Human microvascular endothelial synthesis of interleukin‐8 during in vitro ischemia and reperfusion

Abstract

These studies were undertaken to evaluate human microvascular endothelial cell (MEC) synthesis of interleukin-8 (IL-8), a potent neutrophil chemoattractant, under in vitro conditions of ischemia and reperfusion. IL-8 and other related CXC chemokines are believed to mediate tissue injury in a variety of pathologic conditions in humans. MEC grown on microcarrier beads were exposed to 3 or 6 h of in vitro ischemia followed by 2 h of reperfusion. Conditioned medium, MEC protein, and total RNA extracts were assayed for IL-8 using an ELISA. During ischemia alone, MEC increased intracellular, but not extracellular levels of IL-8 secretion. In contrast, reperfusion markedly stimulated both intracellular and extracellular IL-8 secretion. Neither 3 h of ischemia alone or followed by reperfusion altered steady-state levels of IL-8 mRNA when compared to pre-ischemic levels. In contrast, after 6 h of ischemia alone and ischemia followed by reperfusion, IL-8 mRNA was increased eight- and sixfold, respectively, when compared to pre-ischemic levels. These studies demonstrate an inverse relationship between the rate of IL-8 protein secretion and the steady-state levels of IL-8 mRNA during ischemia and reperfusion. During ischemia and reperfusion both the increase in cell-associated IL-8 protein and the release of IL-8 into the medium is dependent on de novo protein synthesis rather than the intracellular accumulation of IL-8. These experiments indicate that post-ischemic modulation of IL-8 release and synthesis following ischemia reperfusion will require strategies directed towards inhibition of IL-8 transcription and in depth knowledge of the mechanisms regulating IL-8 secretion.