Abstract 667: Endothelial Cell “Memory”: Rapid Recall of Chemokines After Prior Exposure to Inflammatory Stimuli

Abstract

Background: Surprisingly little has been studied on the capacity of cytokine-primed endothelial cells (EC) to respond upon secondary challenge. The principal hypothesis is that EC store and rapidly recall chemokines after upon secondary challenge. Methods: Confluent primary human aortic (AEC), dermal microvascular (DMVEC), umbilical vein (UVEC), and renal glomerular (RGEC) endothelial cells were pre-exposed to TNFα or IL-1β (10ng/mL, 18hr), allowed to rest (4-24hr), and re-challenged with the secretagogues PMA or histamine. The repertoire of rapidly recalled soluble mediators was determined by ELISA, and cell surface P-selectin by cell-based ELISA. Total P-selectin content was measured by Western Blot. Results: Priming with TNFα or IL-1β overnight increased IL-8 secretion by all EC. 4hr after removal of TNFα or IL-1β, IL-8 was still in all supernatants. After 7hr rest, IL-8 was detectable from RGEC but undetectable in other EC. After 24hr, IL-8 was undetectable in the supernatants from all EC. Secondary challenge with secretagogues 24hr after removal of TNFα or IL-1β stimulated rapid secretion of IL-8, but not from unprimed endothelium. P-selectin was rapidly externalized at the cell surface of unprimed cells after 20min exposure to PMA or 10min Histamine. TNFα-primed EC exhibited significantly lower P-selectin induction, mirrored by profoundly reduced total intracellular P-selectin expression. mRNA expression of LAMP3 (CD63, a tetraspanin critical for P-selectin sorting to Weibel-Palade bodies) was highest in microvascular EC and lowest in larger vessel EC. LAMP3 might be important for controlling WPb content as well as durability of exocytosis upon type 1 activation and sorting of newly synthesized type II mediators to WPb for rapid recall. Conclusions: The data confirm that prior exposure of EC to inflammatory stimuli evokes storage of IL-8 that can be rapidly secreted upon exposure to a second stimulus. Moreover, constitutive expression of P-selectin is suppressed by TNFα exposure, but not IL-1β. Thus, EC exhibit a chronic inflammatory phenotype after cytokines have been encountered and removed, due to storage of chemokines within Weibel-Palade bodies or other granules.