CXCR2 is essential for maximal neutrophil recruitment and methacholine responsiveness after ozone exposure
Richard A. Johnston,
Joseph P. Mizgerd, and
Stephanie A. Shore
Physiology Program, Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts
Submitted 22 March 2004
; accepted in final form 7 September 2004
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ABSTRACT
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Ozone (O3), a common air pollutant, induces airway inflammation and airway hyperresponsiveness. In mice, the neutrophil chemokines KC and macrophage inflammatory protein-2 (MIP-2) are expressed in the lungs following O3 exposure. The purpose of this study was to determine whether CXCR2, the receptor for these chemokines, is essential to O3-induced neutrophil recruitment, injury to lungs, and increases in respiratory system responsiveness to methacholine (MCh). O3 exposure (1 ppm for 3 h) increased the number of neutrophils in the bronchoalveolar lavage fluid (BALF) of wild-type (BALB/c) and CXCR2-deficient mice. However, CXCR2-deficient mice had significantly fewer emigrated neutrophils than did wild-type mice. The numbers of neutrophils in the blood and concentrations of BALF KC and MIP-2 did not differ between genotypes. Together, these data suggest CXCR2 is essential for maximal chemokine-directed migration of neutrophils to the air spaces. In wild-type mice, O3 exposure increased BALF epithelial cell numbers and total protein levels, two indirect measures of lung injury. In contrast, in CXCR2-deficient mice, the number of BALF epithelial cells was not increased by O3 exposure. Responses to inhaled MCh were measured by whole body plethysmography using enhanced pause as the outcome indicator. O3 exposure increased responses to inhaled MCh in both wild-type and CXCR2-deficient mice 3 h after O3 exposure. However, at 24 h after exposure, responses to inhaled MCh were elevated in wild-type but not CXCR2-deficient mice. These results indicate CXCR2 is essential for maximal neutrophil recruitment, epithelial cell sloughing, and persistent increases in MCh responsiveness after an acute O3 exposure.
inflammation; KC; lung; macrophage inflammatory protein-2; mouse
IN HUMANS, EXPOSURE TO OZONE (O3), a common air pollutant and powerful oxidant, causes substernal irritation, cough, decrements in pulmonary function, and airway hyperresponsiveness (AHR) to nonspecific bronchoconstricting agonists such as methacholine (MCh) (6, 10, 25, 33). Even O3 concentrations below the current U.S. Environmental Protection Agency standard are sufficient to initiate symptoms in children with asthma (30). For individuals with preexisting respiratory disease, emergency room visits and hospital admissions increase on days of high atmospheric O3 concentrations (14, 65). Thus it is important to understand the mechanisms by which the respiratory system responds to O3.
O3 inhalation causes airway injury and inflammation. Molecular changes include an increase in the production and/or expression of prostaglandins (PGE2, PGF2
, 6-keto-PGF1
, and 8-epi-PGF2
) (3, 32, 33, 36), cytokines (IL-1
, IL-6, and TNF-
) (4, 39, 54, 55, 59, 60), and chemokines [eotaxin, interferon-
-inducible protein (IP-10), monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1
, KC, and MIP-2] (20, 38, 39, 49, 59, 60, 69). Cellular changes include sloughing of epithelial cells and neutrophil emigration into the air spaces (5, 12, 16, 20, 22, 49, 5961, 69). This neutrophil recruitment may contribute to the pathophysiology of O3-induced airway dysfunction. For example, neutrophil depletion ameliorates O3-induced airway injury in rats (8), and in some studies, prevents O3-induced AHR (19, 53). However, other studies report that O3-induced AHR can be neutrophil independent (21, 42, 45, 54, 68). O3-induced recruitment of neutrophils to the air spaces is mediated by multiple regulatory mechanisms. For example, impairment of IL-1 or TNF-
signaling or complement activation significantly attenuates neutrophil recruitment to the air spaces following O3 exposure (7, 13, 16, 54, 55). In addition, blocking antibodies against diverse chemokines, including IP-10, KC, and MCP-3, attenuates O3-induced airway neutrophil recruitment in mice (49).
The chemokine receptor CXCR2 is a member of the G protein-coupled receptor superfamily and is expressed on neutrophils, monocytes, and T cells (52). In humans, IL-8 is one of several Glu-Leu-Arg (ELR)+ CXC chemokines capable of binding to CXCR2. In mice, the ELR+ CXC chemokines KC and MIP-2 serve as inducible CXCR2 ligands (52). CXCR2 mediates neutrophil chemotaxis in response to tissue injury and many types of infections (11, 15, 18, 40, 48, 51, 52, 62, 66). However, there are also pathways for neutrophil recruitment that are CXCR2 independent (2, 35).
The purpose of this study was to test the hypothesis that CXCR2 contributes to the emigration of neutrophils into the lungs following acute O3 exposure in mice. Wild-type and CXCR2-deficient mice, both on a BALB/c background, were exposed to O3 (1 ppm for 3 h). O3-induced injury to the lungs and inflammation were assessed by measurements of protein, epithelial cells, neutrophils, and neutrophil chemotactic factors in bronchoalveolar lavage fluid (BALF) collected 3 or 24 h after the cessation of O3 exposure. Respiratory responses to inhaled MCh were assessed using whole body plethysmography in wild-type and CXCR2-deficient mice following acute O3 exposure.
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MATERIALS AND METHODS
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Animals.
Male wild-type (BALB/cJ) and CXCR2-deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 69 wk of age. CXCR2-deficient mice were backcrossed onto a BALB/cJ background for at least eight generations. All mice were housed in microisolator cages within a rodent barrier facility where they were given food and water ad libitum, exposed to a 12-h light:dark cycle, and acclimated to their new environment for at least 1 wk before entering the experimental protocol at 813 wk of age. The body mass of wild-type mice was 27.6 ± 0.6 g, whereas that of CXCR2-deficient mice was also 27.6 ± 0.6 g. The Harvard Medical Area Standing Committee on Animals approved all of the experimental protocols used in this study.
O3 exposure.
Conscious mice were placed in individual wire mesh cages and exposed to either room air or 1 ppm O3 for 3 h in a 145-l stainless steel chamber with a Plexiglas door. Chamber temperature was 2025°C, and chamber humidity was 5060%. Air within the chamber was renewed at a rate of 16.5 changes/h. O3 was generated by passing oxygen (Airgas East, Salem, NH) through ultraviolet (UV) light that was subsequently mixed with room air in the chamber. The O3 concentration within the chamber was continuously sampled and monitored by a UV photometric O3 analyzer (model 49; Thermo Electron Instruments, Hopkinton, MA) that was calibrated by a UV photometric O3 calibrator (model 49PS; Thermo Electron Instruments).
Blood collection.
Mice were killed with an intraperitoneal injection of pentobarbital sodium, and blood was immediately collected from the heart via cardiac puncture with a heparinized 25-gauge needle attached to a syringe. The red blood cells were lysed with a cell lysis solution, and blood leukocytes were counted with a hemacytometer. Blood smears were prepared and stained with the Diff-Quik Stain Set (Dade Behring, Düdingen, Switzerland) to differentiate leukocytes and determine the number of blood neutrophils. At least 100 cells per mouse were counted under a light microscope for differential leukocyte analysis.
Bronchoalveolar lavage.
The animals were prepared for bronchoalveolar lavage immediately after the collection of blood. After exposing the trachea in situ, a small incision, distal to the larynx, was made in the trachea, and a 20-gauge FEP polymer catheter (Becton Dickinson, Franklin Lakes, NJ) attached to a syringe was inserted. The lungs were lavaged twice with 35 ml/kg of ice-cold lavage buffer, PBS containing 0.6 mM EDTA. During the first lavage, the lavage buffer was instilled and retrieved once, whereas during the second lavage, the lavage buffer was instilled and retrieved twice, pooled with the first lavagate, and stored on ice. Approximately 75% of the instilled lavage buffer was retrieved, and no differences in retrieval were noted between genotypes. Cells were pelleted by centrifugation, and the supernatants were collected and stored at 80°C. The cell pellets were resuspended in Hanks balanced salt solution (Sigma Chemical, St. Louis, MO). The total number of BALF cells was quantified using a hemacytometer. Cells were spun onto glass microscope slides using a Cytospin 3 Cytocentrifuge (Thermo Shandon, Pittsburgh, PA) and stained with Hema 3 (Biochemical Sciences, Swedesboro, NJ). At least 300 cells per mouse were counted under a light microscope for differential cell analysis.
Enzyme-linked immunosorbent and protein assays.
The levels of the neutrophil chemokines KC, MIP-2, IP-10, and JE/MCP-1 were measured using ELISAs according to the manufacturers instructions (R&D Systems, Minneapolis, MN). The manufacturer also indicates that the lower limit of detection ranged from 1.52.2 pg/ml for these ELISAs. Before ELISA analysis, the BALFs were clarified by centrifugation. The total BALF protein concentration was determined spectrophotometrically according to the Bradford protein assay procedure (Bio-Rad, Hercules, CA).
Whole body plethysmography.
Responses to aerosolized acetyl-
-methylcholine chloride (MCh; Sigma-Aldrich, St. Louis, MO) were assessed by whole body plethysmography (Buxco Electronics, Sharon, CT), as described previously (59, 61). The outcome indicator, enhanced pause (Penh), has been shown to correlate with pulmonary resistance and to be reduced by bronchodilators in some studies (17, 31, 63, 64); however, this relationship has been not demonstrated in other studies (1, 9, 24, 47, 50, 56). These studies, although 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.
Mice were placed awake, unrestrained, and uninstrumented into individual whole body plethysmographs. After at least a 20-min acclimation period in air, baseline Penh was recorded every 15 s for 15 min. Next, dose-response curves to inhaled MCh aerosol were generated as follows. First, an aerosol of PBS, as a vehicle control, was delivered to the animal for 1 min. After aerosol delivery ceased, Penh was measured for the next 10 min. Then, aerosols of MCh, in approximate half-log intervals from 0.1 mg/ml to 30 mg/ml, were delivered to the animal for 1 min at 10-min intervals, with Penh being recorded continuously during the interval. Because the animals peak response to PBS and MCh occurred between 3 and 7 min after the cessation of aerosol delivery, the average Penh value recorded during this period was taken as the animals response to that particular aerosolized agent. Aerosols were generated from an acorn nebulizer at an air flow of 10 l/min and introduced through a port at the top of each plethysmograph. Pre-O3 or -air responses to MCh were measured for each mouse
48 h before exposure to O3 or room air. Post-O3 or -air responses were measured 3 or 24 h after the cessation of O3 or room air exposure.
Protocol.
Forty-eight hours before either a 1 ppm O3 or a room air exposure, respiratory responsiveness to MCh was assessed in all wild-type and CXCR2-deficient mice. These animals were subsequently exposed during the morning to either 1 ppm O3 or room air for 3 h. Three hours after the cessation of O3 or room air exposure, respiratory responsiveness to MCh aerosol was assessed via whole body plethysmography. Immediately after MCh responsiveness was assessed, blood was collected, and BAL was performed on each animal. In a separate cohort of mice, MCh responsiveness was assessed, blood was collected, and BAL was performed 24 h after O3 exposure. No wild-type or CXCR2-deficient mice were examined 24 h after the cessation of room air exposure since we have demonstrated previously that there is no difference in MCh responsiveness or BALF profile between mice examined 3 or 24 h after room air exposure (unpublished observations).
Statistical analysis of results.
The effect of CXCR2 deficiency and O3 on BALF and blood parameters was assessed by factorial ANOVA. In these analyses, genotype (wild-type and CXCR2-deficient) and exposure (room air or O3) were the main effects. For the analyses on BALF and blood cells, values were logarithmically transformed to conform to a normal distribution. To determine the effect of O3 and CXCR2 deficiency on responses to MCh aerosol, the significance of changes in Penh was assessed by repeated measures ANOVA. STATISTICA software (StatSoft, Tulsa, OK) was used to perform all statistical analyses. The results are expressed as the arithmetic means ± SE, where n is the number of mice per treatment group. A P value <0.05 was considered significant.
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RESULTS
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Effect of CXCR2 deficiency on O3-induced neutrophil recruitment.
Figure 1A shows the effect of CXCR2 deficiency on the total number of BALF neutrophils 3 and 24 h after the cessation of O3 exposure and 3 h after exposure to room air. Three hours after O3 exposure, there was no difference in the number of BALF neutrophils between wild-type or CXCR2-deficient mice and their respective air-exposed controls, and there were no genotype-related differences in the number of BALF neutrophils. At 24 h post-O3, wild-type and CXCR2-deficient mice had significantly more BALF neutrophils than their air-exposed controls. However, at 24 h post-O3, CXCR2-deficient mice had significantly fewer BALF neutrophils than wild-type mice. At 24 h post-O3, when the numbers of BALF neutrophils were genotype dependent, there were no statistically significant differences between wild-type and CXCR2-deficient mice in the numbers of BALF macrophages (16.0 ± 2.0 and 14.2 ± 2.0 x 105, respectively) or total BALF cells (22.8 ± 4.1 and 16.6 ± 2.5 x 105, respectively). Thus CXCR2 is essential to maximal neutrophil recruitment observed 24 h, but not 3 h, after an acute (3-h) exposure to 1 ppm O3.

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Fig. 1. Total number of bronchoalveolar lavage fluid (BALF) neutrophils (A) and blood neutrophils (B) in wild-type (BALB/c) and CXCR2-deficient mice 3 or 24 h after the cessation of a 3-h exposure to either room air or 1 ppm ozone (O3). *P < 0.01 compared with genotype-matched, air-exposed controls. #P < 0.01 compared with CXCR2-deficient mice within the same exposure group.
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To determine whether or not the differences we observed in the number of BALF neutrophils were due to differences in the number of blood neutrophils, we quantified blood neutrophils in wild-type and CXCR2-deficient mice after room air and O3 exposure. There was no significant effect of genotype or O3 exposure on the total number of blood neutrophils in wild-type and CXCR2-deficient mice (Fig. 1B). Thus the defect in neutrophil recruitment due to CXCR2 deficiency did not result from inadequate delivery of circulating neutrophils to the lungs.
Effect of CXCR2 deficiency on O3-induced chemokine expression.
To determine whether the attenuation in neutrophil recruitment observed in CXCR2-deficient mice 24 h after O3 exposure was due to the inability of these mice to produce a sufficient neutrophil chemotactic signal, the levels of several neutrophil chemokines, including the CXCR2 ligands KC and MIP-2, the CXCR3 and CCR3 ligand IP-10, and the CCR2 ligand JE/MCP-1, were measured in BALF collected 3 and 24 h after exposure to O3 and 3 h after exposure to room air. Little or no chemokine expression was detectable in the BALF of air-exposed mice (Fig. 2). The levels of KC and MIP-2 increased 3 h after cessation of O3 exposure and declined by 24 h, whereas BALF IP-10 and JE/MCP-1 were sustained through 24 h (Fig. 2). There was no effect of genotype on the BALF levels of any chemokine measured.

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Fig. 2. Concentrations of neutrophil chemokines in the BALF of wild-type (BALB/c) and CXCR2-deficient mice 3 or 24 h after the cessation of a 3-h exposure to either room air or 1 ppm ozone (O3). The BALF concentrations of the CXCR2 ligands KC (A) and macrophage inflammatory protein-2 (MIP-2, B), the CXCR3 and CCR3 ligand interferon- -inducible protein-10 (IP-10, C), and the CCR2 ligand JE/monocyte chemotactic protein-1 (JE/MCP-1, D) were determined with enzyme-linked immunosorbent assays. n = 47 for each treatment group. *P < 0.001 compared with genotype-matched, air-exposed controls.
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Effect of CXCR2 deficiency on O3-induced injury to the lungs.
Because neutrophils can contribute to tissue injury (11, 44, 67), we determined whether diminished neutrophil recruitment to the air spaces of CXCR2-deficient mice after O3 exposure influenced the degree of O3-induced injury to the lungs. The numbers of epithelial cells in the BALF from wild-type mice, but not CXCR2-deficient mice, were significantly increased compared with air-exposed controls 24 h post-O3 (Fig. 3A), and there were significantly more BALF epithelial cells recovered from wild-type mice at this time point. The levels of total BALF protein were significantly elevated at 24 h post-O3 in mice of both genotypes, yet no genotype-related differences were observed (Fig. 3B).

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Fig. 3. Total number of BALF epithelial cells (A) and levels of BALF protein (B) from wild-type (BALB/c) and CXCR2-deficient mice 3 or 24 h after the cessation of a 3-h exposure to either room air or 1 ppm ozone (O3). N = 49 for each treatment group. *P < 0.01 compared with genotype-matched, air-exposed controls. #P < 0.05 compared with CXCR2-deficient mice within the same exposure group.
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Effect of CXCR2 deficiency on O3-induced changes in responses to MCh.
Before O3 exposure, there were no differences in Penh responses to inhaled MCh between wild-type and CXCR2-deficient mice (Fig. 4). Exposure to room air did not alter MCh responsiveness in either genotype (data not shown). Three hours after the cessation of O3 exposure, responses to MCh were increased in both wild-type and CXCR2-deficient mice (Fig. 4A), and there was no genotype-related difference in the magnitude of this O3-induced change in MCh responsiveness. However, 24 h after the cessation of O3 exposure, responses to MCh were still elevated in wild-type mice, whereas MCh responsiveness had returned to pre-O3 exposure levels in CXCR2-deficient mice (Fig. 4B). Thus O3-induced increases in Penh measures of MCh responsiveness 24 h after O3 exposure require CXCR2.

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Fig. 4. Enhanced pause (Penh) responses to aerosolized methacholine measured in wild-type (BALB/c) and CXCR2-deficient mice before and again either 3 (A) or 24 (B) h after the cessation a 3 h exposure to 1 ppm ozone (O3). n = 810 for each treatment group. *P < 0.005 compared with pre-O3 values in genotype-matched mice. #P < 0.02 compared with CXCR2-deficient mice within the same exposure group.
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DISCUSSION
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The results of this study indicate that CXCR2, the receptor for the neutrophil chemotactic factors KC and MIP-2, contributes to the majority of neutrophil emigration into the air spaces that occurs over the 24-h period following acute (3-h) exposure to 1 ppm O3. Our results also indicate that CXCR2 signaling, either directly or indirectly, contributes to O3-induced increases in Penh responses to MCh.
The O3 exposure regimen used in this study elicited a statistically significant recruitment of neutrophils to the air spaces of both wild-type and CXCR2-deficient mice 24 h after the cessation of O3 exposure (Fig. 1A). The number of BALF neutrophils was significantly greater in wild-type mice compared with CXCR2-deficient mice. The diminished neutrophil recruitment to the air spaces of CXCR2-deficient mice was not due to a reduction in the number of neutrophils in the blood (Fig. 1B). CXCR2 is a receptor for KC and MIP-2, which are potent neutrophil chemotactic factors (28, 29, 43) that we and others (12, 20, 38, 39, 49, 54, 55, 59, 60) observe to be induced by O3 exposure. In both wild-type and CXCR2-deficient mice, the levels of KC and MIP-2 peak 3 h after the cessation of O3 exposure, although maximal neutrophil recruitment does not occur until 24 h postexposure, demonstrating that the generation of these CXCR2 ligands precedes neutrophil recruitment. Other CXCR2 ligands may also contribute to CXCR2-dependent neutrophil recruitment elicited by O3. Together, these results indicate that CXCR2 signaling, likely from KC and MIP-2, is required for maximal O3-induced emigration of neutrophils to the air spaces.
CXCR2 deficiency did not completely abolish neutrophil recruitment to the air spaces after O3 exposure (Fig. 1A), indicating that CXCR2-independent mechanisms also play a role in O3-induced neutrophil recruitment. Levels of both the CXCR3 and CCR3 ligand IP-10 and the CCR2 ligand JE/MCP-1 were increased in the BALF of wild-type and CXCR2-deficient mice following O3 exposure. Both these chemokines can elicit neutrophil emigration (37, 49). Furthermore, blocking antibodies against IP-10 attenuates O3-induced neutrophil recruitment (49). Together, the results suggest that expression of chemokines, including IP-10, that activate receptors other than CXCR2 may mediate CXCR2-independent neutrophil recruitment to the air spaces of the lungs.
Attenuation of neutrophil recruitment to sites of inflammation has been shown to ameliorate tissue injury induced by some stimuli (8, 11, 44, 67). Upon exposure to O3, wild-type mice manifest significant epithelial cell sloughing and increased lung permeability, as demonstrated by an increase in total BALF protein levels, whereas CXCR2-deficient mice had only a marked increase in lung permeability. Because airway epithelial cells are an effective diffusion barrier when intact (23), it is reasonable to conclude that epithelial cell sloughing would increase lung permeability. However, in this study, increased lung permeability is independent of epithelial cell sloughing in CXCR2-deficient mice. Similar findings were reported from O3-exposed mice genetically deficient in either one or both TNF receptors (TNFR1 and TNFR2) or mice treated with an IL-1 receptor antagonist before O3 exposure (16, 55). Because there is no distinction in the degree of lung permeability between wild-type and CXCR2-deficient mice, it is unlikely that the differences in the number of BALF neutrophils observed between genotypes contribute to this response, a finding consistent with other studies (41, 57). Furthermore, reduced numbers of neutrophils in the air spaces have been associated with decreased epithelial cell sloughing (46). These results suggest that increased lung permeability and epithelial cell sloughing are coincident but independent events with distinct underlying mechanisms after exposure to O3.
The role of neutrophils in the development of O3-induced AHR is controversial. Some investigators observe an association between the number of neutrophils in the lung tissue with the degree of O3-induced AHR (19, 34, 53), whereas others do not (21, 42, 54, 68). Neutrophil depletion by cyclophosphamide, anti-neutrophil antibodies, or a blocking antibody against CD11b/CD18 decreases neutrophil recruitment but not AHR after O3 exposure (45, 54, 68), indicating the maximal neutrophil recruitment is not essential to O3-induced AHR. In contrast, neutrophil depletion by hydroxyurea or anti-neutrophil serum attenuates O3-induced AHR (19, 53), suggesting that neutrophils are essential to this airway pathophysiology.
Our data demonstrate that O3 increases responses to MCh even 3 h after O3 exposure when the number of neutrophils in the air spaces is not yet substantially elevated (Fig. 1) and that this early increase in MCh responsiveness is not affected by CXCR2 deficiency (Fig. 4A). O3-induced increases in response to MCh persist 24 h after O3 exposure in wild-type mice (Fig. 4B), at which time the number of neutrophils in the air spaces of wild-type mice is increased (Fig. 1A). However, at this time, CXCR2 deficiency leads to a reduction in both BALF neutrophils and responses to MCh (Figs. 1A and 4B). We interpret these data as evidence that acute O3 exposure induces an early and transient increase in MCh responsiveness that is independent of CXCR2 or neutrophil recruitment. However, our data suggest that the persistence of this increase in MCh responsiveness depends on CXCR2-mediated neutrophil recruitment. Similarly, a time-dependent role for neutrophils during the induction of allergic airway responses in mice has recently been reported by Taube and colleagues (62a).
Penh, the outcome indicator used in assessing responses to inhaled MCh in this study, is a dimensionless factor that describes the shape of the pressure excursions inside the plethysmograph, largely during expiration. Penh has been shown to correlate with airway resistance during MCh challenge in BALB/c mice and to reproduce changes in airway responsiveness induced by allergen sensitization and challenge in this same strain (1, 31). However, the relationship is strictly an empirical one without theoretical basis (47, 50). Penh can also be influenced by changes in the upper airways and in the pattern of breathing, and it may be that the correspondence between Penh and airway resistance, under certain circumstances, reflects concomitant effects of inhaled MCh on the airway smooth muscle, leading to changes in airway resistance, and on pulmonary receptors whose activation alters the pattern of breathing. Changes in airway resistance and in the pattern of breathing may also be mechanistically linked. Fredberg and colleagues (26, 27) have demonstrated that airway smooth muscle shortening is critically dependent on tidal stretching of the muscle during breathing: the smaller the tidal volume or the lower the breathing frequency, the higher the airway resistance. This link may be particularly important with O3 exposure in rodents, since these animals demonstrate a decrease in tidal volume and breathing frequency in response to O3 that is maintained for hours after cessation of exposure (58, 59, 61). Indeed, the effects of O3 on breathing pattern may account for the amplification of responses to MCh observed 3 h after cessation of O3, whereas other effects, including those dependent on neutrophils, may account for effects observed 24 h later (Fig. 4).
Two other studies have examined the role of neutrophils in O3-induced changes in MCh responsiveness in mice (54, 68). In both of these studies, O3-induced AHR persisted despite depletion of neutrophils using cyclophosphamide, an anti-neutrophil antibody, or anti-neutrophil serum. The investigators used C57BL/6 mice, rather than the BALB/c mice used in this study, and exposed animals to 2 ppm O3 for 3 h as opposed to 1 ppm O3 for 3 h. Together with the results of this study, the data suggest that strain differences and O3 dose, as well as the time course of the response, may determine the role of neutrophils in the development of O3-induced increases in airway responses to bronchoconstrictors. Indeed, the development of O3-induced AHR in mice is known to be strain dependent (68).
In summary, we conclude that in BALB/c mice, the majority of the neutrophil recruitment following acute O3 exposure is CXCR2 dependent. Furthermore, CXCR2 deficiency also attenuates the prolonged increase in MCh responsiveness after acute O3 exposure.
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GRANTS
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This work was supported by the Science to Achieve Results program of the U.S. Environmental Protection Agency (EPA) and National Institutes of Health Grants ES-00002, HL-33009, HL-07118, and HL-68153. 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.
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ACKNOWLEDGMENTS
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The authors thank Michal M. Lupa and Igor N. Schwartzman for excellent technical assistance with this study.
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FOOTNOTES
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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.
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REFERENCES
|
---|
- Adler A, Cieslewicz G, and Irvin CG. Unrestrained plethysmography is an unreliable measure of airway responsiveness in BALB/c and C57BL6 mice. J Appl Physiol 97: 286292, 2004.[Abstract/Free Full Text]
- Ajuebor MN, Zagorski J, Kunkel SL, Strieter RM, and Hogaboam CM. Contrasting roles for CXCR2 during experimental colitis. Exp Mol Pathol 76: 18, 2004.[CrossRef][ISI][Medline]
- Alexis N, Urch B, Tarlo S, Corey P, Pengelly D, OByrne P, and Silverman F. Cyclooxygenase metabolites play a different role in ozone-induced pulmonary function decline in asthmatics compared to normals. Inhal Toxicol 12: 12051224, 2000.[CrossRef][ISI][Medline]
- 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: 6068, 1995.[Abstract]
- Balmes JR, Aris RM, Chen LL, Scannell C, Tager IB, Finkbeiner W, Christian D, Kelly T, Hearne PQ, Ferrando R, and Welch B. Effects of ozone on normal and potentially sensitive human subjects. Part I. Airway inflammation and responsiveness to ozone in normal and asthmatic subjects. Res Rep Health Eff Inst: 137; discussion 8199, 1997.
- Balmes JR, Chen LL, Scannell C, Tager I, Christian D, Hearne PQ, Kelly T, and Aris RM. Ozone-induced decrements in FEV1 and FVC do not correlate with measures of inflammation. Am J Respir Crit Care Med 153: 904909, 1996.[Abstract]
- Barrett EG, Johnston C, Oberdorster G, and Finkelstein JN. Silica-induced chemokine expression in alveolar type II cells is mediated by TNF-
. Am J Physiol Lung Cell Mol Physiol 275: L1110L1119, 1998.[Abstract/Free Full Text]
- Bassett D, Elbon-Copp C, Otterbein S, Barraclough-Mitchell H, Delorme M, and Yang H. Inflammatory cell availability affects ozone-induced lung damage. J Toxicol Environ Health A 64: 547565, 2001.[CrossRef][ISI][Medline]
- Bates JH and Irvin CG. Measuring lung function in mice: the phenotyping uncertainty principle. J Appl Physiol 94: 12971306, 2003.[Abstract/Free Full Text]
- Beckett WS, McDonnell WF, Horstman DH, and House DE. Role of the parasympathetic nervous system in acute lung response to ozone. J Appl Physiol 59: 18791885, 1985.[Abstract/Free Full Text]
- Belperio JA, Keane MP, Burdick MD, Londhe V, Xue YY, Li K, Phillips RJ, and Strieter RM. Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury. J Clin Invest 110: 17031716, 2002.[Abstract/Free Full Text]
- Bhalla DK and Gupta SK. Lung injury, inflammation, and inflammatory stimuli in rats exposed to ozone. J Toxicol Environ Health A 59: 211228, 2000.[CrossRef][ISI][Medline]
- Bhalla DK, Reinhart PG, Bai C, and Gupta SK. Amelioration of ozone-induced lung injury by anti-tumor necrosis factor-
. Toxicol Sci 69: 400408, 2002.[Abstract/Free Full Text]
- Burnett RT, Brook JR, Yung WT, Dales RE, and Krewski D. Association between ozone and hospitalization for respiratory diseases in 16 Canadian cities. Environ Res 72: 2431, 1997.[CrossRef][ISI][Medline]
- Cacalano G, Lee J, Kikly K, Ryan AM, Pitts-Meek S, Hultgren B, Wood WI, and Moore MW. Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog. Science 265: 682684, 1994.[ISI][Medline]
- Cho HY, Zhang LY, and Kleeberger SR. Ozone-induced lung inflammation and hyperreactivity are mediated via tumor necrosis factor-
receptors. Am J Physiol Lung Cell Mol Physiol 280: L537L546, 2001.[Abstract/Free Full Text]
- 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: 163168, 1998.[CrossRef][ISI][Medline]
- Del Rio L, Bennouna S, Salinas J, and Denkers EY. CXCR2 deficiency confers impaired neutrophil recruitment and increased susceptibility during Toxoplasma gondii infection. J Immunol 167: 65036509, 2001.[Abstract/Free Full Text]
- DeLorme MP, Yang H, Elbon-Copp C, Gao X, Barraclough-Mitchell H, and Bassett DJ. Hyperresponsive airways correlate with lung tissue inflammatory cell changes in ozone-exposed rats. J Toxicol Environ Health A 65: 14531470, 2002.[CrossRef][ISI][Medline]
- Driscoll KE, Simpson L, Carter J, Hassenbein D, and Leikauf GD. Ozone inhalation stimulates expression of a neutrophil chemotactic protein, macrophage inflammatory protein 2. Toxicol Appl Pharmacol 119: 306309, 1993.[CrossRef][ISI][Medline]
- 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: 140144, 1988.[ISI][Medline]
- Fedan JS, Millecchia LL, Johnston RA, Rengasamy A, Hubbs A, Dey RD, Yuan LX, Watson D, Goldsmith WT, Reynolds JS, Orsini L, Dortch-Carnes J, Cutler D, and Frazer DG. Effect of ozone treatment on airway reactivity and epithelium-derived relaxing factor in guinea pigs. J Pharmacol Exp Ther 293: 724734, 2000.[Abstract/Free Full Text]
- Fedan JS, Van Scott MR, and Johnston RA. Pharmacological techniques for the in vitro study of airways. J Pharmacol Toxicol Methods 45: 159174, 2001.[CrossRef][ISI][Medline]
- 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: 11291136, 2003.[Abstract/Free Full Text]
- Foster WM, Brown RH, Macri K, and Mitchell CS. Bronchial reactivity of healthy subjects: 1820 h postexposure to ozone. J Appl Physiol 89: 18041810, 2000.[Abstract/Free Full Text]
- Fredberg JJ. Frozen objects: small airways, big breaths, and asthma. J Allergy Clin Immunol 106: 615624, 2000.[CrossRef][ISI][Medline]
- Fredberg JJ, Inouye DS, Mijailovich SM, and Butler JP. Perturbed equilibrium of myosin binding in airway smooth muscle and its implications in bronchospasm. Am J Respir Crit Care Med 159: 959967, 1999.[Abstract/Free Full Text]
- Frevert CW, Farone A, Danaee H, Paulauskis JD, and Kobzik L. Functional characterization of rat chemokine macrophage inflammatory protein-2. Inflammation 19: 133142, 1995.[ISI][Medline]
- Frevert CW, Huang S, Danaee H, Paulauskis JD, and Kobzik L. Functional characterization of the rat chemokine KC and its importance in neutrophil recruitment in a rat model of pulmonary inflammation. J Immunol 154: 335344, 1995.[Abstract/Free Full Text]
- 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: 18591867, 2003.[Abstract/Free Full Text]
- 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: 766775, 1997.[Abstract/Free Full Text]
- Hazbun ME, Hamilton R, Holian A, and Eschenbacher WL. Ozone-induced increases in substance P and 8-epi-prostaglandin F2
in the airways of human subjects. Am J Respir Cell Mol Biol 9: 568572, 1993.[ISI][Medline]
- Hazucha MJ, Madden M, Pape G, Becker S, Devlin R, Koren HS, Kehrl H, and Bromberg PA. Effects of cyclo-oxygenase inhibition on ozone-induced respiratory inflammation and lung function changes. Eur J Appl Physiol 73: 1727, 1996.[CrossRef]
- Holtzman MJ, Fabbri LM, OByrne PM, Gold BD, Aizawa H, Walters EH, Alpert SE, and Nadel JA. Importance of airway inflammation for hyperresponsiveness induced by ozone. Am Rev Respir Dis 127: 686690, 1983.[ISI][Medline]
- Huang C, De Sanctis GT, OBrien PJ, Mizgerd JP, Friend DS, Drazen JM, Brass LF, and Stevens RL. Evaluation of the substrate specificity of human mast cell tryptase
I and demonstration of its importance in bacterial infections of the lung. J Biol Chem 276: 2627626284, 2001.[Abstract/Free Full Text]
- Joad JP, McDonald RJ, Giri SN, and Bric JM. Ozone effects on mechanics and arachidonic acid metabolite concentrations in isolated rat lungs. Environ Res 66: 186197, 1994.[CrossRef][ISI][Medline]
- Johnston B, Burns AR, Suematsu M, Issekutz TB, Woodman RC, and Kubes P. Chronic inflammation upregulates chemokine receptors and induces neutrophil migration to monocyte chemoattractant protein-1. J Clin Invest 103: 12691276, 1999.[Abstract/Free Full Text]
- Johnston CJ, Reed CK, Avissar NE, Gelein R, and Finkelstein JN. Antioxidant and inflammatory response after acute nitrogen dioxide and ozone exposures in C57Bl/6 mice. Inhal Toxicol 12: 187203, 2000.[CrossRef][ISI][Medline]
- 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: 8197, 1999.[CrossRef][ISI][Medline]
- Kielian T, Barry B, and Hickey WF. CXC chemokine receptor-2 ligands are required for neutrophil-mediated host defense in experimental brain abscesses. J Immunol 166: 46344643, 2001.[Abstract/Free Full Text]
- Kleeberger SR and Hudak BB. Acute ozone-induced change in airway permeability: role of infiltrating leukocytes. J Appl Physiol 72: 670676, 1992.[Abstract/Free Full Text]
- 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: 234239, 1997.[Abstract/Free Full Text]
- Lee J, Cacalano G, Camerato T, Toy K, Moore MW, and Wood WI. Chemokine binding and activities mediated by the mouse IL-8 receptor. J Immunol 155: 21582164, 1995.[Abstract]
- Li X, Klintman D, Liu Q, Sato T, Jeppsson B, and Thorlacius H. Critical role of CXC chemokines in endotoxemic liver injury in mice. J Leukoc Biol 75: 443452, 2004.[Abstract/Free Full Text]
- Li Z, Daniel EE, Lane CG, Arnaout MA, and OByrne PM. Effect of an anti-Mo1 MAb on ozone-induced airway inflammation and airway hyperresponsiveness in dogs. Am J Physiol Lung Cell Mol Physiol 263: L723L726, 1992.[Abstract/Free Full Text]
- Longphre M and Kleeberger SR. Susceptibility to platelet-activating factor-induced airway hyperreactivity and hyperpermeability: interstrain variation and genetic control. Am J Respir Cell Mol Biol 13: 586594, 1995.[Abstract]
- Lundblad LK, Irvin CG, Adler A, and Bates JH. A reevaluation of the validity of unrestrained plethysmography in mice. J Appl Physiol 93: 11981207, 2002.[Abstract/Free Full Text]
- Mehrad B, Strieter RM, Moore TA, Tsai WC, Lira SA, and Standiford TJ. CXC chemokine receptor-2 ligands are necessary components of neutrophil-mediated host defense in invasive pulmonary aspergillosis. J Immunol 163: 60866094, 1999.[Abstract/Free Full Text]
- 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: 846852, 2002.[Abstract/Free Full Text]
- Mitzner W and Tankersley C. Interpreting Penh in mice. J Appl Physiol 94: 828831; author reply 831832, 2003.[Free Full Text]
- Moore TA, Newstead MW, Strieter RM, Mehrad B, Beaman BL, and Standiford TJ. Bacterial clearance and survival are dependent on CXC chemokine receptor-2 ligands in a murine model of pulmonary Nocardia asteroides infection. J Immunol 164: 908915, 2000.[Abstract/Free Full Text]
- Murphy PM. Neutrophil receptors for interleukin-8 and related CXC chemokines. Semin Hematol 34: 311318, 1997.[ISI][Medline]
- OByrne 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: 214219, 1984.[ISI][Medline]
- Park JW, Taube C, Joetham A, Takeda K, Kodama T, Dakhama A, McConville G, Allen CB, Sfyroera G, Shultz LD, Lambris JD, Giclas PC, Holers VM, and Gelfand EW. Complement activation is critical to airway hyperresponsiveness following acute ozone exposure. Am J Respir Crit Care Med 169: 726732, 2004.[Abstract/Free Full Text]
- Park JW, Taube C, Swasey C, Kodama T, Joetham A, Balhorn A, Takeda K, Miyahara N, Allen CB, Dakhama A, Kim SH, Dinarello CA, and Gelfand EW. IL-1 receptor antagonist attenuates airway hyperresponsiveness following exposure to ozone. Am J Respir Cell Mol Biol 30: 830836, 2004.[Abstract/Free Full Text]
- 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: 22212230, 2001.[Abstract/Free Full Text]
- Reinhart PG, Bassett DJ, and Bhalla DK. The influence of polymorphonuclear leukocytes on altered pulmonary epithelial permeability during ozone exposure. Toxicology 127: 1728, 1998.[CrossRef][ISI][Medline]
- Shore SA, Abraham JH, Schwartzman IN, Murthy GG, and Laporte JD. Ventilatory responses to ozone are reduced in immature rats. J Appl Physiol 88: 20232030, 2000.[Abstract/Free Full Text]
- 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: 10191028, 2002.[Abstract/Free Full Text]
- Shore SA, Rivera-Sanchez YM, Schwartzman IN, and Johnston RA. Responses to ozone are increased in obese mice. J Appl Physiol 95: 938945, 2003.[Abstract/Free Full Text]
- 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: 602607, 2001.[Abstract/Free Full Text]
- Sue RD, Belperio JA, Burdick MD, Murray LA, Xue YY, Dy MC, Kwon JJ, Keane MP, and Strieter RM. CXCR2 is critical to hyperoxia-induced lung injury. J Immunol 172: 38603868, 2004.[Abstract/Free Full Text]
- Taube C, Nick JA, Siegmund B, Duez C, Takeda K, Rha YH, Park JW, Joetham A, Poch K, Dakhama A, Dinarello CA, and Gelfand EW. Inhibition of early airway neutrophilia does not affect development of airway hyperresponsiveness. Am J Respir Cell Mol Biol 30: 837843, 2004.[Abstract/Free Full Text]
- Tepper JS, Blanchard JD, and Pfeiffer JW. Validation of noninvasive measures of bronchoconstriction in mice (Abstract). Am J Respir Crit Care Med 155: A160, 1997.
- Thorne PS and Karol MH. Assessment of airway reactivity in guinea pigs: comparison of methods employing whole body plethysmography. Toxicology 52: 141163, 1988.[CrossRef][ISI][Medline]
- 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: 798810, 2000.[Abstract]
- Tsai WC, Strieter RM, Mehrad B, Newstead MW, Zeng X, and Standiford TJ. CXC chemokine receptor CXCR2 is essential for protective innate host response in murine Pseudomonas aeruginosa pneumonia. Infect Immun 68: 42894296, 2000.[Abstract/Free Full Text]
- Welborn MB III, Moldawer LL, Seeger JM, Minter RM, and Huber TS. Role of endogenous interleukin-10 in local and distant organ injury after visceral ischemia-reperfusion. Shock 20: 3540, 2003.[CrossRef][ISI][Medline]
- Zhang LY, Levitt RC, and Kleeberger SR. Differential susceptibility to ozone-induced airway hyperreactivity in inbred strains of mice. Exp Lung Res 21: 503518, 1995.[ISI][Medline]
- 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: L39L46, 1998.[Abstract/Free Full Text]