ABSTRACTS: Oral Presentation Abstracts
Introduction: The goal of our study was to evaluate the role of necrotic and apoptotic IEC on the activation of näive DC. We postulate that mode of the cell death can provide DC with additional pro-inflammatory information. DC are major antigen presenting cells in the intestinal mucosa. Their activation is prerequisite for antigen presentation and generation of effector T-cells. In our previous studies we have shown that primary colonic epithelial cells (CEC) and primary small intestinal epithelial cells (SIEC) are able to take up large amounts of complex soluble antigens. IEC can contain high concentration of antigen in the intracellular compartment. IEC have short life span (3–5 days) so antigens can be released from the cells undergoing apoptosis and be available for re-presentation by the DC. IEC can also undergo necrosis when exposed to toxins produced by intestinal bacteria potentially providing pro-inflammatory signaling to antigen presenting cells.
Methods: We used a previously described method of isolating and culturing primary murine IEC. Freshly isolated cells were evaluated for the markers of epithelial cells, viability, metabolic integrity and contamination by hematopoietic cells and mesenchymal cells. Preparations with less than 90% viability or more than 5% contamination by non-epithelial (hematopoietic) cells were discarded. DC were produced from one marrow by stimulation with GM-CSF. Apoptosis of IEC was induced by three separate methods: exposure to IFN- followed by TNF, UV radiation and by separation from the basal membrane. Repeated freezing and thawing induced necrosis. Apoptosis has been confirmed by evaluation of DNA content after staining with the Hochst dye and by measuring caspase levels. Expression of homing receptors: CD11c, CD44, CD56, and co-stimulatory molecules CD40, CD44, CD56 was evaluated by flow cytometry.
Results: In comparison to intact IEC both apoptotic and necrotic IEC induced higher expression of homing receptors: CD11c, CD44, CD56 and co-stimulatory molecules: CD40, CD44, CD56 by the DC. Necrotic IEC are inducing higher expression of co-stimulatory molecule CD86 (B7.2) than apoptotic IEC. The role of the differential expression of co-stimulatory molecules in generation of intestinal inflammation requires further study
Conclusion: Apoptotic and necrotic intestinal cells induce maturation of naive dendritic cells.