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Blockade of airway hyperresponsiveness and inflammation in a murine model of asthma by a prodrug of cysteine, L-2-oxothiazolidine-4-carboxylic acid

YONG CHUL LEE^*,,1, KYUNG SUN LEE^*,, SEOUNG JU PARK^*,, HEE SUN PARK^*,, JAE SUNG LIM^*,, KWANG-HYUN PARK, MIE-JAE IM, IL-WHAN CHOI, HERN-KU LEE and UH-HYUN KIM^,||

^* Department of Internal Medicine;
Research Center for Allergic Immune Diseases;
Department of Biochemistry;
Department of Immunology; and
^|| Institute of Cardiovascular Research, Chonbuk National University Medical School, Jeonju, South Korea

¹Correspondence: Department of Internal Medicine, Chonbuk National University Medical School, 634-18, Keumamdong, Jeonju, 561-712, South Korea. E-mail: leeyc{at}chonbuk.ac.kr

SPECIFIC AIMS

Excess production of reactive oxygen species (ROS) and defective endogenous antioxidant defense mechanisms may play an important role in the pathogenesis of bronchial asthma. In the present study, we used a mouse model for asthma to determine effects of reducing agent L-2-oxothiazolidine-4-carboxylic acid (OTC), a prodrug of cysteine, on allergen-induced bronchial inflammation and airway hyperresponsiveness.

PRINCIPAL FINDINGS

1. ROS generation is increased by ovalbumin (OVA) inhalation and the increase is reduced by administration of OTC
ROS generation in bronchoalveolar lavage (BAL) fluids was increased significantly 72 h after OVA inhalation compared with the levels after saline inhalation. Increased ROS generation was significantly reduced by administration of OTC.

2. Administration of OTC reduces airway inflammation
Numbers of total cells, eosinophils, lymphocytes, and neutrophils in BAL fluids increased significantly 72 h after OVA inhalation vs. after saline inhalation. The increased numbers of these cells were significantly reduced by administration of OTC (Fig. 1 A). Histological analysis revealed typical pathologic features of asthma in OVA-exposed mice. Numerous inflammatory cells including eosinophils infiltrated around the bronchioles (Fig. 1C ) as compared with the control (Fig. 1B ). Mice treated with OTC (Fig. 1D-F ) showed marked reductions in infiltration of inflammatory cells in the peribronchiolar and perivascular region. Scores of peribronchial, perivascular, and total lung inflammation were increased significantly 72 h after OVA inhalation compared with scores after saline inhalation (Fig. 1G ). Increased peribronchial, perivascular, and total lung inflammation after OVA inhalation was significantly reduced by administration of OTC.

View larger version (57K): [in this window] [in a new window]   Figure 1. Effect of OTC on total and differential cellular components of BAL and on pathologic changes in lung tissues of OVA-sensitized and -challenged mice. A) Numbers of each cellular component of BAL from saline-inhaled mice administered saline (SAL+SAL), OVA-inhaled mice administered saline (OVA+SAL), OVA-inhaled mice administered OTC 40 mg/kg (OVA+OTC 40), OVA-inhaled mice administered OTC 80 mg/kg (OVA+OTC 80), and OVA-inhaled mice administered OTC 160 mg/kg (OVA+OTC 160) were counted 72 h after the last challenge. B–F) Representative hematoxylin and eosin-stained sections of the lungs. Sampling was performed 72 h after the last challenge in B) saline-inhaled mice administered saline; C) OVA-inhaled mice administered saline; D) OVA-inhaled mice administered OTC 40 mg/kg; E) OVA-inhaled mice administered OTC 80 mg/kg; and F) OVA-inhaled mice administered OTC 160 mg/kg. Bars indicate scale of 50 µm. G) Peribronchial and perivascular lung inflammation were measured 72 h after the last challenge in SAL+SAL, OVA+SAL, OVA+OTC 40, OVA+OTC 80, and OVA+OTC 160. Bars represent mean ± SE from 6 independent experiments. #P <0.05 vs. SAL+SAL; *P <0.05 vs. OVA+SAL.

3. Administration of OTC reduces airway hyperresponsiveness
Airway responsiveness was assessed as a percent increase of enhanced pause (Penh) in response to increasing doses of methacholine. In OVA-sensitized and -challenged mice, the dose-response curve of percent Penh shifted to the left compared with that of control mice. The percent Penh produced by methacholine administration (at doses from 2.5 mg/mL to 50 mg/mL) increased significantly in OVA-sensitized and -challenged mice compared with controls. OVA-sensitized and -challenged mice treated with OTC showed a dose-response curve of percent Penh that shifted to the right compared with that of untreated mice. The shift was dose-dependent. These results indicate that OTC treatment reduces OVA-induced airway hyperresponsiveness.

4. Levels of NF-B protein in nuclear protein extracts from lung tissues are increased by OVA inhalation and the increase is reduced by administration of OTC
Western blot analysis revealed that levels of NF-B p65 protein in nuclear protein extracts from lung tissues were increased 72 h after OVA inhalation compared with levels in control mice. Increased NF-B p65 levels in nuclear protein extracts from lung tissues 72 h after OVA inhalation were decreased by administration of OTC. In contrast, levels of NF-B p65 protein in cytosolic protein extracts from lung tissues were decreased 72 h after OVA inhalation compared with levels in control mice. Decreased NF-B p65 levels in cytosolic protein extracts from lung tissues 72 h after OVA inhalation were increased by administration of OTC.

EMSA revealed that NF-B-DNA binding in nuclear extracts from lung tissues was increased 72 h after OVA inhalation compared with levels in control mice. Increased NF-B-DNA binding in nuclear extracts from lung tissues 72 h after OVA inhalation was decreased by administration of OTC. Supershift assay confirmed the presence of p50 and p65 subunits of NF-B.

5. Increased IL-4, IL-5, and IL-13 levels in lung tissues and BAL fluids after OVA inhalation are significantly reduced by administration of OTC
Western blot analysis revealed that levels of IL-4, IL-5, and IL-13 protein in lung tissues were increased significantly 72 h after OVA inhalation compared with levels after saline inhalation. Increased levels of these cytokines were significantly reduced by administration of OTC. Consistent with results obtained from Western blot analysis, enzyme immunoassays revealed that levels of IL-4, IL-5, and IL-13 in BAL fluids were also increased significantly 72 h after OVA inhalation compared with levels after saline inhalation. Increased levels of these cytokines were significantly reduced by administration of OTC.

6. Increased ICAM-1, VCAM-1, RANTES, and eotaxin expressions in lungs after OVA inhalation are significantly reduced by administration of OTC
RT-PCR analysis revealed that expression of ICAM-1, VCAM-1, RANTES, and eotaxin mRNA in lung tissues was increased significantly 72 h after OVA inhalation compared with levels after saline inhalation. Increased mRNA expression of these adhesion molecules and chemokines was decreased by administration of OTC. Western blot analysis revealed that levels of ICAM-1, VCAM-1, and eotaxin protein in lung tissues were increased significantly 72 h after OVA inhalation compared with levels after saline inhalation. Increased levels of ICAM-1, VCAM-1, and eotaxin protein were significantly reduced by administration of OTC. Enzyme immunoassays revealed that levels of RANTES in BAL fluids were also increased significantly 72 h after OVA inhalation as compared with levels after saline inhalation. Increased RANTES levels were significantly reduced by administration of OTC.

7. Increased ECP levels BAL fluids after OVA inhalation are significantly reduced by administration of OTC
Fluoroenzyme immunoassays revealed that levels of ECP in BAL fluids were increased significantly 72 h after OVA inhalation compared with levels after saline inhalation. Increased levels of ECP 72 h after OVA inhalation were significantly reduced by administration of OTC.

CONCLUSIONS AND SIGNIFICANCE

Oxidative stress plays an important role in the pathogenesis of bronchial asthma. Our present study with the OVA-induced model of asthma has revealed that ROS production in cells from BAL fluids is increased and that administration of a prodrug of cysteine, OTC, which is readily transportable into cells, converted to L-cysteine and used for glutathione biosynthesis, reduces eosinophilic inflammation and airway hyperresponsiveness as well as OVA-induced ROS generation. Our results also indicate that ROS produced by induction of asthma with OVA up-regulate a transcription factor NF-B activity and that OTC down-regulates activity of this transcription factor NF-B.

ROS released by eosinophils and other leukocytes play an important role in airway tissue injury observed in asthma. Previous reports have shown that release of ROS is increased in airways of OVA-challenged animals. In this study, we found that generation of ROS in BAL fluids is significantly increased after allergen challenge in OVA-induced asthma model and that administration of OTC reduced generation of ROS. Although the lung has a well-developed antioxidant system, overproduction of ROS or depression of the protective system results in epithelial cell damage, cell shedding, and bronchial hyperreactivity. Studies with animal models have indicated that ROS contribute to airway hyperresponsiveness by increasing vagal tone due to damage of oxidant sensitive ß-adrenergic receptors as well as decreasing mucociliary clearance. These findings suggest that ROS induce and maintain pathogenesis of asthma.

The molecular basis of ROS-mediated induction of asthma has not been clearly defined. A recent study has indicated that development of oxidant/antioxidant imbalance in asthma leads to activation of redox-sensitive transcription factor NF-B. NF-B is present in most cell types and is known to play a critical role in immune and inflammatory responses, including asthma. Consistent with these observations, NF-B protein level in nuclear protein extracts of lung tissues is substantially increased in the OVA-induced model of asthma used for the present study. It is known that activation of this transcription factor induces a variety of inflammatory genes that are abnormally expressed in asthma. These genes include cytokines (e.g., IL-4, IL-5, IL-9, IL-15, and TNF-), chemokines (e.g., RANTES, eotaxin, and monocyte chemotactic protein-3), and adhesion molecules (e.g., ICAM-1 and VCAM-1). We also assessed whether these genes are up-regulated in the OVA-induced asthma model. As expected, expression of adhesion molecules (ICAM-1 and VCAM-1), chemokines (RANTES and eotaxin), and cytokines (IL-4, IL-5, and IL-13) was increased significantly after allergen challenge in a murine model of asthma. Administration of OTC results in significant reduction of NF-B translocation into nucleus and of expression of these adhesion molecules, chemokines, and cytokines (Fig. 2 ). These results indicate that OTC inhibits NF-B activity by preventing translocation of this transcription factor into nucleus that is induced by ROS. ROS have been directly implicated as second messengers in the activation of NF-B, based upon the ability of oxidants to activate NF-B by oxidation of its cysteine-SH group or by ubiquitination and proteolysis. Inhibitory B (IB) has 5–7 conserved domains known as ankylin repeats, each consisting of 30 amino acids, forming a unit which is able to interact with NF-B subunits, thus masking nuclear localization signals and preventing activation of NF-B and its translocation to the nucleus. Antioxidants have been known to inhibit NF-B activation by preventing IB degradation in response to various stimuli. We suggest that OTC inhibits NF-B signal transduction pathway by decreasing NF-B binding activity to promoter regions of a large variety of genes which are considered to be involved in airway inflammation in asthma.

View larger version (21K): [in this window] [in a new window]   Figure 2. Schematic diagram of OTC effect on ROS response in asthma. Anti-inflammatory action of OTC is mediated through regulation of NF-B signal transduction pathway. Administration of OTC results in reduction of NF-B translocation into nucleus as well as expression of adhesion molecules, chemokines, and cytokines.

A significant amount of data showing an increase of oxidative stress in asthma and indicating a potential role for oxidants in pathogenesis of the diseases has been accumulated. Based on these observations and in order to find a potential therapeutic strategy for treating asthma, we administered an antioxidant agent OTC to allergen-challenged mice. Administration of OTC results in a significant reduction of all pathophysiological symptoms examined. Our study provides evidence that oxidative stress is one of the important determinants of asthma and that antioxidant treatment such as administration of OTC, which is transportable into cells, may be a recommendable therapeutic strategy.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2212fje;

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