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The bradykinin 1 receptor and bradykinin 2 receptor are G-protein-coupled seven-transmembrane domain receptors. The B2 receptor is constitutively expressed in different organs and mediates most of the known physiological actions of kinins, while the B1 receptor is generally expressed at low levels under normal conditions, but may be rapidly up-regulated during trauma and following various pathological states (Regoli and Barabe, 1980; McEachern et al., 1991; Menke et al., 1994; Marceau et al., 1998; Calixto et al., 2004). The B1 receptor exhibits a higher affinity for BK metabolites, such as des-Arg9-bradykinin and des-Arg10-kallidin, than the B2 receptor, which, in turn, shows higher affinity for BK and Lys-BK (Regoli et al., 1977; Hess et al., 1992; Calixto et al., 2004; Marceau and Regoli, 2004). The first B2 receptor knockout mice (B2−/−) were generated in 1995 (Borkowski et al., 1995), whereas knockout mice (B1−/−) were only generated in 2000 (Pesquero et al., 2000). Since then, both knockout mice have greatly contributed to our understanding of the role of kinin receptors in most physiological and pathological conditions.
B2 receptor activation by BK results in the formation of PGE2 and PGI2 and NO (Marceau and Bachvarov, 1998), and also stimulates intracellular signalling mechanisms, mainly involving MAPK and PI3K, culminating in the activation of the transcriptional NF-κB (Ritchie et al., 1999; Medeiros et al., 2001). Interestingly, in an experimental model of hypertension and in Akita diabetic mice it has been found that in B2 receptor null animals, the B1 receptor is strongly induced and assumes some of the properties of the B2 receptor (Duka et al., 2001; Kakoki et al., 2004), suggesting a compensatory mechanism. The expression of B1 receptors is also induced by a range of inflammatory mediators, namely IL-1β, TNF-α and INF-γ, both in vitro and in vivo (Calixto et al., 2004; Leeb-Lundberg et al., 2005). Moreover, the B1 receptor itself is able to induce its own expression through the NF-κB pathway, resulting in an elevation of IL-1β and TNF-α synthesis (Schanstra et al., 1998).
The two most prevalent inflammatory bowel diseases (IBDs) are Crohn's disease and ulcerative colitis, both of which are a problem worldwide (Baumgart and Carding, 2007). Many factors have been linked to the onset and maintenance of IBDs, such as genetic and environmental factors, as well as disturbances in the immune response, which may alter the integrity of the epithelial mucosa (Baumgart and Carding, 2007). The loss of epithelial mucosa integrity increases the permeability of the intestinal barrier and has been linked to the direct exposure of the immune system to luminal antigens (Schmitz et al., 1999; Edelblum and Turner, 2009). Interestingly, the peptide BK and its signalling cascade have been implicated in the regulation of proteins that are critical for cell–cell adhesion, such as tight junctions (TJs) (Dey et al., 2010). The TJ disruption may induce extensive and unbalanced activation of the mucosal immune system driven by the commensal flora (McGuckin et al., 2009).
Both B1 and B2 receptors are constitutively expressed in the gut and seem to play a fundamental role in water secretion, vasodilatation and capillary permeability (Cuthbert and Margolius, 1982; Bhoola et al., 1992). Additionally, recent studies have demonstrated the involvement of the kinins and their receptors in gut inflammation. Experiments carried out using a B1 or B2 receptor selective antagonist as well as B1−/− or B2−/− mice revealed that the inflammatory parameters of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice were significantly improved (Hara et al., 2007; 2008).
Against this background, in the present study, we sought to investigate, using selective B1 and B2 receptor antagonists and B1−/− mice plus molecular studies, the role and the underlying mechanisms through which the B1 receptor modulates intestinal function and the development of gut inflammation in mice treated with dextran sulfate sodium (DSS).
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Humans with IBD have been widely studied in recent years as they represent an important health problem worldwide and also because an effective and safe therapy is currently lacking (Baumgart and Carding, 2007). There are several murine model of IBD available, each one with different mechanisms of inflammatory induction. Nevertheless, the most widely used murine models of IBD are those induced by administering a toxic chemical, such as TNBS, oxazolone and DSS.
TNBS is believed to haptenize colonic autologous or microbiota proteins, rendering them immunogenic to the host immune system (Wirtz et al., 2007). A single enema of TNBS results in the formation of granulomas associated with the infiltration of inflammatory cells in all layers, strong thickening of the intestinal wall, hyperplasia of the epithelium and ulceration (Neurath et al., 1995). This inflammation is characterized by a dense, transmural infiltration of T cells, mainly of the CD4+ phenotype, which when stimulated in vitro produce high levels of IFN-γ and reduce the amounts of IL-4 compared with intestinal T cells from control mice (Neurath et al., 1995; Elson et al., 1996). For this reason, the TNBS model of inflammation is associated with a Th1-cell-mediated response and is immunologically similar to Crohn's disease, which is also characterized by the predominance of Th1 cells (Fuss et al., 1996; Parronchi et al., 1997).
Another model of mucosal inflammation is DSS-induced colitis, an acute and chronic colitis model that shows disruption of the epithelial cell barrier and, therefore, increased cellular exposure to normal mucosal microflora, resembling human ulcerative colitis (Cooper et al., 1993; Wirtz et al., 2007). One possible or even probable consequence of this change in barrier function is that mucosal phagocytes become subject to activation by substances produced in the mucosal microflora, which, in turn, leads to the non-specific release of pro-inflammatory cytokines, such as TNF-α and IL-1β, and, consequently, gut inflammation. In addition, the disruption of barrier function, as a mechanism in DSS-induced colitis, suggests its relative independence from lymphocyte-mediated responses, because RAG−/− mice, which are deficient in T and B cells, can still develop colitis in response to DSS (Dieleman et al., 1994).
Previous studies suggest that B1 receptors are constitutively expressed in the gut, indicating their possible role in the healthy intestinal physiology (Sawant et al., 2001; Stadnicki et al., 2005; Hara et al., 2008). Hara et al. (2008) reported that TNBS-induced colitis was associated with marked tissue damage, neutrophil infiltration and a time-dependent increase in colon B1 receptor-mediated contraction. The up-regulation of B1 receptors was also confirmed by means of binding studies, while B1 receptor mRNA levels were elevated as early as 6 h after induction of colitis and remained high for up to 48 h. Of note, the same authors also reported that TNBS-evoked tissue damage and neutrophil influx were significantly reduced by treating animals with the selective B1 receptor antagonist, SSR240612, and are also reduced in B1−/− mice. These results provide convincing evidence for the role of B1 receptors in the pathogenesis of colitis and suggest that its blockade represents a new therapeutic option for treating IBD.
In marked contrast with a previous study (Hara et al., 2008), our data show that the deletion of B1 receptors leads to a marked exacerbation of DSS-induced colitis. In line with our current results, Stadnicki et al. (2005) demonstrated that B2 receptors are normally present in the apical region of enterocytes and intracellularly in human IBD patients. In contrast, the B1 receptor was found in the basal area of enterocytes in normal intestine, but in the apical portion of enterocytes in inflamed tissue. Moreover, the B1 receptor was expressed mainly in macrophages at the centre of granulomas in tissue from Crohn's disease patients (Stadnicki et al., 2005). Such data led Stadnicki et al. (2005) to conclude that both B1 and B2 receptors are present in normal and IBD conditions, and to highlight the relevance of B1 and B2 receptors in intestinal physiology. Taken together with our data, these findings suggest that the lack of B1 receptors in the DSS-induced colitis model in mice seems to affect the pathogenesis of this condition in the intestine.
There is a body of evidence implying the involvement of pro-inflammatory cytokines in IBD as they are released in the gut and seem to have different functions in this disease, including cellular adhesion, differentiation and transmigration (Taylor et al., 1998; Marchiando et al., 2010). We thus investigated whether or not pro-inflammatory cytokines could be associated with the exacerbation of DSS-induced colitis in mice lacking B1 receptors. Our data clearly show that pro-inflammatory cytokines such as TNF-α, IL-1β, INF-γ, CXCL1/KC and MIP-2 are significantly higher in the gut of animals lacking B1 receptors and treated with DSS, when compared with WT littermates. Such data strongly suggest that exacerbation of the production and release of pro-inflammatory cytokines in the gut of B1−/− mice treated with DSS is likely to account for the observed exacerbation of colitis in these animals.
In fact, innate immune responses are activated during the progression of IBD and up-regulate the expression of most pro-inflammatory cytokines and chemokines, including IL-1β, IL-6, KC, and MIP-2 (Korzenik and Podolsky, 2006; Berndt et al., 2007). These soluble mediators can subsequently trigger nuclear transcription factors such as NF-κB, which, in turn, stimulate the expression of other mediators relevant to the pathogenesis of IBD, such as B1 receptors, B2 receptors, IL-1β, TNF-α and adhesion molecules, which together influence chemoattraction (Sartor, 2006; Shin and Ha, 2011). In agreement with our data, recent reports suggest that the B1 receptor modulates the production of pro-inflammatory cytokines/chemokines (Schulze-Topphoff et al., 2009; Su et al., 2009; Talbot et al., 2010; Gulliver et al., 2011). Collectively, these results suggest that B1 receptor deficiency contributes directly or indirectly to the up-regulation of chemotactic factors, which, in turn, help attract more phagocytes and, consequently, aggravate inflammation.
An increasing body of evidence has emerged indicating that intestinal epithelial cells are sensitive to BK, which may modulate the opening of TJs (Ma et al., 2011). TJs are essential for epithelial barrier integrity and seem to play a pivotal role in intestinal homeostasis (Turner, 2009). These proteins act as a semipermeable gate and can regulate the passage of molecules between epithelial cells (Schneeberger and Lynch, 2004). It was recently demonstrated that BK is able to mediate opening of the blood–tumour barrier and increase vascular permeability, which was associated with the down-regulation of TJ proteins zonula occludens-1 and occludin, as well as the re-arrangement of F-actin (Liu et al., 2008). Of note, our results showed that B1−/− mice per se exhibited reduced levels of occludin mRNA, which was exacerbated after inflammation, and blockade of B2 receptor partially prevents the occludin mRNA reduction in B1−/− mice after DSS-induced colitis. In sharp contrast, we observed a significant increase in claudin-4 mRNA expression in colonic tissue obtained only from B1−/− mice per se but not after DSS treatment.
Likewise, we also observed an increase in macromolecule permeability in the intestinal mucosa of B1−/− , when compared with WT mice. It is now well established that BK binding to the B2 receptor activates PKC (Tippmer et al., 1994), which can result in the redistribution and phosphorylation of claudins and occludins from TJs (Suzuki et al., 2009; Sjo et al., 2010; Willis et al., 2010), causing epithelial barrier opening. Herein, we demonstrated that the lack of B1 receptors leads to significant changes in TJs, associated with a marked increase in epithelial permeability and, consequently, disruption of the epithelial cell barrier, which seems be related to B2 receptor activation.
Although the B1 and B2 receptors exhibit low sequence homology (about 35%), there is experimental evidence, from both in vitro and in vivo studies, suggesting that these two kinin receptors conduct crosstalk, an effect that could contribute to the regulation of their expression (Campos and Calixto, 1995; Schanstra et al., 1998; Phagoo et al., 2000; Cayla et al., 2002; Barki-Harrington et al., 2003; Xu et al., 2005; Duka et al., 2006). Furthermore, previous findings showed that genetic deletion of a particular receptor may promote a compensatory mechanism in some GPCRs, including between the B1 and B2 receptors (Gan et al., 2004; Xu et al., 2005; Teng et al., 2008). To investigate this further, we examined whether the exacerbation of DSS-induced colitis in B1−/− mice could be a consequence of the compensatory up-regulation of B2 receptors in the gut. Our RT-PCR analysis revealed that both B1 and B2 mRNA are constitutively expressed in WT mice. Of note, B1 receptor mRNA was not detectable in B1 knockout mice, and expression of B2 receptor mRNA was significantly higher in WT and B1−/− mice 5 days after DSS administration, but not on the seventh day in DSS-treated WT mice. This observation could explain the partial protective effect of HOE-140 on DSS-induced colitis in mice (Arai et al., 1999). Such findings suggest that compensatory up-regulation of B2 receptors occurs in mice lacking B1 receptors when treated with DSS.
To explore further the role of B2 receptors in the exacerbation of DSS-induced colitis in B1−/− mice, we treated these animals with the B2 selective antagonist, HOE-140, before administering DSS. Our data clearly show that all the exacerbated parameters observed in B1−/− animals treated with DSS (DAI, body weight reduction, colon length, macroscopic score and MPO activity) were significantly improved or even completely normalized in animals treated with HOE-140. Therefore, it is tempting to speculate that DSS administration in B1−/− mice induces compensatory B2 receptor expression, which could promote the up-regulation of TJs and, thus, increase the cellular permeability, through the action of BK. Another piece of evidence supporting the above conclusion comes from data showing that the systemic treatment of animals with a selective peptide kinin B1 antagonist (DALBK) (Campos et al., 1999; McLean et al., 1999; Calixto et al., 2003; Brunius et al., 2005; Leeb-Lundberg et al., 2005; Hamza et al., 2010) or selective non-peptide kinin B1 antagonist (SSR240612) (Gougat et al., 2004; Campos et al., 2006; Hara et al., 2008) completely failed to protect or exacerbate DSS-induced colitis in WT mice. One could argue that the dose of DALBK or SSR240612 was so low that it was unable to reach the plasma concentration required to prevent colitis. We used a very similar dose and scheme of treatment for DALBK or SSR240612 and observed that this antagonist was highly effective at blocking experimental autoimmune encephalomyelitis in mice (Dutra et al., 2011) and TNBS-induced colitis (Hara et al., 2008).
In summary, our present results demonstrate that genetic deletion of B1 receptors greatly increases the severity of experimental colitis induced by DSS. The susceptibility of B1 receptor-deficient mice to colitis seems to be strongly associated with a compensatory mechanism, leading to the overexpression of B2 receptors in the gut, which modulates TJ expression, mainly occludin protein expression, which, in turn, may contribute to the disruption of the epithelial barrier. The reason for the contrasting effect of B1 receptors in the TNBS (Hara et al., 2008) and DSS model of colitis (present study) remains elusive. However, as discussed earlier, the two models of colitis involve quite different mechanisms, especially regarding activation of the immune system (TNBS), which could explain a large part of this discrepancy.