Markers of Tyrosine Kinase Activity for Diagnosis and Treatment Response in Eosinophilic Esophagitis: A Pilot Study of pERK 1/2 and pSTAT5

Abstract

Introduction: In eosinophilic esophagitis (EoE), food and other allergens appear to activate Th2 cells to release IL-13 and IL-4, cytokines that can induce esophageal squamous epithelial cells to produce the potent eosinophil chemoattractant eotaxin-3. GERD also can cause an eosinophilic infiltration of the esophagus, usually milder than that seen in EoE, through mechanisms that are not clear. In earlier studies, we showed that IL-13 and IL-4 caused a marked increase in eotaxin-3 protein secretion in an esophageal squamous cell line derived from a patient with EoE, but only a minimal increase in eotaxin-3 protein secretion in esophageal squamous cell lines derived from GERD patients. To explore this mechanism further, we studied the effects of IL-13 and IL-4 on transcriptional activation of eotaxin-3 in esophageal squamous cell lines from patients with EoE and GERD. Methods: Two telomerase-immortalized, non-neoplastic esophageal squamous cell lines derived from patients with GERD (NES-B10T & NES-G4T), and one derived from a patient with EoE (EoE1-T) were stimulated with IL-13 (100 ng/ml) or IL-4 (1 ng/ml) for times ranging from 24-48 hours. Eotaxin-3 mRNA expression levels were determined by RT-PCR, and an eotaxin-3 promoter (-800bp) construct attached to a luciferase reporter was used to measure transcriptional activity. Results: In the GERD cells, IL-13 and IL-4 induced only slight increases in eotaxin-3 mRNA expression levels at 24 and 48 hours. In contrast, both cytokines caused a marked increase in eotaxin-3 mRNA expression levels at 24 and 48 hours in the EoE cells. Both IL-13 and IL-4 significantly increased eotaxin-3 promoter activity in all 3 cell lines. There was no significant difference in the degree of eotaxin-3 promoter induction by IL-13 and IL-4 between the two GERD cell lines (NES-G4T cells IL-13: 431 ± 42.5 SEM RLUs % control, IL-4: 279± 38; NES-B10T cells IL-13: 554 ± 109, IL-4: 463 ± 54). In contrast, the cytokine-stimulated increase in eotaxin-3 promoter activity in the EoE1 cells (IL-13: 1522 ± 225, p<0.01; IL-4: 1714 ± 385, p<0.01) was significantly greater than that in the two GERD cell lines. Conclusions: In an EoE esophageal squamous cell line, stimulation with IL-13 and IL-4 caused a far greater increase in eotaxin-3 mRNA expression and promoter transcriptional activity than in esophageal squamous cell lines from GERD patients. These observations suggests that the esophagus of patients with EoE produces more eotaxin3 in response to Th2 cytokine stimulation than the esophagus of patients with GERD, and that differences among patients in the degree of cytokine-stimulated production of eotaxin3 by esophageal squamous cells might underlie the degree of esophageal infiltration by activated eosinophils.