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Association of surfactant protein C with isolated alveolar type II cells
BB,
ELSEVIER
Biochimica et Biophysica Acta 1255 (1995) 16-22
Biochi~ic~a et BiophysicaA~ta
Association of surfactant protein C with isolated alveolar type II cells Ricardo A. Pinto a Samuel Hawgood a,c John A. Clements a,c, Bradley J. Benson d Asha Naidu d, Robert L. Hamilton e, Jo Rae Wright a,b,* a CardiovascularResearch Institute, University of California, San Francisco, CA 94143-0130, USA b Department of Physiology, University of California, San Francisco, CA 94143-0130, USA c Department of Pediatrics, University of California, San Francisco, CA 94143-0130, USA d Scios/Nova, Mountain View, CA 94043, USA e Department of Anatomy, University of California, San Francisco, CA 94143-0130, USA Received 1 April 1994; revised 16 September 1994; accepted 27 October 1994
Abstract
Surfactant protein C (SP-C) is a small hydrophobic protein that is synthesized and secreted by alveolar type II cells. The mechanism of clearance of SP-C from the alveolar airspace is not well understood, although previous studies demonstrated that recombinant SP-C instilled into the lungs of spontaneously breathing anaesthetized rats was taken up by type II cells and incorporated into lamellar bodies. The current investigation was undertaken to characterize the interaction of a complex of SP-C and surfactant-like lipids with freshly isolated rat alveolar type II cells under conditions in which the extracellular milieu can be regulated. SP-C was isolated from alveolar proteinosis lavage fluid and radiolabeled with 125I-Bolton-Hunter reagent. The radiolabeled protein retained its ability to facilitate adsorption of phospholipids to an air/liquid interface. Labeled human SP-C associated with isolated type II cells in a concentration-dependent manner that was also dependent upon temperature and time. The association of labeled SP-C with isolated type II cells did not saturate up to 150 /xg/ml. SP-A significantly enhanced the association of SP-C with isolated type II cells. Under the experimental conditions tested, SP-C was not degraded to TCA-soluble products. These results are consistent with the hypothesis that association or uptake of SP-C by type II cells may be enhanced by SP-A and that like SP-A, SP-C is recycled by type II cells. Keywords: Alveolar type II cell; Surfactant protein C; Surfactant protein A; Phospholipid; Liposome
I. Introduction
Pulmonary surfactant lipids and proteins are synthesized and secreted by the type II cells into the alveoli, where surfactant lipids reduce surface tension at the air/liquid interface. There are at least three processes by which surfactant lipids and proteins are cleared from the alveolar airspace: a degradative pathway in which components are metabolized and removed from the lung, a reutilization pathway in which degraded products are reutilized to synthesize new surfactant, and a recycling pathway in which intact surfactant components are taken up and incor-
Abbreviations: SP-A, surfactant protein A; SP-B, surfactant protein B; SP-C, surfactant protein C; MEM, minimal essential media; TCA, trichloroacetic acid; HPLC, high-performance liquid chromatography. * Corresponding author. Associate Professor of Cell Biology and Medicine. Present address: Box 3709, Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA. Fax: + 1 (919) 681 7978. 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 4 ) 0 0 2 0 5 - 3
porated into lamellar bodies from which they are resecreted (reviewed in Ref. [1]). It was shown that lipid clearance proceeds via each of these pathways although the contribution of each pathway to total clearance varies according to age, species, and other factors. For example, the extent of recycling of lipids has been estimated to range from 23% to 85% in adult rabbits [2-4], up to 85% in 3-day old rabbits [5]. The quantitative aspects of clearance of surfactant proteins have been less well characterized. It has been demonstrated that SP-A binds to high affinity receptors on isolated type II cells and is internalized by a pathway that includes endosomal organelles associated with receptormediated endocytic pathways [6,7]. Using electron microscopic autoradiography, Young and co-workers [8] demonstrated that exogenously administered SP-A is incorporated into lamellar bodies, supporting biochemical studies which demonstrated that intratracheally administered SP-A could be recovered in isolated lamellar bodies [9]. Kalina and co-workers [7,10] also demonstrated that gold-labeled sur-
R.A. Pinto et al. /Biochimica et Biophysica Acta 1255 (1995) 16-22
factant is taken up by isolated type II cells and incorporated into the multivesicular portion of composite bodies. SP-A appears to play a role in feedback regulation of lipid uptake by enhancing lipid uptake [11,12] and inhibiting lipid secretion [13,14] by isolated type II cells. Recently Breslin and colleagues [15] reported that radiolabeled SP-B binds to and is internalized by type II cells, although they concluded that the binding is probably not mediated by a receptor. SP-C, like SP-A and SP-B, also appears to be taken up by type II cells. Baritussio and co-workers reported that SP-C is taken up from the alveolar space and incorporated into lamellar bodies and that SP-C is cleared from the alveolar airspace more quickly than is phosphatidylcholine [16,17]. We recently observed that recombinant radiolabeled SP-C instilled into the airways of anaesthetized rats is taken up by type II cells and incorporated into lamellar bodies [18]. The overall goal of the current study was to characterize further the interaction of SP-C with isolated alveolar type II cells. Specifically, a method of radiolabeling purified native SP-C with high specific activity without altering its functional ability to accelerate adsorption of surfactant lipids to an air/water interface was developed, the association of SP-C with isolated type II cells was characterized, the effects of SP-A on the association of SP-C with type II cells was examined, and the ability of type II cells to degrade SP-C was investigated.
2. Materials and methods
2.1. Materials and animals The following chemicals and reagents were purchased from their respective companies: reverse phase C8 Vydac cartridge (Western Analytical Products, Temecular, CA); elastase (Worthington, Malverne, PA), 125I-Bolton-Hunter Reagent (N-succinim idyl-3-(4-hydroxy-3(125I)iodophenyl)proprionate) (New England Nuclear/DuPont, Wilmington, DE); lipids (Avanti Polar Lipids, Birmingham, AL); minimal essential media (MEM) containing Krebs improved salts [11] (Cell Culture Facility, University of California, San Francisco, CA); all other chemicals (Sigma Chemical, St. Louis, MO). Adult male Sprague-Dawley rats, which were pathogen-free and weighed approx. 180 g, were obtained from Bantin-Kingrnan (Fremont, CA). The experiments were performed under institutional animal care protocols.
2.2. Isolation of cells Type II cells were isolated by techniques that are routine in this laboratory. Briefly, lungs of adult male rats were perfused free of blood and filled with elastase solution [19]. The minced lung tissue was filtered through
17
gauze and contaminating alveolar macrophages were removed by adherence to IgG-coated Petri dishes. Cell yields routinely averaged 20-30.106 cells/animal. All preparations had greater than 95% viability and 85% purity [11].
2.3. Isolation SP-C SP-C was isolated from the lavage fluid of a patient with alveolar proteinosis by previously described procedures [20]. Briefly, a 60 000 X g pellet of lavage fluid was extracted with butanol and the insoluble proteins were removed by centrifugation. The supernatant, which contains SP-B, SP-C, and lipids was chromatographed on a 1.5 X 80 cm LH-60 column in chloroform/methanol, 1:1 (v/v), containing 5% (v/v) 0.1 M HC1. Fractions were analyzed by SDS-PAGE followed by silver staining and those fractions containing proteins of molecular mass of approx. 4000 to 6000 were pooled.
2.4. Radiolabeling of SP-C The Bolton-Hunter reagent (250/~Ci) was dried under a gentle stream of nitrogen. 100/zg of SP-C was dried under a gentle stream of nitrogen and resuspended in 25 /xl of 4% octylglucoside and diluted with 25/~1 of 0.1 M sodium phosphate buffer, pH 8.06. 25 /xl of the above solution was added with a Hamilton syringe to the dried 125I-Bolton-Hunter reagent and incubated 1 h on ice and then overnight at 4°C. The incubation mixture was then diluted with 53/~1 of 95% isopropyl alcohol/5% 50 mM HC1 and injected onto a reverse phase C8 cartridge column on a Hewlett-Packard 1090 liquid chromatograph. The column was eluted with a gradient of 45% isopropanol containing 5% (v/v) 0.1 M HCI to 95% isopropanol containing 5% (v/v) 0.1 M HC1 at a flow rate of 0.4 ml/min. Fractions of 0.4 ml were collected. The optical density at 280 nm was read on-line and radioactivity in the fractions was measured in a gamma counter.
2.5. Preparation of liposomes Liposomes in medium were prepared from commercially available lipids by the method of Hamilton [21] using a French pressure cell. The liposomes consisted of 55% dipalmitoylphosphatidylcholine, 27% egg phosphatidylcholine, 10% egg phosphatidylglycerol and 8% cholesterol (by weight). 4 mg of lipid in solvent and 40/xg of SP-C were combined and dried in a round bottom flask. 4 ml of medium were added and mixed in the presence of small glass beads. The mixture was heated at 57°C for 30 minutes and passed through the French pressure cell 3 times at 20 000 psi.
2.6. Ultrastructure studies Negative staining of SP-C lipid complexes was performed as previously described [21].
18
R.A. Pinto et aL / Biochiraica et Biophysica Acta 1255 (1995) 16-22 4.0 x 105 -
E
Q.. "O
3. Results
3.1. Characteristics of radiolabeled SP-C 3.0 x 10s -
> 2.0 x 105-
rr 1.o x lO5.
'1'o ''o ' ' o ' ' o ','o Fraction Number Fig. 1. Elution profile of radiolabeled SP-C after separation on a C8 column by HPLC. Free 125I-Bolton-Hunter reagent was separated from that bound to protein by HPLC as described in Section 2. Fractions containing protein include 18-40, which were pooled and analyzed by SDS-PAGE (see Fig. 2).
2. 7. Adsorption balance studies The ability of SP-C modified with non-radioactive Bolton-Hunter reagent to accelerate the rate of phospholipid film formation was compared to that of unmodified SP-C in an adsorption chamber, which records the rate at which lipids adsorb to an air/liquid interface and reduce surface tension. SP-C was mixed with phospholipids exactly as previously described [18] and the rate of film formation was measured as described by King and Clements [22].
Radiolabeled SP-C was separated from unbound 1251by high performance liquid chromatography (HPLC). A typical profile of radioactivity from column fractions is shown in Fig. 1. Only approx. 10-15% of the radioactivity in pooled fractions from the first peak (fractions 1-10) was precipitable by TCA and no protein could be detected by SDS-PAGE in these fractions. Since authentic BoltonHunter reagent (not coupled to protein) eluted in these fractions, we assumed that the majority of the radioactivity represented unbound Bolton-Hunter reagent and these fractions were not analyzed further. The fractions were analyzed by SDS-PAGE and those containing SP-C (fractions 18-40) were pooled (Fig. 2). The reason for the broad distribution of recovered protein is not known but may be due to variable states of aggregation, degrees of palmitoylation [23] or peptide length [24]. 97 ___1.5% (n = 3) of the radioactivity in the pooled fractions was precipitable with TCA. The radiolabeled SP-C was diluted with cold SP-C to a final specific activity of approx. 3 • 105 cpm//zg. In order to test if addition of the Bolton-Hunter reagent to SP-C alters its functional properties, the ability of SP-C reacted with non-radioactive Bolton-Hunter reagent to facilitate adsorption of phospholipids to an air/water interface was tested. As shown in Fig. 3, the SP-C labeled with the non-radioactive Bolton-Hunter reagent accelerated lipid adsorption in a manner similar to that of unlabeled (control) SP-C.
A
2.8. Binding/uptake studies Type II cells were resuspended in medium at a concentration of 2.5 • 10 6 cells/ml in microfuge tubes. If SP-A were included in the experiment, it was added prior to the addition of the SP-C-lipid complex. The mixture was incubated for various lengths of time and the ceils were washed by centrifugation as previously described [11]. Radioactivity was analyzed in a Packard Cobra gamma counter. The binding/uptake studies were performed in MEM, which contains approx. 2 /zM calcium, in order to minimize the aggregation of lipids. For degradation studies, samples after centrifugation were resuspended in 200/zl of medium and 200/xl of 5% bovine serum albumin, 200 /xl of water and 200 /xl of 50% trichloroacetic acid (TCA) were added. The samples were allowed to sit on ice for 30 min and then were centrifuged in the cold at top speed in a microfuge. The supernatant was removed and the end of the microfuge tube containing the pellet was cut off with a razor blade and both were analyzed by gamma counting.
Coomassie
I
I
ST
SP-C
I
I
I 12sI-SP-C
I
B Autorad
Q
Fig. 2. SDS-PAGE of pooled fractions from purified SP-C and 125I-labeled SP-C. (A) Gel stained with Coomassie Blue. (B) An autoradiogram of gel shown in A.
R.A. Pinto et al. / Biochimica et Biophysica Acta 1255 (1995) 16-22 7o-
'e
6O
x Z
5O
A
B
19
C
40
g~
20
100
I 0
Time (mJn)
1
Time (min)
Time (min)
Fig. 3. Surface tension reducing properties of SP-C and Bolton-Hunter modified SP-C. (A) Rate of film formation of SP-C and lipids. (B) Rate of film formation of Bolton-Hunter modified SP-C and lipids. (C) Rate of film formation of lipids alone. The rate at which surface pressure was increased was measured in an adsorption chamber. A mixture of SP-C and surfactant lipids (ratio 1:10, weight/weight and final phospholipid concentration 1.25/xg/ml) was injected into the subphase and film formation as a function of time was measured.
The morphology of the SP-C/liposome complex after passage through the French-press was examined by negative staining (Fig. 4). The preparation contained many small disk-like structures; the minimal edge thickness of o the smaller disks measured 55 A, the thickness of a single lipid bilayer of this lipid composition. The larger structures may be a mixture of collapsed vesicles and larger discs. 3.2. Interaction of SP-C with type H cells The time course of association of SP-C with isolated type II cells is shown in Fig. 5. A phase of rapid association of SP-C with isolated type II cells occurred over the first 15 min of incubation followed by a phase of slower
association. All further incubations were carried out over 120 min. We estimate that approx. 8 ng of SP-C was associated with 106 type II cells after 2 h of incubation with 1 /zg of SP-C, assuming that the specific activity did not change during the course of the experiments. The binding of SP-C to isolated type II cells was dependent upon protein concentration (Fig. 6) and increased linearly with increasing concentrations of SP-C. In contrast to previous reports [25,26] characterizing binding of SP-A to isolated type II cells, binding of SP-C was not saturable over the concentration range tested. The interaction of SP-C with type II cells was dependent upon temperature: at 37°C, the association averaged 3954 _ 498 dpm, whereas at 4°C the association averaged
Fig. 4. Negativestain of the French-pressedSP-C/liposome complex.White arrows point to smaller discs that have the appropriatethickness of a single bilayer seen on edge. MagnificationX 180000, white bar indicates 100 nm.
R.A. Pinto et al. / Biochimica et Biophysica Acta 1255 (1995) 16-22
20 3500
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2500
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~
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500
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I
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"lime (rain) Fig. 5. Time-dependent association of SP-C with isolated type II cells. SP-C (1/~g) complexed with lipids (100/zg) was incubated with 2.5.106 type II cells in 1 ml of medium for various times at 37°C. After washing by centrifugation, the cell pellet was analyzed for radioactivity. The maximal binding of 2500 cpm represents approx. 8 ng of SP-C. Values shown are mean _+S.E., n = 3.
1882 + 269 (mean _ S.E., n = 3, P < 0.05). Thus, the association at 37°C was approx. 2-fold greater than association at 4°C. 3.3. SP-A enhances the interaction of SP-C with type H cells
Time (min) Fig. 7. Effects of SP-A on association of SP-C with isolated type II cells. Cells were incubated with 10/~g SP-A, 1 /~g SP-C, and 100/~g lipid/ml for various lengths of time at 37°C. The data shown are representative of 3 experiments in that the association of SP-C with type II cells was greater in the presence than in the absence of SP-A in every experiment at every time point measured.
cells. As shown in Fig. 7, SP-A increased the interaction of SP-C with isolated type II cells at both 30 min and 120 min of incubation. 3.4. Type II cells do not degrade SP-C
We have previously demonstrated that recombinant SPC, when instilled in liposomes into the lungs of spontaneously breathing, anaesthetized rats, is taken up by type II cells and incorporated into lamellar bodies [18]. Because the exogenously administered SP-C could interact with endogenous surfactant lipids and proteins, we examined the effects of SP-A on interaction of SP-C with type II
6000
In order to determine if type II cells degrade SP-C, the apoprotein was incubated in the presence or absence of type II cells and the amount of radioactivity that could be precipitated by TCA was assessed. As shown in Table 1, very little TCA-soluble material could be detected associated with the cells. Similar results were obtained in 2 experiments in which media samples were analyzed (data not shown). Thus, it appears that type II cells do not significantly degrade SP-C as assessed by formation of TCA-soluble radioactivity.
5000
.>_.
n-
4000
Table 1 Degradation of SP-C by isolated type II cells
3000
Time (min)
TCA precipitable radioactivity (total%) SP-C
SP-C + SP-A
0 15 30 60 120
99.3 5:0.3 (3) 99.3 + 0.3 (3) 99 :t: 0 (3) 99, 100 (2) 99, 100 (2)
98.3 + 0.7 (3) 96, 100 (2) 96 + 1.4 (3) 95, 100 (2) 95, 100 (2)
2000
1000
0
0
I
I
I
I
20
40
60
80
I
1O0
I
120
I
140
I
160
SP-C(I.tg/ml) Fig. 6. Concentration-dependence of association of SP-C with isolated type II cells. Cells were incubated for 120 min at 37°C as described in Fig. 3 but with varying amounts of SP-C. Individual values from two separate experiments are shown.
A complex of SP-C (1 /.~g) and 100/~g of liposomes was incubated alone or with 10 /~g SP-A/ml at 37°C for various lengths of time. The cells were washed by centrifugation and the percentage of total cell-associated radioactivity that could be precipitated by tricholoracetic acid (TCA) was measured. The number in the parenthesis indicates the number of experiments. Values are mean + S.E. for 3 experiments or individual values for 2 experiments.
R~A. Pinto et aL /Biochimica et Biophysica Acta 1255 (1995) 16-22
4. Discussion The studies presented in this communication demonstrate that SP-C can be labeled in vitro with 125I-BoltonHunter reagent to a high specific activity and still retain its ability to accelerate the insertion of lipids into an air/water interface. The labeled SP-C interacts with type II cells in a time, temperature, and concentration-dependent manner and the magnitude of interaction of SP-C is enhanced by SP-A. Furthermore, it was demonstrated that SP-C is not degraded by isolated type II cells under the experimental conditions tested. To the best of our knowledge these are the first studies examining the interaction of SP-C with isolated type II cells, although previous studies have investigated metabolism of SP-C in whole animals. Baritussio and co-workers [16] compared the rates of clearance of phosphatidylcholine and metabolically labeled SP-C in adult rabbits and concluded that the turnover rate of SP-C is faster than that of saturated phosphatidylcholine. We have recently reported [18] that exogenously administered recombinant SP-C is taken up from the airspace and incorporated into lamellar bodies in spontaneously breathing anaesthetized rats. These reports are consistent with studies by Baritussio and co-workers [17] and suggest that SP-C, like SP-A [6,8,10] and SP-B [15], may also be internalized by the type II cell and incorporated into lamellar bodies or the multivesicular compartment of composite bodies [10]. It is difficult to determine if the amount of binding we observed is physiologically relevant because there are no reports (of which we are aware) of alveolar type II cell surfactant protein pool size. However, the magnitude of SP-C association with type II cells was similar to that reported for binding of SP-B to type II cells. We found that approx. 8 ng of SP-C associated with 10 6 isolated type II cells and Breslin and Weaver [15] reported binding of approx. 0.08 ng SP-B//xg cell protein. We have previously found that 10 6 cells contain approx. 70-100 /xg protein (unpublished observation). Thus, the magnitude of binding of SP-B and SP-C are comparable: approx. 8 ng/106 cells. In contrast, we previously observed that approx. 30-50 ng of SP-A bound to 1 0 6 freshly isolated type II cells [26]. The association of SP-C with isolated type II cells was greater at 37°C than at 4°C. One interpretation of these results is that the association with cells is dependent upon metabolic energy. However, other factors may account for the increase, such as a temperature-dependent change in the liposome/SP-C complex. Because the exogenously administered complex of SP-C and liposomes might interact with endogenous surfactant components in the alveoli in the whole animal studies, we tested whether SP-A affected the interaction of SP-C with isolated type II cells in vitro. We found that SP-A increases the interaction of SP-C with type II cells. Although investigation of the mechanism of this enhancement is
21
beyond the scope of this study, two possibilities seem likely. First, SP-A may cause aggregation of the SPC/liposome complex [27]. We purposely performed the studies in medium that contained a low concentration of calcium (approx. 2 /zM) to minimize SP-A-induced lipid aggregation. When we examined the effects of SP-A on aggregation of the SP-C-containing liposomes in a balanced salt solution containing millimolar calcium, we found SP-A enhanced the sedimentation of radioactive liposomes. Although we could not detect an increase in sedimentation of radioactivity in the low calcium medium, it is certainly possible that smaller aggregated complexes were formed that were not precipitated by the low speed centrifugation. A second possibility is that SP-A associates with the SP-C/liposome complex and that the entire complex is internalized via the SP-A receptor. Additional studies will be required to investigate these possibilities. Although SP-A appears to interact with isolated type II cells via a saturable high-affinity receptor [6,25,26], our studies suggest that the interaction of SP-C with type II cells is not saturable over a concentration range of 25 to 150 /zg/ml. These studies are consistent with previous work reported by Rice and co-workers [12] in which they reported that SP-C enhanced the association of phospholipids with isolated type II cells and that the effects of SP-C on lipid uptake were not saturable. They suggested that the enhancement of lipid uptake by SP-C was due to protein-mediated alterations in lipid structure rather than a receptor-mediated process. The results of the current study suggest that isolated type II cells do not significantly degrade SP-C, at least under these experimental conditions. It is of interest that type II cells also do not degrade SP-A [25]. Recently we have observed however that SP-A is degraded by alveolar macrophages in vitro (Wright, unpublished observations). Additional studies will be required to characterize the degradative pathways of SP-C. The SP-C used in this study was purified from a patient with alveolar proteinosis. It was necessary to use this protein because of the prohibitive cost of purifying adequate amounts of SP-C from animals. Although we have previously found that SP-A isolated from lavage of patients with alveolar proteinosis is functional in regulating many cellular functions, much less information is available about SP-C purified from alveolar proteinosis lavage fluid. After this study was completed, Voss and co-workers [24] reported that SP-C from alveolar proteinosis patients contained fewer palmitate residues and had more N-terminal proteolytic degradation than SP-C isolated from normal lungs. Therefore, the results of our study should be interpreted with this observation in mind. SP-C is a component of some forms of surfactant replacement therapy used to treat premature infants suffering from Respiratory Distress Syndrome [28]. The results present in this study are consistent with the possibility that exogenously administered surfactant proteins may also be
22
R.A. Pinto et al. / Biochimica et Biophysica Acta 1255 (1995) 16-22
'recycled' into type II cells in patients with Respiratory Distress Syndrome who receive surfactant replacement therapy provided that the cells' internalization processes have become competent. Important future investigations might include studies to determine if SP-C is taken up by type II cells in newborn, especially premature newborn, animals and to determine if SP-C has an effect on synthesis and/or secretion of surfactant components by type II cells. In summary, we have developed a method for in vitro labeling of SP-C with high specific activity and with retention of functional properties. The labeled SP-C associates with isolated type II cells in a time, temperature, and concentration-dependent manner and this association is enhanced by SP-A. These results suggest that the previously observed uptake of radiolabeled recombinant SP-C by type II cells in intact lung does not require, but may be enhanced by, endogenous SP-A. Additional studies will be required to more thoroughly define the mechanism of association and/or uptake of SP-C by type II cells.
Acknowledgements This work was supported in part by National Heart Lung and Blood Institute PPG HL-24075 (JRW, SH, JAC, RH), and a grant from the American Lung Association (RP). JRW is an Established Investigator of the American Heart Association, JAC is a Career Investigator of the American Heart Association, and SH is a Career Investigator of the American Lung Association. The authors also wish to thank Edward Hamilton for editorial assistance and figure preparation and Lysle Buchbinder for excellent technical assistance.
References [1] Wright, J.R. and Clements, J.A. (1989) Lung surfactant turnover and factors that affect turnover, in: Lung Cell Biology, pp. 655-699, Marcel Dekker, New York. [2] Jacobs, H., Jobe, A., Ikegami, M. and Conaway, D. (1983) J. Biol. Chem. 258, 4156-4165. [3] Jaeobs, H.C., Ikegami, M., Jobe, A.H., Berry, D.D. and Jones, S. (1985) Biochim. Biophys. Acta 837, 77-84. [4] Magoon, M.W., Wright, J.R., Baritussio, A., Williams, M.C., Go-
erke, J., Benson, B.J., Hamilton, R.L. and Clements, J.A. (1983) Biochim. Biophys. Acta 750, 18-31. [5] Jacobs, H.C., Jobe, A.H., Ikegami, M. and Jones, S. (1985) Biochim. Biophys. Acta 834, 172-179. [6] Ryan, R.M., Morris, R.E., Rice, W.R., Ciraolo, G. and Whitsett, J.A. (1989) J. Histochem. Cytochem. 37, 429-440. [7] Kalina, M. and Socher, R. (1990) J. Histochem. Cytochem. 38, 483-492. [8] Young, S.L., Fram, E.K., Larson, E. and Wright, J.R. (1993) Am. J. Physiol. (Lung Cell. Mol. Physiol. 9) 265, L19-L26. [9] Young, S.L., Fram, E.K. and Craig, B.L. (1985) Am. J. Anat. 174, 1-14. [10] Kalina, M., McCormack, F.X., Crowley, H., Voelker, D.R. and Mason, R.J. (1993) Journal of Histochemistry and Cytochemistry 41, 57-70. [11] Wright, J.R., Wager, R.E., Hawgood, S., Dobbs, L. and Clements, J.A. (1987) J. Biol. Chem. 262, 2888-2894. [12] Rice, W.R., Sarin, V.K., Fox, J.L., Baatz, J., Wert, S. and Whitsett, J.A. (1989) Biochim. Biophys. Acta 1006, 237-245. [13] Rice, W.R., Ross, G.F., Singleton, F.M., Dingle, S. and Whitsett, J.A. (1987) J. Appl. Physiol. 63, 692-698. [14] Dobbs, L.G., Wright, J.R., Hawgood, S., Gonzalez, R., Venstrom, K. and Nellenbogen, J. (1987) Proc. Natl. Acad. Sci. USA 84, 1010-1014. [15] Breslin, J.S. and Weaver, T.E. (1992) Am. J. Physiol. 262, L699707. [16] Baritussio, A., Benevento, M., Pettenazzo, A., Bruni, R., Santucci, A., Dalzoppo, D., Barcaglioni, P. and Crepaldi, G. (1989) Biochim. Biophys. Acta 1006, 19-25. [17] Baritussio, A., Pettenazzo, A., Benevento, M., Alberti, A. and Gamba, P. (1992) Am. J. Physiol. 263, L607-11. [18] Pinto, R.P., Wright, J.R., Lesikar, D., Benson, B.J. and Clements, J.A, (1993) J. Appl. Physiol. 74, 1005-1011. [19] Dobbs, L.G., Gonzalez, R. and Williams, M.C. (1986) Am. Rev. Respir. Dis. 134, 141-145. [20] Warr, R.G., Hawgood, S., Buckley, D.I., Crisp, T.M., Schilling, J., Benson, B.J., Ballard, P.L., Clements, J.A. and White, R.T. (1987) Proc. Natl. Acad. Sci. USA 84, 7915-7919. [21] Hamilton, R.L, Jr., Goerke, J., Guo, L.S.S., Williams, M.C. and Havel, R.J. (1980) J. Lipid. Res. 21, 981-992. [22] King, R.J. and Clements, J.A. (1972) Am. J. Physiol. 223, 715-726. [23] Curstedt, T., Johansson, J., Persson, P., Eklund, A., Robertson, B., Lfwenadler, B. and JiSrvall, H. (1990) Proc. Natl. Acad. Sci. USA 87, 2985-2989. [24] Voss, T., Schafer, K.P., Nielsen, P.F., Schafer, A., Maier, C., Hannappel, E., Maassen, J., Landis, B., Klemm, K. and M., P. (1992) Biochim. Biophys. Acta 1138, 261-267. [25] Kuroki, Y., Mason, R.J. and Voelker, D.R. (1988) Proc. Natl. Acad. Sci. USA 85, 5566-5570. [26] Wright, J.R., Borchelt, J.D. and Hawgood, S. (1989) Proc. Natl. Acad. Sci. USA 86, 5410-5414. [27] Poulain, F.R., Allen, L., Williams, M.C., Hamilton, R.L. and Hawgood, S. (1992) Am. J. Physiol. 262, L730-L739. [28] Jobe, A. and Ikegami, M. (1987) Am. Rev. Respir. Dis. 136, 1256-1275.
ELSEVIER
Biochimica et Biophysica Acta 1255 (1995) 16-22
Biochi~ic~a et BiophysicaA~ta
Association of surfactant protein C with isolated alveolar type II cells Ricardo A. Pinto a Samuel Hawgood a,c John A. Clements a,c, Bradley J. Benson d Asha Naidu d, Robert L. Hamilton e, Jo Rae Wright a,b,* a CardiovascularResearch Institute, University of California, San Francisco, CA 94143-0130, USA b Department of Physiology, University of California, San Francisco, CA 94143-0130, USA c Department of Pediatrics, University of California, San Francisco, CA 94143-0130, USA d Scios/Nova, Mountain View, CA 94043, USA e Department of Anatomy, University of California, San Francisco, CA 94143-0130, USA Received 1 April 1994; revised 16 September 1994; accepted 27 October 1994
Abstract
Surfactant protein C (SP-C) is a small hydrophobic protein that is synthesized and secreted by alveolar type II cells. The mechanism of clearance of SP-C from the alveolar airspace is not well understood, although previous studies demonstrated that recombinant SP-C instilled into the lungs of spontaneously breathing anaesthetized rats was taken up by type II cells and incorporated into lamellar bodies. The current investigation was undertaken to characterize the interaction of a complex of SP-C and surfactant-like lipids with freshly isolated rat alveolar type II cells under conditions in which the extracellular milieu can be regulated. SP-C was isolated from alveolar proteinosis lavage fluid and radiolabeled with 125I-Bolton-Hunter reagent. The radiolabeled protein retained its ability to facilitate adsorption of phospholipids to an air/liquid interface. Labeled human SP-C associated with isolated type II cells in a concentration-dependent manner that was also dependent upon temperature and time. The association of labeled SP-C with isolated type II cells did not saturate up to 150 /xg/ml. SP-A significantly enhanced the association of SP-C with isolated type II cells. Under the experimental conditions tested, SP-C was not degraded to TCA-soluble products. These results are consistent with the hypothesis that association or uptake of SP-C by type II cells may be enhanced by SP-A and that like SP-A, SP-C is recycled by type II cells. Keywords: Alveolar type II cell; Surfactant protein C; Surfactant protein A; Phospholipid; Liposome
I. Introduction
Pulmonary surfactant lipids and proteins are synthesized and secreted by the type II cells into the alveoli, where surfactant lipids reduce surface tension at the air/liquid interface. There are at least three processes by which surfactant lipids and proteins are cleared from the alveolar airspace: a degradative pathway in which components are metabolized and removed from the lung, a reutilization pathway in which degraded products are reutilized to synthesize new surfactant, and a recycling pathway in which intact surfactant components are taken up and incor-
Abbreviations: SP-A, surfactant protein A; SP-B, surfactant protein B; SP-C, surfactant protein C; MEM, minimal essential media; TCA, trichloroacetic acid; HPLC, high-performance liquid chromatography. * Corresponding author. Associate Professor of Cell Biology and Medicine. Present address: Box 3709, Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA. Fax: + 1 (919) 681 7978. 0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 4 ) 0 0 2 0 5 - 3
porated into lamellar bodies from which they are resecreted (reviewed in Ref. [1]). It was shown that lipid clearance proceeds via each of these pathways although the contribution of each pathway to total clearance varies according to age, species, and other factors. For example, the extent of recycling of lipids has been estimated to range from 23% to 85% in adult rabbits [2-4], up to 85% in 3-day old rabbits [5]. The quantitative aspects of clearance of surfactant proteins have been less well characterized. It has been demonstrated that SP-A binds to high affinity receptors on isolated type II cells and is internalized by a pathway that includes endosomal organelles associated with receptormediated endocytic pathways [6,7]. Using electron microscopic autoradiography, Young and co-workers [8] demonstrated that exogenously administered SP-A is incorporated into lamellar bodies, supporting biochemical studies which demonstrated that intratracheally administered SP-A could be recovered in isolated lamellar bodies [9]. Kalina and co-workers [7,10] also demonstrated that gold-labeled sur-
R.A. Pinto et al. /Biochimica et Biophysica Acta 1255 (1995) 16-22
factant is taken up by isolated type II cells and incorporated into the multivesicular portion of composite bodies. SP-A appears to play a role in feedback regulation of lipid uptake by enhancing lipid uptake [11,12] and inhibiting lipid secretion [13,14] by isolated type II cells. Recently Breslin and colleagues [15] reported that radiolabeled SP-B binds to and is internalized by type II cells, although they concluded that the binding is probably not mediated by a receptor. SP-C, like SP-A and SP-B, also appears to be taken up by type II cells. Baritussio and co-workers reported that SP-C is taken up from the alveolar space and incorporated into lamellar bodies and that SP-C is cleared from the alveolar airspace more quickly than is phosphatidylcholine [16,17]. We recently observed that recombinant radiolabeled SP-C instilled into the airways of anaesthetized rats is taken up by type II cells and incorporated into lamellar bodies [18]. The overall goal of the current study was to characterize further the interaction of SP-C with isolated alveolar type II cells. Specifically, a method of radiolabeling purified native SP-C with high specific activity without altering its functional ability to accelerate adsorption of surfactant lipids to an air/water interface was developed, the association of SP-C with isolated type II cells was characterized, the effects of SP-A on the association of SP-C with type II cells was examined, and the ability of type II cells to degrade SP-C was investigated.
2. Materials and methods
2.1. Materials and animals The following chemicals and reagents were purchased from their respective companies: reverse phase C8 Vydac cartridge (Western Analytical Products, Temecular, CA); elastase (Worthington, Malverne, PA), 125I-Bolton-Hunter Reagent (N-succinim idyl-3-(4-hydroxy-3(125I)iodophenyl)proprionate) (New England Nuclear/DuPont, Wilmington, DE); lipids (Avanti Polar Lipids, Birmingham, AL); minimal essential media (MEM) containing Krebs improved salts [11] (Cell Culture Facility, University of California, San Francisco, CA); all other chemicals (Sigma Chemical, St. Louis, MO). Adult male Sprague-Dawley rats, which were pathogen-free and weighed approx. 180 g, were obtained from Bantin-Kingrnan (Fremont, CA). The experiments were performed under institutional animal care protocols.
2.2. Isolation of cells Type II cells were isolated by techniques that are routine in this laboratory. Briefly, lungs of adult male rats were perfused free of blood and filled with elastase solution [19]. The minced lung tissue was filtered through
17
gauze and contaminating alveolar macrophages were removed by adherence to IgG-coated Petri dishes. Cell yields routinely averaged 20-30.106 cells/animal. All preparations had greater than 95% viability and 85% purity [11].
2.3. Isolation SP-C SP-C was isolated from the lavage fluid of a patient with alveolar proteinosis by previously described procedures [20]. Briefly, a 60 000 X g pellet of lavage fluid was extracted with butanol and the insoluble proteins were removed by centrifugation. The supernatant, which contains SP-B, SP-C, and lipids was chromatographed on a 1.5 X 80 cm LH-60 column in chloroform/methanol, 1:1 (v/v), containing 5% (v/v) 0.1 M HC1. Fractions were analyzed by SDS-PAGE followed by silver staining and those fractions containing proteins of molecular mass of approx. 4000 to 6000 were pooled.
2.4. Radiolabeling of SP-C The Bolton-Hunter reagent (250/~Ci) was dried under a gentle stream of nitrogen. 100/zg of SP-C was dried under a gentle stream of nitrogen and resuspended in 25 /xl of 4% octylglucoside and diluted with 25/~1 of 0.1 M sodium phosphate buffer, pH 8.06. 25 /xl of the above solution was added with a Hamilton syringe to the dried 125I-Bolton-Hunter reagent and incubated 1 h on ice and then overnight at 4°C. The incubation mixture was then diluted with 53/~1 of 95% isopropyl alcohol/5% 50 mM HC1 and injected onto a reverse phase C8 cartridge column on a Hewlett-Packard 1090 liquid chromatograph. The column was eluted with a gradient of 45% isopropanol containing 5% (v/v) 0.1 M HCI to 95% isopropanol containing 5% (v/v) 0.1 M HC1 at a flow rate of 0.4 ml/min. Fractions of 0.4 ml were collected. The optical density at 280 nm was read on-line and radioactivity in the fractions was measured in a gamma counter.
2.5. Preparation of liposomes Liposomes in medium were prepared from commercially available lipids by the method of Hamilton [21] using a French pressure cell. The liposomes consisted of 55% dipalmitoylphosphatidylcholine, 27% egg phosphatidylcholine, 10% egg phosphatidylglycerol and 8% cholesterol (by weight). 4 mg of lipid in solvent and 40/xg of SP-C were combined and dried in a round bottom flask. 4 ml of medium were added and mixed in the presence of small glass beads. The mixture was heated at 57°C for 30 minutes and passed through the French pressure cell 3 times at 20 000 psi.
2.6. Ultrastructure studies Negative staining of SP-C lipid complexes was performed as previously described [21].
18
R.A. Pinto et aL / Biochiraica et Biophysica Acta 1255 (1995) 16-22 4.0 x 105 -
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3. Results
3.1. Characteristics of radiolabeled SP-C 3.0 x 10s -
> 2.0 x 105-
rr 1.o x lO5.
'1'o ''o ' ' o ' ' o ','o Fraction Number Fig. 1. Elution profile of radiolabeled SP-C after separation on a C8 column by HPLC. Free 125I-Bolton-Hunter reagent was separated from that bound to protein by HPLC as described in Section 2. Fractions containing protein include 18-40, which were pooled and analyzed by SDS-PAGE (see Fig. 2).
2. 7. Adsorption balance studies The ability of SP-C modified with non-radioactive Bolton-Hunter reagent to accelerate the rate of phospholipid film formation was compared to that of unmodified SP-C in an adsorption chamber, which records the rate at which lipids adsorb to an air/liquid interface and reduce surface tension. SP-C was mixed with phospholipids exactly as previously described [18] and the rate of film formation was measured as described by King and Clements [22].
Radiolabeled SP-C was separated from unbound 1251by high performance liquid chromatography (HPLC). A typical profile of radioactivity from column fractions is shown in Fig. 1. Only approx. 10-15% of the radioactivity in pooled fractions from the first peak (fractions 1-10) was precipitable by TCA and no protein could be detected by SDS-PAGE in these fractions. Since authentic BoltonHunter reagent (not coupled to protein) eluted in these fractions, we assumed that the majority of the radioactivity represented unbound Bolton-Hunter reagent and these fractions were not analyzed further. The fractions were analyzed by SDS-PAGE and those containing SP-C (fractions 18-40) were pooled (Fig. 2). The reason for the broad distribution of recovered protein is not known but may be due to variable states of aggregation, degrees of palmitoylation [23] or peptide length [24]. 97 ___1.5% (n = 3) of the radioactivity in the pooled fractions was precipitable with TCA. The radiolabeled SP-C was diluted with cold SP-C to a final specific activity of approx. 3 • 105 cpm//zg. In order to test if addition of the Bolton-Hunter reagent to SP-C alters its functional properties, the ability of SP-C reacted with non-radioactive Bolton-Hunter reagent to facilitate adsorption of phospholipids to an air/water interface was tested. As shown in Fig. 3, the SP-C labeled with the non-radioactive Bolton-Hunter reagent accelerated lipid adsorption in a manner similar to that of unlabeled (control) SP-C.
A
2.8. Binding/uptake studies Type II cells were resuspended in medium at a concentration of 2.5 • 10 6 cells/ml in microfuge tubes. If SP-A were included in the experiment, it was added prior to the addition of the SP-C-lipid complex. The mixture was incubated for various lengths of time and the ceils were washed by centrifugation as previously described [11]. Radioactivity was analyzed in a Packard Cobra gamma counter. The binding/uptake studies were performed in MEM, which contains approx. 2 /zM calcium, in order to minimize the aggregation of lipids. For degradation studies, samples after centrifugation were resuspended in 200/zl of medium and 200/xl of 5% bovine serum albumin, 200 /xl of water and 200 /xl of 50% trichloroacetic acid (TCA) were added. The samples were allowed to sit on ice for 30 min and then were centrifuged in the cold at top speed in a microfuge. The supernatant was removed and the end of the microfuge tube containing the pellet was cut off with a razor blade and both were analyzed by gamma counting.
Coomassie
I
I
ST
SP-C
I
I
I 12sI-SP-C
I
B Autorad
Q
Fig. 2. SDS-PAGE of pooled fractions from purified SP-C and 125I-labeled SP-C. (A) Gel stained with Coomassie Blue. (B) An autoradiogram of gel shown in A.
R.A. Pinto et al. / Biochimica et Biophysica Acta 1255 (1995) 16-22 7o-
'e
6O
x Z
5O
A
B
19
C
40
g~
20
100
I 0
Time (mJn)
1
Time (min)
Time (min)
Fig. 3. Surface tension reducing properties of SP-C and Bolton-Hunter modified SP-C. (A) Rate of film formation of SP-C and lipids. (B) Rate of film formation of Bolton-Hunter modified SP-C and lipids. (C) Rate of film formation of lipids alone. The rate at which surface pressure was increased was measured in an adsorption chamber. A mixture of SP-C and surfactant lipids (ratio 1:10, weight/weight and final phospholipid concentration 1.25/xg/ml) was injected into the subphase and film formation as a function of time was measured.
The morphology of the SP-C/liposome complex after passage through the French-press was examined by negative staining (Fig. 4). The preparation contained many small disk-like structures; the minimal edge thickness of o the smaller disks measured 55 A, the thickness of a single lipid bilayer of this lipid composition. The larger structures may be a mixture of collapsed vesicles and larger discs. 3.2. Interaction of SP-C with type H cells The time course of association of SP-C with isolated type II cells is shown in Fig. 5. A phase of rapid association of SP-C with isolated type II cells occurred over the first 15 min of incubation followed by a phase of slower
association. All further incubations were carried out over 120 min. We estimate that approx. 8 ng of SP-C was associated with 106 type II cells after 2 h of incubation with 1 /zg of SP-C, assuming that the specific activity did not change during the course of the experiments. The binding of SP-C to isolated type II cells was dependent upon protein concentration (Fig. 6) and increased linearly with increasing concentrations of SP-C. In contrast to previous reports [25,26] characterizing binding of SP-A to isolated type II cells, binding of SP-C was not saturable over the concentration range tested. The interaction of SP-C with type II cells was dependent upon temperature: at 37°C, the association averaged 3954 _ 498 dpm, whereas at 4°C the association averaged
Fig. 4. Negativestain of the French-pressedSP-C/liposome complex.White arrows point to smaller discs that have the appropriatethickness of a single bilayer seen on edge. MagnificationX 180000, white bar indicates 100 nm.
R.A. Pinto et al. / Biochimica et Biophysica Acta 1255 (1995) 16-22
20 3500
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I
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"lime (rain) Fig. 5. Time-dependent association of SP-C with isolated type II cells. SP-C (1/~g) complexed with lipids (100/zg) was incubated with 2.5.106 type II cells in 1 ml of medium for various times at 37°C. After washing by centrifugation, the cell pellet was analyzed for radioactivity. The maximal binding of 2500 cpm represents approx. 8 ng of SP-C. Values shown are mean _+S.E., n = 3.
1882 + 269 (mean _ S.E., n = 3, P < 0.05). Thus, the association at 37°C was approx. 2-fold greater than association at 4°C. 3.3. SP-A enhances the interaction of SP-C with type H cells
Time (min) Fig. 7. Effects of SP-A on association of SP-C with isolated type II cells. Cells were incubated with 10/~g SP-A, 1 /~g SP-C, and 100/~g lipid/ml for various lengths of time at 37°C. The data shown are representative of 3 experiments in that the association of SP-C with type II cells was greater in the presence than in the absence of SP-A in every experiment at every time point measured.
cells. As shown in Fig. 7, SP-A increased the interaction of SP-C with isolated type II cells at both 30 min and 120 min of incubation. 3.4. Type II cells do not degrade SP-C
We have previously demonstrated that recombinant SPC, when instilled in liposomes into the lungs of spontaneously breathing, anaesthetized rats, is taken up by type II cells and incorporated into lamellar bodies [18]. Because the exogenously administered SP-C could interact with endogenous surfactant lipids and proteins, we examined the effects of SP-A on interaction of SP-C with type II
6000
In order to determine if type II cells degrade SP-C, the apoprotein was incubated in the presence or absence of type II cells and the amount of radioactivity that could be precipitated by TCA was assessed. As shown in Table 1, very little TCA-soluble material could be detected associated with the cells. Similar results were obtained in 2 experiments in which media samples were analyzed (data not shown). Thus, it appears that type II cells do not significantly degrade SP-C as assessed by formation of TCA-soluble radioactivity.
5000
.>_.
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4000
Table 1 Degradation of SP-C by isolated type II cells
3000
Time (min)
TCA precipitable radioactivity (total%) SP-C
SP-C + SP-A
0 15 30 60 120
99.3 5:0.3 (3) 99.3 + 0.3 (3) 99 :t: 0 (3) 99, 100 (2) 99, 100 (2)
98.3 + 0.7 (3) 96, 100 (2) 96 + 1.4 (3) 95, 100 (2) 95, 100 (2)
2000
1000
0
0
I
I
I
I
20
40
60
80
I
1O0
I
120
I
140
I
160
SP-C(I.tg/ml) Fig. 6. Concentration-dependence of association of SP-C with isolated type II cells. Cells were incubated for 120 min at 37°C as described in Fig. 3 but with varying amounts of SP-C. Individual values from two separate experiments are shown.
A complex of SP-C (1 /.~g) and 100/~g of liposomes was incubated alone or with 10 /~g SP-A/ml at 37°C for various lengths of time. The cells were washed by centrifugation and the percentage of total cell-associated radioactivity that could be precipitated by tricholoracetic acid (TCA) was measured. The number in the parenthesis indicates the number of experiments. Values are mean + S.E. for 3 experiments or individual values for 2 experiments.
R~A. Pinto et aL /Biochimica et Biophysica Acta 1255 (1995) 16-22
4. Discussion The studies presented in this communication demonstrate that SP-C can be labeled in vitro with 125I-BoltonHunter reagent to a high specific activity and still retain its ability to accelerate the insertion of lipids into an air/water interface. The labeled SP-C interacts with type II cells in a time, temperature, and concentration-dependent manner and the magnitude of interaction of SP-C is enhanced by SP-A. Furthermore, it was demonstrated that SP-C is not degraded by isolated type II cells under the experimental conditions tested. To the best of our knowledge these are the first studies examining the interaction of SP-C with isolated type II cells, although previous studies have investigated metabolism of SP-C in whole animals. Baritussio and co-workers [16] compared the rates of clearance of phosphatidylcholine and metabolically labeled SP-C in adult rabbits and concluded that the turnover rate of SP-C is faster than that of saturated phosphatidylcholine. We have recently reported [18] that exogenously administered recombinant SP-C is taken up from the airspace and incorporated into lamellar bodies in spontaneously breathing anaesthetized rats. These reports are consistent with studies by Baritussio and co-workers [17] and suggest that SP-C, like SP-A [6,8,10] and SP-B [15], may also be internalized by the type II cell and incorporated into lamellar bodies or the multivesicular compartment of composite bodies [10]. It is difficult to determine if the amount of binding we observed is physiologically relevant because there are no reports (of which we are aware) of alveolar type II cell surfactant protein pool size. However, the magnitude of SP-C association with type II cells was similar to that reported for binding of SP-B to type II cells. We found that approx. 8 ng of SP-C associated with 10 6 isolated type II cells and Breslin and Weaver [15] reported binding of approx. 0.08 ng SP-B//xg cell protein. We have previously found that 10 6 cells contain approx. 70-100 /xg protein (unpublished observation). Thus, the magnitude of binding of SP-B and SP-C are comparable: approx. 8 ng/106 cells. In contrast, we previously observed that approx. 30-50 ng of SP-A bound to 1 0 6 freshly isolated type II cells [26]. The association of SP-C with isolated type II cells was greater at 37°C than at 4°C. One interpretation of these results is that the association with cells is dependent upon metabolic energy. However, other factors may account for the increase, such as a temperature-dependent change in the liposome/SP-C complex. Because the exogenously administered complex of SP-C and liposomes might interact with endogenous surfactant components in the alveoli in the whole animal studies, we tested whether SP-A affected the interaction of SP-C with isolated type II cells in vitro. We found that SP-A increases the interaction of SP-C with type II cells. Although investigation of the mechanism of this enhancement is
21
beyond the scope of this study, two possibilities seem likely. First, SP-A may cause aggregation of the SPC/liposome complex [27]. We purposely performed the studies in medium that contained a low concentration of calcium (approx. 2 /zM) to minimize SP-A-induced lipid aggregation. When we examined the effects of SP-A on aggregation of the SP-C-containing liposomes in a balanced salt solution containing millimolar calcium, we found SP-A enhanced the sedimentation of radioactive liposomes. Although we could not detect an increase in sedimentation of radioactivity in the low calcium medium, it is certainly possible that smaller aggregated complexes were formed that were not precipitated by the low speed centrifugation. A second possibility is that SP-A associates with the SP-C/liposome complex and that the entire complex is internalized via the SP-A receptor. Additional studies will be required to investigate these possibilities. Although SP-A appears to interact with isolated type II cells via a saturable high-affinity receptor [6,25,26], our studies suggest that the interaction of SP-C with type II cells is not saturable over a concentration range of 25 to 150 /zg/ml. These studies are consistent with previous work reported by Rice and co-workers [12] in which they reported that SP-C enhanced the association of phospholipids with isolated type II cells and that the effects of SP-C on lipid uptake were not saturable. They suggested that the enhancement of lipid uptake by SP-C was due to protein-mediated alterations in lipid structure rather than a receptor-mediated process. The results of the current study suggest that isolated type II cells do not significantly degrade SP-C, at least under these experimental conditions. It is of interest that type II cells also do not degrade SP-A [25]. Recently we have observed however that SP-A is degraded by alveolar macrophages in vitro (Wright, unpublished observations). Additional studies will be required to characterize the degradative pathways of SP-C. The SP-C used in this study was purified from a patient with alveolar proteinosis. It was necessary to use this protein because of the prohibitive cost of purifying adequate amounts of SP-C from animals. Although we have previously found that SP-A isolated from lavage of patients with alveolar proteinosis is functional in regulating many cellular functions, much less information is available about SP-C purified from alveolar proteinosis lavage fluid. After this study was completed, Voss and co-workers [24] reported that SP-C from alveolar proteinosis patients contained fewer palmitate residues and had more N-terminal proteolytic degradation than SP-C isolated from normal lungs. Therefore, the results of our study should be interpreted with this observation in mind. SP-C is a component of some forms of surfactant replacement therapy used to treat premature infants suffering from Respiratory Distress Syndrome [28]. The results present in this study are consistent with the possibility that exogenously administered surfactant proteins may also be
22
R.A. Pinto et al. / Biochimica et Biophysica Acta 1255 (1995) 16-22
'recycled' into type II cells in patients with Respiratory Distress Syndrome who receive surfactant replacement therapy provided that the cells' internalization processes have become competent. Important future investigations might include studies to determine if SP-C is taken up by type II cells in newborn, especially premature newborn, animals and to determine if SP-C has an effect on synthesis and/or secretion of surfactant components by type II cells. In summary, we have developed a method for in vitro labeling of SP-C with high specific activity and with retention of functional properties. The labeled SP-C associates with isolated type II cells in a time, temperature, and concentration-dependent manner and this association is enhanced by SP-A. These results suggest that the previously observed uptake of radiolabeled recombinant SP-C by type II cells in intact lung does not require, but may be enhanced by, endogenous SP-A. Additional studies will be required to more thoroughly define the mechanism of association and/or uptake of SP-C by type II cells.
Acknowledgements This work was supported in part by National Heart Lung and Blood Institute PPG HL-24075 (JRW, SH, JAC, RH), and a grant from the American Lung Association (RP). JRW is an Established Investigator of the American Heart Association, JAC is a Career Investigator of the American Heart Association, and SH is a Career Investigator of the American Lung Association. The authors also wish to thank Edward Hamilton for editorial assistance and figure preparation and Lysle Buchbinder for excellent technical assistance.
References [1] Wright, J.R. and Clements, J.A. (1989) Lung surfactant turnover and factors that affect turnover, in: Lung Cell Biology, pp. 655-699, Marcel Dekker, New York. [2] Jacobs, H., Jobe, A., Ikegami, M. and Conaway, D. (1983) J. Biol. Chem. 258, 4156-4165. [3] Jaeobs, H.C., Ikegami, M., Jobe, A.H., Berry, D.D. and Jones, S. (1985) Biochim. Biophys. Acta 837, 77-84. [4] Magoon, M.W., Wright, J.R., Baritussio, A., Williams, M.C., Go-
erke, J., Benson, B.J., Hamilton, R.L. and Clements, J.A. (1983) Biochim. Biophys. Acta 750, 18-31. [5] Jacobs, H.C., Jobe, A.H., Ikegami, M. and Jones, S. (1985) Biochim. Biophys. Acta 834, 172-179. [6] Ryan, R.M., Morris, R.E., Rice, W.R., Ciraolo, G. and Whitsett, J.A. (1989) J. Histochem. Cytochem. 37, 429-440. [7] Kalina, M. and Socher, R. (1990) J. Histochem. Cytochem. 38, 483-492. [8] Young, S.L., Fram, E.K., Larson, E. and Wright, J.R. (1993) Am. J. Physiol. (Lung Cell. Mol. Physiol. 9) 265, L19-L26. [9] Young, S.L., Fram, E.K. and Craig, B.L. (1985) Am. J. Anat. 174, 1-14. [10] Kalina, M., McCormack, F.X., Crowley, H., Voelker, D.R. and Mason, R.J. (1993) Journal of Histochemistry and Cytochemistry 41, 57-70. [11] Wright, J.R., Wager, R.E., Hawgood, S., Dobbs, L. and Clements, J.A. (1987) J. Biol. Chem. 262, 2888-2894. [12] Rice, W.R., Sarin, V.K., Fox, J.L., Baatz, J., Wert, S. and Whitsett, J.A. (1989) Biochim. Biophys. Acta 1006, 237-245. [13] Rice, W.R., Ross, G.F., Singleton, F.M., Dingle, S. and Whitsett, J.A. (1987) J. Appl. Physiol. 63, 692-698. [14] Dobbs, L.G., Wright, J.R., Hawgood, S., Gonzalez, R., Venstrom, K. and Nellenbogen, J. (1987) Proc. Natl. Acad. Sci. USA 84, 1010-1014. [15] Breslin, J.S. and Weaver, T.E. (1992) Am. J. Physiol. 262, L699707. [16] Baritussio, A., Benevento, M., Pettenazzo, A., Bruni, R., Santucci, A., Dalzoppo, D., Barcaglioni, P. and Crepaldi, G. (1989) Biochim. Biophys. Acta 1006, 19-25. [17] Baritussio, A., Pettenazzo, A., Benevento, M., Alberti, A. and Gamba, P. (1992) Am. J. Physiol. 263, L607-11. [18] Pinto, R.P., Wright, J.R., Lesikar, D., Benson, B.J. and Clements, J.A, (1993) J. Appl. Physiol. 74, 1005-1011. [19] Dobbs, L.G., Gonzalez, R. and Williams, M.C. (1986) Am. Rev. Respir. Dis. 134, 141-145. [20] Warr, R.G., Hawgood, S., Buckley, D.I., Crisp, T.M., Schilling, J., Benson, B.J., Ballard, P.L., Clements, J.A. and White, R.T. (1987) Proc. Natl. Acad. Sci. USA 84, 7915-7919. [21] Hamilton, R.L, Jr., Goerke, J., Guo, L.S.S., Williams, M.C. and Havel, R.J. (1980) J. Lipid. Res. 21, 981-992. [22] King, R.J. and Clements, J.A. (1972) Am. J. Physiol. 223, 715-726. [23] Curstedt, T., Johansson, J., Persson, P., Eklund, A., Robertson, B., Lfwenadler, B. and JiSrvall, H. (1990) Proc. Natl. Acad. Sci. USA 87, 2985-2989. [24] Voss, T., Schafer, K.P., Nielsen, P.F., Schafer, A., Maier, C., Hannappel, E., Maassen, J., Landis, B., Klemm, K. and M., P. (1992) Biochim. Biophys. Acta 1138, 261-267. [25] Kuroki, Y., Mason, R.J. and Voelker, D.R. (1988) Proc. Natl. Acad. Sci. USA 85, 5566-5570. [26] Wright, J.R., Borchelt, J.D. and Hawgood, S. (1989) Proc. Natl. Acad. Sci. USA 86, 5410-5414. [27] Poulain, F.R., Allen, L., Williams, M.C., Hamilton, R.L. and Hawgood, S. (1992) Am. J. Physiol. 262, L730-L739. [28] Jobe, A. and Ikegami, M. (1987) Am. Rev. Respir. Dis. 136, 1256-1275.