CD40 ligand induces expression of vascular cell adhesion molecule 1 and E-selectin in orbital fibroblasts from patients with Graves' orbitopathy.

purposeThe aim of this study was to detect the effect of the CD40 ligand (CD40L) on the expression of vascular cell adhesion molecule 1 (VCAM-1) and E-Selectin in orbital fibroblasts (OFs) from patients with Graves' orbitopathy (GO), as well as the signaling pathways involved in this effect.

methodsOFs were isolated from orbital tissues obtained from patients with severe GO who were undergoing orbital decompression surgery. VCAM-1 and E-selectin RNA and protein expression levels were quantified in OFs stimulated with soluble CD40L (sCD40L). RNA and protein quantification was performed with real-time polymerase chain reaction (PCR) and western blot analysis. Cytoplasmic and nuclear fractions were isolated in order to detect the nuclear translocation of nuclear factor-κB (NF-κB). Signaling pathway inhibitors were applied to determine the pathways involved.

resultsCompared to unstimulated OFs, the mRNA and protein levels of VCAM-1 and E-selectin in OFs incubated with sCD40L were significantly increased. This was observed in dose- and time-course experiments, and the inductive effects of sCD40L were much weaker in OFs from healthy donors. At the same time, we observed that CD40L induced nuclear translocation of NF-κB, also in a dose- and time-dependent manner. The up-regulation of VCAM-1 and E-selectin, as well as the NF-κB nuclear translocation induced by CD40L, was significantly attenuated by inhibitors targeting mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K), and NF-κB.

conclusionsCD40L demonstrated the ability to up-regulate the expression of VCAM-1 and E-selectin at the pre-translational level in OFs from patients with GO. The MAPK and PI3K pathways and NF-κB may play important roles in CD40L-induced VCAM-1 and E-selectin expression.