Introduction

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Intrauterine human lung development can be divided into five stages: embryonal (Days 26 to 52), pseudoglandular (Day 52 to 16 wk), canalicular (17 to 28 wk), saccular (29 to 36 wk), and alveolar (36 wk to term) (1). During these stages, the epithelial-mesenchymal interactions play a marked role in the development of conducting airways, acini, and alveoli, and demonstrate a simultaneous decrease in the proportion of mesenchymes. Extracellular matrix proteins have been suggested to be associated with the branching morphogenesis of the lung and the maturation of the fetal lung alveolar epithelium (2, 3). The spatial and temporal patterns of expression of extracellular matrix proteins such as collagens I, III, IV, V, VI, laminin, and fibronectin of lung interstitium during normal human lung development have also been previously described (4). The stage of alveolar development differs between various species, i.e., in mice and rats no alveoli are present at birth, which is notable because most studies on lung development have been performed with animals.

Tenascin is a large extracellular matrix glycoprotein. It is synthesized during embryonic development and expressed in many malignant and benign tumors that are absent or restricted in most adult tissues (5). Five isoforms of tenascin have been characterized, of which tenascin-C is the most studied. In normal adult lung, tenascin-C expression is scant, whereas in adult pulmonary fibrosis, especially in usual interstitial pneumonia (UIP), its expression is increased (6). In early chicken lung buds, tenascin-C messenger RNA (mRNA) is synthesized by epithelia at the sites of active growth of bronchial tubes (7). In rat lung, tenascin-C concentrates at the epithelial-mesenchymal interface during fetal lung development in the period of branching morphogenesis (8), and it is downregulated by dexamethasone in early organogenesis (9). Our recent study showed that the cells expressing tenascin-C mRNA in UIP were mainly myofibroblasts and type II pneumocytes (10), whereas in rat lung, tenascin-C-producing cells are fibroblasts and endothelial cells (11). Transforming growth factor- regulates the expression of tenascin-C alternative-splicing isoforms in fetal rat lung (12), and the expression of TGF- isoforms has also been observed during the first trimester of human embryos (13).

To our knowledge, no studies have been performed on tenascin-C during the morphogenesis of human lung. The aim of this study was to investigate the immunohistochemical distribution and mRNA expression of tenascin-C in developing human lung at various gestational ages, encompassing pseudoglandular, canalicular, saccular, and alveolar periods. The findings were compared with the normal-looking lung tissue of adults.

	Materials and Methods

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Patients and Handling of Specimens

Samples of lung tissue were retrieved from the files of the Department of Pathology, Oulu University Hospital. The study protocol was approved by the Ethical Committee of the Medical Faculty of the University of Oulu. The study material consisted of 14 cases of spontaneous abortion, 10 cases of stillbirth, and 10 cases of autopsied babies who had died within 1 h after birth at Oulu University Hospital between 1988 and 1999. Autopsies had been performed within 3 d. The gestational ages of these infants ranged from 12 to 40 wk corresponding to pseudoglandular (Weeks 12 to 16, 10 cases), canalicular (Weeks 17 to 28, 12 cases), saccular (Weeks 29 to 35, four cases), and alveolar (Weeks 36 to 40, eight cases) periods. None of the cases studied had pneumonia, cardiac or pulmonary abnormalities, infection, congenital malformations, or features of maseration. Causes of death were either hypoxia, prematurity, or remained unknown. Uninvolved peripheral lung tissue from adults, used as a control, was obtained from seven patients operated on for a bronchial carcinoid tumor of the lung.

Lung tissues either from the right or the left lung removed at autopsy were fixed in 10% formalin, then dehydrated, and embedded in paraffin. Four-micron sections were stained with hematoxylin and eosin. The whole material was re-evaluated, and one representative tissue block from each case was selected for immunohistochemical studies in all study material. In 15 cases, one tissue block was selected for tenascin-C mRNA in situ hybridization; in these cases, the autopsy had been performed within 1 d after death. Some mRNAs have been shown to be stable under postmortem conditions, enabling in situ hybridization techniques to be performed on postmortem material (14). To verify the in situ hybridization reaction, a positive control previously demonstrated to express signals for tenascin-C mRNA was used in the experiments (10). In order to identify the phenotype of the tenascin-C-expressing cells, the sections were stained with commercially available antibodies against -smooth muscle actin (clone 1A4 from Sigma BioSciences, St. Louis, MO, at a dilution of 1:50) and CD31 (clone JC/70A from DAKO, Glostrup, Denmark, at a dilution of 1:40). The latter antibody is characterized by its strong reactivity with a formalin-resistant epitope on CD31 in endothelial cells in normal tissues and in benign and malignant proliferations.

Scoring of Tenascin Immunoreactivity

The extent of tenascin immunoreactivity was scored as absent (0), low (+), moderate (++), and strong (+++) in and around different types of pulmonary cells.

Anti-Tenascin Antibodies and Immunohistochemical Staining

A monoclonal antibody (mAb), 143DB7, known to react with the two major isoforms of tenascin-C was used. mAb 143DB7 was developed to detect tenascin-C in formaldehyde-fixed tissue. It has been characterized in detail elsewhere (15) and is available from Locus-Genex Inc. (Helsinki, Finland).

Sections 4 µm thick were deparaffinized in xylene and rehydrated in graded ethanol. Endogenous peroxidase was consumed by incubating the sections in 0.1% hydrogen peroxide in absolute methanol for 20 min. Before immunostaining, the sections were treated with 0.4% pepsin (E. Merck, Darmstadt, Germany) at 37°C for 30 min. For the immunostainings, the avidin-biotin-peroxidase complex method was used as described (6).

mAb 143DB7 with a dilution of 1:1,000 was used as the primary antibody. Sections of lung tissues were incubated with the primary antibody at 4°C overnight, followed by a biotinylated rabbit antimouse secondary antibody (at a dilution of 1:300 for 30 min) and the avidin-biotin-peroxidase complex (both from Dakopatts, Glostrup, Denmark). The color was developed with diaminobenzidine. Sections were counterstained with a light hematoxylin stain and mounted with Eukitt (Kindler, Freiburg, Germany). The negative control consisted of substituting the primary antibody with phosphate-buffered saline (PBS) (pH 7.2) or serum isotype control (Zymed Laboratories Inc., San Francisco, CA).

Preparation of Tissue Sections for In Situ Hybridization

Sections 4 µm thick from paraffin-embedded lung biopsies were collected on clean Superfrost Plus glass slides (Erie Co., Portsmouth, NH), paraffin was removed by xylene, and tissues were rehydrated through a graded ethanol series. After three immersions in PBS, pH 7.2, the sections were treated in 0.2 M HCl for 20 min and twice in PBS for 3 min each followed by proteinase K (100 µg/ml in PBS) treatment for 15 min at 37°C. Then, tissue sections were transferred into 0.025 M glycine for 30 s. Tissues were postfixed in 4% paraformaldehyde (Fluka, Buchs, Switzerland) in PBS, pH 7.2 (all solutions made with 0.1% diethyl pyrocarbonate-treated water). After postfixation, tissue sections were transferred into 0.025 M glycine in PBS for 3 min, and then acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min (16). After that, tissue sections were rinsed in 2 × saline sodium citrate (SSC), dehydrated through a graded ethanol series, and air-dried.

Preparation of RNA Probes

A complementary DNA fragment (bases 814 to 1316) of the full-length human tenascin-C (16) was synthesized by polymerase chain reaction (PCR) from the HT-11 subclone kindly provided by Dr. Luciano Zardi (Instituto Nazionale Per la Ricerca Sul Cancro, Genova, Italy) by using the following primers: 5' CCC TGC AGT GAG GAG CAC GGC ACA 3' and 5' TGC CCA TTG ACA CAG CGG CCA TGG 3'. The 503-bp PCR product containing a specific sequence for tenascin-C was subcloned into a TA vector (TA cloning kit; Invitrogen, San Diego, CA). Sense and antisense RNA probes were generated from a linearized template by using a riboprobe transcription kit (Promega, Madison, WI) and the probes were labeled with ³⁵S[uridine triphosphate] (Amersham, Little Chalfont, UK). The radioactively labeled RNA probes were purified by centrifugation through Bio-Gel P-30 columns (Bio-Spin 30; Bio-Rad, Richmond, CA). Each in vitro transcription reaction yielded RNA probes of high specific activity (typically 4.5 to 6 × 10⁸ dpm/ 1 µg DNA template and 50 to 70% incorporation).

In Situ Hybridization

The hybridization mixture contained the ³⁵S-labeled RNA probe (1.2 × 10⁵ dpm/µl), 50% deionized formamide (BRL, Rockville, MD), 5 mM dithiothreitol, 500 µg/ml yeast transfer RNA (BRL), 2 mg/ml bovine serum albumin (BRL), and 4 × SSC. The samples were hybridized overnight at 50°C while covered with parafilm (18), and then washed three times in 2 × SSC/50% formamide at 52°C and 2 × SSC at room temperature, followed by incubation in an RNase solution (100 µg/ml RNase A; Boehringer Mannheim, Mannheim, Germany) in 2 × SSC at 37°C for 30 min. The tissue sections were subsequently washed in 2 × SSC/50% formamide at 52°C for 5 min and three changes of 2 × SSC at room temperature, dehydrated sequentially in 70, 80, and 95% ethanol for 1 min each with agitation, and air-dried. Autoradiography was performed by dipping them in Kodak NTB-2 nuclear track emulsion (Eastman Kodak, Rochester, NY) diluted 1:1 with sterile distilled water at 42°C. After a week's exposure in the dark at 4°C, the slides were developed in Kodak D-19 developer for 5 min, rinsed in 1% acetic acid in distilled water for 30 s, fixed in a Kodak Agefix for 5 min, rinsed in distilled water, and stained with hematoxylin and eosin.

To evaluate the specificity of the ³⁵S-labeled antisense tenascin-C probes to the tissue sections of lung specimens, control experiments were performed using ³⁵S-labeled sense tenascin-C probes separately for each sample.

The hybridized tissue sections of lung samples were examined by light microscopy, and the number of grains over the cells were evaluated in general and especially at the locations where tenascin-C immunoreactivity was located. Cells or cell groups hybridized with the ³⁵S-labeled antisense tenascin probe were considered positive if they contained more grains than did the corresponding cells and tissue areas that had been hybridized with the ³⁵S-labeled sense tenascin-C probe.

	Results

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Immunohistochemical Findings

Adult lung as a control. Most normal alveolar walls contained no immunohistochemical expression for tenascin-C (Figure 1A). Faint immunoreactivity, however, could occasionally be observed in these areas. Weak immunoreactivity was observed in bronchioli underneath the bronchiolar epithelium and among smooth muscle cells. The intima of veins, and to a lesser degree arteries also, stained positively for tenascin-C. Pleural mesothelium and submesothelial connective tissue were negative.

View larger version (123K): [in this window] [in a new window]   Figure 1.   (A) An immunohistochemical staining in normal adult lung shows no expression for tenascin-C within alveolar walls. Scale bar = 80 µm. (B) An increased immunohistochemical expression for tenascin-C can be seen (arrows) underneath the epithelia of the branching (left) and the simple (right) airway in the developing lung of a fetus of 15 wk gestational age. Scale bar = 160 µm. (C) -Smooth muscle actin-positive cells (arrows) in a serial section from the same area as in B. -Smooth muscle actin positivity indicates a myofibroblast-like phenotype. Scale bar = 160 µm. (D) The same case as in B and C. Immunoperoxidase stain, PBS control with hematoxylin counterstain. Scale bar = 160 µm. (E) Slender and linear tenascin-C-positive fibers can be seen below the developing alveolar epithelium (arrow) in the lung of a fetus of 17 wk gestational age. Scale bar = 80 µm. (F ) -Smooth muscle actin positivity (arrow) in the cells below the developing alveolar epithelium in the same area in a serial section from the same case as in E. Scale bar = 80 µm. (G) The same case as in E and F. Immunoperoxidase stain, PBS control with hematoxylin counterstain. Scale bar = 80 µm. (H ) An increased immunoreactivity for tenascin-C in bronchial cartilages (arrows) from a fetus of 17 wk gestational age. Scale bar = 160 µm.

Pseudoglandular period (Weeks 12 to 16). During this period, the airways are subdividing (i.e., branching morphogenesis). The airways form round glandlike structures, which are lined by pseudostratified epithelia and separated by cellular mesenchymes. The immunohistochemical expression for tenascin-C was strong (+++) below the pseudostratified airway epithelia, especially at the sites of airway subdivision, i.e., branching (Figures 1B and 1C), and also below the epithelia of the bronchi. A strong expression for tenascin-C (+++) was also observed around chondrocytes. The intima of the veins, but not the intima of the arteries, expressed weak (+) positivity. A faint immunoreactivity was detected occasionally in the media of the arteries. The staining in mesothelial cells and in submesothelial connective tissue of pleura was negative.

Canalicular period (Weeks 17 to 28). During the canalicular period, the airways are dividing further, the vascular system is developing, and the amount of mesenchymes is decreasing. The epithelium becomes thinner. From Week 16, pretype II cells appear, and from Weeks 24 to 28, type I and II pneumocytes are appearing. The airways are mostly simple and numerous, but among them there are also branching airways. The immunohistochemical expression for tenascin-C was strong (+++) underneath the airway epithelia in both the simple and the branching airways between Weeks 17 and 23 (Figures 1E and 1G), whereas during Weeks 24 to 28 the immunoreactivity was weak (+) (Figures 2A and 2C). A strong immunopositivity (+++) was shown around, and occasionally within, chondrocytes during the whole canalicular period (Figure 1H). The immunoreactivity for tenascin-C was also strong (+++) in the intima of the veins (Figures 2D and 2F), whereas the intima of the arteries (Figure 2G), pleura, and mesothelia were negative.

View larger version (175K): [in this window] [in a new window]   Figure 2.   (A) A faint and dispersed immunoreactivity for tenascin-C (arrows) can be seen below the alveolar epithelia in the lung of a fetus of 25 wk gestational age. Scale bar = 80 µm. (B) A serial section in the same case as in A demonstrated -smooth muscle actin-positive cells (arrows) in the same location. Scale bar = 80 µm. (C) The same case as in A and B. Immunoperoxidase stain, PBS control with hematoxylin counterstain. Scale bar = 80 µm. (D) A very faint immunohistochemical expression for tenascin-C (arrows) in the walls of alveoli from a fetus of 35 wk gestational age. Scale bar = 80 µm. (E) -Smooth muscle actin-positive cells (arrows) in a serial section in the same case as in D. Scale bar = 80 µm. (F ) The same patient as in D and E. Immunoperoxidase stain, PBS control with hematoxylin counterstain. Scale bar = 80 µm.

Saccular (Weeks 29 to 35) and alveolar (Weeks 36 to 40) periods. The saccular period is characterized by an increase in gas-exchanging surface area, and a decrease of the mesenchymes between the saccules. Small crests appear in the walls of sacculi, finally developing to alveoli from Week 36 onward. The immunohistochemical expression of tenascin-C was low (+) or moderate (++) beneath the bronchiolar and alveolar epithelia (Figures 2D, 2E, 3A, and 3C), and it was moderate (++) around chondrocytes and in the intima of the veins between Weeks 29 and 40. Again, the immunoreactivity in the intima of the arteries, mesothelial cells, and submesothelial connective tissues of pleura was negative.

View larger version (112K): [in this window] [in a new window]   Figure 3.   (A) A very faint immunohistochemical expression for tenascin-C (arrow) in the walls of alveoli from a patient of 37 wk gestational age. Scale bar = 80 µm. (B) -Smooth muscle actin- positive cells (arrow) in a serial section from the same patient as in A. Scale bar = 80 µm. (C) The same patient as in A and B. Immunoperoxidase stain, PBS control with hematoxylin counterstain. Scale bar = 80 µm. (D) Immunoreactivity for tenascin-C (arrow) in the intima of the vein of the lung from a fetus of 17 wk gestational age. Scale bar = 80 µm. (E) -Smooth muscle actin-positive cells (arrow) are detected in the same location as immunoreactivity for tenascin-C. The figure represents a serial section from D. Scale bar = 80 µm. (F ) The same patient as in D and E. Immunoperoxidase stain, PBS control with hematoxylin counterstain. Scale bar = 80 µm. (G) Tenascin-C is negative in the artery of lung from a fetus of 17 wk gestational age. Scale bar = 160 µm. (H) -Smooth muscle actin-positive cells within the media of the artery in the same case as in G in a serial section. Scale bar = 160 µm.

Immunohistochemical findings for -smooth muscle antigen and CD31. In all cases, immunohistochemical staining for -smooth muscle actin showed intracellular positivity in the cells below the airway epithelia, including alveolar and bronchiolar epithelia, although the number of positive cells decreased during the canalicular, saccular, and alveolar periods (Figures 1C, 1F, 2B, 2D, and 3B). Positive cells within the alveolar walls were localized beneath the basement membranes of the alveolar epithelia, corresponding to myofibroblast-type cells. Positive immunoreactivity for -smooth muscle actin was also observed in the media of the arteries and in the intima of the veins (Figures 3E and 3H). A few interstitial cells were faintly positive for -smooth muscle antigen, whereas the submesothelial connective tissue cells of the pleura were negative.

The endothelial cells of both veins and arteries showed an intracellular immunoreactivity for CD31 in all cases, whereas the reactivity was negative in other types of pulmonary cells.

mRNA In Situ Hybridization

The number of mRNA signals was diffusely increased in all 15 cases of tissues hybridized with ³⁵S-labeled antisense tenascin-C RNA probe when compared with those hybridized with ³⁵S-labeled sense RNA probe. The cells underneath the airway epithelia, often at the sites of subdivision, showed increased expression of tenascin-C mRNA (Figures 4A, 4B, 4C, and 4E). Immunohistochemical staining demonstated that these cells were positive for -smooth muscle actin, suggesting a myofibroblast phenotype (Figures 4D and 4F). An expression for tenascin-C mRNA was also detected in the cells of the intima of the veins, which by immunohistochemistry were positive for CD31 and -smooth muscle actin (Figures 4G and 4H). The number of the tenascin mRNA signals was also increased in chondrocytes. In general, the expression of tenascin-C mRNA by in situ hybridization studies corresponded closely to the immunohistochemical findings of the expression of tenascin-C.

View larger version (116K): [in this window] [in a new window]   Figure 4.   (A) In situ hybridization (arrows) showing tenascin-C mRNA below the airway epithelium of a fetus of 15 wk gestational age. Scale bar = 80 µm. (B) High-power field from the same airway as in A. Many cells show signals for tenascin-C mRNA (arrows) beneath the epithelium. Scale bar = 40 µm. (C) The same patient as in B. In situ hybridization with tenascin-C sense RNA probe that is used as a negative control. Scale bar = 40 µm. (D) The same patient as in B. -Smooth muscle actin-positive cells (arrows) are located in the same area as signals for tenascin-C mRNA in B. The figure represents a serial section. Scale bar = 40 µm. (E) Signals for tenascin-C mRNA (arrow) can be seen by in situ hybridization in the cells below the airway epithelium in the lung of a fetus of 15 wk gestational age. Scale bar = 40 µm. (F ) The same patient as in E. -Smooth muscle actin-positive cells (arrow) in the same location as tenascin-C mRNA in E. The figure represents a serial section. -Smooth muscle actin positivity at this location suggests a myofibroblast phenotype. Scale bar = 40 µm. (G) Endothelial cells (arrow) in the intima of a vein show tenascin-C mRNA expression. The case represents lung tissue from a fetus of 15 wk gestational age. Scale bar = 40 µm. (H ) A serial section from the same case as in G. -Smooth muscle actin- positive cells can be seen in the same location as mRNA signals for tenascin-C in G. Scale bar = 40 µm.

	Discussion

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This is the first study on tenascin-C during the organogenesis of human lung. A varying immunohistochemical expression of tenascin-C was visible through all developmental periods, being especially strong at the sites of airway subdivision between Weeks 12 and 23 when it was localized underneath the airway epithelia. Even though there are important differences between the development of human and animal lungs, our results are consistent with previous findings on rat lung, which show that tenascin-C concentrates at the epithelial-mesenchymal interface during the period of branching morphogenesis (8, 9). The amount of the immunohistochemical expression of tenascin-C seemed to decrease within alveolar walls at the end of the canalicular period, when type I and II pneumocytes are also known to differentiate. Interestingly, this decline occurred at the same gestational age when the lungs of preterm infants have reached maturation for survival. Tenascin-C could, however, be detected until the gestational age of 40 wk, i.e., full-term, when its expression was still markedly stronger than that in adult lung. In contrast to many animals, human lung development continues after birth (1). Therefore, it is not surprising that the expression of tenascin-C is higher at the time of birth than in the lungs of adults.

Tenascin-C has been found to be expressed in embryonic connective tissue, at the sites of branching morphogenesis and cell motility, and throughout the developing central nervous system (19, 20). In a recent study by Derr and coworkers (21), the two different domains of amino-acid repeats of tenascin-C were studied in chicken embryonic tissues. They found that tenascin-C with the AD1 repeat has a widespread origin in the early chicken embryo, whereas tenascin-C with the AD2 repeat has origins limited to sites of epithelial-mesenchymal interactions, such as the tips of lung bronchioles and the base of feather buds. In the fibrotic lungs of adults, tenascin-C has been shown to increase during the regeneration of the alveolar epithelia, suggesting that it has a role in the process of epithelial repair (6, 10). In this study, tenascin-C increased during the development of the conducting airway and alveoli simultaneously with the appearance of pretype II, type I and type II pneumocytes, indicating that it may participate in the epithelial-mesenchymal interactions in the morphogenesis of the human lung. Probably other extracellular matrix glycoproteins, such as fibronectin, which has been shown to bind tenascin-C, take part in this process.

The expression of tenascin-C in the arteries and veins was different during all developmental stages, a finding which has not been previously published. Tenascin-C was expressed moderately or strongly in the intima of the veins, whereas it was absent in the intima of the arteries. In the pulmonary vessels of adult lung, however, it is expressed in the intima of both the arteries and the veins. In a previous study performed on rat arteries, tenascin-C was observed in the intimal layer, but not in the media (22). The same study also shows that tenascin-C synthesis is induced concomitantly with changes in the smooth muscle phenotype (22), supporting the findings of our study. We observed that the intima of the veins was positive for -smooth muscle actin, whereas the intima of the arteries was negative for it. Our previous study showed that the cells expressing tenascin-C in adult idiopathic pulmonary fibrosis are mainly -smooth muscle actin-positive myofibroblasts (10). This is in concordance with our present study, which demonstrates that the expression of tenascin-C and -smooth muscle actin colocalize in the walls of bronchioli and alveoli and in the intima of the veins. The media of arteries were positive for -smooth muscle actin because smooth muscle cells exist in the media of arteries. In some cases, however, we occasionally noticed a faint immunoreactivity for tenascin-C in the media of small arteries. Sharifi and coworkers (23) have observed that angiotensin II regulates tenascin-C gene expression in cultured vascular muscular cells. However, the meaning of the findings of tenascin-C expression in the vessels of developing human lung remains to be clarified in the future.

A strong expression for tenascin-C was observed around chondrocytes through all the developmental stages. An intense positivity by immunohistochemical studies and an increased number of signals in mRNA in situ hybridization studies for tenascin-C were also detected in chondrocytes. In previous animal studies, tenascin-C was shown to be expressed in chondrocytes and to associate with articular cartilage development (24, 25). A recent study showed that mechanical loading regulates tenascin-C expression in chondrocytes and fibroblasts of the osteotendinous junction (26). Interestingly, an intermittent mechanical strain has been observed to regulate gene and protein expression of various extracellular matrix molecules, such as collagen I and IV and biclycan, in a study that simulates fetal breathing movements in fetal rat lung cells (27).

In conclusion, there is a versatile expression of tenascin-C in human lung during all developmental periods studied. Its expression is especially strong below airway epithelia during the subdivision of airways in gestational ages of 12 to 23 wk. Tenascin-C seems to be an important extracellular matrix glycoprotein in the morphogenesis of human lung.