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. Author manuscript; available in PMC: 2011 Apr 1.
Published in final edited form as: Lung. 2010 Feb 13;188(2):133–141. doi: 10.1007/s00408-010-9229-4

Crocidolite Induces Prostaglandin I2 Release Mediated by Vitronectin Receptor and Cyclooxygenase-2 in Lung Cells

Francisco J Leyva 1,✉, Kevan Roberts 2
PMCID: PMC2862624  NIHMSID: NIHMS194895  PMID: 20155273

Abstract

Interstitial lung disease (ILD) produces disruption of alveolar walls with loss of functionality and scar tissue accumulation. Asbestosis is the ILD produced by the inhalation of asbestos fibers. This study attempts to elucidate the role of lung epithelial cells in the generation of asbestos-induced ILD. When exposed to crocidolite LA-4 cells had a decrease in viability and an increase in the release of lactate dehydrogenase (LDH) and 6-keto PGF1α, a PGI2 metabolite. PGI2 release was mediated by cyclo-oxygenase-2 (COX-2) and vitronectin receptor (VNR). When LA-4 cells were treated with VNR inhibitors, either RGD (Arg-Gly-Asp) peptide or VNR blocking antibody, a statistically significant decrease in PGI2 metabolite production was observed, but crocidolite-induced cytotoxicity was not prevented. These findings propose that crocidolite is coated by an RGD protein and binds VNR-inducing COX-2 expression and PGI2 release. Moreover, when LA-4 cells were exposed to crocidolite in the presence of reduced serum culture media, PGI2 production was prevented, and when bronchoalveolar lavage fluid (BALF) was added, PGI2 production was rescued. Cytotoxicity did not occur, either in reduced serum culture media or when BALF was added. In conclusion, crocidolite requires the presence of an RGD protein coating the fibers to induce inflammation (PGI2 production) and crocidolite alone cannot induce cytotoxicity in lung cells.

Keywords: Crocidolite, Pulmonary fibrosis, Cyclooxygenase 2, Prostaglandin I2, Vitronectin receptor

Introduction

Asbestos fibers are a naturally occurring hydrated silicate mineral used for commercial applications. It has been reported that over the last century, more than 30 million tons of asbestos have been mined, processed, and applied in the United States [1]. Exposure to asbestos typically occurs during mining and milling of the fibers or during industrial application of asbestos in textiles, insulation, shipbuilding, brake lining mechanics, and other areas. Nonoccupational exposure is usually related to asbestos fibers inadvertently released into the environment and transported by asbestos-contaminated clothing or other materials [2]. Asbestos has two primary classes: serpentine and amphibole. Structurally, serpentine fibers are curly-stranded structures and amphibole fibers are straight, rigid, and needlelike. Three examples of amphibole fibers are crocidolite, tremolite, and amosite [3]. Regardless of their different physical properties, all types of asbestos fibers are considered fibrogenic to the lung, but some of them, including amphibole, are also considered carcinogenic.

Asbestosis is an interstitial lung disease (ILD) produced by the inhalation of asbestos fibers. ILD is a large and heterogeneous group of lower respiratory tract disorders. ILD is also known as “interstitial pulmonary fibrosis” or “diffuse parenchymal lung disease” [4, 5]. The cause of most ILDs is unknown, but it has been suggested that a stimulus (e.g., asbestos, beryllium) produces repeated episodes of lung injury and the damaged alveolar epithelium induces accumulation and activation of immunoinflammatory cells, with subsequent migration and proliferation of fibroblasts and extracellular matrix deposition in the alveolar interstitium leading to fibrosis and loss of lung function [6, 7].

Coating crocidolite asbestos with vitronectin enhances its cell internalization via the alpha V integrin VNR (vitronectin receptor) αvβ5 in mesothelial cells [8]. VNR and other integrins recognize the RGD (Arg-Gly-Asp) motif present in a variety of ligands such as vitronectin, fibronectin, osteopontin, bone sialoprotein, thrombospondin, fibrinogen, von Willebrand factor, tenascin, and agrin [9–12]. Additional studies have reported the involvement of VNR and RGD motif in crocidolite internalization and toxicity [13–15]. In endothelial cells, an increase in the release of PGI2 has been reported after asbestos exposure [16, 17]. It has been suggested that changes in the PGI2 homeostasis could lead to endothelial damage, cell activation, and inflammation contributing to the development of ILD.

After asbestos inhalation, fibers deposited in the lungs typically remain in close contact with lung epithelial cells. Since this fiber-cell interaction could potentially initiate or inhibit cellular functions, the purpose of this study was to characterize the cellular response of lung epithelial cells directly exposed to asbestos fibers.

Methods

Cell Line

LA-4 cells (lung adenoma clone 4) derived from A/He strain mouse, characterized as alveolar type II cell like [18], were cultured in Ham’s F12 K medium (Mediatech, Inc., Herdon, VA) supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B (Mediatech), and 15% fetal bovine serum (FBS) (Hyclone, Logan, UT). Cell cultures were incubated at a temperature of 37°C in an atmosphere of 95% air and 5% CO2 (ThermoForma, Mariette, OH).

Asbestos Exposure

All asbestos exposure experiments were performed using the amphibole crocidolite, which was chosen due to its high toxicity. Adherent cells were trypsinized with 2–3 ml of trypsin/EDTA (Mediatech), counted using a Z1 Coulter Particle Counter (Beckman Coulter, Hialeah, FL), and plated 24 h prior to exposure at 50,000 cells in a volume of 200 μl of medium per well in a 96-well plate. Cell culture medium was changed at exposure time. Crocidolite suspension (10 mg/ml) was freshly made by mixing crocidolite obtained from Research Triangle Institute (Research Triangle Park, NC) with pure water. The suspension was sonicated using a Sonicator Ultrasonic processor XL (Misonix, Farmingdale, NY) for 1 min, mixed with fresh cell culture medium, and added to the 96-well plate. Cells were exposed to crocidolite for 24 h before performing any assessment unless indicated.

Cell Cytotoxicity Assays

Viability Assay

After exposure, culture medium was removed leaving 100 μl of medium in each well and 20 μl of CellTiter96 AQueous One Solution Reagent (Promega, Madison, WI) was added into each well and incubated for 1–4 h at 37°C in a humidified 5% CO2 atmosphere. Optical density (OD) was measured using a microplate reader (Molecular Devices, Sunnyvale, CA) at a wavelength of 490 nm and for reference at 650 nm. For normalizing purposes each control group (nonexposed) was considered to have 100% viability.

Cytotoxicity Assay

Lactate dehydrogenase (LDH) release was measured using the Cytotoxicity Detection Assay (LDH) (Roche Applied Science, Indianapolis, IN). Cells were plated 24 h before exposure as previously described. At exposure time, cell culture medium was replaced with 1% FBS in order to decrease the LDH background absorbance, as recommended by the manufacturer. Cells exposed to Triton X-100 (Sigma-Aldrich Inc., St Louis, MO) at 1% were used as positive control. After exposure, 100 μl of cell culture medium was transferred to a 96-well plate, mixed with 100 μl of reaction mixture as described by manufacturer, and incubated for 30 min at room temperature protected from light. OD was measured at wavelengths of 492 and 650 nm. Cytotoxicity was calculated as a percentage between minimum or 0% (nonexposed) and maximum or 100% (Triton X-100) control values.

6-Keto Prostaglandin F1α Detection Assay

PGI2 is nonenzymatically hydrated to become 6-keto PGF1α, which is a more stable metabolite. In order to detect PGI2, experiments were conducted to measure its metabolite 6-keto PGF1α. Cells were plated at 40,000–50,000 cells/well in a 96-well plate. After 24 h of exposure, as previously described 6-keto PGF1α was detected using an ACE competitive enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) according to the manufacturer’s instructions. For each condition, 50 μl of cell culture medium was transferred to a precoated 96-well plate, mixed with 6-keto PGF1α tracer and antiserum, and incubated for 18 h at 4°C. Plates were developed using Ellman’s reagent (Cayman Chemical, Ann Arbor, MI) and read at a wavelength of 405 nm.

Cyclooxygenase Inhibitors

The COX-1/COX-2 inhibitor indomethacin and the COX-2 inhibitor indomethacin heptyl ester were used at doses 10 and 100 times their respective IC50 [19]. Inhibitors were added at the same time that fresh cell culture medium containing crocidolite was added to the cells as previously described.

Cyclooxygenase-2 mRNA Quantification by Real-time PCR

Two to four hours after crocidolite exposure (10 μg/cm2), LA-4 cells were lysed using Trizol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated following the manufacturer’s instructions. First-strand reverse transcription was performed using RETROscript™ (Ambion Inc., Austin, TX) with oligo-dT primers, and amplification was performed using the Platinum® Quantitative PCR Super-Mix-UDG (Invitrogen) following the manufacturer’s recommendations. Real-time probes and primers from murine COX-2 and GAPDH (endogenous control) were obtained from TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA). Amplification was run and signals detected using an ABI Prism 7700 Sequence Detection System (Applied Biosystems).

Vitronectin Receptor Expression

Three hundred thousand LA-4 cells were labeled with fluorescent murine-specific anti-αv antibody (anti-CD51, RMV-7, Armenian hamster IgG, phycoerythrin-labeled [BD PharMingen, San Jose, CA]). After 20 min of incubation at 4°C, fluorescence was detected on a FACs Aria Flow cytometer using FACs Diva software (Becton Dickson, Franklin Lakes, NJ). In order to exclude dead cells, Hoechst 33258 dye (Invitrogen) was added to a final concentration of 1 μg/ml. Cells that took up the dye were considered dead cells and excluded from the analysis.

Vitronectin Receptor Inhibition

RGDS Peptide

The tetrapeptide Arg-Gly-Asp-Ser or RGDS (Sigma-Aldrich, St Louis, MO) was used as a competitive inhibitor of VNR binding, and the Arg-Gly-Glu-Ser or RGES (Sigma-Aldrich) was used for nonspecific binding control. Peptides were added at a final concentration of 10 and 100 μg/ml simultaneously with crocidolite exposure as previously described.

VNR Blocking Antibody (RMV-7)

The blocking antibody RMV-7 was used to specifically inhibit VNRs. RMV-7 antibody was added at a final concentration of 2 μg/ml at 1 h before crocidolite exposure as previously described.

Bronchoalveolar Lavage Fluid (BALF) Isolation

BALB/c mice were obtained from our animal facility. Mice 6–8 weeks old were used for BALF isolation. All animal procedures were approved by the University of Montana Institutional Animal Care and Use Committee (IACUC). Five female mice were euthanized by a lethal injection of pentothal. Lungs were surgically removed with the heart and lavaged one time only to prevent dilution of the BALF. The lavage was done using 0.9 ml of cold sterile phosphate-buffered saline containing 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B (Mediatech). All samples were mixed and centrifuged at 1500 rpm for 10 min. The BALF was separated and the cell pellet discarded.

Asbestos Exposure in the Presence of BALF

LA-4 cells were trypsinized and plated as previously described. At exposure time the medium was changed to Optimem® I reduced serum medium (Mediatech). Then BALF was added to attain a final concentration of 25%, followed by crocidolite at a final concentration of 10 and 15 μg/cm2. Cells were incubated for 24 h before performing any measurement.

Statistical Analysis

Experiments were repeated at least three times. Results are presented as the mean and SEM unless otherwise indicated. When required, data were normalized with their respective controls in order to exclude differences in background conditions. Data were analyzed using the t test for independent variables, nonparametric statistics methods such as the Mann-Whitney U test when comparing two groups, and the Kruskal-Wallis test when comparing three groups or more. Statistical analyses were performed using GB-Stat version 6.5.6. Alpha error was set at P < 0.05.

Results

Crocidolite Asbestos is Cytotoxic to LA-4 Lung Epithelial Cells

To determine if crocidolite could induce toxicity in LA-4 cells, a viability assay was performed using increasing doses of crocidolite. Three doses of crocidolite were used: 5, 10, and 20 μg/cm2, and Triton X-100 1% was used for positive control. LA-4 cells showed a dose-dependent decrease in viability (Fig. 1a). To confirm these results, a cytotoxicity assay was performed using the crocidolite dose of 20 μg/cm2. The results showed an increase in LDH release in the exposed group, confirming the results of the viability assay (Fig. 1b).

Fig. 1.

Fig. 1

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a Crocidolite-induced cytotoxicity of lung epithelial cells LA-4, dose response. Crocidolite was added at a final concentration of 5, 10, or 20 μg/cm2. After 24 h of exposure, viability was evaluated as described in the Methods section. b LDH release by LA-4 cells after crocidolite exposure. Crocidolite was added at a final concentration of 20 μg/cm2. After 24 h of exposure, cytotoxicity was evaluated by detecting LDH release. c 6-Keto PGF1α production in LA-4 cells after crocidolite exposure. Crocidolite was added at a final concentration of 10 μg/cm2. After 24 h of exposure, 6-keto PGF1α release was measured as described in the Methods section. d 6-Keto PGF1α production in LA-4 cells after crocidolite exposure in the presence of COX inhibitors. COX-1/COX-2 inhibitor indomethacin and specific COX-2 inhibitor indomethacin heptyl ester were added at a dose 10 times their respective IC50 simultaneously with crocidolite (10 μg/cm2). After 24 h of exposure, 6-keto PGF1α release was measured as described in the Methods section

Crocidolite Asbestos Promotes COX-2-dependent PGI2 Release from LA-4 Lung Epithelial Cells

Asbestos is known to induce lung inflammation that progresses to fibrosis. PGI2 release is an indicator of cellular activation and inflammation. Endothelial cells release PGI2 when exposed to asbestos prompting speculation that epithelial cells may respond in a similar way. To address this, it was necessary to verify whether LA-4 cells release PGI2 after crocidolite exposure. PGI2 production was determined by measuring its metabolite 6-keto PGF1α in LA-4 cells after crocidolite exposure at a final concentration of 10 μg/cm2 for 24 h. A statistically significant increase of PGI2 metabolite was observed in the exposed group (Fig. 1c). Since PGI2 synthesis requires cyclooxygenase activity, COX inhibitors were used to determine if PGI2 synthesis was either COX-1-or COX-2-mediated. Indomethacin (COX-1 and COX-2 inhibitor) and indomethacin heptyl ester (COX-2 inhibitor) decreased the production of PGI2 metabolite after crocidolite exposure (Fig. 1d), suggesting that COX-2 was required for the increase in PGI2 production.

Based on these findings using COX inhibitors, COX-2 mRNA was quantified using real-time PCR to determine whether asbestos induced its expression. COX-2 mRNA levels increased more than five times 2 h after exposure, returning to its basal concentration 4 h after exposure (Fig. 2a), suggesting a transitory increase.

Fig. 2.

Fig. 2

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a Cyclooxygenase-2 mRNA expression in LA-4 cells after crocidolite exposure. Crocidolite was added at a final concentration of 10 μg/cm2. After 2 and 4 h of crocidolite exposure, cells were lysed and total RNA isolated. COX-2 mRNA levels were measured by quantitative real-time PCR. b Crocidolite-induced cytotoxicity of LA-4 cells in the presence of indomethacin, a COX-1/COX-2 inhibitor. Indomethacin was added at doses 10 and 100 times its IC50 simultaneously with crocidolite (5, 10, 20 μg/cm2). After 24 h of exposure, viability was evaluated. c Crocidolite-induced cytotoxicity of LA-4 cells in the presence of indomethacin heptyl ester, a COX-2 inhibitor. Indomethacin heptyl ester was added at doses 10 and 100 times its IC50 simultaneously with crocidolite (5, 10, 20 μg/cm2). After 24 h of exposure, viability was evaluated. d Integrin receptors expression in LA-4 cells. Three hundred thousand LA-4 cells were labeled with anti-αv fluorescent-labeled antibody. After 20 min of incubation at 4°C, fluorescence was detected by flow cytometry

Asbestos Toxicity is not Dependent on Cyclooxygenase Activity

To determine whether COX-1 and/or COX-2 contributed to asbestos-induced cytotoxicity, COX inhibitors at increasing doses were used before asbestos exposure. After 24 h of exposure, COX inhibitors did not protect LA-4 cells from asbestos-induced cytotoxicity (Fig. 2b, c). These findings suggest that LA-4 cells have the ability to respond in two different ways after crocidolite exposure: A cytotoxic response, which is COX-independent, and a PGI2 response, which is COX-2-dependent.

The Vitronectin Receptor Plays an Important Role in Asbestos-induced PGI2 Production

As previously described, asbestos coated with vitronectin binds mesothelial cells through the vitronectin receptor (VNR). Therefore, we determined if asbestos-induced cytotoxicity and inflammation required lung epithelial cell VNR ligation. Antibodies recognizing the αv subunit detected VNR expression on the surface of LA-4 cells. This finding verified that LA-4 cells constitutively express the αv subunit, which is the common subunit in all VNR (Fig. 2d). To determine if the PGI2 release was VNR-mediated, inhibitors of VNRs were used. In the groups exposed to crocidolite (20 μg/cm2) and treated with either RGDS peptide or VNR blocking antibody, a statistically significant decrease in PGI2 metabolite production was observed, confirming VNR involvement in the PGI2 release (Fig. 3a, b).

Fig. 3.

Fig. 3

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6-Keto PGF1α production in LA-4 cells after crocidolite exposure in the presence of VNR inhibitors. a RGDS peptide (a VNR inhibitor) or RGES peptide (nonspecific binding control) at a final concentration of 10 μg/ml was added simultaneously with crocidolite (20 μg/cm2). After 24 h of exposure, 6-keto PGF1α release was measured as described in the Methods section. b VNR blocking antibody (anti-αv) was added at a final dose of 2 μg/ml at 1 h before crocidolite exposure (20 μg/cm2). After 24 h of exposure, 6-keto PGF1α release was measured as described in the Methods section

Bronchoalveolar Lavage Fluid Proteins are Required for Asbestos-induced PGI2 Production

In all previous experiments involving VNR it was assumed that the crocidolite fibers were coated with the extracellular matrix (ECM) proteins present in the FBS of the cell culture medium. To verify this assumption and to establish that the required ECM proteins are also present in the BALF, LA-4 cells were exposed to crocidolite in the presence of reduced serum medium and BALF. LA-4 cells in reduced serum medium did not release PGI2 when exposed to crocidolite; however, when BALF was added to the reduced serum medium, LA-4 cells released PGI2 when exposed to crocidolite (Fig. 4a). This finding suggests that proteins present in BALF and also in FBS are required in order to induce the release of PGI2. When viability was evaluated in the presence of reduced serum, LA-4 cells were protected from crocidolite-induced toxicity and the addition of BALF did not increase the toxicity (Fig. 4b), suggesting that factors present in the FBS are required in order to induce cell toxicity of LA-4 cells when exposed to crocidolite.

Fig. 4.

Fig. 4

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Crocidolite-induced response of LA-4 cells in the presence of bronchoalveolar lavage fluid (BALF). Cell culture medium was changed for reduced serum medium and BALF at a final concentration of 25% with crocidolite (10 and 15 μg/cm2). a 6-Keto PGF1α production in LA-4 cells after crocidolite exposure in the presence of BALF. After 24 h of exposure, 6-keto PGF1α release was measured as described in the Methods section. b Crocidolite-induced cytotoxicity in the presence of reduced serum medium and BALF. After 24 h of exposure, viability was evaluated as described in the Methods section

Asbestos Toxicity is not Dependent on VNR

As previously established, the cytotoxic response is COX-independent. To determine whether a protein containing a RGD motif or whether VNR was required for asbestos-induced cytotoxicity in LA-4 cells, cell toxicity assays were performed using RGDS peptide and VNR blocking antibody. Neither RGDS peptide nor VNR blocking antibody prevented crocidolite-induced cytotoxicity (10 μg/cm2) of LA-4 cells (Fig. 5a, b).

Fig. 5.

Fig. 5

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a Crocidolite-induced cytotoxicity of LA-4 cells in the presence of RGDS peptide inhibitor. RGDS peptide (a VNR inhibitor) or RGES peptide (nonspecific binding control) at a final dose of 10 and 100 μg/ml was added simultaneously with crocidolite (5, 10, 20 μg/cm2). After 24 h of exposure, viability was measured as described in the Methods section. b LDH release by LA-4 cells after crocidolite exposure (20 μg/cm2) in the presence of RMV-7, a VNR inhibitor. RMV-7 blocking antibody (anti-αv) was added at a final dose of 2 μg/ml at 1 h before crocidolite exposure (20 μg/cm2 per well). After 24 h of exposure, cytotoxicity was measured by detecting LDH release as described in the Methods section

Discussion

Asbestos is known to induce lung inflammation that progresses to fibrosis. It has been postulated that ILD develops in an inflammatory environment with abnormal repair mechanisms. Crocidolite-induced direct damage to lung cells such as macrophages and epithelial cells (e.g., alveolar type I and type II cells) may generate the conditions required for ILD development. Alveolar type II cells exemplify the importance of epithelial cells in ILD pathogenesis since they secrete pulmonary surfactant (to prevent alveolar collapse), and as a consequence of their stem cell function, they replace the damaged type I cells [20]. Our study used the immortalized murine cell line LA-4, which had previously been reported to be of alveolar type II cell origin [18], to study the role of lung epithelial cells in asbestos-induced response. Our results show that LA-4 cells were susceptible to crocidolite-induced toxicity.

PGI2 release, a product of arachidonic acid metabolism, is an indicator of asbestos-induced cell activation and inflammation [16, 17]. Interestingly PGI2 is highly unstable since it is spontaneously nonenzymatically hydrated to 6-keto PGF1α, a more stable metabolite. In this study 6-keto PGF1α production was considered an indicator of PGI2 synthesis. PGI2 has diverse effects in endothelial cells, epithelial cells, macrophages, and fibroblasts. Curiously, all these different cells are known to play a role in ILD development. This study has shown that the LA-4 epithelial cells produce PGI2 after asbestos exposure. This finding raises the possibility that epithelial production of PGI2 following asbestos exposure may modulate the function of several cell types involved in the development of ILD.

PGI2 synthesis requires the action of the COX enzymes COX-1 (constitutively expressed) and COX-2 (inducible). Several studies have suggested that PGI2 biosynthesis is highly but not solely COX-2-dependent [21, 22]. Lovgren et al. [23] reported that COX-2-dependent PGI2 production played an important role in the development of lung fibrosis in the bleomycin-induced pulmonary fibrosis mouse model. Murakami et al. [24] reported that repeated administration of a long-acting PGI2 agonist attenuated the development of bleomycin-induced pulmonary fibrosis and also improved survival in these mice. The present study showed that PGI2 production, induced by crocidolite exposure, was prevented in the presence of COX-2 inhibitors, suggesting that PGI2 production in LA-4 cells was COX-2-dependent. Moreover, a 2-h crocidolite exposure increased the expression of COX-2 mRNA fivefold in LA-4 cells. This increase was transitory but enough to maintain PGI2 synthesis for a minimum of 24 h. Based on cited reports and our findings, we propose that lung epithelial cells exposed to crocidolite express COX-2 which promotes PGI2 production.

The VNRs are a group of integrins that share a common αv subunit. Murphy et al. [25] reported that engagement of the VNR αvβ3 induces COX-1 and COX-2 expression in endothelial cells. There are five different types of VNRs (based on their β subunit): αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8. VNRs αvβ6 and αvβ8 activate transforming growth factor (TGFβ) in lung and skin by binding TGFβ latency-associated peptide (LAP), and β6-null mice are protected from bleomycin-induced pulmonary fibrosis [26, 27]. As previously described, coating crocidolite asbestos with vitro-nectin enhances its internalization via the αv integrin vitronectin receptor αvβ5 in mesothelial cells [8]. Using flow cytometry, the present study showed that LA-4 cells express the αv integrin subunit. VNR and some other integrins recognize the Arg-Gly-Asp (RGD) motif present in ligands for this receptor, which includes vitronectin. RGD peptides have been used previously as competitive inhibitors for receptors that recognized RGD domain [10, 11]. In this study, RGD peptides reduced PGI2 production by 20% after crocidolite exposure. Although the decrease is statistically significant, it also suggests that other mechanisms that do not depend on the RGD domain recognition are responsible for PGI2 production after crocidolite exposure. Interestingly, it has been proposed that VNR plays a role in TGFβ activation and also that TGFβ plays an important role in the development of pulmonary fibrosis [7, 27–29]. In this study, when the VNRs were blocked with an antibody specific for the αv chain, crocidolite-induced PGI2 production decreased more than 20%, which is consistent with the decrease observed using RGD peptides. These findings are consistent with, and extend, other studies that reported that the VNR αvβ5 enhances internalization of vitronectin-coated crocidolite fibers by mesothelial cells [8, 14]. In all asbestos exposure experiments detailed in this study, it was assumed that the crocidolite fibers were coated with the ECM proteins present in the FBS of the cell culture medium. Since most of the asbestos exposure is typically via inhalation, it was important to demonstrate similar effects at a physiologically relevant concentration of ECM proteins in the BALF. In this study, when LA-4 cells were exposed to crocidolite in the presence of reduced serum culture medium, PGI2 production was prevented, and when BALF was added, PGI2 production was rescued. These findings suggest that one or more factors present in BALF (and in FBS) are necessary to induce PGI2 release in LA-4 cells exposed to crocidolite. Vitronectin is a multifunctional protein with an RGD motif that is recognized by different members of the integrin family, including VNR [30–32]. Vitronectin coats asbestos fibers, enhancing cell internalization. It is a multifunctional adhesive protein present in large concentrations in blood serum [31]. It diffuses into the extravascular tissue where it could bind some other proteins [33]. It has been found in BALF and pleural liquid, and in higher concentrations during inflammatory states [8]. Although this study did not look for the presence of vitronectin in the BALF or FBS, our findings suggest that VNR ligands coat crocidolite fibers, favoring cell interaction and PGI2 release.

Surprisingly, crocidolite cytotoxicity was prevented in reduced serum culture medium, and the addition of BALF did not rescue the cytotoxicity. This finding favors the idea that a factor present in FBS (e.g., ECM proteins) and not in BALF is required to induce cytotoxicity, possibly binding to a receptor other than the VNR (e.g., the fibronectin receptor VLA-4). It is also possible that the same factor is present normally in very low amounts in BALF, but during lung injury and inflammatory states, production of the factor increases allowing crocidolite-induced cytotoxicity. Interestingly, LA-4 cells were also not protected from cytotoxicity by RGD peptides or VNR blocking antibodies. It should be noted that the RGD domain is not the only one recognized by integrins (e.g., CS-1 domain) and that the RGD domains of some ECM proteins involved in cytotoxicity have higher affinity for their respective receptors than the RGD peptides, reducing their ability to compete. The finding that VNR inhibitors did not prevent crocidolite-induced cytotoxicity strengthens the idea that LA-4 cells respond in two different pathways after crocidolite exposure: a PGI2 response, which is COX-2- and at least partly VNR-dependent, and a cytotoxic response, which is COX- and VNR-independent (Fig. 6). Both pathways could be associated with the development of lung fibrosis because they could affect the balance of bound and free LAP. Regardless of the pathway, the free LAP is able to bind other VNRs (such as αvβ6 or αvβ8) expressed in lung epithelial cells such as alveolar type II cells, inducing TGFβ activation and promoting healing and/or fibrosis.

Fig. 6.

Fig. 6

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Lung epithelial cells respond in two different pathways after crocidolite exposure. First, an RGD protein present in fetal bovine serum and BALF coats crocidolite and binds VNR. This interaction induces COX-2 expression and activity promoting PGI2 biosynthesis. Second, crocidolite induces cytotoxicity independently from RGD protein, VNR, COX-1, or COX-2

Because of the inherent difficulties associated with studying murine alveolar type II cells in situ, this study modeled responses using a previously characterized murine alveolar type II tumor cell. Although this approach has its limitations arising from differences in growth rate and contact inhibition, which are important and closely regulated in the alveoli environment, our results provide new insights into the pathogenesis of asbestosis that need to be considered in future studies with primary lung epithelial cells (e.g., alveolar type II cells) or animal studies.

The cause of most ILDs is still unknown. In this study, the environmental/occupational agent crocidolite was studied. This agent can activate inflammation and produce direct injury to the resident pulmonary cells [34]. Crocidolite alone could not induce either inflammation (PGI2 production) or cytotoxicity. The former required the presence of an RGD protein coating the fibers, and the latter required one or more factors present in the fetal bovine serum. Other factors such as ECM proteins (e.g., vitronectin), different cell surface receptors (e.g., integrins), and the cell’s activation status may be required to initiate and maintain the response required to produce ILD.

Acknowledgments

This work was supported by grants from National Heart, Lung, and Blood Institute, National Institutes of Health (NIH) (R01-HL079189-01A1) and from the Centers of Biomedical Research Excellence (P20RR017670). We thank Rex Robinson from the NIH library for editorial assistance. The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Contributor Information

Francisco J. Leyva, Email: fleyva1@jhmi.edu, Division of Lung Diseases, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20824, USA; Division of Clinical Pharmacology, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Osler 527, Baltimore, MD 21287, USA

Kevan Roberts, Center for Environmental Health Sciences, Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA.

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