Involvement of haemoxygenase-1 in ozone-induced airway inflammation and hyperresponsiveness

Abstract

Haemoxygenase catalyses the degradation of haem to bilirubin, and the inducible form of haemoxygenase, haemoxygenase-1, is highly induced in response to oxidative stress in vitro. The effect of haemoxygenase-1 in oxidant stress in vivo is not known. We determined the effect of exposure to ozone on haemoxygenase-1 expression, and the modulation of haemoxygenase-1 expression on ozone-induced lung neutrophilia and bronchial hyperresponsiveness in rats. Ozone caused a significant induction of lung haemoxygenase-1. Pretreatment of rats with haemoglobin, a potent inducer of haemoxygenase-1, resulted in a large induction of haemoxygenase-1 expression, and inhibited ozone-induced neutrophilia and bronchial hyperresponsiveness. Tin protoporphyrin, a competitive inhibitor of haemoxygenase, reduced the expression of haemoxygenase-1 induced by haemoglobin. It enhanced ozone-induced neutrophilia, but not the bronchial hyperresponsiveness, and reduced the protective effect of haemoglobin. Overall, there was an association between bronchial hyperresponsiveness and the neutrophilic response. These data indicate that haemoxygenase-1 plays an important role in modulating the effects of an oxidant, such as ozone in the lungs.

Introduction

Haemoxygenase is an enzyme that catalyses the degradation of haem to form bilirubin and carbon monoxide. Two distinct iso-forms of haemoxygenase called haemoxygenase-1 and haemoxygenase-2 have been described and are the products of two distinct genes Choi and Alam, 1996, Maines, 1988. Whereas, haemoxygenase-2 is constitutively expressed and is found abundantly in brain, testis and liver, haemoxygenase-1 is induced by haem, as well as non-haem agents, such as heavy metals and cytokines. Haemoxygenase-1 is induced by a variety of agents that lead to oxidant stress, including ultraviolet irradiation and hydrogen peroxide Keyse et al., 1990, Lautier et al., 1992, Choi et al., 1996. Induction of haemoxygenase-1 is considered to be part of a generalised response to oxidative stress, manifested by glutathione depletion and oxidative degradation of protein Ewing and Maines, 1993, Applegate et al., 1991. A protective role for haemoxygenase-1 has been postulated since the reactions of haemoxygenase lead to the degradation of a pro-oxidant (haem) and, ultimately, the formation of an antioxidant (bilirubin). This is supported by studies, in which transfection of the haemoxygenase gene led to protection against oxidative stress Dennery et al., 1997, Lee et al., 1996.

One environmental source of oxidative stress is ozone, which is an important component of the photochemical oxidation product of air pollution, involving substrates emitted from automobile engines. Attention has been drawn to its potential adverse effects on respiratory health, because of its toxic effects related to its oxidative properties. Exposure of animals to ozone induces airway and alveolar epithelial necrosis, together with an inflammatory response, characterised by an influx of neutrophils, together with the increased expression of several pro-inflammatory cytokines and enzymes, such as interleukin-1β, tumour necrosis factor-α (TNF-α), cytokine-induced neutrophil chemoattractant (CINC), macrophage inflammatory protein (MIP-2) and inducible nitric oxide synthase Pendino et al., 1994, Punjabi et al., 1994, Haddad et al., 1995a, Haddad et al., 1995b, Haddad et al., 1996. In addition, exposure to ozone leads to an increase in bronchial responsiveness to bronchoconstrictor agents (Tsukagoshi et al., 1995), although the relationship of the inflammatory process to this phenomenon is unclear.

We have determined whether the oxidant stress of breathing ozone can induce the expression of haemoxygenase-1 in the lungs and whether this expression can modulate the various pro-inflammatory effects of ozone and bronchial hyperresponsiveness. We therefore examined the effect of the haemoglobin, an inducer of haemoxygenase (Pimstone et al., 1971), and of the Sn protoporphyrin, an inhibitor of haemoxygenase Anderson et al., 1984, Kappas and Drummond, 1986, in rats exposed to ozone.