Neurokinins and Inflammatory Cell iNOS Expression in Guinea Pigs with Chronic Allergic Airway Inflammation
Carla M. Prado, Edna A. Leick-Maldonado, Vanessa Arata, David I. Kasahara, Mílton A. Martins, Iolanda F.L.C. Tibério
Department of Medicine, University of São Paulo School of Medicine, São Paulo, Brazil
This study was presented in part at the International Meeting of the American Thoracic Society in Atlanta-2002 and Seattle-2003 and at the International Meeting of the European Respiratory Society in Stockholm-2002.
Running head: Nitric oxide and chronic airway inflammation
Address correspondence and request for reprints to:Iolanda F.L.C. Tibério, M.D.Departamento de Clínica MédicaFaculdade de Medicina da Universidade de São PauloAv. Dr. Arnaldo, 455 - Sala 121601246-903 - São Paulo - SP - BrazilFax: 55-11-3083-0827 and 55-11-3085-0992Phone: 55-11-3066-7317e-mail: [email protected]
Supported by the following Brazilian Scientific Agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Programa de Núcleos de Excelência (PRONEX-MCT)
Abstract
Articles in PresS. Am J Physiol Lung Cell Mol Physiol (December 3, 2004). doi:10.1152/ajplung.00208.2004
Copyright © 2004 by the American Physiological Society.
2
In the present study we evaluated the role of neurokinins in the modulation of inducible nitric
oxide synthase (iNOS) inflammatory cell expression in guinea pigs with chronic allergic
airway inflammation. In addition, we studied the acute effects of nitric oxide inhibition on this
response. Animals were anesthetized and pretreated with capsaicin (50 mg.kg-1 sc) or
vehicle 10 days before receiving aerosolized ovalbumin or normal saline twice weekly for
four weeks. Animals were then anesthetized, mechanically ventilated, given normal saline or
NG-nitro-L-arginine methyl ester (L-NAME 50 mg.kg-1, ic) and challenged with ovalbumin.
Pre-challenge exhaled NO increased in ovalbumin-exposed guinea pigs (p < 0.05 compared
to controls) and capsaicin reduced this response (p < 0.001). When compared to animals
inhaled with normal saline, ovalbumin-exposed animals presented increases in respiratory
system resistance and elastance, and numbers of total mononuclear cells and eosinophils,
including those expressing inducible NO synthase (iNOS) (p < 0.001). Capsaicin reduced all
these responses (p < 0.05) except for iNOS expression in eosinophils. Treatment with L-
NAME increased post-antigen-challenge elastance and restored both resistance and
elastance previously attenuated by capsaicin treatment. Isolated L-NAME administration also
reduced total eosinophils and mononuclear cells, as well as those cells expressing iNOS (p <
0.05 compared to ovalbumin alone). Since L-NAME treatment restored lung mechanical
alterations previously attenuated by capsaicin, NO and neurokinins may interact in
controlling airway tone. In this experimental model, NO and neurokinins modulate eosinophil
and lymphocyte infiltration in the airways.
Keywords: Nitric Oxide, Neurokinins, Asthma, Lymphocytes, Eosinophils
3
Introduction
Neurokinins such as substance P and neurokinin A are involved in the modulation of
several aspects of inflammatory responses in the lungs. These peptides induce
airway smooth muscle contraction, peribronchial edema and airway mucous
secretion and are considered mediators of the excitatory nonadrenergic-
noncholinergic response. It has been previously shown that substance P and
neurokinin A play significant roles in priming eosinophils and in lymphocyte
recruitment in allergic lung inflammation (33, 44). In guinea pigs and other mammals,
pretreatment with capsaicin has been used as a research tool to elucidate the
physiologic effects of neurokinins (18, 29). It has been previously show that
administration of high doses of capsaicin reduces tissue concentrations of substance
P and other neurokinins (28, 29).
Nitric oxide (NO), a gaseous molecule that is generated during the conversion of the
amino acid L-arginine to L-citrulline by NO synthase (NOS), is considered a mediator
of the inhibitory nonadrenergic-noncholinergic response (3, 26, 32). The role of NO in
the modulation of allergic inflammation and bronchial hyperresponsiveness is
currently unclear. There is some evidence that NO has both inflammatory and anti-
inflammatory effects in experimental models of airway inflammation (38). The NO
derived from constitutive NO synthase (cNOS) can act as a vasodilator and
bronchodilator. However, some pro-inflammatory cytokines provoke inducible NO
synthase (iNOS) to release large quantities of NO in pathological situations that
contribute to maintaining the inflammatory process (2). Various mammalian cells,
such as endothelial cells (34), neurons (32), epithelial cells (16) and immune cells
4
(24), can produce NO. A variety of immunocompetent cells express iNOS, including
macrophages (17, 49), neutrophils (6, 30, 49) and eosinophils (9, 49). There is
evidence that NO is involved in eosinophil recruitment in various models of lung
inflammation (13, 14). In contrast, some authors have observed that NO derived
through iNOS activation contributes to neutrophil recruitment rather than to
eosinophil recruitment (11).
Since both neurokinins and NO have been considered neurotransmitters of
nonadrenergic-noncholinergic fibers and are involved in the regulation of some
inflammatory responses in the lungs, we reasoned that neurokinins could modulate
inducible NO synthase expression in inflammatory cells. Thus, the purpose of the
present study was to evaluate the effects of neurokinin depletion on iNOS expression
in eosinophils and mononuclear cell recruited in the airways of guinea pigs with
chronic allergic airway inflammation. In addition, we studied the acute effects of NO
inhibition on this response.
5
Material and Methods
All guinea pigs received humane care in compliance with the “Guide for care and use
of laboratory animals” (NIH publication 85-23, revised 1985), and the study was
approved by the institutional review board.
Antigen sensitization: Male Hartley guinea pigs, weighing 300-400 g, were placed in
a Plexiglas box (30 x 15 x 20 cm) coupled to an ultrasonic nebulizer (Soniclear, São
Paulo, Brazil). A solution of ovalbumin (Sigma Chemical Co., St. Louis, MO) diluted
in 0.9% NaCl (normal saline) was prepared. This solution was continuously
aerosolized into the environment until respiratory distress (sneezing, coryza, cough
or retraction of the thoracic wall) occurred, or until 15 minutes had elapsed. The
observer who made the decision to withdraw the guinea pig from the inhalation box
was blinded as to the treatment status of the animal. This protocol was repeated
twice a week for four weeks (23, 44) with increasing concentrations of ovalbumin
(from 1 to 5 mg.ml-1) in order to counteract tolerance. Control animals received
aerosolized normal saline.
Capsaicin treatment: Neurokinin was depleted with a single dose (50 mg.kg-1 sc) of
capsaicin (Spectrum Chemical Corporation, Gardena, CA), as previously described
(28, 29, 44). Each guinea pig received aminophylline (10 mg.kg-1 ip), terbutaline (0.1
mg.kg-1 sc) and was anesthetized with ketamine (50 mg.kg-1 im) and xylazine (0.1
mg.kg-1 im). Capsaicin was suspended in a 50 mg.ml-1 solution consisting of 80%
6
normal saline, 10% ethanol and 10% Tween 80 (Sigma Chemical Co.). Guinea pigs
received supplemental oxygen during anesthesia and recovery.
Experimental groups and pulmonary mechanics evaluation: Eight groups of guinea
pigs were used in the experimental protocol. The first group was submitted to
inhalation of aerosolized normal saline only (NS group, n = 9). The second group
was exposed to aerosolized ovalbumin (OVA group, n = 10). The third group was
treated with capsaicin and exposed to aerosolized normal saline (CAP-NS group, n =
9). The fourth group was treated with capsaicin and exposed to aerosolized
ovalbumin (CAP-OVA group, n = 9). The fifth group was exposed to aerosolized
normal saline and received NG-nitro-L-arginine methyl ester (L-NAME; NS-L group, n
= 10; see below). The sixth group was exposed to aerosolized ovalbumin and
received L-NAME (OVA-L group, n = 10). The seventh group was treated with
capsaicin, submitted to inhalation of aerosolized normal saline and given L-NAME
(CAP-NS-L group, n = 9). The eighth and final group was treated with capsaicin,
exposed to aerosolized ovalbumin and given L-NAME (CAP-OVA-L group, n = 9).
Seventy-two hours after the last inhalation, the guinea pigs were anesthetized with
pentobarbital sodium (50 mg.kg-1, ip). They were then tracheostomized and
mechanically ventilated at 60 breaths.min-1 with a tidal volume of 8 ml.kg-1 using a
Harvard 683 ventilator (Harvard Apparatus, South Natick, MA). Tracheal pressure
(Ptr) was measured with a 142PC05D differential pressure transducer (Honeywell,
Freeport, IL) connected to a side tap in the tracheal cannula. Airflow (V') was
determined using a pneumotachograph (Fleisch 4-0, Richmond, VA) connected to
7
the tracheal cannula and to a Honeywell 163PC01D36 differential pressure
transducer. Changes in lung volume (V) were determined by digital integration of the
airflow signal. Nine to ten respiratory cycles were averaged to provide one data point
(15, 36, 44). After baseline measurements of Ptr and V', guinea pigs received
intracardiac injection of either L-NAME (Sigma Chemical Co.; 50 mg.kg-1) (50) diluted
in normal saline (1 ml), or normal saline in the same volume. Ten minutes after
administration of either L-NAME or vehicle, we performed two one-minute challenges
with either aerosolized ovalbumin (30 mg.ml-1) or normal saline delivered into the
breathing circuit through the air inlet of the ventilator. Measurements of Ptr and V'
were taken 1, 3 and 5 minutes after the beginning of the first challenge (Figure 1).
Respiratory system elastance and resistance were obtained using the equation of
motion of the respiratory system:
Ptr(t) = Ers.V(t) + Rrs.V'(t)
where t is time, Ers is respiratory system elastance and Rrs is respiratory system
resistance.
Concentration of exhaled nitric oxide: Concentrations of ENO were measured by
chemiluminescence using a fast-responding analyzer (NOA 280, Sievers
Instruments, Inc., Boulder, CO). Prior to each measurement, the analyzer was
calibrated with a certified 47 parts per billion (ppb) NO source (White Martins, São
Paulo, Brazil) and zero NO filter (Sievers Instruments, Inc.). In order to avoid
environment contamination, NO filter was attached to the breathing circuit. We
measured ENO levels at the expiratory port of the ventilator using a Mylar bag (31).
ENO was measured before and 15 minutes after L-NAME administration (Figure 1).
8
We measured ENO after L-NAME or vehicle administration and antigen or normal
saline challenge to observe the effect of L-NAME in reducing endogenous NO
production.
Yet under anesthesia and after measurement of pulmonary mechanics and ENO, the
anterior chest wall was opened and lungs were washed with heparinized saline
solution (1:40). Immediately after that, guinea pigs were exsanguinated, a positive
end-expiratory pressure of 5 cm H2O was applied to the respiratory system and the
airways were occluded at the end of expiration. The lungs were removed en bloc for
a) morphometric studies; b) histochemical analysis of eosinophil peroxidase activity;
c) immunohistochemical analysis with monoclonal antibodies against iNOS.
Morphometric Studies: One lung was removed, fixed with 4% buffered
paraformaldehyde for 24 hours and then transferred to 70% ethanol. Sections
representing peripheral areas of the lung were cut and processed for paraffin
embedding. Histological sections (5 µm in thickness) were cut and stained with
hematoxylin and eosin and were evaluated by researchers blinded to the protocol
design. We evaluated mononuclear cells and eosinophils around the airway
(between the bronchial epithelium and the adventitia) employing an integrating
eyepiece (104µm2 of total area). Ten to twenty fields were analyzed per lung at a
magnification of x1000 and expressed as cells/unit area (104µm2).
Eosinophil peroxidase activity: The other lung was inflated via the trachea with 5 ml
of optimum cutting temperature (OCT) compound (Reichert-Jung, Heidelberg,
9
Germany), covered with OCT and cooled in liquid nitrogen. Sections were cut on a
cryostat, mounted on glass slides pre-coated with aminopropyltriethoxysilane (Sigma
Chemical Co.) and fixed in chloroform-acetone (Merck, Rio de Janeiro, Brazil) vol/vol
for 10 minutes at room temperature. A histochemical method for cyanide-resistant
eosinophil peroxidase (EPO) activity employing diaminobenzidine (DAB; Sigma
Chemical Co.), H2O2 and potassium cyanide (Sigma Chemical Co.) was used to stain
eosinophils, as previously described (25, 44, 52). Counterstaining with hematoxylin
was employed in order to reveal cellular nuclei, and counts of positive cells in the
airway walls were performed as previously described for morphometric studies (44).
Ten to twenty fields were analyzed per lung at a magnification of x1000 and
expressed as cell/unit area (104µm2).
Evaluation of iNOS: For iNOS detection, we used the same sections employed for
histochemical evaluation of cells containing eosinophil peroxidase (EPO+ cells).
Immunohistochemistry was performed as previously described (7). Subsequently, the
sections were incubated for 30 minutes at room temperature with a blocking solution
containing normal mouse serum (Dako Corp., Carpinteria, CA). Monoclonal antisera
raised in mouse against iNOS (IgG2a - iNOS/NOS Type II - N32020 – BD
Transduction Laboratories, San Diego, CA) were used as primary antisera
(incubation overnight at room temperature, 1:5 dilution in Tris buffer). After three 5-
minute washes in Tris-buffered saline (TBS), sections were incubated with a
secondary antibody (LSAB+AP Link Universal, Dako Corp.) for 30 minutes at 37°C in
a humid chamber. Slides were given three more 5-minute washes in TBS and were
coverslipped with pre-diluted (for 30 minutes) alkaline phosphatase (LSAB + AP -
10
Streptavidin AP - Dako Corp.) This was followed by incubation with substrate Fast
Red TR (Sigma Chemical Co.) for 6 minutes and light hematoxylin counterstaining
for 1 minute. Ten to twenty fields were analysed per lung at a magnification of x1000
and expressed as cells/unit area (104µm2).
Statistical analysis: Values are expressed as means ± SEM. Statistical analysis was
performed using SigmaStat software (Jandel Scientific, San Rafael, CA). Data were
evaluated by ANOVA. Multiple comparisons were made using Tukey test or
Bonferroni test. A p value of less than 0.05 were considered significant (51).
11
Results
Pulmonary Mechanics Evaluation: There were no significant differences among the
study groups in respiratory system elastance or resistance values measured before
antigen challenge/vehicle administration (data not shown). In addition, L-NAME
administration did not affect respiratory system elastance and resistance values.
Figures 2A and 2B show the maximal increase in respiratory system elastance and
resistance (percentage of baseline) obtained after ovalbumin challenge or normal
saline administration. The OVA group showed increased elastance and resistance of
respiratory system after antigen challenge of (mean ± SEM) 452 ± 46% and 230 ±
29%, respectively (p < 0.001). Considering maximal responses of both elastance and
resistance of respiratory system, the increases in both parameters were more
attenuated in the CAP-OVA group than in the OVA group (p < 0.05).
After L-NAME treatment, there was an increase in maximal values of respiratory
system elastance after antigen challenge in ovalbumin-exposed guinea pigs. In fact,
maximal increase in respiratory system elastance was greater in the OVA-L group
than in the OVA group (p < 0.001). In addition, in the presence of L-NAME, maximal
values of respiratory system resistance and elastance induced by ovalbumin
challenge in sensitized guinea pigs were similar those found for capsaicin-treated
and non-capsaicin-treated guinea pigs (CAP-OVA-L and OVA-L groups,
respectively).
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Concentration of Exhaled Nitric Oxide: Figure 3 shows levels of ENO measured
before and 15 minutes after L-NAME administration. Baseline values of ENO were
higher in the OVA group than in the saline only (NS) group (p < 0.05). Pretreatment
with capsaicin reduced ENO in sensitized guinea pigs (p < 0.05). We observed a
significant reduction in ENO after 15 minutes of L-NAME treatment in all experimental
groups, thereby confirming that L-NAME has an acute effect of inhibiting NO
production.
Morphometric Analysis and Histochemistry for Eosinophils: Figure 4A shows the
numbers of mononuclear cells, eosinophils and EPO+ cells in the distal airways of
the eight experimental groups studied. There were no significant differences among
the four control groups (NS, NS-L, CAP-NS and CAP-NS-L). The number of
mononuclear cells, eosinophils and EPO+ cells in ovalbumin-exposed guinea pigs
was greater than that found in controls (p < 0.001). In ovalbumin-sensitized animals,
capsaicin pretreatment and L-NAME treatment (p < 0.001 and p < 0.01, respectively)
reduced recruitment of mononuclear cells, eosinophils and EPO+ cells. In addition,
the combination of the two treatments in ovalbumin-exposed guinea pigs had no
additional effects over those observed in response to isolated treatment with one
agent alone.
Immunohistochemistry for iNOS Detection: Figure 4B shows the numbers of
mononuclear cells and eosinophils showing iNOS expression and infiltrating the
airways of the experimental groups studied. There were few of these inflammatory
cells in control guinea pigs and no differences were found among the control groups.
13
Ovalbumin-treated animals presented increases in the total numbers of mononuclear
cells and eosinophils showing iNOS expression in the airways compared to controls
(p < 0.001). We noticed that the eosinophil populations were divided between iNOS-
positive and iNOS-negative cells (45.7% and 54.3%, respectively, in the OVA group).
Inhibition of NO production by L-NAME treatment reduced the total number of
eosinophils, but the reduction was primarily within the iNOS-positive subgroup (Table
1). In contrast most (84.3%) of the mononuclear cells were iNOS negative. Although
the total number of mononuclear cells was reduced by L-NAME treatment, we did not
observe any preferential effect in iNOS-positive or iNOS-negative mononuclear cells,
with similar reductions in both subgroups (Table 1).
Although neurokinin depletion by capsaicin treatment reduced the total number of
eosinophils, we observed no reduction in iNOS-positive eosinophils, showing a
preferential effect in the iNOS-negative subgroup (Table 1). Neurokinin depletion by
capsaicin pretreatment reduced the total numbers of both iNOS-positive and iNOS-
negative mononuclear cells (Table 1). The combination of neurokinin depletion and
L-NAME treatment did not modify this response.
Figure 5 shows photomicrographs of the distal airway walls of guinea pigs exposed
to aerosolized ovalbumin highlighting a large amount of EPO+ cells and iNOS-
positive inflammatory cells. In normal saline-exposed animals, we observed low
numbers of inflammatory cells stained for eosinophil peroxidase and iNOS around
distal airway walls.
15
Discussion
In a previous investigation, we evaluated airway inflammation induced by repeated
exposure to aerosolized ovalbumin in guinea pigs and the effects of neurokinin
depletion (by capsaicin pretreatment) on that inflammation in guinea pigs (44). In that
experimental model, we observed peribronchial edema, an increase in lymphocytes
and eosinophils (both in bronchoalveolar lavage fluid and in distal airways) and an
increase in pulmonary responsiveness to methacholine. Immunohistochemistry with
monoclonal antibodies revealed that most mononuclear cells present in the airway
walls are CD4+ T cells. Neurokinin depletion resulted in lower maximal values of
respiratory system elastance and resistance after antigen challenge, less intense
bronchoconstriction and airway edema formation and a decrease in the number of
CD4+ T cells in the airway wall (44).
In the present study, using the same experimental model of chronic allergic airway
inflammation in guinea pigs, we evaluated the effects of neurokinin on iNOS
inflammatory cell expression. In ovalbumin-exposed guinea pigs, in contrast to the
effects of neurokinin depletion, L-NAME administration induced a decrease in NO
production through inhibition of NO synthase, resulting in a significant increase in
respiratory system elastance response to allergen challenge. In addition, the
pulmonary mechanical response to allergen challenge in the CAP-OVA-L group was
greater than that of those in the CAP-OVA group and comparable to that of those in
the OVA group. These results suggest in vivo correlations of the effects of NO and
neurokinins in modulation of proximal and distal airway tone since the attenuation of
16
the acute effects of antigen challenge in animals depleted of neurokinins was
negated by the inhibition of nitric oxide. The inhibitory effect of L-NAME on NO
production was confirmed by the observation that all animals receiving L-NAME
presented a significant decrease in the concentration of ENO (Figure 3).
Our findings are in accordance with previous studies that demonstrated the
bronchoprotective effect of NO. Dupuy et al. (10) showed that inhaled gaseous NO is
a potent bronchodilator in guinea pigs, acting in the distal airways only in high doses.
However, many authors have suggested a more pronounced effect of NO in the
proximal airways (8, 35, 48) and have proposed that this may be due to decreased
nitrergic innervation density in the distal airways (48). Notwithstanding, in the present
study, we observed that the increased respiratory system mechanical response to
ovalbumin challenge in L-NAME treated animals was more evident in respiratory
system elastance than in respiratory system resistance. This suggests that NO
inhibition affects distal airways and lung parenchyma more than it does proximal
airways. One possible explanation is that changes in respiratory system elastance
may also be related to vascular effects in the alveolar wall. We noticed that
sensitized, L-NAME-treated guinea pigs presented extensive blood extravasation in
the alveolar spaces (data not shown) probably resulting from NO modulation of
vascular tone (12, 27, 47).
Endogenously produced NO has been measured in the expired gases of animals
and humans. It has been reported that ENO is a useful marker of airway inflammation
in asthma (1, 21, 22). Asthmatic patients have higher ENO levels than normal
17
subjects do and there is a decline in ENO levels when asthmatic patients receive
treatment with corticosteroids. Increased ENO after antigen challenge in experimental
models of allergic airway inflammation has also been shown (11, 31). However, the
precise source of the increased NO measured in expired air in asthma and
experimental models of allergic airway inflammation remains to be determined. In our
study, we also noticed increased ENO in guinea pigs with chronic airway inflammation
induced by repeated exposure to aerosolized ovalbumin. We observed that
neurokinin depletion in guinea pigs chronically exposed to antigen reduced ENO
levels to values similar to those obtained for guinea pigs not exposed to ovalbumin
(Figure 3). A possible explanation is that in animals sensitized and pretreated with
capsaicin, the reduction in total inflammatory cells could induce some changes in
nitric oxide metabolism, as suggested by Hunt et al. (19).
As a counterpoint to the increased mechanical response to allergen challenge, we
observed that L-NAME treatment reduced numbers of both lymphocytes and
eosinophils in the airway wall. This implies that NO plays an important role in
inflammatory cell recruitment in this animal model of asthma. This role became clear,
even despite the fact that only one dose of NO inhibitor was given to sensitized
animals and the lungs were not excised until after 20 minutes of antigen challenge.
By that time, in sensitized-only guinea pigs, the inflammatory cells had aggregated to
blood vessels, resulting in acute bronchial wall infiltration. As we had expected, the
blockage of NO production in these animals resulted in diminished cell recruitment.
In accordance with these results, some other authors have also observed the acute
18
effect that administration of NO inhibitors has on inflammatory cell recruitment (13,
38).
The lung represents an important target organ for NO generation, and NO is a crucial
mediator of the inflammatory response. It has been previously suggested that NO is
involved in the development of pulmonary eosinophilia (13), and that treatment with
L-NAME may reduce eosinophilic and/or neutrophilic influx, both in vitro and in
models of antigen-induced airway inflammation (5, 11, 14, 20, 38, 39, 43). In
contrast, some authors have found no participation of NO in inflammatory cell
recruitment (46).
This L-NAME-induced anti-inflammatory effect was similar to that observed in
capsaicin-treated guinea pigs, in which there was also a decrease in numbers of both
lymphocytes and eosinophils in the airway wall. However, we did not notice any
additional effects when capsaicin and L-NAME treatments were combined.
Some mechanisms could explain the effects of capsaicin pretreatment and L-NAME
administration in reducing inflammatory cells around airways of sensitized animals.
One possibility is that neurokinins and nitric oxide act in apoptosis of these
inflammatory cells. Some evidence suggested that substance P and neurokinin A
can delay the apoptosis process in some types of cells (4, 37). Considering nitric
oxide, its effect in cell death is unclear. Taylor et al. (41) suggested that NO could
have different effects on inflammatory cell apoptosis (anti- and pro-apoptotic
properties), depending on the concentration and flux of NO, and the source from
19
which NO is derived. Other possibility could be related to the effects of neurokinins
and/or nitric oxide in other mediators that can act directly in the inflammatory cells,
delaying or anticipating apoptosis process.
We also performed immunohistochemical evaluation of the expression of inducible
NO synthase (iNOS) in inflammatory cells surrounding the airway wall. We detected
increases in the total numbers of mononuclear cells and eosinophils showing iNOS
expression in the airways of repeated ovalbumin-exposed animals. Of greater
interest, we demonstrated that there were both iNOS-positive and iNOS-negative
populations of eosinophils (45.7% and 54.3%, respectively, in the OVA group).
Treatment with L-NAME primarily reduced the numbers of the iNOS-positive
eosinophils, whereas neurokinin depletion had a preferential effect in the iNOS-
negative eosinophil subgroup. In light of these various findings, one possible
explanation is that nonadrenergic-noncholinergic actions in eosinophilic recruitment
may be related to autocrine or paracrine effects of NO and neurokinins.
Both neurokinins and NO may contribute to lymphocyte cell recruitment with no
preferential effect in the iNOS-positive or iNOS-negative cell subgroups. The
combination of neurokinin depletion and reduction of NO production did not modify
this response.
Most mononuclear cells infiltrating the airway wall in chronically ovalbumin-exposed
guinea pigs were iNOS negative (84.3%). Although total numbers mononuclear cells
were reduced by L-NAME treatment, we did not observe any preferential effect in the
20
iNOS-positive or iNOS-negative mononuclear cells, with similar reductions in both
subgroups (Table 1). The same behavior was observed with neurokinin depletion by
capsaicin pretreatment, which reduced total numbers of both iNOS-positive and
iNOS-negative mononuclear cells (Table 1). The combination of neurokinin depletion
and reduction of NO production did not modify this response.
Some studies in isolated cells have also shown iNOS expression in inflammatory
cells such as macrophages (17, 40, 49), neutrophils (6, 30, 49) and eosinophils (9,
49). In the present study, a model of chronic allergic inflammation was used for the
first time to show to describe iNOS expression in various subgroups of eosinophils
and lymphocytes. Taylor-Robinson et al. (42) found that cloned murine Th1 cells
activated by antigens or mitogens expressed iNOS and produced substantial
amounts of NO. The authors proposed that both proliferation of Th1 cells and
production of IL-2 and IFN-γ may be inhibited by high concentration of NO, although
Th2 cells neither produced nor were affected by NO. In this animal model, the
majority of lymphocytes in the distal airways are T lymphocytes, mainly of the CD4+
subset (44). We therefore speculate that the decrease in inflammatory cells induced
by either capsaicin pretreatment or L-NAME administration in our experimental
asthma model may be attributed to a reduction in the Th2 helper lymphocyte
population.
In conclusion, even in the case of neurokinin depletion NO modulates airway tone.
Using a model of chronic allergic airway inflammation, we were able to identify
subgroups within the eosinophils and lymphocytes that showed expression of iNOS.
21
Neurokinins and NO amplify recruitment of both iNOS-positive and iNOS-negative
lymphocytes. Neurokinins are involved in iNOS-negative eosinophil recruitment and
NO affects recruitment within the iNOS-positive eosinophil subgroup. In the guinea
pig model NO may have both beneficial and detrimental effects, acting as a
bronchodilator and promoting eosinophil and mononuclear cell response in the distal
airways.
22
Acknowledgments
The authors are grateful to Sandra de Moraes Fernezlian, Esmeralda Meristene,
Adriana Salles Leme and Flávia Tayar Fernandes for skillful technical assistance.
This study was supported by the following Brazilian scientific agencies: Fundação de
Amparo à Pesquisa do Estado de São Paulo (FAPESP) e Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq).
23
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Legends for Illustrations
Figure 1: Time line of the experimental protocol. Capsaicin pretreatment was
performed on Day 0 in groups CAP-NS and CAP-OVA and exposure to ovalbumin
(OVA) or normal saline (NS) aerosols were started on Day 14 in the four
experimental groups (NS, CAP-NS, OVA and CAP-OVA). On Day 36, all animals
were anesthetized, tracheostomized and mechanically ventilated. We collected
values of exhaled NO and mechanical parameters (baseline) and then animals
received either L-NAME (NS-L, OVA-L, CAP-SF-L and CAP-OVA-L) or vehicle (NS,
OVA, CAP-NS and CAP-OVA). Ten minutes after, animals received either ovalbumin
(OVA, OVA-L, CAP-OVA and CAP-OVA-L) or saline (NS, NS-L, CAP-NS and CAP-
NS-L) challenge for two minutes and we performed mechanical evaluation 1, 3 and 5
minutes after the beginning of the antigen challenge. Immediately after the
mechanical evaluation, we measured exhaled nitric oxide in the eight groups studied
to evaluate the efficacy of the L-NAME treatment.
Figure 2. Vertical bars represent mean±SE of maximal increase in respiratory
system (A) elastance (Ers) and (B) resistance (Rrs) (percentage of baseline)
obtained after challenge with ovalbumin or normal saline and acute treatment with L-
NAME or vehicle (i.c.). The sensitization increases both Ers and Rrs responses in
OVA animals compared to controls. CAP pretreatment reduced significantly this
response. There were no differences between CAP-OVA groups and control groups.
L-NAME acute treatment increased only Ers in OVA animals (OVA-L compared to
32
OVA animals), however L-NAME restored capsaicin pretreatment effects in both Ers
and Rrs. * p < 0.01 compared to controls and CAP-OVA group; ** p < 0.05 compared
to OVA group.
Figure 3. Vertical bars represent mean±SE of baseline ENO (after 72 hours of last
inhalation) and after vehicle (Ve) or L-NAME intracardiac treatment (L) in animals
that were exposed to ovalbumin aerosols, with pretreated with capsaicin (CAP-OVA)
or vehicle (OVA), and control animals (NS and CAP-NS). * p < 0.05 compared to
other groups (baseline values) and ** p <0.005 compared to their respective baseline
values and after vehicle treatment.
Figure 4. Vertical bars represent mean±SEM. Results are expressed per unit of area
(104µm2).
(A) Number of total mononucleated cells (MN), eosinophils (EOS) and eosinophils
EPO+ cells (EPO+). * p < 0.05 compared to all other groups, † p <0.05 compared to
other groups (black bars) except for CAP-OVA; and ‡ p < 0.05 compared to controls
(NS, NS-L, CAP-NS and CAP-NS-L);
(B) Number of mononucleated cells (MN-iNOS+) and eosinophils (EOS-iNOS+) that
are positive to iNOS expression present in distal airways of guinea pig from the eight
experimental groups. * p < 0.05 compared to other groups, except for CAP-OVA
EOS-iNOS+ and ** p < 0.05 compared to controls EOS-iNOS+ (black bars).
Figure 5. Photomicrograph of distal wall airway submitted to histochemical and
immunohistochemical techniques:
33
(A) Distal airway of an OVA-sensitized guinea pig demonstrating a great number of
EPO+ cells (arrows) around and in epithelium (400x);
(B) Distal airway of control guinea pig (400X);
(C) Airway wall of an OVA-sensitized guinea pig demonstrating iNOS+ inflammatory
cells (1000X). The arrows identify the positive cells.
(D) Airway wall of normal saline group showing iNOS-negative cells (1000X).
34
TABLE 1Number and percentage of iNOS-positive and iNOS-negative mononucleated cells
and eosinophils in guinea pigs submitted to the protocol of multiple exposures to
aerosols of ovalbumin (OVA), pretreated with capsaicin (CAP-OVA), and treated with
L-NAME (OVA-L and CAP-OVA-L)
Mononucleated cells Eosinophils
Total iNOS-
(%)
iNOS+
(%)
Total iNOS-
(%)
INOS+
(%)
OVA 34.3 28.9
(84.3%)
5.4
(15.7%)
25.8 14.0
(54.3%)
11.8
(45.7%)
CAP-OVA 7.0 4.3
(62.0%)
2.7
(38.0%)
11.6 2.6
(22.4%)
9.0
(77.6%)
OVA-L 19.5 16.6
(85.1%)
2.9
(14.9%)
17.7 12.4
(69.7%)
5.3
(30.3%)
CAP-OVA-L 4.1 2.5
(61.3%)
1.6
(39.7%)
9.5 4.5
(46.9%)
5.0
(53.1%)
The cells were counted at a magnification of X1000 and expressed as cells/unit area
(104µm2).
36
FIGURE 2
NSNS-L
OVAOVA-L
CAP-NS
CAP-NS-L
CAP-OVA
CAP-OVA-L
% m
axim
al in
crea
se o
f E
rs
0
100
200
300
400
500
600
700
800
900
1000* ***
*
**A
B
*
**
NSNS-L
OVAOVA-L
CAP-NS
CAP-NS-L
CAP-OVA
CAP-OVA-L
% m
axim
al in
crea
se o
f R
rs
0
50
100
150
200
250
300
37
FIGURE 3
NS CAP-NS OVA CAP-OVA
Exh
aled
NO
(p
pb
)
0
5
10
15
20
25
30
35 BaselineAfter vehicleAfter L-NAME
*
** ** ** **
*
38
FIGURE 4
A
B
NSNS-L
CAP-NS
CAP-NS-L
OVAOVA-L
CAP-OVA
CAP-OVA-L
Cel
ls /1
04 µm
2
0
5
10
15
20
25
30
35
40*
*
* *†
‡‡
‡
MN cellsEOS cellsEPO+ cells
NSNS-L
CAP-NS
CAP-NS-L
OVAOVA-L
CAP-OVA
CAP-OVA-L
iNO
S +
cel
ls /
104 µ
m2
0
2
4
6
8
10
12
14*
**
*
MN-iNOS+EOS-iNOS+
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