CCSP deficiency does not alter surfactant homeostasis during adenoviral infection

Machiko Ikegami, Kevin S. Harrod, Jeffrey A. Whitsett, and Alan H. Jobe

Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039


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Clara cell secretory protein (CCSP) deficiency in mice is associated with increased susceptibility to pulmonary inflammation after hyperoxia or viral infection. Because adenoviral exposure perturbs pulmonary surfactant homeostasis in vivo, we hypothesized that CCSP deficiency would influence surfactant metabolism after pulmonary infection. Alveolar and total lung saturated phosphatidylcholine pool sizes were similar in CCSP-deficient [CCSP(-/-)] and wild-type [CCSP(+/+)] mice before and 7 days after intratracheal administration of adenovirus. Radiolabeled choline and palmitate incorporation into saturated phosphatidylcholine was similar, and there was no alteration by previous infection 7 days before the incorporation measurements. Furthermore, CCSP deficiency did not influence clearance of [14C]dipalmitoylphosphatidylcholine and 125I-labeled recombinant surfactant protein C. Increased persistence of alveolar capillary leak was observed in CCSP(-/-) mice after adenoviral infection. Surfactant lipid homeostasis was not influenced by CCSP before or after administration of adenovirus to the lung. Persistence of alveolar capillary leak in CCSP(-/-) mice after adenovirus provides further evidence for the role of CCSP in the regulation of pulmonary inflammation.

phosphatidylcholine; transgenic mice; surfactant protein A; surfactant protein C; Clara cells; Clara cell secretory protein


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THE PRESENCE OF PHOSPHOLIPIDS and the autoradiographic concentration of palmitate into the secretory granules of nonciliated bronchiolar epithelial cells (Clara cells) suggested that these cells contributed to surfactant homeostasis in the lung (4, 6, 7). Clara cells also express surfactant protein (SP)-A, SP-B, and SP-D (18, 22). However, the most abundant secretory product of Clara cells is Clara cell secretory protein (CCSP), a 10- to 16-kDa protein that binds xenobiotics and may play a role in the modulation of lung inflammation (3, 20). CCSP-deficient [CCSP(-/-)] mice were more sensitive to oxygen injury (14), and leukocytic infiltration and chemokine and cytokine production were increased in CCSP(-/-) mice after viral infection (8). In normal mice, adenoviral infection altered SP homeostasis, increasing alveolar protein concentration and locally inhibiting SP mRNA expression at the site of inflammation (17, 23). CCSP concentrations are decreased in chronic lung disease such as chronic bronchiolitis and lung fibrosis, and these diseases are associated with changes in the amount of surfactant components in bronchoalveolar lavage (9).

Crystallized CCSP is a dimer with a hydrophobic pocket that binds phosphatidylcholine (PC) and phosphatidylinositol, and these phospholipids copurify with CCSP (21). CCSP also inhibits the activity of phospholipase A2 by binding PC and could modulate surfactant catabolism (19). Given the links of Clara cells and CCSP to the surfactant system and the potential anti-inflammatory role of CCSP in the lungs, we hypothesized that CCSP(-/-) mice would have abnormalities in surfactant metabolism. We also hypothesized that the increased and prolonged inflammatory response resulting from adenoviral exposure of CCSP(-/-) mice would further alter surfactant metabolism. We evaluated metabolic variables of saturated PC (Sat PC) and a recombinant human SP-C (rSP-C) in CCSP(-/-) mice and in CCSP(-/-) mice challenged with replication-deficient adenovirus to induce lung injury.


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Mice. CCSP(-/-) (129J Ola/129J hybrid), generated as previously described (20), and wild-type [CCSP(+/+); 129J, Taconic Farms, Germantown, NY] mice were housed in the Children's Hospital (Cincinnati, OH) Research Foundation vivarium under pathogen-free conditions (8). Groups of 8-10 eight-week-old CCSP(-/-) and CCSP(+/+) mice were used for all the studies.

Intratracheal administration of adenovirus. CCSP(-/-) and CCSP(+/+) mice were randomized to intratracheal adenovirus or saline treatment. Metabolic studies of surfactant were performed 1 day after adenovirus instillation to evaluate the surfactant system during the peak of the early inflammatory response (8). The metabolic studies also were repeated on day 7 because the CCSP(-/-) mice have a prolonged proinflammatory response to adenovirus (8). Mice were anesthetized with methoxyflurane and orally intubated with a 25-gauge animal feeding needle. Each mouse received 50 µl of saline or 50 µl of Av1Luc1 (1 × 109 plaque-forming units), an E1- to E3-deleted replication-deficient adenoviral vector expressing firefly luciferase from the Rous sarcoma virus promoter in buffer (10 mM Tris, 1 mM MgCl2, and 10% glycerol, pH 7.4) (8). The characteristics of the inflammation after exposure of mice to this virus were previously described (8).

Precursor incorporation into Sat PC and secretion. Eight hours before alveolar wash, which was 24 h or 7 days after intratracheal administration of adenovirus or saline, CCSP(+/+) and CCSP(-/-) mice were given an 8 µl/g body wt intraperitoneal injection of 0.1 µCi/g of [3H]choline chloride (DuPont-New England Nuclear, Boston, MA) and 0.2 µCi/g of [14C]palmitic acid (American Radiolabeled Chemicals, St. Louis, MO) (11). The palmitic acid was stabilized in solution with 2.5% human serum albumin. An interval of 8 h between precursor injection and alveolar wash was used because this time provides an accurate estimate of total precursor incorporation into Sat PC (13). At 8 h, the ratio of radiolabeled Sat PC in the alveolar wash to the sum of radiolabeled Sat PC in the alveolar wash and lung tissue was used to estimate net secretion on the linear part of the secretion curve (13).

Clearance of dipalmitoylphosphatidylcholine and rSP-C. CCSP(+/+) and CCSP(-/-) mice were given intratracheal injections of 50 µl of saline that contained 0.3 µCi of [14C]choline-labeled dipalmitoylphosphatidylcholine (DPPC), and 0.15 µCi of 125I-rSP-C. [14C]DPPC was purchased from Amersham (Arlington Heights, IL). The rSP-C (a gift from Byk Gulden, Constance, Germany) is the 34-amino acid human sequence altered by replacement of cystine-3 and -4 with phenylalanine and methionine-32 with isoleucine (10). The rSP-C, iodinated with the 125I-labeled Bolton-Hunter reagent as previously reported (10), was metabolized in rabbit and mouse lungs comparably to the native peptide. [14C]DPPC and 125I-rSP-C were mixed with a small amount of a chloroform-methanol extract of natural mouse surfactant in chloroform, dried under N2, and resuspended in saline by brief sonication. The 50-µl injection volume contained a trace dose of 0.1 µmol/kg of Sat PC relative to the endogenous pool of ~15 µmol Sat PC/kg. The intratracheal injections were given with 30-gauge needles after exposure of the trachea of each anesthetized mouse (13). The interval between intratracheal injection of the radiolabeled surfactant components and alveolar wash was 16 h based on a previous study (13) of clearance of surfactant components in mice.

Protein permeability. The recovery of 125I-albumin in alveolar washes 2 h after intraperitoneal injection of 5 µCi in 100 µl of iodinated bovine serum albumin was used as a measure of lung injury (12).

Alveolar lavage and tissue processing. 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 (~1 ml) and were withdrawn by syringe three times for each aliquot (13). The recovered lavage fluid was pooled, and the volume was measured. The relevant alveolar washes and lung tissue after alveolar wash were counted for 125I to measure recovery of 125I-rSP-C relative to the recovery evaluated in animals killed 10 min after the intratracheal injection. The amount of Sat PC recovered by alveolar wash was measured by extracting the alveolar wash with chloroform-methanol (2:1) followed by treatment of the lipid extract with OsO4 in carbon tetrachloride and silica column chromatography according to Mason et al. (16). Phosphorus in Sat PC was measured with the Bartlett (1) assay. Lung tissue after alveolar wash was homogenized in saline, and an aliquot was extracted and analyzed for Sat PC content.

Data analysis. All values are means ± SE. Differences between groups were tested by two-tailed Student's t-tests. When more than two comparisons were made, analysis of variance followed by the Student-Newman-Keuls multiple comparison procedure was used. Significance was accepted at P < 0.05.


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Protein permeability. Radiolabeled albumin was given by intraperitoneal injection 2 h before the mice were killed for alveolar washes. Adenovirus or vehicle had been given 24 h or 7 days before the alveolar washes. The recovery of 125I-albumin in alveolar washes increased in both CCSP(+/+) and CCSP(-/-) mice 24 h after adenoviral exposure, indicating increased alveolar-capillary permeability (Fig. 1). Recovery of the labeled albumin was not increased 7 days after adenoviral exposure in the CCSP(+/+) mice but remained elevated in the CCSP(-/-) mice, indicating persistent injury.


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Fig. 1.   Recovery of 125I-albumin in alveolar washes. Mice were given intratracheal adenovirus or saline 24 h and 7 days (d) before alveolar wash. Two hours before alveolar wash, animals received 125I-albumin, and percent recovery was measured. Exposure of Clara cell secretory protein (CCSP) wild-type [CCSP(+/+)] mice to adenovirus increased albumin recovery at 24 h. In contrast, exposure of CCSP-deficient [CCSP(-/-)] mice to adenovirus increased albumin recovery at 24 h and 7 days. * P < 0.05 vs. saline control group.

Sat PC pool sizes. The alveolar pool sizes of Sat PC were similar in CCSP(+/+) and CCSP(-/-) mice 24 h after saline or adenoviral administration (Fig. 2). In comparison to the values for saline-instilled animals, both CCSP(+/+) and CCSP (-/-) mice had small increases in alveolar and total lung (alveolar plus tissue) Sat PC 7 days after adenoviral exposure.


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Fig. 2.   Pool sizes of saturated phosphatidylcholine (Sat PC) in alveolar washes and total lungs calculated as Sat PC in alveolar wash plus lung tissue. Mice were given adenovirus or saline by tracheal injection 24 h or 7 d before measurement of Sat PC. Alveolar and total Sat PC pools were similar in CCSP(+/+) and CCSP(-/-) mice, and Sat PC pools increased 7 d after adenoviral exposure. * P < 0.01 vs. 7-d saline control group.

Precursor incorporation into Sat PC. The CCSP(+/+) and CCSP(-/-) mice were given [3H]choline and [14C]palmitic acid 8 h before alveolar wash and 24 h or 7 days after intratracheal challenge with adenovirus or saline. Choline incorporation into total lung Sat PC was not increased after adenoviral exposure in the CCSP(+/+) and CCSP(-/-) mice relative to that in the saline control mice (Fig. 3). There was more variability in the measurements for CCSP(-/-) mice after adenoviral exposure, which did not reach significance except for a higher [3H]choline-labeled Sat PC in alveolar lavage in CCSP(-/-) mice 7 days after adenovirus injection. A similar pattern of responses was observed in experiments with labeled palmitate (Fig. 4). The [14C]palmitic acid-labeled Sat PC tended to be higher in CCSP(-/-) mice 7 days after adenoviral exposure. Percent secretion of [14C]palmitic acid- and [3H]choline-labeled Sat PC in the alveolar washes relative to that in the total lung is shown in Figs. 3 and 4. Percent secretion was higher 24 h after adenoviral exposure for [14C]palmitic acid-labeled Sat PC. Percent secretion also was higher 7 days after adenoviral exposure for [3H]choline-labeled Sat PC in CCSP(-/-) mice compared with that in the saline control group and was somewhat lower in CCSP(-/-) mice 24 h after adenovirus injection. There were no changes in surfactant secretion in CCSP(+/+) mice after adenovirus infection.


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Fig. 3.   Incorporation of [3H]choline into Sat PC in alveolar washes (top) and total lungs (middle) and secretion of Sat PC (bottom). Mice received adenovirus 24 h or 7 d before alveolar wash and 8 h after receiving [3H]choline. Incorporation into total lung Sat PC was similar in all groups at 24 h and 7 d. More labeled Sat PC was recovered in alveolar washes of CCSP(-/-) mice 7 d after adenoviral exposure than from CCSP(+/+) mice. Percent secretion was calculated as radiolabeled Sat PC in alveolar washes divided by that in total lung in CCSP(-/-) mice. * P < 0.05 vs. saline- and adenovirus-exposed CCSP(+/+) mice.



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Fig. 4.   Incorporation of [14C]palmitate into Sat PC in alveolar washes (top) and total lungs (middle) and secretion of Sat PC (bottom). Mice received adenovirus 24 h and 7 d before alveolar wash and 8 h after receiving [14C]palmitate. There were no differences between CCSP(+/+) and CCSP(-/-) mice in incorporation into total lung Sat PC or in recovery in alveolar washes. Secretion was increased at 24 h for CCSP(-/-) mice exposed to adenovirus. * P < 0.05 vs. saline control group.

Recovery of DPPC and rSP-C. The CCSP(-/-) and CCSP(+/+) mice received a trace dose of surfactant radiolabeled with [14C]DPPC and 125I-rSP-C by tracheal injection 16 h before alveolar wash and 24 h or 7 days after intratracheal saline or adenovirus challenge. About 18% of the radiolabeled DPPC was recovered by alveolar wash, and 24% was recovered in the total lung of CCSP(+/+) and CCSP(-/-) mice 24 h and 7 days after intratracheal saline or adenovirus (Fig. 5). The recovery of rSP-C was ~17% in the alveolar washes, and total lung recovery was ~32% in all groups (Fig. 6). Although more rSP-C than DPPC was recovered, there was no effect of CCSP deficiency or adenovirus on the recovery of the surfactant components.


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Fig. 5.   Percent recovery of [14C]dipalmitoylphosphatidylcholine (DPPC) in alveolar washes (top) and total lungs (bottom). Mice received intratracheal saline or adenovirus 24 h or 7 d before alveolar wash and [14C]DPPC 16 h before alveolar wash. Percent recovery was measured as Sat PC from alveolar washes and total lungs. There were no differences between CCSP(+/+) and CCSP(-/-) mice.



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Fig. 6.   Percent recovery of 125I-labeled recombinant human surfactant protein C (rSP-C) in alveolar washes (top) and total lungs (bottom). Mice received intratracheal saline or adenovirus 24 h or 7 d before alveolar wash and 125I-rSP-C 16 h before alveolar wash. Percent recovery was measured from alveolar washes and total lungs. There were no differences between CCSP(+/+) and CCSP(-/-) mice.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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CCSP deficiency did not alter surfactant phospholipid pool sizes, precursor incorporation, or Sat PC secretion and clearance in vivo. Furthermore, pulmonary infection with the replication-deficient adenovirus that caused pulmonary inflammation did not alter surfactant metabolism either acutely at 24 h or more chronically after 7 days (8). Small differences in Sat PC secretion were observed after adenovirus challenge in CCSP(-/-) but not in CCSP(+/+) mice. The persistence of increased alveolar capillary permeability as indicated by the increased recovery of 125I-albumin in the alveolar washes of the CCSP(-/-) mice at 7 days supports an increased duration of lung inflammation in CCSP(-/-) mice. Adenoviral exposure also increased alveolar and total lung Sat PC pool sizes at 7 days in both CCSP(+/+) and CCSP(-/-) mice, indicating the effects of the adenoviral infection on the surfactant system.

The present findings indicate that CCSP does not play a major role in the control of surfactant homeostasis in vivo. Although Clara cells contain SP-A, SP-B, and SP-D mRNAs, it remains unclear whether Clara cells secrete significant amounts of these proteins and whether these SPs play a role in the function of conducting airways. A link between surfactant homeostasis and Clara cell function was suggested by the observations that SP-D deficiency results in large increases in alveolar phospholipid pool sizes (2, 15). The ability of CCSP to inhibit phospholipase A2 activity also suggests a link between CCSP and surfactant (19).

The function(s) of the major secretory product of Clara cells, CCSP, has remained enigmatic. CCSP deficiency in mice results in increased sensitivity to oxygen and persistent inflammation after pulmonary infection with adenovirus (8, 14). Both stresses to the lung result in elevated levels of proinflammatory mediators in CCSP(-/-) mice relative to those in CCSP(+/+) mice. Because surfactant lipid and protein concentrations are altered after hyperoxic and viral-induced lung injury, we hypothesized that CCSP is playing a role in the regulation of inflammation that, in turn, influences surfactant homeostasis. This hypothesis is not supported by the experiments that we performed. The results are of sufficiently high resolution to exclude large effects of CCSP on the surfactant system. Certainly, subtle regional effects in small airways could occur that would not be detected by the techniques used, which emphasize the net metabolic activities of type II cells and alveolar macrophages.

Although Clara cells produce surfactant-specific products and are positioned in the airways in locations that require surfactant to maintain airway patency (5), the major secretory product of these cells seems to have no role in the regulation of surfactant metabolism. The persistent injury resulting from adenoviral exposure of CCSP(-/-) mice also had minimal effects on surfactant metabolism.


    ACKNOWLEDGEMENTS

This work was funded by National Heart, Lung, and Blood Institute Grants HL-28623 and P01-HL-61646 and the Cystic Fibrosis Foundation Research Development Program Center.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. Ikegami, Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: machiko.ikegami{at}chmcc.org).

Received 6 May 1999; accepted in final form 12 July 1999.


    REFERENCES
TOP
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1.   Bartlett, G. R. Phosphorous assay in column chromatography. J. Biol. Chem. 234: 466-468, 1959[Free Full Text].

2.   Botas, C., F. Poulain, J. Akiyama, C. Brown, L. Allen, J. Goerke, J. Clements, E. Carlson, A. M. Gillespie, C. Epstein, and S. Hawgood. Altered surfactant homeostasis and alveolar type II cell morphology in mice lacking surfactant protein D. Proc. Natl. Acad. Sci. USA 95: 11869-11874, 1998[Abstract/Free Full Text].

3.   Crouch, E. C. Collectins and pulmonary host defense. Am. J. Respir. Cell Mol. Biol. 19: 177-201, 1998[Abstract/Free Full Text].

4.   Cutz, E., and P. E. Conen. Ultrastructure and cytochemistry of Clara cells. Am. J. Pathol. 62: 127-141, 1971[Medline].

5.   Enhorning, G., L. C. Duffy, and R. C. Welliver. Pulmonary surfactant maintains patency of conducting airways in the rat. Am. J. Respir. Crit. Care Med. 151: 554-556, 1995[Abstract].

6.   Etherton, J. E., D. M. Conning, and B. Corrin. Autoradiographical and morphological evidence for apocrine secretion of dipalmitoyl lecithin in the terminal bronchiole of mouse lung. Am. J. Anat. 138: 11-35, 1973[Medline].

7.   Etherton, J. E., I. F. Purchase, and B. Corrin. Apocrine secretion in the terminal bronchiole of mouse lung. J. Anat. 129: 305-322, 1979[Medline].

8.   Harrod, K. S., A. D. Mounday, B. R. Stripp, and J. A. Whitsett. Clara cell secretory protein decreases lung inflammation after acute virus infection. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L924-L930, 1998[Abstract/Free Full Text].

9.   Hermans, C., and A. Bernard. Lung epithelium-specific proteins: characteristics and potential applications as markers. Am. J. Respir. Crit. Care Med. 159: 646-678, 1999[Free Full Text].

10.   Ikegami, M., A. D. Horowitz, J. A. Whitsett, and A. H. Jobe. Clearance of SP-C and recombinant SP-C in vivo and in vitro. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L933-L939, 1998[Abstract/Free Full Text].

11.   Ikegami, M., T. R. Korfhagen, M. D. Bruno, J. A. Whitsett, and A. H. Jobe. Surfactant metabolism in surfactant protein A-deficient mice. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L479-L485, 1997[Abstract/Free Full Text].

12.   Ikegami, M., T. R. Korfhagen, J. A. Whitsett, M. D. Bruno, S. E. Wert, K. Wada, and A. H. Jobe. Characteristics of surfactant from SP-A-deficient mice. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L247-L258, 1998[Abstract/Free Full Text].

13.   Ikegami, M., T. Ueda, W. Hull, J. A. Whitsett, R. C. Mulligan, G. Dranoff, and A. H. Jobe. Surfactant metabolism in transgenic mice after granulocyte-macrophage colony-stimulating factor ablation. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L650-L658, 1996[Abstract/Free Full Text].

14.   Johnston, C. J., G. W. Mango, J. N. Finkelstein, and B. R. Stripp. Altered pulmonary response to hyperoxia in Clara cell secretory protein deficient mice. Am. J. Respir. Cell Mol. Biol. 17: 147-155, 1997[Abstract/Free Full Text].

15.   Korfhagen, T. R., V. Sheftelyevich, M. S. Burhans, M. D. Bruno, G. F. Ross, S. E. Wert, M. T. Stahlman, A. H. Jobe, M. Ikegami, J. A. Whitsett, and J. H. Fisher. Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo. J. Biol. Chem. 43: 28438-28443, 1998.

16.   Mason, R. J., J. Nellenbogen, and J. A. Clements. Isolation of disaturated phosphatidylcholine with osmium tetroxide. J. Lipid Res. 17: 281-284, 1976[Abstract].

17.   Pryhuber, G. S., C. Bachurski, R. Hirsch, A. Bacon, and J. A. Whitsett. Tumor necrosis factor-alpha decreases surfactant protein B mRNA in murine lung. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L714-L721, 1996[Abstract/Free Full Text].

18.   Savov, J., J. R. Wright, and S. L. Young. Instilled SP-A is not recycled by rat lung Clara cells (Abstract). Am. J. Respir. Crit. Care Med. 159: A731, 1999.

19.   Singh, G., and S. L. Katyal. Clara cells and Clara cell 10 kD protein (CC10). Am. J. Respir. Cell Mol. Biol. 17: 141-143, 1997[Free Full Text].

20.   Stripp, B. R., J. Lund, G. W. Mango, K. C. Doyen, C. Johnston, K. Hultenby, M. Nord, and J. A. Whitsett. Clara cell secretory protein: a determinant of PCB bioaccumulation in mammals. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L656-L664, 1996[Abstract/Free Full Text].

21.   Umland, T. C., S. Swaminathan, G. Singh, V. Warty, W. Furey, J. Pletcher, and M. Sax. Structure of a human Clara cell phospholipid-binding protein-ligand complex at 1.9 A resolution. Nat. Struct. Biol. 1: 538-545, 1994[Medline].

22.   Voorhout, W. F., T. Veenendaal, Y. Kuroki, Y. Ogasawara, L. M. G. van Golde, and H. J. Geuze. Immunocytochemical localization of surfactant protein-D (SP-D) in type-II cells, Clara cells, and alveolar macrophages of rat lung. J. Histochem. Cytochem. 40: 1589-1597, 1992[Abstract/Free Full Text].

23.   Zsengeller, Z. K., S. E. Wert, W. M. Hull, X. Hu, S. Yei, B. C. Trapnell, and J. A. Whitsett. Persistence of replication-deficient adenovirus-mediated gene transfer in lungs of immune-deficient (nu/nu) mice. Hum. Gene Ther. 6: 457-467, 1995[Medline].


Am J Physiol Lung Cell Mol Physiol 277(5):L983-L987
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