Antenatal ureaplasma infection impairs development of the fetal ovine gut in an IL-1-dependent manner.

Ureaplasma infection of the amniotic cavity is associated with adverse postnatal intestinal outcomes. We tested whether interleukin-1 (IL-1) signaling underlies intestinal pathology following ureaplasma exposure in fetal sheep. Pregnant ewes received intra-amniotic injections of ureaplasma or culture media for controls at 3, 7, and 14 d before preterm delivery at 124 d gestation (term 150 d). Intra-amniotic injections of recombinant human interleukin IL-1 receptor antagonist (rhIL-1ra) or saline for controls were given 3 h before and every 2 d after Ureaplasma injection. Ureaplasma exposure caused fetal gut inflammation within 7 d with damaged villus epithelium and gut barrier loss. Proliferation, differentiation, and maturation of enterocytes were significantly reduced after 7 d of ureaplasma exposure, leading to severe villus atrophy at 14 d. Inflammation, impaired development and villus atrophy of the fetal gut was largely prevented by intra-uterine rhIL-1ra treatment. These data form the basis for a clinical understanding of the role of ureaplasma in postnatal intestinal pathologies.

Antenatal exposure to chorioamnionitis affects lipid metabolism in 7-week-old sheep.

Antenatal exposure of the fetus to inflammation may alter postnatal organ development. In our previous work, we demonstrated that the fetal liver is involved in the systemic inflammation associated with chorioamnionitis, leading to metabolic changes. On the basis of these findings, we hypothesized that chorioamnionitis can lead to postnatal inflammation-related liver injury and disturbed lipid metabolism. Chorioamnionitis was induced in sheep by intra-amniotic injection of lipopolysaccharide (LPS) or saline at 90, 100 and 110 days of gestation. Liver homeostasis and lipid metabolism were analyzed at term and at 7 weeks of age. At term, hepatic T-lymphocytes and apoptotic hepatocytes were increased. In addition, hepatic cholesterol and triglyceride levels were decreased in LPS-exposed animals compared with controls. At 7 weeks of age, no hepatic inflammation could be detected. However, liver triglycerides and plasma cholesterol levels were increased in LPS-exposed animals relative to controls. The changes in lipid levels at 7 weeks of age were associated with increased leptin receptor mRNA levels, increased lipid peroxidation, increased expression of cytochrome c oxidase subunit 4 as a marker for mitochondrial function and increased circulating ceramide levels. These findings demonstrate that chorioamnionitis-mediated antenatal inflammation-related liver disturbances have long-lasting postnatal effects on lipid metabolism.

Contribution of inflammation to lung injury and development.

Inflammation interferes with lung development in model systems and is present chronically in the lungs of preterm infants who develop bronchopulmonary dysplasia (BPD). Antenatal inflammation is very commonly associated with preterm deliveries, but there is generally minimal information about the duration, intensity, or organisms associated with chorioamnionitis. In preterm lamb models, chorioamnionitis causes a lung injury similar to BPD and also causes clinical lung maturation. Continuous exposure of the developing lung before and after delivery to inflammation may be central to the development of BPD.

Dose and time response after intraamniotic endotoxin in preterm lambs.

Intraamniotic endotoxin causes chorioamnionitis, which is followed by improved fetal lung function after 4 d in fetal sheep. We evaluated 0.1 mg, 1 mg, 4 mg, and 10 mg endotoxin for inflammation and lung maturation effects after 7 d. Four and 10 mg endotoxin caused similar lung maturation and inflammation in the lung and chorioamnion. The number of neutrophils in cord blood and the inflammatory cells in alveolar lavage and fetal lung tissue increased in a dose-dependent manner. Lower endotoxin doses induced indicators of chorioamnionitis, lung and systemic inflammation without inducing lung maturation. Therefore, some degree of inflammation can occur without subsequent lung maturation. The inflammatory changes caused by 4 mg endotoxin were assessed after 5 h, 24 h, 72 h, and 7 d to discern local versus systemic inflammation after intraamniotic endotoxin. At 5 h active inflammatory cells were in the airways producing hydrogen peroxide, and interleukin-6 and -8 were increased in the cord blood indicating both lung and systemic responses. Cells recruited into the amniotic fluid produced proinflammatory cytokine mRNA for 7 d with no cytokine mRNA in chorioamnion, lung, or spleen after 72 h. The cells in the amniotic fluid may be a source of prolonged fetal exposure to proinflammatory cytokines.

Effects of ventilation with different positive end-expiratory pressures on cytokine expression in the preterm lamb lung.

Ventilator-induced lung injury increases proinflammatory cytokines in the adult lung. We asked if positive end-expiratory pressure (PEEP) affects proinflammatory cytokine mRNA expression in the preterm lung. Preterm lambs at 129 +/- 3 d gestation were treated with 100 mg/kg recombinant human surfactant protein-C surfactant and ventilated for 2 or 7 h with 0, 4, or 7 cm H(2)O of PEEP. Unventilated fetal lambs were used as controls. Within 2 h of ventilation, alveolar total protein and activated neutrophils were increased and expression of mRNAs for the proinflammatory cytokines interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-alpha) was increased in lung tissue of all ventilated animals relative to unventilated controls. Alveolar protein and neutrophils were higher for 0 and 7 PEEP animals than 4 PEEP animals. IL-1beta, IL-6, and IL-8 mRNAs were significantly elevated in animals ventilated with 0 PEEP compared with 4 PEEP. The percentage fractional area of collapsed alveoli was significantly higher for 0 PEEP compared with 4 and 7 PEEP groups. Mechanical ventilation increased the expression of proinflammatory mediators in surfactant-treated preterm lungs and the use of 4 PEEP minimized this response.

Intra-amniotic endotoxin: chorioamnionitis precedes lung maturation in preterm lambs.

The inflammatory and lung maturational effects of intra-amniotic exposure to endotoxin were assessed in fetal lambs. Five hours to 25 days after intra-amniotic injection of endotoxin, preterm lambs were delivered at 119-125 days gestation. Intra-amniotic endotoxin caused an inflammatory cell infiltration in amnion/chorion at 5 h, which persisted for 25 days. At 5-15 h after endotoxin, amnion/chorion cytokine mRNAs increased [12- to 26-fold for interleukin (IL)-1beta, IL-6, and IL-8 mRNA and 3-fold for tumor necrosis factor-alpha mRNA]. At 1-2 days after endotoxin, lung cytokine mRNAs increased 6- to 49-fold. Endotoxin caused modest changes in peripheral white blood cell counts and no significant cytokine mRNA responses in fetal liver, placenta, or jejunum. Lung maturation, as characterized by increased lung volumes and alveolar saturated phosphatidylcholine, occurred at 7 days and persisted for 25 days after endotoxin. We conclude that exposure to a single dose of intra-amniotic endotoxin causes inflammation and increases in cytokine mRNA in amnion/chorion and the fetal lung before lung maturation, consistent with the hypothesis that proinflammatory cytokines signal lung maturation.

The neural cell adhesion molecule (NCAM) heparin binding domain binds to cell surface heparan sulfate proteoglycans.

The neural cell adhesion molecule (NCAM) has been strongly implicated in several aspects of neural development. NCAM mediated adhesion has been proposed to involve a homophilic interaction between NCAMs on adjacent cells. The heparin binding domain (HBD) is an amino acid sequence within NCAM and has been shown to be involved in NCAM mediated adhesion but the relationship of this domain to NCAM segments mediating homophilic adhesion has not been defined. In the present study, a synthetic peptide corresponding to the HBD has been used as a substrate to determine its role in NCAM mediated adhesion. A neural cell line expressing NCAM (B35) and its derived clone which does not express NCAM (B35 clone 3) adhered similarly to plates coated with HBD peptide. A polyclonal antiserum to NCAM inhibited B35 cell-HBD peptide adhesion by only 10%, a value not statistically different from inhibition caused by preimmune serum. Both these experiments suggested no direct NCAM-HBD interactions. To test whether the HBD peptide bound to cell surface heparan sulfate proteoglycans (HSPG), HSPG synthesis was inhibited using beta-D-xyloside. After treatment, B35 cell adhesion to the HBD peptide, but not to control substrates, was significantly decreased. B35 cell adhesion to the HBD peptide could be inhibited by 10(-7) M heparin but not chondroitin sulfate. Preincubation of the substrate (HBD peptide) with heparin caused dramatic reduction of B35 cell-HBD peptide adhesion whereas preincubation of B35 cells with heparin caused only modest reductions in cell-HBD adhesion. Furthermore, inhibition of HSPG sulfation with sodium chlorate also decreased the adhesion of B35 cells to the HBD peptide. These results strongly suggest that, within the assay system, the NCAM HBD does not participate in homophilic interactions but binds to cell surface heparan sulfate proteoglycan. This interaction potentially represents an important mechanism of NCAM adhesion and further supports the view that NCAM has multiple structurally independent binding sites.