Intra-amniotic endotoxin increases pulmonary surfactant proteins and induces SP-B processing in fetal sheep

Cindy J. Bachurski, Gary F. Ross, Machiko Ikegami, Boris W. Kramer, and Alan H. Jobe

Division of Pulmonary Biology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-1beta , 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-1alpha 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-1beta , 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-alpha 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.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1.   Time-course protocol. Intra-amniotic (IA) injection of 20 mg of Escherichia coli endotoxin (black-down-triangle ) was given on the indicated day (d) of gestational age (GA) to fetal lambs (term is 150 days). Animals that received IA saline (down-triangle) at 118 (n = 6) or 123 days GA (n = 5) were pooled as the control comparison group. All lambs were delivered at 125 days GA. n, No. of animals/group.

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 right-arrow 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 [gamma -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.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   IA endotoxin increased saturated phosphatidylcholine (Sat PC) and improved lung mechanics. Data are plotted as mean values ± SE calculated relative to the mean of control IA saline group for each measurement. Dynamic total thoracic compliance measured after 40 min of ventilation was increased both 7 and 15 days after treatment (15). Mean static lung gas volumes (ml/kg body wt), measured at 40 cmH2O pressure (V40), were significantly increased by 4 days after endotoxin treatment. All animals showed increased lung gas volumes at 7 and 15 days. Alveolar Sat PC increased from a very low value in control animals incrementally up to 26-fold by 15 days. *P < 0.05 vs. control. **P < 0.01 vs. control.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 3.   Surfactant protein (SP) mRNAs were rapidly induced and sustained at increased levels for 15 days after IA endotoxin. A: representative autoradiogram of S1 nuclease protection analysis for SP-A, -B, and -C and ribosomal protein L32 mRNAs performed with 3 µg of total RNA from fetal lungs on the indicated days after IA endotoxin or saline (control) injection. B: protected fragments were quantified by phosphorimaging, and SP mRNAs were normalized relative to L32 as an internal control. The mean of the saline-injected control animals was set to 1, and the relative values of SP-A, SP-B, and SP-C mRNA for each time point are shown as means ± SE and were compared with control values with the 2-tailed t-test; n = 4-8 animals/group. *P < 0.05 vs. control. **P < 0.01 vs. control.

Because very little is known about SP-D mRNA expression during sheep lung development, total RNA from untreated fetal sheep lungs at 118, 125, and 130 days GA was analyzed. SP-D mRNA was low at both 118 and 125 days GA and increased at 130 days (Fig. 4A), suggesting that SP-D expression increases late in normal ovine lung development. SP-D was expressed at very low levels in saline-treated fetal lung and was significantly induced by 1 day after endotoxin (Fig. 4B). Unlike the other surfactant-associated proteins, SP-D mRNA remained at peak induced levels through 7 days (Fig. 4C).


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4.   SP-D mRNA was increased late in gestation and induced by IA endotoxin. Shown are representative autoradiograms of S1 nuclease protection analysis for SP-D and L32 mRNAs in 10 µg of total lung RNA from lambs of the indicated GAs (A) and 125-day GA lambs treated with IA endotoxin (B). C: relative SP-D mRNA was plotted as means ± SE as in Fig. 2; n = 3-5 animals/group. SP-D mRNA was expressed at low levels in control 125-day GA fetal lung, was significantly induced by 1 day, and was sustained at higher levels for 7 days after exposure. **P < 0.01 vs. control.

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).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5.   SPs were increased in the bronchoalveolar lavage fluid (BALF) by 4-7 days after IA endotoxin. SP-A, -C, and -D were detected by Western blotting of BALF samples containing equivalent amounts of Sat PC with rabbit polyclonal antibodies. A: anti-bovine SP-A (R362). B: anti-recombinant human SP-C (R22/96). C: anti-mouse SP-D (11567). Band intensities were quantified by densitometry (ISI digital imaging system), and the amount of each SP was calculated in the total BALF relative to kilograms of body weight. Values are means ± SE relative to those in saline-treated control animals; n = 3 animals/group. *P < 0.05 vs. control.

Western blots that used anti-bovine SP-B antibody R28031, which detects both SP-B proproteins and the mature peptide (21), identified a predominant, partially processed form of SP-B (Mr ~25,000) in the BALF of control 125-day GA fetal sheep (Fig. 6A). Total SP-B levels in the BALF increased more slowly than Sat PC (note low signal in day 4 samples) and were not significantly increased until day 7 (Fig. 6B). Processing to the mature Mr ~16,000 SP-B dimer was induced by IA endotoxin between days 4 and 7 when 95% of the BALF SP-B was processed to the active mature peptide (Fig. 6C). SP-B processing was maintained through 15 days after IA endotoxin.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 6.   SP-B processing was induced by IA endotoxin. A: Western blot for SP-B of BALF samples containing equivalent amounts of Sat PC with rabbit polyclonal anti-bovine SP-B (R28031). The partially processed Mr ~25,000 form of SP-B was detected in control samples (0), and processing to the mature Mr ~16,000 SP-B homodimer was detected between 4 and 7 days. Nos. at left, molecular weight standards. B: mature SP-B was calculated in the BALF relative to body weight and is shown as in Fig. 5. C: relative amount of alveolar SP-B in the mature Mr ~16,000 homodimer form was calculated as a percent of total SP-B (Mr ~25,000 intermediate plus Mr ~16,000 SP-B homodimer) and plotted as means ± SE; n = 3 animals/group. *P < 0.05 vs. control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-1alpha stimulated SP gene expression, whereas similar treatment of lung explants from 27-day fetal or newborn rabbits inhibited SP gene expression (8).

In summary, 125-day GA fetal sheep have an immature surfactant system characterized by deficient alveolar pools of SPs and Sat PC. Not only is the alveolar pool of SP-B deficient, SP-B processing is incomplete. IA endotoxin stimulated a rapid and sustained maturation of the surfactant system, including induction of SP-B processing in premature lamb lungs. Determining the signal that stimulates early type II cell maturation in this model may be useful in designing future therapies for pregnancies at high risk of premature birth.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alcorn, DG, Adamson TM, Maloney JE, and Robinson PM. A morphologic and morphometric analysis of fetal lung development in the sheep. Anat Rec 201: 655-667, 1981[ISI][Medline].

2.   Bry, K, Lappalainen U, and Hallman M. Intraamniotic interleukin-1 accelerates surfactant protein synthesis in fetal rabbits and improves lung stability after premature birth. J Clin Invest 99: 2992-2999, 1997[Abstract/Free Full Text].

3.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

4.   Crouch, E, Rust K, Marienchek W, Parghi D, Chang D, and Persson A. Developmental expression of pulmonary surfactant protein D (SP-D). Am J Respir Cell Mol Biol 5: 13-18, 1991[ISI][Medline].

5.   Deterding, RR, Shimizu H, Fisher JH, and Shannon JM. Regulation of surfactant protein D expression by glucocorticoids in vitro and in vivo. Am J Respir Cell Mol Biol 10: 30-37, 1994[Abstract].

6.   Dulkerian, SJ, Gonzales LW, Ning Y, and Ballard PL. Regulation of surfactant protein D in human fetal lung. Am J Respir Cell Mol Biol 15: 781-786, 1996[Abstract].

7.   Emerson, GA, Bry K, Hallman M, Jobe AH, Wada N, Ervin MG, and Ikegami M. Intra-amniotic interleukin-1 alpha treatment alters postnatal adaptation in premature lambs. Biol Neonate 72: 370-379, 1997[ISI][Medline].

8.   Glumoff, V, Vayrynen O, Kangas T, and Hallman M. Degree of lung maturity determines the direction of the interleukin-1-induced effect on the expression of surfactant proteins. Am J Respir Cell Mol Biol 22: 280-288, 2000[Abstract/Free Full Text].

9.   Griese, M. Pulmonary surfactant in health and human lung diseases: state of the art. Eur Respir J 13: 1455-1476, 1999[Abstract/Free Full Text].

10.   Guttentag, SH, Beers MF, Bieler BM, and Ballard PL. Surfactant protein B processing in human fetal lung. Am J Physiol Lung Cell Mol Physiol 275: L559-L566, 1998[Abstract/Free Full Text].

11.   Hallman, M. Cytokines, pulmonary surfactant and consequences of intrauterine infection. Biol Neonate 76, Suppl 1: 2-9, 1999[ISI][Medline].

12.   Hannaford, K, Todd DA, Jeffery H, John E, Blyth K, and Gilbert GL. Role of Ureaplasma urealyticum in lung disease of prematurity. Arch Dis Child Fetal Neonatal Ed 81: F162-F167, 1999[Abstract/Free Full Text]. (Corrigenda. Arch Dis Child Fetal Neonatal Ed 82: January 2000, p. F78.)

13.   Harrod, KS, Mounday AD, and Whitsett JA. Adenoviral E3-14.7K protein in LPS-induced lung inflammation. Am J Physiol Lung Cell Mol Physiol 278: L631-L639, 2000[Abstract/Free Full Text].

14.   Hull, W, Stahlman M, Gray MP, Wert S, and Whitsett J. Immunolocalization of SP-D in human secretory tissues (Abstract). Am J Respir Crit Care Med 161: A42, 2000.

15.   Jobe, AH, Newnham JP, Willet KE, Moss TJ, Padbury JF, Ervin MG, Sly P, and Ikegami M. Endotoxin-induced lung maturation in preterm lambs is not mediated by cortisol. Am J Respir Crit Care Med 162: 1656-1661, 2000[Abstract/Free Full Text].

16.   Jobe, AH, Newnham JP, Willet KE, Sly P, Ervin MG, Bachurski C, Possmayer F, Hallman M, and Ikegami M. Effects of antenatal endotoxin and glucocorticoids on the lungs of preterm lambs. Am J Obstet Gynecol 182: 401-408, 2000[ISI][Medline].

17.   Jobe, AH, Newnham J, Willet K, Sly P, and Ikegami M. Fetal versus maternal and gestational age effects of repetitive antenatal glucocorticoids. Pediatrics 102: 1116-1125, 1998[Abstract/Free Full Text].

18.  Kallapur SG, Willet KE, Jobe AH, Ikegami M, and Bachurski CJ. Intra-amniotic endotoxin: chorioamnionitis precedes lung maturation in preterm lambs. Am J Physiol Lung Cell Mol Physiol. In press.

19.   Korimilli, A, Gonzales LW, and Guttentag SH. Intracellular localization of processing events in human surfactant protein B biosynthesis. J Biol Chem 275: 8672-8679, 2000[Abstract/Free Full Text].

20.   Lesur, O, Veldhuizen RA, Whitsett JA, Hull WM, Possmayer F, Cantin A, and Begin R. Surfactant-associated proteins (SP-A, SP-B) are increased proportionally to alveolar phospholipids in sheep silicosis. Lung 171: 63-74, 1993[ISI][Medline].

21.   Lin, S, Na CL, Akinbi HT, Apsley KS, Whitsett JA, and Weaver TE. Surfactant protein B (SP-B) -/- mice are rescued by restoration of SP-B expression in alveolar type II cells but not Clara cells. J Biol Chem 274: 19168-19174, 1999[Abstract/Free Full Text].

22.   McIntosh, JC, Swyers AH, Fisher JH, and Wright JR. Surfactant proteins A and D increase in response to intratracheal lipopolysaccharide. Am J Respir Cell Mol Biol 15: 509-519, 1996[Abstract].

23.   Mora, R, Arold S, Marzan Y, Suki B, and Ingenito EP. Determinants of surfactant function in acute lung injury and early recovery. Am J Physiol Lung Cell Mol Physiol 279: L342-L349, 2000[Abstract/Free Full Text].

24.   Pinkerton, KE, Willet KE, Peake JL, Sly PD, Jobe AH, and Ikegami M. Prenatal glucocorticoid and T4 effects on lung morphology in preterm lambs. Am J Respir Crit Care Med 156: 624-630, 1997[Abstract/Free Full Text].

25.   Pryhuber, GS, Bachurski C, Hirsch R, Bacon A, and Whitsett JA. Tumor necrosis factor-alpha decreases surfactant protein B mRNA in murine lung. Am J Physiol Lung Cell Mol Physiol 270: L714-L721, 1996[Abstract/Free Full Text].

26.   Ross, GF, Ikegami M, Steinhilber W, and Jobe AH. Surfactant protein C in fetal and ventilated preterm rabbit lungs. Am J Physiol Lung Cell Mol Physiol 277: L1104-L1108, 1999[Abstract/Free Full Text].

27.   Sugahara, K, Iyama K, Sano K, Kuroki Y, Akino T, and Matsumoto M. Overexpression of surfactant proteins SP-A, SP-B, and SP-C mRNA in rat lungs with lipopolysaccharide-induced injury. Lab Invest 74: 209-220, 1996[ISI][Medline].

28.   Tan, RC, Ikegami M, Jobe AH, Yao LY, Possmayer F, and Ballard PL. Developmental and glucocorticoid regulation of surfactant protein mRNAs in preterm lambs. Am J Physiol Lung Cell Mol Physiol 277: L1142-L1148, 1999[Abstract/Free Full Text].

29.   Watterberg, KL, Demers LM, Scott SM, and Murphy S. Chorioamnionitis and early lung inflammation in infants in whom bronchopulmonary dysplasia develops. Pediatrics 97: 210-215, 1996[Abstract].

30.   Weaver, TE. Synthesis, processing and secretion of surfactant proteins B and C. Biochim Biophys Acta 1408: 173-179, 1998[ISI][Medline].

31.   Wong, CJ, Akiyama J, Allen L, and Hawgood S. Localization and developmental expression of surfactant proteins D and A in the respiratory tract of the mouse. Pediatr Res 39: 930-937, 1996[Abstract].

32.   Zsengellér, ZK, Wert SE, Bachurski CJ, Kirwin KL, Trapnell BC, and Whitsett JA. Recombinant adenoviral vector disrupts surfactant homeostasis in mouse lung. Hum Gene Ther 8: 1331-1344, 1997[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 280(2):L279-L285
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society