Eotaxin and FGF enhance signaling through an extracellular signal-related kinase (ERK)-dependent pathway in the pathogenesis of Eosinophilic esophagitis

Abstract

Background: Eosinophilic esophagitis (EoE) is characterized by the inflammation of the esophagus and the infiltration of eosinophils into the esophagus, leading to symptoms such as dysphagia and stricture formation. Systemic immune indicators like eotaxin and fibroblast growth factor were evaluated for possible synergistic pathological effects. Moreover, blood cells, local tissue, and plasma from EoE and control subjects were studied to determine if the localized disease was associated with a systemic effect that correlated with presence of EoE disease.

Method: Real-time polymerase chain reaction from peripheral blood mononuclear cells (PBMC), immunohistochemistry from local esophageal biopsies, fluid assays on plasma, and fluorescence-activated cell sorting on peripheral blood cells from subjects were used to study the systemic immune indicators in newly diagnosed EoE (n = 35), treated EoE (n = 9), Gastroesophageal reflux disease (GERD) (n = 8), ulcerative colitis (n = 5), Crohn's disease (n = 5), and healthy controls (n = 8).

Result: Of the transcripts tested for possible immune indicators, we found extracellular signal-regulated kinase (ERK), Bcl-2, bFGF (basic fibroblast growth factor), and eotaxin levels were highly upregulated in PBMC and associated with disease presence of EoE. Increased FGF detected by immunohistochemistry in esophageal tissues and in PBMC was correlated with low levels of pro-apoptotic factors (Fas, Caspase 8) in PBMC from EoE subjects. Plasma-derived bFGF was shown to be the most elevated and most specific in EoE subjects in comparison to healthy controls and disease control subjects.

Conclusion: We describe for the first time a possible mechanism by which increased FGF is associated with inhibiting apoptosis in local esophageal tissues of EoE subjects as compared to controls. Eotaxin and FGF signaling pathways share activation through the ERK pathway; together, they could act to increase eosinophil activation and prolong the half-life of eosinophils in local tissues of the esophagus in EoE subjects.

Figure 1

a-f The fold expression of immune indicators bFGF, FGF-R, Eotaxin-3, CCR3, IL-5, IL-13, ERK, JNK, Bcl-2, Capase 8, and Fas. a) EoE subjects statistically significant increases in levels of bFG and FGFR2 (p < 0.001) There was an 8-fold increase in the bFGF levels in EoE subjects as compared to HC subjects and a 4-fold increase compared to GERD; b)There was a 6-fold increase in eotaxin-3 comparing EoE subjects to HC and a 2-fold increased when compared to GERD subjects. However, this was not statistically significant. CCR3 levels were consistent among all subject groups; c) There was a statistically significant increase in the amount of IL-5 in subjects with EoE as compared to GERD (p < 0.01). Increase in IL-13 in EoE was statistically significant compared to all other subject groups (p < 0.001); d) ERK was high increased compared to other subjects groups (p < 0.001) while JNK levels remained consistent; e) EoE subjects had a 3-fold increase of Bcl-2 and a 3-fold decrease in caspase 8 expression compared to treated EoE subjects (p < 0.05); f) Fas expression was down-regulated by 3-fold in EoE subjects as compared to treated EoE subjects (p < 0.05). EoE: Eosinophilic Esophagitis; GERD: gastroesophageal reflux disease; HC: healthy control; UC: ulcerative colitis; CD: Crohn's disease.

Figure 2

a There is a positive correlation between the fold expression of bFGF and ERK, suggesting that the upregulation of bFGF may also influence the upregulation of ERK. Figure 2b: There is a positive correlation between the fold expression of ERK and eotaxin-3.

Figure 3

a) EoE subjects had an elevated level of bFGF in comparison (p < 0.05) to those of HC, GERD, and treated EoE subjects (same subjects as EoE subject but now on therapy); b)IL-5 was also elevated in EoE subjects compared to treated EoE subjects (p < 0.05) and gastroesophageal reflux disease and healthy control subjects (p < 0.05). (*p < 0.05).

Figure 4

The number of cells that are FGF positive cells are significantly greater in EoE subjects than in HC (p < 0.05) or GERD (p < 0.05). (* p < 0.05).

Figure 5

IHC at 400× with FGF staining (representative EoE subject).

Figure 6

IHC at 400× with FGF staining (representative GERD subject).