Membrane depolarization is induced in tolerant B lymphocytes by stimulation with antigen

Membrane depolarization is one of the earliest events in activation of cells by ligand receptor interaction. It is known that crosslinking of antigen-specific Ig receptors on B cells by antigen can induce membrane depolarization and subsequent Ia antigen expression on the cell surface. To determine whether a tolerance-inducing form of the antigen can also induce membrane depolarization after Ig receptor binding we used splenic B cells enriched for dinitrophenyl (DNP)-specific cells and determined relative membrane potential in these cells after binding of DNP-murine IgG2a (MGG) (tolerogen) or antigens (DNP-keyhole limpet hemocyanin (KLH) and DNP-Ficoll). Relative membrane potential was determined by loading the cells with the dye, 3.3-dipentyloxacarboxyanine (DiOC5(3)) after 2 hr incubation with ligand and determining relative fluorescence intensity on the fluorescence-activated cell sorter (FACS). Carriers alone did not depolarize these normal cell populations, but 100% of DNP-specific cells were depolarized by DNP-KLH and DNP-MGG while 85% were depolarized by DNP-Ficoll. To determine if tolerant B cells could be depolarized by antigen we induced tolerance in vitro or in vivo with DNP-MGG and measured the depolarization of DNP-specific B cells in response to antigens and tolerogen. DNP-specific B cells made tolerant by DNP-MGG underwent membrane depolarization when incubated with either DNP-KLH, DNP-MGG, or DNP-Ficoll but not with carriers alone. These data suggest that tolerogen induces membrane depolarization equally as well as antigen in normal cells. In addition, tolerant cells can be depolarized by Ig receptor crosslinking with either antigen or tolerogen. Thus, tolerance does not block the early membrane events induced by antigen in B cells.