The changing role of eosinophils in long-term hyperreactivity following a single ozone exposure
Bethany L. Yost,1
Gerald J. Gleich,4,5
David B. Jacoby,2,3 and
Allison D. Fryer1,3
Departments of 1Environmental Health Sciences and 2Medicine, Johns Hopkins University Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland; 3School of Medicine, Oregon Health and Science University, Portland, Oregon; 4Departments of Immunology and Medicine, Mayo Clinic and Foundation, Rochester, Minnesota; and 5Departments of Dermatology and Medicine, University of Utah Health Sciences Center, Salt Lake City, Utah
Submitted 8 October 2004
; accepted in final form 25 May 2005
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ABSTRACT
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Ozone hyperreactivity over 24 h is mediated by blockade of inhibitory M2 muscarinic autoreceptors by eosinophil major basic protein. Because eosinophil populations in the lungs fluctuate following ozone, the contribution of eosinophils to M2 dysfunction and airway hyperreactivity was measured over several days. After one exposure to ozone, M2 function, vagal reactivity, smooth muscle responsiveness, and inflammation were measured in anesthetized guinea pigs. Ozone-induced hyperreactivity to vagal stimulation persisted over 3 days. Although hyperreactivity one day after ozone is mediated by eosinophils, AbVLA-4 did not inhibit either eosinophil accumulation in the lungs or around the nerves or prevent hyperreactivity at this time point. Two days after ozone, eosinophils in BAL, around airway nerves and in lungs, were decreased, and neuronal M2 receptor function was normal, although animals were still hyperreactive to vagal stimulation. Depleting eosinophils with AbIL-5 prevented hyperreactivity, thus eosinophils contribute to vagal hyperreactivity by mechanisms separate from M2 receptor blockade. Three days after ozone, vagal hyperreactivity persisted, eosinophils were again elevated in BAL in lungs and around nerves, and M2 receptors were again dysfunctional. At this point, airway smooth muscle was also hyperresponsive to methacholine. Eosinophil depletion with AbIL-5, AbVLA-4, or cyclophosphamide protected M2 function 3 days after ozone and prevented smooth muscle hyperreactivity. However, vagal hyperreactivity was significantly potentiated by eosinophil depletion. The site of hyperreactivity, muscle or nerve, changes over 3 days after a single exposure to ozone. Additionally, the role of eosinophils is complex; they mediate hyperreactivity acutely while chronically may be involved in repair.
M2 muscarinic receptors; vagus nerves
EXPOSURE TO OZONE IS ASSOCIATED with inflammation and with exacerbations of asthma in children (51) and adults (45). However, the mechanism by which airway inflammation mediates ozone-induced hyperresponsiveness remains unclear. Although much of the research on ozone-induced inflammation and hyperreactivity has been directed at neutrophils (2, 42, 54), eosinophils are also increased in the lungs and nasal lavage of human subjects following exposure to ozone (34, 46). Eosinophils are increased in the lungs of animals exposed to ozone, and they appear in the lavage fluid within 1 h of ozone exposure and decline between 48 and 72 h later (29, 43, 58).
Ozone-exposed animals are hyperreactive to electrical stimulation of the vagus nerves. However, ozone does not induce hyperresponsiveness to intravenous methacholine in animals that have been vagotomized to block reflex bronchoconstriction (27, 49, 62). Thus, 24 h after ozone exposure, airway hyperreactivity occurs at the level of the airway parasympathetic nerves.
Release of acetylcholine from the parasympathetic nerves is limited by M2 muscarinic receptors on the nerves. These inhibitory M2 receptors normally provide powerful control over acetylcholine release since stimulation with agonists, such as pilocarpine, decreases bronchoconstriction by 7080%. Conversely, blockade of M2 receptor function with selective antagonists potentiates vagally induced bronchoconstriction 5- to 10-fold (24, 25). Decreased function of the neuronal M2 receptors is associated with airway hyperreactivity in virus-infected (26), antigen-challenged (22), ozone-exposed (49), and organophosphate-exposed animals (23, 36) and in humans with asthma (3, 41).
One of the mechanisms for loss of M2 receptor function is eosinophil activation. Eosinophil major basic protein (MBP) is an endogenous antagonist for M2 receptors (15, 31). We have demonstrated that depletion of eosinophils with an antibody to IL-5 or selective blockade of eosinophil MBP protected the function of the inhibitory M2 receptors and prevented airway hyperreactivity to vagal stimulation 24 h after exposure to ozone (62). Furthermore, acutely restoring M2 receptor function by neutralizing eosinophil MBP with heparin simultaneously reverses airway hyperreactivity (62). Thus eosinophil activation with release of MBP and subsequent blockade of neuronal M2 receptors is one mechanism of ozone-induced airway hyperreactivity 24 h after exposure to ozone.
The presence of eosinophils in the lungs appears to vary over hours and days following either antigen challenge or ozone exposure. After ozone exposure of guinea pigs, eosinophils decline over 14 h then significantly increase over 48 h and remain elevated at least 4 days (43, 58). In monkeys, the ozone-induced influx of eosinophils into the lungs also waxes and wanes over 3 days following ozone exposure (29). This is similar to the waxing and waning of eosinophils over 10 h after nasal antigen challenge of patients with allergic rhinitis (55) and over 72 h after Sephadex-induced hyperreactivity in guinea pigs (39).
These experiments were designed to test whether eosinophil populations in guinea pig lung fluctuate after exposure to ozone and to test whether changing eosinophil populations influence M2 receptor function and hyperreactivity after ozone exposure. We therefore examined airway reactivity 1, 2, and 3 days after a single exposure to ozone to test whether eosinophils, MBP, and neuronal M2 muscarinic receptors still mediate ozone-induced hyperreactivity over this longer time period.
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METHODS
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Specific pathogen-free female guinea pigs (350400 g) were shipped from Hilltop Lab Animals (Scottsdale, PA) in filtered crates, housed in high-efficiency particulate filtered air, and fed a normal diet. All protocols were approved by The Johns Hopkins University and Oregon Health and Science University Animal Care and Use Committee. Guinea pigs were exposed either to ozone (2 ppm) or to filtered air for 4 h as described previously (62). Airway physiology, M2 receptor function, airway inflammation of the lungs, and the number of eosinophils in the lungs and around the nerves were measured 1, 2, or 3 days after a single exposure to ozone.
Antibodies and drugs.
Antibodies and drugs directed at inhibiting eosinophils and their proteins [antibody to IL-5 (AbIL-5), antibody to very late activating antigen-4 (AbVLA-4), antibody to MBP (AbMBP), and cyclophosphamide] were all administered before and during ozone exposure. Heparin was administered acutely, during the physiological measurements. Eosinophils were inhibited or depleted from the lungs by pretreatment with AbIL-5 (Pharmingen, San Diego, CA; 240 µg/kg, ip 3 days before ozone exposure), AbVLA-4 (4 mg/kg ip, 2 h before ozone exposure and once daily thereafter), or cyclophosphamide (30 mg/kg ip every other day for 7 days before exposure to ozone). All these treatments either deplete eosinophils or inhibit eosinophil migration into the lungs as previously described (13, 20, 27). Eosinophil MBP was inhibited by pretreatment with AbMBP (1 ml ip, 2 h before ozone exposure and daily thereafter) (15) or removed by heparin (2,000 U/kg iv) 20 min before physiological measurements (21).
Anesthesia and measurement of pulmonary inflation pressure.
Guinea pigs were anesthetized with 1.5 g/kg urethane ip. The jugular veins were cannulated for administration of drugs. Both vagus nerves were cut, and the distal ends were placed on platinum electrodes under liquid paraffin. Animals were tracheostomized, ventilated with a positive pressure (constant volume 1 ml/100 g body wt and 100 breaths/min), and paralyzed with a constant infusion of succinylcholine (10 µg/kg iv). Blood pressure and heart rate were measured from a carotid artery cannula. Pulmonary inflation pressure (Ppi) was measured at the trachea. Bronchoconstriction was measured as the increase in Ppi over the pressure produced by the ventilator as previously described (12).
Measurement of vagally induced bronchoconstriction.
All animals were pretreated with guanethidine (10 mg/kg iv) to deplete noradrenaline 25 min before the start of the experiment. Electrical stimulation of both vagi (0.2-ms pulse width, 10 V, 125 Hz, 5-s duration at 2-min intervals) produced frequency-dependent bronchoconstriction and bradycardia due to release of acetylcholine onto muscarinic receptors since both responses could be abolished by atropine (1 mg/kg iv).
Measurement of neuronal M2 muscarinic receptor function.
Pilocarpine is a muscarinic agonist with some selectivity for neuronal overpostjunctional receptors (25, 35). Neuronal M2 muscarinic receptor function was measured as the ability of the agonist pilocarpine to inhibit vagally induced bronchoconstriction in a dose-dependent manner (25). Electrical stimulation of both vagus nerves (2 Hz, 0.2 ms, 520 V, 44 pulses/train at 1-min intervals) produced transient bronchoconstriction. Before administering pilocarpine, we chose voltages so that vagally induced bronchoconstriction was the same in all groups. To obtain matched bronchoconstrictions (24 ± 7 mmH2O in controls), lower voltages were used for ozone-exposed animals (6.7 ± 0.6 V) compared with control animals (10.3 ± 1.0 V), although these differences were not significant. The effect of pilocarpine on vagally induced bronchoconstriction was measured as the ratio of bronchoconstriction in the presence of pilocarpine to bronchoconstriction in the absence of pilocarpine.
Measurement of smooth muscle M3 muscarinic receptor function.
Methacholine (110 µg/kg iv)-induced bronchoconstriction was measured in vagotomized guinea pigs to assess M3 muscarinic receptor function on airway smooth muscle.
Histological evaluation of inflammatory cells.
At the end of each experiment, the lungs were lavaged via the tracheal cannula with four 10-ml aliquots of saline, and inflammatory cells were assessed in this bronchoalveolar lavage (BAL) fluid as previously described (20). The lungs were removed and fixed in 4% formalin in 0.1 M phosphate buffer. Transverse sections from each lobe were embedded in paraffin, and 6-µm sections were mounted on glass microscope slides. Nerves were stained with a rabbit polyclonal antibody to protein gene product 9.5 (PGP9.5; Biogenesis), and eosinophils were stained with a 1% solution of chromotrope 2R as previously described (10). The numbers of eosinophils within the walls of four different cartilaginous airways per animal were counted within a x40 lens width (Olympus B-2 microscope) around each airway starting at an obvious visual landmark on the slide (10). In addition, eosinophils within 810 µm of an airway nerve (
1 eosinophil width) were also counted. Thus the number of eosinophils per mm2 could be calculated for each treatment group, and the number of eosinophils associated with airway nerves could be derived.
Antibodies and drugs.
Mouse IgG1 anti-human VLA-4 monoclonal antibody that is cross-reactive with guinea pig VLA-4 was generated endotoxin free as described previously (60) and was a gift from Dr. Roy R. Lobb (Biogen, Cambridge, MA). Purified rabbit anti-guinea pig MBP was the same reagent as described previously (37). Chromotrope 2R, atropine, guanethidine, cyclophosphamide, pilocarpine, methacholine, succinylcholine, and urethane were purchased from Sigma (St. Louis, MO). Heparin was purchased from Elkins-Sinn (Cherry Hill, NJ). All drugs were dissolved and diluted in 0.9% NaCl except heparin, AbMBP, and AbVLA-4, which were used undiluted.
Statistics.
All data are expressed as means ± SE. Methacholine, frequency, and pilocarpine responses were analyzed using a two-way analysis of variance (ANOVA) for repeated measures. Baseline heart rates, blood pressures, Ppi, changes in Ppi (before pilocarpine administration), voltages used, and BAL were analyzed ANOVA (Statview 4.5; Abacus Concepts, Berkley, CA). A P value of 0.05 was considered significant.
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RESULTS
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Exposure to 2 ppm ozone for 4 h significantly increased baseline Ppi from 86 ± 6 mmH2O in air-exposed controls to 169 ± 15 mmH2O 1 day after ozone, 147 ± 6 mmH2O 2 days after ozone, and 125 ± 7 mmH2O 3 days after ozone. Treatment with AbIL-5, AbVLA-4, or cyclophosphamide did not affect the ozone-induced increase in Ppi at any time point. Resting heart rate (289 ± 7 beats/min in controls) and blood pressure (55 ± 3/28 ± 1 systolic/diastolic in mmHg in controls) were not affected by ozone at any time point or by treatment with AbIL-5, AbVLA-4, AbMBP, or cyclophosphamide.
Airway reactivity.
Electrical stimulation of the vagus nerves (125 Hz) caused a frequency-dependent bronchoconstriction that was significantly greater than control 1, 2, and 3 days after a single exposure to ozone (Fig. 1, left). The maximum increase in vagally induced bronchoconstriction occurred 1 day after ozone exposure. However, vagally induced bronchoconstriction was still significantly potentiated above control 2 and 3 days after exposure to ozone. The magnitude of the potentiation diminished over time.
In contrast, bronchoconstriction induced by intravenous methacholine was only potentiated 3 days after ozone (Fig. 1, right). Thus ozone-induced hyperreactivity is mediated only at the level of the parasympathetic nerves 1 and 2 days after ozone, whereas by 3 days after ozone, airway smooth muscle is also hyperresponsive.
M2 receptor function.
Neuronal M2 muscarinic receptors were tested using the agonist pilocarpine. In air-exposed controls, pilocarpine (1100 µg/kg iv) dose dependently inhibited vagally induced bronchoconstriction, demonstrating that the neuronal M2 receptors were functional (Fig. 2). One and 3 days after ozone, the ability of pilocarpine to inhibit vagally induced bronchoconstriction was completely abolished (Fig. 2), indicating that the M2 muscarinic receptors were no longer responding to agonists. However, 2 days after ozone exposure, the dose-response curve to pilocarpine was unchanged from air-exposed controls (Fig. 2). Thus neuronal M2 receptor function fluctuates over 3 days after a single ozone exposure.
Role of eosinophils in neuronal M2 dysfunction.
To test whether eosinophil recruitment is required for renewed loss of M2 function 3 days after ozone, animals were treated with an antibody to the adhesion molecule VLA-4. One day after ozone, treatment with AbVLA-4 failed to prevent M2 muscarinic receptor dysfunction (Fig. 3, left). However, this antibody also failed to decrease eosinophils in the BAL (data not shown), and histological examination of the lungs confirmed that the number of eosinophils in the conducting airways or around the airway nerves was not significantly inhibited by pretreatment with the VLA-4 antibody 1 day after ozone (see Fig. 10). Because we have already demonstrated that hyperreactivity and M2 dysfunction are mediated by eosinophils at this time point (62), it may be that acute hyperreactivity is mediated by eosinophils that are already resident in the lungs before exposure to ozone.

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Fig. 10. Eosinophils in the airways and around the nerves fluctuate over 3 days after exposure to ozone. Total eosinophils in the airways were measured in tissue sections of bronchi from guinea pigs. There were significantly more eosinophils in the airways (A) and around the nerves (B) of ozone-exposed guinea pigs (black columns, n = 57) compared with controls (white columns, n = 5) on days 1 and 3 after ozone. AbIL-5 (light gray columns; n = 5) inhibited the number of eosinophils in the airways and around the nerves of ozone-exposed guinea pigs, whereas AbVLA-4 (dark gray columns; n = 5) only inhibited eosinophil accumulation in the lungs and around the nerves 3 days after ozone. Data are expressed as the number of eosinophils per mm2 ± SE; *significantly different from control; significantly different from ozone exposed.
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In contrast to 1 day after ozone, histology demonstrated that the AbVLA-4 significantly decreased eosinophils in the airways and around the airway nerves on the third day after ozone exposure (see Fig. 10). This AbVLA-4-mediated decrease in eosinophils was sufficient to protect M2 receptor function 3 days postozone (Fig. 3, right). The role of eosinophils in renewed loss of M2 receptor function 3 days after ozone was confirmed since depletion of eosinophils with AbIL-5 or administration of heparin, which we have shown binds eosinophil MBP, protected or restored M2 receptor function, respectively (Fig. 4). Thus M2 dysfunction 3 days after ozone appears to follow a second wave of eosinophil influx into the lung.
Role of eosinophils in ozone-induced airway hyperreactivity.
Although hyperreactivity 2 days after ozone (Fig. 1) occurs independently of M2 receptor dysfunction (Fig. 2), it is mediated by eosinophils since depletion of eosinophils with AbIL-5 before ozone prevented development of vagal hyperreactivity. Furthermore, heparin acutely reversed vagal hyperreactivity 2 days postozone (Fig. 5, left). Thus at 1 and 2 days after ozone, depleting eosinophils or their proteins prevented or reversed hyperreactivity. Three-day postozone vagal hyperreactivity is mediated by positively charged proteins since heparin, which neutralizes charged proteins, acutely reversed hyperreactivity (Fig. 5, right). However, at this time point, eosinophils appear to have a novel role in that they are beneficial rather than the cause of hyperreactivity (Figs. 5 and 6).

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Fig. 5. Vagally induced bronchoconstriction (controls, ) was significantly increased 2 (left, circles) and 3 (left, triangles) days after a single exposure to ozone. Two days postozone exposure (left), pretreatment with AbIL-5 ( ) prevented, and heparin (crossed circles) reversed, vagal hyperreactivity. In contrast, whereas heparin still reversed hyperreactivity 3 days after ozone (right, crossed circles), depletion of eosinophils with AbIL-5 significantly potentiated vagally induced hyperreactivity (right, ). Control and untreated ozone groups are from Fig. 1. Each point is the mean ± SE of 5 animals; *significantly different from control.
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Three days after ozone exposure, depleting eosinophils significantly potentiated vagally induced hyperresponsiveness. Vagal hyperresponsiveness was significantly increased 3 days after exposure to ozone regardless of whether or not eosinophils were depleted with AbIL-5 (Fig. 5, right), AbVLA-4, or cyclophosphamide (Fig. 6).
Increased hyperreactivity in the absence of eosinophils must be mediated at the level of the nerves, since methacholine-induced bronchoconstriction was inhibited, not potentiated, by depletion of eosinophils (with AbIL-5). For these experiments, only an Ab to guinea pig MBP was used to test specifically the role of MBP. Since the Ab inhibited hyperreactivity, this would suggest that hyperreactivity at the airway smooth muscle was specifically mediated by MBP (AbMBP, Fig. 7).
BAL.
After ozone, there was a significant increase in neutrophils and eosinophils in BAL 1 and 3 days, but not 2 days, postozone exposure (Fig. 8). The eosinophil increase in lavage fluid 1 and 3 days postozone was significantly inhibited by pretreatment with AbIL-5 (Fig. 9). In contrast, treatment with AbVLA-4 did not inhibit the increase in eosinophils seen in BAL 3 days postozone, although histological analysis demonstrated a significant decrease in tissue eosinophils. Cyclophosphamide treatment significantly decreased macrophages, eosinophils, and neutrophils recovered in the lavage 3 days postozone. No treatment affected the number of lymphocytes in lavage fluids.

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Fig. 8. Eosinophils and neutrophils in the bronchoalveolar lavage were increased 1 day postozone (light gray columns) compared with control (white columns). Two days postozone, all inflammatory cells were decreased (dark gray columns). Three days postozone (black columns), there is a second significant increase in eosinophils and neutrophils in bronchoalveolar lavage. Each point is the mean ± SE of 58 animals; *significantly different from control.
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Fig. 9. Three days postozone, both eosinophils and neutrophils in the bronchoalveolar lavage were increased (black columns) compared with control (white columns). Effects of pretreatment with AbIL-5 (light gray columns), AbVLA-4 (medium gray columns), and cyclophosphamide (dark gray columns) in the 3-day postozone animals are shown. Each point is the mean ± SE of 58 animals; *significantly different from control; significantly different from 3-day postozone. Cycloph, cyclophosphamide.
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Histology.
Ozone induced a significant increase in eosinophils in the lungs at 1 and 3 days, but not at 2 days, postexposure (Fig. 10A). Similarly, eosinophils were also increased around the airway nerves at 1 and 3 days, but not at 2 days (Fig. 10B). AbIL-5 depleted eosinophils in the lung and around the airway nerves. AbVLA-4, however, did not significantly inhibit eosinophil recruitment into either the whole lung or airway nerves at 1 day postozone (Fig. 10). The AbVLA-4 was effective at inhibiting eosinophil recruitment to the lung and nerves at 3 days postozone.
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DISCUSSION
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This study demonstrates that following ozone, eosinophil number in the lung fluctuates. One day following ozone, eosinophils are increased in the BAL and in the lung tissues. Eosinophil numbers decline on the second day and are significantly increased in the BAL, airways, and around the airway nerves. Similar ozone-induced increases in eosinophils in the BAL have been previously reported (11, 29, 43, 50, 58).
This study confirms that ozone-induced hyperreactivity 1 day after ozone occurs at the level of the vagus nerves and is associated with loss of M2 receptor function mediated by eosinophils (27, 49, 62). Furthermore, these data show that the eosinophils mediating loss of M2 receptor function 1 day after ozone are not sensitive to VLA-4 since the AbVLA-4 did not decrease eosinophil number in the lungs or around the nerves or protect M2 receptor function. The effects of activated eosinophils last <48 h since 2 days postozone, eosinophils are no longer elevated in the lungs and neuronal M2 receptors are fully functional. Thus either eosinophil MBP has been metabolized or new M2 receptors have been synthesized between 24 and 48 h after ozone exposure. Eosinophils reappear in the lungs 3 days after ozone. Treatment with AbVLA-4, which prevents eosinophil migration into the lungs (20), was effective at inhibiting ozone-induced eosinophilia at this time point.
In contrast to vagally induced hyperreactivity, which declines over 3 days, the smooth muscle increases in its response to muscarinic agonists. One and 2 days postozone, there was no change in methacholine-induced bronchoconstriction in vagotomized guinea pigs (Fig. 1), whereas by 3 days postozone, bronchoconstriction induced by methacholine was significantly potentiated in vagotomized animals. The potentiation of methacholine is sufficient to account for the potentiation of vagally induced bronchoconstriction at day 3. Therefore, airway hyperreactivity postozone primarily resides in the vagus nerves 1 and 2 days postozone and primarily in the smooth muscle 3 days postozone.
The mechanisms underlying vagal hyperreactivity 2 and 3 days after ozone are no longer associated with loss of neuronal M2 receptor function. However, they are eosinophil mediated since depletion of eosinophils with AbIL-5 inhibited vagal hyperreactivity. That neutralization of charged substances with intravenous heparin (Fig. 5) acutely reversed hyperreactivity indicates that a charged product must be involved. Whether that product is eosinophil MBP is not known.
Hyperreactivity 3 days postozone is predominantly mediated at the level of the airway smooth muscle. Bronchoconstriction induced by intravenous methacholine, in animals vagotomized to eliminate the vagal reflex (4, 59), is potentiated 3 days postozone. This hyperreactivity to methacholine appears to be mediated directly by eosinophil MBP, since depletion of eosinophils and specific blockade of MBP prevent airway smooth muscle hyperreactivity 3 days postozone.
The mechanisms by which eosinophils cause smooth muscle hyperreactivity are unknown. MBP is cytotoxic to the airway epithelium (18). Furthermore, application of MBP to airway epithelium increases airway smooth muscle responsiveness to exogenous acetylcholine in vivo (7, 17) and in situ (61). Furthermore, eosinophil MBP causes direct, dose-related contraction of the airway smooth muscle (61). Therefore, by 3 days postozone, eosinophil MBP is augmenting the response of the airway smooth muscle to muscarinic agonists. These data demonstrate a physiological role of eosinophil MBP in vivo 3 days after ozone exposure, confirming the pharmacological observations that MBP increases smooth muscle responsiveness to muscarinic agonists (7, 61).
However, although eosinophils are increasing smooth muscle responsiveness, their major role 3 days postozone actually appears to be protective. Preventing the second influx of eosinophils significantly enhanced vagally induced hyperreactivity beyond that occurring in the presence of eosinophils. Depleting eosinophils with AbIL-5, preventing eosinophil migration into the lungs with AbVLA-4, or depleting granulocytes with the cytotoxic agent cyclophosphamide each worsened vagally induced bronchoconstriction beyond that seen with ozone alone. This increased vagal hyperreactivity with eosinophil depletion would have been even greater but for the fact that these treatments also decreased airway smooth muscle responsiveness and restored M2 receptor function. Thus, although eosinophils cause hyperreactivity immediately after ozone, they appear to be beneficial in maintaining or repairing the parasympathetic nerves.
It has long been noted that eosinophils are present in the airways of hyperreactive animals and of asthmatics (6, 9, 10), and it has been assumed that they are mediating hyperreactivity. However, repeated administration of polymyxin B to guinea pigs induces airway hyporesponsiveness that is associated with an increase in eosinophils in the lavage fluid (30). The data presented here demonstrate that eosinophils have multiple functions in the lungs, some of which are beneficial.
Recruitment of eosinophils to the nerves may be necessary for the normal growth, repair, or survival of the parasympathetic nerves. We have shown that the parasympathetic nerves express chemotactic factors for eosinophils, including eotaxin (14) and adhesion molecules VCAM and ICAM (47, 48). Eosinophils are clustered along the airway nerves in humans dying of asthma and in antigen-challenged animals (10). They are found associated with nerves in other chronic inflammatory conditions such as inflammatory bowel disease and multiple sclerosis (40, 53). With the exception that eosinophils are able to block neuronal M2 receptor function, their function at the nerves is largely unknown. Eosinophils produce nerve growth factor (52), which is a neurotrophic polypeptide essential for the development, survival, and function of neurons (8, 56). Production and release of nerve growth factor are enhanced in eosinophils by exposure to proinflammatory cytokines including IL-5 (33, 57). Nerve growth factor serum levels are elevated in patients with allergic disease (5, 57), possibly from eosinophils that are also elevated. Therefore, nerve growth factor released by eosinophils may play an important role in repair following allergic inflammation.
Eosinophils are not normally thought to contribute to ozone-induced hyperreactivity. However, ozone exposure increases expression of eotaxin (which is chemotactic for eosinophils) in rat lung (32, 44) and recruits eosinophils to the lungs and nose of humans exposed to ozone (34, 46). Furthermore, the concentration of eosinophil cationic protein in lavage fluid of asthmatic (28) and healthy individuals (19) is increased following ozone exposure, suggesting that, in humans, eosinophils are not only recruited but are also activated by ozone. Blocking ICAM, which we have demonstrated is key to the interaction of eosinophils with nerves (16, 47), decreases eosinophilia and inhibits ozone-induced hyperreactivity (38), possibly by preventing the interaction of eosinophils with the nerves. Finally, we have previously demonstrated that ozone-induced hyperreactivity can be prevented if eosinophils are depleted before exposure to ozone or if eosinophil products are neutralized with heparin 24 h after administration of ozone (62). Thus there is evidence that eosinophils are associated with ozone exposure and may contribute to ozone-induced airway hyperreactivity.
The data presented here show that the presence of eosinophils in the lungs changes over 3 days after ozone. Hyperreactivity is initially mediated by the eosinophils that are resident within the lungs. Over 3 days, these eosinophils have less effect and new eosinophils enter the lungs. These eosinophils induce a small degree of hyperreactivity at the level of the airway smooth muscle, which is masking a largely protective effect at the level of the airway nerves. Thus depleting eosinophils significantly exacerbated ozone-induced hyperreactivity 3 days after exposure. It might be telling that the protective effects of eosinophils assert themselves days after the initial injury. In another chronic model of inflammation, viral infection, eosinophils appear to have additional beneficial effects in that they are antiviral (1). It is possible that eosinophils contribute to normal lung function and that their presence, although harmful in the short term, is beneficial to a repair process in the long run.
Thus the effects of eosinophils on airway physiology after ozone exposure are complex. It should not come as a surprise that a cell as plentiful as the eosinophil in the airways of patients with asthma may have beneficial as well as deleterious effects. These data suggest that targeting specific eosinophil products, such as MBP, may be a more successful therapeutic strategy than approaches aimed at depleting eosinophils, which would deplete potentially beneficial neurotrophic factors critical to repair mechanisms.
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GRANTS
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This work was funded by National Institutes of Health Grants RO1-HL-55543 (A. D. Fryer), RO1-HL-54659 (D. B. Jacoby), HL-61013 (D. B. Jacoby), and AI-09728 (G. J. Gleich). The animals were exposed in a facility funded by the National Institute of Environmental Health Sciences (ES-03819).
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ACKNOWLEDGMENTS
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We acknowledge the generous donation of the antibody to very late activating antigen-4 from Dr. Roy R. Lobb (Biogen, Cambridge, MA).
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FOOTNOTES
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Address for reprint requests and other correspondence: A. D. Fryer, Dept. Physiology and Pharmacology, Oregon Health and Science Univ., 3181 SW Sam Jackson Park Road, Portland, OR 97034 (e-mail: fryera{at}ohsu.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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