Clearance of SP-C and recombinant SP-C in vivo and in vitro

Machiko Ikegami, Ann D. Horowitz, Jeffrey A. Whitsett, and Alan H. Jobe

Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Surfactant protein (SP) C metabolism was evaluated in vivo by measurements of the clearance of bovine native SP-C (nSP-C) and a recombinant SP-C (rSP-C) in rabbits and mice and in vitro by the uptake into MLE-12 cells. rSP-C is the 34-amino acid human sequence with phenylalanine instead of cysteine in positions 4 and 5 and isoleucine instead of methionine in position 32. Alveolar clearances of iodinated SP-C and rSP-C after tracheal instillation were similar and slower than those for dipalmitoyl phosphatidylcholine (DPC) in the rabbit. nSP-C and rSP-C were cleared from rabbit lungs similarly to DPC, each with a half-life (t1/2) of ~11 h. In mice, the clearance of rSP-C from the lungs was slower (t1/2 28 h) than the clearance of DPC (t1/2 12 h). Liposome-associated dinitrophenyl-labeled rSP-C was taken up by MLE-12 cells, and the uptake was inhibited by excess nSP-C. The pattern of inhibition of dinitrophenyl-rSP-C uptake by SP-B, but not by SP-A, was similar to that previously reported for nSP-C. Clearance kinetics of nSP-C were similar to previous measurements of pulmonary clearance of SP-B in rabbits and mice. rSP-C has clearance kinetics and uptake by cells similar to those of nSP-C.

surfactant protein C; lung; dipalmitoyl phosphatidylcholine; rabbits; mice

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

SURFACTANT PROTEIN (SP) C is the most lipophilic of the three SPs SP-A, SP-B, and SP-C. SP-C is synthesized as a 21-kDa primary translation product that is cleaved to form an exceptionally hydrophobic protein of 3.5 kDa. The active SP-C peptide is not immunogenic, is difficult to separate from SP-B and lipids, and is not reliably measured with standard protein assays (5). Antibodies to the proSP-C translation products were used to demonstrate its translocation into "prelamellar body" organelles (6, 31), and mature SP-C is present in high concentration in lamellar bodies (33). Intracellular processing of de novo synthesized SP-B and SP-C follows a similar path, and inactivation of the SP-B gene disrupts the secretory pathway for surfactant lipids and results in abnormal processing of SP-C (7), suggesting interactions between proSP-B and proSP-C within the biosynthetic pathway. Although the biophysical properties of SP-C have been studied extensively in vitro (5), the functions of SP-C within the air space are poorly understood. SP-C facilitates surface adsorption of phospholipids (25, 32) and alters phospholipid organization in surface films to yield small condensed domains (21). The protein stimulates liposomal fusion in vitro (24) and enhances the binding of lipid vesicles to cell membranes in association with the endocytosis of the lipids (16, 26). SP-C is found in tubular myelin and large lipid arrays in alveolar surfactant, but it is not detected in the small vesicles thought to be catabolic forms of surfactant lipids that are cleared from the air space primarily by type II cells and macrophages (1, 33). SP-C is taken up from the air spaces in vivo and subsequently concentrates in lamellar bodies (3, 23). Recent studies with fluorescently labeled SP-C or dinitrophenyl (DNP)-SP-C conjugates (DNP-SP-C) demonstrate that SP-C augments the uptake of liposomes by MLE-12 and type II cells (15). Although SP-B has been considered essential for surfactant function, surfactants containing only SP-C or SP-C analogs and lipids can improve lung function of surfactant-deficient preterm animals or lung-injured adult animals (11, 12). The kinetics of alveolar and lung clearance of native SP-C (nSP-C) are not well characterized, and there is no information about the metabolism of human recombinant SP-C (rSP-C) analogs of potential clinical utility. Therefore, we have combined in vivo measurements of SP-C clearance in two species with in vitro assessments of the uptake of rSP-C by MLE-12 cells to begin to evaluate the metabolism of SP-C.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

SPs. nSP-C was purified from lipid extracts of bovine lung lavage by chromatography on C-8 silica and Sephadex LH-60, with quantification by amino acid compositional analysis as previously described (15). rSP-C (Byk Gulden, Constance, Germany) is the 34-amino acid human SP-C sequence altered by the replacement of cysteine by phenylalanine in positions 4 and 5 and of methionine by isoleucine in position 32. This rSP-C, expressed in bacteria and purified, was designed to circumvent the aggregation characteristics of SP-C while maintaining the physical properties of the dipalmitoylated form of nSP-C. This rSP-C has excellent surfactant function in vitro and in surfactant-deficient animals (8). SP-B was purified from the same organic solvent extract of bovine lung lavage used for the isolation of nSP-C. SP-B was separated from SP-C and lipids by LH-60 chromatography (13). SP-A was isolated from human alveolar proteinosis lavage fluid according to Haagsman et al. (10) and was a gift from Dr. Gary Ross (Children's Hospital Medical Center, Cincinnati, OH).

Iodination of SP-C. nSP-C was iodinated with 125I-labeled Bolton-Hunter reagent (ICN, Irvine, CA) by adding 260 µg of nSP-C in 500 µl of methanol to 120 µl of 0.3 M phosphate buffer, with pH adjusted to 8.5 with NaOH. The 125I-nSP-C was purified by extensive dialysis against chloroform-methanol (2:1, vol/vol). The radiolabeled SP-C was run on a polyacrylamide-SDS gel, and an autoradiograph had one band at the appropriate molecular weight. When the gel was cut and the radioactivity was measured, 90.4% of the radioactivity on the gel was associated with the band. rSP-C (300 µg in 450 µl of propanol) was iodinated similarly, with 91.5% of the radioactivity associated with the SP-C band after gel electrophoresis. The iodinated proteins were mixed with dipalmitoyl phosphatidylcholine (DPC) and stored in chloroform-methanol (2:1, vol/vol) until used. For intratracheal injection, 125I-nSP-C or 125I-rSP-C and [3H]choline-labeled DPC (American Radiolabeled Chemicals, St. Louis, MO) were mixed in chloroform, dried on a round-bottom flask, and resuspended in 0.15 M NaCl with glass beads (30). [3H]DPC was used to verify that the clearance of DPC was similar to clearances measured previously with either radiolabeled DPC alone or when mixed with surfactant lipids (17, 27, 29).

Procedures with rabbits. Groups of five to seven rabbits were randomized to receive intratracheal injections with 125I-SP-C and [3H]DPC followed by measurement of the radiolabels at preselected times based on previous measurements for DPC, SP-A, and SP-B in the same model (29, 30). The rabbits were sedated with a CO2-O2 gas mixture to facilitate passing a 3.5-Fr catheter through the cords with direct visualization using a modified bronchoscope (Olympus, Melville, NY) (29). Each rabbit then was given 2 ml of a 0.15 M NaCl solution containing 0.5 mg of DPC, 4.5 µCi of [3H]DPC, a trace amount of SP-C or rSP-C, and 0.5 µCi of 125I-nSP-C or 125I-rSP-C via a catheter measured to be just proximal to the carina. The amount of DPC was ~3% of the alveolar DPC pool size. At preselected times after injection, each rabbit was killed with 100 mg/kg of pentobarbital sodium given by intravascular injection and exsanguinated by cutting the abdominal aorta. The chest was opened, the lungs were filled with a 0.15 M NaCl solution at 4°C, and the fluid was withdrawn and reinfused three times by syringe and recovered. This procedure was repeated five times, the recovered lavage fluid was pooled, and the volume was measured. Aliquots were used to measure the recoveries of the radiolabels. Alveolar lavages also were used to isolate macrophages by centrifugation using sucrose step gradients (29). The lungs were removed and homogenized in a 0.15 M NaCl solution. To measure recovery of [3H]DPC, aliquots of alveolar lavages and lung homogenates were extracted with chloroform-methanol (2:1, vol/vol) and treated with osmium tetroxide in carbon tetrachloride followed by alumina column chromatography to recover saturated phosphatidylcholine (Sat PC), which was used for measuring 3H and phosphorus assay (4, 20).

Procedures with mice. NIH black Swiss mice (7-9 wk old) were randomized to groups of eight or nine animals for measurement of the clearances of [3H]DPC and 125I-rSP-C at preselected times to 40 h after intratracheal injection on the basis of previous measurements for DPC, SP-A. and SP-B in mice (17, 19). Each mouse was given, by intratracheal injection, 50 µl of saline that contained 5 µg of DPC, 0.25 µCi of [3H]DPC, and 0.025 µCi of 125I-rSP-C. This amount of DPC is ~2% of the alveolar pool of Sat PC in mice. For a tracheal injection, the mice were sedated with intraperitoneal ketamine (50 mg/kg). The trachea was exposed through a 0.5-cm midline skin incision in the neck, and the isotope mixture was injected with a 30-gauge needle (17). Each mouse was killed with intraperitoneal pentobarbital sodium (100 mg/kg), the distal aorta was cut to exsanguinate the animal, and the chest was opened. A 20-gauge blunt needle was tied into the trachea, and a 0.15 M NaCl solution at 4°C was instilled into the lungs to achieve full inflation and then withdrawn from the lungs three times. The procedure was repeated five times. The recovered lavage fluids were pooled, the volume was measured, and the lungs were removed. 125I radioactivity was measured for the alveolar lavage fluid and lung tissue after alveolar lavage. The lungs were homogenized in 0.15 M NaCl, and aliquots were subsequently used to isolate Sat PC.

rSP-C uptake by MLE-12 cells. rSP-C was labeled with DNP [6-(2,4-dinitrophenyl)aminohexanodic acid, succinimidyl ester; Molecular Probes, Eugene, OR] to produce DNP-rSP-C as previously described for bovine SP-C (15). DNP was an accessible antigen that permitted measurement of rSP-C. DNP-rSP-C was purified by Sephadex LH-60 chromatography. Successful labeling was verified by SDS-PAGE followed by immunoblotting with anti-DNP antibody. The amount of labeling was quantitated by the absorbance of DNP at 349 nm. The conjugation of SP-C with DNP did not interfere with the surface properties of SP-C (15). The DNP moiety was detected by incubation with 1:10,000 rabbit anti-DNP-keyhole limpet hemocyanin IgG followed by 1:10,000 horseradish peroxidase-labeled goat anti-rabbit IgG.

Multilamellar liposomes for uptake experiments were prepared with a lipid mixture that approximates the acyl chain and lipid composition of pulmonary surfactant [1:5.5:3.3:0.24 (wt/wt) dioleoylphosphatidylglycerol-egg PC-DPC-cholesterol] (15). The lipids or the lipids mixed with DNP-rSP-C and/or SP-B and nSP-C in chloroform were dried under a stream of nitrogen, with heating to 50°C. After removal of residual solvent under vacuum at 50°C, vesicles were formed by rehydration in 20 mM HEPES, pH 7.4, and 0.15 M NaCl. Multilamellar vesicles were formed by vortexing for 30 s and sonicating in a bath-type sonicator for 30 s. The vesicles were diluted in RPMI 1640 medium without phenol red and buffered with 20 mM HEPES, pH 7.6 (H-RPMI), before incubation with the cells. SP-A was added to the vesicles diluted in H-RPMI at room temperature before incubation with the cells.

The mouse lung epithelial cell line MLE-12 was grown in supplemented RPMI 1640 medium on glass coverslips coated with bovine type I collagen (Sigma). The cell monolayer was washed two times with H-RPMI and was incubated in H-RPMI for 30 min at 37°C before the addition of liposomes. Immunocytochemical detection of bound and internalized DNP-rSP-C was performed exactly as described previously for DNP-nSP-C (15). The procedure utilizes rabbit anti-DNP-keyhole limpet hemocyanin IgG as the primary antibody, followed by horseradish peroxidase-labeled goat anti-rabbit IgG. Diaminobenzidine and hydrogen peroxide were used as substrates, producing a dark precipitate in the presence of horseradish peroxidase. The total area of each image occupied by the cells and the area of the cells occupied by stain for DNP-rSP-C were measured from transmitted light micrographs analyzed with Meta Morph software (15). Each experimental condition was performed on duplicate coverslips. Five separate images were analyzed from each coverslip.

Data analysis. All values are means ± SE. The method we used to inject the radiolabels into the rabbit lungs avoided visible loss of material via the upper airways. However, some of the material may remain in the upper airways and may be cleared soon after injection. Clearances from adult rabbit lungs were calculated from the slopes of semilog regression curves corrected such that the total amount of radiolabeled DPC and nSP-C or rSP-C recovered in the total lung (alveolar lavage fluid + lung tissue) at zero time was equal to 100%. The intercepts at zero time were ~82% of the estimates of the amount of the radiolabels given to the animals. The clearance data for the mice were calculated similarly.

    RESULTS
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Methods
Results
Discussion
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Clearance from rabbit lung. Aliquots of alveolar lavage fluid from the rabbits studied at times up to 16 h after intratracheal injection with nSP-C were lyophilized and run on polyacrylamide gels. Autoradiographs demonstrate that most of the 125I label remaining in the alveolar lavage fluid ran at the molecular mass of SP-C (Fig. 1).


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Fig. 1.   Autoradiograph of 125I-labeled native surfactant protein (nSP) C given to rabbits (lane 1) and from alveolar lavages at 0.5 (lane 2), 2 (lane 3), 5 (lane 4), and 16 (lane 5) h after injection. Nos. at right, molecular mass.

The percent recoveries of radiolabeled nSP-C, rSP-C, and DPC in alveolar lavage fluid, postlavage lung tissue, and the total lung compartments from the rabbits are shown in Fig. 2. The clearance data for the two measurements of DPC made with the measurements for labeled nSP-C and labeled rSP-C were similar and were combined. nSP-C and rSP-C were cleared from alveolar lavages with similar kinetics. Less nSP-C and rSP-C than DPC were recovered at 30 min and at later times up to 16 h (P < 0.05); however, the differences were not large. The losses of nSP-C, rSP-C, and DPC from the total lung were best fit by exponential curves (Fig. 3). The half-life (t1/2) values for nSP-C (12.3 h), rSP-C (11.4 h), and DPC (10.6 h) from the rabbit lung were not different within the resolution of these experiments. The amount of labeled nSP-C in macrophages recovered from alveolar lavage was <0.5% of the total radioactivity recovered in the lavage at each time.


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Fig. 2.   Percent recoveries of 125I-nSP-C (A), 125I-recombinant SP (rSP)-C (B), and [3H]dipalmitoyl phosphotidylcholine (DPC; C) measured in total lung (TL; sum of alveolar lavage fluid and lung tissue), alveolar lavage fluid (Lavage), and lung tissue after alveolar lavage (Tissue) from rabbits. Clearance of each iodinated protein was measured with [3H]DPC in separate experiments, and data for DPC were combined to yield composite curves.


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Fig. 3.   Clearance curves fit by linear regression, regression coefficients (r), and half-life (t1/2) values for loss of labeled DPC, nSP-C, and rSP-C from rabbit lungs (A) and ratios of recovery of 125I-nSP-C to [3H]DPC and of 125I-rSP-C to [3H]DPC (B). Biological t1/2 values for curves are not different for the 3 data sets. Ratios of recovery were normalized to isotope ratios in mixtures of isotopes given to rabbits, which were given a value of 1.0. There were no changes in ratios with time, indicating similar clearances from lungs for proteins and DPC.

A more sensitive method for evaluating differences in the rates of clearance of radiolabeled surfactant components is to compare the ratios of recovery of the components in each animal, avoiding differential losses in processing. The ratios of nSP-C to DPC and rSP-C to DPC, normalized to the ratio at zero time, did not change over the 16-h study period (Fig. 3). This result demonstrates proportional losses of nSP-C, rSP-C, and DPC from rabbit lungs.

Clearance of rSP-C and DPC from mouse lung. 125I-rSP-C was lost from the total lung of mice exponentially, with a t1/2 of 27.3 h (Fig. 4). The exponential clearance of DPC from the lungs of mice was more rapid (t1/2 of 12.2 h) than was the loss of rSP-C. The loss of DPC from the alveolar lavage fluid was also more rapid than that for rSP-C. At 40 h, 6.7 ± 0.9% of rSP-C and 4.5 ± 0.7% of DPC were recovered by alveolar lavage.


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Fig. 4.   Percent recoveries of 125I-rSP-C (A) and [3H]DPC (B) from TL, Lavage, and Tissue for mice lungs. C: clearance curves fit by linear regression, r, and t1/2 values for loss of 125I-rSP-C and [3H]DPC from lungs of mice. t1/2 values were different, P < 0.05.

Uptake of DNP-rSP-C by MLE-12 cells. Representative images demonstrating DNP-rSP-C uptake by MLE-12 cells are shown in Fig. 5, and quantification of the uptake is shown in Fig. 6. Incubation of MLE-12 cells with DNP-rSP-C-containing vesicles resulted in intense staining of large perinuclear inclusions (Fig. 5A). When the cells were incubated with a 10-fold excess of liposomes containing 10% nSP-C, almost no endocytosis of DNP-rSP-C was observed (Fig. 5B). A 10-fold excess of liposomes containing no SPs did not inhibit DNP-rSP-C uptake (Fig. 5C). Incubation with vesicles containing both 10% SP-B and 10% DNP-rSP-C resulted in less intense staining than in vesicles containing DNP-rSP-C alone (Fig. 6). When vesicles containing 10% DNP-rSP-C and 10% SP-B were mixed with 10% SP-A, the amount of DNP-rSP-C endocytosed by MLE-12 cells was reduced relative to DNP-rSP-C vesicles, and an increase in large extracellular aggregates containing DNP-rSP-C was observed binding to the cell surface in the presence of SP-A and SP-B together. The amount of DNP-rSP-C uptake in the presence of 10% SP-B and 10% SP-A was also less than that detected in the presence of 10% SP-B. Addition of 10% SP-A to DNP-rSP-C-containing vesicles did not decrease the endocytosis of DNP-rSP-C.


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Fig. 5.   Representative images of dinitrophenyl (DNP)-rSP-C uptake by MLE-12 cells. MLE-12 cells were incubated with 10 mg/ml of lipid vesicles containing 10% (wt/wt) DNP-rSP-C (A), 10% (wt/wt) DNP-rSP-C plus a 10-fold excess of lipid vesicles containing 10% (wt/wt) nSP-C (B), DNP-rSP-C plus a 10-fold excess of lipid vesicles (C), or no vesicles (control; D). Bar, 50 µm.


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Fig. 6.   Image analysis of DNP-rSP-C uptake by MLE-12 cells. MLE-12 cells were incubated with 10 mg/ml of lipid vesicles containing SPs. Immunologic product was not detected in control cells. Percent cell area containing stain was 5.2 ± 0.3% when cells were exposed to DNP-rSP-C and decreased when a 10-fold excess of SP-C in lipid vesicle was added with DNP-rSP-C vesicles (P < 0.05). Liposomes containing lipids only had no effect on DNP-rSP-C uptake by cells. Lipid vesicles containing 10% DNP-rSP-C and 10% SP-B had decreased uptake (P < 0.05). Addition of 10% SP-A to vesicles that contained DNP-rSP-C and SP-B further decreased uptake (P < 0.05). There was no effect of 10% SP-A alone when combined with 10% DNP-rSP-C in lipid vesicles.

    DISCUSSION
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These experiments have assessed the kinetics of clearance of SP-C and rSP-C in two species and the reuptake behavior of rSP-C in vitro. In the adult rabbit, the clearance kinetics of SP-C from the lungs are essentially identical to those of DPC. The clearance of rSP-C was similar to that of nSP-C. The amount of nSP-C associated with macrophages was <0.5% of the 125I-nSP-C recovered by alveolar lavage, indicating either that macrophages are not large factors in the catabolism of SP-C or that once SP-C is associated with macrophages, it is rapidly degraded and does not accumulate. The latter situation applies to DPC (27, 28). Pinto et al. (22) could not detect degradation of SP-C by type II cells. The sites of SP-C catabolism are not known, although the overall catabolic rate is similar to that for DPC. Although the kinetics of the clearance of SP-C and DPC are similar in the rabbit, these findings do not imply equivalence of their metabolic pathways.

In previous short-term experiments, SP-C was shown to be taken up from the air spaces into lamellar bodies (3, 23). Baritussio et al. (2) found a more rapid loss of SP-C than of DPC from the air spaces 4 h after administration to newborn rabbits. Pinto et al. (23) reported similar losses of alveolar DPC and 125I-SP-C and associations with lamellar bodies at 4 h in adult rats. Our data demonstrate a more rapid association of SP-C than of DPC with the lung tissue (an inability to recover SP-C by alveolar lavage) soon after tracheal administration of the tracers in both rabbits and mice. Therefore, all studies consistently show a more rapid loss of SP-C than of DPC from the air spaces soon after tracheal administration. We did not isolate lamellar bodies because the association of SP-C with lamellar bodies was shown previously (2, 23). In the rabbit, the recovery of SP-C from alveolar washes also was lower than that of DPC at 8 and 16 h, perhaps suggesting a somewhat less efficient recycling of SP-C than of DPC.

The precise role of SP-C in surfactant function and homeostasis remains poorly characterized primarily because its physical properties hinder experimental procedures and quantification (5). The biophysical effects of SP-C on lipid mixtures are less striking than and overlap the effects of SP-B (25, 32). The protein is expressed only in type II cells of all mature mammals that have been studied to date, and its amino acid sequence is remarkably conserved (5). As perhaps the most hydrophobic protein described, the full range of its contributions to surfactant has not been identified. The protein is processed by sequential cleavage in the trans-Golgi apparatus, and the mature 3.5-kDa protein concentrates in lamellar bodies and is presumably cosecreted with phospholipids and SP-B (6, 31). Its concentration, as best as can be estimated from the staining of gels, is highest in tubular myelin and the large lipid arrays that comprise the most biophysically active forms of surfactant (33). However, the small vesicles that are derived from the monolayer and account for ~50% of the alveolar phospholipid pool contain very little SP-A, SP-B, and SP-C. These vesicles are preferentially taken up by type II cells and are thought to be the primary form for surfactant phospholipids that are either catabolized or recycled (14). Once SP-B becomes dissociated from the lipids in vivo, dense protein aggregates that are relatively enriched in SP-B have been tentatively identified (1). The forms or associations of SP-C after it is lost from lipoprotein complexes and/or the surfactant film are not known. Therefore, the clearance pathways for SP-C and DPC are distinct even though kinetic measurements for the two components are similar.

In this study, we were able to efficiently label nSP-C with the Bolton-Hunter reagent. Pinto et al. (22) iodinated nSP-C with the same reagent under different conditions. The labeled nSP-C did not aggregate, and the radiolabel that was recovered by alveolar lavage was the same size as the 125I-nSP-C and 125I-rSP-C administered to the animals. Although the iodine on nSP-C could alter function, the present findings were consistent with the clearance data of Baritussio et al. (2) with 35S-labeled SP-C. Pinto et al. (23) found no effect of iodination on the biophysical properties of nSP-C.

nSP-C is difficult to study because it aggregates and becomes insoluble when separated from lipids (5). We therefore studied an rSP-C because the amino acid substitutions prevent aggregation while preserving biophysical function, making this rSP-C attractive for developing a synthetic SP-C-based surfactant for clinical use. Surfactant treatment of infants with respiratory distress syndrome is effective, in part, because the phospholipid and protein components are recycled by the immature lung (18). Therefore, it is important to know whether the metabolism of the rSP-C analog is similar to that of nSP-C. We studied SP-C clearance in two species because most of the information concerning surfactant component catabolism and clearance is in the rabbit and information in mice is useful because of newly available transgenic mice with abnormalities in the surfactant system (17, 29, 30). In the rabbit, we could not distinguish between total lung clearance curves for nSP-C or rSP-C and DPC. Although the clearance of SP-B was slightly faster than that of DPC in the rabbit (29), the overall conclusion is that the lung clearance of the hydrophobic proteins is similar in the rabbit. In contrast to the rabbit, the clearance of rSP-C was slower than that of DPC in the mouse. Although there are no studies of nSP-C clearance from the mouse, the clearance of SP-B was also slower than that of DPC (t1/2 of 10 h for DPC and 28 h for SP-B) (17). The t1/2 of 28 h for rSP-C was similar to that of SP-B, indicating that catabolic rates for both hydrophobic surfactant proteins are similar and relatively slow in the mouse. Although there are differences in the rates of clearance of SP-B and SP-C relative to that of DPC in the two species, the two hydrophobic proteins have similar clearance rates in each species. This observation is consistent with the possibility that they are catabolized by common pathways. In the rabbit, 20-30% of Sat PC is catabolized by alveolar macrophages, and the majority of the rest of the Sat PC is taken up into type II cells for recycling or catabolism (28). Similar measurements of the relative importance of type II cells and macrophages for catabolism and recycling have not been made in the mouse, although recycling efficiency for Sat PC is similar in mice and rabbits (9, 27). An explanation for the differences in hydrophobic protein catabolic rates will depend on future measurements of specific catabolic and recycling pathways in type II cells and alveolar macrophages.

The characteristics of uptake of DNP-rSP-C by MLE-12 cells were similar to those recently described for nSP-C (15). nSP-C stimulates the uptake of liposomes by type II cells and MLE-12 cells in vitro. The uptake of DNP-labeled SP-C is competitively inhibited by nSP-C but not by liposomes that contain no SPs (15). The uptake is also inhibited by SP-B and further inhibited when liposomes contain SP-B and SP-A together with nSP-C. The rSP-C was labeled with DNP with the same technique as for nSP-C to make immunologic detection possible. DNP-rSP-C was readily taken up by MLE-12 cells, and uptake was inhibited by nSP-C but not by lipids alone. Intracellular DNP-rSP-C was detected in relatively large cytoplasmic inclusions in a pattern similar to that of nSP-C.

The responses of rSP-C uptake after the addition of the other SPs were also similar to those reported for nSP-C. Therefore, cellular uptake mechanisms appear to be similar for nSP-C and rSP-C. These results together with the clearance measurements in animals indicate that rSP-C has metabolic characteristics very similar to those of nSP-C. The present studies on SP-C clearance complete measurements of the clearance characteristics of the major surfactant components DPC, SP-A, SP-B, and SP-C in rabbits (29, 30). Although there are small differences in clearance, the overall pattern is similar for each of the surfactant components, each having similar alveolar and total lung clearance kinetics. The precise cellular pathways and mechanisms regulating this complex process of conservation and catabolism of surfactant components remain to be elucidated.

    ACKNOWLEDGEMENTS

This work was supported primarily by National Institute of Child Health and Development Grant HD-11932. Partial support was from the Cystic Fibrosis Foundation Research Development Program; National Heart, Lung, and Blood Institute Grant HL-51832; and Byk Gulden, Constance, Germany.

    FOOTNOTES

Address for reprint requests: M. Ikegami, Children's Hospital Medical Center, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039.

Received 25 September 1997; accepted in final form 13 February 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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