Diversity in the oesophageal phenotypic response to gastro-oesophageal reflux: immunological determinants

Abstract

Background and aims: Approximately 10% of adults experience gastro-oesophageal reflux symptoms with a variable oesophageal response. A total of 60% have no endoscopic abnormality, 30% have oesophagitis, and 10% have Barrett's oesophagus. We investigated whether the inflammatory cell infiltrate and cytokine profiles of these clinical phenotypes merely vary in severity or are fundamentally different.

Methods: Patients with reflux symptoms and a normal oesophagus (n=18), oesophagitis (n=26), and Barrett's oesophagus (n=22 newly diagnosed, n=28 surveillance) were recruited. Endoscopic and histopathological degrees of inflammation were scored. Cytokine expression was determined by competitive reverse transcriptase-polymerase chain reaction and immunohistochemistry.

Results: In oesophagitis, endoscopic and histopathological grades of inflammation correlated highly. mRNA expression of proinflammatory interleukin (IL)-1beta, IL-8, and interferon gamma (IFN-gamma) were increased 3-10-fold compared with non-inflamed squamous or Barrett's oesophageal samples. There was a modest increase in anti-inflammatory IL-10 but no increase in IL-4. In Barrett's oesophagus, 29/50 had no endoscopic evidence of inflammation and histopathological inflammation was mild in 17/50 and moderate in 24/50, independent of acid suppressants. Expression of IL-1beta, IL-8, and IFN-gamma was similar to non-inflamed squamous mucosa. IL-10 was increased 1.6-fold similar to oesophagitis. IL-4 was increased fourfold, with 100-fold increase in IL-4/T cell receptor expression, compared with squamous oesophagus or oesophagitis.

Conclusions: Barrett's oesophagus is characterised by a distinct Th-2 predominant cytokine profile compared with the proinflammatory nature of oesophagitis. The specific oesophageal immune responses may influence disease development and progression.

Figures

Figure 1

In patients with Barrett's oesophagus, the relationships between histopathological grade (Gd) of inflammation and the degree of intraepithelial neutrophil (IEN) infiltration (A, n=50), intraepithelial eosinophil (IEE) infiltration (B, n=50), and the CD-45 count (C, n=20) are demonstrated. Diffuse IEN, diffuse IEE infiltration, and the CD-45 count exhibited a positive correlation with the degree of light microscopic inflammation (p

Figure 2

Number of molecules of mRNA per μg total RNA for T cell receptor (TCR) (A) and the three proinflammatory cytokines interleukin (IL)-1β (B), IL-8 (C), and interferon γ (IFN-γ) (D). The patient groups examined were: non-inflamed squamous oesophagus (NO), oesophagitis (O), and Barrett's oesophagus (BO). Samples from non-inflamed gastric antrum (GA) and duodenum (D) from each of the three groups were taken as within patient controls. Note that the scale is different for IFN-γ due to the lower levels of cytokine expression. Sample number was 10–20 for all tissue groups studied. *p

Figure 3

Representative gels for T cell receptor (TCR) (A) and the three proinflammatory cytokines interleukin (IL)-1β (B), IL-8 (C), and interferon γ (IFN-γ) (D). In each case one band represents the synthetic/standard RNA in reducing quantities and the other band represents the target cytokine specific RNA. The point of approximate equal competition is indicated by an arrow. The more abundant the cytokine being analysed the greater the amount of synthetic RNA being competed for and hence the point of equal competition is further to the left hand side of the gel. NO, non-inflamed squamous oesophagus; O, oesophagitis; BO, Barrett's oesophagus.

Figure 4

Levels of proinflammatory cytokine mRNA expression (T cell receptor (TCR), interleukin (IL)-1β, and IL-8) as a function of Barrett's oesophagus patient medication (PPI, proton pump inhibitor). The corresponding cytokine levels in oesophagitis patients are shown for comparison. Sample number was at least eight for all tissue groups studied. *p

Figure 5

Number of molecules of mRNA per μg total RNA for interleukin (IL)-10 (A) and IL-4 (B) for three patient groups: non-inflamed squamous oesophagus (NO), oesophagitis (O), and Barrett's oesophagus (BO). Gastric antrum (GA) and the duodenum (D) were taken as within patient controls. Sample number was at least 10 for all tissue groups studied. *p

Figure 6

Sample gels for interleukin (IL)-10 (A) and IL-4 (B). The amounts of synthetic RNA added to each reaction are shown at the top of the gel and the arrow represents the approximate point of equal competition. NO, non-inflamed squamous oesophagus; O, oesophagitis; BO, Barrett's oesophagus.

Figure 7

Number of molecules of interferon γ (IFN-γ) and interleukin (IL)-4 as a ratio of T cell receptor (TCR) expression for each of the following tissue samples: non-inflamed squamous oesophagus (NO), oesophagitis (O), Barrett's oesophagus (BO), gastric antrum (GA), and duodenum (D). **p

Figure 8

Representative examples of the cellular source of cytokine protein expression is shown by immunohistochemistry. Positive cells stain brown. The counterstain is haematoxylin and alcian blue staining has been used to highlight goblet cells in Barrett's oesophagus (C panels). (A) Expression of the proinflammatory cytokines (interleukin (IL)-1β, IL-8, and interferon γ (IFN-γ)) in oesophagitis (original magnification ×10). (B) High power views (×100) of these same cytokines in oesophagitis. (C) Expression of these cytokines in Barrett's oesophagus (×100). IL-8 was expressed by epithelial cells as well as by inflammatory cells in the lamina propria.

Figure 9

Representative immunohistochemical staining for interleukin (IL)-4 and IL-10. (A) Negative control (original magnification ×40). (B) Positive staining for IL-10 by epithelial and inflammatory cells in oesophagitis (×100). (C) Epithelial cytoplasmic cell expression of IL-10 in Barrett's oesophagus (×40). (D) Inflammatory cell staining for IL-10 in the lamina propria of Barrett's oesophagus. (E, F) Paucity of IL-4 expression in oesophagitis (E ×10 and F ×100 magnification of the same field). (G, H) Inflammatory cell expression of IL-4 in Barrett's oesophagus within the intraepithelial layer (G) and the lamina propria (H) (both ×100).