Introduction

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Although antioxidant systems play an important part in adaptation of the lung to elevated oxygen tensions, it is increasingly clear that other stress-induced proteins and responses may also be critical components. Among these are changes in pulmonary surfactant. During exposure to lethal hyperoxia (100% O₂), adult rabbits develop multiple defects in their pulmonary surfactant system (diminished phosphatidylcholine synthesis [60% decreased], cell lipid content, and activities of related enzymes) in less than 3 d (1). This hyperoxic lung injury could be ameliorated by lung supplementation with native surfactant (2), which replenishes depleted endogenous pools (5, 6). In addition, adult rabbits in whom tolerance to hyperoxia was induced by exposure to sublethal hyperoxia followed by a recovery period in air before re-exposure had a three-fold increase in lung phospholipid recovered by lavage after re-exposure. However, there was no change in any of the lung antioxidant enzymes preceding re-exposure (7). These findings indicate that the surfactant system could play an important role in adaptation to hyperoxia.

Among adult animals, rats have a remarkable capability to adapt to relatively high concentrations of oxygen (85%). In association with this adaptation, marked changes occur in the expression of pulmonary surfactant proteins (8, 9). Newborn rats born at term gestation have the capability to adapt to even higher concentrations of oxygen for similar durations, and this ability is shared by term newborns of many other species. The expression of surfactant proteins in this adaptive process has not been examined. The role of surfactant proteins, especially surfactant proteins (SP) A and D, was of interest to us because inflammation is thought to be a key factor in the evolution of acute lung disease in the neonate, and SP-A and SP-D are believed to be potentially anti-inflammatory. In addition, they may have antioxidant properties. Herein, we report that considerable upregulation of expression of SP-A, SP-B, SP-C, and SP-D messenger RNAs (mRNAs) and SP-A and SP-D proteins occurs in a time-dependent fashion, despite the well-documented decreases in lung growth and development that occur during hyperoxic exposure in the early postnatal period.

	Materials and Methods

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Exposures of Newborn Rats to Hyperoxia

All animal care procedures were performed according to the National Research Council's Guide for the Care and Use of Laboratory Animals. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the National Jewish Medical and Research Center (Denver, CO). Timed-pregnant Sprague-Dawley rats were obtained from Charles River (Wilmington, MA) and acclimated to Denver altitude beginning on Day 13 of the pregnancy. Newborn exposures began at 48 h of age and continued for eight more days. Exposures were in Plexiglas chambers (0.28 cubic meters) to > 99% oxygen at a flow rate of 10 liters/min humidified oxygen (relative humidity, 60 to 70%; temperature, 22 to 24°C; barometric pressure, 755 mm Hg [approximately sea level]). Two litters were housed in each cage, and dams were rotated from air to hyperoxia and vice versa every 24 h in order to minimize the development of oxygen toxicity in them. Dams were first rotated at 72 h of age in the newborns. When dams were rotated, newborns were handled with latex gloves that were soaked in warm tap water and rubbed briefly in the bedding of the dams' former cage just before the dam was introduced into the new cage. With handling in this manner, mortality was minimal (< 3%) in litters both in air and hyperoxia. On Days 1, 2, 4, 6, and 8, all of the litters, both in air and hyperoxia, were reduced successively by an additional 20% of their original size when animals were removed for experiments. During the time at which newborns were removed, approximately 30 min on Days 1, 2, 4, 6, and 8, cages were held in isolettes with hand ports that were flushed with > 99% oxygen, humidified, at Denver pressure. Anesthesia of animals before thoracotomy was with pentobarbital (130 mg/kg, intraperitoneal).

Isolation of RNA

Excised lungs were immediately homogenized in chilled 4 M guanidinium isothiocyanate, 0.5% laurylsarcosine, 0.1 M -mercaptoethanol in 25 mM sodium citrate buffer with a Polytron tissue homogenizer (Brinkman Instruments, Westbury, NY) and stored at 70°C until use. Total RNA from whole lung tissue was isolated by ultracentrifugation through a 5.7-M CsCl cushion at 150,000 × g for 18 h at 20°C.

Northern Blot Analysis

Northern blot analysis was performed as described previously (10). Briefly, total cellular RNA was electrophoresed through a 1% agarose gel under denaturing conditions, then blotted onto a nylon membrane (Nytran; Schleicher and Schuell, Keene, NH) by capillary action. The isolation and characterization of complementary DNAs (cDNAs) for rat SP-A, SP-B, SP-C, and SP-D have been described previously (11). The cDNAs for rat CC-10, a protein specific for Clara cells, and rat thyroid transcription factor (TTF)-1 (15) were provided by Dr. Arun Rishi (Boston University, Boston, MA) and Dr. Lyn Thet (University of Wisconsin), respectively. cDNAs were labeled to high specific activity by random priming using [³²P]deoxycytidine triphosphate and a commercially available kit (both from Amersham Life Science, Arlington Heights, IL). Membranes were prehybridized, hybridized, washed, and autoradiographed on Kodak XAR5 film (Eastman Kodak, Rochester, NY) as previously described (16). Hybridized blots were placed on a phosphorus screen for a direct quantitation of radioactive counts using a Molecular Dynamics Storm instrument and Molecular Dynamics ImageQuant software, version 3.3 (Molecular Dynamics, Sunnyvale, CA). Results were normalized based on loading similar amounts of total lung RNA, which was confirmed by examination of the ethidium bromide-stained gel.

Measurement of SP-A and SP-D in Bronchoalveolar Lavage by Enzyme-Linked Immunosorbent Assay

After general anesthesia with pentobarbital (130 mg/kg injected intraperitoneally), a tracheostomy was placed with a 22-gauge catheter. Normal saline (five instillations, 1 ml each) was infused and removed gently with a tuberculin syringe. Lavage fluid was centrifuged (300 × g, 10 min, 4°C) to sediment cells and cell debris, and the supernatant was recovered. Aliquots of cell-free supernatant were taken for SP-A and SP-D assays. SP-A and SP-D were quantitated with a standard enzyme-linked immunosorbent assay as reported elsewhere (17, 18). In brief, microtiter plates were coated overnight at room temperature with capture antibody diluted in 0.1 M NaHCO₃ (pH 8.3) to a final concentration of 10 µg/ml. The plates were washed three times with buffer A (phosphate-buffered saline [PBS]/3% nonfat dry milk/1% Triton-X). Native rat SP-A or recombinant rat SP-D expressed in Chinese hamster ovary cells was employed as standard. Bronchoalveolar lavage (BAL) samples were prepared by adding 1% Triton-X, probe sonicating on ice for 5 s, diluted in buffer A, and added to the wells. The plates were incubated at 37°C for 2 h, washed three times, and then incubated with polyclonal rabbit antirat SP-A or polyclonal antibodies raised against recombinant rat SP-D. After washing three times, the wells were incubated with peroxidase-conjugated goat antirabbit immunoglobulin G (10 µg/ml). Color was developed using orthophylene diamine substrate system. Samples were measured in triplicate dilutions.

In Situ Hybridization

Excised lungs were instilled with freshly prepared 4% paraformaldehyde in RNase-free PBS (pH 7.4). The instilled lungs were then immersed in 4% paraformaldehyde in PBS and fixed overnight at 4°C. The fixed lungs were cut into blocks, washed in PBS, then stored in 70% ethanol until paraffin embedding. In situ hybridization was performed as previously described (19). Tissue sections (4 to 6 µm) were mounted on Super Frost II glass slides (Fisher) and hybridized with ³³P-labeled sense or antisense RNA probes transcribed from full-length rat cDNAs for SP-A, SP-B, SP-C, SP-D, CC-10, and TTF-1. After a series of high stringency washes, the slides were dipped in Kodak NTB-2 nuclear track emulsion. Autoradiograms were exposed in light-tight boxes for 3 to 14 d at 4°C, developed, and then counterstained with hematoxylin.

Statistics

Significant treatment effects were identified by using two-way analysis of variance procedures provided by the JMP software package (20). Linear contrasts were used to identify significant gas effects within each time group when a significant time by gas interaction was found. Individual comparisons also were made at given time points using Student's t test with Bonferroni correction. Significance was accepted in all comparisons when P was  0.05.

	Results

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Survival and Gross and Microscopic Lung Appearance

Animal survival in > 99% oxygen was 82% through 8 d. Survival in air-exposed newborns was 98% over that interval. No mortality was observed in the hyperoxia-exposed group through Day 4. Mortality was 6% through Day 6 in hyperoxia, with an additional 12% mortality between Days 6 and 8. Mortality in the air-exposed group was minimal and was noted only between Days 0 through 2, apparently due to feeding difficulties.

Among the surviving animals, a few areas of confluent lung edema, involving two or fewer lobes, could be observed grossly on Day 6 and thereafter at necropsy (Day 8 of exposure). These areas were observed in 14% of animals necropsied on Day 6, and in 27% of those killed on Day 8. Grossly edematous lobes were excluded from further study.

Microscopically, lungs were generally free of edema during hyperoxic exposure except for occasional focal edema in some of the lungs from Days 6 and 8 of exposure. During the first 4 d of exposure, the appearance of lungs in the two groups were remarkably similar. However, by Day 8 distinct differences were apparent, such that lungs from animals in the hyperoxia-exposed group showed alveolar simplification, that is, lungs with fewer and larger alveoli.

mRNA Expression for Surfactant Proteins, CC-10, and TTF-1

mRNAs for SP-A, -B, -C, and -D, CC-10, and TTF-1 were analyzed by Northern blot analysis (Figure 1G), then quantitated by phosphorimaging (Figures 1A to 1F). SP-A mRNA expression did not change significantly in the lungs of air-exposed newborns during the interval studied. In addition, newborn lung SP-A mRNA expression was not different between air- and hyperoxia-exposed newborns on exposure Days 1 and 2 (P > 0.05). By contrast, SP-A mRNA expression was progressively greater in hyperoxia than in air on exposure Days 4 through 8 (P < 0.05, < 0.001, and < 0.000001 on Days 4, 6, and 8, respectively). A significant gas-time interaction was identified, indicating that the magnitude of the effect of oxygen exposure was dependent on the duration of that exposure (Figure 1A).

View larger version (25K): [in this window] [in a new window]   View larger version (68K): [in this window] [in a new window]   Figure 1.   Effect of hyperoxia on whole lung expression of mRNAs encoding SP-A, -B, -C, and -D, CC-10, and TTF-1. Graphic representation of data presented in (A-F ). A representative Northern blot analysis of mRNA isolated from newborn rats exposed to air or oxygen (O2) on Days 1, 2, 4, 6, and 8 of life are shown (G). The ethidium bromide-stained gel demonstrating 18S and 28S ribosomal RNA bands is shown below the various mRNAs. Data is normalized based on loading of similar amounts of total lung RNA. *A significant difference between air- and hyperoxia-exposed groups on a given day.

Although SP-B followed a similar pattern of expression to that seen for SP-A, the divergence in expression between air- and hyperoxia-exposed lungs began earlier for SP-B (Figure 1B). SP-B expression showed a slight, but progressive, decline in the lungs of air-exposed newborns during the study period. After 24 h of hyperoxic exposure (exposure Day 1, Day 3 of life), lung SP-B mRNA was not different in air- and hyperoxia-exposed newborns. By contrast, SP-B mRNA expression was greater in these animals on exposure Days 2 through 8 within a given day. Here, although the gas-dependent and combined gas- and time-dependent effects were significant, the time effect alone was not.

For SP-C, there was a gradual decline in mRNA expression in the lungs of air-exposed newborns during the study period. In addition, SP-C mRNA expression was significantly greater in the lungs of hyperoxia-exposed, compared with air-exposed, newborns throughout the exposure period (Figure 1C). Both gas- and time-dependent effects were significant independently (P < 0.0001) and when combined (P = 0.05).

SP-D mRNA followed a pattern of expression similar to that for SP-C (Figure 1D). For SP-D, there also was a gradual decline in lung mRNA expression in air-exposed newborns during the study period. In addition, SP-D mRNA expression was significantly greater in the lungs of hyperoxia-exposed, compared with air-exposed, newborns throughout the exposure period at each level of time (Figure 1D). Both gas- and time-dependent effects were significant independently and when combined (P = 0.02).

The pattern of expression for TTF-1 mRNA resembled that for SP-C and SP-D mRNAs in that TTF-1 mRNA expression tended to decline over time in the lungs of air- exposed newborns and was significantly greater in the lungs of hyperoxia- than air-exposed newborns at all time points (Figure 1E). Although there was a significant gas effect, there was no time-dependent effect nor combined effect of gas and time.

The pattern of expression for CC-10 mRNA was unlike that seen for any of the surfactant protein or TTF-1 mRNAs (Figure 1F). CC-10 mRNA was significantly greater in hyperoxia- than in air-exposed newborn lungs on Day 4 (P < 0.0001) and on Day 6 (P = 0.05), but not different on Days 1, 2, and 8 of exposure.

To summarize, increases in SP-A and SP-B mRNAs in hyperoxia relative to air were progressive, whereas those for SP-C and SP-D were more consistently maintained. SP-A and SP-B mRNAs increased progressively during exposure to hyperoxia, and the greatest differences in expression of these surfactant protein mRNAs were apparent on Day 8. The elevation of lung mRNAs encoding SP-C and SP-D in hyperoxia were more consistent, occurring at each time point. By Day 4 of exposure, both SP-C and SP-D mRNAs had already achieved maximal difference in the lungs of hyperoxia-exposed, relative to air-exposed, newborns. However, in the case of SP-D, this maximal difference was maintained on Day 8. In contrast to surfactant protein mRNAs, CC-10 mRNA expression was increased maximally in the lungs of hyperoxia-exposed newborns on Day 4 relative to those from air-exposed newborns. Thereafter, these differences became less apparent. There appeared to be more individual variation in TTF-1 mRNA expression within groups, although TTF-1 mRNA was increased in hyperoxia-exposed, relative to air-exposed, newborn rats on each day examined. This pattern resembled that seen for SP-A and SP-B mRNAs.

Expression of SP-A and SP-D in BAL

The results of the analysis of SP-A and SP-D levels in BAL of air- and hyperoxia-exposed rats revealed that neither was normally distributed. Therefore, all of the data were log-transformed, which normalized the data, before being subjected to two-way analysis of variance. SP-A levels were greater in the BAL fluid (BALF) of hyperoxia-exposed, than air-exposed, newborns at all time points (Figure 2A). BAL SP-D levels did not increase in hyperoxia-exposed newborn lungs until Days 6 and 8 (Figure 2B).

View larger version (13K): [in this window] [in a new window]   Figure 2.   Effect of hyperoxia on the expression of SP-A and SP-D in BAL from newborn rats on Days 1-8 of hyperoxic exposure (Days 3-10 of life). (A) SP-A; (B) SP-D. *A significant difference between air- and hyperoxia-exposed groups on a given day.

For SP-A measurements, there were significant time- and gas-dependent effects, although there was no significant interaction between these. The pattern of SP-A expression over time was the same in the air- and oxygen-exposed groups. SP-A levels in BAL were slightly higher on Day 6 versus Day 1, and even more so on Day 8 versus Day 1, in both air- and hyperoxia-exposed groups. In addition, the effect of oxygen exposure was the same across time. That is, BAL SP-A levels were significantly greater in hyperoxia than in air at each time point (Day 1 through 8).

The results of the analysis for BAL SP-D protein were considerably different. Here, there was a significant interaction between the effects of time and of oxygen exposure (gas). The effect of oxygen exposure was slightly less significant than that of time. BAL SP-D levels were the same in air- and hyperoxia-exposed newborns for the first 4 d of exposure, and then they diverged on Days 6 (P = 0.05) and 8 (P = 0.0001).

In summary, the patterns of mRNA and protein expression for SP-A and SP-D were relatively different. SP-A mRNA expression in hyperoxia progressively increased over time, whereas BALF SP-A protein was consistently increased in hyperoxia exposure relative to air exposure. Nonetheless, the magnitude of that increase became greater by Day 8. For SP-D, the situation was almost opposite to that for SP-A. Specifically, SP-D mRNA expression was consistently greater in hyperoxia than in air from the earliest time points examined. The maximal difference was apparent by Day 4 and no greater by Day 8. By contrast, SP-D protein in the lavage was not different in the lungs of air- versus hyperoxia-exposed newborns on Days 1 through 4, but thereafter it was progressively increased with hyperoxia exposure relative to air exposure.

Expression of mRNAs for Surfactant Proteins, CC-10, and TTF-1 by In Situ Hybridization

At the alveolar level, SP-A and SP-D mRNAs showed the greatest differential expression between the lungs of air- and hyperoxia-exposed newborns. Lung sections were examined by in situ hybridization at time points at which maximal statistical differences in expression of the various mRNAs were present. For all of the surfactant protein mRNAs, this was done on Day 8 of exposure. As seen in Figure 3, message intensity for SP-A mRNA was increased in alveolar walls in the lungs of hyperoxia-exposed newborns (Figure 3D) as compared with that in lungs from air-exposed newborns (Figure 3B). This occurred despite the finding that large alveoli with diminished septation were present in the lungs from hyperoxia-exposed animals (Figure 3C) relative to those in lungs from air-exposed animals (Figure 3A). Increased SP-A appeared to have a patchy distribution within the peripheral lung (Figure 3D).

View larger version (153K): [in this window] [in a new window]   Figure 3.   Effect of hyperoxia on the expression of proteins in terminal airways and alveolar regions in the lungs of newborn rats on Day 8 of exposure (Day 10 of life). Dark-field images from distal lung regions of air-exposed (B, F, J, N, R, and V ) and hyperoxia-exposed newborns (D, H, L, P, T, and X ) are compared using the in situ hybridization technique as described in MATERIALS AND METHODS for SP-A, SP-B, SP-C, SP-D, CC-10, and TTF-1. Bright field images (hematoxylin and eosin-stained sections: A, C, E, G, I, K, M, O, Q, S, U, W) appear to the immediate left of each dark field image (original magnification: ×20).

Changes in SP-D mRNA expression were similarly striking. Thus, there was a diffuse and homogenous increase in SP-D distributed throughout the peripheral lung tissue of hyperoxia-exposed, relative to air-exposed, newborns (Figures 3M through 3P).

The intensities of SP-B hybridization in autoradiograms for sections from air- (Figure 3F) and hyperoxia-exposed lungs (Figure 3H) were minimally different. The same could be said of SP-C in hyperoxia (Figure 3L) relative to air (Figure 3J). Although much less striking than for SP-A or SP-D, focal punctate areas of increased SP-C mRNA density were present in the lung periphery in the lungs of hyperoxia-exposed, relative to air-exposed, newborns.

For each of the respective surfactant protein mRNAs examined, there appeared to be little difference in the intensity of mRNA signal present in the more proximal airways in lungs of hyperoxia-exposed newborns compared with air-exposed newborns.

CC-10 message was localized primarily to the airways in both air- and hyperoxia-exposed newborns' lungs. Peripheral lung tissue was virtually devoid of detectable CC-10 mRNA. There appeared to be no difference in mRNA intensity in air versus hyperoxia.

In contrast to all of the other mRNAs studied, TTF-1 appeared to be evenly distributed both within distal lung and more proximal airways. Little, if any, difference could be appreciated in TTF-1 mRNA detected in the lungs of air-exposed, compared with hyperoxia-exposed, newborns.

At the level of the distal airways, CC-10 mRNA was strongly expressed and SP-D and SP-B mRNAs were moderately expressed. Expression of SP-A and TTF-1 mRNAs appeared more patchy or less intense, though still detectable, whereas SP-C mRNA was not detected in distal small airways. At the level of the small airways, none of the mRNAs for these proteins appeared to show increased expression in the lungs of hyperoxia-exposed, as compared with air-exposed, newborns.

	Discussion

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We found striking increases in SP-A and SP-D mRNAs in whole lung, as detected by Northern blot analysis, and in alveolar regions, as indicated by in situ hybridization studies, despite a pattern of alveolar simplification, in the lungs of newborn rats exposed to hyperoxia relative to those exposed to normoxia. During exposure on Days 3 through 10 of life, whole lung mRNAs for SP-A and SP-B gradually and progressively increased, relative to levels in age-matched, air-exposed newborns. In contrast, whole lung SP-C and SP-D mRNAs were greater in hyperoxia than in air at all time points examined, with the greatest differences occurring on Day 4 and thereafter. Lung mRNA for CC-10, a protein specific for Clara cells, was greater in hyperoxia than in air controls on Day 4, but not at other times during exposure. Lung mRNA for TTF-1 was increased throughout exposure, being most significantly increased on Day 8.

In situ hybridization studies demonstrated increases in SP-A and SP-D in peripheral lung tissues from oxygen- exposed newborns. The findings from these studies indicate that regional increases in at least some of these surfactant protein mRNAs may have exceeded those measured in whole lung. Both SP-A and SP-D proteins were increased in lung lavage samples taken from hyperoxia-exposed newborns, relative to those taken from air-exposed controls, with the greatest increases occurring on Days 6 and 8 of exposure. Expression of the mRNAs for each of these also was most different on Day 8, suggesting that increased protein expression was dependent, at least in part, on increased expression of the mRNAs. Taken together, these data indicate that specific surfactant proteins were upregulated, at least in part, at the pretranslational level in distal lung epithelium during adaptation to hyperoxia in the newborn rat. However, because the patterns of mRNA expression and protein expression in BAL differed both for SP-A and for SP-D, the results suggest that additional levels of regulation of protein synthesis, stability, and/or secretion may be affected by exposure to hyperoxia.

Previous studies by others in adult rats adapted to hyperoxia for up to 7 d have shown that the alveolar lavage content of SP-B and SP-C were increased up to five-fold, with maximal increases after 5 d, then declining again. Lung contents of both these mRNAs increased about two- to three-fold at this time (8). All the mRNAs (SP-A, -B, and -C) increased during 1 wk of exposure, with SP-C mRNA being increased two- to three-fold after 1 wk, whereas SP-A and SP-B mRNAs increased more than ten-fold during that time. Two SP-A mRNA transcripts were measured (1.0 and 1.7 kb), with both increasing progressively. In this model, lavage SP-A increased to > 20-fold above control, and with a > ten-fold increase in lung SP-A content after 7 d (9). In isolated rat type II cells, SP-A synthesis was increased two- to three-fold and secretion by six- to seven-fold after this duration in hyperoxia. The prominence of the SP-A response among the various surfactant proteins in adult rats was similar to what we found in newborns. The magnitude of the increases in surfactant protein mRNAs that were reported in lung lavage and in whole lung in adult rats contrasts sharply with the considerably smaller changes that we found in the newborn. This might appear paradoxical given the greater resistance of the newborn to hyperoxia. There are at least two possible explanations for this difference. One is that even though the newborn is more resistant, the animals in our study were exposed to > 99% oxygen, whereas those reported in the adult rat study were exposed to 85% oxygen. Adult rats in 85% oxygen are truly adapted; that is, there is very little death of these animals even when such exposure is sustained continuously for up to 2 wk (21). Adaptation of newborn rats to pure oxygen, on the other hand, is relative. That is, mortality begins to occur after approximately 1 wk and is progressive when exposure is sustained for another week. This may, of course, be related to nutritional problems that can develop in addition to the cumulative toxicity of oxygen.

There may be another more straightforward explanation for this difference. All of our comparisons with the hyperoxia-exposed newborn rat were made with normally developing air-exposed controls. In adult rats, there is no decline in the number of lung cells nor in any of the alveolar epithelial cells during adaptation to 85% oxygen as compared with air-exposed controls (21). The newborn rat lung is in a period of remarkably rapid growth between birth and Day 10 of life. In particular, the process of alveolarization, critical to increase the internal surface area of the lung, is occurring during this time and is severely compromised by exposure to hyperoxia during this period (22). For example, in this model Frank (22) found a 34% increase in mean air space diameter and a > 20% decline in lung internal surface area caused by early exposure to hyperoxia relative to the norms for air-exposed newborns. In addition, Shaffer and coworkers (23) found that the mean linear intercept increased by 50%, internal surface area decreased by 25%, alveolar number was decreased by one-third, and small arteries were decreased by 40% after hyperoxic exposure for the first 8 d of life. Furthermore, a markedly decreased lung DNA content resulted from early exposure to hyperoxia both in newborn rats and mice (24). Thus, the decline in alveolar constituent cells may contribute to the apparently smaller magnitude response of surfactant proteins in the newborn relative to the adult during oxygen adaptation.

Although lung growth in general is suppressed in the newborn by hyperoxia, the possibility that proliferation of certain cell types might lead to the increased expression of specific proteins during adaptation to hyperoxia remains a consideration. In particular, an increase in multiple surfactant proteins might be attributable to proliferation of alveolar type II cells. SP-C is generally regarded as the surfactant protein most specifically expressed in type II cells, whereas SP-B is specifically expressed in type II and Clara cells. Among the four surfactant protein mRNAs studied by in situ hybridization, the mRNA encoding SP-C was the least affected with respect to extent of distribution at the alveolar level. SP-B mRNA appeared to be the second least affected. On the other hand, those mRNAs encoding the other surfactant proteins, namely the collectins SP-D and SP-A, were most greatly affected with respect to both signal intensity and extent of distribution. Thus, upregulation of these proteins appears relatively selective, or at least preferred, at the alveolar level. Therefore, it appears unlikely that changes in surfactant protein mRNA and protein expression are the result of type II cell proliferation.

Studies in the Newborn

Term newborns of numerous species, including rats, rabbits, and mice, have been found to be more resistant to O₂-induced lethality due to pulmonary toxicity than are their adult counterparts (25). Based on the survival data, newborn rabbits are more susceptible than newborn rats to oxygen toxicity. In newborn rabbits, SP-A mRNA was identified as one of the three induced messages in hyperoxia, with a three-fold elevation measured after 4 d in hyperoxia (100% O₂) (26). After more prolonged exposure to hyperoxia (> 95% O₂ for 8 d followed by 60% O₂ for 22 to 36 d), considerable lethality (> 50% at Day 8) followed by lung fibrosis occurred. After 4 d in this model, whole lung SP-A mRNA content increased two-fold, confirming the previous finding, whereas SP-B and SP-C mRNAs were unchanged. Thereafter (8, 22, and 36 d), there were no increases in whole lung SP-A, SP-B, or SP-C mRNA contents. In situ hybridization at 4 d showed increased SP-A mRNA in type II cells and terminal bronchioles. Lesser elevations in SP-B and SP-C were manifested later (8 d) and were more prominent in areas of inflammation. Of interest and relevance to the current study, no increases in whole lung mRNAs for any of the surfactant proteins were noted during the second 4 d of lethal (LD50) oxygen nor during the more prolonged exposure to lesser, adaptive concentrations of oxygen (26). Hence, oxygen exposure at lethal or nearly lethal (LD50) levels did not result in sustained or progressive measurable increases in whole lung SP-A mRNA or mRNA for other surfactant proteins in newborn rabbits. This is a different temporal pattern of expression than what we found in term neonatal rats adapting to hyperoxia through 10 d of age, even at sea level pressure. In these animals, expression of most of the surfactant protein mRNAs and proteins was progressively increased or sustained, being greatest after 8 d of hyperoxic exposure.

Studies in the Premature

In lambs and primates (baboons), as in rats, whole lung SP mRNAs increased progressively during the final third of gestation, demonstrating their developmental regulation. In very premature lambs of 120 d gestation, during ventilation with 100% oxygen, with or without treatment with artificial surfactant, no increases were noted in SP-A, -B, or -C mRNAs after ventilation for 5 h (0.75 h without surfactant). In less premature lambs (132 and 139 d, ventilated for 5 or 10 h, respectively), lung SP-A mRNA content increased with or without surfactant treatment. Only in the most mature (139 d) group did SP-B mRNA increase, and to a much smaller extent, in both ventilated groups. Lung SP-C mRNA did not increase at any gestational age, in any group (27). These data demonstrate the relative delay in the elevation of expression of surfactant proteins with exposure to hyperoxia and ventilation in the more prematurely born.

In premature humans, immunostaining for SP-A, SP-B, and SP-C was less intense in both premature and more mature infants dying of respiratory distress syndrome (RDS) compared with the findings in the lungs of control newborns dying of other causes. In addition, these investigators noted a lack of tubular myelin in the lungs of infants dying of RDS (28, 29). In baboons, Minoo and colleagues (30) found that interruption of gestation by premature birth, coupled with exposure to elevated oxygen tension, resulted in increased lung expression of three surfactant protein mRNAs (SP-A, -B, and -C). SP-B and SP-C mRNAs increased promptly (within 24 h) and surpassed levels found in the full-term fetus by 48 h, whereas increases in SP-A mRNA occurred more slowly and never exceeded those in the full-term fetus. The effect of hyperoxia was greater on expression of SP-B and SP-C mRNAs than it was on that of SP-A mRNA in this model (140-d gestational age baboon). Specifically, animals given 100% oxygen had greater SP-B and SP-C mRNA contents than those given PRN oxygen (as needed; generally < 50% O₂ by this age) on Day 6. By Day 10, SP-B and SP-C mRNA levels were similar in the two groups. Such a difference was never present for SP-A mRNA, suggesting that it may not be as responsive to hyperoxia in the premature (30). In further studies of SP-A expression in this model, 140-d animals given 100% O₂ for 11 d followed by 5 d of PRN oxygen (as needed) developed chronic lung disease (bronchopulmonary dysplasia [BPD]), and their lungs showed decreased expression of SP-A mRNA relative to age-matched fetal controls or animals ventilated for 16 d with PRN oxygen only. A similar SP-A mRNA deficiency was found in BPD animals that were given pneumonia by intratracheal instillation of bacteria on Day 11 (31). In more recent studies in this model, there was a considerable increase in lung tissue SP-A and SP-D during hyperoxic ventilation. However, there was not a parallel rise in these surfactant proteins in BALF, suggesting decreased secretion and/or increased degradation in that compartment. BAL SP-D was not reduced to the extent of SP-A, with levels in the BALF of hyperoxic premature newborns approximating those in adults (32). In human adults, Greene and coworkers (33) found low SP-A, but not SP-D, levels in the BALF of patients developing respiratory distress syndrome, and SP-A and SP-D levels in BALF were related to the risk for development of the syndrome. Taken together, these studies have demonstrated decreased expression of SP-A, and to a lesser extent SP-D, mRNA and protein in premature primates and humans with respiratory distress syndromes. Such individuals generally are ventilated with concentrations of oxygen greater than 21%. These findings are opposite to those for SP-A and SP-D, which we found to be prominent in term newborn rats adapting to hyperoxia.

Why Are the Collectins Selectively Elevated?

The basis for the relatively selective increases in SP-A and SP-D (calcium-dependent lectins or collectins) is not apparent. However, there are several potential reasons that such increases could be important. First, both SP-A and SP-D have important roles in the initiation and propagation of inflammation, having important interactions both with the monocyte/macrophage and neutrophil (34). Both neutrophilic and macrophage infiltration into lung occur during hyperoxic lung injury (40), and their modulation could play a role in adaptation. In addition, it is clear that the injured lung, including the hyperoxia-injured lung, has increased susceptibility to infection (41). SP-A and SP-D appear to have important roles in preventing lung infection by a variety of organisms, as demonstrated in knockout mice (34, 35). In addition, both these surfactant proteins may have important roles in the uptake and recycling of surfactant phospholipid, processes which appear to be important in hyperoxic lung injury (4, 5, 7). Although these potential roles have been minimized by recent studies in knockout animals, these studies may not be altogether pertinent in the context of acute lung injury.

Recently, it was shown that SP-A and SP-D can directly protect surfactant phospholipids and macrophages from oxidative damage (42). In those studies, it was found that both SP-A and SP-D can inhibit copper-induced oxidation of surfactant lipids or low-density lipoprotein particles via a mechanism that does not appear to involve metal chelation or oxidative modification of these proteins. Although the antioxidant action of SP-A has been partially mapped within the protein, the mechanism of action of these proteins in inhibiting lipid oxidation is poorly understood. Nonetheless, the increases in SP-A and SP-D during hyperoxic adaptation are more readily understood in this context.

In conclusion, newborn rats exposed to levels of hyperoxia that are normally lethal for adults have increased expression of lung mRNAs for SP-A, SP-B, SP-C, and SP-D when compared to age-matched, air-exposed newborns. In addition, SP-A and SP-D proteins increased in lung lavage samples taken from hyperoxia-exposed newborns, relative to those taken from air-exposed controls, with the greatest increases occurring on the later days of exposure. In situ hybridization studies demonstrated increases in SP-A and SP-D mRNAs at the lung periphery in oxygen-exposed newborns. The patterns of increased expression of these mRNAs and their corresponding proteins differed. These data indicate that surfactant proteins, especially the collectins SP-A and SP-D, are upregulated in distal lung epithelium during adaptation to hyperoxia in the newborn rat. The mechanisms responsible appear to be complex and to involve both pre- and post-translational events.