Stimulation of resting B lymphocytes with antibodies to surface unoglobulin (sIgD or sIgM) induces protein tyrosine phosphorylation, implicating one or more Bcell protein-tyrosine kinases (PTKs) in sIg signal transduction.We have evaluated whether members of the src family of PTKs are involved in this process. (12)(13)(14)(15). Of the enzymes that may be involved in B-cell tyrosine phosphorylation signaling, members of the src family of protein-tyrosine kinases (PTKs) need to be considered as direct or indirect slg signaling components, based on previous evidence implicating p56lck and p60tf" in T-cell proliferation and activation events (refs. 16-18; reviewed in refs. 19 and 20). Moreover, the recent description of a B-cell-specific member of the src family, blk (21), suggests that this group of PTKs may be enzymes recruited in the B-cell activation cascade.

It has been found that the principal biochemical pathway activated in B cells stimulated by antigen-or anti-immunoglobulin-mediated crosslinking of surface immunoglobulin is that resulting in hydrolysis of phosphatidylinositol bisphosphate with generation ofdiacylglycerol and inositol trisphosphate. Recent evidence suggests that surface immunoglobulin-mediated B-cell activation can proceed without detectable increases in the concentration of either diacylglycerol or intracellular Ca2+ concentration, implicating involvement of other non-protein-kinase-C/Ca2+-dependent signal-transduction pathways. Therefore, we sought evidence for activation of a signalin pathway that is associated with growth regulation in other cell types-i.e., the protein-tyrosine kinases. We now show that crosslinking of membrane immunoglobulin by mitogenic antibodies leads to rapid tyrosine phosphorylation of several cellular substrates, consistent with the induction of a tyrosine kinase activity. This increase in tyrosine phosphorylation is weakly (if at all) stimulated by other B-cell mitogens, including phorbol esters and ionophores, and does not require the presence of detectable protein kinase C. Furthermore, inhibition ofanti-immunglobulin-stimulated phosphatidylinositol bisphosphate hydrolysis does not inhibit activation of this tyrosine kinase-dependent pathway. These findings suggest that occupancy of the membrane immunglobulin receptor may induce multiple pathways of activation.Antigen interaction with the B-cell surface immunoglobulin (sIg) receptor stimulates resting B cells to proliferate and, in the presence of T cells or T cell-derived lymphokines, to secrete immunoglobulin (1)(2)(3)(4). While the biochemical mechanisms that underlie B-cell activation are beginning to be unraveled, there is one pathway of activation that has received considerable attention. Many investigators have demonstrated that crosslinking of sIg leads to hydrolysis of phosphatidylinositol bisphosphate (Ptdlns-P2) with the generation of diacylglycerol and inositol trisphosphate (InsP3) (5-9). The released diacylglycerol activates protein kinase C (PKC) (10), and inositol trisphosphate leads to a rise in intracellular calcium ([Ca2+Ji) (11). This pathway was thought to be the major, if not only, sIg-mediated signaling pathway in B cells. We and others have demonstrated that antiimmunoglobulin (anti-Ig)-mediated B-cell stimulation can proceed in the absence of cellular PKC, implicating a role for a non-PtdIns-P2-dependent pathway of sIg activation (12, 13). More recently, we have shown that dextran-conjugated anti-Ig at pg/ml concentrations stimulates B-cell proliferation in the absence ofdetectable PtdIns-P2 hydrolysis or increases in [Ca2+], (14,15), thus also raising the possibility that other PtdIns-P2-independent pathways are being recruited during sIg-mediated signaling. A third line of evidence supporting this possibility was our recent finding that inhibition of anti-Ig-stimulated PtdIns-P2 hydrolysis by PKC activators enhanced rather than...

SummarySurface immunoglobulin (slg)-mediated stimulation of B lymphocytes induces a tyrosine kinase-dependent sequence of events leading to rapid and large elevations in intracellular ionized calcium ([Ca2+]i). These early biochemical events do not necessarily lead to proliferation of B cells, however, and conversely, the absence of or inhibition of these events does not necessarily prevent cellular proliferation. We now show by digital image analysis of single B cells that conditions which lead to B cell proliferation are associated with low-level but persistent sustained or cyclic elevations in [Ca 2 + ]i. In marked contrast, early and nonsustained elevations in [Ca 2 § ]i are induced in B cells by stimuli that lead to G1 transition but fail to progress to DNA synthesis. Thus, when B cells were stimulated with mitogenic and nonmitogenic anti-IgD antibodies, both of which induce entry of cells into G1 and early calcium transients of comparable magnitude, persistent low-level calcium elevations were only detected in cells stimulated with the mitogenic antibody. Furthermore, persistent calcium elevations were also seen when B cells were stimulated with a multivalent dextran-anti-Ig conjugate which induced very high levels of B cell proliferation in the absence of detectable phosphatidylinositol 4,5-biphosphate hydrolysis or elevations in [Ca2+]i as detected by flow cytometry. Finally, B cells from X-linked B cell-defective mice, which do not proliferate in response to anti-Ig antibody, show marked and early increases in [Ca2+]i, but do not show persistent calcium elevations. These data suggest that the rapid and large increases of [Ca 2 + ]i seen in lymphocytes within seconds after antigen receptor ligation may be associated with entry in G1, whereas low-level but persistent elevations may be the hallmark of a cell destined to synthesize DNA. kinase (8-10), all of which are intimately involved in cell growth of B cells as well as other cell types. Although it is clear that the summation of these events reflects an early response to sIg-mediated signal transduction in B cells which will lead to cell growth, the specific role of each event in regulating entry of the cell into G1 or S of the cell cycle is not known. Experiments both in human and in murine systems suggest that there is no correlation between the ability of an antigen or of anti-Ig antibody to induce phosphatidylinositol 4,5-biphosphate (PIP2) activation or tyrosine kinase activation and its ability to induce B cell DNA synthesis (11)(12)(13)(14). Thus, for example, inhibition of sIg-mediated PiP2 hydrolysis does not prevent B cell proliferation (15). Furthermore, stimulation of these early events by anti-Ig antibodies does not necessarily lead to B cell proliferation (13,16,17).

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...

In order to determine the carrier nature of lipopolysaccharide from Brucella abortus (LPS-BA) in evoking humoral responses, normal and immunodeficient mice were immunized with trinitrophenyl (TNP)-conjugated LPS-BA (TNP-LPS-BA) and the responses were compared with those to known T-dependent and T-independent antigens. TNP-LPS-BA, like T-independent type 1 (TI-1) antigens such as TNP-BA and TNP-LPS from Escherichia coli (TNP-LPS-EC), generated anti-TNP responses in BALB/c, athymic BALB/c nulnu, and CBAIN mice. In contrast, N-2,4-dinitrophenyl-j8-alanylglycylglycyl-substituted keyhole limpet hemocyanin, a typical T-dependent antigen, was not immunogenic in athymic mice, and TNP-Ficoll (T-independent type 2) was ineffective in eliciting humoral responses in CBA/N mice. These results indicate that LPS from B. abortus acts as a TI-i carrier in generating antibody responses. In C3H/HeJ mice, TNP-LPS-BA generated higher-titer immunoglobulin Gi (IgGI), IgG2a, and IgG2b anti-TNP antibodies than TNP-LPS-EC. Compared with those from BALB/c mice, pure resting B cells isolated from C3H/HeJ mice exhibited a 30-fold lower proliferative response to LPS-EC, whereas the LPS-BA response was reduced to a lesser extent (5-fold). This suggests that the disparity observed in antibody titers was due to different abilities of LPS from B. abortus and E. coli to stimulate C3H/HeJ B cells. The ability of LPS from B. abortus to act as a carrier in generating humoral immune responses indicates that LPS-BA can be substituted for whole B. abortus organisms in vaccine development. * Corresponding author. polysaccharides such as TNP-Ficoll are TI-2 antigens. Typically, TI-1 and TI-2 antigens are able to induce IgM antibody production in athymic nude mice, whereas a TD antigen, e.g., DNP-KLH, will induce little or no antibody