One of the first steps during Ig heavy chain isotype switching to IgE is the IL-4 induced synthesis of IgE germline transcripts. To further characterize the molecular mechanism of the IL-4 action, the regulatory DNA elements involved in the control of expression of these transcripts were analyzed. Transient transfection of a B cell tumor line revealed the presence of a 15 bp IL-4 responsive cis-acting element (IL-4RE) highly homologous to an IL-4 response element in the human CD23b promoter. An IL-4 induced DNA binding protein specifically interacted with this sequence. Point mutations within that sequence not only abolished IL-4 inducibility of reporter constructs but also prevented binding of the nuclear factor to the mutated sequence. A stretch of 16 nucleotides just upstream of the IL-4RE contributed to IL-4 inducibility and formed nucleoprotein complexes with constitutive factors. All reporter constructs containing the functional IL-4RE were transcriptionally very weak but could be readily activated upon IL-4 induction. Transfection of constructs containing the mutated IL-4RE or plasmids lacking that sequence displayed a high constitutive promoter activity and were IL-4 unresponsive. These data suggest that in the absence of the cytokine the activity of the IgE germline promoter is actively repressed through the action of the IL-4RE. The same sequence appears to be critically involved in the IL-4 induced activation of the promoter via the binding of a cytokine induced transcription factor.

Based on THP1, U937 and HL60 and their alpha-chain transfectants we present a model system for the study of Fc epsilonRI regulation and signalling on human cells. THP1 in particular, due to its responsiveness to both ligand and IL-4, even without prior manipulation, is ideally suited to address questions on Fc epsilonRI modulation in an 'allergic environment'.

Ig heavy chain class switching is directed by cytokines inducing transcription from unrearranged CH genes. Subsequently, such primed cells can undergo switch recombination to express the selected new isotype. In the case of IgE class switching, IL-4 activates the IgE germline promoter by inducing the interaction of the transcription factor STAT6 (IL-4STAT) with a responsive DNA element in the proximal region of the promoter. This study describes the characterization of two additional cis-acting elements that interact with members of the NF kappa B/rel transcription factor family in an IL-4-independent fashion. Electrophoretic mobility shift assays show that the nucleoprotein complex formed on the upstream site (NF kappa B1) contains the classical p50/p65 heterodimer. The complex on the proximal site (NF kappa B2) appears to be composed of p50 and relB. IgE germline promoter reporter gene constructs carrying point mutations in the NF kappa B2 site were largely unresponsive to IL-4 stimulation in transient transfection experiments, while plasmids with similar mutations in the NF kappa B1 site responded to cytokine stimulation better than the wild-type promoter. The NF kappa B2 effect was dependent on the presence of the STAT6 binding site, demonstrating that the NF kappa B2 motif is necessary but not sufficient for mediating cytokine up-regulation. In addition, the combination of a NF kappa B/rel binding site and the STAT6 response element conferred IL-4 inducibility to a heterologous minimal promoter, while the individual sites had no effect. The available data suggest that the NF kappa B2 nucleoprotein complex may cooperate with DNA-bound STAT6 to achieve IL-4-dependent activation of the human IgE germline gene.

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