Vol. 276, Issue 3, L522-L529, March 1999
Mediators of anaphylaxis but not activated neutrophils augment
cholinergic responses of equine small airways
Michal A.
Olszewski1,
N. Edward
Robinson1,
Feng-Xia
Zhu1,
Xiang-Yang
Zhang1, and
Patricia K.
Tithof2
1 Department of Large Animal
Clinical Sciences and 2 Department
of Pharmacology and Toxicology, Michigan State University, East
Lansing, Michigan 48824
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ABSTRACT |
Neutrophilic inflammation in small airways (SA)
and bronchospasm mediated via muscarinic receptors are features of
chronic obstructive pulmonary disease in horses (COPD). Histamine,
serotonin, and leukotrienes (LTs) are reported to be involved in the
exacerbation of COPD, and currently, histamine has been shown to
increase tension response to electrical field simulation (EFS) in
equine SA. We tested the effects of these mediators and the effects of
activated neutrophils on the cholinergic responses in SA. Histamine,
serotonin, and LTD4 had a
synergistic effect on EFS responses and only an additive effect on the
tension response to exogenous ACh or methacholine. Atropine and TTX
entirely eliminated the EFS-induced tension response in the presence of
all three inflammatory mediators, indicating that augmentation of the
EFS response applies only to the endogenous cholinergic response.
Neutrophils isolated from control and COPD-affected horses were
activated by zymosan, producing 18.1 ± 2.3 and 25.0 ± 2.3 nmol
superoxide · 106
cells
1 · 30 min1, respectively.
However, in contrast to the profound effect of mediators, incubation of
SA for over 1 h in a suspension of up to 30 × 106 zymosan-treated neutrophils/ml
did not significantly affect EFS responses of SA isolated from either
control or COPD-affected horses. We conclude that in equine SA
1) the endogenous cholinergic responses are subject to strong facilitation by inflammatory mediators; 2) activated neutrophils do not
affect cholinergic responses in SA; and
3) in acute bouts of equine COPD,
histamine, LTD4, and serotonin
(mediators primarily associated with type I allergic reaction) rather
than mediators derived from neutrophils most likely contribute to
increased cholinergic airway tone.
airway smooth muscle; chronic obstructive pulmonary disease; inflammatory mediators; neutrophil activation; zymosan
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INTRODUCTION |
CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) in horses
or "heaves" is a naturally occurring syndrome sharing multiple
features with human asthma and COPD (25). Exposure of COPD-susceptible animals to natural (hay and straw) antigens precipitates an
inflammatory response in the airways, with airway hyperresponsiveness
and bronchospasm leading to severe airway obstruction (25). A
cholinergic mechanism of airway obstruction in horses with COPD has
been clearly demonstrated in several studies (6, 24), but the origin of
increased cholinergic tone in the airways remains largely unknown. In
vivo, horses with COPD demonstrate airway hyperresponsiveness to a
variety of spasmogens, and airway obstruction can be resolved by the
use of antimuscarinic drugs (1, 6, 20). In contrast, the isolated
airway responses to ACh and electrical field stimulation (EFS) are
decreased (18, 36), and ACh release from airway parasympathetic nerves,
measured in vitro, is not elevated in horses with COPD (31).
Inhalation of allergens by heavey horses leads to several inflammatory
events in the airways, such as neutrophil recruitment and activation
(12, 21), changes in lymphocyte populations, release of histamine from
airway mast cells (19), and activation of the arachidonic acid cascade
in the airway mucosa, with a significant shift in the lipid mediator
profile (14). The latter results in a decrease in mucosal
PGE2 production and an increase in
proinflammatory lipid mediators such as thromboxane,
15-hydroxyeicosatetraenoic acid (HETE), cysteinyl leukotrienes (LTs),
and platelet-activating factor (10, 13, 14). We propose that
inflammatory mediators released in response to antigen challenge are
responsible for the altered cholinergic responses of the airways in
COPD. In this context, the discrepancy between in vitro tissue behavior
and in vivo airway responses could be caused by washout of these
mediators from the tissues in vitro before measurement of tension or
ACh release. Additionally, earlier studies were
conducted on tracheae and bronchi (18, 36), whereas in COPD, the most
predominant inflammatory response (retention of mucopurulent
secretion and airway wall infiltration) occurs in peripheral airways.
Therefore, if inflammation is the source of altered cholinergic tone in
the airways, detectable changes in cholinergic responses may be limited to small airways (SAs).
Some of these issues were addressed in a previous study by Olszewski et
al. (22) that confirmed that peripheral airways from
horses, in vitro, produce entirely cholinergic contractions in response
to nerve stimulation by EFS. These contractions were increased by
cyclooxygenase blockade and application of histamine, indicating that
inflammatory mediators can exert a profound effect on the responses to
nerve stimulation in equine SAs (22). In the present study, we extended
our research to further investigate the effects of inflammation on
cholinergic mechanisms in equine SAs. Several approaches were used to
reach our goal. We used SAs from both control and acutely heavey
animals to compare their responses to EFS. Because histamine had quite
dramatic effects on the EFS response and virtually no effect on the
methacholine (MCh) concentration-response curve, we examined whether
cholinergic or other mechanisms are responsible for the effects of
histamine on EFS response. We also tested the effect of other
inflammatory mediators reported to be involved in COPD, namely
LTD4 and serotonin [5-hydroxytryptamine (5-HT)], on cholinergic SA responses.
Finally, we were particularly interested in the effects of
neutrophil-derived mediators on cholinergic airway responses for many
reasons. First, in contrast to other inflammatory cells, the number of
neutrophils in the airways consistently increases during an
exacerbation of COPD (8, 12, 29). Neutrophil recruitment takes place
within a few hours of natural antigen challenge and, in most cases,
parallels early changes in lung function (12); neutrophils washed from airways of heavey horses are strongly activated (21); and, as shown in
multiple studies (32, 33), the number of neutrophils in bronchoalveolar
fluid generally increases with the severity of airway obstruction.
Additionally, it has been shown in dog airways that neutrophils are
critical to the development of both ozone- and intravenous
platelet-activating factor-induced hyperresponsiveness (5). Thus, in
our final approach, we used isolated and activated neutrophils to test
whether mediators released acutely from these cells activated in
proximity of SAs are capable of altering their cholinergic responses.
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MATERIALS AND METHODS |
Animals
Control horses and horses with COPD were tissue donors for this in
vitro study, which was approved by the All-University Committee on
Animal Use and Care at Michigan State University (East Lansing). The
control group consisted of 17 geldings and mares of various breeds, 7.3 ± 0.8 yr old, weighing 901.4 ± 20.2 kg, and free of signs of
respiratory disease. At 48 h before euthanasia, animals were brought
from the pasture into the barn and kept on hay and straw.
To induce airway obstruction in horses with a history of COPD, 10 horses (geldings and mares of various breeds, 13.3 ± 3.5 yr old, weighing 1,034.8 ± 37.2 kg) were brought from the pasture into the barn and kept on hay and straw until significant clinical signs of airway obstruction had developed (on average 1 wk). We assessed the severity of airway obstruction on a daily basis by means
of a clinical score system that is tightly correlated with changes in
pulmonary functions (26). As blood donors, we used two horses with
heaves that consistently developed severe airway obstruction within
1-2 days of stabling and four control horses stabled in the barn.
Tissue Collection
The animals were killed by an intravenous injection of pentobarbital
sodium; the rib cage was opened; and the heart, lung, and trachea were
excised. Immediately after death, the cardiac region of the right lung
was collected and suspended in Krebs-Henseleit (KH) solution
(composition in mM: 118.4 NaCl, 25.0 NaHCO3, 11.7 dextrose, 4.7 KCl,
2.6 CaCl2 · 2H2O,
1.19 MgSO4 · 7H2O,
and 1.16 KH2PO4)
saturated with 95% O2-5%
CO2. During dissection and
experimental protocols, the tissues were kept in the KH solution that
was continuously gassed with 95%
O2-5%
CO2.
Tissue Preparation
SA preparations were isolated from the peripheral part of the cardiac
region following procedures described previously (22). In this anatomic
location, 1- to 2-mm OD horse airways represent generations 12-16
and are the smallest airways that still contain some cartilaginous
elements. Dissected SA preparations were placed in a 2-ml tissue bath
(Radnoti Glass Technology) filled with KH solution (38°C), which
was replaced every 15 min and bubbled with 95%
O2-5%
CO2 during the entire experiment.
The lower end of the SA preparation was fixed with surgical silk ties
to the glass tissue holder, which secured the tissue at the bottom of
the tissue bath. The upper tie was hooked to a force transducer (model
FT03, Grass Instruments, Quincy, MA) installed on a tension
manipulator. Isometric force of the tissue preparations was recorded on
a polygraph (model 7D or 7E, Grass Instruments). In this setup, tissues
were suspended in the middle of the tissue bath between two platinum wire electrodes built vertically in the wall of tissue bath. During a
2-h equilibration period, optimal passive tension was determined by
gently stretching the tissue and using 127 mM KCl to induce contraction, followed by two applications of EFS (1 Hz, 20 V, 0.5 ms)
at 30-min intervals. Optimal tension was in the range of 2-2.2 g.
Square-wave EFS impulses were generated by a stimulator (model S88,
Grass Instruments) and delivered to the electrodes via a stimulus power
booster (Stimu-Splitter II, Med Lab Instrument, Loveland, CO). After
equilibration, we treated the tissues with KCl for a second time to
determine the maximal response (100% KCl). The experimental protocols
were conducted in eight muscle baths.
Blood Collection and Neutrophil Isolation
For protocols that required neutrophils, peripheral blood (60 ml from
each horse) was collected into EDTA-containing Vacutainer tubes via
jugular venipuncture from an acutely heavey and a control horse. A
two-step isolation method was used. Buffy coat was collected from blood
tubes after centrifugation (TJ-6R, Beckman Instruments, Palo Alto, CA)
at 1,500 rpm for 15 min and gently layered on the surface of a density
gradient of 59 and 75% isosmotic Percoll solutions. After 45 min of
centrifugation at 3,000 rpm and 14°C, the neutrophils accumulated
in the form of a cloudy band at the gradient interface. After
aspiration, the neutrophils were suspended in Ca-, Mg-, and phenol-free
Hanks' balanced salt solution (HBSS) and spun and washed two times.
Cell count, purity, and viability were assessed, and activity of the
neutrophils stimulated with serum-treated zymosan (STZ) was determined
by measurement of superoxide (O2·)
production. For that purpose, we used the superoxide dismutase
(SOD)-inhibitable ferricytochrome c
reduction assay according to method developed by Babior et al. (2) and
adapted by Tithof et al. (28), who provided its detailed description in
their papers.
Protocols
We designed two major groups of protocols to test the effects of
inflammation on SA responses.
Inflammatory mediators. In the first
subset of experiments, we tested the effects of inflammatory mediators
on SAs from either control or COPD horses. In all protocols, one of the
tissues was not treated with any inflammatory mediator and served as a
time control. Depending on the protocol, other tissues were each
treated with one concentration of mediator: histamine (3 µM),
LTD4 (0.3, 1, 3, or 10 nM), or
5-HT (0.01, 0.1, 1, or 10 µM). After 15 min of incubation, EFS
frequency-response curves were created by the application of increasing
EFS frequencies (0.05-32 Hz). Frequency was increased when the
response to the lower frequency reached a plateau. To confirm that
mediators affected exclusively the endogenous cholinergic response to
EFS (neurally released ACh), two additional tissues were treated: one
with the sodium-channel blocker tetrodotoxin (TTX; 3 µM) and the
other with the muscarinic-receptor antagonist atropine (Atr; 3 µM).
The latter tissues were treated before EFS with a concentration of
inflammatory mediator that, in a pilot study, had the greatest effect
on the EFS response.
After the EFS-response curves were obtained, the tissues were washed
thoroughly with fresh buffer (with and without inhibitor) and rested
for 30 min. The same concentrations of inflammatory mediator were then
added to each tissue bath, and after a 15-min incubation period, the
concentration-response curves to ACh or MCh (depending on the protocol)
were created.
Effects of neutrophils. In a second
subset of experiments, we compared responses to EFS in control and
heavey horse SAs and tested the effects of activated neutrophils on
these responses. Because neutrophils of horses with COPD may have
different mediator profiles or, alternatively, the sensitivity of SAs
to neutrophil-derived mediators in these horses could be different, we
applied a crossover design with four combinations of neutrophils and
tissues isolated from both COPD-affected and control horses. In this
protocol, untreated control tissue and tissue treated solely with STZ
were included in addition to six tissue baths in which SAs were treated with a mixture of neutrophils and STZ (3 mg/ml). Each time, we used
neutrophils isolated from both control and heavey horses, and SAs were
incubated with 3, 10, or 30 × 106 neutrophils/ml. During
experiments with neutrophils, the first EFS frequency-response curve
was created before incubation with the neutrophils; the second and
third EFS curves were created at 30 and 60 min of incubation,
respectively, in the presence of STZ-activated neutrophils.
Before the series of experiments with SAs, we performed pilot studies
in which we measured O2·
production in response to chemotactic ligands
(formyl-methionyl-leucyl-phenylalanine, human recombinant
C5a, and LTB4) and STZ in the
neutrophils isolated from peripheral blood of both control and heavey
horses. The purpose of these studies was to select the optimal
neutrophil activator and to compare the responses of neutrophils
isolated from control and heavey horses. Additionally, to confirm that
neutrophils maintained their activity at the time of incubation,
activity of the neutrophils was tested. We used a fraction of the
neutrophils isolated for our tissue experiments, treated them with STZ,
and measured O2· production by
means of the cytochrome c reduction assay.
Agents
On the day of the experiments, acetylchloride hydrochloride, atropine
sulfate, histamine hydrochloride, 5-hydroxytryptamine hydrochloride,
MCh, and TTX (all from Sigma, St. Louis, MO) were dissolved in
deionized water to obtain stock solutions (10 or 100 mM) as needed.
LTD4 (Calbiochem) was diluted in
KH solution to 10 µM and frozen in portions that were diluted for use
shortly before addition to the tissue baths. Stock solutions of Atr and TTX were directly mixed into the KH solution; other compounds were
serially diluted in the KH solution, and each concentration was added
to the muscle baths in a volume of 1%. The concentrations of all
substances are expressed as their final bath concentration. Cytochrome
c,
formyl-methionyl-leucyl-phenylalanine, HBSS, Percoll, and SOD were all
from Sigma. Sterile Percoll, after adjustment of osmolality and pH by
addition of 10× HBSS and 1 N HCl, was diluted to 59 and 75%
solutions in sterile Ca-, Mg-, and phenol red-free HBSS and carefully
layered in 50-ml tubes as a discontinuous gradient. SOD and cytochrome
c were dissolved in sterile HBSS without phenol red. All solutions were prepared directly before use.
Zymosan (Sigma) was prepared according to the manufacturer's guidelines and opsonized in equine serum. Small portions of STZ were
frozen and stored at 
20°C, and each portion was brought to
room temperature directly before use.
Statistics
Tension study data (means ± SE) are expressed as a percentage of
the response to 127 mM KCl-substituted KH solution, and
n represents the number of horses used
in each protocol. To determine drug effects, we applied between-bath
comparisons of treated and control tissues. This excluded any effects
of time or tachyphylaxis. Data were calculated and analyzed (Excel 7.0, Microsoft, and SSPS for Windows 7.0, SSPS, on a Gateway 2000 P5-133
computer) by means of paired or unpaired
t-test, one-way ANOVA, or mixed-design
factorial ANOVA as appropriate. A post hoc Dunnett's test was used to
compare means between treatment and control values. Means were accepted to be significantly different at P 
0.05.
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RESULTS |
SA Responses to EFS
Just as in larger airways, SAs isolated from horses with COPD produced
weaker responses to EFS than those from control animals (Fig.
1), although the responses to KCl were
identical in both groups of tissues.
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Fig. 1.
Comparison of electrical field stimulation (EFS)-induced tension
responses in small airways isolated from horses with chronic
obstructive pulmonary disease (COPD; heavey) and control horses
(n = 5/group). Responses are
expressed as a percentage of tissue contraction evoked by 127 mM KCl
(%KCl). * Significantly different from control value.
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Inflammatory Mediators
Effect of LTD4.
LTD4 contracted the airway in a
concentration-dependent manner (Fig. 2);
however, there was a great deal of variability among individual tissue
responses to LTD4. The response to
EFS was augmented by 0.3-3 nM
LTD4 (Fig.
3A).
The increase in the response to EFS was greatest when the tissues were
slightly contracted by LTD4; however, elevation of baseline tension was not absolutely necessary for
this augmentation to occur. Both TTX and Atr completely blocked the
responses to EFS in the presence of
LTD4 (Fig.
3B). The response to MCh was
additive with the LTD4-induced
contraction but was not synergistic (Fig.
3C). Similar to control tissues, in
SAs from COPD horses, LTD4
augmented the response to EFS (Fig.
4A), whereas the MCh concentration-response curve was only slightly affected
(data not shown).
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Fig. 3.
Effects of LTD4 on cholinergic
small-airway responses. EFS of control horse tissues treated with
LTD4 was carried out in absence
(A; n = 5 animals) and presence (B;
n = 5 animals) of atropine (Atr) or
TTX. Methacholine (MCh) was used as exogenous cholinergic stimulus
(C; n = 5 animals). Results with 10 nM
LTD4 are not shown because they
overlap with those with 3 nM. * Significantly different from
control value (A) and 3 × 109 M
LTD4
(B).
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Fig. 4.
Effect of LTD4
(A; n = 5 animals), 5-HT (B;
n = 5 animals), and histamine
(C; n = 5 animals) on responses of small airways from COPD horses to EFS.
* Significantly different from control value.
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Effect of 5-HT. 5-HT (0.01-10
µM) contracted only some of the SA preparations, and the magnitude of
the 5-HT-induced contraction was generally small (Fig. 2). Much greater
than the effect on the baseline tension was the dose-dependent increase
in SA responses to EFS in the presence of 5-HT (Fig.
5A).
Maximal augmentation was observed at 1 µM 5-HT. In the presence of
Atr and TTX, tissues treated with 1 µM 5-HT did not respond to EFS
(Fig. 5B). 5-HT had no effect on the
response to exogenous ACh (Fig. 5C).
In SAs from COPD horses, 5-HT produced an effect similar to that in
normal tissue (Fig. 4B).
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Fig. 5.
Effects of 5-HT on cholinergic small-airway responses. EFS of tissues
treated with 5-HT was carried out in absence
(A; n = 5 animals) and presence (B;
n = 5 animals) of Atr or TTX, and ACh
was used as exogenous cholinergic stimulus
(C; n = 5 animals). Results with 10 µM 5-HT are not shown because they
overlap with those with 1 µM. * Significantly different from
control value (A) and
106 M 5-HT
(B).
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Effect of histamine. Consistent with
previous data (22), histamine (3 µM) induced a small
contraction of SAs and dramatically augmented the responses to EFS
(Fig. 6). In the presence of Atr, responses
to EFS in the presence of histamine were abolished (Fig. 6), indicating
that augmentation of the EFS response by histamine was due to an
increased cholinergic response and not by activation of other
mechanisms. Similar augmentation in response to histamine was also
observed in SAs from the COPD group (Fig.
4C) but not in their response to MCh
(data not shown).
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Fig. 6.
EFS responses of small airways incubated with 3 µM histamine in
presence and absence of Atr (n = 4 animals). * Significantly different from 3 µM histamine.
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Neutrophils
For each experiment, we were able to isolate consistently a
sufficient number of neutrophils (1-4 × 108), with both purity and
viability > 98%, to perform the tissue bath experiments with
neutrophils from both control and heavey horses. We selected STZ as the
best neutrophil activator because it does not affect SA responses to
EFS, and in contrast to chemotactic ligands that activate neutrophils
only for a 5-min period, the STZ-induced respiratory burst lasted over
the period of 1 h (data not shown). Neutrophils isolated from heavey
horses stimulated with STZ produced significantly more
O2· than those from control horses
(Fig. 7).
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Fig. 7.
Production of superoxide by peripheral blood neutrophils treated with
serum-treated zymosan (STZ; 1 mg/ml). * Significantly different
from control value.
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Even though a sufficient number of neutrophils were isolated and
these cells were strongly activated by STZ during each experiment, we
did not observe any effect of neutrophils on the EFS response in SAs
(Fig. 8). Coincubation of SAs with
neutrophils over the period of 30 (data not shown) and 60 min (Fig. 8)
neither increased baseline tension nor significantly affected the
response to EFS.
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Fig. 8.
Effect of neutrophils on small-airway responses to EFS. Small airways
(n = 5 animals) were incubated for 60 min with STZ-activated neutrophils. A:
control small airways and control neutrophils.
B: control small airways and
neutrophils from COPD horses. C: small
airways from COPD horses and control neutrophils.
D: small airways and neutrophils from
COPD horses. There was no significant effect of neutrophils compared
with tissue treated only with STZ.
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DISCUSSION |
In a variety of airway diseases and animal models of airway
obstruction, the inflammatory response has been shown to be the crucial
event leading to bronchospasm and airway hyperresponsiveness (27). In
equine COPD, airway inflammation and cholinergically mediated
bronchospasm are also associated, but the role of inflammatory response
and the mechanisms by which it might affect airway tone remain
obscure. Additionally, as previously shown in larger airways (36)
and presently in peripheral ones (Fig. 1), tissues isolated from horses with COPD are not hyperresponsive. Paradoxically, they
produce weaker responses to cholinergic stimulation. This apparent
discrepancy between the in vitro and in vivo airway responses provides
important information about the mechanism of cholinergic bronchospasm
in COPD. Rather than being caused by some chronic changes in nerve
terminals or smooth muscle itself, e.g., by upregulation of
M3 muscarinic receptors on airway
smooth muscle (ASM) or decreased acetylcholinesterase
activity, the increase in cholinergic airway tone is most likely caused
by factors that, when present in the airways, facilitate either local
ACh release or the response of smooth muscle to ACh released by nerves.
Because COPD is an inflammatory disease in which both airway cytology
and autacoid profile change rapidly in response to antigen challenge,
we reasoned that inflammatory mediators may be responsible for altered
cholinergic responses of horses with COPD. Several inflammatory
mediators are known to cause bronchospasm not only via a direct
contractile effect on ASM but also by more complex interactions with
mechanisms of airway control. Previously, Olszewski et al. (22) showed
that histamine has synergistic effects with SA responses to EFS in vitro. To further investigate the effects of mediators on cholinergic airway response, we applied two experimental models. In the first, we
tested the effect of several inflammatory mediators implicated in the
pathogenesis of COPD. In the second approach, we used activated inflammatory cells.
In response to natural antigen challenge, all three inflammatory
mediators, histamine, LTD4, and
5-HT, are reported to increase in respiratory secretion, urine, and
plasma of COPD horses (10, 11, 19). In our experiments, treatment of
SAs from either control or COPD horses with any of these mediators
caused a quite dramatic leftward shift of the frequency-response curve
to EFS. Considering the possibility of interactions of these mediators with the airway responses in vivo, particularly interesting is the
large (often manyfold) augmentation of SA contraction at the lower
range of EFS frequencies. These are the physiological frequencies at
which postganglionic parasympathetic nerves are thought to periodically
fire in the airways. With regard to the mechanism, we further confirmed
that all mediator effects on the EFS responses were exclusively due to
modulation of the cholinergic activity because Atr and TTX (blockade of
either muscarinic receptors or neuronal fast sodium channels) abolished
the responses to EFS in the presence of all three inflammatory
mediators. This is in contrast to dog airways where histamine unmasks
otherwise absent 
-adrenergic contractions in atropine-treated
tissues (3).
Even though the maximal synergistic effect of the mediators was
observed when tissues were contracted by the mediators up to the level
of 10-20% of their maximal response, the effects of the mediators
on the EFS response are not just related to the contractile status of
the tissue. As we observed, particularly with the lower concentrations
of mediators, baseline elevation was not necessary to produce a quite
impressive increase in tissue response to EFS. We also observed that
the effect of inflammatory mediators on the EFS response was more long
lasting than their direct effect on tissue tension. Although responses
to smaller concentrations of mediators started to decrease or even
waned after several minutes (compare the maximal responses of
LTD4 in Fig. 2 with the baseline
representing remaining tension response after 15 min of incubation in
Fig. 3), the increased response to EFS was present for a long period of
time and, in the case of histamine, persisted for up to 30 min after
the washout (Olszewski, personal observations). The
response to exogenous cholinergic stimulation with either ACh or MCh
was not subject to similar synergism and was simply additive. This
observation indicates that the effects of histamine, LTD4,
and 5-HT are not mediated by an alteration at the level of muscarinic
receptors on ASM or by a change in the mechanical properties of the
tissue due to elevation of the baseline tension by these mediators. The
large effect of the mediator on the EFS response and lack of a similar effect on the exogenous ACh-response curve may provide evidence of
prejunctional modulation of ACh release from parasympathetic nerve
terminals. To make a firm conclusion, measurements of ACh release from
SA cholinergic nerves in the presence of inflammatory mediators is
required. We attempted to measure ACh release from SAs in the presence
and absence of histamine utilizing HPLC coupled with electrochemical
detection. Even though this method is very well established in our
laboratory for measurement of ACh release in both bronchi and the
trachea (31, 37), we could not determine the release of ACh from equine
SAs because the amount of ACh released was very small and below the
level of detection. Regardless of the mechanism by which inflammatory
mediators exerted their effect on the EFS response, we have shown that
inflammatory autacoids may greatly influence the magnitude of the
endogenous, cholinergic response in equine terminal airways, and in
this respect, several mediators may exert a similar effect. This strong
synergism between mediator-induced airway contraction in response to
nerve stimulation at a physiological range of frequencies is consistent
with the hypothesis that inflammatory mediators released in response to antigen challenge are responsible for the increased cholinergic tone of
the airways in horses with COPD. Noteworthy, all of these mediators
could produce effects of similar magnitude. In the clinical course of
COPD, where many inflammatory mediators act in concert, this may be
responsible for the relatively low therapeutic efficacy of compounds
that block the effects of singular mediators (e.g., antihistamines) in
contrast to very efficient glucocorticosteroids that blunt all of the
inflammatory process (4, 17).
In our second approach, we re-created the milieu of neutrophilic
inflammation through direct contact of SA preparations with activated
neutrophils. Neutrophils are implicated in the pathogenesis of COPD
because 1) they are consistently
recruited into the airways and their increase in bronchoalveolar lavage
is one of the classic clinical findings in COPD (25);
2) neutrophils isolated from peripheral blood of heavey horses produced more
O2· in response to activation with
chemotactic ligands and STZ (Fig. 7) and neutrophils in the airways of
horses with COPD are strongly activated (21); and
3) in other species, neutrophils or
neutrophil-derived inflammatory mediators such as reactive oxygen
species or thromboxane A2 have
been shown to contract smooth muscle or affect their responses (7, 15,
35). Even though indirect evidence supports it, the exact role of the
neutrophil in the pathogenesis of COPD is not very clear. On one hand,
antigen challenge-induced recruitment of neutrophils into the airways
of COPD-susceptible horses is generally concurrent with the first
changes in lung function, which argues for a strong association between
these two events. On the other hand, in some exceptional individuals,
changes in lung function precede neutrophil recruitment or the
recruitment appears earlier than the alterations in pulmonary function
(12). These last few pieces of evidence indicate that even though the association between neutrophil recruitment and changes in lung function
may exist, neutrophils are neither sufficient nor necessary for airway
obstruction to occur. Our data clarify this even further. Coincubation
of SAs with a large number of strongly activated neutrophils over a
period of 1 h did not significantly alter the SA responses. In fact,
there was a tendency to decrease the EFS response rather than to cause
augmentation. Also, smaller numbers of neutrophils, unactivated
neutrophils, or neutrophils activated by stimuli other than STZ failed
to alter in vitro responses of SAs or equine tracheae (personal
observations). Lack of a synergistic effect of neutrophils was not
entirely surprising and parallels some other observations. For example,
hydrogen peroxide, one of the reactive oxygen species produced by
activated neutrophils, decreases cholinergic responses in equine
tracheae (23), and the neutrophil-derived enzyme elastase, in
concentrations present in respiratory secretion, decreases tension
responses of rabbit trachealis (7).
Our observation changes our view of the pathogenesis of COPD by
suggesting that neutrophils may not be as important in the pathogenesis
of COPD as previously postulated and that the cholinergic component of
airway obstruction in horses is most likely "neutrophil independent." However, the role of the neutrophil cannot be entirely excluded based on our data. Neutrophil products may exert some long-term effects on airways, e.g., promote inflammation, edema formation, and mucus secretion, and in this way contribute to airway
obstruction by mechanisms not directly related to neuromuscular regulation of airway tone. In contrast to neutrophil-derived mediators, effects of histamine, LTD4, or
5-HT may explain the mechanisms of increased cholinergic airway tone in
COPD. These mediators are traditionally associated with a type I
allergic reaction (mast cell derived), and in this respect, our data
support some current reports (9, 16, 19, 30, 34) favoring a type I
reaction as an important mechanism in the development of airway
obstruction in COPD.
| |
ACKNOWLEDGEMENTS |
We thank Cathy Berney and Deborah Boehler for technical assistance
and Victoria Hoelzer-Maddox and MaryEllen Shea for manuscript preparation.
| |
FOOTNOTES |
We are grateful to Bayer (Leverkusen, Germany), and the Matilda R. Wilson Fund for the support for these studies.
Address for reprint requests and other correspondence: N. E. Robinson,
Dept. of Large Animal Clinical Sciences, Michigan State Univ., East
Lansing, MI 48824-1314.
Received 1 August 1997; accepted in final form 23 November 1998.
| |
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