Division of Pulmonary Biology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229
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ABSTRACT |
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Intra-amniotic (IA) endotoxin induces lung maturation within 6 days in fetal sheep of 125 days gestational age. To determine the early fetal lung response to IA endotoxin, the timing and characteristics of changes in surfactant components were evaluated. Fetal sheep were exposed to 20 mg of Escherichia coli 055:B5 endotoxin by IA injection from 1 to 15 days before preterm delivery at 125 days gestational age. Surfactant protein (SP) A, SP-B, and SP-C mRNAs were maximally induced at 2 days. SP-D mRNA was increased fourfold at 1 day and remained at peak levels for up to 7 days. Bronchoalveolar lavage fluid from control animals contained very little SP-B protein, 75% of which was a partially processed intermediate. The alveolar pool of SP-B was significantly increased between 4 and 7 days in conjunction with conversion to the fully processed active airway peptide. All SPs were significantly elevated in the bronchoalveolar lavage fluid by 7 days. IA endotoxin caused rapid and sustained increases in SP mRNAs that preceded the increase in alveolar saturated phosphatidylcholine processing of SP-B and improved lung compliance in prematurely delivered lambs.
lipopolysaccharide; surfactant protein B; gene regulation
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INTRODUCTION |
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PULMONARY SURFACTANT is a complex mixture of lipids, primarily saturated phosphatidylcholine (Sat PC) and surfactant proteins (SPs) A, B, C, and D, that is synthesized and secreted by alveolar type II cells (9). Sat PC synthesis and SP gene expression normally increase late in gestation in preparation for the transition to air breathing at birth. The hydrophobic SP-B and SP-C organize surfactant lipids and lower surface tension at the air-liquid interface. Lack of adequate surfactant components leads to respiratory distress syndrome in premature infants. Inherited SP-B deficiency in full-term babies or SP-B gene-targeted mice results in perinatal death due to respiratory failure.
SP-B and SP-C are translated as preproproteins that are processed to surface-active airway peptides and stored with surfactant phospholipids in lamellar bodies for secretion. In type II cells, the SP-B proprotein is proteolytically processed in at least two stages (30). Cleavage of the amino-terminal propeptide to generate a processing intermediate [relative molecular weight (Mr) ~25,000] is followed by cleavage of the carboxy-terminal propeptide to produce the Mr ~8,000 hydrophobic mature peptide, which homodimerizes (Mr ~16,000). A second processing intermediate (Mr ~9,000) has been identified in human fetal lung (10, 19). Only the Mr ~25,000 processing intermediate is detectable in Western blots of human fetal lung tissue at 24 wk gestational age (GA), suggesting that SP-B processing is induced late in development (10).
Recent epidemiologic evidence suggests that very low birth weight
babies exposed to chorioamnionitis or colonized with Ureaplasma urealyticum, a common cause of chorioamnionitis, may have a
decreased incidence of respiratory distress syndrome (12,
29). Chorioamnionitis is associated with increased
proinflammatory cytokine levels in the amniotic fluid, including
interleukin (IL)-6, IL-1, and IL-8, which are risk factors for the
development of bronchopulmonary dysplasia. Nevertheless, these
cytokines may also be regulators of lung maturation. Intra-amniotic
(IA) administration of IL-1
induces lung maturation in preterm
rabbits and lambs (2, 7, 11), but the mechanism of this
effect is not understood. We recently developed an IA endotoxin
model of amnionitis-induced lung maturation in fetal lambs
(16). The fetal lamb lung has immature type II cells at 125 days GA
(term is 150 days) and very little surfactant in the alveolar pool,
which results in poor compliance and difficulty in ventilating these
lungs without exogenous surfactant treatment. Lambs delivered
at the same GA have improved postnatal lung function and increased
surfactant lipids by 7 days after IA endotoxin exposure (15,
16). Increased expression of mRNAs for the
proinflammatory cytokines IL-1
, IL-6, and IL-8 was detected in the
lung 5-15 h after IA endotoxin administration, and peak levels
were measured at 1-2 days (18). These results are
consistent with the hypothesis that a proinflammatory stimulus to the
fetus accelerates lung maturation.
Type II cell expression of the hydrophobic SPs in the adult lung is
inhibited by infection or intratracheal instillation of tumor necrosis
factor- or endotoxin (13, 25, 32). The initial inhibition of SP gene expression is followed by increased expression in
areas of repair (27, 32). In adult rats, intratracheal endotoxin induces expression of the pulmonary collectins SP-A and SP-D
(22). The early effects of IA endotoxin on surfactant components are unknown. A time-course study was designed to determine the kinetics of the fetal lung maturation response to a proinflammatory stimulus by varying the interval between administration of IA endotoxin
and preterm delivery.
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MATERIALS AND METHODS |
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Animals.
Animal studies were performed in Western Australia with the approval of
the animal care and use committees from Children's Hospital Medical
Center (Cincinnati, OH) and the Western Australia Department of
Agriculture. Merino ewes with singleton gestations were randomized to
one of six treatment groups (see Fig. 1).
IA injection of 20 mg of Escherichia coli 055:B5 endotoxin
(Sigma, St. Louis, MO) in saline was performed on the indicated day of gestation. Dosing experiments that used between 1 and 100 mg of IA
endotoxin showed similar lung maturational responses (15); therefore, the 20-mg dose was used to compare with previous
observations by Jobe et al. (16). At 15, 7, 4, 2, or 1 day
after IA injection, lambs were delivered by cesarean section (125 days
GA) as previously described (15). The control group
contained 11 ewes randomized and delivered concurrently 7 or 2 days
after receiving IA saline.
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Lung physiology and bronchoalveolar lavage. Lambs were delivered at 125 days GA and ventilated for 40 min for assessment of lung function (15). Values for lung compliance and lung gas volume, measured by static inflation of the lungs to 40 cmH2O, are reported as relative changes from the mean value of saline-treated control animals to permit comparison with changes in surfactant components.
Lungs were removed from the chest and weighed. The left lung was lavaged five times with cold saline as described (15). Bronchoalveolar lavage fluid (BALF) samples from each animal were pooled, volume was measured, and aliquots were frozen for SP and Sat PC quantification. Sat PC measurements on these samples are reported in Ref. 15, and the relative change from control samples was calculated for each animal and plotted as means ± SE. A portion of the right lower lobe was snap-frozen for RNA analysis.RNA analysis.
Total lung RNA was purified by a modification of the acid-phenol
extraction method (3) with Phase Lock gels (5 Prime 3 Prime, Gaithersburg, MD) and quantified by optical density at 260 nM.
SP-A, -B, and -C mRNAs were quantified by S1 nuclease protection assay
with ribosomal protein L32 mRNA as an internal control, essentially as
described (15). SP-D mRNA was quantified in separate
hybridization reactions with 10 µg of total RNA and L32 probe as a
control. The plasmid pGEMshSPD containing a portion of the sheep SP-D
cDNA (GenBank accession no. AJ133002) was kindly provided by Mikko
Hallman (University of Oulu, Oulu, Finland). The SP-D S1 probe was
generated by linearization of pGEMshSPD with MSC I and
dephosphorylation with calf intestinal alkaline phosphatase. The 151-bp
S1 nuclease protected fragment encodes a portion of the carbohydrate
recognition domain of SP-D. All probes were end labeled with
[
-32P]ATP (DuPont NEN, Boston, MA), combined as
indicated, and hybridized overnight with 10 µg of total lung RNA to
detect SP-D or 3 µg of RNA to detect the other SPs. S1 nuclease
protected fragments were resolved on 8 M urea-6% polyacrylamide gels,
visualized by autoradiography, and quantified by phosphorimage analysis
with the Storm system and ImageQuant software (Molecular Dynamics).
Western immunoblotting. BALF samples containing equivalent amounts of Sat PC were concentrated and separated by electrophoresis under reducing conditions on SDS-polyacrylamide gradient gels (8-16%) in Tris-glycine buffer for SP-A and SP-D and on 10-20% gels in tricine buffer (NOVEX, San Diego, CA) for SP-C. Samples were separated under nonreducing conditions on 10-20% gradient gels in tricine buffer for SP-B detection. SPs were detected by Western blotting with the following rabbit polyclonal antibodies: anti-bovine SP-A (R362) (20), anti-recombinant human SP-C (R22/96) (26), anti-bovine SP-B (R28031) (2, 21), and anti-mouse SP-D (11567) (14). Dr. Wolfram Steinhilber (Byk Gulden, Constance, Germany) provided the SP-C antibody, and Dr. Jeffrey Whitsett (Children's Hospital Medical Center, Cincinnati, OH) provided all other primary antibodies for this study. Horseradish peroxidase-conjugated goat anti-rabbit IgG (Calbiochem, La Jolla, CA) was used as a second antibody. Western blots were developed with the enhanced chemiluminescence system (Amersham, Arlington Heights, IL), and band intensities were quantified by densitometry (ISI100 digital imaging system, Alpha Innotech, San Leandro, CA). The Mr ~25,000 SP-B precursor and Mr ~16,000 mature SP-B dimer bands were quantified both together and separately to determine percent mature SP-B. Single bands at the expected sizes for mature SP-A, -C, and -D were quantified. The relative amount of each SP was then calculated in the total BALF and normalized to kilograms of body weight. Values were plotted relative to those of the saline-treated control samples as means ± SE (n = 3-5 animals/group as indicated in Figs. 1 and 4-6).
Statistical analysis.
The number of animals in each group ranged from 6 to 11. Data for all
animals are included in Fig. 2. Three to
five animals with Sat PC values in the alveolar wash close to the mean
for each group were selected for analysis of SPs and mRNAs. Comparisons between control and treatment intervals were by ANOVA with Dunnett's test to identify significance at P < 0.05. Selected comparisons were made by two-tailed t-tests.
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RESULTS |
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Lung function and surfactant lipids. Pregnant ewes were randomized to receive IA saline 118 or 123 days into pregnancy (the control group) or IA endotoxin at the indicated times (from 110 to 124 days into pregnancy), and all lambs were delivered by cesarean section at 125 days GA (Fig. 1). Antenatal IA endotoxin did not alter birth weights, cord blood pH, or cord plasma cortisol levels from control values (15). Mean compliance values indicated a progressive improvement from 4 to 15 days after endotoxin exposure (Fig. 2). Total lung compliance increased approximately twofold by 7 days after endotoxin exposure to ~70% of term values (17). Mean lung gas volumes at 40 cmH2O pressure were increased significantly, by approximately twofold, by 4 days and showed large improvements by 7 and 15 days to ~60% of term values (17). The changes in lung mechanics were accompanied by a progressive increase in Sat PC to 26-fold by 15 days after IA endotoxin.
Induction of SP mRNAs.
Total RNA from fetal lungs was analyzed for SP mRNA abundance by S1
nuclease protection assays with the ribosomal protein L32 mRNA as an
internal control (Fig. 3A). In
saline-treated control animals, mRNAs for SP-A, -B, and -C were very
low, and 7-day endotoxin-treated animals had increased SP mRNAs,
consistent with a previous study from our laboratory (16).
SP-A and SP-B mRNAs were significantly elevated by 1 day, and SP-A, -B,
and -C mRNAs all peaked 2 days after endotoxin exposure. SP-A mRNA was
induced to the greatest extent, by 8- to 10-fold, at 2 days (Fig.
3B). All three SP mRNAs remained significantly elevated
through 7-15 days postexposure.
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SPs in the BALF.
SPs were detected by Western blot analysis of BALF samples containing
equivalent amounts of Sat PC. The calculated alveolar pools of SP-A and
SP-C were significantly increased by 4 days after IA endotoxin.
SP-A had increased >100-fold by 15 days (Fig. 5A), whereas mature SP-C was
increased ~60-fold by 7 days and maintained a high level through 15 days (Fig. 5B). SP-D protein in the BALF was very low in
control animals and was significantly increased (~100-fold) 7 and 15 days after IA endotoxin (Fig. 5C).
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DISCUSSION |
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IA endotoxin induced maturation of the pulmonary surfactant system in 125-day GA fetal sheep within 4-7 days after exposure. Peak levels of SP-A, -B, and -C mRNAs were detected 2 days after IA endotoxin, whereas SP-D mRNA remained at peak levels for at least 7 days. SPs and Sat PC accumulated to high levels in the BALF from 4 to 15 days after IA endotoxin. As expected, all SPs were present at low levels in the BALF of control animals. Partially processed SP-B was the predominant form in the alveolar pool of ventilated 125-day fetal sheep, indicating that SP-B is not only deficient in these lungs, it is in an inactive form (21). SP-B processing was induced in endotoxin-treated animals (60% mature by 4 days and 95% mature by 7 days). Improved physiological lung function and maturation of the surfactant system occurred 4-7 days after IA endotoxin-induced chorioamnionitis and was maintained for at least 15 days.
SP-B processing. Processing of SP-B is induced late in human fetal lung development (10). Low levels of proSP-B are detectable in Western blots of human fetal lung tissue starting at 19 wk, and the Mr ~25,000 processing intermediate is detected at 24 wk (10). We now show that BALF of 125-day GA fetal lambs, which have a surfactant system maturation similar to that of 22- to 24-wk humans, primarily contains the Mr ~25,000 SP-B processing intermediate. Specific processing intermediates of SP-A and SP-C were not detected in BALF at any time point. SP-A and SP-C were increased concomitantly with Sat PC, even though the maximal induction of SP-A and SP-C surpassed that of Sat PC. Increased production of SP-B and processing to the mature peptide was delayed relative to that of the other surfactant components. By 7 days after IA endotoxin, mature SP-B had increased 100-fold in the BALF, concomitant with large improvements in lung physiology measurements. The apparent maturation of type II cell markers in this model suggests that all measures of type II cell maturation are likely to be increased by IA endotoxin.
Induction of SP-D. SP-D expression is increased in late gestation in the rat (4), mouse (31), and human (6) lung and is induced by dexamethasone treatment of fetal lung explants in culture (5, 6). We now show that SP-D is increased late in gestation in fetal sheep lung as well. SP-D was induced by IA endotoxin with different kinetics than the other SPs, suggesting that independent regulatory pathways may be involved. The sustained approximate fourfold elevation in SP-D mRNA resulted in an ~100-fold elevation in SP-D protein in the BALF, demonstrating a striking amplification of the extracellular pool size of this protein by IA endotoxin exposure.
IA endotoxin induces the surfactant system, whereas glucocorticoid induces structural changes in the lung. Antenatal betamethasone (Beta) treatment improves postnatal lung function within 15-24 h in sheep, primarily as a result of acute thinning of the alveolar wall (24). Tan et al. (28) showed that Beta also induces a transient increase in SP mRNAs of up to threefold at 1-2 days, with a subsequent return to baseline values. Whereas the glucocorticoid effect is fully reversible by 7 days after treatment, IA endotoxin increased mRNAs for surfactant components to higher levels that remained elevated for up to 15 days. Assuming that relative SP mRNA levels in 125-day GA control fetuses in this study were similar to those calculated by Tan et al., IA endotoxin induced SP-A mRNA to peak levels approximately twofold greater than those in term fetuses and SP-B and SP-C to peak levels equivalent to term. Alveolar Sat PC increased by 20-fold over that in control animals to ~25% of near-term values (17) 7 days after IA endotoxin, and the extracellular SP pools increased 80- to 100-fold, demonstrating an augmented effect on the protein components of surfactant. In contrast, Beta induces more modest changes in Sat PC and SP mRNA and protein levels that are of similar magnitude (28).
A single dose of IA endotoxin induced maturation of the surfactant system that was maintained for at least 15 days posttreatment. A single dose of antenatal Beta had minimal effects on surfactant when given 14 days before preterm delivery of sheep (28). IA endotoxin-treated animals did not have increased plasma cortisol levels and were not growth restricted like fetal lambs treated with maternal glucocorticoid (16). Unlike Beta treatment, IA endotoxin induced an inflammatory response in the amnion/chorion and lung that persisted through 15 days (16, 18), suggesting that not all responses to IA endotoxin may be beneficial. Although endotoxins should not be considered for use in humans, a long-term goal of these studies is to define the signaling pathway responsible for induction of type II cell maturation in this model to determine whether a specific inducer of lung maturation can be developed for clinical use.Fetal lung response to inflammatory mediators is different from
that in the adult.
Intratracheal instillation of endotoxin or proinflammatory cytokines
into the adult lung caused transient inhibition of the hydrophobic SPs
followed by increased SP gene expression in areas of remodeling and
repair (13, 23, 25, 27). The fetal sheep lung is in the
saccular/early alveolar stage of development at 125 days GA, and SP
mRNAs are expressed at very low levels in immature type II cells
(1, 28). IA endotoxin induced all SP mRNAs in the fetal
lung by 1-2 days after treatment. These results suggest that
either the immature type II cell response to inflammatory mediators is
somehow different or IA endotoxin does not induce the same inflammatory
mediators when delivered to the amniotic fluid as when directly
instilled in the adult lung. The observed increase in SP gene
expression is consistent with recent in vitro data from the
fetal rabbit (8). Treatment of immature 19-day GA fetal
rabbit lung explants with high-dose IL-1 stimulated SP gene
expression, whereas similar treatment of lung explants from 27-day
fetal or newborn rabbits inhibited SP gene expression (8).
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ACKNOWLEDGEMENTS |
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We thank Drs. W. Steinhilber and J. Whitsett for providing antibodies, Dr. M. Hallman for providing the sheep SP-D cDNA clone, and Kathryn Foss for expert technical assistance.
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FOOTNOTES |
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This research was supported by National Heart, Lung, and Blood Institute Grants HL-60907 (C. J. Bachurski) and HL-65397 (A. H. Jobe).
Address for reprint requests and other correspondence: C. J. Bachurski, Children's Hospital Research Foundation, Division of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 25229-3039 (E-mail: cindy.bachurski{at}chmcc.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 June 2000; accepted in final form 7 September 2000.
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