The B lymphocyte calcium response to anti-Ig is diminished by membrane immunoglobulin cross-linkage to the Fc gamma receptor.
We report the cytosolic free calcium, [Ca2+]i, responses of single murine B lymphocytes to whole and F(ab')2 fragments of anti-Ig measured in the flow cytometer with indo-1, a new fluorescent chelator of calcium. The principle advantages of this recording system are these: Indo-1 is highly fluorescent; hence, loading concentrations that introduce artifacts in the reported [Ca2+]i signal may be avoided. The measurement of [Ca2+]i by fluorescence ratio corrects for nonuniform dye uptake, making possible quantitative estimates of [Ca2+]i in single cells and an assessment of the variability of population responses. Baseline recordings of unstimulated lymphocytes indicated a narrow, stable range of [Ca2+]i (75 to 125 nM). The [Ca2+]i rise induced by various anti-Ig preparations exhibited considerable heterogeneity. The initial mean value for F(ab')2 anti-Ig-stimulated cells peaked above 1 microM and was due only to the release of Ca2+ from intracellular stores. A steady state elevation of [Ca2+]i was reached by 5 min and persisted for hours. Cells stimulated with intact anti-Ig reached similar initial peak [Ca2+]i values, but then declined toward baseline. This difference was due to membrane Ig-IgG Fc receptor (mIg-Fc gamma R) cross-linkage, because blocking the Fc gamma R with a monoclonal antibody made the [Ca2+]i responses to F(ab')2 and intact anti-Ig identical. The attenuation of the [Ca2+]i signal by mIg-Fc gamma R cross-linkage is proceeded by a corresponding Fc gamma-mediated reduction in anti-Ig-induced inositol trisphosphate elevation. These findings outline a biochemical basis for mIg- and Fc gamma R-mediated activation and regulation intrinsic to the B cell, and demonstrate the advantages of indo-1 over quin2 for fluorescent measurement of [Ca2+]i in small cells.
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D001402:B-Lymphocytes / Q000502:physiology*; D002118:Calcium / Q000502:physiology*; D003432:Cross-Linking Reagents; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007138:Immunoglobulin delta-Chains / Q000276:immunology; D007148:Immunoglobulin mu-Chains / Q000276:immunology; D007295:Inositol Phosphates / Q000378:metabolism; D051379:Mice; D011947:Receptors, Antigen, B-Cell / Q000502:physiology*; D011961:Receptors, Fc / Q000502:physiology*; D017452:Receptors, IgG
D000888:Antibodies, Anti-Idiotypic; D003432:Cross-Linking Reagents; D007140:Immunoglobulin Fab Fragments; D007138:Immunoglobulin delta-Chains; D007148:Immunoglobulin mu-Chains; D007295:Inositol Phosphates; D011947:Receptors, Antigen, B-Cell; D011961:Receptors, Fc; D017452:Receptors, IgG; D002118:Calcium