Absence of demonstrable phospholipid turnover in B cells stimulated by low mitogenic concentrations of dextran‐anti‐immunoglobulin conjugates

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Abstract
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 × 10−4 μg/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 μg/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.