T-helper 2 cytokines, transforming growth factor β1, and eosinophil products induce fibrogenesis and alter muscle motility in patients with eosinophilic esophagitis

Abstract

Background & aims: Patients with eosinophilic esophagitis (EoE) often become dysphagic from the combination of organ fibrosis and motor abnormalities. We investigated mechanisms of dysphagia, assessing the response of human esophageal fibroblasts (HEFs), human esophageal muscle cells (HEMCs), and esophageal muscle strips to eosinophil-derived products.

Methods: Biopsy specimens were collected via endoscopy from the upper, middle, and lower thirds of the esophagus of 18 patients with EoE and 21 individuals undergoing endoscopy for other reasons (controls). Primary cultures of esophageal fibroblasts and muscle cells were derived from 12 freshly resected human esophagectomy specimens. Eosinophil distribution was investigated by histologic analyses of full-thickness esophageal tissue. Active secretion of EoE-related mediators was assessed from medium underlying mucosal biopsy cultures. We quantified production of fibronectin and collagen I by HEF and HEMC in response to eosinophil products. We also measured the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by, and adhesion of human eosinophils to, HEFs and HEMCs. Eosinophil products were tested in an esophageal muscle contraction assay.

Results: Activated eosinophils were present in all esophageal layers. Significantly higher concentrations of eosinophil-related mediators were secreted spontaneously in mucosal biopsy specimens from patients with EoE than controls. Exposure of HEFs and HEMCs to increasing concentrations of eosinophil products or co-culture with eosinophils caused HEFs and HEMCs to increase secretion of fibronectin and collagen I; this was inhibited by blocking transforming growth factor β1 and p38 mitogen-activated protein kinase signaling. Eosinophil binding to HEFs and HEMCs increased after incubation of mesenchymal cells with eosinophil-derived products, and decreased after blockade of transforming growth factor β1 and p38 mitogen-activated protein kinase blockade. Eosinophil products reduced electrical field-induced contraction of esophageal muscle strips, but not acetylcholine-induced contraction.

Conclusions: In an analysis of tissues samples from patients with EoE, we linked the presence and activation state of eosinophils in EoE with altered fibrogenesis and motility of esophageal fibroblasts and muscle cells. This process might contribute to the development of dysphagia.

Keywords: Immune Regulation; Primary Human Cells; Swallowing; Transmural Disease.

Figure 1. Eosinophil infiltration and activation state of in all layers of a surgically resected full thickness human EoE specimen compared to those in the normal esophagus

Histologic evaluation of hematoxylin & eosin (H&E; left side of each panel) and eosinophil peroxidase (EPX; right side of each panel) stained esophageal tissue. In the EoE esophagus (Panel A and C) activated eosinophils were found scattered throughout the esophageal wall with the highest density in the epithelium. The normal control esophagus (Panel B and D) was essentially devoid of eosinophils, even in the deeper tissue layers. Panel A and B: 50X magnification; Panel C and D: 160X magnification.

Figure 2. Secretion of ECM by HEF and HEMC in response to cytokines and co-culture with human eosinophils

2A and B: HEF and HEMC were cultured for 72 h with or without the presence of cytokines known to be involved in the pathogenesis of EoE. FN secretion (2A) and PINP (2B) were measured using ELISA and RIA, respectively. TGF-β1 enhanced both FN and PINP secretion in HEF and HEMC, while IL-4 and IL-5 increased only PINP in HEF. N=5–8 for FN and PINP. *p<0.05, **p<0.01. 2C and D: HEF and HEMC were co-cultured with the human eosinophil cell line AML14.3D10 for 72h with and without eosinophil activation by IL-3, IL-5 and GM-CSF. With increasing eosinophil numbers HEF and HEMC increased secretion of FN (2C) and PINP (2D). Eosinophil activation enhanced matrix secretion by HEF and HEMC, and inhibition of direct contact in a transwell system reduced the matrix secretion. N=4–6 for FN and PINP. *p<0.05, **p<0.01 compared to no eosinophils; #p<0.05 for activated versus non-activated eosinophils; §p<0.05 for transwell versus no transwell.

Figure 3. Secretion of ECM by HEF and HEMC in response to eosinophil sonicates

HEF and HEMC were co-cultured for 72 h with sonicates of the human cell line AML14.3D10. With increasing concentrations of the eosinophil sonicates HEF and HEMC increased secretion of FN (3A) and PINP (3B). N=4–8 for FN and PINP. *p<0.05 compared to untreated. 3C: immunoblot showing that incubation with eosinophil sonicates enhances the intracellular amounts of FN in HEF in a dose-dependent manner. Figure representative of 3 experiments. 3D compares the effect of AML14.3D10 cells on HEF and HEMC with sonicates of human peripheral blood T-cells (PBT), the T-cell line MOLT4, human cord blood derived mast cells (HCMC) and peripheral blood monocytes (PBM). N=4–5 for HEF and HEMC. *p<0.05, **p<0.01 compared to untreated HEF and HEMC.

Figure 4. Activation and blockade of signaling pathways in HEF and HEMC in response to TGF-β1 and eosinophil sonicates

4A: HEF and HEMC were incubated with TGF-β1 or eosinophil sonicates for 0 to 60 minutes. Both TGF-β1 and eosinophil sonicates activate SMAD2, SMAD3 and p38MAPK signaling. Figure representative of 8 experiments for both HEF and HEMC. 4B: Blockade of p38MAPK and ALK5 signaling reduces the FN secretion by HEF and HEMC in response to TGF-β1 and eosinophil sonicates. HEF and HEMC were stimulated for 72 h with TGF-β1 or eosinophil sonicates; p38MAPK was inhibited by SB203580 and ALK5 by SB431542. Inhibition of both pathways, alone or combined, reduced FN secretion by HEF and HEMC in response to TGF-β1 or eosinophil sonicates. N=7–8 for HEF and HEMC. *p<0.05, **p<0.01, *p<0.001 compared to no inhibitors. Comparable results found by adding TGF-β1 neutralizing antibodies to the eosinophil sonicates (4C). N=3 for HEF. *p<0.05.

Figure 5. Adhesion of eosinophils to HEF and HEMC in response to cytokines

5A: HEF and HEMC were cultured for 72 h with or without cytokines involved in the pathogenesis of EoE, and adherence of AML14.3D10 cells to HEF or HEMC was measured using fluorescent labeling with calcein. IL-4, IL-6, IL-13 and TGF-β1 markedly enhanced the adherence of eosinophils to HEF and HEMC. N=8–9 for HEF and HEMC. *p<0.05, **p<0.01, ***p<0.001 compared to untreated HEF or HEMC. 5B and C: Eosinophil sonicates and human MBP increased adherence of eosinophils to HEF and HEMC. N=8–9 for HEF and HEMC for the eosinophil sonicates; N=2 combined HEF and HEMC for the MBP. *p<0.05, ***p<0.001 compared to untreated HEF or HEMC. 5D depicts representative brightfield images of human monocytes (arrows) adherent to untreated and treated HEF monolayers. Magnification 100x.

Figure 6. Expression of VCAM-1 by HEF and HEMC in response to cytokines and eosinophil sonicates and after blockade of signaling pathways

6A: HEF and HEMC were cultured for 72 h with or without cytokines involved in the pathogenesis of EoE or eosinophil sonicates, and expression of VCAM-1 was measured by flow cytometry. IL-4 and IL-13 enhanced the expression of VCAM-1 in HEF and HEMC, while TGF-β1 decreased the amount of VCAM-1 positive HEF. N=5 for HEF and HEMC. *p<0.05, **p<0.01 compared to untreated HEF or HEMC. 6B: HEF and HEMC were plated on glass slides and incubated as for 6A. VCAM-1 expression was assessed via fluorescent microscopy after staining with a specific anti-VCAM-1 antibody. IL-4 and IL-13 increased the expression of VCAM-1 in HEF and HEMC. Figure representative of 6 experiments. Magnification 100x. 6C: Blockade of p38MAPK and ALK5 signaling reduces eosinophil adhesiveness of HEF and HEMC in response to TGF-β1 and eosinophil sonicates. HEF and HEMC were stimulated as for 6A, and p38MAPK inhibited by SB203580 and ALK5 by SB431542. Inhibition of both pathways, alone or combined, reduced the adhesion of AML14.3D10 cells to HEF and HEMC in response to TGF-β1 or eosinophil sonicates. N=5–7 for HEF and HEMC. *p<0.05 compared to no signaling inhibitor.

Figure 7. Effect of cytokines, growth factors and primary esophageal cell culture supernatants on esophageal smooth muscle contraction

Cat esophageal circular muscle strips were incubated with recombinant cytokines (7A), eosinophil sonicates or human MBP (7B) as well as supernatants from co-cultures of HEF and HEMC with human eosinophils (7C). In response to electrical stimulation IL-6, IL-13, eotaxin-3 and TGF-β1 reduced the amplitude of contraction field, while IL-5 and eotaxin-1 had no effect. Eosinophil sonicates and MBP induced a decrease in muscle cell contraction in response to electrical stimulation. Incubation with supernatants of eosinophils or HEMC alone, but not HEF alone, caused a mild to moderate reduction in the contractile response. In contrast, supernatants of the eosinophil and HEF/HEMC co-cultures almost entirely abolished muscle contraction. Contraction of all muscle strips returned to normal after wash out of the supernatants. N=6–9 for cytokines and sonicates, N=2 for MBP and N=3 for co-culture supernatants. *p<0.05, **p<0.01, ***p<0.001 compared to untreated.