Elsevier

Biochemical Pharmacology

Volume 84, Issue 9, 1 November 2012, Pages 1174-1185
Biochemical Pharmacology

Evidence for ligand-specific conformations of the histamine H2-receptor in human eosinophils and neutrophils

https://doi.org/10.1016/j.bcp.2012.08.014Get rights and content

Abstract

The histamine H2-receptor (H2R) couples to GS-proteins and induces adenylyl cyclase-mediated cAMP accumulation. In human neutrophils and eosinophils, the H2R reduces chemotactic peptide-stimulated superoxide anion (O2) formation. However, pharmacological characterization of the H2R in these cells is far from being complete. The aim of this study was to provide a comprehensive profiling of the H2R in neutrophils and eosinophils. Histamine inhibited O2 formation in human neutrophils more effectively than in eosinophils. H2R agonists mimicked the effects of histamine and H2R antagonists blocked the effects of histamine. We noticed multiple discrepancies in the potencies and efficacies of H2R agonists with respect to cAMP accumulation and inhibition of O2 formation in both cell types. There were also differences in the antagonist profiles between cAMP accumulation and inhibition of O2 formation in neutrophils. Moreover, the pharmacological profile of the recombinant H2R did not match the H2R profile in native cells. The H2R sequence identified in human neutrophils corresponds to the published H2R sequence, excluding the exclusive expression of a new H2R isoform as explanation for the differences. Very likely, the differences between ligands are explained by the existence of ligand-specific receptor conformations with unique affinities, potencies and efficacies. Thus, our data provide evidence for the notion that the concept of ligand-specific receptor conformations can be extended from recombinant systems to native cells.

Graphical abstract

In this paper, we show dissociations in the effects of H2R agonists and antagonists in native cells versus recombinant systems. These data support the concept of ligand-specific receptor conformations.

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Introduction

Neutrophil and eosinophil granulocytes are cells of the innate immune system and pivotal in host defence against microbes and viruses [1], [2]. Both cell types are specialized in repelling distinct invading organisms. Neutrophils are crucial for the protection against bacteria, viruses, fungi, and tumour cells [1], [3] and are able to phagocytose invading or altered cells. Eosinophils play an important role in the defence against parasitic infections [2]. However, both neutrophils and eosinophils are also involved in the pathogenesis of chronic inflammatory diseases [4], [5], [6], [7]. In the course of an infection, eosinophils and neutrophils, upon proper stimulation, can migrate in response to chemotactic factors [8], [9] to the focus of infection and release a variety of cytotoxic enzymes, cytokines and reactive oxygen species (ROS) [10], [11]. The NADPH oxidase is one of the most important prerequisites for the production of superoxide anions (O2) and is found in plasma and phagosome membranes [12], [13], [14]. This enzyme catalyses the reduction of oxygen (O2) to O2 with NADPH serving as electron donor.

Activation of neutrophils and eosinophils occurs by exposure to several proinflammatory mediators such as cytokines, chemokines, immunoglobulins and bacterial formyl peptides [2], [15], [16]. Binding of the formyl peptide N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP) to the Gi-protein-coupled formyl peptide receptor (FPR) [17], [18] activates phospholipase C (PLC) resulting in the cleavage of phosphatidylinositol-4,5-bisphosphate into 1,2-diacylglycerol and inositol-1,4,5-trisphosphate. DAG activates protein kinase C (PKC) which leads to an activation of NADPH oxidase via phosphorylation and finally to an increase of O2 production in granulocytes [3], [19], [20].

fMLP-induced O2 production by granulocytes is inhibited by increases in intracellular cyclic adenosine 3′:5′ monophosphate (cAMP) concentration [12]. Various substances induce cAMP accumulation, such as prostaglandins, the unspecific inhibitor of phosphodiesterases 3-isobutyl-1-methylxanthine (IBMX), the cell-permeable cAMP analogue dibutyryl–cAMP, as well as agonists of the GS-protein-coupled β2-adrenergic receptor [16], [21], [22], [23], [24]. The mechanisms involved in cAMP-mediated inhibition of fMLP-induced O2 production are only incompletely understood, but the phosphorylation of FPRs, Gi-proteins, PLC, and NADPH oxidase by cAMP-dependent protein kinase [12], [25], [26] or the inhibition of the extracellular-signal regulated kinase phosphorylation [27] may play important roles.

Neutrophils and eosinophils both express the H2R, a seven-transmembrane receptor coupled to Gs-proteins [28]. Activation of H2R leads to adenylyl cyclase activation and elevation of intracellular cAMP concentration [29], [30], [31]. Furthermore, an additional Gq-protein-mediated pathway has been described [32], [33]. Numerous compounds which agonize or antagonize the H2R have been synthesized [34] and H2R antagonists are used for the treatment of acid-related gastrointestinal diseases [35]. A H2R-mediated increase of intracellular cAMP in neutrophils has been described [36]. However, since previously cAMP concentrations were mainly measured by a rather unselective and insensitive immunoassay, these data should be interpreted with caution. Information about H2R-mediated effects in human eosinophils is also sparse. Some studies indicate H2R-mediated increases in intracellular cAMP accumulation [37], [38] or the involvement of the H2R in the induction of O2 formation [11]. Finally, the use of H2R agonists in the treatment of acute myeloid leukaemia is a focus of research with promising results [39].

Activation of H2R results in inhibition of neutrophil effector functions like O2 release [10], [36], platelet-activating-factor-induced chemotaxis [40] and leukotriene biosynthesis [26]. Moreover, eosinophil functions are inhibited by H2R activation as follows: histamine (HA) via the H2R diminishes eosinophil peroxidase release [41] and, at high concentrations, inhibits eosinophil chemotaxis [29], [42]. Additionally, we have recently reported the H2R antagonist-induced enhancement of HA-stimulated eosinophil chemotaxis [28].

Early data dealing with the characterisation of H2R agonists in neutrophils indicated that fMLP-induced O2 formation is effectively inhibited by various guanidine-derived compounds [16]. Additionally, differences in H2-agonistic activity of H2R agonists monitored in neutrophils and guinea pig atrium were observed [16]. H2R ligands were also extensively characterized in Sf9 cell membranes [43], [44], [45], but a comprehensive comparison of the properties of the H2R in native cells versus recombinant systems has not yet been performed. To conclude, data about the pharmacological characterisation of the H2R in primary human cells such as neutrophils or eosinophils are scarce, but H2R agonists could be promising drug candidates for the treatment of inflammatory diseases.

We, therefore, used various selective H2R ligands for the pharmacological characterisation of the human H2R on neutrophils and eosinophils and compared the data with the results obtained with recombinant H2R expressed in Sf9 cells. Both neutrophils and eosinophils are granulocytes and related to each other with a high physiological relevance. O2 release and intracellular cAMP accumulation were chosen as readouts. While cAMP elevation is a rather upstream event in the signal transduction of the H2R, O2 release in response to FPR activation is more distal and could be affected by different signalling pathways. However, such modulations of ligand-induced distal cell responses are of great interest in clinical research as they may be responsible for unwanted drug effects.

Section snippets

Materials

Anti-CD16 micro beads and MACS running buffer were obtained from Miltenyi Biotech (Bergisch Gladbach, Germany), Percoll was from Biochrom (Berlin, Germany) and the phycoerythrin (PE)-labelled CD16b antibody was from Immunotools (Altenoythe, Germany). The H2R ligands (Fig. 1) HA, amthamine (AM), dimaprit (DI), 5-methylhistamine (5-MHA), zolantadine (ZOL) and tiotidine (TIO) were purchased from Tocris Bioscience (Bristol, UK). Impromidine (IM) was a gift from Dr. A. Buschauer (Dept. of

Isolation of neutrophils and eosinophils from peripheral blood

As discussed recently in detail [28] cell purity and viability is an underestimated problem when using eosinophils for functional assays. Therefore, we analysed each isolated granulocyte cell population carefully with respect to purity and viability as described in Sections 2.2 Isolation of human neutrophils, 2.3 Isolation of human eosinophils, and demonstrated in detail in Reher et al. [28]. Nearly all cells other than granulocytes were discarded after density gradient centrifugation and

Discussion

H2R antagonists such as famotidine and ranitidine have been used in the therapy of acid-related gastrointestinal disorders for many years [58]. Recently, HA-dihydrochloride (Ceplene®) has been approved for treating patients suffering from acute myeloid leukaemia [59] and H2R agonists are in focus of research for the treatment of acute myeloid leukaemia [39], [60]. Moreover, H2R agonists are promising drug candidates for the treatment of inflammatory diseases [10], [16], [61]. Neutrophils and

Acknowledgements

The authors thank the Deutsche Forschungsgemeinschaft (DFG) for financial support (Graduate Training Programs 761 and 1441), Dr. Heike Burhenne for performing the HPLC–MS analyses, and Mrs. Solveig Kälble for excellent technical assistance. Thanks are due to the reviewers for their helpful comments.

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