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. 2015 Feb 27;10(2):e0114831.
doi: 10.1371/journal.pone.0114831. eCollection 2015.

Altered esophageal histamine receptor expression in Eosinophilic Esophagitis (EoE): implications on disease pathogenesis

Jamie Merves  1 , Prasanna Modayur Chandramouleeswaran  2 , Alain J Benitez  2 , Amanda B Muir  1 , Anna J Lee  2 , Diana M Lim  2 , Kara Dods  2 , Isha Mehta  2 , Eduardo D Ruchelli  3 , Hiroshi Nakagawa  4 , Jonathan M Spergel  5 , Mei-Lun Wang  1
Affiliations

Altered esophageal histamine receptor expression in Eosinophilic Esophagitis (EoE): implications on disease pathogenesis

Jamie Merves et al. PLoS One. .

Abstract

Eosinophilic Esophagitis (EoE) is a chronic allergic disorder, whose pathobiology is incompletely understood. Histamine-producing cells including mast cells and basophils have been implicated in EoE. However, very little is currently known about the role of histamine and histamine receptor (HR) expression and signaling in the esophageal epithelium. Herein, we characterized HR (H1R, H2R, H3R, and H4R) expression in human esophageal biopsies and investigate the role of histamine signaling in inducible cytokine expression in human esophageal epithelial cells in vitro. HR expression was quantified in esophageal biopsies from non-EoE control (N = 23), inactive EoE (<15 eos/hpf, N = 26) and active EoE (>15 eos/hpf, N = 22) subjects using qRT-PCR and immunofluorescent localization. HR expression and histamine-mediated cytokine secretion were evaluated in human primary and telomerase-immortalized esophageal epithelial cells. H1R, H2R, and H4R expression were increased in active EoE biopsies compared to inactive EoE and controls. H2R was the most abundantly expressed receptor, and H3R expression was negligible in all 3 cohorts. Infiltrating eosinophils expressed H1R, H2R, and H4R, which contributed to the observed increase in HR in active subjects. H1R and H2R, but not H3R or H4R, were constitutively expressed by primary and immortalized cells, and epithelial histamine stimulation induced GM-CSF, TNFα, and IL-8, but not TSLP or eotaxin-3 secretion. Epithelial priming with the TLR3 ligand poly (I:C) induced H1R and H2R expression, and enhanced histamine-induced GM-CSF, TNFα, and IL-8 secretion. These effects were primarily suppressed by H1R antagonists, but unaffected by H2R antagonism. Histamine directly activates esophageal epithelial cytokine secretion in vitro in an H1R dependent fashion. However, H1R, H2R and H4R are induced in active inflammation in EoE in vivo. While systemic antihistamine (anti-H1R) therapy may not induce clinical remission in EoE, our study suggests that further study of histamine receptor signaling in EoE is warranted and that targeting of additional histamine receptors may lead to novel treatment strategies for this important disease.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. H1R and H2R are expressed in human esophageal mucosal biopsies.
Quantification of HR expression in esophageal biopsies from nonEoE control biopsies (A). Kruskal wallis test and Dunn’s multiple test was used to determine significance between groups. Horizontal lines represent medians of each data set (***p<0.001). Immunofluorescence for H1R (B), H2R (C), and H4R (D) in esophagus of non-EoE control, subjects (original magnification 200x).
Fig 2
Fig 2. Expression of H1R, H2R, and H4R 4 are increased in esophageal biopsies in actively inflamed EoE.
Comparison of H1,H2, H3, and H4 receptor expression in non-EoE control, inactive EoE and active EoE biopsies, using Mann-Whitney test (A-D). Horizontal bars represent median of each data set. Horizontal lines represent median of the data set.
Fig 3
Fig 3. Infiltrating eosinophils express HR and correlate with mucosal HR expression in active EoE.
Immunofluorescent localization of MBP and H1R (A-C), H2R (D-F) and H4R (G-I) in esophageal biopsies from active EoE subject (original magnification 200x). Arrows indicate MBP-HR colocalization. Representative cells with MBP-HR colocalization are magnified in inserts. Correlation of H1R (J), H2R (K), and H4R (L) mRNA in human esophageal biopsies to peak tissue eosinophil count per hpf, using Spearman correlation and linear regression analyses.
Fig 4
Fig 4. Primary and immortalized esophageal epithelial cells express H1R and H2R.
HR mRNA expression in human esophageal epithelial cell line, EPC2-hTERT (A) and primary esophageal epithelial cell lines derived from non-EoE control (B,D,E) and EoE (C,D,E) subjects. A: Data represent mean +/- SEM for n = 3. B-E: horizontal bars represent median with IQR. Mann-Whitney test was employed to quantify significance. *p<0.05, ns = not significant.
Fig 5
Fig 5. Esophageal epithelial cells produce cytokines in response to histamine stimulation in vitro.
Quantification of GM-CSF, TNFα, and IL-8 (pg/mL) secretion by histamine stimulated EPC2-hTERT cells (100uM histamine for 24 hrs). Two-tailed student t-test was used for statistical analysis. **p<0.005,***<0.0005. Effect of pretreatment with H1R antagonist pyrilamine or H2R antagonists (cimetidine and ranitidine) prior to histamine stimulation upon cytokine secretion in EPC2-hTERT cells (B-D). One way ANOVA with Bonferroni post-test was used for statistical analysis. *p<0.05, **p<.01,****p<0.0001. ns = not significant. Data are represented as the mean +/- SEM of n = 6.
Fig 6
Fig 6. Toll-like receptor agonist poly (I:C) induces expression of H1R and H2R and enhances epithelial histamine responsiveness in EPC2-hTERT cells.
(A) Expression of H1R, H2R, H3R, and H4R following stimulation with poly (I:C) (10ug/ml) for 24 hrs. A Student t-test was used to calculate significance **p<0.005,***p<0.0005, ND = not detected. (B) Effect of poly(I:C) priming upon histamine-induced cytokine expression in EPC2-hTERT cells. One way ANOVA with Bonferroni post test was used for statistical analysis. ***p<0.001, ****p<0.0001. ns = not significant. Data represented as mean +/- SEM of n = 3. (C-E) Poly (I:C)—histamine induced cytokine secretion in EPC2-hTERT cells with/without H1R, H2R antagonism. One way ANOVA with Bonferroni post test was used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001****p<0.0001. ns = not significant. Data represented as mean +/- SEM of n = 6.
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