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Induction And Regulation of Endogenous DARC Expression in Primary Human Endothelial Cells

The Duffy antigen receptor for chemokines (DARC, also known as atypical chemokine receptor 1) is a blood group antigen associated with susceptibility to the parasite causing malaria. Despite homology to other chemokine receptors, DARC lacks the intracellular DRY motif present on most G‐protein‐coupled receptors that is required for signaling. Human biopsy and animal studies have indicated the presence of DARC on the endothelium of post‐capillary venules where its function and regulation are unclear. The lack of literature on endothelial DARC is attributed in part to the fact that its expression disappears in cultured endothelial cells within hours of isolation from tissue. Thus, almost all studies on endothelial DARC have required in vivo approaches. However, while the in vivo work is intriguing, the ability to study endogenous endothelial DARC in vitro will be necessary to elucidate its regulation and true physiologic function. We have discovered that incubation with human whole blood for 24 hours leads to induction of DARC on cultured primary human pulmonary microvascular endothelial cells (HPMEC). DARC induced in this manner is appropriately targeted to the cell surface, as it renders endothelial cells sensitive to an extracellular leukocidin. Induction of DARC on the endothelium was sensitive to cycloheximide, implicating de novo protein synthesis, and chromatin immunoprecipitation (ChIP) assays for RNA polymerase II also confirm transcription of the gene. While exposure to whole blood for 24 hours induced endogenous endothelial DARC expression, exposure to plasma had no effect. Transwell experiments indicated that physical contact with cellular elements in blood was necessary for induction of DARC. After induction of DARC and removal of blood, we observed rapid loss of DARC protein and mRNA within 24 hours. Our preliminary experiments suggest that endothelial DARC is rapidly degraded at least in part by matrix metalloproteinases. In conclusion, DARC expression on endothelial cells is regulated by cues from the microenvironment. DARC may play an important role in the immune system and its regulation is likely to have implications for our understanding of how cultured cells maintain or lose their in situ tissue phenotype.