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

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-kappaB to the nucleus. In this paper, we provide evidence that CpG-ODN-induced cell cycle entry and apoptosis protection are blocked by SN50 or gliotoxin and thus require NF-kappaB activation. NF-kappaB 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-kappaB inducers, CpG-ODN caused an early IkappaBalpha and IkappaBbeta degradation plus cleavage of the p50 precursor and subsequent NF-kappaB nuclear translocation. A group of closely related S-ODN, which specifically blocked CpG-induced B cell activation at submicromolar concentrations, also prevented NF-kappaB 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-kappaB DNA binding but prevented CpG-induced NF-kappaB nuclear translocation of p50, p65, and c-Rel and blocked p105, IkappaBalpha, and IkappaBbeta degradation, we concluded that their putative target must lie upstream of inhibitory kinase (IKK) activation.

Effect of Suppressive DNA on CpG-Induced Immune Activation

“…3 and 4). Consistent with these findings, suppressive motifs were recently shown to down-regulate CpG-dependent NF-B and AP-1 induction (17,18). These observations suggest that CpG motifs must continuously signal receptive cells for triggering to persist.…”

Influence of stimulatory and suppressive DNA motifs on host susceptibility to inflammatory arthritis

“…TCR ϭ T cell receptor. activation and may thus limit the pathologic changes caused by CpG-driven immune responses (10,13,25).…”

“…The mechanism by which suppressive ODNs inhibit the activity of CpG DNA is a matter of considerable interest. Recent studies suggest that suppressive ODNs interfere with CpG-mediated cell signaling upstream of nuclear factor B activation (11,25). However, suppressive ODNs do not block the cellular uptake of CpG DNA or binding to Toll-like receptor 9 (13).…”

The Molecular Basis for the Lack of Immunostimulatory Activity of Vertebrate DNA

“…AO was used in double-stranded form by mixing with an equimolar amount of reverse activating ODN (RAO), heating to 65°C, and cooling slowly to room temperature. 2114L and 2088L are based on 2114 and 2088 (16), but lengthened and made as PO-ODN. LPS from Salmonella minnesota Re595 (Sigma-Aldrich) was dissolved in 0.1% triethylamine and sonicated.…”

“…Although a number of studies have shown an inhibitory action of viral (12) and vertebrate (13)(14)(15) DNA or specific ODN (12-14, 16 -20), there is no consensus in the literature about the nature of such motifs. The suggestions for inhibitory motifs include GC-rich sequences such as CCGG (12), GC-rich sequences when linked closely to CpG (21), methylated CpG motifs (15), certain G-rich sequences such as GGAGGGG (16,17), and deoxyguanosine (dG) homopolymer (13,14,22). In none of these cases is it known how an inhibitory effect is mediated, and competition for CpG DNA uptake has not always been eliminated as a mechanism.…”