1 Division of Clinical Sciences, Center for Child Health Research, 2 University Department of Obstetrics and Gynecology, University of Western Australia, Perth, Australia; and 3 Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio, 45229
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ABSTRACT |
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Antenatal
inflammation may be an important triggering event in the pathogenesis
of bronchopulmonary dysplasia but may also accelerate fetal lung
maturation. We examined the effects of intra-amniotic (IA) interleukin
(IL)-1 and IL-1
on maturation of the fetal sheep lung. These
cytokine effects were compared with IA endotoxin, a potent
proinflammatory stimulus that accelerated lung maturation. Date-bred
ewes received 15 or 150 µg recombinant ovine IL-1
or IL-1
or 10 mg Escherichia coli endotoxin by IA injection at 118 days
gestation (term = 150 days), and fetuses were delivered at 125 days. IL-1
and IL-1
improved lung function and increased alveolar
saturated phosphatidylcholine (Sat PC) and surfactant protein mRNA
expression at the higher dose. The maturation response to IL-1
was
greater than that to IL-1
, which was similar to endotoxin response.
Inflammation was also more pronounced after IL-1
treatment. Only
endotoxin animals had residual inflammation of the fetal membranes at 7 days. Lung compliance, lung volume, and alveolar Sat PC were positively
correlated with residual alveolar wash leukocyte numbers 7 days after
IL-1 treatment, suggesting a link between lung inflammation and maturation.
respiratory distress syndrome; bronchopulmonary dysplasia; chorioamnionitis; surfactant; cytokines
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INTRODUCTION |
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INTERLEUKIN
(IL)-1 and IL-1
are distinct proteins that are encoded by
different genes but that bind to the same receptors and share the same
spectrum of biological activities (10). Although IL-1
is mainly active as a cytosolic precursor and a membrane-associated protein, IL-1
is active only when secreted in its mature form. IL-1
plays an important role in the development of the acute inflammatory response by mediating its own production and by stimulating the synthesis of other cytokines such as IL-6, IL-8, and tumor necrosis factor (TNF)-
(11, 21, 45). Endotoxin, a
lipopolysaccharide component of gram-negative bacteria, is a potent
inducer of many cytokines, including IL-1
and IL-1
(7,
18). Many of the pathophysiological effects of endotoxin are
similar to those induced by IL-1, and the two share a common
intracellular signaling pathway (14). We recently found
that intra-amniotic (IA) injection of endotoxin, a potent
proinflammatory stimulus, improved lung function, increased production
and secretion of surfactant components, and caused structural changes
in fetal sheep lungs (23, 54). Expression of mRNA
for IL-1
and other cytokines in the chorioamniotic membranes increased within 5 h of endotoxin injection, with a subsequent inflammatory response in the lungs (25). Our findings in
preterm sheep are consistent with clinical observations of a decreased risk of respiratory distress in infants exposed prenatally to chorioamnionitis (53). In the present study we
hypothesized that the maturational effects of endotoxin on the fetal
sheep lung are mediated by IL-1 and that antenatal exposure to IL-1
or to IL-1
would result in changes similar to those seen after IA
endotoxin exposure. We report on the response of the fetal sheep to IA
injection of recombinant ovine IL-1
or IL-1
and compare that
response to IA endotoxin.
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METHODS |
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Preparation of recombinant ovine IL-1 and IL-1
.
Nucleotides corresponding to the mature polypeptides (amino acid
113-268) of IL-1
(13) and amino acids 114-266
of IL-1
(12) were cloned into the LIC site of vector
pET-30 Xa/LIC (Novagen, Madison, WI). The recombinant proteins were
custom expressed (Protein Express, Cincinnati, OH) in Escherichia
coli BL21(DE3) and initially purified by metal chelate
chromatography using Ni-NTA agarose (Qiagen, Valencia, CA). Purified
fusion proteins were subsequently cleaved with Factor Xa and
recombinant IL-1
and IL-1
then collected from the unbound
fraction of Ni-NTA columns. IL-1
was further purified to homogeneity
by gel filtration chromatography (Sephacryl S-200, Pharmacia,
Piscataway, NJ) and IL-1
by cation exchange chromatography (SP
Sepharose, Pharmacia). Purified proteins were passed over polymixin B
agarose columns (Pierce, Rockford, IL) for removal of endotoxin and
were quantified by bicinchoninic acid protein assay (Pierce) using BSA
as a standard.
Fetal treatments.
Protocols were approved by the Animal Ethics Committees at the
Children's Hospital Medical Center in Cincinnati and the Western Australian Department of Agriculture. Date-bred Merino ewes were randomized to one of six groups: control (n = 12), 15 µg IL-1 (n = 6), 150 µg IL-1
(n = 7), 15 µg IL-1
(n = 6), 150 µg IL-1
(n = 7), and 10 mg endotoxin (E. coli 055:B5, Sigma Chemical, St. Louis, MO; n = 7). Treatments were administered by ultrasound-guided IA injection at
118 days gestation. To verify the IA rather than allantoic location of
each injection, Na+ and Cl
concentrations
were determined on samples of fluid (22). All fetuses were
delivered by cesarean section at 125 days gestation (term = 150 days).
Delivery and postnatal measurements. For delivery each ewe was sedated with ketamine (1 g im) and xylazine (25 mg im) followed by spinal anesthesia (2% lidocaine, 3 ml). The fetal head was exposed through midline abdominal and uterine incisions, and the fetus was sedated (10 mg/kg ketamine). After a local anesthetic (2% lidocaine sc) was administered, a tracheotomy was performed and a 4.0-mm endotracheal tube was secured in place. Lung liquid was removed by suction through the endotracheal tube. Animals were delivered, and the umbilical cord was cut. After delivery, lambs were weighed, dried, and covered with plastic wrap to minimize heat loss. Temperature was maintained at 39°C with an overhead warmer. Animals were placed on an infant ventilator (Bournes BP200) set to deliver 100% oxygen at a rate of 40 breaths per min, inspiratory time 0.75 s, and positive end-expiratory pressure (PEEP) 3 cmH2O. Peak inspiratory pressure (PIP) was initially set at 35 cmH2O. Tidal volume and arterial carbon dioxide partial pressure (PaCO2) were monitored closely, and PIP was adjusted to maintain adequate ventilation. No other ventilator setting was altered during the study. To minimize the risk of ventilator-induced lung injury, PIP was not permitted to exceed 40 cmH2O and tidal volume was kept <10 ml/kg. An arterial catheter was advanced to the level of the descending aorta via an umbilical artery, and lambs were anesthetized by slow arterial infusion of pentobarbital sodium (15 mg/kg).
A pressure transducer (model 8507C-2, Endevco, San Juan Capistrano, CA) and pneumotachograph (model 35-597, Hans Rudolph) were placed between the tracheotomy tube and the ventilator to measure tracheal pressure and flow, respectively. Volume was obtained by integrating flow. Compliance was calculated by dividing tidal volume by ventilatory pressure (PIP-PEEP) and then normalized to body weight in kilograms (24). Arterial oxygen partial pressure, PaCO2, and pH were measured at 10-min intervals. The target PaCO2 was 45-50 mmHg; however, animals were permitted to become hypercarbic when the target PaCO2 was not able to be attained at maximum PIP (40 cmH2O, V40). Ventilation efficiency index (VEI), an index that integrates ventilation with respiratory support, was calculated according to the formula VEI = 3,800/(P × f × PaCO2), where 3,800 ml · mmHg · kgSurfactant lipid and protein mRNA measurements. The lungs were removed from the chest, each lung was weighed, and the left lung was lavaged five times by infusing and withdrawing a sufficient volume of saline at 4°C to fully distend the lungs (24). The five lavages were pooled, the total volume was measured, and lipids were extracted with chloroform:methanol. Saturated phosphatidylcholine (Sat PC) was isolated from lipid extracts by neutral alumina column chromatography after exposure to osmium tetroxide (32). Sat PC was quantified by phosphorus assay (3). The relative abundance of surfactant protein (SP) mRNA was measured using S1 nuclease protection assays as previously described (2). Briefly, an excess of linearized probes for SP-A, SP-B, SP-C, and L32 that were 5'-end [32P] labeled were hybridized at 56°C with 3 µg of total RNA from lung tissue. SP-D was detected in a separate hybridization using 10 µg RNA and L32 as an internal control (2). After incubation with S1 nuclease, the protected fragments were resolved on 6% polyacrylamide 8-mol urea sequencing gels, visualized by autoradiography, and quantified on a phosphorimager (ImageQuant software, Molecular Dynamics, Sunnyvale, CA). Hybridization of messenger RNA was normalized to L32, a ribosomal protein mRNA. Values for control animals were standardized to a mean value of 1.
Inflammation and markers of cellular activation. Aliquots of amniotic fluid and alveolar wash fluid were centrifuged at 500 g for 10 min, and the pellets resuspended in PBS. After total cell counts with trypan blue, differential cell counts were performed on cytospin preparations after staining with Diff-Quick (Scientific Products, McGaw Park, IN). Activation state of cells in alveolar wash fluid was assessed by measuring hydrogen peroxide using an assay based on the oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+) by hydrogen peroxide under acidic conditions (Bioxytech H2O2-560 assay, OXIS International, Portland, OR).
Aliquots of alveolar cells were incubated on ice with monoclonal antibodies (primary antibody) against ovine CD11b (Statistical analyses. Unless otherwise stated, values are given as group means ± SE. Where data were normally distributed, control and treated groups were compared by one-way ANOVA, and post hoc pairwise comparisons were made using Dunnett's procedure. Where data were not normally distributed, global comparisons were made by Kruskal-Wallis ANOVA on ranks, and post hoc pairwise comparisons were made using Dunn's procedure. The association between indexes of inflammation (amniotic fluid cell count, alveolar wash cell count, and lung inflammation score) and indexes of functional maturity (lung compliance, lung volume, and alveolar Sat PC pool size) were examined by backward stepwise multiple linear regression analysis. Statistical significance was accepted at P < 0.05.
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RESULTS |
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Delivery and ventilatory characteristics.
There were no differences in birth weight or in cord blood pH
values between control and treated animals (not shown). Ventilatory and
blood gas values in the low-dose IL-1 and IL-1
groups were also
comparable to control animals at 40 min (Table
1). Lambs exposed to the high dose of
IL-1
or IL-1
had significantly lower ventilatory pressures and
higher tidal volumes at 40 min than controls. Lambs given 150 µg
IL-1
also had significantly improved 40-min PaCO2
and pH compared with control lambs. Lambs exposed to 10 mg endotoxin
were ventilated at lower peak pressures, but other ventilatory
characteristics were not different from control lambs. Lung
weight-to-body weight ratios were similar in all groups.
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Lung function.
Lung compliance values at 40 min were improved after high-dose IL-1,
IL-1
, or endotoxin (Fig. 1). VEI more
than doubled in each group compared with control. Animals receiving
high-dose IL-1
, IL-1
, or endotoxin also had large increases in
maximal lung volumes and in volumes at low transpulmonary pressures.
For all three indexes of lung function, high-dose IL-1
induced a greater improvement than either high-dose IL-1
or endotoxin. Lung
compliance, VEI, and lung volumes in low-dose IL-1
and IL-1
groups were similar to control.
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Surfactant pool size and SP mRNA expression.
Alveolar wash Sat PC pool size averaged 0.4 ± 0.1 µmol/kg in
control animals, increasing 23-, 9-, and 8-fold in 150 µg IL-1, 150 µg IL-1
, and endotoxin groups, respectively (Fig.
2). Sat PC pool size was not different
from control values after 15 µg IL-1
or IL-1
. Lung tissue SP
mRNA expression was determined in control, 150 µg IL-1
, 150 µg
IL-1
, and 10 mg endotoxin animals (Fig. 2). SP-A, SP-B, SP-C, and
SP-D mRNA expression was increased after exposure to 150 µg IL-1
.
IL-1
exposure also resulted in increases in all except SP-B. Lambs
exposed to endotoxin had increased expression of SP-A, SP-B, and SP-C.
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Inflammation and cellular activation.
Total peripheral white cell count increased significantly in animals
exposed to 150 µg IL-1 or to endotoxin (Table
2). Other cytokine groups also had
slightly elevated white cell counts, although the differences were not
statistically significant. The increase in total white cell count was
predominantly due to an increase in neutrophils, which increased by up
to 27-fold (150 µg IL-1
vs. control). Platelet count was also
elevated in the low-dose IL-1
, high-dose IL-1
, and endotoxin
groups. There were no differences in lymphocyte or monocyte counts
between control and treated groups.
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White cell counts as predictors of lung maturation indexes.
We asked whether indexes of inflammation (total alveolar wash
cell count, total amniotic fluid cell count, air space inflammatory cell score, and fetal membranes inflammatory score) correlated with
indicators of lung maturation [increased lung compliance, lung volume
(V40), and increased alveolar wash Sat PC pool size] 7 days after exposure to IL-1. We pooled the data from control and
IL-1-treated animals (n = 31) and examined the
predictive ability of indexes of inflammation by multiple linear
regression analysis. For each index of lung maturation, a linear model
including all predictors was fitted and nonsignificant terms were
sequentially removed from the model until only significant predictors
remained. Total alveolar wash cell count was found to be a strong
predictor of compliance (r = 0.78, P < 0.0005), Sat PC pool size (r = 0.76, P < 0.0005), and V40 (r = 0.70, P < 0.0005), accounting for 61, 57, and 49% of
variability in these maturation indexes, respectively (Fig.
5). In all cases, the relationship was
well described by a linear model. Inclusion of amniotic fluid cell
count improved the fit of the regression model for Sat PC pool size
(r = 0.79, P < 0.0005), but not for
compliance or V40. Sat PC, V40, and compliance were also highly correlated with one another: Sat PC vs.
V40 (r = 0.694, P < 0.0005); Sat PC vs. compliance (r = 0.735, P < 0.0005); compliance vs. V40
(r = 0.762, P < 0.0005).
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DISCUSSION |
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We examined the effects of in utero exposure to IL-1 and
IL-1
on postnatal lung function in preterm lambs and compared the responses to the effects of endotoxin, a proinflammatory stimulus that
accelerated fetal lung maturation (23). Preterm lambs
given an IA injection of 150 µg IL-1
or IL-1
7 days before
preterm delivery had increased lung compliance and lung volumes,
improved gas exchange, and greater ventilatory efficiency. Both
cytokines also caused a significant increase in alveolar Sat PC pool
size and in SP mRNA expression. IL-1
had a greater effect than
IL-1
on lung volume, efficiency of ventilation, and on surfactant
components. Both cytokines were associated with a fetal inflammatory
response, characterized by an increase in peripheral leukocyte count,
an influx of leukocytes into lung tissue and air spaces, and an
increase in cellular activation. As with the lung maturational changes, leukocyte migration and activation appeared to be more pronounced in
animals exposed to IL-1
than to IL-1
. Total leukocyte count in
alveolar wash was a strong predictor of lung compliance, lung volume,
and Sat PC pool size, suggesting a link between the magnitude of
residual inflammation in the lungs and maturational changes.
Intrauterine infection has long been recognized as an important cause
of preterm labor. It is believed that the majority of preterm infants
born before 30 wk gestation were exposed in utero to low-grade
ascending infection (17). IL-1 was implicated as a signal
for the onset of parturition in the setting of infection (41). Both IL-1 and IL-1
are produced by human
decidual explants in response to bacterial endotoxin (44),
and these cytokines stimulate prostaglandin production by the amnion
and decidua (33, 42). Romero and colleagues
(43) reported that amniotic fluid IL-1
and IL-1
concentrations were low throughout pregnancy (range 0-0.2 ng/ml)
but were elevated in women with preterm labor and microbial invasions.
Median concentrations of IL-1
and IL-1
were 3.5 and 10 ng/ml,
respectively in women with preterm labor and microbial invasions. In
our study, IL-1
/
concentrations after 150-µg injections would
be in the order of 0.3 µg/ml, based on an estimated amniotic fluid
volume of ~500 ml. These concentrations are 30- to 90-fold higher
than the levels reported by Romero's group, and yet none of the fetal
sheep exposed to 150 µg IL-1
or IL-1
aborted.
Watterberg et al. (53) reported that infants exposed to
histological chorioamnionitis, although smaller and less mature than
infants not exposed to chorioamnionitis, had a decreased incidence of
respiratory distress syndrome. These same infants, however, had higher
levels of the inflammatory mediators IL-1, leukotriene B4,
and thromboxane B2 in tracheal aspirates shortly after
birth and were more likely to develop brochnopulmonary dysplasia (BPD) (53). Yoon et al. (55) found that
elevated levels of the proinflammatory cytokines IL-1, IL-6, IL-8,
and TNF-
were associated with a significantly increased risk of
developing BPD. This group more recently reported that elevated cord
blood IL-6 concentration was a better predictor of BPD than was
elevated amniotic fluid IL-6 concentration, suggesting that development of a systemic fetal inflammatory response may be important
(56). Overall, the evidence suggests that in utero
inflammation may offer short-term benefits to the preterm neonate but
may also lead to arrested alveolarization and progressive lung injury.
Our findings are in agreement with those of previous studies that have
examined the lung maturational effects of IL-1. Bry and colleagues
(5) reported an increase in lung compliance, Sat PC, and
in SPs in fetal rabbits delivered on day 27 of gestation, 40 h after IA injection of recombinant human IL-1
. More
recently Glumoff et al. (16) reported that the effect of
IL-1
on SP mRNA expression in cultured fetal rabbit lung explants
varied with gestational age and was also time and dose dependent.
Although SP-A and SP-B mRNA expression increased substantially in
explants from 19-day fetuses, SP-B and SP-C expression decreased in
explants from term or near-term animals. Our study is unique insofar as there are no other studies of the direct inflammatory effects of IA
IL-1
or IL-1
.
Hybertson et al. (20) recently examined alterations in the
surfactant system in a rat model of IL-1-induced acute lung injury.
Intratracheal instillation of IL-1
caused neutrophil accumulation
and increased phospholipid levels in bronchoalveolar lavage. However,
electron microscopic examination of the lungs revealed evidence of
alveolar type II cell abnormalities, including apical membrane
disruption, abnormal extrusion of lamellar bodies into the airspaces,
increased cytoplasmic deposition of H2O2, and
increased
-glutamyl transferase activity (a marker of oxidative stress). The authors speculated that increased phospholipid release may
be a consequence of alveolar type II cell injury as a result of
neutrophil-mediated oxidative stress. It should be emphasized that
these observations were made in adult animals, and their relevance to
the preterm lung is unclear. Of note, 150 µg IL-1
caused
inflammatory cells in the lungs to produce H2O2
7 days after IA injection, an observation that is consistent with
oxidative stress. However, SP mRNA levels increased after IL-1
and
IL-1
, indicating upregulated surfactant synthesis, an effect that is not typical of oxidative stress in the adult lung. Furthermore, endotoxin increases antioxidant enzymes in the fetal lung
(48).
Despite effects on cellular proliferation and on surfactant production,
studies in knockout mice suggest that IL-1 is unlikely to be an
important modulator of lung development during normal pregnancy. Mice
deficient in IL-1 receptor type I (IL-1R I), the transmembrane receptor
that mediates all known biological activities of IL-1 and IL-1
,
have phenotypically normal lungs and do not appear to exhibit
respiratory dysfunction (28), although detailed functional
and morphometric assessments have not been made. Similarly, mice
deficient in IL-1
-converting enzyme, a cysteine protease that
converts the biologically inactive precursor of IL-1
to its active
form, also appear to have structurally normal lungs (29).
Further support for the redundancy of IL-1 in normal lung development
comes from the study by Bry et al. (5), examining SP
expression in rabbits. Although IL-1 upregulated SP expression both in
vitro and in vivo, IL-1 receptor antagonist (IL-1ra) failed to modify
the expression of SP-A, SP-B, or SP-C mRNA when either added to culture
medium of fetal lung explants or given in excess to fetal rabbits by IA injection.
We found that IL-1 induced more pronounced inflammatory and
maturational changes than IL-1
did in the fetal lamb. Differences in
half-life could contribute to the observed difference, although the
authors are unaware of any pharmacokinetic studies comparing the
elimination half-lives of IL-1
and IL-1
after IA injection. The
two cytokines appear to have similar plasma distribution half-lives after intravenous administration in rats (38, 40) and are metabolized predominantly by the kidney. Differences in receptor-ligand binding affinities are unlikely to account for the apparent difference in potencies of IL-1
and -
in our preterm lamb model, as evidence in the literature suggests that IL-1
binds to IL-1R I with
approximately equal or greater affinity than IL-1
(8, 26,
27) and is as potent, if not more potent than IL-1
, at
inducing proinflammatory events in target cells (8, 36,
52). A second type of IL-1 receptor, IL-1R II, binds both
IL-1
and -
but does not transduce a signal, thus acting as a
"decoy" receptor, inhibiting the bioactivity of IL-1. IL-1R II
binds IL-1
with greater affinity than IL-1
(4).
Differences in the ability of IL-1R II to sequester IL-1
and IL-1
may be the most plausible explanation for the disparity in biological
activity in the present study.
IL-1 is a potent inducer of chemokine synthesis from monocytes,
fibroblasts, and endothelial cells and, next to TNF-, is the most
potent inducer of endothelial cell adhesion molecule expression
(10). IL-1 also stimulates synthesis of
platelet-activating factor and prostaglandin E2 from
endothelial and other cell types (1, 6), induces
neutrophil degranulation and stimulates neutrophil/dependent oxygen
metabolism (46), inhibits lung fibroblast proliferation
(49), stimulates airway epithelial cell proliferation (34), and differentially regulates fibroblast-derived
extracellular matrix components such as collagens and proteoglycans
(49). Many of the biological effects of IL-1 are similar
to those of bacterial endotoxin, and numerous cell types release IL-1
when stimulated by endotoxin (9, 30, 31, 47). Migration of neutrophils into the lung after intratracheal injection of endotoxin is
greatly inhibited by concurrent administration of IL-1ra
(50), suggesting a significant role for IL-1 in the acute
inflammatory response to endotoxin. Recent studies implicate Toll-like
receptor 4 (TLR4), a transmembrane receptor with a cytosolic domain
that shows significant homology to IL-1R I, in endotoxin signaling (15, 19, 37, 39). Ligand binding to IL-1R/TLR initiates a
common intracellular signaling pathway that culminates in activation of
nuclear factor-
B and subsequent gene transcription. We found no
remarkable difference in the fetal lung response to IL-1 or endotoxin,
a result consistent with equivalent signaling of the fetal lung by IL-1
and endotoxin. This result could mean that the IL-1 induced by
endotoxin is the mediator of the lung effects. However, both IL-1 and
endotoxin induce chorioamnionitis, which may indirectly cause the lung
response by as yet unidentified mediators.
The inflammatory responses to IL-1 and endotoxin differed in several
respects. An abundance of inflammatory cells was seen in fetal
membranes from endotoxin but not from IL-1- or IL-1
-treated animals at the time of delivery. Furthermore, the numbers of
neutrophils, lymphocytes, and macrophages were 5- to 10-fold higher in
amniotic fluid from endotoxin-treated animals compared with those
exposed to IL-1 7 days before evaluation. These observations suggest
that endotoxin induced a more marked and/or more prolonged inflammation of the amniotic cavity than did IL-1. By contrast, the inflammatory response in the fetal lung was more prolonged and/or more pronounced after IL-1. Animals exposed to 150 µg IL-1
had elevated levels of
H2O2 in alveolar wash fluid at the time of
delivery, indicating the presence of activated inflammatory cells.
These observations are consistent with the finding in IL-1-treated
animals that lung inflammation was a stronger predictor of lung
maturation than was inflammation of the amniotic cavity. Although the
greatest improvement in lung function was found in those animals with
the greatest inflammatory response in the lung, it should be emphasized that there is a temporal dissociation between the peak inflammatory and
maturational responses. The inflammatory cell response to endotoxin has
decreased 7 days after exposure, with the residual inflammation
characterized by cells in the lung and chorioamnion that no longer
produce cytokines or H2O2 (25).
The maturational response is evident at 4-7 days and persists to
25 days and is preceded by elevated cytokine mRNA levels between 5 and
15 h in chorioamniotic membranes, between 24 and 48 h in the
fetal lung, and by cellular infiltration between 5 h and 25 days
in the chorioamniotic membranes and between 15 h and 7 days in the
lung (25).
In a previous study, IA endotoxin induced changes in both lung
structure and in the surfactant system (23, 54). Six days after exposure, surfactant pool size increased ~10-fold. Structural changes at this time point included a 20-50% decrease in the
volume of interstitial tissue and a 10% decrease in alveolar wall
thickness, both of which could impact on lung function. Although we did
not undertake a morphological assessment in the present study, we would
speculate that, like endotoxin, IL-1 and IL-1
improve lung
function through effects on both lung structure and the surfactant system.
We do not know how IA endotoxin/cytokines signal the fetal lung to mature, although we do know that the response to endotoxin is not mediated by cortisol (22). The lung maturation response to endotoxin is preceded by an increase in cells in the amnion/chorion and in the amniotic fluid and by a decrease in peripheral white cell count (25). The amniotic cavity is tolerant of endotoxin that is lethal when given directly to the fetus at doses of 1,000- to 10,000-fold lower (23), suggesting that endotoxin is signaling the fetus indirectly and not by systemic absorption. There are a number of ways that endotoxin/cytokines may signal the fetal lung: 1) an inflammatory response in the membranes/placenta/amniotic fluid results in mediator release into the fetal circulation; 2) an inflammatory response activates white blood cells or other elements in the fetus, which then generate a secondary fetal response; 3) the fetus swallows large volumes of endotoxin/cytokine, and the gut responds by signaling the lungs; 4) endotoxin/cytokine and/or other mediators in the amniotic cavity directly target the fetal lung during fetal breathing movements. Our results from a previous study did not distinguish between the maturation responses to endotoxin exposure via the amnion/chorion, fetal lung fluid, or gastrointestinal tract (Newnham J, Moss T, and Jobe A, unpublished data).
It is tempting to speculate that the strong association between inflammatory cells in alveolar wash and accelerated lung maturation provides evidence of a cause-effect relationship between the two and that influx of inflammatory cells per se is an essential prerequisite for accelerated lung maturation. However, it would be unwise to preclude the possibility that accelerated maturation results from chorioamnionitis or noninflammatory effects of IL-1 that we did not measure. Further studies will be required to determine whether IL-1 induced inflammatory and maturational changes are linked or independent of one another.
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ACKNOWLEDGEMENTS |
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Supported by National Heart, Lung, and Blood Institute Grant HL-65397 and the Women and Infants Research Foundation, Perth, Western Australia.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. H Jobe, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229 (E-mail:jobea0{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.
10.1152/ajplung.00097.2001
Received 20 March 2001; accepted in final form 13 June 2001.
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