br Funding This work was supported by grants to

Funding This work was supported by grants to A.S. from the Swedish Cancer Foundation, Sweden (Grant number: CAN, 2015/637), the Swedish Medical Council, Sweden (Grant number: 2017- 01274), the Foundations at Skåne University Hospital, Sweden and to KB, SRS, NC, BCS, and SS from the Royal Physiographic Society in Lund, Sweden.
Author contributions
Conflicts of interest
Acknowledgments
Introduction Cysteinyl leukotrienes (CysLTs, such as LTC4, LTD4, and LTE4) are inflammatory lipid mediators involved, via activation of CysLT1 receptors, in various pathophysiological conditions, including airway smooth muscle constriction, vascular hyperpermeability, mucus secretion, and inflammatory cell migration [1], [2], [3], [4], [5]. CysLT1 receptor antagonists, such as pranlukast, montelukast and zafirlukast are widely used as therapeutic agents for the treatment of bronchial 182 8 [6]. However, the efficacy of these agent is limited in almost half of asthma patients [7], [8]. Previous studies have shown that beside CysLT1 receptors, CysLT2 receptors are also expressed on the bronchial epithelium, smooth muscle and inflammatory leukocytes [9], [10], [11]. We have also reported that not only CysLT1 receptors, but also CysLT2 receptors are expressed in airway tissue isolated from bronchial asthma subjects [12]. Although the exact role of CysLT2 receptors in asthma pathogenesis is still unclear, evidence has shown that CysLT2 receptors are involved in the induction of asthma [13], [14]. It is therefore expected that CysLT2 / CysLT1/2 receptor antagonists provide beneficial effects in the treatment of asthma. Guinea pigs are widely used as asthma experimental models, because their airway smooth muscle responds well to CysLTs stimulation. Guinea pig CysLT2 receptors are activated mainly by LTC4, but have only weak binding affinity for LTD4[15]. In contrast, human CysLT2 receptors are equally stimulated by both LTC4 and LTD4[16]. Because LTC4 is rapidly metabolized to LTD4 by γ-glutamyl transpeptidase (GTP) [17], [18] in any species, LTC4-induced bronchoconstriction in guinea pigs is preferentially mediated via LTD4, which activates CysLT1 receptors. Thus, in order to elicit asthmatic response via CysLT2 receptors both in vitro and in vivo, it is necessary to inhibit LTC4 metabolism to LTD4[19]. We have previously reported that guinea pigs pre-treated with S-hexyl GSH, a synthetic substrate of γ-GTP, exhibit LTC4- or OVA-induced bronchoconstriction, airway vascular hyperpermeability and/or lung air-trapping via not only CysLT1 but also CysLT2 receptors activation [20], [21], [22]. However, it is still unclear whether treatment with S-hexyl GSH in guinea pigs alters antigen-induced systemic anaphylaxis to CysLT2 receptor-mediated response. In addition, the decline in CysLT2 receptor-mediated asthmatic response has not been pharmacologically validated. In this study, we describe the development of a new animal model that can be used to pharmacologically evaluate the efficacy of CysLT1 and CysLT2 receptors antagonists on anaphylactic response in vivo. In our experiments, we examined whether antigen-induced anaphylaxis in sensitized guinea pigs is altered to a partially CysLT2 receptor-mediated response by treatment with S-hexyl GSH. We also examined whether S-hexyl GSH actually inhibits the conversion of LTC4 to LTD4 in guinea pig peripheral blood and lung tissue. Finally, we pharmacologically validated antigen-induced anaphylactic response using two anti-asthma drugs, i.e. dexamethasone, a corticosteroid, and salmeterol, an adrenergic β2 receptor agonist.
Materials and methods
Results
Discussion In this study, we developed a new CysLT1 and CysLT2 receptors-mediated anaphylaxis guinea pig model for screening both CysLT2 receptor and CysLT1/2 receptor antagonists. Unlike human CysLT2 receptors, which are equally stimulated by both LTC4 and LTD4[9], [24], guinea pig CysLT2 receptors are activated mainly by LTC4[15]. However, because LTC4 is rapidly metabolized to LTD4 by γ-GTP, we used S-hexyl GSH [19], a synthetic substrate of γ-GTP to mediate guinea pigs anaphylactic response via CysLT2 receptors.