Histamine H and H receptors are considered as low affinity
Histamine H1 and H2 receptors are considered as low affinity receptors (pKi of 4.2 and 4.3 respectively), whereas histamine H3 and H4 receptors as a high affinity receptors (pKi of 8 and 8.2 respectively) (Schreeb et al., 2013). Therefore it might well be that other histamine receptors might be involved in eosinophils adhesion at higher histamine concentration. Nevertheless experiments on selected histamine receptors antagonists in the presence of 100µM histamine concentration, confirmed single histamine H4 receptor involvement in eosinophils adhesion. Histamine concentration in serum ranges from 1nM up to 10nM (Ling et al., 2004). Taking into the consideration that EC50 for histamine in given experimental setup was 1.36µM, it is unlikely that histamine itself is able to induce eosinophils migration from blood into tissue. However in physiological conditions, histamine may act synergistically with other eosinophilic activators like eotaxin, C5a and platelet-activating factor, all of them reported to increase eosinophils adhesive properties (Burke-Gaffney and Hellewell, 1998). The importance of histamine H4 receptor in eosinophils recruitment into the site of inflammation was also reported in vivo TLR (Toll-like Receptor) pathways dependent mouse Atovaquone models, in which addition of H4 antagonists in LPS induced inflammation reduced the observed number of eosinophils (Cowden et al., 2013). Based on that study it could be hypothesized that the decreased number of eosinophils could be due to the adhesion blockade. Currently the mechanism by which the H4 receptor modulates histamine-induced cellular adhesion is not completely understood. The adhesive interactions between eosinophils and endothelial cells is dependent on the upregulation of adhesion molecules expression on cell surface such as: CD11a, CD11b, CD11c, CD18, VLA-4, ICAM-1, VCAM-1 and others (Rosenberg et al., 2013). At present only one paper has reported that histamine induced upregulation of cell surface proteins CD11b/CD18 (Mac-1) and CD54 on human eosinophils. These effects were histamine H4 receptor-dependent (Ling et al., 2004). Histamine is known to affect not only eosinophils but also endothelial cells, through its vasoactive and microvascular permeability effect, involving H1 receptor (Zampeli and Tiligada, 2009). Moreover it was recently reported that functional histamine H3 and H4 receptors are expressed on rat brain-endothelial cells, which could point out to the major role of endothelium in histamine-induced adhesion cascade (Karlstedt et al., 2013). Accordingly, we cannot rule out that histamine effects on human eosinophils adhesion to endothelium is dependent on both cell types rather than on eosinophils alone. The studies on human eosinophils are hampered by limited cell availability (5–7% of all leukocytes), not-optimal eosinophils isolation protocols, low viability of isolated eosinophils and rapid deterioration of their functionality after isolation (Kita, 2011). Here we provide detailed information on human eosinophils isolation from the peripheral blood as well as validation of isolated cell viability and functionality. In the past isolation of human eosinophils was mainly achieved through discontinuous percoll gradient centrifugation (Raible et al., 1994). This method resulted in low eosinophils purity and cellular viability (Reher et al., 2012). Nowadays the immunomagnetic cell separations methods are widely used in immunological research therefore few eosinophils separation protocols have been introduced so far (Akuthota et al., 2014, Hansel et al., 1991). Still many of the eosinophil's research was conducted without reliable assessment of cellular purity and viability. In our experiments, only the method 3 yielded the eosinophils population with sufficient quantity and quality, for reliable studies in our adhesion assay. Rapid time dependent loss of eosinophils viability after isolation from blood is a known phenomenon (Percopo et al., 2010). In the present study eosinophilic viability lost was observed both with trypan blue staining and MTS assay, which measures cellular mitochondrial oxidative activity (Cory et al., 1991). The number of eosinophils in the blood and tissues is balanced between cell maturation, trafficking and removal during the apoptosis process (Kankaanranta et al., 2005). When isolated eosinophils were examined for apoptosis 4h after isolation, more than half of the cells were marked with apoptotic signals, including annexin V staining. Based on our experiments we suggest to use flow cytometry as a routine check-point for apoptosis of isolated eosinophils before conducting functional studies on human eosinophils. All experiments needs to be done immediately after cellular isolation as marked by other authors (Reher et al., 2012). In our experiments the functionality of eosinophils was confirmed based on eosinophilic degranulation assays in which isolated eosinophils were responsive to fMLP activation, as evidenced by eosinophilic peroxidase (EPO) assay. Taking into consideration the challenges to carry our functional studies on eosinophils that we did overcome using modified protocol of isolation of eosinophils and testing their post-isolation functional integrity we claim that H4 receptor dependent regulation of eosinophils adhesion to endothelium, demonstrated in the present work in vitro set-up, may bear pathophysiological relevance.