SPECIAL TOPIC
Pre- and Postnatal Lung Development, Maturation, and Plasticity
Intra-amniotic injection of IL-1 induces inflammation and maturation in fetal sheep lung

Karen E. Willet1, Boris W. Kramer3, Suhas G. Kallapur3, Machiko Ikegami3, John P. Newnham2, Timothy J. Moss2, Peter D. Sly1, and Alan H. Jobe3

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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)-1alpha and IL-1beta 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-1alpha or IL-1beta or 10 mg Escherichia coli endotoxin by IA injection at 118 days gestation (term = 150 days), and fetuses were delivered at 125 days. IL-1alpha and IL-1beta improved lung function and increased alveolar saturated phosphatidylcholine (Sat PC) and surfactant protein mRNA expression at the higher dose. The maturation response to IL-1alpha was greater than that to IL-1beta , which was similar to endotoxin response. Inflammation was also more pronounced after IL-1alpha 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INTERLEUKIN (IL)-1alpha and IL-1beta 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-1alpha is mainly active as a cytosolic precursor and a membrane-associated protein, IL-1beta 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)-alpha (11, 21, 45). Endotoxin, a lipopolysaccharide component of gram-negative bacteria, is a potent inducer of many cytokines, including IL-1alpha and IL-1beta (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-1beta 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-1alpha or to IL-1beta 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-1alpha or IL-1beta and compare that response to IA endotoxin.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of recombinant ovine IL-1alpha and IL-1beta . Nucleotides corresponding to the mature polypeptides (amino acid 113-268) of IL-1alpha (13) and amino acids 114-266 of IL-1beta (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-1alpha and IL-1beta then collected from the unbound fraction of Ni-NTA columns. IL-1alpha was further purified to homogeneity by gel filtration chromatography (Sephacryl S-200, Pharmacia, Piscataway, NJ) and IL-1beta 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-1alpha (n = 6), 150 µg IL-1alpha (n = 7), 15 µg IL-1beta (n = 6), 150 µg IL-1beta (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 · kg-1 · min-1 is a carbon dioxide production constant, P is ventilatory pressure, and f is the ventilation rate (35). At 40 min postdelivery, animals were deeply anesthetized with pentobarbital sodium (30 mg/kg iv). The lungs were degassed by compression of the thoracic cavity then by clamping the endotracheal tube, allowing any remaining oxygen to be absorbed over a 5-min period. The lamb was exanguinated, the chest was opened, and a deflation pressure-volume curve was obtained as previously described (24).

Surfactant 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 (alpha M-subunit of integrin CR3) and CD44 (proteoglycan link protein). The cell pellet was washed twice with PBS to remove unbound antibody and incubated with phycoerythrin labeled F(ab')2 anti-IgG fragments (secondary antibody) in the dark on ice. Cells were washed twice, resuspended in PBS, kept on ice, and immediately analyzed on a FACS Calibur flow cytometer (Becton Dickinson, Mountain View, CA). Control staining was performed with isotype antibodies and with secondary antibody alone to obtain background fluorescence. All antibodies were purchased from SEROTEC (Raleigh, NC). Apoptotic cells were detected by annexin V and propium iodide staining (51) (Trevigen, Gaithersburg, MD). Apoptotic and necrotic cells expose the inner cell membrane to the outside, allowing annexin V to bind to phosphatidylserine. Cell aliquots were stained with fluorescent labeled annexin V and counterstained with propium iodide to detect necrotic cells.

A sample of fetal membrane (~2 × 2 cm) was collected at time of delivery, folded into a roll, and fixed in 10% phosphate-buffered formalin. The membrane roll was cut into 3-4 slices before embedding. A single 5-µm section through the 3-4 pieces of membrane was graded in a blinded fashion. The degree of inflammation was designated as 0 (no inflammatory cell influx), 1 (minimal cellular influx), 2 (moderate cellular influx), or 3 (extensive cellular influx). The right cranial lobe from each animal was inflation fixed with 10% phosphate-buffered formalin at a distending pressure of 30 cmH2O. The degree of lung inflammation was also scored for inflammatory cells using the same scale on three 5-µm sections from each animal. Average scores for airspaces and lung tissue were calculated for each animal.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-1alpha and IL-1beta groups were also comparable to control animals at 40 min (Table 1). Lambs exposed to the high dose of IL-1alpha or IL-1beta had significantly lower ventilatory pressures and higher tidal volumes at 40 min than controls. Lambs given 150 µg IL-1alpha 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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Table 1.   Characteristics of preterm lambs

Lung function. Lung compliance values at 40 min were improved after high-dose IL-1alpha , IL-1beta , or endotoxin (Fig. 1). VEI more than doubled in each group compared with control. Animals receiving high-dose IL-1alpha , IL-1beta , 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-1alpha induced a greater improvement than either high-dose IL-1beta or endotoxin. Lung compliance, VEI, and lung volumes in low-dose IL-1alpha and IL-1beta groups were similar to control.


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Fig. 1.   Lung function. Lung compliance (A) and ventilation efficiency index (B) increased in 150 µg interleukin (IL)-1alpha , 150 µg IL-1beta , and 10 mg endotoxin (ENDO) groups compared with control. C: deflation pressure-volume curves. Control (), 15 µg IL-1alpha (open circle ), 150 µg IL-1alpha (black-down-triangle ), 15 µg IL-1beta (down-triangle), 150 µg IL-1beta (), 10 mg endotoxin (). Volumes at 0 cmH2O (V0) and at 40 cmH2O (V40) increased in 150 µg IL-1alpha , 150 µg IL-1beta , and 10 mg ENDO groups compared with control. Group means ± SE shown. *P < 0.05, **P < 0.01 vs. control group.

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-1alpha , 150 µg IL-1beta , and endotoxin groups, respectively (Fig. 2). Sat PC pool size was not different from control values after 15 µg IL-1alpha or IL-1beta . Lung tissue SP mRNA expression was determined in control, 150 µg IL-1alpha , 150 µg IL-1beta , 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-1alpha . IL-1beta 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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Fig. 2.   Surfactant components. A: alveolar wash saturated phosphatidylcholine (Sat PC) pool size increased in 15 µg IL-1alpha , 150 µg IL-1beta , and 10 mg ENDO groups compared with control. B: surfactant protein (SP) mRNA expression in lung tissue. SP-A (open bars), SP-B (hatched bars), SP-C (gray bars), SP-D (black bars). Bars represent change in mRNA expression relative to control. IL-1alpha increased expression of all surfactant protein mRNAs, whereas IL-1beta increased expression of all but SP-B mRNA expression. ENDO exposure increased expression of SP-A, SP-B, and SP-C mRNA. SP-D mRNA expression was not measured in these animals. Group means ± SE shown. *P < 0.05, **P < 0.01 vs. control group.

Inflammation and cellular activation. Total peripheral white cell count increased significantly in animals exposed to 150 µg IL-1alpha 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-1alpha vs. control). Platelet count was also elevated in the low-dose IL-1alpha , high-dose IL-1beta , and endotoxin groups. There were no differences in lymphocyte or monocyte counts between control and treated groups.

                              
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Table 2.   Peripheral blood cell counts

Amniotic fluid neutrophil, lymphocyte, and monocyte counts were elevated in lambs exposed to 15 or 150 µg IL-1alpha compared with controls (Fig. 3). Although cell counts were also slightly higher in IL-1beta groups, the only statistically significant increase was in lymphocyte count after 150 µg IL-1beta . There was no apparent effect of cytokine dose on the degree of cellular influx in amniotic fluid. Endotoxin also induced an influx of white cells into amniotic fluid. The response was 5- to 10-fold greater than for the cytokine-treated groups: neutrophil, macrophage, and lymphocyte counts in endotoxin-treated animals were significantly higher than in 150-µg IL-1beta animals (P < 0.05 by Mann-Whitney Rank Sum tests), and macrophage and lymphocyte counts were higher than in 150-µg IL-1alpha animals. White cell counts were also higher in alveolar washes of endotoxin- and cytokine-treated animals, although the pattern of increase was different from that seen in amniotic fluid (Fig. 3). First, by contrast to amniotic fluid, white cell counts in alveolar wash fluid were of a similar magnitude in endotoxin and high-dose cytokine groups. Second, there was an apparent effect of cytokine dose on alveolar wash cell count. Macrophage and lymphocyte counts were higher in both 150-µg IL-1alpha and IL-1beta animals than in 15 µg IL-1alpha and IL-1beta animals (P < 0.05 by Mann-Whitney rank sum tests).


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Fig. 3.   Amniotic fluid and alveolar wash white cell counts. A: amniotic fluid neutrophils (open bars), lymphocytes (hatched bars), and monocytes (black bars) increased after 15 or 150 µg IL-1alpha or 10 mg ENDO. Lymphocyte count increased after 150 µg IL-1beta . B: alveolar wash neutrophils increased after 15 or 150 µg IL-1alpha or 10 mg ENDO. Lymphocyte count increased in 150 µg IL-1alpha , 150 µg IL-1beta , and ENDO groups. Monocyte count increased in 15 and 150 µg IL-1alpha , 150 µg IL-1beta , and ENDO groups. Group means ± SE shown. *P < 0.05, **P < 0.01 vs. control group.

Five-micron sections of lung tissue and chorioamniotic membranes were scored for white cell influx (Fig. 4). Inflammatory cell scores were significantly greater for lung tissue and airspaces after 150 µg IL-1alpha , 150 µg IL-1beta , or 10 mg endotoxin. There was also evidence of increased cellular influx for the lower-dose cytokine groups, although this was less pronounced than in the other treatment groups and was not significantly different to controls. Only animals exposed to endotoxin 7 days before delivery had significantly more white cells in their chorioamniotic membranes than controls.


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Fig. 4.   Lung and fetal membrane inflammatory cell scores. Air space (A) and lung (B) inflammatory cell scores increased in 150 µg IL-1alpha , 150 µg IL-1beta , and 10 mg ENDO groups. Fetal membrane inflammatory cell score (C) increased in ENDO group only. Median, interquartile range (box) and 95% confidence interval (whiskers) are shown. *P < 0.05, **P < 0.01 vs. control group.

Peroxide (H2O2) concentration, a marker for cellular activation, was determined for cells recovered from alveolar wash fluid (Table 3). Alveolar wash H2O2 concentration was greater than control levels in the 150-µg IL-1alpha group only, indicating persistent inflammatory cell activation only for the 150-µg IL-1alpha group 7 days after IA treatment. The adhesion molecules CD11b and CD44 also provide an indication of cellular recruitment. The percentage of CD11b-positive cells increased significantly in all but the low-dose IL-1beta group. A greater proportion of cells were also CD44 positive in both high-dose IL-1 groups and in the endotoxin group. Mean fluorescence of CD11b and CD44 cells was also greater in treated compared with control animals. The total number of apoptotic and necrotic cells increased with exposure to cytokines or endotoxin, but the proportion of apoptotic and necrotic cells was highly variable, and there were no significant differences between groups. The apoptotic cells indicate an ongoing process of resolution of inflammation 7 days after the proinflammatory treatments.

                              
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Table 3.   Markers of inflammation and cellular activation in alveolar wash fluid

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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Fig. 5.   Association between lung inflammation and maturation. Alveolar wash total white cell count vs. lung compliance (A), lung volume (B), and Sat PC pool size (C). Total cell count accounted for 61% of variability in lung compliance (r = 0.78, P < 0.0005), 49% of variability in lung volume (r = 0.70, P < 0.0005), and 57% of variability in Sat PC pool size (r = 0.76, P < 0.0005).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the effects of in utero exposure to IL-1alpha and IL-1beta 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-1alpha or IL-1beta 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-1alpha had a greater effect than IL-1beta 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-1alpha than to IL-1beta . 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-1alpha and IL-1beta 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-1alpha and IL-1beta 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-1alpha and IL-1beta were 3.5 and 10 ng/ml, respectively in women with preterm labor and microbial invasions. In our study, IL-1alpha /beta 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-1alpha or IL-1beta 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-1beta , IL-6, IL-8, and TNF-alpha 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-1alpha . 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-1alpha . More recently Glumoff et al. (16) reported that the effect of IL-1alpha 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-1alpha or IL-1beta .

Hybertson et al. (20) recently examined alterations in the surfactant system in a rat model of IL-1alpha -induced acute lung injury. Intratracheal instillation of IL-1alpha 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 gamma -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-1alpha 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-1alpha and IL-1beta , 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-1alpha and IL-1beta , 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-1beta -converting enzyme, a cysteine protease that converts the biologically inactive precursor of IL-1beta 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-1alpha induced more pronounced inflammatory and maturational changes than IL-1beta 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-1alpha and IL-1beta 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-1alpha and -beta in our preterm lamb model, as evidence in the literature suggests that IL-1beta binds to IL-1R I with approximately equal or greater affinity than IL-1alpha (8, 26, 27) and is as potent, if not more potent than IL-1alpha , at inducing proinflammatory events in target cells (8, 36, 52). A second type of IL-1 receptor, IL-1R II, binds both IL-1alpha and -beta but does not transduce a signal, thus acting as a "decoy" receptor, inhibiting the bioactivity of IL-1. IL-1R II binds IL-1beta with greater affinity than IL-1alpha (4). Differences in the ability of IL-1R II to sequester IL-1alpha and IL-1beta 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-alpha , 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-kappa 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-1alpha - or IL-1beta -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-1alpha 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-1alpha and IL-1beta 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.


    ACKNOWLEDGEMENTS

Supported by National Heart, Lung, and Blood Institute Grant HL-65397 and the Women and Infants Research Foundation, Perth, Western Australia.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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