Development of Histamine H4 Receptor Target Engagement Assays in Immune Cell Populations Involved in Atopic Dermatitis Pathophysiology
By Eugenia Sergeev et al. | Dec 18, 2019
The British Pharmacological Society
15-17 December 2019
Eugenia Sergeev, Research Scientist, Molecular Pharmacology, attended Pharmacology 2019 organised by the British Pharmacological Society held on 15-17 December in Edinburgh, UK to present on Development of Histamine H4 Receptor Target Engagement Assays in Immune Cell Populations Involved in Atopic Dermatitis Pathophysiology.
Atopic dermatitis (AD) is a chronic inflammatory skin condition with limited options for long-term disease management. Leukocytes in inflamed skin tissue are involved in the pathophysiology of AD and several populations express the histamine H4 receptor (H4R)1. H4R activation exacerbates pro-inflammatory processes, hence H4R antagonists are being considered as AD therapeutics1. With the aim to develop a clinically translatable target engagement assay in healthy human blood, we assessed the effect of H4R ligands on eosinophil shape change, a process underlying eosinophil migration2, and the secretion of IL-31, a cytokine linked to pruritus3, from T helper type 2 (Th2) cells.
The eosinophil shape change assay was performed as described4. Data were analysed using a 4-parameter sigmoidal concentration-response model to determine IC50, EC50 and maximum response (Emax). For the IL-31 assay, peripheral blood mononuclear cells (PBMCs) were isolated from human blood by density gradient centrifugation on LymphoprepTM and processed using the EasySepTM Naïve CD4+ T Cell Isolation Kit. Naïve CD4+ T cells were polarised using the ImmunoCultTM Th2 Differentiation Supplement Protocol and polarisation was confirmed by a CD4+CCR4+CD45RO+IL‑4+/CD45RA-IFN-γ- expression profile. Th2 cells were treated with 10nM-10µM H4R ligands for 16h, or positive control 25ng/mL PMA and 1µg/mL ionomycin for 2h, with 5µg/mL Brefeldin A added during the last 2h. IL-31 expression was evaluated by flow cytometry.
H4R agonist histamine induced eosinophil shape change, albeit with a lower potency and efficacy than the positive control, CCR3 agonist eotaxin. Increasing concentrations of selective H4R antagonists resulted in inhibition of eosinophil shape change following histamine challenge and pIC50 values obtained are consistent with literature data showing approximately 10-fold higher affinity of ZPL-3893787 for H4R compared to toreforant5.
Table 1. Eosinophil shape change in response to H4R ligand treatment
Emax (% Change)
Histamine (Mean ± SD; n=3)
7.5 ± 0.3
22 ± 6
8.4 ± 0.4
Eotaxinb (Mean ± SD; n=3)
9.7 ± 0.5
32 ± 5
7.6 ± 0.4
aInhibition of histamine EC80 challenge concentration determined for each donor (68 – 198 nM)
bCCR3 agonist used as positive control
Naïve CD4+ T cells from healthy donors were successfully polarised into Th2 cells, however neither treatment with non-specific agonist histamine, H4R-specific agonist 4-methylhistamine nor positive control PMA/Ionomycin were able to induce a consistent increase in IL-31 expression across five donors.
The eosinophil shape change assay produced useful potency data and would be clinically translatable. Indeed, it has been applied to characterise a H4R antagonist in phase I trials6. The ability of H4R agonists to increase IL-31 expression in Th2 cells from healthy donors appears donor-to-donor dependent. However, H4R agonists have been shown to be more efficacious in inducing IL-31 expression in PBMCs from AD patients compared to healthy volunteers7, which could be an area to explore in the future.
1) Schaper-Gerhardt, K. (2018). The role of the histamine H4 receptor in atopic dermatitis and psoriasis. British Journal of Pharmacology, Epub ahead of print. DOI: 10.1111/bph.14550.
2) Willetts, L. (2014). Eosinophil shape change and secretion. Methods in Molecular Biology, 1178, 111-28.
3) Gibbs, B. F. (2019). Role of the Pruritic Cytokine IL-31 in Autoimmune Skin Diseases. Frontiers in Immunology, 10, 1383.
4) Ling, P. (2004). Histamine H4 receptor mediates eosinophil chemotaxis with cell shape change and adhesion molecule upregulation. British Journal of Pharmacology, 142(1), 161-71.
5) Paul Chazot, P. (2019). Histamine receptors: H4 receptor. IUPHAR/BPS Guide to PHARMACOLOGY. Retrieved from http://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=265#
6) Thurmond, R. L. (2014). Clinical and preclinical characterization of the histamine H(4) receptor antagonist JNJ-39758979. Journal of Pharmacology and Experimental Therapeutics, 349(2), 176-84.
7) Gutzmer, R. (2009). The histamine H4 receptor is functionally expressed on T(H)2 cells. Journal of Allergy and Clinical Immunology, 123(3), 619-25.