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THE EXTRACELLULAR ATP RECEPTOR P2X7 IS INCREASED IN MURINE TNBS COLITIS
RN Rabie, S Lourenssen, MJ Beyak
Gastrointestinal Diseases Research Unit, Queen’s University, Kingston, Ontario
The P2X7 receptor is a ligand gated ion channel that responds to the presence of extracellular ATP. It is found primarily on immune cells such as macrophages and is a key mediator of the release of cytokines such as IL-1beta. Therefore we hypothesized that P2X7 receptor expression would be increased in the colon of mice with TNBS colitis.
METHODS: Colitis was induced with 4% TNBS in 30% ethanol intracolonically (n=4). Controls were saline injected (n=5). After 5-7 days, colons were harvested, fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose and sectioned. Primary rabbit anti human P2X7 antiserum (Alomone) was used (1/250), with a goat anti rabbit secondary antibody conjugated to Alexa-488 (1:1000).
RESULTS: In control colon sections, P2X7 immunoreactivity was visualized just at the apical surface in the epithelium with occasional P2X7 positive cells in the mucosa, which appeared to be intraepithelial immune cells. Positive cells were also visualized in the submucosal and myenteric plexuses, as well as occasional cells in the smooth muscle layer. Compared to controls, colonic sections from TNBS treated animals exhibited extensive ulceration, crypt elongation, wall thickening and a dense inflammatory infiltrate in the lamina propria In TNBS sections, there was dense P2X7 staining in the lamina propria, with a marked increase (16.8 ± 1.5 control vs. 56.0 ± 5.0 TNBS, p<0.0001 unpaired t test) in the number of P2X7 positive immune cells. In TNBS treated colons, where the mucosa was intact there was in increase in intraepithelial P2X7 positive immune cells, with a relative reduction of staining of colonocytes along the apical surface.
CONCLUSION: In normal colon, P2X7 staining was present in the myenteric plexuses as well at the apical surface of the colonic mucosa. In TNBS animals there was a marked increase in P2X7 positive cells, most of them in the inflammatory infiltrate. These findings are consistent with the known role for the P2X7 receptor in other inflammatory states. Increased expression and activation of the P2X7 receptor may be involved in cytokine release in colitis. This suggests a possible role for the P2X7 receptor in the pathophysiology of colonic inflammation.