The overall objective of this proposal is to delineate the B-cell mechanisms that regulate the production of IgE antibody; antibody which is implicated in atopic diseases which affects nearly 20% of the population. Initially we'll complete delineation of the sequence of novel human epsilon mRNAs not predicted from other species. Formed by alternative splicing of membrane sequence, these mRNAs are produced by human IgE producing B-cells both in continuous culture and when fresh B-cells are stimulated by IL-4 + CD40. Sequencing will provide data about the predicted proteins. This is particularly important in relationship to membrane IgE (mlgE) as our PCR data shows that all forms of mlgE contain >60 amino acids between the end CHepsilon4 and prior to the transmembrane region. Thus we will develop antibody reagents to be used both to detect/quantitate specifically these mlgEs and to experimentally test the role of mlgE forms in modulating B cell IgE production. With this information, we will compare and contrast the induction/production of IgE from normal B cells when driven via two separate signaling pathways; IL-4 + CD40 stimulation vs IL-4 + T-cell contact + IL-6. This approach will be extended to purified resting as well as in vivo IgE committed B-cells from atopic subjects as our hypothesis is that the IL-4/CD40 system is a highly epsilon specific alternative pathway preferentially used in atopic individuals. We will determine the role of two isotype specific B-cell membrane molecules, membrane IgE and the low affinity IgE receptor (FcepsilonR11, CD23), in the regulation of IgE from 1) resting cells of normals and atopic persons; 2) spontaneous IgE producing cells from atopic individuals; and 3) cells in continuous culture making IgE. We'll test the prediction that both binding to the unique membrane domains of the membrane IgE molecule(s) and FcepsilonR11 can modulate IgE production but each will have differing effects depending on whether the cells were driven to produce IgE via CD40 vs T cell contact. Outcomes may also be different in atopic versus normal subjects. Finally, we will undertake studies to elucidate the mechanism for isotype specificity of the CD40/IL-4 system. We will test the hypothesis that CD40 provides an isotype specific switch event as well as a signal affecting epsilon gene transcription. Initially we will perform gel shift assays using ~250 base pair fragments across the epsilon switch region up to the initiation site(s) of germ line transcription, predicting that CD40 induces an epsilon DNA binding protein(s) targeted to the epsilon switch region. This will be followed by DNAase footprinting. Finally, we'll examine the effects of CD40 stimulated cell extracts on constructs containing epsilon sequences so identified along with the IgH enhancer to determine whether, following epsilon germ line transcription, CD40 only stimulates specific DNA binding protein(s) that direct production of the mature epsilon tran...