DNP‐phycobiliproteins, fluorescent antigens to study dynamic properties of antigen‐IgE‐receptor complexes on RBL‐2H3 rat mast cells

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Abstract
In RBL-2H3 rat mucosal mast cells, the crosslinking of cell-surface IgE-receptor complexes by multivalent antigens initiates a sequence of responses leading to degranulation. We have developed a family of dinitrophenol (DNP)-conjugated fluorescent antigens to study dynamic membrane events associated with these responses. Lysyl groups on the phycobiliproteins, B-phycoerythrin and C-phycocyanin, were labelled with DNP, yielding fluorescent conjugates that cause the release of [3H]serotonin from anti-DNO-IgE-primed RBL-2H3 cells. The binding of these antigens to IgE-receptor complexes was observed by fluorescence microscopy and quantified by flow cytometry. Incubation with 1 μg/ml DNP42-B-phycoerythrin stimulates maximum degranulation from IgE-saturated cells. Under these conditions, approximately 26 × 103 molecules of DNP42-B-phycoerythrin are bound per cell at equilibrium. The rate and extent of antigen binding and of antigenstimulated mediator release decrease in parallel as the concentration and DNP:protein ratio of the fluorescent conjugates is reduced. Secretion stops immediately when the nonfluorescent monovalent antigen, DNP-lysine, is added to degranulating cell suspensions. DNP-lysine also displaces surface-bound antigen when added during the first minutes after multivalent antigen. However, the ability of DNP-lysine to displace surface-bound DNP42-B-phycoerythrin from IgE-receptor complexes decreases progressively with time. Treatment with dihydrocytochalasin B and several analogs that prevent antigen-stimulated F-actin assembly enhances secretion and delays the transition of antigen to its DNP-lysine-resistant form. Cytochalasin treatment also permits the long-range movement of antigen into surface caps. Based on these data, we propose that secretion is triggered by the act of IgE-receptor crosslinking or by a shortlived excited state of the crosslinked antigen-IgE-receptor complex. We propose further that antigen-stimulated F-actin assembly contributes to the transition of antigen-IgE-receptor complexes to a DNP-lysine-resistant form that does not trigger secretion. Two posible mechanisms for the transition to DNP-lysine resistance are discussed.