Squamous Tissue Lymphocytes in the Esophagus of Controls and Patients with Reflux Esophagitis and Barrett’s Esophagus Are Characterized by a Non-Inflammatory Phenotype

<div><p>Background and Objective</p><p>Reflux esophagitis (RE) is characterized by inflammation of the squamous epithelium (SQ) of the esophagus and may progress to Barrett’s esophagus (BE) characterized by intestinal metaplasia. The role of inflammation in this transition has been postulated but lacks experimental evidence. Here, the inflammatory responses in the esophagus of these patients were investigated.</p><p>Patients and Methods</p><p>Fifty-one esophageal biopsies from with patients BE (n = 19), RE (n = 8) and controls (n = 23) were analyzed. T-cells were analyzed before and after <i>ex vivo</i> expansion (14 days) by multicolor flow cytometric analysis. The following markers were studied: CD3, CD4, CD8 (T-cell markers), Granzyme B (marker of cytotoxicity), CD103 (αE/epithelial integrin) and NKg2a (inhibitory receptor on T-cells and NK-cells).</p><p>Results</p><p>Analysis of <i>ex vivo</i> cultures from normal looking SQ from controls, RE patients, and BE patients revealed no significant differences in the number and phenotypes of T-cells. In contrast, tissue from RE was different to normal SQ in four aspects: 1) higher percentages of CD3⁺CD4⁺-cells (72±7% vs 48±6%, p = 0.01) and 2) CD8⁺GranzymeB⁺ -cells (53±11% vs 26±4%, p<0.05), while 3) lower percentages of CD4⁺CD103⁺-cells (45±19% vs 80±3%, p = 0.02) and 4) CD8⁺NKg2a⁺- cells (31±12% vs 44±5%).</p><p>Conclusion</p><p>Despite the fact that both tissues are exposed to the same reflux associated inflammatory triggers, the immune response observed in RE is clearly distinct from that in SQ of BE. The differences in immune responses in BE tissue might contribute to its susceptibility for transformation into intestinal metaplasia.</p></div

Similar percentage of CD8⁺CD103⁺ and CD8⁺NKg2a⁺-cells in RE and other squamous esophageal biopsies.

<p>Panel A depicts percentage of CD103⁺-cells percentage of the population of CD8⁺-cells (CD8⁺CD103⁺) in 14 days <i>ex vivo</i> culture of squamous epithelial biopsies from controls (C SQ 5 cm: 5 cm above the Z-line), RE patients and (RE 5 cm: 5 cm above the inflamed RE tissue) and BE patients (BE SQ 5 cm: 5 cm above the BE tissue). Panel B represents CD8⁺CD103⁺- percentage in 14 days <i>ex vivo</i> culture of normal looking distal squamous epithelial biopsies of controls (C SQ 2 cm: 2 cm above the Z-line) and inflamed esophageal tissue of RE-patients (RE). Panel C depicts percentage of NKg2a⁺-cells of the population of CD8⁺-cells (CD8⁺NKg2a⁺) in 14 days <i>ex vivo</i> culture of squamous epithelial biopsies from controls (C SQ 5 cm: 5 cm above the Z-line), RE patients and (RE 5 cm: 5 cm above the inflamed RE tissue) and BE patients (BE SQ 5 cm: 5 cm above the BE tissue. Panel D represents CD8⁺NKg2a⁺-percentage in 14 days <i>ex vivo</i> culture of squamous epithelial biopsies of controls (C SQ 2 cm: 2 cm above the Z-line) and inflamed esophageal tissue of RE-patients (RE). Panels A and C: Kruskal-Wallis test. Panels B and D: Mann-Whitney test. Mean value and SEM are calculated.</p

Higher percentage of CD8⁺GranzymeB⁺-cells in <i>ex vivo</i> biopsies from RE compared to <i>ex vivo</i> biopsies from normal looking squamous esophagus.

<p>Panel A depicts percentage of GranzymeB⁺-cells of the CD8⁺-population (CD8⁺Granzyme B⁺-cells) in 14 days <i>ex vivo</i> culture of squamous epithelial biopsies from controls (C SQ 5 cm: 5 cm above the Z-line) and BE patients (BE SQ 5 cm: 5 cm above the BE tissue. Panel B represents CD8⁺GranzymeB⁺-percentage in 14 days <i>ex vivo</i> culture of normal looking squamous epithelial biopsies of controls (C SQ 2 cm: 2 cm above the Z-line) and inflamed esophageal tissue of RE-patients (RE). Mann-Whitney test was used (*p<0.05). Mean value and SEM are calculated.</p

Higher percentage of CD8⁺GranzymeB⁺CD94⁺-cells in <i>ex vivo</i> cultures of RE compared to <i>ex vivo</i> cultures of proximal esophagus.

<p>Panel A depicts percentage of GranzymeB⁺CD94⁺-cells of the percentage of CD8⁺-cells (CD8⁺GranzymeB⁺CD94⁺) in 14 days <i>ex vivo</i> culture of normal looking proximal squamous epithelial biopsies from controls (C SQ 5 cm: 5 cm above the Z-line) and BE patients (BE SQ 5 cm: 5 cm above the BE tissue. Panel B represents CD8⁺ GranzymeB⁺CD94⁺ percentage in 14 days <i>ex vivo</i> culture of normal looking distal squamous epithelial biopsies of controls (C SQ 2 cm: 2 cm above the Z-line) and inflamed esophageal tissue of RE-patients (RE). Mean value and SEM are calculated.</p

Patients and Controls characteristics.

<p>Patients and Controls characteristics.</p

Normal looking squamous esophageal epithelium is characterized by higher percentage of CD4⁺CD103⁺-cells.

<p>Panel A depicts percentage of CD103⁺-cells of the population of CD4⁺-cells (CD4⁺CD103⁺) in 14 days <i>ex vivo</i> culture of normal looking squamous epithelial biopsies from controls (C SQ 5 cm: 5 cm above the Z-line), RE patients and (RE 5 cm: 5 cm above the inflamed RE tissue) and BE patients (BE SQ 5 cm: 5 cm above the BE tissue. Panel B represents CD4⁺CD103⁺-percentage in 14 days <i>ex vivo</i> culture of normal looking squamous epithelial biopsies of controls (C SQ 2 cm: 2 cm above the Z-line) and inflamed esophageal tissue of RE-patients (RE). Panel A: Kruskal-Wallis test. Panel B: Mann-Whitney test. Mean value and SEM are calculated.</p

More CD3⁺CD4⁺-cells in <i>ex vivo</i> cultures of RE biopsies, compared to normal looking squamous esophageal tissue biopsies.

<p>Panel A depicts percentage of CD4⁺-cells of the population of CD3⁺-cells (CD3⁺CD4⁺) in 14 days <i>ex vivo</i> culture of normal looking squamous epithelial biopsies from controls (C SQ 5 cm: 5 cm above the Z-line), RE patients and (RE 5 cm: 5 cm above the inflamed RE tissue) and BE patients (BE SQ 5 cm: 5 cm above the BE tissue. Panel B represents CD3⁺CD4⁺-percentage in 14 days <i>ex vivo</i> culture of normal looking squamous epithelial biopsies of controls (C SQ 2 cm: 2 cm above the Z-line) and inflamed esophageal tissue of RE-patients (RE). In panel A Kruskal-Wallis test was used. In panel B, a Mann-Whitney test was used. Between panels A and B a paired t-test was used to compare CD3⁺CD4⁺-percentage in <i>ex vivo</i> cultures from RE tissue to normal looking squamous esophageal epithelium taken 5 cm above the gastroesophageal junction from the same RE patients (*p<0.05, **p<0.005). Mean value and SEM are calculated.</p

Illustration of the location of the taken biopsies.

<p>Arrows indicate the locations of the biopsying in controls, RE (Reflux Esophagitis) patients and BE (Barrett’s esophagus) patients.</p

mRNA and protein expression of factors associated with EMT upon BMP4 or Noggin incubation.

<p>a. Q-RT-PCR was performed to determine mRNA expression of SNAIL2 and its target genes, CDH1, CDH2 and Vimentin. B2M was used for normalization. Data are relative to the mean ΔCt of cells incubated with Noggin and are expressed as mean±SEM. *p<0.05. b. Western blot analysis of BAR-T and OE33 cells showed that SNAIL2 expression was upregulated and CDH1 expression was downregulated in cells incubated with BMP4 compared to cells incubated with Noggin. Actin was used as loading control. Pictures are representative of three independent experiments.</p

Expression of BMP4, BMP4 pathway associated molecules and the downstream target ID2.

<p>a. Q-RT-PCR was used to determine mRNA expression of BMP4, its downstream target, ID2, BMP4 associated receptors and SMAD molecules in SQ, BE and EAC biopsy specimens. B2M and GAPDH were used for normalization. Data are relative to the mean ΔCt of SQ biopsies and are expressed as box plots, representing the mean with the minimum and maximum values. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. b. Western blot analysis in SQ, BE and EAC biopsy specimens showed BMP4, and ID2 expression and phosphorylation of SMAD1/5/8 in BE and EAC. Actin was used as loading control. Biopsy samples from 6 EAC patients were used. Representative pictures are shown. c. IHC showed nuclear and cytoplasmic expression of SMAD4 in 10 of 13 BE (arrowhead) and 11 out of 13 EAC tissue sections. EAC* represents a biopsy specimen with positive SMAD4 staining, EAC** represents a biopsy specimen with negative SMAD4 staining, stromal cells are SMAD4 positive and serve as internal control. Haematoxylin counterstain was used. Representative pictures are shown.</p