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1Department of Physiology, Michigan State University, East Lansing, Michigan; and 2Servicio de Neumologia, Institut Clinic del Tórax, Hospital Clinic, Universidad de Barcelona; 3Servicio de Anatomía Patológica, and 4Departamento de Prologia Experimental, Institut de Investigaciones Biomèdiques, Consejo Superior de Investigaciones Cientificas, Barcelona, Spain
Submitted 10 October 2005 ; accepted in final form 19 June 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-smooth muscle actin (-SMA) MAb] and an in situ DNA end labeling (ISEL) method to detect apoptosis. In normal human lung, IHC detected AGT protein in smooth muscle underlying normal bronchi and vessels, but not elsewhere. Real-time RT-PCR and Western blotting revealed that AGT mRNA and protein were 21-fold and 3.6-fold more abundant, respectively, in IPF lung biopsies relative to biopsies of normal human lung (both P < 0.05). In IPF lung, both AGT protein and mRNA were detected in AEC that double-labeled with MAb MNF-116 and with ISEL, suggesting AGT expression by apoptotic epithelia in situ. AGT protein and mRNA also colocalized to myofibroblast foci detected by -SMA MAb, but AGT mRNA was not detected in smooth muscle. These data are consistent with earlier data from isolated human lung cells in vitro and bleomycin-induced rat lung fibrosis models, and they suggest that apoptotic AEC and myofibroblasts constitute key sources of locally derived ANG peptides in the IPF lung. lung fibrosis; myofibroblast; alveolar epithelial cells; apoptosis
Several lines of evidence suggest that the source(s) of precursor for the ANG II synthesis that drives fibrogenesis in the lung is generated locally, i.e., within the lung tissue itself. Cultured AEC of either human or rat origin synthesize angiotensinogen (AGT) mRNA and secrete ANG peptides on exposure to proapoptotic stimuli such as Fas ligand (24), TNF- (21), or bleomycin (10). ANG II itself also induces apoptosis of AEC through ANG receptor AT1 (25). In addition, myofibroblasts isolated from the lungs of patients with idiopathic pulmonary fibrosis (IPF) also synthesize AGT mRNA constitutively and secrete ANG peptides (23). The notion that local pulmonary synthesis of ANG II de novo, i.e., from the precursor AGT within the lung, is required for lung fibrogenesis is supported by the recent demonstration that intratracheal administration of antisense oligonucleotides against AGT mRNA could prevent bleomycin-induced lung fibrosis in rats without affecting circulating levels of AGT protein (9).
To date, the evidence in support of local pulmonary synthesis of AGT protein de novo is derived from either animal models (8, 9) or indirect studies of primary cells isolated from fibrotic human lung (23). In the present study it was therefore of interest to directly examine fibrotic human lung tissue in an attempt to identify local tissue sources of ANG peptide generation. We report here the findings of ANG peptide expression in at least two cell types, apoptotic AEC and myofibroblasts, in lung tissue from patients with IPF.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-smooth muscle actin (-SMA; clone 1A4), propidium iodide (PI), purified AGT from human serum, and biotinylated 2'-deoxyuridine 5'-triphosphate (Bio-dUTP) were obtained from Sigma (St. Louis, MO). Anti-cytokeratin monoclonal antibody (MAb) MNF-116 was purchased from Dako Cytomation (Carpinteria, CA). Antibodies recognizing AGT and ANG peptides were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). DNA polymerase I was obtained from Promega (Madison, WI). Biotinylated oligonucleotides for in situ hybridization (ISH) were obtained from Invitrogen (Carlsbad, CA). Solutions A and B of the Vectastain ABC Elite formulation were purchased from Vector Laboratories (Burlingame, CA). All other materials were of reagent grade. Tissue samples and handling. Human lung tissue was obtained by open lung biopsy or video-assisted thoracoscopic surgery performed at Instituto del Tórax, Hospital Clínic de Barcelona. Fibrotic lung tissue was obtained from 12 patients with IPF; biopsies were obtained from more than one lung lobe. All patients had clinical, functional, and radiological features that fulfill the diagnostic criteria for an interstitial lung disease (ILD) (5). Briefly, all had progressive dyspnea, bilateral reticulonodular images on chest roentgenogram, restrictive lung functional impairment with decreased lung volumes and reduced single-breath carbon monoxide diffusing capacity, and hypoxemia at rest that worsened with exercise. Patients with IPF had neither antecedents of any occupational or environmental exposure nor any other known cause of ILD. None of the IPF patients had received steroids or other immunosupressant therapy at the time of clinical sample collection. Normal human lung tissue was obtained from individuals undergoing surgical treatment for spontaneous pneumothorax with no history of pulmonary disease. No histopathological evidence of disease was found in these tissue samples. Written informed consent was obtained from the patients according to institutional guidelines, and the study was approved by the Ethics Committee of Hospital Clínic de Barcelona.
All tissue was fixed in 10% neutral buffered formalin for 16 h and embedded in paraffin. Sections were cut at 5.0-µm thickness and mounted on glass coverslips. Human lung tissues designated for RNA isolation were immediately immersed in ice-cold TRI reagent (Molecular Research Center, Cincinnati, OH) after excision and were processed immediately. Lung tissues designated for analysis of proteins were flash-frozen in liquid nitrogen and stored at –80°C until protein isolation as described below.
In situ end labeling of fragmented DNA.
Tissue sections were deparaffinized by passing through xylene, xylene-alcohol (1:1), 100% alcohol, and 70% alcohol for 10 min each. In situ end labeling (ISEL) of fragmented DNA was conducted with a modification of the method of Mundle et al. (16) performed as described previously in IPF lung tissue (18). Briefly, ethanol was removed by rinsing in distilled water for at least 10 min. The slides were then placed in 0.23% periodic acid (Sigma) for 30 min at 20°C. Samples were rinsed once in water and three times in 0.15 M phosphate-buffered saline (PBS) for 4 min each and were then incubated in saline sodium citrate solution (0.3 M NaCl and 30 mM sodium citrate in water, pH 7.0) at 80°C for 20 min. After four rinses in PBS and four rinses in buffer A [50 mM Tris·HCl, 5 mM MgCl, 10 mM 
-mercaptoethanol, and 0.005% bovine serum albumin (BSA) in water, pH 7.5], the slides were incubated at 18°C for 2 h with ISEL solution (1.0 µM Bio-dUTP, 20 U/ml DNA polymerase I, and dATP, dCTP, and dGTP each at 0.01 mM in buffer A). Afterward the sections were rinsed thoroughly five times with buffer A and three additional times in 0.5 M PBS. For detections based on diaminobenzidine (DAB), the tissue was then incubated at 20°C with a solution consisting of 80 µl each of reagents A (avidin solution) and B (biotin-peroxidase solution) of the Vectastain Elite Kit (Burlingame, CA) in 3.84 ml of buffer B (1% BSA and 0.5% Tween 20 in 0.5 M PBS). After 30 min, the sections were washed four times in PBS and were then immersed for 10 min in a solution of 0.25 mg/ml DAB in 0.05 M Tris·HCl, pH 7.5, containing 0.01% hydrogen peroxide. Alternatively, detection of incorporated dUTP was achieved with a Fast Blue chromogen system. The tissues were rinsed in distilled water three times and mounted under Fluoromount solution (Southern Biotechnology Associates, Birmingham, AL).
Immunohistochemistry and in situ hybridization.
Immunohistochemistry (IHC) for ANG peptides, type II cell-specific cytokeratins, and -SMA was performed with anti-ANG peptide antibody (Santa Cruz Biotechnology; 1:50 dilution), anti-cytokeratin antibody MNF-116 (Dako; 1:50 dilution), and an -SMA-specific MAb (Sigma; 1:100 dilution). Deparaffinized lung sections were blocked with a solution of 3% BSA in PBS for 1 h; the primary antibody was then applied overnight at 4°C in 3% BSA-PBS. After a wash in PBS, the antibody was detected with a biotin-conjugated secondary antibody and avidin-linked chromogen system. Chromogens were either DAB (brown) or nitro blue tetrazolium (NBT; dark gray or black). For double labeling for ANG peptides and ISEL, ISEL was performed first as described above, and the next day ANG peptide primary antibody was applied for 2 h followed by detection with DAB chromogen. For double labeling with ANG peptide antibody and MNF-116 antibody, both primary antibodies were applied together overnight, and differential detection was achieved with anti-goat-horseradish peroxidase (HRP) secondary antibody (ANG peptide) or anti-mouse-AP (MNF-116) secondary antibody. Negative controls were obtained by completing the same procedure described above, but with omission of the primary antibody from the 3% BSA-PBS solution.
ISH was performed essentially as described previously (24). Deparaffinized slides were hybridized with digoxigenin-labeled antisense oligonucleotide DNA probes specific for AGT, which were detected with an amplified biotin-avidin system linked to NBT chromogen (purple). The digoxigenin-labeled probes used were 5'-AGGGTGGGGGAGGTGCTGAACAGC-3', as described by Lai et al. (7). A digoxigenin-labeled probe of the same base composition, but with scrambled sequence, was used as the control.
Microscopy and image acquisition.
The prepared lung sections were photographed under transmitted or epifluorescent light on an Olympus BH2 epifluorescence microscope fitted with a SPOT Slider digital camera. Images of green fluorescence (-SMA-FITC) were acquired through a 520-nm band-pass filter, and images of red fluorescence (PI) were acquired through a >570-nm long-pass filter.
RNA isolation and reverse transcriptase polymerase chain reaction.
Reverse transcriptase polymerase chain reaction (RT-PCR) was performed as described previously (21, 24), and real-time RT-PCR was performed in the Physiology Department of Michigan State University. The annealing temperatures for PCR reactions were optimized for each primer by preliminary trials. The identity of the PCR products was determined by expected size in 1.6% agarose gels and by DNA sequencing of the PCR product excised from agarose gels (not shown). Total RNA was extracted from biopsies with TRI reagent (Molecular Research Center) according to the manufacturer's protocol. First-strand cDNA was synthesized from 1 µg of total RNA with Superscript II reverse transcriptase (Invitrogen) and oligo (dT)12–18. Real-time RT-PCR was performed with cDNA synthesized from 50 ng of total RNA, SYBR Green PCR core reagents (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions, and 0.2 µM specific primers for human AGT (forward 5'-GAG CAA TGA CCG CAT CAG-3' and reverse 5'-CAC AGC AAA CAG GAA TGG-3') and -actin (forward 5'-AGG CCA ACC GCG AGA AGA TGA CC-3' and reverse 5'-gaa gtc cag ggc gac gta gc-3'), which produce PCR products of 151 and 332 bp, respectively. The PCR thermal profile started with 10-min activation of Taq polymerase at 95°C followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 37 s, and extension at 72°C for 37 s, ending with dissociation curve analysis to validate the specificity of the PCR products. Reactions were performed in a Mx3000P machine (Stratagene, La Jolla, CA) and threshold cycle (CT) data were collected with MxPro-Mx3000P software version 3.0. The relative AGT expression was normalized to -actin and calculated with the comparative CT method of 2–
CT.
Western blotting. Protein was extracted from biopsy samples by tissue homogenization in ice-cold Tris-buffered saline pH 8.0, supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany) and tributylphosphine. Soluble protein extracts (10 µg) were diluted 1:2 in Laemmli sample buffer (Bio-Rad, Hercules, CA), loaded on 10% Tris·HCl polyacrylamide gels, separated by SDS-PAGE, and then transferred to Immun-Blot polyvinylidene difluoride membrane (Bio-Rad) in Towbin buffer. Blotting membrane was blocked by 5% nonfat dry milk in 0.1% Tween 20 in Tris-buffered saline. Western blot analysis of AGT was performed with anti-ANG peptide antibody (1:400 dilution; Santa Cruz Biotechnology). Bands were visualized by HRP-conjugated donkey anti-goat secondary antibody (1:2,000 dilution; Santa Cruz Biotechnology) and the chemiluminescent substrate Super Signal West Femto Maximum Sensitivity (Pierce, Rockford, IL). Images of the chemiluminescence-exposed film were analyzed for band intensity with Scion Image software (release beta 4.0.2) and normalized to total protein band intensities obtained by silver staining of SDS-polyacrylamide gels of replicate biopsy extracts. Silver staining was performed with a commercially available kit (Silver Stain Plus, Bio-Rad) according to the manufacturer's instructions.
Cell culture. The human lung cell line A549 was cultured as described previously (23–25). The primary human lung fibroblast strain N13, isolated from normal human lung, was recovered from cryostorage and cultured as described previously (23). Before analysis, cells were switched from growth medium containing fetal bovine serum to serum-free medium (Ham's F-12) for at least 2 days before harvesting. Immunoreactive AGT was detected by Western blotting of cells lysed in buffer containing NP-40 detergent and a commercially available protease inhibitor cocktail.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Fig. 3. As shown in Fig. 1, normal human lung (Fig. 1, A–C) showed immunoreactivity (dark brown color) in smooth muscle underlying airways (arrowheads) and occasional alveolar wall cells (arrow) but did not label in the absence of the primary antibody (Fig. 1C). Smooth muscle underlying large vessels of normal lung also was immunoreactive for ANG peptides (Fig. 1B, brown). In the human IPF lung (Fig. 1D), intense immunoreactivity was observed in numerous areas resembling fibroblastic foci throughout the parenchyma (arrows) and in cells resembling cuboidal epithelia within the surfaces of air spaces (arrowheads).
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-actin mRNAs on total RNA isolated from lung biopsies from five IPF patients and four patients without fibrotic lung disease. In Fig. 2, AGT mRNA was found to be 21-fold more abundant in IPF lung tissue relative to the control specimens of human lung (P < 0.05). Figure 3 shows quantitation of immunoreactive AGT in total protein extracts from a panel of lung biopsy specimens and purified human lung cells. By Western blotting with the same antibodies used for IHC, two bands of differing molecular mass, which represent the two isoforms of human AGT, were detected in a commercially available purified AGT standard isolated from human serum (Fig. 3, lane 1) and in IPF lung (Fig. 3, lanes 8–12). In human serum, the higher (
61 kDa)- and lower (58 kDa)-molecular-mass isoforms of AGT were detected in apparently similar abundance. Cultured human lung epithelial cells (A549; Fig. 3, lane 2) and primary human lung fibroblasts (N13; Fig. 3, lane 3) expressed exclusively the 61-kDa isoform of AGT. Lung biopsies from both normal lung (Fig. 3, lanes 4–7) and IPF lung (Fig. 3, lanes 8–12) contained primarily the higher-molecular-mass isoform of AGT expressed by the isolated lung cells, although IPF biopsies did contain some of the low-molecular-mass isoform. Densitometric quantitation of the higher-molecular-mass (61 kDa) isoform of AGT only relative to total protein revealed 3.6-fold higher levels of the 61-kDa isoform of AGT in IPF lung biopsies compared to nonfibrotic lung (P < 0.05; Fig. 3). Because of the severely altered structure of IPF lung, the determination of cell type in tissue sections is difficult if based on morphology alone. To begin identifying the cell types labeled by the ANG peptide antibodies in Fig. 1, we subjected lung tissue from a patient with IPF to IHC with an antibody that recognizes type II pneumocytes (MAb MNF-116; Ref. 4), applied alone or together with the ANG peptide antibody. In Fig. 4, immunoreactivity for MAb MNF-116 (Fig. 4A, black) was observed in cuboidal epithelial cells that were usually located in air space corners, or occasionally in attenuated cells in the air space surfaces (arrowhead). In Fig. 4B, double labeling with both MAb MNF-116 and ANG peptide MAb revealed ANG peptide immunoreactivity (brown) within MNF-116-positive cells (black). Figure 4C shows ANG peptide reactivity detected with an NBT-based chromogen system (dark gray) in cuboidal epithelial cells that appeared to contain lamellar bodies (arrows). In some regions, cells with the morphology of alveolar macrophages showed positive labeling with anti-ANG peptide antibody but negative reactivity with MNF-116 (not shown).
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-SMA and ANG peptide. In Fig. 6, moderate immunoreactivity with -SMA and ANG peptide antibodies (green and brown, respectively) was found in smooth muscle underlying vessels (paired white and black arrowheads, Fig. 6, A and B), but intense ANG peptide immunoreactivity was found in myofibroblast foci (paired arrows, Fig. 6, A and B). In Fig. 6, C and D, higher magnification revealed precise colocalization of ANG peptide immunoreactivity (D) with small microfoci of myofibroblasts (C), denoted by paired arrows. In Fig. 6D, the arrowhead denotes ANG peptide labeling within nearby epithelium.
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-SMA (Fig. 7C) applied to adjacent serial sections. In contrast, no evidence for AGT mRNA was detected in the smooth muscle underlying airways (Fig. 7D) or large vessels (Fig. 7E), nor was label deposited by scrambled-sequence control oligonucleotides (Fig. 7F). Little positive ISH signal was detected in normal lung (not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Determinations of the mechanisms by which AGT expression is regulated in lung cells are the subject of ongoing investigations in this laboratory. Cultured AEC have been shown to synthesize ANG II in response to many proapoptotic stimuli in vitro, including Fas ligation (24), TNF- (21), and bleomycin (10). For this reason we sought to determine whether ANG peptide expression might be found in AEC together with markers of apoptosis within the IPF lung. The localization of ANG peptide immunoreactivity in cells that bind anti-cytokeratin antibody MNF-116 and contain putative lamellar bodies (Fig. 4) supports the interpretation that at least some of the cells expressing ANG peptides are type II pneumocytes. Moreover, the finding of positive labeling by ISH for AGT mRNA in both cuboidal epithelia and -actin-positive foci (Figs. 7, A–C) supports the contention that the ANG peptides are being synthesized de novo in AEC and myofibroblasts rather than being sequestered, for example, from other sources such as the blood. On the other hand, negative labeling for AGT mRNA in the smooth muscle underlying airway and vessel walls (Fig. 7, D and E) suggests that the positive IHC labeling of these cells (Fig. 1) may reflect uptake of ANG peptides rather than de novo synthesis.
Some cells with the morphology of alveolar macrophages and negative immunoreactivity to MNF-116 also were found to label with the ANG peptide antibody and by ISH for AGT mRNA (not shown), but positive markers of macrophage phenotype were not available for positive phenotype analyses. Although far from definitive, these preliminary observations are consistent with the studies of normal human alveolar macrophages by Dezso et al. (3) and suggest that alveolar macrophages may also be a source of ANG peptides in fibrotic human lung. The possibility of ANG peptide expression by alveolar macrophages will be an interesting topic for further study.
The ANG peptide antibody used for these studies, which was derived against the peptide ANG I, recognizes ANG I, ANG II, and the two isoforms of AGT found in serum (Fig. 3). Given that the 58- and 61-kDa isoforms of AGT are both found in human serum, whereas isolated lung cells express only the 61-kDa isoform (Fig. 3), the finding of both isoforms in IPF lung biopsies suggests that some of the increase in lung tissue AGT in the IPF specimens may be due to serum-derived AGT. On the other hand, the observation that the 61-kDa isoform expressed by isolated lung cells was more highly abundant than the 58-kDa form in both normal and IPF lung supports the theory that most of the increase in lung tissue AGT in IPF occurs through de novo synthesis of AGT within the lung.
Because of the reactivity of the ANG peptide antibodies to AGT, ANG I, and ANG II, it is not possible to determine from the IHC studies alone whether the labeled regions contain primarily precursor AGT or the processed ANG peptides ANG I and ANG II. On the other hand, in vitro experiments with human lung myofibroblasts isolated from IPF biopsies (23) or cultured human or rat AEC (21, 24) show that both myofibroblasts and apoptotic lung epithelial cells can constitutively convert newly synthesized AGT to ANG II, apparently by autocrine mechanisms. Thus it seems likely that at least some of the immunoreactivity observed within foci of apoptotic AEC and myofibroblasts consisted of the processed peptides ANG I and ANG II. Determinations of the abundance of these processed peptides in human lung and the kinetics of their appearance will be interesting topics for future investigations.
Regardless, the detection of ANG peptide immunoreactivity in epithelial cells that simultaneously labeled positively for fragmented DNA by ISEL (Fig. 5B) and also exhibited chromatin condensation and margination against the nuclear envelope (Figs. 5, C and D) strongly suggests ANG peptide expression by apoptotic AEC in IPF lung in situ. Thus these findings are consistent with earlier studies of cultured AEC exposed to proapoptotic stimuli in vitro (21, 24). Nonetheless, the stimuli that caused the apoptosis of AEC detected in the IPF lung biopsies studied here are unknown. The observation that occasional epithelial cells in the normal human lung were found to be ANG peptide positive but ISEL negative (Fig. 5A) might be explained by the kinetics of apoptosis; the DNA fragmentation detected by ISEL is a relatively late downstream event in apoptosis (1), whereas AGT expression occurs within 2–3 h (9). Thus, depending on the timing of tissue fixation, not all ANG-positive cells would be expected to colabel by ISEL, and the images of fibrotic lung are consistent with that interpretation (Fig. 5B). Regardless, the findings herein also are consistent with an earlier investigation of IPF lung biopsies that revealed ISEL within epithelial cells adjacent to foci of myofibroblasts and heavy collagen deposition (7).
The findings that foci of -SMA-positive cells also label heavily with the ANG peptide antibodies (Fig. 6) and AGT mRNA (Fig. 7, B and C) are consistent with other investigations showing ANG peptide expression by myofibroblasts of the fibrosing heart (26), kidney (6), and liver (27). They are also consistent with our earlier study (23) of myofibroblasts isolated from IPF lung biopsies, which synthesize and secrete ANG peptides in culture. Given that primary cultures of AEC undergo apoptosis on exposure to purified ANG II (25), TNF- (21), or Fas ligand (24), it is difficult to know which of these proapoptotic factors were responsible for the induction of DNA fragmentation in AEC within the IPF biopsies studied here (Fig. 5).
In animal models, both ACEI (15, 22) and ANG receptor AT1 antagonists (8, 14, 20) have been shown to prevent radiation- and chemical-induced experimental lung fibrosis; furthermore, knockout mice deficient in ANG receptor AT1a are resistant to bleomycin-induced lung fibrosis (17). On that basis, it is speculated that the production of ANG peptides by apoptotic AEC and myofibroblasts is an important component of the molecular mechanisms that maintain a profibrotic environment within the IPF lung. Experiments are in progress to determine whether blockade of ANG receptors in short-term explant cultures of IPF lung tissue can reduce the expression of profibrotic genes.
In summary, immunolabeling and ISH studies of normal and fibrotic human lung have identified at least two extravascular sources, myofibroblasts and apoptotic AEC, that synthesize ANG peptides de novo in the IPF lung. These results confirm earlier investigations of cultured human AEC and myofibroblasts isolated from IPF tissue and are consistent with reports of local ANG-generating systems in other fibrosing organs. Given the roles of ANG II in stimulating collagen deposition in the lungs (8, 12) and other organs (6, 26, 27), it is theorized that the ANG peptides produced by apoptotic AEC and myofibroblasts contribute to the fibrogenic response in IPF lung. To evaluate this theory in human lung tissue, experiments designed to test for reduction of -I-collagen mRNA in IPF lung explants by ANG receptor antagonists are currently under way.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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1 crosstalk in pulmonary fibrosis: autocrine mechanisms in myofibroblasts and macrophages. Curr Pharm Des. In Press.This article has been cited by other articles:
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