DNP-phycobiliproteins, fluorescent antigens to study dynamic properties of antigen-IgE-receptor complexes on RBL-2H3 rat mast cells.
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-DNP-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 microgram/ml DNP42-B-phycoerythrin stimulates maximum degranulation from IgE-saturated cells. Under these conditions, approximately 26 X 10(3) molecules of DNP42-B-phycoerythrin are bound per cell at equilibrium. The rate and extent of antigen binding and of antigen-stimulated 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.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000936:Antigen-Antibody Complex / Q000032:analysis*; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D004140:Dinitrophenols; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D007073:Immunoglobulin E / Q000032:analysis*; D007700:Kinetics; D045342:Light-Harvesting Protein Complexes; D008407:Mast Cells / Q000276:immunology; D008856:Microscopy, Fluorescence / Q000379:methods; D045524:Phycobilisomes; D010940:Plant Proteins; D051381:Rats; D011961:Receptors, Fc / Q000032:analysis*; D017455:Receptors, IgE; D011971:Receptors, Immunologic / Q000032:analysis*
D000936:Antigen-Antibody Complex; D004140:Dinitrophenols; D005456:Fluorescent Dyes; D045342:Light-Harvesting Protein Complexes; D045524:Phycobilisomes; D010940:Plant Proteins; D011961:Receptors, Fc; D017455:Receptors, IgE; D011971:Receptors, Immunologic; D007073:Immunoglobulin E