B cell activation. VI. Effects of exogenous diglyceride and modulators of phospholipid metabolism suggest a central role for diacylglycerol generation in transmembrane signaling by mIg

Previous evidence indicates that in vitro activators of protein kinase C, such as phorbol myristate acetate (PMA), are able to induce early activation events in murine B cells, including membrane depolarization and increased I-A antigen expression. These same events are induced by specific antigen and anti-receptor antibody. This evidence suggests that protein kinase C activation may be an important intermediary event in mIg-mediated transmembrane signaling. Previously, investigators have suggested that protein kinase C activation is regulated by a novel second messenger, diacylglycerol (DG), and DG is generated by phosphatidylinositol (PI) hydrolysis after receptor-ligand interaction in many systems. In view of this concept, we examined the effects of nonspecific activators and inhibitors of DG production and DG itself on membrane potential and levels of I-A antigen expression in murine B cells. Our results indicate that exposure to DG, or induction of DG production by treatment of B cells with exogenous phospholipase C, results in depolarization and increased I-A antigen expression similar to that induced by anti-receptor antibody and specific antigen. Furthermore, we demonstrate that depolarization and increased I-A expression induced by anti-receptor antibody is blocked under conditions in which DG production is inhibited. As expected, based on its direct activation of protein kinase C, PMA stimulation is unaffected by this inhibition. These results support our earlier hypothesis that occupancy of antigen receptors on B cells is linked to subsequent activation events by PI hydrolysis, DG generation, and protein kinase C activation.     

B cell activation. VII. Independent and synergistic effects of mobilized calcium and diacylglycerol on membrane potential and I-A expression

Although cross-linking of murine B cell membrane Ig (mIg) has been shown to induce a rapid increase in intracellular free calcium [Ca++)i), both the source and the function of the Ca++ in lymphocyte activation is unclear. Toward elucidation of its function, we investigated the relationship between the initial (Ca++)i response and other cell physiologic changes that occur early after mIg cross-linking, apparently as a linear cascade, leading to increased membrane I-A expression. Results suggest that the (Ca++)i response results from polyphosphoinositol hydrolysis induced by mIg cross-linking. The (Ca++)i response cannot be induced by activation of protein kinase C (PKC) with phorbol diesters (e.g., PMA) or synthetic diacylglycerol (DAG), suggesting that this response precedes the PKC activation. However, inhibition of phosphatidylinositol turnover by exposure of cells to dbcAMP during anti-Ig stimulation significantly inhibits the (Ca++)i response, suggesting that phosphatidylinositol turnover may be causally related to Ca++ mobilization. The ability of exogenous phospholipase C to induce the (Ca++)i response also supports this conclusion. Of the products of mono- and poly-phosphatidylinositol hydrolysis, the inositol phosphates (InsP, InsP2, InsP3) are implicated as promoters of Ca++ mobilization, because exogenous synthetic diacylglycerol is without effect on (Ca++)i. In light of recent evidence obtained with other systems, we suggest that InsP3 is responsible for mIg cross-linking-induced Ca++ mobilization from intracellular stores in B lymphocytes. Both depolarization and increased I-A expression are induced by increasing (Ca++)i with the Ca++ ionophores A23187 and ionomycin. These events can also be induced by the activation of PKC with high doses of PMA. When suboptimal doses of both A23187 and PMA are present, these reagents synergize in the induction of depolarization. This suggests that one role for the initial rise in (Ca++)i is to act with the DAG liberated from PtdIns turnover, possibly by enhancing translocation of cytosolic PKC to the plasma membrane, and thereby promote changes in ion transport that are apparent as a decrease in the membrane potential.     

Translocation of protein kinase C during membrane immunoglobulin-mediated transmembrane signaling in B lymphocytes

Previous studies have implicated a role for protein kinase C (PKC) in transmembrane signal transduction by B cell surface immunoglobulin (Ig). Specifically, the pharmacologic PKC activator phorbol myristate acetate mimics the biologic effects of mIg cross-linking ligands, and cross-linking of membrane Ig (mIg) induces polyphosphoinositide hydrolysis generating diacylglycerol, a potent activator of PKC. Studies described here additionally implicate PKC in mIg-mediated signaling by demonstrating rapid translocation of activatable PKC (PKCa) from cytosol to Triton-soluble membrane fractions after cross-linking of cell surface IgM or IgD. This response, which is also induced by phorbol myristate acetate and lipolysaccharide, is detectable within 1 min of mIg cross-linking and is followed within 4 min by additional translocation of PKCa to a Triton-insoluble particulate compartment. The ability of dbcAMP plus theophylline to inhibit polyphosphoinositide hydrolysis, PKCa translocation, and the B cell's subsequent biological response suggests that these events may be causally related.     

B cell activation. IV. Induction of cell membrane depolarization and hyper-I-A expression by phorbol diesters suggests a role for protein kinase C in murine B lymphocyte activation

Analysis of the effects of phorbol diesters on mouse B lymphocyte kinase C activity, membrane potential, mI-A expression, and cell cycle state are reported. Results indicate that the phorbol diesters PMA and 4 beta-PDD, which are potent tumor promoters, activate partially purified B cell protein kinase C and stimulate B cell membrane depolarization and increased mI-A expression. The analog 4 alpha-PDD has none of these effects. Similarly, none of the phorbol diesters tested promoted G0 to G1 transition of B lymphocytes. Results are consistent with the possibility that the transmembrane signal transduction mediated by cell membrane immunoglobulin, which results in membrane depolarization and increased I-A antigen expression, operates via activation of protein kinase C.     

Generation of phosphatidic acid and diacylglycerols following ligation of surface immunoglobulin in human B lymphocytes: potential role in PKC activation.

We have examined signal transduction via membrane IgM (mIgM) in resting and cycling human B cells. Crosslinking mIgM on all of the cell types studied transduced a signal through the phosphatidylinositol pathway, producing inositol 1,4,5-trisphosphate and release of intracellular free calcium. These second messengers were formed regardless of quantitative or qualitative differences in the surface expression of mIgM: cells that had low levels of surface IgM (T-51) or had no light chain associated with surface heavy chain (DB) signaled phosphatidylinositol pathway activation after mIgM crosslinking. Production of specific lipid products in nonquiescent B cells differed from that in normal resting cells. Ligation of surface immunoglobulin on resting B cells resulted in sustained increases of both diacylglycerol and phosphatidic acid, two lipids that can influence PKC activation. Whereas PKC was strongly activated in normal tonsillar B cells, several cell lines had reduced PKC activation following crosslinking of mIgM. The reduction in protein kinase C activation correlated with the absence or reduced levels of phosphatidic acid or diacylglycerol following stimulation: protein kinase C translocated and was activated only in cells that had elevated levels of both diacylglycerides and phosphatidic acid. Anti-IgM-induced phosphorylation of a protein kinase C substrate protein CD20, also increased in those cells having PKC activation and not in cells in which kinase activity was reduced. CD20 phosphorylation also increased following the direct addition of exogenous phosphatidic acid to resting B cells. Together, these observations show that the generation of lipid products following mIgM crosslinking in resting cells can vary from that in cycling cells and may relate to the different levels of PKC activation. In a companion study we report that ligation of surface IgM activates both an acyltransferase and phospholipase D to form phosphatidic acid.     

Constitutive turnover of inositol-containing phospholipids in B220+ T cells from autoimmune-prone MRL-lpr/lpr mice.

B220+ T cells from mice that are homozygous for the lpr gene exhibit profound defects in their capacity to produce and respond to IL-2 and provide a cellular model for investigating the basic requirements for effective transmembrane signal transduction in immunologically normal T cells. A correlation between defective lectin-stimulated proliferation and deficient hydrolysis of inositol-containing phospholipids (PI) has recently been demonstrated in B220+ T cells. The finding has been postulated to explain abnormal expression of protein kinase C (PKC) activity in these cells. In a previous study, we found that the constitutive turnover of [3H]arachidonyl-PI was markedly increased in B220+ T cells from lpr-bearing MRL mice relative to that in controls. This observation suggested that an inability to metabolize PI and to generate second messengers putatively necessary for transmembrane signaling might not be responsible for aberrant PKC activity in B220+ T cells. To clarify this issue, the constitutive turnover of phosphoinositides in B220+ T cells from autoimmune-prone MRL-lpr/lpr mice was investigated. We found that in the absence of stimulation with exogenous Ag, B220+ T cells exhibited greatly increased 1) incorporation of labeled myoinositol into PI, 2) production of inositol phosphates in cells prelabeled with [3H]myoinositol, and 3) formation of diacylglycerol in [3H]arachidonic acid-labeled cells. Increased spontaneous PI turnover in B220+ cells was associated with normal phosphatidyl inositol-4,5-biphosphate-phospholipase C activity in membrane homogenates, normal levels of membrane PI, and normal resting and mitogen-stimulated levels of intracellular free-ionized Ca2+. The results suggest that an incomplete form of the PI cycle, one unassociated with PKC activation, is constitutively expressed in B220+ T cells.     

Polyclonal activation of the murine immune system by an antibody to IgD. XI. Contribution of membrane IgD cross-linking to the generation of an in vivo polyclonal antibody response

The injection of mice with a foreign, polyclonal antibody to IgD sequentially induces: 1) activation of B cells by cross-linking of their cell membrane (m) IgD; 2) B cell processing and presentation of the bound anti-IgD antibody to T cells; 3) activation of these T cells; and 4) T-dependent stimulation of B cell differentiation into IgG1 secreting cells. To determine whether the cross-linking of B cell membrane IgD and/or the resulting B cell activation that follows contribute to the generation of the polyclonal IgG1 response, we examined the abilities of three sets of anti-delta mAb or mAb fragments to stimulate polyclonal IgG1 production. Within each set mAb were matched for species and Ig isotypic determinants, but differed in avidity for IgD or in ability to cross-link IgD. In addition, experiments were performed to determine whether the anti-delta mAb had to be foreign to the immunized mouse to stimulate an IgG1 response. Results of these experiments indicate that: 1) recognition of the injected anti-delta antibody as foreign is required for the induction of a polyclonal IgG1 response; 2) the cross-linking of B cell membrane Ig, which directly activates B cells, can contribute considerably to the generation of in vivo IgG1 production; and 3) that even relatively weak cross-linking of membrane Ig by ligands that bind it with low avidity can make this contribution.     

Translocation of the B cell antigen receptor into lipid rafts reveals a novel step in signaling

The cross-linking of the B cell Ag receptor (BCR) leads to the initiation of a signal transduction cascade in which the earliest events involve the phosphorylation of the immunoreceptor tyrosine-based activation motifs of Ig alpha and Ig beta by the Src family kinase Lyn and association of the BCR with the actin cytoskeleton. However, the mechanism by which BCR cross-linking initiates the cascade remains obscure. In this study, using various A20-transfected cell lines, biochemical and genetic evidence is provided that BCR cross-linking leads to the translocation of the BCR into cholesterol- and sphingolipid-rich lipid rafts in a process that is independent of the initiation of BCR signaling and does not require the actin cytoskeleton. Translocation of the BCR into lipid rafts did not require the Ig alpha/Ig beta signaling complex, was not dependent on engagement of the FcR, and was not blocked by the Src family kinase inhibitor PP2 or the actin-depolymerizing agents cytochalasin D or latrunculin. Thus, cross-linking or oligomerization of the BCR induces the BCR translocation into lipid rafts, defining an event in B cell activation that precedes receptor phosphorylation and association with the actin cytoskeleton.     

Up-regulation of c-fos expression is a component of the mIg signal transduction mechanism but is not indicative of competence for proliferation

Control of entry into and progression through the early phases of cell cycle in B lymphocytes is poorly understood at the molecular level. Products of the c-fos proto-oncogene have been implicated in regulation of G0 to G1 cell cycle phase transition and cell proliferation in other systems. In view of these observations, the relationship between signals generated through receptor Ig which alter the B cells position in cell cycle and relative level of c-fos expression was investigated. Not unexpectantly, anti-Ig under conditions which promote G0-G1 and G1-S phase transition was observed to selectively up-regulate expression of c-fos. More interestingly, however, anti-Ig-induced cross-linking of surface Ig on the WEHI-231 B lymphoma also caused rapid and transient up-regulation of c-fos mRNA levels although it was associated with inhibition of proliferation of these cells. These results are important because they show that 1) c-fos expression is inducible in both normal and transformed B lymphocytes as a consequence of signals generated through receptor Ig, and 2) up-regulation of c-fos expression is not positively linked to B cell proliferation but rather appears to be a component of the surface Ig signal transduction mechanism. Finally, studies utilizing phorbol diesters suggest that pathways leading through protein kinase C are involved in both the growth inhibition and c-fos expression WEHI-231 following membrane-associated Ig cross-linking.     

Modeling of T cell contact-dependent B cell activation. IL-4 and antigen receptor ligation primes quiescent B cells to mobilize calcium in response to Ia cross-linking.

The generation of antibody secretory cells from resting B lymphocytes after immunization with most protein Ag requires B cell signaling by Ag, direct Th cell contact and lymphokines. Previous studies suggest that cell contact-mediated signals may be transduced by Ia after Ia binding by alpha beta TCR and/or CD4. Seemingly inconsistent with this concept are findings that cross-linking of Ia molecules on quiescent B cells leads to cAMP generation that is antagonistic for B cell mitogenesis. Here we show that ligand binding to IL-4 and Ag receptors on quiescent B cells induce transition of these cells into a competent state in which Ia molecules transduce signals via a distinct mechanism. This mechanism involves the tyrosine kinase-dependent activation of phospholipase C leading to Ca2+ mobilization from intracellular stores and the extracellular space. This competence, which is seen within 4 h of priming, is not simply a function of increased Ia expression by the B cell because the response can be induced by cross-linking of less than 5% of cell surface Ia molecules on primed cells. Finally, cross-linking of Ia molecules leads to more than fivefold greater increase in [Ca2+]i than is induced by membrane Ig ligation. These findings are consistent with alpha beta TCR/CD4 delivery via Ia of proliferative signals mediated by tyrosine kinase activation, phosphoinositide hydrolysis and Ca2+ mobilization.     

Vav-dependent and vav-independent phosphatidylinositol 3-kinase activation in murine B cells determined by the nature of the stimulus

We show in this study that B cell activation following high avidity ligation of IgM or coligation of membrane Ig with CD19 elicits similar levels of Ca(2+) flux using different mechanisms. Each form of activation requires the function of Vav and PI3K. However, Vav regulates Ca(2+) flux independently of PI3K following anti-IgM cross-linking. By contrast, Vav function is essential for PI3K activation following membrane Ig (mIg)/CD19 coligation. Inhibition of PI3K revealed anti-IgM-stimulated Ca(2+) flux has a PI3K-independent component, while Ca(2+) flux following mIg/CD19 coligation is totally PI3K dependent. The p85alpha and p110delta subunits of PI3K both participate in anti-IgM and mIg/CD19 coligation-induced Ca(2+) flux, although the defects are not as severe as observed after pharmacological inhibition. This may reflect the recruitment of additional PI3K subunits, as we found that p110alpha becomes associated with CD19 upon B cell activation. These data show that the nature of the Ag encountered by B cells determines the contribution of Vav proteins to PI3K activation. Our results indicate that the strong signals delivered by multivalent cross-linking agents activate B cells in a qualitatively different manner from those triggered by coreceptor recruitment.     

Antigen receptor-induced signal transduction imbalances associated with the negative selection of immature B cells

Signals transduced through the B cell Ag receptor (BCR) drive B cell development. However, BCR-induced responses are developmentally regulated; immature B cells are tolerized following antigenic exposure while mature B cells are triggered to proliferate and differentiate. This differential responsiveness allows for the negative selection of self-reactive immature B cells while simultaneously allowing for clonal expansion of mature B cells in response to foreign Ags. Intrinsic differences in BCR-induced signal transduction at various stages of development may account for this functional dichotomy. We had previously demonstrated that the BCR-induced proliferation of mature B cells is accompanied by an increase in intracellular calcium levels and polyphosphoinositide bis phosphate (PIP2) hydrolysis. In contrast, immature B cells that undergo BCR-induced apoptosis increase intracellular calcium in the relative absence of PIP2 hydrolysis. Since PIP2 hydrolysis leads to the generation of diacylglycerol, a cofactor for protein kinase C (PKC) activation, these data suggested that an "imbalance" in BCR-induced signal transduction resulting from a relative inability to activate PKC may play a role in the susceptibility of immature B cells to BCR-induced apoptosis. In support of this hypothesis, we demonstrate that PKC activation can rescue immature B cells from BCR-induced apoptosis. Furthermore, the susceptibility of immature B cells to BCR-induced apoptosis is recapitulated in mature B cells that are either PKC depleted or are stimulated in the presence of PKC inhibitors, suggesting that an uncoupling of PKC activation from BCR-induced signaling is responsible for the apoptotic response of immature B cells.     

Membrane IgM-mediated signaling of human B cells. Effect of increased ligand binding site valency on the affinity and concentration requirements for inducing diverse stages of activation.

The potential for ligand-initiated signal transduction through B cell membrane IgM is assessed in terms of ligand concentration, binding site valency, and binding site affinity for membrane Ig. Estimates of the physicochemical requirements for achieving G0* enhancement of class II MHC expression, G1 entry, and S phase entry in human B cells were made by comparing the stimulatory effects of three affinity-diverse anti-Cmu2 mAb when in bivalent (unconjugated) form, or as mAb-dextran conjugates with low binding site valency (oligovalent ligands) or high binding site valency (multivalent ligands). An increase in binding site number (and concomitant molecular mass) caused a profound reduction in both the minimal concentration and affinity requisites for B cell activation. The enhancing effect of increased binding site valency was most evident for the signaling of those most distal stages in B cell activation, i.e., G1 and S phase, which were difficult to induce with bivalent ligands. The results suggest that highly multimeric TI-2 Ag may be good immunogens because they are able to elicit a full activation response not only from infrequent high affinity B cells, but also from a substantial proportion of the many lower affinity Ag-specific B cells in virgin B cell populations. Interestingly, the activation of B cells by ligands with binding sites of high intrinsic affinity (Ka = 5 x 10(8) M-1) was less influenced by increases in binding site valency than was B cell activation by ligands with intermediate binding site affinity (Ka = 2 x 10(7) M-1). This suggests that the minimal epitope valency requirement for T cell-independent B cell activation by mIg cross-linking Ag may be dependent on the intrinsic affinity with which membrane Ig molecules on a given B cell interact with the redundantly expressed epitopes.     

EGF-dependent cell cycle progression is controlled by density-dependent regulation of Akt activation

The normal human breast epithelial cell line, MCF10A, was used to investigate the mechanism by which high-density inhibits EGF-dependent cell cycle progression. EGF-dependent Akt activation was found to be transient in high-density cells and sustained in low-density cells. High-density cells also showed decreased EGF receptor (EGFR) autophosphorylation, decreased retinoblastoma protein phosphorylation, and increased p27 protein expression. Although EGFR activation was decreased in the high-density cells, the activation was sufficient to stimulate EGFR substrates comparable to low-density cells. EGF-dependent activation of the Erk1/2 pathway and the upstream activators of Akt (Gab1, erbB3, PI3 kinase, and PDK1) showed no density dependency. Antagonists of Akt activity provided further evidence that regulation of Akt activation is the critical signal transduction step controlling EGF-dependent cell cycle progression. Both adenovirus-mediated expression of dominant-negative Akt and inhibition of PI3 kinase-mediated Akt activation with LY294002 blocked cell cycle progression of low-density cells. In summary, we report the novel finding that high-density blocks EGF-dependent cell cycle progression by inhibiting EGF signaling at the level of EGF-dependent Akt activation rather than at the level of EGFR activation.     

Signaling through surface IgM in tolerance-susceptible immature murine B lymphocytes. Developmentally regulated differences in transmembrane signaling in splenic B cells from adult and neonatal mice.

During the course of B lymphocyte development, newly emerging surface Ig+ B cells pass through a stage when Ag-Ag receptor interactions lead not to immune responsiveness but to a state of functional tolerance. We have explored the molecular basis of antigenic nonresponsiveness and tolerance susceptibility using tolerance-susceptible surface Ig+ splenic B lymphocytes from neonatal mice and anti-mu chain antibodies as a polyclonal ligand. In this population of cells, surface IgM is uncoupled from the inositol phospholipid (PI)-hydrolysis pathway at a point proximal to the receptor; anti-mu antibodies did not stimulate inositol phosphate generation despite the fact that PI-hydrolysis was observed after treatment with A1F4, implicating the existence of a functional G protein and phospholipase C. Further evidence for a difference early in the signal transduction pathway stems from the finding that anti-mu stimulation does not induce the expression of two immediate/early PKC-linked genes egr-1 and c-fos. This appears to be the primary signaling difference between the mature and immature B cells from the neonatal mouse splenic population, as these cells undergo a G0-G1 cell cycle phase transition when surface IgM is bypassed using phorbol diester and calcium ionophore. Interestingly, despite undetectable levels of PI-hydrolysis, we observed equivalent receptor-mediated changes in intracellular calcium when comparing the immature and mature populations. These results indicate incomplete coupling of surface IgM to the signal transduction machinery operative in mature, immunocompetent B cells and suggests a molecular mechanism accounting for the differential processing of surface IgM signals into activation vs tolerogenic responses observed in these two stages of B cell development.     

The role of tyrosine phosphorylation in signal transduction through surface Ig in human B cells. Inhibition of tyrosine phosphorylation prevents intracellular calcium release.

Cross-linking surface Ig on human B cells, or the TCR complex on T cells leads to the rapid appearance of newly tyrosine phosphorylated proteins. This is associated with inositol phospholipid turnover and a rise in intracellular calcium. Incubation of human B or T lymphocytes with the tyrosine kinase inhibitors, herbimycin and genistein, inhibits new tyrosine phosphorylation after receptor-linked activation. This is associated with complete abrogation of the increase in intracellular calcium in these lymphocytes and inhibition of inositol phospholipid turnover. Herbimycin- and genistein-treated lymphocytes are nevertheless still capable of responding to aluminum fluoride with a rise in intracellular calcium. These data support the contention that a B cell-associated protein tyrosine kinase regulates signal transduction via phospholipase C. CD45, the membrane associated protein tyrosine phosphatase, and PMA that activates protein kinase C, both inhibit the calcium response in B lymphocytes induced by receptor cross-linking. PMA and cross-linking CD45 both induced the appearance of tyrosine phosphorylated proteins in human B cells, although the pattern is quite distinct from that seen when surface lg is cross-linked. However, the induction of new tyrosine phosphorylation by anti-mu does not appear to be affected by these reagents. Although this may reflect an insensitivity of the tyrosine phosphorylation assay, it could indicate that regulation of the calcium response and regulation of the tyrosine kinase can be independent processes.     

B cell activation. II. Receptor cross-linking by thymus-independent and thymus-dependent antigens induces a rapid decrease in the plasma membrane potential of antigen-binding B lymphocytes

We report the specific induction of B cell plasma membrane depolarization with the use of thymus-dependent and -independent antigens. We have utilized various trinitrophenol-carrier conjugates for the stimulation of isolated trinitrophenol-binding mouse B cells. Membrane depolarization was assessed by flow cytometric analysis of 3-3'-pentyloxacarbocyanine iodide (DiOC5[3])-stained cells. Entry into the cell cycle was determined by flow cytometric analysis of acridine orange-stained cells. The results indicate that polyvalent antigens, but not free hapten, induce B cell membrane depolarization by a large proportion of antigen-binding cells within 2 hr of stimulation. Although all polyvalent antigens induce membrane depolarization, only thymus-independent antigens induce the subsequent G0 to G1 transition, suggesting that the membrane Ig cross-linking signal alone, although sufficient to induce membrane depolarization and subsequent increased IA expression, is insufficient to drive the entry of B cells into the cell cycle. The G0 to G1 transition appears to be dependent on a second signal, perhaps mediated by the thymus-independent carrier or antigen-specific, Ia-restricted T cell helper.