A Noninflammatory Interleukin-1 Fragment Stimulates Fetal Lung Fluid Absorption in Guinea Pigs
چکیده
We have previously demonstrated that full-length interleukin (IL)-1 can induce and stimulate lung fluid absorption in nearterm guinea pig fetuses via stimulation of fetal cortisol synthesis and release. To develop a potentially clinically useful drug, we tested the hypothesis that maternal administration of a noninflammatory IL-1 -fragment (IL-1 Fr) induced cortisol synthesis and stimulated lung fluid absorption in preterm fetuses. IL-1 Fr was administered s.c. daily to timed-pregnant guinea pigs for 3 days with and without simultaneous cortisol synthesis inhibition by metyrapone. Fetuses were obtained by abdominal hysterotomy at 61 and 68 days gestation and instilled with isosmolar 5% albumin into the lungs, and lung fluid absorption was measured over 1 h by mass balance. Lung fluid absorption was induced at 61 days and stimulated at 68 days gestation by IL-1 Fr, which both were attenuated by cortisol synthesis inhibition. Moreover, induction of labor by oxytocin stimulated lung fluid absorption at 61 days but had no stimulatory effect at 68 days gestation when given with the IL-1 Fr. Plasma adrenocorticotropin and cortisol concentrations were increased by IL1 Fr at 61 days gestation and remained high but unstimulated by IL-1 Fr at 68 days gestation, and metyrapone always reduced cortisol concentrations. Prenatal lung fluid absorption, when present as well as IL-1 Fr-induced, was always propranololand amiloride-sensitive, suggesting that -adrenoceptor stimulation and the epithelial Na channel (ENaC) were critical for the induced/stimulated lung fluid absorption. ENaC expression was increased by IL-1 Fr and attenuated by cortisol synthesis inhibition. Thus, our results suggest a potential clinical use of IL-1 Fr therapeutically to induce lung fluid absorption in fetuses at risk of preterm delivery. Experimental and clinical findings support the notion that rapid preand postnatal lung fluid absorption is critical for establishment of normal pulmonary gas exchange immediately after birth (Kitterman et al., 1979; Hooper and Harding, 1995; Norlin and Folkesson, 2001; Ye et al., 2004; Nair et al., 2005). Rising endogenous epinephrine and cortisol concentrations near term contribute to the decrease in fetal lung fluid volume (Barker et al., 1990; Finley et al., 1998; Norlin and Folkesson, 2001; Ye et al., 2004). Fetal lung fluid is absorbed by lung epithelia after an osmotic gradient, generated by active Na absorption (Finley et al., 1998) via alveolar epithelial apical amiloride-sensitive ENaC channels (Finley et al., 1998) and basolateral Na ,K -ATPases (Nici et al., 1991). ENaC is considered important for lung fluid absorption at birth, because mice made to be ENaC-deficient died within 40 h after birth, possibly because of insufficient lung fluid absorption (Hummler et al., 1996). Cytokines, such as interleukin (IL)-1, may be important during pregnancy, because IL-1 can signal parturition onset (Romero et al., 1989) and is important in preterm labor in which high amniotic fluid IL-1 concentrations have been detected (Taniguchi et al., 1991). Recent studies in adult lungs have suggested that elevated IL-1 levels may lead to development of acute lung injury (Roux et al., 2005). However, in pregnant guinea pigs, IL-1 has been proposed to stimulate synthesis and release of plasma cortisol (Ye et al., 2004), which is important for maintaining alveolar fluid balance in both adult and newborn animals (Liggins, 1994; Norlin et al., 1999). It was in fact demonstrated that IL-1 induced hypothalamic CRF release and activated the adrenocortical axis with ACTH secretion from the pituitary gland, which led to increased cortisol synthesis and secretion from the adrenal glands (Sapolsky et al., 1987). By this pathway, full-length IL-1 can induce lung maturation and reduce development of RDS. A high number of preterm infants have infections and RDS, both of which are important reasons for preterm infant mortality. It has been proposed that incomplete conversion of lung fluid secretion to fluid This work was supported by Research Grant 6-FY03-64 from the March of Dimes Birth Defects Foundation. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.111369. ABBREVIATIONS: ENaC, epithelial Na channel; IL, interleukin; CRF, corticotrophin-releasing factor; ACTH, adrenocorticotropic hormone, RDS, respiratory distress syndrome; Fr, fragment; AR, -adrenoceptor; CPAP, continuous positive airway pressure; RT-PCR, reverse transcriptasepolymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance. 0022-3565/07/3202-877–884$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 320, No. 2 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 111369/3172620 JPET 320:877–884, 2007 Printed in U.S.A. 877 at A PE T Jornals on O cber 8, 2017 jpet.asjournals.org D ow nladed from absorption may cause RDS (Barker et al., 1997). Meanwhile, experimental data suggested that proinflammatory cytokines may signal lung maturation (Bry et al., 1997; Willet et al., 2002; Ye et al., 2004; Nair et al., 2005). We previously demonstrated that IL-1 augmented lung maturation and that oxytocin-induced labor was additive in stimulation of lung fluid absorption (Ye et al., 2004; Nair et al., 2005); these findings led to our hypothesis that maternal IL-1 pretreatment would produce preterm lung maturation and a more complete transition of lung fluid secretion to fluid absorption. However, because IL-1 is a proinflammatory cytokine, it is unlikely to be useful clinically. Therefore, our aims for the current investigation were to 1) compare maternal IL-1 pretreatment with maternal pretreatment with a noninflammatory IL-1 fragment (IL-1 Fr) (Antoni et al., 1986; Nencioni et al., 1987) with respect to induction of lung fluid absorption in fetal lungs, 2) study the effects of the combination of IL-1 Fr and the clinically used labor-inducting agent, oxytocin, on lung fluid absorption in fetal lungs, and 3) study the mechanisms for IL-1 Fr stimulation of fetal lung fluid absorption. Plasma cortisol is important for maintenance of lung fluid absorption in both adult and newborn animals (Liggins, 1994; Norlin et al., 1999; Ye et al., 2004), and by this pathway maternal IL-1 pretreatment induces lung maturation (Ye et al., 2004). Therefore, we hypothesized that maternal IL-1 Fr pretreatment mediated its effects on lung fluid absorption at least partly via stimulation of maternal and/or fetal cortisol synthesis and release via the adrenocortical axis. Materials and Methods Animals. Preterm Dunkin-Hartley guinea pigs (Hilltop Laboratory Animals, Inc., Scottdale, PA) were used (n 107 divided on 30 litters). Timed-pregnant guinea pigs were kept at a 12:12-h day/ night rhythm and had free access to food (Purina standard guinea pig chow; Copley Feed, Copley, OH) and tap water. The Institutional Animal Care and Use Committee at the Northeastern Ohio Universities College of Medicine approved this study. Injection Solutions. The IL-1 pretreatment solution was prepared by dissolving 10 g of rat recombinant IL-1 (Sigma-Aldrich, St. Louis, MO) in 0.1% bovine serum albumin in 0.9% NaCl. The dissolved IL-1 was then aliquoted into vials, each containing 500 ng, and stored frozen at 20°C until used. The noninflammatory IL-1 Fr 163–171 (Antoni et al., 1986; Nencioni et al., 1987) pretreatment solution was prepared by dissolving 500 g of human IL-1 Fr (Sigma-Aldrich) in 0.1% bovine serum albumin in 0.9% NaCl. The dissolved IL-1 Fr was then aliquoted into vials, each containing 500 ng, and stored frozen at 20°C until used. The metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone; Sigma Chemical) pretreatment solution was prepared by dissolving 62.5 mg/ml metyrapone in 24% ethanol in 0.9% NaCl. Injection of 24% ethanol in 0.9% NaCl had earlier been shown to not affect lung fluid absorption in guinea pigs (Norlin et al., 1999). Instillation Solutions. A 5% albumin solution was prepared by dissolving 50 mg/ml bovine serum albumin (Calbiochem-Novabiochem, La Jolla, CA) in 0.9% NaCl. In some studies, the Na channel inhibitor amiloride (10 3 M; MP Biomedicals Inc., Aurora, OH) or the general AR antagonist, propranolol (10 4 M; Sigma Chemical) was added to the instillate. To study the direct effects from the IL-1 Fr on lung fluid absorption, 50 ng of IL-1 Fr/ml was added to the instillate. IL-1 Fr and Full-Length IL-1 Pretreatment. Timed-pregnant guinea pigs of 59 and 66 days gestation were injected s.c. on the dorsal neck once daily with 500 ng/kg IL-1 Fr or 250 ng/kg IL-1 for 3 days. Control timed-pregnant guinea pigs were given injections of 0.9% NaCl. Lung fluid absorption experiments were performed on the morning of the last pretreatment day. Metyrapone Pretreatment. Pretreatment with metyrapone was performed over 3 days simultaneously with administration of IL1 Fr or IL-1 . Subcutaneous metyrapone injections were given twice daily in the morning and in the evening (25 mg/kg to reach a total daily dose of 50 mg/kg) to guinea pigs of 59 and 66 days gestation. On the morning of the day of the lung fluid absorption experiment, one half of the daily metyrapone dose was given. The metyrapone dose was adapted from its higher ranges of clinical dosage. Oxytocin Pretreatment. Labor was induced by s.c. oxytocin injections (1 mg/kg; Phoenix Pharmaceuticals, St. Joseph, MO) every 15 min for 45 min (Norlin and Folkesson, 2001). Fetuses were delivered by abdominal hysterotomy (see Abdominal Hysterotomy) after 45 min, if normal vaginal delivery did not occur. Timed-pregnant guinea pigs with 1 day of gestation left usually delivered their fetuses vaginally within 45 min after oxytocin, whereas fetuses of timed-pregnant guinea pigs with 8 days of gestation left were delivered by abdominal hysterotomy. The uterus was significantly contracted upon post-mortem examination in guinea pigs that received oxytocin without delivering the fetuses vaginally, i.e., 61-day gestation animals. This was taken as evidence that oxytocin had induced labor in the 61-day gestation guinea pigs. The oxytocin dose was adapted from the clinical dose for labor induction in humans (Brindley and Sokol, 1988). Abdominal Hysterotomy. Timed-pregnant guinea pigs were anesthetized by i.p. pentobarbital sodium injections (120 mg/kg; Abbott Laboratories, Chicago, IL) and euthanized by direct intracardiac injections of 60 mg of pentobarbital sodium. A laparotomy was rapidly done, and the fetuses were carefully delivered. The umbilical cord was ligated to prevent bleeding. The fetuses were immediately euthanized by i.p. sodium pentobarbital (12 mg) with 500 IU of heparin (Elkins-Sinn, Cherry Hill, NJ). Fetal Surgery. After euthanasia, an endotracheal tube (PE-190; Clay Adams, Parsippany, NJ) was inserted through a tracheotomy. The animals were immediately connected to a constant O2 flow (FIO2 1.0; Praxair, Akron, OH), and the lungs were expanded by adjusting the O2 flow to a constant positive airway pressure (CPAP) of 5 cm H2O. The surgical procedure after euthanasia required 5 min. The animals were placed in between heating pads to maintain their body temperature during the experiments. A temperature probe measured incubation temperature, and heating was adjusted to maintain the temperature at 37 to 38°C. Airway pressure was continuously monitored by calibrated pressure transducers (ADInstruments, Grand Junction, CO) and analog-to-digital converters and amplifiers (ADInstruments). Lung Fluid Absorption Experiments. After surgery and connection to CPAP, the albumin solution (10 ml/kg) was instilled into the lungs through the endotracheal tube as had been done in earlier studies (Norlin and Folkesson, 2001; Ye et al., 2004; Nair et al., 2005). Total protein concentrations in instillates and initial and final solutions were determined spectrophotometrically (Labsystems Multiscan Microplate Reader; Labsystems, Helsinki, Finland) by the method of Lowry et al. (1951) adapted for microtiter plates. Lung fluid absorption or secretion was calculated from the change in protein concentration over 1 h. This was possible because the alveolar epithelium is relatively impermeable to large molecules, such as albumin (mol. wt. 67,000). Therefore, water movement (absorption or secretion) will change the airspace protein concentration. Lung fluid absorption (AFC) and secretion (AFS) were calculated from eq. 1: AFC or AFS ((Vinst Cinst)/Cinit) (Vinit Cinit)/Cfin))/(Vinst Cinst)/Cinit 100 (1) where Vinst and Vinit are volumes of instillate and initial solutions, respectively, and Cinst, Cinit, and Cfin are protein concentrations in corresponding solutions. 878 Li et al. at A PE T Jornals on O cber 8, 2017 jpet.asjournals.org D ow nladed from To ascertain that lung fluid absorption proceeded at similar rates in our in situ lungs as in in vivo lungs, we compared our control lung fluid absorption with values for absorption published previously in ventilated and in CPAP-oxygenated fetal and newborn guinea pigs according to previous protocols. Lung fluid absorptions in ventilated (Finley et al., 1998), earlier in situ CPAP animals (Norlin and Folkesson, 2001; Ye et al., 2004; Nair et al., 2005) and in our in situ CPAP animals were not significantly different (data not shown). Moreover, it has been demonstrated that there is no additional vascular leak of fluid and protein in this model compared with anesthetized, ventilated animals (Nair et al., 2005). Specific Protocols. Guinea pig fetuses at 61 and 68 days gestation (term 69 days gestation) were studied. Day of conception was set to the day when timed-pregnant guinea pigs gave birth to their earlier litter, because guinea pigs enter estrus immediately after birth. All groups contained fetuses from at least two litters, and all fetuses were surgically prepared as described under Abdominal Hysterotomy and Fetal Surgery and studied on 5 cm H2O CPAP for 1 h after fluid instillation. Lung fluid absorption was measured as described under Lung Fluid Absorption Experiments. Plasma and lung tissue was collected from each group, snap-frozen in liquid N2, and stored at 80°C. IL-1 Fr. Preterm guinea pig fetuses of 61 and 68 days gestation were delivered by abdominal hysterotomy from timed-pregnant guinea pigs that were pretreated with IL-1 Fr. In some studies, 10 4 M propranolol ( AR blocker) was added to the 5% albumin instillate. In other studies, 10 3 M amiloride (Na channel blocker) was added to the 5% albumin instillate. In parallel to the IL-1 Fr studies, preterm guinea pig fetuses of 61 and 68 days gestation were delivered by abdominal hysterotomy from dams pretreated with saline injections once daily and were used as control animals without and with amiloride and propranolol. Amiloride at 10 3 M was used because a large fraction becomes protein-bound and another significant fraction rapidly leaves the airspaces because of its low molecular weight (Yue and Matalon, 1997); therefore, the active alveolar amiloride concentration was probably lower. In addition, the same amiloride concentration had been used in several earlier studies of lung fluid absorption (Finley et al., 1998; Norlin and Folkesson, 2001; Ye et al., 2004; Nair et al., 2005). IL-1 Fr and oxytocin. Guinea pig fetuses from oxytocin-injected timed-pregnant guinea pigs of 61 and 68 days gestation from both control and IL-1 Fr-pretreated animals were given the 5% albumin instillate. Labor was induced by giving timed-pregnant guinea pigs oxytocin. In some studies, 10 4 M propranolol was added to the 5% albumin instillate. Separate sets of guinea pig fetuses at 61 and 68 days gestation were obtained from dams injected with the vehicle, 0.9% NaCl, every 15 min during 45 min to control for stress from the injections per se. Guinea pig fetuses from NaCl-injected timed-pregnant guinea pigs of 61 (n 4) and 68 days (n 4) gestation were also surgically prepared and instilled with a 5% albumin instillate containing 10 4 M oxytocin to control for direct oxytocin effects. IL-1 . Preterm guinea pig fetuses of 61 and 68 days gestation were delivered by abdominal hysterotomy from timed-pregnant guinea pigs that were pretreated with IL-1 . Cortisol inhibition. Preterm guinea pig fetuses of 61 and 68 days gestation with or without maternal IL-1 Fr pretreatment were delivered by abdominal hysterotomy from guinea pigs pretreated with metyrapone for 3 days. Lung fluid absorption and plasma cortisol levels were measured in these guinea pig fetuses. Direct effects of IL-1 Fr on lung fluid absorption. Preterm guinea pig fetuses of 68 days gestation were delivered by abdominal hysterotomy. Lung fluid absorption was measured after instillation of the 5% albumin instillate containing 50 ng of IL-1 Fr/ml. Control 68-day gestation fetuses received an instillation of the 5% albumin instillate without the IL-1 Fr. RT-PCR. RNA was extracted from lung tissue with a Versagene tissue total RNA isolation kit (Gentra Systems, Inc., Minneapolis, MN). RNA yield and purity were determined spectrophotometrically at 260/280 nm, and RNA integrity was verified by agarose gel electrophoresis. A competitive RT-PCR was carried out using the OneStep RT-PCR kit from EMD Biosciences (San Diego, CA) in a volume of 25 l containing 50 ng of total RNA, 1 PCR buffer, 0.2 mM concentrations of each dNTP, 2.5 mM MgSO4, 0.1 M concentrations of each primer, and 1.5 U of rTth DNA polymerase, under optimized reaction conditions (Li and Folkesson, 2006): 60°C for 30 min for reverse transcription, followed by 40 cycles at 94°C for 45 s, 60°C for 2 min, and final extension for 7 min at 60°C. Two pairs of primer sets ( , sense; , antisense) were derived from GenBank sequences and synthesized for competitive RT-PCR: guinea pig ENaC (AF071230), ENa : 5 -CATGATGTACTGGCAGTTCGC-3 (830–850), ENa : 5 TCAGGGACAGACCATTGTTGA-3 (1507–1527); and GAPDH (AB060340), GAPD : 5 -ACCACAGTCCATGCCATCAC-3 (510–529), GAPD : 5 -TCCACCACCCTGTTGCTGTA-3 (942–961). Amplification of the competitive RT-PCR yields a 698-base pair ENaC fragment and a 452-base pair GAPDH fragment (internal control for equal loading). RT-PCR products were resolved in 1.5% agarose gels stained with ethidium bromide, and gels were scanned by a Typhoon 8610 Scanner (GE Healthcare, Piscataway, NJ). Western blot. Western blots were carried out following the protocols in the earlier reports (Ye et al., 2004; Nair et al., 2005). Protein concentrations of the samples were measured to ensure equal loading in each lane of the electrophoresis gel. AntiENaC antibodies were purchased from Alpha Diagnostics International (San Antonio, TX) and directed against 20 residues near the N terminus. These antibodies specifically recognize membrane proteins of appropriate sizes (85–90 kDa for ENaC) in rats. We tested for cross-reactivity with guinea pig and found bands specifically labeled for both subunits. Monoclonal rabbit anti-human GAPDH antibodies were obtained from Cell Signaling Technology Inc. (Danvers, MA). These antibodies specifically recognize a protein of appropriate size (37 kDa) in rats and humans and were tested for cross-reactivity with guinea pig and found to be cross-reactive. ELISA Analyses. Plasma was collected from guinea pigs in all experimental groups and gestation ages (n 4–5/experimental group) and was immediately frozen in liquid N2 after centrifugation (3000g, 5 min) and stored at 80°C until analysis. Plasma epinephrine concentrations were measured using a commercially available ELISA (CatCombi; IBL, Hamburg, Germany). The assay had a sensitivity of 12 pg/ml and intraand interassay variabilities of 5 and 12%, respectively. Plasma cortisol and ACTH concentrations were measured using commercially available ELISAs (ALPCO Diagnostics, Windham, NH). The assays had a sensitivity of 0.3 g/dl (cortisol) and 0.46 pg/ml (ACTH) and intraand interassay variabilities of 4.4 and 6.7% for cortisol and 3.6 and 6.0% for ACTH, respectively. Statistics. Values are presented as means S.D. Statistical analysis was carried out with one-way analysis of variance (ANOVA) with Tukey’s test post hoc or Student’s t test when appropriate. Differences were considered statistically significant when P 0.05. The number of fetuses per group is given in the legends to the figures when data are presented as a figure and in the text otherwise.