Pulmonary surfactant secretion in briefly cultured mouse type II cells

Laurice I. Gobran and Seamus A. Rooney

Division of Perinatal Medicine, Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510

Submitted 17 September 2003 ; accepted in final form 10 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
There is little information on the regulation of surfactant secretion in mouse type II cells. We isolated type II cells from C57BL/6 and FVB mice, cultured them overnight, and then examined their response to known surfactant secretagogues. Secretion of phosphatidylcholine, surfactant protein (SP)-B and SP-C was stimulated by terbutaline, 5'-N-ethylcarboxyamidoadenosine (NECA), ATP, UTP, TPA, and ionomycin. Phosphatidylcholine secretion was increased approximately twofold by all agonists in both strains of mice. The response to terbutaline and NECA is the same as in rat type II cells, whereas the response to ATP, UTP, TPA, and ionomycin is considerably less. Secretion of SP-B and SP-C was increased sevenfold by terbutaline and threefold by ATP, effects similar to those in rat type II cells. The response to terbutaline was significantly decreased in type II cells from {beta}2-adrenergic receptor null mice. These data establish that briefly cultured type II cells provide a suitable model for investigation of surfactant secretion in normal and genetically altered mice.

phosphatidylcholine; surfactant protein B; surfactant protein C; exocytosis; {beta}2-adrenergic receptor null mice


LUNG SURFACTANT IS A COMPLEX of lipids and proteins that are synthesized in the type II pneumocyte (33). Surfactant lipids are largely phospholipids, of which phosphatidylcholine is by far the most abundant component (33). Surfactant proteins consist of surfactant protein (SP)-A, SP-B, SP-C, and SP-D (37). SP-A and SP-D are hydrophilic glycoproteins that have a role in innate lung immunity and host defense (24), whereas SP-B and SP-C are small hydrophobic proteins that, together with phospholipids, are involved in the biophysical functions of surfactant (19).

Surfactant phospholipids are synthesized in the endoplasmic reticulum, stored in lamellar inclusion bodies, the secretory organelle characteristic of the type II cell (36), and finally secreted into the alveolar lumen by the process of regulated exocytosis (22, 31). The phospholipid composition of isolated lamellar bodies is virtually identical to that of surfactant obtained by lung lavage (29), and there is abundant evidence that surfactant phospholipids are secreted together with lamellar body contents (3, 22, 31, 39). Lamellar bodies are enriched in SP-B and SP-C (25), and it has been established that these hydrophobic proteins are secreted together with the phospholipids (36) and by the same regulated process (11). In contrast, secretion of SP-A and SP-D occurs independently of lamellar bodies and is either not regulated or regulated by a different mechanism (11, 21).

Secretion of phosphatidylcholine has been extensively investigated in isolated rat type II cells (3, 22, 31, 39). There is considerable evidence that it is regulated by at least three distinct signaling mechanisms (31). Surfactant phospholipid secretagogues include terbutaline and 5'-N-ethylcarboxyamidoadenosine (NECA), agents that activate {beta}-adrenergic and adenosine A2B receptors, respectively, as well as ATP and UTP, agonists that activate P2Y2 receptors (31). Other surfactant secretagogues include agonists, such as TPA, that directly activate protein kinase C, and ionophores, such as ionomycin, that increase intracellular Ca2+ levels (31). All of these secretagogues also stimulate SP-B and SP-C secretion in rat type II cells (11).

Surfactant secretion has been much less extensively studied in type II cells from other species, although secretion of phosphatidylcholine has been investigated in type II cells isolated from rabbit and human lungs (30). Methods for isolation of type II cells from the mouse have been developed (6, 26, 34), but there is little information on surfactant secretion in mouse type II cells. Terbutaline and TPA were recently reported to stimulate surfactant secretion in mouse type II cells that were cultured for 7 days under conditions that maintained their characteristic phenotype (26). However, the effects of these secretagogues on mouse type II cells that were cultured for a brief period have not previously been reported. Similarly, there is no information on the influence of NECA, ATP, UTP, and ionomycin on surfactant secretion in mouse type II cells. In view of the availability of transgenic and knockout mice with alterations in the surfactant system, a model in which mouse surfactant secretion can easily be examined is highly desirable. Consequently, the goal of this investigation was to examine the influence of known surfactant secretagogues on surfactant secretion in freshly isolated mouse type II cells cultured for <24 h. As activation of {beta}-adrenergic receptors is considered to be an important signaling mechanism in the physiological regulation of surfactant secretion (30, 31), an additional goal was to investigate surfactant secretion in type II cells from {beta}-receptor null mice.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Chemicals and reagents. Anti-mouse CD45 and CD32 monoclonal antibodies were obtained from BD Biosciences (San Diego, CA); Matrigel base membrane matrix, rat tail type 1 collagen, and dispase from BD Biosciences (Bedford, MA); human recombinant keratinocyte growth factor (KGF) from PeproTech (Rocky Hill, NJ); bronchial epithelial cell growth medium (BEGM) from Cambrex Bioscience (Walkersville, MD); collagenase (type I), DNase I, NECA, ATP, UTP (sodium salts), and horseradish peroxidase-conjugated goat anti-rabbit IgG from Sigma (St. Louis, MO); TPA and ionomycin from Calbiochem (San Diego, CA); terbutaline sulfate (Brethine) from Novartis (East Hanover, NJ); and [methyl-3H]choline chloride and Western Blot Chemiluminescence Reagent from Perkin-Elmer Life Sciences (Boston, MA). FBS was obtained from Hyclone (Logan, UT) and was treated with dextran-coated charcoal (Sigma). Rabbit polyclonal antibodies against SP-A, SP-B, and SP-D (4, 38) were obtained from Dr. Samuel Hawgood (University of California, San Francisco, CA), and a similar antibody against mature SP-C (35) from Byk Gulden (Konstanz, Germany).

Secretagogue concentrations were as follows: 3.5 x 10-5 M terbutaline, 10-5 M NECA, 10-3 M ATP and UTP, 10-7 M TPA, and 2.5 x 10-8 M ionomycin. TPA and ionomycin were dissolved in DMSO. The maximum DMSO concentration was 0.005%, and this amount was also added to the corresponding control cultures. None of the secretagogues were cytotoxic as determined by lactate dehydrogenase (LDH) release (10). The rate of LDH release was 1.23 ± 0.18% (means ± SE, n = 4) per hour in the control and 1.32 ± 0.11 (n = 12) in the agonist-treated cells.

Animals. Female C57BL/6 and FVB mice were obtained from Charles River Laboratories. The {beta}2-adrenergic receptor knockout mice in the FVB background (5) were obtained from Dr. Brian K. Kobilka (Stanford University, Palo Alto, CA). Both males and females were used for type II cell isolation as there was no sex difference in basal or agonist-stimulated phosphatidylcholine secretion. Procedures involving animals were reviewed and approved by the Yale University Institutional Animal Care and Use Committee.

Type II cell isolation. Type II cells were isolated from 7- to 8-wk-old mice as described by Rice et al. (26). The method involves perfusion of the lungs via the pulmonary artery with 0.9% NaCl, instillation of agar via the trachea, digestion of the lungs with dispase, and sequential filtration of the resulting cell suspension through 100-µm and 40-µm cell strainers (Falcon; Becton-Dickinson, Franklin Lakes, NJ) and 20-µm Nitex gauze (Sefar America, Kansas City, MO). The cells were pelleted by centrifugation at 130 g for 8 min and suspended in DMEM containing 25 mM HEPES buffer, 10% charcoal-stripped FBS, gentamicin (10 µg/ml), and amphotericin (2.5 µg/ml). They were then plated on 100-mm diameter tissue culture dishes (Falcon, one dish per mouse) that had been coated with CD45 and CD32 antibodies (26) and washed three times with PBS and once with DMEM. After incubation in 10% CO2 at 37°C for 2 h, the type II cells were gently panned from the dishes and collected by centrifugation at 130 g for 8 min. The cell yield at this stage was 3-8.5 x 106 per mouse. The cells (1-1.4 x 106) were plated on 12-well tissue culture dishes (Falcon) coated with a mixture (70:30 by vol) of Matrigel (10.5 mg/ml) and rat tail collagen (3.25 mg/ml) and cultured for 18-20 h in 10% CO2 at 37°C in 0.6 ml of BEGM without hydrocortisone or epinephrine but containing KGF (10 ng/ml) and 5% charcoal-stripped FBS. The cells were finally washed three times with DMEM. At that stage, type II cell purity was 97.4% ± 0.9 (means ± SE, n = 3) as determined by tannic acid staining (23). Viability was 96.7 ± 0.2% (n = 4) as determined by trypan blue exclusion.

Phosphatidylcholine secretion. [3H]Choline chloride (3 µCi per well, 60-90 Ci/mmol) was included in the medium during the overnight culture period. The cells were washed three times with DMEM at 37°C; fresh DMEM with or without secretagogues was added, and the incubation was continued for 3 h except in time course experiments when the total period of incubation varied from 0 to 240 min. The medium was then removed and centrifuged at 130 g for 8 min. The matrices were digested with a 250-µl mixture of dispase (10 units) and collagenase (0.2 mg) for 1 h at 37°C, after which the cells were lysed with ice-cold 0.05% Triton X-100. The cells and medium were extracted with chloroform and methanol (2), and total lipid radioactivity was measured by liquid scintillation counting (10). When the lipids were fractionated by thin-layer chromatography (20), phosphatidylcholine was found to account for 95-96% of the radioactivity in choline containing lipids in both the cells and media. Fractionation of the lipids was therefore not necessary, and secretion is expressed as the amount of radioactivity in the medium as percentage of the total in cells and medium combined.

Secretion of surfactant proteins. Secretion of SP-A, SP-B, SP-C, and SP-D was measured as described for phosphatidylcholine except that [3H]choline was not included during the overnight culture period, and DMEM without phenol red was used. After incubation with secretagogues for 3.5 h, the medium was centrifuged at 130 g for 8 min. The supernatant was then further centrifuged at 20,000 g for 1 h (for SP-B and SP-C analysis) or treated with cold 5% TCA (SP-A and SP-D). The protein pellets were suspended in NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA) containing 10% dithiothreitol, heated at 37°C for 15 min (SP-B and SP-C) or at 95°C for 5 min (SP-A and SP-D), and subjected to electrophoresis in MES (SP-B and SP-C) or MOPS (SP-A and SP-D) buffer on NuPAGE 12% Bis-Tris gels (Invitrogen), after which the resolved proteins were electrotransferred to nitrocellulose membranes in NuPAGE transfer buffer (Invitrogen). The membranes were then sequentially incubated in Trisbuffered saline containing Tween 20 and nonfat dry milk for 2 h (13), 1:1,000 (SP-B and SP-C) or 1:2,000 (SP-A and SP-D) dilution of primary antibody for 1 h, 1:10,000 dilution of peroxidase-conjugated goat anti-rabbit IgG for 1 h, and Western Blot Chemiluminescence Reagent for up to 1 min. The blots were exposed to X-ray film, and the autoradiographs were quantified by scanning densitometry as previously described (13). At least two exposures of each blot were scanned, and the values were averaged. Some SP-C blots were stripped (13) and reprobed with the SP-B antibody. The SP-D blots were similarly reprobed for SP-A.

Data analysis and statistics. Type II cells isolated from three to eight mice were pooled in each experiment and distributed among the various control and treated groups. Two wells were used for each group in the phosphatidylcholine secretion experiments. Each well was processed separately, and the values were averaged to give a single data point per group per experiment. Sixteen wells were generally used for each group in the surfactant protein experiments. Data from several experiments were averaged, and the groups were compared statistically with Student's two-tailed t-test or ANOVA followed by the Newman Keuls test. P <= 0.05 was considered significant.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The response of type II cells from C57BL/6 mice to known surfactant secretagogues is shown in Table 1. We used agonist concentrations that optimally stimulate phosphatidylcholine secretion in rat type II cells (10, 12). Terbutaline, NECA, ATP, UTP, TPA, and ionomycin all significantly increased phosphatidylcholine secretion. The extent of the increase ranged from 66 to 94%, and the effects of the different secretagogues were not significantly different (one-way ANOVA). The extent of the increase in response to terbutaline and NECA in the mouse cells was similar to that previously reported for rat type II cells, approximately twofold (10). However, ATP, UTP, TPA, and ionomycin were less stimulatory in the mouse cells. They increased phosphatidylcholine secretion by 66-88% in mouse cells compared with two- to threefold for ionomycin (10, 15, 16) and UTP (12), three- to fourfold for ATP (10, 12, 15, 16), and four- to fivefold for TPA (10, 15, 16) in rat type II cells. The smaller response in mouse cells is not due to an insufficient intracellular pool of surfactant, as the rate of phosphatidylcholine secretion was linear with time for at least 4 h in the presence of selected agonists (terbutaline or TPA), as well as in the absence of any agonist (Fig. 1). The extent of the increase in phosphatidylcholine secretion in response to TPA in the briefly cultured cells (Table 1) was similar to that reported for C57BL/6 mouse type II cells that were cultured for 7 days (26). However, the basal rate of secretion was higher in the latter study, 2.7% vs. 1.1-1.4% in the current study (Table 1).


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Table 1. Effect of agonists on phosphatidylcholine secretion in type II cells isolated from C57BL/6 mice

 


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Fig. 1. Time course of phosphatidylcholine secretion in type II cells from C57BL/6 mice. The cells were cultured with terbutaline (3.5 x 10-5 M), TPA (10-7 M), or no agonist (control) for the times indicated after which phosphatidylcholine secretion was measured as described in Table 1. The data are means ± SE (bars) from 3 control, 3 terbutaline, and 2 TPA experiments.

 

The effects of selected agonists on SP-B and SP-C secretion are shown in Figs. 2 and 3, respectively. Terbutaline and ATP both significantly stimulated secretion of the hydrophobic surfactant proteins. Secretion of both SP-B and SP-C was increased sevenfold by terbutaline and threefold by ATP. Although the response to terbutaline tended to be greater than to ATP, such differences were not significant (t-test). The three- to sevenfold increase in SP-B and SP-C secretion in mouse type II cells is similar to the three- to fivefold increase reported for rat type II cells (11). Thus in contrast to the lipids, secretion of surfactant hydrophobic proteins is stimulated similarly in both rat and mouse type II cells.



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Fig. 2. Effects of terbutaline and ATP on surfactant protein (SP)-B secretion in type II cells from C57BL/6 mice. The cells were isolated from a pool of 8 mice and cultured for 18-20 h with serum and keratinocyte growth factor (KGF). They were then cultured in DMEM containing terbutaline (3.5 x 10-5 M), TPA (10-7 M), or no agonist (control) for 3.5 h, after which the amount of SP-B in the medium was measured by Western blotting and densitometry. Protein derived from equal numbers of cells was applied to each lane. Aliquots of rat lung lavage were also applied. A: typical Western blot. B: quantitative data. The data are means ± SE from 5-6 preparations of type II cells and were analyzed statistically with the paired t-test. The terbutaline (P < 0.02) and ATP (P < 0.03) values were significantly different from the corresponding controls.

 


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Fig. 3. Effects of terbutaline and ATP on SP-C secretion in type II cells from C57BL/6 mice. Experimental details are as described in Fig. 2. The quantitative data are means ± SE from 4 preparations of type II cells. The terbutaline (P < 0.05) and ATP (P < 0.03) values were significantly different from the corresponding controls.

 

As previously reported for rat type II cells (11), NECA, ATP, and TPA did not stimulate SP-A or SP-D secretion in mouse type II cells (Fig. 4). However, in contrast to the rat (11), terbutaline had a small stimulatory effect (Fig. 5). It increased SP-A and SP-D secretion by 49 and 36%, respectively. The effect of terbutaline on SP-A secretion was statistically significant, whereas that on SP-D was not. Whether the small terbutaline-induced increase in SP-A secretion in mouse type II cells is of any biological significance is doubtful. The increase in SP-A secretion is negligible compared with the increase in SP-B and SP-C secretion in response to the same agonist: almost sevenfold in mouse type II cells (Figs. 2 and 3) and approximately fourfold in rat type II cells (11). Furthermore, terbutaline either alone (21) or in combination with other surfactant secretagogues (9, 11, 32) did not stimulate SP-A secretion in rat type II cells.



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Fig. 4. Effect of 5'-N-ethylcarboxyamidoadenosine (NECA), ATP, and TPA on SP-A and SP-D secretion in type II cells from C57BL/6 mice. The cells were isolated from a pool of 8 mice and cultured for 18-20 h with serum and KGF. They were then cultured in DMEM containing NECA (10-5 M), ATP (10-3 M), TPA (10-7 M), or no agonist (control) for 3.5 h, after which the amounts of SP-A (A) and SP-D (B) in the medium were measured by Western blotting. Protein derived from equal numbers of cells was applied to each lane. Aliquots of rat lung lavage were also applied.

 


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Fig. 5. Effect of terbutaline on SP-A and SP-D secretion in type II cells from C57BL/6 mice. The cells were cultured with terbutaline (3.5 x 10-5 M) or no agonist (controls). Other experimental details are as described in Fig. 4. The data are means ± SE from 5 SP-A and 4 SP-D experiments. The terbutaline value was significantly different from the control in the SP-A (P = 0.0095) but not in the SP-D (P = 0.3595) experiments (paired t-test).

 

We considered the possibility that the relatively small increase in phosphatidylcholine secretion in response to ATP, UTP, TPA, and ionomycin (Table 1) was strain dependent. We therefore examined phosphatidylcholine secretion in response to agonists in type II cells isolated from a different mouse strain (FVB). The basal rates of phosphatidylcholine secretion in type II cells from FVB (1.54 ± 0.14% in 3 h, n = 6) and C57BL/6 mice (1.29 ± 0.06, n = 17) were not significantly different (unpaired t-test). Likewise, the response to secretagogues was similar (Fig. 6). The FVB and C57BL/6 % stimulation data were not significantly different (unpaired t-test). Although the response of type II cells from FVB mice to terbutaline, NECA, and ATP tended to be less, and the response to UTP, TPA, and ionomycin tended to be greater than in the cells from C57BL/6 mice, such differences were not significant. These data show that the relatively small increase in phosphatidylcholine secretion in mouse type II cells compared with those from the rat is not strain dependent. Whether it is due to the different cell isolation and culture conditions used for the rat and mouse cells or to other factors remains to be established.



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Fig. 6. Phosphatidylcholine secretion in response to agonists in type II cells from wild type and {beta}2-receptor null FVB mice. The cells were cultured with terbutaline (3.5 x 10-5 M), NECA (10-5 M), ATP (10-3 M), UTP (10-3 M), TPA (10-7 M), ionomycin (2.5 x 10-8 M), or no agonist (control) for 3 h, after which phosphatidylcholine secretion was measured as described in Table 1. The data are means ± SE from 4-6 preparations of type II cells from wild-type mice and 5-10 preparations from knockout mice and were analyzed statistically with Student's t-test for paired samples. The effects of all agonists were statistically significant (P < 0.05 to <0.0001) when the % secretion values in the treated groups were compared with the corresponding controls. Except for terbutaline, the wild-type and knockout data were not significantly different. *P = 0.0052 vs. wild type (unpaired t-test).

 

Of the three {beta}-adrenergic receptor subtypes (1), the {beta}3-receptor is not expressed in the lung (1), whereas both the {beta}1- and {beta}2-subtypes are expressed in type II cells (13, 17). In general, pharmacological data suggest that it is the {beta}2-subtype that regulates surfactant secretion (7, 8). If that were true, type II cells from mice lacking the {beta}2-receptor would not be expected to have increased surfactant secretion in response to terbutaline. We therefore examined phosphatidylcholine secretion in type II cells from {beta}2-adrenergic receptor null FVB mice (5). The basal rates of phosphatidylcholine secretion were the same in cells from both the wild-type and knockout mice: 1.54 ± 0.14% (n = 6) and 1.60 ± 0.10% (n = 10), respectively. However, as shown in Fig. 6, the response to terbutaline was significantly lower in type II cells from the knockout mice compared with those from wild-type FVB animals. Whereas terbutaline increased secretion by 60% in type II cells from wild-type FVB mice, it increased secretion in the cells from the knockout mice by only 27%, a difference that was statistically significant. There was no difference between cells from the knockout and wild-type mice in the response to NECA, ATP, or UTP (Fig. 6).

The decreased response to terbutaline in the knockout mice is consistent with involvement of the {beta}2-receptor in the terbutaline signal transduction mechanism. The fact that terbutaline had a small but significant stimulatory effect on surfactant secretion in the cells from the {beta}2-receptor knockout mice suggests that the {beta}1-receptor is also involved in the signaling mechanism. However, the possible involvement of an atypical {beta}-receptor (14) cannot be excluded. Type II cells from {beta}1-knockout (28) or {beta}1/{beta}2-double knockout mice (27) might help clarify that issue. In this context, it is noteworthy that, although pharmacological data generally suggest that it is the {beta}2-receptor that is involved in the regulation of surfactant secretion (7, 8), there is one pharmacological report that also suggests the involvement of both {beta}1- and {beta}2-receptors (18). Thus both genetic and pharmacological data suggest involvement of both {beta}1- and {beta}2-receptors in regulation of surfactant secretion.

In summary, we have shown that briefly cultured mouse type II respond to surfactant secretagogues. The increase in phosphatidylcholine secretion in response to terbutaline and NECA is similar to that previously reported for rat type II cells, but the response to ATP, UTP, TPA, and ionomycin is less in the mouse cells. The increase in SP-B and SP-C secretion in response to terbutaline and TPA is similar to that reported for rat type II cells. Phosphatidylcholine secretion in response to terbutaline is reduced but not eliminated in type II cells from {beta}2-receptor null mice, suggesting that another subtype of the {beta}-receptor may also have a role in mediating the effect of terbutaline. Freshly isolated type II cells cultured for <24 h provide a useful system for the investigation of surfactant secretion in genetically altered mice.


    ACKNOWLEDGMENTS
 
We thank Dr. Samuel Hawgood, University of California, San Francisco, CA, and Dr. Wolfram Steinhilber, Byk Gulden, Konstanz, Germany, for providing the antibodies and Dr. Brian K. Kobilka, Stanford University, Palo Alto, CA, for providing the {beta}2-adrenergic receptor null mice.

A preliminary report of these data was presented at the Experimental Biology annual meeting in San Diego, CA, 11-15 April 2003. An abstract has been published (FASEB J 17: A81-A82, 2003).

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-43320.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. A. Rooney, Dept. of Pediatrics, Yale Univ. School of Medicine. PO Box 208064, New Haven, CT 06520-8064 (E-mail: Seamus.Rooney{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.


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 RESULTS AND DISCUSSION
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