Persistence of Serotonergic Enhancement of Airway Response in a Model of Childhood Asthma

Article (PDF Available)inAmerican Journal of Respiratory Cell and Molecular Biology 51(1) · January 2014with52 Reads
Impact Factor: 3.99 · DOI: 10.1165/rcmb.2013-0387OC · Source: PubMed
Abstract
Rationale: Persistence of airway hyperresponsiveness and serotonergic enhancement of airway smooth muscle contraction induced by ozone plus allergen has not been evaluated. If this mechanism persists after a prolonged recovery, it would indicate that early-life exposure to ozone plus allergen induces functional changes predisposing allergic individuals to asthma-related symptoms throughout life, even in absence of environmental insult. A persistent serotonergic mechanism in asthma exacerbations may offer a novel therapeutic target, widening treatment options for asthmatics. Objectives: To determine if previously documented airway hyperresponsiveness and serotonin-enhanced airway smooth muscle contraction in allergic monkeys exposed to ozone plus house dust mite allergen persist after prolonged recovery. Methods: Infant rhesus monkeys sensitized to house dust mite allergen were exposed to filtered air (n=6) or house dust mite allergen plus ozone (n=6) for 5 months. Monkeys were then housed in a filtered air environment for 30 months. At 3 years, airway responsiveness was assessed. Airway rings were then harvested and airway smooth muscle contraction was evaluated using electrical field stimulation with and without exogenous serotonin and serotonin subtype receptor antagonists. Measurements and Main Results: Ozone plus house dust mite allergen exposed animals exhibited persistent airway hyperresponsiveness. Serotonin exacerbated the airway smooth muscle contraction in the exposure group, but not in the filtered air group. Serotonin subtype receptors 2, 3, and 4 appear to drive the response. Conclusions: Airway hyperresponsiveness and serotonin-dependent exacerbation of cholinergic-mediated airway smooth muscle contraction induced by early-life exposure to ozone plus allergen persist for at least 2.5 years and may contribute to a persistent asthma phenotype.

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Available from: Brian D Moore, Jul 10, 2014
ORIGINAL RESEARCH
Persistence of Serotonergic Enhancement of Airway Response
in a Model of Childhood Asthma
Brian D. Moore
1
, Dallas M. Hyde
2
, Lisa A. Miller
2
, Emily M. Wong
2
, and Edward S. Schelegle
2
1
University of the Pacific, Stockton, California; and
2
California National Primate Research Center, Respiratory Diseases Unit, University of
California Davis, Davis, California
Abstract
The persistence of airway hyperresponsiveness (AHR) and
serotonergic enhancement of airway smooth muscle (ASM)
contraction induced by ozone (O
3
) plus allergen has not been
evaluated. If this mechanism persists after a prolonged recovery, it
would indicate that early-life exposure to O
3
plus allergen induces
functional changes predisposing allergic individuals to asthma-
related symptoms throughout life, even in the absence of
environmental insult. A persistent serotonergic mechanism in
asthma exacerbations may offer a novel therapeutic target, widening
treatment options for patients with asthma. The objective of this
study was to determine if previously documented AHR and
serotonin-enhanced ASM contraction in allergic monkeys exposedto
O
3
plus house dust mite allergen (HDMA) persist after prolonged
recovery. Infant rhesus monkeys sensitized to HDMA were exposed
to ltered air (FA) (n = 6) or HDMA plus O
3
(n = 6) for 5 months.
Monkeys were then housed in a FA environment for 30 months. At
3 years, airway responsiveness was assessed. Airway rings were then
harvested, and ASM contraction was evaluated using electrical eld
stimulation with and without exogenous serotonin and serotonin-
subtype receptor antagonists. Animals exposed to O
3
plus HDMA
exhibited persistent AHR. Serotonin exacerbated the ASM
contraction in the exposure group but not in the FA group.
Serotonin subtype receptors 2, 3, and 4 appear to drive the response.
Our study shows that AHR and serotonin-dependent exacerbation of
cholinergic-mediated ASM contraction induced by early-life
exposure to O
3
plus allergen persist for at least 2.5 years and may
contribute to a persistent asthma phenotype.
Keywords: serotonin; ozone; antigens; hyperresponsiveness;
Macaca mulatta
Clinical Relevance
Airway hyperresponsiveness and serotonin-dependent
exacerbation of cholinergic-mediated airway smooth muscle
contraction induced by early-life exposure to ozone plus allergen
persist for at least 2.5 years and may contribute to a persistent
asthma phenotype. These ndings substantiate the need to
minimize exposure of young individuals to known environmental
contributors to asthma during critical periods of lung maturation
because damage inicted during these times can contribute to
prolonged asthma symptoms. The identication of a persistent
5-HTmediated enhanced airway smooth muscle contraction
mayidentifynoveltherapeutic targets for pharmacological
intervention in the treatment of childhood asthma.
Asthma is one of the most common chronic
childhood conditions in the United States.
In 2011, z 7 million children suffered from
asthma (1). The most frequent reason for
school absences is asthma, accounting for
one third of school days missed, and the
severity of symptoms is negatively
correlated with achievement (2).
The link between ozon e (O
3
)andhouse
dust mite allergen (HDMA) exposure and
childhood asthma has been supported by
a wealth of research (39). In the latest State
of the Air report, almost half of United States
citizensover 148 million peoplelive in
areas with unhealthy O
3
levels (6).
Although the mechanisms responsible
for asthma symptoms are debatable,
research shows that O
3
and HDMA
( Received in original form September 6, 2013; accepted in final form January 23, 2014 )
This research was supported by a National Institute of Health Sciences Program Project grant P01ES000628–33.
Author Contributions: Conception and design: D.H., L.M., E.S., B.M. Analysis and interpretation: E.S., B.M., E.W. Drafting the manuscript for important
intellectual content: B.M., E.S.
Correspondence and requests for reprints should be addressed to Edward Schelegle, Ph.D., Anatomy, Physiology & Cell Biology, University of California–Davis,
Davis, CA 95616. E-mail: esschelegle@ucdavis.edu
This article has an online supplement, which is acces sible from this issue’s table of contents at www.atsjournals.org
Am J Respir Cell Mol Biol Vol 51, Iss 1, pp 77–85, Jul 2014
Copyright © 2014 by the American Thoracic Society
Originally Published in Press as DOI: 10.1165/r cmb.2013-0387OC on January 31, 2014
Internet address: www.atsjournals.org
Moore, Hyde, Miller, et al.: Persistent Response to Allergen and O
3
77
Page 1
exposure leads to functional, structural,
neural, vascular, and immunological
alterations in airways (1016). This airway
remodeling indicates that environmental
insults early in life can have life-long
deleterious effects on lung function and
may lead to chronic asthma symptoms.
Airway hyperresponsiveness (AHR),
a functional hallmark of asthma, is assessed
with a bronchoprovocation test (17). The
presence of AHR after O
3
and HDMA
exposure indicates that airway function has
been compromised. In humans, nonhuman
primates, and other models of asthma, O
3
and HDMA exposure has been shown to
increase AHR (1821).
Recent literature suggests that
serotonin (5-HT) plays a role in the asthma
response (2224). Animal studies show that
5-HT increases airway resistance and O
3
1
HDMA exposure of infant monkeys results
in 5-HTpositive cells within airway
epithelia (2527). Patients with asthma
have higher 5-HT plasma levels, which are
inversely correlated with lung function, and
drug treatments that lower plasma 5-HT
decrease symptom severity and improve
lung function (6, 28, 29). Our lab has
shown that exposure of O
3
1 HDMA
induces AHR and exacerbates 5-
HTmediated airway smooth muscle
(ASM) contraction in a model of childhood
asthma (18) and that the combined
exposure of O
3
1 HDMA results in
alterations in 9 of 10 immune, structural,
and functional end points, with six of the
end points demonstrating greater than
additive effects of O
3
or HDMA exposure
alone (10). Studies have epidemiologically
conrmed asthma persistence from
childhood through adulthood, linking
persistent symptoms to atopy, smoking, air
pollution (including O
3
), early age at onset of
asthma, and airway remodeling (3034).
To date, no study has examined the
persistence of AHR and 5-HT enhancement
of ASM contraction in a controlled setting
using a model of childhood asthma. The
aims of this project are (1) to determine
if AHR and 5-HTenhanced ASM
contraction induced by O
3
1 HDMA
exposure persist after a prolonged recovery
period in a ltered air (FA) environment
and (2) to identify which 5-HT subtype
receptors are responsible for driving the 5-
HT response. Conrming the persistence of
AHR caused by early-life exposure to O
3
1
HDMA will help guide environmental
policy and substantiate the need to mitigate
exposure, especially in young populations.
Associating a 5-HTmediated mechanism
with persistent AHR offers a novel
therapeutic target for asthma treatment.
Rhesus monkeys were used because
they have similar lung cellular morphology,
airway architecture, and immunology
and undergo a similar extensive period
of postnatal development compared
with humans (3539). In addition to
possessing all of the components of the
intrapulmonary conducting airways that
are altered in humans with asthma, rhesus
monkeys display a similar progression of
asthma pathophysiology and symptoms
(11, 40). The sensitization protocol used
induces the functional, immunologic,
histological, and clinical characteristics that
are used to diagnose allergic asthma (40).
Materials and Methods
Care of animals complied with the Institute
of Laboratory Animal Resources and the
American Association for Accreditation of
Laboratory Animal Care (AAALAC).
Procedures were approved by the University
of California - Davis Institutional Animal
Care and Use Committee (41). The
University of California - Davis and the
California National Primate Research
Center are accredited by AAALAC.
General Protocol
Twelve 30-day-old, captive-born rhesus
monkeys were randomly assigned to one of
two groups: FA or O
3
plus HDMA (O
3
1
HDMA). All animals were sensitized to
HDMA and exposed to 11 episodes of FA
or O
3
1 HDMA as previously described
(10, 18) (Figure 1). Exposures had a HDMA
mass concentration averaging 7.05 6
0.73 mg/m
3
and a mean O
3
concentration of
0.500 6 0.005 ppm. Monkeys were killed
with sodium pentobarbital (15 ml/kg). A
distal tracheal portion was harvested and
placed in modied Krebs solution.
Airway Responsiveness Testing
At 3 years of age, airway resistance (R
aw
)
was measured during a histamine challenge
and expressed as the concentration of
histamine causing a 200% increase in Raw
(EC200R
aw
) (40).
Electrical Field Stimulation
Airway rings were suspended between
platinum wire electrodes in tissue baths
(Myobath; WPI Inc., Sarasota, FL) as
previously described (18). Tension was
measured via Fort 10 g transducers (WPI
Inc.) and recorded with Powerlab Chart 5.1
software (ADInstruments, Colorado
Springs, CO). Monophasic square-wave
impulses (50 V, 4 Hz, 0.5 ms) were
delivered for 30 seconds every 4 minutes
until three consecutive stable responses
were observed. Pulses were induced via S88
Stimulators (Grass Technologies, West
Warwick, RI).
5-HT Concentration-Response
Curves
Six rings from each animal were used to
perform 5-HT concentration-response
curves during electric eld stimulation
(EFS)-induced contractions.
Antagonist Concentration-
Response Curves
5-HT concentration-response curves were
performed in the presence of antagonists
(Table 1).
The Effect of 5-HT
1A
R Activation
Previous research identied an inhibitory
pathway mediated through 5-HT
1A
receptors (18). These analyses were
reproduced.
Baseline Responses
Acetylcholine (ACh) concentration-
response curves were performed (one
control and one preincubated with 10 mM
5-HT). Voltage-response curves and
frequency-response curves were performed
on tracheal rings.
Concluding Experiments
Airway rings were exposed to 10 mM ACh
to compare tension with the initial ACh
concentration-response curves. The effect of
atropine (1 mM) was evaluated to ensure
muscarinic-mediated contractions. To
verify that the responses were neurogenic,
tissue was incubated in 3 mM tetrodotoxin
before EFS. All drugs were purchased from
Sigma-Aldrich Co. (St. Louis, MO).
Statistical Analysis
Results are expressed as mean 6 SEM.
Airway responsiveness data; between-group
EC
50
,EV
50,
EF
50
; and 5-HT direct effect
values were analyzed using Students t tests.
Within-group 5-HT direct effects and the
direct effect of 8-OH-DPAT were analyzed
with paired-samples t tests.
ORIGINAL RESEARCH
78 American Journal of Respiratory Cell and Molecular Biology Volume 51 Number 1
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Concentration-responses were compared
using repeated-measures ANOVAs. Tukey
post hoc testing was used to identify the source
of signicance. The a level was set at 0.05.
Signicance was based on the adjusted
P value. A one-way t test was used to assess
the direct effect of 5-HT on ASM
contraction based on previous ndings that
5-HT constricts ASM.
Results
Airway Responsiveness
O
3
1 HDMA exposure induced
a signicant increase in airway
responsiveness when compared with FA
controls, even with a prolonged 2.5-year
recovery period in FA (EC200R
aw
FA =
14.43 6 3.89 mg/ml; O
3
1 HDMA = 4.88 6
0.60 mg/ml; P = 0.04) (Figure 2).
5-HT Concentration Response
O
3
1 HDMA exposure resulted in
enhanced airway contractility in the
presence of 5-HT, indicated by signicantly
increased EFS-induced ASM tension
production when compared with FA
animals at 100 mM 5-HT (FA = 308.5 6
39.0%; O
3
1 HDMA = 930.3 6 97.4%; P =
0.008). All 5-HT concentrations in the
O
3
1 HDMA group produced signicantly
greater tension than the EFS-induced
tension produced during their control
response (EFS-induced contraction in the
absence of 5-HT; P , 0.05), which was not
the case in the FA group. In the FA group,
none of the 5-HT concentrations elicited
a contraction signicantly greater than its
control response (P . 0.05) (Figure 3).
There was also an overall group effect, with
the O
3
1 HDMA group producing a
signicantly higher mean tension (as % of
control EFS response) than the FA group
(O
3
1 HDMA = 378.0%; FA = 214.3%; P ,
0.006).
Antagonist Concentration Response
In the O
3
1 HDMA group, incubation with
increasing concentrations of 5-HT
2A
(ketanserin), 5-HT
3
(ondansetron), or
5-HT
4
(GR 113808) subtype receptor
antagonists attenuated the tension induced
by EFS at all 5-HT concentrations,
indicating that these three receptors are
involved in the ASM response to 5-HT
(Figure 4).
The Effect of 5-HT
1A
Receptor Activation
Addition of the 5-HT
1A
receptor agonist
8-OH-DPAT signicantly attenuated
EFS-induced ASM contraction in
a concentration-dependent manner
(Figure 5A). This effect was seen in both
the FA and O
3
1 HDMA groups,
indicating that exposure had no effect. The
direct effect of 5-HT
1A
receptor activation
on ASM tension induced by 100 mM
exogenous ACh was also evaluated.
Concentrations of 10 and 100 mM 8-OH-
DPAT signicantly attenuated ACh-
induced tension in both the FA and O
3
1
HDMA groups. There was no between-
group difference in the response, indicating
that exposure had no effect on 5-HT
1A
receptor activation (Figure 5B).
Direct Effect of 5-HT on ASM Tension
The addition of 10 mM 5-HT produced
a small, but consistent, increase in ASM
tension in the FA and O
3
1 HDMA
groups. In the FA group, tension increased
from 0.992 6 0.007 g to 1.204 6 0.229 g
(P = 0.037). This increase was just over 6%
of the maximal response to ACh. In the
O
3
1 HDMA group, 10 mM 5-HT caused
a tension increase amounting to 12.75% of
the maximal ACh response (Figure 6A).
When comparing the change in absolute
tension between the FA and O
3
1 HDMA
groups, O
3
1 HDMA exposure produced
a signicantly greater 5-HTinduced
tension increase compared with the FA
group (0.549 6 0.146 g versus 0.211 6
0.094 g) (Figure 6B). This indicates that 5-
HT directly induces ASM contraction and
that O
3
1 HDMA exposure exacerbates
this response.
Baseline ASM Response
Before 5-HT addition, frequency-response
and voltage-response curves were
performed. There was no difference in the
voltage needed to induce 50% of maximum
EFS tension (EV
50
) between groups (EV
50
,
FA = 41.6 6 2.5 V; O
3
1 HDMA = 42.5 6
4.4 V; P . 0.05). There was no signicant
difference in the frequency necessary to
induce 50% of maximum EFS tension
(EF
50
) between groups (EV
50
, FA = 9.6 6
2.4 Hz; O
3
1 HDMA = 7.1 6 0.7 Hz;
Figure 1. Timeline of exposure protocol. FA, filtered air; HDMA, house dust mite allergen.
Table 1. Summary of Antagonists
Drug Receptor Concentration Range (mM) References
Ketanserin 5-HT2A 1, 10, 100 61, 62
Ondansetron 5-HT3 1, 10, 100 63, 64
GR 113808 5-HT4 1, 10, 100 65, 66
ORIGINAL RESEARCH
Moore, Hyde, Miller, et al.: Persistent Response to Allergen and O
3
79
Page 3
P . 0.05). This indicates that without
exogenous 5-HT added to the tissue baths,
the airway rings from the O
3
1 HDMA
animals and the FA animals responded
similarly to EFS.
Concluding Experiments
Tissue response to 10 mM ACh at the
conclusion of the experiment produced over
93% (1.442 g versus 1.541 g) of the tension
seen at the beginning of the protocol,
indicating adequate tissue viability
throughout the testing. Atropine and
tetrodotoxin completely attenuated EFS
response, conrming that EFS-induced
contractions were neurogenic of origin and
induced by activation of cholinergic
receptors on the ASM.
Discussion
Although persistent asthma symptoms have
been linked to environmental O
3
and
allergen exposure, no study to date has
examined the persistent effect of O
3
1
HDMA exposure in a model of childhood
asthma. Our previous research showed that
cyclical exposure to O
3
1 HDMA from 1 to
6 months of life in allergic rhesus monkeys
results in a hyperresponsive airway and in
a 5-HTmediated enhancement of ASM
contraction (18). With the wealth of
epidemiologic data supporting the negative
effects of early-life exposure to O
3
and
allergens, it makes sense to question
whether or not the functional decrements
seen with O
3
1 HDMA exposure in our
model of childhood asthma would persist if
the animals were allowed a prolonged
recovery in a FA environment (6, 4244).
This study conrms that O
3
1 HDMA
exposure induces persistent AHR and
exacerbated 5-HTmediated ASM
contraction, even after a prolonged recovery
period, in a model of childhood asthma.
Airway Responsiveness
AHR is a functional indicator of asthma.
When comparing EC200R
aw
between the
O
3
1 HDMA exposure and FA groups, the
exposure group required a signicantly
lower dose (Figure 2). The AHR seen in the
O
3
1 HDMA group closely resembles that
of our previous work using the same
exposure protocol that did not allow for
a prolonged recovery (18). This signies
that the functional decrement induced by
O
3
1 HDMA exposure seen after 5 months
of exposure persists even after 2.5 years
of recovery in a FA environment. The
persistence of AHR after a prolonged
recovery period underscores the deleterious
effects of early-life exposure to O
3
1
HDMA and that such exposure not only
leads to acute pulmonary dysfunction in
allergic individuals but induces chronic
changes in airway function that remain
after a long recovery period even when the
environmental insult is no longer present.
Unlike previous studies (25, 45, 46) in
which sensitivity to HDMA was maintained
in the O
3
1 HDMA group during recovery,
persistent functional alterations were found
in this study even though there was no
attempt to ensure maintained HDMA
sensitivity and the recovery period was
extended from 6 to 30 months. The impact
of this observation is even more signicant
when one considers that the exposure
occurred during a period of rapid postnatal
lung development and that the detrimental
effects were still present at an age
equivalent to preadolescence in humans.
This reinforces the need to minimize
childrens exposures to air pollution and
allergens during the extended postnatal
maturation of the lungs, otherwise risking
decrements in lung function lasting into
adulthood, regardless of the presence of
environmental insults.
5-HT Concentration-Response
Curves
Not only did the pulmonary functional
decrements induced by O
3
1 HDMA
exposure persist after the prolonged
recovery p eriod, but the 5-HTmediated
exacerbation of ASM contraction did
as well (Figure 3 ). The persistence of
a 5-HT mechanism with the functional
decrements indi cates that altered
serotonergic signaling at the postganglionic
nerveinnervatingASMmayplayarole
in the persistent AHR induced by O
3
1
HDMA exposure. 5-HT has been
implicated in asthma from clinical,
inammatory, immunologic, and
neurogenic points of view (23, 28, 29,
4752). It is well subst antiated that
5-HT can enhance the neuronal release
of ACh at nerve endings (53). Mechanistically,
it is possible that an increase in the
presence of 5-HT or an up-regulation
of 5-HT receptors at the postganglionic
nervecouldinduceexacerbated5-
HTmediated ASM contraction, leading
to AHR and contribut ing to chronic
asthma symptoms. 5-HT has bee n shown
to up-regulate thromboxane release,
leadingtoAHR(22).ExposuretoO
3
1
HDMA induces the proliferation of
5-HTcontaining cells in the airway
epithelia (25), and 5-HT levels are
increased in the bronchoalveolar lavage
uid of patients with asthma after allergen
challenge (23). In mice, allergen challenge
induces 5-HT release from platelets
(23). These studies indicate a plausible
mechanism for 5-HT contributing to AHR
Figure 2. Airway responsiveness during
histamine challenge. *The O
3
1 HDMA group
exhibited a significant increase in airway
responsiveness when compared with FA (P =
0.04; n = 6). EC200R
aw
is the effective
concentration of histamine needed to induce
a 200% increase in airway resistance. The lower
the effective concentration, the more responsive
the airway.
Figure 3. Effect of serotonin (5-HT)
concentration on airway contractility during
electric field stimulation (EFS). *Tension
production in O
3
1 HDMA group is significantly
greater than in the FA group at 100 mM 5-HT.
In
the O
3
1 HDMA group, tension production at
each 5-HT concentration was greater than the
control response. Control response is defined as
the amount of tension produced via EFS before
addition of 5-HT. There was no within-group
effect seen in the FA group (P . 0.05; n = 6).
ORIGINAL RESEARCH
80 American Journal of Respiratory Cell and Molecular Biology Volume 51 Number 1
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and offer possible sources of 5-HT in the
asthmatic airway.
Antagonist Concentration-
Response Curves
To identify the specic 5-HT subtype
receptors involved in the 5-HT response in
the O
3
1 HDMA group, 5-HT
concentration response curves where
conducted in the presence of 5-HT
2A
,
5-HT
3
, and 5-HT
4
subtype receptor
antagonists. These receptors were targeted
due to previous experiments run by this
group and an extensive review of literature
(18, 27, 49, 54, 55). Separate incubation
with 5-HT
2A
, 5-HT
3
, and 5-HT
4
subtype
receptor antagonists signicantly
attenuated the ASM contractile response
to 5-HT, indicating that these receptors
play a prominent role in the O
3
1
HDMAinduced enhancement of ASM
contraction.
The Effect of 5-HT
1A
Receptor
Activation
As noted in our previous exposure study,
a counterbalancing inhibitory 5-HT effect
was seen, mediated through 5-HT
1A
receptors (18). The 5-HT
1A
receptor
agonist 8-OH-DPAT attenuated any tissue
response to EFS and was able to diminish
the tension produced by exogenous ACh,
indicating that these receptors exert their
effect at the ASM, as opposed to inducing
postganglionic neural inhibition. This
inhibitory effect was seen in both the FA
and O
3
1 HDMA groups, with exposure
having no effect. Although dysregulation of
an inhibitory pathway could lead to AHR
and enhanced ASM contraction, these
results suggest that increased ASM
contraction with 5-HT is due to up-
regulation of an excitatory pathway rather
than the down-regulation of an inhibitory
pathway.
Direct Effect of 5-HT on ASM Tension
O
3
1 HDMA exposure enhanced the direct
effect of 5-HT on ASM (Figure 6). 5-HT
has been shown to directly contract ASM
in multiple species (56, 56 58). The
Figure 4. Effect of 5-HT concentration on airway contractility during EFS in the presence of 5-HT subtype receptor antagonists. (A) The 5-HT
2A
receptor
antagonist ketanserin (Ket). (B) The 5-HT
3
receptor antagonist ondansetron (Ond). (C) The 5-HT
4
receptor antagonist, GR 113808. *All concentrations
of the receptor antagonist induced a significant reduction in tension at each 5-HT concentration when compared with concentration-respon se curve with
5-HT alone.
Administration of 10 and 100 mM of antagonist significantly reduced tension compared with the concentration-response curve with 5-HT
alone. “% of control” response is defined as the amount of tension produced via EFS before addition of 5-HT or 5-HT receptor antagonist (P , 0.05; n = 6).
ORIGINAL RESEARCH
Moore, Hyde, Miller, et al.: Persistent Response to Allergen and O
3
81
Page 5
enhancement of 5-HTs ability to contract
ASM is consistent with recent research
identifying that a similar exposure protocol
in rhesus monkeys induces an up-
regulation of 5-HT receptor expression
on ASM (59).
Although a functional study of this
nature using EFS on whole excised airway
tissue allows insight into the role of 5-HT in
ASM function and AHR in a model of
childhood allergic asthma, the limitations of
such an experimental preparation must be
acknowledged. Pharmacologic identication
of receptor subtypes is common practice,
but one must be careful when drawing
conclusions. We attempted to use the most
selective antagonists available, but, due to
the variance of published receptor afnities,
quantitative rank-order comparison of the
contributions of each receptor subtype could
not be established. Therefore, it was deemed
imprudent to make assumptions regarding
the relative contribution of each identied
5-HT receptor subtype (24) that was
shown to be involved in the serotonergic
enhancement of ASM contraction in
the exposed animals. Current
immunohistochemical studies are
underway to identify which receptor
subtypes are present at the ganglia and
terminal axon of cholinergic nerves in
FA and exposed monkeys.
This study focused on a conducting
airway site located in the middle to lower
trachea. Previous research shows that
vascular remodeling in HDMA-exposed
airways is generation specic (60). It is
possible that O
3
1 HDMA exposure may
have differential effects along segments
of the tracheobronchial tree. Studies
evaluating ASM function at alternate
airway generations could assess whether
Figure 5. (A) Effect of the 5-HT
1A
agonist 8-OH-DPAT on airway contractility during EFS. *EFS-induced tension is significantly less than within-group
EFS control contraction before agonist addition. “% of control response” is defined as the amount of tension produced via EFS before addition of 5-HT
or 5-HT receptor agonist. There was no difference in response between groups. (B) Direct effect of 8-OH-DPAT on airway smooth muscle (ASM)
precontracted with 100 mM ACh. *ASM tension is significantly less than within-group control after 10 minutes of incubation with agonist.
Tension at
100 mM 5-HT is significantly less than within-group tension at 10 mM 5-HT. There was no difference in ASM tension between groups (P . 0.05; n = 6).
Figure 6. Direct effect of 5-HT on ASM tension. (A) *Addition of 10 mM 5-HT induced a significant increase in ASM compared with the within-group
baseline tension. PRE 5-HT, tissue at baseline tension of 1.0 g before addition of 5-HT; POST 5-HT, tissue tension after addition of 10 mM 5-HT. (B)
5-HT–induced tension increase was significantly greater in the O
3
1 HDMA group than in the FA group (P , 0.05; n = 6).
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82 American Journal of Respiratory Cell and Molecular Biology Volume 51 Number 1
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exposure effects are widespread and
consistent throughout conducting airways.
Also, studies examining the intensity and
longevity of the persistence of AHR and
enhanced ASM contraction at different
time points of exposure and recovery could
help identify a critical window during
postnatal lung development in which the
airway is most susceptible to environmental
toxicant damage.
This study did not use strategies to
differentiate between responses due to
HDMA-associated immune responses or
reactive oxygen species formation via O
3
exposure. Previous research has shown
a synergistic effect of O
3
1 HDMA
exposure, with O
3
amplifying the allergic,
structural, and neural remodeling effects of
HDMA sensitization and inhalation (10, 14,
25). Also, previous work has shown that
immediately after 6 months of exposure
to HDMA, O
3
,orO
3
1 HDMA, similar
5-HTinduced increases in neurally
mediated ASM contractions were seen in all
the groups (18). The common factor in all
of these exposures is inammation, whether
it was induced via an HDMA-driven
immune response or reactive oxygen
species production via O
3
exposure. This
experimental design did not allow us to
differentiate between immune versus
oxidant-induced inammation, but this is
a viable avenue for future research.
Further evaluation linking histological
and structural changes to alterations in
airway function and 5-HT handling will
bridge a critical gap in our proposed model.
Future research could also evaluate the
effectiveness of 5-HT receptor antagonists at
reversing AHR in whole animal studies. This
study focused on the persistence of the
combined effect of O
3
1 HDMA exposure
on AHR and 5-HT enhancement of ASM
contraction and did not investigate the
effect of exposure to each environmental
insult separately, so the individual
contributions of O
3
or HDMA to
persistence cannot be addressed.
In conclusion, this study veries for the
rst time that combined exposure to two
recognized environmental contributors to
asthma, O
3
and HDMA, induce prolonged
decrements in lung function and lead to
a 5-HTmediated exacerbation of ASM
contraction in a model of childhood
asthma. These hallmarks of asthmaAHR
and enhanced ASM contractionpersisted
even after exposure to O
3
1 HDMA had
been discontinued for 2.5 years. This study
also identied three 5-HT subtype receptors
that contribute to the enhanced ASM
contractile response (5-HT
2A
, 5-HT
3
, and
5-HT
4
). These ndings substantiate the
need to minimize exposure of young
individuals to known environmental
contributors to asthma during critical
periods of lung maturation because
damage inicted during these times
can contribute to prolonged asthma
symptoms. The identication of
a persistent 5-HTmediated enhanced
ASM contraction may identify novel
therapeutic targets for pharmacological
intervention in the treatment of childhood
asthma. n
Author disclosures are available with the text
of this article at www.atsjournals.org.
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  • [Show abstract] [Hide abstract] ABSTRACT: In rhesus macaques, previous studies have shown that episodic exposure to allergen alone or combined with ozone inhalation during the first 6months of life results in a condition with many of the hallmarks of asthma. This exposure regimen results in altered development of the distal airways and parenchyma (Avdalovic et al., 2012). We hypothesized that the observed alterations in lung parenchyma would be permanent following a long-term recovery in filtered (FA) housing. Forty-eight infant rhesus macaques (30days old) sensitized to house dust mite (HDM) were treated with two week cycles of FA, house dust mite allergen (HDMA), ozone (O3) or HDMA/ozone (HDMA+O3) for five months. At the end of the five months, six animals from each group were necropsied. The other six animals in each group were allowed to recover in FA for 30 more months at which time they were necropsied. Design-based stereology was used to estimate volumes of lung components, number of alveoli, size of alveoli, distribution of alveolar volumes,interalveolar capillary density . After 30months of recovery, monkeys exposed to HDMA, in either group, had significantly more alveoli than filtered air. . These alveoli also had higher capillary densities as compare with FA controls. These results indicate that early life exposure to HDMA alone or HDMA+O3 alters the development process in lung alveoli. Copyright © 2014. Published by Elsevier Inc.
    Article · Dec 2014