CROSS-LINKING OF IGG RECEPTORS INHIBITS MEMBRANE IMMUNOGLOBULIN-STIMULATED CALCIUM INFLUX IN B-LYMPHOCYTES

By cross-linking membrane immunoglobulins (mIg), the antigenic stimula tion of B lymphocytes induces an increase in intracellular free calciu m levels ([Ca2+]i) because of a combination of release from intracellu lar stores and transmembrane influx. It has been suggested that both e vents are linked, as in a number of other cases of receptor-induced in crease in [Ca2+]i. Conversely, in B lymphocytes, type II receptors for the Fc fragment of IgG (FcgammaRII) inhibit mIg-mediated signaling. T hus, we have investigated at the level of single cells if these recept ors could act on specific phases of mIg Ca2+ signaling. Lipopolysaccha ride-activated murine B splenocytes and B lymphoma cells transfected w ith intact or truncated FcgammaRII-cDNA were used to determine the dom ains of FcgammaRII implicated in the inhibition of the Ca2+ signal. [C a2+]i was measured in single fura-2-loaded cells by microfluorometry. The phases of release from intracellular stores and of transmembrane i nflux were discriminated by using manganese, which quenches fura-2, in the external medium as a tracer for bivalent cation entry. The role o f membrane potential was studied by recording [Ca2+]i in cells voltage -clamped using the perforated patch-clamp method. Cross-linking of mIg M or mIgG with F(ab')2 fragments of anti-Ig antibodies induced a susta ined rise in [Ca2+]i due to an extremely fast and transitory release o f Ca2+ from intracellular stores and a long lasting transmembrane Ca2 influx. The phase of influx, but not that of release, was inhibited b y membrane depolarization. The increase in [Ca2+]i occurred after a de lay inversely related to the dose of ligand. Co-cross-linking mIgs and FcgammaRII with intact anti-Ig antibodies only triggered transitory r elease of Ca2+ from intracellular stores but no Ca2+ influx. even when the cell was voltage-clamped at negative membrane potentials. These t ransitory Ca2+ rises had similar amplitudes and delays to those induce d by cross-linking mIgs alone. Thus, our data show that FcgammaRII doe s not mediate an overall inhibition of mIg signaling but specifically affects transmembrane Ca2+ influx without affecting the release of Ca2 + from intracellular stores. Furthermore, this inhibition is not media ted by cell depolarization. Thus, FcgammaRII represents a tool to diss ociate physiologically the phases of release and transmembrane influx of Ca2+ triggered through antigen receptors.