B cell tolerance induction by cross-linking of membrane IgM, but not IgD, and synergy by cross-linking of both isotypes

Previous studies in our laboratory demonstrated that overnight exposure of adult splenic B cells to anti-Ig resulted in an unresponsive state characterized by decreased antibody synthesis but normal mitogen-driven proliferation (i.e., anergy). Because both anti-F(ab′)₂ and anti-μ were equally effective at inducing tolerance, it was important to determine whether cross-linking of IgD together with or separately from IgM influenced the induction of unresponsiveness. Although anti-μ induced significant unresponsiveness, treatment of adult splenic B cells with anti-δ alone generally failed to reduce the subsequent response to either LPS or fluoresceinated Brucella abortus. Interestingly, anti-δ synergized with suboptimal concentrations of anti-μ to induce tolerance. Synergy could be observed in this system when anti-δ was added either simultaneously with or before (but not after) anti-μ; moreover, anti-δ was effective in a pretreatment (wash-out) protocol. To investigate the role of protein tyrosine kinase (PTK) activity in tolerance induction, splenic B cells were treated with tyrphostin before treatment with either anti-μ or anti-δ. We found that pretreatment with tyrphostin for 2 h before the addition of anti-μ prevented the induction of unresponsiveness with this antibody, whereas this PTK inhibitor facilitated tolerance when used with anti-δ treatment only. We propose that cross-linking of surface IgM directly or indirectly invokes a tyrphostin-sensitive, PTK-dependent pathway leading to the early events in tolerance induction, which can be augmented under limiting conditions by anti-IgD. Because cross-linking of either receptor initiates several common pathways, simultaneous cross-linking can lead to synergy and a dominance of the IgM signal. In contrast, IgD alone may fail to elicit tolerance because this isotype may also be associated with different PTK that cause positive signaling.