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Published ahead of print on January 12, 2004, doi:10.1165/rcmb.2002-0249OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 816-822, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2002-0249OC

Interferon-{gamma} Inhibits Transforming Growth Factor-ß Production in Human Airway Epithelial Cells by Targeting Smads

Fu-Qiang Wen, Xiangde Liu, Tetsu Kobayashi, Shinji Abe, Qiuhong Fang, Tadashi Kohyama, Ronald Ertl, Yusuke Terasaki, Lidia Manouilova and Stephen I. Rennard

Department of Respiratory Medicine, West China Hospital, West China Medical School, Sichuan University, Chengdu, Sichuan, China; Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska; and Department of Respiratory Medicine, University of Tokyo, Tokyo, Japan

Address correspondence to: Stephen I. Rennard, M.D., University of Nebraska Medical Center, 985885 Nebraska Medical Center, Omaha, NE 68198–5885. E-mail:srennard{at}unmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Because interferon (IFN)-{gamma} may attenuate pulmonary fibrosis, we hypothesized that IFN-{gamma} may regulate transforming growth factor (TGF)-ß production by airway epithelial cells. Human bronchial epithelial cells (HBECs) were incubated with IFN-{gamma} ± TGF-ß1, -ß3, or interleukin (IL)-1ß, platelet-derived growth factor (PDGF), epidermal growth factor, and IL-4. TGF-ß2 protein was measured by enzyme-linked immunosorbent assay and mRNA expression for TGF-ß2, Smad 2, 3, 4, and 7 was evaluated by real-time reverse transcriptase–polymerase chain reaction. Localization of Smads 2, 3, 4, and 7 was evaluated by immunostaining. Exogenous TGF-ß1 and 3, IL-1ß, PDGF, and IL-4 enhanced TGF-ß2 release by HBECs (P < 0.01). IFN-{gamma} reduced basal and TGF-ß or IL-4–augmented TGF-ß2 release, but had little effect on IL-1ß– or PDGF-augmented TGF-ß2 release. IFN-{gamma} stimulated Smad 7 protein and mRNA expression. Smad 7–specific siRNA decreased Smad 7 protein expression both in control and IFN-{gamma}–treated cells. The inhibitory effect of IFN-{gamma} on TGF-ß2 production was abrogated when the HBECs were treated with Smad 7 siRNA. These results suggest that IFN-{gamma} downregulates TGF-ß2 production by HBECs by regulating Smad 7. Through this mechanism, IFN-{gamma} may play an important role in tissue remodeling.

Abbreviations: chronic obstructive pulmonary disease, COPD • epidermal growth factor, EGF • enzyme-linked immunosorbent assay, ELISA • human bronchial epithelial cells, HBEC • interferon-{gamma}, IFN-{gamma} • interleukin, IL • lactate dehydrogenase, LDH • polymerase chain reaction, PCR • platelet-derived growth factor, PDGF • reverse transcriptase, RT • transforming growth factor-ß, TGF-ß


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Members of the transforming growth factor (TGF)-ß family are expressed in a variety of cell types, including endothelial, epithelial, hematopoietic, mesenchymal, and neuronal cells (1). TGF-ß mediates a diverse range of cellular responses, including cell proliferation and differentiation, embryonic development, wound healing, and angiogenesis (2). Overexpression of TGF-ß is believed to contribute to numerous disease states, including atherosclerosis and fibrotic disease of the kidney, liver, and lung. TGF-ß likely plays an important role in asthma and chronic obstructive pulmonary disease (COPD), in which chronic inflammation and injury of both the airways and lung parenchymal structures are observed, and peribronchiolar fibrosis and subepithelial fibrosis are often seen (3–5). Interferon (IFN)-{gamma}, which is produced by T-helper 1 (Th1) lymphocytes and natural killer cells, plays a major role in the biology of monocytes/macrophages as a primary macrophage-activating factor. IFN-{gamma} stimulates tumor cell cytotoxicity and antimicrobial activity, and also downregulates TGF-ß and procollagen I and III gene expression in the bleomycin-mouse model of lung fibrosis (6, 7). The level of IFN-{gamma} is decreased in bronchoalveolar lavage fluid (BALF) from patients with asthma, suggesting that its absence may contribute to airway remodeling in asthma (8). Although IFN-{gamma} is observed to inhibit TGF-ß signaling in transfected cancer cells by activation of Stat 1 (9), how IFN-{gamma} modulates the TGF-ß signaling in human lung cells remains to be determined.

Airway epithelial cell damage and repair are believed to play important roles in the tissue remodeling that characterizes both asthma and COPD (5). TGF-ß has been suggested to play an important role in mediating these processes (10). The current study, therefore, was designed to address the question of whether IFN-{gamma} is capable of modulating TGF-ß production by airway epithelial cells in response to various kinds of stimuli. In addition, the signaling mechanisms by which IFN-{gamma} modulates TGF-ß production were explored.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant human IFN-{gamma}, TGF-ß1, -ß2, and -ß3, interleukin (IL)-1ß, IL-4, epidermal growth factor (EGF), PDGF-AA and -BB, mouse anti-human TGF-ß2 (clone: 8,607.211) monoclonal antibodies (used for capture in enzyme-linked immunosorbent assay [ELISA]), and biotinylated anti-human TGF-ß2 antibodies (for detection) were purchased from R&D Systems Inc. (Minneapolis, MN). Tetramethylbenzidine dihydrochloride was purchased from Sigma Chemical Co. (St. Louis, MO).

Cell Culture
Normal human bronchial epithelial cells (HBECs) were obtained by the explant method (11). After cells grew to confluence, they were trypsinized and passaged onto Vitrogen 100 (Cohesion Technologies, Palo Alto, CA)–coated dishes in LHC-9/RPMI (Invitrogen Life Technologies, Grand Island, NY). Third-passage cultures were stored in liquid N2. Cells between passages 4 and 9 were used for experiments. BEAS-2B cells were purchased from the American Type Culture Collection (ATCC; Rockville, MD), and cultured with LHC-9/RPMI, as were HBECs.

Experimental Protocol
To evaluate the effect of IFN-{gamma} on basal release of TGF-ß2 by human bronchial epithelial cells, 1 x 105 HBECs or BEAS-2B cells were seeded in LHC-9/RPMI in 12-well plates (FALCON; Becton Dickinson, Franklin Lakes, NJ) and grown for 3–4 d to reach confluence. After washing with LHC basal media (LHC-B/RPMI; 1:1 mixture), fresh media containing various concentrations of IFN-{gamma} were added and incubated for 24 h.

To examine the effect of IFN-{gamma} on TGF-ß1– or -ß3–induced production of TGF-ß2 by HBECs, cells were incubated with 2.5 ng/ml of TGF-ß1 or -ß3, together with various concentrations of IFN-{gamma} for 24 h.

To compare the effect of IFN-{gamma} on TGF-ß2 production in response to other cytokines, cells were incubated with 10 ng/ml of IL-1ß, EGF, PDGF-AA and -BB, IL-4 alone and together with IFN-{gamma} (200 U/ml) for 24 h.

At the indicated times, all culture media were harvested and stored at –80°C until ELISA for TGF-ß2 was performed.

Measurement of TGF-ß2 by ELISA
TGF-ß2 concentration in post-culture media was determined by ELISA (12). The assays are isoform-specific and detect an epitope present in the active form of TGF-ß2, and there is no cross-reaction with TGF-ß1 or TGF-ß3. The results are expressed as amount per million cells based on cell number when harvested in the wells.

RNA Preparation
To determine whether changes in TGF-ß2 or Smad 3 or 7 mRNA levels were present, real-time reverse transcription (RT)–polymerase chain reaction (PCR) was done. Cells were cultured until confluence and exposed to 2.5 ng/ml of TGF-ß1 or -ß3 in the absence or presence of 200 U/ml of IFN-{gamma} for 24 h as described above. Total RNA was isolated by a single-step guanidinium-thiocyanate-phenol-chloroform extraction procedure described by Chomczynski (13) and treated with RNase-free DNase I (Invitrogen). After denaturation of the freshly prepared RNA at 65°C for 10 min and 95°C for 5 min, a single-strand cDNA was produced by RT.

Quantitative Real-Time RT-PCR
The PCR primers and Taqman probes for human TGF-ß2, Smad 2, 3, 4, 7, and glyceraldehyde-3-phosphate dehydrogenase were designed using the Primer Express program and synthesized by Applied Biosystems (Foster City, CA). The sequences used were for TGF-ß2 and glyceraldehyde-3-phosphate dehydrogenase as previously described (12) and for Smads: Smad 3 forward, 5'-TCA CCA CGC AGA ACG TCA A-3'; reverse, 5'-GGC GGC AGT AGA TGA CAT GA-3'; probe, 6-FAM TGC ATC ACC ATC CCC AGG TCC CT TAMRA. Smad 7 forward, 5'-TGC TGT GAA TCT TAC GGG AAG AT-3'; reverse, 5'-CTC TAG TTC GCA GAG TCG GCT AA-3'; probe, 6-FAM AGC TGG TGT GCT GCA ACC CCC A TAMRA.

RT and PCR were performed using GeneAmp RNA PCR Kit and Taqman Universal PCR Master Mix (Applied Biosystems) according to manufacturer's specifications (14). Each sample was run in duplicate, and threshold cycle (Ct) values were averaged from each reaction. Data were analyzed using a Sequence Detector V1.6 program (Applied Biosystems).

Immunocytochemical Staining
Human bronchial epithelial cells were cultured in LHC9/RPMI in eight-well chamber glass slides, coated with Vitrogen. When cells reached subconfluence, cells were washed twice with fresh LHC-B/RPMI (1:1 mixture). Immediately before each experiment, fresh LHC-B/RPMI containing TGF-ß1 or -ß3, or IFN-{gamma} alone and in combination were added. After incubation for 24 h, slides were fixed with 4% buffered formaldehyde followed by treatment with 0.5% Triton X 100 and blocking serum. Slides were then used for staining with anti-Smad 2, 3, 4, or 7 (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C, followed by incubation with fluorescein-conjugated secondary antibody (1:1,000 dilution) for 1 h. After washing with PBS, cover slips were mounted with Vector shield. Cellular localization of fluorescence was examined by fluorescence or confocal microscopy and photographed with a confocal microscope.

Cell Viability and Growth Kinetics Assays
To determine cell viability, the effect of IFN-{gamma}, other growth factors and cytokines on lactic dehydrogenase (LDH) activity of epithelial cells was evaluated. LDH in the supernatant from cytokine-treated cells was measured with an LDH kit (Cat #500; Sigma) following the manufacturer's instructions. To evaluate the growth kinetics of epithelial cells, MTT assay was performed according to the published method (15). Briefly, cells (2 x 104 cells/well) were cultured for 24 h on 96-well collagen-coated tissue culture plates in the absence or presence of IFN-{gamma}, other growth factors, and cytokines. The culture media were then replaced with 200 µl of MTT labeling reagent (0.5 mg/ml), and incubated for 4 h at 37°C to yield a dark blue formazan product. After washing with PBS, DMSO (100 µl/well) was added and shaken for 20 min. Absorbance at wavelength of 540 nm was determined with microplate reader. Absolute OD value was obtained and expressed as percent of control. Both LDH and MTT assay showed no significant evidence of cytotoxicity in comparison to controls (data not shown).

Smad 7 siRNA Transfection and Immunoblot
HBECs were plated in type I collagen precoated tissue culture plates in LHC-9/RPMI with antibiotics and fungizone and cultured overnight. The medium was then changed to LHC-9/RPMI without antibiotics and fungizone and cultured for 6 h. After washing once with DPBS, cells were then transfected with Smad 7 siRNA using TransIT-TKO (Mirus, Madison, WI) in LHC-D/RPMI without antibiotics following manufacturer's instruction and a previously reported method (16). Smad 7 siRNA was designed by the investigators (5'-AA GCU CAA UUC GGA CAA CAA G-dTdT-3'; GI: 2460041; Accession #: AF015261) and synthesized by Dharmacon Inc. (Lafayette, CO). The cells were treated with siRNA for 24 h. After that, cells were treated with TGF-ß1 ± IFN-{gamma} for additional 24 h in LHC-D/RPMI without antibiotics. Following this, medium was harvested to measure TGF-ß2 production by ELISA and cells were lysed with cell lysis buffer (35 mM Tris-HCl, pH 7.4, 0.4 mM EGTA, 10 mM MgCl2, 100µg/ml aprotinin, 1µM phenylmethylsulfonyl fluoride, 1µg/ml leupeptin, and 0.1% Triton X-100) to perform immunoblotting. For this, 10µg of total proteins were loaded in 10% polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membrane. After blocking with 5% dry milk, anti-Smad 7 antibody (sc-9183), anti–Smad 6 antibody (sc-6034; Santa Cruz Biotechnology, Santa Cruz, CA) or anti–pan-cytokeratin (NCL-L-PAN-CK; Novocastra, Newcastle, UK) were used. After applying appropriate secondary antibodies, targeted proteins were detected with an enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech, Little Chalfont, UK).

Statistical Analysis
Each condition in every experiment included three replicate dishes. Each experiment was repeated on multiple occasions, and each figure represents composite data from these experiments as indicated in the figure legends. Data were evaluated by one-way ANOVA. If the overall F statistic was significant at 0.05, subsequent intergroup significance testing was assessed post hoc by Scheffe's F-test. Student's t test was also performed to compare paired samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of IFN-{gamma} on TGF-ß2 Release by HBECs and BEAS-2B Cells Treated with or without TGF-ß1 or -ß3
Figure 1 shows the release of TGF-ß2 by HBECs and BEAS-2B that were treated with or without 2.5 ng/ml of TGF-ß1 or -ß3 in the presence of various concentrations of IFN-{gamma} for 24 h. In the absence of TGF-ßs, IFN-{gamma} moderately but significantly suppressed the release of TGF-ß2 by both HBECs (Figure 1A) and BEAS-2B cells (Figure 1B) in a concentration-dependent manner (P < 0.01). In the presence of 2.5 ng/ml of TGF-ß1 or TGF-ß3, the concentrations of TGF-ß2 released in culture of HBECs and BEAS-2B significantly exceeded those observed in the absence of TGF-ß1 or TGF-ß3 (P < 0.01). Addition of IFN-{gamma} to the media containing TGF-ß1 or TGF-ß3 significantly suppressed the production of TGF-ß2 from HBECs (Figure 1A), as well as from BEAS-2B cells (Figure 1B) in a concentration-dependent manner in all cases (P < 0.01). As seen in Figure 1, 200 U/ml of IFN-{gamma} reduced TGF-ß2 release by TGF-ß1–stimulated HBECs and BEAS-2B by 43% and 50%, respectively. Similarly, IFN-{gamma} reduced TGF-ß2 release by 35% in HBECs and 46% in BEAS-2B cells treated with TGF-ß3.



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  		Figure 1. Effect of IFN-{gamma} on TGF-ß2 release by human bronchial epithelial cells treated with or without exogenous TGF-ßs. HBECs (A) and BEAS-2B cells (B) were incubated for 24 h with LHC-B/RPMI containing various concentrations of IFN-{gamma} ± 2.5 ng/ml TGF-ß1 or -ß3. TGF-ß2 was quantified by specific ELISA. Vertical axes: TGF-ß2 production (pg/d/106 cells); horizontal axes: conditions. Results are pooled from nine samples in three different experiments. Open bars, TGF-ßs (–); striped bars, TGF-ß1 (+); filled bars, TGF-ß3 (+). P < 0.05 as analyzed by Scheffe's test, compared with control without TGF-ßs (*) or with TGF-ßs (#).

 
Effect of IFN-{gamma} on TGF-ß2 Release by Cells Treated with IL-1ß, EGF, PDGF, or IL-4
To compare the effect of other cytokines on TGF-ß production and its modulation by IFN-{gamma}, cells were cultured for 24 h in media containing IL-1ß, EGF, PDGF, IL-4 alone or together with IFN-{gamma}. The addition of IL-1ß, PDGF and IL-4 resulted in a significant increase in TGF-ß2 release (Table 1) (P < 0.01). The addition of IFN-{gamma} in combination with IL-1ß, or PDGF had little effect on the release of TGF-ß2. The release of TGF-ß2 in cells treated with EGF was not affected by the addition of IFN-{gamma}. IFN-{gamma} significantly reduced IL-4-augmented TGF-ß2 release as observed before (12).


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  		TABLE 1. Effect of IFN-{gamma} on TGF-ß2 production stimulated by cytokines

 
TGF-ß2 mRNA Expression and its Modulation by IFN-{gamma}
To determine whether IFN-{gamma} regulates TGF-ß2 production by altering mRNA expression, real-time RT-PCR analysis was done. TGF-ß1 or TGF-ß3 treatment resulted in a 2- to 6-fold increase in TGF-ß2 mRNA expression in HBECs (Figure 2A) and BEAS-2B cells (Figure 2B) after 24 h incubation (P < 0.01). IFN-{gamma} treatment significantly inhibited not only the spontaneous TGF-ß2 mRNA expression, but also TGF-ß1– and -ß3–stimulated TGF-ß2 mRNA expression in HBECs and BEAS-2B cells (P < 0.01).



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  		Figure 2. TGF-ß2 mRNA expression in human bronchial epithelial cells and its modulation by IFN-{gamma}. HBECs (A) and BEAS-2B cells (B) were incubated for 24 h with LHC-B/RPMI containing 2.5 ng/ml TGF-ß1 or -ß3, 200 U/ml IFN-{gamma} alone or in combinations. TGF-ß2 mRNA was quantified as described in MATERIALS AND METHODS. Vertical axes: TGF-ß2 mRNA; horizontal axes: conditions. Values are from triplicate cultures from a single experiment. Similar results were obtained in two other experiments. Student t test, compared with control (*) or TGF-ß1– or -ß3–stimulated cells (#) (both P < 0.01).

 
Effect of IFN-{gamma} on Cellular Localization of Smads
Immunostaining was done to test whether IFN-{gamma} could affect nuclear staining of receptor-regulated Smads 2, 3, and 4, the intracellular signaling components for TGF-ß (Table 2). In HBECs and BEAS-2B cells without exposure to TGF-ßs, there were a few cells with nuclear staining for Smads 2, 3, or 4. Upon TGF-ß exposure, most of the cells appeared with Smad 2, 3, and 4 associated intranuclear fluorescence and a strong paranuclear staining for Smad 3 was also noted in HBECs (Figure 3A). The increased staining of Smad 2, 3, and 4 induced by TGF-ßs was markedly reduced by the addition of IFN-{gamma}.


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  		TABLE 2. IFN-{gamma} effect on Smads immunostaining in airway epithelial cells

 



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  		Figure 3. Cellular localization of Smad 3 and 7 in HBECs. HBECs were incubated for 24 h with LHC-B/RPMI containing 2.5 ng/ml TGF-ß1or -ß3, or 200 U/ml IFN-{gamma} alone or in combination before immunostaining for Smad 3 (A) and 7 (B). The slides were then photographed by confocal microscope. Representative microscopic fields are shown. Cytokine additions are indicated on the figure.

 
Immunostaining for Smad 7 was done to determine whether IFN-{gamma} could induce this inhibitory Smad to interrupt TGF-ß signaling. In untreated HBECs, immunofluorescence associated with Smad 7 was not noted, whereas a very weak cytoplasmic staining was observed in cells treated with TGF-ßs. Upon IFN-{gamma} exposure, most cells appeared with Smad 7–associated fluorescence in the paranuclear area together with less prominent cytoplasmic staining (Figure 3B).

Smads mRNA Expression and their Modulation by IFN-{gamma}
Figure 4 shows the effect of IFN-{gamma} on Smad 3 mRNA expression. In HBECs (Figure 4A), TGF-ß1 or TGF-ß3 treatment resulted in decreased expression of Smad 3 mRNA. IFN-{gamma} also reduced Smad 3 mRNA expression in HBECs whether untreated or treated with TGF-ß1 or -ß3. In BEAS-2B cells (Figure 4B), TGF-ß1 or TGF-ß3 treatment resulted in ~ 3-fold increase in Smad 3 mRNA expression (P < 0.01). IFN-{gamma} treatment significantly reduced Smad 3 mRNA expression in both untreated and TGF-ß–treated cells.



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  		Figure 4. Smad 3 mRNA expression in human bronchial epithelial cells and its modulation by IFN-{gamma}. HBECs (A) and BEAS-2B cells (B) were incubated for 24 h with LHC-B/RPMI containing 2.5 ng/ml TGF-ß1 or -ß3, or 200 U/ml IFN-{gamma} alone or in combination. Vertical axes: Smad 3 mRNA expression; horizontal axes: conditions. Values are from triplicate separate cultures from a single experiment. Similar results were obtained in two other experiments. *,#P < 0.05; **,##P < 0.01, Student t test, compared with control (*) or TGF-ß1– or -ß3–stimulated cells (#).

 
Figure 5 shows the effect of IFN-{gamma} on Smad 7 mRNA expression. TGF-ß1 or TGF-ß3 treatment resulted in a 3- and a 5- to 10-fold increase in Smad 7 mRNA expression in HBECs (Figure 5A) and BEAS-2B cells (Figure 5B), respectively, after 24 h incubation (P < 0.01). In HBECs, IFN-{gamma} treatment significantly induced Smad 7 mRNA expression and further augmented Smad 7 mRNA expression stimulated by TGF-ß1 or -ß3 (150% and 50%, respectively; P < 0.01). In BEAS-2B cells, IFN-{gamma} had little effect on basal Smad 7 mRNA expression, but significantly attenuated TGF-ß1– or -ß3–stimulated Smad 7 mRNA expression (P < 0.01).



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  		Figure 5. Smad 7 mRNA expression in human bronchial epithelial cells and its modulation by IFN-{gamma}. HBECs (A) and BEAS-2B cells (B) were incubated for 24 h with LHC-B/RPMI containing 2.5 ng/ml TGF-ß1 or -ß3, or 200 U/ml IFN-{gamma} alone or in combination. Vertical axes: Smad 7 mRNA expression; horizontal axes: conditions. Values are means of triplicate cultures from a single experiment. Similar results were obtained in two other separate experiments. P < 0.01 by Student t test, compared with control (*), or TGF-ß1– or -ß3–stimulated cells (#).

 
In addition, the effect of IFN-{gamma} on Smad 2 and Smad 4 mRNA expression in HBECs and BEAS-2B cells was also evaluated. Smad 2 mRNA was not affected by treatment with IFN-{gamma} or TGF-ß1 alone in HBECs or BEAS-2B cells. However, attenuated Smad 2 mRNA expression was observed when cells were treated with both TGF-ß1 and IFN-{gamma} (data not shown). TGF-ß1 treatment resulted in increased expression of Smad 4 mRNA in HBECs, and this was blocked by IFN-{gamma} (data not shown).

Effect of Smad 7 mRNA Silencing on TGF-ß2 Production by HBECs
To further explore the mechanism of IFN-{gamma} inhibition on TGF-ß2 production by HBECs, the role of Smad 7 was evaluated using RNA interference. Smad 7 siRNA treatment resulted in a significant decrease of Smad 7 protein, whereas Smad 6 and cytokeratin were not affected (Figure 6A). Consistently, TGF-ß2 production was slightly increased by IFN-{gamma} in the HBECs treated with Smad 7 siRNA whereas it was decreased in control cells (Figure 6B). Addition of TGF-ß1 resulted in further stimulation of TGF-ß2 production in Smad 7 siRNA treated cells (Figure 6B).




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  		Figure 6. Smad 7 siRNA transfection in human bronchial epithelial cells and its effect on TGF-ß2 production. HBECs were transfected with Smad 7–specific siRNA as described in MATERIALS AND METHODS. (A) Immunoblot showing decreased Smad 6, Smad 7, and keratin with and without IFN-{gamma} and siRNA treatment. (B) TGF-ß2 production by HBECs after Smad 7 siRNA treatment. Open bars, cells cultured with LHC-D/RPMI only; striped bars, cells treated with 200 U/ml IFN-{gamma}; dotted bars, cells treated with 2.5 ng/ml TGF-ß1; filled bars, cells treated with IFN-{gamma} + TGF-ß1. Vertical axis: TGF-ß2 production (pg/d/106 cells); horizontal axis: siRNA treatment. *P < 0.05 or **P < 0.01 by Student t test, compared with control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that in human bronchial epithelial cells, IFN-{gamma} suppresses basal and TGF-ß1–, -ß3–, or IL-4–augmented TGF-ß2 release, but has little effect on IL-1ß- or PDGF-stimulated TGF-ß2 release. This effect was due, in part, to a decrease in TGF-ß2 mRNA levels. Correspondingly, IFN-{gamma} decreased Smad 3 nuclear staining and mRNA expression in the presence or absence of TGF-ß1 or -ß3. IFN-{gamma} also induced the expression of Smad 7 protein and mRNA in HBECs but not in BEAS-2B cells. Smad 7 siRNA transfection resulted in less Smad 7 protein and abrogated the inhibitory effect of IFN-{gamma} on TGF-ß2 production by HBECs. These results, therefore, suggest that IFN-{gamma} can modulate TGF-ß–augmented production of TGF-ß2 in human airway epithelial cells by targeting TGF-ß signaling proteins, Smad 3 and Smad 7. Remodeling of the bronchial wall is believed to be a major determinant of morbidity in asthma. An increased number of myofibroblasts beneath the bronchial epithelial basement membrane has been described in asthma (3, 17). The production of mediators by epithelial cells in close proximity to myofibroblasts during epithelial repair is one of the possible mechanisms leading to airway remodeling. In this regard, epithelial cells produce factors that can drive fibroblast recruitment, proliferation, matrix production, and matrix remodeling (1). TGF-ß, believed to modulate tissue repair in many settings, appears to play a key role in mediating several of these functions (5, 18, 19).

Using a three-dimensional co-culture system in which myofibroblasts derived from human bronchial wall were maintained in collagen gels and a human bronchial epithelial cell line, 16HBE14o-, grown on the surface of the gels, Zhang and coworkers found that myofibroblasts in the co-cultures showed significantly enhanced proliferation after poly-L-arginine–induced epithelial damage (20). Conditioned media from mechanically damaged epithelial cells also increased fibroblast proliferation. After epithelial perturbation, increased levels of growth factors, including TGF-ß2, PDGF, IGF-1, and endothelin-1, were found in supernatant culture media. Blockade of these growth factors inhibited fibroblast proliferation by 76% after epithelial injury, which suggests liberation and activation of potent cytokines like TGF-ß by epithelial cells in the process of tissue remodeling.

The level of the Th1 cytokine IFN-{gamma} has been found to be decreased in the BALF from patients with asthma (8). IFN-{gamma} downregulates the induction of IgE-secreting B cells. It is also a negative growth factor for Th2 lymphocytes and thus counteracts Th2-mediated allergic reactions (6). Loss of IFN-{gamma} action, therefore, has been suggested to contribute to the altered immune state characterizing asthma. Several lines of evidence suggest that decreased IFN-{gamma} could also alter tissue remodeling in asthma (21). In the bleomycin-mouse model of lung fibrosis, IFN-{gamma} downregulates TGF-ß gene expression and suppresses both the proliferation of fibroblasts and collagen synthesis (7). In patients with idiopathic pulmonary fibrosis, 6-mo treatment using IFN-{gamma} with a low dose of prednisolone has been reported to reduce the levels of transcription of both TGF-ß1 and connective tissue growth factor (CTGF) genes, and to improve lung function and blood gases (22).

There are three isoforms of TGF-ß: TGF-ß1, -ß2, and -ß3, themselves members of a large family of signaling molecules. The TGF-ßs are believed to regulate cell proliferation, differentiation, and, in particular, cell matrix interactions both during development and repair following injury (2). By acting on the TGF-ß R2, TGF-ßs lead to phosphorylation and activation of TGF-ß R1, which in turn leads to phosphorylation of Smad 2 and Smad 3. These molecules, termed receptor-activated Smads, then dimerize or bind to Smad 4 and enter the nucleus, where they can modulate gene expression. Smad 3, in particular, is believed to play a key role in TGF-ß–induced production of TGF-ß isoforms (23).

Inhibitory Smads can interfere with TGF-ß signaling. By binding to the TGF-ß receptor, they can prevent phosphorylation of the receptor-activated Smads. The inhibitory Smad, Smad 7, has been suggested to play an important role in mediating IFN-{gamma} interference with TGF-ß signaling (24, 25). Induction of Smad 7 expression has been reported by Ulloa and colleagues in U4A/Jak1 cells, a transfected cancer cell line (9). In addition, TGF-ß may induce autoregulatory inhibition by inducing the nuclear cytoplasmic transport of Smad 7, although this may vary with cell type (26).

The current study supports and extends a role for Smad 7 in modulating IFN-{gamma} regulation of TGF-ß activity, as well as provides evidence for cell specificity in these mechanisms. Normal HBECs demonstrated IFN-{gamma} induction of Smad 7. In contrast, the widely used human bronchial epithelial cell line, BEAS-2B, demonstrated suppression of Smad 7 in response to IFN-{gamma}. In contrast to these opposing effects with regard to Smad 7, very similar effects of IFN-{gamma} were observed on Smad 3. IFN-{gamma} reduced Smad 3 expression as reflected by both immunofluorescence and mRNA, and also reduced Smad 3 nuclear localization. Similar effects of a smaller magnitude were observed on Smad 4, whereas very modest effects were observed on Smad 2.

TGF-ß and IFN-{gamma} signal through distinct signaling pathways. The present study demonstrates mechanisms for cross-talk between these pathways. In HBECs, IFN-{gamma} may decrease TGF-ß induction of TGF-ß isoform production by at least two mechanisms: first, by decreasing Smad 3 levels; and second, by increasing Smad 7. In contrast, IFN-{gamma} decreases TGF-ß production in BEAS-2B cells by interfering with Smad 3 signaling and decreasing Smad 3 levels. In this context, reduction in Smad 3 signaling has also been suggested to be a feedback control mechanism controlling TGF-ß–induced fibrosis in vivo (27). Interestingly, the inhibition of TGF-ß production demonstrates some specificity. The augmented TGF-ß production induced by EGF, IL-1ß, and PDGF was unaffected by IFN-{gamma}. Finally, under the experimental conditions used, neither IFN-{gamma} nor TGF-ß affected Smad 2 expression. However, together the cytokines inhibited Smad 2 expression. Although the mechanism for this apparent synergy is unexplained, the synergistic increase in Smad 7 induced by the two cytokines together is one possible mechanism.

In summary, the normal airway has considerable capacity for repair. TGF-ß is believed to play an important role both in normal airway repair and in the remodeling processes that characterize airway disease (28–30). Inflammation, however, is a characteristic feature of both airway diseases such as asthma and chronic bronchitis, and also of the normal airway following injury (29–31). The current study suggests that interactions among mediators present in the inflammatory milieu, such as IFN-{gamma}, have the capacity for modulating TGF-ß response. These results raise the possibility that these mediators may play key roles in regulating the repair and remodeling responses. In diseases where these mediators are present either in excess or are deficient, abnormal repair and remodeling may result.


   Acknowledgments
 
The authors thank Ms. Lillian Richards for secretarial support and Ms. Mary Tourek for editorial assistance. This work was supported by grant RO1-HL64088 from National Heart Lung and Blood Institute and by the Larson Endowment, University of Nebraska Medical Center, Omaha, Nebraska.

Received in original form November 13, 2002

Received in final form December 24, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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