Cell-specific expression of fibronectin and EIIIA and EIIIB splice variants after oxygen injury

William M. Maniscalco, Richard H. Watkins, Patricia R. Chess, Robert A. Sinkin, Stuart Horowitz, and Liana Toia

Division of Neonatology, Department of Pediatrics, Strong Children's Research Center, University of Rochester School of Medicine, Rochester, New York 14642

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Cellular fibronectin (cFN) expression is characteristic of injured tissues. Unlike plasma FN, cFN mRNA often contains the EIIIA or EIIIB domains. We examined the lung cell-specific expression of total cFN mRNA and the EIIIA and EIIIB splice variants in rabbits after acute oxygen injury. By in situ hybridization, control lung had low cFN mRNA. After exposure to >95% oxygen, mRNAs for total cFN and EIIIA were noted primarily in alveolar macrophages and large-vessel endothelial cells. By 3-5 days recovery, cFN and EIIIA mRNA abundance was increased in alveolar septal cells (i.e., alveolar epithelial, interstitial, or endothelial cells) and in some large-vessel endothelial cells but was low in bronchial epithelial cells. During recovery, EIIIB mRNA was low in alveolar septal cells but was noted mainly in chondrocytes. Immunostaining for EIIIA increased during recovery, paralleling the in situ hybridizations. Because FN may modulate alveolar type II cell phenotype, we investigated type II cell cFN mRNA expression in vivo. During recovery, neither isolated type II cells nor cells with surfactant protein C mRNA in vivo contained FN mRNA. In summary, these data suggest that cFN with the EIIIA domain has a role in alveolar cell recovery from oxygen injury and that type II cells do not express cFN during recovery.

alveolar type II cells; endothelial cells; surfactant protein C

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

FIBRONECTIN (FN) is an adhesive extracellular matrix glycoprotein with many putative functions, including regulation of cell proliferation, differentiation, and migration (16). A large, multidomain heterodimer, FN has multiple sites for binding to other extracellular matrix constituents and to signal-transducing cellular integrins. Two major isoforms of FN, plasma FN (pFN) and cellular FN (cFN), result from alternative splicing of the primary mRNA transcript, which is the product of a single gene (28). pFN is synthesized by hepatocytes, is soluble in blood plasma, and lacks sequences coded by two alternatively spliced exons, EIIIA and EIIIB. cFN, which is less soluble, frequently contains either or both of the EIIIA and EIIIB domains. Many cell types can express cFN mRNA; however, its expression is often greatest in developing and injured tissues (4, 17, 24, 31). The biological functions of cFN and the various splice variants are incompletely understood, but the EIIIA and EIIIB domains have been implicated in formation of pericellular matrices and modulation of cell migration and differentiation (10, 11).

In normal adult lung, transcripts for FN can be found in alveolar interstitial cells, endothelial cells, chondrocytes, and vascular smooth muscle cells (31). Normal air space epithelial cells are devoid of FN message. cFN mRNA and FN protein are more abundant in developing lung than in mature lung (31, 32). Normal adult lung expresses cFN mRNA primarily with the EIIIA domain and with little EIIIB (22). In fibrotic lung, several studies have documented increased FN mRNA or protein, especially in fibrotic areas (3, 18, 25). Although cFN probably has roles in repair of injured tissues, limited data are available on cFN expression during repair of acute lung injury (6, 8). In acute, severe oxygen injury, we documented that whole lung cFN mRNA abundance increased during a 5-day recovery in air (22). A role for FN in lung repair is also suggested by its in vitro effects on cell phenotype, including alveolar epithelial type II cells (26, 29, 33, 34), which probably migrate, proliferate, and differentiate into type I cells after severe oxidant injury (1). In vitro studies show that cFN is expressed by migrating airway epithelial cells and that exogenous FN accelerates type II cell migration and closure of an artificial epithelial wound (13, 14, 19). These data suggest that cFN has a role in repair of oxidant injury, but in vivo data are lacking.

In vitro, many lung cell types can express FN, including alveolar epithelial type II cells (7, 21, 27), bronchial epithelial cells (20), endothelial cells (17), and other mesenchymal cells (6). However, type II cell FN mRNA expression in vitro, including the EIIIA and EIIIB variants, is associated with decreased surfactant protein C (SP-C) expression (21, 22). Because SP-C is specific to differentiated type II cells, this observation suggests that type II cells might not express cFN in vivo. In support of this hypothesis, we found that type II cells isolated from normal lung have low cFN mRNA abundance (21). Alveolar macrophages express FN during recovery from oxidant lung injury (30), but the cell-specific expression of cFN by resident lung cells, including type II cells, during recovery has not been investigated. cFN expression by recovering alveolar cells would support the speculation that cFN has a role in alveolar repair. The objective of the current study was to determine the cell-specific expression of total cFN (without regard to splicing) and the EIIIA and EIIIB splice variants during acute oxygen injury and recovery. Our findings show that total cFN mRNA and the EIIIA variant increased in alveolar septal cells, alveolar macrophages, and large-vessel endothelial cells during recovery. cFN transcripts were rare in bronchial epithelial cells, SP-C-expressing cells, or freshly isolated type II cells during recovery. Immunostaining for EIIIA followed temporal and spatial patterns similar to mRNA. Together, these data support the speculation that expression of cFN and the EIIIA splice variant by alveolar and endothelial cells contributes to repair after severe oxygen injury.

    MATERIAL AND METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals, hyperoxia exposures, and recovery. Hyperoxic exposures were conducted as previously described (23). Adult male rabbits were placed in Plexiglas chambers with constant oxygen flow (7 l/min) for 64 h. Oxygen concentration was checked twice daily and was always >95%. This treatment causes severe alveolar injury as determined by increased alveolar proteins (15). Some animals were allowed to recover in air. At 0, 1, 3, 5, and 7 days of recovery, animals were killed by intravenous injection of pentobarbital sodium. Control animals were maintained in standard vivarium conditions. Both groups were fed standard rabbit chow and were supplied with water. Two separate hyperoxia experiments were performed, which resulted in a total of two control animals and two animals at each time point. Use of animals for these experiments was approved by the University Committee on Animal Resources.

Tissue preparation. To preserve tissue architecture, phosphate-buffered 10% Formalin was instilled in situ into the trachea at a pressure of 20 cmH2O for 20-25 min. The trachea and lungs were then removed together and immersed in Formalin. Tracheal instillation of fixative continued for 2 h at a pressure of 20 cmH2O. The lungs were cut into cubes and embedded in paraffin for later sectioning.

Alveolar type II cell isolation. Alveolar type II epithelial cells were isolated from rabbits recovering in air for 3 days using methods previously described (23). Briefly, the lungs were perfused in situ with saline, removed, and lavaged with protease solution (trypsin, DNase, and elastase). The lungs were minced and filtered, and type II cells were purified on a discontinuous Percoll gradient. The cells were sedimented on slides and prepared for in situ hybridization.

Riboprobe preparation. Rat cDNAs for total cFN (SR270) and the EIIIA and EIIIB exons were gifts from R. O. Hynes (11). The SR270 probe hybridizes to sequences in the 10th and 11th type I repeats, which are not alternatively spliced. It hybridizes to all known cFN mRNA variants. The EIIIA and EIIIB probes are exon specific. The rat cDNAs for total cFN, EIIIA, and EIIIB are 270, 213, and 255 bp, respectively. For double in situ hybridization for cFN and SP-C, we used a rabbit cDNA (2.5 kb) for total FN (30) and a rabbit cDNA (0.85 kb) for SP-C (a gift from F. Possmayer, Univ. of Western Ontario). Sense and antisense complementary RNA (cRNA) probes for rat cFN, the EIIIA and EIIIB variants, and rabbit SP-C were synthesized and labeled with 35S to a specific activity of ~2 × 109 dpm/µg. The rabbit FN probe was synthesized using digoxigenin 11-UTP. The rabbit probes were hydrolyzed to a length of ~200 bp by alkaline hydrolysis.

In situ hybridization. In situ hybridization was performed as described previously (23). Briefly, lung tissue sections of 4-µm thickness were cut, placed on N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES)-treated slides, and dried, and the paraffin was removed. Cytospun type II cells on TES-treated slides were fixed in buffered Formalin and rinsed in SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0). They were then dehydrated and dried for storage at -70°C. Before hybridization, the tissue sections were treated with proteinase K. All slides were then equilibrated with 100 mM triethanolamine-HCl (pH 8.0) and treated with 0.25% acetic anhydride. The slides were then washed in SSC, dehydrated, and dried. Prehybridization was at 55°C for 2 h. The slides were then rinsed in SSC and dehydrated. Hybridization was performed for 16 h at 53°C using a hybridization solution containing 30 ng · kb-1 · ml probe-1. After hybridization, slides were rinsed, digested with RNases A and T1, and rinsed in RNase buffer. The slides were then rinsed in 0.2× SSC at 60°C and in 0.1× SSC at room temperature. The slides were dehydrated, dipped in a 1:1 dilution of NTB-2 emulsion (Eastman Kodak, Rochester, NY), exposed at 4°C for 3 wk, developed, fixed, and stained with hematoxylin and eosin.

Double in situ hybridization. Double in situ hybridization was performed as detailed previously (23). Hybridization with the radiolabeled SP-C probe was conducted as in In situ hybridization until the RNase digestion step. The slides were not digested with RNases but were washed in 0.5× SSC for 1 h. The slides were then rinsed in 2× SSC and 0.1× SSC, followed by dehydration, dipping in emulsion, exposure, development, and fixation as above. Slides for double in situ hybridization were not counterstained but were dehydrated, and the hybridization was repeated as above using hybridization solution containing 150 ng · kb-1 · ml digoxigenin-labeled probe-1 for rabbit FN. Washes were as above, including RNase digestion. After the washes, the double in situ hybridization slides were treated with anti-digoxigenin antibody-alkaline phosphatase conjugate and then incubated for the chromogen reaction as previously described (23). These slides were not counterstained but were rinsed in water and coverslipped using Aqua Polymount (Polysciences, Warrington, PA).

Immunohistochemistry for total FN and EIIIA. Goat polyclonal anti-rabbit FN antibody was used to detect total FN (Cappel, Durham, NC). This antibody detects both pFN and cFN. After deparaffinization and rehydration, slides were placed in 0.3% H2O2 in methanol for 30 min and rinsed in Tris-buffered saline (TBS) and in TBS containing 0.05% Triton X-100. Slides were then blocked with 3% BSA in TBS for 45 min and incubated overnight at 4°C with the primary antibody diluted 1:2,000 with TBS and 0.1% BSA. (Purified goat IgG diluted to the same concentration was used as a negative control.) The slides were rinsed in TBS for 15 min, and then biotinylated rabbit anti-goat IgG antibody diluted 1:200 was applied for 1 h. After a rinsing in TBS, avidin-biotinylated horseradish peroxidase complex (ABC Elite) reagent (Vector Laboratories, Burlingame, CA) was applied for 30 min. Slides were rinsed in TBS, and 3,3'-diaminobenzidine substrate was applied for 10 min. The slides were rinsed and lightly counterstained with 0.01% methylene blue.

Mouse monoclonal anti-human cFN (DH1) was used to detect cFN containing the EIIIA domain (ICN Biochemicals, Costa Mesa, CA). This antibody does not cross-react with pFN. Antigen retrieval was necessary to unmask the EIIIA epitope on Formalin-fixed, paraffin-embedded tissues. After deparaffinization and rehydration, the slides were boiled by microwave in 100 mM citric acid, pH 2.0, for 10 min. The immunostaining was performed as for total FN, except slides were blocked with 3% horse serum, and the primary antibody was diluted 1:150. (Mouse monoclonal IgG1 antibody against Aspergillus niger glucose oxidase diluted 1:150 was used as a negative control.) The secondary antibody was biotinylated horse anti-mouse IgG diluted 1:200.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Expression of total cFN mRNA increases in alveolar and endothelial cells during recovery from acute oxygen injury. The rat probes for total cFN and the splice variants used in the current investigations detect appro-priately sized transcripts in rabbit tissues (22). Lung tissue sections from control and oxygen-injured rabbits were hybridized with the rat cRNA probe for total cFN, which detects all FN splice variants. The results from two separate experiments were nearly identical. In control lung, little or no total cFN mRNA was detected in alveolar septal cells (i.e., alveolar epithelial, interstitial, or endothelial cells), bronchial epithelial cells, large-vessel endothelial cells, or loose adventitial fibroblasts (Fig. 1, A and B). Weak hybridization was noted in vascular smooth muscle cells (not shown). Hybridization with sense-strand probe gave very low background (Fig. 1C). After 64 h of hyperoxia with no recovery, the alveolar septa were thickened, and inflammatory cells were abundant. Overall, cFN mRNA signal intensity appeared increased, due mostly to cells located in the alveolar spaces (Fig. 1D). Higher magnification indicated that these cells were alveolar macrophages (Fig. 1E). Polymorphonuclear cells had no cFN mRNA, and little cFN message was noted in alveolar septal cells. Chondrocytes and endothelial cells in muscularized arteries had increased cFN mRNA after injury (Fig. 1F), but little or no signal was noted in bronchial epithelial cells.


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Fig. 1.   Cell-specific expression of cellular fibronectin (cFN) mRNA during recovery from acute hyperoxic injury. Adult rabbits were exposed to 100% oxygen for 64 h and allowed to recover in air for up to 5 days. Lung tissue sections were hybridized with a complementary RNA (cRNA) probe for total cFN (SR270). In control lung, low (a)- and high-power (b) magnifications show low basal levels of cFN transcripts. Sense-strand control gave low background (c). After 64 h of hyperoxia, cFN transcripts (arrows) were noted in alveolar macrophages (d and e). These animals also had cFN transcripts in endothelial cells of muscularized arteries ( f; arrow) and in chondrocytes ( f; arrowhead). At 3 days of recovery, increased cFN mRNA was detected in alveolar septal cells ( g and h) and in endothelial cells (i). At 5 days of recovery ( j-l), cFN mRNA was still abundant in alveolar septal cells and endothelial cells. Bar = 100 µm in a, c, d, f, g, j, l; bar = 25 µM in b, e, h, k; bar = 50 µm in i.

After 1 day of recovery in air (not shown), alveolar septal thickness was decreased, and the number of inflammatory cells was reduced. cFN mRNA hybridization intensity in alveolar septal cells was modestly increased compared with the nonrecovered animals, and a subset of macrophages with high signal intensity was apparent. Some large-vessel endothelial cells continued to have cFN mRNA (not shown). By 3 days of recovery (Fig. 1, G-I), the gross morphology of the alveolar regions appeared nearly normal. However, cFN mRNA hybridization in alveolar septal cells was increased substantially compared with controls (Fig. 1G). On higher magnification (Fig. 1H), cFN mRNA was detected in many alveolar cells, but the precise identity of these cells could not be determined by light microscopy. Alveolar macrophages appeared decreased, but some that remained had abundant cFN mRNA. cFN mRNA was rarely detected in bronchial epithelial cells, but endothelial cells in some large vessels had substantial message (Fig. 1I). Compared with controls, message in chondrocytes remained increased. At 5 days of recovery, cFN message was abundant in alveolar septal cells and endothelial cells of muscularized arteries, similar to 3 days of recovery (Fig. 1, J-L). Adventitial cells surrounding vessels, but not conducting airways, also had increased signal, but bronchial epithelial cells rarely had detectable message. Lymphoid cells in all conditions had relatively low levels of FN transcripts (not shown). Recovery for 7 days, which was allowed in one experiment, resulted in low cFN mRNA hybridization and was similar to controls (not shown).

FN EIIIA and EIIIB mRNA expression. In situ hybridization using the exon-specific rat probe for the EIIIA splice variant gave results that were similar to total cFN. Relatively low levels of EIIIA mRNA were detected in control lung (Fig. 2A). Alveolar macrophages and endothelial cells of muscularized vessels in the 64-h hyperoxic lung had abundant EIIIA mRNA (Fig. 2B). Unlike total cFN, EIIIA transcripts were not seen in chondrocytes. Sense-strand hybridization gave low signal (Fig. 2C). At 1 day of recovery, hybridization was somewhat increased compared with controls, and signal intensity increased further in alveolar septal cells at 3 and 5 days of recovery (Fig. 2, D-I). Similar to total cFN, EIIIA message was also detected in large-vessel endothelial cells and adventitial cells in the recovering animals but was not abundant in bronchial epithelial cells. In situ hybridization using the EIIIB probe gave a pattern that differed from EIIIA. EIIIB mRNA was very low in all cells of the control lung; after 64 h of hyperoxia without recovery, some signal was detected in alveolar macrophages but not in endothelial cells (not shown). Unlike EIIIA, EIIIB mRNA was abundant in chondrocytes of oxygen-injured animals (Fig. 3). During recovery, EIIIB mRNA remained low in alveolar septal cells compared with EIIIA but remained abundant in chondrocytes (not shown).


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Fig. 2.   cFN mRNA with EIIIA sequence increases in alveolar septal cells during recovery from acute hyperoxic injury. In situ hybridization using an exon-specific probe for EIIIA mRNA in lung sections from rabbits exposed to 100% oxygen for 64 h and allowed to recover for up to 5 days. Control lung had low EIIIA mRNA abundance (a). After 64 h of hyperoxia (b), EIIIA mRNA was detected in alveolar macrophages (arrowhead) and endothelial cells (arrow). Sense-strand control gave minimal background (c). EIIIA mRNA was slightly increased in alveolar septal cells at 1 day of recovery (d and g). At 3 (e and h) and 5 days ( f and i) of recovery, EIIIA mRNA increased in alveolar septal cells. Bar = 100 µm in a-f and 25 µm in g-i.


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Fig. 3.   EIIIB mRNA is restricted mainly to chondrocytes. In situ hybridization with exon-specific probes for EIIIA (a) and EIIIB (b) after 64 h of hyperoxia without recovery. Chondrocytes contain abundant EIIIB but not EIIIA mRNA. Neither splice variant was detected in bronchial epithelial cells. Bar = 100 µm.

cFN mRNA is not expressed in isolated type II cells or SP-C-expressing cells in recovering lung. The identity of the recovering alveolar septal cells with total cFN or EIIIA messages could not be completely discerned by light microscopy of the tissue sections. Alveolar type II cells identified by morphological criteria in fibrotic lung have been reported to have FN mRNA in vivo (3), and type II cells can express FN in vitro (7, 21, 22). To determine whether type II cells express cFN during recovery, we performed in situ hybridization for total cFN on cytospin preparations of freshly isolated type II cells from 3-day recovering animals. These cells rarely had significant cFN mRNA signal (Fig. 4A). As a positive control, we analyzed cytospin preparations of alveolar macrophages isolated from recovering lung. These cells had abundant FN mRNA (Fig. 4B).


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Fig. 4.   Alveolar type II cells isolated from 3-day recovering rabbits do not have cFN mRNA. Adult rabbits were exposed to 100% oxygen for 64 h and allowed to recover in air for 3 days. Type II cells (a) and alveolar macrophages (b) were isolated and hybridized with a probe for cFN. Bar = 50 µm.

To confirm lack of FN mRNA in type II cells in vivo, we performed double-label in situ hybridization for total cFN and SP-C on the same tissue sections (Fig. 5). Cells with cFN message were identified with dark staining, and SP-C mRNA was noted as white grains. The control animals had SP-C-expressing cells only in the alveolar regions, and little or no signal for cFN was noted (Fig. 5A). The animals exposed to oxygen for 64 h but not allowed to recover had decreased SP-C-positive cells (Fig. 5B). Alveolar macrophages in these animals contained abundant cFN message, but little cFN message was noted in alveolar septal cells. At 1 day of recovery, cFN mRNA was noted in some alveolar septal cells. SP-C-positive cells appeared increased compared with the nonrecovered animals. However, congruent expression of cFN and SP-C mRNA in the same cells was not observed (Fig. 5C). At 3 days of recovery, cFN message appeared increased in alveolar septal cells, but these cells rarely had SP-C message (Fig. 5D). These data suggest that differentiated type II cells in lungs recovering from severe oxygen injury do not express cFN.


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Fig. 5.   Surfactant protein C (SP-C)-expressing cells do not contain cFN mRNA. Double in situ hybridization was performed using probes for SP-C and cFN on same tissue sections. SP-C probe was radiolabeled, and signal is indicated by white grains (arrows). cFN probe was digoxigenin labeled, and signal is indicated by dark staining (arrowheads). a: control lung had SP-C mRNA-positive cells but very little cFN mRNA. b: 64-h hyperoxic lung had alveolar macrophages with cFN mRNA. c: 1-day recovering lung had increased SP-C mRNA-positive cells compared with 64-h animals, and cFN mRNA signal was detectable in alveolar septal cells. Fibronectin (FN)-positive cells did not have SP-C mRNA. d: 3-day recovering lung had increased cFN mRNA but not in SP-C-positive cells. Bar = 50 µm.

Immunolocalization of total FN and the EIIIA isoform. The antibody used to immunolocalize total FN recognizes all forms of FN including pFN. Control lung had immunostaining noted primarily around blood vessels. Little or no staining was detected in airway epithelial cells or in the alveolar septa (Fig. 6A). After 64 h of hyperoxia without recovery, staining intensity increased around blood vessels, and increased staining was noted in alveolar septa (Fig. 6B). At 1 day of recovery, substantial immunostaining was present in the alveolar septa (Fig. 6C) and was also noted in chondrocytes (Fig. 6D). Debris in the alveolar spaces was also positively stained. No staining was noted in airway epithelial cells. By 3 and 5 days of recovery, alveolar septal staining was reduced compared with 1-day recovery but was still noted around blood vessels (Fig. 6, E and F). By 7 days of recovery (Fig. 6G), immunostaining was at relatively low levels in the alveolar septa compared with 1-day recovery. Nonimmune control antibody had no staining (Fig. 6H).


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Fig. 6.   Immunostaining for total FN [plasma fibronectin (pFN)+cFN]. Lung sections were immunostained with a polyclonal antibody that detects all forms of FN including pFN and cFN. a: control lung had minimal immunostaining in alveolar septa, but staining was noted around blood vessels. b: 64-h hyperoxic lung without recovery had increased FN protein detected in alveolar septa and around blood vessels. c: 1-day recovering lung had increased immunostaining in alveolar septa and in debris in alveolar spaces. d: chondrocytes but not airway epithelial cells stained for total FN in 1-day recovering lung. e: 3-day recovering lung had decreased immunostaining in alveolar septa. f: 5-day recovering lung also had decreased immunostaining compared with 1 day of recovery. g: 7-day recovering lung had weak immunostaining for total FN. h: nonimmune control IgG antibody. Bar = 100 µm.

The antibody used to immunolocalize cFN recognizes an epitope on EIIIA and does not detect pFN. The time course of maximal EIIIA immunostaining differed from total FN (Fig. 7). Control lung had minimal EIIIA immunostaining of the parenchyma, with some signal noted around blood vessels (Fig. 7A). Unlike total FN, EIIIA immunostaining remained relatively low after 64 h of hyperoxia with no recovery (Fig. 7B). At 1 day of recovery, when total FN immunostaining was most intense, EIIIA was slightly increased in some alveolar septa (Fig. 7C). Unlike total FN, no EIIIA protein was detected in chondrocytes (Fig. 7D), similar to the in situ hybridizations. Whereas total FN immunostaining peaked at 1 day of recovery and then decreased, EIIIA protein remained increased in the alveolar septa during recovery (Fig. 7, E-G), generally paralleling the EIIIA mRNA expression. The protein was localized mainly in alveolar septal regions and blood vessels, with little or no EIIIA protein detected in airway epithelial cells. Unlike the EIIIA in situ hybridizations, EIIIA immunostaining was still prominent at 7 days of recovery (Fig. 7G). Negative control antibody gave no signal (Fig. 7H).


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Fig. 7.   Immunostaining for EIIIA-containing cFN. Monoclonal antibody used detects only EIIIA-containing cFN and not pFN. a: control lung had little EIIIA immunostaining in alveolar septal regions, but some weak staining was noted surrounding blood vessels. b: after 64 h of hyperoxia with no recovery, EIIIA protein remained low in alveolar septal regions. c: at 1 day of recovery, increased EIIIA was noted in some alveolar septa. d: neither chondrocytes nor airway epithelial cells stained for EIIIA at 1 day of recovery. e: at 3 days of recovery, some alveolar septal regions had intense EIIIA immunostaining. f: EIIIA immunostaining was still increased but more uniform in alveolar regions at 5 days of recovery. g: at 7 days of recovery, there continued to be abundant EIIIA immunostaining in alveolar septa. h: nonimmune control antibody. Bar = 100 µm.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Acute hyperoxic lung injury is characterized by destruction of alveolar cells, particularly endothelial and alveolar type I cells, infiltration of inflammatory cells, and increased capillary permeability, which results in accumulation of serum proteins such as pFN. Repair of tissue injuries, including hyperoxic alveolar damage, is associated with cell migration, proliferation, differentiation, and production of provisional extracellular matrix. Each of these cellular functions can be regulated by FN (16). A role for cFN in repair of various tissue injuries is suggested by several in vitro and in vivo studies (4, 14, 17, 24). The major findings of the current investigation are 1) cFN mRNA and immunostaining for EIIIA increased in alveolar septal cells during recovery from acute, severe oxygen injury, consistent with a role for cFN in alveolar repair; 2) the patterns of EIIIA and EIIIB mRNA expression differ, suggesting lung cell-specific cFN alternative splicing and different functions of the EIIIA and EIIIB variants; and 3) neither isolated alveolar type II cells nor SP-C-expressing cells in vivo had cFN mRNA during recovery.

Total FN protein, detected by immunostaining, increases in various experimental and human lung injuries, including the current study (3, 8, 18). Much of the FN protein in acute lung injury is probably pFN, resulting from capillary leakage. Increased cFN mRNA and protein during recovery of injured lung, as documented in this study, imply a role for cFN that may be separate from pFN. The molecular differences between pFN and cFN are known (16, 28), but the functional differences between these isoforms or among the splice variants of cFN are just emerging. In general, cFN is expressed at low levels in adult tissues but is increased in injury, coincident with cell migration and proliferation (9, 11). For example, in an in vitro model of airway epithelial injury, cFN and the alpha 5beta 1-integrin were expressed by migrating cells (14). Some injured tissues reexpress fetal variants of cFN that contain EIIIA or EIIIB domains (4, 11, 24). cFN with these domains is often located in injured tissues where cells are migrating or proliferating (9).

The present study found a restricted pattern of cFN expression during recovery from oxygen injury. Confirming previous work (30), we noted that alveolar macrophages had abundant FN mRNA, but polymorphonuclear cells and lymphoid cells did not. Similar to injured skin and kidney (2, 4), cFN expression by infiltrating macrophages preceded cFN expression by resident cells. Limited data in fibrotic human lung suggested that resident pulmonary parenchymal cells have cFN mRNA and protein (3, 18). In the present study of recovery from severe hyperoxic injury, cFN mRNA in resident lung cells was restricted mainly to alveolar septal cells, endothelial cells of muscularized arteries, and chondrocytes. The limited resolution of light microscopy precluded exact identification of the alveolar septal cells with cFN mRNA. Little cFN message and protein were detected in bronchial epithelial cells. Increased endothelial cell cFN expression has been noted during repair of endothelium (17). Both alveolar septal cells and endothelial cells are injured in oxygen toxicity (12). The finding of increased cFN in these cells during recovery suggests a role for cFN in repair of hyperoxic injury.

The cell-specific patterns of EIIIA and EIIIB mRNA expression differed in recovering lung: EIIIB mRNA was limited largely to chondrocytes, whereas EIIIA mRNA was detected in alveolar septal cells, alveolar macrophages, and endothelial cells but not in chondrocytes. A predominance of EIIIA mRNA in infiltrating macrophages has also been noted in injured kidney and peritoneal macrophages, whereas dermal macrophages had both EIIIA and EIIIB mRNA (2, 4). Lack of EIIIA immunostaining in chondrocytes confirmed the mRNA findings. Preferential expression of EIIIB by chondrocytes and EIIIA by endothelial cells has been noted in other organs (9). These data suggest that FN splicing is lung cell-type specific.

The cell-specific roles of different cFN splice variants are not yet known. cFN with the EIIIA domain promotes cellular adherence and stress fiber formation in the presence of pFN (35). EIIIA may be vectorially secreted and incorporate efficiently into pericellular matrices. Thus EIIIA expression by cells such as alveolar macrophages and resident lung cells may modify the composition and adhesive qualities of the provisional matrix after acute lung injury. EIIIB-containing cFN has been implicated in regulating cell proliferation (9). The EIIIB domain may alter the three-dimensional conformation of FN, which would modulate interactions with other matrix components and integrins (9). For example, inclusion of the EIIIB domain exposed a previously cryptic FN epitope, suggesting that the tertiary structure of FN was altered (9). Expression of EIIIB by damaged cartilage underscores its role in chondrocyte response to injury (5), but its unique functions have not been elucidated.

In previous work using Northern hybridizations, we found that fetal rabbit lung had both EIIIA and EIIIB mRNA (22). The finding of increased EIIIA and EIIIB mRNA in recovering lung in the current studies supports the general concept that a fetal pattern of FN expression is reestablished during lung repair. However, several factors limit the conclusion that recovering lung recapitulates fetal patterns of cFN mRNA. First, the cell-specific expression of FN splice variants in fetal lung is not known; therefore, it is not clear that repairing lung cells reexpress variants characteristic of that cell type in development. Second, we found EIIIA mRNA by Northern hybridization in normal adult lung in previous studies (22). Thus the increase in EIIIA mRNA found in the current study may represent increased cFN mRNA abundance rather than a change in FN splicing to reflect fetal patterns.

The identity of cFN-expressing alveolar septal cells is particularly interesting because FN has substantial effects on type II cell phenotype in vitro. Culture of type II cells on an FN-rich matrix promotes loss of type II cell characteristics and acquisition of a spread, flattened type I cell-like phenotype (26, 29, 34) similar to the changes in type II cell phenotype during repair of the alveolar epithelium. FN also stimulates type II cell motility in vitro (19), which may be important in alveolar repair. The finding that isolated type II cells from recovering lung did not have cFN mRNA is similar to findings in cells isolated from normal lung (21). Type II cells in vitro can express cFN mRNA, but this occurred when SP-C mRNA was decreased (21, 22). Similarly, the current study found that SP-C-positive cells rarely had FN mRNA in vivo. These findings suggest that differentiated type II cells do not express FN mRNA in vivo in either normal or recovering lung. A lack of markers for type I cells or for type II cells in transition to type I cells precludes identification of either of these cells as putative FN-expressing cells. Nevertheless, it is unlikely that differentiated type II cells contribute cFN to provisional matrix during alveolar repair of acute oxygen injury.

The temporal and spatial patterns of EIIIA immunostaining generally paralleled the EIIIA message detected by in situ hybridization. EIIIA protein was detected in alveolar septal regions and blood vessels but not in bronchial epithelial cells or chondrocytes. EIIIA immunostaining was greatest during recovery. In contrast, immunostaining for total FN, probably mainly pFN, increased during injury and declined during 7 days of recovery. It is likely that staining for total FN detected abundant pFN in extravasated serum proteins during injury and early recovery. The finding of differing patterns of pFN and EIIIA immunostaining implies differing functions of these molecules in lung injury. Some differences between EIIIA immunostaining and in situ hybridizations were noted. For example, EIIIA mRNA returned to control levels by 7 days of recovery, whereas immunostaining for the protein remained elevated at this time, possibly reflecting protein accumulation during recovery.

These findings have certain limitations. In previous work (22), we found that EIIIB mRNA increased on Northern hybridization during recovery. Although this may be due to increased EIIIB in chondrocytes, it is possible that other cell types had EIIIB mRNA that was below the sensitivity of in situ hybridization. We noted occasional SP-C-positive cells in vivo that also had FN mRNA. This finding is likely due to overlapping cells in the tissue section. We have documented previously that type II cells may have variable SP-C abundance after hyperoxic injury (23). Thus our double in situ hybridization data do not exclude the possibility that low SP-C-expressing type II cells may also express cFN. However, the finding that isolated type II cells had minimal cFN mRNA supports our speculation that these cells do not express cFN in vivo. The methods we used to immunodetect total FN and EIIIA were optimized for the individual antibodies, including the tissue preparation and the concentration of antibody. This allows comparing the patterns of antibody staining over time, but it precludes comparing the abundance of total FN and EIIIA at any particular time point.

In summary, cFN and EIIIA mRNA and immunostaining increase in alveolar septal cells and some large-artery endothelial cells during recovery from acute, severe hyperoxic injury. This pattern is consistent with a role for cFN in repair of alveolar septal and endothelial cells, both of which are damaged by hyperoxia. The paucity of cFN mRNA in type II cells during recovery from injury suggests that putative effects of cFN on type II cell phenotype are mediated by other cell types. The divergent patterns of EIIIA and EIIIB mRNA expression imply lung cell-type specific regulation of cFN alternative splicing.

    ACKNOWLEDGEMENTS

We thank R. O. Hynes and F. Possmayer for providing cDNA probes.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-54632 and Specialized Center of Research Grant HL-365443.

Present address of S. Horowitz: CardioPulmonary Research Institute, Winthrop Univ. Hospital, State Univ. of New York at Stony Brook, Mineola, NY 11501.

Address for reprint requests: W. M. Maniscalco, Box 651, Dept. of Pediatrics, Univ. of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642.

Received 17 March 1997; accepted in final form 5 January 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 274(4):L599-L609
1040-0605/98 $5.00 Copyright © 1998 the American Physiological Society




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