Upregulated eotaxin expression and T cell infiltration in the basal and papillary epithelium in cows' milk associated reflux oesophagitis

Abstract

Background: Cows' milk sensitive reflux oesophagitis is an emerging clinical entity in children, normally indistinguishable from primary gastro-oesophageal reflux (GOR) apart from the response to dietary antigen exclusion. It is conjectural whether a tendency towards mucosal eosinophilia distinguishes this group from primary GOR.

Aims: To determine whether there may be differences in the mucosal lesion within the oesophagus in those children with reflux in association with cows' milk induced small bowel pathology, particularly in relation to the eosinophil chemokine eotaxin.

Methods: A total of 29 children underwent endoscopic assessment, including nine with cows' milk sensitive enteropathy (CMSE) and associated GOR, seven histologically normal controls, six with primary GOR, and seven disease controls. Oesophageal biopsy specimens were examined immunohistochemically for the chemokines eotaxin and MCP-2, and T cell lineage and activation markers.

Results: Strong upregulation of eotaxin expression, limited to basal and papillary epithelium, occurred in all CMSE patients. By contrast, weak expression was seen in a minority of controls and in 50% of primary GOR patients. Infiltration of CD3, CD4, and CD8 lymphocytes occurred in similar distribution in CMSE patients, significantly increased above controls. Significant upregulation of activation markers (CD25, HLA-DR) was also seen in the CMSE group within basal and papillary epithelium compared to controls and primary GOR.

Conclusion: Basal and papillary epithelial eotaxin expression, with focal lymphocyte activation, was seen in infants with CMSE associated GOR. This preliminary study provides early evidence to suggest a pathogenesis distinct from primary GOR, in which specific recruitment of T cells and eosinophils may contribute to oesophageal dysmotility.

Figure 1

Eotaxin expression within the basal oesophageal epithelium in CMSE, histologically normal controls, primary reflux oesophagitis (PRO), and disease control (DC) groups. Epithelial staining, graded 0–4, as determined by intensity of staining (see Methods).

Figure 5

Histological and immunohistological findings in CMSE associated oesophageal reflux. (A) Dark staining lymphocytes within core of papilla, which is in continuity with the lamina propria (H&E). (B) Normal control showing few intraepithelial or subepithelial CD3⁺ T cells (red staining). (C) Contrasting findings in milk allergic reflux, of dense subepithelial and basal epithelial CD3⁺ infiltration. (D) CD4⁺ cells localised to basal epithelium and elongated papillae in another case of milk allergic reflux. The specimen has curled during freezing, leaving surface epithelium on top and at bottom right. (E) Serial section showing CD8⁺ cells with similar distribution. Smaller numbers of CD25⁺ cells were seen in identical positions on further serial section. (F) Strong HLA-DR immunoreactivity in basal epithelium and papillae, as well as on submucosal vascular endothelium, in milk allergic reflux. (G,H) Dense eotaxin immunoreactivity within basal (G) and papillary (H) epithelium in two cases of milk allergic reflux (substantially increased compared to controls and primary mechanical reflux).

Figure 2

Pattern of eotaxin expression; proportion of patients with "positive staining" (staining score >1) of basal (closed bars) and papillary (open bars) oesophageal epithelium. Patient groups as in fig 1.

Figure 3

Density of (A) CD4 and (B) CD25 cell infiltrates with (C) intensity of HLA-DR expression within basal oesophageal epithelium in the comparative groups. Symbols as in fig 1.

Figure 4

Proportion of patients with cell density score >1 for (A) CD3 and (B) CD4 cells within the basal (closed bars) and papillary (open bars) oesophageal epithelium in the comparative groups.