YOKOCHI Takashi Aichi Med. Univ., Dept. of Microbiol. & Immunol., Professor, 医学部, 教授 (20126915)
IWASAKI Hiromichi Uviv. of Fukui, 1^<st> Dept. of Int. Med., Lecturer, 医学部附属病院, 講師 (10242588)
|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2003: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥1,800,000 (Direct Cost: ¥1,800,000)
CpG DNA induces Th1-dominant immune responses in vertebrate. The activity of CpG DNA is dependent on both the base sequences and species in mammalian. In this study, we identify the base sequence of CpG DNA that activates human plasmacytoid cells (PDC), and examine its activation mechanisms.
Poly G-flanked palindrome CpG DNA, GGGGGGGGGG-GACGATCGTC-GGGGGGGGGG(G10GACGA), was identified as an active form of CpG DNA for PDC to induce IFN-α/IP-10/MIP-1α. The activity of G10GACGA is dependent on the sequence of GACGATCGTC, and endosomal maturation is required for the induction of IFN-α/IP-10/MIP-1α. No activity is observed in TLR9-KO mice. In G10GACGA-treated PDC, phosphorylation of p38 MAPK is induced in a manner independent of autocrine IFN-α/β signaling, and this pathway is essential for the induction of IFN-α/IP-10/MIP-1α. PDC constitutively express both IRF-3 and IRF-7, and G10GACGA enhances the expression of IRF-7 but not of IRF-3. Prior to the increase of IRF-7, STAT1 is phosphorylated
dependently on p38 MAPK, and ISGF3 component (STAT1, STAT2, and IRF-9) translocates to the nuclei. PDC also constitutively express NF-κB p65 and p50 in the nuclei. The activities increase by the treatment with G10GACGA, but not in the presence of p38 MAPK inhibitor. The NF-κB inhibitors, PDTC, MG-132, Dexamethasone, and CAPE, suppress G10GACGA-induced IRF-7/IFN-α/IP-10/MIP-1α expression. In late phase, the autocrine activation via IFNAR takes part in PDC.
From these results, we propose that poly-G-flanked palindrome CpG DNA up-taken in PDC activates both STAT1 and NF-κB through the processes of endosomal maturation and p38 MAPK activation. These pathways would cause, respectively or cooperatively, transcription of the genes, in the promoters of which ISRE and/or κB site exists, such as IRF-7/IP-10 and IRF-7/IP-10/MIP-1α, thereby inducing IFN-α/IP-10/MIP-1α in a manner independent of autocrine activation of IFNAR. The consecutive activation of IFNAR-signaling loop produces a large amount of IFN-α/IP-10/MIP-1α in PDC. Less