Nitrosative events in atopic asthma pathogenesis
The correlation between high exhaled nitric oxide levels and eosinophilic-mediated airway inflammation in patients with atopic asthma has been well documented. This generates prerequisites that a regulatory feedback mechanism exists between them. Therefore, the paper briefly describes evidence imple...
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Parilova, O.O. Volodina, T.T. Shandrenko, S.G. 2019-06-12T16:13:43Z 2019-06-12T16:13:43Z 2015 Nitrosative events in atopic asthma pathogenesis / O.O. Parilova, T.T. Volodina, S.G. Shandrenko // Вiopolymers and Cell. — 2015. — Т. 31, № 6. — С. 405-416. — Бібліогр.: 98 назв. — англ. 0233-7657 1993-6842 DOI: http://dx.doi.org/10.7124/bc.0008FD https://nasplib.isofts.kiev.ua/handle/123456789/152698 616.248 The correlation between high exhaled nitric oxide levels and eosinophilic-mediated airway inflammation in patients with atopic asthma has been well documented. This generates prerequisites that a regulatory feedback mechanism exists between them. Therefore, the paper briefly describes evidence implementing biosynthesis, enzyme structural features, expression regulation of its isoforms and effects of nitric oxide, which have helped elucidate molecular mechanisms by which nitric oxide selectively promotes asthma exacerbation. In previous study we have demonstrated that airway infiltrate of immune cells contributes to NO synthesis in the respiratory tract during allergic inflammation under guinea pig model of acute asthma with multiple challenges. On the basis of these findings the authors posits that nitric oxide represents an additional signal of the induction of Th2 subset response and be considerably involved in the complex network of immune regulation distinctive for atopic asthma phenotype. Кореляцію між високим рівнем оксиду азоту, що видихається та еозинофіл-опосередкованим запаленням дихальних шляхів у пацієнтів з атопічною бронхіальною астмою добре доведено. Це створює передумови існування регуляторного механізму зворотнього зв’язку між ними. Тому стаття стисло наводить свідчення стосовно біосинтезу, структурних особливостей ензиму, регуляції експресії його ізоформ та дії оксиду азоту, що допомагає з’ясувати молекулярні механізми завдяки яким оксид азоту сприяє загостренню перебігу захворювання. В попередній експериментальній роботі ми продемонстрували, що інфільтрат імунних клітин, які персистують в дихальних шляхах, робить внесок у синтез оксиду азоту в респіраторному тракті під час пізньої алергічної реакції за моделі гострої бронхіальної астми мурчаків з множинними провокаціями алергену. На основі цих даних автори констатують, що оксид азоту є додатковим сигналом індукції відповіді Th2 ланки та залучений в складну мережу імунної регуляції, характерної для фенотипу атопічної астми. Корреляция между высоким уровнем оксида азота в выдыхаемом воздухе и эозинофил-опосредованным воспалением дыхательных путей у пациентов с атопической бронхиальной астмой хорошо обоснована. Это порождает предпосылку, что существует механизм регулирования обратной связи между ними. Таким образом, статья кратко описывает данные касающиеся биосинтеза, структурных особенностей энзима, регулирования экспрессии его изоформ и воздействия оксида азота, которые помогают выяснить молекулярные механизмы, благодаря чему оксид азота селективно содействует обострению астмы. В предыдущей экспериментальной работе мы показали, что инфильтрат иммунных клеток в дыхательных путях дополняет синтез оксида азота в респираторном тракте во время аллергического воспаления на модели острой бронхиальной астмы морских свинок с множественными провокациями аллергена. На основании этих данных авторы констатируют, что оксид азота представляет собой дополнительный сигнал индукции ответа Th2 звена и значительно вовлечен в сложную сеть иммунной регуляции, отличительной для фенотипа атопической астмы. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Reviews Nitrosative events in atopic asthma pathogenesis Нітрозативні події в патогенезі атопічної астми Нитрозативные события в патогенезе атопической астмы Article published earlier |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
| title |
Nitrosative events in atopic asthma pathogenesis |
| spellingShingle |
Nitrosative events in atopic asthma pathogenesis Parilova, O.O. Volodina, T.T. Shandrenko, S.G. Reviews |
| title_short |
Nitrosative events in atopic asthma pathogenesis |
| title_full |
Nitrosative events in atopic asthma pathogenesis |
| title_fullStr |
Nitrosative events in atopic asthma pathogenesis |
| title_full_unstemmed |
Nitrosative events in atopic asthma pathogenesis |
| title_sort |
nitrosative events in atopic asthma pathogenesis |
| author |
Parilova, O.O. Volodina, T.T. Shandrenko, S.G. |
| author_facet |
Parilova, O.O. Volodina, T.T. Shandrenko, S.G. |
| topic |
Reviews |
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Reviews |
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2015 |
| language |
English |
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Вiopolymers and Cell |
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Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Нітрозативні події в патогенезі атопічної астми Нитрозативные события в патогенезе атопической астмы |
| description |
The correlation between high exhaled nitric oxide levels and eosinophilic-mediated airway inflammation in patients with atopic asthma has been well documented. This generates prerequisites that a regulatory feedback mechanism exists between them. Therefore, the paper briefly describes evidence implementing biosynthesis, enzyme structural features, expression regulation of its isoforms and effects of nitric oxide, which have helped elucidate molecular mechanisms by which nitric oxide selectively promotes asthma exacerbation. In previous study we have demonstrated that airway infiltrate of immune cells contributes to NO synthesis in the respiratory tract during allergic inflammation under guinea pig model of acute asthma with multiple challenges. On the basis of these findings the authors posits that nitric oxide represents an additional signal of the induction of Th2 subset response and be considerably involved in the complex network of immune regulation distinctive for atopic asthma phenotype.
Кореляцію між високим рівнем оксиду азоту, що видихається та еозинофіл-опосередкованим запаленням дихальних шляхів у пацієнтів з атопічною бронхіальною астмою добре доведено. Це створює передумови існування регуляторного механізму зворотнього зв’язку між ними. Тому стаття стисло наводить свідчення стосовно біосинтезу, структурних особливостей ензиму, регуляції експресії його ізоформ та дії оксиду азоту, що допомагає з’ясувати молекулярні механізми завдяки яким оксид азоту сприяє загостренню перебігу захворювання. В попередній експериментальній роботі ми продемонстрували, що інфільтрат імунних клітин, які персистують в дихальних шляхах, робить внесок у синтез оксиду азоту в респіраторному тракті під час пізньої алергічної реакції за моделі гострої бронхіальної астми мурчаків з множинними провокаціями алергену. На основі цих даних автори констатують, що оксид азоту є додатковим сигналом індукції відповіді Th2 ланки та залучений в складну мережу імунної регуляції, характерної для фенотипу атопічної астми.
Корреляция между высоким уровнем оксида азота в выдыхаемом воздухе и эозинофил-опосредованным воспалением дыхательных путей у пациентов с атопической бронхиальной астмой хорошо обоснована. Это порождает предпосылку, что существует механизм регулирования обратной связи между ними. Таким образом, статья кратко описывает данные касающиеся биосинтеза, структурных особенностей энзима, регулирования экспрессии его изоформ и воздействия оксида азота, которые помогают выяснить молекулярные механизмы, благодаря чему оксид азота селективно содействует обострению астмы. В предыдущей экспериментальной работе мы показали, что инфильтрат иммунных клеток в дыхательных путях дополняет синтез оксида азота в респираторном тракте во время аллергического воспаления на модели острой бронхиальной астмы морских свинок с множественными провокациями аллергена. На основании этих данных авторы констатируют, что оксид азота представляет собой дополнительный сигнал индукции ответа Th2 звена и значительно вовлечен в сложную сеть иммунной регуляции, отличительной для фенотипа атопической астмы.
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0233-7657 |
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https://nasplib.isofts.kiev.ua/handle/123456789/152698 |
| citation_txt |
Nitrosative events in atopic asthma pathogenesis / O.O. Parilova, T.T. Volodina, S.G. Shandrenko // Вiopolymers and Cell. — 2015. — Т. 31, № 6. — С. 405-416. — Бібліогр.: 98 назв. — англ. |
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405
O. O. Parilova, T. T. Volodina, S. G. Shandrenko
© 2015 O. O. Parilova et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
UDC 616.248
Nitrosative events in atopic asthma pathogenesis
O. O. Parilova, T. T. Volodina, S. G. Shandrenko
Palladin Institute of Biochemistry, NAS of Ukraine
9, Leontovycha Str., Kyiv, Ukraine, 01030
elenparil@gmail.com
The correlation between high exhaled nitric oxide levels and eosinophilic-mediated airway inflammation in
patients with atopic asthma has been well documented. This generates prerequisites that a regulatory feedback
mechanism exists between them. Therefore, the paper briefly describes evidence implementing biosynthesis,
enzyme structural features, expression regulation of its isoforms and effects of nitric oxide, which have helped
elucidate molecular mechanisms by which nitric oxide selectively promotes asthma exacerbation. In previous
study we have demonstrated that airway infiltrate of immune cells contributes to NO synthesis in the respira-
tory tract during allergic inflammation under guinea pig model of acute asthma with multiple challenges. On
the basis of these findings the authors posits that nitric oxide represents an additional signal of the induction of
Th2 subset response and be considerably involved in the complex network of immune regulation distinctive
for atopic asthma phenotype.
K e y w o r d s: nitric oxide, atopic asthma, nitric oxide synthases, allergic airway inflammation, eosinophilia.
Introduction
Asthma is a chronic inflammation of the airways that
involves airflow obstruction and increased airway
responsiveness to a variety of stimuli, which leads to
the symptoms of recurrent coughing and dyspnea [1].
Albeit the understanding of disease pathogenesis has
progressed enormously in the last several decades
[1–3], asthma is still a major public health problem,
affecting 300 million people worldwide and rapidly
increasing in all age groups [4, 5]. Clinically, several
distinct phenotypes are recognized, but the therapy
and research have been basically focused on allergic
(atopic) asthma [1–3].
Currently, the routine asthma treatment relies on
suppressing the inflammation (glucocorticoids, leu-
kotriene inhibitors as additional medication) as well
as removal of bronchoconstriction (β agonists, anti-
cholinergics) [6, 7]. The symptom-based guidance
of treatment could predispose to an increased num-
ber of exacerbations. Thus, asthmatics might benefit
from the inflammatory marker-addressed therapy [1,
8]. Although many physiologic processes have been
implicated in asthma pathogenesis, the range of evi-
dence supports the key roles of endogenous nitric
oxide (NO) and NO-derived reactive nitrogen spe-
cies (RNS) in modulating either a normal airway
function [8, 9] or inflammation in asthma [10, 11].
Therefore, NO has received a tremendous amount of
attention in the medical community as a selective
pulmonary disorder marker [8, 12, 13].
NO is an ubiquitous messenger that regulates var-
ious biological functions — either at low concentra-
tions as a signal molecule in many physiological
processes, or at high concentrations as cytotoxic and
cytostatic defensive mechanisms against tumors and
pathogens [9, 11]. Also it is thought to be a marker of
lung inflammation. NO is detected in exhaled breath
[8, 12, 13]. Fractional exhaled NO (FeNO) levels
were shown to correlate with sputum eosinophil
Reviews ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2015. Vol. 31. N 6. P 405–416
doi: http://dx.doi.org/10.7124/bc.0008FD
406
O. O. Parilova, T. T. Volodina, S. G. Shandrenko
count, airway hyperresponsiveness, bronchodilator
response, serum IgE levels, allergen skin prick test-
ing, asthma symptoms, and lung function. This led
to the general belief that atopy is associated with
higher levels of exhaled NO (eNO) [11].
From this background, the current article deals
with the consideration of the source of NO formation
and its triggers in asthma pathogenesis. We aim to
discuss the cases, when NO becomes a marker of al-
lergic inflammation. Simultaneously, we present our
data that revealed the contribution of immune cells
settling down the airways to the respiratory NO gen-
eration.
1. Biosynthesis and enzyme structure basis
The role of NO in the human body is under the scope
of intensive research. NO is generated by oxidation
of L-arginine; this reaction is typically catalyzed by
the nitric oxide synthases (NOS) [9] in the presence
of several co-factors: flavones (FAD, FMN), tetrahy-
drobiopterin (BH4) and NADPH [14]. NOS are ste-
reospecific and active as homodimers [14]. The en-
zyme exists in three isoforms: neuronal NOS (nNOS/
NOS1), inducible NOS (iNOS/NOS2) and endothe-
lial NOS (eNOS/NOS3) [15–18].
nNOS and eNOS are constitutively expressed iso-
forms (cNOS) in cells as the preformed proteins that
are activated in response to the cell-specific stimuli
through the elevation of intracellular Ca2+ concentra-
tions and the binding of calmodulin. iNOS form is
generally associated with the immune system and
produces NO for prolonged periods of time in a cal-
cium-independent manner [9–11, 14–22]. iNOS is
activated by bacterial lipopolysaccharides and pro-
inflammatory cytokines such as inteleukin-1, TNF-α,
IFN-γ [15]. However, IFN-γ is the most potent and
prevailing inducer of iNOS in vivo [23]. As
T-lymphocytes and NK-cells are the main source of
IFN-γ production, the iNOS induction is mediated
by them. The endothelial cell derived factor, endo-
thelin-1 (ET-1), has been shown to promote iNOS
expression [24, 25]. Ultimately, iNOS incorporates a
calmodulin binding site to which the calmodulin is
tightly bound independently of a calcium signal –
this is thought to be responsible for the continuous
activity of the enzyme [15–17]. Up to 1000 times
higher levels of NO can be generated by iNOS com-
pared to cNOS [11, 19].
The NOS enzyme functions as a dimer consisting
of two identical monomers, which can be function-
ally and structurally divided into two major domains:
a C-terminal reductase domain and an N-terminal
oxygenase (catalytic) domain. All NOS isoforms are
members of the cytochrome P450 enzyme group
owing to the presence of a haem complex [20]. Thus
the mammalian NOS share similar domain architec-
ture, with an N-terminal catalytic domain containing
a haem active site, a nearby cofactor site for BH4
and a C-terminal reductase domain consisting of
FMN, FAD and NADPH binding sites. We visual-
ized catalytic domain of human NOS enzymes in
three-dimensional form using PDB files via Cn3D
viewer from MMDB (Fig. 1). The macromolecular
3D structures display the evidence, that eNOS and
iNOS isoforms are very similar in the overall mo-
lecular shape, relative orientation of cofactors and
stereochemistry within the catalytic centre. As the
catalytic sites of iNOS and eNOS are so similar it is
not obvious from a structural starting point how the
selective inhibitors may be designed [26].
2. Localization of nitric oxide synthase iso-
forms in respiratory tract and its biological
role
In the respiratory tract all isoforms have been identi-
fied [20, 21]. cNOS-derived NO along with other
NO adduct molecules are involved in maintaining
normal metabolic functions, such as airway and pul-
monary vascular tone, intracellular signaling, immu-
nity (platelet aggregation, leukocyte adhesion) and
neurotransmission [7, 22]. By contrast to this, iNOS-
derived NO seems to play a pro-inflammatory pri-
mary role in the cytokine dependent processes [19].
Albeit, agonists such as sheer stress, bradykinin,
acetylcholine and histamine still may activate cNOS,
resulting in the release of NO within seconds [20].
eNOS is constitutively expressed in both the en-
dothelial cells of the bronchial and pulmonary circu-
407
Nitrosative events in atopic asthma pathogenesis
lation and the alveolar endothelial cells, and airway
epithelial cells throughout the respiratory tract [27].
The traces of NO from eNOS activity have been iso-
lated in the airway epithelium and found to partici-
pate in ciliary movement and mucus ejection [28,
29]. Although eNOS is present in airway epithelium,
iNOS is considered to be a predominant form in it
[19]. The recent evidence has demonstrated a link
between certain eNOS gene polymorphisms and in-
creased serum IgE levels in patients with allergic
asthma [30]. Functionally, nNOS mediates neuronal
bronchodilation in the skeletal muscles and choliner-
gic airway nerves [27]. When allergic asthma was
being launched, the increased arginase activity at-
tenuates the nNOS-catalyzed synthesis of NO and
induces neural bronchoconstriction [31]. In this way
cNOS isoforms reveal an indirect involvement in
asthma pathophysiology.
In general, iNOS regulates approximately 200
genes that are relevant to inflammation, infection or
apoptosis [21]. In the airways iNOS is expressed by
epithelial and endothelial cells [23, 32], smooth mus-
cle cells [18, 32, 33], lung fibroblasts [34]. In contrast
to other cell types that do not express NOS2 unless
induced by cytokines, the NOS2 mRNA expression
and NOS2 protein were detected in epithelial cells of
normal, noninflamed upper and lower airways and in
pulmonary myocytes of large hilar veins by a variety
of techniques, including Northern blotting, in situ hy-
bridization, Western blotting, and immunohisto-
chemistry [23, 35]. Additionally, iNOS-derived NO
is produced by the genuine immune-system cells in-
cluding monocytes and macrophages [16, 17, 36],
eosinophils [37, 38], neutrophils [39, 40], dendritic
cells [41]. The reports about the NOS expression in
the mast cell population appear to be ambiguous. The
data presented in numerous papers clearly indicate
distinct differences in diversity and localization of
NOS in a variety of mast cell populations expressed
in human and rodents [42–44]. Notably, the iNOS ex-
pression and NO generation are up-regulated by the
antigen stimulation FcɛRI engagement in the differ-
ent subsets of mast cells [44]. Additionally, the NO
production prevents mitochondrial integrity collapse,
thereby protecting mast cells against antigen-induced
apoptosis [45]. Previously several reports have con-
firmed that the rat mast cells produce iNOS mRNA
and the protein upon treatment [46–48]. However
subsequent studies have demonstrated that some
types of human mast cell were positive for eNOS but
showed variable expression of nNOS and no detect-
able iNOS [42, 43]. Together these findings suggest
the important differences between the rat and human
mast cells in the NO production and NO-mediated
regulation of mast cell function [42]. NK cells ex-
press constitutive eNOS mRNA and protein, howev-
er the levels of iNOS are not detectable. The endog-
enous NO production is involved in protection of NK
cells from activation of apoptosis induced by CD16
cross-linking, thereby maintaining NK activity [49].
This is the further evidence that, unlike NO produced
by iNOS, cNOS-derived NO is critical for normal
physiology.
Farther, the biological effect of NO ultimately de-
pends on its concentration and interaction with other
bioreactive molecules and proteins [50]. NO can be
converted to NO2, NO2–, NO3– and other reactive
nitrogen intermediates (RNI), among which the most
important are S-nitrosothiols (S-NO), peroxynitrite
(ONOO–) and nitrosyl-metal complexes, which are
directly implicated in the RNI-mediated free radical
reactions (S-nitrosylation of cysteines, nitration of
tyrosines, and nitrosylation of prosthetic groups, re-
spectively) [51]. In summary, the excessive NO-
adduct molecules namely ONOO–, nitrogen dioxide
(NO2), dinitrogen trioxide (N2O3), and higher ox-
ides of nitrogen can provoke detrimental post-trans-
lational modifications [13, 50, 52].
3. Nitric oxide formation as a marker
of Th2-mediated infllammation
There is rising evidence for an important immuno-
regulatory role of NO in the development of the
adaptive immune responses associated with autoim-
mune and allergic diseases. Increased levels of ex-
haled NO [53–56] along with an up-regulation of
iNOS in asthmatic airways [57] confirm that this
radical should be involved in asthma. It has been
408
O. O. Parilova, T. T. Volodina, S. G. Shandrenko
demonstrated, that, in humans, a higher than normal
NO concentration in exhaled breath is closely asso-
ciated with the enlarged transcriptional activation of
NOS2 gene [58].
Nevertheless, the previous studies, using murine
and guinea pig models of asthma and iNOS inhibitor
treatment or iNOS-deficient animals, have generated
controversial results. In two different studies, iNOS
deficiency [59] or iNOS inhibitor treatment [60] had
no effect on the lung inflammation, whereas in other
study the airway inflammation was inhibited in the
iNOS deficient mice [61]. Such a discrepancy was
attributed to significant differences in the immuniza-
tion and challenge protocols [59].
Further investigations have shown that inhibited
iNOS activity correlates with a decrease of nitroty-
rosine and, more importantly, ameliorates FEV1 and
airway responsiveness to histamine [14]. However,
the different subgroups analysis of atopic individu-
als now suggests that it is the inflammation in atopic
individuals with clinical manifestations of airway
disease, rather than atopy itself, that accounts for the
increased production of NO by iNOS [62]. eNO was
commonly regarded qua a marker of eosinophilic in-
flammation [63–65]. In addition, the 2011 American
Thoracic Society (ATS) guideline for the use of
FeNO in clinical practice characterized FeNO as an
indicator of eosinophilic airway inflammation [12].
However, the recent studies have indicated that
FeNO is more representative of a Th2-driven local
inflammation, specifically of the bronchial mucosa,
rather than general eosinophilic inflammation, as
measured by blood or induced sputum [66]. The dis-
connect between FeNO and eosinophilic inflamma-
tion has been highlighted by two separate studies
with monoclonal antibodies (mAb) against IL-5 and
IL-13, which indicate that treatment with mepoli-
zumab, an anti-IL-5 mAb, significantly reduces
blood and sputum eosinophils without affecting
FeNO levels [67] whereas treatment with lebriki-
zumab, an anti-IL-13 mAb, significantly reduces
FeNO levels without reducing blood eosinophils
[68]. Another research has shown that, in asthmatic
children but not in controls, eNO levels and BAL
fluid eosinophil percentages correlate well regard-
less of the methods used to measure eNO [69]. Thus,
eNO levels interconnect better with bronchial eo-
sinophils than with sputum eosinophils.
Based on the findings of the bronchoalveolar la-
vage fluid (BALF) studies and the lung tissue histo-
logical analysis, the mechanisms for late allergic re-
sponsiveness (LAR) are considered to be causally
related to the infiltration of eosinophils and other
inflammatory cells into the bronchial mucosa [70]. It
has been suggested that NO play an important role in
LAR [10, 71]. In the airways of asthmatic patients
[57] or in rodent lung after allergen challenge [61,
72], iNOS expression and/or enzymatic activity are
increased.
To give insight into the role of nitrosative stress
established by immune cells infiltrating the airways
we examined the NO formation in BAL cells under
the guinea pig model of acute ovalbumin (OVA)-
induced asthma with repeated challenges following
32 weeks after sensitization [73]. This model mimics
those seen in humans exposed to an allergen a long
period after acquired hypersensitivity. A suggested
protocol reflects the allergen-driven pathway of
asthma reproducing several characteristic features,
such as airways infiltration by inflammatory cells,
early allergic responsiveness (EAR) and LAR, AHR.
Flow cytometry analysis using DAF-2DA dye
showed, that multiple allergen challenge exposures
of sensitized guinea pigs were associated with an ex-
cessive level of the intracellular NO generation in
BAL cells (Figure 2). Hence, OVA aerosol provoca-
tions of guinea pigs with sensitization resulted in a
substantial growth of BAL cells containing NO in
comparison with sensitized group (33.50[27.80–
45.80] % versus 6.30[2.20–7.82] %; p=0.0001). Our
results are in line with the data confirming that al-
lergic inflammation is accompanied by NO.
Moreover, there is apparent evidence that iNOS in-
hibition, during the challenge period, markedly re-
duced the development of the inflammatory process
in the OVA-induced murine model after allergen
challenge through down-regulation of chemokine
expression [74].
409
Nitrosative events in atopic asthma pathogenesis
Taking into account that BAL explores large ar-
eas of the alveolar compartment providing cellular
constituents from the lower respiratory tract, we
identified the alterations in cellular composition of
BAL as indicated in FACS density graphs (Fig. 3).
OVA provocations in sensitized animals were
shown to cause a strong inflammation in lungs.
This led to the redistribution of immune subpopula-
Fig. 1. The structures of oxygenase do-
main of human NOS isoforms.
A – dimeric unit of eNOS haem domain
with L-arginine bound (PBD ID:4D1O).
B – tetrameric unit of nNOS haem domain
with L-arginine bound (PBD ID: 4D1N).
C – tetrameric unit of iNOS with Zn-bound
and L-arginine complex (PBD ID: 1NSI).
The enzyme structures show a ball and
stick backbone in a trace shape, no side
chains to alignment protein views, and sol-
id objects – condensed lines and massive
cylinders with arrows – to represent strands
and helices. The arrows on helix cylinders
point in the N-to-C direction. The extent of
each monomer element in macromolecule
is designated by color (blue, violet, green
and brown). Haem, BH4 and arginine,
glycerol are shown in a tube representation.
The association into a dimer involves a
large interface, which includes the binding
site for BH4 and helps to structure the ac-
tive-site pocket containing the haem and
the L-arginine binding site
Fig. 2. NO content in BAL cells 18–20 h after induction of allergic lung inflammation: morphological flow cytometric parameters of
cell types concerning size (FS) and granularity (SS). Panel A: BAL cells derived from sensitized guinea pigs with allergen chal-
lenges (II coumn) display augment of NO generation compared to sensitized animals (I column). Panel B: fractions of BAL cells
incorporating NO from two compared groups (I and II columns) are also demonstrated in the individual dot plots. Green-colored cells
include NO molecules (adopted from [73] and supplemented in this paper)
410
O. O. Parilova, T. T. Volodina, S. G. Shandrenko
tions in the general pool of cells infiltrating respira-
tory tract.
In addition to quantitative assessment of NO for-
mation, the allocation of intracellular NO content in
BAL suspension was analyzed depending on the size
and granularity (Fig. 4, Fig. 5) [73].
In view of these findings we infer that in asthma,
due to increased iNOS protein expression and ac-
tivity, the excessive NO generation is activated in
lung tissue cells (mainly airway epithelial cells),
but also in multi-cellular airway infiltrate recruited
to trigger organ during allergic inflammation. High
BAL levels of NO may reflect circulating nitrosa-
tive stress in respiratory tract, when exacerbation in
asthma is present. Indeed inhibition of iNOS-de-
rived NO attenuates antigen-induced airway con-
striction, inflammatory, and reduces collagen and
elastic fiber deposition in a guinea pig model of al-
lergic asthma [37].
4. Immunoregulatory cross-talk of nitric
oxide in asthma pathogenesis
The network of molecules involved in the allergic
inflammatory processes becomes more complex
since NO derivatives have been demonstrated to
play a role in these reactions. The allergic disorder
pathogenesis is modulated by NO at the level of im-
mune system [20, 32]. In turn, iNOS transcription is
regulated by a number of pathways, including the
JNK, JAK-STAT, and p38 MAPK pathways, which
are largely activated through the cytokine induction
Fig. 3. Distinction of BAL cellular compostion of sensitized
guinea pigs (I column) compared to sensitized animals, that re-
ceived allergen challenges (II column). Subpopulation content
and ratio in BAL suspensions based on the morphological flow
cytometric parameters side scatter (SS) versus forward scatter
(FS) are illustrated in density plots
Fig. 4. NO formation as a function of cellular size in BALF sub-
populations derived from OVA sensitized guinea pigs (I column)
and OVA sensitized animals treated with OVA aerosol exposures
(II column). DAF-2DA stained cells are colored in green (ad-
opted from [73] and supplemented here)
Fig. 5. NO generation in accordance with the complexity of
BALF cell subpopulations of OVA sensitized guinea pigs (I col-
umn) and of OVA sensitized animals treated with OVA aerosol
exposures (II column). DAF-2DA stained cells are colored in
green (adopted from [73] and supplemented here)
411
Nitrosative events in atopic asthma pathogenesis
[75]. In allergic asthma the immune response to in-
haled antigens results from the activation of mast
cells and antigen-specific Th2 cells, followed by the
production of cytokines, including interleukin IL-4,
IL-5 and IL-13 [3]. Further, IL-4 and IL-13 cause
lung epithelial expression of iNOS to be upregulated
via STAT-6, a process which is corticosteroid sensi-
tive [76]. Moreover, the group of researchers pro-
vided evidence, that IL-13 robustly induces the ex-
pression of an active dimeric iNOS enzyme in pri-
mary HAEC (human airway epithelial cells) main-
tained in air–liquid interface culture, consistent with
its expression in relation to Th2 inflammation [77].
Therefore, eNO is a direct signal of the Th2-
mediated, pro-inflammatory cytokine mechanisms
of central importance in the pathophysiology of al-
lergic airway inflammation. At present, there is open
debate about whether NO exacerbates or reduces au-
toimmune and allergic chronic inflammation.
The detrimental impact of NO is implemented due
to its ability to launch a hyperinflammatory response,
leading to tissue damage. Two mechanisms are pri-
marily responsible for inducing NO-mediated dam-
age: action of NO-induced inflammatory mediators
and apoptosis of NO-targeted cells. NO modulates
the production and function of cytokines released by
immune cells, chemokines, and growth factors [78].
NO-mediated apoptosis represents one of the key
factors contributing to enhanced inflammation and
tissue damage during noninfectious respiratory dis-
eases, such as asthma [79]. The recent study has re-
vealed that NO induces eosinophil apoptosis in a
mechanism mediated via ROS, JNK, and later mito-
chondrial permeability transition [80].
iNOS-derived NO plays an immunomodulatory
role, as it may shift the Th1/Th2 balance in favor of
Th2, thus promoting IgE-mediated allergy [75, 81].
Th1 cells secrete IFN-γ, TNF-α, and IL-2 and pro-
mote cellular immune responses against intracellular
antigens, whereas Th2 cells secrete IL-4, IL-5, IL-
10, and IL-13, induce IgG1- and IgE-mediated hu-
moral responses, and are important for the elimina-
tion of large extracellular parasites such as helmin-
thes and nematodes [82]. The molecular mechanisms
of Th1 versus Th2 regulation rely on the counterbal-
ance between expression of the IL-12 receptor β2-
chain (IL-12Rβ2)
3 on Th1 cells and GATA-3 tran-
scription in Th2 cells. IL-4 induced GATA-3 allows
the stable commitment to the Th2 phenotype through
promotion of Th2 cytokine production, such as IL-4,
IL-5, IL-10 and IL-13 [83, 84]. IL-10 produced by
Th2 cells further suppresses the Th1 development by
inhibiting the secretion of IFN-γ and IL-12 and may
also directly inhibit the induction of iNOS [81].
Moreover, NO markedly inhibits the induction of
IL-2 promoter, which could account for most of the
reduction in IL-2 production, and increases the pro-
duction of IL-4 by effector T cells [85]. To conclude,
NO can inhibit Th1 lymphokines but has no direct
impact on the Th2 lymphokines production [78, 81].
As the continuous expression of iNOS in the normal
epithelium is maintained mainly by IFN-γ [19, 21,
23], the present observations indicated that NO se-
lectively inhibits the expansion of Th1 cells by a
negative feedback mechanism. In contrast, low con-
centrations of NO stimulate T cells to express IL-
12R and promote Th1 differentiation [86].
The counter-evidence of the absence of a direct
effect of NO on Th2 cells was found since the NO
donors inhibit the proliferation of Th1 and Th2 pop-
ulations. The NO donors SIN-1 (3-morpholino-syd-
nonimine) and SNAP (S-nitroso-N-acetyl-DL-
penicillamine) suppress the proliferation of anti-
CD3 or mitogen activated human peripheral Th1 and
Th2 cell clones, and prevent the release of IFN-γ,
IL-2, IL-4, IL-5 and IL-10 [87]. Additionally it has
also been reported that NO inclusion to human bron-
chial epithelial cells reversibly inhibits the prolifera-
tion of activated Th1 and Th2 CD4+ T cells in atopic
asthma [88].
IgE-dependent synthesis of NO by various cell
populations bearing low-affinity IgE receptors
(FceRII; CD23) implicates in local allergic and in-
flammatory reactions, including macrophages, eo-
sinophils and keratinocytes [89]. NO is also an im-
portant agent in eosinophil migration and infiltra-
tion. Several reports demonstrated that the acute
treatment with the non-selective inhibitors of NO
412
O. O. Parilova, T. T. Volodina, S. G. Shandrenko
reduced allergen-induced eosinophilia, showing that
NO is involved in the inflammatory cell recruitment
[44, 90, 91]. Furthermore, it has been found that the
l-NAME (N ω-nitro-l-arginine methyl ester) treat-
ment decreases the number of eosinophils positive
for both nNOS and iNOS, whereas the treatment
with 1400W, a highly selective iNOS inhibitor, de-
creases only the iNOS-positive eosinophils, reduc-
ing the eosinophil density in alveolar septa of aller-
gen-sensitized animals [92]. In addition to these
findings, NO is crucial to the numerous mast cell
functions, including degranulation, adhesion, and
protease release, as well as the production and secre-
tion of chemokines and cytokines [42]. NO also con-
trols the processes in immune system through regu-
lation of the chemokine expression. Among the
asthma pathophysiology processes, involving NO,
are modulation of the co-stimulatory and adhesion
molecules, such as VCAM-1 (vascular cell adhesion
molecule-1), ICAM-1 (intercellular adhesion mole-
cule-1), CD62E (E-selectin) and CD62P (P-selectin),
synthesis and deposition of the extracellular material
components via TGF-β [93].
The anti-inflammatory effects of NO may be me-
diated by several mechanisms including the inhibi-
tion of gene expression and the secretion of proin-
flammatory cytokines [94, 95], reduction of the pro-
inflammatory effects of cytokines such as IL-8
through the covalent modification by nitration of ty-
rosine [96], or by protection against the programmed
cell death through inactivation of the proteolytic en-
zymes responsible for apoptosis [97, 98]. Thus, the
endogenous NO synthesis in asthma, may be an im-
portant early physiological defense mechanism
against the injury and inflammation. Nevertheless,
persistently elevated levels of the NO formation can
deepen the tissue injury over time.
Conclusions
The presented evidence posits the nitrosative stress
as a relevant pathogenic mechanism underlying
atopic asthma. The diversity of NOS enzymes pro-
duces different profiles of NO activities as well as of
its derivatives facilitating and broadening the func-
tional versatility of nitrosative events. Many studies
and clinical observations are bringing to light a mul-
titude of different mechanisms through which NO
operates in respiratory tract and mediates dysfunc-
tion in the disease. Mounting evidence of a link be-
tween the NO synthesis and the allergic inflamma-
tion raises the possibilities for therapeutic interven-
tion. Two major challenges currently hinder the
progress towards this aim; on one hand the multi-
plicity of asthma nature with a complex immunoge-
netic basis and a strong environmental contribution;
on the other hand the likelihood of mimicking the
full spectrum of responses to the allergen exposures
and the tested pharmaceuticals by in vivo and in vitro
models seems fragile. Hence, despite the significant
advances in our understanding, the effective marker-
addressed therapy strategies and reliable approaches
for the precise diagnostics and monitoring are cur-
rently still limited.
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Нітрозативні події в патогенезі атопічної астми
О. О. Парилова, Т. Т. Володіна, С. Г. Шандренко
Кореляцію між високим рівнем оксиду азоту, що видихається
та еозинофіл-опосередкованим запаленням дихальних шляхів
у пацієнтів з атопічною бронхіальною астмою добре доведено.
Це створює передумови існування регуляторного механізму
зворотнього зв’язку між ними. Тому стаття стисло наводить
свідчення стосовно біосинтезу, структурних особливостей ен-
зиму, регуляції експресії його ізоформ та дії оксиду азоту, що
допомагає з’ясувати молекулярні механізми завдяки яким
оксид азоту сприяє загостренню перебігу захворювання. В по-
передній експериментальній роботі ми продемонстрували, що
інфільтрат імунних клітин, які персистують в дихальних шля-
хах, робить внесок у синтез оксиду азоту в респіраторному
тракті під час пізньої алергічної реакції за моделі гострої брон-
хіальної астми мурчаків з множинними провокаціями алерге-
ну. На основі цих даних автори констатують, що оксид азоту є
додатковим сигналом індукції відповіді Th2 ланки та залуче-
ний в складну мережу імунної регуляції, характерної для фено-
типу атопічної астми.
Ключов і слова: оксид азоту, атопічна астма, синтази
оксиду азоту, алергічне запалення дихальних шляхів, еозино-
філія.
Нитрозативные события в патогенезе атопической
астмы
Е. А. Парилова, Т. Т. Володина, С. Г. Шандренко
Корреляция между высоким уровнем оксида азота в выдыхае-
мом воздухе и эозинофил-опосредованным воспалением ды-
хательных путей у пациентов с атопической бронхиальной
астмой хорошо обоснована. Это порождает предпосылку, что
существует механизм регулирования обратной связи между
ними. Таким образом, статья кратко описывает данные касаю-
щиеся биосинтеза, структурных особенностей энзима, регули-
рования экспрессии его изоформ и воздействия оксида азота,
которые помогают выяснить молекулярные механизмы, благо-
даря чему оксид азота селективно содействует обострению
астмы. В предыдущей экспериментальной работе мы показа-
ли, что инфильтрат иммунных клеток в дыхательных путях
дополняет синтез оксида азота в респираторном тракте во вре-
мя аллергического воспаления на модели острой бронхиаль-
ной астмы морских свинок с множественными провокациями
аллергена. На основании этих данных авторы констатируют,
что оксид азота представляет собой дополнительный сигнал
индукции ответа Th2 звена и значительно вовлечен в сложную
сеть иммунной регуляции, отличительной для фенотипа ато-
пической астмы.
Ключевые слова: оксид азота, атопическая астма, синта-
зы оксида азота, аллергическое воспаление дыхательных пу-
тей, эозинофилия.
Received 29.09.2015
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