Urpo Lappalainen

Intraamniotic interleukin-1 accelerates surfactant protein synthesis in fetal rabbits and improves lung stability after premature birth.

Intraamniotic infection is associated with increased IL-1 activity in amniotic fluid, increased incidence of preterm labor, and with decreased incidence of respiratory distress syndrome in infants born prematurely. We hypothesized that an elevated IL-1 in amniotic fluid promotes fetal lung maturation. On day 23 or 25 of gestation (term 31 d), either IL-1alpha (150 or 1,500 ng per fetus) or its antagonist IL-1 receptor antagonist (IL-1ra, 20 microg) was injected to the amniotic fluid sacs in one uterine horn, whereas the contralateral amniotic sacs were injected with vehicle. Within 40 h, IL-1alpha caused a dose-dependent increase in surfactant protein-A (SP-A) and SP-B mRNAs (maximally, fivefold), without affecting lung growth or increasing inflammatory cells in the lung. Both genders, and upper and lower lung lobes were similarly affected. IL-1ra did not modify SP-A, -B, or -C mRNA. IL-1 increased the intensity of staining of alveolar type II cells for SP-B, and the concentrations of SP-B, -A, and disaturated phosphatidylcholine in bronchoalveolar lavage. The dynamic lung compliance and the postventilatory expansion of lungs were increased two- to fourfold after IL-1alpha treatment. In fetal lung explants, IL-1alpha increased the expression of SP-A mRNA. IL-1 in amniotic fluid in preterm labor may promote lung maturation and thus be part of a host-defense mechanism that prepares the fetus for extrauterine life.

Inhaled Nitric Oxide Decreases Hyperoxia-Induced Surfactant Abnormality in Preterm Rabbits

Inhaled nitric oxide (iNO) is a specific pulmonary vasodilator. By serving as a pro-oxidant or antioxidant, iNO may influence other pulmonary functions as well. This study was designed to test the hypothesis that iNO affects the alveolar lining after premature birth. Preterm rabbits (gestation 29 d, term 31 d) were nose-only exposed NO (14 ppm) and 98% O2 for 20 h. The others were exposed to either 98% O2 or air. In another experiment, premature rabbits were exposed to either NO in air or to air. After the exposure, bronchoalveolar lavage (BAL) was performed and the surfactant aggregates were isolated. The surfactant components and surface activity were analyzed. In total, 144 animals were studied. There were no significant differences in the number, distribution, or respiratory burst activity of cells recovered by BAL. Neither brief hyperoxia nor iNO increased plasma-derived proteins in BAL. Exposure to O2 decreased large surfactant aggregates, surface activity, and the content of surfactant protein B in BAL, whereas iNO prevented completely or partially these effects of acute hyperoxia on surfactant. Hyperoxia increased the content of malondialdehyde and decreased glutathione in epithelial lining fluid. iNO decreased malondialdehyde (p < 0.05) and tended to increase glutathione (p = 0.06) in animals breathing O2. Nitrotyrosine was not detectable in BAL, and NO2 was low in the breathing area. In room air, iNO had no significant effect on surfactant. According to the present results, a brief period of hyperoxia causes an oxidant stress and decreases the surface activity of alveolar surfactant in premature rabbits. In contrast, a low dosage of iNO decreased or prevented the O2-induced detrimental effects on alveolar surfactant and alleviated the oxidant stress.

Matrix metalloproteinase-9 deficiency worsens lung injury in a model of bronchopulmonary dysplasia.

Increased activity of matrix metalloproteinase (MMP)-9 is associated with the development of bronchopulmonary dysplasia (BPD) in newborn infants, but the role of MMP-9 in the pathophysiology of BPD is unclear. We have shown that perinatal expression of interleukin-1 beta (IL-1 beta) in the lung is sufficient to cause a BPD-like illness in infant mice. To study the hypothesis that MMP-9 is an important downstream mediator in IL-1 beta-induced lung injury in the newborn, we compared the effects of IL-1 beta on fetal and postnatal lung inflammation and development in transgenic mice with regulatable pulmonary overexpression of human mature IL-1 beta with wild-type (IL-1 beta/MMP-9(+/+)) or null (IL-1 beta/MMP-9(-/-)) MMP-9 loci. IL-1 beta increased the expression of MMP-9 mRNA and amount of MMP-9 protein in the lungs of MMP-9(+/+) mice. IL-1 beta/MMP-9(-/-) mice had fewer neutrophils but more macrophages in the lungs than did IL-1 beta/MMP-9(+/+) mice. MMP-9 deficiency increased pulmonary cell death and macrophage clearance of dying cells in IL-1 beta-expressing mice. IL-1 beta/MMP-9(-/-) mice had more severe alveolar hypoplasia than IL-1 beta/MMP-9(+/+) mice, implying that IL-1 beta-induced lung disease was worsened in the absence of MMP-9. These results suggest that MMP-9 activity in the inflamed neonatal lung protects the lung against injury.

Pulmonary toxicity associated with nitric oxide in term infants with severe respiratory failure.

We prospectively analyzed airway specimens from 24 newborn infants. Inhaled nitric oxide (< or = 20 ppm for 1 to 4 days to 12 infants) did not affect the concentrations of the lipid peroxidation product, the surface activity, or the cytokines (interleukin-1, granulocyte-macrophage colony-stimulating factor, interleukin-1 receptor antagonist). Nitrotyrosine was detected after 10 days of life in the two infants requiring prolonged ventilation, suggesting toxicity of endogenous nitric oxide.

Intra-amniotic endotoxin accelerates lung maturation in fetal rabbits

The hypothesis that endotoxin in amniotic fluid accelerates fetal lung maturation was tested. On day 25 of gestation, LPS (5 μg/fetus) was injected intra‐amniotically into one uterine horn of eight New Zealand white rabbits, whereas the contralateral amniotic sacs were injected with saline vehicle. The fetuses were delivered 48 h after LPS administration and their lungs were studied. One dam went into premature labor prior to the 48 h time point and was excluded from the study. Mean white cell counts in amniotic fluid and bronchoalveolar lavage fluid from LPS‐treated fetuses were increased 3.2‐fold (p= 0.04) and 9.9‐fold (p= 0.04), respectively. Fetal weights and lung weights were not affected by LPS. Surfactant protein SP‐A and SP‐B mRNA expressions in LPS‐treated fetuses were increased 2.3‐fold (p= 0.03) and 1.4‐fold (p= 0.04), respectively. Static lung compliance was increased in animals treated with LPS (p= 0.001). Lungs from LPS‐treated animals had better aeration than those of controls. Mean volume of inflation‐fixed lungs of LPS‐treated fetuses was 1.7 times greater than that of controls (p= 0.03).

Granulocyte-Macrophage Colony-Stimulating Factor in Amniotic Fluid and in Airway Specimens of Newborn Infants

Granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that promotes white cell maturation, participates in the metabolism of pulmonary surfactant. Little is known on the production of GM-CSF during pregnancy or the neonatal period. We studied how the concentrations of GM-CSF in amniotic fluid (AF) or in tracheal aspirates (TA) of newborn infants are influenced by length of gestation, postnatal age, as well as conditions affecting the mother or the fetus. One hundred and forty-three AF samples from 143 pregnant patients (gestational age range, 28-42 wk) and 202 TA samples from 82 neonates(gestational age, 24-42.5 wk, postnatal age 0.2 d to 4 wk) were analyzed for GM-CSF using ELISA. In patients with intact membranes, AF GM-CSF increased as a function of gestational age; the concentrations were below 7.5 ng/L(detection limit of the assay) (n = 5), 18.6 ± 2.3 ng/L(n = 56), and 56.7 ± 7.9 ng/L (n = 58) at gestational ages between 28 and 32 wk, between 32 and 37 wk, and in term patients, respectively (linear regression: r = 0.404, p = 0.001). Among patients at less than 33 wk of gestation, those with intact membranes had a median AF GM-CSF concentration under the detection limit(n = 7), whereas in those with preterm premature rupture of membranes, the concentration was 50.1 ± 22.2 ng/L (n = 16)(p = 0.002). Among term patients, those in labor had higher AF GM-CSF than those without signs of labor. TA GM-CSF at less than 12 h of age correlated with gestational age (r = 0.654, p = 0.0002,n = 28); thereafter, TA GM-CSF increased, and gestation dependence disappeared. We conclude that GM-CSF in AF and in fetal lung liquid is developmentally regulated and GM-CSF production increases in inflammatory conditions during pregnancy.

Developmental Stage Is a Major Determinant of Lung Injury in a Murine Model of Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD) is a common inflammatory lung disease in premature infants. To study the hypothesis that the sensitivity of the lung to inflammatory injury depends on the developmental stage, we studied postnatal lung development in transgenic mice expressing human IL-1β (hIL-1β) in the lungs during the late canalicular-early saccular, saccular, or late saccular-alveolar stage. Overexpression of hIL-1β in the saccular stage caused arrest in alveolar development, airway remodeling, and goblet cell hyperplasia in the lungs as well as poor growth and survival of infant mice. Overexpression of hIL-1β during the late canalicular-early saccular stage did not adversely affect lung development, growth, or survival of the pups. Mice expressing hIL-1β from the late saccular to alveolar stage had smaller alveolar chord length, thinner septal walls, less airway remodeling and mucus metaplasia, and better survival than mice expressing hIL-1β during the saccular stage. Human IL-1β overexpression in the saccular stage was sufficient to cause a BPD-like illness in infant mice, whereas the lung was more resistant to hIL-1β-induced injury at earlier and later developmental stages.

Cimetidine Does Not Prevent Lung Injury in Newborn Premature Infants

Animal studies have shown that induction of cytochrome P450 (CYP) in the lung by oxygen exposure may result in the release of free radical oxidants and arachidonic acid metabolites, which can cause lung injury that is reduced by treatment with cimetidine, a CYP inhibitor. To determine whether cimetidine would reduce lung injury in human infants at risk for chronic lung disease, we conducted a randomized clinical trial in which we administered either cimetidine or a placebo for 10 d beginning <24 h after birth to 84 newborn infants weighing ≤1250 g who were receiving O2 and mechanical ventilation. Cimetidine had no significant effect on severity of respiratory insufficiency assessed at 10 d postnatal age. F2-isoprostane levels (a marker of oxidant injury) in tracheal aspirates were significantly higher in the cimetidine group at 4 d and at 10 d. There were no significant differences between the groups in tracheal aspirate levels of inflammatory markers (leukotriene B4, IL-8, and nucleated cell count) or arachidonic acid metabolites. We conclude that cimetidine does not reduce lung injury in newborn premature infants receiving O2 and mechanical ventilation. It is possible that cimetidine was not an adequate CYP inhibitor in this context.

Interleukin-4 and transforming growth factor-β1 modulate the production of interleukin-1 receptor antagonist and of prostaglandin E2 by decidual cells

OBJECTIVE Increased production of prostaglandins is associated with parturition. The production of interleukin-1 is increased in preterm labor occurring in the setting of infection. Interleukin-1 receptor antagonist prevents the effects of interleukin-1. Interleukin-4 and transforming growth factor-beta inhibit the production of prostaglandin E2 by monocytes and amnion cells, respectively. In addition, these cytokines enhance the production of interleukin-1 receptor antagonist by monocytes. We investigated whether the production of prostaglandin E2 and interleukin-1 receptor antagonist by decidual cells is modulated by interleukin-4 and transforming growth factor-beta 1. STUDY DESIGN Human decidual cells in monolayer culture were treated for 44 hours with interleukin-4 (1 to 100 ng/ml), transforming growth factor-beta 1 (1 to 10 ng/ml), the combination of these cytokines, or vehicle. Production of prostaglandin E2 and interleukin-1 receptor antagonist was measured by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. The Wilcoxon signed-rank test was used. RESULTS Both interleukin-4 and transforming growth factor-beta 1 inhibited prostaglandin E2 production by decidual cells (p < 0.01). Decidual cells produced interleukin-1 receptor antagonist at a basal rate of 26.4 +/- 6.7 pg/micrograms protein per 44 hours (n = 13). Interleukin-4 stimulated the production of interleukin-1 receptor antagonist by decidual cells (p < 0.01). Transforming growth factor-beta 1 potentiated the stimulatory effect of interleukin-4 on decidual cell interleukin-1 receptor antagonist production (p < 0.05). CONCLUSION By suppressing the production of prostaglandin E2 and enhancing the production of interleukin-1 receptor antagonist by decidual cells, interleukin-4 and transforming growth factor-beta may have a role in inhibiting preterm labor in the setting of infection.

β6 Integrin Subunit Deficiency Alleviates Lung Injury in a Mouse Model of Bronchopulmonary Dysplasia

Pulmonary inflammation is associated with the development of bronchopulmonary dysplasia in premature infants. We have previously shown that perinatal pulmonary expression of human IL-1beta is sufficient to cause a lung disease similar to bronchopulmonary dysplasia, characterized by inflammation, impaired alveolarization, poor postnatal growth, and increased mortality in infant mice. The alphavbeta6 integrin plays a critical role in regulating inflammation in the adult lung. To study the role of the beta6 integrin subunit in neonatal inflammatory lung disease, we compared the pulmonary development in IL-1beta-expressing infant mice with wild-type or null beta6 integrin loci. Absence of the beta6 integrin subunit decreased the mortality and improved the postnatal growth of IL-1beta-expressing pups. The disrupted alveolar development of IL-1beta-expressing mice was improved by beta6 integrin deficiency. IL-1beta-expressing beta6(-/-) pups had shorter alveolar chord length and thinner alveolar walls than IL-1beta-expressing beta6(+/+) pups. In addition, the absence of the beta6 integrin subunit reduced IL-1beta-induced neutrophil and macrophage infiltration into the alveolar spaces. beta6 integrin subunit deficiency suppressed inflammation and goblet cell hyperplasia in the airways and alleviated airway remodeling in IL-1beta-expressing mice. The expression of the chemoattractant proteins, keratinocyte-derived chemokine, macrophage-inflammatory protein-2, calgranulin A, and calgranulin B, of osteopontin, and of the chitinase-like lectins, Ym1 and Ym2, was lower in IL-1beta-expressing beta6(-/-) than in IL-1beta-expressing beta6(+/+) mice. We conclude that absence of the beta6 integrin subunit protects the infant murine lung against IL-1beta-induced inflammation and injury.