1 Department of Environmental Health Sciences, School of Hygiene and Public Health, and 2 Division of Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, Johns Hopkins University, Baltimore, Maryland 21205; and 3 Departments of Immunology and Medicine, Mayo Clinic, Rochester, Minnesota 55905
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Neuronal muscarinic (M2) receptors inhibit release of acetylcholine from the vagus nerves. Hyperreactivity in antigen-challenged guinea pigs is due to blockade of these M2 autoreceptors by eosinophil major basic protein (MBP) increasing the release of acetylcholine. In vivo, substance P-induced hyperactivity is vagally mediated. Because substance P induces eosinophil degranulation, we tested whether substance P-induced hyperreactivity is mediated by release of MBP and neuronal M2 receptor dysfunction. Pathogen-free guinea pigs were anesthetized and ventilated. Thirty minutes after intravenous administration of [Sar9,Met(O2)11]- substance P, guinea pigs were hyperreactive to vagal stimulation and M2 receptors were dysfunctional. The depletion of inflammatory cells with cyclophosphamide or the administration of an MBP antibody or a neurokinin-1 (NK1) receptor antagonist (SR-140333) all prevented substance P-induced M2 dysfunction and hyperreactivity. Intravenous heparin acutely reversed M2 receptor dysfunction and hyperreactivity. Thus substance P releases MBP from eosinophils resident in the lungs by stimulating NK1 receptors. Substance P-induced hyperreactivity is mediated by blockade of inhibitory neuronal M2 receptors by MBP, resulting in increased release of acetylcholine.
muscarinic receptors; eosinophil major basic protein; antigen challenge
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN THE LUNGS, THE DOMINANT control of airway smooth muscle is provided by the parasympathetic fibers of the vagus nerves (39). These fibers release acetylcholine onto postjunctional muscarinic (M3) receptors to cause airway smooth muscle contraction (43). The release of acetylcholine by airway parasympathetic nerves is inhibited by the activation of neuronal M2 receptors in an autocrine manner (19). In antigen-challenged animals, these neuronal M2 receptors are dysfunctional (20), resulting in increased acetylcholine release (3) and airway hyperreactivity (9, 17). However, M3 receptor function remains unaltered because no change in airway smooth muscle sensitivity to acetylcholine occurs (16).
Inhalation of antigen causes an influx of eosinophils into the airways (11, 13, 44) where they accumulate around airway nerves (11, 15, 16). Blockade of eosinophil accumulation in the airways of antigen-challenged guinea pigs by inhibition of interleukin-5 or very late activation antigen-4 prevents airway hyperreactivity and loss of M2 receptor function (15, 17, 36, 46).
During antigen-induced airway inflammation, eosinophils release major basic protein (MBP) (22, 30). MBP is an allosteric antagonist of M2 receptors in vitro, which can be removed with heparin (29). Treatment of antigen-challenged guinea pigs with heparin restores M2 receptor function (18). Specific removal of MBP with an antibody prevents antigen-induced hyperreactivity via protection of M2 receptor function (16). Thus eosinophil recruitment to the airway nerves and the subsequent release of MBP cause hyperreactivity by blocking inhibitory neuronal M2 receptors in antigen-challenged guinea pigs.
Substance P, which binds neurokinin (NK) receptors, induces vagal reflex-mediated airway hyperreactivity (40). In antigen-challenged guinea pigs, NK1 receptor, but not NK2 receptor, antagonists prevent loss of M2 receptor function and hyperreactivity without affecting eosinophil recruitment (11). Substance P stimulates degranulation of eosinophils in vitro via NK1 receptor stimulation (31). To define the role of substance P in airway hyperreactivity, these studies were designed to test whether intravenous substance P induces airway hyperreactivity via eosinophil degranulation and subsequent M2 receptor dysfunction.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals. Specific pathogen-free female Dunkin-Hartley guinea pigs (250-350 g; supplied by Hilltop Animal Farms, Scottsdale, PA) were used. 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 United States Animal Welfare Acts set forth in the 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.
Measurements of pulmonary inflation pressure and effects of [Sar9,Met(O2)11]substance P. Guinea pigs were anesthetized with urethan (2.0 g/kg ip). This dose produces a deep anesthesia lasting 8-10 h (23), although none of these experiments lasted for more than 3 h. Both jugular veins were cannulated for the administration of drugs. One internal carotid artery was cannulated for measurement of blood pressure with a DTX pressure transducer (Viggo-Spectramed, Oxnard, CA), and the heart rate was derived from the blood pressure tracing with a tachograph.
Animals were ventilated through a tracheal cannula with a positive-pressure, constant-volume rodent respirator (Harvard Apparatus, South Natick, MA) at a tidal volume of 10 ml/kg and a respiratory rate of 100 breaths/min. Succinylcholine (10 mg·kgStudies of vagal hyperresponsiveness. Anesthetized, ventilated, and paralyzed pathogen-free guinea pigs were used. Both vagi were cut, and the distal ends were placed on platinum-stimulating electrodes. Electrical stimulation of both vagus nerves produced frequency-dependent bronchoconstriction (measured as an increase in Ppi above baseline) and bradycardia. The vagus nerves were stimulated at frequencies ranging from 2 to 15 Hz for 5 s at 120-s intervals, keeping both pulse duration (0.1 ms) and voltage (10 V) constant among groups. Increases in Ppi were recorded on a Grass polygraph as described above. At the end of experiments, vagally induced bronchoconstriction was abolished by administration of atropine (1 mg/kg iv), demonstrating that these responses were mediated via release of acetylcholine.
To test whether a cationic substance increased responsiveness to vagal stimulation, some animals were treated with heparin. Animals were treated with SM-substance P, and 30 min later, a frequency-response curve was generated. Heparin was then administered (3,000 U/kg iv), and 20 min later, the frequency-response curve was repeated. To ensure that there was no tachyphylaxis to repeated vagal nerve stimulation, some animals were treated with heparin without first generating a frequency response as described above. The frequency-response curves in these animals were identical to the data obtained in animals stimulated before and after heparin, and these data have been combined.Studies of neuronal M2 receptor function. Anesthetized, ventilated, and paralyzed pathogen-free guinea pigs (as described above) were used. Both vagus nerves were cut, and the distal ends were placed on platinum-stimulating electrodes. The effects of muscarinic agonists are more apparent at lower frequencies of stimulation because fewer agonist binding sites are occupied by endogenous acetylcholine (19); thus the effect of pilocarpine on vagally induced bronchoconstriction was tested at 2 Hz. Before administration of pilocarpine, baseline responses to electrical stimulation (2 Hz, 0.1 ms, 2-45 V, for 45 pulses/train at 50-s intervals) of the vagus nerves were obtained. Electrical stimulation of the vagus nerves at these parameters induced bronchoconstriction and bradycardia, which were rapidly reversed on cessation of stimulation. The voltage (mean, 13 ± 2 V) was varied at the beginning of each experiment to give a mean bronchoconstriction of 24.2 ± 1.1 mmH2O that was reproducible over 5-10 stimulus trains. Once set, the voltage was not altered within each experiment. Cumulative doses of pilocarpine (0.1-100 µg/kg iv) were administered, and the affect on vagally induced bronchoconstriction was measured. The results are expressed as a ratio of bronchoconstriction in the presence of pilocarpine to bronchoconstriction in the absence of pilocarpine.
Pilocarpine (30-100 µg/kg) produced a small, transient bronchoconstriction (which was not significantly different among treatment groups; data not shown). Therefore, the effect of these doses of pilocarpine on vagally induced bronchoconstriction was measured after the Ppi had returned to baseline. At the end of the pilocarpine dose-response tests, heparin (2,000 U/kg iv) was administered to animals treated with SM-substance P to test whether a cationic substance blocked neuronal M2 receptor function (18). The results were recorded 15-20 min after administration of heparin.Studies of M3 receptor function. After the pilocarpine dose- response and frequency-response experiments, the sensitivity of airway smooth muscle to acetylcholine (1-10 µg/kg iv) was tested. In vagotomized guinea pigs, acetylcholine-induced bronchoconstriction is due to M3 receptor stimulation (4, 43).
Bronchoalveolar lavage. At the end of the experiment, bronchoalveolar lavage was performed via the tracheal cannula. The lungs were lavaged with five aliquots of 10 ml of phosphate-buffered saline (PBS). The recovered lavage fluid (35-45 ml) was centrifuged. Total cells were counted under a Neubauer Hemocytometer (Hausser Scientific, Hoarsham, PA). Aliquots of the cell suspension were cytospun onto glass slides, stained with Diff-Quik (Baxter Healthcare, McGaw Park, IL), and counted.
Drugs and reagents. Acetylcholine, cyclophosphamide, guanethidine, PBS, pilocarpine, SM-substance P, sodium chloride, succinylcholine, and urethan were purchased from Sigma (St. Louis, MO). Rabbit anti-guinea pig MBP antibody was produced as described previously (35, 48). The NK receptor antagonists SR-140333 and SR-48968 were the generous gifts of Dr. Xavier Emonds-Alt (Sanofi Reserche, Montpellier, France). All drugs were dissolved and diluted in 0.9% NaCl or PBS with the exception of SR-140333, which was dissolved in DMSO (10 mg/ml stock solution) and then diluted in PBS. Appropriate controls with this vehicle were carried out, and they demonstrated that DMSO had no effect on any of the parameters measured here.
Statistics.
All data are expressed as means ± SE. Acetylcholine, frequency,
and pilocarpine responses were analyzed using two-way analyses of
variance for repeated measures. Baseline heart rates, blood pressures,
Ppi, and changes in Ppi (before
pilocarpine administration), and bronchoalveolar lavage were analyzed
using analysis of variance. The effect of the NK2
antagonist on vagal simulation in pathogen-free guinea pigs was
analyzed using paired Student's t-test (Statview 4.5;
Abacus Concepts, Berkeley, CA; P 0.05 was considered significant).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the absence of pilocarpine, electrical stimulation of both vagus nerves caused bronchoconstrictions, which were not significantly different among groups (control, 22.0 ± 2.1; SM-substance P, 21.6 ± 1.8; SM-substance P with cyclophosphamide pretreatment, 28.9 ± 4.6; SM-substance P with antibody to MBP pretreatment, 26.0 ± 2.0; SM-substance P with NK1 antagonist pretreatment, 24.8 ± 2.6; and NK2 antagonist treatment, 19.3 ± 3.2 mmH2O). In the absence of pilocarpine, the voltages used to obtain the above bronchoconstrictions were variable but not significantly different among groups (control, 18.3 ± 7.7; SM-substance P, 16.6 ± 4.6; SM-substance P with cyclophosphamide pretreatment, 7.7 ± 4.3; SM-substance P with antibody to MBP pretreatment, 9.9 ± 2.2; SM-substance P with NK1 antagonist pretreatment, 10.5 ± 0.9; and NK2 antagonist treatment, 19.3 ± 7.1 V). Vagal stimulation also caused transient bradycardia, which was not significantly altered by SM-substance P (data not shown). Responses to electrical stimulation of the vagus nerves were cholinergic because they were abolished by administration of atropine (1 mg/kg).
In control guinea pigs, increasing doses of pilocarpine (0.1-100
µg/kg) inhibited vagally induced bronchoconstriction in a dose-dependent manner, demonstrating the presence of functional M2 receptors (Fig. 1).
However, in guinea pigs treated with SM-substance P, pilocarpine did
not significantly inhibit vagally induced bronchoconstriction, indicating neuronal M2 receptor dysfunction (Fig. 1). The
highest dose of pilocarpine (100 µg/kg) only inhibited vagally
induced bronchoconstriction by 15% in the SM-substance P-treated
animals compared with a greater than 60% inhibition of vagally induced bronchoconstriction in controls. Heparin acutely restored
M2 receptor function in SM-substance P-treated animals,
such that the effect of 100 µg/kg pilocarpine was similar to that in
control animals (Fig. 1).
|
Depletion of inflammatory cells by pretreatment of pathogen-free guinea
pigs with cyclophosphamide (30 mg/kg ip) prevented SM-substance
P-induced loss of neuronal M2 receptor function (Fig. 1).
Similarly, pretreatment with either the antibody to MBP (Fig. 2) or the NK1 antagonist
(Fig. 3A), but not the
NK2 antagonist (Fig. 3B), also prevented
SM-substance P-induced M2 receptor dysfunction. In these
experiments, neither the antibody to MBP (data not shown; n = 2) nor the NK1 antagonist SR-140333
(Fig. 3A) affected M2 receptor function in
control guinea pigs.
|
|
In control guinea pigs, electrical stimulation of the distal ends of
the cut vagi at increasing frequencies (2-15 Hz, 0.1 ms, 10.0 V,
5 s at 120-s intervals) produced frequency-dependent bronchoconstriction, measured as an increase in Ppi.
SM-substance P significantly potentiated vagally induced
bronchoconstriction compared with that in control animals (Fig.
4). Administration of heparin after
SM-substance P reversed the SM-substance P-induced potentiation of
vagally induced bronchoconstriction (Fig. 4A). Heparin acutely reduced vagally induced bronchoconstriction below control levels; this difference was not statistically significant (P = 0.6240).
|
Pretreatment with antibody to MBP prevented potentiation of vagally
induced bronchoconstriction in SM-substance P-treated guinea pigs (Fig.
4B). Likewise, pretreatment with the NK1
receptor antagonist (Fig. 5) also
prevented potentiation of vagally induced bronchoconstriction in
SM-substance P-treated guinea pigs. In control guinea pigs, neither
antibody to MBP (Fig. 4B) nor SR-140333 (Fig. 5) affected
airway reactivity to vagal stimulation. We attempted to test the
effects of the NK2 antagonist SR-48968 on SM-substance P-induced potentiation of vagally induced bronchoconstriction. In
animals given the NK2 antagonist (1 mg/kg ip), the
responses to vagal stimulation were markedly inhibited so that we were
unable to measure the reactivity of the vagus nerves after SM-substance P (data not shown).
|
We subsequently tested the effect of the NK2 antagonist on
airway responsiveness to vagal stimulation in control guinea pigs. In
these guinea pigs, repeated vagal stimulation at 10 Hz, 10 V, 0.1 ms,
for 5 s at 120-s intervals caused a baseline change in
Ppi of 98.3 ± 3.9 mmH2O
(n = 4). Once a baseline response to vagal
stimulation was achieved, the NK2 antagonist SR-48968 was given (1 mg/kg iv). This dose caused a 79% inhibition of vagally induced bronchoconstriction over a 12-min period of repeated
stimulation at the same pulse parameters and intervals (Fig.
6A). There was not, however, a
change in vagally induced bradycardia at this time (Fig.
6B).
|
Intravenous acetylcholine (1-10 µg/kg) induced dose-dependent
bronchoconstriction in all groups regardless of treatment. In all of
the guinea pigs, both vagi were cut to eliminate any possible vagal
reflex. There were no significant differences in the response to
intravenous acetylcholine in vagotomized guinea pigs treated with the
NK2 antagonist vs. animals not receiving the
NK2 antagonist (Fig. 7).
Additionally, in vagotomized animals treated with intravenous SM-substance P, there were no significant differences in
acetylcholine-induced bronchoconstriction among groups (Fig.
8).
|
|
Administration of SM-substance P did not significantly alter the number
of inflammatory cells recovered from bronchoalveolar lavage fluid
of pathogen-free guinea pigs (Table
1). Cyclophosphamide treatment caused a marked decrease in the total numbers of leukocytes returned in bronchoalveolar lavage. Eosinophils, neutrophils, and
lymphocytes comprised less than 2% of the returned cells in the
cyclophosphamide-treated animals compared with 25% in control animals
(n = 2; data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intravenous SM-substance P did not significantly alter heart rate, systolic and diastolic blood pressure, or baseline Ppi. Likewise, SM-substance P did not cause any significant differences among the populations of inflammatory cells returned in bronchoalveolar lavage fluid (Table 1). In addition, substance P did not change the responsiveness of the airway smooth muscle to acetylcholine (Fig. 6). Although intravenous muscarinic agonists can elicit a vagal reflex (4), there was no involvement of the vagus nerves here because both vagus nerves were cut.
In the heart, vagally induced bradycardia is mediated by M2 receptors (32). Unlike vagally induced bronchoconstriction in the lungs, SM-substance P did not significantly affect vagally induced bradycardia in the heart compared with that in control animals (data not shown). Preliminary histological studies have shown that there are no eosinophils resident in the heart (data not shown); thus the lack of effect of SM-substance P on vagally induced bradycardia is probably due to a lack of eosinophils in the heart.
Neuronal M2 receptors were functioning to inhibit release of acetylcholine in control guinea pigs because the muscarinic agonist pilocarpine inhibited vagally induced bronchoconstriction in a dose-dependent manner. In contrast, in animals treated with intravenous SM-substance P, pilocarpine no longer inhibited vagally induced bronchoconstriction (Fig. 1). Thus SM-substance P causes dysfunction of neuronal M2 receptors in the lungs.
Cyclophosphamide pretreatment prevented SM-substance P-induced loss of M2 receptor, suggesting inflammatory cell involvement (Fig. 1). The lungs of pathogen-free guinea pigs contain resident eosinophils (10, 11, 15, 17). We have previously shown that eosinophils cause dysfunction of neuronal M2 receptors by releasing MBP (9, 16, 29). Heparin reversed SM-substance P loss of neuronal M2 receptor function, demonstrating a reversible role of cationic proteins in tachykinin-mediated loss of M2 receptor function (Fig. 1; Ref. 10). In addition, specific inhibition of eosinophil MBP also protected M2 receptor function in the SM-substance P-treated guinea pigs, indicating that the positively charged MBP released by eosinophils is the cause of neuronal M2 receptor dysfunction (Fig. 2). Because none of these treatments affect M2 receptor function in control animals (Fig. 2; Refs. 18, 21), SM-substance P-induced loss of neuronal M2 receptor function must be mediated via release of eosinophil MBP.
Intravenous SM-substance P also causes hyperreactivity to electrical stimulation of the vagus nerves. Vagally induced bronchoconstriction is significantly potentiated in the animals treated with SM-substance P compared with control guinea pigs (Fig. 4). Because there is no change in the sensitivity of the airway smooth muscle to acetylcholine, SM-substance P-induced hyperreactivity is mediated entirely through the vagus nerves (Fig. 6). These data agree with a study by Omini et al. (40) showing that substance P-induced hyperreactivity is vagally mediated.
SM-substance P-induced hyperreactivity is mediated by the release of MBP. Pretreatment with antibody to eosinophil MBP prevented SM-substance P-induced hyperreactivity, and administration of heparin to SM-substance P-treated guinea pigs acutely reversed vagal hyperreactivity (Fig. 4). Neither the antibody to MBP (Fig. 4B) nor heparin (18) affects reactivity in control animals. Treatments that protect or restore neuronal M2 receptor function after SM-substance P also prevent or reverse vagally mediated hyperreactivity in the absence of any change in airway smooth muscle sensitivity to acetylcholine. Thus loss of M2 receptor function is the cause of hyperreactvity in SM-substance P-treated guinea pigs. Both of these effects are dependent on release of MBP from eosinophils.
After treatment with SM-substance P, the response to high-frequency vagal stimulation was markedly increased (Fig. 4), whereas the response to low-frequency stimulation was not. This further suggests that M2 receptor dysfunction is responsible for the hyperresponsiveness. Inhibition of acetylcholine release by neuronal M2 receptors is much greater at high-frequency nerve stimulation since there is more acetylcholine released to stimulate the receptors (19, 20). Therefore, increased bronchoconstriction due to loss of neuronal M2 receptor function is more apparent at higher frequencies than at lower frequencies. Conversely, the effects of exogenous agonists on M2 receptors (as in our pilocarpine experiments) are more readily apparent at lower frequencies when they are not competing for receptors with endogenous acetylcholine.
SM-substance P-induced loss of neuronal M2 receptor function was prevented by pretreatment with a NK1 receptor antagonist (Fig. 3A) but not with a NK2 antagonist (Fig. 3B). In addition, the NK1 receptor antagonist prevented SM-substance P-induced hyperreactivity (Fig. 5). We were not able to test the effect of the NK2 antagonist on hyperreactivity since the NK2 antagonist blocked vagally induced bronchoconstriction in the absence of SM-substance P (Fig. 6A). Neither NK antagonist treatment affected the sensitivity of airway smooth muscle to acetylcholine (Figs. 7 and 8).
Because the NK2 receptor antagonist inhibited vagally induced bronchoconstriction in the absence of exogenous SM-substance P, it appears that the endogenous tachykinins enhance vagal neurotransmission in pathogen-free guinea pigs via NK2 receptors (26, 49). This may explain the conflicting results over the roles of NK1 and NK2 receptors found in models of hyperreactivity (6, 45). It has been suggested that NK2 receptors mediate inflammation, whereas our data suggest that tachykinins directly enhance neurotransmission via NK2 receptors independently of inflammation and presumably also independently of antigen challenge.
Substance P can initiate degranulation of eosinophils (31) and does so by stimulating NK1 receptors (14). Recovery of eosinophils in the bronchoalveolar lavage of control guinea pigs indicates that they have eosinophils resident within their airways (Table 1). Eosinophil MBP is an endogenous allosteric antagonist for M2 receptors in vitro (29). Although, substance P can induce eosinophil degranulation directly via the NK1 receptors (14), an indirect pathway that may include the release of mediators from other inflammatory cells, such as mast cells, macrophages, or neutrophils (33, 42), cannot be excluded.
In total, these studies provide a potential in vivo mechanism for
substance P-induced hyperreactivity. It is the result of M2
receptor blockade by MBP, and the release of MBP by eosinophils is
NK1 receptor mediated. Loss of M2 receptor
function increases the release of acetylcholine in response to
electrical stimulation of the vagus nerves (3,
28), which increases bronchoconstriction (4,
16, 17). Thus SM-substance P-induced
hyperreactivity is the direct result of increased release of
acetylcholine due to loss of neuronal M2 receptor function
(Fig. 9).
|
In antigen-challenged guinea pigs, hyperreactivity to vagal stimulation (10, 16, 17) and to histamine (9, 10) is mediated entirely via increased release of acetylcholine from the vagus nerves. This increased release of acetylcholine from the vagi is due to loss of neuronal M2 receptor function via blockade of the M2 receptors by eosinophil MBP (9, 15-17). A role for tachykinins in this pathway has been suggested by the finding that NK1 antagonists prevent hyperreactivity and loss of neuronal M2 receptor function in antigen-challenged guinea pigs without inhibiting eosinophil influx into the airways (10).
It has generally been assumed that the absence or presence of eosinophils in the lungs is enough to implicate a role for eosinophils in a particular response such as hyperreactivity. However, this is clearly not the case. Although eosinophils were present in tachykinin antagonist-treated antigen-challenged guinea pigs, neuronal M2 receptors were functional and the animals were not hyperreactive (10). Eosinophil MBP had been demonstrated previously to be the mechanism for loss of M2 receptor function in antigen-challenged guinea pigs (16). Therefore, the conclusion from these studies could not be that eosinophils were not important to antigen-induced airway hyperreactivity induced by M2 receptor dysfunction. Rather, it is activation and not the mere presence of eosinophils that is important. It was demonstrated previously in primates that although antigen-induced hyperreactivity was associated with a decrease in eosinophils, extracellular eosinophil peroxidase was increased, demonstrating that the eosinophils had been activated (24). In the experiments reported here, SM-substance P-induced hyperreactivity and M2 receptor dysfunction were also not associated with a change in eosinophil number (Table 1). However, the eosinophils were activated as indicated by the ability of the antibody to MBP to inhibit hyperreactivity (Fig. 4). Thus the number of eosinophils present in the lungs is less important than the activation state of the eosinophils (Fig. 9).
There are several points to consider based on the observation that activation is more important than the presence of eosinophils. First, the experiments described here were carried out in pathogen-free guinea pigs. Although guinea pigs have been described as hypereosinophilic, the presence of eosinophils in the lungs does not contribute to hyperreactivity unless the eosinophils are activated, in this case by SM-substance P. Second, although antigen challenge of sensitized, pathogen-free guinea pigs causes recruitment of additional eosinophils into the lungs, there are already enough eosinophils resident in pathogen-free guinea pigs to cause the same degree of M2 receptor dysfunction and hyperreactivity as in the antigen-challenged animals (Figs. 1 and 5; Ref. 16).
In humans, administration of substance P induces hyperreactivity to inhaled methacholine (8). Methacholine-induced bronchoconstriction has been assumed to be due to a direct effect on the airway smooth muscle. However, it has been demonstrated that there is a vagal reflex component to methacholine (4, 47). Neuronal M2 receptors inhibit release of acetylcholine from the vagal parasympathetic nerves in humans (37) and are dysfunctional in some patients with asthma (2, 38). There are significant numbers of eosinophils in the airways of asthmatics (25, 27), and we have demonstrated an association of eosinophils with airway nerves in patients who have died of asthma (11). Therefore, the potentiation of agonist-induced bronchoconstriction by substance P in humans with asthma may also be mediated by eosinophil MBP and M2 receptors.
In conclusion, SM-substance P-induced hyperreactivity in pathogen-free guinea pigs is mediated by release of eosinophil MBP and subsequent loss of neuronal M2 receptor function. In addition, we have demonstrated that it is not the presence of eosinophils, but the release of eosinophil MBP, that is critical to the development of hyperreactivity.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was funded by National Institutes of Health Grants ROI-HL-55543 and POI-HL-10342 to A. D. Fryer, ROI-HL-61013 and ROI-HL-54659 to D. B. Jacoby, and ROI-AI-09728 and ROI-AI-34577 to G. J. Gleich; by the American Heart Association to A. D. Fryer; and by the Center for Indoor Air Research to D. B. Jacoby.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: A. D. Fryer, Dept. of Environmental Health Sciences, School of Hygiene and Public Health, Johns Hopkins Univ., 615 N. Wolfe St., Baltimore, MD 21205 (E-mail:afryer{at}jhsph.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. §1734 solely to indicate this fact.
Received 4 June 1999; accepted in final form 29 March 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Amann, R,
Schuligoi R,
Holzer P,
and
Donnerer J.
The non peptide NK1 receptor antagonist SR140333 produces long-lasting inhibition of neurogenic inflammation, but does not influence acute chemo- or thermonociception in rats.
Naunyn Schmiedebergs Arch Pharmacol
352:
201-205,
1995[ISI][Medline].
2.
Ayala, LE,
and
Ahmed T.
Is there loss of a protective muscarinic receptor in asthma?
Chest
96:
1285-1291,
1989[Abstract].
3.
Baker, D,
Don H,
and
Brown J.
Direct measurement of acetylcholine release in guinea pig trachea.
Am J Physiol Lung Cell Mol Physiol
263:
L142-L147,
1992
4.
Belmonte, KE,
Fryer AD,
and
Costello RW.
Role of insulin in antigen-induced airway eosinophilia and neuronal M2 muscarinic receptor function.
J Appl Physiol
88:
1708-1718,
1998.
5.
Blaber, LC,
Fryer AD,
and
Maclagan J.
Neuronal muscarinic receptors attenuate vagally-induced contraction of feline bronchial smooth muscle.
Br J Pharmacol
86:
723-728,
1985[Abstract].
6.
Boichot, E,
Biyah K,
Germain N,
Emonds-Alt X,
Lagente V,
and
Advenier C.
Involvement of tachykinin NK1 and NK2 receptors in substance P-induced microvascular leakage and airway hyperresponsiveness in guinea-pigs.
Eur Respir J
9:
1445-1450,
1996
7.
Buckner, C,
Liberati N,
Dea D,
Lengel D,
Stinson-Fisher C,
Campbell J,
Miller S,
Shenvi A,
and
Krell R.
Differential blockade by tachykinin NK1 and NK2 receptor antagonists of bronchoconstriction induced by direct-acting agonists and the indirect-acting mimetics capsaicin, serotonin and 2-methyl-serotonin in the anesthetized guinea pig.
J Pharmacol Exp Ther
267:
1168-1175,
1993[Abstract].
8.
Cheung, D,
van der Veen H,
den Hartigh J,
Dijkman JH,
and
Sterk PJ.
Effects of inhaled substance P on airway responsiveness to methacholine in asthmatic subjects in vivo.
J Appl Physiol
77:
1325-1332,
1994
9.
Costello, RW,
Evans CM,
Yost BL,
Belmonte KE,
Gleich GJ,
Jacoby DB,
and
Fryer AD.
Antigen induced hyperreactivity to histamine. The role ot the vagus nerves and eosinophils.
Am J Physiol Lung Cell Mol Physiol
276:
L709-L714,
1999
10.
Costello, R,
Fryer A,
Belmonte K,
and
Jacoby D.
Effects of tachykinin NK1 receptor antagonists on vagal hyperreactivity and neuronal M2 muscarinic receptor function in antigen challenged guinea pigs.
Br J Pharmacol
124:
267-276,
1998[Abstract].
11.
Costello, R,
Schofield B,
Kephart G,
Gleich G,
Jacoby D,
and
Fryer A.
Localization of eosinophils to airway nerves and the effect on neuronal M2 muscarinic receptor function.
Am J Physiol Lung Cell Mol Physiol
273:
L93-L103,
1997
12.
Dixon, WE,
and
Brody TG.
Contributions to the physiology of the lungs. Part 1, the bronchial muscles and their innervation and the action of drugs upon them.
J Physiol (Lond)
29:
97-173,
1903.
13.
Dunn, CJ,
Elliott GA,
Oostveen JA,
and
Richards IM.
Development of a prolonged eosinophil rich inflammatory leukocyte infiltration in the guinea pig asthmatic response to ovalbumin inhalation.
Am Rev Respir Dis
137:
541-547,
1988[ISI][Medline].
14.
El-Shazly, AE,
Masuyama K,
and
Ishikawa T.
Mechanisms involved in activation of human eosinophil exocytosis by substance P: an in vitro model of sensory neuroimmunomodulation.
Immunol Invest
26:
615-629,
1997[ISI][Medline].
15.
Elbon, CL,
Jacoby DB,
and
Fryer AD.
Pretreatment with an antibody to interleukin-5 prevents loss of pulmonary M2 muscarinic receptor function in antigen challenged guinea-pigs.
Am J Respir Cell Mol Biol
12:
320-328,
1995[Abstract].
16.
Evans, CM,
Jacoby DB,
Gleich GJ,
Fryer AD,
and
Costello RW.
Pretreatment with antibody to eosinophil major basic protein protects M2 receptor function in antigen challenged guinea pigs in vivo.
J Clin Invest
100:
2254-2262,
1997
17.
Fryer, AD,
Costello RW,
Yost BY,
Lobb RR,
Tedder TF,
Steeber AS,
and
Bochner BS.
Antibody to VLA-4, but not to L-selectin, protects neuronal M2 muscarinic receptors in antigen challenged guinea pig airways.
J Clin Invest
99:
2036-2044,
1997
18.
Fryer, AD,
and
Jacoby DB.
Function of pulmonary M2 muscarinic receptors in antigen challenged guinea-pigs is restored by heparin and poly-L-glutamate.
J Clin Invest
90:
2292-2298,
1992[ISI][Medline].
19.
Fryer, AD,
and
Maclagan J.
Muscarinic inhibitory receptors in pulmonary parasympathetic nerves in the guinea-pig.
Br J Pharmacol
83:
973-978,
1984[Abstract].
20.
Fryer, AD,
and
Wills-Karp M.
Dysfunction of M2 muscarinic receptors in pulmonary parasympathetic nerves after antigen challenge in guinea pigs.
J Appl Physiol
71:
2255-2261,
1991
21.
Gambone, LM,
Elbon CL,
and
Fryer AD.
Ozone-induced loss of neuronal M2 muscarinic receptor function is prevented by cyclophosphamide.
J Appl Physiol
77:
1492-1499,
1994
22.
Gleich, GJ,
Loegering DA,
and
Maldonado JE.
Identification of a major basic protein in guinea pig eosinophil granules.
J Exp Med
137:
1459-1471,
1973[ISI][Medline].
23.
Green, CJ.
Animal anaesthesia.
In: Laboratory Animal Handbooks. London: Laboratory Animals, 1982, vol. 8, p. 81-82.
24.
Gundel, RH,
Wegner CD,
and
Letts LG.
Antigen-induced acute and late phase responses in primates.
Am Rev Respir Dis
146:
369-373,
1992[ISI][Medline].
25.
Haley, K,
Sunday M,
Wiggs B,
Kozakewich H,
Reilly J,
Mentzner S,
Sugarbaker D,
Doerschuk C,
and
Drazen J.
Inflammatory cell distribution within and along asthmatic airways.
Am J Respir Crit Care Med
158:
565-572,
1998
26.
Hall, AK,
Barnes PJ,
Meldrum LA,
and
Maclagan J.
Facilitation by tachykinins of neurotransmission in guinea-pig pulmonary parasympathetic nerves.
Br J Pharmacol
97:
274-280,
1989[Abstract].
27.
Hamid, Q,
Song Y,
Kotsimbos T,
Minshall E,
Bai T,
Hegele R,
and
Hogg J.
Inflammation of small airways in asthmatics.
J Allergy Clin Immunol
100:
44-51,
1997[ISI][Medline].
28.
Jacoby, DB,
Xiao H-Q,
Lee NH,
Chan-Li Y,
and
Fryer AD.
Virus and interferon-gamma induced loss of inhibitory M2 muscarinic receptor function and gene expression in cultured airway parasympathetic neurons.
J Clin Invest
102:
242-248,
1998
29.
Jacoby, DB,
Gleich GJ,
and
Fryer AD.
Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.
J Clin Invest
91:
1314-1318,
1993[ISI][Medline].
30.
Kita, H,
Weiler DA,
Abu-Ghazaleh R,
Sanderson CJ,
and
Gleich GJ.
Release of granule proteins from eosinophils cultured with Il-5.
J Immunol
149:
629-635,
1992
31.
Kroegel, C,
Giembycz M,
and
Barnes P.
Characterization of eosinophil cell activation by peptides. Differential effects of substance P, melittin, and FMLP.
J Immunol
145:
2581-2587,
1990
32.
Kubo, T,
Sukuda K,
Mikami A,
Maeda A,
Takahashi H,
Mishina M,
Haga T,
Haga K,
Ichiyama A,
Kangawa K,
Kojima M,
Matsuo H,
Hirose T,
and
Numa S.
Cloning, sequencing, and expression of complementary DNA encoding the muscarinic acetylcholine receptor.
Nature
323:
411-416,
1988.
33.
Lazarus, SC,
Borson DB,
Gold WM,
and
Nadel JA.
Inflammatory mediators, tachykinins and enkephalinase in airways.
Int Arch Allergy Appl Immunol
82:
372-376,
1987[ISI][Medline].
35.
Lewis, DM,
Loegering DA,
and
Gleich GJ.
Antiserum to the major basic protein of guinea pig eosinophil granules.
Immunochemistry
13:
743-746,
1976[ISI][Medline].
36.
Mauser, PJ,
Pitman AM,
Fernandez X,
Foran SK,
Adams GK,
Kreutner W,
Egan RW,
and
Chapman RW.
Effects of an antibody to interleukin-5 in a monkey model of asthma.
Am J Respir Crit Care Med
152:
467-472,
1995[Abstract].
37.
Minette, P,
and
Barnes PJ.
Prejunctional inhibitory muscarinic receptors on cholinergic nerves in human and guinea pig airways.
J Appl Physiol
64:
2532-2537,
1988
38.
Minette, PJ,
Lammers JWJ,
Dixon CMS,
McCusker MT,
and
Barnes PJ.
A muscarinic agonist inhibits reflex bronchoconstriction in normal but not asthmatic subjects.
J Appl Physiol
67:
2461-2465,
1989
39.
Nadel, JA.
Autonomic control of airway smooth muscle and airway secretions.
Am Rev Respir Dis
115:
S117-S126,
1977.
40.
Omini, C,
Brunelli G,
Hernandez A,
and
Daffonchio L.
Bradykinin and substance P potentiate acetylcholine-induced bronchospasm in guinea-pig.
Eur J Pharmacol
163:
195-197,
1989[ISI][Medline].
41.
Regoli, D,
Boudon A,
and
Fauchere J.
Receptors and antagonists for substance P and related peptides.
Pharmacol Rev
46:
551-599,
1994[ISI][Medline].
42.
Reynolds, PN,
Holmes MD,
and
Scicchitano R.
Role of tachykinins in bronchial hyper-responsiveness.
Clin Exp Pharmacol Physiol
24:
273-280,
1997[ISI][Medline].
43.
Roffel, AF,
Elzinga CRS,
and
Zaagsma J.
Muscarinic M3 receptors mediate contraction of human central and peripheral airway smooth muscle.
Pulm Pharmacol
3:
47-51,
1990[Medline].
44.
Rossi, GA,
Crimi E,
Lantero S,
Gianioro P,
Oddera S,
Crimi P,
and
Brusasco V.
Late-phase asthmatic reaction to inhaled allergen is associated with early recruitment of eosinophils in the airways.
Am Rev Respir Dis
144:
379-383,
1991[ISI][Medline].
45.
Van Oosterhout, AJ,
van Ark I,
van der Linde HJ,
Fattah D,
and
Nijkamp FP.
Role of interleukin-5 and substance P in development of airway hyperreactivity to histamine in guinea-pigs.
Eur Respir J
9:
493-499,
1996
46.
Van Oosterhout, AJM,
Ladenius ARC,
Savelkoul HFJ,
van Ark I,
Delsman KC,
and
Nijkamp FP.
Effect of anti-IL-5 and IL-5 on airway hyperreactivity and eosinophils in guinea pigs.
Am Rev Respir Dis
147:
548-552,
1993[ISI][Medline].
47.
Wagner, EM,
and
Jacoby DB.
Methacholine causes reflex bronchoconstriction.
J Appl Physiol
86:
294-297,
1999
48.
Wassom, DL,
Loegering DA,
and
Gleich GJ.
Measurement of guinea pig eosinophil major basic protein by radioimmunoassay.
Mol Immunol
16:
711-719,
1979[ISI][Medline].
49.
Watson, N,
Maclagan J,
and
Barnes PJ.
Endogenous tachykinins facilitate transmission through parasympathetic ganglia in guinea-pig trachea.
Br J Pharmacol
109:
751-759,
1993[Abstract].