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Chorioamnionitis, a risk factor for bronchopulmonary dysplasia in preterm infants, causes an influx of inflammatory cells into the fetal lung. Using a fetal sheep model, we evaluated the time course of activation, functional maturity, and apoptosis of the leukocytes recruited to the fetal air spaces by lipopolysaccharide (LPS). Time-mated sheep were given intra-amniotic injections with 10 mg of Escherichia coli LPS or saline 2 or 7 days before preterm delivery at 124 days of gestation (term is 150 days). Both neutrophils and monocytes in bronchoalveolar lavage fluid (BALF) had activated NF-[kappa]B after 2- and 7-day LPS exposures. These neutrophils and monocytes expressed the activation factor CD 11b and the maturation factor PU.1 at 2 days, and increased PU.1 expression was detected in macrophages at 7 days. Leukocyte oxidative burst activity was greatest at 7 days. BALF lipid peroxidation increased fivefold at 2 days, while protein carbonyls increased eightfold at 7 days. Nitrative stress was not detected in the BALF, but leukocytes in the lung expressed nitric oxide synthase (NOS)II (inducible NOS). BALF leukocytes expressed the antioxidant peroxiredoxin V. Lung glutathione peroxidase was also increased with LPS exposure. There was minimal apoptosis of airway and lung leukocytes assessed by caspase-3 activation. Intra-amniotic LPS recruits leukocytes to the fetal air space that have a persistent activation. These results have implications for the pathogenesis of lung inflammatory disorders in the preterm. bronchopulmonary dysplasia; nuclear factor-[kappa]B; oxidative stress; preterm lung; PU.1

The fetal lung normally contains immature monocytes and very few mature macrophages. The chorioamnionitis frequently associated with preterm birth induces monocyte influx into the fetal lung. Previous studies demonstrated that monocytes in the developing lung can mediate lung injury responses that resemble BPD in humans. We hypothesized that chorioamnionitis would induce maturation of immature monocytes in the fetal lung. Groups of three to seven time-mated ewes received saline or 10 mg of endotoxin (Escherichia coli 055:B5) in saline by intra-amniotic injection for intervals from 1 to 14 days before operative delivery at 124 days of gestational age. Monocytic cells from lung tissue were recovered using Percoll gradients. Monocytic cells consistent with macrophages were identified morphologically and by myosin heavy chain class II expression. An increase in macrophages was preceded by induction of granulocyte-macrophage colony-stimulating factor in the lung and subsequent activation of the transcription factor PU.1. The production of IL-6 by monocytes/macrophages in response to endotoxin challenge in vitro increased 7 and 14 days after exposure to intra-amniotic endotoxin. Recombinant TNF-[alpha] induced IL-6 production by lung monocytic cells exposed to intra-amniotic endotoxin but not in control cells. Monocytic phagocytosis of apoptotic neutrophils also increased 7 and 14 days after exposure to intra-amniotic endotoxin. Intra-amniotic endotoxin induced lung monocytes to develop into functionally mature cells consistent with macrophages. These findings have implications for lung immune responses after exposure to chorioamnionitis. macrophage; fetal immunity; bronchopulmonary dysplasia; chorioamnionitis; granulocyte-macrophage colony-stimulating factor; PU.1

Chorioamnionitis is frequently associated with preterm birth and increases the risk of adverse outcomes such as bronchopulmonary dysplasia (BPD). Transforming growth factor (TGF)-[beta]1 is a key regulator of lung development, airway remodeling, lung fibrosis, and regulation of inflammation, and all these processes contribute to the development of BPD. Connective tissue growth factor (CTGF) is a downstream mediator of some of the profibrotic effects of TGF-[beta]1, vascular remodeling, and angiogenesis. TGF-[beta]1-induced CTGF expression can be blocked by TNF-[alpha]. We asked whether chorioamnionitis-associated antenatal inflammation would regulate TGF-[beta]1, the TGF-[beta]1 signaling pathway, and CTGF in preterm lamb lungs. Fetal sheep were exposed to 4 mg of intraamniotic endotoxin or saline for 5 h, 24 h, 72 h, or 7 days before preterm delivery at 125 days gestation (full term = 150 days). Intra-amniotic endotoxin increased lung TGF-[beta]1 mRNA and protein expression. Elevated TGF-[beta]1 levels were associated with TGF-[beta]1-induced phosphorylation of Smad2. CTGF was selectively expressed in lung endothelial cells in control lungs, and intra-amniotic endotoxin caused CTGF expression to decrease to 30% of control values and TNF-[alpha] protein to increase. The antenatal inflammation-induced TGF-[beta]1 expression and Smad signaling in the fetal lamb lung may contribute to impaired lung alveolarization and reduced lung inflammation. Decreased CTGF expression may inhibit vascular development or remodeling and limit lung fibrosis during remodeling. These effects may contribute to the impaired alveolar and pulmonary vascular development that is the hallmark of the new form of BPD. chorioamnionitis; bronchopulmonary dysplasia; Smad signaling; fibrosis

Surfactant protein A (SP-A) and transforming growth factor-[beta]1 (TGF-[beta]1) have been shown to modulate the functions of different immune cells and specifically to inhibit T lymphocyte proliferation. The aim of the present study was to elucidate whether the Smad signaling pathway, which is activated by TGF-[beta]1, also plays a role in SP-A-mediated inhibition of [CD4.sup.+] T lymphocyte activation. Recombinant human SP-A1 expressed in Chinese hamster ovary cells [rSP-A1m (mammalian)], but not recombinant Baculovirus-derived rSP-[A1.sup.hyp] (hydroxyproline-deficient), suppressed T lymphocyte proliferation and IL-2 mRNA expression. To test whether SP-A induced Smad signaling, a Smad3/4-specific reporter gene was transfected in primary human [CD4.sp.+] T lymphocytes. Only rSP-A1m, but not [rSP-A1.sup.hyp], induced Smad-specific reporter genes, Smad2 phosphorylation, and Smad7 mRNA expression. The effect of rSP-A1m was mediated through the TGF-[beta]RII and could he antagonized by anti-TGF-[beta]1 neutralizing antibodies and sTGF-[beta]RII. Western blot and ELISA analysis revealed that rSP-A1m, but not rSP-[A1.sup.hyp], contained TGF-[beta]1. TGF-[beta]1 was responsible for the differences in inhibition of [CD.sup.4+] T lymphocyte proliferation and activation of the Smad signaling pathway between rSP-A1m and [rSP-A1.sup.hyp]. After acidification, native SP-A, obtained from patients with alveolar proteinosis, also induced Smad signaling in human [CD.sup.4+] T lymphocytes leading to an increased inhibition of T lymphocyte proliferation, thus indicating the presence of inactive, latent TGF-[beta]1 in native SP-A samples. Association between SP-A and latent TGF-[beta]1 provides a possible novel mechanism to regulate TGF-[beta]1-mediated inflammation and fibrosis reactions in the lung but also leads to possible misinterpretation of immune-modulator functions of SP-A. Monitoring of SP-A preparations for possible TGF-[beta]1 is essential. Smad signaling; acidification; surfactant protein A; transforming growth factor-[beta]1

Chronic early gestational chorioamnionitis is associated with development of bronchopulmonary dysplasia in preterm infants. A single intra-anmiotic exposure to endotoxin decreased alveolarization and reduced expression of endothelial proteins in 125-day gestational age preterm lambs. We hypothesized that prolonged exposure to intra-amniotic endotoxin would cause progressive lung inflammation and inhibit alveolar and pulmonary vascular development. Endotoxin (1 mg/day) or saline was administered via an intra-amniotic osmotic pump from 80 to 108 days of gestational age (continuous pump) or by four weekly 10-mg intraanmiotic endotoxin rejections starting at 100 days of gestational age (multiple dose). Lung morphometry, lung inflammation, vascular effects, and lung maturation were measured at delivery. The continuous pump lambs delivered at 100 days (~70% of total endotoxin exposure) had lung inflammation, fewer saccules, and decreased endothelial proteins endothelial nitric oxide synthase and VEGF receptor 2 expression compared with controls. The continuous pump (delivered at 138 days) and multiple dose lambs (delivered at 130 and 145 days) had mild persistent lung inflammation and no significant differences in lung morphometry or expression of endothelial proteins compared with controls. Surfactant saturated phosphatidylcholine pool sizes were increased in all endotoxin-exposed groups, but lung function was not changed relative to controls. Contrary to our hypothesis, a prolonged fetal exposure to intra-amniotic endotoxin caused mild persistent inflammation but did not lead to progressive structural abnormalities in lungs of near-term gestation lambs. chorioamnionitis; bronchopulmonary dysplasia; respiratory distress syndrome lung maturation- surfactant

TNF-[alpha] has been associated with chorioamnionitis and the subsequent development of bronchopulmonary dysplasia in preterm infants. We asked whether bioactive recombinant ovine TNF-[alpha] could induce chorioamnionitis, lung inflammation, lung maturation, and systemic effects in fetal sheep. We compared the responses to IL-1[alpha] a cytokine known to induce these responses in preterm sheep. Intra-amniotic TNF-[alpha] caused no chorioamnionitis, no lung maturation, and a very small increase in inflammatory cells in the fetal lung after 5 h, 2 days (d), and 7 d. In contrast, IL-1[alpha] induced inflammation and lung maturation. TNF-[alpha] given into the airways at birth increased granulocytes in the bronchoalveolar lavage fluid of ventilated preterm lungs and decreased the mRNA for surfactant protein C but did not adversely effect postnatal lung function. An intravascular injection of IL-1[alpha] caused a systemic inflammatory response in fetal sheep, whereas there was no fetal response to intravascular TNF-[alpha]. Fetal and newborn preterm sheep are minimally responsive to TNF-[alpha]. Therefore, the presence of a mediator such as TNF-[alpha], in a developing animal does not necessarily mean that it is causing the responses anticipated from previous results in adult animals. inflammation; fetal maturation; cytokines; interleukin-1[alpha]; chorioamnionitis; endotoxin

Antenatal betamethasone (Beta) is widely used in women with asymptomatic chorioamnionitis at risk for preterm delivery, but its effects on fetal inflammation are unstudied. Groups of ewes at 109 [+ or -] 1 days of gestation received the following treatments: intra-amniotic (IA) saline (control), 0.5 mg/kg intramuscular Beta, 10 mg IA endotoxin (Endo), and Beta + 2 h later Endo (Beta + Endo). Beta suppressed Endo-induced lung inflammation at 1 day. However, compared with Endo 5 days after treatment, Beta + Endo lambs had increased alveolar neutrophils, proinflammatory cytokine mRNA expression, and serum amyloid A3 (SAA3) mRNA expression. IL-1[beta] mRNA expression was localized to the inflammatory cells, whereas SAA3 mRNA expression was induced in the bronchial epithelium and the inflammatory cells. Compared with Endo, Beta + Endo lambs had increased lung inflammation but equivalent lung volumes 15 days after treatment. The late increase in inflammation in the Beta + Endo animals suggests that glucocorticoids impair the ability of the preterm lung to downregulate Endo-induced inflammation after fetal clearance of the glucocorticoids. These results have implications for lung inflammation and bronchopulmonary dysplasia in preterm infants exposed to chorioamnionitis and maternal glucocorticoids. bronchopulmonary dysplasia; acute-phase reactant; proinflammatory cytokines; endotoxin tolerance; chorioamnionitis

Chorioamnionitis is frequent in preterm labor and increases the risk of bronchopulmonary dysplasia. We hypothesized that intra-amniotic endotoxin injures the lung in utero, causing a sequence of inflammation and tissue injury similar to that which occurs in the injured adult lung. Preterm lamb lungs at 125 days gestational age were evaluated for indicators of inflammation, injury, and repair 5 h, 24 h, 72 h, and 7 days after 4 mg of intra-amniotic endotoxin injection. At 5 h, the epithelial cells in large airways expressed heat shock protein 70, and alveolar interleukin-8 was increased. Surfactant protein B (SP-B) decreased in alveolar type II cells at 5 h, and SP-B in lung tissue and alveolar lavage fluid increased by 72 h. By 24 h, neutrophils were recruited into the large airways, and cell death was the highest. Alveolar type II cells decreased by 25% at 24 h, and proliferation was highest at 72 h, consistent with tissue remodeling. Intraamniotic endotoxin caused surfactant secretion, inflammation, cell death, and remodeling as indications of lung injury. The recovery phase was accompanied by maturational changes in the fetal lung. surfactant; lung maturation; alveolar type II cells; respiratory distress syndrome; chorioamnionitis

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[alpha] and IL-1[beta] 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 [micro]g recombinant ovine IL-1[alpha] or IL-1[beta] 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[alpha] and IL-1[beta] 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[alpha] was greater than that to IL-1[beta], which was similar to endotoxin response. Inflammation was also more pronounced after IL-1[alpha] 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