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Published ahead of print on February 5, 2003, doi:10.1164/rccm.200112-132OC
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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 1264-1270, (2003)
© 2003 American Thoracic Society

Surfactant Homeostasis Is Maintained In Vivo during Keratinocyte Growth Factor–induced Rat Lung Type II Cell Hyperplasia

Antonia Fehrenbach, Christoph Bube, Jens M. Hohlfeld, Paul Stevens, Thomas Tschernig, Heinz G. Hoymann, Norbert Krug and Heinz Fehrenbach

Division of Electron Microscopy, Center of Anatomy, University of Göttingen, Göttingen; Fraunhofer Institute for Toxicology and Experimental Medicine and Departments of Respiratory Medicine and Functional and Applied Anatomy, Medical School of Hannover, Hannover; Clinic of Neonatology, University Children's Hospital Charité, Humboldt-University, Berlin; and Clinical Research Group "Chronic Airway Diseases", Department of Internal Medicine (Respiratory Medicine), Philipps-University, Marburg, Germany

Correspondence and requests for reprints should be addressed to H. Fehrenbach, Ph.D., Department of Internal Medicine (Respiratory Medicine), Philipps-University, Baldingerstrasse, D-35043 Marburg, Germany. E-mail: heinz.fehrenbach{at}mailer.uni-marburg.de


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Keratinocyte growth factor (KGF) induces transient proliferation of alveolar type II cells (AEII) associated with surfactant alterations. To test the hypothesis that homeostasis of intracellular phospholipid stores is maintained under KGF-induced hyperplasia, we (1) collected tissue from adult rat lungs, fixed for light and electron microscopy 3 days after intratracheal instillation of 5 mg recombinant human (rHu) KGF/kg body weight or phosphate-buffered saline (PBS), and from untreated control animals (five animals/group) for design-based stereology of AEII and lamellar body (LB) ultrastructure; and (2) we analyzed uptake and distribution of instilled radiolabeled phospholipids. After rHuKGF, AEII-coverage of alveolar walls (PBS:8.3 ± 3.0%; rHuKGF:30.6 ± 4.8%) and number of AEII/ml lung volume (PBS:28.5 ± 6.5 x 106; rHuKGF:48.2 ± 5.8 x 106) were increased (p < 0.008). Number (PBS:97 ± 25; rHuKGF:54 ± 7) and volume (PBS:45.3 ± 13.8 µm3; rHuKGF:21.0 ± 4.7 µm3) of LBs per cell were decreased (p < 0.008), but not total amount/ml lung volume (PBS:128 ± 46. 4 x 107 µm3; rHuKGF:103 ± 34. 7 x 107 µm3). This was paralleled by a shift to larger LBs. After rHuKGF, radiolabeled phospholipids accumulated in whole lung tissue relative to lavage fluid (p < 0.01). However, less radiolabel was incorporated per cell (p < 0.01). We conclude that under rHuKGF-induced AEII proliferation intracellular surfactant was decreased per single cell, whereas a constant amount was maintained per unit lung volume. We suggest that surfactant homeostasis is regulated at the level of phospholipid transport processes, for example, secretion and reuptake.

Key Words: surfactant • growth factors • proliferation • homeostasis

Keratinocyte growth factor (KGF), a heparin-binding member of the fibroblast growth factor family (1), promotes proliferation of rat alveolar epithelial Type II cells in vitro (2, 3) and in vivo (4, 5). Alveolar type II cells (AEII) are the source of pulmonary surfactant, which regulates alveolar surface tension, fluid balance, and host defense (as reviewed in References [6–8]).

AEII proliferation is required for re-epithelization after acute lung injury (9). Impairment of alveolar surfactant function (10) and changes in the profiles of the fatty acids of different surfactant phospholipid classes (11) have been documented in the acute respiratory distress syndrome. However, the therapeutic potential of KGF in acute lung disease is yet to be fully explored. Recently, increased levels of KGF in the airways of premature infants were found to be associated with an absence of bronchopulmonary dysplasia suggesting that KGF may reduce the risk of bronchopulmonary dysplasia by preventing epithelial injury and/or enhancing cell repair (12). The protective effect of exogenous KGF was first reported in an animal model of acute lung injury by Panos and coworkers (13) and has since been shown to be protective in a variety of other lung injury models (as reviewed in Reference 9).

KGF stimulates surfactant phospholipid synthesis but does not induce proliferation in Type II cells of the premature rabbit in vivo (14) or in fetal rat lung Type II cells in vitro (15). Application of recombinant human (rHu) KGF in vitro was reported to increase gene expression of surfactant proteins (15–17). Enhancement of surfactant protein gene expression in vivo and of the amount of surfactant protein A and D in the lavage fluid of lungs from adult rats has recently been reported by Yano and coworkers (18). A striking result of their study was that, although the amount of total lung surfactant protein messenger RNA was increased after rHuKGF treatment, the quantity of specific messenger RNA of the four surfactant proteins per individual Type II cell was reduced. Thus, the in vivo effects of KGF on the surfactant metabolism of Type II cells appear to depend on the stage of lung maturity and may therefore lead to different outcomes in fetal or adult animal models.

So far, nothing is known about the effects of KGF on the amount of intracellular surfactant phospholipids in lungs of adult animals. We, therefore, sought to determine the effects of intratracheal instillation of rHuKGF on the intracellular surfactant phospholipid stores in normal adult rat lungs using light and electron microscopy.

Lamellar bodies (LBs) represent the intracellular storage form of surfactant (19). Because phospholipid composition was shown to correlate with structural parameters, ultrastructural methods can also be used to quantitatively determine intracellular surfactant (20). Fixation and visualization of pulmonary surfactant at its sites of synthesis, storage, secretion, and action are based on established methods in morphology, which allow for the structural analysis as well as the quantification of intracellular and intra-alveolar surfactant in the organ (20–23). The amount and size of LBs were quantified per single Type II cell using design-based stereology (24).

Lung ventilation was shown to influence the release of surfactant from AEII (as reviewed in Reference 25). Ventilation of lung parenchyma might be restricted by the marked alterations in parenchymal architecture of rHuKGF-treated lungs, which are characterized by micropapillary and cuboidal epithelial cell hyperplasia (4, 5). To exclude ventilatory effects on surfactant release, alveolar micromechanics were evaluated indirectly by estimating the size of alveoli and alveolar ducts using the established stereological method of point-sampled intercepts (26, 27). In a separate set of experiments, the effect of rHuKGF treatment on surfactant recycling was investigated after intratracheal instillation of radioactively labeled surfactant phospholipids. Its pattern of distribution in the lungs was analyzed as described previously (28, 29).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Design
Male Brown Norway rats (Charles River, Germany; mean body weight 212 ± 6.7SEM g) received a single intratracheal bolus of rHuKGF or vehicle. Animals were killed on Day 3 after treatment. To determine the relative coverage of the alveolar surface with AEII, immunohistochemistry for surfactant protein D was performed on paraffin sections of 10 lungs (5 + rHuKGF, 5 + phosphate-buffered saline [PBS]), which had been fixed by instillation of paraformaldehyde. Another 15 lungs (5 + rHuKGF, 5 + PBS, 5 uninstilled), fixed by vascular perfusion with a mixture of paraformaldehyde and glutardialdehyde, were embedded in a methacrylate resin and served to study alterations in parenchymal architecture. The number of AEII as well as the content of LBs was estimated using light and electron microscopy. In addition, effects of rHuKGF on surfactant recycling were studied on adult rats at Day 3 after treatment with rHuKGF (n = 20) or PBS (n = 17) at different time points (0, 1, and 4 hours) after instillation of radioactively labeled surfactant phospholipids prepared from whole surfactant of pooled rat lung lavages. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85–23, revised 1985). The experiments have been approved by the regional government.

Intratracheal Instillation of rHuKGF
After intubation of the trachea via the oral cavity, 5 mg/kg body weight rHuKGF or vehicle (PBS) (both at a volume of 1.25 ml/kg body weight) were instilled under short halothane anesthesia. rHuKGF was prepared, characterized, and tested at Amgen Inc. (Thousand Oaks, CA) (30).

Fixation, Tissue Sampling, and Processing
Surgery, fixation by vascular perfusion, and tissue sampling as well as tissue processing were described previously (22, 31) (for details, see online supplement).

Stereological Analysis
The sections were analyzed by established stereological methods (32) using light and electron microscopy. Intersection counting was used to determine the relative alveolar epithelial surface coverage with AEII and the apical (secretory) fraction of the AEII cell surface. Distension of alveoli and alveolar ducts, number and volume of AEII as well as number, size, and volume of LBs were quantified using established design-based stereological means (33, 34) (see also online supplement). Light microscopical analysis was performed on a computer-based system (Cast-Grid 2.00; Olympus, Denmark).

Surfactant Preparation and Instillation
Whole surfactant was prepared from pooled lung lavages from adult rats by sodium bromide density gradient according to Hawgood and coworkers (35). Surfactant labeling and instillation as well as tissue preparation and measurement of radioactivity, protein and lipid content were performed according to standard protocols as described previously (36, 37) (for details, see online supplement).

Statistical Analysis
Differences between experimental groups were tested for significance (p < 0.05) with parametric one-way analysis of variance followed by Tukey test or by Kruskal–Wallis one-way analysis of variance on ranks followed by Dunn's multiple comparisons procedure. If only two groups were to be tested, the nonparametric Mann–Whitney rank sum test was used. Differences in the distribution of radioactively labeled phospholipids were tested for significance (p < 0.05) by two-way analysis of variance because the experimental groups differed with respect to treatment (rHuKGF, PBS) and incubation time (0, 1, and 4 hours), which results in a 2 x 3 matrix. Mean values are given ± SEM. Analyses and graphic presentations were performed using the SigmaStat 2.0 and Sigmaplot 3.0 software programs (Jandel Scientific, Erkrath, Germany).


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Proliferation/Hypertrophy
On Day 3 after rHuKGF instillation, the lungs exhibited marked changes in a number of histological and ultrastructural parameters (Table 1) . The fraction of alveolar epithelial surface covered by surfactant protein D immunoreactive AEII was significantly increased (30.6 ± 4.8%) compared with lungs treated with PBS alone (8.3 ± 3.0%, p < 0.008). At this time point, the total number of AEII/ml lung volume had nearly doubled (PBS: 28.5 ± 6.5 x 106; rHuKGF: 48.2 ± 5.8 x 106) and the cell volume of AEII was slightly increased in rHuKGF-treated lungs (PBS: 328.8 ± 52.7 µm3; rHuKGF: 370.4 ± 30.0 µm3). Groups of AEII either appeared as cuboidal monolayer or as micropapillae (Figures 1, 2B, and 2C) . The apical (secretory) surface of AEII was reduced (PBS: 34.6 ± 3.9%; rHuKGF: 27.3 ± 5.7%; p < 0.05) (Table 1).


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  		TABLE 1. Effect of recombinant human keratinocyte growth factor on characteristics of alveolar type II cells

 


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  		Figure 1. Immunohistochemistry for surfactant protein D (SP-D). (A) Phosphate-buffered saline (PBS)–instilled lung on Day 3 with single Type II cells in the alveolar corners (arrowhead). (B) marked proliferation of alveolar type II cells (AEII) on Day 3 after treatment with recombinant human keratinocyte growth factor (rHuKGF). Changes exhibit the characteristic hyperplasia in the form of micropapillae of epithelial cells (arrow) or monolayers of cuboidal epithelial cells lining the alveolar septa (double arrow).

 



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  		Figure 2. Electron micrographs showing AEII on Day 3 after PBS (A) and rHuKGF (B and C). (A) Single cell with numerous lamellar bodies (Lb). (B) Micropapillary proliferation with few but larger lamellar bodies. (C) Cuboidal monolayer lining the alveolar wall. Al = alveolus; Ca = capillary; Lb = lamellar body; N = nucleus; Nu = nucleolus.

 
Intracellular Surfactant
Three days after rHuKGF treatment, the amount of intracellular surfactant phospholipids per individual cell was significantly (p < 0.008) reduced (Table 2 and Figure 2). The number (PBS: 97 ± 25; rHuKGF: 54 ± 7) and total volume (PBS: 45.3 ± 13.8 µm3; rHuKGF: 21.0 ± 4.7 µm3) of LBs per single AEII, as well as the relative volume density of LBs referred to the AEII volume (PBS: 13.7 ± 2.3%; rHuKGF: 5.7 ± 1.2%) were decreased to approximately 50% of the control values. However, the LBs in rHuKGF-treated lungs appeared to be larger as compared with the PBS-treated control animals (Figure 2). This was supported quantitatively by an increase in the volume-weighted mean volume (V) of LBs after rHuKGF-induced AEII hyperplasia (PBS: 0.9 ± 0.6 µm3; rHuKGF: 1.5 ± 0.5 µm3) as determined by the method of point-sampled intercepts (Table 2 and Figure 3) . Despite this marked decrease in surfactant phospholipids per cell, the total amount of LBs per unit lung volume remained unchanged (PBS: 128 ± 46.4 x 107 µm3; rHuKGF:103 ± 34.7 x 107 µm3) as a consequence of the increased number of AEII (Table 2).


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  		TABLE 2. Effect of recombinant human keratinocyte growth factor on characteristics of lamellar bodies

 


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  		Figure 3. In vivo size distribution of lamellar bodies on Day 3 after PBS and rHuKGF as determined by the method of point-sampled intercepts (PSI). Sizes increase with increasing number of class.

 
Parenchymal Architecture
Neither rHuKGF nor PBS instillation resulted in alterations in alveolar and alveolar duct size on Day 3. Estimation of the volume-weighted mean volumes (V) of alveoli (rHuKGF: 4.2 ± 0.6 x 105 µm3; PBS: 4.1 ± 0. 5 x 105 µm3) and alveolar ducts (rHuKGF: 2.1 ± 0.6 x 107 µm3; PBS: 2.6 ± 0.5 x 107 µm3) revealed size characteristics which were comparable to those of uninstilled control lungs fixed in situ (alveoli: 3.7 ± 0.6 x 105 µm3; alveolar ducts: 2.0 ± 0.06 x 107 µm3) (Figures 4–6) . The values (mean ± SEM) obtained are in the range of the dimensions of alveoli (2.0 ± 0.13 x 105 µm3) and of alveolar ducts (1.5 ± 0.6 x 107 µm3) as measured by serial section analysis (38).



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  		Figure 4. Methacrylate sections (1 µm) of lung parenchyma showing alveoli (Al) and alveolar ducts (Du) in an untreated control lung (A) and 3 days after treatment with either rHuKGF (B) or diluent (PBS) (C). Without rHuKGF, Type II pneumocytes appeared as single cells whereas, after rHuKGF treatment, the alveolar wall exhibited numerous groups of proliferated Type II cells (arrows).

 


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  		Figure 6. Volume-weighted mean volumes (V) and size distribution of alveolar ducts in lungs of the different experimental groups were determined by the method of PSI. Differences among the tested groups are not significant (p > 0.2).

 


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  		Figure 5. Volume-weighted mean volumes (V) and size distribution of alveoli in lungs of the different experimental groups were determined by the method of PSI. Differences among the tested groups are not significant (p > 0.2).

 
Surfactant Recycling
Specific lung weight as well as protein and phospholipid amounts of the tissue homogenate were increased after rHuKGF treatment (p < 0.001), which is indicative of Type II cell hyperplasia in these lungs, too (see online supplement). Bronchoalveolar lavage volume was similar in both groups at all three time points (Table 3) . Loss of instilled counts from total lung increased significantly with time (p < 0.01) but was not affected by rHuKGF treatment (9 vs. 10% loss of label/hour in PBS vs. rHuKGF). However, there was a relative accumulation of radiolabel from intratracheally instilled phospholipids in the tissue homogenate at the expense of label in the bronchoalveolar lavage fluid of rHuKGF compared with PBS-treated lungs. Notably, less radiolabel was incorporated per microgram tissue phospholipid or protein in rHuKGF than in PBS-treated lungs (p < 0.001), i.e., the uptake of phospholipid per cell was significantly decreased.


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  		TABLE 3. Effect of recombinant human keratinocyte growth factor on the distribution of intratracheally instilled [3h]-dppc

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The purpose of this study was to investigate the effect of Type II cell proliferation on the intracellular surfactant phospholipid stores in vivo. We used a model of rHuKGF-induced transient Type II cell hyperplasia in the rat, which has been described previously (4, 5). Our hypothesis was that homeostasis of the intracellular surfactant phospholipid pool was maintained under rHuKGF-induced AEII hyperplasia in the adult lung. As proposed, we found a marked decrease in surfactant phospholipids per cell, whereas the total amount of LBs per unit lung volume did not change as a consequence of the increased number of AEII due to rHuKGF treatment. In addition, there were fewer but larger LBs per cell of which the apical membrane surface was reduced after rHuKGF treatment. This was accompanied by a relative accumulation of instilled radiolabeled phospholipid in the tissue homogenate at the expense of label in the lavage fluid. Again, on a per cell basis, the amount of radiolabel incorporated by lung tissue was reduced. This indicates that surfactant reuptake by the individual Type II cell was decreased during AEII hyperplasia.

Using a tetrazolium salt–based proliferation assay and a cell counting method by standard hemocytometer, Abatai and coworkers (39) found that on Day 2 after rHuKGF instillation, the number of Type II cells had nearly doubled, which is in accordance with the increase in Type II cell numbers we estimated on Day 3 after rHuKGF using stereology. Young and coworkers (20) demonstrated a correlation between biochemical and morphometric parameters in the quantification of intracellular surfactant, i.e., of LBs, so that we can assume that the amount of LBs corresponded to the biochemical surfactant phospholipid pool in the cells. Recently, Ochs and coworkers (24) have developed a novel approach, which combines established stereological methods, to determine the number and volume of LBs per cell more precisely. On the basis of this method, our results (97 ± 11.2SEM LBs per cell in the control animals) corroborate with the data obtained by three-dimensional reconstruction on serial sections of AEII (95 ± 22SD per cell) (40). In contrast to serial section analysis, with which only very low numbers of cells can be studied (40), our method is highly efficient because it allows for evaluation of many cells from many tissue samples in a much shorter period of time. By isotropic uniform random sampling, tissue samples were collected with a uniform probability in three-dimensional space (27). This also takes into account that the normal pattern of surfactant production/secretion as well as effects of rHuKGF treatment may be heterogeneously distributed in the lungs. The technique we used is independent of assumptions on size and shape of the LBs, and our counts were below the numbers (150 ± 30SD per cell) estimated by an earlier assumption-based morphometrical approach assuming LBs to be spheres (20). Otherwise, the total number of LBs per cell has been extremely underestimated (approximately 12 per cell) due to inadequate morphometry (41, 42).

Several mechanisms may be involved in the changes of the intracellular surfactant phospholipid stores after rHuKGF treatment: (1) segregation of LBs among the dividing cells, (2) upregulation of surfactant secretion, (3) downregulation of surfactant reuptake, and (4) downregulation of surfactant phospholipid biosynthesis.

When differentiated cells divide, their specific functions are assumed to be temporarily suspended and a cell-cycle–dependent loss of LBs, specifically in injury-induced mitosis after injection of butulated hydroxytoluene, has been suggested (42). In vitro studies in a mouse lung epithelial cell line, that lacks lamellar body secretion, indicated that phosphatidylcholine synthesis peaked during cell cycle arrest (G0/G1), declined during transition to G1/S and remained low during S and G2/M (43). These alterations were primarily a consequence of change in activity, phosphorylation, and membrane affinity of cytidine triphosphate:phosphocholine cytidylyltransferase, a key enzyme of phosphatidylcholine synthesis (43). On the contrary, very recent data indicate that KGF may increase gene expression as well as messenger RNA levels of certain lipogenic enzymes in isolated rat AEII and stimulate incorporation of acetate into phospholipids (44, 45). Thus, even if we assume that rHuKGF-induced proliferation resulted in a mere mitosis-related segregation of LBs, rHuKGF may be able to counteract these mechanisms at the metabolic level. However, the homeostatic mechanisms involved may be indirect effects of rHuKGF and are likely to be complex. Yano and coworkers (18) previously showed that the bromodeoxyuridine-labeling index of Type II cells peaked on Day 2 after rHuKGF instillation. Therefore, although in our study mitotic figures were very rare and nuclei contained heterochromatin as well as condensated single nucleoli, we cannot unequivocally exclude that the reduced number of LBs we found in our study might have been, in fact, due to a homogeneous segregation of LBs among the daughter cells during mitosis. However, if the reduced number of LBs were a mere effect of segregation, the histograms showing LB size distributions should have been equivalent in both groups. In contrast, we observed a shift to larger LBs in the rHuKGF-treated lungs.

The increase in lamellar body size but not number is an indicator for reduced surfactant secretion. In line with this, a relative accumulation of radioactively labeled phospholipids in the tissue at the expense of label in the lavage fluid was observed. Surfactant is released into the alveolar space after fusion of the secretory granule membrane with the cell surface (46). These cell surface patches were shown to be retrieved back to existing LBs by a pathway different from that of bulk membrane, which was proposed to be one pathway for surfactant endocytosis (47). In the present study, the apical (secretory) surface of Type II cells from rHuKGF-treated lungs was smaller than in the control animals, which may be related to reduced incorporation of lamellar body membrane into the apical cell surface due to reduced surfactant secretion, and which in turn may also affect surfactant reuptake (see below). Notably, the release of surfactant from Type II cells does not seem to be mechanically impeded because epithelial cell proliferation does not show any effect on parenchymal distension as there were no changes in the size of alveoli and alveolar ducts under our highly standardized settings for lung fixation by vascular perfusion.

The reduced amount of radiolabeled phospholipid per cell in rHuKGF compared with PBS-treated lungs indicates that surfactant reuptake by the individual Type II cell was decreased as well. Assuming that surfactant reuptake did not depend on the number of cells present, the amount of radiolabel incorporated per cell should have been equivalent in rHuKGF- and PBS-treated lungs. A combined decrease in secretion and reuptake of surfactant is in agreement with previous studies showing that exocytosis and endocytosis of surfactant by Type II cells were strongly coupled processes (47, 48).

Our findings demonstrate that the total intracellular surfactant phospholipid pool remains unchanged in rHuKGF-treated compared with control lungs, whereas the surfactant phospholipid store of the individual Type II cell is reduced. We suggest that this is achieved by (direct or indirect) mechanisms, which downregulate surfactant secretion and reuptake in relation to the number of Type II cells. The maintenance of pulmonary surfactant homeostasis under rHuKGF-induced Type II cell proliferation is probably an integrative result of the well-balanced regulation of exocytosis, transport, and endocytosis.


   Acknowledgments
 
The authors gratefully acknowledge the expert technical assistance of M. Fathollahy and R. Korolewitz (both Hannover, Germany), A. Apel, S. Freese, A. Gerken, and H. Hühn (all Göttingen, Germany), M. Braun and H. Kemmer (Berlin, Germany). They also thank I. Peterson (TU Dresden, Germany) for performing the immunohistochemistry, M. Glauche (TU Dresden, Germany) for artwork, and Prof. Dr. J. Richter and Dr. M. Ochs (both Göttingen, Germany) for kindly supporting the stereological evaluation. The authors also thank Cyrilla Maelicke (Göttingen, Germany) for checking the English version of the manuscript. rHuKGF was a generous gift from Dr. Th. Ulich (Amgen Inc.).


   FOOTNOTES
 
This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Received in original form December 16, 2001; accepted in final form January 15, 2003


   REFERENCES
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 ABSTRACT
 METHODS
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 DISCUSSION
 REFERENCES
 

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