Calcitonin gene-related peptide (CGRP) induces interleukin-6 (IL-6) production by microvascular endothelial cells (ECs)

Summary: Abstract Body: Exposure of murine dermal microvascular ECs to CGRP endows them with the ability, acting as bystanders, to bias the outcome of antigen presentation by Langerhans cells (LCs), or bone marrow-derived dendritic cells, to responsive T cells away from Th1-type immunity and toward Th17-type immunity. We have previously demonstrated that treatment of ECs with small siRNA to IL-6 partially inhibits this CGRP effect. Furthermore, when IL-6 knockout ECs are used in the system, the CGRP effect is lost. We have now directly examined the ability of CGRP to induce IL-6 production by microvascular ECs. CD31+, CD45- ECs were obtained from the dermis of wild-type C57BL/6 mice and IL-6 knockout mice (C57BL/6 background) by magnetic antibody techniques. Twenty-five thousand ECs per ml were cultured in 12-well plates in complete endothelial cell growth medium for 24 hours. Then, complete medium was replaced with basal medium plus only 2% FBS and 100 U/ml penicillin/100 μg/ml. CGRP 0, 10 or 100 nM CGRP was added to wells. After 24 hours of culture, supernatants were harvested and IL-6 content assessed by ELISA: 0 nM CGRP – 9.7+/- 3.8 (SEM) pg/ml, 10 nM CGRP – 312.7+/- 47.0 (SEM) pg/ml, 100 nM CGRP 480.0+/- 41.5 (SEM) pg/ml [N=3; p<0.001 for 0 nM vs. 10 nM, p<0.001 for 0 nM vs. 100 nM]. These results are in accord with earlier experiments and definitively confirm that CGRP induces IL-6 production by microvascular ECs. Wanhong Ding¹, Lori Stohl¹, Richard D. Granstein¹ 1. Weill Cornell Medicine, New York, NY, United States. Adaptive and Auto-Immunity