Mikko Hallman

Transforming growth factor-β2 prevents preterm delivery induced by interleukin-α and tumor necrosis factor-α the rabbit

Objectives: The purpose of the study was to determine whether preterm parturition in the rabbit can be induced by intraamniotic injection of proinflammatory cytokines, interleukin-α and tumor necrosis factor-β2, and whether transforming growth factor-p2, an inhibitor of the cytokine-induced prostaglandin synthesis, modifies the effect of these cytokines. Study Design: New Zealand White rabbits were injected in each amniotic cavity on day 24 of gestation with one of the following: a combination of interleukin-α (150 µg) and tumor necrosis factor-α (1.25 µg), 50 ng of transforming growth factor-β2 concomitantly with interleukin-α and tumor necrosis factor-α, or vehicle. In the first study the animals were observed for signs of delivery until day 29 of gestation. In the second study the effect of transforming growth factor-β2 (50 ng/fetus) on the rate of premature delivery was evaluated. In the third study the concentrations of prostaglandin E 2 and 13,14-dihydro-15-keto-prostaglandin F2α. were measured in the amniotic fluid on day 27 of gestation. The statistics used were Fishers exact test, the X 2 test, and the Mann-Whitney U test. Results: Altogether 61 of 191 fetuses (32%) were born prematurely in the interleukin-1 α-tumor necrosis factor-a group, whereas only two of 161 fetuses (1.2%) ( p = 0.0001) and one of 159 (0.6%) ( p = 0.0001) were born prematurely in the interleukin-1a tumor necrosis factor-α-transforming growth factor-β2 group and in the control group, respectively. Of the 23 animals injected with interleukin-1 α and tumor necrosis factor-α, six (26%) delivered all of their fetuses prematurely versus none in the other groups ( p = 0.02). None of the 88 fetuses in the transforming growth factor-α group were born prematurely. The prostaglandin E 2 concentrations in the amniotic fluids were higher in the interleukin-1a-tumor necrosis factor-α group than in the interleukin-lα-tumor necrosis factor-α-transforming growth factor-β2 group ( p = 0.05) or in the control group ( p = 0.02). Conclusions: Preterm parturition can be provoked in the rabbit by intraamniotic injections of interleukin-la and tumor necrosis factor-a. Transforming growth factor-P2 prevents the cytokine-induced increase in premature delivery.

The Fate of Exogenous Surfactant in Neonates with Respiratory Distress Syndrome

SummaryRespiratory distress syndrome (RDS) in newborn neonates is characterised by deficient secretion of surfactant from type III alveolar cells. Administration of surfactant to airways acutely decreases the degree of respiratory failure and increases the survival rate in neonates with RDS. Clinically available surfactants are lipid extracts derived from animal lung lavage or from whole lung. Synthetic surfactants contain phospholipids or additional spreading agents. An optimal exogenous surfactant would be efficacious, nontoxic and nonimmunogenic, resistant to oxidants and proteolytic agents, widely available at reasonable cost and manufactured with little batch-to-batch variability.Surfactant has been instilled into the airways as a bolus infusion through the endotracheal tube. In addition, surfactant may be given by aerosolisation or continuous infusion into the airways. Suggested dosages range from 50 to 200 mg/kg. Exogenous surfactant is cleared from the epithelial lining fluid (ELF) mainly by alveolar epithelial cells, although alveolar macrophages and the central airways may also contribute to clearance of the drug. Only small quantities of surfactant actually enter the blood stream. A significant fraction of surfactant is taken up, processed, and secreted back into the alveolar space by type II alveolar cells. This process is termed recycling.Phosphatidylglycerol, given to small premature neonates as a component of exogenous human surfactant, has an apparent pulmonary half-life of 31 ± 3 hours (n=11). The apparent pulmonary half-life of the main surfactant component dipalmitoyl phosphatidylcholine is 45 hours (n=3) and that of surfactant protein A is about 9 hours (n=4). A relationship between the dose of exogenous surfactant and its concentration in the ELF has been demonstrated.Some neonates with RDS respond poorly to surfactant therapy. The reasons for this include insufficient levels of surfactant in the ELF, uneven distribution of exogenous surfactant, inability of exogenous surfactant to enter the metabolic pathways, inhibition of surface activity by plasma-derived proteins, or inactivation of surfactant as a result of proteases, phospholipases, or oxygen free radicals. In addition, surfactant therapy may be ineffective in neonates with respiratory failure caused by factors other than surfactant deficiency. The efficacy of exogenous surfactant can be improved by increasing the dosage of surfactant and by administration of surfactant very early in respiratory failure.

Nitric oxide and lung surfactant

Inhalation of nitric oxide (NO) is an experimental treatment for severe pulmonary hypertension. Being rapidly metabolized by hemoglobin, inhaled NO causes selective vasodilation in the pulmonary vascular bed. In addition to the vascular smooth muscle, other pulmonary structures are exposed to inhaled NO, resulting in suppression of NO synthesis in a variety of pulmonary cells and in potential toxicity. NO is a free radical that interacts with a number of proteins, particularly metalloproteins. Together with superoxide radical, it rapidly forms highly toxic peroxynitrite. Peroxynitrite is involved in the killing of microbes by activated phagocytosing macrophages. In severe inflammation, peroxynitrite may be responsible for damaging proteins, lipids, and DNA. Peroxynitrite added to surfactant in vitro is capable of decreasing the surface activity, inducing lipid peroxidation, decreasing the function of surfactant proteins, SP-A and SP-B, and inducing protein-associated nitro-tyrosine. Exposure of animals for prolonged periods (48 to 72 hours) to inhaled NO (80 to 120 ppm) has been associated with a decrease in surface activity. This is caused by binding of surfactant to iron-proteins that are modified by NO (particularly methemoglobin), or by peroxynitrite induced damage of surfactant. In contrast, exposure of isolated surfactant complex to NO during surface cycling strikingly decreases the inactivation of surfactant, preventing the conversion of surfactant to small vesicles that are no longer surface-active, and preventing lipid peroxidation. This finding is consistent with the function of NO as a lipid-soluble chain-braking antioxidant. It is possible that this lipophilic gas has as yet undefined roles in regulation of surfactant metabolism and maintenance of surface activity. Deficiency in pulmonary NO may be present during the early neonatal period in respiratory distress syndrome and in persistent fetal circulation. The premature lung is likely to be sensitive to NO toxicity that may include lung damage, abnormal alveolarization, and mutagenicity. Defining of the indications, the dosage, and the toxicity of inhaled NO therapy remains the challenge for experimental and clinical research.

lnterleukin-1α Upregulates the Expression of Surfactant Protein-A in Rabbit Lung Explants

Interleukin-1 (IL-1) is an important participant in infectious and inflammatory conditions. Interleukin-1 receptor antagonist (IL-1 ra) prevents the effect of IL-1. We have shown that injection of interleukin-1 α (IL-1α) into the amniotic fluid of pregnant rabbits stimulates the expression of surfactant protein-A (SP-A) in the lungs of the fetuses. We hypothesized that IL-1α similarly enhances the expression of SP-A in rabbit lung explants in vitro. Explants obtained from 22-day fetal rabbit lungs were cultured in Waymouth’s medium on a rotating platform in the presence or absence of IL-1α (5.7–570 ng/ml) or IL-Ira (1–10 μg/ml). Dibutyryl cAMP (1 mM) served as a positive control. After 3 days in culture, the explants were harvested and Northern analysis of SP-A was performed using the 1.9-kb rabbit SP-A cDNA probe. IL-1α and dibutyryl cAMP increased the expression of SP-A twofold, as judged by video densitometry. IL-1ra did not change SP-A expression as compared with controls, suggesting that endogenous IL-1 activity was not responsible for the basal level of SP-A expression in the explants. Dibutyryl cAMP increased the expression of SP-B mRNA, whereas IL-1 α had no effect on SP-B mRNA concentration. We conclude that inflammatory mediators interact with lung cells to alter synthesis of important components of the surfactant system.

Closure of Patent Ductus arteriosus Decreases Pulmonary Myeloperoxidase in Premature Infants with Respiratory Distress Syndrome

The aim of the study was to determine whether in premature infants with respiratory distress syndrome (RDS), an interrelationship existed between patent ductus arteriosus (PDA) and pulmonary neutrophil content. Thirteen premature infants with PDA (gestational age 26.9 +/- 2.4 weeks, birth weight 903 +/- 300 g) were pair-matched with infants without PDA (gestations age 27.1 +/- 2.5 weeks, birth weight 1,041 +/- 430 g) with regard to gestational age, birth weight and severity of RDS. The myeloperoxidase (MPO) contents of tracheal aspirates were analyzed before (age 2.8 +/- 1.7 days, MPO I) and after closure of PDA with indomethacin (5.5 +/- 1.9 days, MPO II), and at corresponding time points in controls (2.6 +/- 1.6 and 4.8 +/- 1.8 days). In PDA patients closure of PDA decreased MPO from 14.7 (range 2.1-27.4) to 4.6 (1.2-8.0) micrograms/mg protein (p = 0.0008), whereas in controls it remained unchanged: 5.4 (2.9-7.8) and 5.3 (2.9-7.8; p = 0.58). A significant difference existed in the change in MPO contents between the infants with PDA and the controls. In premature infants with RDS, closure of PDA decreases the neutrophil content of the lungs. The effect may be caused by closure of PDA as such, or by indomethacin. The high pulmonary neutrophil content in PDA may exacerbate inflammatory processes and contribute to the development of chronic pulmonary disease.

Variable oxygenation response to inhaled nitric oxide in severe persistent pulmonary hypertension of the newborn.

The causes of variable responsiveness to inhaled nitric oxide (NO) in Persistent Pulmonary Hypertension of the Newborn (PPHN) are unknown. The changes in the severity of respiratory failure after the onset of inhaled NO (maximal dose 20ppm) were studied in 13 consecutive neonates with severe PPHN. Response was defined as a sustained decrease of alveolar‐arterial oxygen gradient (AaD02) by > 20%, or a decrease in oxygenation index (OI) by > 40%. Six neonates had a rapid response within 30min, three had an intermediate response within 8h, and three had a delayed response within 12 h after the onset of NO. Three infants with birth asphyxia responded rapidly to inhaled NO. One infant with sepsis did not respond, and two with suspected sepsis had a delayed response. The infants with Meconium Aspiration Syndrome and idiopathic PPHN had a variable response time. Twelve neonates required 4 to 14 days of mechanical ventilation and survived. Infants with PPHN may benefit from a trial of inhaled NO therapy that exceeds 30min. The variability of the response time to inhaled NO is likely to be multifactorial and dependent on the disease process associated with PPHN.

Interleukin-1 binding and prostaglandin E2 synthesis by amnion cells in culture: regulation by tumor necrosis factor-α, transforming growth factor-β, and interleukin-1 receptor antagonist

Proinflammatory cytokines may promote preterm labor in the setting of intrauterine infection. Tumor necrosis factor (TNF) and interleukin-1 (IL-1) synergistically stimulate the production of prostaglandin E2 (PGE2) by amnion cells. Transforming growth factor-beta (TGF-beta) inhibits the cytokine-stimulated PGE2 production. In the present study, we investigated the binding of IL-1 beta on human amnion cells in culture. Untreated amnion cells possessed 540 +/- 60 IL-1 receptors per cell, with a dissociation constant of 1.4 +/- 0.4 nM. Cells treated with TGF-beta 1 (10 ng/ml) had 570 +/- 110 receptors per cell. TNF-alpha (50 ng/ml) increased the number of IL-1 receptors to 2930 +/- 590. TGF-beta 1 inhibited the receptor upregulation by TNF-alpha. Cells treated with TGF-beta 1 and TNF-alpha expressed 1140 +/- 590 receptors per cell. The binding affinity was not changed by the cytokines. IL-1 receptor antagonist (IL-1ra) inhibited the stimulation of amnion cell PGE2 production by IL-1 beta, but not by TNF-alpha. Amnion cells secreted large amounts of IL-1ra (1.1 +/- 0.3 ng/10(5) cells). Treatment of the cells with TGF-beta 1 or TNF-alpha did not affect the release of IL-1ra. We conclude that IL-1 receptor expression is an important step in the regulation of the effects of cytokines on amnion cell PGE2 production.

Cytokines and production of surfactant components

The production of pulmonary surfactant, a complex of lipids and proteins that reduces surface tension at the alveolar air-liquid interface, is developmentally regulated. Several hormones, most notably glucocorticoids, are known to accelerate maturation of the surfactant system. Cytokines are polypeptides that act mostly in a paracrine fashion and possess a wide spectrum of activities on multiple types of cells. Many cytokines are produced by different lung cells a various stages of fetal development or under pathological conditions affecting the fetus. In addition, cytokines present in amniotic fluid or in the blood stream may reach the fetal lungs. Some cytokines, including epidermal growth factor, transforming growth factor-alpha, and interferon-gamma have been shown to stimulate the production of surfactant components. On the other hand, tumor necrosis factor and transforming growth factor-beta downregulate the production of surfactant lipids and proteins. We have recently shown that the proinflammatory cytokine interleukin-1 (IL-I) enhances the expression of surfactant protein A (SP-A) in fetal rabbit lung explants. In addition, injection of IL-I into the amniotic fluid of fetal rabbits enhances the expression of surfactant proteins and improves the lung compliance of preterm animals. Preterm delivery is often associated with subclinical intraamniotic infection. In these cases, amniotic fluid concentrations of IL-I are often elevated. We propose that this cytokine accelerates maturation of the surfactant system in fetal lungs and thus prepares the fetus for extrauterine life.

Interleukin-1 receptor antagonist in the fetomaternal compartment.

Interleukin‐1 (IL‐1) is a major mediator in infections and inflammation. Interleukin‐1 receptor antagonist (IL‐1ra) opposes the actions of IL‐1. IL‐1ra is present in exceptionally high concentrations in third trimester amniotic fluid. We studied IL‐1ra in amniotic fluid, fetal serum and newborn urine. The concentrations of IL‐1ra in amniotic fluid at mid‐trimester and at 25‐41 gestational weeks were 6.6 ± 0.5ng/ml (n = 30) and 100 ± 4ng/ml (n = 202), respectively. At mid‐trimester, amniotic fluid IL‐1ra was not dependent on fetal gender, whereas during the third trimester IL‐1ra was higher in female‐ than in male‐bearing gestations. Urine of normal term newborns during the first day of life contained a very high concentration of IL‐1ra (125 ± 16ng/ml, n= 50). Urinary concentration in female newborns was significantly higher than that in male newborns (202 ± 19ng/ml, n = 25 versus 49 ± 14ng/ml, n = 25). IL‐1ra concentration in fetal serum at 22‐36 gestational weeks was 0.50 ± 0.07ng/ml (n= 31) and at term 1.5 ± 0.3ng/ml (n= 17). Serum concentrations were not gender‐dependent. The gender differences in IL‐1ra concentrations may in part explain the lower susceptibility of female fetuses to infection.

Cytokines released by granulocytes and mononuclear cells stimulate amnion cell prostaglandin E2 production

Preterm labor is associated with histologic chorioamnionitis and intraamniotic infection. Chorioamnionitis is characterized by infiltration of the fetal membranes by granulocytes. In intraamniotic infection, white cells accumulate in amniotic fluid. Granulocytes and mononuclear cells have been shown to release products that stimulate prostaglandin E2 (PGE2) production by amnion cells. The aim of the present study was to identify some of these products. Cell-free supernatant obtained from formylmethionyl-leucyl-phenylalanine (FMLP)-activated granulocytes was subjected to size exclusion chromatography. The fractions obtained were analyzed for their stimulatory effect on PGE2 production by human amnion cells in culture. Two peaks of PGE2-stimulatory activity were found. The activity present in one of these was synergistic with interleukin-1 beta (IL-1 beta) in stimulating amnion cell PGE2 production and was found to contain mainly transforming growth factor-alpha (TGF-alpha) immunoreactivity. This cytokine originated at least in part from eosinophils. The PGE2-stimulatory activity present in the other peak contained IL-1 beta, epidermal growth factor (EGF), and tumor necrosis factor-alpha (TNF-alpha). The release of IL-beta, TNF-alpha, TGF-alpha, and EGF by granulocytes differed from that of mononuclear cells. We have previously shown that TGF-alpha and EGF are synergistic with IL-1 and TNF-alpha in enhancing amnion cell PGE2 production. The different pattern of prostaglandin-stimulatory cytokines released from different types of white cells suggest that the cells may potentiate each others effects.