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: iocalvo@uol.com.br

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.

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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%

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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

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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.

14

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).

35

FIGURE 1.

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+

39

FIGURE 5

A

B

C

D