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Published ahead of print on June 10, 2004, doi:10.1165/rcmb.2003-0420OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 344-350, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0420OC

Elastase Mediates the Release of Growth Factors from Lung In Vivo

Jo Ann Buczek-Thomas, Edgar C. Lucey, Phillip J. Stone, Chia L. Chu, Celeste B. Rich, Isabel Carreras, Ronald H. Goldstein, Judith A. Foster and Matthew A. Nugent

Department of Biochemistry and the Pulmonary Center at Boston University School of Medicine, and the VA Boston Healthcare System, Boston, Massachusetts

Address correspondence to: Matthew A. Nugent, Ph.D., Department of Biochemistry, K420, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118. E-mail: nugent{at}biochem.bumc.bu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Uncontrolled elastase activity is involved in the development of several types of lung disease. Previous reports demonstrated that growth factors are liberated from pulmonary matrix storage sites by elastase; however, release of these entities in vivo is not well defined. In the present study, we investigated the release of fibroblast growth factor-2 (FGF-2) and transforming growth factor-ß (TGF-ß), after intratracheal instillation of porcine pancreatic elastase into mice. We found that elastase promoted a time-dependent release of FGF-2 and TGF-ß1 from the lung into bronchoalveolar lavage (BAL) fluid. A large fraction of the TGF-ß1 in BAL fluid was in the active form (~ 60%), suggesting that elastase might participate in the activation of TGF-ß1 from its latent form. Analysis of the levels of FGF-2 and TGF-ß1 in mouse blood indicated that the growth factors in BAL fluid were not entirely derived from blood. Moreover, elastase treatment of pulmonary fibroblasts cultures caused the release of TGF-ß1, suggesting that the TGF-ß1 in BAL fluid could have come from lung cells/matrix. Additional in vitro studies also indicated that TGF-ß1 plays a role in upregulating elastin mRNA levels. These data suggest that elastase releases growth factors from lung that participate in elastolytic injury responses.

Abbreviations: bronchoalveolar lavage, BAL • bovine serum albumin, BSA • chronic obstructive pulmonary disease, COPD • diisopropyl fluorophosphate, DFP • extracellular matrix, ECM • enzyme-linked immunosorbent assay, ELISA • fibroblast growth factor, FGF • glycosaminoglycan, GAG • heparan sulfate proteoglycan, HSPG • phosphate-buffered saline, PBS • porcine pancreatic elastase, PPE • transforming growth factor-ß, TGF-ß


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Uninhibited elastase activity has been identified as a key underlying cause of elastic fiber degradation that occurs during the onset and progression of certain types of chronic obstructive pulmonary disease (COPD) (1, 2). Neutrophil elastase has been shown to disrupt the integrity of the microvascular barrier in acute respiratory distress syndrome (3, 4), and has been implicated in the development of other types of pulmonary vascular disease including pulmonary hypertension (5). Direct infusion of elastase has been shown to contribute to the development of pulmonary edema (6). Macrophage elastase participates in the development of emphysema in mice that results from chronic cigarette smoke inhalation (7). Although the net effect of uncontrolled elastase activity has been correlated strongly with the development of several types of lung disease, the initial molecular events that underlie the development of these elastase-mediated pathologies are poorly understood.

The extracellular matrix (ECM) of the lung is composed mainly of structural proteins and proteoglycans and exhibits unique physiologic properties. Collagen and elastin confer the primary structural and resiliency features to the lung and make up the majority of the tissue's interstitial mass (8). Proteoglycans, which comprise less than 1% of the lung's tissue mass, represent an important family of macromolecules that participate in maintaining the structural integrity of the lung and also play a central role in regulating biological processes which range from cell adhesion, proliferation, and migration to ECM deposition (9, 10). Moreover, heparan sulfate proteoglycans (HSPG), present at the cell surface and within the extracellular matrix, have been shown to modulate the activity and transport of FGF-2 and other heparin-binding growth factors under normal and pathologic conditions (11–14). Although the vast majority of studies investigating elastase-mediated lung injury have focused on direct enzymatic damage to the elastin and collagen fibers (15, 16), more recent studies have begun to elucidate the effects of elastase on other pulmonary extracellular matrix components. Several laboratories have shown that heparan sulfate and chondroitin sulfate proteoglycans are targets for elastolytic release from pulmonary matrices both in vivo (6, 17) and in vitro (18, 19). Studies from our laboratory have further demonstrated a direct correlation between elastase-mediated proteoglycan release and the release of active fibroblast growth factor-2 (FGF-2) from cultured pulmonary fibroblasts (18, 20). FGF-2 released by elastase has been shown to downregulate elastin gene transcription (21). Although these studies have investigated various aspects of elastolytic injury in vitro, little is known about the acute effects of elastolytic injury in vivo.

In the present study, we investigated the elastase-mediated release of growth factors and proteoglycan fragments within the lung in vivo. For these studies, a single dose of porcine pancreatic elastase (PPE) or saline vehicle was instilled into the lungs of FVB mice and the kinetics of the release of FGF-2 and transforming growth factor (TGF)-ß1 and -ß2 into bronchoalveolar lavage (BAL) fluid were measured. Results from these studies indicated that elastase promoted a time-dependent release of active TGF-ß1 and FGF-2 that reached maximal levels at 1–2 h after treatment. Elastase instillation did not result in the release of significant levels of TGF-ß2 into BAL fluids relative to saline-instilled animals. In vitro studies indicated that growth factors present in the BAL fluids from elastase-treated animals were not entirely derived from blood but were released from pulmonary cells and their surrounding matrix. Studies with pulmonary fibroblast cell cultures demonstrated that TGF-ß1 plays a role in upregulating elastin mRNA levels. Collectively, these data demonstrate that elastase causes the acute release of active growth factors into soluble form within the lung in vivo, suggesting that these effector substances may play important roles in mediating the initial response of the lung to elastase-mediated lung injury.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Porcine pancreatic elastase was purchased from Elastin Products (Owensville, MI). Female FVB mice and fresh harvest, whole mouse blood were purchased from Taconic Farms (Germantown, NY). Quantikine immunoassay kits and the pan-specific anti–TGF-ß rabbit polyclonal antibody were purchased from R&D Systems (Minneapolis, MN). TGF-ß1 was purchased from Roche Diagnostics Corporation (Indianapolis, IN). All other chemicals were reagent grade products obtained from commercial sources.

Animals
All animals were used and maintained in accordance with the recommendations of the National Institutes of Health and the Animal Care and Use Committee of Boston University.

In Vivo Elastase Administration and BAL
The mice were arbitrarily divided into saline-treated control groups (n = 11) and elastase treatment groups (n = 22). Animals in the control groups were killed at 0.5 h (n = 2), 2 h (n = 7), and 4 h (n = 2) after saline instillation. Animals in the elastase treatment group were also subdivided into smaller groups based on time of killing at 0.5 h (n = 5), 1 h (n = 5), 1.5 h (n = 4), 2 h (n = 4), and 4 h (n = 4) after elastase administration. The mice were initially anesthetized with methoxyflurane and received a single intratracheal instillation of 50 µg of porcine pancreatic elastase in 0.1 ml of sterile saline solution (0.9% sodium chloride) or 0.1 ml of saline alone. At the indicated times, the animals were anesthetized with sodium pentobarbital and exsanguinated by severing the abdominal aorta. The larynx was exposed and cannulated. The lungs were lavaged with 1 ml saline and the BAL fluid was collected and placed on ice. The ice-cold BAL fluids were centrifuged and the supernatant and pellets were frozen at –80°C for subsequent analyses. Upon thawing, 1 µM diisopropyl fluorophosphate was added to the BAL fluids to inhibit any residual enzymatic activity before PG and growth factor assays. After BAL collection, the pulmonary vessels were perfused with paraformaldehyde and the lungs were prepared for tissue fixation.

Isolation of Mouse Blood Fractions
Whole mouse blood was obtained from animals by cardiac puncture immediately after asphyxiation with carbon dioxide. The mouse blood was shipped on ice and the fractionation occurred within 24 h of harvest. Mouse serum was prepared by allowing whole mouse blood to clot by adding calcium chloride (20 mM) for 2 h at 37°C. The blood was then centrifuged at 1,000 x g for 15 min at 4°C to remove the clot and the blood cells. The supernatant was retained as the serum fraction. Mouse plasma and platelet poor serum fractions were prepared by centrifuging whole mouse blood at 1,000 x g for 15 min at 4°C to remove the blood cells. An aliquot of the supernatant was retained as the plasma fraction. The remaining plasma was incubated with 20 mM calcium chloride and allowed to clot for 2 h at 37°C. The clotted plasma was centrifuged at 12,000 x g for 15 min at 4°C. The supernatant was retained as the platelet poor serum fraction.

Isolation and Elastase Treatment of Pulmonary Fibroblast Cell Cultures
Primary cultures of neonatal rat pulmonary fibroblasts were isolated using established protocols (22). For the TGF-ß release studies, the cells were treated with elastase 14 d after the time of second passage. At the onset of the experiment, the cell cultures were rinsed once with phosphate-buffered saline (PBS) and twice with 44 mM sodium bicarbonate buffer, pH 7.4. Pulmonary fibroblasts were treated with sodium bicarbonate buffer alone (mock) or with bicarbonate buffer supplemented with porcine pancreatic elastase (0.1–10 µg/ml) for 15 min or with 0.5 µg/ml porcine pancreatic elastase for 0–30 min at 37°C. The elastase and bicarbonate solutions were removed from the cell layers and diisopropyl fluorophosphate (DFP) was added to the digest fractions at a final concentration of 1 µM to inhibit residual elastase activity. The digest fractions were stored at –80°C before assay. Cells for the RNA analyses were plated at 2 x 104/cm2 in 75 cm2 flasks and were maintained for 2–8 d in Dulbecco's Modified Eagle's Medium supplemented with 5% fetal bovine serum until harvesting. Specified amounts of TGF-ß1 and the TGF-ß blocking antibodies were added and the cells were harvested for RNA as indicated.

Quantitation of Total Glycosaminoglycans
Total sulfated glycosaminoglycan content in BAL fluids was determined by spectrophotometric analysis using the dimethyl methylene blue assay (23). Unknown concentrations of GAG were determined from standard curves of chondroitin sulfate prepared in phosphate buffered saline or sodium bicarbonate buffer.

Quantitation of Growth Factors
FGF-2, TGF-ß1, and TGF-ß2 levels were quantitated in BAL fluid, mouse blood fractions and pulmonary fibroblast elastase supernatants using the respective Quantikine immunoassay kits. The FGF-2 immunoassay kit recognizes active FGF-2. Both active and latent forms of TGF-ß1 and TGF-ß2 were assayed according to the manufacturer's recommended acid activation and neutralization protocols. Assay sensitivity limits were less than 3 pg/ml for FGF-2 and 7 pg/ml for the detection of TGF-ß1 and TGF-ß2. No significant cross-reactivity or interference with other TGF-ß family members was observed with the respective enzyme-linked immunosorbent assay (ELISA) kits.

Heme Measurements
Pellets from centrifuged BAL fluids were thawed from –80°C and were resuspended in 1 ml water and were kept on ice for 1 h. The fractions were diluted 1:50 with water and the absorbance was read in a Spectronic Instruments (Rochester, NY) Genesys spectrophotometer at 412 nm. Concentrations of blood present in the BAL fluids were determined by linear regression analysis from a standard curve of 412 nm absorbance of serially diluted whole mouse blood prepared in water.

Isolation and Analysis of RNA
Elastin mRNA levels were measured by Northern blotting using a cDNA probe for tropoelastin as previously described (20). The filters were exposed to X-ray film and the exposed images were scanned and processed using Adobe Photoshop software (Adobe Systems Inc., San, Jose, CA). The mouse histone H3.2 plasmid was provided by Dr. W. F. Marzluff at the University of North Carolina (Chapel Hill, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Elastase Causes the Release of FGF-2 In Vivo
Previous studies have shown that proteoglycans and growth factors stored in the extracellular matrix are targets for elastolytic release both in vivo (17) and in vitro (18). Our previous studies with pulmonary fibroblast cell cultures have shown that elastase-mediated proteoglycan release is accompanied by the release of active growth factors from matrix storage sites in pulmonary fibroblast cultures (18, 20). Thus, in the present study we wanted to determine if acute elastase exposure caused the release of fibroblast growth factor-2 in vivo. For these studies, BAL fluids from saline- and elastase-treated FVB mice were assayed for the presence of FGF-2 using a commercially available ELISA kit. As shown in Figure 1A, elastase treatment resulted in a time-dependent increase in FGF-2 accumulation with respect to saline controls. Within 30 min of elastase instillation, FGF-2 levels were nearly 10-fold higher with respect to control animals treated with saline. Maximal FGF-2 release was observed at 1.5 h after PPE instillation, and FGF-2 levels present in BAL fluid had declined within 4 h of elastase treatment. These results indicated that elastase caused the release of FGF-2 in vivo. We also assessed whether proteoglycans were released from pulmonary matrix stores under these conditions by assaying the BAL fluids from elastase-treated animals for sulfated proteoglycan content. Sulfated proteoglycan levels were measured in BAL fluid by spectrophotometric analysis, and the results are presented in Figure 1B. In contrast to our in vitro findings, total sulfated proteoglycan levels in BAL fluids derived from elastase-treated animals were lower than the respective saline controls at 0.5, 2, and 4 h after elastase administration. Hence, elastase treatment does not stimulate the release of GAG above that caused by the hydraulic edema induced by saline loading (6). However, it is important to note that this analysis did not distinguish between specific GAG types or proteoglycans. Thus, elastase might specifically degrade and release FGF-2 binding HSPG, which do not constitute a significant portion of the total GAG present in BAL fluid.



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  		Figure 1. Elastase-mediated release of FGF-2 and glycosaminoglycan (GAG) in BAL fluid. FVB mice were instilled with porcine pancreatic elastase or saline and the lungs were subject to BAL at the times indicated. The BAL fluids were centrifuged and the supernatants were assayed for fibroblast growth factor-2 and total sulfated GAG content. In A, the BAL supernatants from elastase-treated (filled circles) and control (open circles) animals were assayed for FGF-2 content using a commercially available ELISA. The results are expressed as the mean ± SEM for all treatment groups where n >= 4. For the 0.5 and 4 h saline controls (n = 2), the data ranged from 0–0.8 pg/ml. A Welch-Satterthwaite t test analysis was conducted to determine if there was a difference in the levels of FGF-2 in the BAL from the PPE-treated animals (n = 22) compared with the saline controls (n = 11). The difference was significant (P = 0.0088). A linear regression was run with time as the independent variable to predict FGF-2, and showed that there were no significant differences in the levels of FGF-2 with time after PPE treatment (P = NS). In B, BAL fluids from elastase-treated (filled circles) and control (open circles) animals were assayed for total sulfated GAG using the spectrophotometric dimethyl methylene blue assay. The plotted values represent the average GAG content ± SEM for each treatment groups where n >= 4. For the 0.5 and 4 h saline controls (n = 2), the data ranged from 4.20–6.31 µg/ml. A t test was conducted indicating that the differences between GAG levels in the BAL fluid from PPE- and saline-treated animals were significant (P = 0.0015), however, a linear regression analysis indicated that these levels did not vary significantly with time (P = NS).

 
Elastase Causes the Release of TGF-ß1
TGF-ß can mediate elastin gene transcription by affecting message stability (24) and has been identified as a central mediator in certain types of acute lung injury (25). Thus, we wanted to assess whether TGF-ß was also released by elastase from pulmonary storage sites in vivo. For these studies, active and total (active plus latent) TGF-ß1 and TGF-ß2 levels were measured in BAL fluids from saline and PPE-treated animals. As shown in Figure 2A, elastase instillation caused a significant release of total TGF-ß1. Within 30 min of PPE instillation, there was a 5-fold increase in total (active plus latent) TGF-ß1 (240 pg/ml) with respect to saline controls (47 pg/ml). Within 1–2 h of elastase administration, TGF-ß1 levels had increased 25- to 57-fold over 2 h saline control animals (678 and 1,534 pg/ml, respectively, versus 26 pg/ml). Four hours after PPE administration, total TGF-ß1 levels present in the BAL fluid were still increased more than 57-fold over 4 h, saline control animals (1,888 pg/ml versus 32 pg/ml, respectively).



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  		Figure 2. Elastase-mediated release of total and active TGF-ß1 into BAL fluid supernatants. BAL samples from PPE-treated and control animals were assayed for TGF-ß1 content by ELISA. In A, BAL supernatants were subject to acid activation and neutralization to determine the total level (active plus latent) of TGF-ß1 present in the BAL fluids from elastase-treated (filled circles) and control animals (open circles). The data are expressed as the mean ± SEM for each treatment groups where n >= 4. For the 0.5 and 4 h saline controls, the data ranged from 3.24–10.99 pg/ml. In B, the BAL supernatants from elastase-treated (filled circles) and control (open circles) animals were assayed without acid activation for active TGF-ß1 and the results are expressed as the mean ± SEM for treatment groups where n >= 4. For the 0.5 and 4 h saline controls, the data ranged from 31.73–52.89 pg/ml. Statistical analysis of these data is presented in the legend to Table 1.

 

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  		TABLE 1. TGF-ß1 levels in BAL fluid supernatants

 
The amount of active TGF-ß1 present in BAL fluid from elastase-treated animals increased as a function of time and the results are summarized in Figure 2B. Within 30 min of PPE instillation, the amount of active TGF-ß1 present in the BAL fluids was 14-fold greater than in saline-treated animals. The relative amounts of active TGF-ß1 continued to increase significantly with time up to 4 h after enzyme administration. As presented in Table 1, 29–68% of the total TGF-ß1 released into BAL fluids from PPE-instilled animals was in the active form for up to 4 h.

BAL fluids from saline and elastase-treated animals were also assayed for active and total TGF-ß2 levels. In contrast to the significant levels of TGF-ß1 released by elastase, only minimal amounts of TGF-ß2 were detected in BAL fluids from saline and PPE-treated animals. Total TGF-ß2 levels in BAL fluids from control animals were 39.4 and 40.3 pg/ml at 30 min and 2 h after saline instillation. Within 4 h of saline instillation, total TGF-ß2 levels present in BAL fluid declined to 4.9 pg/ml. Thirty minutes after PPE administration, total TGF-ß2 levels present in the BAL fluids were below the detection limits of the assay (7 pg/ml). One hour after PPE instillation, average TGF-ß2 levels were 22.9 pg/ml, and by 4 h after PPE instillation total TGF-ß2 levels declined to 7.4 pg/ml. Collectively, these data indicate that elastase exposure results in the time-dependent release of large quantities of TGF-ß1 but does not result in the release of significant amounts of TGF-ß2 relative to saline controls in vivo.

Heme and Blood Volume Determination in BAL Fluids after Elastase Challenge
Because elastase injury can affect the alveolar microvasculature to enable blood infiltration into the lung (3), we analyzed BAL fluid cell pellets from saline- and PPE-treated animals for heme content. Heme absorbance (A412nm) of these samples was compared with standard curves generated with whole mouse blood. Whereas BAL fluid from saline-treated animals showed very low levels of blood contamination (< 1.3 µl/ml), those from PPE-treated animals showed a time-dependent increase (3–51 µl/ml; Table 2). To determine whether this level of blood contamination in the BAL fluid samples could contribute significant amounts of blood-derived FGF-2, we measured the level of FGF-2 in mouse serum and found it to be very low (6 pg/ml), indicating that blood-derived FGF-2 in the BAL samples would be insignificant (0.002–0.31 pg/ml). Although the low levels of FGF-2 in blood were expected based on previous findings, blood has been shown to be a significant source of TGF-ß1 (26); consequently, a more detailed analysis of the possible contribution of blood-derived TGF-ß1 was conducted.


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  		TABLE 2. Measurement of heme absorbance and determination of blood volume present in BAL fluids

 
Determination of TGF-ß1 Levels in Mouse Blood Fractions
To evaluate the possible contribution of blood-derived TGF-ß1 to that measured in BAL fluid samples, we determined the levels of active and latent TGF-ß1 in mouse blood. For these studies, mouse serum, plasma, and platelet-poor serum fractions were isolated from whole blood and were assayed for TGF-ß1 under acid activating (total TGF-ß1) and nonactivating (active TGF-ß1) conditions, and the results are presented in Table 3. Mouse serum contained the greatest amount of TGF-ß1 of all blood fractions assayed. Total TGF-ß1 (active plus latent) present in serum was 29,783 pg/ml, whereas the amount of active TGF-ß1 present in the serum fraction was 465 pg/ml or ~ 1.6% of the total. Mouse plasma and the platelet poor-serum fractions contained significantly less TGF-ß1 than was present in the serum fraction. The amount of total TGF-ß1 measured in mouse plasma was 8,544 pg/ml with 3.96% or 338 pg/ml present as active TGF-ß1. Platelet-poor serum contained the least amount of TGF-ß1.


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  		TABLE 3. TGF-ß1 levels in mouse blood fractions

 
Based on the values of TGF-ß1 in plasma and serum, we extrapolated the data to calculate the potential, maximal amounts of active and total TGF-ß1 in the BAL fluid that could be accounted for by blood contamination of the samples (Table 4). If the blood that was present in the BAL fluid was solely attributed to the presence of serum, at 30 min after PPE administration, the amount of active TGF-ß1 derived from serum infiltrating into the BAL is estimated to be 1.42 pg. Because the amount of blood infiltrating into the BAL fluids increased as a function of time after PPE instillation (Table 2), the amount of active TGF-ß1 that could be contributed from serum would also increase as a function of time to a maximum of 24.16 pg, 4 h after PPE instillation. This represents less than 5% of the amount of active TGF-ß1 detected within the BAL fluid 4 h after enzyme administration. With respect to total TGF-ß1 present in BAL fluids from PPE-treated animals, the potential amount of total TGF-ß1 derived from infiltrating serum would have a more dramatic impact on the levels of total TGF-ß1 present in the BAL fluids. Serum infiltration into the BAL fluids from PPE-treated animals could account for 90 pg or 37% of total TGF-ß1 present in the BAL fluids within 30 min of PPE administration. Within 1 h of enzyme administration, the amount of total TGF-ß1 potentially derived from serum would increase to 537 pg or to 79% of the amount of TGF-ß1 released. At 4 h after PPE instillation, the amount of total TGF-ß1 that could be contributed by infiltrating serum would increase to 1,546 pg or 81% of the amount of TGF-ß1 present in the BAL fluid. With respect to the plasma and platelet-poor serum fractions, the potential contribution of active and total TGF-ß1 would be less significant (8–23% of the total TGF-ß1). Although this analysis indicates that infiltration of blood into BAL fluid could influence the amount of TGF-ß1 present, it suggests that it is unlikely that the TGF-ß1 present in the BAL fluid samples was entirely derived from blood. Hence, these data suggest that elastase instillation causes the release and activation of TGF-ß1 from lung tissue itself.


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  		TABLE 4. Potential contribution of TGF-ß1 present in mouse blood fractions to TGF-ß1 levels present in BAL fluid

 
Determination of TGF-ß1 Release by Elastase from Pulmonary Fibroblast Cell Cultures
A possible source of the TGF-ß1 in the BAL fluid from elastase-treated mice is the lung extracellular matrix. TGF-ß1 has previously been demonstrated to be stored in the ECM bound to proteoglycan sites (26, 27). Thus, elastase could mediate the release of both FGF-2 and TGF-ß1 from ECM storage sites by targeting PG core proteins for degradation.

To determine if lung fibroblasts are capable of synthesizing and depositing sufficient levels of TGF-ß1 into the matrix to account for the levels observed in the BAL fluids, we evaluated the levels of elastase-releasable TGF-ß1 from primary pulmonary fibroblast cell cultures in vitro. As shown in Figure 3, elastase treatment of pulmonary fibroblast cell cultures resulted in the release of significant quantities of TGF-ß1 that were consistent with the levels of TGF-ß1 released into the BAL fluids by elastase in vivo. As shown in Figure 3A, treatment of cell cultures with 0.5 µg/ml porcine pancreatic elastase caused more than 2,000 pg/ml of total (active plus latent) TGF-ß1 to be released within 5 min of enzyme addition. Treatment of fibroblast cultures with elastase for additional times up to 30 min did not result in the release of significantly greater amounts of TGF-ß1. The percentage of active TGF-ß1 present in the pulmonary fibroblast elastase supernatants was less than 5% at all time points assayed. As shown in Figure 3B, relatively low concentrations of elastase were required to promote the release of TGF-ß1 from fibroblast matrices. As shown in Figure 3B, 0.1 and 0.25 µg/ml elastase caused the release of large amounts of total TGF-ß1 (1,493.83 and 1,922.55 pg/ml, respectively) within 15 min of elastase addition, and the elastase mediated-release of total TGF-ß1 was similar with the addition of elastase concentrations up to 2.5 µg/ml. Treatment of fibroblast cell cultures with 5 and 10 µg/ml elastase for 15 min resulted in the detection of diminished amounts of total TGF-ß1 (1,215.94 and 1,199.96 pg/ml, respectively), which may be, in part, due to elastase-mediated proteolysis of TGF-ß1 upon release from extracellular storage sites. The percentage of active TGF-ß1 present in the elastase supernatants from cultures treated with 0.1–10 µg/ml elastase was below 3%. These findings indicate that elastase caused the release of significant amounts of TGF-ß1 from pulmonary fibroblast cell cultures. Furthermore, these studies collectively suggest that a large fraction of TGF-ß1 released as a consequence of elastase action in vivo could result from growth factor liberation from pulmonary extracellular storage sites.



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  		Figure 3. Elastase-mediated TGF-ß1 release from pulmonary fibroblast cell cultures. Pulmonary fibroblasts were maintained for 14 d after the time of second passage before elastase treatment. In A, the cells were treated with 0.5 µg/ml porcine pancreatic elastase for up to 30 min, and in B the cells were treated with increasing elastase concentrations for 15 min at 37°C. The elastase supernatants were assayed for active (filled circles) and total (active plus latent) (open circles) TGF-ß1 using a commercially available ELISA kit. The data are expressed as the mean ± SEM from triplicate samples. Values of active TGF-ß1 (filled circles) ranged from 20–100 pg/ml for the elastase time course study and from 0–43.5 pg/ml for the elastase dose response study.

 
TGF-ß1 Upregulates Elastin Expression in Pulmonary Fibroblast Cell Cultures
Because we have demonstrated that TGF-ß1 is released as a consequence of elastase exposure both in vivo and in vitro, we wanted to assess the potential role of endogenous TGF-ß1 on elastin gene expression. Two approaches were taken to ascertain the possible involvement of TGF-ß in this process. Initially, pulmonary fibroblasts were maintained for up to 8 d in both the absence and presence of 0.5 µg/ml TGF-ß blocking antibody (blocking TGF-ß1, -ß2, and -ß3) before RNA harvest. As we have previously observed with this cell culture model, elastin message levels in control cells increased in a time-dependent fashion beginning at Day 3 and continued to increase until Day 8 in culture (Figure 4A) (28). In cells that were pretreated with TGF-ß–blocking antibody before RNA harvest, elastin message levels were reduced with respect to control cells. The decrease in elastin message level in response to the TGF-ß blocking antibody was shown to be dose-dependent, as illustrated in Figure 4B. In addition, to determine whether TGF-ß could directly upregulate elastin message levels, 1 and 10 ng/ml TGF-ß1 were added to fibroblast cultures and were maintained for 3 d before harvest. As shown in Figure 4B, addition of exogenous TGF-ß1 resulted in a dose-dependent increase in elastin message levels. Collectively, these findings suggest that TGF-ß1 plays a role in upregulating elastin message levels in pulmonary fibroblasts. Additional studies are necessary to identify the mechanisms underlying the TGF-ß1–mediated increase in elastin mRNA.



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  		Figure 4. Northern blot analysis of subconfluent and confluent primary pulmonary fibroblast cultures treated with TGF-ß1–blocking antibody and TGF-ß1. Total RNA was extracted from pulmonary fibroblast cell cultures on the indicated days after treatment without or with the indicated concentrations of TGF-ß1–blocking antibody or TGF-ß1. In A, pulmonary fibroblasts were maintained for up to 8 d after second passage in the absence or presence of 0.5 µg/ml TGF-ß–blocking antibody (blocking TGF-ß1, -ß2, and -ß3) before RNA harvest. Northern blot analysis was performed on 20 µg of total RNA and was probed for elastin, histone H3.2, and actin mRNA levels as indicated. rRNA was stained with methylene blue as indicated. In B, pulmonary fibroblasts were seeded and on Day 2, increasing concentrations of the TGF-ß–blocking antibody or TGF-ß were added and the cells were maintained until Day 5 before RNA harvest. Northern blot analysis was performed and was probed for elastin, histone, and actin message levels and the corresponding rRNA was stained as indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Elastase activity has been identified as an underlying theme in the development of several types of pulmonary disease. Elastolytic injury results in direct damage to the structural components and organization of the pulmonary matrix, and also results in the release of active growth factors and proteoglycan fragments from extracellular matrix storage sites that may participate in cellular injury and repair responses (18, 21). In the present study, we have shown that intratracheal elastase administration caused the time-dependent release of active FGF-2 and TGF-ß1 within the lung in vivo within 30 min of enzyme instillation. These findings are consistent with our previous work investigating FGF-2 and proteoglycan release by elastase in vitro (18, 20), and adds biological relevance in support of the hypothesis that pulmonary cell function is regulated by the presence of active growth factors and potentially proteoglycan fragments released as a consequence of elastolytic injury.

The release of active growth factors by elastase both in vivo and in vitro represents a potentially important regulatory mechanism in controlling cellular responses to elastolytic injury. The elastase-mediated release of proteoglycan fragments from the matrix would be expected to directly impact growth factor diffusion and availability at the site of injury (12), as well as growth factor–dependent signaling pathways which use heparan sulfate and chondroitin sulfate proteoglycans in co-receptor capacities (27, 29, 30). The elastase-mediated displacement of growth factors from proteoglycan storage sites within the matrix also represents an important mechanism for regulating growth factor release, recruitment, and activation in response to protease injury. For example, FGF-2 released by elastase downregulates elastin gene transcription (21, 31), whereas active TGF-ß1 upregulates elastin production and other matrix molecules in pulmonary fibroblasts (32, 33). Sustained TGF-ß1 release from proteoglycan binding sites within the ECM has also been shown to be a key mediator in the development of tissue fibrosis (27, 30). Although this work has identified some of the possible initial mediators of acute elastase injury in the lung, the precise role of growth factors as biological effectors of these processes remains to be more clearly defined.

An intriguing finding in this report was the magnitude of TGF-ß1 released by elastase both in vitro and in vivo. Elastase administration caused the release of large quantities (ng amounts) of TGF-ß1 from pulmonary cell storage sites under relatively mild treatment conditions in vitro (Figure 3) and the levels of active TGF-ß1 present in BAL fluid were not entirely attributable to blood contamination (Table 4). In addition, we observed that a significant proportion of the TGF-ß1 released into BAL fluid after elastase treatment was present in the active state (29–68%; Table 1), whereas less than 5% of the TGF-ß1 derived from elastase treatment of fibroblast cell cultures was active (Figure 3). The difference in the relative percentages of active TGF-ß1 under in vivo and in vitro conditions suggests that elastase may play a direct role not only in the release of TGF-ß1 from its storage sites in vivo, but may also participate in its activation from the latent form. The extent to which TGF-ß1 is activated in vivo likely results from the complexity of the elastolytic injury response. For example, elastase might activate factors (e.g., MMPs) within the lung that directly mediate activation of TGF-ß1 in vivo. Although activated TGF-ß1 might play important roles in injury repair through its ability to stimulate matrix synthesis (25, 34), excessive levels of active TGF-ß1 might also play a direct role in the development of pulmonary emphysema by altering MMP-12 expression (35). Additional studies to define mechanisms responsible for the activation of latent TGF-ß1 under normal and elastolytic injury conditions in vivo warrant further investigation.

In summary, we report the acute release of active growth factors into mice bronchoalveolar lavage fluid after elastase instillation. These studies have identified the release of FGF-2 and TGF-ß1 as an acute consequence of elastolytic injury in vivo. These studies further suggest that these elastase-released entities may play important roles in the normal tissue response to injury, that when excessive, might also participate in the development and progression of chronic obstructive pulmonary disease.


   Acknowledgments
 
The authors thank Mrs. Valerie Verbitzki and the Department of Biochemistry Cell Culture Core Facility for providing the pulmonary fibroblast cell cultures. The authors also thank Dr. Adrienne Goerges for her assistance on preparing the Photoshop figures in this manuscript. They gratefully acknowledge Gerald Coffman for his expert statistical analysis. This work was supported by NIH Program Project Grant P01 HL 46902 and by NIH NRSA HL 10332 to J.B.T.

Received in original form November 20, 2003

Received in final form April 29, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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