Surface immunoglobulin-mediated B-cell activation in the absence of detectable elevations in intracellular ionized calcium: a model for T-cell-independent B-cell activation.

Abstract

We recently showed that anti-Immunglobulin conjugated to high molecular weight dextran is 1000-fold more mitogenic for B cells than unconjugated anti-immunoglobulin. This system serves as a model for T-cell-independent ype 2 antigens such as haptenated Ficoll, dextran, and bacterial polysaccharides, which can also stimulate B-cell proliferation and antibody production at low concentrations. We show here that conjugated anti-immunoglobulin, at concentrations that stimulate significant increases in expression of major histocompatibility complex class II molecules and incorporation of thymidine into DNA, does not induce detectable modulation of surface inmunoglobulin. These results indicate that the facilitated T-cell-independent B-cell activation by polysaccharide antigens may result from inability to modulate surface immunoglobulin, possibly resulting in persistent and/or repetitive signaling. Early large increases in Ca2+ and breakdown of inositol phospholipids presently thought to be involved in transduction of the mitogenic signal are not detectable at low concentrations of conjugated anti-immunoglobulin, raising the possibility that these biochemical events may not in fact be central to this signaling pathway.Crosslinking of B-cell-surface immunoglobulin by antigen or anti-Ig antibody has been demonstrated to induce phospholipase C-dependent phosphatidylinositol bisphosphate (PIP2) hydrolysis (1-6). The second messengers thereby generated, diacylglycerol (DAG) and inositol trisphosphate (IP3), lead to activation of protein kinase C (PKC) and mobilization of intracellular ionized calcium ([Ca2]jD (reviewed in refs. 7, 8) Recently we showed that when anti-Ig antibody is conjugated to high molecular weight dextran (anti-Ig-dex) it stimulates B-cell proliferation at concentrations 1000-fold lower than that stimulated by unconjugated anti-Ig antibody (18). Furthermore, at these low concentrations anti-Ig-dex induces greater B-cell proliferation than that induced by unconjugated anti-Ig but induces no modulation of slg and very low levels of binding to slg. This suggests that sIg-modulated B-cell activation could be stimulated despite low-level receptor occupancy and led us to investigate whether these conjugates stimulate B-cell activation via stimulation of PIP2 hydrolysis similar to that induced by high concentrations of unconjugated anti-Ig antibody. The data show that antiIg-dex stimulates greater accumulation ofinositol phosphates and greater increases in [Ca2+]i as compared to comparable concentrations of anti-Ig. However, at lower concentrations that remain fully mitogenic we can detect no such increases. Antibody and Other Reagents. Anti-T-cell antibodies for B-cell purification were anti-Thyl.2 (clone 30-H12), anti-L3T4 (clone GK1.5), anti-Lyt-2 (clone 53-6.7), and rat antimouse immunoglobulin K chain (clone MAR18.5). Other antibodies were anti-Fc-y receptor (clone 24G2), anti-IgD of a allotypes (clone H5a/1 and clone FF1), fluoresceinated anti-Iad (clone MKD6), fluoresceinated anti-IgD of the a a...

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Substrate stiffness regulates B‐cell activation, proliferation, class switch, and T‐cell‐independent antibody responses in vivo

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