Photobleaching recovery studies of antigen-specific mouse lymphocyte stimulation by DNP-conjugated polymerized flagellin.
We have used fluorescence photobleaching recovery (FPR) to study the lateral diffusion of antigen-receptor complexes during stimulation of DNP-specific mouse B cells by the T-independent antigens DNP-polymerized flagellin (DNP-POL). Depending on epitope density and dose, these antigens behave either as immunogens or tolerogens. Lymphocyte DNP receptors binding DNP0.5 flagellin monomer show a diffusion constant D of 2.2 X 10(-10) cm2 sec-1 and ca 50% fluorescence recovery after bleaching. For DNP-POL bound to DNP-specific lymphocytes, the observed diffusion constants decrease monotonically with increased antigen dose and epitope density. Under optimally immunogenic conditions of DNP2.3-POL at 1 micrograms/ml, D = 1.5 X 10(-11) cm2 sec-1, some 14-fold less than for a single DNP receptor. Under tolerogenic conditions lower diffusion constants approaching 0.8 X 10(-11) cm-2 sec1 are observed. The fraction of aggregates mobile on the time scale of the experiment remains constant at about 50 to 60% in all immunogenic situations, but falls abruptly to about 24 to 32% in precisely those situations where the antigen/dose combination is tolerogenic. This might support the hypotheses that there exist critical epitope densities above which antigens and receptors form rigidly cross-linked aggregates that bring about B cell tolerance. The mobility of DNP0.5 flagellin monomer bound to receptors left unoccupied after treatment with various doses and batches of DNP-POL is independent of DNP-POL presence. Receptor aggregate diffusion is unaffected by treatment with colchicine or cytochalasin B.
D000818:Animals; D000941:Antigens; D001426:Bacterial Proteins / Q000276:immunology*; D001665:Binding Sites; D003078:Colchicine / Q000494:pharmacology; D003571:Cytochalasin B / Q000494:pharmacology; D004136:Dinitrobenzenes / Q000276:immunology*; D004306:Dose-Response Relationship, Immunologic; D000939:Epitopes; D005260:Female; D005408:Flagellin / Q000276:immunology*; D008213:Lymphocyte Activation; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation; D009038:Motion; D009578:Nitrobenzenes / Q000276:immunology*; D011971:Receptors, Immunologic; D012235:Rhodamines / Q000276:immunology; D013861:Thiocyanates / Q000276:immunology
D000941:Antigens; D001426:Bacterial Proteins; D004136:Dinitrobenzenes; D000939:Epitopes; D009578:Nitrobenzenes; D011971:Receptors, Immunologic; D012235:Rhodamines; D013861:Thiocyanates; D005408:Flagellin; D003571:Cytochalasin B; C009434:tetramethylrhodamine isothiocyanate; D003078:Colchicine