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CHO cells permanently transfected with mouse FcΥRI αchain were prepared and used as a model to polyclonally activate murine B cells. The transfected CHO cells were treated with mitomycin C and placed into culture with varying quantities of anti-IgD. Using this model, murine splenic B cells (from BALB/c or C57Bl/6) were activated by mouse IgG2a-anti-IgD (10.4.22 or AF3.33) in a manner that is analogous to the activation of B cells seen with highly polyvalent anti-IgD (HΥa/1) prepared by chemical cross-linking to dextran. Efficient B cell activation was seen with nanogram quantities of anti-IgD. In the presence of IL-4 and IL-5, IgG1 production levels were equivalent to or better than seen when stimulation was with Hda/1-dextran; however, Ig;E induction was not seen in either situation. The Ig production capacity was compared to that seen when B cells were activated with CD40L, using either CD40L-transfected CHO or a soluble CD40L construct. In the presence of IL-4 and IL-5, once a critical threshold of B cells was present, IgE and to a lesser extent IgG1 production was inversely proportional to B cell number when CD40L was the activating agent. In contrast, with FcgRI-anti-IgD, IgM and IgG1 production was directly proportional to B cell number, while IgE production was never seen. Finally, when B cells were co-activated with immobilized anti- IgD and CD40L simultaneously, the IgE production from B cells induced by CD40L was strongly inhibited, while IgG1 and IgM production were not affected. Since B cell co-activation via sIg; and CD40L would be a common scenario in secondary follicles, this inhibition of IgE production may be one of the reasons why serum IgE levels are much below IgG in normal immune situations. [ABSTRACT FROM AUTHOR]

To determine the role of germline ∈ transcription in IgE synthesis, the effects of cytokines on germline ∈ RNA synthesis in IL-4 dependent ∈ switching in B cells was investigated. Induction of germline ∈ transcription in highly purified B cells seems to be a specific property of IL-4, since none of the other cytokines tested [IL-1α, β, IL-2, IL-3, IL-5, IL-6, IL-7, IL-9, IL-10, G-CSF, GM-CSF, M-CSF, IFN-γ, IFN-α, tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β] were effective. TGF-β, IFN-γ, and IFN-α inhibit IL-4 dependent IgE synthesis, but only TGF-β blocked germline ∈ RNA synthesis in purified B cells, indicating that this may be the mechanism by which TGF-β inhibits IgE synthesis, and that IFN-γ and IFN-α act on other stages of the regulatory process resulting In IgE production. IL-5, IL-6, and TNF-α enhance IL-4 dependent IgE synthesis, but only TNF-α enhanced IL-4 induced germline ∈ RNA synthesis. Finally, anti-CD40 mAbs and the non-IL-4 producing CD4 T cell clone A3, which in the presence of IL-4 induce IgE synthesis by purified B cells, both strongly enhanced germline ∈ transcription. These data, together with the observation that ∈ switching in cultures initiated with single slgM, slgE B cells in all instances was preceded by germline ∈ RNA synthesis, indicate that there is a strong relationship between germline ∈ transcription and IgE synthesis. [ABSTRACT FROM PUBLISHER]