help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on April 22, 2004, doi:10.1164/rccm.200304-478OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
200304-478OCv1
170/2/188    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bechara, R. I.
Right arrow Articles by Guidot, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bechara, R. I.
Right arrow Articles by Guidot, D. M.
American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 188-194, (2004)
© 2004 American Thoracic Society

Original Article

Transforming Growth Factor ß1 Expression and Activation Is Increased in the Alcoholic Rat Lung

Rabih I. Bechara, Lou Ann S. Brown, Jesse Roman, Pratibha C. Joshi and David M. Guidot

Section of Pulmonary and Critical Care Medicine, Atlanta VA Medical Center, Decatur; and Division of Pulmonary, Allergy and Critical Care Medicine and Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia

Correspondence and reprint requests should be addressed to David M. Guidot, M.D., Atlanta VAMC, (151-P), 1670 Clairmont Road, Decatur, GA 30033. E-mail: dguidot{at}emory.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Alcohol abuse increases the incidence of acute respiratory distress syndrome more than threefold in patients with septic shock. We have shown that chronic ethanol ingestion in a rat model impairs alveolar epithelial barrier function and enhances lung injury during sepsis. We speculated that transforming growth factor ß1 (TGFß1), a pluripotent cytokine implicated in models of epithelial barrier disruption and lung injury, could mediate alveolar epithelial injury in the alcoholic lung. We report that chronic ethanol ingestion (6 weeks) in rats increased both TGFß1 mRNA and protein tissue expression (p < 0.05), but alone did not induce the release of TGFß1 into the alveolar space. However, during endotoxemia, ethanol-fed rats released fivefold more TGFß1 protein (by ELISA, p < 0.05) into the alveolar space than control-fed rats. Furthermore, lung lavage fluid from endotoxemic, ethanol-fed rats had more biologically active TGFß1 protein than control-fed rats (p < 0.05), as reflected by anti-TGFß1 antibody-inhibitable induction of permeability in rat alveolar epithelial monolayers in vitro. We conclude that chronic ethanol ingestion increases lung expression of TGFß1, which, during endotoxemia, is released and activated in the alveolar space in which it can disrupt the normally tight epithelial barrier. We speculate that this mechanism could contribute to the increased risk of acute respiratory distress syndrome in alcoholic patients.

Key Words: acute respiratory distress syndrome • alcoholism • alveolar type II cell • epithelium • sepsis

Acute respiratory distress syndrome (ARDS) is a common and devastating form of acute lung injury that occurs in response to a variety of pulmonary and extrapulmonary insults, which affects as many as 75,000–100,000 individuals per year in the United States (1). A cardinal feature of ARDS is epithelial cell dysfunction with disruption of the normally tight alveolar epithelial barrier that leads to flooding of the airspaces with exudative fluid (1–3). A seminal observation by Moss and colleagues in 1996 implicated chronic alcohol abuse as an independent, comorbid variable that significantly increases the risk of developing ARDS (4). A subsequent prospective clinical epidemiological study by our group confirmed that a history of alcohol abuse independently increases the risk of developing ARDS approximately threefold in patients with sepsis (5). Compounding this connection is the well known association between alcohol abuse and many of the critical illnesses that predispose patients to developing ARDS, including gastric aspiration and trauma.

We have determined in previous studies that chronic ethanol ingestion in a rat model impairs alveolar epithelial barrier function both in vitro and in vivo (6). We have also determined that chronic ethanol ingestion markedly depletes the alveolar epithelium of the critical antioxidant, glutathione, and, in parallel, renders the alveolar epithelial type II cell vulnerable to apoptosis in response to diverse proinflammatory mediators, including tumor necrosis factor-{alpha} and hydrogen peroxide (7–9). Furthermore, chronic ethanol ingestion impairs surfactant production by alveolar epithelial type II cells in vitro (7, 10), and increases surfactant dysfunction and acute lung injury during sepsis in vivo (11). Importantly, chronic ethanol ingestion alone does not cause lung injury in our rat model, and there is no evidence that alcohol abuse alone in humans causes clinically significant lung dysfunction. However, both the animal models and the epidemiologic studies clearly demonstrate that excessive alcohol use predisposes the lung to acute epithelial barrier disruption in response to inflammatory stresses, such as sepsis. Therefore, we have sought to identify mechanisms by which chronic ethanol ingestion potentiates alveolar epithelial barrier dysfunction and consequent sepsis-mediated lung injury.

Recent experimental evidence suggests a role for transforming growth factor ß1 (TGFß1) as a mediator of acute lung injury (12). TGFß1 is a pluripotent cytokine with multiple potential effects on tissue injury and repair during lung injury. It appears to mediate cell death (13), glutathione depletion (14), and disruption of epithelial integrity (12, 13) in experimental models, effects that parallel our findings in the lungs of ethanol-fed rats. Therefore, we questioned whether chronic ethanol ingestion could alter the expression and/or activation of TGFß1 in the lung. In this study, we report that ethanol significantly increases lung expression of TGFß1, and, during endotoxemia, increases the release and activation of TGFß1 within the alveolar airspace in which it can disrupt the alveolar epithelial barrier.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ethanol Feeding
All work was performed with the approval of the Atlanta VAMC Institutional Care and Use of Animals Committee. Male Sprague-Dawley rats (200–250 g) were fed the standard Lieber-DeCarli liquid diet (Research Diets, New Brunswick, NJ) (6, 8–11, 15) with either ethanol or an isocaloric carbohydrate substitution (control diet). Ethanol-fed rats were furnished 1/3 strength ethanol diet (12% total calories as ethanol) for 1 week, 2/3 strength ethanol diet (24% total calories as ethanol) for 1 week, and then full-strength ethanol diet for 4 weeks, for a total of 6 weeks of ethanol ingestion.

Lung Lavage and Tissue Preparation for Determination of TGFß1 Expression and Biological Activity
Rats were killed under pentobarbital anesthesia. Some rats were given Salmonella typhimurium endotoxin (2 mg/kg) intraperitoneally 2 hours before killing, as described previously (9, 15). Freshly excised lungs were perfused blood-free with saline via the pulmonary artery, then lavaged with 5 ml of saline (three times) with an average total recovery of 12 ± 1 ml. The lavage fluids were centrifuged at 500 x g for 10 minutes, and the supernatants were concentrated approximately 10-fold using a Centricon 10,000 molecular weight filter (Millipore, Billerica, MA) and stored at –70°C for subsequent analysis of TGFß1 protein levels and biological activity. The lungs were excised free from other tissues; one piece of the right upper lobe was placed in RNAzol solution for analysis of TGFß1 mRNA expression by PCR, and the remainder of the right lung tissue was excised and frozen at –70°C for analysis of TGFß1 protein expression by ELISA and Western blot. In selected experiments, lung tissue was placed in formalin for immunohistochemistry of TGFß1 protein.

Determination of Lung TGFß1 mRNA Expression
Briefly, lung tissue mRNA was isolated and cDNA synthesized using reverse transcriptase (Promega, Madison, WI). Primers and probes for rat hypoxanthine phosphoribosyltransferase and TGFß1 mRNA were constructed at Emory University (Atlanta, GA) based on sequences obtained from GenBank and synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX). The relative expression of TGFß1 mRNA compared with hypoxanthine phosphoribosyltransferase mRNA was determined by a semi-quantitative luminescence method we have used previously (16).

ELISA for TGFß1
For the tissue TGFß1 assays, 0.5 g of frozen rat lung were added to 2 ml of cold acid-ethanol (93% ethanol, 2% concentrated HCL), 85 µg/ml phenylmethylsulfonyl fluoride, and 5 µg/ml pepstatin A, and homogenized 1–2 minutes with a polytron homogenizer. The samples were extracted overnight at 4°C by gentle rocking followed by centrifugation at 10,000 x g for 10 minutes. The supernatants were dialyzed against 4 mM HCL using 3,500 molecular weight cutoff dialysis tubing. Samples were again centrifuged at 13,000 x g for 10 minutes and the supernatants stored at –70°C until the time of analysis. For the lung lavage fluid TGFß1 assays, the lavage supernatants were not acidified prior to performing the ELISA. We determined by multiple testing of samples with and without acidification that TGFß1 protein levels in the lavage fluid were not affected by acidification, indicating that all of the TGFß1 protein released into the alveolar space was in the free homodimer (i.e., active) form. Levels of TGFß1 in the lung lavage fluid and the prepared lung tissue were determined with a commercial ELISA kit (R&D Systems, Minneapolis, MN) that detects only the free or active TGFß1 homodimer. Absorbance was read at 450 nm and quantified using a standard curve. The amount of TGFß1 in the lung lavage fluid and lung tissue was expressed per mg of protein in each sample.

Western Blot Analysis of Lung Tissue for TGFß1 Protein Expression
Frozen lung tissue was homogenized in phosphate-buffered saline (PBS). Thirty micrograms of protein from each sample were loaded onto a 12% acrylamide gel and separated by electrophoresis at 160 volts for 90 minutes. The isolated proteins were transferred to a 0.45 µM polyvinylidine difluoride membrane at 15 volts for 70 minutes. The membrane was maintained at room temperature for 1 hour in TRIS-buffered saline (TBS) with 0.2% Tween 20 containing 5% nonfat dry milk and probed overnight at 4°C with a primary antibody (diluted 1:200 in 5% nonfat dry milk in TBS with 0.2% Tween 20) that detects both latent and active forms of TGFß1 protein (Santa Cruz Biotechnology, Santa Cruz, CA). The membrane was incubated at room temperature with horseradish peroxidase-labeled anti-rabbit IgG secondary antibody (Santa Cruz Biotechnology) at a dilution of 1:400 in 5% nonfat dry milk in TBS with 0.2% Tween 20 for 2 hours. Protein bands on the membrane were enhanced by chemiluminescence (ECL; Amersham, Arlington Heights, IL) and detected using a BioRad Imaging System (BioRad, Hercules, CA).

Immunohistochemistry for TGFß1 in Lung Tissue
Briefly, 5-µm sections of fixed, paraffin-embedded lung tissue were placed on glass slides, deparaffinized, and washed twice in TBS. Endogenous peroxidase activity was quenched using 1.6% H2O2 in TBS for 30 minutes. Slides were rinsed twice in TBS containing 0.1% Triton X-100. Nonspecific immunoglobulin binding was blocked by incubation of the sections in 10% goat serum in TBS with 0.1% Triton X-100 for 1 hour. Slides were drained and TGFß1 primary antibody (Santa Cruz Biotechnology) diluted 1:400 in DAKO antibody diluent (DAKO Corporation, Carpinteria, CA) or control rabbit IgG (Sigma, St. Louis, MO) were added. After incubation overnight at 4°C, slides were washed twice for 5 minutes in TBS containing 0.1% Triton X-100. Biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA), diluted 1:200 in DAKO antibody diluent, was placed on the slides, which were incubated at room temperature for 1 hour and washed twice with TBS. Slides were incubated with Vectastain ABC Reagent (Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature, washed twice with TBS, and incubated with DAB (DAB Peroxidase Substrate Kit [SK-4100]; Vector Laboratories) for 10 minutes. Slides were washed in H2O for 5 minutes and counterstained with hematoxylin, dehydrated in graded alcohols and xylene, and mounted.

Formation of Alveolar Epithelial Monolayers for TGFß1 Bioassay
Type II alveolar epithelial cells were isolated using a standard protocol (17). Cells obtained by this method contain approximately 90% type II cells (17) that are > 90% viable by Trypan blue dye exclusion. These cells were plated onto Transwell plates and cultured until intact monolayer formation (8 days), as reported previously (6).

Assessment of TGFß1 Activity Using an Alveolar Epithelial Monolayer Permeability Bioassay
A reference assay was performed by adding recombinant rat TGFß1 to the apical surface of alveolar epithelial monolayers derived from control-fed rats at concentrations of 0.5 or 10 ng/ml, with or without a 100x concentration of mouse IgG2B monoclonal anti-TGFß1 antibody (R&D Systems). These concentrations were chosen because the estimated levels of TGFß1 in the approximately 10x-concentrated lavage fluids were in the range of 0–6 ng/ml. The monolayers were incubated overnight, and barrier function was assessed by placing [14C]sucrose and [3H]inulin on the apical side of each monolayer and measuring the passage of [14C]sucrose and [3H]inulin to the basolateral surface after 2 hours using a scintillation counter. TGFß1-induced permeability determined in the reference assay was the same by [14C]sucrose and [3H]inulin clearance. Concentrated lung lavage fluids from endotoxemic control- and ethanol-fed rats were added to the apical surface of epithelial monolayers. In some wells, PBS or lung lavage fluid from control-fed rats not treated with endotoxin was added as a baseline control. In some of the wells in which lavage fluid from endotoxemic rats was added, mouse IgG2B monoclonal anti-TGFß1 antibody or mouse IgG2B isotype control antibody (R&D Systems) was added with the lavage fluid. Figure 1 shows the general scheme of the TGFß1 bioassay developed for this study.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 1. Schematic illustration of TGFß1 bioassay developed for this study. Lung lavage fluids were assayed for the ability to produce a TGFß1-specific permeability change in a naive target epithelium derived from freshly isolated rat alveolar epithelial type II cells (see METHODS for complete details).

 
Assessment of TGFß1 Activity Using Mink Lung Epithelial Cells Transformed With a Plasminogen Activator Inhibitor-1 Inhibitor/Luciferase Construct
Mink lung epithelial cells transfected with a plasminogen activator inhibitor-1/luciferase construct (28) were a generous gift from Dr. D. B. Rifkin. Cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells (1.6 x 104/well) were plated in a 96-well plate and incubated for 16 hours with 10x lavage fluid from either control- or ethanol-fed animals (with or without prior endotoxemia, as above), medium alone (no lavage fluid), or recombinant human TGFß1 (10 ng/ml; R&D Systems) ± 100x concentration of mouse IgG2B monoclonal anti-TGFß1 antibody. Luciferase activity in cell lysates was assayed using a luciferase assay kit (Promega, Madison, WI), analyzed using a microplate luminometer, and expressed as relative light units.

Statistical Analyses
Values shown represent the mean ± SEM. Values were compared using one-way analysis of variance and corrected by Student-Newman-Keuls test for differences between groups. A p value of < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Chronic Ethanol Ingestion on Lung TGFß1 Expression
We first determined that TGFß1 mRNA expression was significantly (p < 0.05) increased (~ ninefold) in the lungs of ethanol-fed rats compared with control-fed rats (Figure 2A) . In parallel, we determined that TGFß1 protein expression was significantly (p < 0.05) increased (twofold) in the lungs of ethanol-fed rats compared with control-fed rats by quantitative ELISA (Figure 2B). Furthermore, the TGFß1 protein in the lung tissue of both control- and ethanol-fed rats appeared to be bound to its latency-associated peptide and, therefore, in the inactive form. As shown in the representative Western blot images in Figure 3 , although there was more TGFß1 protein in the lung tissue of ethanol-fed rats (consistent with the ELISA findings), the majority of TGFß1 protein in both control- and ethanol-fed rat lungs was approximately 40 kD in size, consistent with the TGFß1–latency-associated peptide complex. In contrast, a relatively small fraction of the TGFß1 protein was approximately 23 kD in size, consistent with the free (active) homodimer. Finally, increased TGFß1 protein expression in the lungs of ethanol-fed rats was evident in the alveolar epithelium and macrophages when lung sections were immunostained for TGFß1 protein and examined by light microscopy. Figure 4 shows representative digitized immunohistochemistry images from the lung of a control- and ethanol-fed rat, demonstrating increased immunostaining (brown staining) for TGFß1 protein in the alveolar epithelium and macrophages in ethanol-fed rats compared with control-fed rats.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 2. Effects of chronic ethanol ingestion on TGFß1 mRNA and protein expression in whole lungs of rats fed either a liquid control diet (open bars) or an isocaloric liquid diet containing ethanol (filled bars). (A) TGFß1 messenger RNA levels measured by a semi-quantitative PCR and expressed in normalized light units. (B) TGFß1 protein levels measured by ELISA. Each value represents the mean ± SEM of six or more determinations. *p < 0.05 compared with control diet.

 


View larger version (39K):
[in this window]
[in a new window]
  		Figure 3. Western blot analysis for the relative expression of latent versus active TGFß1 protein expression in whole lungs of rats fed either a liquid control diet or an isocaloric liquid diet containing ethanol. Shown are representative determinations from three control-fed rat lungs (lanes 1–3) and three ethanol-fed rat lungs (lanes 4–6). Although there was some variability among the samples, the predominant form of TGFß1 protein in each lung tissue was the 40-kD form, consistent with the TGFß1–latency-associated protein complex. The minor form in each tissue was the 23-kD form, which is the known size of the active TGFß1 homodimer protein.

 


View larger version (95K):
[in this window]
[in a new window]
  		Figure 4. Representative immunohistochemistry of lung sections from a control- and ethanol-fed rat stained with anti-TGFß1 primary antibody (A and B, respectively), or stained with a control rabbit IgG antibody (C and D, respectively).

 
Effect of Chronic Ethanol Ingestion on the Release of TGFß1 Protein into the Alveolar Airspace in Rats before and after Acute Endotoxemia
We next examined the release of TGFß1 into the alveolar airspaces of control-fed versus ethanol-fed rats, both at baseline and in response to endotoxemia. As shown in Figure 5 , lung lavage fluid recovered from either nonendotoxin-treated control or ethanol-fed rats (i.e., no endotoxin treatment in either group) had no detectable levels of TGFß1 protein, indicating that even though chronic ethanol ingestion increased TGFß1 protein expression in the lung tissue (Figures 2– 4), TGFß1 protein was not released into the alveolar epithelial lining fluid in the absence of an acute inflammatory stress. In contrast, we determined that endotoxemia caused a measurable release of TGFß1 protein into the alveolar epithelial lining fluid of both control- and ethanol-fed rats (Figure 5). Importantly, ethanol-fed rats had significantly higher levels of TGFß1 protein (approximately fivefold; p < 0.05) in their lung lavage fluid following endotoxemia than similarly treated control-fed rats (Figure 5). Furthermore, as discussed in METHODS, the levels of TGFß1 protein in the lavage fluids, as measured by ELISA, were the same in both acid-treated and untreated samples (data not shown), suggesting that all of the released TGFß1 protein in both control- and ethanol-fed rats was in the free (active) form.



View larger version (12K):
[in this window]
[in a new window]
  		Figure 5. Effects of chronic ethanol ingestion on TGFß1 protein levels in the lung lavage fluid. TGFß1 protein levels were determined by ELISA in lung lavage fluid recovered from ethanol- and control-fed rats with and without two hours of endotoxemia (induced with Salmonella typhimurium LPS, 2 mg/kg intraperitoneally), and expressed per mg of total protein. Each value represents the mean ± SEM of six or more determinations. *p < 0.05 compared with control + LPS.

 
Biological Activity of TGFß1 Protein in Lung Lavage Fluid Using an Alveolar Epithelial Monolayer Permeability Assay
To determine whether ethanol ingestion increased the biological activation of TGFß1 protein in (as well as its release into) the alveolar space during endotoxemia, we assayed lung lavage fluid from endotoxemic control- and ethanol-fed rats for the ability to induce a permeability defect in a target epithelial monolayer, and to determine if this permeability could be prevented by treatment with anti-TGFß1 antibodies. We first determined in a reference assay that recombinant rat TGFß1 protein produced a concentration-dependent permeability in a target alveolar epithelial monolayer, and that this permeability was completely inhibited by anti-TGFß1 antibody. As shown in Figure 6 , TGFß1 at concentrations of 5 and 10 ng/ml produced a progressive and significant (p < 0.05) increase in epithelial permeability to both [14C]sucrose and [3H]inulin, and that even at a concentration of 10 ng/ml, this permeability was completely inhibited by treatment with a 100x concentration of an anti-TGFß1 antibody. As discussed in METHODS, these concentrations were chosen because the estimated concentrations of TGFß1 protein in the concentrated lavage fluids were in the range of 0–6 ng/ml. Also evident in Figure 6 is that TGFß1-induced permeability was the same when determined by either [14C]sucrose or [3H]inulin flux at 2 hours. Thus, we used only [14C]sucrose flux in the bioassay to determine the TGFß1-specific permeability induced by lung lavage fluid obtained from control-and ethanol-fed rats after 2 hours of endotoxemia. As shown in Figure 7 , lung lavage fluid from both endotoxemic control and ethanol-fed rats induced a permeability change in the target epithelial monolayers as compared with monolayers that were treated with either buffered saline or lung lavage fluid obtained from untreated control-fed rats. Furthermore, this permeability defect appeared to be TGFß1-specific, as treatment with the anti-TGFß1 antibody, but not the isotype-matched control antibody (anti-IgG), prevented (p < 0.05) a permeability change in both the control-and ethanol-fed groups (Figure 7). However, consistent with the findings in Figure 5, the lung lavage fluid from endotoxemic ethanol-fed rats induced significantly greater (p < 0.05) TGFß1-specific permeability in epithelial monolayers than did lung lavage fluid from endotoxemic control-fed rats. To compare these findings in the permeability bioassay with a previously published TGFß bioassay, we analyzed the lung lavage fluids using a mink lung epithelial cell assay in which TGFß bioactivity is correlated to luciferase activity via activation of a plasminogen activator inhibitor-1 inhibitor/luciferase reporter construct. As shown in Figure 8 , relative TGFß1 bioactivity in each of the four experimental groups in this luciferase reporter assay correlated with the permeability assay above. Specifically, lung lavage fluids from control- and ethanol-fed rats, in the absence of prior endotoxin treatment, had no detectable TGFß1 bioactivity compared with medium alone (no lavage fluid fluid). In contrast, lung lavage fluids from control and ethanol-fed rats that were treated with endotoxin had detectable TGFß1 bioactivity (p < 0.05) compared with rats not treated with endotoxin, but this bioactivity was greater (p < 0.05) in the lung lavage fluids of ethanol-fed, endotoxemic rats (Figure 8). Also shown in Figure 8 is the relative TGFß1 bioactivity of the recombinant TGFß1 (10 ng/ml), as well as the near complete elimination of this bioactivity by the anti-TGFß1 antibody.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 6. Reference assay for the TGFß1 bioassay used in Figure 7. Recombinant rat TGFß1 at concentrations of 0, 5, and 10 ng/ml was added to the culture medium of naive alveolar epithelial monolayers derived from control-fed rats and cultured for 8 days. Permeability was assessed 18 hours later, by determining the flux of both [14C]sucrose (gray bars) and [3H]insulin (black bars) across the monolayer in 2 hours. In parallel determinations, either anti-TGFß1 antibody or an isotype-matched control antibody (anti-IgG) was added with the recombinant TGFß1. Each value represents the mean ± SEM of four determinations. *p < 0.05 compared with 0 ng/ml; **p < 0.05 compared with 5 ng/ml.

 


View larger version (34K):
[in this window]
[in a new window]
  		Figure 7. Effects of chronic ethanol ingestion on TGFß1 biological activity in the lung lavage fluid, as reflected by alveolar epithelial permeability. TGFß1 biological activity was assayed by incubating naive alveolar epithelial monolayers (derived from rats fed a standard chow diet) with the lung lavage fluid recovered from control- and ethanol-fed rats after 2 hours of endotoxemia (induced with Salmonella typhimurium lipopolysaccharides LPS, 2 mg/kg intraperitoneally). The ability of the fluid to induce a permeability defect in the target epithelial monolayer was determined by measuring the percent clearance of [14C]sucrose across the monolayer in 2 hours. The specificity of this TGFß1 activity was determined by simultaneous assays of each lavage fluid, but with an anti-TGFß1 antibody added. An isotype-matched control antibody (anti-IgG) had no inhibitory effect on the TGFß1 activity in any of the groups. Each value represents the mean ± SEM of six or more determinations. *p < 0.05 compared with none; **p < 0.05 compared with control + LPS.

 


View larger version (15K):
[in this window]
[in a new window]
  		Figure 8. Effects of chronic ethanol ingestion on TGFß1 biological activity in the lung lavage fluid, as reflected by induction of plasminogen activator inhibitor-1inhibitor/luciferase reporter activity in transformed mink lung epithelial cells. Luciferase activity in mink cell lysates is expressed as relative light units (RLU). Mink cell lysates with no lavage fluid (none); 10x lavage fluid from rats on control-fed diet ± LPS, as indicated (control); 10x lavage fluid from rats on ethanol-fed diet ± LPS (ethanol); and recombinant TGFß1 ± 100x anti-TGFß1, as indicated (TGFß1). *p < 0.05 compared with none; **p < 0.05 compared with control + LPS.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The primary objective of the present study was to determine whether the release during endotoxemia of TGFß1 into the alveolar space and its activation could explain, at least in part, why the alcoholic lung is more vulnerable to acute barrier disruption and subsequent flooding with exudative fluid, the fundamental defect in ARDS. We first determined that chronic ethanol ingestion increases the expression of TGFß1 in the lung. However, consistent with the known biology of TGFß1, it appears to be expressed primarily in a tissue-bound latent form with no detectable TGFß1 protein in the alveolar airspace in the absence of an acute inflammatory stress. However, chronic ethanol ingestion significantly enhanced the release of active TGFß1 protein into the alveolar space during endotoxemia. This was determined by both a standard ELISA and a bioassay that we developed for this study in which the ability of lung lavage fluid to produce a TGFß1-specific increase in permeability of naive alveolar epithelial monolayers was assessed in vitro. Taken together, this study provides the novel observations that chronic ethanol ingestion induces aberrant TGFß1 expression in the lung, and that activation of latent TGFß1 during acute inflammatory stresses, such as endotoxemia, can mediate alveolar epithelial barrier dysfunction.

This extends our previous studies that examined potential mechanisms underlying chronic ethanol ingestion and the susceptibility to acute lung injury. A cardinal finding is the depletion of the antioxidant, glutathione, within the alveolar epithelial cells and adjacent lining fluid, which is accompanied by markedly elevated levels of oxidized glutathione, indicating previously unrecognized oxidative stress within the alveolar space (7). Ethanol-induced oxidative stress in the alveolar epithelium leads to increased sensitivity to apoptosis (8, 9), decreased surfactant production (10), abnormal barrier function both in vitro and in vivo (6), and increased lung injury in response to sepsis in vivo (11). The clinical relevance of these experimental findings is illustrated by our finding that otherwise healthy alcoholic subjects have low levels of glutathione and high levels of oxidized glutathione in their lung lavage fluid (18). However, alcohol abuse alone does not cause clinically apparent alveolar epithelial permeability and/or lung injury, and the specific molecular trigger(s) that produces excessive alveolar epithelial barrier dysfunction in the alcoholic lung during sepsis remained undefined. In this context, TGFß1 emerged as a possible candidate. TGFß1 is a pluripotent cytokine that decreases glutathione in the lung epithelial cell line A549 in vitro (14), and recently has been implicated mechanistically in a mouse model of acute lung injury induced by either bleomycin or endotoxin (12). Although TGFß1 expression and/or activity was not evaluated in the in vivo model in that study, but, instead, was inferred indirectly (12), that same study showed that TGFß1 added directly to alveolar epithelial monolayers in vitro decreased cellular glutathione levels and caused an acute increase in epithelial monolayer permeability (12), consistent with our findings here.

TGFß1 and related isoforms, TGFß2, and TGFß3 (19–21), are induced in a wide array of pathologic conditions, and diverse factors, including oxidative stress, can induce aberrant expression in diseased tissues (13, 19–21). Although we do not yet know how chronic ethanol ingestion induces TGFß1 expression in the lung, the previously unrecognized chronic oxidative stress we have identified in the alcoholic lung could play an important role. TGFß1 exerts diverse effects on target tissues by binding to specific receptors on the surface of target cells. However, its biological function is regulated at the level of activation from a latent form. Specifically, TGFß1 is synthesized and secreted as an approximately 23 kD homodimer. The homodimer is noncovalently associated with a latency-associated peptide that is part of the originally synthesized propeptide that undergoes proteolytic cleavage (20). The TGFß1–latency-associated peptide complex is approximately 40 kD in size and called the small latent complex. This complex is usually bound to a larger peptide of variable length called latent TGFß1 binding protein, of which four family members have been identified (20, 21). The latent TGFß1 binding protein is covalently bound to the latency-associated peptide, and the entire complex of TGFß1, latency-associated peptide, and latent TGFß binding protein form the large latent complex. TGFß1 can be released and activated from the latency-associated peptide by multiple factors, including oxidants (22, 23), nitric oxide, and/or reactive nitrogen species (24), at cell surfaces by thrombospondin-1 from platelets (23) or by cell-associated plasmin (20), and via interactions with specific integrins, such as {alpha}vß6 (12) or matrix glycoproteins (25, 26). Upon activation, TGFß1 can interact with specific receptors on the surface of its target cells (13, 20). The subsequent intracellular signaling cascade involves phosphorylation of a unique family of intracellular molecules, known as Smads (13, 19), that ultimately affect transcription of a wide array of TGFß1-responsive genes. The end result of TGFß1 signaling can be proliferative or antiproliferative depending on the target cell and the local conditions. The assessment of latent versus active TGFß1 in biological samples can be made in several ways. One is to perform a standard ELISA with and without acidification of the samples, as acidification releases the homodimer from the latency peptide and enables it to bind to the conjugated antibody in the assay. Therefore, the relative amount of active protein in the nonacidified sample compared with the total protein in the acidified sample has been touted as one method of determining the relative amount of active TGFß1 in a given fluid or tissue (27). In this study, we determined that acidification of the lung lavage fluid had no affect on the measured TGFß1 protein levels, suggesting that all of the protein released into the alveolar space was in the free or active form. To provide further evidence that there was increased biologically active TGFß1 protein in the alveolar space capable of inducing epithelial permeability, we developed a relevant bioassay in which TGFß1-specific ability to induce a permeability defect in a target rat alveolar epithelial monolayer was determined. This assay correlated well with the TGFß bioassay developed by Rifkin and colleagues in which mink lung epithelial cells express a plasminogen activator inhibitor-1 promoter–luciferase construct in proportion to the amount of active TGFß protein present in a sample (28). Our bioassay also uses a lung epithelial cell as the target, but we reasoned that it was necessary to determine TGFß1 bioactivity by an assay that is more relevant to this particular study. Specifically, in our permeability assay, the targeted epithelial cells are primary isolates derived from the rats used in the study (rather than transformed cells from a different species), and the biological endpoint of epithelial permeability reflects the cardinal feature of the pathophysiology of ARDS being examined, namely acute alveolar epithelial barrier dysfunction.

Our experimental findings in this model are consistent with the evolving recognition of how TGFß1 is involved in both acute and chronic forms of tissue injury, although to our knowledge this is the first study demonstrating that TGFß1 is released and activated in the alveolar airspace during acute inflammatory stress. In the chronic alcoholic lung, it appears that although TGFß1 is overexpressed, it remains tissue bound, and therefore, in a latent form. However, during endotoxemic stress when oxidants and proteolytic enzymes are increased, TGFß1 is released and activated in the alveolar space in which it can induce permeability defects in the alveolar epithelium. Although the correlation between TGFß1 protein concentration (by ELISA) and TGFß1 bioactivity (by the permeability assay) was not linear, the two assessments were nevertheless in parallel. This is consistent with the understanding that the net effect of TGFß1 (and other biological mediators) on a target tissue depends on complex signaling events, and not simply on protein concentrations. In this regard, the deleterious effects of TGFß1 in the patient with chronic alcohol abuse may be even more devastating than suggested by this study, as the target alveolar epithelium within the alcoholic lung has profound glutathione depletion and epithelial barrier dysfunction even before it is exposed to activated TGFß1 and other damaging molecules during acute inflammatory stress.

In summary, we conclude that chronic ethanol ingestion increases lung expression of TGFß1 that is released and activated in the alveolar space during endotoxemia where it can disrupt the normally tight epithelial barrier. The relevance of our results to acute lung injury and ARDS in humans still remains an open question. However, our data suggest that activation of latent TGFß1 and its direct effects on the alveolar epithelium could play an important role in producing the alveolar flooding with proteinaceous fluid that characterizes ARDS. We speculate that targeting the processes that activate TGFß1, particularly in patients with a significant history of alcohol abuse who are, therefore, at a threefold risk of developing the syndrome, could limit the high morbidity and/or mortality currently associated with ARDS.


   FOOTNOTES
 
Supported by National Institutes of Health grants P50 AA013757 and R01 AA12197, and by a VA Merit Review (D.M.G.), a Department of Defense Grant (D.M.G. and J.R.), and a VA Career Development Award (R.I.B.).

Conflict of Interest Statement: R.I.B. does not have a financial relationship with a commercial entity that has a financial interest in the subject of this article; L.A.S.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; J.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; P.C.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; D.M.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this article.

Received in original form April 3, 2003; accepted in final form April 16, 2004


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med 2000;342:1334–1349.[Free Full Text]
  2. Harris JB, Chang LY, Crapo JD. Rat lung alveolar type I epithelial cell injury and response to hyperoxia. Am J Respir Cell Mol Biol 1991;4:115–125.
  3. Tate RM, Repine JE. Neutrophils and the adult respiratory distress syndrome. Am Rev Respir Dis 1983;128:552–559.[Medline]
  4. Moss M, Bucher B, Moore FA, Moore EE, Parsons PE. The role of chronic alcohol abuse in the development of acute respiratory distress syndrome in adults. JAMA 1996;275:50–54.[Abstract/Free Full Text]
  5. Moss M, Parsons PE, Steinberg KP, Hudson LD, Guidot DM, Burnham EL, Cotsonis GA. Chronic alcohol abuse is associated with an increased incidence of acute respiratory distress syndrome and severity of multiple organ dysfunction in patients with septic shock. Crit Care Med 2003;31:869–877.[CrossRef][Medline]
  6. Guidot DM, Modelska K, Lois M, Jain L, Moss IM, Pittet J-F, Brown LAS. Ethanol ingestion via glutathione depletion impairs alveolar epithelial barrier function in rats. Am J Physiol Lung Cell Mol Physiol 2000;279:L127–L135.[Abstract/Free Full Text]
  7. Holguin F, Moss IM, Brown LS, Guidot DM. Chronic ethanol ingestion impairs alveolar type II cell glutathione homeostasis and function and predisposes to endotoxin-mediated acute edematous lung injury in rats. J Clin Invest 1998;101:761–768.[Medline]
  8. Brown LAS, Harris FL, Guidot DM. Chronic ethanol ingestion potentiates TNF-alpha–mediated oxidative stress and apoptosis in rat alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 2001;281:L377–L386.[Abstract/Free Full Text]
  9. Brown LAS, Harris FL, Bechara R, Guidot DM. Effect of chronic ethanol ingestion on alveolar type II cell glutathione and inflammatory mediator-induced apoptosis. Alcohol Clin Exp Res 2001;25:1078–1085.[CrossRef][Medline]
  10. Guidot DM, Brown LAS. Mitochondrial glutathione replacement restores surfactant synthesis and secretion in alveolar epithelial cells of ethanol-fed rats. Alcohol Clin Exp Res 2000;24:1070–1076.[CrossRef][Medline]
  11. Velasquez A, Bechara R, Lewis JF, Malloy J, McCaig L, Brown LAS, Guidot DM. Glutathione replacement preserves the functional surfactant phospholipid pool size and decreases sepsis-mediated lung dysfunction in ethanol-fed rats. Alcohol Clin Exp Res 2002;26:1245–1251.[CrossRef][Medline]
  12. Pittet J-F, Griffiths MJD, Geiser T, Kaminski N, Dalton SL, Huang X, Brown LAS, Gotwals PJ, Koteliansky VE, Matthay MA, Sheppard D. TGF-beta is a critical mediator of acute lung injury. J Clin Invest 2001;107:1537–1544.[Medline]
  13. Hartsough MT, Mulder KM. Transforming growth factor-beta signaling in epithelial cells. Pharmacol Ther 1997;75:21–41.[CrossRef][Medline]
  14. Arsalane K, Dubois CM, Muanza T, Begin R, Boudreau F, Asselin C, Cantin AM. Transforming growth factor-beta 1 is a potent inhibitor of glutathione synthesis in the lung epithelial cell line A549: transcriptional effect on the GSH rate-limiting enzyme gamma-glutamylcysteine synthetase. Am J Respir Cell Mol Biol 1997;17:599–607.[Abstract/Free Full Text]
  15. Lois M, Brown LAS, Moss M, Roman J, Guidot DM. Ethanol ingestion increases activation of matrix metalloproteinases in rat lungs during acute endotoxemia. Am J Respir Crit Care Med 1999;160:1354–1360.[Abstract/Free Full Text]
  16. Perez RL, Roman J, Roser S, Little C, Hunter R, Actor J. Cytokine message and protein expression during lung granuloma formation and resolution induced by the mycobacterial cord factor trehalose-6, 6'-dimycolate. J Interferon Cytokine Res 2000;9:795–804.
  17. Dobbs LG, Gonzalez R, Williams MC. An improved method for isolating Type II cells in high yield and purity. Am Rev Respir Dis 1986;134:141–145.[Medline]
  18. Moss M, Guidot DM, Wong-Lambertina M, Ten Hoor T, Perez RL, Brown LAS. The effects of chronic alcohol abuse on pulmonary glutathione homeostasis. Am J Respir Crit Care Med 2000;161:414–419.[Abstract/Free Full Text]
  19. Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor beta in human disease. N Engl J Med 2000;342:1350–1358.[Free Full Text]
  20. Munger JS, Harpel JG, Gleizes P-E, Mazzieri R, Nunes I, Rifkin DB. Latent transforming growth factor-beta: structural features and mechanism of activation. Kidney Int 1997;51:1376–1382.[Medline]
  21. Oklu R, Hesketh R. The latent transforming growth B binding protein (LTBP) family. Biochem J 2000;352:601–610.
  22. Barcellos-Hoff MH, Dix TA. Redox-mediated activation of latent transforming growth factor-beta 1. Mol Endocrinol 1996;10:1077–1083.[Abstract/Free Full Text]
  23. Bellocq A, Azoulay E, Marullo S, Flahault A, Fouqueray B, Philippe C, Cadranel J, Baud L. Reactive oxygen and nitrogen intermediates increase transforming growth factor-beta1 release from human epithelial alveolar cells through two different mechanisms. Am J Respir Cell Mol Biol 1999;21:128–136.[Abstract/Free Full Text]
  24. Vodovotz Y, Chesler L, Chong H, Kim S-J, Simpson JT, DeGraff W, Cox GW, Roberts AB, Wink DA, Barcellos-Hoff MH. Regulation of transforming growth factor beta 1 by nitric oxide. Cancer Res 1999;59:2142–2149.[Abstract/Free Full Text]
  25. Kolb M, Margetts PJ, Sime PJ, Gauldie J. Proteoglycans decorin and biglycan differentially modulate TGF-beta-mediated fibrotic responses in the lung. Am J Physiol Lung Cell Mol Physiol 2001;280:L1327–L1334.[Abstract/Free Full Text]
  26. Noble NA, Harper J, Border WA. In vivo interactions of TGF-beta and extracellular matrix. Prog Growth Factor Res 1992;4:369–382.[CrossRef][Medline]
  27. Brenchley PE, Williams S, Coupes B, Short CD. Strategies for meaningful measurement of circulating levels of transforming growth factor beta 1 in clinical samples. Transplantation 2001;71:1355–1356.[CrossRef][Medline]
  28. Abe M, Harpel JG, Metz CN, Nines I, Loskutoff DJ, Rifkin DB. An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. Anal Biochem 1994;216:276–284.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
D. M. Boe, R. W. Vandivier, E. L. Burnham, and M. Moss
Alcohol abuse and pulmonary disease
J. Leukoc. Biol., November 1, 2009; 86(5): 1097 - 1104.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
P. O. Mitchell and D. M. Guidot
Alcohol Ingestion by Donors Amplifies Experimental Airway Disease after Heterotopic Transplantation
Am. J. Respir. Crit. Care Med., December 1, 2007; 176(11): 1161 - 1168.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
P. C. Joshi and D. M. Guidot
The alcoholic lung: epidemiology, pathophysiology, and potential therapies
Am J Physiol Lung Cell Mol Physiol, April 1, 2007; 292(4): L813 - L823.
[Abstract] [Full Text] [PDF]

Home page
 Nephrol Dial TransplantHome page
A. S. Gueye, M. Chelamcharla, B. C. Baird, C. Nguyen, H. Tang, A. L. Barenbaum, J. K. Koford, F. Shihab, and A. S. Goldfarb-Rumyantzev
The association between recipient alcohol dependency and long-term graft and recipient survival
Nephrol. Dial. Transplant., March 1, 2007; 22(3): 891 - 898.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
S. C. Wesselkamper, L. M. Case, L. N. Henning, M. T. Borchers, J. W. Tichelaar, J. M. Mason, N. Dragin, M. Medvedovic, M. A. Sartor, C. R. Tomlinson, et al.
Gene Expression Changes during the Development of Acute Lung Injury Role of Transforming Growth Factor {beta}
Am. J. Respir. Crit. Care Med., December 1, 2005; 172(11): 1399 - 1411.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
R. I. Bechara, A. Pelaez, A. Palacio, P. C. Joshi, C. M. Hart, L. A. S. Brown, R. Raynor, and D. M. Guidot
Angiotensin II mediates glutathione depletion, transforming growth factor-{beta}1 expression, and epithelial barrier dysfunction in the alcoholic rat lung
Am J Physiol Lung Cell Mol Physiol, September 1, 2005; 289(3): L363 - L370.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
J. Roman, J. D. Ritzenthaler, R. Bechara, L. A. Brown, and D. Guidot
Ethanol stimulates the expression of fibronectin in lung fibroblasts via kinase-dependent signals that activate CREB
Am J Physiol Lung Cell Mol Physiol, May 1, 2005; 288(5): L975 - L987.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
B. Nemery, W. W. Yew, R. Albert, C. Brun-Buisson, W. MacNee, F. J. Martinez, D. C. Angus, and E. Abraham
Tuberculosis, Nontuberculous Lung Infection, Pleural Disorders, Pulmonary Function, Respiratory Muscles, Occupational Lung Disease, Pulmonary Infections, and Social Issues in AJRCCM in 2004
Am. J. Respir. Crit. Care Med., March 15, 2005; 171(6): 554 - 562.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
200304-478OCv1
170/2/188    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bechara, R. I.
Right arrow Articles by Guidot, D. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bechara, R. I.
Right arrow Articles by Guidot, D. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2004 American Thoracic Society