Bacterial DNA triggers B-cell proliferation and induces immunoglobulin secretion. Chromatin-IgG complexes activate autoreactive B cells by co-engaging B-cell receptor (BCR) and TLR-9, thus suggesting a role for innate signaling in systemic autoimmunity. Spleen cells from lupus prone Palmerston North (PN) mice produce several fold less IL-12p40 than controls in response to CpG-oligodeoxynucleotides (ODNs). Here we show that B cells are primarily responsible for this abnormality. The removal of B cells from PN cultures markedly increased IL-12p40. Moreover, the addition of purified B cells back to PN splenocyte cultures resulted in a B-cell number dependent/ IL-10-mediated suppression of IL-12p40. The B cells were the major source of IL-10. In response to CpG, B cells from several lupus strains produced twice as a much IL-10 as controls, but failed to produce IL-10 when stimulated through BCR or CD40. PN and control mice expressed IL-10R similarly, and the difference in IL-10 secretion remained when anti-IL-10R blocking antibodies were used. IFN-gamma and IL-4 regulated CpG-induced IL-10 secretion in opposite directions. The abnormal IL-10 response in lupus mice was derived from B cells with the marginal zone phenotype, and could be downregulated with inhibitory ODNs. We hypothesize that TLR-9 activated lupus B cells can modulate T-cell mediated inflammatory responses through IL-10 production. Therefore, B cells may contribute to the lupus pathogenesis in many different ways: as antigen-presenting cells for self antigens, as effector cells for autoantibody production, and as IL-10 secreting regulatory cells.

Reaction to certain motifs in bacterial DNA is an important function of natural immunity. For example, single stranded oligonucleotides (ODN) containing the motif "not C, unmethylated C, G, not G" are powerful mitogens and apoptosis inhibitors for mouse spleen B cells. But replacing GCGTT or ACGTT with GCGGG or ACGGG converted a stimulatory 15‐mer ODN into an inhibitory ODN. All inhibitory ODN had three consecutive G, and a fourth G increased inhibitory activity, but a deazaguanosine substitution to prevent planar stacking did not affect activity. Inhibitory ODN blocked apoptosis protection and cell‐cycle entry induced by stimulatory ODN, but not that induced by lipopolysaccharide, anti‐CD40 or anti‐IgM+IL‐4. ODN‐driven up‐regulation of cyclin D2, c‐Myc, c‐Fos, c‐Jun and BclXL and down‐regulation of cyclin kinase inhibitor p27kip1 were all blocked by inhibitory ODN. The relative potency of a series of stimulatory and inhibitory ODN was the same for all readouts measured. Interference with uptake of stimulatory ODN could not account for their inhibitory effects. Even if addition of inhibitory ODN was delayed several hours, partial inhibition of stimulatory ODN effects occurred. Inhibitory ODN hold potential as antidotes for excessive ODN stimulation in the clinical setting and provide an important tool for studying ODN recognition.

Introduction B cells have many different roles in systemic lupus erythematosus (SLE), ranging from autoantigen recognition and processing to effector functions (for example, autoantibody and cytokine secretion). Recent studies have shown that intracellular nucleic acid-sensing receptors, Toll-like receptor (TLR) 7 and TLR9, play an important role in the pathogenesis of SLE. Dual engagement of rheumatoid factor-specific AM14 B cells through the B-cell receptor (BCR) and TLR7/9 results in marked proliferation of autoimmune B cells. Thus, strategies to preferentially block innate activation through TLRs in autoimmune B cells may be preferred over non-selective B-cell depletion.

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.