Expression of epidermal growth factor and surfactant proteins during postnatal and compensatory lung growth

David J. Foster1, Xiao Yan1, Dennis J. Bellotto2, Orson W. Moe1,3, Herbert K. Hagler2, Aaron S. Estrera4, and Connie C. W. Hsia1

Departments of 1 Internal Medicine, 2 Pathology, and 4 Cardiovascular and Thoracic Surgery, University of Texas Southwestern Medical Center, and 3 Medical Service, Department of Veteran Affairs Medical Center, Dallas, Texas 75390-9034


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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We examined whether lung growth after pneumonectomy (PNX) invokes normal signaling pathways of postnatal development. We qualitatively and quantitatively assessed the immunoexpression of epidermal growth factor (EGF), its receptor (EGFR), surfactant proteins (SP) [SP-A and -D and surfactant proproteins (proSP)-B and -C] and proliferating cell nuclear antigen (PCNA) in immature and mature dog lung. We also assayed these proteins in lungs of immature dogs 3 wk or 10 mo after they underwent right PNX compared with simultaneous matched sham controls. During maturation, alveolar cell proliferation is regionally regulated in parallel with EGF and EGFR levels and inversely correlated with SP-A and proSP-C levels. In contrast, post-PNX lung growth is not associated with EGF or EGFR upregulation but with markedly increased SP-A level and moderately increased SP-D level; proSP-B and proSP-C levels did not change. We conclude that 1) signaling of EGF axis and differential regulation of SPs persist during postnatal lung development, 2) post-PNX lung growth is not a simple recapitulation of maturational responses, and 3) SP-A and SP-D may modulate post-PNX lung growth.

immunohistochemistry; immunogold; immunoassay; lung resection; pneumonectomy


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

COMPENSATORY LUNG GROWTH FOLLOWING pneumonectomy (PNX) is vigorous in immature dogs. Within 8 wk, the volume of extravascular septal tissue in the remaining lung increases to that expected in two normal lungs (41). When raised to maturity after PNX, septal cellular volumes and surface areas remain completely normal, associated with normal gas exchange up to peak exercise (42). In contrast, compensatory lung growth in adult dogs occurs only after resection of at least 55% of lung, suggesting that a threshold exists for growth initiation (13). Long-term structural and functional lung capacity is incompletely restored, remaining at 70-80% of that in control animals (13, 14). The primary signal initiating compensatory lung growth involves alveolar septal strain. However, elimination of mechanical strain of the remaining lung after PNX does not completely eliminate the compensatory response (15, 49). Thus other factors, including endothelial shear stress produced by elevated blood flow to the remaining lung and release of soluble growth factors, must also play a role.

Post-PNX lung growth consists of early cellular proliferation (3) and thickening of alveolar septa, with interstitial cell volume increasing to a greater extent than epithelial or endothelial volume (13). Subsequently, remodeling occurs associated with thinning of the septa and declining volume of the interstitial matrix (13), eventually resulting in balanced growth of septal cellular components. The events leading to normalization of septal architecture after PNX parallel those seen during postnatal maturation, suggesting that compensatory lung growth may occur via reactivation of normal developmental pathways. Thus we hypothesized that growth-factor signaling systems known to be important during normal maturation could be persistently activated or reactivated after PNX.

The signaling of epidermal growth factor (EGF) via its receptor (EGFR) plays a crucial role in lung development (32, 40). EGF stimulates the cytodifferentiation of alveolar type II cells and biosynthesis of surfactant precursor proteins (8, 12). We, therefore, tested our hypothesis on the EGF axis, as well as the surfactant protein (SP) system in the dog lung. We assayed EGF, EGFR, SP-A, SP-D, and the proproteins for SP-B (proSP-B) and SP-C (proSP-C) in dog lung tissue obtained at two time points (3 wk and 10 mo) after right PNX and from simultaneous litter-matched control animals. We also measured the level of proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation. Results were compared with the corresponding developmental patterns observed in normal immature and mature dogs.


    MATERIALS AND METHODS
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Animal procedures and tissue collection. Studies were approved by the Institutional Animal Care and Research Advisory Committee. Dogs reach somatic maturation by 9-12 mo of age. To examine postnatal lung growth, lung tissue was obtained during thoracotomy from peripheral and central locations within the right upper lobe of normal foxhounds at 3 or 12 mo of age (n = 6 per group). To examine compensatory lung growth, litter- and sex-matched immature foxhounds underwent either right PNX or thoracotomy without PNX (sham) at ~9 wk of age. Under isoflurane anesthesia, the right lung was exposed via a lateral thoracotomy through the 5th intercostal space. The lobar arteries and veins were individually ligated and cut between ligatures. The right main stem bronchus was stapled and cut. The stump was immersed under saline to check for leaks. After hemostasis was ensured, the thorax was closed in five layers. Residual air within the thorax was aspirated. Sham animals underwent right thoracotomy only without lung resection. At 3 wk (n = 6 per group) or 10 mo (n = 5 per group) after surgery, corresponding to 3 and 12 mo of age, respectively, animals were deeply anesthetized with intravenous pentobarbital and mechanically ventilated. Via a left thoracotomy, the left upper lobe was removed. Lung tissue samples were taken from the peripheral and central regions of the lobe, rinsed with saline, flash frozen in liquid nitrogen, and stored at -70°C for later protein analysis. Additional tissue samples were immersed in Bouin's solution for 4-5 h, washed with 70% ethanol, and embedded in paraffin for immunohistochemical analysis. For immunogold labeling, tissue was fixed in a mixture of 0.5% glutaraldehyde plus 2% paraformaldehyde for 4 h, cut into 1.0-mm pieces, and infused overnight with a mixture of 2.3 M sucrose + 20% (wt/vol) polyvinylpyrrolidone in 0.1 M phosphate buffer. Tissue blocks were then placed on aluminum pins and frozen in liquid nitrogen.

Immunohistochemistry. Paraffin-embedded blocks were sectioned at 4-µm thickness. Affinity-purified rabbit polyclonal IgG antibodies (5 µg/ml) to human recombinant EGF and human EGFR (Oncogene Science, Cambridge, MA) were used. Normal rabbit IgG (5 µg/ml) was substituted for the primary antibody to EGF or EGFR as a negative control. A specific control for EGFR immunostaining utilized a 10-fold higher concentration of the competitive peptide antigen (Oncogene Science) to block antibody-antigen interaction. PCNA was detected by using monoclonal mouse anti-PCNA clone PC 10 (DAKO, Carpinteria, CA) at 1 µg/ml. Bound primary antibody was detected by using the avidin-biotin complex kit (Vector Laboratories, Burlingame, CA).

Immunogold labeling. After the Tokuyasa thawed cryosectioning method (11), ultrathin sections (80 nm) from four dog lungs were stained with primary antibodies to EGF and EGFR, as shown above, at a concentration of 20 µg/ml. The primary antibodies were bound to protein-A gold probe (10 nm, from G. Posthuma, Dept. of Cell Biology, Utrecht University, the Netherlands) and mounted on copper grids. Grids were qualitatively examined by a transmission electron microscope (JEOL EXII) at ×5,000 to ×25,000 magnification.

Immunoassay. Lung tissue (50-100 mg wet wt) was minced on ice and homogenized by polytron in a buffer containing 300 mM sucrose, 20 mM Tris, pH 8.0, 10 mM HEPES, 5 mM EGTA, 2 mM beta -mercaptoethanol, and protease inhibitors aprotinin (5 µg/ml), pepstatin A (1.5 µM), leupeptin (100 µM), trypsin inhibitor (5 µg/ml), p-aminobenzamidine (200 µM), and phenylmethylsulfonyl fluoride (1 mM). The homogenates were centrifuged at 15,000 g, and the supernatants containing crude tissue lysate were removed to new tubes. The nuclear pellets were resuspended in radioimmunoprecipitation assay buffer containing 150 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholic acid, and protease inhibitors aprotinin (5 µg/ml), pepstatin A (1.5 µM), leupeptin (5 µg/ml), and phenylmethylsulfonyl fluoride (1 mM). The radioimmunoprecipitation assay suspensions were pelleted at 15,000 g to remove insoluble matter, and the detergent-soluble proteins in the supernatant served as the source for PCNA analysis. The crude tissue lysates were centrifuged at 100,000 g to yield membrane pellets and soluble supernatants. Membrane pellets were resuspended in buffer containing 5 mM Tris, pH 6.8, 10% glycerol, 1% beta -mercaptoethanol, and 1% SDS. This membrane fraction was analyzed for EGFR, and the soluble supernatant was analyzed for EGF, SP-A, SP-D, proSP-B, and proSP-C.

Total protein content in each fraction was quantified by Bradford assay (Bio-Rad Laboratories, Hercules, CA). Proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The blots were blocked in Blotto-Tween solution (5% nonfat dry milk, 0.05% Tween in PBS) for 1 h and then incubated in Blotto-Tween with primary antibody for a minimum of 2 h. Rabbit anti-human polyclonal EGFR antiserum (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-human PCNA mouse monoclonal antibody (CalBiochem-Novabiochem, San Diego, CA) were used at 1-2 µg/ml. Polyclonal anti-human SP-A antiserum was generously provided by Dr. Carole R. Mendelson (Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX) and was diluted 1:100. SP-D goat polyclonal anti-human antiserum (Santa Cruz Biotechnology) was used at 2 µg/ml. ProSP-B and proSP-C rabbit polyclonal antibodies (Chemicon International, Temecula, CA) were diluted 1:1,000. Labeled protein was visualized by using a chemiluminescence detection system (ECL, Amersham, Piscataway, NJ) and quantified by densitometry. EGF levels were assayed by using a competitive ELISA kit for human EGF (Chemicon International). Extract volumes were reduced by vacuum centrifugation and assayed in duplicate. EGF concentrations were determined with reference to a standard curve of known EGF quantities and then normalized per unit protein present in the extracts.

Data analysis. For quantitation of immunohistochemical labeling within the septum, one section per animal was selected at random and analyzed. Starting with a random microscopic field, 20 non-overlapping fields (10 in the subpleural alveolar region and 10 in deep alveolar region) were sampled systematically and examined at ×600 magnification. A test grid was laid over each field, and the volume density of septum was estimated by point counting. The number of positive staining septal cells within each field was counted and divided by the volume density of the septum. Nonseptal structures (bronchioles and blood vessels exceeding 20 µm in diameter) and blood cells within vessels were excluded from analysis. Results between groups and between regions were compared by two-way ANOVA.

At least three immunoblots were prepared that compared immature and mature dogs or sham and PNX dogs at each time point. Each assay utilized independent tissue samples obtained from different locations in five or six dog lungs per group. The chemiluminescent signal intensities on each blot were expressed as a percentage of the mean signal intensity of the respective control group (mature or sham) obtained from the same blot. The percent values of signal intensity from three replicate assays were combined and compared by unpaired t-test (n = 15 or 18 independent observations per group). A separate assay was also performed for SP-A expression in which triplicate samples of lung extract from sham and PNX dogs at each time point (n = 66 independent observations) were resolved and transferred simultaneously and then immunostained together. In this case, signal intensities per unit protein were compared by two-way ANOVA and Student Newman-Keuls multiple-comparison tests. EGF levels in triplicate samples from sham and PNX dogs at each time point were expressed per unit protein present in each extract and were compared in a similar way. A P value <0.05 was considered significant.


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PCNA expression. In all animals, the bronchiolar epithelium stained strongly for PCNA. Labeling within the septum was more extensive in immature than mature animals (Fig. 1). Semiquantitative analysis of immunolabeling confirmed the qualitative assessment (Table 1). Because lung samples were immersion fixed, the alveoli were not fully inflated. However, volume density of alveolar septum in lung was similar between immature and mature animals and between deep and subpleural regions. The number of labeled septal cells per unit septal volume was significantly greater in the immature than mature dog lung (P < 0.01) and greater in the subpleural than deeper region in both immature (P < 0.05) and mature animals (P < 0.02). Immunoblot analyses corroborated the immunohistochemical labeling. PCNA levels were greatly enhanced in immature lungs (2,392% above mature lungs, Fig. 2A; P < 0.0005). These parallel findings indicate vigorous cellular proliferation during postnatal lung maturation, especially in the lung periphery.


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Fig. 1.   Representative photomicrographs of immunolabeling for proliferating cell nuclear antigen (PCNA), epidermal growth factor (EGF), and EGF receptor (EGFR) in subpleural and deep septal regions of immersion-fixed immature and mature dog lung. Bar = 100 µm.


                              
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Table 1.   Number of labeled cells normalized by volume density of septum



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Fig. 2.   PCNA expression (means ± SE). Representative immunoblots are shown in insets. Signal intensity, normalized for protein loading, is expressed as a percentage of the mean intensity obtained in the mature or sham group in each of triplicate assays (15-18 independent observations per group). A: postnatal maturation; n = 6 animals/group. B: after pneumonectomy (PNX); n = 6 animals/group at 3 wk; n = 5 animals/group at 10 mo after surgery.

At 3 wk after PNX, the PCNA level significantly increased (by 79%) above age-matched sham levels (P < 0.0025; Fig. 2B). Thus the normally high levels of PCNA in immature dog lungs were further augmented by PNX. At 10 mo after PNX, the PCNA level was still 43% above age-matched sham levels, but the difference was no longer statistically significant (Fig. 2B). Results show further enhancement of cell proliferation in the remaining lung during the early weeks after PNX.

SP expression. SP-A and proSP-C levels were significantly lower in immature lungs compared with mature lungs, by 79% and 62%, respectively (Fig. 3A; P < 0.0005). SP-D expression was slightly but significantly lower in the mature lung by 24% (Fig. 3A; P < 0.0025). ProSP-B level did not change significantly with maturation. Thus there was differential SP expression during postnatal maturation, and SP-A and proSP-C expression were inversely related to cellular proliferation.


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Fig. 3.   Surfactant protein (SP) expression. Values are means ± SE. Representative immunoblots are shown in insets. A: SP-A, surfactant proprotein (proSP)-B, proSP-C, and SP-D during maturation. Signal intensity, normalized for protein loading, is expressed as a percentage of the mean intensity in mature animals. Triplicate assays were performed; n = 6 animals/group (18 independent observations). B: SP-A expression after PNX. Triplicate lung samples were taken from each animal (3 wk, n = 6 animals/group; 10 mo, n = 5 animals/group; total of 66 independent observations). Signal intensity is expressed as a percentage of the mean intensity in the sham group 3 wk after surgery. C: SP-D expression after PNX. Signal intensity is expressed as a percentage of the mean intensity in the respective sham group. Triplicate assays were performed; n = 6 animals/group at 3 wk; n = 5 animals/group at 10 mo after surgery (15 or 18 independent observations/group).

In contrast to the maturational pattern, tissue SP-A level in the proliferating lungs 3 wk after PNX markedly increased to 468% of that in controls (Fig. 3B; P < 0.01) and then subsided by 10 mo to 295% above 3-wk control levels as proliferation slowed. The magnitude of SP-A increase early after PNX relative to sham was similar to that observed in normal mature dogs relative to immature dogs (Fig. 3), but SP-A expression decreased with age after PNX. SP-D levels were modestly increased 3 wk after PNX to 138% of sham value (P < 0.0005; Fig. 3C) but fell to 65% of sham levels 10 mo after surgery (P < 0.0005). ProSP-B and proSP-C levels did not change after PNX (data not shown); signal intensities normalized for protein loading were 119 and 98% (proSP-B) and 105 and 101% (proSP-C) of control values at 3 wk and 10 mo after PNX, respectively (P > 0.05). Instead of the increasing SP-A and proSP-C levels expected during normal maturation, post-PNX lung growth is associated with an early exaggerated SP-A response, whereas proSP-C did not increase above control levels.

EGF expression. In immature lungs, EGF immunostaining was more intense than in mature lungs, with scattered staining found throughout the parenchyma and within alveolar macrophages (Fig. 1). Semiquantitative analysis (Table 1) indicated significantly greater numbers of labeled cells within the alveolar septum per unit septal volume in immature compared with mature lung (P < 0.0001) and in subpleural regions of both immature (P < 0.01) and mature animals (P < 0.0005). EGF protein measured by ELISA was modestly but significantly enhanced in immature lungs by 27% (P < 0.02) (Fig. 4A). Thus results were similar, regardless of whether we examined whole lung tissue or the septal tissue selectively. These parallel findings indicate that vigorous septal cellular proliferation during postnatal lung maturation is associated with upregulated EGF expression, especially in the lung periphery.


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Fig. 4.   EGF protein levels, determined by ELISA expressed as mean (±SE). Triplicate assays were performed (15-18 independent observations per group). A: postnatal maturation; n = 6 animals/group. B: after PNX; n = 6 animals/group at 3 wk; n = 5 animals/group at 10 mo after surgery.

EGF level was modestly but significantly lower (by 34%; P < 0.001) in the proliferating lungs 3 wk after PNX, compared with sham controls, but returned to sham levels 10 mo after PNX (Fig. 4B). Thus, in contrast to maturational lung growth, early post-PNX cellular proliferation was associated with mildly reduced EGF expression.

EGFR expression. Similar to PCNA and EGF, EGFR labeling was more intense in the subpleural than deeper septa and more intense in immature than mature lungs (Fig. 1). Results were corroborated by semiquantitative analysis (Table 1). EGFR levels in whole lung homogenates were also modestly but significantly enhanced in immature lungs by 40% (Fig. 5A; P < 0.005). Thus EGFR levels also correlated with cellular proliferation during postnatal lung maturation.


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Fig. 5.   EGFR expression. Representative immunoblots are shown in insets. Signal intensity is normalized for protein loading and expressed as a percentage of the mean (±SE) intensity in the mature or sham group. Triplicate assays were performed (15-18 independent observations/group). A: postnatal maturation; n = 6 animals/group. B: after PNX; n = 6 animals at 3 wk; n = 5 animals at 10 mo after surgery.

EGFR protein level was slightly but significantly lower (by 13%; P < 0.05) at 3 wk after PNX, compared with sham controls but was similar to controls at 10 mo after PNX (Fig. 5B). As with EGF, post-PNX cellular proliferation was associated with mildly reduced EGFR expression.

Localization of EGF and EGFR. Immunogold labeling of EGF was concentrated in the cytoplasmic granules of septal interstitial cells, which were frequently situated next to collagen bundles and morphologically resemble fibroblasts (Fig. 6A); scattered labeling was also found within the cytoplasm of alveolar epithelial cells. Immunogold labeling of EGFR was concentrated along the inner surface of the lamellar body membrane of type II alveolar epithelial cells, with scattered labeling along the cell membrane of alveolar epithelial cells (Fig. 6B).


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Fig. 6.   Immunogold localization of EGF and EGFR within alveolar septum. Boxes (×10,000) in top show the area being magnified in bottom (×25,000). A: EGF was found within cytoplasmic granules of interstitial cells (arrowheads). These cells were frequently situated next to collagen bundles. Scattered cytoplasmic labeling was also found within epithelial cells. B: EGFR was found along the inner surface of lamellar body membrane (arrows) of type II cells, with scattered labeling along the cytoplasmic membrane of epithelial cells.


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Summary of results. We demonstrate for the first time the differential regional expression of growth factors in alveolar tissue and the response of EGF axis and SPs during postnatal and post-PNX lung growth in the dog, summarized in Fig. 7. During postnatal maturation, cell growth is more active in subpleural lung regions than in deeper regions. EGF and EGFR expression parallels that of cell proliferation, whether analyzed with respect to whole lung tissue or septal tissue selectively. In contrast, the further accelerated cell proliferation occurring in the first few weeks after PNX does not entail increased EGF or EGFR; in fact, both proteins were mildly suppressed. Whereas cell proliferation during maturation is inversely related to SP-A and proSP-C levels, early post-PNX proliferation is associated with greatly enhanced SP-A level but no change in proSP-C level. Modest changes in SP-D level correlated with proliferation, whereas proSP-B levels did not change during maturation or after PNX. These data show that 1) the EGF axis continues to regulate lung maturation after birth, 2) SPs continue to be differentially regulated after birth, and 3) post-PNX lung growth is not a simple recapitulation of maturational processes.


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Fig. 7.   Summary of PCNA, EGF axis, and SP expression during postnatal and post-PNX lung growth (3 wk). Arrow direction represents relative change in protein quantity compared with mature animals (postnatal growth) or age-matched sham animals (post-PNX growth). No. of arrows indicates magnitude of change observed: left-right-arrow, nonsignificant change; up-arrow , statistically significant change <50%; up-arrow up-arrow , 50-100%; up-arrow up-arrow up-arrow , 100-1,000%; up-arrow up-arrow up-arrow up-arrow , >1,000%.

EGF axis during maturation. Previous work clearly demonstrated the importance of EGF-EGFR signaling in embryonic lung development. The mRNA and protein of EGF and EGFR are detected in human fetal lung beginning at gestational week 10 (35). Both EGF and EGFR colocalize to embryonic distal airway epithelium, suggesting an autocrine regulatory loop (45). EGF stimulates the biosynthesis of surfactant precursor molecules (8, 12) and surfactant-associated proteins (47), induces branching morphogenesis in fetal organotypic lung cultures (25), and accelerates fetal lung maturation (40). Offspring of rats with EGF deficiency show reduced lung weights and morphological features of respiratory distress syndrome (32). EGFR knockout mice show impaired epithelial development of various organs, including lung, resulting in perinatal lethality (24).

The role of EGF in postnatal lung development is less well documented. Lung growth and maturation continue after birth in the dog. We now show that, during the first year, EGF and EGFR levels correlate to a proliferative index. From 3 to 12 mo of age, PCNA decreased dramatically by 24-fold, but EGF and EGFR levels fell only modestly (by 27 and 40%, respectively), suggesting that the EGF axis mediates functions other than proliferation. EGF signaling mediates alveolar type II cell differentiation (31, 51). Exogenous EGF administration accelerates cytodifferentiation of type II cells in fetal primates, indicated by increased SP-A concentration and number of lamellar bodies (31). The number of lamellar bodies is reduced after administration of EGF antiserum to fetal mice (51). Continuing EGF signaling is likely required for the maintenance of a differentiated type II phenotype, and EGF may mediate epithelial cell turnover throughout life. At the opposite end of the developmental spectrum, senescent cultures of lung fibroblasts become less responsive to the mitogenic effects of EGF (2) associated with a loss of the autophosphorylating activity of EGFR (4). It is not known whether in vivo EGF and EGFR expression might decline progressively with advancing age.

Regional expression of PCNA, EGF, and EGFR. Massaro and Massaro (22) reported a faster clearance rate of ingested silver nitrate particles in subpleural alveolar regions than in deeper regions, implying more active cellular growth in the peripheral than central lung. Their data are consistent with our observation of more intense EGF, EGFR, and PCNA immunolabeling in the subpleural region, suggesting that postnatal alveolar growth occurs by the addition of new gas exchange tissue from the periphery. We had expected equally minimal immunoactivity in all regions once the animal reached maturity. The persistent differential regional proliferative index and EGF and EGFR levels into adulthood indicate chronically elevated cell turnover in peripheral lung, even when maturation has completed.

Localization of EGF and EGFR. Aida et al. (1) localized EGF immunoreactivity to secretory granules and endoplasmic reticulum of serous acinar cells of adult human bronchial glands. EGFR immunoreactivity was detected at the cell membrane of the basal cells and the bronchial surface of nonciliated bronchial and bronchiolar cells (Clara cells), as well as type II alveolar epithelial cells. Their results suggest that EGF may be secreted into the lumen from bronchial glands and stimulate proliferation and/or differentiation of basal cells of the bronchus. EGF and EGFR colocalize to airway epithelium of normal fetal and postnatal human lung, as well as to scattered alveolar epithelial cells in postnatal human lung (39). EGF detected in lung interstitium may reside in macrophages (20). We observed that interstitial cells possessing EGF immunoreactivity were located in close proximity to collagen fibers, suggesting that some of these cells may in fact be fibroblasts.

The strong EGFR immunoreactivity that we observed along the inner membrane of lamellar bodies of type II alveolar epithelial cells is a novel finding that supports a direct role of the EGF axis in regulating SP metabolism. Newborn rats deficient of EGF show reduced immunostaining for SP-A (32). In human fetal lung explants, type II cell differentiation is associated with increased SP-A content, as well as EGFR immunostaining and mRNA levels (17), with EGFR immunoreactivity found along cell membranes of epithelial cells lining the prealveolar ducts. Antisense inhibition of EGFR directly reduces SP-A gene expression in cultured lung explants (18). Regulation of surfactant metabolism by EGFR within lamellar bodies may, in turn, affect the action of EGFR at the epithelial cell surface. Because EGF-EGFR binding leads to phosphorylation of EGFR and endocytosis of the ligand-receptor complex (38), EGF signaling requires continual replenishment of EGFR at the cell surface. The rate of exocytosis of lamellar bodies and their fusion with the epithelial membrane could be one mechanism that determines EGFR availability at the cell surface.

EGF axis after PNX. Our results clearly show that post-PNX lung growth does not mimic the developmental pattern of growth factor expression. The increased PCNA reactivity after PNX is associated with a twofold increase in morphometric indexes of cell growth reported by our laboratory (13, 42); the volume of interstitium increased disproportionately by more than threefold early after PNX and then subsided (13). Unexpectedly, active cell proliferation after PNX is associated with mildly reduced EGF and EGFR levels; one possible explanation is the disproportionate expansion of interstitial tissue volume, which may contain a lower concentration of EGF and EGFR than epithelial tissue. Hence, tissue EGF and EGFR levels may have been diluted when normalized with respect to total lung protein. We examined whether this dilution effect can account for our findings by normalizing EGF and EGFR signal intensities by the average relative volume change of septal epithelial cells measured by morphometry in a separate group of PNX animals, but the results did not alter our conclusions (data not shown). Thus EGF and EGFR synthesis were suppressed, or their degradation accelerated, in the remaining lung at 3 wk after PNX.

Our data do not preclude the possibility that exogenous EGF may facilitate post-PNX lung growth via interaction with other mediators. Systemic EGF administration modestly enhances lung size in normal minipigs (44) and increases lung weight and volume and EGFR levels in adult rats after PNX (16). However, response to PNX in the rat differs from that in larger mammals. Resection of even a small amount of lung (<45%) induces vigorous compensatory growth in the adult rat (33, 34) but not in the adult dog. An important difference is that rat epiphyses do not close, and their lung weight and volume continue to increase in proportion to somatic growth throughout life (7). The continually growing adult rat lung is much more susceptible to further growth stimulation than larger adult mammals whose rib cage has attained a maximum size and effectively imposes an upper limit to lung growth.

Surfactant-associated proteins during maturation. In the rat, SP-A production increases during late gestation, falls off slightly after birth, and then increases again to maximum levels in the adult (37). The twofold higher SP-A level in the adult rat corresponds to a twofold increase in the number of type II alveolar epithelial cells during maturation. However, SP-A content increased 4.5-fold during maturation in the dog lung, far in excess of the twofold increase in septal tissue volume, indicating either a greater capacity for SP-A production by differentiated type II cells in dogs than in rats or increased production of SP-A in dog lungs by other cell types such as Clara cells. SP-D, like SP-A, is a member of the collectin family of proteins (29) that mediate surfactant homeostasis (10), as well as native lung immunity (6). SP-D mRNA and protein appear during late gestation in the rat and reach adult levels in early postnatal life (5, 28). In the dog, SP-D levels are high at 3 mo of age and decline with maturation. Unlike the collectins, we found little change in proSP-B content during lung development, which is consistent with other work in rats showing that adult SP-B levels are achieved by gestation day 20 (30, 37). ProSP-C levels, however, were markedly enhanced in mature dog lungs, in contrast to the rat, which achieves peak levels of SP-C during gestation (37). Therefore, postnatal SP expression is differentially regulated in a species-specific manner.

Surfactant-associated proteins after PNX. Rat hypertrophic type II cells isolated after PNX show similar intracellular contents of phosphatidylcholine as normotrophic type II cells obtained from control rats (43). In addition, tritiated choline incorporation does not differ between cells isolated from either source, suggesting that PNX does not stimulate surfactant phospholipid production. The disproportionate increase in SP-A and mild increase in SP-D early after PNX probably mediates functions other than lowering surface tension. Transgenic mice lacking a functional SP-A gene demonstrate no obvious abnormalities in respiratory function or surfactant metabolism but show impaired bacterial clearance and more severe lung inflammation (19, 21). In contrast, targeted disruption of the SP-D gene results in marked pulmonary lipoidosis that is corrected by lung-specific SP-D expression (10), suggesting an important role of SP-D in surfactant homeostasis. Elevated SP-A and SP-D levels following rat hyperoxic lung injury (27, 46) also implicate these proteins in lung protection or repair, perhaps via modulation of cytokine and free-radical production (48), as SP-A can inhibit activation of lung macrophages in a mouse model of idiopathic pneumonia syndrome (50). Although the remaining lung after PNX is not "inflamed" in the classical sense, there is an early influx of leukocytes and circulatory proteins associated with abundant macrophages and increased insulin-like growth factor levels in the bronchoalveolar lavage fluid (23). Recruitment of cytokine-laden cells to the remaining lung could be a crucial process underlying the post-PNX mitogenic activity.

Our finding of a greatly enhanced SP-A and a modestly enhanced SP-D level after PNX is consistent with strain-triggered differential regulation of SPs. Mechanical lung strain has been shown to induce surfactant secretion and type II cell differentiation, as well as apoptosis (9). In cultured lung epithelial cells, cyclic mechanical stretch increases SP-A and SP-B mRNA levels (36). In organotypic culture of fetal rat lung, mechanical strain increases SP-C mRNA but has no effect on SP-A mRNA (26). The reason for the different in vitro results is unexplained; their in vivo significance is also unclear. It is possible that the greater mechanical strain on the remaining lung following PNX accelerates type II cell differentiation. Alternatively, upregulation of SP-A and SP-D may be necessary for the enhancement of lung growth after PNX. These possibilities need to be examined in future studies.

We conclude that, during postnatal lung maturation, regional septal cell proliferation, EGF axis activation, and surfactant-associated protein expression remain differentially regulated. Post-PNX lung growth is associated with a different pattern of SP expression, and the EGF axis is not preferentially activated. Thus post-PNX lung growth does not simply represent an exaggeration or reactivation of normal maturational responses. During somatic maturation, the superimposition of large, sustained mechanical lung strain by PNX accelerates growth of the remaining lung via different biochemical mediators and/or interactions than during maturation alone. In particular, selective enhancement of SP-A and SP-D production or release after PNX may play an important role in the modulation of compensatory lung growth.


    ACKNOWLEDGEMENTS

We thank Heather L. Stanley, Richard T. Hogg, and Debbie C. Tuttle for expert animal care, as well as technical assistance with animal procedures, and the staff of the Animal Resources Center for veterinary assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants RO1-HL-40070, HL-54060, HL-45716, and HL-62873; National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-48482 and DK-54396; as well as the Department of Veteran Affairs Research Service.

Address for reprint requests and other correspondence: C. C. W. Hsia, Dept. of Internal Medicine, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9034.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

July 12, 2002;10.1152/ajplung.00053.2002

Received 6 February 2002; accepted in final form 27 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 283(5):L981-L990