Impaired Intracellular Calcium Mobilization and NFATc1 Availability in Tolerant Anti-Insulin B Cells

Abstract

B lymphocytes that recognize soluble self-Ags are routinely found in normal individuals in a functionally inactive or anergic state. Current models indicate that this tolerant state is maintained by interactions with self-Ags that uncouple the BCR from downstream signaling pathways and increase levels of free calcium. Contrary to this expectation, B cells that harbor anti-insulin Ig transgenes (125Tg) are maintained in a tolerant state even though free calcium levels remain normal and tyrosine kinase substrate phosphorylation is preserved following BCR stimulation. Under basal conditions, intracellular levels of inositol 1,4,5-trisphosphate are increased and NFATc1 levels are reduced in 125Tg B cells. The 125Tg B cells are markedly impaired in their ability to mobilize calcium upon stimulation with ionomycin, and BCR-induced calcium mobilization from internal stores is decreased. In contrast, poisoning intracellular calcium pumps with thapsigargin increases calcium mobilization in 125Tg B cells. Changes in calcium signaling are accompanied by a failure of 125Tg B cells to translocate NFATc1 into the nucleus following stimulation with either anti-IgM or ionomycin. Thus, disassociation of BCR from multiple signaling pathways is not essential for maintaining tolerance in anti-insulin 125Tg B cells. Rather, BCRs that are occupied by autologous insulin deliver signals that induce changes in intracellular calcium mobilization and maintain tolerance by preventing activation of key transcription factors such as NFAT.

Coronin 1 Is Essential for IgM-Mediated Ca2+ Mobilization in B Cells but Dispensable for the Generation of Immune Responses In Vivo

“…As shown in Fig. 1A, BCR stimulation resulted in similar induction of phosphotyrosine, including phosphorylation of a 55 kDa ‫)ء(‬ and 70 kDa ‫)ءء(‬ protein, suggesting phosphorylation of Lyn (55kDa) and Syk (70 kDa) (21)(22)(23), and confirming that early signaling events are not affected in B cells lacking coronin 1. Similarly, phosphorylation of phospholipase C␥2 that is required for the generation of InsP 3 generation was independent of coronin 1.…”

B Cell Receptor Affinity and B Cell Subset Identity Integrate to Define the Effectiveness, Affinity Threshold, and Mechanism of Anergy

“…2-12H/V4 FO B cells undergo Tyr, Syk, and ERK phosphorylation in response to BCR cross-linking as well or better than non-Tg FO B cells (Fig. 4C), indicating that, similar to anti-insulin B cells (44), the failure to survive BCR cross-linking (Fig. 4A) is not due to a gross failure of BCR signaling.…”

B-cell anergy: from transgenic models to naturally occurring anergic B cells?

“…For example, highavidity polyclonal IgM-specific antibodies induce robust tyrosine phosphorylation and intracellular Ca 2+ mobilization in insulin-specific B cells (from V H 125 × V κ 125 mice). This is somewhat paradoxical because in this model, immunization with antigen fails to elicit antibody production 61 . This may simply reflect the ability of high-avidity crosslinkers (such as polyclonal IgM-specific antibody) to overcome tolerance induced by the low-avidity antigen-BCR inter action.…”