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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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In antigen-challenged guinea pigs, airway hyperreactivity is due to
recruitment of eosinophils to the airway nerves and dysfunction of
M2 muscarinic receptors. M2 receptor dysfunction is caused by
eosinophil major basic protein, which is an allosteric antagonist at
the receptor. Because glucocorticoids inhibit airway hyperreactivity in humans and in animal models of asthma, we tested whether
dexamethasone treatment (6 µg · kg
1 · d1 for 3 d, intraperitoneal) before antigen challenge prevents M2 receptor dysfunction
and airway hyperreactivity. Guinea pigs were sensitized to ovalbumin via intraperitoneal injections, and were challenged with ovalbumin via inhalation. Twenty-four hours later, hyperreactivity and
M2 receptor function were tested. Antigen-challenged animals
were hyperreactive to vagal stimulation, and demonstrated loss of
M2 receptor function. Dexamethasone pretreatment prevented hyperreactivity and M2 receptor dysfunction in antigen-challenged guinea pigs. Antigen challenge resulted in recruitment of
eosinophils to the airways and to the airway nerves. Dexamethasone prevented recruitment of eosinophils to the airway nerves
but did not affect total eosinophil influx into the airways. These
results demonstrate that dexamethasone prevents antigen-induced
hyperreactivity by protecting neuronal M2 muscarinic receptors
from antagonism by eosinophil major basic protein, and this protective mechanism appears to be by specifically inhibiting eosinophil recruitment to the airway nerves.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Vagal parasympathetic neurons provide the dominant autonomic control of airway smooth muscle contraction (1). These fibers release acetylcholine (ACh) onto postjunctional M3 muscarinic receptors to cause smooth muscle contraction (2) and bronchoconstriction.
Antigen inhalation in sensitized animals results in airway hyperreactivity that is vagally mediated (3). Antigen-challenged guinea pigs (6) and rats (9) are hyperreactive to electrical stimulation of the vagus nerves. They are also hyperreactive to intravenous histamine when the vagus nerves are intact (5). Administering atropine, or cutting the vagus nerves, prevents hyperreactivity to histamine, indicating that the direct response of airway smooth muscle to histamine is unchanged by antigen challenge (5). Thus, antigen-induced airway hyperreactivity is mediated by the vagus nerves.
Release of ACh from the parasympathetic nerves is controlled by prejunctional M2 muscarinic autoreceptors located on the postganglionic parasympathetic nerves (10). These M2 receptors inhibit release of ACh (11), thus functioning as a negative feedback mechanism. The function of these neuronal M2 receptors can be tested with agonists (10). Stimulating these inhibitory receptors decreases ACh release and inhibits vagally induced bronchoconstriction. Conversely, blocking M2 receptors with selective antagonists such as gallamine removes the negative feedback control these receptors provide, thereby increasing ACh release. In pathogen-free guinea pigs gallamine potentiates vagally induced bronchoconstriction in a dose-related manner, increasing bronchoconstriction by as much as 10-fold, demonstrating that M2 receptors are functional (10).
In antigen-challenged guinea pigs, the M2 receptors are dysfunctional (7, 8, 12). Loss of M2 function is characterized by the inability of agonists to inhibit vagally induced bronchoconstriction and by inability of the ability of gallamine to potentiate bronchoconstriction (12). Antigen challenge does not, however, change the sensitivity of airway smooth muscle to ACh (8). Treatments that prevent M2 receptor dysfunction also prevent hyperreactivity (5, 7, 8, 14). Thus, hyperreactivity in antigen-challenged guinea pigs is due to loss of neuronal M2 muscarinic receptor function, and increased ACh release by the vagus nerves (11).
Antigen-induced loss of M2 receptor function is mediated by eosinophils. Antigen challenge results in an inflammatory response characterized by marked eosinophil influx (15, 16). In antigen-challenged guinea pigs and in humans who died during an asthma exacerbation, eosinophils are recruited to the airway nerves (8, 16). Depletion of eosinophils with an antibody to interleukin 5 (13) or inhibition of eosinophil migration into the airways with an antibody to very late activation antigen 4 (VLA-4 [7]) protects M2 receptor function in antigen-challenged guinea pigs and prevented hyperreactivity.
Eosinophils release the protein contents of their cytosolic granules in the airways of antigen-challenged guinea pigs (17, 18) and patients with asthma (16, 19, 20). Among these proteins, eosinophil major basic protein (MBP) is an allosteric antagonist for M2 muscarinic receptors in vitro (21). In vivo, blockade of MBP with a specific antibody prevents airway hyperreactivity (5, 8, 18) by protecting neuronal M2 receptor function (8). Thus, antigen-induced hyperreactivity is due to antagonism of neuronal M2 muscarinic receptors by MBP released from eosinophils.
Glucocorticoid treatment prevents airway hyperreactivity in antigen-challenged animals (22, 23) and in humans with asthma (24). In monkeys and in humans, in vivo prevention of hyperreactivity by glucocorticoids is associated with decreased eosinophil proteins in lavage fluid (23, 24). Because eosinophil recruitment, MBP release, and subsequent M2 receptor dysfunction are required for airway hyperreactivity in antigen-challenged guinea pigs (8, 13, 14), these studies were designed to test whether the glucocorticoid dexamethasone prevents hyperreactivity by protecting M2 receptor function in antigen-challenged guinea pigs.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
Specific pathogen-free female Dunkin-Hartley guinea pigs (250-300 g) were purchased from Hilltop Animal Farms (Scottsdale, PA). All animals were shipped in filtered crates and kept in high-efficiency particulate-filtered air. Guinea pigs were fed a normal diet (Prolab; Agway, Syracuse, NY) and were handled in accordance with the standards established by the U.S. Animal Welfare Acts set forth in National Institutes of Health guidelines and the "Policy and Procedures Manual" published by the Johns Hopkins University School of Hygiene and Public Health Animal Care and Use Committee.
Sensitization and Challenge with Antigen
On arrival, guinea pigs were divided into four groups (control, control
dexamethasone-pretreated, antigen-challenged, and antigen-challenged dexamethasone-pretreated groups). Guinea pigs were sensitized by injection with ovalbumin every other day for a total of three
injections (10 mg · kg1 · d1, intraperitoneal). Three weeks after the
last ovalbumin injection, sensitized animals were challenged with an
aerosol of 5.0% ovalbumin for 5 min or until signs of respiratory distress appeared, in which case antigen challenge was immediately
stopped. Some ovalbumin-sensitized guinea pigs were pretreated for 3 d
with dexamethasone (6 µg · kg1 · d1, intraperitoneal) with the last
dose administered 1 h before antigen challenge. Some nonsensitized
guinea pigs were also treated with dexamethasone (6 µg · kg1 · d1,
intraperitoneal) daily for 3 d (control dexamethasone group). Other
investigations in our laboratory have demonstrated that a larger dose
of dexamethasone (100 µg · kg1 · d1, intraperitoneal, for 2 d) suppresses vagal responsiveness and M2 receptor function in pathogen-free (control) guinea pigs (27). The dose of dexamethasone used in
these experiments was chosen on the basis of preliminary data demonstrating that it had no effect on vagal responsiveness in control
guinea pigs.
At the end of the experiments, sensitization was confirmed in
some randomly chosen animals by injecting ovalbumin (1.0 mg · kg1,
intravenous). This dose of ovalbumin caused acute anaphylaxis in all
sensitized animals regardless of dexamethasone pretreatment, but it
had no effect in nonsensitized controls.
Measurements of Pulmonary Inflation Pressure
Experiments were conducted 18-24 h after antigen challenge. The
guinea pigs were anesthetized with urethane (1.8 g · kg1, intraperitoneal). This dose produces a deep anesthesia lasting 8-10 h (28), although none of these experiments lasted for longer than 4 h.
The jugular veins were cannulated bilaterally for the administration of drugs. One internal carotid artery was cannulated for measurement of blood pressure, using a DTX pressure transducer (Viggo-Spectramed, Oxnard, CA). The heart rate was monitored via a tachograph
connected to the transducer used for the blood pressure measurements. The trachea was cannulated and the animals were ventilated
by a positive pressure, constant volume rodent respirator (Harvard
Apparatus, South Natick, MA). The animals were ventilated at a respiratory rate of 100 breaths · min1 with the tidal volume adjusted to
approximately 10 ml · kg1. The animals were paralyzed by continuous succinylcholine infusion (10 µg · kg1 · min1, intravenous). Pulmonary inflation pressure (Ppi) was measured via a sidearm off the
tracheal cannula, using a DTX pressure transducer (Viggo-Spectramed). A positive pressure of 102 ± 3 mm H2O (range, 80-150 mm
H2O) was needed to ventilate the animals. All signals were recorded
on a polygraph (Grass Instruments, Quincy, MA). Vagally induced
bronchoconstriction was measured as the increase in Ppi above the
basal inflation pressure produced by the ventilator (29). The sensitivity of this method was increased by taking the output Ppi signal from
the driver of one channel to the input of the preamplifier of a separate
channel on the polygraph. Using this method baseline Ppi was recorded on one channel and increases in Ppi above the baseline were
recorded on a separate channel, such that increases in Ppi as small as
2-3 mm H2O could be accurately recorded.
All animals were chemically sympathectomized with guanethedine
(30 mg · kg1, intravenous) in order to deplete norepinephrine stores
(30). Guanethedine produces a temporary tachycardia and hypertension that returns to baseline 20 min later. Experiments testing reactivity of the vagus nerves and function of the neuronal M2 muscarinic receptors were conducted after heart rate and blood pressure had returned to baseline.
Tests of Vagal Responsiveness
Anesthetized, ventilated, and paralyzed guinea pigs were vagotomized. The distal ends of the cut vagi were placed on platinum stimulating electrodes, which were attached to an electrical stimulator (model SD9; Grass Instruments). Electrical stimulation of the vagus nerves (2-15 Hz, 10 V, 0.1-ms pulse duration, for 5 s at 90-s intervals) produced frequency-dependent bronchoconstriction and bradycardia that recover on cessation of stimulation. Vagal reactivity was measured as an increase in Ppi in response to electrical stimulation of the nerves at increasing frequencies. Bronchoconstriction in response to these stimulus parameters was completely abolished by atropine (data not shown), confirming that vagally induced bronchoconstriction in these experiments was cholinergic.
Tests of Neuronal M2 Muscarinic Receptor Function
The cut vagi of anesthetized, ventilated, and paralyzed guinea pigs were stimulated electrically (15 Hz, 0.1-ms pulse duration, for 3 s at 60-s intervals). The voltage of electrical stimulation in each experiment (range, 3-25 V; mean, 9.3 ± 1.1 V) was adjusted in order to produce repeated bronchoconstrictions of similar magnitude (range, 8-24 mm H2O; mean 13.3 ± 0.8 mm H2O) before administration of the muscarinic antagonist gallamine. Stimulation of the vagi at these parameters caused endogenous activation of neuronal M2 muscarinic receptors by ACh released from the nerves.
Cumulative doses of gallamine (0.03-10.0 mg · kg1, intravenous)
were then administered, and the effects on vagally induced bronchoconstriction were measured on the polygraph. Gallamine blocked the
endogenous activation of M2 receptors by ACh, thereby potentiating
vagally induced bronchoconstriction by removing their inhibitory
function. The degree by which gallamine potentiated vagally induced
bronchoconstriction is therefore a measure of M2 receptor function.
These data are presented graphically as a ratio of bronchoconstriction
in the presence of increasing doses of gallamine over bronchoconstriction in the absence of gallamine.
Measurement of M3 Receptor Function
To test the sensitivity of airway smooth muscle to ACh, increasing
doses of ACh (1-10 µg · kg1, intravenous) were administered to vagotomized animals. ACh stimulates M3 muscarinic receptors on the airway smooth muscle to induce bronchoconstriction. The vagus nerves
were cut to eliminate any possible reflex induced by ACh (9, 31).
Bronchoalveolar Lavage
At the end of the experiments, guinea pigs were killed by an overdose of pentobarbital injected intravenously. The lungs were lavaged with five 10-ml aliquots of phosphate-buffered saline (PBS). The aliquots were combined to provide a final returned volume of 40-45 ml and centrifuged at 300 × g at 4° C for 10 min. The cells were resuspended in 10 ml of PBS and counted on a hemocytometer (Hausser Scientific, Hoarsham, PA). Aliquots of the cell suspension were also cytospun onto glass slides, stained with Diff-Quik (Baxter Healthcare, McGaw Park, IL), and counted.
Immunohistochemical Detection of Airway Nerves
After performing bronchoalveolar lavages, the lungs were excised from the thoracic cavity, rinsed briefly in PBS, and fixed in 4% formalin in 0.1 M phosphate buffer at 4° C overnight. The lungs were then rinsed in 0.1 M phosphate buffer (pH 7.0). Transverse sections 0.5 cm thick were cut from the proximal regions of each lobe, embedded in paraffin, and sectioned. Six-micrometer sections were mounted onto poly-L-lysine-coated glass microscope slides.
Airway nerves were detected immunohistochemically by using a rabbit polyclonal antibody to protein gene product 9.5 (PGP 9.5) as previously described (8, 16). Slides were dewaxed in xylene, and rehydrated through serial washings in graded alcohols, then soaked in PBS for 5 min. To obtain specific staining of PGP 9.5 in guinea pig tissues an antigen retrieval step must be performed. The tissues were treated with target unmasking fluid (TUF) equilibrated to 90° C in a water bath for 10 min and then allowed to cool to room temperature for 10 min. The slides were then washed in PBS at room temperature for 5 min. Endogenous peroxidase was quenched with 3% H2O2 (diluted in H2O) for 5 min. The sections were rinsed in PBS for 5 min, blocked with 10% normal goat serum-1% bovine serum albumin in PBS for 30 min, and rinsed briefly with PBS. The primary antibody to PGP 9.5 was diluted 1:4,000 in 1% bovine serum albumin (BSA) in PBS, and then overlaid onto the tissue sections and incubated at 4° C for 48 h. After incubation with the primary antibody, the sections were rinsed with PBS (3 × 5 min). The secondary antibody, biotinylated goat anti-rabbit IgG, was diluted 1:100 and incubated on the slides for 30 min at room temperature. The slides were rewashed with PBS (3 × 5 min) and incubated with a streptavidin-linked horseradish peroxidase substrate. The PGP 9.5 staining was visualized by incubating tissues with chromagen SG, which stains airway nerves blue-gray. In these studies, omission of the incubation step with the primary antibody served as a negative control.
The slides were incubated in a 1% solution of chromotrope 2R, which stains eosinophils red, for 30 min at room temperature. The slides were then washed in tap water (2 × 5 min) and were then dehydrated in graded ethanol solutions, defatted in xylene, and permanently mounted.
Histopathological Evaluation
In lung sections stained with antibody to PGP 9.5 and chromotrope 2R, the numbers of eosinophils within the walls of four different cartilaginous airways per animal were counted, using five animals per treatment group. The airways were first photographed with a video camera attached to an Olympus BH-2 microscope, and the diameters and the areas of the airways were measured with the Image Pro Plus software package (Media Cybernetics, Del Mar, CA). Under oil immersion, the numbers of eosinophils within the airway walls and below the basement membrane were then counted in 10 consecutive high-power fields, starting at an obvious visual landmark on the slide. Total eosinophils were counted. In addition, eosinophils within 10 µm of an airway nerve (approximately one eosinophil width) were also counted. Thus, the number of eosinophils per mm2 could be calculated for each treatment group, and the proportions of eosinophils associated (i.e., within 10 µm of a nerve) or not associated with airway nerves could be derived. These studies were performed by two investigators, and they were confirmed by a third, blinded investigator.
The 10-µm limit used to define eosinophil association with nerves is approximately the average diameter of a normodense eosinophil (32). We have previously shown that eosinophil recruitment to the airway nerves occurs in antigen-challenged guinea pigs and in patients who have died of asthma (8, 16). The method for measuring eosinophil association with airway nerves has been previously described in detail by Costello and coworkers (16). Released MBP is unlikely to diffuse far once in the extracellular matrix, because eosinophil MBP is cationic (pI 10.9), and it contains five unpaired cysteine residues per molecule, which readily form disulfide bonds (33). We therefore chose a distance to measure eosinophil association with airway nerves that is less than or equal to the width of an eosinophil.
Drugs and Chemicals
ACh chloride, atropine, bovine serum albumin, chromotrope 2R, dexamethasone (water soluble), gallamine, guanethedine, succinylcholine chloride, and urethane were all purchased from Sigma (St. Louis, MO). Ethanol, formalin solution, and xylene were purchased from Fisher Scientific (Suwanee, GA). Biotinylated goat anti-rabbit IgG, normal goat serum, ABC complex, and chromagen SG were all purchased from Vector Laboratories (Burlingame, CA). TUF was purchased form Signet Laboratories (Dedham, MA). Antibody to PGP 9.5 was purchased from Biogenesis (Sandown, NH).
Statistical Analyses
All data are expressed as means ± SEM. ACh, frequency, and gallamine response curves were analyzed by two-way analyses of variance (ANOVA) for repeated measures, and a p value < 0.05 was considered significant. Baseline heart rates, blood pressures, pulmonary inflation pressures, baseline changes in pulmonary inflation pressure (before treating with gallamine), and bronchoalveolar lavage were analyzed by two-way ANOVA with the Bonferroni-Dunn correction, and a p value of 0.0083 was considered significant. Histological counts and measurements were analyzed by two-way ANOVA, and a p value of 0.0167 was considered significant. All statistical analyses were made with the software package Statview 4.5 (Abacus Concepts, Berkley, CA).
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RESULTS |
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Baseline heart rates among groups were as follows: control, 273 ± 13 beats/min; control dexamethasone pretreated, 287 ± 8 beats/min; antigen challenged, 261 ± 7 beats/min; antigen challenged dexamethasone pretreated, 245 ± 5 beats/min. Baseline blood pressures (systolic/diastolic) among groups were as follows: control, 50/28 ± 1/3 mm Hg; control dexamethasone pretreated, 54/25 ± 3/3 mm Hg; antigen challenged, 46/24 ± 1/1 mm Hg; antigen challenged dexamethasone pretreated, 42/22 ± 0/2 mm Hg. Baseline Ppi values were as follows: control, 94 ± 3 mm H2O; control dexamethasone pretreated, 95 ± 3 mm H2O; antigen challenged, 117 ± 5 mm H2O; antigen challenged dexamethasone pretreated, 95 ± 5 mm H2O. There was a small, but statistically significant increase in baseline Ppi in antigen-challenged (untreated) guinea pigs compared with control, control dexamethasone-treated, and antigen-challenged dexamethasone-treated animals.
Effects of Dexamethasone on Airway Hyperreactivity
Electrical stimulation of the vagus nerves at increasing frequencies caused frequency-dependent bronchoconstriction (measured as increases in Ppi) in control animals (Figure 1). Antigen challenge resulted in a significant potentiation of vagally induced bronchoconstriction compared with controls (Figure 1A), demonstrating that antigen challenge causes airway hyperreactivity. Dexamethasone pretreatment prevented airway hyperreactivity in antigen-challenged guinea pigs (Figure 1A), because the responses were significantly different from those of antigen-challenged (untreated) guinea pigs (p < 0.0001) and not significantly different from control guinea pigs (p = 0.42). Dexamethasone treatment of control guinea pigs did not affect airway reactivity to vagal stimulation compared with untreated controls (Figure 1A; p = 0.96). There were no differences in vagally induced bradycardia among groups (Figure 1B).
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Effects of Dexamethasone on Neuronal M2 Muscarinic Receptor Function
For experiments testing M2 receptor function, the vagus nerves were stimulated electrically (15 Hz, 0.1-ms pulse duration, for 3 s) at 1-min time intervals. The voltages used in these experiments did not differ among groups (control, 10.1 ± 1.8 V; control dexamethasone pretreated, 8.5 ± 0.9 V; antigen challenged, 8.6 ± 2.1 V; antigen challenged dexamethasone pretreated, 9.5 ± 3.2 V). In the absence of gallamine, vagally induced bradycardia (control, 19.2 ± 8.4 beats/min; control dexamethasone pretreated, 20.0 ± 5.0 beats/min; antigen challenged, 24.3 ± 7.8 beats/min; antigen challenged dexamethasone pretreated, 25.8 ± 8.5 beats/min) and vagally induced bronchoconstriction (control, 12.1 ± 1.2 mm H2O; control dexamethasone pretreated, 13.2 ± 1.0 mm H2O; antigen challenged, 14.0 ± 2.2 mm H2O; antigen challenged dexamethasone pretreated, 14.0 ± 1.3 mm H2O) were also not significantly different among groups.
In untreated, pathogen-free guinea pigs, gallamine potentiated vagally induced bronchoconstriction in a dose-dependent
manner, with a maximal potentiation of approximately 7-fold
at 10 mg · kg1, intravenous (Figure 2A), demonstrating that control animals had a functional neuronal M2 muscarinic receptor.
In antigen-challenged guinea pigs, however, the ability of gallamine to potentiate vagally induced bronchoconstriction was
significantly reduced compared with controls (Figure 2A). In
the antigen-challenged animals gallamine (10 mg · kg1) potentiated vagally induced bronchoconstriction only 3-fold, demonstrating loss of M2 receptor function after antigen challenge (Figure 2A). Pretreating antigen-challenged guinea pigs
with dexamethasone prevented M2 receptor dysfunction, because the ability of gallamine to potentiate vagally induced
bronchoconstriction was the same as in controls (Figure 2A;
maximal effect approximately 7.5-fold increase). Dexamethasone pretreatment in control pathogen-free animals did not
affect M2 receptor function, because the effect of gallamine
on vagally induced bronchoconstriction was identical to control (untreated) animals (Figure 2A; maximal effect approximately 7-fold increase).
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Stimulation of the vagus nerves caused bradycardia via stimulation of cardiac M2 receptors. In all groups gallamine caused a dose-dependent decrease in vagally induced bradycardia. The effect of gallamine on vagally induced bradycardia did not differ among groups (Figure 2B).
Effects of Dexamethasone on Responsiveness of Airway Smooth Muscle to Acetylcholine
ACh causes dose-dependent bronchoconstriction in control, vagotomized animals by stimulating M3 muscarinic receptors on airway smooth muscle. There were no significant differences among ACh dose-response curves in control, antigen-challenged animals, and antigen-challenged animals pretreated with dexamethasone (Figure 3). In control guinea pigs treated with dexamethasone, bronchoconstriction induced by intravenous ACh was also not different from controls (n = 2, data not shown).
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Effects of Dexamethasone on Recovery of Inflammatory Cells from Bronchoalveolar Lavage Fluid
Antigen challenge resulted in a significant increase in the total number of cells recovered from bronchoalveolar lavage (BAL) compared with controls (Figure 4). Dexamethasone treatment before antigen challenge did not reduce this increase in inflammatory cells. In addition, dexamethasone did not alter the total number of cells in BAL in control (nonchallenged) animals compared with control (untreated) animals. Antigen challenge resulted in an increase in all cell types (macrophages, lymphocytes, eosinophils, and neutrophils) recovered from BAL (Figure 4). Dexamethasone pretreatment did not reduce the number of any of these cell types recovered from BAL.
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Effects of Dexamethasone on Eosinophil Recruitment to the Airways and Airway Nerves
In sections of guinea pig airways, nerves were stained blue-gray immunohistochemically for PGP 9.5, and eosinophils were stained red with chromotrope 2R. Cartilaginous airways of similar size were chosen under low magnification for further analysis. The sizes of these airways were as follows: control, 912 ± 115 µm in diameter, 248 ± 40 µm2 in wall thickness; antigen challenged, 954 ± 111 µm in diameter, 305 ± 38 µm2 in wall thickness; antigen challenged dexamethasone pretreated, 988 ± 86 µm in diameter, 325 ± 47 µm2 in wall thickness. Neither the diameter nor the thickness of the airways measured in these experiments differed among treatment groups.
In control guinea pigs, there was a resident population of eosinophils within the airway wall (Figures 5 and 6). After antigen challenge, the number of eosinophils within the airway wall increased significantly (Figures 5 and 6). Pretreating guinea pigs with dexamethasone did not affect the antigen induced influx of eosinophils into the airways (Figures 5 and 6).
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In antigen-challenged guinea pigs, eosinophils accumulated around the nerves in the airways compared with controls (Figures 6 and 7). However, dexamethasone pretreatment inhibited the recruitment of eosinophils to the airway nerves of antigen-challenged guinea pigs (Figures 6 and 7). In control guinea pigs, 29% of the resident eosinophils within the airway wall were associated with airway nerves, whereas in antigen-challenged guinea pigs, 42% of the eosinophils in the airway wall were associated with the nerves. In dexamethasone-pretreated antigen-challenged guinea pigs, only 20% of the eosinophils within the airways were associated with the airway nerves.
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significantly different from antigen-challenged dexamethasone-treated animals.
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Antigen challenge of sensitized guinea pigs causes airway hyperreactivity and neuronal M2 muscarinic receptor dysfunction (Figures 1 and 2). The airways of antigen-challenged guinea pigs are hyperreactive to electrical stimulation of the vagus nerves (Figure 1A). In addition, in antigen-challenged guinea pigs the ability of gallamine to potentiate vagally induced bronchoconstriction is markedly suppressed compared with controls, indicating that neuronal M2 muscarinic receptors are dysfunctional (Figure 2A). These data confirm previous studies of antigen-challenged guinea pigs (7, 8, 12).
Dexamethasone pretreatment (3 d, 6 µg · kg1 · d1) does
not inhibit frequency responses in control guinea pigs, but it
does inhibit frequency responses in antigen-challenged guinea
pigs (Figure 1A). Therefore, dexamethasone prevents airway
hyperreactivity, confirming previous studies in antigen-challenged guinea pigs (34), monkeys (23), dogs (22), mice (35),
and humans (26, 36, 37).
Dexamethasone does not inhibit vagally induced bronchoconstriction via a decrease in airway reactivity at the level of M3 muscarinic receptors (Figure 3). The ability of intravenous ACh to cause bronchoconstriction in vagotomized guinea pigs is unaltered by antigen challenge (Figure 3), confirming previous studies (8). Dexamethasone treatment of control or of antigen-challenged guinea pigs also does not affect the responsiveness of the airways to ACh (Figure 3). Thus, neither antigen challenge nor dexamethasone alters the responses mediated by M3 muscarinic receptors on airway smooth muscle to ACh.
Dexamethasone pretreatment prevents neuronal M2 muscarinic receptor dysfunction in antigen-challenged guinea pigs (Figure 2A). The ability of gallamine to potentiate vagally induced bronchoconstriction in dexamethasone-pretreated antigen-challenged guinea pigs is protected, and it is not different from controls (Figure 2A). Dexamethasone has no effect on gallamine dose-response curves in control (nonchallenged) guinea pigs (Figure 2A). Therefore, dexamethasone protects neuronal M2 muscarinic receptor function in antigen-challenged guinea pigs.
The dose of dexamethasone used in these experiments, 6 µg · kg1 · d1 for 3 d, did not change vagal responses or M2 muscarinic receptor function in the lungs (Figures 1A and 2A). Interestingly, a higher dose of dexamethasone (100 µg · kg1 · d1, intraperitoneal, for 2 d) decreases vagal responsiveness
by increasing M2 receptor function in pathogen-free (control)
guinea pigs (27). Thus, it appears that dexamethasone can alter M2 muscarinic receptor function by multiple mechanisms.
Vagal responses and M2 muscarinic receptor function in the heart are unaltered by antigen challenge, as previously demonstrated (7, 8, 12). M2 muscarinic receptor function in the heart is also unaffected by dexamethasone because there are no differences in vagal responses (Figure 1B) or in the ability of gallamine to block cardiac M2 muscarinic receptors among treatment groups (Figure 2B).
Antigen-induced hyperreactivity is vagally mediated in guinea pigs (5) and in rats (9), and it is the result of neuronal M2 muscarinic receptor dysfunction (5, 7). In antigen-challenged guinea pigs, hyperreactivity and loss of M2 muscarinic receptor function are due to blockade of M2 receptors by the endogenous antagonist eosinophil MBP (8, 21).
Eosinophils are recruited into the airways and to the airway nerves of antigen-challenged guinea pigs (7, 13, 14, 16). Recruitment of eosinophils to the airways is required for antigen-induced M2 receptor dysfunction, because inhibition of eosinophil influx into the airways protects M2 receptor function and prevents airway hyperreactivity in antigen-challenged guinea pigs (7, 13).
Dexamethasone prevents M2 receptor dysfunction (Figure 2A) and airway hyperreactivity (Figure 1A) after antigen challenge. This protective effect is not accompanied by any change in the total numbers of eosinophils in the lungs of dexamethasone-pretreated antigen-challenged guinea pigs as measured in BAL (Figure 4) or by histology (Figure 5). However, recruitment of eosinophils to the airway nerves in antigen-challenged guinea pigs is markedly reduced by dexamethasone (Figures 6 and 7). In antigen-challenged guinea pigs, there are more eosinophils in the airways (Figure 5), and nearly half of them are associated with airway nerves (Figures 6 and 7). In contrast, although dexamethasone does not prevent total eosinophil influx into the airways after antigen challenge (Figure 5), only 20% of the eosinophils are associated with airway nerves (Figure 7). Therefore, the mechanism by which dexamethasone prevents M2 muscarinic receptor dysfunction and hyperreactivity in antigen-challenged guinea pigs may be that dexamethasone inhibits recruitment of eosinophils to the airway nerves.
These studies also show that total eosinophil numbers as measured in BAL or even by histology do not necessarily reflect the mechanism of airway hyperreactivity (38). Blocking MBP (8) or its release by eosinophils (14) prevents M2 receptor dysfunction and hyperreactivity without inhibiting eosinophil recruitment to the lungs. Thus, using BAL as a measure of antigen challenge can be misleading unless further measures confirm changes in the distribution or the activation of eosinophils in the tissues.
The exact mechanism of eosinophil recruitment to the airway nerves of antigen-challenged guinea pigs is not known.
Because eosinophil recruitment to the airway nerves also occurs in antigen-challenged rats (9), as well as in humans who
died of asthma (16), it is likely that there is a common mechanism across species. Guinea pig airway parasympathetic nerves
grown in culture express eotaxin (39), which is a potent and
selective eosinophil chemokine (40). In antigen-challenged
guinea pigs, a large dose of dexamethasone (40 mg · kg1, intraperitoneal) inhibits eosinophil recruitment but not eotaxin expression in the whole lung (41). However, nanomolar concentrations of dexamethasone suppress eotaxin expression in
the A549 bronchial epithelial cell line in vitro (42) and in human rhinitis patients topical beclomethasone (400 µg · d1)
suppresses eotaxin expression measured in nasal biopsies (43). It is conceivable, then, that additional studies may demonstrate that the low dose of dexamethasone used in these studies (6 µg · kg1 · d1, intraperitoneal, for 3 d) does inhibit eotaxin expression in the lungs of antigen-challenged guinea
pigs. Alternatively, a role for eosinophil adhesion molecule
expression by airway parasympathetic nerves has been suggested. Intercellular adhesion molecule 1 (ICAM-1) is expressed by guinea pig tracheal parasympathetic nerves in culture (44). In accordance with the effects of dexamethasone observed in these experiments (see Figures 6 and 7), there may be specific regulation of eotaxin or ICAM-1 in the airway
nerves of antigen-challenged guinea pigs in response to glucocorticoid treatment rather than a gross antiinflammatory effect as is seen with much larger doses.
We have previously demonstrated that eosinophils are recruited to the airway nerves in antigen-challenged guinea pigs, where they cause hyperreactivity by releasing the M2 muscarinic receptor antagonist MBP (5, 8, 21). Studies from our laboratory have shown that substance P, which is released by tachykinergic nerves running adjacent to the parasympathetic nerves in the airways, induces eosinophil degranulation and subsequent M2 receptor dysfunction via NK1 receptor stimulation (14, 38). Because eosinophil MBP is a strongly charged and highly reactive molecule that does not diffuse freely, the eosinophils are required to transport MBP to the nerves. Neurotransmitters, namely substance P, then activate the eosinophils, causing the release of MBP onto neuronal M2 muscarinic receptors. Thus, eosinophil recruitment to the airway nerves of antigen-challenged guinea pigs appears to be a required step for neuronal M2 muscarinic receptor dysfunction and airway hyperreactivity.
In conclusion, dexamethasone prevents airway hyperreactivity in antigen-challenged guinea pigs, by inhibiting eosinophil recruitment and, therefore, protecting neuronal M2 muscarinic receptor function. The protection of M2 receptor function by dexamethasone appears to be due to specific inhibition of eosinophil recruitment to the nerves of antigen-challenged guinea pigs and not merely to inhibition of eosinophil influx into the airways. Because dexamethasone prevents antigen-induced airway hyperreactivity without affecting the total number of eosinophils in the airways, the presence of eosinophils in the airways of antigen-challenged guinea pigs is not sufficient to cause hyperreactivity, unless the eosinophils are recruited to the nerves.
Glucocorticoids have long been used as treatments for
asthma (25, 26, 36, 37). Their effect has been attributed mainly to their antiinflammatory effects in humans (25, 26) and in animal models (22, 23, 45). These studies demonstrate that the
protective mechanism of a low dose of dexamethasone (6 µg · kg1 · d1 for 3 d) in antigen-challenged guinea pigs occurs by
preventing neuronal M2 muscarinic receptor dysfunction. This
is mediated by disruption of eosinophil recruitment to the
nerves and by prevention of MBP release onto M2 receptors.
Eosinophils are associated with the airway nerves of humans
who have died of asthma (16), and they may, therefore, play
an important role in the severity of the disease. The ability of
dexamethasone to prevent eosinophil recruitment to the airway nerves and protect M2 receptor function may therefore
provide valuable insight into the roles of eosinophils and the
parasympathetic nerves in the pathogenesis and treatment of asthma.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Allison D. Fryer, M.D., Division of Physiology, Department of Environmental Health Sciences, Johns Hopkins University School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205. E-mail: afryer{at}jhsph.edu
(Received in original form July 10, 2000 and in revised form November 7, 2000).
Funded by the following grants: NIEHS Center grant ES-03819; by NIH grants HL-54659 and HL-61013 (Dr. Jacoby); by NIH grants P01-HL-10342 and HL-55543 (Dr. Fryer); and by a grant from the American Heart Association (Dr. Fryer).Acknowledgments: The authors thank Bethany Yost and Brian Schofield for technical assistance.
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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