Role of nitric oxide in allergic inflammation and bronchial hyperresponsiveness

doi:10.1016/S0014-2999(02)02237-9

European Journal of Pharmacology

Volume 452, Issue 1, 27 September 2002, Pages 123-133

https://doi.org/10.1016/S0014-2999(02)02237-9 Get rights and content

Abstract

The role of nitric oxide (NO) in allergic inflammation and bronchial hyperresponsiveness is unclear. We studied a selective prodrug nitric oxide synthase (NOS)-2 inhibitor, l-N⁶-(1-iminoethyl)lysine 5-tetrazole amide (SC-51). In ovalbumin-sensitized and challenged rats, exhaled NO levels increased by 3 h following challenge (3.73±0.74 ppb; P<0.05), peaking at 9 h (11.0±2.75; P<0.01) compared to saline controls (1.87±0.26; P<0.05 and 2.81±0.18; P<0.01). Immunoreactive lung NOS2 expression was increased in ovalbumin-challenged rats compared with ovalbumin-sensitized, saline-challenged rats at 8 h post-challenge. SC-51 (10 mg/kg; p.o.) inhibited allergen-induced increase in exhaled NO levels to 1.3±0.17 ppb. SC-51 inhibited bronchial hyperresponsiveness in ovalbumin-sensitized and challenged rats (P<0.05). In sensitized non-exposed rats, SC-51 increased bronchial responsiveness (P<0.05). SC-51 reduced the allergen-induced increase in bronchoalveolar lavage neutrophils, but caused a nonsignificant reduction in bronchial mucosal eosinophil numbers. NO generated through NOS2 contributes to allergen-induced bronchial hyperresponsiveness but not to bronchial eosinophilia, indicating that these are independently expressed.

Introduction

Endogenous nitric oxide may play an essential role in the physiological regulation of airway function and has been implicated in the pathogenesis of airway diseases, such as bronchial asthma (Barnes, 1995). Asthma is a chronic disease of the airways characterized by airway inflammation and bronchial hyperresponsiveness, and is associated with increased concentrations of nitric oxide (NO) in the expired air Alving et al., 1993, Kharitonov et al., 1995b. Nitric oxide is synthesized by a variety of cell types from the amino acid, l-arginine, by the action of enzymes, nitric oxide synthases (NOS) (Moncada et al., 1991). Three main isoforms of NOS have been characterized, each the product of genes located on different chromosomes, with each isoform differing in structural and biochemical properties. Neuronal NOS or NOS1 and endothelial NOS or NOS3 are constitutively expressed isoforms activated by depolarization- or agonist-induced intracellular calcium changes, resulting in the generation of small (picomolar) amounts of NO that serves as a diffusible signaling molecule mediating intracellular processes. NOS1-derived NO may be involved in neurotransmission (Synder and Fleisch, 1989), and NOS3-derived NO may be important for the relaxation of vascular smooth muscle and in the regulation of systemic and pulmonary blood pressure. In contrast, the inducible isoform of NOS or NOS2 is expressed via prolonged calcium-independent mechanisms, leading to the production of relatively large (nanomolar) amounts of NO, which may not only activate soluble guanylyl cyclase, but may additionally have cytostatic and cytotoxic effects Nathan, 1992, Liew, 1994.

NO is detected in the exhaled air of humans and various experimental animals and is increased in the exhaled air of asthmatic patients Kharitonov et al., 1995b, Alving et al., 1993. Increased expression of NOS2 has been detected in the bronchial epithelium of asthmatic patients (Hamid et al., 1993), and following allergen challenge in sensitized rats, NOS2 is expressed mainly by alveolar macrophages and to a lesser extent in the airway epithelium (Liu et al., 1997). After bronchial provocation with specific allergen, atopic asthmatic patients demonstrate an increase of exhaled NO (Kharitonov et al., 1995a). In sensitized rats (Yeadon and Price, 1995) and guinea pigs (Yan et al., 1995), allergen exposure results in enhanced endogenous NO production during the late phase, together with an induction of NOS2 in the lungs (Liu et al., 1997).

The precise role of NOS2-derived NO in allergic inflammation and in bronchial hyperresponsiveness remain unclear. In in vivo studies of mice rendered deficient of NOS2, there was a reduction in pulmonary eosinophilia following allergen exposure of sensitized mice (Xiong et al., 1999), while another study showed no effects on pulmonary eosinophilia (De Sanctis et al., 1999). However, in both studies, allergen-induced bronchial hyperresponsiveness was fully expressed despite the absence of NOS2. Because unconditional gene disruption may not fully reveal the role played by NOS enzymes in view of potential effects to the development of innate immunity, pharmacological isoenzyme inhibition using highly selective inhibitors may be a better way of investigating the role of NOS enzymes.

We examined the role of endogenously produced NO in a rat model of allergic asthma. We examined the levels of exhaled NO and NOS isoform protein expression, and determined the effect of NOS inhibitor, l-N⁶-(1-iminoethyl)lysine 5-tetrazole amide (SC-51), which is a prodrug of l-NIL, l-N⁶-(1-iminoethyl lysine 5-tetrazole amide) (Hallinan et al., 2002), in allergic inflammation and bronchial hyperresponsiveness following allergen challenge in sensitized Brown–Norway rats. This inhibitor has some selectivity against NOS2.

Section snippets

Animals, sensitization procedures and allergen-exposure

Pathogen-free male Brown–Norway rats (weighing 200–280 g, 9–13 weeks old; Harlan Olac, Bicester, UK) were sensitized on days 1, 2 and 3 using 1 mg/kg intraperitoneal injections of ovalbumin (grade V, salt-free, Sigma, Dorset, UK) in 0.9% (w/v) sterile sodium chloride solution containing 100 mg aluminium hydroxide as adjuvant. Three weeks later, rats were exposed to ovalbumin aerosol (1% w/v, 20 min) or saline (0.9% w/v, 20 min) with the use of a 6.5-l Plexiglas acrylic plastic connected to an

Exhaled NO levels

Exhaled NO following ovalbumin challenge of ovalbumin-sensitized rats was significantly increased by 3 h (3.73±0.74 ppb) when compared to ovalbumin-sensitized and saline-exposed rats (1.87±0.26; P<0.05). This allergen-induced increase in exhaled NO remained significantly elevated for 12 h, reaching a maximum at around 9 h (11.0±2.75), when compared to ovalbumin-sensitized and saline-exposed rats (2.81±0.18; P<0.01), returning to baseline at 24 h (2.51±0.35). There was no significant difference

Discussion

We have used a relatively selective inhibitor of inducible nitric oxide synthase, SC-51, in order to examine the role played by endogenous nitric oxide production under the stimulation of NOS2 in allergic inflammation and allergen-induced bronchial hyperresponsiveness. We showed that nitric oxide in exhaled air of allergen-provoked rats is increased over the ensuing 9 h, and that these levels are suppressed by the NOS2 inhibitor, indicating that this increase in exhaled NO is derived from NOS2.

Acknowledgements

We thank Mark Currie and Pamela Manning of Pharmacia for their support of this study and for the provision of SC-51.

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Role of nitric oxide in allergic inflammation and bronchial hyperresponsiveness

Abstract

Introduction

Section snippets

Animals, sensitization procedures and allergen-exposure

Exhaled NO levels

Discussion

Acknowledgements

J. Allergy Clin. Immunol.

Pulm. Pharmacol. Ther.

Immunol. Lett.

J. Allergy Clin. Immunol.

Am. J. Pathol.

Increased amount of nitric oxide in exhaled air of asthmatics

Eur. Respir. J.

Measurement of nitric oxide in biological models

FASEB J.

Nitric oxide and asthma

Res. Immunol.

Exogenous nitric oxide elicits chemotaxis of neutrophils in vitro

J. Cell. Physiol.

Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide

Proc. Natl. Acad. Sci. U. S. A.

Inhibitors of nitric oxide synthase attenuate human neutrophil chemotaxis in vitro

J. Lab. Clin. Med.

Identification of T-lymphocytes, macrophages and activated eosinophils in the bronchial mucosa of intrinsic asthma: relationship to symptoms and bronchial hyperresponsiveness

Am. Rev. Respir. Dis.

Dual action of nitric oxide on airway plasma leakage

Am. J. Respir. Crit. Care Med.

Enhanced superoxide production by alveolar macrophages and air-space cells, airway inflammation, and alveolar macrophage density changes after segmental antigen bronchoprovocation in allergic subjects

Am. Rev. Respir. Dis.

Role played by inflammation in the hyperreactivity of the airways in asthma

Thorax

Contribution of nitric oxide synthases 1, 2, and 3 to airway hyperresponsiveness and inflammation in a murine model of asthma

J. Exp. Med.

Bronchodilator action of inhaled nitric oxide in guinea pigs

J. Clin. Invest.

Role of nitric oxide on eosinophilic lung inflammation in allergic mice

Am. J. Respir. Cell Mol. Biol.

Expression of Th-2 cytokines IL-4 and IL-5 and of Th-1 cytokine IFN-γ in ovalbumin-exposed sensitised Brown–Norway rats

Immunology