CpG Stimulation of Primary Mouse B Cells Is Blocked by Inhibitory Oligodeoxyribonucleotides at a Site Proximal to NF-κB Activation

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Abstract
Bacterial DNA and CpG-oligodeoxyribonucleotides (ODN) are powerful B cell activators, inducing apoptosis protection, cell cycle entry, proliferation, costimulatory molecule expression, immunoglobulin (Ig) and interleukin-6 (IL-6) secretion. However, proximal events in B cell activation by ODN are only partially characterized, including the translocation of NF-κB to the nucleus. In this paper, we provide evidence that CpG-ODNinduced cell cycle entry and apoptosis protection are blocked by SN50 or gliotoxin and thus require NF-κB activation. NF-κB activation occurred within 30 minutes of stimulation of murine B cells with a phosphorothioate (S) CpG-ODN and persisted for up to 40 hours, with p50, p65, and c-Rel as the major components. Similar to other NF-κB inducers, CpG-ODN caused an early IκBα and IκBβ degradation plus cleavage of the p50 precursor and subsequent NF-κB nuclear translocation. A group of closely related S-ODN, which specifically blocked CpG-induced B cell activation at submicromolar concentrations, also prevented NF-κB DNA binding and transcriptional activation. These inhibitory S-ODN differed from stimulatory S-ODN by having 2-3 G substitutions in the central motif. As inhibitory S-ODN did not directly interfere with the NF-κB DNA binding but prevented CpG-induced NF-κB nuclear translocation of p50, p65, and c-Rel and blocked p105, IκBα , and IκBβ degradation, we concluded that their putative target must lie upstream of inhibitory kinase (IKK) activation.