CXCR2 is essential for maximal neutrophil recruitment and methacholine responsiveness after ozone exposure

Richard A. Johnston, Joseph P. Mizgerd, Stephanie A. Shore

Abstract

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, PGF, 6-keto-PGF, and 8-epi-PGF) (3, 32, 33, 36), cytokines (IL-1β, IL-6, and TNF-α) (4, 39, 54, 55, 59, 60), and chemokines [eotaxin, interferon-γ-inducible protein (IP-10), monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1α, 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-α 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.

MATERIALS AND METHODS

Animals.

Male wild-type (BALB/cJ) and CXCR2-deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 6–9 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 8–13 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 20–25°C, and chamber humidity was 50–60%. 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 manufacturer’s instructions (R&D Systems, Minneapolis, MN). The manufacturer also indicates that the lower limit of detection ranged from 1.5–2.2 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-β-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 animal’s 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 animal’s 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.

RESULTS

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 × 105, respectively) or total BALF cells (22.8 ± 4.1 and 16.6 ± 2.5 × 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.

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.

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.

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-γ-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 = 4–7 for each treatment group. *P < 0.001 compared with genotype-matched, air-exposed controls.

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).

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 = 4–9 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.

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.

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 = 8–10 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.

DISCUSSION

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.

GRANTS

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.

Acknowledgments

The authors thank Michal M. Lupa and Igor N. Schwartzman for excellent technical assistance with this study.

Footnotes

  • 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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View Abstract