Human IgE synthesis in vitro: A reassessment

Since conflicting data have emerged from several laboratories regarding the kinetics of in vitro IgE production by human peripheral blood mononuclear cells (MNC) and the effects of pokeweed mitogen on cells from various types of donors, we re-examined factors influencing supernatant IgE concentrations. In experiments involving a total of 30 donors, we evaluated the effects of freezing and thawing and of inhibitors of protein and DNA synthesis on supernatant IgE concentrations. These studies clearly demonstrated that baseline quantities of IgE cannot be accurately assessed by measuring IgE in day 0 supernatants, since mean supernatant IgE concentrations of cells frozen and thawed on day 0 and incubated for 7 to 10 days were 2- to 3-fold higher. This slowly released IgE represented 40 to 50% of peak quantities detected in cultures of atopic patients' MNC and 100% of those of normal controls. When intact or lysed MNC were incubated in the presence of protein synthesis inhibitors, mean supernatant IgE values were the same, indicating that both such approaches can be used to measure essentially all preformed IgE. Supernatant IgE concentrations in 10-day cultures of atopic patients' intact MNC were higher than those in cultures frozen and thawed on day 0 and incubated for 10 days (p = <0.02), indicating true IgE synthesis in those cultures; this did not occur in parallel cultures containing puromycin or cycloheximide. Pretreatment of atopic patients' MNC with irradiation or mitomycin C failed to abrogate the IgE synthesis seen in cultures of nonpretreated MNC. This indicates that virtually all of the incremental increase in IgE by atopic MNC is contributed by an in vivo activated terminally differentiated cell, which does not undergo replication and may be largely impervious to regulatory influences of other cell types in in vitro studies. When supernatant IgE concentrations from parallel experiments with 12 normal control donors were analyzed, there was no evidence of significant IgE production. Thus, these studies emphasize that claims of IgE 'synthesis' in all previously published studies must be viewed with caution and that much of the work reported to date must necessarily be repeated in light of these observations.