H4R activation utilizes distinct signaling pathways for the production of RANTES and IL-13 in human mast cells.

CONTEXT: The histamine H4 receptor functionally expressed on human mast cells and their signaling pathways for the production of IL-13 and RANTES have never been analyzed side by side in a directly comparable manner. OBJECTIVE: Therefore, the aim of the study was to investigate signaling transduction pathways of H4R via ERK1/2, Akt and NFκB leading to the induction of inflammatory cytokine expression. MATERIALS AND METHODS: In the present study, HMC-1 cells and CBMCs were pretreated individually with H4R antagonist JNJ7777120, H1R antagonist mepyramine and signaling molecule inhibitors PD 98059, LY294002, Bay 117082 followed by stimulation was done with or without histamine or 4-MH. Furthermore, the siRNA mediated H4R gene silencing effects are studied at the H4R protein expression level and also signal transduction level. RESULTS: We found that the pretreatment with JNJ7777120 and H4R gene silencing decreased histamine, 4-MH induced phosphorylation of ERK1/2, Akt and NFκB-p65. Moreover, PD 98059, LY294002 and Bay 117082, which respectively inhibited the histamine and 4-methylhistamine induced phosphorylation of ERK1/2, Akt and NFκB-p65 respectively. We also found that the activation of H4R caused the release of IL-13 and RANTES on human mast cells. The MEK inhibitor PD98059 blocked H4R mediated RANTES/CCL5 production by 20.33 pg/ml and inhibited IL-13 generation by 95.71 pg/ml. In contrast, PI3 kinase inhibitor LY294002 had no effect on 4-MH induced RANTES/CCL5 production but blocked IL-13 generation by 117.58 pg/ml. DISCUSSION AND CONCLUSION: These data demonstrate that the H4R activates divergent signaling pathways to induce cytokine and chemokine production in human mast cells.

Stimulation of the histamine 4 receptor upregulates thymic stromal lymphopoietin (TSLP) in human and murine keratinocytes.

The cytokine thymic stromal lymphopoietin (TSLP) is involved in the development and the progression of allergic diseases. It is mainly released by epithelial cells at barriers such as skin and gut in response to danger signals. Overexpression of TSLP in keratinocytes (KC) can provoke the development of a type 2 inflammatory response. Additionally, TSLP directly acts on sensory neurons and thereby triggers itch. Since histamine is also increased in lesions of inflammatory skin diseases, the aim of this study was to investigate possible effects of histamine as well as different histamine receptor subtype agonists and antagonists on TSLP production in KC. We therefore stimulated human KC with histamine in the presence or absence of the known TSLP-inductor poly I:C and measured TSLP production at protein as well as mRNA level. Histamine alone did not induce TSLP production in human KC, but pre-incubation with histamine prior to challenge with poly I:C resulted in a significant increase of TSLP production compared to stimulation with poly I:C alone. Experiments with different histamine receptor agonists (H1R: 2-pyridylethylamine; H2R: amthamine; H2R/H4R: 4-methylhistamine (4MH)) revealed a dominant role for the H4R receptor, as 4-MH in combination with poly I:C displayed a significant increase of TSLP secretion, while the other agonists did not show any effect. The increase in TSLP production by 4MH was blocked with the H4R antagonist JNJ7777120. This effect was reproducible also in the murine KC cell line MSC. Taken together, our study indicates a new role for the H4 receptor in the regulation of TSLP in keratinocytes. Therefore, blocking of the H4R receptor in allergic diseases might be promising to alleviate inflammation and pruritus via TSLP.

Structure-based prediction of subtype selectivity of histamine H3 receptor selective antagonists in clinical trials.

Histamine receptors (HRs) are excellent drug targets for the treatment of diseases, such as schizophrenia, psychosis, depression, migraine, allergies, asthma, ulcers, and hypertension. Among them, the human H(3) histamine receptor (hH(3)HR) antagonists have been proposed for specific therapeutic applications, including treatment of Alzheimer's disease, attention deficit hyperactivity disorder (ADHD), epilepsy, and obesity. However, many of these drug candidates cause undesired side effects through the cross-reactivity with other histamine receptor subtypes. In order to develop improved selectivity and activity for such treatments, it would be useful to have the three-dimensional structures for all four HRs. We report here the predicted structures of four HR subtypes (H(1), H(2), H(3), and H(4)) using the GEnSeMBLE (GPCR ensemble of structures in membrane bilayer environment) Monte Carlo protocol, sampling ∼35 million combinations of helix packings to predict the 10 most stable packings for each of the four subtypes. Then we used these 10 best protein structures with the DarwinDock Monte Carlo protocol to sample ∼50 000 × 10(20) poses to predict the optimum ligand-protein structures for various agonists and antagonists. We find that E206(5.46) contributes most in binding H(3) selective agonists (5, 6, 7) in agreement with experimental mutation studies. We also find that conserved E5.46/S5.43 in both of hH(3)HR and hH(4)HR are involved in H(3)/ H(4) subtype selectivity. In addition, we find that M378(6.55) in hH(3)HR provides additional hydrophobic interactions different from hH(4)HR (the corresponding amino acid of T323(6.55) in hH(4)HR) to provide additional subtype bias. From these studies, we developed a pharmacophore model based on our predictions for known hH(3)HR selective antagonists in clinical study [ABT-239 1, GSK-189,254 2, PF-3654746 3, and BF2.649 (tiprolisant) 4] that suggests critical selectivity directing elements are: the basic proton interacting with D114(3.32), the spacer, the aromatic ring substituted with the hydrophilic or lipophilic groups interacting with lipophilic pockets in transmembranes (TMs) 3-5-6 and the aliphatic ring located in TMs 2-3-7. These 3D structures for all four HRs should help guide the rational design of novel drugs for the subtype selective antagonists and agonists with reduced side effects.

Identification and characterization of surface receptors for histamine in the human promyelocytic leukemia cell line HL-60. Comparison with human peripheral neutrophils.

The magnitude, the potency, the duration, and the specificity of histamine-induced cyclic AMP formation has been compared in human promyelocytic leukemic HL-60 cells and in human peripheral neutrophils. In HL-60 cells incubated at 37 degrees in the absence of phosphodiesterase inhibitor, histamine caused a 20-fold stimulation of basal cyclic AMP levels, with an EC50 of 5 X 10(-6) M. Typical H2 receptors were involved as shown by the relative potencies of the H1-selective agonists, 2-(2-pyridyl)ethylamine (PEA) and 2-(2-amino-ethyl)thiazole (AET), and the H2-selective agonists, impromidine and 4-methyl)histamine(4-MH): impromidine greater than histamine greater than 4-MH greater than AET greater than PEA. In this system, impromidine had mixed agonist-antagonist properties as shown by the rightward shift of the concentration-response curve of histamine (EC50 = 2 X 10(-3) M histamine in the presence of 10(-4) M impromidine). Histamine stimulation was competitively inhibited by the furane derivative ranitidine (Ki = 0.16 X 10(-6) M) as well as by the imidazole analogues oxmetidine (Ki = 0.48 X 10(-6) M) and cimetidine (Ki = 0.65 X 10(-6) M), whereas the H1 antagonist diphenhydramine inhibited histamine action at about 100-300 times higher concentrations (Ki = 51 X 10(-6) M). Prostaglandin E1 (PGE1) also stimulated cyclic AMP levels (50-fold increase) in HL-60 cells; half-maximal activation by PGE1 occurred at 3.2 X 10(-6) M. Our results indicate, first, that prostaglandin and histamine H2 receptors are present and functional at an early stage during myeloid differentiation; second, that there is no substantial difference between the pharmacological properties of the histamine H2 receptors in HL-60 cells and in mature human peripheral neutrophils; third, that the remarkable capacity for cyclic AMP formation noted in HL-60 leukemic cells after cell surface interaction by histamine or prostaglandin suggests that cyclic AMP and agents which increase its formation may have a role in the regulation of proliferation and/or differentiation of human myeloid progenitor cells.

Relationship between occupation of cerebral H1-receptors and sedative properties of antihistamines. Assessment in the case of terfenadine.

Alpha-[4(1,1-Dimethylethyl)phenyl]-4-(hydroxydiphenylmethyl)-1- piperidinebutanol (terfenadine, RMI 9918, Triludan, Teldane, resp.) inhibits the in vitro binding of [3H]mepyramine to H1-receptors in cerebral membranes with an apparent dissociation constant in good agreement with that found for antagonism of histamine-induced contractions of guinea pig ileum. In contrast, administration of terfenadine in therapeutic dosage does not result in the occupation of cerebral H1-receptors in the living mouse, as observed for most H1-antihistamines. A poor access of the drug to cerebral H1-receptors might account for the absence of sedative side effects.