Cross-linking of IgG receptors inhibits membrane immunoglobulin-stimulated calcium influx in B lymphocytes.
By cross-linking membrane immunoglobulins (mIg), the antigenic stimulation of B lymphocytes induces an increase in intracellular free calcium levels ([Ca2+]i) because of a combination of release from intracellular stores and transmembrane influx. It has been suggested that both events are linked, as in a number of other cases of receptor-induced increase in [Ca2+]i. Conversely, in B lymphocytes, type II receptors for the Fc fragment of IgG (Fc gamma RII) inhibit mIg-mediated signaling. Thus, we have investigated at the level of single cells if these receptors could act on specific phases of mIg Ca2+ signaling. Lipopolysaccharide-activated murine B splenocytes and B lymphoma cells transfected with intact or truncated Fc gamma RII-cDNA were used to determine the domains of Fc gamma RII implicated in the inhibition of the Ca2+ signal. [Ca2+]i was measured in single fura-2-loaded cells by microfluorometry. The phases of release from intracellular stores and of transmembrane influx were discriminated by using manganese, which quenches fura-2, in the external medium as a tracer for bivalent cation entry. The role of membrane potential was studied by recording [Ca2+]i in cells voltage-clamped using the perforated patch-clamp method. Cross-linking of mIgM or mIgG with F(ab')2 fragments of anti-Ig antibodies induced a sustained rise in [Ca2+]i due to an extremely fast and transitory release of Ca2+ from intracellular stores and a long lasting transmembrane Ca2+ influx. The phase of influx, but not that of release, was inhibited by membrane depolarization. The increase in [Ca2+]i occurred after a delay inversely related to the dose of ligand. Co-cross-linking mIgs and Fc gamma RII with intact anti-Ig antibodies only triggered transitory release of Ca2+ from intracellular stores but no Ca2+ influx, even when the cell was voltage-clamped at negative membrane potentials. These transitory Ca2+ rises had similar amplitudes and delays to those induced by cross-linking mIgs alone. Thus, our data show that Fc gamma RII does 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 mediated by cell depolarization. Thus, Fc gamma RII represents a tool to dissociate physiologically the phases of release and transmembrane influx of Ca2+ triggered through antigen receptors.
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Laboratoire de Neurobiologie Cellulaire, Institut National de la Santé de la Recherche Médicale (INSERM) U261, Institut Pasteur, 75724 Paris, France.
Partiseti
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Amigorena
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Bonnerot
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Fridman
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Korn
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D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000494:pharmacology; D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000494:pharmacology; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008815:Mice, Inbred Strains; D011940:Receptor Aggregation / Q000502:physiology*; D017452:Receptors, IgG / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects
D000888:Antibodies, Anti-Idiotypic; D007140:Immunoglobulin Fab Fragments; D017452:Receptors, IgG; D002118:Calcium