Marked deposition of eosinophil-derived neurotoxin in adult patients with eosinophilic esophagitis

Abstract

Objectives: Eosinophilic esophagitis (EoE) is characterized by infiltration of eosinophils into esophageal epithelium. Blood levels of an eosinophil granule protein, eosinophil-derived neurotoxin (EDN), have been proposed as a biomarker for EoE. However, information regarding localization of EDN in the diseased tissues has not been available. The goal of this study was to evaluate the magnitude and distribution of EDN deposition in tissue specimens from the esophagus of EoE patients.

Methods: We studied specimens from 10 adult EoE patients and eight histologically normal controls (three under age 17). Sections from mid-esophageal biopsy specimens were stained for EDN by immunofluorescence, using a polyclonal rabbit antibody to EDN. Cellular staining (i.e., infiltration of intact eosinophils) and extracellular staining (i.e., deposition of released EDN) were scored in a blinded manner on an established 7-point scale.

Results: Esophageal biopsy specimens from histologically normal controls showed no or few intact eosinophils and no or minimal extracellular EDN deposition. In contrast, EDN staining was clearly observed in specimens from all EoE patients. In some EoE patients, marked extracellular EDN deposition was observed despite relatively small numbers of intact eosinophils. Overall, there was no correlation between the eosinophil infiltration and the extracellular EDN staining scores.

Conclusions: Marked tissue deposition of extracellular EDN is present in the esophagus of EoE patients. Tissue eosinophil counts may underestimate how extensively eosinophils are involved, particularly in individuals with marked eosinophil degranulation. Evaluation of EDN staining in esophageal biopsy specimens may be useful to diagnose and manage patients with EoE.

Figure 1

Comparison of EDN and MBP staining in an EoE patient esophageal biopsy specimen. Serial sections of a specimen from Patient 6 (Table 1) were stained as follows: A, rabbit anti-human EDN; B, affinity-purified rabbit anti-human MBP; C, H&E; and D, NRS as a negative control. Original magnification, X160.

Figure 2

Localization of EDN in histologically-normal control esophageal biopsy specimens. The sections from 3 histologically-normal controls are stained with anti-EDN. A, C, and E, rabbit anti-human EDN; B, D, and F, H&E. The arrowheads in A identify intact eosinophils. Original magnification, X160.

Figure 3

Localization of EDN in EoE patient esophageal biopsy specimens. A, C, E, and G, sections stained with anti-EDN. B, D, F, and H, sections stained with H&E. Panel B is an H&E counterstain of the same section shown in Panel A.; therefore, the black arrows in B identify the same eosinophils shown by the white arrows in A. D, F, and H, serial sections to C, E, and G, respectively. G and H are a higher magnification of E and F, respectively; the arrowheads identify an area of intense extracellular EDN deposition. Original magnification, X160.

Figure 4

Heterogeneity of EDN staining in an EoE patient esophageal biopsy specimen. A and B, section from Patient 5 (Table 1) stained with anti-EDN. B is one X160 field to the right of A; the arrows in A and B point to the same cell. The arrowheads in B identify areas of EDN deposition. Original magnification, X160.

Figure 5

Scoring of eosinophil infiltration and extracellular EDN deposition in esophageal biopsy specimens. A–F, sections stained with anti-EDN. A, B, and C show representative examples of eosinophil infiltration scored as 1, 2, or 3, respectively. D, E, and F show representative examples of extracellular EDN deposition scored as 1, 2, or 3, respectively. Original magnification, X160.

Figure 6

Correlation between eosinophil infiltration scores and extracellular EDN deposition scores in EoE patients. Each dot represents a mean score from two independent observers for each biopsy.