Adenovirus Vector-Induced Expression of the C-X-C Chemokine IP-10 Is Mediated through Capsid-Dependent Activation of NF-κB

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Abstract
The use of adenovirus vectors for gene therapy has been limited by well-defined cellular and humoral immune responses. We have previously shown that adenovirus vectors rapidly induce the expression of the C-X-C chemokine, interferon-inducible protein 10 (IP-10), in vivo. Various first-generation, type 5 adenovirus vectors, including adCMVβgal and UV-psoralen-inactivated adenovirus, equally induced the expression of IP-10 mRNA as early as 3 h following infection in mouse renal epithelial cells (REC). Luciferase reporter experiments using deletional mutants of the murine IP-10 5′-flanking region revealed that transcriptional activation of the IP-10 promoter by adCMVβgal was dependent on the −161- to −96-bp region upstream of the transcription start site. In electrophoretic mobility shift assays, adCMVβgal, adCMV-GFP, FG140, and transcription-defective adenovirus induced protein binding to oligonucleotides containing a consensus sequence for NF-κB at position −113 of the IP-10 promoter. Supershift assays confirmed an increase in binding activity of NF-κB p65 but not p50 or cRel in REC cells infected with various replication-deficient adenoviruses. Coinfection of REC cells with adCMVβgal and an adenoviral vector expressing IκBα resulted in suppression of adCMVβgal-induced expression of IP-10 at 6 and 16 h, further strengthening the conclusion that adenovirus-induced activation of IP-10 is dependent on NF-κB. The induction of IP-10 appeared to be direct because infection with adenovirus vectors failed to induce the expression of the potent IP-10 stimulators, interferon gamma and tumor necrosis factor alpha. Together, these findings demonstrate that adenovirus vectors directly induce the expression of IP-10 through capsid dependent activation of NF-κB.

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