The Acute Respiratory Distress Syndrome A Role for Transforming Growth Factor-1 Ruairi J. Fahy, Frank Lichtenberger, Christine B. McKeegan, Gerard J. Nuovo, Clay B. Marsh, and Mark D. Wewers Division of Pulmonary and Critical Care Medicine, Dorothy M. Davis Heart and Lung Research Institute; and Department of Pathology, The Ohio State University, Columbus, Ohio
The acute respiratory distress syndrome (ARDS) remains a major cause of morbidity and mortality. Enhanced fibrosis and elevated procollagen III levels have been linked to increased mortality. We hypothesized that transforming growth factor (TGF)-1 may play an important role in ARDS, given its role in stimulating fibrosis. Using reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) and immunohistochemistry, we analyzed lung tissue from four fibroproliferative ARDS cases and control subjects. We also compared active TGF-1 levels in the bronchoalveolar lavage (BAL) fluid of 13 de novo ARDS cases, and 7 normal control subjects. RT in situ PCR showed TGF-1 mRNA expression in fibroproliferative ARDS cases. Immunohistochemistry confirmed protein expression in these samples. Controls were negative for both techniques. In the newly enrolled ARDS cases, TGF-1 levels, as measured by luciferase assay, were elevated in the 11 of 13 samples, averaging 98 ⫾ 40 pg/mg protein. Controls had no detectable TGF-1 activity. These data suggest that activation of TGF-1 may be important in the early phases of acute lung injury in addition to driving fibroproliferation. These data may lead to new therapeutic approaches in ARDS through more targeted inhibition of fibrosis.
Since its description in 1967, the adult respiratory distress syndrome (ARDS) has remained a major medical challenge (1). ARDS is defined as the acute onset of lung injury, bilateral infiltrates on chest radiography, a PaO2/FiO2 ratio of ⬍ 200, and a pulmonary artery occlusion pressure of ⭐ 18 mm Hg (2). Historically, mortality rates of 40–60% have been described (3), with rates dropping to 30–40% in more recent years (4, 5). Although a majority of deaths are due to factors other than primary respiratory failure, such as sepsis, failure to improve respiratory function predicts a poor outcome (6). Three phases of ARDS have been described: (i ) an early exudative phase of edema and inflammation; (ii) a proliferative phase with pneumocyte hyperplasia and myofibroblast
(Received in original form June 25, 2002 and in revised form November 13, 2002) This article has an online data supplement, which is accessible from this issue’s table of contents online at www.atsjournals.org Address correspondence to: Ruairi J. Fahy, 201 The Davis Heart & Lung Research Institute, 473 West 12th Avenue, Columbus, OH 43210-1252. E-mail:
[email protected] Abbreviations: acute respiratory distress syndrome, ARDS; bronchoalveolar lavage, BAL; fetal bovine serum, FBS; mink lung epithelial cell, MLEC; matrix metalloproteinase, MMP; reverse transcriptase in situ polymerase chain reaction, RT in situ PCR; transforming growth factor-1, TGF-1. Am. J. Respir. Cell Mol. Biol. Vol. 28, pp. 499–503, 2003 DOI: 10.1165/rcmb.2002-0092OC Internet address: www.atsjournals.org
proliferation; and (iii) a fibrotic phase with collagen deposition, progressive lung fibrosis, and pulmonary microvasculature obliteration (7). When persistent inflammation fails to resolve, fibroproliferation, a stereotypic response to tissue injury, ensues (8). Although traditionally thought to occur after the first week, recent studies suggest that increased collagen turnover is an early event in ARDS (9–12). The fibroproliferative response, if excessive, can be problematic, impairing the lung’s primary function of gas exchange. Clinically, this is seen as progressive hypoxia, decreased lung compliance, progression of chest radiograph infiltrates, and increasing pulmonary vascular resistance (13, 14). In fatal cases, lung collagen content can increase 2- to 3-fold, after 2 wk (15). A correlation has also been found between pulmonary fibrosis and fatality in established cases of ARDS (16). Many inflammatory cytokines are involved in the pathogenesis of ARDS, and some have prognostic significance (17, 18). An important role has been suggested for cytokines that regulate mesenchymal cell proliferation and fibrosis, such as transforming growth factor (TGF)-␣ and TGF-, in view of the association between mortality and pulmonary fibrosis in established ARDS (16). TGF-␣ is a member of the epidermal growth factor peptide family. TGF-␣ levels are elevated in the bronchoalveolar lavage (BAL) fluid from patients with ARDS (19). In vitro, TGF-␣ causes enhanced alveolar epithelial repair (20). In a murine in vivo transgenic model, the overexpression of TGF-␣ decreased inflammation and edema (21). These studies support a role for TGF-␣ in the repair of lung injury, perhaps focused at epithelial repair. However, the precise role played by TGF-␣ is still under investigation. In contrast, TGF-1 is more directly associated with fibrosis and wound repair. The TGF-1 gene is upregulated in response to tissue injury, and TGF-1 is implicated in the fibrosis and wound repair process (22). Given that fibroproliferative ARDS is a pathologic form of normal tissue repair, TGF-1 might be considered a potential key participant in ARDS in general, and of progressive lung fibrosis in particular. Animal models of acute lung injury support a significant role for TGF-1 in ARDS (23, 24). With the recent finding of increased collagen turnover and fibroproliferation within 24 h of acute lung injury (12), and the known role of TGF-1 in inducing procollagen gene expression (25, 26), we postulated that TGF-1 may play an important role in both the early acute injury and the fibroproliferative phase of ARDS. Therefore, in this study, we sought to document the presence of TGF-1 in fibroproliferative ARDS lung tissue
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using both immunohistochemical and reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) techniques. As a control, normal lung tissue was subject to similar analysis. To assess the potential role for TGF-1 in the early stages of ARDS, we sought to document the presence of functionally active TGF-1 in human BAL fluid obtained within 24 h of the diagnosis of ARDS.
Materials and Methods Patient Selection All patients admitted to The Ohio State University Medical Intensive Care Unit who met the American-European Consensus definition for ARDS were enrolled (2). Informed consent was obtained from patients or surrogates. The protocol was approved by the Institutional Review Board of the university. Details on the inclusion/exclusion criteria are available in the online data supplement.
This complex was detected using the Super Sensitive peroxidase based kit (Biogenex, San Roman, CA).
Measurement of TGF-1 by Mink Lung Epithelial Cell Luciferase Assay As detailed in the online data supplement, this technique utilizes mink lung epithelial cells (MLECs; a gift from D. B. Rifkin, New York University School of Medicine, New York, NY) transfected with a TGF-1–sensitive plasminogen activator inhibitor promoter luciferase construct (29). ARDS or control BAL fluid was added to the MLEC for 16–20 h at 37⬚C. Following the addition of reporter lysis buffer (Promega, Madison, WI), cell lysates were centrifuged and the supernatant collected. Light emission was measured on cell extracts (Lumat LB 9507; Berthold Technologies, Oak Ridge, TN). A monoclonal human anti–TGF-1 antibody (R&D Systems, Minneapolis, MN) at a concentration of 10 g/ml was used in blocking experiments.
BAL Protocol As detailed in the online data supplement, BAL was performed on healthy volunteer control subjects, and within 24 h of onset of ARDS in patients with ARDS. Fluid was analyzed for total cell count and cytomorphology. The remaining fluid was spun at 1,200 rpm for 10 min at 4⬚C, aliquoted, and stored at ⫺80⬚C.
Statistical Analysis Data are expressed as mean ⫾ SEM. Data were compared using Student’s t test (JMP 4.0; SAS Institute Inc., Cary, NC). Statistical significance was defined as P ⬍ 0.05
Results Measurement of BAL Fluid Protein Levels
Patient Characteristics
Aliquots of BAL fluid were analyzed for total protein content using a modified Bradford assay (BioRad, Hercules, CA).
The four cases of fibroproliferative ARDS, evaluated by immunohistochemistry and RT in situ PCR, had a mean age of 62 yr, one being male and three female. Pneumonia was the cause of ARDS in three cases, with pneumonia/ sepsis accounting for the fourth. TGF-1 measurements in BAL fluid from patients with ARDS and from control subjects were prospectively collected. During the study period, 13 patients and 7 control subjects were analyzed. The age of the patients (mean ⫾ SEM) was 44 ⫾ 3.5 yr. There were eight whites, four African Americans, and one Native American. A majority of cases were smokers (10/13). The A-a O2 gradient (mean ⫾ SEM) in patients with ARDS was 525 ⫾ 28 mm Hg, and the mean APACHE II score was 29 ⫾ 2.1. The mean ARDS BAL neutrophil percentage was 76 ⫾ 5%. Pneumonia was the etiologic factor in 11 of the 13 patients with ARDS. Three patients died within 30 d of hospitalization (mortality 23%). Seven of 13 patients had placement of a Swan-Ganz catheter.
Lung Samples for RT in situ PCR and Immunohistochemistry Paraffin-embedded tissue, obtained over the previous 5 yr from the Department of Pathology archives, was reviewed. Four cases who had a pathologic diagnosis of fibroproliferative ARDS, and no precedent clinical lung pathology, were identified. Normal lung tissue, from four patients undergoing lung resection, was also evaluated.
RT in situ PCR As previously described (27, 28) optimal protease digestion of lung tissue was followed by incubation in RNase free-DNase and one step RT/PCR using the rTth system and digoxigenin dUTP. The primer sequence for TGF-1 mRNA was previously described (Stratagene, La Jolla, CA). The digoxigenin-labeled target-specific cDNA was detected using the antidigoxigenin–alkaline phosphatase conjugate (Boehringer Mannheim, Indianapolis, IN) followed by exposure to the chromogens nitroblue tetrazolium and bromochloroindolyl phosphate (NBT/BCIP) (Enzo Biochemicals, Farmingdale, NY). Nuclear fast red, which stains negative cells pink, served as the counterstain.
Immunohistochemistry Paraffin-embedded lung biopsy tissue, from fibroproliferative ARDS cases and normal controls was evaluated. An anti–TGF-1 (TGF-1 [v] antibody; Santa Cruz Biotechnology, Santa Cruz, CA) was incubated with the samples. A secondary biotinylated goat antirabbit antibody was followed with peroxidase-labeled streptavidin.
Fibroproliferative ARDS Samples Show TGF-1 Activity by RT in situ PCR Four archival non-ARDS lung samples, in addition to four lung samples showing evidence of fibroproliferative ARDS, were evaluated by RT in situ PCR. Three of the four ARDS lung samples demonstrated signal for TGF-1 mRNA (Figure 1). Overall, ⵒ 10% of the cells in the area of inflammation/active fibrosis were positive for TGF- mRNA. Signal intensity was absent in regions of established fibrosis, but was evident in areas adjacent to fibrotic foci. Positive staining cells included fibroblasts, macrophages, alveolar epithelial cells, and occasional endothelial cells. Non-ARDS lung
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Figure 1. TGF-1 RT in situ PCR lung sample from fibroproliferative ARDS and normal lung tissue. A, B, and C represent increasing magnification of TGF-1 nuclear staining, and D shows the no DNase control. Overall, ⵒ 10% of the cells were positive for TGF-1 mRNA. Positive signal was evident adjacent to fibrotic foci, but not in areas of established fibrosis. Alveolar epithelial cells, macrophages, fibroblasts, and occasionally endothelial cells showed positive signal. E and F show TGF-1 RT in situ PCR of normal lung tissue. In this example of two normal lung samples TGF-1 mRNA was not detected.
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Figure 2. Detection of TGF-1 by immunohistochemistry. Lung sections from a subject with fibroproliferative ARDS (A and B ) show TGF-1 immunoreactivity with a polyclonal anti–TGF-1 antibody. Two normal control lung sections are shown in the bottom panels (C and D). In this representative ARDS sample, strong signal was evident in areas adjacent to established fibrotic foci. Alveolar epithelial cells, macrophages, fibroblasts, and occasional intra-vascular mononuclear cells demonstrated positive signal (arrows). Normal lung tissue stained negative.
fluid, samples were expressed as pg of TGF-1/mg protein (Figure 3). When referenced to protein, ARDS BAL samples had a value of 98 ⫾ 40 pg/mg protein (mean ⫾ SEM), whereas control subjects had unmeasurable amounts of TGF-1 (P ⬍ 0.0001) To document the specificity of the MLEC assay, BAL fluid from seven normal volunteers and from six patients
samples showed no evidence of TGF- mRNA signal (Figures 1E and 1F). Immunohistochemistry for TGF-1 Four fibroproliferative ARDS samples and four control lung samples, subject to RT in situ PCR, were also used for immunohistochemical detection of TGF-1. A strong signal was detected in the ARDS tissue samples (Figures 2A and 2B). As in the RT in situ PCR samples, areas adjacent to fibrotic foci showed the most signal intensity. Little signal was seen in areas of established fibrosis. Alveolar epithelial cells, macrophages, mononuclear cells, and fibroblasts stained positive for immunoreactive TGF-1. No signal was seen in the non-ARDS control samples (Figures 2C and 2D). Measurement of Active TGF-1 by MLEC Luciferase Assay A total of 13 cases and 7 control subjects were evaluated by luciferase assay. In ARDS cases, samples were obtained within 24 h of the diagnosis of ARDS. Based on the TGF-1 standard curve, relative light units were expressed as pg/ml of BAL fluid. Following standardization for protein in BAL
Figure 3. Active TGF-1 levels in BAL samples in ARDS cases (n ⫽ 13) and controls (n ⫽ 7) standardized to BAL fluid protein. Mean and SEM are shown. *Significantly greater than control (P ⬍ 0.0001).
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Figure 4. Specificity of TGF-1 luciferase activity. A blocking anti–TGF-1 antibody was used to determine the specificity of the luciferase activity. BAL fluid from both control and ARDS samples was subject to luciferase assay in the absence (black bars) or presence (gray bars) of antibody. Mean and SEM are shown. Controls had no detectable signal in both instances. ARDS samples showed inhibition of the TGF-1 signal with the addition of antibody. *Significantly less than ARDS samples without antibody.
with ARDS was evaluated by luciferase assay, before and after the addition of a blocking anti–TGF-1 antibody (10 g/ml). BAL samples from patients with ARDS showed elevated TGF-1 activity compared with control subjects. The addition of a neutralizing antibody to active TGF-1 resulted in loss of the luciferase activity, indicating the specificity of the assay for active TGF-1 (Figure 4). Values are expressed in pg of active TGF-1 per ml of BAL fluid. Paraffin-embedded lung tissue, used for TGF-1 immunohistochemistry, was also subject to staining for TGF- receptor I and II expression (see online data supplement for Methods and Figure E1). Positive staining for both receptors was evident in type two alveolar epithelial cells in ARDS lung tissue. Normal lung tissue had fewer type two epithelial cells and little to no expression.
Discussion Although a role for TGF-1 has been defined in fibrotic conditions in the lung (30), clinical studies of its role in human ARDS are lacking. In support for the concept that TGF-1 plays a critical role in the pathogenesis of ARDS, we have: (i ) documented the presence of TGF-1 mRNA in ARDS lung tissue; (ii) demonstrated the presence of TGF-1 protein in lung tissue from patients with fibroproliferative ARDS; (iii) identified functionally active TGF-1 in BAL fluid from patients with ARDS within the first 24 h of diagnosis, which was not evident in normal control subjects; and (iv) inhibited the functional activity of TGF-1 with a blocking antibody, thus highlighting the specificity of the assay. By RT in situ PCR and immunohistochemistry, TGF-1 signal localized to alveolar epithelial cells, macrophages, and fibroblasts. Conversely, minimal localization was seen in areas of established fibrosis. Normal lung tissue had no evidence of TGF-1 by either method. To our
knowledge, this is the first documentation of elevated, functionally active TGF-1 in human ARDS. TGF-, a multifunctional cytokine, exists in three isoforms. Of these, TGF-1 is the most abundant, the most upregulated in response to tissue injury, and the most implicated in fibrosis (22). TGF-1, applied topically, can accelerate wound healing (22), but in excess can cause organ and tissue fibrosis (31). TGF-1 is produced by a variety of cells, including epithelial cells. Latent TGF-1 resides at the cell surface and in extracellular matrix in an inactive precursor form. It can be activated by a number of stimuli, including plasmin, metalloproteinases, (32) reactive oxygen species, radiation, and thrombospondin (33). Spatially restricted activation of TGF-1 occurs by binding to the integrin ␣v6. ␣v6, expressed at low levels in healthy cells, is upregulated by injury and inflammation. (34) TGF-1 signals through a transmembrane complex, which has two distinct transmembrane proteins. Ligand binding results in their association and activation. The signal is transduced to the nucleus via SMAD proteins, though other pathways may also exist (35). The significance of finding active TGF-1 in the BAL fluid in early ARDS is twofold. First, it suggests that the early increase in collagen turnover in ARDS, as indicated by elevated procollagen levels, may be driven by activation of latent TGF-1. Second, active TGF-1 may, in addition, have effects on epithelial permeability in humans, enhancing pulmonary edema, as was demonstrated in a murine model of acute lung injury (24). Whether active TGF-1 levels correlate with the development of fibroproliferative ARDS has yet to be determined. In this particular study, we did not find a correlation between TGF-1 levels from one time point and time on the ventilator or mortality. A larger cohort of patients with multiple TGF-1 measurements will be needed to fully address this issue. The precise mechanism of TGF-1 activation in ARDS is under investigation. Binding to ␣v6 can cause spatially restricted activation of TGF-1 (36). Proteases such as neutrophil elastase and matrix metalloproteinases (MMP) are elevated in patients with ARDS (37, 38). Furthermore, MMP-2, -3, and -9 have been shown to activate latent TGF-1 (39). Although upregulation of TGF- receptor mRNA and protein has been identified in animal models of lung inflammation, the regulation of receptor expression and the pattern of receptor expression in ARDS is unknown. In summary, we have documented elevated TGF- levels within 24 h of the diagnosis of ARDS. We have also demonstrated TGF-1 activity in archived fibroproliferative ARDS tissue samples. These findings have important implications for understanding the pathogenesis of ARDS. Acknowledgments: This research was supported by National Institutes of Health Grants HL 69899, HL 40871, HL 62054, and HL 66108.
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