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Insulin-Like Growth Factor-Binding Protein-5 Induces Pulmonary Fibrosis and Triggers Mononuclear Cellular Infiltration

2006; Elsevier BV; Volume: 169; Issue: 5 Linguagem: Inglês

10.2353/ajpath.2006.060501

1525-2191

Hidekata Yasuoka, Zhihong Zhou, Joseph M. Pilewski, Tim D. Oury, Augustine M.K. Choi, Carol Feghali‐Bostwick,

Chronic Obstructive Pulmonary Disease (COPD) Research

We have recently shown that insulin-like growth factor-binding protein (IGFBP)-5 is overexpressed in idiopathic pulmonary fibrosis lung tissues and increases collagen and fibronectin deposition. Here, we further examined the effect of IGFBP-5 in vivo by intratracheal administration of replication-deficient adenovirus expressing human IGFBP-5 (Ad5), IGFBP-3 (Ad3), or no cDNA (cAd) to wild-type mice. Increased cellular infiltration and extracellular matrix deposition were observed in mice after Ad5 administration compared with Ad3 and cAd. Mononuclear cell infiltration consisted predominantly of T lymphocytes at day 8. By day 14, the number of infiltrating T cells decreased, whereas that of B cells and monocytes/macrophages increased. IGFBP-5 also induced migration of peripheral blood mononuclear cells in vitro, suggesting that in vivo mononuclear cell infiltration may be the direct result of IGFBP-5 expression. α-Smooth muscle actin and Mucin-1 co-localized in cells of mice treated with Ad5, suggesting that IGFBP-5 induced epithelial-mesenchymal transition. In addition, exogenous IGFBP-5 induced α-smooth muscle actin expression in primary fibroblasts and epithelial-mesenchymal transition of pulmonary epithelial cells in vitro. In conclusion, our results suggest that overexpression of IGFBP-5 in mouse lung results in fibroblast activation, increased extracellular matrix deposition, and myofibroblastic changes. Thus, the IGFBP-5-induced fibrotic phenotype in vivo may represent a novel model to better understand the pathogenesis of fibrosis. We have recently shown that insulin-like growth factor-binding protein (IGFBP)-5 is overexpressed in idiopathic pulmonary fibrosis lung tissues and increases collagen and fibronectin deposition. Here, we further examined the effect of IGFBP-5 in vivo by intratracheal administration of replication-deficient adenovirus expressing human IGFBP-5 (Ad5), IGFBP-3 (Ad3), or no cDNA (cAd) to wild-type mice. Increased cellular infiltration and extracellular matrix deposition were observed in mice after Ad5 administration compared with Ad3 and cAd. Mononuclear cell infiltration consisted predominantly of T lymphocytes at day 8. By day 14, the number of infiltrating T cells decreased, whereas that of B cells and monocytes/macrophages increased. IGFBP-5 also induced migration of peripheral blood mononuclear cells in vitro, suggesting that in vivo mononuclear cell infiltration may be the direct result of IGFBP-5 expression. α-Smooth muscle actin and Mucin-1 co-localized in cells of mice treated with Ad5, suggesting that IGFBP-5 induced epithelial-mesenchymal transition. In addition, exogenous IGFBP-5 induced α-smooth muscle actin expression in primary fibroblasts and epithelial-mesenchymal transition of pulmonary epithelial cells in vitro. In conclusion, our results suggest that overexpression of IGFBP-5 in mouse lung results in fibroblast activation, increased extracellular matrix deposition, and myofibroblastic changes. Thus, the IGFBP-5-induced fibrotic phenotype in vivo may represent a novel model to better understand the pathogenesis of fibrosis. Lung fibrosis in idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) is characterized by excessive fibrosis attributable to fibroblast proliferation and activation resulting in excessive production of extracellular matrix (ECM).1American Thoracic Society Idiopathic pulmonary fibrosis: diagnosis and treatment. International Consensus Statement. American Thoracic Society and the European Respiratory Society.Am J Respir Crit Care Med. 2000; 161: 646-664Crossref PubMed Scopus (981) Google Scholar However, the pathogenic mechanisms of fibrosis are still unknown. Fibrotic changes result in structural changes and lead to alveolar destruction and progressive restrictive lung disease.1American Thoracic Society Idiopathic pulmonary fibrosis: diagnosis and treatment. International Consensus Statement. American Thoracic Society and the European Respiratory Society.Am J Respir Crit Care Med. 2000; 161: 646-664Crossref PubMed Scopus (981) Google Scholar, 2Gross TJ Hunninghake GW Idiopathic pulmonary fibrosis.N Engl J Med. 2001; 345: 517-525Crossref PubMed Scopus (868) Google Scholar In IPF and SSc these changes result in significant morbidity and mortality.3Bjoraker JA Ryu JH Edwin MK Myers JL Tazelaar HD Schroeder DR Offord KP Prognostic significance of histopathologic subsets in idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 1998; 157: 199-203Crossref PubMed Scopus (968) Google Scholar In SSc, pulmonary involvement is currently the leading cause of death.4Steen VD Medsger Jr, TA Severe organ involvement in systemic sclerosis with diffuse scleroderma.Arthritis Rheum. 2000; 43: 2437-2444Crossref PubMed Scopus (738) Google Scholar There is no effective treatment to halt the progression of pulmonary fibrosis in IPF and SSc, and the only current life-prolonging intervention is lung transplantation.5Douglas WW Ryu JH Schroeder DR Idiopathic pulmonary fibrosis: impact of oxygen and colchicine, predonisone, or no therapy on survival.Am J Respir Crit Care Med. 2000; 161: 1172-1178Crossref PubMed Scopus (241) Google Scholar The pathological features of IPF are injury and activation of alveolar epithelial cells in a heterogeneous subpleural distribution, the distinctive presence of fibroblastic foci, and excessive deposition of ECM proteins including collagen and fibronectin by resident fibroblasts.1American Thoracic Society Idiopathic pulmonary fibrosis: diagnosis and treatment. International Consensus Statement. American Thoracic Society and the European Respiratory Society.Am J Respir Crit Care Med. 2000; 161: 646-664Crossref PubMed Scopus (981) Google Scholar, 6Katzenstein AL Myers JL Idiopathic pulmonary fibrosis: clinical relevance of pathologic classification.Am J Respir Crit Care Med. 1998; 157: 1301-1315Crossref PubMed Scopus (1167) Google Scholar Thus, fibroblasts and epithelial cells play an important role in the pathogenesis of pulmonary fibrosis.In addition, inflammation may also contribute to the pathogenesis of lung fibrosis.7Kamp DW Idiopathic pulmonary fibrosis: the inflammation hypothesis revisited.Chest. 2003; 124: 1187-1190Crossref PubMed Scopus (28) Google Scholar Increased numbers of lymphocytes have been detected in the bronchoalveolar lavage fluid of SSc patients with lung involvement.8Frigieri L Mormile F Grilli N Mancini D Ciappi G Pagliari G Magaro M Flamini G Bilateral bronchoalveolar lavage in progressive systemic sclerosis: interlobar variability, lymphocytes subpopulations, and functional correlations.Respiration. 1991; 58: 132-140Crossref PubMed Scopus (18) Google Scholar Inflammatory cells not only release cytokines, chemokines, and growth factors during the immunoinflammatory phase of the disease but also regulate the fibrogenic phase of the disease process through direct interaction with fibroblasts, thus resulting in the development of fibroproliferative lesions.9Bombara MP Webb DL Conrad P Marlor CW Sarr T Ranges GE Aune TM Greve JM Blue ML Cell contact between T cells and synovial fibroblasts causes induction of adhesion molecules and cytokines.J Leukoc Biol. 1993; 54: 399-406PubMed Google Scholar, 10Burger D Rezzonico R Li JM Modoux C Pierce RA Welgus HG Dayer JM Imbalance between interstitial collagenase and tissue inhibitor of metalloproteinases 1 in synoviocytes and fibroblasts upon direct contact with stimulated T lymphocytes: involvement of membrane-associated cytokines.Arthritis Rheum. 1998; 41: 1748-1759Crossref PubMed Scopus (146) Google Scholar Recent studies indicate that some chemokines recruit not only inflammatory cells but also fibrocytes, which are associated with ECM production and induction of fibrosis.11Phillips RJ Burdick MD Hong K Lutz MA Murray LA Xue YY Belperio JA Keane MP Strieter RM Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis.J Clin Invest. 2004; 114: 438-446Crossref PubMed Scopus (911) Google Scholar, 12Moore BB Kolodsick JE Thannickal VJ Cooke K Moore TA Hogaboam C Wilke CA Toews GB CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury.Am J Pathol. 2005; 166: 675-684Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar To date, there are no reports of a single molecule that can simultaneously explain both resident fibroblast activation and recruitment of inflammatory cells.We previously reported increased insulin-like growth factor-binding protein (IGFBP)-5 mRNA and protein levels in lung tissues of patients with lung fibrosis and in primary fibroblasts cultured from lung tissues of patients with IPF.13Pilewski JM Liu L Henry AC Knauer AV Feghali-Bostwick CA Insulin-like growth factor binding protein 3 and 5 are overexpressed in idiopathic pulmonary fibrosis and contribute to extracellular matrix deposition.Am J Pathol. 2005; 166: 399-407Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar In addition, we have shown that overexpression of IGFBP-5 in normal primary fibroblasts increases collagen and fibronectin deposition in the extracellular milieu.13Pilewski JM Liu L Henry AC Knauer AV Feghali-Bostwick CA Insulin-like growth factor binding protein 3 and 5 are overexpressed in idiopathic pulmonary fibrosis and contribute to extracellular matrix deposition.Am J Pathol. 2005; 166: 399-407Abstract Full Text Full Text PDF PubMed Scopus (173) Google ScholarIn this study, we report the in vivo effects of human IGFBP-5 expression in the mouse. Expression of IGFBP-5 induced prominent inflammatory changes and fibrosis resembling human fibrotic disease states. IGFBP-5 confers chemoattractant activity and can induce production and deposition of ECM through fibroblast activation and myofibroblast differentiation. IGFBP-5 also induced myofibroblastic changes of type II alveolar epithelial cells in vivo. These data suggest that IGFBP-5 is sufficient to initiate the histopathological changes characteristic of IPF and SSc, and in conjunction with our prior observations, strongly implicate IGFBP-5 in the pathogenesis of human IPF and SSc. In addition, the overexpression of IGFBP-5 provides a novel murine model to study the pathogenesis of human pulmonary fibrotic diseases such as IPF and SSc.Materials and MethodsAdenovirus Construct PreparationAdenovirus constructs were obtained as previously reported.13Pilewski JM Liu L Henry AC Knauer AV Feghali-Bostwick CA Insulin-like growth factor binding protein 3 and 5 are overexpressed in idiopathic pulmonary fibrosis and contribute to extracellular matrix deposition.Am J Pathol. 2005; 166: 399-407Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar Briefly, the full-length cDNAs of human IGFBP-3 and −5 were obtained by reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from primary human lung fibroblasts. The cDNAs were subcloned into the shuttle vector pAdlox and used for the preparation of replication-deficient adenovirus serotype 5 expressing IGFBP-3 (Ad3), IGFBP-5 (Ad5), or no cDNA (cAd) in the Vector Core Facility at the University of Pittsburgh.Mice and Adenoviral AdministrationEight-week-old wild-type C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). All studies and procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Mice were intratracheally injected with phosphate-buffered saline (PBS), or 109 plaque-forming units (PFU) of cAd, Ad3, or Ad5 in a 55-μl volume. Mice were sacrificed on days 8 and 14 after adenoviral administration. Harvested lung tissues were inflated and embedded in paraffin.Culture of Primary Mouse Lung FibroblastsPrimary mouse fibroblasts from lung tissues of C57BL/6J mice were cultured in Dulbecco's modified Eagle's medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO), penicillin, streptomycin, and anti-mycotic agent (Invitrogen Life Technologies), as previously described.14Feghali CA Wright TM Identification of multiple, differentially expressed mRNAs in dermal fibroblasts from patients with systemic sclerosis.Arthritis Rheum. 1999; 42: 1451-1457Crossref PubMed Scopus (58) Google Scholar Cells were used in early passage (passages 3 to 6).Western Blot AnalysisCulture supernatants, ECM, and cellular lysates were obtained from cultured fibroblasts or the type II alveolar epithelial cell line A549, as previously described.15Zheng B Clemmons DR Methods for preparing extracellular matrix and quantifying insulin-like growth factor-binding protein binding to the ECM.Methods Mol Biol. 2000; 139: 221-230PubMed Google Scholar In brief, 2.0 × 105 primary fibroblasts or A549 cells were cultured in 35-mm wells in the absence or presence of 500 ng/ml of recombinant human IGFBP-5. In parallel wells, fibroblasts were infected with cAd, Ad3, or Ad5 at a multiplicity of infection of 50. Culture supernatants, ECM, and cellular lysates were harvested at 48 hours. Culture supernatants were centrifuged for 10 minutes at 4°C to pellet cell debris. ECM was harvested as we have previously described.13Pilewski JM Liu L Henry AC Knauer AV Feghali-Bostwick CA Insulin-like growth factor binding protein 3 and 5 are overexpressed in idiopathic pulmonary fibrosis and contribute to extracellular matrix deposition.Am J Pathol. 2005; 166: 399-407Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar Samples were analyzed by Western blot analysis using one of the following antibodies: anti-human IGFBP-3 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-IGFBP-5 (Gropep Ltd., Thebarton, Australia), anti-fibronectin (Santa Cruz Biotechnology), or anti-α-smooth muscle actin (α-SMA) (Sigma-Aldrich). Signals were detected after incubation with horseradish peroxidase-conjugated secondary antibody and chemiluminescence (Perkin Elmer Life Sciences, Inc., Boston, MA).Hydroxyproline AssayHydroxyproline incorporation was assayed as previously described.16Oury TD Thakker K Menache M Chang LY Crapo JD Day BJ Attenuation of bleomycin-induced pulmonary fibrosis by a catalytic antioxidant metalloporphyrin.Am J Respir Cell Mol Biol. 2001; 25: 164-169Crossref PubMed Scopus (127) Google Scholar, 17Woessner Jr, JF The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid.Arch Biochem Biophys. 1961; 93: 440-447Crossref PubMed Scopus (3346) Google Scholar In brief, lung tissues were dried, acid-hydrolyzed, lyophilized, and assayed for hydroxyproline content using chloramine-T.Histological Staining for CollagenFor the detection of collagen fibers, 6-μm sections of paraffin-embedded skin tissues were deparaffinized and stained using the Chromaview Masson-trichrome stain kit (Richard-Allan Scientific, Kalamazoo, MI) following the manufacturer's recommendations.ImmunohistochemistrySix-μm sections of paraffin-embedded skin tissues were deparaffinized, and endogenous peroxidase and biotin were quenched using 10% H2O2 and a biotin blocking kit (Dakocytomation, Carpinteria, CA), respectively. The sections were blocked with 5% serum and incubated with one of the following primary antibodies: polyclonal anti-IGFBP-3, anti-IGFBP-5 (Gropep Ltd.), polyclonal anti-vimentin (LabVision Corp., Fremont, CA), polyclonal anti-proliferating cell nuclear antigen (Santa Cruz Biotechnology), polyclonal anti-fibronectin (Santa Cruz Biotechnology), monoclonal anti-α-SMA (Sigma-Aldrich), monoclonal anti-CD3 (LabVision Corp.), monoclonal anti-B220 (BD Pharmingen, San Diego, CA), monoclonal anti-Mac-1 (Chemicon, Temecula, CA), and polyclonal anti-Mucin-1 (Fujirebio Diagnostics, Malvern, PA) antibodies. Sections were washed and incubated with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA). Bound secondary antibody was detected using the AEC Red kit (Zymed, San Francisco, CA) or fluorescence detection kit (Zymed). A light hematoxylin counterstain was used to identify nuclei using Hematoxylin QS (Vector Laboratories). Images were taken on a Nikon Eclipse 800 microscope (Nikon Instruments, Inc., Huntley, IL) or Olympus Fluoview 1000 (Olympus America Inc., Melville, NY) using identical camera settings.Chemotaxis AssayChemoattractant activity was examined by trans-well migration assay using human peripheral blood mononuclear cells (PBMCs). Chemoattractant activity was assessed in a 24-well trans-well cell culture dish with 5-μm-pore-size polycarbonate filters (Costar, Cambridge, MA). Human PBMCs were obtained from heparinized venous blood using Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden) gradient centrifugation. PBMCs were resuspended in RPMI 1640 supplemented with 1% bovine serum albumin, and 2 × 105 of PBMCs in 0.1 ml of medium were added to the upper compartment of each chamber. Recombinant IGFBP-3, IGFBP-5, RANTES, or vehicle was diluted in RPMI 1640 supplemented with 1% bovine serum albumin and added to the lower compartment. After a 4-hour-incubation at 37°C, cells were harvested from both upper and lower chambers and manually counted under a phase contrast microscope. The percentage of migrated cells was calculated as a ratio of the cell count of the upper chamber to the total cell count of the upper and lower chambers.Statistical AnalysisStatistical comparisons were performed using the Mann-Whitney U-test or Student's t-test as appropriate.ResultsIn Vitro Expression of Human IGFBPs in Primary Mouse FibroblastsWe had previously shown that replication-deficient adenovirus serotype 5 can be used for efficient overexpression of IGFBP-3 and −5 in primary human lung fibroblasts.13Pilewski JM Liu L Henry AC Knauer AV Feghali-Bostwick CA Insulin-like growth factor binding protein 3 and 5 are overexpressed in idiopathic pulmonary fibrosis and contribute to extracellular matrix deposition.Am J Pathol. 2005; 166: 399-407Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar To confirm the expression of adenovirally expressed human IGFBP in mouse fibroblasts, we infected primary mouse lung fibroblasts cultured from lung tissues of wild-type C57BL/6J mice with replication-deficient adenovirus expressing human IGFBP-3 or-5 (Ad3 and Ad5, respectively) or no cDNA (cAd). Human IGFBP-3 and −5 were secreted by mouse fibroblasts as detected by Western blot analysis of culture supernatants. No human IGFBP-3 or IGFBP-5 expression was detected in fibroblasts infected with a control adenovirus (cAd) (Figure 1A).Detection of IGFBP-Expressing Cells In VivoHuman IGFBP-expressing cells were detected in mouse lung tissues harvested 8 days after adenoviral administration. As shown in Figure 1B, immunohistochemical analysis showed increased expression of human IGFBP-5 in Ad5-treated mice (Figure 1B, c and d) compared with Ad3- and cAd-treated mice (Figure 1B, b and a, respectively). Weak expression of IGFBP-5 was detected in the Ad3-treated lung (Figure 1Bb). The anti-IGFBP-5 antibody, although directed against the human protein, may display some cross-reactivity with endogenous mouse IGFBP-5 because of the high degree of homology between human and mouse proteins. IGFBP-3 was detected in Ad3-treated lungs (Figure 1Bf) and to a lesser extent in Ad5-treated lungs (Figure 1Bg). In Ad5-treated lung, IGFBP-5 expression was primarily localized to scattered cells in the alveolar lumen and wall (Figure 1Bh).Fibrotic and Proinflammatory Effects of IGFBP-5To analyze the in vivo effects of human IGFBP-5 expression in mouse lung, histopathological changes were analyzed using hematoxylin and eosin (H&E) staining and Masson's trichrome staining. As shown in Figure 2A, cellular infiltration and deposition of collagen were prominent, especially around airways, in Ad5-treated mice (Figure 2Ad) compared with PBS- (Figure 2Aa), Ad3- (Figure 2Ab), and cAd-treated (Figure 2Ac) mice at 14 days after adenoviral administration. In Figure 2B, ECM deposition (homogenous-acidic deposits) was observed in Ad5-treated mice at high magnification (arrows in Figure 2Bc) compared with cAd (Figure 2Ba) and Ad3 (Figure 2Bb) mouse lung tissues. Masson's Trichrome stain demonstrated increased deposition of collagen in Ad5 lung tissues (Figure 2Bf) compared with cAd (Figure 2Bd) and Ad3 (Figure 2Be) lungs.Figure 2In vivo effects of IGFBP-3 and IGFBP-5 overexpression in mouse lung. A: Increased collagen deposition in IGFBP-5-expressing lungs. Phosphate-buffered saline (PBS) (a), 109 PFU control adenovirus (cAd) (c), or IGFBP-3- and IGFBP-5-expressing adenovirus [Ad3 (b) and Ad5 (d), respectively] were administered intratracheally to mice. Mice were sacrificed 2 weeks later, and lung tissue sections were stained with Masson's Trichrome. B: Mononuclear cell infiltration and ECM deposition in IGFBP-5-expressing lungs. Lung sections from cAd-, Ad3-, and Ad5-treated mice were stained with H&E (a–c) or Masson Trichrome staining (d–f). Original magnifications: ×20 (A); ×200 (Ba–Bc); ×100 (Bd–Bf).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Characterization of Infiltrating Cells in IGFBP-5-Expressing LungsImmunochemistry was performed to characterize the infiltrating mononuclear cells in Ad5-treated lungs. As shown in Figure 3A, on day 8 T cells were the predominant cell type in the alveolar area (Figure 3Aa). Some scattered B cells (Figure 3Ab) and several monocytes/macrophages (Figure 3Ac) were also observed around the vessels at day 8 after adenoviral administration. At day 14, the number of T cells decreased (Figure 3, Ad and B) (104.80 ± 39.95/HPF versus 22.65 ± 22.07/HPF, P = 5.5 × 10−9) and the number of B cells (Figure 3Ae) and monocytes/macrophages (Figure 3Af) increased (50.30 ± 30.64/HPF versus 107.60 ± 83.02/HPF and 17.4 ± 10.81/HPF versus 57.55 ± 30.36/HPF, P = 0.008, and P = 9.8 × 10−6, respectively) (Figure 3B). Moreover, B cells appeared to form germinal center-like clusters (not shown). Infiltrating mononuclear cells were rare in cAd-treated lungs (day 8: T cells, 5.50 ± 2.43/HPF; B cells, 2.67 ± 1.15/HPF; monocytes/macrophages, 2.57 ± 2.44/HPF; day 14: T cells, 5.11 ± 2.57/HPF; B cells, 4.17 ± 3.13/HPF; monocytes/macrophages, 1.75 ± 1.48/HPF).Figure 3Characterization of infiltrating immune cells. A: Immunohistochemistry for the detection of T lymphocytes, B lymphocytes, and monocytes/macrophages. Mice were infected with 109 PFU control-, IGFBP-3-, or IGFBP-5-expressing adenovirus (cAd, Ad3, and Ad5, respectively), and lung samples were analyzed at 8 days and 14 days after injection. Solid arrows point to representative positive cells. B: Number of T cells, B cells, and monocytes/macrophages in mouse lungs. Two representative samples were randomly selected from each group of mice and the number of each cell subset was counted in 10 random fields/sample at ×400 magnification. Statistical analysis was done using Student's t-test. Each bar denotes the mean of each group. C: PBMC migration assay. Purified human PBMCs were used in a migration assay with vehicle control, recombinant IGFBP-3 (BP3), recombinant human IGFBP-5 (BP5), or RANTES as a positive control. Statistical analysis was done using the Mann-Whitney U-test. *P < 0.007. Original magnifications, ×400.View Large Image Figure ViewerDownload Hi-res image Download (PPT)IGFBP-5 Has Chemoattractant Activity for PBMCsTo determine whether IGFBP-5 can directly recruit immune cells, we examined the chemoattractant activity of IGFBP-5 using transwell migration assays. As shown in Figure 3C, 500 ng/ml of recombinant IGFBP-5 induced significant migration of human PBMCs compared with vehicle control or recombinant IGFBP-3 at the same concentration (19.40 ± 4.87% versus 3.22 ± 1.46% or 4.90 ± 2.13%; P < 0.007 and P < 0.007, respectively). IGFBP-5 concentrations used are within physiological range 200 to 800 ng/ml.18Mohan S Libanati C Dony C Lang K Srinivasan N Baylink DJ Development, variation, and application of radioimmunoassay for insulin-like growth factor binding protein-5 in human serum and other biological fluids.J Clin Endocrinol Metab. 1995; 80: 2638-2645Crossref PubMed Google Scholar, 19Baxter RC Meka S Firth SM Molecular distribution of IGF binding protein-5 in human serum.J Clin Endocrinol Metab. 2002; 87: 271-276Crossref PubMed Scopus (38) Google Scholar, 20Rooman RP De Beeck LO Martin M van Doorn J Mohan S Du Caju MV Ethinylestradiol and testosterone have divergent effects on circulating IGF system components in adolescents with constitutional tall stature.Eur J Endocrinol. 2005; 152: 597-604Crossref PubMed Scopus (30) Google ScholarQuantitation of Collagen Deposition in IGFBP-5-Expressing LungsTo quantify the collagen levels deposited in mouse lung tissues, the amount of hydroxyproline incorporation in lungs harvested at day 14 after adenoviral administration was measured. As shown in Figure 4A, deposition of collagen was significantly increased in lung tissues of Ad5-treated mice compared with those of Ad3- or cAd-treated mice (597.11 ± 60.64 μg versus 518.58 ± 100.73 μg or 516.38 ± 88.10 μg; P < 0.03 and P < 0.03, respectively).Figure 4A: Collagen production and deposition in lung tissues. The amount of collagen was measured in lungs injected with 109 PFU control adenovirus-, IGFBP-3-, or IGFBP-5-expressing adenoviruses (cAd, Ad3, and Ad5, respectively) using a hydroxyproline assay. The bar in each group denotes mean collagen amount. Statistical analysis was done using the Mann-Whitney U-test. *P < 0.03. B: Expression of fibronectin and α-SMA in mouse lungs 14 days after injection. Expression of fibronectin and α-SMA was examined in mouse lung samples infected with 109 PFU control adenovirus (cAd), IGFBP-3- or IGFBP-5-expressing adenovirus (Ad3 and Ad5, respectively). Fibronectin (a–c) and α-SMA (d–f) were detected by immunohistochemistry. Solid arrows point to representative positive cells. C: Detection of fibronectin and α-SMA by Western blot analysis. Primary mouse lung fibroblasts were incubated with vehicle control or recombinant human IGFBP-5 (rIGFBP-5). ECM and lysates were harvested after 48 hours and analyzed by Western blot for fibronectin and α-SMA. β-Actin was detected as a loading control. Each experiment was done in duplicate. Original magnifications: ×200 (a–f); ×1000 (inset).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Comparison of Fibronectin Levels and Fibroblast PhenotypeThe deposition of fibronectin was examined by immunohistochemistry. As shown in Figure 4B, fibronectin staining was increased around airways and blood vessels in Ad5 lung tissues (Figure 4Bc) compared with cAd (Figure 4Ba) and Ad3 (Figure 4Bb) lung tissues. To characterize the fibroblast phenotype, α-SMA expression was examined. As shown in Figure 4B, α-SMA-positive fibroblasts were more abundant in Ad5 lung tissues (Figure 4Bf) compared with cAd (Figure 4Bd) and Ad3 lungs (Figure 4Be). To determine whether IGFBP-5 can also induce fibronectin and α-SMA expression in vitro, primary mouse lung fibroblasts were incubated with a physiological concentration of recombinant IGFBP-5. As shown in Figure 4C, expression of fibronectin and α-SMA increased in the presence of exogenous recombinant IGFBP-5 compared with vehicle control. Thus, IGFBP-5 induces increased deposition of fibronectin and transformation of lung fibroblasts into a myofibroblastic phenotype.Epithelial-Mesenchymal Transition of Type II Alveolar Epithelial Cells Is Induced by IGFBP-5We further examined the effect of IGFBP-5 on type II alveolar epithelial cells. As shown in Figure 5A, type II epithelial cells expressing Mucin-1 (Figure 5A, b and e) and α-SMA (Figure 5A, a and d) were increased in Ad5-treated lungs (Figure 5A, d–f) compared with cAd-lungs (Figure 5A, a–c). Increased numbers of cells expressing both Mucin-1 and α-SMA were detected in Ad5-treated mice (Figure 5, Af and Bb), suggesting that IGFBP-5 results in α-SMA expression in vivo. To examine the effect of IGFBP-5 on α-SMA expression by type II alveolar epithelial cells in vitro, A549 cells were cultured with various dilutions of cAd- or Ad5-infected primary fibroblast culture supernatants. Culture supernatants of fibroblasts infected with Ad5 secrete abundant levels of human IGFBP-5 as shown in Figure 1. As shown in Figure 5C, culture supernatants of Ad5-infected fibroblasts induce expression of α-SMA in A549 cells in a dose-dependent manner (Figure 5Ca). When 500 μl of culture supernatant was added to A549 cells (25% of the final volume), α-SMA expression was up-regulated in A549 cells incubated with supernatants from Ad5-infected fibroblasts compared with cAd-infected fibroblasts (Figure 5Cb). Thus, IGFBP-5 overexpression can induce epithelial-mesenchymal transition of type II alveolar epithelial cells.Figure 5Characterization of alveolar epithelial cells. A: Immunohistochemistry for α-SMA and Mucin-1. Mice were infected with 109 PFU control adenovirus (cAd) or IGFBP-5-expressing adenovirus (Ad5). Lung tissues were harvested 14 days after injection. Cells expressing α-SMA (green, a and d), Mucin-1 (red, b and e), and both α-SMA and Mucin-1 [yellow (merge), c and f] are detected in IGFBP-5-expressing lung (white arrows). Solid arrows point to representative positive cells. Red blood cells had high levels of autofluorescence even in the absence of antibody. The broken arrow with asterisk (c) shows representative autofluorescence of red blood cells (shown in yellow)