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ACTIVATION OF INTESTINAL EPITHELIAL CELL P2Y NUCLEOTIDE RECEPTORS STIMULATES CYTOKINE EXPRESSION AND INCREASE MACROPHAGES ADHESION

C Langlois, D Grbic, É Degagné, AA Dupuis, FP Gendron
INTRODUCTION: Intestinal epithelial cells (IECs) express multiple cytokines and adhesion molecules required for the recruitment and adhesion of immune cells to the epithelium monolayer. Extracellular nucleotides, such as adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP), are a new class of immune-active molecules that activate specific receptors known as P2Y G-protein-coupled receptors and P2X ligand-gated ion channels. We thus investigated the role of extracellular nucleotides and associated P2Y receptors in the inflammatory responses of IECs and more particularly looked at the up-regulation of macrophages adhesion to IECs.

METHODS: Human colon adenocarcinoma Caco-2/15 and HT-29 cells and untransformed rat intestinal epithelial IEC-6 cells were stimulated with 100µM ATP, ADP, UTP or UDP in a time-dependent manner. Cytokines expression was measured by RT-PCR and cytokine antibody array. Effect of an inflammatory insult on P2Y receptor expression was measured in vivo by RT-PCR from colonic tissues obtained from DSS-induced colitis in mice and in vitro by RT-PCR and Western blot following stimulation of IECs with IL-1beta. Monocytic U-937 cells were differentiated to macrophages-like cells by an 18h treatment with 100ng/ml of PMA. Cells were then labeled with the green fluorescent dye PKH2 and added (1x10⁶ cells/well) to a monolayer of IECs previously stimulated for 8h with 100µM of ATP, ADP, UTP or UDP. The cells were washed in PBS, and adherent U-937 cell numbers were determined by microscopy using fluorescein isothiocyanate illumination.
RESULTS: Pro-inflammatory insult increase P2Y₂ receptor expression. Increase P2Y₂ expression is regulated at the transcriptional level. Stimulation of IECs by extracellular nucleotides resulted in an increase expression of pro-inflammatory cytokines, namely: TNF-alpha, GM-CSF, IL-8 and IL-12. Adhesion of macrophage-like U-937 cells to a monolayer of IECs stimulated by extracellular nucleotides is increased by more than a 3 fold factor as compared to control. Hence, preliminary results show enhance adhesion of U-937 to IECs correlate with increase expression of ICAM-1 and CD47 on IECs.
CONCLUSION: These results are clearly demonstrating the implication of extracellular nucleotides and P2Y receptors in the recruitment of immune cells, such as macrophages, to the epithelial barrier and the importance of extracellular nucleotide signaling in the modulation of IECs immune response in inflammatory bowel diseases.
This work was supported by grants from the CCFC, NSERC and the FRSQ

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