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Bronchial hyper-responsiveness and inflammation are characteristic of acute asthma and serve to trigger events of adverse airway remodelling, which include loss of airway epithelium, goblet cell and smooth muscle cell hyperplasia, and fibrosis (Rennard, 1996). Within the complex framework of mechanisms involved in the pathogenesis of asthma, a key role for oxidative/nitrosative stress, epithelial cell apoptosis and airway inflammation has been well documented (Andreadis et al., 2003). Besides the classical allergen-dependent pathways, reactive oxygen and nitrogen species produced during airway inflammation activate neutrophils, eosinophils and mast cells to release histamine, prostanoids and cytokines, which exacerbate airway hyperresponsiveness and favour the transition to subacute/chronic disease (Comhair et al., 2005).
The inflammatory responses resulting from the release of histamine have, for many years, been thought to be mediated by histamine H1 receptors (receptor nomenclature follows Alexander et al., 2011). Consequently, H1 receptor antagonists have been used as major components of the treatment of allergies. Recent evidence suggests that histamine can play diverse roles in inflammation and immune responses through the activation of another histamine-activated GPCR, the H4 receptor, expressed on dendritic and inflammatory cells (Thurmond et al., 2008).
The discovery of the histamine H4 receptors has given investigators new tools to further address the function of histamine and its receptors in allergic and inflammatory processes. Thus, histamine, acting through H4 receptors, induces chemotaxis of murine mast cells in vitro (Hofstra et al., 2003), and leads to changes of their tissue localization in vivo (Thurmond et al., 2004). Both effects are consistent with the previously reported distribution of mast cells in the epithelial lining of nasal mucosa in patients with rhinitis induced by allergen (Slater et al., 1996). Activation of histamine H4 receptors also induces chemotaxis of human eosinophils, enhances the effect of chemotactic agents and stimulates up-regulation of adhesion molecules (Ling et al., 2004). Indeed, H4 receptor expression and function on mast cells, eosinophils, basophils, dendritic and T-cells suggests that this receptor may play a role in allergen-induced asthmatic responses.
In a murine model of ovalbumin (OA)-induced asthma-like reaction, H4 receptors were involved in the activation of CD4+ cells by dendritic cells (Dunford et al., 2006). The administration of JNJ7777120 (JNJ), a novel selective H4 receptor antagonist, showed significant anti-inflammatory effects during both the sensitization and effector phases. These findings indicate that H4 receptors are also involved in the initial priming of the immune system after allergen challenge (Thurmond et al., 2008).
Indeed, the poor clinical efficacy of H1 receptor blockers in asthma would suggest the possible involvement of other histamine receptors in this disease and H4 receptor-deficient mice show reduced lung inflammation and Th2-derived cytokine production upon allergen challenge (Dunford et al., 2006). Moreover, the recent finding that resolution of an inflammatory reaction is an active phenomenon brought out by endogenous anti-inflammatory mediators, such as lipocortin-1 (LC-1), also known as annexin-A1, has opened new pathways to understand the mechanism of action of anti-inflammatory drugs (D'Acquisto et al., 2008). LC-1, a glucocorticoid-modulated protein, was initially characterized by its ability to inhibit prostanoid release (Cirino et al., 1987). Further studies indicated that LC-1 also inhibited leukocyte migration, reduced fluid extravasation and exhibited anti-nociceptive activity (Cirino et al., 1989; Perretti and Flower, 1993; Ayoub et al., 2008). The allergic guinea pig model is an old model, still largely used to reproduce the different syndromes presented by human asthma. The animals are sensitized to OA and the bronchial responsiveness, as well as the cough reflex response, is measured after antigen inhalation and this model has been widely used to evaluate the anti-asthmatic effect of class of compounds (Masini et al., 2005; 2007; Giannini et al., 2008; Cinci et al., 2010; Evans et al., 2012).
In this study we investigate the effects of a selective histamine H4 receptor antagonist in reducing airway inflammation and hyper-responsiveness, oxidative stress and epithelial cell apoptosis and the possible interactions with LC-1.
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The results of this study show that JNJ, a selective antagonist of the histamine H4 receptor, blocked the functional, histopathological and biochemical changes induced by OA challenge in an in vivo model of allergic asthma in guinea pigs and suggest that these effects are mediated by down-regulation of key pro-inflammatory cytokines, such as TNF-α and IL-4 and, at least in part, up-regulation of LC-1. This last protein, also known as annexin A1, was originally identified as a corticosteroid-regulated protein and involved in the anti-inflammatory effects of the corticosteroids (D'Acquisto et al., 2008). However, more recent observations have shown that its anti-inflammatory actions are much broader. In fact, LC-1 inhibits the activity of cytoplasmic phospholipase A2, thus playing a key role in down-regulation of inflammatory lipid mediators such as PGs and LTs, and inhibits the extravasation of leukocytes and release of inflammatory mediators (D'Acquisto et al., 2008). Further studies are required to unveil the exact mechanisms linking blockade of H4 receptors by JNJ and LC-1 up-regulation, but it is possible that JNJ could reduce proteolytic cleavage of LC-1 induced by allergen challenge (Chung et al., 2004), thereby extending LC-1 bioactivity. Mast cells contains abundant LC-1 located in, or on, the α-granules and these cells are an active site of LC-1 synthesis (Oliani et al., 2000). Moreover, mast cells in the LC-1 null animal exhibit histological signs of constitutive activation being partially degranulated and there is evidence that LC-1 inhibits mast cell granule release in vitro or in models of allergic inflammation (Bandeira-Melo et al., 2005). Given that histamine H4 receptors are expressed by various immune and inflammatory cells, such as dendritic cells, CD4+ and CD8+ T-cells (Zampeli and Tiligada, 2009), it seemed logical to assume that JNJ could reduce the complex cytokine interplay involved in the pathogenesis of allergic asthma. Asthma is characterized by infiltration of leukocytes, especially eosinophils, in the lung tissue, which produce large amounts of superoxide anion and other harmful mediators, which are responsible for bronchoconstriction and airway remodelling (Jarjour and Calhoun, 1994). In fact, the forced expiratory volume is inversely correlated with the increased production of superoxide by leukocytes (Jarjour and Calhoun, 1994). In addition, superoxide and peroxynitrite can promote the expression of genes encoding pro-inflammatory cytokines, IL-1, TNF-α and IL-6, which can spark endothelial cell damage (Ndengele et al., 2005). We have previously shown that superoxide and peroxynitrite promote mast cell degranulation and histamine release (Masini et al., 2005), as well as activation of COX and PG production (Mollace et al., 2005).
The present results show that treatment with JNJ significantly reduced overall leukocyte infiltration in the lung tissue, measured as MPO activity and particularly eosinophils, measured as eMBP-positive cells. It also reduced the release of proinflammatory cytokines and prostanoids, for example TNF-α, IL-4, PGD2 and LTB4, in the BAL fluid. These results are in agreement with previous data showing marked anti-inflammatory properties of JNJ in vitro and in vivo models (Thurmond et al., 2004; 2008). As mentioned earlier, eosinophils also play a key role in the pathogenesis, symptoms and severity of allergic asthma (Fujimoto et al., 1997; Bousquet et al., 2000). Eosinophils are involved in adverse airway remodelling through the release of TGF-β, which shifts stromal cells towards the myofibroblastic pro-fibrotic phenotype (Kisseleva and Brenner, 2008). Eosinophils, together with mast cells, also play a key role in angiogenesis (Nissim Ben Efraim and Levi-Schaffer, 2008). In particular, stimulation of H4 receptors on eosinophils triggers cellular changes required for chemotaxis, actin polymerization, shape changes and expression of adhesion molecules involved in cell migration such as CD11b and ICAM-1 (Ling et al., 2004). Treatment with JNJ effectively reduced eosinophil chemotaxis, confirming the notion mentioned earlier that that this process involves histamine H4 receptors (Ling et al., 2004; Zampeli and Tiligada, 2009).
It is well known that mast cells play a crucial role in the pathogenesis of allergic asthma in humans. Mast cell-released mediators are implicated in the mechanisms of bronchoconstriction, smooth muscle cell proliferation and inflammatory cell recruitment, which in turn maintain and amplify the inflammatory signalling (Wardlaw et al., 1988). Of note, treatment with JNJ prevented activation of lung mast cells, as demonstrated by the significant reduction of anaphylactic granule discharge.
Our results show that JNJ was able to counteract the allergic inflammatory process through the up-regulation of LC-1. In turn, the reduction of the lung inflammatory response was associated with diminished oxidative damage, as assessed by measurements of the tissue levels and activity of MDA, 8-OHdG and Mn-SOD. Moreover, the decreased lung inflammatory response and oxidative injury induced by JNJ treatment resulted in a decreased activity of caspase-3, the key enzyme of cell apoptosis, thus decreasing the conditions for airway remodelling.
It is currently believed that allergic asthma is a multifactorial process in which multiple inflammatory and immune cells cooperate through reciprocal interactions. The expression of H4 receptors by many of these cells indicates its involvement in the modulation of allergic inflammatory lung response and suggests that targeting this receptor could be a novel causative therapeutic strategy for asthmatic and allergic diseases (Zhang et al., 2006).
In conclusion, this study provides further support to the concept that histamine H4 receptors can play a major role in the modulation of immune and inflammatory response in allergic asthma and suggests that H4 receptor antagonists can be viewed as novel drugs to down-regulate eosinophil and mast cell activation and the downstream events leading to airway dysfunction and remodelling.