Asthma Research Group, Departments of Medicine and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
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ABSTRACT |
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We investigated allergen-induced airway hyperresponsiveness (AH) in bronchial tissues obtained from dogs that inhaled Ascaris suum leading to AH (RESP) in vivo or that exhibited no change (NON-RESP) as well as from dogs that inhaled saline (SHAM). RESP tissues were not hyperresponsive to KCl or to carbachol, whereas contractions to electrical field stimulation (EFS) were reduced. This reduction was reversed partially by indomethacin and completely by replacement of the bathing fluid. Radioimmunoassay revealed marked elevation of prostaglandin (PG) E2 generation in RESP tissues compared with SHAM and NON-RESP tissues. EFS-evoked contractions were often followed by a slowly developing secondary contraction in RESP tissues but not in SHAM or NON-RESP tissues. However, indomethacin unmasked such secondary contractions in many SHAM and NON-RESP tissues and markedly enhanced those in RESP tissues, whereas L-655,240 (thromboxane A2/PGD2 receptor antagonist) abolished such contractions in all groups. We were unable to detect thromboxane using radioimmunoassay. We conclude that allergen-induced AH involves altered generation of cyclooxygenase metabolites of arachidonic acid (particularly PGE2) as well as of a nonprostanoid inhibitory factor; as such, the responsiveness of the tissue in vitro is dependent on the relative levels of inhibitory and excitatory metabolites.
prostaglandin E2; thromboxane A2; cholinergic innervation
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INTRODUCTION |
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ASTHMA IS CHARACTERIZED by bronchoconstriction and hyperresponsiveness to various spasmogens. In early attempts to elucidate the pathophysiological mechanisms underlying asthma, attention was initially focused on the regulation of the airway smooth muscle (ASM) per se. It is now recognized that asthma is secondary to airway inflammation, accompanied by an influx of inflammatory cells and elevated levels of proinflammatory mediators and cytokines in the airways. Changes in other components of the airway wall participate in reversible airway narrowing. A number of models of asthma have been developed that involve induction of an inflammatory response in the airways. For example, dogs exhibit airway bronchoconstriction, airway inflammation, and hyperresponsiveness to inhaled spasmogens after exposure to the aerosolized allergen Ascaris suum (4, 10, 14, 20). The respiratory response was thought to be due to release of bronchoconstrictor inflammatory mediators after cross-reactivity of the A. suum antigen with a parasitic nematode, Toxocara canis, commonly found in dogs. This response is believed to be immunoglobulin E (IgE) mediated, as indicated by a positive skin test (i.e., wheal), although IgE levels have not yet been measured directly. Since then, many studies have been carried out using allergen inhalation to elucidate the mechanisms underlying airway hyperreactivity (5, 6, 8, 15, 16, 19). Nonetheless, the mechanisms underlying allergen-induced airway hyperresponsiveness (AH) are still not completely understood. Many studies indicate the involvement of several arachidonic acid metabolites in the responses to inhalation of allergen. For example, allergen-induced AH is accompanied by elevation of prostaglandin (PG) D2 levels (11) and is antagonized by blockade of thromboxane (Tx) A2 receptors (6, 11) or activation of PGE2 receptors (21).
Another model of asthma involves induction of an inflammatory response by exposure to ozone (12). Like asthma and allergen-induced AH, the ozone-induced inflammatory response is associated with AH to spasmogens in vivo and, in this case, also in vitro; however, this model differs from asthma and allergen-induced AH in that it is not IgE mediated. Ozone-induced AH seems to involve decreased levels of an inhibitory cyclooxygenase metabolite(s) (likely PGE2) and possibly also increased levels of an excitatory cyclooxygenase metabolite(s) (possibly TxA2; see Ref. 12). There are a variety of in vitro data which show that PGE2 in dogs can affect airway responsiveness by either inhibiting release of acetylcholine (ACh) from airway nerves or inhibiting the responsiveness of the airway muscle (2, 18).
In the present study, we investigated whether allergen-induced AH developed in vivo could also be demonstrated in isolated tissues in vitro and examined the roles of various arachidonic acid metabolites in the changes in airway responsiveness. Bronchial airway tissues were used, since these are primarily responsible for determining peripheral resistance to airflow. The data suggest that metabolism of arachidonic acid is markedly altered during allergen-induced inflammation. This manifests primarily as increased generation of PGE2 (as well as a nonprostanoid inhibitory factor) accompanied by reduced mechanical responses; however, there is also evidence that production of an excitatory autacoid is simultaneously increased, leading to a contraction that develops and resolves slowly.
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MATERIALS AND METHODS |
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In vivo measurements and inhalation of
allergen. Dogs (15-30 kg; either sex) were
anesthetized using intravenous pentobarbital sodium (30 mg/kg) to
induce surgical anesthesia; this level of anesthetization was
maintained during the course of the in vivo study by additional
injections as required. An endotracheal tube and an esophageal balloon
catheter were inserted. The endotracheal tube was connected to a
constant-volume ventilator set at a tidal volume of 10 ml/kg and
frequency of 30 min1. The
esophageal balloon catheter and a port at the equipment end of the
endotracheal tube were connected to a differential pressure transducer
(Hewlett-Packard 267B; Hewlett-Packard, Waltham, MA) and pressure
amplifier (Hewlett-Packard 8805C) to monitor transpulmonary pressure.
Measurements of peripheral resistance (RL) were obtained at
constant volume using techniques described previously, and airway
responsiveness to aerosolized ACh was assessed 30 min after induction
of anesthesia using methods that have been described previously (10,
12, 25). Dose-response curves to ACh were constructed by plotting the
baseline and peak values of
RL after each
concentration of ACh aerosol delivered. From each curve, an
ACh-provocative concentration
(PC5.0, which is the concentration
of ACh that increased
RL by 5.0 cmH2O · l
1 · s
above the baseline value) was calculated by interpolation as previously
described (10, 12, 25).
Dogs were then exposed to allergen (A. suum in 0.9% saline) or saline alone. During this
one-time challenge, the concentration of allergen was increased from
105 M in 10-fold increments
until RL was elevated
10 cmH2O · l
1 · s
above baseline, after which the dogs were ventilated with air until
RL returned to
baseline. Later (24 h), dogs were anesthetized as described above, and
the airway responsiveness to ACh was again assessed, after which they
were euthanized with pentobarbital sodium (100 mg/kg). Dogs were
defined as being hyperresponsive when there was a decrease in the ACh
PC5.0 of twofold or more (10, 12,
25).
Tissue dissection and organ bath
studies. After euthanasia, pulmonary lobes were excised
and were pinned out in physiological solution, and the overlying
parenchymal tissue and vasculature were dissected away, thereby
exposing the bronchiolar tree from which ring segments were excised
(5-10 mm wide; 2-10 mm outer diameter), as described
previously (12). Ring segments were mounted vertically in 3-ml organ
baths using platinum hooks inserted through the lumen (taking care to
not damage the epithelium); one of the platinum hooks was fastened to a
force displacement transducer while the other served as an anchor.
Throughout the studies, tissues were bathed in Krebs-Ringer buffer
(KRB) bubbled with 95% O2-5%
CO2; in some cases, KRB also
included either indomethacin (IDM;
105 M) or
3-[1-(4-chlorobenzyl)-5-fluoro-3-methyl-indol-2-yl]2,2-dimethylpropanoic acid (L-655,240; 10
6 M; see
Ref. 9). Preload tension was maintained at
1.25 g (determined
previously to allow maximal responses). Electrical stimulation (60 V;
0.5-ms pulse duration) was supplied via the platinum hook, which served
as the anchor, and another platinum pole not in direct contact with the
tissue (the 2 poles were 2 cm apart). Isometric changes in tension in
response to various agonists and conditions were recorded on an
ink-writing polygraph.
Experimental protocol. Tissues from
each dog were studied using one of two protocols. One set of eight
tissues was mounted in the muscle baths containing standard KRB or KRB
plus IDM (105 M) or
L-655,240 (10
6 M). Tissues
were then electrically stimulated (20 pulses at 20 Hz) at 20-min
intervals for 140 min, each time preceded by a wash and preload
adjustment. Immediately after the final stimulation at 140 min, tissues
were washed with standard KRB or KRB containing IDM
(10
5 M) or L-655,240
(10
6 M), and the
frequency-response relationship of the tissues was examined: 20 pulses
were delivered at 5-min intervals at frequencies of 0.5-20
pulses/s (pps), without any wash or preload adjustment between
stimulations. In some cases, the twitch contraction evoked by
electrical field stimulation [EFS; which is known to be mediated by release of ACh (12)] was followed by a slowly developing secondary contraction that did not always resolve fully before the
subsequent response was elicited. Next, the response to KCl (100 mM
added hypertonically) was assessed. Finally, the tissues that had been
exposed to IDM were washed and allowed to equilibrate for
60 min,
after which cumulative concentration-response curves for carbachol
(CCh) were generated in the presence or absence of nifedipine
(10
7 M; added
20 min
before CCh).
A second set of eight tissues from each dog was set aside at room
temperature in standard KRB (bubbled with 95%
O2-5%
CO2) until the conclusion of the
experiments described above (5 h after euthanasia). Tissues were
mounted in the organ baths, washed with standard KRB or KRB containing
IDM (10
5 M) at 37°C,
and then allowed to equilibrate for
45 min before obtaining a
contractile response to KCl (60 mM). Next, the bath fluid surrounding
the tissues was collected before and after three series of EFS
stimulations for quantitation of
PGE2 released by the tissues (see
below). Each series of EFS involved trains of pulses (10-s train
duration) with frequencies ranging from 0.1 to 30 pps, without any wash
or preload adjustment between stimulations; some tissues were not
electrically stimulated during this 40-min period to serve as time
controls. Finally, after the second collection of bath fluid for
radioimmunoassay, cumulative concentration-response curves for CCh were
generated.
In all cases, tissues were saved and dried for standardization of contractile responses.
Radioimmunoassay. Samples of organ
bath fluid were collected immediately after the tissues were washed of
KCl as well as after the third series of EFS trains. Samples were
collected using plastic syringes and then were immediately frozen and
stored at 70°C. On the day of radioimmunoassay, samples were
thawed on the bench top and then were immediately analyzed for
PGE2 or
TxB2 content using a commercially
prepared kit from Advanced Magnetics (Cambridge, MA). The
radioimmunoassay is based on competition of the PG with radioactively
labeled PG for a number of sites on the specific antibody.
Antibody-bound PG was separated from unbound with magnetic dextran-coated charcoal through magnetic separation. The counting rate
was correlated with concentration via a standard curve. The sensitivity
of this assay is 2.5 pg/0.1 ml.
Solutions and chemicals. Experiments
were conducted using standard KRB containing (in mM): 116 NaCl, 4.2 KCl, 2.5 CaCl2, 1.6 NaH2PO4,
1.2 MgSO4, 22 NaHCO3, and 11 D-glucose. Chemicals were obtained from Sigma Chemical. All
agents were prepared as neutral aqueous solutions, with the exceptions
of IDM and L-655,240 (both in 22 mM
) and nifedipine (absolute
ethanol).
Statistical analysis. The concentration of CCh required to evoke a half-maximal response (EC50) was derived from the concentration-response curve of each tissue. Contractile responses were standardized using tissue dry weight. Data are reported as means ± SE. Statistical comparisons were made using analysis of variance with corrections for multiple comparisons using the Bonferroni technique. The sources of significant differences were determined using a Student's t-test, with P values <0.05 being considered significant.
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RESULTS |
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In vivo measure of airway responsiveness. Inhalation of allergen (A. suum) caused an immediate bronchoconstrictor response in all 20 dogs assayed, whereas inhalation of vehicle did not. When assayed 24 h later, 10 of the allergen-exposed animals were found to be hyperresponsive to ACh (i.e., exhibited more than a 2-fold decrease in ACh PC5.0; RESP), whereas the other 10 dogs did not (NON-RESP). The mean magnitudes of the change in PC5.0 (i.e., PRE and POST) were 0.84 ± 0.17 in dogs exposed to vehicle (SHAM), 1.29 ± 0.17 in NON-RESP dogs, and 3.56 ± 0.38 in RESP dogs. Figure 1 indicates the range of changes in sensitivity to ACh in these animals. None of the eight SHAM dogs exhibited AH to inhaled ACh.
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In vitro responses to exogenously added KCl or cholinergic agonist. To determine if hyperresponsiveness measured in vivo could also be detected in vitro, we examined the contractile responses to KCl (60 mM) or the cholinergic agonist CCh. Although the former acts exclusively through electromechanical coupling mechanisms, the latter acts through both electromechanical and pharmacomechanical coupling mechanisms.
In the absence of IDM, there were no significant differences between
the three groups with respect to the contractile response to KCl (Fig.
2), the maximal response to CCh (i.e.,
104 M), or the CCh
EC50 (Table
1). IDM had no effect on the KCl responses
in any of the three groups (Fig. 2) but displaced the CCh
concentration-response curves
0.5 log units to the left (Table 1);
there were no significant differences between the maximal responses or
the EC50 values for CCh in SHAM,
NON-RESP, and RESP tissues exposed to IDM (Table 1). Preincubation of
the tissues with nifedipine
(10
7 M; in the maintained
presence of IDM) to eliminate the electromechanical component of the
CCh contraction caused the concentration-response curves to be shifted
slightly to the right (Table 1) and caused a small but statistically
insignificant reduction in the maximal response to CCh; the CCh
responses (EC50 values and maxima)
in the presence of nifedipine were not significantly different between the three groups.
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In vitro responses to nerve-released cholinergic agonist. The frequency-response characteristics of the tissues were examined using trains of pulses with frequencies ranging from 0.1 to 30 pps delivered at 5-min intervals (Fig. 3); tissues were electrically stimulated over prolonged periods without replacement of bath fluid between stimulations. In tissues stimulated in this way in the absence of IDM, there were no statistically significant differences between SHAM and NON-RESP, whereas those in RESP tissues were significantly reduced (Fig. 3A). IDM potentiated the responses in all three groups, particularly at low EFS frequencies, consistent with its prevention of the inhibitory effect of one or more cyclooxygenase metabolites on neurotransmission in this tissue (2). Nonetheless, the responses in RESP tissues exposed to IDM were still significantly smaller than those in the other two groups (Fig. 3B), suggesting that an inhibitory factor unrelated to cyclooxygenase metabolism is generated in RESP tissues.
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We obtained somewhat different results when the tissues were washed
immediately before electrical stimulation to wash out any accumulated
autacoids. In this case, we used trains of pulses (20 pulses at 20 Hz)
delivered at 20-min intervals in standard KRB or KRB containing IDM
(105 M) or L-655,240
(10
6 M;
TxA2/PGD2
receptor antagonist), and the bathing fluid was replaced
5 min
before each response. In the absence of pharmacological agents, EFS
responses within each group increased during the first 60 min and then
decreased over the next 80 min (Fig.
4A).
There was no significant difference in the responses at each time point between the three groups. In the presence of IDM
(10
5 M), the EFS responses
at 20 min were not significantly different from those obtained in its
absence (compare points at time = 20 min in Fig. 4,
A and
B), but all subsequent responses
within each group showed a progressive increase in magnitude (Fig.
4B). Again, there were no
significant differences between the groups at each time point in the
presence of IDM. In tissues pretreated with L-655,240, the EFS
responses obtained at 20 min in each group were not significantly
different from each other (Fig. 4C)
or from those obtained in the same group in the absence of L-655,240 (compare Fig. 4, A and
C). In SHAM and NON-RESP tissues,
the magnitudes of responses to successive electrical stimulations
remained relatively constant (as opposed to the progressive changes in
control and IDM-treated tissues described above), whereas RESP tissues
showed a progressive and marked decrease such that the response at 140 min was almost negligible (Fig. 4C).
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In some cases, primarily among tissues from responders, the EFS-evoked
twitch contraction was followed by a smaller contraction that developed
relatively slowly, reaching a peak ~1-2 min after EFS and then
relaxing partially during the period between stimulations (Fig.
5A).
These were more prevalent and notably larger in RESP than in NON-RESP
or SHAM tissues (Fig. 5B). IDM
(105 M) unmasked small
secondary contractions in some SHAM and NON-RESP tissues and markedly
enhanced the prevalence, magnitude, and duration of such secondary
contractions in RESP tissues (Fig.
5B). L-655,240 (10
6 M) did not
significantly alter the initial twitch contractions but eliminated the
secondary contractions in all three groups (Fig.
5B).
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Radioimmunoassay for PGE2 and
TxB2.
Levels of PGE2 in the bathing
medium before EFS were less than the detection limit (i.e., 2.5 pg/0.1
ml) in SHAM and NON-RESP groups but were markedly elevated to 1
ng/ml in RESP (Fig. 6). The concentration
of PGE2 in the latter case (i.e.,
10
9 M) has previously
been shown to be sufficient to mediate prejunctional inhibition of
cholinergic neurotransmission (2); the concentration of
PGE2 within the tissue is likely
to be even higher. PGE2 levels were increased by 2-3 ng/ml in all three groups over the
subsequent 90 min (Fig. 6). This accumulation was not significantly
increased in tissues that were electrically stimulated (Fig. 6).
PGE2 accumulation in the
electrically stimulated tissues as well as in the time controls was
greatly reduced, if not abolished, by IDM (data not shown). Levels of
TxB2 never exceeded the detection
limit of the assay.
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DISCUSSION |
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Allergen-induced AH has been used in many studies as a model to
elucidate the mechanisms underlying asthma. However, these mechanisms
remain poorly understood, in part because the majority of the studies
were carried out using in vivo preparations in which the various
effects of allergens on the nervous, immune, and vascular systems in
the airways confound interpretation of the data. In the present study,
we used isolated airway tissues in which these effects are absent or
controlled. We also used CCh as a cholinergic agonist rather than ACh,
as was done in earlier studies (11), since ACh is susceptible to
degradation by cholinesterases, and it is possible that cholinesterase
activity is altered during AH; for example, in a previous study of a
canine model of asthma (11), ACh had no effect over the concentration
range 107 to
10
6 M, whereas these
concentrations are effective in nonhyperresponsive dogs. CCh, on the
other hand, is not susceptible to cholinesterase activity. In contrast
to our expectations, we found that bronchial tissues isolated from
hyperresponsive dogs demonstrated no change in responses to exogenously
added spasmogens and reduced responses to electrical stimulation (this
reduction was reversed partially by IDM or by replacement of the
bathing medium to remove any inhibitory factors in the bathing medium).
The data are consistent with allergen exposure having enhanced
generation of inhibitory arachidonic acid metabolites from
cyclooxygenase, as well as of a possible nonprostanoid inhibitory
factor. Therefore, these data suggest that the responsiveness of the
airway tissues in vitro is determined by the relative levels of
inhibitory and excitatory factors elicited by exposure to allergen, as
discussed below.
Changes in myogenic mechanisms? Contraction of ASM is a Ca2+-dependent event, primarily involving activation of myosin light chain kinase in response to an elevation in cytosolic Ca2+ concentration ([Ca2+]i). The latter is a result of agonist-induced release of internally sequestered Ca2+ and/or depolarization-induced opening of voltage-dependent Ca2+ channels. A number of Ca2+ homeostatic mechanisms contribute to restoring [Ca2+]i to basal levels and thereby mediate recovery from excitation, including uptake into the internal store and extrusion from the cell. It has been suggested that AH may involve changes in the function of the ASM per se, such as changes in Ca2+ handling or increased sensitivity to Ca2+ (14, 15, 24). We did not obtain any evidence for such changes under the present experimental conditions. For example, there seems to be no change in electromechanical coupling mechanisms (i.e., voltage-dependent Ca2+ influx), since we found no difference between SHAM and RESP tissues with respect to the sensitivities or magnitudes of responses to CCh in the absence of nifedipine (Table 1) or to KCl (Fig. 2). The lack of a significant difference between the CCh responses in the presence or absence of nifedipine, as well as the KCl responses, also suggests that there is no change in the sensitivity of the contractile apparatus to Ca2+ after exposure to allergen. Likewise, there seems to be no change in agonist-induced release of internally sequestered Ca2+, since there was no change in the maximal contractile response or in the EC50 for CCh (Table 1). Measurements of ionic currents in single cells also did not reveal any difference between SHAM and RESP tissues with respect to membrane currents at rest or during cholinergic stimulation (Janssen, unpublished observation), consistent with our claim that basal [Ca2+]i is not elevated in these tissues and that agonist-induced release of internal Ca2+ is not altered.
Airway inflammation may involve a change in airway tissue elastance
(via lymphocyte-mediated destruction of the matrix), which would result
in a change in the optimal preload for these tissues. If this occurred
in the present experiments, our use of the same preload tension
(1.25 g) for all tissues could account for the observed decrease in
responses in allergen-exposed tissues. However, there was no difference
in the magnitude of responses between the three groups when tissues
were washed before stimulation (to remove any inhibitory substances;
Fig. 4A), which contraindicates an
allergen-induced change in the optimal preload. It is worth reiterating
that, when this type of experiment is repeated without a wash
before each stimulation, there is a marked difference between the
three groups (Fig. 3A).
Changes in neurogenic mechanisms? In canine ASM the excitatory innervation is almost exclusively cholinergic in nature. We have demonstrated above that the myogenic response to cholinergic stimulation is unaltered. However, it is possible that allergen-induced airway inflammation may result in changes in the release of ACh from the nerve endings, leading to exaggerated neurogenic excitation. For example, Elbon et al. (8) found that allergen-induced AH in guinea pigs involves release of eosinophil-derived mediators that downregulate prejunctional inhibitory muscarinic receptors, leading to increased cholinergic neurotransmission. Similarly, Mitchell et al. (19) have shown that basal and histamine-induced release of ACh is increased in ragweed-sensitized dogs, suggesting that immune sensitization facilitates the release of the neurotransmitter from postganglionic parasympathetic nerves. In a different canine model of AH [that induced by inhalation of ozone (12)], we have found that AH seems to involve decreased prejunctional inhibition (likely due to decreased generation of PGE2) and possibly also increased prejunctional excitatory input (perhaps due to increased generation of TxA2).
Surprisingly, we found that tissues from dogs that had demonstrated AH in vivo after allergen exposure were not more excitable to EFS than control tissues. In fact, there was a significant decrease in the neurogenic responses in RESP tissues compared with SHAM and NON-RESP tissues. This reduction of the neurogenic responses was reversed partially by IDM (Fig. 3) and completely by replacement of the bathing medium to remove any inhibitory factors (Fig. 4). Thus allergen-induced inflammation may be accompanied by generation of inhibitory factors for mediator release. Radioimmunoassay and the effect of IDM on mechanical responses indicate that one of these factors is PGE2 (discussed in more detail below); however, the inability of IDM to completely reverse the hyporesponsiveness suggests that a nonprostanoid inhibitory factor is also produced. Studies of epithelium-dependent inhibition of canine trachealis are also consistent with involvement of both PGE2 and a nonprostanoid factor (McGrogan and Daniel, unpublished observation).
Changes in cyclooxygenase metabolites? Our observations and those of others suggest that allergen-induced airway inflammation is accompanied by marked changes in the metabolism of arachidonic acid.
First, we found that basal levels of
PGE2 were markedly increased in
RESP tissues (Fig. 6A). The overall
bath concentration of PGE2 reached
10
9 M (the effective
concentration within the tissue is likely to be higher); this
concentration of PGE2 has been
shown to mediate marked inhibition of neurogenic responses, but not
those to exogenously added ACh, in canine bronchial smooth muscle (2).
Thus PGE2 accumulation could
explain the sensitivity of responses in allergen-exposed animals to
neurally released cholinergic agonist but not those to cholinergic
agonist added exogenously. The accumulation of PGE2 concentration was sensitive
to the cyclooxygenase antagonist IDM. Similarly, Itabashi et al. (11)
have shown that inhalation of allergen by dogs is accompanied by
significant accumulation of another prostanoid
(PGD2), although this was not
prevented by IDM.
Second, we found that the responses to EFS in SHAM and NON-RESP tissues were not significantly altered by L-655,240, whereas those in RESP tissues were markedly reduced in the presence of this Tx/PG receptor antagonist (Fig. 4). Itabashi et al. (11) also found that blockade of Tx receptors (using an antagonist distinct from the one used in our study) had no effect on the cholinergic responses in control and nonresponder groups but markedly antagonized those in the hyperresponder group. Similarly, Chung et al. (6) have shown that the late phase of allergen-induced AH in dogs is antagonized by yet another TxA2 receptor antagonist.
Third, the slowly developing secondary contractions that followed the initial EFS-evoked twitch contractions were larger and much more prevalent in RESP tissues than in SHAM or NON-RESP tissues, and these were enhanced in all three groups by IDM but were eliminated by L-655,240 (Fig. 5). Similar secondary contractions have been shown to be induced by leukotrienes and to be sensitive to inhibition by PGE2, although the mechanism involved is as yet unclear (1).
These observations are consistent with allergen exposure initiating changes in the generation of both excitatory and inhibitory arachidonic acid metabolites via cyclooxygenase. As a result, the effect of allergen-induced inflammation on airway responsiveness depends on the relative levels of these metabolites as well as on the sensitivity of the tissue to them (e.g., density of receptors for the metabolites within the particular tissue). For example, allergen causes airway responsiveness to be increased in the small airways (5, 11), decreased in the trachealis (5, 18), and unchanged in the larger bronchi (this study and Ref. 11), perhaps reflecting the relative levels of arachidonic acid metabolites and their receptors throughout the airways.
Distinct mechanisms underlying different models of airway inflammation. The epithelium is the major source of PGE2 and of a nonprostanoid inhibitory factor (17, 18, 23). Ozone-induced inflammation in canine ASM is accompanied by destruction of the epithelium and increased responsiveness of isolated bronchial tissues, the latter of which seems to be secondary to decreased generation of an inhibitory cyclooxygenase metabolite, likely PGE2 (12). In the present study, tissues excised from dogs exhibiting allergen-induced hyperresponsiveness demonstrated reduced in vitro mechanical responses (Fig. 3) accompanied by increased levels of PGE2 (Fig. 6); electron-microscopic examination of the tissues showed no appreciable damage of the epithelium in tissues from hyperresponsive dogs compared with their control counterparts (Daniel, unpublished observation). Thus the mechanisms underlying ozone-induced and allergen-induced AH seem to differ markedly, and this could account for the contrasting observations (i.e., hyperresponsiveness versus hyporesponsiveness) made in excised tissues from these two models. Decreased ASM responsiveness is also seen in the mouse after Toxocara-induced eosinophilic inflammation (5) as well in the Basenji-Greyhound model of asthma (even though the tissues were excised from dogs that demonstrated hyperresponsiveness in vivo; see Ref. 7). In the rat, induction of an airway inflammatory response causes increased generation/release of nitric oxide from the airway epithelium (22) and hyporesponsiveness of the smooth muscle, which is secondary to the effects of nitric oxide on the smooth muscle (16). In the present study, NG-nitro-L-arginine (a blocker of nitric oxide synthesis) had no effect on the magnitudes of contractions evoked by EFS (Janssen, unpublished observation).
The mechanisms underlying other models of asthma may also differ from those described above. For example, in canine airway tissues that have been sensitized to allergen but that are not actually inflamed at the time of study, there are changes in shortening velocity that were interpreted to reflect increased myosin light chain kinase levels within the smooth muscle, although maximum force generation is not increased (14). Moreover, studies of the changes in airway function after acute exposures to allergen or ozone do not take into account the structural changes that accompany repeated/prolonged exposures (which are also seen in the airways of asthmatics). Thus the relationship between these various animal models of asthma and the clinical (i.e., human) condition is equivocal. Studies in humans vary with respect to whether or not smooth muscle force generation is increased; we are not aware of any reports of decreased responsiveness of human ASM after inflammation or in asthma.
We conclude that allergen-induced changes in airway responsiveness studied in vitro reveal altered metabolism of arachidonic acid by cyclooxygenase, leading to increased generation of the inhibitory metabolite PGE2 and possibly other excitatory prostanoids. In addition, a nonprostanoid inhibitory factor may also be produced. As such, the responsiveness of the tissue is dependent on the relative levels of inhibitory and excitatory autacoids.
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ACKNOWLEDGEMENTS |
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This study was supported by grants from the Medical Research Council and the Ontario Thoracic Society.
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FOOTNOTES |
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These data have been presented in abstract form (Am. J. Respir. Crit. Care Med. 153: A741, 1996).
Address for reprint requests: L. J. Janssen, Dept. of Medicine, McMaster University, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5.
Received 22 November 1996; accepted in final form 26 August 1997.
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