Migration chemotaxis and trafficking of leukocytes including

Migration, chemotaxis and trafficking of leukocytes, including eosinophils from the peripheral blood into the site of inflammation requires their adhesion to endothelial (+)-Usniacin sale (Ley et al., 2007). Despite ongoing research, the histamine effect on eosinophils adhesion to endothelium cells has not been investigated yet. This may be important as eosinophils are considered pleiotropic, multifunctional, end-stage inflammatory granulocytes involved in allergy and asthma. During this inflammatory conditions the increased number of blood and tissue eosinophils are observed, contributing to the disease pathogenesis. Therefore several new strategies in treatment of eosinophilic disorders are developed that are aimed at blocking specific steps involved in eosinophil migration and adhesion (Rosenberg et al., 2013, Rothenberg and Hogan, 2006). Here for the first time the role of histamine receptors in histamine-induced eosinophils interaction with endothelial cells was investigated. We provided detailed information on human eosinophils isolation from peripheral blood, using modified immunomagnetic separation technique, which represents combination of known cellular separation methods. In order to assure the reliable investigation, the purity, viability and functionality of human eosinophils was validated in detail. The involvement of histamine receptors in adhesion was studied using selective compounds (Fig. 1): H1 antagonist/inverse agonist-diphenhydramine, mepyramine (Hill et al., 1997); H2 antagonists-famotidine, ranitidine (Mahdy and Webster, 2014, Takagi et al., 1982); H3 antagonist-pitolisant (Dauvilliers et al., 2013); H4 antagonists-JNJ7777120 and thioperamide (Jablonowski et al., 2003, Liu et al., 2001). Additionally selective H4 agonist 4-methylhistamine was also studied (Lim et al., 2005).
Materials and methods
Results
Discussion Recruitment of leukocytes into the site of inflammation is a critical event in the inflammatory response. In presented study role of histamine in human eosinophils adhesion to endothelial cells was investigated. Up to date several research groups have demonstrated that histamine via activation of histamine H4 receptor induces cytoskeleton and eosinophils shape change, adhesion protein upregulation, and eosinophils chemotaxis (Buckland et al., 2003, Ling et al., 2004, Reher et al., 2012). This implicates role of histamine in eosinophils trafficking into the site of inflammation, which requires highly dynamic multistep process of cellular interaction with endothelial cells (Ley et al., 2007). However, the role of histamine in the regulation of eosinophils adhesion to endothelium is not completely understood. Furthermore to the best of our knowledge, this is the first study to comprehensively demonstrate concentration-dependent activation of human eosinophils adhesion to endothelium by histamine. In order to determine the functional role of histamine receptor subtypes in the adhesion process, the panel of selective histamine receptor antagonists/inverse agonists was used. Only JNJ7777120 compound (the selective histamine H4 antagonist) and thioperamide (dual H3/H4 antagonist) had significant effect on eosinophils adhesion, suggesting histamine H4 receptor to be involved in the histamine-induced adhesion. Furthermore experiments with the use of selective histamine H4 receptor agonist 4-methylhistamine, which caused dose dependent increase in eosinophils adhesion, that was inhibited by JNJ7777120 support that hypothesis. Interestingly, JNJ7777120 has been reported to exhibit a paradoxical effects apart from full antagonistic properties that refers to ligand functional selectivity (Seifert, 2013). This include partial inverse agonistic properties and biased activity in β-arrestin pathway activation (Nijmeijer et al., 2013, Seifert et al., 2011). However, in our experiments JNJ7777120 as well as thioperamide decreased the number of eosinophils that adhered to endothelium only in the presence of histamine, exhibiting purely antagonistic effects. Similar no-paradoxical stimulatory effects of JNJ7777120 have been observed on several other eosinophils activation markers including chemotaxis or intracellular calcium increase assays (Reher et al., 2012). This however cannot exclude the existence of functional selectivity of the histamine H4 receptors, taking into the consideration the great differences in the potencies of JNJ777120, histamine and 4-methylhistamine for various parameters tested in native human eosinophils (Seifert, 2013). To prove that hypothesis further studies are required using different agonists and antagonists and multiple cell parameters assays.