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The Journal of Immunology, 2007, 178: 4993-5000.
Copyright © 2007 by The American Association of Immunologists, Inc.

Increased Hyperoxia-Induced Mortality and Acute Lung Injury in IL-13 Null Mice1

Vineet Bhandari*, Rayman Choo-Wing*, Robert J. Homer{ddagger} and Jack A. Elias2,{dagger}

* Division of Perinatal Medicine, Yale University School of Medicine, Department of Pediatrics, Children’s Hospital, New Haven, CT 06520; {dagger} Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, Department of Internal Medicine, New Haven, CT 06520; and {ddagger} Yale University School of Medicine, Department of Pathology, New Haven, CT 06520


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-13 is a critical effector at sites of Th2 inflammation and remodeling. As a result, anti-IL-13-based therapies are being actively developed to treat a variety of diseases and disorders. However, the beneficial effects of endogenous IL-13 in the normal and diseased lung have not been adequately defined. We hypothesized that endogenous IL-13 is an important regulator of oxidant-induced lung injury and inflammation. To test this hypothesis, we compared the effects of 100% O2 in mice with wild-type and null IL-13 loci. In this study, we demonstrate that hyperoxia significantly augments the expression of the components of the IL-13R, IL-13R{alpha}1, and IL-4R{alpha}. We also demonstrate that, in the absence of IL-13, hyperoxia-induced tissue inflammation is decreased. In contrast, in the IL-13 null mice, DNA injury, cell death, caspase expression, and activation and mortality are augmented. Interestingly, the levels of the cytoprotective cytokines vascular endothelial cell growth factor, IL-6, and IL-11 were decreased in the bronchoalveolar lavage fluid. These studies demonstrate that the expression of the IL-13R is augmented and that the endogenous IL-13-IL-13R pathway contributes to the induction of inflammation and the inhibition of injury in hyperoxic acute lung injury.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Interleukin-13 is a 12-kDa cytokine product of a gene on chromosome 5 at q31 that is produced in large quantities by Th2 cells and lesser quantities by Th1 cells, macrophages, mast cells, and eosinophils (1). Early studies of IL-13 focused on its IL-4-like properties including its ability to stimulate IgE production and endothelial VCAM expression (2, 3). However, it has subsequently become clear that IL-13 and IL-4 have only partially overlapping effector profiles, and that IL-13 may be the major effector at sites of Th2-induced inflammation and tissue remodeling (4, 5, 6). In keeping with this concept, IL-13 is felt to be a critical mediator of these responses in diseases as diverse as asthma, hepatic cirrhosis, and idiopathic pulmonary fibrosis (IPF)3 (5, 6), and significant effort has been directed at the generation and characterization of anti-IL-13-based therapies for these and other disorders. Surprisingly, the toxicities that might arise from these interventions are poorly understood. This is due, in great extent, to our inadequate understanding of the beneficial roles of endogenous IL-13 in healthy and diseased tissues.

Oxidant injury is a major cause of morbidity and mortality. This effect is easily appreciated in the lung where reactive oxidant species (ROS) contribute to the pathogenesis of diseases as diverse as asthma, chronic obstructive lung disease, IPF, and acute lung injury syndromes such as that caused by prolonged hyperoxia (hyperoxic acute lung injury; HALI) (7, 8, 9, 10). In the normal and injured lung, the severity and consequences of oxidant injury are modulated by generalized and tissue-specific protective mechanisms. Previous studies from our laboratory and others demonstrated that exogenously administered cytokines, including IL-13, confer tissue cytoprotection in HALI (11, 12, 13, 14). Surprisingly, the role(s) that endogenous IL-13 plays in the control of these oxidant-induced responses has not been adequately defined.

We hypothesized that endogenous IL-13 is an important regulator of tissue oxidant injury. To test this hypothesis, studies were undertaken to determine whether IL-13 or its receptor are altered in hyperoxia, and the tissue responses that were induced by hyperoxia in mice with wild-type (WT) and null (–/–) IL-13 loci were compared. These studies demonstrate that, although IL-13 was not induced by 100% O2, hyperoxia did stimulate the expression of the components of the IL-13R (IL-13R{alpha}1 and IL-4R{alpha}). They also demonstrate that, in the absence of IL-13, hyperoxia-induced DNA injury, cell death, caspase expression and activation, and mortality were increased, and the levels of the cytoprotective cytokines VEGF, IL-6, and IL-11 in BAL fluids were decreased.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

IL-13 null (IL-13–/–) mice (C57BL/6 strain) were a gift from A. Mackenzie (Cambridge, U.K.). Unless otherwise indicated, WT (IL-13+/+) and heterozygous (IL-13+/–) littermates were used as controls. OVA sensitization and challenge of WT mice were done as described previously (15). Mice were sacrificed 48 h after aerosol exposure and lungs removed for RNA analysis. All animal work was approved by the Institutional Animal Care and Use Committee at the Yale University School of Medicine.

Bronchoalveolar lavage (BAL)

Mice were euthanized, the trachea was isolated by blunt dissection, and a small-caliber tube was inserted into the airway and secured. Two volumes of 1 ml of PBS with 0.1% BSA were instilled and gently aspirated and pooled (BAL fluid). Samples were then centrifuged at 1250 x g for 5 min to recover cells, and the supernatants were collected and stored at –70°C for further analysis. Cell pellets were resuspended in PBS, and total cell counts were determined using a hemocytometer. Aliquots were cytospun onto microscope slides and stained for cellular differentials. VEGF, IL-6, IL-11, and IL-13 levels were measured using ELISA kits (R&D Systems), as per the manufacturer’s instructions.

Histology

Tissues were fixed overnight in 10% buffered formalin. After washing in fresh PBS, fixed 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), deparaffinized, and stained with H&E.

Analysis of mRNA

Mice were anesthetized, and the lungs were rapidly removed and frozen on liquid nitrogen. RNA was isolated from frozen lungs using TRIzol Reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. RNA samples were then DNase treated and subjected to semiquantitative RT-PCR. The following primers were used for semiquantitative RT-PCR: 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'; Bcl-2, 5'-CTGGCATCTTCTCCTTCCAG-3', 5'-GACGGTAGCGACGAGAGAAG-3'; Bcl-xL, 5'-TTCGGGATGGAGTAAACTGG-3', 5'-TGGATCCAAGGCTCTAGGTG-3'; protein kinase C (PKC)-{delta}, 5'-TACCGGGCTACGTTTTATGC-3', 5'-CCAGGAGGGACCAGTTGATA-3'; A1, 5'-CAGGGAAGATGGCTGAGTCT-3', 5'-TTCTGCCGTATCCATTCTCC-3'; BID, 5'-TCCACAACATTGCCAGACTA-3', 5'-CACTCAAGCTGAACGCAGAG-3'; BAX, 5'-CTGCAGAGGATGATTGCTGA-3', 5'-GAGGAAGTCCAGTGTCCAGC-3'; BAK, 5'-CCAACATTGCATGGTGCTAC-3', 5'-AGGAGTGTTGGGAACACAGG-3'; and beta-actin, 5'-GTGGGCCGCTCTAGGCACCA-3', 5'-TGGCCT TAGGGTTCAGGGGG-3'.

Real-time RT-PCR was done as described previously (16).

Oxygen exposure

Four- to 6-wk-old mice were placed in cages in an airtight Plexiglas chamber (55 x 40 x 50 cm), as described previously (11, 12, 13). Throughout the experiment, they were given free access to food and water. Oxygen levels were constantly monitored by an oxygen sensor, which 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.

TUNEL staining

End labeling of exposed 3'-OH ends of DNA fragments was undertaken with the TUNEL in situ cell death detection kit AP (Roche Diagnostics) as described by the manufacturer. We used the NBT/5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt (BCIP) blue stain and a nuclear red counterstain. The TUNEL index was calculated by randomly selecting multiple high-powered fields on each slide, counting 200 cells in each area and expressing the number of TUNEL-positive cells as a percentage.

Measurement of activity of caspase-3

Lung homogenates were prepared and commercial kits were used to assess the activity of caspase-3 (Promega) according to the manufacturer’s instructions.

OVA sensitization and challenge

Sensitization and challenge with the aeroallergen OVA was undertaken as described by our laboratory (15).

Statistical analyses

Values are expressed as means ± SEM. Groups were compared with the Student’s two-tailed unpaired t test, one-way ANOVA analysis, or the log-rank test, using GraphPad Prism 3.0 (GraphPad), as appropriate. A value of p ≤ 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effects of hyperoxia on IL-13 and its receptors

To begin to understand the importance of IL-13 in oxidant-induced injury states, we compared the expression of IL-13 and its receptors in mice breathing room air and 100% O2 (Fig. 1 and data not shown). Samples from OVA-sensitized and challenged mice were used as positive controls for the expression of IL-13 and IL-13 R{alpha}2 in these experiments (Fig. 1B). These studies demonstrate that hyperoxia did not alter the expression (Fig. 1A) or production of IL-13. In contrast, hyperoxia caused impressive alterations in the expression of IL-13R{alpha}1 and IL-4R{alpha} that make up the multimeric IL-13R complex. These alterations were most prominent after 72 h of exposure to 100% O2, at which time hyperoxia caused a 69 ± 12 and 37 ± 10% increase in the levels of mRNA encoding IL-13R{alpha}1 and IL-4R{alpha}, respectively (p < 0.001 and p < 0.03, respectively). These studies demonstrate that although hyperoxia does not alter the production of IL-13, it does enhance the expression of the IL-13R in the murine lung.


Figure 1
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  		FIGURE 1. Effect of hyperoxia on the expression of IL-13 and its receptors. Mice were exposed to room air or 100% O2 for 72 h. Pulmonary mRNA for IL-13, IL-13R{alpha}1, IL-13R{alpha}2, and IL-4{alpha} were assessed using RT-PCR (A), and those showing an increase were confirmed by real-time RT-PCR (C and D). B, The induction of IL-13 and IL-13R{alpha}2 mRNA after OVA sensitization and challenge. beta-Actin is used as a loading control. The noted values represent assessments in a minimum of five animals. **, p < 0.01; #, p < 0.03.

 
Role of IL-13 in HALI

To address the role of IL-13 in the pathogenesis of HALI, we compared the survival of IL-13+/+ and IL-13–/– mice in 100% oxygen. In accord with studies from our laboratory and others, WT mice died after 3–6 days of exposure to 100% O2 (11, 12, 13, 14, 16). In this setting, 50% of WT mice were dead after 4.5 days and 100% had died by day 5.5 (Fig. 2A). In contrast, this response was accelerated in mice that were deficient in IL-13. In these experiments, 50% of the IL-13–/– mice were dead after 4 days and 100% were dead after 4.5 days of exposure to 100% O2 (Fig. 2A). In the absence of IL-13, hyperoxia-induced inflammation was also enhanced, but less than that seen in WT mice. This response was readily seen in comparisons of IL-13–/– and WT mice lungs exposed to 100% O2 for 72 h (Fig. 2B). These studies demonstrate that endogenous IL-13 plays a protective role in HALI.


Figure 2
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  		FIGURE 2. A, Survival in hyperoxia. IL-13+/+ and IL-13–/– mice were exposed to 100% O2 and survival was assessed. The noted values represent assessments in a minimum of nine animals in each group. **, p < 0.01. Histology (H&E stain) of the lung of the IL-13+/+ and IL-13–/– mice exposed to room air or 100% O2 for 72 h is shown in B. Photographs in the upper panels taken at x10, lower panels at x40.

 
Role of IL-13 in hyperoxia-induced inflammation

Studies were next undertaken to define the role(s) of IL-13 in the hyperoxia-induced inflammatory response. In WT mice, 100% oxygen caused significant inflammatory cell accumulation with enhanced BAL macrophage, lymphocyte, and neutrophil recovery (Fig. 3) after 72 h of exposure. IL-13 and the IL-13R played a significant role in these responses because, in the absence of IL-13, BAL total cell, macrophage, lymphocyte and neutrophil recovery were significantly decreased (Fig. 3). Thus, the IL-13 pathway plays an important role in the pathogenesis of hyperoxia-induced pulmonary inflammation.


Figure 3
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  		FIGURE 3. Effect on hyperoxia on BAL cell cellularity. IL-13+/+, IL-13+/–, and IL-13–/– mice were exposed to 100% O2 for 24, 48, and 72 h. BAL total cell recovery (A) and differential cell recovery (B, macrophages; C, lymphocytes; D, neutrophils) were assessed. The noted values represent assessments in a minimum of five animals. *, p < 0.0001; **, p ≤ 0.01; #, p ≤ 0.03.

 
Role of endogenous IL-13 in hyperoxia-induced cell death

Because oxidant-induced DNA injury and cell death are felt to play important roles in the pathogenesis of HALI (12, 16, 17), the role(s) of IL-13 in these responses was evaluated. In accord with this conceptualization, 100% O2 caused DNA injury and cell death that manifest as enhanced pulmonary tissue TUNEL staining in WT mice (Fig. 4A) after 72 h of exposure. IL-13 appeared to be a critical regulator of this response because the levels of TUNEL staining was significantly increased in comparisons of hyperoxia-challenged IL-13–/– and IL-13+/+ animals (Fig. 4B). These studies demonstrate that endogenous IL-13 is a critical inhibitor of hyperoxia-induced DNA injury and cell death.


Figure 4
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  		FIGURE 4. Role of IL-13 in hyperoxia-induced DNA injury and cell death. IL-13+/+ and IL-13–/– mice were exposed to room air or 100% O2 for 24, 48, or 72 h, and TUNEL evaluations were undertaken. A, A NBT/BCIP blue stain and a nuclear red counterstain of lungs from animals exposed to room air or 100% O2 for 72 h. The percentage of TUNEL (+) cells is illustrated in B. The noted values represent assessments in a minimum of four animals. *, p < 0.0001; #, p ≤ 0.02.

 
Endogenous IL-13 modulation of apoptosis regulators and caspases

To gain insights into the mechanisms that IL-13 might use to regulate tissue cell death responses, we compared the expression of apoptosis regulators and caspases and the levels of caspase activity in lungs from IL-13+/+ and IL-13–/– mice in room air and 100% oxygen. In the WT mice in room air, mRNA encoding A1, Bax, Bak, Fas, and PKC{delta} were readily apparent, whereas the levels of mRNA encoding Bcl-2, Bcl-xL, Bid, and Fas ligand (FasL) were below the limits of detection of our assays (Fig. 5A). Similarly, the levels of mRNA encoding caspase-3, -6, -8, and -9 were below the limits of detection of our assay and only modest levels of caspase-3 bioactivity could be appreciated (Fig. 5). Interestingly, in IL-13–/– mice in room air, the levels of mRNA encoding Bak and FasL were increased, whereas the expression of the other regulators and the levels of caspase-3 bioactivity were not altered (Fig. 5). In contrast, hyperoxia increased the levels of mRNA encoding A1, Bcl-2, Bcl-xL, Bax, Bak, FasL, and PKC{delta} (Fig. 5A). The levels of mRNA encoding caspase-3, -6, -8, and -9, and the levels of caspase-3 bioactivity were similarly increased (Fig. 5). In the absence of IL-13, the induction of A1, Bcl-2, Bcl-xL, Bax, and PKC{delta} by hyperoxia were not altered (Fig. 5). However, the levels of mRNA encoding Bid, Fas, FasL, and caspase-3, -6, -8, and -9, and the levels of caspase-3 bioactivity were increased compared with IL-13+/+ mice experiencing a similar hyperoxic challenge (Fig. 5). These studies demonstrate that, at baseline, the IL-13 pathway inhibits the expression of Bak and FasL in the murine lung. They also demonstrate that, in hyperoxia, endogenous IL-13 inhibits the expression of Bid, Fas, FasL, and caspase-3, -6, -8, and -9, and inhibits caspase-3 bioactivity.


Figure 5
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  		FIGURE 5. Role of IL-13 in hyperoxia-induced alterations in apoptosis regulators and caspases. A, The levels of mRNA encoding A1, Bcl-2, Bcl-xL, Bax, Bak, Bid, Fas, Fas-L, PKC-{delta}, and caspase-3, -6, -8, and -9 in IL-13+/+ and IL-13–/– mice exposed to room air or 100% O2 for 72 h are shown. This figure is illustrative of a minimum of four experiments. B, The levels of caspase-3 bioactivity were assessed in IL-13+/+, IL-13+/–, and IL-13–/– mice exposed to room air or 100% O2 for 72 h. The values are the mean ± SEM of evaluations in a minimum of four mice. **, p < 0.01; ##, p ≤ 0.05.

 
Endogenous IL-13 modulation of IL-13Rs

To determine whether IL-13 regulates the expression of its receptors in hyperoxia, we compared the expression of IL-13R{alpha}1 and IL-4R{alpha} in lungs from IL-13+/+ and IL-13–/– mice in room air and 100% oxygen. As shown in Fig. 6, hyperoxia caused a marked increase in the mRNA of IL-13R{alpha}1 and IL-4R{alpha}. IL-13 did not play a major role in this response because the magnitude of this induction was similar in the IL-13+/+ and IL-13–/– mice.


Figure 6
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  		FIGURE 6. Effect of hyperoxia on the expression of IL-13Rs in IL-13 null mice. Mice were exposed to room air or 100% O2 for 72 h. Pulmonary mRNA for IL-13R{alpha}1, and IL-4{alpha} were assessed. beta-Actin served as a control. This figure is illustrative of a minimum of four experiments.

 
Role of IL-13 in the regulation of the levels of BAL VEGF, IL-6, and IL-11

Previous studies from our laboratory demonstrated that exogenously administered IL-13 has cytoprotective effects in HALI and that this effect is mediated, at least in part, by VEGF (14). Our studies also demonstrated that exogenously administered IL-6 and IL-11 confer cytoprotection in this setting (11, 12). Thus, studies were undertaken to determine whether alterations in the production and or expression of any these mediators might be contributing to the effects of endogenous IL-13 in our experimental system. This approach was done by comparing the levels of these mediators in BAL fluids from IL-13+/+ and IL-13–/– mice in room air or hyperoxia for 72 h. As shown in Fig. 7, A and B, the levels of BAL fluid VEGF and IL-6 in WT mice breathing room air were low and increased significantly after hyperoxic exposure. In room air, the levels of VEGF and IL-6 in IL-13–/– mice were also comparable to those in IL-13+/+ animals. In contrast, after hyperoxic exposure, the levels of VEGF and IL-6 in BAL fluids from IL-13–/– mice were significantly lower than the levels in BAL fluids from similarly challenged IL-13+/+ controls (Fig. 7, A and B). Interestingly, IL-11 followed a slightly different pattern because it was detected in BAL fluids from WT mice in room air and did not increase significantly in hyperoxia. In the absence of IL-13, however, the levels of BAL IL-11 were decreased in mice breathing 100% O2 compared with mice undergoing room air exposures (Fig. 7C). When viewed in combination, these studies demonstrate that in the setting of hyperoxic exposure, IL-13–/– mice have lower levels of cytoprotective cytokines including VEGF, IL-6, and IL-11 in their lungs than IL-13+/+ controls.


Figure 7
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  		FIGURE 7. Role of IL-13 in the regulation of BAL VEGF, IL-6, and IL-11. IL-13+/+, IL-13+/–, and IL-13–/– mice were exposed to room air or 100% O2 for 72 h. BAL levels of VEGF (A), IL-6 (B), and IL-11 (C) were assessed by ELISA, as described in Materials and Methods. The noted values represent assessments in a minimum of five animals. *, p ≤ 0.0001; **, p ≤ 0.01; #, p ≤ 0.03.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
To further understand the biology of IL-13, studies were undertaken to test the hypothesis that IL-13 is an important endogenous regulator of the oxidant-induced pulmonary responses that occur during HALI. These studies demonstrate that hyperoxia stimulates the expression of IL-13R. They also demonstrate that hyperoxia-induced mortality and DNA injury and cell death are enhanced and tissue inflammation is decreased in the absence of IL-13. When viewed in combination, these studies demonstrate that the expression of the IL-13R is augmented and that the endogenous IL-13-IL-13R pathway contributes to the induction of tissue inflammation and inhibition of injury in HALI.

Supplemental oxygen is commonly administered to patients with serious pulmonary or cardiac disorders to enhance tissue O2 delivery. However, high concentrations of O2 (fractional inspired concentrations ≥50%), when administered for prolonged periods, cause HALI characterized by endothelial and epithelial injury (11, 12, 16, 18, 19, 20, 21). Studies of this response have led to the free radical theory, which suggests that, in 100% O2, lung cells poison themselves by producing an excess of ROS (16, 20, 22). Recent studies from our laboratory and others have added to this pathogenic paradigm by demonstrating that ROS mediate their effects, in part, by inducing an endothelial and epithelial cell death response with features of apoptosis and necrosis (11, 12, 16, 17, 20, 21, 23) and that a variety of exogenously administered regulators including IL-11, VEGF, and IL-13 inhibit these toxic events by regulating local cell death responses (12, 14, 16). Despite the obvious importance of pathways that inhibit these toxic responses, and the well-documented variations in the ability of inbred mice to withstand exposure to 100% O2 (11, 12, 16, 24), the endogenous mechanisms that contribute to the control of these responses have not been adequately defined. The present studies, however, shed light on this issue by demonstrating, for the first time, that the endogenous IL-13-IL-13R pathway is induced and feeds back to inhibit tissue injury during HALI.

A variety of lines of evidence suggests that the consequences of hyperoxia are mediated and regulated, at least in part, by the ability of 100% O2 to induce a variety of cytokine-receptor pathways (14, 25, 26, 27, 28, 29, 30, 31). In the majority of these studies, the cytokine ligand is induced and is presumed to mediate its effects by binding to its unaltered receptor (14, 25, 26, 27, 28, 29, 31). Interestingly, our studies of IL-13 highlight a different paradigm with 100% O2 stimulating the expression of both the IL-4R{alpha} and IL-13R{alpha}1 subunits of the IL-13R without altering the expression of IL-13. These findings are in accord with studies from our laboratory and others that demonstrated that IL-4R{alpha} and IL-13R{alpha}1 are impressively regulated by Th2 and Th1 cytokines (4, 32, 33) and extend our knowledge of this regulatory ability to oxidant-induced injury states. It is tempting to speculate from these findings that similar oxidant-induced mechanisms contribute to the exaggerated expression of IL-13R{alpha}1 and the remodeling responses that have been noted in fibrotic disorders such as IPF (34, 35, 36). Because IL-13R{alpha}1 and IL-4R{alpha} also make up the type II IL-4R, one can easily envision a mechanism by which this receptor regulation could contribute to IL-4-induced responses in diseases like asthma, and pulmonary fibrosis that are characterized by oxidant injury and dysregulated IL-4 expression (37, 38, 39).

In animal models of HALI, inflammation and lung injury are frequently juxtaposed. This process has led to studies investigating the mechanisms of hyperoxia-induced inflammation and the relationship between injury and inflammation in this disorder (17, 40, 41, 42). Our studies add to the former by demonstrating, for the first time, that the IL-13 pathway is activated during and plays an important role in the pathogenesis of the inflammatory response that is seen in HALI. Our studies also demonstrate that null mutations of IL-13, while diminishing hyperoxia-induced inflammation, increase hyperoxia-induced DNA injury and cell death. This dissociation demonstrates that HALI-induced tissue injury cannot be attributed solely to local tissue inflammation. In addition, although structural cell apoptosis (such as that seen in HALI) can stimulate tissue inflammation (43, 44), these studies also demonstrate that hyperoxia-induced inflammation cannot be attributed solely to the nearby cell death response.

Under physiologic conditions, tissue homeostasis is controlled by the tight regulation of apoptosis and necrosis. This effect is accomplished by the continuous integration of pro- and anticell death signals. In many cases stimuli that induce cell death interact with the cell membrane. This membrane (extrinsic) pathway triggers cell surface "death receptors" such as Fas, which binds FasL, and TNFR1, which binds TNF and lymphotoxin and activate caspase 8 (45, 46). Many of the other stimuli use mitochondrial dysfunction to signal the death response. In this intrinsic pathway, BH3-only domain family members such as Bid are activated to truncated Bid and interact with Bax-type proteins to form and interact with mitochondrial pores, release cytochrome C, activate caspase 9, and induce cell death (16, 47, 48, 49). In accord with prior studies of HALI (16, 23, 50), our studies demonstrate that hyperoxia stimulates both the extrinsic and intrinsic cell death responses in the murine lung. They also highlight the induction of the antiapoptotic Bcl-2 family proteins A1 and Bcl-2, which are presumed to represent protective responses to oxidant injury (16). The present studies add to our knowledge of the processes that regulate these critical responses by demonstrating that, in the absence of IL-13, hyperoxia stimulates Fas, FasL, and Bid and induces caspases-3, -8, and -9 expression and activates caspase-3 in an exaggerated fashion. Previous studies from our laboratory and others have demonstrated that Bcl-2 and A1 inhibit oxidant-induced death receptor and mitochondrial cell death pathway activation and caspase activation in the lung and other organs (16, 51, 52, 53). Our studies also demonstrated that the induction of A1 is partially responsible for the cytoprotective effects of IL-11, VEGF, and IL-13 in HALI (16). Interestingly, A1 and Bcl-2 were not regulated by endogenous IL-13 in the 100% O2-exposed murine lung. In contrast, the levels BAL VEGF, IL-11, and IL-6, all of which have cytoprotective effects in HALI (11, 12, 14), were diminished in the absence of IL-13. When viewed in combination, these studies demonstrate that endogenous IL-13 inhibits the ability of hyperoxia to activate the death receptor and mitochondrial cell death pathways and that this inhibition is mediated by A1- and Bcl-2-independent mechanisms that may involve VEGF, IL-11, and/or IL-6. It is important to point out, however, that alterations in the clearance of dead and dying cells could also contribute to these findings. Additional experiments will be required to define the effects of IL-13 on efferocytosis in this experimental system.

Oxidative injury is a key element in the pathogenesis of a wide variety of diseases and disorders. This is nicely illustrated in the lung where hyperoxia leads to acute and chronic injuries like bronchopulmonary dysplasia in the newborn and adult respiratory distress syndrome in adults (54, 55). Oxidant injury also plays a major role in the pathogenesis of interstitial lung diseases, asthma, and chronic obstructive lung disease and can worsen the effects of pulmonary infections (7, 8, 9, 56, 57). Our studies demonstrate that the expression of the IL-13R is enhanced and that the IL-13-IL-13R pathway plays an important role in the regulation of oxidant-induced inflammation and cell death responses. These observations suggest that IL-13-dependent pathways may be able to be manipulated to control oxidant-induced pulmonary responses. They also suggest that genetic polymorphisms, environmental exposures, or pharmacologic interventions that alter the IL-13-IL-13R pathway can have major effects on an individual’s ability to tolerate an oxidative load and thus could contribute to the severity and/or natural history of these and other disorders. This result highlights areas where appropriate caution is required in the development of IL-13-IL-13R-based pathway inhibitors.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported in part by Grants HL-74195 (to V.B.), HL-64642, HL-61904, and HL-56389 (to J.A.E.) from the National Heart, Lung, and Blood Institute of the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Jack A. Elias, Yale University School of Medicine, Department of Internal Medicine, 1 Gilbert Street, New Haven, CT 06520. E-mail address: jack.elias{at}yale.edu Back

3 Abbreviations used in this paper: IPF, idiopathic pulmonary fibrosis; ROS, reactive oxidant species; HALI, hyperoxic acute lung injury; WT, wild type; BAL, bronchoalveolar lavage; PKC, protein kinase C; BCIP, 5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt; FasL, Fas ligand. Back

Received for publication July 21, 2006. Accepted for publication January 29, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Minty, A., S. Asselin, A. Bensussan, D. Shire, N. Vita, A. Vyakarnam, J. Wijdenes, P. Ferrara, D. Caput. 1997. The related cytokines interleukin-13 and interleukin-4 are distinguished by differential production and differential effects on T lymphocytes. Eur. Cytokine Netw. 8: 203-213. [Medline]
  2. Bochner, B. S., D. A. Klunk, S. A. Sterbinsky, R. L. Coffman, R. P. Schleimer. 1995. IL-13 selectively induces vascular cell adhesion molecule-1 expression in human endothelial cells. J. Immunol. 154: 799-803. [Abstract]
  3. Punnonen, J., G. Aversa, B. G. Cocks, A. N. McKenzie, S. Menon, G. Zurawski, R. de Waal Malefyt, J. E. de Vries. 1993. Interleukin 13 induces interleukin 4-independent IgG4 and IgE synthesis and CD23 expression by human B cells. Proc. Natl. Acad. Sci. USA 90: 3730-3734. [Abstract/Free Full Text]
  4. Zheng, T., Z. Zhu, W. Liu, C. G. Lee, Q. Chen, R. J. Homer, J. A. Elias. 2003. Cytokine regulation of IL-13R{alpha}2 and IL-13R{alpha}1 in vivo and in vitro. J. Allergy Clin. Immunol. 111: 720-728. [Medline]
  5. Wills-Karp, M.. 2004. Interleukin-13 in asthma pathogenesis. Immunol. Rev. 202: 175-190. [Medline]
  6. Wynn, T. A.. 2003. IL-13 effector functions. Annu. Rev. Immunol. 21: 425-456. [Medline]
  7. Andreadis, A. A., S. L. Hazen, S. A. Comhair, S. C. Erzurum. 2003. Oxidative and nitrosative events in asthma. Free Radic. Biol. Med. 35: 213-225. [Medline]
  8. Rahman, I.. 2003. Oxidative stress, chromatin remodeling and gene transcription in inflammation and chronic lung diseases. J. Biochem. Mol. Biol. 36: 95-109. [Medline]
  9. Saleh, D., P. J. Barnes, A. Giaid. 1997. Increased production of the potent oxidant peroxynitrite in the lungs of patients with idiopathic pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 155: 1763-1769. [Abstract]
  10. Zhang, X., P. Shan, M. Sasidhar, G. L. Chupp, R. A. Flavell, A. M. Choi, P. J. Lee. 2003. Reactive oxygen species and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase mediate hyperoxia-induced cell death in lung epithelium. Am. J. Respir. Cell Mol. Biol. 28: 305-315. [Abstract/Free Full Text]
  11. Ward, N. S., A. B. Waxman, R. J. Homer, L. L. Mantell, O. Einarsson, Y. Du, J. A. Elias. 2000. Interleukin-6-induced protection in hyperoxic acute lung injury. Am. J. Respir. Cell Mol. Biol. 22: 535-542. [Abstract/Free Full Text]
  12. Waxman, A. B., O. Einarsson, T. Seres, R. G. Knickelbein, J. B. Warshaw, R. Johnston, R. J. Homer, J. A. Elias. 1998. Targeted lung expression of interleukin-11 enhances murine tolerance of 100% oxygen and diminishes hyperoxia-induced DNA fragmentation. J. Clin. Invest. 101: 1970-1982. [Medline]
  13. Wang, J., Q. Chen, J. Corne, Z. Zhu, C. G. Lee, V. Bhandari, R. J. Homer, J. A. Elias. 2003. Pulmonary expression of leukemia inhibitory factor induces B cell hyperplasia and confers protection in hyperoxia. J. Biol. Chem. 278: 31226-31232. [Abstract/Free Full Text]
  14. Corne, J., G. Chupp, C. G. Lee, R. J. Homer, Z. Zhu, Q. Chen, B. Ma, Y. Du, F. Roux, J. McArdle, et al 2000. IL-13 stimulates vascular endothelial cell growth factor and protects against hyperoxic acute lung injury. J. Clin. Invest. 106: 783-791. [Medline]
  15. Wang, J., R. J. Homer, L. Hong, L. Cohn, C. G. Lee, S. Jung, J. A. Elias. 2000. IL-11 selectively inhibits aeroallergen-induced pulmonary eosinophilia and Th2 cytokine production. J. Immunol. 165: 2222-2231. [Abstract/Free Full Text]
  16. He, C. H., A. B. Waxman, C. G. Lee, H. Link, M. E. Rabach, B. Ma, Q. Chen, Z. Zhu, M. Zhong, K. Nakayama, et al 2005. Bcl-2-related protein A1 is an endogenous and cytokine-stimulated mediator of cytoprotection in hyperoxic acute lung injury. J. Clin. Invest. 115: 1039-1048. [Medline]
  17. Mantell, L. L., S. Horowitz, J. M. Davis, J. A. Kazzaz. 1999. Hyperoxia-induced cell death in the lung: the correlation of apoptosis, necrosis, and inflammation. Ann. NY Acad. Sci. 887: 171-180. [Abstract/Free Full Text]
  18. Crapo, J. D.. 1986. Morphologic changes in pulmonary oxygen toxicity. Annu. Rev. Physiol. 48: 721-731. [Medline]
  19. Barazzone, C., S. Horowitz, Y. R. Donati, I. Rodriguez, P. F. Piguet. 1998. Oxygen toxicity in mouse lung: pathways to cell death. Am. J. Respir. Cell Mol. Biol. 19: 573-581. [Abstract/Free Full Text]
  20. Barazzone, C., C. W. White. 2000. Mechanisms of cell injury and death in hyperoxia: role of cytokines and Bcl-2 family proteins. Am. J. Respir. Cell Mol. Biol. 22: 517-519. [Free Full Text]
  21. O’Reilly, M. A., R. J. Staversky, H. L. Huyck, R. H. Watkins, M. B. LoMonaco, C. T. D’Angio, R. B. Baggs, W. M. Maniscalco, G. S. Pryhuber. 2000. Bcl-2 family gene expression during severe hyperoxia induced lung injury. Lab. Invest. 80: 1845-1854. [Medline]
  22. Freeman, B. A., J. D. Crapo. 1982. Biology of disease: free radicals and tissue injury. Lab. Invest. 47: 412-426. [Medline]
  23. Wang, X., S. W. Ryter, C. Dai, Z. L. Tang, S. C. Watkins, X. M. Yin, R. Song, A. M. Choi. 2003. Necrotic cell death in response to oxidant stress involves the activation of the apoptogenic caspase-8/bid pathway. J. Biol. Chem. 278: 29184-29191. [Abstract/Free Full Text]
  24. Paine, R., III, S. E. Wilcoxen, S. B. Morris, C. Sartori, C. E. Baleeiro, M. A. Matthay, P. J. Christensen. 2003. Transgenic overexpression of granulocyte macrophage-colony stimulating factor in the lung prevents hyperoxic lung injury. Am. J. Pathol. 163: 2397-2406. [Abstract/Free Full Text]
  25. Johnston, C. J., T. W. Wright, C. K. Reed, J. N. Finkelstein. 1997. Comparison of adult and newborn pulmonary cytokine mRNA expression after hyperoxia. Exp. Lung Res. 23: 537-552. [Medline]
  26. Jensen, J. C., H. W. Pogrebniak, H. I. Pass, C. Buresh, M. J. Merino, D. Kauffman, D. Venzon, H. N. Langstein, J. A. Norton. 1992. Role of tumor necrosis factor in oxygen toxicity. J. Appl. Physiol. 72: 1902-1907. [Abstract/Free Full Text]
  27. Piedboeuf, B., S. Horowitz, C. J. Johnston, M. Gamache, S. Belanger, P. E. Poubelle, S. E. Welty, R. H. Watkins. 1998. Interleukin-1 expression during hyperoxic lung injury in the mouse. Free Radic. Biol. Med. 24: 1446-1454. [Medline]
  28. Johnston, C. J., B. R. Stripp, B. Piedbeouf, T. W. Wright, G. W. Mango, C. K. Reed, J. N. Finkelstein. 1998. Inflammatory and epithelial responses in mouse strains that differ in sensitivity to hyperoxic injury. Exp. Lung Res. 24: 189-202. [Medline]
  29. Pryhuber, G. S., D. P. O’Brien, R. Baggs, R. Phipps, H. Huyck, I. Sanz, M. H. Nahm. 2000. Ablation of tumor necrosis factor receptor type I (p55) alters oxygen-induced lung injury. Am. J. Physiol. 278: L1082-L1090.
  30. Chen, Q., L. Rabach, P. Noble, T. Zheng, C. G. Lee, R. J. Homer, J. A. Elias. 2005. IL-11 receptor {alpha} in the pathogenesis of IL-13-induced inflammation and remodeling. J. Immunol. 174: 2305-2313. [Abstract/Free Full Text]
  31. Watkins, R. H., C. T. D’Angio, R. M. Ryan, A. Patel, W. M. Maniscalco. 1999. Differential expression of VEGF mRNA splice variants in newborn and adult hyperoxic lung injury. Am. J. Physiol. 276: L858-L867. [Medline]
  32. Rahaman, S. O., P. Sharma, P. C. Harbor, M. J. Aman, M. A. Vogelbaum, S. J. Haque. 2002. IL-13R{alpha}2, a decoy receptor for IL-13 acts as an inhibitor of IL-4-dependent signal transduction in glioblastoma cells. Cancer Res. 62: 1103-1109. [Abstract/Free Full Text]
  33. Daines, M. O., G. K. Hershey. 2002. A novel mechanism by which interferon-{gamma} can regulate interleukin (IL)-13 responses: evidence for intracellular stores of IL-13 receptor {alpha}-2 and their rapid mobilization by interferon-{gamma}. J. Biol. Chem. 277: 10387-10393. [Abstract/Free Full Text]
  34. Jakubzick, C., E. S. Choi, B. H. Joshi, M. P. Keane, S. L. Kunkel, R. K. Puri, C. M. Hogaboam. 2003. Therapeutic attenuation of pulmonary fibrosis via targeting of IL-4- and IL-13-responsive cells. J. Immunol. 171: 2684-2693. [Abstract/Free Full Text]
  35. Kolodsick, J. E., G. B. Toews, C. Jakubzick, C. Hogaboam, T. A. Moore, A. McKenzie, C. A. Wilke, C. J. Chrisman, B. B. Moore. 2004. Protection from fluorescein isothiocyanate-induced fibrosis in IL-13-deficient, but not IL-4-deficient, mice results from impaired collagen synthesis by fibroblasts. J. Immunol. 172: 4068-4076. [Abstract/Free Full Text]
  36. Jakubzick, C., S. L. Kunkel, R. K. Puri, C. M. Hogaboam. 2004. Therapeutic targeting of IL-4- and IL-13-responsive cells in pulmonary fibrosis. Immunol. Res. 30: 339-349. [Medline]
  37. Chatila, T. A.. 2004. Interleukin-4 receptor signaling pathways in asthma pathogenesis. Trends Mol. Med. 10: 493-499. [Medline]
  38. Bryborn, M., M. Adner, L. O. Cardell. 2004. Interleukin-4 increases murine airway response to kinins, via up-regulation of bradykinin B1-receptors and altered signalling along mitogen-activated protein kinase pathways. Clin. Exp. Allergy 34: 1291-1298. [Medline]
  39. Baecher-Allan, C. M., R. K. Barth. 1993. PCR analysis of cytokine induction profiles associated with mouse strain variation in susceptibility to pulmonary fibrosis. Reg. Immunol. 5: 207-217. [Medline]
  40. Bustani, P., S. Kotecha. 2003. Role of cytokines in hyperoxia mediated inflammation in the developing lung. Front. Biosci. 8: S694-S704. [Medline]
  41. Lian, X., Y. Qin, S. A. Hossain, L. Yang, A. White, H. Xu, J. M. Shipley, T. Li, R. M. Senior, H. Du, C. Yan. 2005. Overexpression of Stat3C in pulmonary epithelium protects against hyperoxic lung injury. J. Immunol. 174: 7250-7256. [Abstract/Free Full Text]
  42. Thiel, M., A. Chouker, A. Ohta, E. Jackson, C. Caldwell, P. Smith, D. Lukashev, I. Bittmann, M. V. Sitkovsky. 2005. Oxygenation inhibits the physiological tissue-protecting mechanism and thereby exacerbates acute inflammatory lung injury. PLoS Biol. 3: e174[Medline]
  43. Henson, P. M.. 2005. Dampening inflammation. Nat. Immunol. 6: 1179-1181. [Medline]
  44. Serhan, C. N., J. Savill. 2005. Resolution of inflammation: the beginning programs the end. Nat. Immunol. 6: 1191-1197. [Medline]
  45. Benedict, C. A., P. S. Norris, C. F. Ware. 2002. To kill or be killed: viral evasion of apoptosis. Nat. Immunol. 3: 1013-1018. [Medline]
  46. De Paepe, M. E., Q. Mao, Y. Chao, J. L. Powell, L. P. Rubin, S. Sharma. 2005. Hyperoxia-induced apoptosis and Fas/FasL expression in lung epithelial cells. Am. J. Physiol. 289: L647-L659.
  47. Joza, N., G. Kroemer, J. M. Penninger. 2002. Genetic analysis of the mammalian cell death machinery. Trends Genet. 18: 142-149. [Medline]
  48. Werner, A. B., E. de Vries, S. W. Tait, I. Bontjer, J. Borst. 2002. Bcl-2 family member Bfl-1/A1 sequesters truncated bid to inhibit is collaboration with pro-apoptotic Bak or Bax. J. Biol. Chem. 277: 22781-22788. [Abstract/Free Full Text]
  49. Schuchmann, M., P. R. Galle. 2001. Apoptosis in liver disease. Eur. J. Gastroenterol. Hepatol. 13: 785-790. [Medline]
  50. Budinger, G. R., M. Tso, D. S. McClintock, D. A. Dean, J. I. Sznajder, N. S. Chandel. 2002. Hyperoxia-induced apoptosis does not require mitochondrial reactive oxygen species and is regulated by Bcl-2 proteins. J. Biol. Chem. 277: 15654-15660. [Abstract/Free Full Text]
  51. Liu, C. Y., P. I. Chuang, C. L. Chou, S. M. Lin, H. C. Chen, P. Chou, Y. H. Liu, C. T. Yu, C. H. Wang, H. C. Lin, H. P. Kuo. 2004. Cytoprotective response of A1, a Bcl-2 homologue expressed in mature human neutrophils and promyelocytic HL-60 cells, to oxidant stress-induced cell death. J. Biomed. Sci. 11: 214-227. [Medline]
  52. Lee, Y. J., J. C. Chen, A. A. Amoscato, J. Bennouna, D. R. Spitz, M. Suntharalingam, J. G. Rhee. 2001. Protective role of Bcl2 in metabolic oxidative stress-induced cell death. J. Cell Sci. 114: 677-684. [Abstract]
  53. Kim, H., Y. N. Kim, H. Kim, C. W. Kim. 2005. Oxidative stress attenuates Fas-mediated apoptosis in Jurkat T cell line through Bfl-1 induction. Oncogene 24: 1252-1261. [Medline]
  54. Bhandari, A., V. Bhandari. 2003. Pathogenesis, pathology and pathophysiology of pulmonary sequelae of bronchopulmonary dysplasia in premature infants. Front. Biosci. 8: e370-e380. [Medline]
  55. Matthay, M. A., G. A. Zimmerman. 2005. Acute lung injury and the acute respiratory distress syndrome: four decades of inquiry into pathogenesis and rational management. Am. J. Respir. Cell Mol. Biol. 33: 319-327. [Free Full Text]
  56. Tateda, K., J. C. Deng, T. A. Moore, M. W. Newstead, R. Paine, III, N. Kobayashi, K. Yamaguchi, T. J. Standiford. 2003. Hyperoxia mediates acute lung injury and increased lethality in murine Legionella pneumonia: the role of apoptosis. J. Immunol. 170: 4209-4216. [Abstract/Free Full Text]
  57. Tuder, R. M., L. Zhen, C. Y. Cho, L. Taraseviciene-Stewart, Y. Kasahara, D. Salvemini, N. F. Voelkel, S. C. Flores. 2003. Oxidative stress and apoptosis interact and cause emphysema due to vascular endothelial growth factor receptor blockade. Am. J. Respir. Cell Mol. Biol. 29: 88-97. [Abstract/Free Full Text]



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