Lymphoma models for B-cell activation and tolerance. VII. Pathways in anti-Ig-mediated growth inhibition and its reversal

WEHI-231, CH33, and CH31 are B-cell lymphomas that are inhibited in their growth by crosslinking of surface Ig receptors during early G1. This "negative signaling" process can be prevented or reversed under certain conditions. In the present paper, we use a cell synchronization procedure to demonstrate that activation of protein kinase C (PKC) is not involved in the negative signal for growth, but rather that PKC activation prevents growth inhibition when present early in the cell cycle. Indeed, the prevention of negative signaling is only accomplished by active phorbol esters. Moreover, phorbol esters and a calcium ionophore fail to deliver a negative signal under conditions in which anti-Ig can significantly prevent cell cycle progression into S phase, thereby ruling out synergy between PKC and calcium in growth inhibition. Whether phorbol esters reverse negative signaling by preventing internalization of the immune complex or phosphorylation of a critical intracellular protein is discussed.     

Lymphoma models for B-cell activation and tolerance. IX. Efficient reversal of anti-Ig-mediated growth inhibition by an activated TH2 clone.

We have utilized several B-cell lymphomas that are growth inhibited by anti-Ig reagents as models for tolerance induction. In a previous communication, we demonstrated that the growth inhibition by anti-Ig can be partially prevented by the recombinant lymphokine, IL-4. In this paper, we report that complete protection of B lymphomas from anti-Ig was provided by a type 2 helper cell clone, D10.G4, when these T cells were activated by monoclonal anti-CD3. Conditioned medium from anti-CD3-stimulated D10.G4 cells also provided protection from anti-Ig. In contrast, little protection was observed with activated cells from a type 1 T-cell clone, A.E7. Furthermore, we show that combinations of IL-4 and tumor necrosis factors (both TNF alpha and TNF beta), as well as IL-4, effected partial protection by themselves and enhanced the activity of the other lymphokine if used in a pretreatment protocol. However, anti-cytokine antibodies were ineffective at reversing the T-cell-mediated protection. The possibility that direct T:B-cell contact mediates part of the protective signal is discussed.     

Lymphoma models for B cell activation and tolerance. I. Conditions for the anti-mu-dependent stimulation of growth in NBL, a nude B cell lymphoma

NBL is a spontaneous B cell lymphoma that originated in NIH Swiss nude mouse, and has been maintained as an in vitro line for 4 yr in our laboratory. It is surface IgM positive and expresses several B cell markers including Fc receptors, as well as Ly-1. Although clones of NBL will grow in serum-containing medium, this cell line enters a quiescent state in serum-free culture. However, in the presence of affinity-purified or monoclonal anti-mu reagents, NBL increases its rate of proliferation as measured by thymidine incorporation or in absolute cell numbers. This stimulation is specific for mu-chain, because it does not occur with anti-beta 2 microglobulin, irrelevant nonbinding antibodies, or with monoclonal anti-B cell lineage markers. Bivalent anti-mu is required, and no consistent Fc-mediated inhibition of growth has been detected. Stimulation of NBL occurs optimally at critical cell densities (greater than 3 X 10(3)/well) in the absence of serum. Therefore, we reasoned that NBL either produced or was receptive to known B cell growth factors. Although no classic IL 1 was detected in NBL supernatants, some BCGF-I-like activity was found. Finally, in the presence of LPS, both spontaneous and anti-mu-stimulated NBL growth was inhibited, a result suggesting maturation of this lymphoma. These results suggest that NBL represents an excellent model to study the growth and differentiation of B cell subsets.     

Lymphoma models for B cell activation and tolerance. VIII. Cross-desensitization by sIgM and sIgD and its effects on growth regulation by anti-isotype antibodies

ECH408-1 is a murine B cell lymphoma expressing idiotypically and allotypically distinguishable transfected and endogenous IgD. Previously, we demonstrated that this cell line was not growth inhibited by antibodies directed at membrane IgD, but could be inhibited by antibodies which crosslink membrane IgM. Herein, we demonstrate that both anti-mu and anti-delta will cause calcium mobilization in this transfected cell line; this is followed by a period during which antibodies against the alternative isotype are unable to induce significant increases in intracellular calcium concentrations. This phenomenon, called "desensitization," is short-lived, lasting 20 min. We further demonstrate that acute desensitization of these cells by anti-delta has no effect on immediate growth inhibition which is elicited by anti-mu. These data confirm our earlier proposal that the rapid, initial calcium response seen in these lymphomas is not required for the negative signal for growth. Moreover, we also demonstrate that pretreatment of these lymphoma cells with phorbol myristate acetate (PMA) also renders these lymphoma cells temporarily incapable of manifesting a significant calcium signal. Nonetheless, PMA-pretreated B lymphoma cells are not altered in their subsequent sensitivity to anti-mu growth inhibition, nor are they affected in their resistance to inhibition by anti-delta. Our data confirm the proposal that neither the calcium signal nor protein kinase-C activation is involved in the modulation of B lymphoma growth.     

Coordinate regulation of immune and inflammatory responses by T cell-derived lymphokines

In response to antigenic stimulation, helper T cells secrete a set of protein mediators called lymphokines that regulate proliferation, differentiation, and maturation of lymphocytes and hemopoietic cells. Because all known lymphokines are composed of a single polypeptide chain, their coding sequences can be isolated by functional expression in appropriate host cells. Based on this expression cloning protocol, a number of T cell lymphokine genes have been isolated, their primary structure has been determined, and biological properties of their recombinant products have been examined. These studies revealed the existence of a regulatory network between lymphoid cells and hemopoietic cells mediated by the actions of multiple pleiotropic lymphokines produced by activated T cells. Because all or a part of this network can be activated in different ways by unique combinations of lymphokines, it is clear that T cells can play a vital role in coordinating the function of different body compartments in the immune and inflammatory responses. The activation of lymphokine genes in T cells by antigen is rapid and temporal. Therefore, an inflammatory response that involves proliferation and maturation of target cells may be restricted to the site of lymphokine production. This inducible hemopoiesis appears to be differentially regulated from the steady state or constitutive hemopoiesis that occurs in the bone marrow microenvironment in the absence of immunological stimuli.     

Effects of cyclosporine on responses of murine B cells to T cell-derived lymphokines

Cyclosporine (CS) inhibits the stimulation of both T and B lymphocytes by certain agents, but not by others. Here we have studied the effects of the drug on the responses of murine B cells to T cell-derived B cell growth and differentiation factors. We show that activation of resting B cells by B cell-stimulatory factor-1 (BSF-1) is resistant to CS, whereas stimulation by anti-Ig antibodies is not, which is in agreement with earlier findings. Furthermore, B cell proliferation elicited by co-stimulation with anti-Ig plus BSF-1 remains drug susceptible. In contrast, the stimulation of large (presumably preactivated) B cells by B cell growth factor II to synthesize DNA or to secrete Ig is inhibited by low concentrations of CS. These results therefore contrast with earlier findings with human B cells, and with those using T cells from various species, which showed that the responses of preactivated cells to growth factors are resistant to the drug. It thus appears that in the mouse CS can affect all stages of B cell stimulation.     

Suppression of B cell MIF production by T cells and soluble T cell-derived factors.

Purified populations of guinea pig B cells from nonimmunized animals may be stimualted by PPD or LPS to produce MIF. Unfractionated lymphocyte suspensions from these animals do not produce MIF under these conditions. Reconstitution of B cells with T cells abolishes their ability to generate detectable MIF. A soluble factor obtained from stimulated T cell cultures (MIFIF) is also capable of suppressing this B cell activity. Thus suppressor T cells can interfere with lymphokine production by B cells and this effect is mediated at least in part by a soluble factor. This previously undescribed capacity of T cells may provide an explanation for the fact that B cells do not appear to play a role in reactions of cell-mediated immunity in vivo.     

Induction of peripheral T cell tolerance by antigen-presenting B cells. II. Chronic antigen presentation overrules antigen-presenting B cell activation

Ag presentation in the absence of danger signals and Ag persistence are the inductive processes of peripheral T cell tolerization proposed so far. Nevertheless, it has never been definitively shown that chronic Ag presentation per se can induce T cell tolerance independent of the state of activation of APCs. In the present work, we investigated whether chronic Ag presentation by either resting or activated B cells can induce tolerance of peripheral Ag-specific T cells. We show that CD4(+) T cells that re-encounter the Ag for a prolonged period, presented either by resting or activated Ag-presenting B cells, become nonfunctional and lose any autoimmune reactivity. Thus, when the main APCs are B cells, the major mechanism responsible for peripheral T cell tolerization is persistent Ag exposure, independent of the B cell activation state.     

Activation of the gamma 1 gene by lipopolysaccharide and T cell-derived lymphokines containing a B cell differentiation factor for IgG1 (BCDF gamma)

By using both pulse labeling of nascent RNA chains and lactoperoxidase-catalyzed cell surface radioiodination, we examined both the de novo synthesis of mRNA for gamma-chains and the expression of membrane IgG (mIgG) on cells which had been stimulated with LPS plus a T cell supernatant (SN) containing a B cell differentiation factor for IgG1 (BCDF gamma). Our results show that neither nascent mRNA for gamma 1 chains nor mIgG1 can be detected in B lymphocytes until they have been stimulated by both LPS and BCDF gamma-containing T cell SN, and suggest that cell surface expression and secretion of IgG1 are coordinately controlled.     

Separation of events mediating B cell proliferation and Ig production by using T cell membranes and lymphokines

The initiation by Th cells of B cell proliferation and differentiation to produce Ig involves both cell contact- and lymphokine-mediated signals. Plasma membrane-enriched fractions from stimulated, but not unstimulated, Th cells induced Ag nonspecific and MHC unrestricted proliferation of 60 to 70% of small dense B cells. Induction of stimulatory membrane activity was inhibited by cycloheximide, and the activity was eliminated by both protease and heat treatment of membranes. Membrane-stimulated B cells did not differentiate to secrete Ig; however, addition of a lymphokine-containing supernatant from activated Th cells or the combination of IL-4 and IL-5 resulted in substantial Ig production, predominantly of the IgM, IgG1, IgA, and IgE isotypes. The quantity and isotype distribution of the antibodies secreted were similar to those produced after B cell activation by the intact Th cells and Ag. Therefore, membranes from activated Th cells in combination with lymphokines normally secreted by such cells can replace intact Th cells and provide a defined system to identify molecular events important for B cell activation.     

T helper cell-dependent B cell activation.

Small, resting B lymphocytes are driven into the cell cycle as a consequence of receiving multiple signals from elements found within their local environment. The first of these signals results from the binding of specific antigen to membrane immunoglobulin (mIg) receptors on the B cells. Pursuant to antigen binding, signals are transduced and the B cell commences to endocytose and degrade the antigen. Fragments of the antigen are expressed on the B cell surface in noncovalent association with class II major histocompatibility complex (MHC) molecules. The antigen-class II MHC complex serves as a recognition complex for CD4+ helper T cells (Th). As a consequence of recognition, Th form stable physical conjugates with the B cells. Over an extended period of time the Th and B cells bilaterally signal one another. This interchange of signals results in the growth and differentiation of both cells. This review will discuss the sequence of events that culminate in the growth and differentiation of B lymphocytes to antibody-producing cells.     

Protein kinase C activation in B cells by indolactam inhibits anti-Ig-mediated phosphatidylinositol bisphosphate hydrolysis but not B cell proliferation

In order to examine the role of phosphatidylinositol bisphosphate (PIP2) hydrolysis in B cell activation, we studied the effect of various classes of protein kinase C (PKC) activators on anti-Ig-mediated B cell stimulation. Anti-Ig-stimulated PIP2 hydrolysis, elevations in [Ca2+]i, and induction of DNA synthesis were inhibited by PMA (a phorbol ester) as previously reported. In contrast, indolactam (an alkaloid PKC activator) inhibited PIP2 hydrolysis and elevations in [Ca2+]i, but stimulated rather than inhibited cellular proliferation. In order to examine whether the binding avidity of the PKC activators to PKC played a role in determining their activity to stimulate or inhibit B cell activation, we studied two other PKC activators, bryostatin and mezerein. Again, both inhibited anti-Ig mediated PIP2 hydrolysis and elevations in [Ca2+]i, whereas only the former inhibited induction of DNA synthesis. These data suggest that a) high levels of PIP2 hydrolysis and elevations in [Ca2+]i are not essential for anti-Ig-mediated induction of B cell DNA synthesis and b) activation of PKC may induce both stimulatory and inhibitory pathways of B cell activation, and whether stimulation or inhibition of cell activation is observed may depend on the combined intensity of these two signals.     

Effect of T cell-derived lymphokines containing B cell differentiation factor(s) for IgG (BCDF gamma) on gamma-specific mRNA in murine B cells

Cytoplasmic RNA was isolated from cells cultured with LPS and LPS plus a T cell-derived supernatant (SN) (PK 7.1) containing B cell differentiation factors. The steady state levels of isotype-specific mRNA were assessed by Northern blot analysis with gamma-specific CH3 probes. It was demonstrated that the SN induces an increase in the level of mRNA for gamma 1 and a concomitant decrease in the levels of mRNA for gamma 2b and gamma 3.     

Unique signaling properties of B cell antigen receptor in mature and immature B cells: implications for tolerance and activation

Immature B cells display increased sensitivity to tolerance induction compared with their mature counterparts. The molecular mechanisms underlying these differences are poorly defined. In this study, we demonstrate unique maturation stage-dependent differences in B cell Ag receptor (BCR) signaling, including BCR-mediated calcium mobilization responses. Immature B cells display greater increases in intracellular calcium concentrations following Ag stimulation. This has consequences for the induction of biologically relevant responses: immature B cells require lower Ag concentrations for activation than mature B cells, as measured by induction of receptor editing and CD86 expression, respectively. BCR-induced tyrosine phosphorylation of CD79a, Lyn, B cell linker protein, and phospholipase Cgamma2 is enhanced in immature B cells and they exhibit greater capacitative calcium entry in response to Ag. Moreover, B cell linker protein, Bruton's tyrosine kinase, and phospholipase Cgamma2, which are crucial for the induction of calcium mobilization responses, are present at approximately 3-fold higher levels in immature B cells, potentially contributing to increased mobilization of calcium. Consistent with this possibility, we found that the previously reported lack of inositol-1,4,5-triphosphate production in immature B cells may be explained by enhanced inositol-1,4,5-triphosphate breakdown. These data demonstrate that multiple mechanisms guarantee increased Ag-induced mobilization of calcium in immature B cells and presumably ensure elimination of autoreactive B cells from the repertoire.     

Superantigen-induced CD4 memory T cell anergy. I. Staphylococcal enterotoxin B induces Fyn-mediated negative signaling

Memory CD4 T cells must provide robust protection for an organism while still maintaining self-tolerance. Superantigens reveal a memory cell-specific regulatory pathway, by which signaling through the TCR can lead to clonal tolerance (anergy). Here we show that the src kinase Fyn is a critical regulator of anergy in murine memory CD4 T cells induced by the bacterial superantigen staphylococcal enterotoxin B (SEB). Exposure to SEB results in impaired TCR signaling due to failed CD3/ZAP-70 complex formation. Further, signal transduction through the TCR remains similarly blocked when anergic memory cells are subsequently exposed to agonist peptide antigen. Pharmacological inhibition or genetic elimination of Fyn kinase reverses memory cell anergy, resulting in SEB-induced cell proliferation. The mechanism underlying impaired TCR signaling and subsequent memory cell anergy must involve a Fyn signaling pathway given that the suppression of Fyn activity restores CD3/ZAP-70 complex formation and TCR proximal signaling.     

Lymphoma models for B-cell activation and tolerance. II. Growth inhibition by anti-mu of WEHI-231 and the selection and properties of resistant mutants

Regulation of the growth of murine B-cell lymphomas has been used as a model for tolerance induction. The inhibition by anti-immunoglobulin reagents of the growth of WEHI-231 and several variant clones has now been studied. The parental line is exquisitely sensitive to growth inhibition by heterologous or monoclonal anti-mu or anti-k reagents and ceases to incorporate thymidine within 24-48 hr of exposure to anti-immunoglobulin reagents. Growth inhibition is initially reversible, but prolonged exposure to anti-mu results in cell death. This inhibition is specific for immunoglobulin light and heavy chains since growth is not inhibited by antibodies directed at either class I or class II histocompatibility antigens. In order to study the mechanism of growth inhibition, we have mutagenized WEHI-231 with ethylmethane sulfonate and cloned the surviving colonies in the presence of anti-mu. Such variants, which have been repeatedly recloned, are able to grow normally in the presence of anti-mu up to 100 micrograms/ml. These "resistant" clones, while expressing amounts of surface IgM similar to that observed on WEHI-231, do not differ markedly in their ability to cap their immunoglobulin receptors compared to the parental line but appear to have lost class II antigens. Cell cycle analysis revealed that anti-mu causes a block in the transition of WEHI-231 from G1 to S phase. The relevance of these processes to models of B-cell tolerance induction are discussed.     

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.