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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Fragment Stimulates Fetal Lung Fluid Absorption in Guinea Pigs
Department of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, Ohio
Received July 25, 2006; accepted November 13, 2006.
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Abstract |
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can induce and stimulate lung fluid absorption in near-term 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-1Fr) induced cortisol synthesis and stimulated lung fluid absorption in preterm fetuses. IL-1Fr 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-1Fr, 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-1Fr. Plasma adrenocorticotropin and cortisol concentrations were increased by IL-1Fr at 61 days gestation and remained high but unstimulated by IL-1Fr at 68 days gestation, and metyrapone always reduced cortisol concentrations. Prenatal lung fluid absorption, when present as well as IL-1Fr-induced, was always propranolol- and 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-1Fr and attenuated by cortisol synthesis inhibition. Thus, our results suggest a potential clinical use of IL-1Fr therapeutically to induce lung fluid absorption in fetuses at risk of preterm delivery.
; 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 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-1Fr) (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-1Fr and the clinically used labor-inducting agent, oxytocin, on lung fluid absorption in fetal lungs, and 3) study the mechanisms for IL-1Fr 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-1Fr 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.
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Materials and Methods |
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Injection Solutions. The IL-1 pretreatment solution was prepared by dissolving 10 µg 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-1Fr 163–171 (Antoni et al., 1986; Nencioni et al., 1987) pretreatment solution was prepared by dissolving 500 µg of human IL-1Fr (Sigma-Aldrich) in 0.1% bovine serum albumin in 0.9% NaCl. The dissolved IL-1Fr 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-1Fr on lung fluid absorption, 50 ng of IL-1Fr/ml was added to the instillate.
IL-1Fr 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-1Fr 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 IL-1Fr 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:
 | (1) |
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-1Fr. 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-1Fr. 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-1Fr 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-1Fr and oxytocin. Guinea pig fetuses from oxytocin-injected timed-pregnant guinea pigs of 61 and 68 days gestation from both control and IL-1Fr-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-1Fr 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-1Fr 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-1Fr/ml. Control 68-day gestation fetuses received an instillation of the 5% albumin instillate without the IL-1Fr.
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 One-Step RT-PCR kit from EMD Biosciences (San Diego, CA) in a volume of 25 µl containing 50 ng of total RNA, 1x PCR buffer, 0.2 mM concentrations of each dNTP, 2.5 mM MgSO4, 0.1 µM 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
[GenBank]
), ENa+: 5'-CATGATGTACTGGCAGTTCGC-3' (830–850), ENa–: 5'-TCAGGGACAGACCATTGTTGA-3' (1507–1527); and GAPDH (AB060340
[GenBank]
), 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. Anti-ENaC 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 intra- and 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 µg/dl (cortisol) and 0.46 pg/ml (ACTH) and intra- and 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.
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Results |
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Fr and Induction of Lung Fluid Absorption. Lung fluid absorption was studied in fetal guinea pigs (61 and 68 days gestation) after maternal IL-1Fr pretreatment and compared with control lung fluid absorption and lung fluid absorption after IL-1 pretreatment. At 61 days gestation, control fetal lungs were still secreting fluid (Fig. 1). Maternal IL-1Fr pretreatment induced (converted secretion into absorption) lung fluid absorption at 61 days gestation (Fig. 1) more effectively than maternal full-length IL-1 pretreatment. At 68 days gestation, control fetal lungs had begun to absorb lung fluid (Fig. 1). Maternal IL-1Fr pretreatment failed to stimulate existing lung fluid absorption in contrast with the situation after maternal full-length IL-1 pretreatment (Fig. 1).
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Direct instillation of the IL-1Fr-containing instillate into the lungs of 68-day gestation fetal guinea pigs did not increase lung fluid absorption compared with normal 68-day gestation control fetal guinea pigs [9 ± 3% (n = 6) and 8 ± 4% (n = 9), respectively; Student's t test].
We then tested whether IL-1Fr pretreatment involved activation of amiloride-sensitive Na+ transport and lung fluid absorption. In 61-day gestation guinea pig fetuses, only IL-1Fr-induced lung fluid absorption was blocked by amiloride (Fig. 2A), whereas control lung fluid secretion was unaffected. In 68-day gestation fetuses (Fig. 2A), both control and IL-1Fr-stimulated lung fluid absorption were blocked by amiloride.
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We then determined whether ENaC mRNA expression increased in general with age and lung development (Fig. 2B). In 61-day gestation fetal lungs, ENaC mRNA expression was low and increased significantly during lung development until 68 days gestation. Maternal IL-1Fr pretreatment significantly increased ENaC mRNA expression at 61 days compared with control fetuses but did not change ENaC mRNA expression at 68 days gestation (Fig. 2B). Maternal labor induction by oxytocin did not affect ENaC mRNA expression at either 61 or 68 days gestation. Combined IL-1Fr and oxytocin pretreatment also increased ENaC mRNA expression at 61 days gestation, but not more than IL-1Fr alone did. The results were different in 68-day gestation fetuses; here this combination pretreatment did not affect ENaC mRNA expression (Fig. 2B).
We also studied whether IL-1Fr up-regulated ENaC expression in lung epithelia. ENaC expression increased in general with age and lung development until 68 days gestation (Fig. 2C). In 61-day gestation fetal lungs, maternal IL-1Fr pretreatment significantly increased ENaC expression compared with control fetuses (Fig. 2C). The results were different in 68-day gestation fetuses; here maternal IL-1Fr pretreatment did not affect ENaC expression (Fig. 2C). GAPDH was used as a control for differences in membrane transfer and loading. There were no differences in GAPDH in any group or between any of the lanes of the Western blot membranes as can be seen in Fig. 2C.
We also investigated whether direct instillation of IL-1Fr affected ENaC mRNA expression at both 61 and 68 days gestation. Direct IL-1Fr instillation into the developing fetal lung airspaces did not affect ENaC mRNA expression [61 days gestation (n = 4), 1.00 ± 0.12; 68 days gestation (n = 4), 0.97 ± 0.05, relative OD to 61 days gestation].
We then tested whether maternal IL-1Fr pretreatment led to AR-stimulated lung fluid absorption in fetal lungs. At 61 days gestation, only IL-1Fr-induced lung fluid absorption was blocked by propranolol (AR antagonist) (Fig. 3), whereas control lung fluid secretion was unaffected. At 68 days gestation (Fig. 3), both control and IL-1Fr-stimulated lung fluid absorptions were blocked by propranolol.
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Fr Pretreatment and Oxytocin-Induced Labor. We know that 1) maternal IL-1 pretreatment increases AR expression in fetal lungs (Ye et al., 2004), 2) oxytocin by itself increases plasma epinephrine levels and lung fluid absorption (Norlin and Folkesson, 2001), and 3) IL-1 and oxytocin given together stimulate lung fluid absorption more than each given separately (Nair et al., 2005). Thus, we determined whether oxytocin-induced preterm labor after maternal IL-1Fr pretreatment additively affected lung fluid absorption. Lung fluid absorption was measured in fetal guinea pigs (61 and 68 days gestation) after maternal IL-1Fr pretreatment with and without induction of labor by oxytocin. Oxytocin alone converted 61-day gestation fetal lungs from lung fluid secretion to fluid absorption (Fig. 4A) and had a tendency to stimulate lung fluid absorption at 68 days gestation. Oxytocin-induced labor in 61-day gestation IL-1Fr-exposed guinea pigs also increased lung fluid absorption (Fig. 4A). The stimulatory effects from oxytocin and IL-1Fr were always blocked by propranolol (61 days, –1 ± 5% versus 21 ± 6%; 68 days, –12 ± 9 versus 12 ± 10%). Oxytocin, when instilled directly into fetal lungs, lacked a stimulatory effect: –11 ± 3% (61 days) and 4 ± 4% (68 days). Fetal plasma epinephrine concentrations were significantly increased after induction of preterm labor by oxytocin independently of pretreatment with IL-1Fr or not (Fig. 4B).
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Fr Induction/Stimulation of Lung Fluid Absorption. To test whether maternal IL-1Fr pretreatment mediated induction of lung fluid absorption by stimulation of maternal and/or fetal plasma cortisol, we measured plasma cortisol concentrations after maternal IL-1Fr pretreatment and with and without cortisol synthesis inhibition. As shown in Fig. 5A, fetal plasma cortisol concentrations were low at 61 days and increased at 68 days gestation. At 61 days, plasma cortisol concentrations were increased after IL-1Fr pretreatment, whereas no further change was observed at 68 days gestation (Fig. 5A). Maternal plasma cortisol concentrations were equal to or lower than fetal cortisol levels in both age-matched groups after IL-1Fr pretreatment (maternal cortisol levels at 61 days: control, 5964 ± 218 nM; IL-1Fr, 5143 ± 112 nM; and at 68 days: control, 8511 ± 110 nM; IL-1Fr, 8673 ± 120 nM). This suggested that fetal plasma cortisol synthesis was stimulated and not dependent on maternal plasma cortisol crossing the placenta. Metyrapone pretreatment lowered maternal and fetal plasma cortisol concentrations in IL-1Fr-pretreated timed-pregnant guinea pigs (Fig. 5A).
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Lung fluid absorption after concomitant IL-1Fr and metyrapone pretreatment was also measured. As shown in Fig. 5B, concomitant IL-1Fr and metyrapone administration attenuated IL-1Fr-induced/stimulated lung fluid absorption at both 61 and 68 days gestation. At 61 days gestation, no changes were observed in control lung fluid secretion compared with metyrapone pretreatment (Fig. 5B). Once plasma cortisol began to rise, i.e., at 68 days gestation, metyrapone blocked lung fluid absorption in unstimulated control fetuses (Fig. 5B).
Plasma ACTH. Plasma ACTH concentrations were increased (P < 0.05) by maternal IL-1Fr pretreatment at both 61 and 68 days gestation compared with age-matched control fetuses (Fig. 6). ACTH concentrations were increased by IL-1Fr in the maternal plasma also. In contrast to plasma cortisol concentrations, maternal plasma ACTH concentrations were higher than those in age-matched fetal groups (maternal ACTH levels at 61 days: control, 76.1 ± 4.3 pg/ml; IL-1Fr, 146.1 ± 7.7 pg/ml; and at 68 days: control, 98.7 ± 7.0 pg/ml; IL-1Fr, 105.0 ± 12.7 pg/ml). This suggested that plasma ACTH may have crossed the placenta, and therefore, the fetuses may have produced their own cortisol in response to ACTH.
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induced lung maturation via cortisol synthesis stimulation (Ye et al., 2004), 2) induction of preterm labor by oxytocin rapidly matured the lung (within 45–60 min) by elevating plasma epinephrine levels (Norlin and Folkesson, 2001), and 3) combined exposure to IL-1 and oxytocin additively stimulated lung fluid absorption (Nair et al., 2005). These observations are interesting and encouraging from a treatment standpoint, but the use of IL-1 as an inducing agent would be unlikely, as it is a proinflammatory cytokine. We therefore reasoned that if we could use a noninflammatory IL-1 fragment, it may still work. Using the noninflammatory IL-1Fr 163–171 (Antoni et al., 1986; Nencioni et al., 1987), we tested the hypothesis that this fragment would act similarly to full-length IL-1. The initial observations suggested that our hypothesis was correct, but with a limitation in that IL-1Fr failed to stimulate lung fluid absorption at 68 days gestation. One potential reason for this difference could be that full-length IL-1 binds to and activates the IL-1 receptor differently from IL-1Fr. We have, however, not seen such evidence in the published literature. Another potential reason is that IL-1Fr failed to stimulate cortisol synthesis and release at 68 days gestation. In fact, IL-1Fr did not, in contrast with full-length IL-1, stimulate cortisol synthesis and release at 68 days gestation, thus potentially explaining the difference between full-length IL-1 (Ye et al., 2004) and IL-1Fr at 68 days gestation. However, the fact that IL-1Fr failed to stimulate lung fluid absorption at 68 days gestation may not be limiting, because the lung is already absorbing fetal lung fluid and responds to epinephrine by increasing lung fluid absorption. Our earlier study (Ye et al., 2004) demonstrated that IL-1 stimulated cortisol synthesis, which increased lung Na+ transport protein and AR expression. Because preterm babies may experience respiratory distress in part related to immaturities in lung ion transport (Barker et al., 1997), we hypothesized that as the lung seemed more mature after IL-1Fr pretreatment, labor, if delivery is nonpreventable, could be beneficial to the outcome. In this study, induction of labor by oxytocin improved IL-1Fr-induced lung fluid absorption, thus providing opportunities to clear lung fluid rapidly from fetal preterm lungs at delivery.
Molecular mechanisms for fetal lung fluid absorption include apical ENaCs (O'Brodovich et al., 1990) and basolateral Na+,K+-ATPases (Ingbar et al., 1997; Sznajder et al., 2002). We found a significant increase in ENaC expression after IL-1Fr administration at 61 days gestation. No changes in ENaC expression were observed at 68 days gestation. Thus, molecular evidence correlated with lung fluid absorption induction and supported our hypothesis that IL-1Fr could up-regulate lung fluid absorption. Functional ENaC channels were evaluated by amiloride inhibition. Previous studies (Finley et al., 1998; Norlin and Folkesson, 2001; Ye et al., 2004; Nair et al., 2005) demonstrated that before birth, amiloride sensitivity is close to 100%, because there are few or no other pathways for Na+ absorption. Simultaneously with birth, alternative amiloride-insensitive pathways (possibly cyclic nucleotide-gated channels) may develop (Norlin et al., 2001). Both types of channels are suggested to remain in postnatal lungs. Amiloride did not affect lung fluid secretion at 61 days gestation, but completely inhibited IL-1Fr-induced lung fluid absorption. Supporting this observation, 61-day gestation fetuses have less ENaC; both ENaC mRNA and protein expression were significantly up-regulated after IL-1Fr pretreatment. At 68 days gestation, ENaC mRNA and protein expression were elevated compared with 61-day gestation fetal lungs but were not further elevated by IL-1Fr. Thus, molecular evidence correlated with changes in the rate of lung fluid absorption and further supported our hypothesis that IL-1Fr up-regulated ion transport across lung epithelia. In a study such as this one, it is impossible to deduce whether IL-1Fr increased ENaC open probability and/or increased Na+,K+-ATPase activity. However, up-regulation of ENaC provided evidence that IL-1Fr could induce ENaC expression.
Plasma epinephrine is an important regulator of lung fluid absorption (Walters and Olver, 1978; Matthay et al., 2002) because it can enhance active Na+ transport (Walters and Olver, 1978; Finley et al., 1998). AR stimulation increases transepithelial Na+ transport by stimulating ENaC at the apical membrane of alveolar epithelial cells (Baxendale-Cox, 1999; Borok et al., 1999; Johnson et al., 2006) and basolateral Na+,K+-ATPase number and activity (Saldías et al., 1999). Consequently, endogenous AR stimulation by labor-released epinephrine accelerates lung fluid absorption near term (Finley et al., 1998). The effect of endogenous AR stimulation on lung fluid absorption during normal gestation can be studied by propranolol and measurement of plasma catecholamines. We added propranolol to study whether endogenous AR stimulation affected lung fluid absorption after IL-1Fr. At 61 days gestation, IL-1Fr-induced lung fluid absorption was propranolol-sensitive, whereas lung fluid secretion was propranolol-insensitive. At 68 days gestation, propranolol inhibited both control and IL-1Fr-stimulated lung fluid absorption. We measured fetal plasma epinephrine with or without IL-1Fr pretreatment at 61 and 68 days gestation. In both age groups, plasma epinephrine was unaffected by IL-1Fr and similar to that previously reported in guinea pigs (Norlin and Folkesson, 2001; Ye et al., 2004). Because IL-1 is important for parturition onset and may stimulate prostaglandin biosynthesis, which is important for labor onset (Romero et al., 1989), IL-1Fr may affect AR sensitivity of alveolar epithelial cells. In an earlier study (Stern and Kunos, 1988) and in our earlier study (Ye et al., 2004), IL-1 acted synergistically with cortisol to increase AR density on lung cells. There are multiple studies displaying either up-regulation (Szentendrei et al., 1992; Bin et al., 2001), down-regulation (Mak et al., 2002), or no change (Szentendrei et al., 1992) in AR expression. Because epinephrine stimulates lung fluid absorption by AR activation and cortisol may affect AR density, we tested these effects by inducing preterm labor by oxytocin. Labor induction by oxytocin demonstrated that both the increased plasma epinephrine levels and the IL-1Fr pretreatment stimulated lung fluid absorption and lung maturation at 61 days gestation. At 68 days gestation there was again no effect from IL-1Fr when given with oxytocin. Thus, a combination treatment strategy may prove successful for infants born preterm.
Maternal IL-1Fr injections induced lung fluid absorption associated with amiloride-sensitive Na+ transport and increased ENaC expression. Because cytokines often act as classic hormones, they bind to specific receptors on target cells and exert biological effects via second messengers. However, as the scenario here is transfer of the signal from mother to offspring, there has to be an extracellular mediator. So, what may act as an extracellular mediator between mother and offspring for IL-1Fr to trigger downstream cascade events in fetal lungs? It may, of course, be IL-1Fr itself if it crossed the placental barriers. However, in our earlier study, measurements of maternal and fetal plasma IL-1 levels suggested that only minute amounts of IL-1 actually crossed the placental barriers (Ye et al., 2004). Thus, it was not likely that IL-1 itself was the extracellular mediator. We could not measure IL-1Fr because there is no assay for it. It had been reported earlier that IL-1 acts directly on the hypothalamus to stimulate CRF release, thus activating the hypothalamus-pituitary-adrenal gland axis with ACTH and plasma cortisol release (Sapolsky et al., 1987; Ye et al., 2004). Thus, we propose that IL-1Fr acts similarly. Because cortisol is a well-established lung maturation factor and can stimulate lung fluid absorption, plasma cortisol was hypothesized to be a likely target candidate. We measured fetal and maternal plasma ACTH and cortisol concentrations at both gestation ages and found that both hormones were stimulated by IL-1Fr at 61 and 68 days gestation. ACTH concentrations were higher in maternal than in fetal plasma, whereas cortisol concentrations were higher in fetal than in maternal plasma, suggesting that ACTH crossed the placenta into the fetal blood circulation and stimulated cortisol synthesis. Thus, IL-1Fr, similarly to IL-1, activates the hypothalamus-pituitary-adrenal gland or the pituitary gland directly.
How important was plasma cortisol for IL-1Fr induction of fetal lung fluid absorption? To answer this question, we studied whether 11-hydroxylase inhibition by metyrapone attenuated IL-1Fr induction of lung fluid absorption and transporter expression. Earlier it was shown that 2 days of metyrapone pretreatment inhibited cortisol synthesis and that this affected baseline lung fluid absorption (Norlin et al., 1999). Here, metyrapone administration reduced fetal plasma cortisol concentrations simultaneously with attenuation of amiloride and propranolol sensitivity of induced fetal lung fluid absorption. These results reiterate a central and important role of cortisol for IL-1Fr modulation of lung maturation. Previous studies have shown important roles of cortisol for lung maturation with respect to surfactant synthesis (Liggins, 1994) and have provided evidence that ENaC possesses glucocorticoid regulatory elements (Renard et al., 1995) and that glucocorticoids can stimulate lung fluid absorption (Folkesson et al., 2000). Our results demonstrate that endogenous plasma cortisol modulates ENaC expression in fetal lungs after maternal IL-1Fr pretreatment.
In this study, it was shown that maternal IL-1Fr participates in lung maturation by affecting multiple cellular and organism systems responsible for stimulation of lung fluid absorption near term. Our results indicate that ACTH and cortisol synthesis and release induced by IL-1Fr, may be useful as a potential treatment for babies delivered preterm to give them better resistance against development of severe RDS.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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 transcriptase-polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance.
Address correspondence to: Dr. Hans G. Folkesson, Professor, Department of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, 4209 State Route 44, P.O. Box 95, Rootstown, OH 44272-0095. E-mail: hgfolkes{at}neoucom.edu x100
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, P < 0.05, compared with age-matched control; 
, P < 0.05, compared with age-matched IL-1
, control; 
, oxytocin (OT)]. Oxytocin pretreatment always increased plasma epinephrine levels (P < 0.05, compared with baseline; ANOVA with Tukey's test post hoc) at each condition and gestation age. Plasma epinephrine levels were also increased at 68 days gestation compared with 61 days gestation (P < 0.05; ANOVA with Tukey's test post hoc).
and 
, confidence intervals of the data; 