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Am J Physiol Lung Cell Mol Physiol 288: L390-L397, 2005. First published October 29, 2004; doi:10.1152/ajplung.00007.2004
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Role of interleukin-6 in murine airway responses to ozone

Richard A. Johnston, Igor N. Schwartzman, Lesley Flynt, and Stephanie A. Shore

Physiology Program, Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts

Submitted 13 January 2004 ; accepted in final form 22 October 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study sought to examine the role of interleukin-6 (IL-6) in ozone (O3)-induced airway injury, inflammation, and hyperresponsiveness (AHR). Subacute (72 h) exposure to 0.3 ppm O3 significantly elevated bronchoalveolar lavage fluid (BALF) protein, neutrophils, and soluble TNF receptors (sTNFR1 and sTNFR2) in wild-type C57BL/6 (IL-6+/+) mice; however, all four outcome indicators were significantly reduced in IL-6-deficient (IL-6–/–) compared with IL-6+/+ mice. Acute O3 exposure (2 ppm for 3 h) increased BALF protein, KC, macrophage inflammatory protein(MIP)-2, eotaxin, sTNFR1, and sTNFR2 in IL-6+/+ mice. However, MIP-2 and sTNFR2 were not significantly increased following O3 exposure in IL-6–/– mice. Increases in BALF neutrophils induced by O3 (2 ppm for 3 h) were also significantly reduced in IL-6–/– vs. IL-6+/+ mice. Airway responsiveness to methacholine was measured by whole body plethysmography before and following acute (3 h) or subacute (72 h) exposure to 0.3 ppm O3. Acute O3 exposure caused AHR in both groups of mice, but there was no genotype-related difference in the magnitude of O3-induced AHR. AHR was absent in mice of either genotype exposed for 72 h. Our results indicate that IL-6 deficiency reduces airway neutrophilia, as well as the levels of BALF sTNFR1 and sTNFR2 following acute high dose and/or subacute low-dose O3 exposure, but has no effect on O3-induced AHR.

airway responsiveness; inflammation; injury; neutrophil; soluble tumor necrosis factor receptor

EXPOSURE TO OZONE (O3) causes lung injury and inflammation. The inflammatory response includes the generation of numerous cytokines and chemokines, as well as an influx of polymorphonuclear leukocytes (3, 8, 17, 20, 22, 25, 31, 33, 41, 48). O3 also causes airway hyperresponsiveness (AHR) (10, 22, 27, 28, 33, 35, 47). AHR is a defining feature of asthma, and hospital admissions for asthma increase on days of high ambient O3 concentrations (11, 40). In children, O3 causes increases in asthmatic symptoms even at concentrations below the U.S. Environmental Protection Agency standard (13). O3-induced AHR and/or inflammation are likely to contribute to the ability of O3 to trigger asthmatic episodes. As such, it is important to understand the mechanistic basis for these responses.

There is reason to believe that interleukin-6 (IL-6) may modify O3-induced AHR and/or inflammation. In many species, IL-6 is released into bronchoalveolar lavage fluid (BALF) upon acute exposure to O3 (31, 33, 41). There is also increased IL-6 mRNA in the lungs of mice exposed to O3 (17). Both macrophages and epithelial cells synthesize IL-6 in response to O3 (3, 8). IL-6 is a pleiotropic cytokine, which has been reported to have both pro- and anti-inflammatory effects (18, 38). In the lungs, transgenic overexpression of IL-6 results in a decrease in airway responsiveness (9, 23). Overexpression of IL-6 also protects against the acute lung injury induced by chronic hyperoxia (43), whereas mice genetically deficient in IL-6 have a reduced acute-phase response to tissue injury (21). Together, these results suggest that O3-induction of IL-6 may serve to ameliorate both the AHR and the airway injury induced by O3. However, Yu et al. (46) reported a decrease in peripheral airway injury in IL-6-deficient compared with wild-type mice following exposure to O3 (0.5 ppm for 24 h), although they did not assess AHR, suggesting that IL-6 contributes to rather than protects against O3-induced injury to the lungs.

The purpose of this study was to examine the role of IL-6 in O3-induced AHR and injury and inflammation to the lungs. To that end, we measured airway responsiveness and airway injury and inflammation to the lungs in wild-type C57BL/6 mice (IL-6+/+) and mice genetically deficient in IL-6 (IL-6–/–) following acute (3 h) and subacute (72 h) O3 exposures. As indexes of O3-induced injury and inflammation to the lungs, we used BALF protein, epithelial cells, and neutrophils as well as BALF IL-6, eotaxin, KC, and macrophage inflammatory protein-2 (MIP-2). Finally, because IL-6 has been reported to increase soluble tumor necrosis factor receptor 1 (sTNFR1) in the blood (39) and because we have reported previously that TNFR2 is required for O3-induced AHR (35), we also determined the concentration of sTNFR1 and sTNFR2 in the BALF.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male and female mice genetically deficient in IL-6 (IL-6–/–) were purchased from The Jackson Laboratory (Bar Harbor, ME). Sex- and age-matched IL-6+/+ mice served as controls since the IL-6–/– mice have been backcrossed onto a C57BL/6J background for at least 11 generations. Mice used in this study were at least 8 wk old. The Harvard Medical Area Standing Committee on Animals approved all of the experimental protocols used in this study.

O3 exposure. For acute (3 h) O3 exposures, conscious mice were placed in individual wire mesh cages and set inside a 145-l stainless steel chamber with a Plexiglas door. We used 0.3 ppm as well as 2 ppm O3 to assess the effect of IL-6 deficiency on acute O3-induced injury and inflammation to the lungs, because we were interested in cytokine/chemokine generation and neutrophil emigration into the air spaces, and these did not occur following acute exposure to 0.3 ppm O3 for 3 h (see RESULTS). For subacute (72 h) exposures to 0.3 ppm O3, the entire mouse cage, with the exception of the microisolator top, was placed inside the exposure chamber. During subacute exposures, the mice had continuous access to food and water. The doses of O3 and exposure times were chosen to allow comparison with other studies of responses to O3 in C57BL/6 mice (6, 19, 48). For exposures to room air, the experimental protocol was identical to the corresponding O3 exposure except that a separate and identical exposure chamber dedicated to room air exposure was used. Finally, because there were no differences in any of the outcome indicators examined in mice exposed to room air for 3 vs. 72 h, the data for all air-exposed mice were pooled.

O3 was generated by passing oxygen (Airgas East, Salem, NH) through ultraviolet (UV) light, which was subsequently mixed with room air in the chamber. By the constant drawing of a sample of the chamber atmosphere through a sampling port, the O3 concentration within the chamber was monitored constantly by a UV photometric O3 analyzer (model 49; Thermo Electron Instruments, Hopkinton, MA), which was calibrated by a UV photometric O3 calibrator (model 49PS, Thermo Electron Instruments).

Whole body plethysmography. Airway responsiveness to inhaled, acetyl-{beta}-methylcholine chloride (methacholine; MCh) (Sigma-Aldrich, St. Louis, MO) was assessed by whole body plethysmography (Buxco Electronics, Sharon, CT), as described previously (33, 35). The outcome indicator, enhanced pause (Penh), has been shown to correlate with pulmonary resistance and to be reduced by bronchodilators in some studies (7, 14, 36, 37); however, this relationship has been not demonstrated in other studies (1, 4, 12, 24, 26, 29). These studies, while not demonstrating the validity of Penh as an independent marker of lower airway obstruction, do suggest that Penh does provide information regarding the integrated ventilatory and mechanical responses of the entire respiratory tract.

On the day before O3 exposure (day 0), conscious mice were placed unrestrained and uninstrumented into individual whole body plethysmographs. After a 30-min acclimation period in air, baseline Penh was recorded every 15 s for 4 min. Dose-response curves to MCh aerosol were generated as follows. First, an aerosol of phosphate-buffered saline (PBS) was delivered to the animal for 1 min. After aerosol delivery ceased, Penh was measured every 15 s for 9 min afterwards. Next, aerosols of MCh, in approximate half-log intervals from 1 to 100 mg/ml, were delivered to the animal for 1 min at 9-min intervals, with Penh being recorded every 15 s during the interval. The average Penh value recorded during the 9-min period following the cessation of delivery of PBS or each MCh dose was taken as the animal’s response to that particular aerosolized agent.

On day 1, the mice were exposed to either 0.3 ppm O3 or room air for 3 or 72 h. Three hours following the 3-h O3 exposure, airway responsiveness to inhaled MCh was again measured. For those animals exposed to 0.3 ppm O3 for 72 h, airway responsiveness was assessed immediately upon removal from the exposure chamber on day 4.

BAL. For acute O3 exposures, BAL was performed 4 h after cessation of O3 exposure to match the time at which measurements of airway responsiveness were assessed. The latter procedure was begun 3 h after cessation of exposure and took about 1 h to complete. For subacute O3 exposures, BAL was performed immediately after MCh challenge on day 4.

Immediately before BAL, the animal was killed with an overdose of pentobarbital sodium. For BAL, the trachea was exposed in situ, a small incision was made in the trachea, and a plastic catheter attached to a syringe was inserted. The lungs were lavaged twice with 1 ml of ice-cold lavage buffer, PBS containing 0.6 mM EDTA. The resulting lavagates were pooled and stored on ice until further use. The lavagate was spun at 4°C for 10 min at 2,000 rpm, the supernatant was collected and stored at –80°C until further use, and the remaining cell pellet was resuspended in 1 ml of Hanks’ balanced salt solution (Sigma). The total number of BALF cells was determined by counting the number of cells in a 10-µl aliquot of the resulting cell suspension with a hemacytometer. Next, ~2.5 x 104 cells were spun onto glass microscope slides at 800 rpm for 10 min at room temperature using a Cytospin 3 Cytocentrifuge (Thermo Shandon, Pittsburgh, PA). Afterwards, the slides were air-dried and stained with Hema 3 (Biochemical Sciences, Swedesboro, NJ), and at least 300 cells were counted under a light microscope (x250 magnification) for differential cell analysis.

Protein and enzyme-linked immunosorbent assays. The total BALF protein concentration was determined spectrophotometrically according to the Bradford protein assay procedure (Bio-Rad Laboratories, Hercules, CA). The concentration of BALF IL-6, KC, MIP-2, sTNFR1, sTNFR2, and eotaxin was determined with enzyme-linked immunosorbent assay (ELISA) kits (Endogen, Woburn, MA for IL-6; R&D Systems, Minneapolis, MN for all others) according to the manufacturer’s instructions. Before the ELISAs, the BALF samples were spun at 40,000 rpm for 30 min at 4°C.

RNA extraction and real-time reverse transcription-PCR. Lungs were homogenized using a PowerGen 125 homogenizer (Fisher Scientific International, Hampton, NH) at full speed in 3 ml of TRIzol reagent (Invitrogen, Carlsbad, CA) for 3 min. Total RNA was then extracted in accordance with the manufacturer’s instructions and stored at –80°C. To ensure RNA purity following extraction, the samples were run through the RNeasy miniprotocol for RNA cleanup as instructed by the manufacturer (Qiagen, Valencia, CA). Reverse transcription (RT) was performed with 2 µg total RNA, 1 µg random hexamer primers (Invitrogen), 50 nmol dNTPs, 25 U RNaseOUT, an RNase inhibitor (Invitrogen), and 200 U M-MLV reverse transcriptase (Promega, Madison, WI) in a total volume of 25 µl consisting of 50 mM Tris·HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 10 mM DTT at 37°C for 60 min. RT reactions were diluted with 30 µl of water and stored at –80°C. Quantitative real-time RT-PCR was performed using an iCycler iQ Real Time Detection System and iQ SYBR Green Supermix in accordance with the manufacturer’s instructions (Bio-Rad Laboratories). Primer sets for murine IL-6 were the same as those reported by Huang and colleagues (16): forward, 5'-ATG AAG TTC CTC TCT GCA AGA GAC T-3' and reverse, 5'-CAC TAG GTT TGC CGA GTA GAT CTC-3' (638 bp). Murine {beta}-actin was the housekeeping gene with the following sequence: forward, 5'-AGA GGG AAA TCG TGC GTG AC-3' and reverse, 5'-CAA TAG TGA TGA CCT GGC CGT-3' (148 bp). PCR products of cDNA from the lungs of O3 exposed (2 ppm for 3 h) ob/ob mice were gel purified and cloned into TOPO-TA vectors (Invitrogen), which were used as a positive control to construct standard curves for real-time RT-PCR since ob/ob mice have robust responses to O3 (34). For each set of primers, melting curve analysis yielded a single peak consistent with one PCR product. Changes in IL-6 mRNA transcript copy number were assessed relative to changes in {beta}-actin mRNA transcript copy number.

Statistical analysis of results. The effect of O3 and IL-6 deficiency on BALF parameters was assessed by factorial ANOVA. In these analyses, IL-6 (sufficient or deficient) and exposure (air or O3) were the main effects. In assessing BALF cell differentials and IL-6 mRNA expression, we performed the statistics on the common logarithm of the values, since the data were not normally distributed. Differences in IL-6 mRNA expression following room air or O3 exposure was determined by ANOVA. To determine the effect of O3 and IL-6 deficiency on airway responsiveness, we assessed the significance of changes in Penh by repeated-measures ANOVA. Statistica software (StatSoft, Tulsa, OK) was used to perform these analyses. The results are expressed, except were indicated, as the means ± SE, where n is the number of animals per treatment group. A P value <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
O3 exposure increases IL-6 mRNA expression in the lungs. Real-time RT-PCR was used to determine IL-6 mRNA expression in the lungs of IL-6+/+ mice exposed to room air or 0.3 ppm O3 (3 or 72 h). IL-6 mRNA was detectable even in room air-exposed mice, and both O3 exposure regimens resulted in a significant increase in IL-6 mRNA expression compared with room air-exposed mice (Fig. 1).



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  		Fig. 1. IL-6 mRNA expression in wild-type C57BL/6 (IL-6+/+) mice following the cessation of exposure to room air or ozone (O3, 0.3 ppm) for 3 h or 72 h. IL-6 mRNA transcript copy number was normalized to {beta}-actin transcript copy number. Results are expressed as a percentage of the IL-6 mRNA in air-exposed controls (n = 8–10 mice for each group). *P < 0.05 compared with air-exposed controls.

 
Effect of IL-6 deficiency on airway responses to acute and subacute 0.3 ppm O3 exposure. Four hours following acute O3 exposure (0.3 ppm for 3 h), the levels of BALF protein, sTNFR1, and sTNFR2 were significantly increased in both IL-6+/+ and IL-6–/– mice compared with their air-exposed controls (Fig. 2). There was no effect of IL-6 deficiency on the levels of BALF protein among the genotypes within each exposure group. The levels of BALF sTNFR1 were significantly less in IL-6–/– mice than IL-6+/+ mice following room air exposure. For BALF sTNFR2, levels were significantly reduced in IL-6–/– mice compared with IL-6+/+ mice following both room air and O3 exposure (Fig. 2). In contrast, acute O3 exposure (0.3 ppm for 3 h) did not increase the percentage of BALF cells that were neutrophils and did not result in detectable O3-induced increases in BALF IL-6 and KC (data not shown). No other chemokines were measured. The observation that IL-6 mRNA expression was increased by this exposure suggests that the cytokine was released but that the levels were below the limits of detection of the assay.



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  		Fig. 2. The total protein concentration (A) and the soluble tumor necrosis factor receptors (sTNFRs, B and C) in the bronchoalveolar lavage fluid (BALF) of IL-6+/+ and IL-6-deficient (IL-6–/–) mice following exposure to either room air or 0.3 ppm O3 for 3 h. BAL was performed immediately after the generation of post-O3 methacholine dose-response curves or room air exposure (n = 6–9 mice for each group). *P < 0.05 compared with genotype-matched, air-exposed controls; #P < 0.05 compared with IL-6–/– mice with an identical exposure.

 
Immediately following the cessation of subacute O3 exposure (0.3 ppm for 72 h), the levels of BALF protein, sTNFR1, and sTNFR2 were increased in both IL-6+/+ and IL-6–/– mice compared with their respective air-exposed controls (Fig. 3). In mice of both genotypes, O3 exposure also increased the percentage of BALF cells that were neutrophils. However, all four outcome indicators were significantly less in IL-6–/– mice than IL-6+/+ mice. There were no genotype-related differences in the total number of BALF cells following O3 exposure (14.1 ± 1.2 and 16.1 ± 2.3 x 105, respectively, for IL-6+/+ and IL-6–/– mice). IL-6 and KC were undetectable in the BALF of IL-6+/+ mice exposed to O3 subacutely (data not shown). Thus no other chemokines were measured.



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  		Fig. 3. The total protein concentration (A), percentage of neutrophils (B), and levels of sTNFRs (sTNFR1, C; sTNFR2, D) in the BALF of IL-6+/+ (solid bars) and IL-6–/– (open bars) mice following exposure to either room air or 0.3 ppm O3 for 72 h. BAL was performed immediately after the generation of post-O3 methacholine dose-response curves or room air exposure. The data shown here from air-exposed mice are identical to those found in Fig. 2 and are included here to demonstrate the effect of O3 on our outcome indicators (n = 5–11 mice for each group). *P < 0.05 compared with genotype-matched, air-exposed controls; #P < 0.05 compared with IL-6–/– mice with an identical exposure.

 
There was no significant difference in baseline airway responsiveness between IL-6+/+ and IL-6–/– mice before O3 exposure (Fig. 4). Exposure to room air did not alter airway responsiveness in either genotype (data not shown). Three hours following an acute O3 exposure (0.3 ppm for 3 h), baseline Penh was elevated in IL-6+/+ and IL-6–/– mice (1.88 ± 0.07 vs. 1.38 ± 0.52, respectively) compared with pre-exposure values (0.38 ± 0.04 vs. 0.33 ± 0.01, respectively); however, there were no genotype-related differences either pre- or post-O3 exposure. Furthermore, at 3 h post-O3, there was a leftward shift of the MCh dose-response curve in both IL-6+/+ and IL-6–/– mice (Fig. 4A), indicative of AHR. There was, however, no difference in the magnitude of the changes in Penh induced by MCh between the genotypes. In contrast to the increase in airway responsiveness that was observed 3 h following an acute O3 exposure in IL-6+/+ mice (0.3 ppm for 3 h) (Fig. 4A), airway responsiveness was not different from pre-O3 values immediately after the cessation of a subacute O3 exposure (0.3 ppm O3 for 72 h) (Fig. 4B). Similar results were obtained in IL-6–/– mice.



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  		Fig. 4. Responsiveness to aerosolized methacholine measured in IL-6+/+ and IL-6–/– mice before and following the cessation of exposure to O3 (0.3 ppm) for 3 h (A) or 72 h (B). End-expiratory pause (EEP) measurements in IL-6+/+ and IL-6–/– mice before and following the cessation of a 3-h exposure to 0.3 ppm O3 (C). EEP were measured between 0.5 and 3 h after the cessation of O3 exposure. Pre-O3 exposure values (time = 0) were measured on the day before O3 exposure (n = 5–9 for each treatment group). Penh, enhanced pause. *P < 0.05 compared with pre-O3 values in genotype-matched mice.

 
In rodents, exposure to O3 leads to prolonged apneas or end-expiratory pauses (EEP) (32, 33, 35). We have reported previously that this change in breathing pattern is dependent upon TNF signaling (35). Because the levels of BALF sTNFRs were significantly lower in IL-6–/– than IL-6+/+ mice, we measured EEP in the 3-h period following the cessation of acute exposure to O3 (0.3 ppm for 3 h) but before measurement of airway responsiveness (Fig. 4C). In both IL-6+/+ and IL-6–/– mice there was an increase in EEP from pre-O3 exposure values that persisted for 3 h following the cessation of O3 exposure. However, there were no statistically significant genotype-related differences in EEP following acute exposure to either O3 concentration. We also measured EEP just after cessation of O3 exposure but before MCh challenge in mice exposed subacutely (0.3 ppm for 72 h). In IL-6+/+ mice, EEP was elevated significantly over baseline EEP after subacute O3 exposure (28 ± 3 vs. 58 ± 12 ms, pre- and post-O3 respectively). In contrast, there was no significant effect of subacute O3 exposure on EEP in IL-6–/– mice (34 ± 3 vs. 30 ± 5 ms, pre- and post-O3 respectively). The data suggest that IL-6 contributes to O3-induced changes in the pattern of breathing after subacute but not acute O3 exposure.

Effect of IL-6 deficiency on airway responses to acute 2 ppm O3 exposure. Because acute exposure to O3 (0.3 ppm for 3 h) resulted in a detectable increase in BALF protein but no detectable increase in neutrophils or chemokines, we used a higher O3 concentration (2 ppm) to examine the role of IL-6 in acute inflammatory responses to O3. This is a concentration chosen by many other investigators examining acute inflammatory responses to O3 in mice (6, 44, 48). Figure 5 shows the effect of O3 exposure (2 ppm for 3 h) on BALF IL-6, protein, sTNFR, and chemokine levels in IL-6+/+ and IL-6–/– mice. Note that IL-6 was undetectable in BALF from all IL-6–/– mice, regardless of exposure. Four hours after cessation of O3 exposure, all BALF inflammatory parameters increased in the IL-6+/+ mice compared with their air-exposed controls. In IL-6–/– mice there were also significant increases in BALF protein, eotaxin, KC, and sTNFR1, whereas sTNFR2 and MIP-2 did not increase significantly with O3 exposure in these mice. The magnitude of the increase in BALF protein, eotaxin, KC, and sTNFR1 was not different between IL-6+/+ and IL-6–/– mice.



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  		Fig. 5. The concentration of IL-6 (A), total protein (B), sTNFRs (sTNFR1, C; sTNFR2, D), and chemokines [macrophage inflammatory protein (MIP)-2, E; KC, F; eotaxin, G] in the BALF of IL-6+/+ and IL-6–/– mice 4 h following a 3-h exposure to either room air or 2 ppm O3. The data shown here from air-exposed mice are identical to those found in Fig. 2 and are included here to demonstrate the effect of O3 on our outcome indicators (n = 5–7 mice for each group). *P < 0.05 compared with genotype-matched, air-exposed controls; #P < 0.05 compared with IL-6–/– mice with an identical exposure.

 
O3-induced changes in BALF leukocytes and epithelial cells are shown in Table 1. O3 (2 ppm for 3 h) did not have any significant effect on the total number of cells recovered from the BALF in either IL-6+/+ or IL-6–/– mice (Table 1). However, there were changes in the percentage of neutrophils, epithelial cells, and eosinophils. Four hours after cessation of O3 exposure, the percentage of cells in the BALF that were neutrophils, epithelial cells, and eosinophils increased in the IL-6+/+ mice compared with their air-exposed controls. BALF neutrophils were significantly lower in IL-6–/– mice than IL-6+/+ mice following O3 exposure, whereas the magnitude of the increase in epithelial cells and eosinophils was not different between IL-6+/+ and IL-6–/– mice.


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  		Table 1. Effect of IL-6 deficiency on BALF cell numbers and differentials following O3 exposure (2 ppm for 3 h)

 
We also measured baseline Penh for 2.5 h after the cessation of acute exposure to 2 ppm O3. Baseline Penh was substantially elevated from pre-exposure values in both IL-6+/+ and IL-6–/– mice following 2 ppm O3 exposure (9.13 ± 0.36 vs. 12.15 ± 0.62, respectively). As was the case following exposure to 0.3 ppm O3, no genotype-related differences in baseline Penh existed. Because baseline Penh was so high following exposure to 2 ppm O3, airway responsiveness to MCh was not assessed in this instance since any further increases in Penh induced by MCh could not be measured accurately.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our results indicate that IL-6 deficiency reduces airway neutrophilia and sTNFR1 and/or sTNFR2 expression following either acute exposure to a high concentration of O3 (2 ppm) or more prolonged exposure to a lower concentration (0.3 ppm). In contrast, IL-6 deficiency has no effect upon O3-induced AHR at least at 0.3 ppm O3.

Our data indicate robust expression of IL-6 in the BALF following acute exposure to 2 ppm O3, consistent with other studies using the same or similar O3 concentrations and exposure times (17, 33, 34), whereas IL-6 was not detectable in IL-6–/– mice. IL-6 was not detectable by ELISA in the BALF of IL-6+/+ mice exposed to 0.3 ppm O3 for either 3 or 72 h. However, IL-6 mRNA expression was significantly increased following exposure to 0.3 ppm O3 for either 3 or 72 h, suggesting that the protein was released but that the levels in BALF were below the limit of detection of the ELISA.

Ward et al. (43) have reported previously that hyperoxia-induced alveolar protein leak, endothelial and epithelial cell membrane injury, and lung lipid peroxidation are decreased while survival is increased in transgenic mice overexpressing IL-6 in the lungs. On the basis of these observations, we hypothesized that IL-6 might also be protective in the context of O3-induced injury to the lungs. Our results suggest that this is not the case, since O3-induced BALF protein, which has been proposed as a sensitive marker of O3-induced lung injury (2), was not different in IL-6+/+ and IL-6–/– mice following acute exposure at high concentrations (Fig. 5) and was actually significantly reduced in IL-6–/– mice following subacute O3 exposure (Fig. 3). Thus, in contrast to hyperoxia-induced lung injury (43), these results suggest that IL-6 promotes rather than ameliorates O3-induced injury to the lungs, suggesting that the role of IL-6 is critically dependent upon the precise inflammatory stimulus used. Similarly, Yu et al. (46) observed reduced epithelial injury in IL-6–/– mice compared with IL-6+/+ mice following O3 exposure, albeit using a different exposure regimen (0.5 ppm for 24 h). Our results also indicate different roles for IL-6 in the O3-induced injury to the lungs, as assessed by BALF protein, during acute and subacute O3 exposures: IL-6 plays no role during acute exposure to O3 but promotes O3-induced injury to the lungs during subacute exposure, at least as assessed by BALF protein. We did not perform a detailed histological assessment of the lungs and so cannot be certain whether the changes in protein are indeed reflective of epithelial injury under these circumstances. The importance of the precise O3 exposure protocol used is also underscored by the results of Yu and colleagues (46), who found that IL-6 deficiency had no effect upon the levels of BALF protein using an O3 exposure regimen that lies between the two we used (0.5 ppm for 24 h).

We observed reduced BALF neutrophils in the IL-6–/– mice compared with the IL-6+/+ mice following either acute (3 h) exposure to a high concentration of O3 (2 ppm) (Table 1) or subacute exposure to lower concentrations of O3 (0.3 ppm) administered for a longer period of time (72 h) (Fig. 3). It is possible that the decrease in airway neutrophilia observed in IL-6–/– mice exposed to acute high-dose O3 was related to effects of IL-6 deficiency on expression of the neutrophil chemotactic factor MIP-2, since this chemokine increased significantly in response to O3 in IL-6+/+ mice but not IL-6–/– mice (Fig. 5). These data also suggest that IL-6 may regulate the expression of MIP-2 during this exposure regimen. In contrast, the BALF levels of KC, another neutrophil chemokine known to mediate O3-induced airway neutrophilia (25), were not different between IL-6+/+ and IL-6–/– mice, suggesting that KC does not play role in the diminished neutrophil emigration observed in IL-6–/– mice. It is possible that IL-6 also influences the expression of other neutrophil chemotactic factors, such as IFN-{gamma}-inducible protein-10, monocyte chemotactic protein (MCP)-1, MCP-3, and MIP-1{alpha}, which were not measured. In addition, there may be effects of IL-6 on O3-induced expression of the adhesion molecules involved in neutrophil migration, which could explain the decreased airway neutrophilia observed in IL-6–/– mice. We do not know whether the mechanism for decreased neutrophil recruitment in IL-6–/– mice following acute and subacute O3 exposures is the same. We could not detect chemokines in the BALF of IL-6+/+ mice immediately after the cessation of subacute O3 exposure (0.3 ppm for 72 h), but it is possible that chemokines were released, but below the limit of detection of the assay. It is also possible that chemokines were released earlier during the exposure and then declined.

Our results demonstrate that exposure to O3 causes an increase in the amount of sTNFR1 and sTNFR2 released into the BALF. To our knowledge, this is the first report of such effects of O3 in any species, although another stimulus that induces oxygen radicals, hydrogen peroxide, has been shown to cause shedding of TNF receptors from pulmonary epithelial cells in culture (15). sTNFR1 and sTNFR2 are the extracellular domains of the p55 and p75 TNF receptors that are released by proteolytic cleavage. Cho et al. (6) have reported an increase in the mRNA expression of TNFR1 and TNFR2 in the lungs of O3-exposed mice, and it is possible that the increased presence of the soluble forms of these receptors following O3 exposure simply reflects their increased synthesis. It is interesting to note that following room air or O3 exposure, BALF sTNFR2 was lower in IL-6–/– than IL-6+/+ mice regardless of the exposure regimen. sTNFR1 was also reduced in IL-6–/– compared with IL-6+/+ mice following room air and subacute O3 exposure (Fig. 3). It is possible that with acute 0.3 ppm O3 exposure, the lower levels of sTNFR1 and sTNFR2 in IL-6–/– compared with IL-6+/+ mice are simply a reflection of baseline (air-exposed) differences in sTNFR expression, since the magnitude of the genotype-related differences are about the same under each condition. However, with subacute O3 exposure (0.3 ppm for 72 h), the magnitude of the difference in sTNFR expression between IL-6+/+ and IL-6–/– mice is much greater than that observed in air-exposed controls, suggesting that IL-6 may be involved in regulating the expression of the sTNFRs, especially during prolonged O3 exposure. In support of this hypothesis, Tilg et al. (39) reported an increase in sTNFR1 in patients treated with exogenous IL-6 by intravenous infusion. We do not yet know the functional significance of the changes in sTNFRs observed in response to O3; however, one possibility is that IL-6 modulates the expression and/or activity of TNF-{alpha} converting enzyme, the enzyme responsible for cleaving TNFR1 and TNFR2 from the plasma membrane, and/or the expression of the receptors themselves.

In mice, transgenic overexpression of IL-6 in the lungs results in reduced airway responsiveness both at baseline and following antigen sensitization and challenge (9, 42). Conversely, AHR induced by antigen sensitization and challenge is enhanced in IL-6–/– mice. Together, these results suggest that IL-6 has the capacity to modulate airway responsiveness. Because IL-6 has been shown by many investigators, including ourselves, to be induced by O3 (3, 8, 17, 31, 33, 34, 41), we reasoned that IL-6 might be acting to attenuate O3-induced AHR. Our results indicate that this is not the case, since baseline airway responsiveness and O3-induced AHR were not different in IL-6+/+ and IL-6–/– mice, even though IL-6 mRNA was detectable following both room air and O3 exposure. In any event, it would not be surprising if IL-6 played a different role in allergen-induced vs. O3-induced AHR, since the role IL-6 in inflammatory or immune responses in the lung appears to depend critically on the precise stimulus used. For example, IL-6 plays an anti-inflammatory role in the lung response to Aspergillus fumigatus (5, 45), whereas it is required for optimal immune cell responses to viral infection (21, 30).

In addition to causing airway inflammation and AHR, exposure to O3 causes changes in the pattern of breathing. In rodents, one of the most profound aspects of this change in breathing pattern is an increase in EEP (32, 33, 35). We have previously reported that in mice, TNF receptors are required for O3-induced changes in EEP (35). Because we observed differences in sTNFR1 and sTNFR2 in IL-6–/– vs. IL-6+/+ mice following O3 exposure, we reasoned that IL-6 might also modify O3-induced changes in EEP. Our results indicated no significant genotype-related difference in EEP following acute O3 exposure, but a reduction in O3-induced changes in EEP in IL-6–/– mice following subacute exposure. These results indicate that IL-6 plays a role in O3-induced changes in breathing pattern following subacute but not acute O3 exposure. Whether or not IL-6-related changes in sTNFRs play a role in these events remains to be determined.

In summary, our results demonstrate that IL-6 promotes O3-induced injury and inflammation to the lungs, especially during subacute O3 exposure. In contrast, IL-6 does not play a role in O3-induced AHR, at least in response to 0.3 ppm O3.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Science to Achieve Results program of the U.S. Environmental Protection Agency (EPA) and NIH Grants HL-33009, ES-00002, and HL-07118. This research has not been subjected to any EPA review and therefore does not necessarily reflect the views of the Agency, and no official endorsement should be inferred.


   ACKNOWLEDGMENTS
 
The authors thank Dr. G. G. Krishna Murthy for assistance with the O3 exposure system. In addition, the authors thank Fallon A. Mattis and Todd A. Theman for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Johnston, Bldg. 1, Rm. 1304A, Physiology Program, Dept. of Environmental Health, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115-6021 (E-mail: rajohnst{at}hsph.harvard.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adler A, Cieslewicz G, and Irvin CG. Unrestrained plethysmography is an unreliable measure of airways responsiveness in BALB/c and C57BL/6 mice. J Appl Physiol 97: 286–292, 2004.[Abstract/Free Full Text]
  2. Agency, U.S. Environmental Protection. Air Quality Criteria for Ozone and Other Photochemical Oxidants. Research Triangle Park, NC: US EPA, 1986, vol. IV.
  3. Arsalane K, Gosset P, Vanhee D, Voisin C, Hamid Q, Tonnel AB, and Wallaert B. Ozone stimulates synthesis of inflammatory cytokines by alveolar macrophages in vitro. Am J Respir Cell Mol Biol 13: 60–68, 1995.[Abstract]
  4. Bates JH and Irvin CG. Measuring lung function in mice: the phenotyping uncertainty principle. J Appl Physiol 94: 1297–1306, 2003.[Abstract/Free Full Text]
  5. Cenci E, Mencacci A, Casagrande A, Mosci P, Bistoni F, and Romani L. Impaired antifungal effector activity but not inflammatory cell recruitment in interleukin-6-deficient mice with invasive pulmonary aspergillosis. J Infect Dis 184: 610–617, 2001.[CrossRef][ISI][Medline]
  6. Cho HY, Zhang LY, and Kleeberger SR. Ozone-induced lung inflammation and hyperreactivity are mediated via tumor necrosis factor-{alpha} receptors. Am J Physiol Lung Cell Mol Physiol 280: L537–L546, 2001.[Abstract/Free Full Text]
  7. Chong BT, Agrawal DK, Romero FA, and Townley RG. Measurement of bronchoconstriction using whole-body plethysmograph: comparison of freely moving versus restrained guinea pigs. J Pharmacol Toxicol Methods 39: 163–168, 1998.[CrossRef][ISI][Medline]
  8. Devlin RB, McKinnon KP, Noah T, Becker S, and Koren HS. Ozone-induced release of cytokines and fibronectin by alveolar macrophages and airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 266: L612–L619, 1994.[Abstract/Free Full Text]
  9. DiCosmo BF, Geba GP, Picarella D, Elias JA, Rankin JA, Stripp BR, Whitsett JA, and Flavell RA. Airway epithelial cell expression of interleukin-6 in transgenic mice. Uncoupling of airway inflammation and bronchial hyperreactivity. J Clin Invest 94: 2028–2035, 1994.[ISI][Medline]
  10. Evans TW, Brokaw JJ, Chung KF, Nadel JA, and McDonald DM. Ozone-induced bronchial hyperresponsiveness in the rat is not accompanied by neutrophil influx or increased vascular permeability in the trachea. Am Rev Respir Dis 138: 140–144, 1988.[ISI][Medline]
  11. Fauroux B, Sampil M, Quenel P, and Lemoullec Y. Ozone: a trigger for hospital pediatric asthma emergency room visits. Pediatr Pulmonol 30: 41–46, 2000.[CrossRef][ISI][Medline]
  12. Flandre TD, Leroy PL, and Desmecht DJ. Effect of somatic growth, strain, and sex on double-chamber plethysmographic respiratory function values in healthy mice. J Appl Physiol 94: 1129–1136, 2003.[Abstract/Free Full Text]
  13. Gent JF, Triche EW, Holford TR, Belanger K, Bracken MB, Beckett WS, and Leaderer BP. Association of low-level ozone and fine particles with respiratory symptoms in children with asthma. JAMA 290: 1859–1867, 2003.[Abstract/Free Full Text]
  14. Hamelmann E, Schwarze J, Takeda K, Oshiba A, Larsen GL, Irvin CG, and Gelfand EW. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am J Respir Crit Care Med 156: 766–775, 1997.[Abstract/Free Full Text]
  15. Hino T, Nakamura H, Abe S, Saito H, Inage M, Terashita K, Kato S, and Tomoike H. Hydrogen peroxide enhances shedding of type I soluble tumor necrosis factor receptor from pulmonary epithelial cells. Am J Respir Cell Mol Biol 20: 122–128, 1999.[Abstract/Free Full Text]
  16. Huang YF, Harrison JR, Lorenzo JA, and Kream BE. Parathyroid hormone induces interleukin-6 heterogeneous nuclear and messenger RNA expression in murine calvarial organ cultures. Bone 23: 327–332, 1998.[Medline]
  17. Johnston CJ, Stripp BR, Reynolds SD, Avissar NE, Reed CK, and Finkelstein JN. Inflammatory and antioxidant gene expression in C57BL/6J mice after lethal and sublethal ozone exposures. Exp Lung Res 25: 81–97, 1999.[CrossRef][ISI][Medline]
  18. Jones KD, Aoki Y, Chang Y, Moore PS, Yarchoan R, and Tosato G. Involvement of interleukin-10 (IL-10) and viral IL-6 in the spontaneous growth of Kaposi’s sarcoma herpes virus-associated infected primary effusion lymphoma cells. Blood 94: 2871–2879, 1999.[Abstract/Free Full Text]
  19. Kleeberger SR, Levitt RC, and Zhang LY. Susceptibility to ozone-induced inflammation. I. Genetic control of the response to subacute exposure. Am J Physiol Lung Cell Mol Physiol 264: L15–L20, 1993.[Abstract/Free Full Text]
  20. Kleeberger SR, Levitt RC, Zhang LY, Longphre M, Harkema J, Jedlicka A, Eleff SM, DiSilvestre D, and Holroyd KJ. Linkage analysis of susceptibility to ozone-induced lung inflammation in inbred mice. Nat Genet 17: 475–478, 1997.[CrossRef][ISI][Medline]
  21. Kopf M, Baumann H, Freer G, Freudenberg M, Lamers M, Kishimoto T, Zinkernagel R, Bluethmann H, and Kohler G. Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature 368: 339–342, 1994.[CrossRef][Medline]
  22. Koto H, Salmon M, Haddad el-B, Huang TJ, Zagorski J, and Chung KF. Role of cytokine-induced neutrophil chemoattractant (CINC) in ozone-induced airway inflammation and hyperresponsiveness. Am J Respir Crit Care Med 156: 234–239, 1997.[Abstract/Free Full Text]
  23. Kuhn C III, Homer RJ, Zhu Z, Ward N, Flavell RA, Geba GP, and Elias JA. Airway hyperresponsiveness and airway obstruction in transgenic mice. Morphologic correlates in mice overexpressing interleukin (IL)-11 and IL-6 in the lung. Am J Respir Cell Mol Biol 22: 289–295, 2000.[Abstract/Free Full Text]
  24. Lundblad LK, Irvin CG, Adler A, and Bates JH. A reevaluation of the validity of unrestrained plethysmography in mice. J Appl Physiol 93: 1198–1207, 2002.[Abstract/Free Full Text]
  25. Michalec L, Choudhury BK, Postlethwait E, Wild JS, Alam R, Lett-Brown M, and Sur S. CCL7 and CXCL10 orchestrate oxidative stress-induced neutrophilic lung inflammation. J Immunol 168: 846–852, 2002.[Abstract/Free Full Text]
  26. Mitzner W and Tankersley C. Interpreting Penh in mice. J Appl Physiol 94: 828–831, 2003.[Free Full Text]
  27. Noviski N, Brewer JP, Skornik WA, Galli SJ, Drazen JM, and Martin TR. Mast cell activation is not required for induction of airway hyperresponsiveness by ozone in mice. J Appl Physiol 86: 202–210, 1999.[Abstract/Free Full Text]
  28. O’Byrne PM, Walters EH, Gold BD, Aizawa HA, Fabbri LM, Alpert SE, Nadel JA, and Holtzman MJ. Neutrophil depletion inhibits airway hyperresponsiveness induced by ozone exposure. Am Rev Respir Dis 130: 214–219, 1984.[ISI][Medline]
  29. Petak F, Habre W, Donati YR, Hantos Z, and Barazzone-Argiroffo C. Hyperoxia-induced changes in mouse lung mechanics: forced oscillations vs. barometric plethysmography. J Appl Physiol 90: 2221–2230, 2001.[Abstract/Free Full Text]
  30. Ramsay AJ, Husband AJ, Ramshaw IA, Bao S, Matthaei KI, Koehler G, and Kopf M. The role of interleukin-6 in mucosal IgA antibody responses in vivo. Science 264: 561–563, 1994.[Abstract/Free Full Text]
  31. Samet JM, Hatch GE, Horstman D, Steck-Scott S, Arab L, Bromberg PA, Levine M, McDonnell WF, and Devlin RB. Effect of antioxidant supplementation on ozone-induced lung injury in human subjects. Am J Respir Crit Care Med 164: 819–825, 2001.[Abstract/Free Full Text]
  32. Shore SA, Abraham JH, Schwartzman IN, Murthy GG, and Laporte JD. Ventilatory responses to ozone are reduced in immature rats. J Appl Physiol 88: 2023–2030, 2000.[Abstract/Free Full Text]
  33. Shore SA, Johnston RA, Schwartzman IN, Chism D, and Krishna Murthy GG. Ozone-induced airway hyperresponsiveness is reduced in immature mice. J Appl Physiol 92: 1019–1028, 2002.[Abstract/Free Full Text]
  34. Shore SA, Rivera-Sanchez YM, Schwartzman IM, and Johnston RA. Responses to ozone are increased in obese mice. J Appl Physiol 95: 938–945, 2003.[Abstract/Free Full Text]
  35. Shore SA, Schwartzman IN, Le Blanc B, Murthy GG, and Doerschuk CM. Tumor necrosis factor receptor 2 contributes to ozone-induced airway hyperresponsiveness in mice. Am J Respir Crit Care Med 164: 602–607, 2001.[Abstract/Free Full Text]
  36. Tepper JS, Blanchard JD, and Pfeiffer JW. Validation of noninvasive measures of bronchoconstriction in mice. Am J Respir Crit Care Med 155: A160, 1997.
  37. Thorne PS and Karol MH. Assessment of airway reactivity in guinea pigs: comparison of methods employing whole body plethysmography. Toxicology 52: 141–163, 1988.[CrossRef][ISI][Medline]
  38. Tilg H, Dinarello CA, and Mier JW. IL-6 and APPs: anti-inflammatory and immunosuppressive mediators. Immunol Today 18: 428–432, 1997.[CrossRef][ISI][Medline]
  39. Tilg H, Trehu E, Atkins MB, Dinarello CA, and Mier JW. Interleukin-6 (IL-6) as an anti-inflammatory cytokine: induction of circulating IL-1 receptor antagonist and soluble tumor necrosis factor receptor p55. Blood 83: 113–118, 1994.[Abstract/Free Full Text]
  40. Tolbert PE, Mulholland JA, MacIntosh DL, Xu F, Daniels D, Devine OJ, Carlin BP, Klein M, Dorley J, Butler AJ, Nordenberg DF, Frumkin H, Ryan PB, and White MC. Air quality and pediatric emergency room visits for asthma in Atlanta, Georgia, USA. Am J Epidemiol 151: 798–810, 2000.[Abstract/Free Full Text]
  41. Vincent R, Vu D, Hatch G, Poon R, Dreher K, Guenette J, Bjarnason S, Potvin M, Norwood J, and McMullen E. Sensitivity of lungs of aging Fischer 344 rats to ozone: assessment by bronchoalveolar lavage. Am J Physiol Lung Cell Mol Physiol 271: L555–L565, 1996.[Abstract/Free Full Text]
  42. Wang J, Homer RJ, Chen Q, and Elias JA. Endogenous and exogenous IL-6 inhibit aeroallergen-induced Th2 inflammation. J Immunol 165: 4051–4061, 2000.[Abstract/Free Full Text]
  43. Ward NS, Waxman AB, Homer RJ, Mantell LL, Einarsson O, Du Y, and Elias JA. Interleukin-6-induced protection in hyperoxic acute lung injury. Am J Respir Cell Mol Biol 22: 535–542, 2000.[Abstract/Free Full Text]
  44. Watkinson WP, Highfill JW, Slade R, and Hatch GE. Ozone toxicity in the mouse: comparison and modeling of responses in susceptible and resistant strains. J Appl Physiol 80: 2134–2142, 1996.[Abstract/Free Full Text]
  45. Xing Z, Gauldie J, Cox G, Baumann H, Jordana M, Lei XF, and Achong MK. IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J Clin Invest 101: 311–320, 1998.[ISI][Medline]
  46. Yu M, Zheng X, Witschi H, and Pinkerton KE. The role of interleukin-6 in pulmonary inflammation and injury induced by exposure to environmental air pollutants. Toxicol Sci 68: 488–497, 2002.[Abstract/Free Full Text]
  47. Zhang LY, Levitt RC, and Kleeberger SR. Differential susceptibility to ozone-induced airways hyperreactivity in inbred strains of mice. Exp Lung Res 21: 503–518, 1995.[ISI][Medline]
  48. Zhao Q, Simpson LG, Driscoll KE, and Leikauf GD. Chemokine regulation of ozone-induced neutrophil and monocyte inflammation. Am J Physiol Lung Cell Mol Physiol 274: L39–L46, 1998.[Abstract/Free Full Text]



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