Absence of demonstrable phospholipid turnover in B cells stimulated by low mitogenic concentrations of dextran-anti-immunoglobulin conjugates.
Previously we have demonstrated that when anti-immunoglobulin (Ig) is conjugated to high molecular weight dextran (Dex) it stimulates B cell activation at pg/ml concentrations in the absence of detectable phosphoinositide hydrolysis or increases in intracellular ionized calcium. To study carefully whether anti-Ig-Dex recruited a phosphoinositide-dependent pathway of activation, we stimulated B cells that were labeled with 32P and [3H]glycerol with anti-Ig-Dex conjugates at concentrations ranging from 1-1 x 10(-4) micrograms/ml. Thirty seconds to thirty minutes after stimulation lipids were extracted and analyzed by thin layer chromatography and spots correlating with known lipid standards were isolated and counted. There was a four- and tenfold increase in the ratio of 32P/3H incorporated into phosphatidic acid (a metabolite of diacylglycerol) and phosphatidylinositol, respectively, when cells were stimulated with 0.1-1.0 microgram/ml of anti-Ig-Dex for 30 min. Below 1 ng/ml there was no detectable increase in the turnover of these metabolites despite the fact that in parallel cultures B cells were stimulated to proliferate by this concentration of anti-Ig-Dex. To determine whether a cAMP-dependent pathway was recruited by low concentrations of conjugates, we evaluated cAMP levels from B cells that were stimulated with anti-Ig-Dex for 5-60 min using a radioimmunoassay. While cholera toxin stimulated a 50-100-fold increase in the levels of cAMP, we observed no alteration in cAMP in anti-Ig-stimulated cells. These results support and extend our previous findings by demonstrating that B cell activation that is induced by cross-linking of surface Ig may not stimulate phosphoinositide-dependent or cAMP-dependent pathways of activation. Possible alternative mechanisms of activation will be discussed.
Division of Rheumatology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799.
M
M
Brunswick
E
E
Bonvini
M
M
Francis
C C
CC
Felder
T
T
Hoffman
J
J
Mond
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000032:analysis; D003911:Dextrans / Q000276:immunology*; D005260:Female; D008213:Lymphocyte Activation; D051379:Mice; D008811:Mice, Inbred DBA; D010716:Phosphatidylinositols / Q000378:metabolism*
D000888:Antibodies, Anti-Idiotypic; D001095:Arachidonic Acids; D003911:Dextrans; D010716:Phosphatidylinositols; D016718:Arachidonic Acid; D000242:Cyclic AMP