Volume 137, Issue 5, Pages 1776-1784 (November 2009) Gastroesophageal Reflux Might Cause Esophagitis Through a Cytokine-Mediated Mechanism Rather Than Caustic Acid Injury  Rhonda F. Souza, Xiaofang Huo, Vivek Mittal, Christopher M. Schuler, Susanne W. Carmack, Hui Ying Zhang, Xi Zhang, Chunhua Yu, Kathy Hormi–Carver, Robert M. Genta, Stuart J. Spechler  Gastroenterology  Volume 137, Issue 5, Pages 1776-1784 (November 2009) DOI: 10.1053/j.gastro.2009.07.055 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Photomicrograph of the distal esophagus of a control rat, 8 weeks after sham surgery (H&E magnification, 20×). Gastroenterology 2009 137, 1776-1784DOI: (10.1053/j.gastro.2009.07.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 (A) Composite inflammation scores (all inflammatory cells) in layers of the distal esophagus at various time points after esophagoduodenostomy. The bar graphs depict the mean + standard error of the mean SEM. *First time point at which the P value was less than .05 compared with day 3. (B) Photomicrograph of the distal esophagus 3 days after esophagoduodenostomy. Lymphocytes can be seen, primarily in the submucosa (arrows) (H&E magnification, 40×). Gastroenterology 2009 137, 1776-1784DOI: (10.1053/j.gastro.2009.07.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Lymphocytic infiltration scores in layers of the distal esophagus at various time points after esophagoduodenostomy. The bar graphs depict the mean + standard error of the mean. *First time point at which the P value was less than .05 compared with sham controls. Gastroenterology 2009 137, 1776-1784DOI: (10.1053/j.gastro.2009.07.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 (A) Photomicrograph of the distal esophagus 3 weeks after esophagoduodenostomy, immunohistochemical staining for CD3 (magnification, 20×). (B) Distal esophagus 1 week after esophagoduodenostomy. Note the hyperplasia of the basal cell zone. Inflammatory cells comprise lymphocytes predominantly, but neutrophils can be seen as well (arrows) (H&E magnification, 40×). (C) Distal esophagus 3 weeks after esophagoduodenostomy. Note the prominent hyperplasia of the basal cell zone and the papillae despite the absence of surface erosions. (H&E magnification, 10×). (D) Distal esophagus 8 weeks after esophagoduodenostomy showing prominent inflammation, hyperplasia of the basal cell zone and papillae, and surface erosions (arrows) (H&E magnification, 20×). Gastroenterology 2009 137, 1776-1784DOI: (10.1053/j.gastro.2009.07.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Degree of (A) basal cell hyperplasia, (B) papillary hyperplasia, and (C) surface erosions in the distal esophagus at various time points after esophagoduodenostomy. The bar graphs depict the mean + standard error of the mean. Gastroenterology 2009 137, 1776-1784DOI: (10.1053/j.gastro.2009.07.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 (A) Photomicrograph of the distal esophagus in sham-operated animals, immunohistochemical staining for IL-8. Note that IL-8 expression is limited to rare lymphocytes (arrows) in the submucosa. (B) Distal esophagus 2 weeks after esophagoduodenostomy. Note the expression of IL-8 in the submucosa (bottom), the lamina propria (white arrows), and within the squamous epithelium, where it is most evident in the intercellular spaces of its upper third (black arrow). (C) Distal esophagus 5 weeks after esophagoduodenostomy. Note strong IL-8 expression in the lamina propria (white arrows), which is expanded by inflammatory cells and edema, and in the epithelium, particularly in the intercellular spaces in the intermediate zone (black arrow). (D) Distal esophagus 7 weeks after esophagoduodenostomy. Note the IL-8 immunoreactivity in the cytoplasm of squamous cells (black arrows). Gastroenterology 2009 137, 1776-1784DOI: (10.1053/j.gastro.2009.07.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Results of exposing NES-B3T and NES-B10T cells to acidic bile salt media on the secretion of (A) IL-8 and (B) IL-1β. The bar graphs depict the mean + standard error of the mean of at least 2 individual experiments. IL-8: *P < .05 compared with control; +P < .05 compared with day 1; #P < .05 compared with day 2; @P < .05 compared with day 3; &P < .05 compared with day 4. IL-1β: *P < .05 compared with control; +P < .05 compared with day 4. Gastroenterology 2009 137, 1776-1784DOI: (10.1053/j.gastro.2009.07.055) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 Migration rate of (A) T cells and (B) neutrophils after exposure to conditioned media (cm) collected from NES-B10T cells on day 4 in controls and on days 4 and 5 after exposure to acidic bile salt media (a+b); to control media lacking IL-8 or IL-1β (c), to IL-8; to IL-1β, or to a combination of IL-8 and IL-1β. The bar graphs depict the mean + standard error of the mean of at least 2 individual experiments. (A) T-cell migration: *P < .05 compared with corresponding control; +P < .05 compared with CM day 4; @P < .05 compared with CM day 5. (B) Neutrophil migration: *P < .05 compared with corresponding control; ns, nonsignificant. Migration rate of (C) T cells and (D) neutrophils after exposure to conditioned media (cm) collected from NES-B10T cells on day 4 in controls and on days 4 and 5 after exposure to acidic bile salt media (a+b), with and without the addition of an IL-8 blocking antibody. (C and D) *P < .05 compared with corresponding control. Gastroenterology 2009 137, 1776-1784DOI: (10.1053/j.gastro.2009.07.055) Copyright © 2009 AGA Institute Terms and Conditions