We have previously shown that the neuropeptide CGRP influences the inflammatory and immune functions of primary dermal microvascular ECs (pDMECs). To expand this line of inquiry, we examined the ability of CGRP to influence EC expression of IL-23p19. IL-23 is a heterodimeric cytokine, composed of p19 and p40 subunits. The murine BALB/c EC line bEND.3 was employed as a surrogate for pDMECs. bEnd.3 cells were plated in 12-well flat bottom plates at 2.5 x 105 cells/well and incubated overnight. Culture media was replaced with fresh media the following day and cells cultured in 0 nM, 0.1nM, 1.0nM, 10nM or 100nM of CGRP. A positive control group was treated with 2 μg/mL of LPS. Cells were collected at various timepoints to quantify mRNA levels using real-time PCR. A significant increase in expression of IL-23p19 mRNA was seen at 8 hours. IL-23p40 subunit mRNA was undetectable; this is in accordance with findings by others that ECs do not express IL-23p40. Supernatant IL-23p19 was not detected by ELISA, also in accordance with findings by others. Western blot of cell contents demonstrated greater quantities of intracellular IL-23p19 at both 12 and 24 hours in the CGRP- and LPS-treated groups when compared with untreated cells. The function of intracellular IL-23p19 is not well understood, but it is a member of the IL-6 cytokine family and has been shown to function similarly to IL-6 in some aspects; both bind gp130 and activate STAT3. IL-23p19 expression in ECs has been associated with enhanced cell surface expression of ICAM-1 and VCAM-1, which could augment leukocytes attachment and transendothelial migration. However, in preliminary experiments, exposure of bEND.3 cells to CGRP reduced, rather than enhanced, VCAM-1 and ICAM-1 expression, perhaps because of diverse signaling pathways induced by CGRP. If dermal ECs also respond to CGRP by increasing IL-23p19 expression, neuronal release of CGRP at dermal blood vessels may provide a previously unidentified biologically important signal to dermal ECs.
© 2017 Published by Elsevier Inc.