The immune cell composition in Barrett's metaplastic tissue resembles that in normal duodenal tissue

Abstract

Background and objective: Barrett's esophagus (BE) is characterized by the transition of squamous epithelium into columnar epithelium with intestinal metaplasia. The increased number and types of immune cells in BE have been indicated to be due to a Th2-type inflammatory process. We tested the alternative hypothesis that the abundance of T-cells in BE is caused by a homing mechanism that is found in the duodenum.

Patients and methods: Biopsies from BE and duodenal tissue from 30 BE patients and duodenal tissue from 18 controls were characterized by immmunohistochemistry for the presence of T-cells and eosinophils(eos). Ex vivo expanded T-cells were further phenotyped by multicolor analysis using flowcytometry.

Results: The high percentage of CD4(+)-T cells (69±3% (mean±SEM/n = 17, by flowcytometry)), measured by flowcytometry and immunohistochemistry, and the presence of non-activated eosinophils found in BE by immunohistochemical staining, were not different from that found in duodenal tissue. Expanded lymphocytes from these tissues had a similar phenotype, characterized by a comparable but low percentage of αE(CD103) positive CD4(+)cells (44±5% in BE, 43±4% in duodenum of BE and 34±7% in duodenum of controls) and a similar percentage of granzyme-B(+)CD8(+) cells(44±5% in BE, 33±6% in duodenum of BE and 36±7% in duodenum of controls). In addition, a similar percentage of α4β7(+) T-lymphocytes (63±5% in BE, 58±5% in duodenum of BE and 62±8% in duodenum of controls) was found. Finally, mRNA expression of the ligand for α4β7, MAdCAM-1, was also similar in BE and duodenal tissue. No evidence for a Th2-response was found as almost no IL-4(+)-T-cells were seen.

Conclusion: The immune cell composition (lymphocytes and eosinophils) and expression of intestinal adhesion molecule MAdCAM-1 is similar in BE and duodenum. This supports the hypothesis that homing of lymphocytes to BE tissue is mainly caused by intestinal homing signals rather than to an active inflammatory response.

Figure 1. Similar morphology of eosinophils in Barrett's esophagus (BE) and duodenum.

Immunohistochemical staining with anti-MBP (eosinophil marker) of duodenum from 6 controls (black bars, panel A) and BE biopsies from 12 BE patients (light grey bars, panel B). Eosinophils were counted in lamina propria and intestinal epithelium separately. DUO: duodenum, LP: lamina propria. Each bar represents mean values±SEM and expressed as MBP-positive cells/mm² (** p<0.005, *** p<0.0005).

Figure 2. Immunohistochemical analysis of inflammatory cells in BE and duodenum.

Panels A–C Barrett's esophagus (BE). Immunohistochemical staining for CD3⁺(panel A), CD8⁺(panel B) and CD4⁺-cells (panel C) was performed in BE biopsies from 6 BE patients. Cells were counted in lamina propria (LP) and epithelium separately (light grey bars, panels A and B). Each bar represents mean value±SEM and the numbers of CD3⁺, CD4⁺- or CD8⁺ are expressed as cells/mm²(n = 6). Panels D–F Duodenum. Immunohistochemical staining for CD3⁺(panel D), CD8⁺ (panel E) and CD4⁺-cells (panel F) was performed in duodenal biopsies from 5 controls. Cells were counted in lamina propria (LP) and epithelium separately (black bars, panels A and B).Each bar represents mean value±SEM, and the numbers of CD3⁺, CD4⁺- or CD8⁺-are expressed as cells/mm² (*p<0.05, ** p<0.005, ***p<0.0005, ns not significant) (n = 5).

Figure 3. Similar percentage of CD3⁺CD4⁺-cells in BE and duodenal ex vivo cultures.

(A) Comparison of the percentage of CD3⁺CD4⁺-cells in collagenated BE biopsies and ex vivo expanded T-cells. The percentage of CD3⁺CD4⁺-cells was determined in paired biopsies from 5 BE patients treated by either collagenase or used for ex vivo expansion. Each bar represents the mean value±SEM of the percentage of CD3⁺CD4⁺-cells in the CD3⁺population (light grey bar: BE T-cells expanded for 14 days ex vivo, light grey with stripes: T-cells from collagenated BE biopsies). (B) Percentage of CD3⁺CD4⁺-cells in ex vivo cultures of BE and duodenum. The percentage of CD3⁺CD4⁺-cells from ex vivo cultures of 17 BE segments and 15 duodenal biopsies from 17 BE patients (DUO BE) and 11 duodenal biopsies from 11 controls (DUO control) was determined by flowcytometry. Each bar represents the mean value±SEM of the percentage of CD3⁺CD4⁺-cells in the CD3⁺ population (black bar: DUO control, dark grey bar: DUO BE, light grey bar: BE) (ns not significant).

Figure 4. Low percentage of CD4⁺CD103⁺-cells in ex-vivo cultures of BE and duodenal tissue.

The percentage of CD4⁺CD103⁺-cells in ex vivo cultures from 14 BE segments and 13 duodenal tissues from 14 BE patients (DUO BE) and 9 duodenal tissues from 9 controls (DUO control) was determined by flowcytometry (ns not significant). Each bar represents mean value±SEM, percentage of CD4⁺CD103⁺-cells in the CD3⁺CD4⁺-cells population, (black bar: DUO control, dark grey bar: DUO BE, light grey bar: BE).

Figure 5. High percentage of CD8⁺Granzyme B^high cells in ex vivo cultures of BE and duodenal tissue.

The percentage of Granzyme B^high -cells (from CD8⁺ cells) was measured by an intracellular FACS-staining in ex vivo cultures of 16 BE segments and 14 duodenal tissues from 16 BE patients (DUO BE) and duodenum from 11 controls (DUO controls). Each bar represents mean value±SEM of the percentage CD8⁺Granzyme B^high cells in the CD3⁺CD8⁺ population (black bar: DUO control, dark grey bar: DUO BE, light grey bar: BE) (ns not significant).

Figure 6. Absence of IL-4 positive lymphocytes in BE cultures determined by intracellular FACS-staining.

(1) Intracellular staining for IFN-γ and IL-4. Intracellular staining for IFN-γ and IL-4 in T-cells was determined after stimulation with PMA (20 ng/ml) and ionomycin (1 µM) for 6 hrs. Hereafter, CD4 ⁺ and CD8⁺ cells from ex-vivo cultures of BE and duodenum from BE (DUO BE) and duodenum from controls (DUO control) were analysed. Each bar represents mean value±SEM of the percentage of IFN-γ cells inside CD3 ⁺CD4⁺-population (panel A), percentage of IFN-γ cells inside CD3⁺CD8⁺-population (panel B), percentage of IL-4 cells inside CD3⁺CD4⁺-population (panel C) and percentage of IL-4 cells inside CD3⁺CD8⁺-population (panel D) (black bar: DUO control, dark grey bar: DUO BE, light grey bar: BE) (ns not significant) (BE, n = 6,DUO BE, n = 5, DUO control, n = 4). (2) Representative flowcytometry plots for IFN-γ and IL-4 Representative flowcytometry plots of a representative staining of lymphocytes from a duodenal culture ex vivo. Gates for CD4 and CD8 were set and positive cells for IL-4 and IFN-γ were determined.

Figure 7. Comparable integrin expression on T-cells from BE and duodenal tissue.

The expression was analysed of α4 (panel A) and β7 (panel B) (arbitrary units, mean fluorescence) by CD3⁺ cells in ex vivo cultures of BE and duodenum of BE patients (DUO BE) and controls (DUO control) (black bar: DUO control, dark grey bar: DUO BE, light grey bar: BE). Also, the percentages of CD3⁺α4⁺-cells (panel C) and CD3⁺α4β7⁺-T-cells (panel D) of the CD3⁺-population in ex-vivo cultures of BE and duodenum from both groups were determined (ns not significant). There was no difference in expression of α4 and β7 and percentage of α4⁺ and α4⁺β7⁺ cells between duodenum of BE patients (DUO BE, n = 7) and duodenum of controls (DUO control) (n = 5).

Figure 8. MAdCAM-1 mRNA expression is similar in BE and duodenal tissue from BE patients and controls.

Real-time quantitative polymerase chain reaction RNA assays were performed on total BE biopsies (BE, n = 12), duodenal biopsies from BE patients (DUO BE, n = 10) and duodenal biopsies from controls (DUO controls, n = 5) (black bar: DUO control, dark grey bar: DUO BE, light grey bar: BE). Expression of MAdCAM-1, corrected for GAPDH, 2^−ΔCT±SEM, is represented on the y-axis (ns: not significant).