Enhancement of fibronectin expression in rat lung by ozone and an inflammatory stimulus

S. K. Gupta, P. G. Reinhart, and D. K. Bhalla

Department of Occupational and Environmental Health Sciences, Wayne State University, Detroit, Michigan 48202

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study investigated the relationship of fibronectin expression and induction of pulmonary inflammation by ozone (O3). Rats were exposed to 0.8 parts/million O3 to induce lung inflammation. A second inflammatory stimulus, rabbit serum, was applied intratracheally to augment O3-induced inflammation. Bronchoalveolar lavage fluid (BALF) and lung tissues were analyzed for fibronectin protein and mRNA expression. Blood plasma was analyzed to investigate the potential of a minimally invasive procedure in predicting lung inflammation and fibronectin levels. Significant increases in the levels of fibronectin protein in the BALF and lung tissue after O3 exposure were further enhanced by pretreatment with normal serum. An increase in fibronectin mRNA following O3 exposure was also enhanced by serum pretreatment, which by itself had no effect on lung fibronectin mRNA expression. Plasma fibronectin levels were comparable in air-PBS and O3-PBS groups but increased in the O3-serum group. The results suggest leakage of fibronectin from blood plasma into the lung following intratracheal application of rabbit serum and upregulation of local synthesis following O3 exposure.

inflammation; bronchoalveolar lavage

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

FIBRONECTINS ARE LARGE GLYCOPROTEINS consisting of two 225-kDa subunits crosslinked by disulfide bonds and found in most body fluids, in the extracellular matrix, and in association with basement membranes (17, 25, 34). These are described as multifunctional proteins with binding sites for different cells and molecules, including collagen, fibrin, heparin, and cell surfaces (8). The plasma contains a large concentration of fibronectin and serves as a source of some fibronectin in tissues, where it is likely to support tissue repair after an injury (10). By providing a site for cell attachment, fibronectin promotes cell adhesion, cell migration, and wound repair (18, 26). Its capacity to mediate adhesion in cell-cell and cell-substratum interactions is also recognized (23). Although fibronectin is an essential component of the interstitial matrix and its expression increases after tissue injury, the relative contributions of plasma and cellular synthesis are not entirely clear. In lung, fibronectin is actively expressed during development, but it decreases to low levels in the adult (8, 35) and can be recovered from terminal airways and alveoli by bronchoalveolar lavage. Enhanced fibronectin expression is noted in a variety of lung disorders, including acute respiratory distress syndrome, bronchiolitis obliterans organizing pneumonia, and idiopathic pulmonary fibrosis (24). Increased release of fibronectin by alveolar macrophages and increased concentration of fibronectin in the bronchoalveolar lavage fluid (BALF) may also reflect enhanced activity of the inflammatory cells in interstitial lung disease (32).

Ozone (O3), a major oxidant pollutant in photochemical smog, is a known pulmonary toxicant. Its deleterious effects in the lung include airway inflammation (15, 20), epithelial damage (1), and increased epithelial and vascular permeability (3, 4, 19). The inflammatory response induced by O3 exposure is associated with an increase in macrophage adhesion (27), release of inflammatory mediators (11, 14, 21), and recruitment of polymorphonuclear leukocytes (PMNs) from the blood (5). Although a role for fibronectin has been speculated in the cascade of events leading to inflammation in general, the present study explored its relationship to lung inflammation induced by O3. This study provides a detailed analysis of fibronectin expression in rat lung tissue, blood plasma, and BALF following exposure to O3 [0.8 part/million (ppm)], both alone and in combination with another inflammatory stimulus, normal rabbit serum. The selection of O3 concentration and of exposure duration in this study was based on our past experience (5, 27) and represents conditions that do not cause massive injury but are sufficient to induce reproducible inflammatory changes in the lung. Fibronectin protein levels in the BALF, blood plasma, and lung tissue were analyzed by ELISA and Western blotting, and fibronectin mRNA expression in the lung was examined by RNase protection assay.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals

Virus-free male Sprague-Dawley rats, 6-8 wk of age (250-275 g), were purchased from Harlan Sprague Dawley (Indianapolis, IN) and shipped in filtered containers to minimize exposure to particulate pollutants. The animals were housed in polycarbonate cages and kept in a laminar flow chamber until used for the study. The animals were quarantined for 1 wk before use. Rats were maintained in accordance with the guidelines of the University Committee for Animal Care and received food and water ad libitum.

Experimental Design

Rats were anesthetized with metofane (Pitman Moore, Mundelein, IL) and intratracheally instilled (200 µl) with either sterile PBS, pH 7.4, or 1% rabbit serum (Sigma, St. Louis, MO) ~3 h before exposure. The rats were exposed to either filtered air or O3 (0.8 ppm) for 3 h in stainless steel whole body chambers. After exposure, rats were returned to their cages and were killed 8-12 h later. The lungs were lavaged, cells were separated, and the BALF was analyzed for fibronectin. At the same time, blood samples were collected in tubes containing the anticoagulant ethylenediaminetetraacetic acid (EDTA). From these samples, plasma was separated and analyzed for fibronectin content. For tissue analysis, the animals were exsanguinated, and the lungs were rapidly removed, frozen in liquid nitrogen, and stored at -70°C.

Exposure System

The animals were exposed for 3 h to either O3 (0.8 ppm) or filtered air in stainless steel whole body chambers. The chamber atmosphere temperature and relative humidity were maintained at 23.2 ± 1.2°C and 48%, respectively. The chambers were supplied with air that had been passed through a coarse particulate filter, charcoal filter, and a high- efficiency particulate filter. O3 was generated by passing dry air through an electrical O3 generator (Orec, Phoenix, AZ) and injecting it into the airstream. The O3 concentration was continuously monitored with an ultraviolet light absorption monitor (Mast, Reno, NV), and data were recorded on a single-channel strip chart.

Bronchoalveolar Lavage

Bronchoalveolar lavage was performed 8-12 h following O3 exposure as described previously (5). Briefly, the animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg). A polyethylene catheter (ID 0.05 in.; OD 0.09 in.) was placed in the trachea and secured in place. Animals were exsanguinated via the abdominal aorta, and an incision in the diaphragm was made to allow the lungs to expand during lavage. The lungs were lavaged with 7 ml of phosphate-buffered saline (PBS, without calcium and magnesium, pH 7.4, prewarmed at 37°C). The fluid was infused and recovered four times, and on the fourth recovery, the fluid was collected and placed immediately on ice. The BALF was centrifuged at 168 g for 10 min at 4°C, and the cell-free supernatant was aliquoted and stored at -70°C.

Collection of Blood Plasma for Fibronectin Estimation

Blood was drawn by cardiac puncture and collected in tubes containing potassium-EDTA as an anticoagulant. Plasma samples were prepared by centrifuging the blood samples at 1,500 g for 10 min at 4°C and were stored at -20°C until they were assayed for fibronectin.

Extraction of Fibronectin From Lung Tissue

Fibronectin from lung tissue was extracted by the method described by Bray et al. (6). In brief, lung tissue samples (~500 mg) were homogenized in 50 mM sodium phosphate buffer (pH 6.0) containing 2 M urea, 2 mM phenylmethylsulfonyl fluoride, and 5 mg/ml of heparin. The homogenate was stirred magnetically for 4 h and centrifuged at 8,000 g for 20 min. The clear supernatant was collected and stored at -20°C.

Fibronectin Analyses

ELISA. The fibronectin analyses in blood plasma, BALF, and lung tissue were performed as described by Gomez-Lechon and Castell (17). Briefly, the samples were diluted with sodium bicarbonate buffer (50 mM, pH 9.6), and 100 µl of either diluted samples or standards were added to the appropriate wells in a 96-well plate. Standards consisted of rat plasma fibronectin in the range of 100-1,000 ng/ml, whereas diluent buffer served as a control. The samples were incubated for 1 h at 37°C in a humidified chamber. After incubation, the wells were washed with PBS containing 0.1% Tween 20 (PBS-T) and incubated with goat anti-rat fibronectin (Calbiochem, San Diego, CA) for 1 h at 37°C. The plate was washed three times with PBS-T and incubated with rabbit anti-goat IgG conjugated with peroxidase (Calbiochem) for 1 h at 37°C. Finally, the plate was washed three times with PBS-T, and the color was developed by adding o-phenylenediamine (0.1 mg/ml in citrate-phosphate buffer, pH 5.0) and 0.003% H2O2. The plate was incubated for 30 min at 37°C, and the reaction was stopped by the addition of 4 N H2SO4. The optical density was measured at 492 nm, and the fibronectin was quantitated from the standard curve.

Western blotting. Western blots were done using 10 µl of BALF from each group. The BALF was mixed with sample buffer and separated on an SDS-10% polyacrylamide gel according to the procedure of Laemmli (22). After separation, the proteins were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA) with a Bio-Rad transfer unit for 2 h at 100 V. Transfer of proteins was confirmed by staining the blot with Ponceau S (Sigma). The nonspecific binding sites were blocked with 1% bovine serum albumin in PBS overnight at 4°C. After being washed three times with PBS-T, the blot was incubated with primary antibody (goat anti-rat fibronectin diluted in PBS-T; Calbiochem) for 2 h at room temperature. The blot was washed three times with PBS-T and incubated with anti-mouse IgG conjugated with peroxidase (diluted in PBS-T) for 1 h at room temperature. The blot was washed again as above, and the fibronectin binding bands were visualized by enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL) as per manufacturer's instructions.

Isolation of Total RNA and Detection of Fibronectin mRNA Expression

Total RNA was isolated according to the procedure of Chomczynski and Sacchi (7). In brief, frozen lung tissue (80-100 mg) from each group was homogenized in Tri Reagent (Sigma), which is a mixture of guanidine thiocyanate and phenol in a monophase solution; RNA was extracted with chloroform at 4°C and precipitated with isopropanol. The RNA pellet was washed with 75% ethanol, air-dried, and resuspended in diethylpyrocarbonate-treated water. The quality and quantity of isolated total RNA were examined by measuring the optical density at 260/280 nm. An RNase protection assay was used to determine the fibronectin mRNA in various samples, using an assay kit, RPA II (Ambion, Austin, TX). In brief, the [alpha -32P]UTP-labeled RNA probe was prepared by T7 transcription of plasmid containing 270-bp cDNA for fibronectin (gift from Dr. R. O. Hynes, Massachusetts Institute of Technology, Cambridge, MA).

Equal amounts of total RNA (20 µg) were used and mixed with radiolabeled probe [~1 × 106 counts/min (cpm)]. Samples were ethanol precipitated, air-dried, and mixed with 20 µl of hybridization buffer. Hybridization was carried out at 45°C for 16-18 h. After hybridization, the unprotected RNA fragments were digested with a mixture of RNase A and RNase T1 at 37°C for 30 min. Finally, the protected fragments were isolated and separated, along with the undigested probe, on a 5% polyacrylamide-8 M urea denaturing gel. The gel was dried and exposed for autoradiography on Kodak Biomax film to detect the expression of fibronectin mRNA. The procedure was repeated three times using different lung samples to confirm the results.

Statistical Analysis

Data were derived from a total of five rats per group for BALF, lung tissue, and blood plasma fibronectin analyses by ELISA. Results were compared with a one-way analysis of variance. Statistical significance was considered at P <=  0.05. Group comparisons were made with Tukey's test.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

A single 3-h exposure of rats to 0.8 ppm of O3 resulted in a significant increase in the BALF fibronectin concentration. Figure 1 shows the effects of O3 and O3 plus normal serum on the fibronectin levels in BALF. The data revealed a statistically significant increase (106%) in fibronectin in rats exposed to O3 compared with control rats exposed to air only. A similar increase (88%) in fibronectin was noted in the O3-serum group compared with the air-serum group. The combined effect of O3 and serum on the fibronectin content was an increase of 250% in comparison with the air-PBS group and was significantly greater than the O3-PBS group. There was no significant difference in fibronectin concentration in BALF recovered from the O3-PBS and air-serum groups.


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Fig. 1.   Effects of ozone (O3) exposure and serum instillation on fibronectin levels in bronchoalveolar lavage fluid (BALF). Results are group means ± SE. *Significant difference (P <=  0.05) between air-PBS (solid bar) and O3-PBS (open bar) or air-serum (hatched bar) and O3-serum (crosshatched bar).

The effects of O3 with and without normal serum pretreatment on fibronectin expression in rat lung tissue are shown in Fig. 2. These data demonstrate a statistically significant increase in lung tissue fibronectin in rats exposed to O3 (65% increase) or treated with serum (106% increase) compared with control rats treated with PBS and exposed to air. A significant increase (31%) in fibronectin was also noted in the O3-serum group, compared with the air-serum group. The exposure of serum-pretreated animals to O3 resulted in a 171% greater increase in lung tissue fibronectin in comparison with the air-PBS group. The fibronectin increase in the O3-serum group was also significantly greater in comparison with the O3-PBS group. These results of lung tissue analysis demonstrate a pattern of O3- and O3-serum-induced changes comparable to those in BALF.


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Fig. 2.   Effect of O3 exposure and serum instillation on levels of fibronectin in lung tissue. Results are group means ± SE. *Significant difference (P <=  0.05) between air-PBS (solid bar) and O3-PBS (open bar) or air-serum (hatched bar) and O3-serum (crosshatched bar).

The data in Fig. 3 show that there is no significant difference in the blood plasma fibronectin levels between the O3-PBS and air-PBS groups. In contrast, there was a slight but statistically significant increase (13%) in the fibronectin levels of the O3-serum group, compared with the air-serum group. In blood plasma, neither O3 exposure nor serum treatment alone caused a change in the fibronectin concentration, but an increase in fibronectin after the combined exposure to O3 and serum demonstrates an interplay between the two inducers.


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Fig. 3.   Effect of O3 exposure and serum instillation on fibronectin levels in blood plasma. Results are group means ± SE. *Significant difference (P < 0.05) between air-serum (hatched bar) and O3-serum (crosshatched bar). Solid bar, air-PBS; open bar, O3-PBS.

Fibronectin expression in various groups was confirmed by Western blotting. BALF analysis revealed an increase in expression of fibronectin in the O3-PBS group (Fig. 4, lane 2) compared with the air-PBS group (Fig. 4, lane 1). A similar increase in fibronectin expression was also observed in the O3-serum group (Fig. 4, lane 4) compared with that in the air-serum group (Fig. 4, lane 3). These results revealed an enhancement of both A and B chains following O3 and O3-serum exposures.


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Fig. 4.   Fibronectin expression in BALF samples by Western blotting. Bands represent A and B chains. Lane 1, air-PBS; lane 2, O3-PBS; lane 3, air-serum; lane 4, O3-serum. Nos. on left, molecular mass in kDa.

The expression of fibronectin mRNA in lung tissue was assessed in different groups to determine whether the increase in fibronectin levels was the result of de novo synthesis or leakage of protein from the blood. The results of RNase protection assay are presented in Fig. 5. Lane 1 shows an increase in expression of fibronectin mRNA in O3-PBS group compared with air-PBS (lane 2). Serum treatment alone did not cause an increase in fibronectin mRNA expression (lane 3), but an increase in fibronectin mRNA occurred in the animals treated with serum and then exposed to O3 (O3-serum group; lane 4). This increase was substantially greater in com-parison with all other groups. Densitometric quantitation of mRNA blots revealed a 5.8-fold increase in fibronectin mRNA in the O3-serum group in comparison with the air-PBS or air-serum group and a 2.2-fold increase in comparison with the O3-PBS group.


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Fig. 5.   Expression of fibronectin mRNA by RNase protection assay. Lane 1, O3-PBS; lane 2, air-PBS; lane 3, air-serum; lane 4, O3-serum; lane 5, undigested fibronectin probe.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Fibronectin plays an important role in biological processes such as cell adhesion, motility, and cell stimulation (37). A widely postulated function of fibronectin is its role in tissue repair resulting from recruitment of cells to sites of tissue injury and promotion of wound healing. The role of fibronectin in cell recruitment and tissue repair is supported by studies (9, 16, 32) demonstrating its ability to recruit fibroblasts in vivo and to cause enhanced migration of monocytes and PMNs in vitro. The present study was undertaken to establish a relationship between O3-induced airway inflammation and induction of lung fibronectin. This study also investigated the potential of plasma analysis as a minimally invasive tool for predicting lung fibronectin and inflammatory status. The levels of fibronectin were quantified in BALF, lung tissue extract, and blood plasma by ELISA. The BALF levels of fibronectin were confirmed by Western blotting, whereas the fibronectin mRNA expression in the lung was detected by RNase protection assay. Because of the previously observed augmentation of O3-induced inflammation in the lung by intratracheally delivered heterologous serum, we also examined the effect of serum-induced neutrophilia prior to O3 exposure on fibronectin expression. This allowed us to compare changes associated with PMN recruitment in response to O3 and PMN recruitment in response to an unrelated stimulus. The reasoning behind this experimental design stems from our assumption that the neutrophil functions depend on specific inducers. In support of this assumption, a recent study by Reinhart et al. (30) demonstrated that whereas O3-induced neutrophilia is associated with tissue injury, as reflected by an increase in airway epithelial permeability, the serum-induced neutrophilia does not adversely affect the epithelial functions.

The results of this study demonstrate that exposure of rats to O3 upregulates the expression of fibronectin in BALF and lung tissue. Despite a significant increase in the lung fibronectin content, O3 by itself did not alter the blood plasma fibronectin levels. However, the plasma fibronectin levels were significantly higher in rats treated with normal serum prior to O3 exposure. Since the liver, a primary source of fibronectin in the blood, is less likely to be affected by O3 inhalation, the lack of increase in plasma fibronectin is not surprising. The observed lack of O3 effect on plasma fibronectin content, however, does not completely rule out the possibility that O3 exposure alone can also cause an increase in blood fibronectin. The existence of a large fibronectin pool in the blood raises the possibility that a small increase in fibronectin level over a large background noise is not detected by the currently available techniques. Nonetheless, the results support the utility of a minimally invasive procedure of blood analysis for identification of changes in lung fibronectin under conditions of extensive inflammation, even though the blood analysis has a limited value in detecting O3-induced changes.

A similar limitation of blood analysis is also apparent in other models of lung injury. Rennard and Crystal (31) reported an increase in the amount of fibronectin in BALF in people with interstitial lung disease compared with normal individuals, but no major difference was observed in the level of fibronectin in blood plasma in the two groups. Despite the limited direct effect of O3 on plasma fibronectin, the large baseline fibronectin levels make plasma a potential source for fibronectin accumulation in the lung following tissue injury. Limper and Roman (24) have suggested that flooding of air spaces by plasma during acute lung injury could cause an elevation in plasma-derived fibronectin in the lung. The results of this study suggest that the contribution of plasma to fibronectin levels in the lung are dependent on the mode of injury and inflammatory stimulus. Thus an increase in fibronectin protein was observed by ELISA in lungs from animals treated with rabbit serum, but the elevation in mRNA expression occurred only after O3 exposure. The fibronectin protein levels in the lungs of rats receiving serum treatment before air exposure (air-serum group) represented a two-fold increase over the air-PBS group, and the levels in O3-PBS group represented a 1.6-fold increase over the air-PBS group. The 2.7-fold increase in the O3-serum group over the air-PBS group suggests an augmented effect of the two insults, i.e., O3 and rabbit serum. The lung tissue analysis by RNase protection assay, however, shows a change in mRNA expression after O3 exposure but not after serum treatment alone. These data suggest leakage of fibronectin from the blood after treatment with rabbit serum but de novo synthesis within the lung after O3 exposure. It is apparent from these data that neutrophils recruited in response to serum treatment alone do not induce fibronectin synthesis, but the substantially higher mRNA expression in the serum-O3 group over that in the O3-PBS group suggests that the neutrophils recruited by serum can be stimulated by O3 to induce increased fibronectin synthesis.

Increased fibronectin levels in the lung can occur after an acute lung injury caused by a chemically generated oxidant (29), after inhalation exposure to a pulmonary toxicant such as asbestos (2), and in association with inflammatory and fibroproliferative lung disorders such as acute respiratory distress syndrome, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis (24, 36). Because of the comparability of the results of this study to the fibronectin increases in association with inflammation and chronic lung disorders, we regard the elevation in lung fibronectin in our animal model as an indication of inflammatory lung injury produced by O3 exposure. The results of this study complement the previously reported biochemical changes in humans exposed to O3 (11) and reveal fibronectin expression at both mRNA and protein levels. Although higher levels of mRNA expression do not always result in higher levels of protein, the O3 exposure in our system caused an induction of both transcription and translation. Because macrophages represent the majority of the inflammatory cells in the lower respiratory tract, they constitute a major potential source of fibronectin in the lung. Several studies have implicated macrophages in the production of fibronectin following tissue injury. Increases in fibronectin production by alveolar macrophages occur in humans and animals exposed to asbestos, nickel, cobalt chloride, and cadmium chloride (13, 33). When macrophages were isolated from rats exposed to 2 ppm O3 and placed in culture, the cells from exposed animals produced more fibronectin than the cells from air-exposed controls (28).

Although a time-related increase in the production of fibronectin by macrophages from both air- and O3-exposed rats was observed, the increase in fibronectin production by macrophages from O3-exposed rats occurred at 2 and 48 h, but not at 18 h, after placement of the cells in culture. In contrast, the increase in fibronectin protein in BALF and lung tissue and the increase in mRNA expression in lungs in the present study were observed at ~12 h after the end of exposure, suggesting that cell activation occurs fairly early and does not necessarily require a lag phase of 48 h as noted by Pendino et al. (28). Also, in contrast to the 2- and 18-h samples of the culture medium in the macrophage study, both A and B chains were detected by Western blotting of BALF in our 12-h samples from the air-exposed animals; O3 exposure caused an increase in both A and B chains. Although these effects were produced by O3 concentrations considerably lower than those used in the macrophage study (28), the results suggest contribution of cells other than macrophages in increased fibronectin production at 12 h postexposure. This interpretation is consistent with the reports describing other cellular sources of fibronectin in acute lung injury and disease states. For instance, the baseline fibronectin production was found to be much greater in isolated human alveolar macrophages than in an epithelial cell line (BEAS-2B) of human bronchial origin, but an in vitro exposure of macrophages to O3 did not cause an increase in the production of fibronectin (12). However, an increase in fibronectin production occurred on exposure of the epithelial cells. These results suggest that macrophages are capable of producing fibronectin, but they are not necessarily stimulated for fibronectin production as a consequence of O3 exposure. In addition to macrophages, fibronectin is produced by endothelial cells, alveolar type II cells, bronchial epithelial cells, and PMNs, which accumulate in the lung under acute inflammatory conditions and injury (24, 38). A correlation of fibronectin levels with the magnitude of neutrophilia following O3-serum exposure points to PMNs as a potential source of fibronectin under our experimental conditions. It is important to note, however, that the increase in fibronectin synthesis did not occur simply as a result of neutrophilia, as in the case of the air-serum group, but an activation signal in the form of O3 was necessary for the induction of fibronectin synthesis.

In summary, the results of fibronectin analysis in BALF, lung tissue extract, and blood plasma by ELISA and further confirmation by Western blots and RNase protection assay demonstrate that O3, at a moderate concentration, stimulates fibronectin expression in the lung. The elevated fibronectin production is attributed to multiple sources, including plasma leakage into the lung after treatment with rabbit serum and local synthesis following O3 exposure. The O3-induced increase in fibronectin expression is further enhanced in animals pretreated with another inflammatory stimulus, which by itself induces neutrophilia but does not significantly alter fibronectin mRNA expression in the lung. It is concluded that O3 exposure causes activation of lung components for fibronectin production and that a simple increase in PMN number, as that produced by serum instillation alone, is associated with increased fibronectin leakage from plasma into the lung, but it does not result in increased fibronectin synthesis.

    ACKNOWLEDGEMENTS

This work was made possible by National Institute of Environmental Health Sciences Grant ES-03521.

    FOOTNOTES

Address for reprint requests: D. K. Bhalla, Dept. of Occupational and Environmental Health Sciences, Wayne State Univ., 628 Shapero Hall, Detroit, MI 48202.

Received 11 December 1997; accepted in final form 21 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(2):L330-L335
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