To do this, transient transfection assays were performed using either one of the two human CCL20 promoter-luciferase constructs: pCCL20 c/EBPmut, containing the full-length human CCL20 promoter bearing the mutated c/EBP site; and the pCCL20 NF-κBmut, containing the full-length CCL20 promoter bearing the mutated NF-κB site 14. As shown in Fig. SCH 900776 in vivo 4, site-specific mutation of the single NF-κB responsive motif almost completely blocked the ability of IFI16 to trigger luciferase activity. In contrast,
mutation of the single C/EBP site only slightly decreased luciferase activity compared with the wild-type CCL20 promoter. In order to provide definitive evidence supporting the role of NF-κB as the mediator of CCL20 promoter activation by IFI16, HUVEC were transfected with the indicator plasmid 5× NF-κB luc 15, infected thereafter with AdVIFI16 or AdVLacZ and reporter gene activity subsequently measured 24 h later. As shown in Fig. 4, overexpression
of IFI16 significantly increased NF-κB transactivation of the reporter gene although at levels lower than those observed with the endogenous CCL20 promoter. Altogether, these results demonstrate that IFI16 interacts with NF-κB in order to trigger CCL20 promoter activity, in line with the results obtained from the ICAM-1 promoter analysis. However, NF-κB does not appear to be the only transactivator GSK126 research buy stimulated by IFI16 in order to trigger CCL20 promoter. The ligand–receptor pair CCL20-CCR6 is believed to be responsible for the chemoattraction of CD34-derived immature DC, Langerhans DC (L-DC), effector/memory T cells and B cells, and it plays a role at skin and mucosal surfaces under homeostatic and inflammatory conditions 16, 17. If this is the case, it is important to verify a functional link between the ability of IFI16 to trigger CCL4, CCL5 and CCL20 release by HUVEC
Dimethyl sulfoxide and DC and B-lymphocyte chemoattraction. Using a transwell migration assay, we demonstrate that both L-DC and B cells migrate to a significantly greater degree in response to the supernatants from IFI16-infected HUVEC compared with the supernatants from LacZ-infected HUVEC (Fig. 5). This migration was significantly reduced by pre-incubation with the anti-CCL4, anti-CCL5 and anti-CCL20 mAb, but only when added to the supernatants from IFI16-infected HUVEC. In contrast, addition of an unrelated mAb of the same isotype, used as an internal control, did not influence cell migration (data not shown). These results confirm that the secretion of CCL4, CCL5 and CCL20 by IFI16-infected HUVEC is functional and important for inducing L-DC and B-cell migration into the mucosa and skin where these cells are particularly abundant.