Implanted IgE-Fc_ϵR Complexes Elicit IgE-Mediated Activation of RBL-2H3 Cells

The high-affinity receptor for IgE (Fc_ϵR) is the cellular trigger of the antigen-induced activation of mast cells and basophils. To examine the functional integrity of Fc_ϵR, we have adopted a protein implantation procedure whereby the purified receptor complex was coreconstituted with Sendai virus envelopes. The latter promoted fusion of the hybrid vesicles with recipient cells such as rat basophilic leukemia, RBL-2H3, thus serving as a vehicle for the receptor. The implanted Fc_ϵR was complexed with ¹²⁵I-labeled mouse IgE (anti-DNP) to permit receptor quantification as well as specific triggering by DNP₂₀BSA. Implantation in the presence of unlabeled rat IgE, which blocked the native receptors on the recipient RBL-2H3 cells, resulted in incorporation of up to 15 ng of receptor-bound IgE/10⁶ cells. This was roughly equivalent in amount to 10-20% of the native receptors on such cells. The exocytosis which was triggered in the recipient cells by reagents that specifically recognized the implanted IgE reached between 15 and 50% of the maximal response. Various treatments that interfered with the activities of the viral envelopes reduced both receptor incorporation (3-5-fold) and cell degranulation (3-10-fold). These included separation of the receptor from the reconstituted envelopes, addition of serum to the incubation mixture (to inhibit vesicle-cell binding), and trypsinization of the virus (to inhibit vesicle-cell fusion). Poly (ethylene glycol) 8000 (4%) enhanced both the incorporation of the receptor and its functional responses. These treatments distinguished between real incorporation of IgE-Fc_ϵR complexes and other mechanisms of ¹²⁵I-IgE association with the recipient cells. The results indicate that the amount of IgE that was exchanged between the implanted and native receptors was minor. This conclusion was confirmed by use of an unstable variant of RBL-2H3 which expressed 10-fold less receptor on the cell surface. ⁴⁵Ca uptake was also elicited by implanted receptors. Sendai envelope treated cells show a reduced uptake when triggered via their native receptors, as compared to that of untreated cells. However, ~80% of this control response was attained by aggregation of implanted IgE-Fc_ϵR complexes. We thus conclude that under the employed conditions the purified receptor appears to be intact with respect to the obligatory functions necessary for the exocytotic process.