1 Department of Pathology, Yale University School of Medicine, New Haven 06520; 2 Pathology and Laboratory Medicine Service, Veterans Affairs Connecticut Healthcare System, West Haven 06516; 3 Section of Pulmonary and Critical Care Medicine, Department of Internal Medicine and 5 Division of Perinatal Medicine, Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510; and 4 Section of Pulmonary and Critical Care Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
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ABSTRACT |
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Interleukin (IL)-13, a key mediator of Th2-mediated immunity, contributes to the pathogenesis of asthma and other pulmonary diseases via its ability to generate fibrosis, mucus metaplasia, eosinophilic inflammation, and airway hyperresponsiveness. In these studies, we compared surfactant accumulation in wild-type mice and mice in which IL-13 was overexpressed in the lung. When compared with littermate controls, transgenic animals showed alveolar type II cell hypertrophy under light and electron microscopy. Over time, their alveoli also filled with surfactant in a pulmonary alveolar proteinosis pattern. At the same time, prominent interstitial fibrosis occurs. Bronchoalveolar lavage fluid from these mice had a three- to sixfold increase in surfactant phospholipids. Surfactant proteins (SP)-A, -B, and -C showed two- to threefold increases, whereas SP-D increased 70-fold. These results indicate that IL-13 is a potent stimulator of surfactant phospholipid and surfactant accumulation in the lung. IL-13 may therefore play a central role in the broad range of chronic pulmonary conditions in which fibrosis, type II cell hypertrophy, and surfactant accumulation occur.
asthma; type 2 pneumocytes; pulmonary fibrosis; interleukin-13
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INTRODUCTION |
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INTERLEUKIN (IL)-13 is a pleiotropic 12-kDa protein that is produced in large quantities by appropriately stimulated CD4+ Th2 cells (18, 22). It has a variety of effects that are relevant to asthma and other Th2-dominated inflammatory disorders, including the ability to induce IgE production, CD23 expression, and endothelial cell vascular cell adhesion molecule-1 expression and activate and inhibit the apoptosis of eosinophils. The exaggerated production of IL-13 is well documented in atopic and nonatopic asthma, atopic dermatitis, allergic rhinitis, and chronic sinusitis. IL-13 also plays an important role in the pathogenesis of type II granulomatous responses and has been implicated in protease-mediated destructive emphysema (Refs. 48 and 50 and references within). The in vivo effector functions of IL-13 in these settings, however, are poorly understood.
IL-13 and IL-4 are closely related cytokines with overlapping effector
profiles due in part to the shared use of essential receptor
components. Despite this close relationship, there are a number of
important functional differences between IL-4 and IL-13. They include
differences in the ability of these cytokines to drive the
differentiation of naive cord blood T cells to a Th2 phenotype, support
the in vitro proliferation of activated human or mouse T cells,
regulate prostaglandin biosynthesis, induce interferon- production,
contribute to nematode-irradicating tissue inflammation, regulate
epithelial electrolyte secretion, prolong eosinophil survival, and
stimulate eosinophil chemotaxis. In addition, IL-13 and IL-4 can be
produced by different cells and are differentially regulated by
mediators such as interferon-
(see Refs. 48 and 50 and references therein). Thus a complete understanding
of the relative roles of these cytokines mandates a comprehensive comparison of their biological activities.
Surfactant is a mixture of phospholipids and proteins produced principally by type II alveolar epithelial cells. Its major role has traditionally been considered the maintenance of alveolar and bronchiolar patency through reduction of surface tension. More recently, the importance of certain surfactant proteins (SPs) in host defense has come to be appreciated (7, 29). SP-A and -D have been shown to participate in non-antigen-specific host defenses against microorganisms, including viruses, bacteria, and fungi and a variety of inflammatory and immune modulating activities. In keeping with the importance of the effector functions of surfactant, its production is exquisitely regulated in the developing and adult lung. Interestingly, IL-4 has been shown to have strong regulatory effects on surfactant in vivo (25). The effects of IL-13 on surfactant homeostasis have not been evaluated.
To characterize the in vivo effector functions of IL-13, we used the Clara cell 10-kDa protein promoter to overexpress IL-13 in the airway. As previously reported, these mice show eosinophil-, lymphocyte-, and macrophage-rich inflammation, mucus metaplasia, protease-dependent acquired emphysema, increased fibrosis, and airway hyperresponsiveness to inhaled methacholine (48, 50). To further understand the effects of IL-13, studies were undertaken to determine if IL-13 also regulated surfactant in lungs from these animals. These studies demonstrate that IL-13 is a potent in vivo stimulator of type II cell hypertrophy and surfactant lipid and protein accumulation.
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METHODS |
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Animals. Transgenic mice were produced as previously described using the murine IL-13 gene under the control of the CC10 promoter (50). These mice were originally made on a (C57BL/6xCBA) F2 background and have been maintained by backcrossing since 1998 onto C57BL/6. The animals used in this study were typically 3-4 mo of age.
Histology and immunohistochemistry. Lungs were inflated to a pressure of 25 cm water through the trachea with Streck's tissue fixative (Streck Laboratories, Omaha, NE). Lungs were then dehydrated via graded alcohols and embedded in paraffin. Coronal sections (5 µm) that included both lungs were cut, deparaffinized, quenched with 2% hydrogen peroxide in 30% methanol, washed with water, and blocked with normal goat serum. Slides were subsequently immunostained using rabbit antisera to SP-B, SP-D, tenascin (all from Chemicon, Temecula, CA), pro-SP-B, pro-SP-C (gift of J. Whitsett, Cincinatti, OH; see Ref. 49), or CC10 (gift of William Philbrick, Yale University, New Haven, CT; see Ref. 3). The SP-B antibody was raised against purified human SP-B and was characterized by the manufacturer as not reacting with other components of human lavage fluid, liver homogenate, or serum. Antibodies were applied at 1:100 (SP-B and SP-D) or 1:1,000 (pro-SP-B and pro-SP-C) and were incubated overnight at 4°. Slides were then washed in PBS-0.5% Tween 20, incubated with biotinylated goat anti-rabbit antiserum (Kirkegaard and Perry, Gaithersburg, MD) for 30 min, washed, incubated with streptavidin-horseradish peroxidase (Zymed, San Francisco, CA) for 15 min, washed, incubated with liquid diaminobenzidine (Vector Laboratories, Burlingame, CA), and then counterstained with hematoxylin. Frozen sections were used for SP-D immunostaining by inflating lungs with a 1:1 ratio of PBS/optimum-cutting temperature compound. Frozen sections were cut, air-dried, and fixed in cold acetone. Tissue was blocked with normal goat serum, and antibody was applied for 1 h and developed with biotinylated goat anti-rabbit followed by alkaline phosphatase-streptavidin (Vector) and Vector Red substrate (Vector). Negative controls consisted of normal rabbit Ig used at a concentration similar to that of the other primary antisera (Zymed). No staining was ever observed with the negative control.
Electron microscopy. Lungs were fixed in 3% gluteraldehyde, postfixed in osmium tetroxide, and embedded in Epox 812. Tissue was then cut onto grids, stained with uranyl acetate and lead nitrate, and examined using a Philips 300 microscope.
RNA analysis. mRNA levels were assessed using RT-PCR as described previously by our laboratories (12). Briefly, total cellular RNA from lungs of IL-13 mice (2-3 mo old) were obtained using Trizol reagent (GIBCO-BRL, Grand Island, NY) as per the manufacturer's instructions. RNA samples were reverse transcribed, and gene-specific primers were used to amplify selected regions of each target. Primers were used as follows: SP-B sense, GAC CTG TGC CAA GAG TGT GA; SP-B antisense, GGC ATA GCC TGT TCA CTG GT; SP-C sense, GCA AAG AGG TCC TGA TGG AG; SP-C antisense, GCC CGT AGG AGA GAC ACC TT; SP-D sense, CTC TCG CAG AGA TCA GTA CC; SP-D antisense, GGA AAG CAG CCT TGT TGT GG. All analyses were done using 35 cycles with an annealing temperature of 60°C. Bands were seen at the expected sizes of 746, 794, and 853 bp for SP-A, SP-B, and SP-C, respectively.
Phospholipid analysis. The mice were killed with an intraperitoneal injection of pentobarbital sodium, and bronchoalveolar lavage (BAL) was performed using four 1-ml aliquots of ice-cold 0.9% saline. Each aliquot was flushed in and withdrawn three times. Lung lavage fluid was centrifuged at 200 g for 10 min to remove any cellular material, and lipids were extracted with a mixture of chloroform and methanol, as described previously (38). The phospholipids were fractionated by thin-layer chromatography (14) and quantified by phosphorus assay (1), as described previously (38). The lavaged lungs were lyophilized, and dry weights were obtained.
Analysis of SPs. Rabbit anti-sheep polyclonal antibodies that have been characterized against murine SP-A (45), SP-B (5), and SP-D (45) were obtained from Dr. Samuel Hawgood (University of California, San Francisco). A polyclonal antibody against mature SP-C was obtained from Byk Gulden (Konstanz, Germany). It was raised in rabbits against human recombinant mature SP-C in which the cysteines in positions 4 and 5 are replaced by phenylalanine and the methionine in position 32 is replaced by isoleucine (17, 39). It has been successfully used to measure SP-C in rabbit alveolar lavage (39) and rat type II cell secretions (15). For analysis of SP-A and SP-D, the 200-g lung lavage supernatant was concentrated 10-fold using Vivaspin concentrator tubes with a 10,000 molecular weight cut off (Vivascience, Lincoln, UK), suspended in Laemmli sample buffer (28), boiled for 5 min, and subjected to electrophoresis under reducing conditions (28) on 12% Tris-glycine gels with a 4% stacking gel overlay (Bio-Rad, Hercules, CA). For analysis of the hydrophobic proteins, the 200-g lung lavage supernatant was centrifuged at 20,000 g for 1 h. For analysis of SP-B, the pellet was suspended in NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA), heated at 37°C for 15 min, and subjected to electrophoresis under reducing conditions on Novex 16% Tricine gel (Invitrogen). SP-C was similarly analyzed by suspending the 20,000-g pellet in Laemmli (28) sample buffer and running the electrophoresis on NuPAGE 12% Bis-Tris gels (Invitrogen) in NuPAGE MES SDS buffer (Invitrogen). The resolved proteins were electrotransferred to either nitrocellulose (SP-A, SP-B, and SP-D) or polyvinylidene difluoride (SP-C) membranes in 25 mM Tris containing 192 mM glycine and 20% methanol or NuPAGE transfer buffer (Invitrogen), respectively. The membranes were then sequentially incubated in Tris-buffered saline containing Tween 20 and nonfat dry milk for 2 h (47), 1:2,000 (SP-A and SP-D) or 1:1,000 (SP-B and SP-C) dilution of primary antibody for 1 h, 1:20,000 dilution of peroxidase-conjugated goat anti-rabbit IgG for 1 h, and Western Blot Chemiluminescence Reagent (DuPont New England Nuclear, Boston, MA) for 1 min. The blots were exposed to X-ray film for 15-30 s, and the autoradiographs were quantified by scanning densitometry as previously described (47). Each protein was analyzed on at least two blots with two to three different exposures of each, and the data were averaged to yield a single value.
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RESULTS |
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Morphological analysis of pulmonary type II cells in IL-13
transgenic mice.
In the course of analyzing the role of IL-13 in allergic lung
inflammation, we produced mice in which IL-13 was overexpressed in the
lung using the CC10 promoter (CC10-IL-13 mice). We previously demonstrated that these mice have airway mucus metaplasia, airway epithelial cell hypertrophy, airway fibrosis, and airway
hyperresponsiveness on methacholine challenge (50).
Comparison of 3- to 4-mo-old transgenic and wild-type littermates also
revealed enlarged granular epithelial cells in the alveoli of
transgenic animals compared with controls (Fig.
1, A and B).
Eventually, proteinaceous material, similar to that seen in alveolar
proteinosis, accumulated in the alveoli of the transgenic animals (Fig.
1C). Electron microscopy demonstrated that the type II
pneumocytes from these mice were massively distended with lamellar
bodies compared with type II pneumocytes in littermate controls (Fig.
1, D and E). The intra-alveolar material had the
ultrastructural appearance of surfactant, since lamellar bodies and
tubular myelin figures were readily apparent (Fig. 1F).
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Immunohistochemical analysis of SPs in IL-13 transgenic mice. Immunohistochemical evaluation of wild-type littermate control mice showed scattered cells in the pulmonary parenchyma that stained with the SP-B antibody, indicative of type II cell differentiation (Fig. 1G). Consistent with the light and electron microscopy findings, numerous large SP-B-positive cells were apparent in the lungs from transgenic animals using identical staining conditions (Fig. 1H). This staining pattern had a granular pattern consistent with the intracellular secretory bodies seen by electron microscopy. The proteinaceous material seen in the alveoli was also strongly positive for SP-B (Fig. 1I). These findings indicate accumulation of SP-B in type II cells and in alveolar spaces of IL-13 transgenic animals.
The protein that accumulated in the alveoli of the transgenic mice could be the result of an increase in surfactant secretion by type II cells or a decrease in surfactant catabolism. Alternatively, it could be a nonspecific finding resulting from type II cell rupture. To address this issue, we determined the immunohistochemical distribution of an epitope on the pro-SP-C molecule that is not secreted during normal surfactant biosynthesis (Fig. 1, J and K). Staining of wild-type and transgenic animals with this antibody showed normal and enlarged type II cells, respectively, similar to that seen with the SP-B antibody. The extracellular material seen in transgenic animals was completely negative, however. This implies that the proteinosis was not occurring because of simple breakdown of the type II cells. Alternatively, the epitope may have been destroyed by proteolysis within the alveolus. In addition to alveolar type II cells, airway Clara cells also produce certain SPs. To address Clara cell-specific SP expression, we analyzed immunostains of airways using a pro-SP-B antibody. As expected, lungs from wild-type littermate controls showed intense staining of airway epithelium (Fig. 1L). In transgenic animals, in contrast to the effect of IL-13 on type II cells, airway epithelial cells showed decreased expression of pro-SP-B (Fig. 1M). Similar, but less intense, results were seen with antibodies against SP-B (data not shown). We previously reported that there is an increase in the numbers of mucus-producing cells in airways from IL-13 transgenic mice (50). Part of the decrease in pro-SP-B expression may therefore be because of replacement of Clara cells by mucus cells. This is supported by the finding of weak staining for CC10, a marker of Clara cell differentiation, in the mucus cells of transgenic animals (data not shown).Analysis of surfactant phospholipids in IL-13 transgenic mice.
There was a sixfold increase in the amount of total phospholipid in BAL
fluid from the IL-13 transgenic mice compared with littermate controls
when the data were expressed per unit body weight (Table
1). The increase was threefold if the
data were expressed per mass of lung dry weight. There were minor
differences in surfactant phospholipid composition between the control
and IL-13 mice (Table 1). There was significantly less
phosphatidylglycerol and more phosphatidylcholine and
phosphatidylinositol in the IL-13 animals. An inverse relationship
between the amounts of phosphatidylglycerol and phosphatidylinositol in
surfactant has previously been reported (37).
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Analysis of SPs and mRNA in IL-13 transgenic mice.
To determine if IL-13 altered the accumulation of SPs and surfactant
phospholipids, quantitative Western analysis was employed. These
studies showed that the amounts of all four SPs were significantly increased in lung lavage from the IL-13 transgenic mice compared with
the controls (Fig. 3). Densitometric
analysis showed a 3-fold increase in SP-A content, an ~2-fold
increase in SP-B and SP-C, and a 70-fold increase in SP-D (Table
2). In addition, there was an additional
high-molecular-weight SP-A band in the lavage sample from the IL-13
mice that was not present in lavage from control mice and rats (Fig.
3). The significance of this band is unknown at the present time.
Together, the results show that IL-13 caused a modest increase in SP-A,
-B, and -C and a striking increase in SP-D. Immunohistochemical
staining for SP-D in wild-type mice shows expression in bronchiolar
cells and, to a lesser degree, expression in type II cells, as
previously reported (45). Transgenic mice have a similar
pattern of expression but with more intense staining, especially in
bronchiolar cells (data not shown).
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DISCUSSION |
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Surfactant is a complex mixture of phospholipids and proteins, including SP-A, SP-B, SP-C, and SP-D. SP-A, SP-B, and SP-C regulate various aspects of surfactant phospholipid metabolism and surfactant film formation and stability. SP-A and SP-D also function in various aspects of innate and acquired immunity (7, 11, 29). The principal site of surfactant synthesis is the alveolar type II cell, although macrophages and type II cells are critically important in the catabolism and recycling of surfactant. In humans, defects in SP-B and granulocyte-macrophage colony-stimulating factor function lead to alveolar proteinosis in children and adults, respectively (10, 27, 36). Multiple other stimuli, including lipopolysaccharide and cytokines, have been reported to affect surfactant biosynthesis and catabolism (2). To further understand the processes that might regulate surfactant biosynthesis at sites of Th2 inflammation, we characterized the type II cells and BAL surfactant levels in mice in which IL-13 was overexpressed in the lung/airways. These studies demonstrate, for the first time, that IL-13 has potent effects on the surfactant system. Changes induced by chronic in vivo IL-13 exposure include type II cell hypertrophy, surfactant lipid and protein accumulation, and increased mRNA expression for SP-B, -C, and -D. The levels of IL-13 seen in the BAL fluid of our mice (~2 ng/ml) are comparable with those seen in bronchoalveolar fluid after antigen challenge of human asthmatics and are less than that seen in BAL fluid of patients with pulmonary fibrosis syndromes (18, 22).
IL-4 and IL-13 are believed to have arisen from a genetic duplication event (8). In keeping with this hypothesis, the two cytokines are known to have partially overlapping effector profiles. In particular, IL-4 has similar effects on the surfactant system, as we show here for IL-13, in that mice that overexpress IL-4 have increased BAL phospholipid content, a striking increase in BAL SP-D, and a lesser increase in SP-A (24, 25). In both lines of mice, the decrease in SP-B accumulation in airway cells was accompanied by an increase in cells that express both mucus and low levels of CC10, indicating a global effect on Clara cell differentiation. There are some minor differences as well, since we show increased mRNA for SP-B and SP-C, whereas the IL-4 mice are reported to show decreased or unchanged mRNA levels for SP-B and SP-C. Finally, although we show increased SP-B and SP-C protein levels, these were not examined in the IL-4 mice.
The mechanism whereby these changes occur is not completely defined. In theory, an increase in surfactant accumulation could be the result of an increase in synthesis (either per cell or because of an increase in the number of cells synthesizing surfactant), a decrease in catabolism, or both. The increased mRNA levels in our mice suggest an increase in protein synthesis. The IL-4 mice have both an increased production and reduced rate of clearance of surfactant phospholipids, although SP-A clearance appears normal. We have not examined phospholipid or SP metabolism in our mice. It is impossible to determine in these transgenic systems if the surfactant-altering effects of IL-13 or IL-4 are direct effects of these cytokines or are indirectly caused by other induced mediators.
The physiological consequences of the dysregulation of surfactant phospholipids reported here are uncertain. In rodent models, acute antigen challenge reduces surfactant phospholipid synthesis by type II cells (32). Human asthmatics have also been reported to have a defect in surfactant activity during acute exacerbations, and some work, both in experimental animals and humans, suggests that exogenous surfactant is actually therapeutic after allergen challenge (20, 21, 26). It is thus plausible to suggest that the increase in surfactant phospholipids in our mice represents a counterregulatory mechanism that can overcome the inhibition of surfactant production and functional activity seen during an acute inflammatory response. In addition, surfactant phospholipids are generally considered anti-inflammatory. Thus the enhanced surfactant production in our transgenic animals may also represent a counterregulatory mechanism that inhibits the inflammation caused by IL-13 and other mediators in Th2-dominated pulmonary inflammation.
The physiological consequences of the overexpression of SPs are also uncertain. It is reasonable to speculate, however, that there are important immune effects, since SP-A and SP-D are established regulators of innate and allergic immunity (7, 11, 29). In this regard, it is particularly interesting that SP-A and SP-D protect mice against an Aspergillus-induced hypersensitivity reaction (33). When viewed in combination, these studies suggest that the enhanced protein production that is present in our mice may act to diminish both infection and Th2-mediated tissue inflammation. Of note, it has recently been reported that human asthmatics have increased SP-A and -D at baseline (4).
A striking finding in our studies is the ability of IL-13 to disproportionally increase SP-D relative to the other SPs. SP-A and SP-D have similar but not identical functions. SP-D, in contrast to SP-A, is widely synthesized outside of the lung. This suggests that SP-D has a generalized role in mucosal immunity (23, 34). The exact nature of this role, however, is difficult to discern, since in vitro and in vivo studies have conflicted (31, 42, 44).
A central process in interstitial lung injury of many types is that of type I epithelial cell injury with basement membrane denudation and serum exudation, followed by type II cell hypertrophy and hyperplasia. The type II cells differentiate toward type I cells, which then cover the denuded basement membrane. Furthermore, in many chronic inflammatory states such as pneumocystis infection, pulmonary fibrosis syndromes, silica exposure, and radiation injury, there is an increase in surfactant pool size (9, 19, 35, 40, 46). The mediators that are responsible for the type II pneumocyte, fibrotic, and surfactant abnormalities in these various settings have not been well characterized. However, Th2 cytokine excess has been implicated in the pathogenesis of pulmonary fibrosis syndromes both in animals and in humans (41). It is noteworthy in this regard that the IL-13 but not the IL-4 mice show an overall increase in lung collagen content (A. Temann, personal communication and Ref. 30). A dominant role of IL-13 over IL-4 in promoting tissue fibrosis has previously been shown in hepatic schistosomiasis (6, 13). Together, these studies suggest that Th2 cytokines, including IL-13, play an important role not merely in the fibrosis seen in these disorders but also in the associated surfactant and type II cell abnormalities.
We have previously reported that these mice develop destructive emphysema (48). Although it may be superficially paradoxical that these mice develop both emphysema and pulmonary fibrosis, this can be readily understood in terms of kinetics, since the emphysema takes place over weeks while the fibrosis takes months to fully develop.
In summary, these studies demonstrate that IL-13 causes type II alveolar cell hypertrophy and an alveolar proteinosis-like tissue response associated with impressive increases in surfactant phospholipid, mRNA, and protein. Because these changes occur in the setting of pulmonary fibrosis, IL-13 may play a role in the pathogenesis of type II epithelial cell hypertrophy, surfactant overproduction, and fibrosis seen in a broad spectrum of pulmonary disorders.
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ACKNOWLEDGEMENTS |
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We thank the institutions and investigators that provided reagents that were employed. We owe special thanks to Lillemore Wallmark for outstanding assistance with electron microscopy performed at the West Haven Veterans Affairs Medical Center.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants P50-HL-56389, RO1-HL-61904, RO1-HL-64242, RO1-HL-31175, and RO1-HL-43320.
Address for reprint requests and other correspondence: R. J. Homer, Dept. of Pathology, Yale Univ. School of Medicine, P.O. Box 208023, New Haven, CT, 06520-8023 (E-mail: Robert.Homer{at}yale.edu).
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.
First published February 15, 2002;10.1152/ajplung.00438.2001
Received 11 November 2001; accepted in final form 12 February 2002.
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