Our previous work has shown that adult mice with overexpression of IL-6 and IL-13 in the lung have enhanced survival in hyperoxia associated with reduced hyperoxia-induced lung injury and cell death. We hypothesized that there are developmental differences in these responses in the adult vs. the newborn (NB) animal, and these responses have clinical relevance in the human NB. We compared the responses to 100% O2 of NB IL-6 and IL-13 transgenic mice with wild-type littermate controls by evaluating mortality, lung tissue TUNEL staining, and mRNA expression using RT-PCR. We used ELISA to measure IL-6 levels in tracheal aspirates from human neonates. Our results show that, in contrast to the cytoprotective effects in mature mice, IL-6 caused significantly increased mortality, DNA injury, caspases, cell death regulator and angiogenic factor expression in hyperoxia in the NB. Furthermore, tracheal aspirate levels of IL-6 were significantly increased in premature neonates with respiratory distress syndrome who had an adverse outcome (bronchopulmonary dysplasia/death). In contrast to the protective effects in adults, there was no survival advantage to the NB IL-13 mice in hyperoxia. These findings imply that caution should be exercised in extrapolating results from the adult to the NB.
hyperoxia-induced acute lung injury (HALI) is complex and multifactorial and characterized by severe endothelial and epithelial cell injury/death, an influx of inflammatory cells, and enhanced pulmonary permeability (8, 38, 39). Some of these effects are orchestrated by interleukins (IL), which are a group of immune mediators. We have previously demonstrated that adult transgenic (TG) mice in which IL-6 (37) and IL-13 (8) were overexpressed in a lung-targeted manner manifest enhanced survival in 100% oxygen. This survival advantage was associated with reduced HALI and significant diminution in hyperoxia-induced cell death (8, 37).
The response of the newborn (NB) to hyperoxia is significantly different from that of the adult animal (5, 10, 15, 16, 23, 36). NB animals of several species survive twice as long as adults in hyperoxia and have a significantly later onset of injury (5, 10, 15, 23, 36, 42, 43). Neonatal responses are unique, probably because injury occurs during the period of alveolar development (38). Studies of hyperoxic injury in NB animals have shown morphological changes similar to those seen in human bronchopulmonary dysplasia (BPD) (7, 12).
Thus we hypothesized that there are developmental differences in HALI and cell death responses in the NB TG mice, and these responses have clinical relevance in the human NB. To test this hypothesis, we compared the responses to 100% O2 of NB IL-6 and IL-13 TG mice with wild-type (WT) littermate controls. Our results show that, in contrast to the cytoprotective effects in mature mice, IL-6 caused significantly increased mortality, DNA injury, and caspases and cell death regulator expression in hyperoxia in the NB. In addition, there was enhanced expression of angiogenic mediators in the NB IL-6 mice lungs exposed to hyperoxia. Furthermore, tracheal aspirate (TA) levels of IL-6 were significantly increased in premature neonates with respiratory distress syndrome (RDS) who had an adverse outcome (BPD/death). In contrast to the protective effects in adults, there was no survival advantage to the NB IL-13 TG mice in hyperoxia compared with controls. These findings highlight the important developmental differences in the responses to hyperoxia in adult vs. NB animals.
IL-6 (13, 37) and IL-13 (8, 13, 37) TG mice were generated as previously reported; WT littermates were used as controls. In brief, IL-6 TG mice were produced on a CBA × C57BL/6 background. In these mice, the Clara cell 10-kDa protein promoter (CC10) was used to overexpress IL-6 in a lung-specific fashion (in Clara cells, and, to a lesser degree, alveolar epithelial cells). In a similar fashion, the CC10 promoter was used to target the expression of IL-13 to the lung/airway. In these TG animals, IL-6 and IL-13 are constitutively expressed with levels of IL-6 and IL-13 being detectable in the bronchoalveolar lavage (BAL) fluid. These mice were then backcrossed for 10 generations on the C57BL/6 background and then used for the experiments.
All animal work was approved by the Institutional Animal Care and Use Committee at the Yale University School of Medicine.
For the exposure to hyperoxia, NB mice (along with their mothers) were placed in cages in an airtight Plexiglas chamber (55 × 40 × 50 cm) as described previously (8, 37). Exposure to oxygen was initiated on day 1 of life. Two lactating dams were used. They were alternated in hyperoxia and room air every 24 h. The litter size was kept limited to 12 pups to control for the effects of litter size on nutrition and growth. Throughout the experiment, they were given free access to food and water. Oxygen levels were constantly monitored by an oxygen sensor that was connected to a relay switch incorporated into the oxygen supply circuit. The inside of the chamber was kept at atmospheric pressure, and mice were exposed to a 12-h light-dark cycle. The survival experiments were done for 6 days. Separate experiments were conducted subsequently with exposure to hyperoxia in the NB IL-6 TG mice for 48 h followed by death and harvesting of lungs.
Mice were anesthetized, and the lungs were rapidly removed and frozen in liquid nitrogen. Lungs were fixed overnight in 10% buffered formalin. From separate experiments, RNA was isolated from frozen lungs using TRIzol reagent (Life Technologies, Grand Island, NY) according to the manufacturer's instructions.
After being washed in fresh PBS, fixed lung tissues were dehydrated, cleared, and embedded in paraffin by routine methods. Sections (5 μm) were collected on Superfrost Plus positively charged microscope slides (Fisher Scientific, Houston, TX), deparaffinized, and stained with hematoxylin and eosin.
TdT-mediated dUTP nick end labeling staining.
End labeling of exposed 3′-OH ends of DNA fragments was undertaken with the TdT-mediated dUTP nick end labeling (TUNEL) in situ cell death detection kit AP (Roche Diagnostics) as described by the manufacturer. The TUNEL index was calculated by randomly selecting 3 high-power fields in each slide, counting 200 cells in each area, and expressing the number of TUNEL-positive cells as a percentage.
Analysis of mRNA.
RNA samples were DNase treated and subjected to semiquantitative RT-PCR. The primers used for semiquantitative RT-PCR were: mouse angiopoietin-1 (Ang-1), 5′-AGGCTTGGTTTCTCGTCAGA-3′, 5′-TCTGCACAGTCTCGAAATGG-3′, mouse Ang-2, 5′-GAACCAGACAGCAGCACAAA-3′, 5′-AGTTGGGGAAGGTCAGTGTG-3′, Ang-4, 5′-CCAGCTTAACAGCCTCCAAG-3′, 5′-CTCTGCACAGTCCTGGAACA-3′, caspase-3, 5′-AGTCTGACTGGAAAGCCGAA-3′, 5′-AAATTCTAGCTTGTGCGCGT-3′, caspase-6, 5′-TTCAGACGTTGACTGGCTTG-3′, 5′-TTTCTGTTCACCAGCGTGAG-3′, caspase-8, 5′-GCTGGAAGATGACTTGAGCC-3′, 5′-CGTTCCATAGACGACACCCT-3′, caspase-9, 5′-CCTGCTTAGAGGACACAGGC-3′, 5′-TGGTCTGAGAACCTCTGGCT-3′, Fas, 5′-ATGCACACTCTGCGATGAAG-3′, 5′-TTCAGGGTCATCCTGTCTCC-3′, Fas-L, 5′-CATCACAACCACTCCCACTG-3′, 5′-GTTCTGCCAGTTCCTTCTGC-3′, Bax, 5′-CTGCAGAGGATGATTGCTGA-3′, 5′-GAGGAAGTCCAGTGTCCAGC-3′, Bak, 5′-CCAACATTGCATGGTGCTAC-3′, 5′-AGGAGTGTTGGGAACACAGG-3′, Bim, 5′-GCCAAGCAACCTTCTGATGT-3′, 5′-CATTTGCAAACACCCTCCTT-3′, Bid, 5′-TCCACAACATTGCCAGACTA-3′, 5′-CACTCAAGCTGAACGCAGAG-3′, mouse VEGF, 5′-CAGGCTGCTGTAACGATGAA-3′, 5′-AATGCTTTCTCCGCTCTGAA-3′, β-actin, 5′-GTGGGCCGCTCTAGGCACCA-3′, 5′-TGGCCTTAGGGTTCAGGGGG-3′.
Immunohistochemical staining for Ang-2 and total caspase-3 protein.
Paraffin-embedded tissues were cut, exposed to three changes of xylene, rehydrated in a series of graded alcohols, and rinsed; endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide for 5 min and blocked with avidin and biotin (Biotin Blocking kit, Dako). The slides were incubated for 1 h at room temperature with either a 1:300 dilution of a murine IgG2 MAb that recognized Ang-2 or total caspase-3 or a 1:300 dilution of a nonspecific anti-mouse IgG2 control antibody (R&D Systems). To prevent nonspecific binding to mouse tissue, the antibodies were previously biotinylated and blocked with nonspecific mouse serum using a commercially available kit (Animal Research kit, Dako). After incubation with antibody, the slides were incubated with a streptavidin-peroxidase enzyme conjugate (Dako) for 15 min followed by 3,3′-diaminobenzidine-tetrahydrochloride (Dako) for 7 min. The slides were counterstained with hematoxylin, dehydrated in a series of graded alcohols, and cleared with xylene.
Lung IL-6 and IL-13 levels.
Human IL-6 levels in the murine lung lysates/human TA and murine IL-13 BAL samples were measured using ELISA (R&D Systems) as per the manufacturer's instructions.
Collection of TA samples and patient characteristics.
All human studies were approved by the Human Investigational Committee at the Yale University School of Medicine.
The TA samples were collected from neonates admitted to the Yale-New Haven Children's Hospital Newborn Special Care Unit. TA samples were only collected if the infant had a clinically indicated endotracheal tube in place. TA samples were obtained by instilling 0.5 ml of normal saline into the infant's endotracheal tube and suctioning the residue with a 5F suction catheter after two or three ventilator breaths. The suction catheter was passed to a standardized length of 0.5–1 cm beyond the tip of the endotracheal tube. This method of collection is used widely in neonates to collect TA samples (19, 29, 34), and it is well tolerated by even the most critically ill neonates. The procedure was repeated four times, and replicates were pooled. The suction catheter was flushed with 0.5 ml of normal saline after each suctioning episode to collect the residual sample in the catheter. The samples were immediately transported to the laboratory on ice and processed within a half an hour in the laboratory. The samples were centrifuged at 4°C for 10 min at 300 g. The supernatant was collected, divided into aliquots, and stored at −70°C for future use. Total protein concentration was measured in each TA sample by the Bradford assay (Bio-Rad, Richmond, CA) to correct for dilution during the lavage procedure. The level of IL-6 was expressed as pg/mg of protein.
There were no statistically significant differences between the no BPD (n = 4) vs. BPD/death (n = 5; 2 deaths) groups in gestational age (means ± SE, 26.3 ± 1.1 vs. 26.3 ± 0.6 wk) or birth weight (868 ± 13 vs. 783 ± 53 g). All infants had RDS and were intubated, administered at least one dose of natural surfactant, and ventilated for treatment, as per standard nursery guidelines. At the time of TA sampling, the babies who developed BPD or died were receiving significantly higher FiO2 compared with those who did not develop BPD (0.62 ± 0.06 vs. 0.37 ± 0.08, P = 0.03). TA samples were collected in the first 12 h of life. BPD was defined as the need for supplemental oxygen at 36 wk postmenstrual age, along with characteristic radiographic features (33).
Values are expressed as means ± SE. As appropriate, groups were compared with the Student's two-tailed unpaired t-test, Mann-Whitney test, or the log-rank test, using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA). A P < 0.05 was considered statistically significant.
Survival of NB IL-6 TG mice in hyperoxia.
We compared the effects of 100% O2 exposure in NB IL-6 TG mice with the WT littermate controls. NB IL-6 TG mice have normal survival, similar to WT mice, in room air (RA). As shown in Fig. 1, exposure to 100% O2 resulted in the death of all IL-6 TG animals by postnatal (PN) day (d) 5, with 23% of the WT littermates being alive at the end of PN d6 (P < 0.01). These studies demonstrate that presence of excess IL-6 in the developing PN lung plays a critical role in the pathogenesis of HALI-induced mortality.
Mechanism of HALI in NB IL-6 TG mice.
Since oxidant-induced DNA injury and cell death are felt to play important roles in the pathogenesis of HALI (20), the role of IL-6 in these responses was evaluated. In accord with this conceptualization, 100% O2 caused DNA injury and cell death that was manifest as enhanced pulmonary tissue TUNEL staining (Fig. 2, A and B). In RA, NB IL-6 TG lungs had significantly increased TUNEL-positive cells compared with NB WT. IL-6 also played a critical role in the pathogenesis of TUNEL staining in hyperoxia, as it was significantly increased in comparisons of hyperoxia-challenged WT littermate mice (Fig. 2, A and B). Thus presence of excess IL-6 in the developing PN lung is a critical mediator of hyperoxia-induced DNA injury and cell death.
Effect on angiogenic factors and caspases in HALI in NB IL-6 TG mice.
We evaluated the role of IL-6 in the regulation of angiogenic mediators as well as critical components of the extrinsic and intrinsic apoptosis/cell death pathways. mRNA encoding Ang-1, -2, and -4 as well as VEGF were not readily apparent in RA in the WT mice but increased significantly when the animals were exposed to 100% O2 for 48 h (Figs. 3 and 4). Interestingly, the presence of IL-6 resulted in an impressive increase in the mRNA encoding Ang-1 and -4 and VEGF in RA (Figs. 3 and 4). Hyperoxia exposure led to increased Ang-2 expression, similar to that noted in the WT hyperoxia-challenged mice pups, as was also the case with VEGF. Ang-1 expression was similarly decreased in the IL-6 TG and WT hyperoxia-exposed animals. However, Ang-4 showed a marked upregulation in the IL-6 TG hyperoxia-exposed mice compared with the similarly exposed WT controls.
mRNA encoding caspases-3, -6, -8, and -9 were not readily apparent in RA in the WT mice but increased significantly when the animals were exposed to 100% O2 for 48 h (Figs. 3 and 4). Interestingly, the presence of IL-6 resulted in an impressive increase in the mRNA encoding caspases-3, -6, -8, and -9 in RA (Figs. 3 and 4). Hyperoxia exposure led to significantly increased caspases-3, -8, and -9 expression, similar to that noted in the WT hyperoxia-challenged mice pups. In addition, caspases-3, -6, -8, and -9 showed a significant upregulation in the IL-6 TG hyperoxia-exposed mice compared with the similarly exposed WT controls.
Thus hyperoxia stimulates and activates angiogenic mediators as well as critical initiator and effector caspases via a mechanism that is, at least partially, IL-6 dependent.
Effect on cell death regulators in HALI in NB IL-6 TG mice.
We next evaluated the role of IL-6 in the regulation of the extrinsic and intrinsic cell death pathways. mRNA encoding BH3-only proteins Bid, Bim, Bak, and Bax as well as Fas/Fas-L were not readily apparent in lungs from WT mice breathing RA but increased significantly when the animals were exposed to 100% O2 for 48 h (Figs. 5 and 6), except for Bid and Fas-L. Importantly, the NB IL-6 TG mice lungs had increased expression of the above cell death regulators in RA compared with WT mice lungs (Figs. 5 and 6), except Fas-L. Interestingly, hyperoxia exposure led to further increased expression of Bim, Fas, and Fas-L. Importantly, the hyperoxia-challenged IL-6 TG mice lungs had increased expression of Bim, Fas, and Fas-L mRNA compared with the similarly exposed WT controls.
Thus hyperoxia stimulates and activates the cell death regulators via mechanisms that are, at least partially, IL-6 dependent.
Effect of hyperoxia on Ang-2 and total caspase-3 protein.
Immunohistochemical (IHC) analysis demonstrated increased levels of Ang-2 protein in airway and alveolar epithelial cells after 48 h of 100% O2 exposure in NB WT mice lungs (Fig. 7A). In accord with the mRNA data, NB IL-6 TG mice lungs had more prominent Ang-2 staining in RA and hyperoxia compared with controls (Fig. 7A). Similarly, NB IL-6 TG mice lungs had more prominent total caspase-3 staining in RA and hyperoxia compared with controls (Fig. 7B). These evaluations were Ang-2 and total caspase-3 specific because IHC staining was not noted in the absence of primary antibody and was competed away by peptide excess (data not shown). Thus IL-6 is a potent inducer of Ang-2 and total caspase-3 protein in NB pulmonary airway and alveolar epithelial cells, which are further enhanced in the presence of hyperoxia.
Survival of NB IL-13 TG mice in hyperoxia.
We compared the effects of 100% O2 exposure in NB IL-13 TG mice with the WT littermate controls. NB IL-13 TG mice have normal survival, similar to WT mice, in RA. As shown in Fig. 8, exposure to 100% O2 resulted in no significant difference in survival of NB IL-13 TG animals and WT littermate controls. These studies demonstrate that presence of excess IL-13 in the developing PN lung does not appear to play a critical role in the pathogenesis of HALI-induced mortality.
Effect on IL-6 and IL-13 production in NB IL-6 and IL-13 TG mice lungs.
To quantify the levels of IL-6 protein in the NB mice lungs, we compared the levels of transgenic IL-6 in lung lysates from WT and TG mice. Levels of IL-6 were significantly increased in the NB IL-6 TG mice lungs (Fig. 9A). In a similar fashion, BAL levels of IL-13 were similarly increased in NB IL-13 TG mice (Fig. 9B). Thus the above results are an effect of the increased IL-6 and IL-13 levels expressed in the developing PN lung.
IL-6 levels in human neonates with RDS.
The studies noted above demonstrate that IL-6 is an important mediator of HALI that regulates oxidant-induced pulmonary cell death. To evaluate the human disease relevance of these findings, studies were undertaken to determine if lung IL-6 levels had a role in neonates. IL-6 was readily apparent in TA from premature babies being ventilated for RDS. A subset of neonatal RDS patients develop HALI and pulmonary edema, subsequently leading to BPD and even death. Interestingly, the levels of TA IL-6 measured in the first 12 h of life were significantly higher in babies that subsequently developed BPD or died (P < 0.02) (Fig. 10). Subsequent measurements of IL-6 were not significantly different (data not shown). These studies demonstrate that IL-6 levels are elevated early in conditions characterized by exposure to high concentrations of oxygen and acute lung injury in neonatal patients.
The most prominent source of IL-6 appears to be stimulated monocytes/macrophages, fibroblasts, and epithelial and endothelial cells (9). There is evidence that IL-6 acts as an autocrine, paracrine, and exocrine inflammatory hormone (9). Interestingly, IL-6 has also been shown to have anti-inflammatory effects by inhibiting neutrophil influx in a model of acute lung inflammation (35). It is obvious that the timing and intensity of the effects of IL-6 are carefully controlled to elicit the appropriate effect needed in the inflammatory response (9).
Adult mice exposed to hyperoxia for 3 days had increased lung mRNA for IL-6 compared with lungs of mice in room air (21, 23). In different strains of adult mice exposed to hyperoxia, death was accompanied by an increase in IL-6 mRNA (22). Using the tetracycline-inducible, lung-specific TG system, we have reported that adult mice overexpressing IL-6 in the lung had significantly increased survival in hyperoxia (37). The adult IL-6 TG mice had a marked diminution in TUNEL-positive cells in the lungs of adult IL-6 TG mice exposed to hyperoxia (37). Furthermore, higher levels of Bcl-2, but not Bax, mRNA, and protein, were noted in lung tissue compared with WT littermate controls (37).
Neonatal mice exposed to hyperoxia had an eightfold increase in lung mRNA for IL-6 after 7 days of exposure in contrast to the adult mice showing an increase after only 3 days in hyperoxia (23). NB rats exposed to 100% O2 (for 9 days) had significant pulmonary edema and increased cellularity on days 1 and 3, which resolved by days 6 and 9 (1). IL-6, primarily of non-macrophage origin, was detected in the BAL on days 6 and 9, but not earlier (1). NB rats exposed to 48 h of hyperoxia (95% O2) had increased levels of IL-6 mRNA (28).
Surprisingly, despite this variability in the response of IL-6 to hyperoxia in adult and NB animals, this phenomenon has not been studied in detail. We report, for the first time, that in contrast to the cytoprotective effects in adult mice, IL-6 caused significantly increased mortality, DNA injury, caspases, cell death regulator and angiogenic factor expression in hyperoxia in the NB.
Acute pulmonary injury secondary to hyperoxia is complex and multifactorial (38). The acute lung injury caused by hyperoxia is characterized by severe endothelial damage, alveolar epithelial injury, and increased pulmonary permeability (8, 39). These alterations can result in impressive increases in alveolar capillary permeability and pulmonary edema.
Our data show an impressive increase in Ang-2 and -4 and VEGF with a decrease in Ang-1 in the hyperoxia-exposed NB IL-6 TG mice lung. Since an excess of Ang-2 can enhance the vascular effects of VEGF (30) while inhibiting the action of Ang-1 (17), it can be envisioned that a simultaneous increase in Ang-2 with a decrease in Ang-1, in the presence of increased VEGF, can result in pulmonary edema and contribute to HALI in the NB IL-6 TG pups. This is in accord with recent data implicating increased Ang-2 contributing to vascular leak in acute lung injury in a sepsis model (32) and in HALI (3). Interestingly, our data also show an impressive increase in Ang-4, which has also been recently implicated in a sepsis model of acute lung injury (25). Recent work from our laboratory (3) has delineated the role of Ang-2 in hyperoxia-induced oxidant injury, cell death, inflammation, permeability alterations, and mortality. Together, our data would suggest that the balance of angiogenic factors favors enhancement of lung edema that could contribute, at least in part, to the increased mortality in the NB TG IL-6 mice.
Furthermore, in HALI there is an influx of inflammatory cells that increases lung damage (38). This inflammatory cell influx is orchestrated and amplified by chemotactic factors, including IL (14). Since reactive oxygen species (ROS) are said to play a major role in HALI and cell death, one possibility would be to see if the exposure to similar oxygen concentrations is leading to enhanced cell injury/death in the NB IL-6 TG and WT mice, which can be detected by TUNEL staining. In accord with our previous adult TG mice data (37), studies using cell cultures (27, 40) or organotypic lung slices from adult IL-6-deficient mice (27) have shown that IL-6 protected against damage by ROS on exposure to hydrogen peroxide. In contrast, we have shown that in NB IL-6 TG, compared with WT mice, there is significantly enhanced positive TUNEL staining (Fig. 2, A and B). Pulmonary parenchymal as well as inflammatory cells were noted to be TUNEL positive (Fig. 2A). These data are in accord with our earlier observation that exposure to hyperoxia in a developmentally appropriate cell culture model led to enhanced IL-6 release and cell death (18). Further research is required to understand the mechanism of cell death in the individual cell types of the lung. Together, this would suggest that presence of excess IL-6 results in increased lung cell death in hyperoxia in the NB, which can explain, at least in part, the increased mortality of the NB IL-6 TG mice.
Activation of key caspases and components of the extrinsic/death receptor and intrinsic/mitochondrial cell death pathways appear to underlie the molecular mechanisms of HALI and cell death (3). The mitochondrial-dependent pathway is probably more relevant in this scenario with other key regulators including the many members of the Bcl-2 gene family, which can be divided into three groups: anti-apoptotic Bcl-2 and Bcl-xL, proapoptotic Bax-type proteins, and proapoptotic BH3 domain-only family members (20, 31).
Thus to further understand the mechanism of cell death in the hyperoxia-exposed NB IL-6 TG mice, we evaluated the activation of key caspases and cell death pathway regulators at the mRNA level. We found that there was significantly increased activation of the key caspases and cell death regulators in the hyperoxia-exposed NB IL-6 TG mice lungs compared with WT mice (Figs. 3–6). Interestingly, the Fas/Fas-L mRNA were most prominently increased in the hyperoxia-exposed NB IL-6 TG mice lungs. The Fas/Fas-L system has recently been implicated to be a critical mediator in hyperoxia-induced cell death in a developmentally appropriate cell culture model (11). Thus increased cell death in NB IL-6 TG mice exposed to hyperoxia is accompanied by increased expression of key caspases and regulators that are critical components of the cell death pathways.
The human disease relevance of our findings was exemplified by the significantly increased IL-6 TA levels in premature neonates who had an adverse outcome (BPD/death). Our TA data is in accord with two previous reports (6, 24), although in contrast to another study (26).
The IL-13 gene is located on human chromosome 5q31 or mouse chromosome 11; the human protein product is a 17-kDa glycoprotein cloned from activated T cells (41). Although IL-13 is predominantly produced by Th2-polarized CD4+ T cells, it is also produced by a variety of cell types including Th1 CD4+ T cells and CD8+ T cells (41). Similar to IL-6, IL-13 also has a variety of pro- as well as anti-inflammatory effects (8).
Work from our laboratory has shown that TG adult mice that overexpress IL-13 had significantly increased survival in hyperoxia compared with the WT mice (8). In the present study, in sharp contrast to the findings in mature mice, presence of excess IL-13 in the NB TG mice did not offer any survival advantage in hyperoxia.
To summarize, IL-6 caused significantly increased mortality, DNA injury, caspase and cell death regulator expression in hyperoxia in the NB. In addition, there was enhanced expression of angiogenic factors in the NB IL-6 mice lungs exposed to hyperoxia. Furthermore, TA levels of IL-6 were significantly increased in premature neonates with an adverse outcome (BPD/death). In contrast to the protective effects in adults, there was no survival advantage to the NB IL-13 TG mice in hyperoxia.
In conclusion, these studies highlight the significant developmental differences that exist in the pulmonary response to hyperoxia and imply caution in extrapolating data from adult studies to the neonate (2, 4).
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-74195 (V. Bhandari), HL-64642, HL-61904, and HL-56389 (J. A. Elias).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society