Role of Vasoactive Intestinal Peptide in Promoting the Pathogenesis of Eosinophilic Esophagitis (EoE).

Eosinophilic esophagitis (EoE) is associated with eosinophil and mast cell accumulation, which promotes dysphagia and esophageal dysmotility.1, 2 Cytokines and chemokines implicated in eosinophil-, mast cell–, and basophil-mediated EoE pathogenesis include interleukin (IL)-5, IL-13, thymic stromal lymphopoietin, and eotaxin-3.3, 4, 5, 6 A correlation between IL-5 and eotaxin-3 expression and eosinophil infiltration has been observed. Nevertheless, it is not clear if eotaxin-3 is the only chemokine, or one of several, that directs eosinophil accumulation in EoE. Herein, we present evidence that a neuroimmune pathway is involved in eosinophil and mast cell accumulation and degranulation in human EoE.

Morphologically, eosinophils accumulate near nerves within the muscular mucosa of the esophagus (Figure 1A). This spatial association suggests that nerve cells may release chemoattractants in EoE. We hypothesize that one such chemoattractant might be vasoactive intestinal peptide (VIP), which has been implicated in eosinophil recruitment during allergic disease (eg, asthma). VIP expression was low in control esophageal biopsy specimens (ie, without eosinophils) (Figure 1B–D). In contrast, eosinophils accumulated adjacent to VIP-expressing nerve cells in EoE biopsy specimens (Figure 1E–G).

Figure 1

Eosinophils accumulate adjacent to nerve cells in the muscular mucosa in patients with a gastrointestinal disorder. (A) Eosinophil accumulation in the esophageal muscularis propria of an undiagnosed human autopsy sample. (B–D) Anti-major basic protein–positive eosinophils (red) were not detected in control esophageal biopsy specimens, and VIP expression (green) in nerves was low. (E–G) Increased eosinophil numbers and VIP expression were observed in EoE. Arrows indicate anti-major basic protein– and anti-VIP– immunostained eosinophils and nerve cells, respectively. DAPI, 4′,6-diamidino-2-phenylindole. n = 1 for esophageal autopsy, n = 4 for control, and n = 6 for EoE patient biopsies.

VIP performs its immunologic functions via binding to 3 receptors: vasoactive intestinal peptide receptor 1 (VPAC-1), VPAC-2, and chemoattractant receptor homologous molecule expressed on Th2 (CRTH2) lymphocytes.8, 9 Our in vitro analyses indicated that eosinophils mainly express CRTH2 receptor, not VAPC1, VAPC2, and VIP (Supplementary Figure 1A–D). Furthermore, eosinophil motility in response to VIP is comparable with that induced by eotaxin (Supplementary Figure 1E), and that anti-CRTH2 pretreatment restricted human eosinophil motility (Supplementary Figure 1F). In EoE biopsy specimens, we show that CRTH2-expressing eosinophils accumulated in the epithelial mucosa (Supplementary Figure 2A–F). Furthermore, mast cell contribution to the pathogenesis of EoE also has been reported, but the mechanism of mast cell recruitment to the esophagus is undefined. Immunofluorescence analyses confirmed CRTH2 receptor on tryptase-positive mast cells in the esophageal mucosa of EoE patients (Supplementary Figure 2G–L). These findings suggest that, similar to eosinophils, mast cells accumulate via interaction of the CRTH2 receptor with neurally derived VIP. The details of control and EoE patient clinical characteristics are provided in Supplementary Table 1.

Supplementary Figure 1

Eosinophils highly express the VIP-receptor CRTH2 compared with VPAC-1 or VPAC-2. An earlier report indicated that intestinal eosinophils produce VIP; therefore, we examined whether blood eosinophils of EoE patients also produce VIP and express VIP-specific receptors. Accordingly, human eosinophils were examined for the expression of VIP and VIP-associated receptors using anti-VIP, anti-VAPC1, anti-VACP2, and anti-CRTH2 antibodies. (A–C) The flow cytometer analysis indicated none to very low expression of VIP and other VIP receptors VPAC-1 and VPAC-2 on eosinophils. (D) However, our analysis detected highly expressed VIP-receptor CRTH2 on eosinophils. Furthermore, we tested the hypothesis of whether the CRTH2 receptor has a critical role in VIP-induced eosinophil motility. Notably, eotaxins and the interaction of its receptor CCR3 are reported to be major motility factor for eosinophils.11, 12 Accordingly, we kinetically examined human eosinophil motility in response to different concentrations of VIP recombinant protein (0, 1, 10, 100, and 500 ng/mL) using Transwell chemotactic chambers for 4 hours. Established eosinophil chemokine eotaxin-2 (200 ng/mL) recombinant protein was used as a positive control. (E) The analysis indicated that VIP indeed has eosinophil chemoattraction activity similar to eotaxin-2. The critical role of CRTH2 in eosinophil motility was established by examining anti-CRTH2 pretreated eosinophil motility in response to VIP. The eosinophils were treated with anti-CRTH2 for 1 hour, and both treated and nontreated eosinophil motility was again examined using Transwell chambers in response to VIP protein (500 ng/mL) and eotaxin-2 protein (200 ng/mL). (F) Analysis indicated that anti-CRTH2 antibody blocks in vitro migration of eosinophils in response to VIP; whereas, anti-CRTH2 treatment did not restrict eosinophil motility in response to eotaxin-2. The analysis establishes the critical role of CRTH2 and VIP interaction in eosinophil motility. Data are expressed as means ± SD (n = 3 experiments).

Supplementary Figure 2

CRTH2 receptor is expressed on the eosinophils and tissue-accumulated mast cells in the esophageal biopsy specimen of human EoE. The in vitro analysis indicated that VIP-receptor CRTH2 is critical for eosinophil motility. Therefore, we examined whether tissue eosinophils in human EoE similarly express CRTH2 receptors. Accordingly, immunofluorescence analysis was performed using anti-CRTH2 receptor on anti-major basic protein–positive tissue eosinophils in human EoE biopsy specimens. (A and B) The anti-major basic protein– and (C and D) anti-CRTH2–expressed eosinophils are detected in the biopsy specimens of human EoE (A–D, original magnification, 400× and 1000×). The merged photomicrograph of anti-major basic protein– and anti-CRTH2–stained and 4′,6-diamidino-2-phenylindole (DAPI)-mounted tissue sections show that tissue eosinophils expressed VIP-receptor CRTH2 in the esophageal biopsy specimens of human EoE patients (E and F, original magnification, 400× and 1000×). Furthermore, induced mast cell numbers and their role in the pathogenesis of EoE is well established.4, 13 However, it is not clear which chemokine receptors are responsible for mast cell recruitment in human EoE. Therefore, we examined VIP-receptor CRTH2 expression on the tissue-accumulated mast cells in the esophageal biopsy specimens of human EoE by performing immunofluorescence analyses using antitryptase and anti-CRTH2 antibodies. We showed the presence of antitryptase-positive mast cells in the (G and H) esophageal biopsy specimens and (I and J) CRTH2-stained receptors. (K and L) A DAPI-mounted merged tissue section detected the co-localized tryptase-expressed mast cell expressing CRTH2 receptor in esophageal biopsy specimens of EoE patients. The arrows indicate tissue-accumulated mast cells and CRTH2 receptors on mast cells in respective photomicrographs. The photomicrographs presented are (A, C, and E) 400× and (B, D, and F) 1000× of the original magnification photomicrographs presented. Data are expressed means ± SEM (n = 6–7).

Supplementary Table 1

Patient Clinical and Pathologic Characteristics

Patients	Age, y	Sex	Esophageal disease	Allergic diseases	Other diseases	Eos/HPF	Current treatment	Steroids
1	9	M	NL	None	None	0		-
2	11	F	NL	None	None	0		-
3	12	F	NL	None	None	0		-
4	9	F	NL	None	None	0		-
5	4	M	NL	None	None	0		-
6	13	M	NL	None	None	0	Elimination diet	Nasocort, Flovent
7	11	M	NL	None	None	0	Food trial	-
8	2	M	EoE	None	None	35 eos/HPF	Elimination	-
9	9	M	EoE	None	None	40 eos/HPF	Elimination	-
10	8	M	EoE	None	None	31 eos/HPF	Ad libitum	-
11	10	M	EoE	None	None	41 eos/HPF	Ad libitum	Flovent (GlaxoSmithKline, Brentford, UK)
12	12	M	EoE	None	None	63 eos/HPF	Elimination	Nasocort (Chattem, Inc, Chattanooga, TN)
13			EoE	None	None	30 eos/HPF	-	Flovent
14	3	M	EoE	None	None	30 Eos/HPF	Ad libitum	Pulmicort (AstraZeneca, Cambridge, UK)
15	5	M	EoE with dysphagia	None	None	63 eos/HPF	-	Flovent
16	10	F	EoE with dysphagia	None	None	80 eos/HPF	Ad libitum	-
17	6	M	EoE with dysphagia	None	None	64 eos/HPF	-	Rhinocort (AstraZeneca), Flovent
18	7	M	EoE with dysphagia	None	Nonspecific colitis with focal cryptitis	73 eos/HPF	Elimination	Flonase (GlaxoSmithKline)
19	15	M	EoE with dysphagia	None	None	70 eos/HPF	Elimination	-
20	8	M	EoE with dysphagia	None	None	50 eos/HPF	Ad libitum	-

eos, eosinophils; F, female; M, male; NL, normal; HPF, high-power field.

Previous clinical trials have shown a reduction of peak eosinophil levels in adult EoE patients treated with a CRTH2 antagonist. However, the effect of the CRTH2 antagonist on mast cells that may be critical to the esophageal functional abnormalities observed in EoE was not examined. Therefore, to better assess the potential therapeutic utility of in vivo CRTH2 blockade, we initially assessed eosinophil and mast cell distributions in mice after induction of experimental EoE (Supplementary Figure 3). As in the human study, eosinophil infiltration in each segment of the esophagus was reduced in CRTH2 antagonist-treated mice (Figure 2A–D). This was paralleled by reduced mast cell numbers (Figure 2E–H). Morphometric quantification analysis indicated that CRTH2 antagonist treatment significantly reduced the number of both eosinophils and mast cells (Supplementary Figure 4B and C).

Supplementary Figure 3

Accumulation of eosinophils and mast cells in muscular mucosa after the induction of experimental EoE. Induced eosinophils and mast cell accumulation is implicated in the induction of esophageal functional abnormalities, including stricture and motility dysfunction in human and experimental EOE. However, the accumulation and mechanism of eosinophils and mast cells beyond the epithelial mucosa has not been examined. Because it is difficult to obtain deep mucosal biopsy specimens in human EoE, we examined the accumulation of eosinophils and mast cells in each segment of the mouse esophagus in experimental EoE. The mouse esophageal tissue sections were examined for eosinophils and mast cells after anti-MBP and chloroacetate esterase staining, respectively. Both eosinophils and mast cells were detected in each segment of mouse esophagus after the induction of experimental EoE. (A) A number of eosinophil accumulations in low and high magnification are shown in the epithelial mucosa, lamina propria, and muscular mucosa. Yellow arrows indicates accumulation of eosinophils. (B) Similarly, mast cell accumulation in low and high magnification is shown mostly in the lamina propria and muscular mucosa. Black arrows indicates accumulation of mast cells. Photomicrographs presented are 100× and 400× original magnification, respectively.

Figure 2

CRTH2 antagonist pretreatment reduces the accumulation of both eosinophils and mast cells in . CRTH2 antagonist pretreatment significantly reduced the number of (D) eosinophils and (H) mast cells relative to (C and H) Aspergillus-challenged mice that were not pretreated. (A and B) No eosinophils and few (E and F) baseline mast cells were detected in sections from mice treated with saline or saline + CRTH2 antagonist. Data are expressed as means ± SD (n = 8–10 mice/group). EP, epithelial mucosa; LP, lamina propria; LU, lumen; MP, muscularis propria.

Supplementary Figure 4

(. Morphometric quantification indicated that CRTH2-antagonist treatment reduced the (B) number of eosinophils and (C) mast cells that accumulate in the esophagus of Aspergillus-challenged mice relative to the untreated Aspergillus-challenged control mice. The levels of eosinophils and mast cells in the esophageal sections are expressed as eosinophils/mm2 and mast cells/mm2, respectively. Data are expressed as means ± SD (n = 8–10 mice/group).

Taken together, the current studies identify a novel and important chemoattractant role for VIP in the accumulation of eosinophils and mast cells in the pathogenesis of EoE. Moreover, we suggest that inhibiting the VIP–CRTH2 axis may ameliorate the dysphagia, stricture, and motility dysfunction of chronic EoE.