INTRODUCTION

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PULMONARY FIBROSIS is the penultimate outcome of pathological responses of the lung to injury resulting from a variety of pathological conditions (19). Among the etiologies are drugs, pulmonary hypertension, hyperoxia, autoimmune diseases, and inhalation of nondegradable fibers and particles (7, 8). Bleomycin, an antineoplastic antibiotic, is known to produce dose-dependent lung parenchymal injury and pulmonary fibrosis in humans and in animal models (14, 21). The lung injury produced by bleomycin is rapid in onset, with an initial alveolitis phase consisting of macrophages, granulocytes, and lymphocytes (31). The administration of bleomycin to rodents has been widely used as an animal model of pulmonary fibrosis, and bleomycin-induced pulmonary fibrosis resembles chronic human fibrotic lung disease histologically and physiologically (21, 22, 28), although the tendency for bleomycin-induced lung fibrosis in rats to resolve over time is unlike the idiopathic human disease.

Regardless of its etiology, pulmonary fibrosis is characterized by proliferation of interstitial cells and accumulation of various proteins of the extracellular matrix (ECM) within the alveolar septa (8). Excessive ECM in the alveolar wall impedes gas exchange between air and blood and results in a stiff or noncompliant lung. Increased synthesis and accumulation of several ECM components, including fibronectin (FN), collagens, and elastin, have been demonstrated in the fibrotic lungs of animals with bleomycin-induced pulmonary fibrosis (13, 25, 34). Those studies indicate that ECM expression is a late phenomenon in pulmonary fibrosis. It is unclear if ECM components are involved in the early lung injury and the pulmonary inflammatory response that lead to fibrosis.

Tenascin (TN) is an ECM glycoprotein consisting of six similar subunits joined together at their NH₂-terminal ends by disulfide bonds. Each subunit contains a series of structural domains, including an NH₂-terminal cysteine-rich region, epidermal growth factor-like repeats, FN type III repeats, and a COOH-terminal fibrinogen-like domain. TN shares similar structural features with FN; it has both cell adhesion and anti-adhesion properties. TN has been shown to interfere with attachment and spreading of a number of cell types in culture by either promoting or inhibiting cell adhesion (2, 6, 16, 29). Tissue culture studies have shown that TN has multiple functions, including promotion of cell attachment and detachment, promotion and inhibition of neural crest cell migration, and stimulation of cell growth (4). TN is expressed with a restricted tissue distribution in both embryonic and adult tissue (1, 11). Two major TN isoforms are expressed during rat lung development and are regulated by transforming growth factor (TGF)- (38). TN is abundantly present during lung organogenesis, but its expression is reduced greatly in adults (33, 37).

In the present study, the potential for a role for TN in bleomycin-induced pulmonary injury and fibrosis was investigated. Expression of TN was remarkably induced during the inflammatory phase of lung injury generated by intratracheal bleomycin instillation. TN was spatially restricted in alveolar septal walls and secondary septal tips in the areas of tissue damage. The development of fibrosis was found to be associated with a decrease in the level of TN but an increase of collagen type III (COL III). These studies demonstrated that TN was a unique ECM protein that constitutes a part of the early inflammatory response to pulmonary injury induced by bleomycin.

	MATERIALS AND METHODS

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Bleomycin instillation. Pathogen-free adult Sprague-Dawley rats weighing 250-310 g were purchased from Charles River (Raleigh, NC). Rats were lightly anesthetized with halothane. Bleomycin (Blenoxane; Bristol-Myers Squibb, Princeton, NJ) at doses of 9 U/kg body wt was reconstituted in sterile 75 mM NaCl solution, and 0.5-ml volumes were instilled intratracheally into rats. Control animals received sterile 75 mM NaCl. Animals were killed at 3, 8, and 12 days postbleomycin administration. Lungs were removed while the animals were under phenobarbital sodium (50 mg/kg) anesthesia. Lung tissues were frozen in liquid nitrogen for RNA preparation or were fixed in ice-cold 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated through graded ethanol, and embedded in paraffin. Five-micrometer-thick sections were cut and mounted on glass slides treated with 3-aminopropyltriethoxysilane (Sigma, St. Louis, MO).

Northern blot analysis. Total RNA was isolated from lung tissues by the guanidine thiocyanate-cesium chloride method. The pellets were extracted with phenol-chloroform and followed by ethanol precipitation. Poly(A)⁺ RNAs were selected by a poly ATtract mRNA isolation kit (Promega, Madison, WI). Rat TN cDNA was subcloned into pGEM-7Zf, which was kindly provided by Dr. Ikramuddin Aukhil from the University of North Carolina at Chapel Hill. Rat COL III cDNA was isolated from rat lungs by using an RT-PCR cloning technique, and the identity of the cDNA was confirmed by partial sequence analysis as described (12). [³²P]cDNA probes were prepared using a random-priming cDNA labeling kit (Boehringer Mannheim, Indianapolis, IN). mRNA was fractionated on an agarose gel, transferred to a Nytran nylon membrane, and fixed by a Stratalinker ultraviolet cross-linker. Filters were hybridized at 42°C in 50% formamide solution containing 5× saline-sodium phosphate-EDTA buffer, 1× Denhart's solution, 0.5% SDS, and 0.2 mg/ml of denatured and sonicated fish sperm DNA with 10⁶ counts/min (cpm) of ³²P-labeled TN and COL III cDNA probes. Filters were washed and autoradiographed. The membranes were reprobed with a rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe.

In situ hybridization. Sections of paraformaldehyde-fixed and paraffin-embedded rat lungs were subjected to in situ hybridization with ³⁵S-cRNA. Sense and antisense cRNA riboprobes were generated from rat TN cDNA subcloned into pGEM-7Zf. The plasmid was linearized with restriction enzymes. cRNA was obtained in the sense and antisense orientation by transcribing from T7 and SP6 promoters, respectively, using the Riboprobe transcription kit (Stratagene, La Jolla, CA). Riboprobes were labeled with ³⁵S-UTP (Amersham, Arlington, IL) and were reduced to an average fragment length of ~200 base pairs by alkaline hydrolysis. Sections were rehydrated, fixed in 4% paraformaldehyde in PBS, and treated with proteinase K. The sections were treated again with 4% paraformaldehyde in PBS and acetylated with acetic anhydride. After dehydration through ethanol, the sections were prehybridized for 2 h at 50°C in hybridization solution (containing 50% formamide, 0.3 M NaCl, 20 mM Tris · HCl, pH 8.0, 5 mM EDTA, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin, 10 mM NaH₂PO₄, 500 µg/ml tRNA, and 100 mM dithiothreitol). Hybridization was performed with 2-5 × 10⁷ cpm/ml ³⁵S-cRNA probe in hybridization solution (with 10% dextran sulfate) and incubated overnight at 55°C. The slides were washed with 5× SSC (1× SSC is 0.15 M NaCl-15 mM trisodium citrate) containing 10 mM dithiothreitol at 37°C, followed by washing with 2× SSC at 65°C and then treated with RNase A. Washing was continued with 2× SSC at 65°C and then 0.1× SSC at 37°C. Sections were next dehydrated, dried, and dipped in Kodak NTB-3 autoradiographic emulsion. After exposure at 4°C for 1-2 wk, the slides were developed and counterstained with hematoxylin and eosin.

Immunohistochemistry. Sections of rat lung tissues were deparaffinized, rehydrated, and digested briefly with trypsin. The slides were then blocked with 5% normal goat serum and 1% BSA for 1 h and incubated with primary anti-TN antiserum from Telios Pharmaceuticals (San Diego, CA) overnight at 4°C. The endogenous peroxidase was suppressed with 6% H₂O₂ in methanol for 30 min. The sections were incubated with horseradish peroxidase-conjugated secondary antibody and visualized using the metal-enhanced 3,3'-diaminobenzidine kit (Pierce, Rockford, IL) as chromogen.

	RESULTS

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Temporal changes of TN mRNA in rat lung during bleomycin-induced injury. Lung injury and parenchymal fibrosis were induced by intratracheal instillation of bleomycin. To determine the dynamic change in TN expression during bleomycin-induced lung injury, mRNAs isolated from rat lungs at various times after intracheal instillation of bleomycin were examined by Northern blot analysis (Fig. 1). There was a barely detectable level of TN message in control animals. After the initial lung injury induced by intratracheal bleomycin instillation, two TN isoforms with sizes of 7.3 and 6.4 kb were remarkably induced. TN peaked at day 3 postbleomycin administration and then declined after 12 days of repair. COL III mRNAs were also induced after the initial lung injury induced by intratracheal bleomycin instillation, and the temporal pattern of COL III induction was distinct from that of TN during bleomycin-induced injury. COL III mRNA started to increase gradually at 8 days after bleomycin instillation and further increased 12 days after bleomycin instillation. The results show that TN was primarily induced during the inflammatory injury and was decreased as the development of fibrosis proceeded, whereas the increase in COL III synthesis continued during the reparative phase.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Northern analysis of tenascin (TN) mRNA expression in rat lungs during bleomycin-induced lung injury. Poly(A)+ RNA isolated from lungs was subjected to electrophoresis through a formaldehyde denaturing agarose gel and, after Northern blotting, hybridized sequentially with radiolabeled TN, collagen type III (COL III), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. Lane 1, control, 3 days of 75 mM NaCl administration; lane 2, 3 days of bleomycin administration; lane 3, 8 days of bleomycin administration; and lane 4, 12 days of bleomycin administration.

Morphological evaluation of bleomycin-induced injury and parenchymal fibrosis lesion of rat lungs. To determine if the extent of inflammation and fibrosis correlated with the induction of TN, the morphological changes in the lungs of rats after intratracheal instillation of bleomycin were examined. Figure 2 shows the histology of rat lungs from animals treated with bleomycin. Extensive inflammation was evident at 3 days after bleomycin administration (Fig. 2, A and B). The inflammation was centered around the bronchiole in a proximal-distal pattern. There was significant infiltration by neutrophils, macrophages, and lymphocytes in peribronchiolar and perivascular areas within the interstitial septa and the alveolar space. Some perivascular edema was present. At 8 days after bleomycin administration, there was apparent hyperplasia in alveolar septa in the area of injury (Fig. 2, C and D). The interstitial infiltrates were associated with prominence of fibroblasts and beginning deposition of interstitial ECM, resulting in distorted alveolar walls. The inflammation was decreased, and the fraction of abnormal lung was reduced. Most regions of the lung showed no abnormality. At 12 days, collagenous scars were formed, and patches of fibrosis were scattered through the lung (Fig. 2, E and F). Control lung sections exhibited no notable morphological changes (Fig. 2, G and H).

View larger version (136K): [in this window] [in a new window]   Fig. 2.   Histological changes of rat lungs after intratracheal instillation of bleomycin. Inflammation was evident at 3 days after bleomycin administration. There was significant infiltration by inflammatory cells and lymphocytes in the alveolar space and the interstitium. Edema was present in the damaged pulmonary vasculature (A and B). Hypercellular activity and deposition of extracellular matrix in the interstitium with the decreased inflammation was noted in lung section from rat 8 days after bleomycin instillation (C and D). At 12 days, the collagenous scars were formed, and patchy fibrosis was apparent (E and F). Lung sections from control rats showed no notable morphological changes (G and H). A, C, E, and G are at the same magnification, bar in A = 25 µm; B, D, F, and H are at the same magnification, bar in B = 5 µm.

Localization of TN mRNA by in situ hybridization in rat lungs undergoing bleomycin-induced pulmonary injury. To determine the localization of TN mRNA in rat lung tissue, we hybridized rat lung sections with an antisense ³⁵S-cRNA probe. At 3 days after bleomycin administration, an intense hybridization signal of TN mRNA was detected in the injured areas in a pattern similar to that of the inflammatory lesions (Fig. 3, A-D). Distribution of TN mRNA was diffused and nonuniform. TN was spatially restricted to the areas of damaged lung. Distal regions of the lung unaffected by the injury showed no induction of TN, which was comparable to that observed in normal lungs. TN mRNA was expressed by interstitial cells within alveolar septal walls, and a strong hybridization with TN probes was observed at secondary septal tips. No significant labeling was seen in the regions of the airway epithelium. The absence of labeling with a sense probe synthesized from the same construct confirmed the specificity of this hybridization pattern (data not shown).

View larger version (106K): [in this window] [in a new window]   View larger version (105K): [in this window] [in a new window]   View larger version (82K): [in this window] [in a new window]   Fig. 3.   In situ hybridization of TN mRNA to rat lungs undergoing bleomycin-induced lung injury and pulmonary fibrosis. TN mRNA was focally induced and was spatially restricted in the areas of tissue inflammation 3 days after bleomycin treatment, and strong labeling was seen in the alveolar septal walls and secondary septal tips of bleomycin-injured lungs (A-D). At 8 days after bleomycin treatment, TN expression was decreased as inflammation was reduced, and TN mRNA was preferentially expressed in alveolar septal walls and secondary septal tips of bleomycin-induced lungs (E-H). TN mRNA was barely detected in the areas of developing fibrosis in lung sections from rats 12 days after bleomycin instillation, and it significantly declined as development of fibrosis proceeded (I-L). A, B, E, F, I, and J are at the same magnification, bar in A, E, and I = 25 µm; C, D, G, H, K, and L are at the same magnification, bar in C, G, and K = 5 µm.

In situ hybridization analysis revealed a substantial induction of TN mRNA along alveolar septal walls and at secondary septal tips in the inflammatory and fibrotic areas 8 days after bleomycin administration (Fig. 3, E-H). The distribution of TN mRNA in rat lung 8 days after bleomycin administration showed a pattern similar to that in rat lungs after 3 days of bleomycin administration. The intensity of TN mRNA labeling in the alveolar septa of lung section at this time point was higher than that recorded at 3 days after bleomycin instillation, but the extent of TN induction was reduced, correlating with the smaller areas of inflammation. At 12 days after bleomycin administration, fibrosis was becoming apparent as indicated by the increased deposition of collagens and distortion of lung parenchyma. Although expression of TN mRNA was greatly reduced, a low level of TN mRNA was still detectable in the area of fibrosis (Fig. 3, I-L). TN mRNA was barely detectable in control rat lung tissues (Fig. 4). The finding is in agreement with the results of Northern analysis (Fig. 1) in which TN message was nearly absent in adult control rat lungs. There was a progressive decrease in the expression of TN mRNA during the development of pulmonary fibrosis.

View larger version (78K): [in this window] [in a new window]   Fig. 4.   In situ hybridization of TN cRNA to control rat lungs. A: rat lung tissues were hybridized with 35S-UTP-labeled antisense TN cRNA probes, and sections were counterstained with hematoxylin and eosin. B: dark-field photomicrograph of A. C: higher power view of the alveolar septal walls and secondary septal tips of bleomycin-induced lungs. D: dark-field photomicrograph of C. TN was barely detectable in lung tissues from normal animals. A and B are at the same magnification, bar in A = 25 µm; C and D are at the same magnification, bar in C = 5 µm.

Immunolocalization of TN protein in rat lungs of bleomycin-induced lung injury. The localization of TN mRNA was compared with that of immunoreactive TN protein. Figure 5 shows the distribution of TN protein in rat lungs of bleomycin-induced lung injury. TN protein was detected along the basement membrane and in the tips of alveolar septa at the site of an inflammatory lesion 3 days after bleomycin instillation (Fig. 5, A and B). Immunoreactive TN protein was spatially distributed throughout the parenchymal interface in the areas of tissue damage, which was compatible with expression of TN messages. However, the distribution of TN protein was more diffuse and extensive than that of TN mRNA. A significant amount of TN was detected in the areas of injured lung tissues from rats 8 days after bleomycin instillation (Fig. 5, C and D). TN immunoreactivity was concentrated on the walls of the thickened alveolar septa. TN was detected in the areas of developing fibrosis in lung sections from rats 12 days after bleomycin instillation (Fig. 5, E and F), but the intensity of TN immunoreactivity was decreased. Lung sections from normal rats showed barely detectable TN (Fig. 5, G and H).

View larger version (129K): [in this window] [in a new window]   Fig. 5.   Immunolocalization of TN protein in rat lungs undergoing bleomycin-induced lung injury and pulmonary fibrosis. Lung sections from rats after intratracheal bleomycin instillation were stained with anti-TN antibody. TN protein was detected along the basement membrane and in the tips of alveolar septa at the site of an inflammatory lesion 3 days after bleomycin instillation (A and B). A significant amount of TN immunostaining was distributed in the areas of injured lung tissues from rats 8 days after bleomycin instillation, and TN immunoreactivity was concentrated on the walls of the thickened alveolar septa (C and D). TN was detected in the areas of developing fibrosis in lung sections from rats 12 days after bleomycin instillation, but the intensity of TN immunoreactivity was decreased (E and F). Lung sections from normal rat lungs showed barely detectable TN (G and H). A, C, E, and G are at the same magnification, bar in A = 25 µm; B, D, F, and H are at the same magnification, bar in B = 5 µm.

	DISCUSSION

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Lung injury induced by bleomycin is accompanied with early inflammation and followed by a complex process of repair initiated at the site of injury. Enhanced ECM biosynthesis is almost always preceded by an increase in the influx of activated inflammatory cells (5). It has been suggested that a variety of cytokines elaborated by the inflammatory cells recruited in response to tissue injury may contribute significantly to the early events of lung inflammation and may initiate the fibrotic process (5, 24-26, 35, 36). However, the mechanism governing the cellular and molecular interactions responsible for the excessive synthesis and accumulation of ECM in the alveolar walls has not been completely understood. This study describes the induction of ECM protein TN expression in rat lungs during bleomycin-induced lung injury. TN was induced during the inflammatory phase of lung injury. Induction of TN was spatially restricted in alveolar septal walls and secondary septal tips in the areas of tissues injured. TN mRNA and protein were decreased as development of fibrosis proceeded. The results of these studies demonstrate that TN was a uniquely early-response ECM component to bleomycin-induced pulmonary injury and provided information on sites of gene activation in the lungs developing fibrotic lesions. The observation raised the possibility that ECM protein, such as TN, might play a role in the inflammatory events of bleomycin-induced lung injury.

FN and collagens were the most extensively analyzed ECM in pulmonary fibrosis. The increase in FN and COL III synthesis in rat lungs undergoing bleomycin-induced injury has been reported previously (17, 21, 27, 32). Although all three representatives of ECM were found to be elevated after bleomycin treatment, there was a significant temporal difference in induction of TN, FN, and COL III mRNAs after the initial lung injury induced by intratracheal bleomycin instillation. TN was actively induced, being an early response during the inflammatory injury, and then decreased 12 days after bleomycin instillation. The increase in FN and COL III synthesis continued during the reparative phase. The reparative phases of lung injury involved new matrix deposition and tissue remodeling. Increased synthesis and excessive deposition of collagen and FN by fibroblasts were the progressive nature of pulmonary fibrosis (20). The induction of TN was distinct from those of FN and COL III and was uniquely regulated during the inflammation phase of injury. Our findings indicated that TN behaved, at least in part, as an acute-phase protein and that it was involved in the inflammatory response of bleomycin-induced lung injury.

The earliest events in the process of injury were an influx of inflammatory cell neutrophils and monocytes and lymphocytes into the alveolar space and the interstitium of the inflammatory lesions within the lung (31). The infiltration of inflammatory cells occurred 3-4 days after bleomycin instillation (30). TN mRNA was focally induced, and TN protein was distributed accordingly in the early stage of lung injury. TN mRNA was produced at the sites where the tissue inflammation occurred and TN immunoreactivity was detected at the inflammatory lesions. The level of TN immunoreactivity that appeared in damaged areas coincided with the invasion of the lesions by neutrophils and monocytes. We speculate that the induction of TN could provide invading cells with migratory pathways that ensure the initiation of the reparative phase of lung injury.

Bleomycin produced diffused lung injury that was manifested by perivascular edema and interstitial cell infiltration, with subsequent intra-alveolar and alveolar wall fibrosis (5, 21). TGF-1 (18) and interleukin-4 (IL-4) receptors (3) were upregulated during bleomycin-induced lung injury and have been implicated in the pathogenesis of fibrosis because they are capable of regulating the inflammatory responses that contribute to the fibrotic response. These cytokines would exert their effects by regulating cell adhesion and substrate molecules. Our in situ hybridization analysis indicated that interstitial cells at the inflammatory lesions were the source of TN production. It has previously been demonstrated that TN was regulated by TGF-1 (37) and by IL-4 (23). Cytokine-mediated activation of TN gene expression may constitute an important part of the early inflammatory response in bleomycin-induced lung injury.

The results of this study demonstrate a strong correlation between the sites and temporal expression of TN and the tissue inflammation. The timing and localization of TN expression in injured lungs indicate that TN was involved in the inflammatory events and place this protein in a functionally interesting position as a regulatory molecule rather than a structural constitution of lung repair. TN can participate in tissue inflammation and remodeling by at least two potential mechanisms: modulation of cell migration and cell proliferation. TN might act as a modulator of tissue inflammation and remodeling in lung repair by mediating cell adhesion and cell migration, thus regulating mesenchymal-epithelial interactions. TN interferes with cell-FN interactions and promotes the motility of many cell types including lymphocytes (6, 9). In addition to its effect on cell adhesion and cell migration, TN might act as a modulator of cell growth. TN has been shown to have a growth factor-like activity (10) and to regulate cellular gene expression (15). A combination of TN and other cytokines may act synergistically to modulate the growth and behavior of different cell types during inflammation and remodeling of lung repair.