Mice that express interleukin (IL)-4 in Clara cells (CCSP-IL-4) develop chronic airway inflammation and an alveolar proteinosis-like syndrome. To identify the role of IL-4 in surfactant homeostasis, we measured lipid and protein metabolism in the lungs of CCSP-IL-4 mice in vivo. Alveolar saturated phosphatidylcholine (Sat PC) pools were increased 6.5-fold and lung tissue Sat PC pools were increased 4.8-fold in the IL-4 transgenic mice. Whereas surfactant protein (SP) A was increased proportionately to Sat PC, SP-D was increased approximately 90-fold in the IL-4 mice compared with wild-type mice and was associated with 2.8-fold increase in SP-D mRNA. The incorporation of palmitate and choline into Sat PC was increased about twofold in CCSP-IL-4 mice. Although trace doses of radiolabeled Sat PC were cleared from the air spaces and lungs of CCSP-IL-4 mice more slowly than in wild-type mice, net clearance of Sat PC from the lungs of CCSP-IL-4 mice was sixfold higher in the IL-4 mice than in wild-type mice because of the larger Sat PC pool sizes. Expression of IL-4 in Clara cells increased surfactant lipid synthesis and clearance, establishing a new equilibrium with increased surfactant pools and an alveolar proteinosis associated with a selective increase in SP-D protein, demonstrating a previously unexpected effect of IL-4 in pulmonary surfactant homeostasis.
- transgenic mice
- saturated phosphatidylcholine
- surfactant protein A
- surfactant protein D
- alveolar proteinosis
pulmonary surfactant pool sizes are precisely regulated in the mammalian lung by linked processes of synthesis, storage, secretion, recycling, and catabolism for both lipid and proteins (8,26, 30). Surfactant pool sizes are increased in various chronic lung diseases, although the mechanisms regulating the disruption of surfactant homeostasis are poorly understood (10). Alveolar lipoproteinosis in humans has been associated with abnormalities in the regulation or function of granulocyte-macrophage colony-stimulating factor (GM-CSF) (7). In mice, alveolar lipoproteinosis-like syndromes were described in severe combined immunodeficiency, surfactant protein (SP) D deficiency, GM-CSF deficiency, and interleukin (IL)-4 overexpression (14, 16-18). In the most extensively evaluated animal model, GM-CSF deficiency or GM-CSF receptor inactivation causes a pulmonary alveolar lipoproteinosis (PAP) in mice that results from impaired surfactant lipid and protein catabolism (14, 25). Surfactant phospholipid homeostasis is also altered by SP-D because there are marked increases in alveolar and lung surfactant lipid pool sizes but without parallel increases in surfactant proteins (2, 18).
IL-4 is a TH2 cytokine with pleiotropic effects on various immune effector cells. IL-4 is thought to regulate protective immune responses, regulate IgE synthesis, and modulate airway inflammation in asthma (3). To investigate the contribution of IL-4 to airway inflammation, transgenic mice were made to selectively express IL-4 in the airways using the Clara cell secretory protein (CCSP) specific promotor (24). CCSP-IL-4 transgenic mice developed airway epithelial cell hypertrophy and increased numbers of alveolar macrophages and leukocytes in the lung parenchyma (24). An unanticipated histological finding was alveolar proteinosis (16). Concentrations of SP-A and SP-B are increased in lung homogenates and alveolar washes of CCSP-IL-4 mice. To identify the mechanisms by which IL-4 alters surfactant metabolism, surfactant lipid homeostasis was assessed in CCSP-IL-4 mice in vivo. Because airway expression of IL-4 is associated with inflammation, the changes in surfactant metabolism resulting in alveolar proteinosis may contribute to an understanding of the clinical associations of inflammation and alveolar proteinosis (3,6, 11).
The construction and original descriptions of the mice expressing IL-4 in the airway epithelium under control of the rat CCSP promoter were reported previously (15, 16, 24). CCSP-IL-4 mice were bred through subsequent generations in the FVB/N strain of mice in filtered cages in the vivarium at Children's Hospital Medical Center in viral-free conditions. All mice were studied at 7–9 wk of age. Wild-type mice were generated in the FVB/N strain for comparison.
Alveolar lavage and surfactant measurements.
Mice were given intraperitoneal pentobarbital sodium to achieve deep anesthesia, and the distal aorta was cut to exsanguinate each animal. The chest of the animal was opened, a 20-gauge blunt needle was tied into the proximal trachea, and five aliquots of 0.9% NaCl were flushed into the lungs to achieve full inflation (about 1 ml) and were withdrawn by syringe three times each for each aliquot (14). The recovered lavage was pooled, and the volume was measured. Lung tissue after alveolar wash was homogenized in 4 ml of saline. Saturated phosphatidylcholine (Sat PC) in lung tissue and in alveolar wash was recovered following chloroform-methanol (2:1) extraction and treatment of lipid extracts with OsO4 in carbon tetrachloride by silica column chromatography according to Mason et al. (20). Phosphorus in Sat PC was measured by the Bartlett assay (1).
Surfactant in alveolar washes was separated into large- and small-aggregate fractions by centrifugation (13). The alveolar washes were centrifuged at 40,000 g for 15 min, and the pellet was suspended in normal saline and centrifuged at 40,000 g over a 0.8 M sucrose cushion for 15 min. The pellet was recovered as the cell fraction. The large-aggregate surfactant was then collected from the interface, diluted with 0.9% NaCl and centrifuged again at 40,000g for 15 min.
Precursor incorporation into Sat PC and secretion.
Eight wild-type and eleven CCSP-IL-4 mice were given a 13 μl/g body wt intraperitoneal injection containing 0.5 μCi/g [3H]choline chloride (DuPont-NEN, Boston, MA) and 0.1 μCi/g [14C]palmitic acid (American Radiolabeled Chemicals, St. Louis, MO) (12). The palmitic acid was stabilized in solution with 5% human serum albumin. Eight hours after isotope injection, the mice were killed and alveolar washes and lung homogenates were used to isolate Sat PC. The Sat PC was divided for measurements of phosphorus and radioactivity. Percent secretion at 8 h was the amount of radioactive Sat PC in the alveolar wash divided by the sum of the radioactive Sat PC in the alveolar wash and lung tissue.
Clearance of dipalmitoylphosphatidylcholine and SP-A.
Wild-type and CCSP-IL-4 mice were given intratracheal injections with 50 μl of saline that contained 0.5 μCi [14C]choline-labeled dipalmitoylphosphatidylcholine (DPPC), and 0.1 μCi125I-SP-A. [14C]DPPC was purchased from Amersham (Arlington Heights, IL). SP-A was isolated from large-aggregate surfactant from alveolar washes of mice with octylglucopyranoside according to Hawgood et al. (9). The purified SP-A was iodinated with Bolton-Hunter reagent (Amersham) as described previously (14, 29). The injection mixture was prepared by first drying the labeled phospholipids with an unlabeled chloroform-methanol extract of mouse surfactant on a round-bottom flask by rotary evaporation. The phospholipids were resuspended with glass beads in 0.9% NaCl to achieve a final Sat PC concentration of 0.1 μmol/ml.125I-SP-A was then added just before intratracheal injection (14). Mice were anesthetized with isoflurane, and the trachea of each mouse was exposed through a 0.5-cm midline skin incision in the neck. The isotope mixture was injected using a 30-gauge needle attached to an insulin syringe. Three minutes after injection, four CCSP-IL-4 and four wild-type mice were killed with intraperitoneal pentobarbital sodium, and alveolar washes were collected. Other groups of four to six wild-type and CCSP-IL-4 mice received alveolar lavages 0.5, 8, 24 and 40 h after tracheal injection. The 125I in alveolar washes and lung homogenates was measured. The alveolar washes and lung homogenates subsequently were used for lipid extraction, Sat PC isolation, and quantification of the recoveries of [14C]DPPC in association with the Sat PC. The recoveries were normalized to the mean total recoveries for the mice that were killed 3 min after the tracheal injections.
Type II cell counts.
Lungs were inflation-fixed at 25 cmH2O pressure with 4% paraformaldehyde, and type II cells were counted using paraffin sections (5 μm) immunostained with antibodies to proSP-C as described previously (11). Cells on ten consecutive fields of 2 × 104μm2 were counted in each of five lobes (50 fields counted per mouse). The average number of type II cells on a 2 × 104-μm2field was determined for wild-type (n= 4) and CCSP-IL-4 mice (n = 4). Type II cells were also estimated as the percent of cells in the same sections by counting 500 nuclei per mouse.
RNA was isolated by a modification of the guanidinium thiocyanate method of Chomczynski and Sacchi (4). Total lung RNA (10 μg) was hybridized in solution at 55°C for 18 h with an excess of32P-end-labeled probes in 400 mM NaCl, 2 mM EDTA, 40 mM PIPES, pH 6.6, and 80% formamide. Each sample was digested with S1 nuclease at room temperature for 1 h in 100 mM NaCl, 15 mM sodium acetate, pH 4.5, 2.5 mM ZnSO4, and 25 ng of denatured salmon sperm DNA with 110 U of S1 nuclease (GIBCO BRL, Gaithersburg, MD). Protected fragments were resolved on a 6% polyacrylamide-8 M urea gel and visualized by autoradiography. Plasmids containing probe sequences for SP-A, SP-B, SP-C, and L32, a ribosomal protein used as an internal control, were described previously (22). The SP-D S1 probe was prepared from an EcoR 1-Kpn I fragment of genomic DNA containing mouse SP-D exons 3–6 and intervening sequences subcloned into pBluescript KS− (Stratagene, La Jolla, CA). A 452-bp PCR product was generated using M13 forward primer and 5′-GTTCTCCCTTTGGTCCAGGTTC-3′ as a reverse primer. The thermocycler program was 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min for a total of 35 cycles. The PCR product was purified by agarose gel electrophoresis prior to end labeling. The predicted size of the SP-D S1-protected fragment was 106 nucleotides.
Alveolar wash samples from three mice for each genotype, containing equal amounts of Sat PC, were electrophoresed on SDS-polyacrylamide 10–27% gradient gels as previously described using antibodies to SP-A and SP-D (23, 27). Immunoreactive bands were identified using enhanced chemiluminescence substrates (Amersham) and exposing blots to XAR film (Kodak, Rochester, NY). Relative band intensity was determined using an ISI00 Digital Imaging System (Alpha Innotech, San Leandro, Ca).
All values are given as means ± SE. Differences between groups were tested by two-tailed Student'st-tests. Where more than two comparisons were made, analysis of variance followed by the Student-Newman-Keuls multiple comparison procedure was used. Curves were fit using the regression programs supplied with SigmaPlot. Significance was accepted at P < 0.05.
Sat PC pool sizes.
The amount of Sat PC in alveolar washes was increased 6.5-fold in CCSP-IL-4 mice compared with wild-type FVB/N mice (Fig.1). The percentage of alveolar Sat PC recovered in the large aggregates was 48.4 ± 1.7% for wild-type (n = 7) and 36.3 ± 1.8% for CCSP-IL-4 mice (n = 5;P < 0.01), demonstrating a difference in the distribution of alveolar forms. The total pool of large-aggregate surfactant was therefore markedly increased to 25.5 μmol/kg Sat PC in CCSP-IL-4 mice compared with 5.2 μmol/kg in wild-type mice (P < 0.01). The pellets recovered from alveolar lavages were analyzed for the amount of cell-associated Sat PC relative to the alveolar Sat PC pool. The cell-associated surfactant was 1.2 ± 0.3% of the total pool in wild-type and 6.6 ± 2.2% of the total pool in CCSP-IL-4 mice (P < 0.01). The Sat PC in lung tissue from CCSP-IL-4 mice was increased 4.8-fold, and total lung Sat PC was increased 5.5-fold.
Incorporation of phospholipid precursors and secretion.
Eight hours after intraperitoneal injection of [14C]palmitate, total lung Sat [14C]PC was 2.7-fold higher in CCSP-IL-4 than in wild-type mice (Fig.2). There was no significant difference in the amount of radiolabeled Sat PC recovered by alveolar wash from the CCSP-IL-4 mice compared with that from wild-type mice. The percent of Sat PC secreted was less in CCSP-IL-4 (9.2 ± 1.1%) compared with wild-type mice (19.5 ± 0.7%). Incorporation of [3H]choline into total lung Sat PC was 3.4-fold higher in CCSP-IL-4 than in wild-type mice, and percent secretion of labeled Sat PC was 10.0 ± 1.2% in lungs of CCSP-IL-4 mice and 20.2 ± 0.9% in wild-type control mice. The incorporation of [3H]choline into Sat PC and secretion of Sat [3H]PC was the same as that obtained with [14C]palmitate. To evaluate whether the increased incorporation of phospholipid precursors resulted from increased numbers of type II cells, the numbers of type II cells were estimated. A modest but statistically significant increase in type II cells was observed in CCSP-IL-4 (13.5 ± 0.1 cells/2 × 104μm2) vs. wild-type (9.8 ± 0.1 cells/2 × 104μm2) mice (P < 0.05). By counting nuclei, type II cells were 23 ± 1% of parenchymal cells in CCSP-IL-4 mice and 17 ± 1% of parenchymal cells in wild-type lungs (P < 0.05). There were no differences in lung weights between the two groups of animals.
Clearance of [14C]DPPC and125I-SP-A.
Radiolabeled DPPC was cleared from the air spaces exponentially, with recoveries at 40 h of 4.7 ± 0.3% for wild-type and 13.9 ± 2.7% for CCSP-IL-4 mice (P < 0.01; Fig.3). The loss of radiolabeled DPPC from the lungs also followed exponential kinetics. Percent clearance and catabolism of Sat PC were decreased in CCSP-IL-4 (21.3 ± 4.2% recovery at 40 h) compared with wild-type mice (6.7 ± 0.4% recovery at 40 h; P < 0.01). However, because of the increased pool size of Sat PC, the total amount of lipid clearance was increased in lungs from CCSP-IL-4 mice. The clearance kinetics of 125I-SP-A from alveolar wash and total lung did not follow simple first-order exponential curves in wild-type or CCSP-IL-4 mice (Fig.4). In contrast to the observed increased clearance of DPPC from lungs of CCSP-IL-4 mice, clearance of SP-A from alveolar wash or lung was not different from that in control mice. The alveolar recovery of SP-A was only 2% at 40 h, and total lung recovery was 11% at 40 h for both wild-type and CCSP-IL-4 mice.
The mRNAs for the surfactant proteins were measured for five CCSP-IL-4 mice relative to the values for five wild-type mice. The values relative to the wild-type mice were 1.7 ± 0.3 for SP-A mRNA (P < 0.01), 0.6 ± 0.1 for SP-B mRNA (P < 0.01), and 0.9 ± 0.2 for SP-C mRNA. SP-D mRNA increased 2.8-fold in lungs from CCSP-IL-4 mice above the normalized steady-state amount in wild-type mice (P < 0.01; Fig.5). The amounts of SP-A and SP-D in alveolar washes were compared by scanning densitometry of Western blots prepared for the samples normalized for the amount of Sat PC. The ratio of SP-A to Sat PC was similar in wild-type and CCSP-IL-4 mice (Fig.6). The alveolar washes of the CCSP-IL-4 mice contained 6.4-fold more Sat PC (Fig. 1) and therefore contained about six times more SP-A than alveolar washes from wild-type mice. In contrast, in the CCSP-IL-4 mice, SP-D was increased disproportionately to Sat PC because SP-D concentration was increased approximately 15-fold. This represents a net increase in SP-D in alveolar pool of about 90-fold over that measured in wild-type mice.
Expression of IL-4 in nonciliated epithelial cells in the conducting airways of mice resulted in a 6.5-fold increase in Sat PC, a 15-fold increase in SP-A, and a 90-fold in SP-D in the alveolar pool relative to wild type. These IL-4 mediated changes in steady-state alveolar pool sizes resulted from multiple alterations in surfactant metabolism. Precursor incorporation into Sat PC using radiolabeled palmitate and choline was increased about threefold, and type II cell numbers in the CCSP-IL-4 mice were increased by 27% based on cell counts. The metabolic measurements must be interpreted relative to the changes in pool size of the surfactant components. With the assumption that plasma concentrations, cellular uptake, and intracellular pools of the lipid precursors were similar in lungs of wild-type and CCSP-IL-4 mice, the increased precursor incorporation into Sat PC reflected an increased rate of Sat PC synthesis of approximately twofold per cell. Therefore, part of the lipid accumulation caused by IL-4 results from increased synthesis of Sat PC.
The percent of the radiolabeled Sat PC that accumulated in the alveolar wash over 8 h was a measure of net secretion (12). For the present study, measurements were obtained at 8 h because secretion of surfactant remains linear for about 12 h in mice (8). The percent radiolabeled Sat PC secreted by the CCSP-IL-4 mice was about 50% that of wild-type mice. With the assumption that the de novo synthesized Sat PC mixed uniformly with the entire tissue Sat PC pool, then 10% of the tissue pool of 101 μmol/kg or 10 μmol/kg was secreted over 8 h in CCSP-IL-4 mice. Similarly, 20% of the 20.5 μmol/kg Sat PC tissue pool or 4 μmol/kg was secreted by wild-type mice. Although newly synthesized Sat PC is thought to preferentially localize to lamellar bodies, there is a considerable amount of Sat PC that is not associated with the intracellular surfactant pathways in the normal lung (30). It is not known which cellular and intracellular compartments contain the increased Sat PC in the lungs of CCSP-IL-4 mice. Whereas the present measurements do provide an accurate estimate of percent secretion for Sat PC, it is not possible to determine a secretory rate from the present experiments.
Although the percent clearance of radiolabeled Sat PC was decreased in CCSP-IL-4 mice, total clearance and catabolism of Sat PC were increased when pool sizes are considered. Radiolabeled surfactant components were mixed with mouse surfactant prior to tracheal injection. We assumed that the tracer mixed uniformly with the endogenous pool, and the tracer was a valid probe for measuring net clearance from the alveolar pool (12, 21, 26). Because the radiolabeled probe then associated with the lung, the second assumption was that the radiolabeled surfactant component mixed uniformly with the lung pool and was a probe of net lung clearance and catabolism. Recovery of Sat PC at 40 h in alveolar washes of CCSP-IL-4 mice was 13.9%. This represents a clearance of 60 μmol of Sat PC because the alveolar Sat PC pool was 70 μmol/kg. In contrast, in wild-type mice recovery was 4.7% of a pool size of 10.8 μmol/kg at 40 h, which is a clearance of 10.3 μmol/kg. Net alveolar clearance was increased sixfold in CCSP-IL-4 mice. Actual alveolar clearances would likely be higher because recycling of the Sat PC also occurs.
The loss of the radiolabeled surfactant components from the lung measures primarily catabolic activity because very little intact Sat PC or SP-A leaves the lung (21, 29). In the normal rabbit, 20–30% of the catabolism of Sat PC can be attributed to macrophages and the rest is catabolized via lysosomal pathways by type II cells (26). The catabolic contribution of macrophages has not been established in mice. The Sat PC recovery was 6.7% in lungs of wild-type mice, representing the catabolism of 29 μmol of Sat PC over 40 h. The recovery of 21.3% of the radiolabeled Sat PC in the lungs of CCSP-IL-4 mice results in a net catabolism of 134 μmol of Sat PC or about 4.6-fold more than in the wild type. The 10-fold increase in the number of alveolar macrophages in the lungs of CCSP-IL-4 mice may account for the observed increased catabolic activity.
SP-A was reported previously to be increased 13-fold in lung tissue, and SP-B was increased 5.7-fold in alveolar washes from CCSP-IL-4 mice. (16). We used Western blots to estimate that SP-A in alveolar wash was increased about sixfold, which was comparable to the increase in Sat PC. The alveolar and total lung clearances of radiolabeled SP-A were equivalent in CCSP-IL-4 and wild-type mice, but the higher SP-A levels represent a sixfold increase in net clearance and catabolism of the protein in the CCSP-IL-4 mice. The metabolism of SP-A appears to be altered in proportion to changes in metabolism of Sat PC in CCSP-IL-4 mice.
Whereas lung tissue SP-D mRNA was increased about 2.8-fold, alveolar SP-D pools were increased about 90-fold in CCSP-IL-4 mice. SP-D is a member of the collectin family that is produced by both Clara cells and type II cells in the lung (5). Based on its structure and lack of close association with surfactant lipids, SP-D has been considered primarily a host-defense protein (19). However, SP-D deficiency in mice caused increased alveolar lipid pools, suggesting that SP-D may be a regulator of surfactant metabolism (2, 18). It is unclear whether the increase in SP-D is related to direct or indirect effects of IL-4 on production or catabolism of SP-D. An interesting possibility is that the increased Sat PC pools stimulate SP-D production to promote normalization of alveolar pool sizes.
In GM-CSF-deficient mice, alveolar proteinosis results from markedly decreased catabolism of Sat PC and surfactant proteins (14). Although pool sizes of surfactant lipids and proteins increased in the alveolar proteinosis caused by CCSP-IL-4, the finding that net surfactant catabolism is increased differs strikingly from the lack of measurable catabolism of Sat PC and poor clearances of SP-A and SP-B in GM-CSF-deficient mice (14). The increased surfactant pool sizes in the CCSP-IL-4 mouse occur because a new equilibrium is established that results from the combined effects of a small increase in the numbers of type II cells and increased precursor incorporation into Sat PC that is not offset by increased net Sat PC catabolism. Whether the effects of IL-4 on surfactant metabolism and type II cell numbers represent a direct effect of the cytokine on epithelial cell function or are mediated indirectly, perhaps related to inflammatory cell infiltration, is unclear. There is no previous information about effects of IL-4 on surfactant other than the observations that IL-4 expression in Clara cells results in alveolar proteinosis with increased levels of the surfactant proteins (15, 16). The mice were generated to evaluate IL-4 effects on airways inflammation, and effects of IL-4 expression in cell types other than Clara cells remain to be explored. Jain-Vora et al. (15) recently demonstrated that these IL-4-overexpressed mice clearedPseudomonas aeruginosa more effectively than did wild-type mice and speculated that the enhanced clearance might be explained by the increased amounts of SP-A and/or SP-D in the CCSP-IL-4 mice. The levels of IL-4 were reported previously to be in the range of 300 pg/ml in lungs of CCSP-IL-4 mice, and IL-4 was not detectable in wild-type mice (15). The measurement was repeated for nine mice and was similar (269 ± 16 pg/ml lung homogenate). IL-4 was not detectable for the nine wild-type mice.
This model of alveolar proteinosis differs from observations in GM-CSF deficiency or chronic glucocorticoid exposure that are associated with decreased surfactant catabolism (14, 31). The present findings demonstrate that IL-4 expression in Clara cells perturbs surfactant metabolism by altering Sat PC synthesis and catabolism and SP-D levels. The adaptations of the lung to chronic IL-4 exposure may represent a general characteristic of the responses of the lung to other injuries likely to be associated with prolonged proinflammatory stimuli. For example, surfactant pool sizes are increased in pneumocystis infection and pulmonary fibrosis syndromes in humans and with silica exposure and radiation injury in animal models (6, 10, 28). Nongenetic cases of alveolar proteinosis in humans that resolve may represent the transient response of the lungs to an unidentified proinflammatory stimulus.
This work was funded by the National Institutes of Health Grants HD-11932, HL-61646, and HL-28623.
Address for reprint requests and other correspondence: M. Ikegami, Div. of Pulmonary Biology, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229–3039 (E-mail:).
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