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

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. Copyright © 1987 Wiley‐Liss, Inc.