Interleukin-4 (IL-4) is known to promote the expression of immunoglobulin E (IgE) in humans and both IgG and IgE in mice, but the mechanism of these effects is unknown. The expression of these isotypes is mediated by a DNA recombination event which occurs between """"""""switch"""""""" regions upstream of the heavy chain constant region genes; the mechanism of these recombination events is also unknown. To better study this recombination and its modulation by IL-4, we have developed two assays, based on the polymerase chain reaction (PCR), that enable us to detect isotype switch recombination at the DNA level in bulk cultures of B lymphoid cells treated with IL-4. Using one assay, we can detect mu-epsilon switching in human peripheral blood lymphocytes treated in vitro with IL-4 and Epstein- Barr virus. We have demonstrated that some of these switching events involve successive switch recombination leaving remnants of gamma switch regions between the mu and epsilon sequences. One such mu-gamma-epsilon switch junction has been cloned from a human cell line and sequenced. We are currently attempting to identify which of the four human gamma genes is involved by completing the sequence analysis of the four human germline switch regions. In a related collaborative project, we have used a different PCR strategy to follow switch rearrangements of IL-4-treated murine spleen cells to gamma-1. In this system, most of the switching occurs between 2.5 and 3.5 days in culture; we have shown that a pulse of the DNA synthesis inhibitor aphidicolin administered at this time blocks the IL-4-induced expression of gamma-1, consistent with a replication- dependent mechanism for the switch event. Another collaborative project involves the analysis of human epsilon transcripts induced in vitro by treatment of B cells with IL-4 plus anti-CD40; using reverse transcriptase-PCR, we have identified several unexpected alternatively spliced transcripts.
