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REGULATION OF LEUKOCYTES TRAFFICKING TO THE INTESTINAL EPITHELIUM BY EXTRACELLULAR NUCLEOTIDES
C Langlois, FP Gendron
CIHR Team on the Digestive Epithelium, Department of Anatomy and Cell Biology, Université de Sherbrooke, Quebec
Aims: Extracellular nucleotides, such as adenosine 5’-triphosphate (ATP) and uridine 5’-triphosphate (UTP), are active immune molecules that stimulate specific receptors known as P2Y. We thus investigated the role of extracellular nucleotides and associated P2Y receptors in the inflammatory responses of intestinal epithelial cells (IECs) and more particularly in the upregulation of macrophages adhesion and neutrophils transepithelial migration.
METHODS: Monocytic U-937 cells were differentiated to macrophages-like cells by an 18h treatment with 100ng/ml of PMA. U-937 cells were then labelled with the green fluorescent dye PKH67 and added (1×106 cells/well) to a monolayer of IECS previously stimulated for 3h with 100µM of ATP, ADP, UTP or UDP. The cells were washed, and adherents U-937 cells counted under FITC illumination. Similarly, PKH67-labelled neutrophils-like PLB-985 cells (nlPLB) were added to the basal side of an inverted monolayer of Caco-2 cells grown on an 8µm pore insert membrane. Caco-2 cells were then stimulated apicaly with 100µM of ATP or UTP in a time-dependent manner and nlPLB having migrated through the Caco-2 monolayer counted. We also investigated the cellular mechanisms by which extracellular nucleotides stimulate the transepithelial migration of neutrophils and leukocytes adhesion to IECs. IECs were stimulated with 100µM ATP and UTP, in a time-dependent manner, and RNA and proteins extracted and assays for ICAM-1, RAGE, cd11B expression. Effect of an inflammatory insult on ICAM-1 expression was observed in vivo by immunofluorescence from colonic tissues isolated from DSS-treated mice.
RESULTS: Adhesion of macrophage-like U-937 cells to a monolayer of IECS stimulated by ATP is increased by more than a 3 fold or compared to control. Transepithelial migration of nlPLB cells through the monolayer was increased in nucleotides stimulated cells compared to control. The increased adhesion of U-937 to IECs correlates with increased expression of ICAM-1 following nucleotide. In addition, expression of ICAM-1 is increased in DSS treated mouse compared to control mouse
CONCLUSION: These results are clearly demonstrating the implication of extracellular nucleotides and ICAM-1 in the recruitment of immune cells, such as macrophages and neutrophils to the epithelial barrier.
This work was supported by the CCFC and a FRSQ scholarship to FP Gendron.