ~g/ml and allowed to react at 4 0 C overnight. The next day, the tissues were washed 4 times in PBS/O.1 % BSA for 3 min and then incubated for 60 min at room temperature with biotinylated swine anti-rabbit secondary antibody (1:200 dilution; DAKO) in PBS/O.1 % BSA. After three washes in PBS/O.1 % BSA for 3 min, the sections were exposed to avidin-biotin (DAKO) complex for 60 min at room temperature. The slides were washed 3 times in PBS/0.1% BSA for 3 min and reacted with 3 % amino-ethyl carbazole (AEC; Biomeda Corp.) for 10 min. Sections were stained in hematoxylin. Controls for the immunohistochemistry included (1) using no first antibody and (2) using normal rabbit IgG in the place of primary antibody.

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Figure 2. Northern blot analysis of RNA in neonatal adult mouse lungs. The ages of the lungs are given above each lane as newborn (Nb), days post pactum (dpp), weeks (wk), and adult (Ad). Three micrograms of poly/A)" RNA was analyzed and exposure time for each probe was 7 days. Equal amounts of RNA were loaded as shown by ethidium bromide staining of the gel (data not shown). The sizes of the transcripts in kilobases are shown on the right.

Results Detection of TGF-{jt, TGF-{jh and TGF-{j3 mRNA Transcripts by Northern Blot Hybridization Using 32P-Iabeled antisense riboprobes for each isoform of TGF-/3, the Northern blot analysis demonstrates that TGF-/3 mRNA transcripts are present at each stage of mouse lung development from birth until 2 wk post partum (Figure 2). During this time period, transcripts for TGF-/31, TGF-/32, and TGF-/33 are differentially expressed. The highest levels of TGF-/31 mRNA were seen at 2 wk after birth, whereas TGF-/32 mRNA peaked at 3 days post partum and TGF-/33 at 8 days post partum. As previously reported, at least four transcripts (6.0, 5.0, 4.0, and 3.5 kb) for TGF-/32 are seen. In contrast to the differential expression of TGF-/3 isoforms during neonatal lung development, the levels of TGF-/3 mRNA in the adult lung are approximately equal. The unique pattern of expression with each probe indicates that the probes used in these experiments are able to discriminate between transcripts of individual TGF-/3 species and do not exhibit any cross-hybridization. Localization of lGF-{j mRNA and Protein Expression in the Adult Mouse Lung by In Situ Hybridization and Immunohistochemistry To more specifically localize the cell type(s) expressing the mRNAs and proteins for each TGF-/3 isoform, in situ hybridization and immunohistochemical analysis on serial sections of adult lungs were performed. Figure 3 demonstrates that

TGF-/31, TGF-/32' and TGF-/33 mRNAs are expressed in similar patterns in the adult lung. At low magnification, the hybridization signal for all three TGF-/3 species appears to outline bronchioles (Figure 3, arrows). Immunostaining with isoform-specific TGF-/3 antibodies reveals that patterns of TGF-/31' TGF-/32, and TGF-/33 immunoreactivity in the lung are strikingly similar to the in situ hybridization patterns (Figure 4). However, TGF-/3 immunostaining is expressed primarily in the bronchiolar epithelial cells of the large conducting airways whereas the mRNA are found in the smooth muscle and connective tissue cells subjacent to the epithelium (compare Figures 3, 4, and 5). Although differences in staining intensities between antibodies for TGF-/31' TGF-/32' and TGF-/33 are seen in the epithelial cells, there are no differences in staining patterns, i.e., all three antibodies stain bronchiolar epithelial cells (Figure 4). At higher power, the specific cellular distribution of mRNA transcripts for TGF-/33 is evident in the connective tissue fibroblasts and smooth muscle cells on the basal side of the bronchiolar epithelium (for example, Figure 5). No transcripts for any of the TGF-/3 genes are found in the bronchiolar epithelium (Figures 5A, 5B, 5D, and 5E). Identical results are obtained with the riboprobes for TGF-/31 and TGF-/32 (data not shown). In contrast, immunostaining with antibodies to TGF-/32 reveals staining in epithelial cells located directly adjacent to the smooth muscle surrounding the airway (Figures 5C and 5F). Antibodies to

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Figure 3. In situ hybridizations showing the expression of TGF-.Bl. TGF-.B2. and TGF-.B3 mRNA in adult mouse lung. Serial but nonadjacent sections of mouse lung hybridized to radiolabeled antisense riboprobes for each of the TGF-.Bl. TGF-.B2. and TGF-.B3 transcripts were photographed at a low-power magnification with brightfield (panels A through C) and corresponding darkground (panels D through F) optics to illustrate the similarity of expression patterns for each of these transcripts. Each section shows a region through one bronchiole and its surrounding alveoli. The hybridization signal in each case outlines the bronchiole (arrow) and is absent from the alveoli. Bar = 100 JLm. bel = bronchiole; alv = alveoli. Counterstain = toluidine blue (nuclear stain).

TGF-/31 and TGF-/33 give almost identical staining patterns although some differences in staining intensity do exist (Figure 4 and data not shown). The expression level for all three TGF-/3 proteins remains high in the proximal conducting airways but decreases distally (Figure 51). Similarly, as the terminal bronchioles split into respiratory bronchioles and lose their connective tissue/smooth muscle sheath, the expression of mRNAs for all three genes rapidly dissipates (Figures 5G and 5H). Thus, expression of transcripts for TGF-/3I' TGF/32, and TGF-/33 in the adult lung appears to be associated with the smooth muscle cells and connective tissue fibroblasts in the bronchioles and not with cells in the alveoli or the capillaries of the distal airways. In addition to the bronchiolar expression of transcripts for TGF-/3h TGF-/320 and TGF-/33, very intense levels of hybridization signal for each of the TGF-/3 genes are seen in the vasculature of the adult lung. Like that seen in the airways, the pattern of hybridization in the blood vessel walls is similar with all three TGF-/3 probes. Figures 6A and 6B show the TGF-/33 mRNA expression pattern in the wall of a large blood vessel in the lung. Identical patterns are seen with riboprobes to TGF-/31 and TGF-/32 (data not shown). The endothelial lining of the vessels does not show a hybridization signal (Figures 6A and 6B, arrows). Unlike in the respiratory tree, the localization ofTGF-/3 mRNA and proteins was nearly identical in the vasculature (Figure 6C). Moreover, it appears that while the TGF-/32 and TGF-/33 antibodies react strongly with the vascular tree, the TGF-/31 antibodies do not (Figure 6C and data not shown). Immunostaining for TGF-/32 (Figure 6C) and TGF-/33 is seen in smooth muscle

cells of the large blood vessels in the lung, but the endothelium of vessels does not exhibit any TGF-/3. As the blood vessels get smaller and start to lose their smooth muscle sheath, the TGF-/32 and TGF-/33 immunostaining decreases.

Discussion To investigate the possibility that the /3-type TGFs play a role in the mammalian lung, we have examined the temporal and spatial gene expression of TGF-/3h TGF-/32' and TGF-/33 in mouse lung tissue. These studies demonstrate that both mRNA and protein of the /3-type TGFs are expressed in the murine lung in a highly restricted pattern. Earlier reports have suggested that TGF-/3 activity in the lung is derived primarily from alveolar macrophages (28, 36). However, the more extensive analysis described here indicates that endogenous production by cells of the bronchiolar and vascular tree of the lung may be the primary sources ofTGF-/3 activity in this organ. Thus, the high levels of TGF-/3 activity present in lymph draining from the ovine lung are probably due to secretion by bronchiolar and vascular cells rather than recruitment of inflammatory cells (27). In addition, these are the first studies to report on the expression of TGF-/32 and TGF-/33 mRNA and protein in the adult mouse lung. These data suggest that, like TGF-/3h TGF-/32 and TGF-/33 may regulate a wide variety of pulmonary cellular processes. In the adult lung, approximately equal levels of TGF-/31' TGF-/320 and TGF-/33 mRNAs are found by Northern blot analysis, consistent with the equal intensities of hybridization signal seen in our in situ hybridization analysis. In addi-

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Figure 4. Immunohistochemistry showing the expression of TGF-{3, , TGF-{32' and TGF-{33 proteins in the mouse lung. Sections were incubated with affinity-purified polyclonal antibodies that are specific for either TGF-{3t, TGF-{32' or TGF-{33 and photographed at low magnification (compare with Figure 3). All three antibodies stain the bronchiolar epithelium but with differing degrees of intensity. In general, the TGF-{32 antibodies stain the darkest and the TGF-{33 antibodies the lightest. bel = bronchiole; alv = alveoli. Counterstain = hematoxylin.

Figure 5. Localization of TGF-{33 mRNA and TGF-{32 protein in adult mouse bronchioles. Sections were analyzed by in situ hybridization for TGF-{33 mRNA (left and middle columns) or TGF-{32 protein by immunohistochemistry (right column). Sections hybridized to TGF-{3, and TGF-{32 riboprobes or with TGF-{3, and TGF-{33 antibodies show essentially identical expression patterns to the data seen here (see Figures 3 and 4). Panels A and B: Cross section through a bronchiole to demonstrate that the TGF-{33 transcripts are found in the smooth muscle and connective tissue cells surrounding the bronchiole. No signal is found in the bronchiolar epithelium (arrowheads). Bar = 50 ~m. Panel C: Cross section showing that TGF-{32 immunoreactivity is found at highest levels not in the smooth muscle but in the bronchiolar epithelium (arrowheads). Panels D and E: Longitudinal section through a bronchiole again demonstrating that TGF-{33 transcripts are found in the region underlying the bronchiolar epithelium. Note that the large dark dots at the base of the bronchiolar epithelium are the nuclei stained by toluidine blue. Bar = 25 ~m. Panel F: Higher power of a longitudinal section showing that slight staining can be seen in the smooth muscle lying subjadent to the bronchiolar epithelium, but highest levels of TGF-{32 antibody reactivity are seen in the epithelium itself. Panels G and H: Section through a terminal bronchiole as it gives rise to respiratory bronchioles. Note that at the point of transition (arrowheads), the TGF-{33 mRNA signal stops. This corresponds with the cessation of the bronchiolar epithelium and its supporting layers of smooth muscle cells and connective tissue. Panel I: Similar section to that seen in panels G and H showing that the immunostaining for TGF-{32 in the airways ceases in the bronchiolar epithelium of the terminal bronchioles. be = bronchiolar epithelium; bel = bronchiole; smc = smooth muscle cells; tb = terminal bronchiole; rb = respiratory bronchiole. Counterstain = toluidine blue (in situ hybridization) or hematoxylin (immunohistochemistry).

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Figure 6. Expression of TGF-/33 mRNA and TGF-/32 protein in the vasculature of the adult mouse lung. Panels, A and B: Cross section through a blood vessel demonstrating in situ hybridization signal (arrowhead) in the smooth muscle cells just under the endotheliallayer. Note that the endothelial cells (the arrow points to a layer of endothelial cells artifactually separated from the vessel wall) do not express TGF-/33 mRNA. Bar = 50 JLm. Panel C: Similar section to panels A and B showing staining for TGF-/32 in the smooth muscle sheath surrounding the endothelium of a blood vessel in the lung. The endothelium shows no staining. end = endotheliallayer; rbc = red blood cells; smc = smooth muscle cells. Counterstain = toluidine blue (in situ hybridization) or hematoxylin (immunohistochemistry).

tion, Northern blot analysis of developing neonatal lungs shows different temporal patterns of mRNA expression for each of the lGF-,Bs. lGF-,B2 mRNA expression peaks at 3 days post partum, lGF-,B3 at 8 days post partum, and lGF-,B1 is highest at 2 wk post partum. At least four lGF,B2 transcripts are differentially expressed during this time period. Although the significance of this differential temporal expression is not known, it may reflect differences in activities of these molecules in vivo that are not reflected by the in vitro assays used to date. Significant growth and development of mammalian lungs occurs well after birth, and these observations raise the possibility that lGF-,B1o lGF-,B2' and lGF-,B3 participate in normal developmental changes that emerge in the neonatal lung (37-39). Further studies correlating the patterns of expression with morphometric studies may provide more insight into the role of the lGF-,Bs in lung development. The localization of lGF-,Bs to the smooth muscle cells and connective tissue cells in the tissues that support the bronchial epithelium is consistent with the role of the lGF-,Bs as inducers of ECM synthesis and deposition by lung fibroblasts. The production of lGF-,B mRNAs by cells subjacent to the epithelium and localization of lGF-,B proteins in the bronchiolar epithelial cells suggests that lGF-,B proteins act in a paracrine fashion on these cells in vivo. This is in agreement with data showing that the lGF-,Bs are secreted proteins and have a range of effects on bronchial epithelial cells in vitro (4). We are not the first to suggest that the lGF-,Bs may act through paracrine mechanisms. Based on a comparison of their lGF-,B1 in situ hybridization data with lGF-,B1 immunohistochemistry, Lehnert and Akhurst proposed that in the embryo, lGF-,B1 uses both paracrine and autocrine mechanisms (29). An equally important finding of these studies is the localization of lGF-,B mRNAs and protein to the smooth muscle cells found in the blood vessel walls. Previous reports, based on Northern blot data, have suggested that lGF-,B1 mRNA is produced by the smooth muscle cells but not the endothelial cells of the rat aorta (40). The in situ hybridization data presented here support these findings and extend them to include the presence of lGF-,B2 and lGF-,B3 mRNA in these cells. Moreover, our immunohistochemical data show that the lGF-,B proteins found in these cells are primarily lGF-,B2 and lGF-,B3. Immunostaining is not seen in the smooth muscle cells of the vasculature with antibodies to lGF-,BI' These observations are significant because in the past it has been assumed that the main in vivo source of lGF-,B proteins affecting the vascular smooth muscle cells originates from the circulating platelets (41, 42). Evidence presented here indicates that endogenous production by the vascular smooth muscle cells themselves may be the principal source of lGF-,B proteins in the lung vasculature in vivo. Furthermore, these data are consistent with the hypothesis that lGF-,Bs play a role in vascular remodeling in systemic and pulmonary hypertension (27, 40). It is not known whether lGF-,B1o lGF-,B20 and lGF-,B3 have different effects in vivo. Previous studies have shown that mRNAs for lGF-,B1o lGF-,B2' and lGF-,B3 are found in different regions of the embryonic murine lung (26-28). In

Pelton, Johnson, Perkett et al.: Expression of l3-type Transforming Growth Factors in the Lung

contrast, in the adult murine lune, lGF-131' lGF-132' and lGF-133 mRNAs are found in the same cell types in almost identicaldistributions. This "switching" from different mRNA expression patterns in the embryo to similar patterns of hybridizationfor all three mammalianisoformsoflGF-13 in the adult is not without precedent. We have previously reported that in the immature hair follicle, lGF-131' lGF-132' and lGF-133 mRNAs are localized to different cell types but showidenticalpatterns of expressionin the mature hair follicle (43). Additionally, in agreementwith our present results, Heine and associates have observed a change in lGF-131 protein localization in the lung during embryonic development (32). Although lGF-I3, is found in both parenchymal and mesenchymal cells of the l l-day mouse lung, by day 18 the expressionis restricted to the subepithelialcells supporting the major airways. The factors regulating this switching are not well understood but may reflect different functions of the lGF-13 proteins in the embryoand adult. This phenomenon has been observed for another member of the lGF-13 family, activin A. This protein regulates follicle-stimulating hormone production in the adult animal but has recently been shownto induce mesoderm formationduring early embryogenesis (44). Hence, in light of the fact that few qualitative differences have been demonstrated for lGF-I3,, lGF132, and lGF-133 in vitro, the differential expression of lGF13 mRNA between embryo and adult may be significant. In conclusion, we demonstrate the localization of TGF-131' lGF-132' and lGF-133 mRNAs and proteins to specific cell types in the adult murine lung. Transcripts for each of these genes are restricted to the smoothmuscle and connectivetissue fibroblasts supportingthe large airways and blood vessels in the adult mouse lung. Moreover, polyclonal antibodies specific for each of the TGF-13 isoforms react with antigens in the bronchial epithelial cells directly adjacent to the fibroblasts and smooth muscle cells in the bronchioles, suggesting a paracrine mode of action for these proteins. Antibodies to lGF-132 and lGF-133' but not lGF-l3h react with the smooth muscle cells of the vasculature. The endothelium does not react with the antibodies. These data, considered together with the previously reported in vitro studies of lGF-13 effects on lung-derived cell types, suggest that endogenously produced TGF-l3h lGF-132' and lGF-133 play an important role in the normal physiology of the lung. Acknowledgments: This work was supported by National Institutes of Health Grants CA 42572-05 (to Dr. Moses), CA 49507-02 (to Dr. Gold), HL 02776 (to Dr. Perkett), and T32-GM07347 for the medical scientist training program (to Dr. Pelton).

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