Cross-linking Of Surface Ig Research Articles

Engagement of CD40 lowers the threshold for activation of resting B cells via antigen receptor.

Cross-linking of surface Ig (sIg) on resting B cells can generate intracellular signals; however, for T-dependent antigens to promote growth and differentiation additional surface receptors must be engaged. Ligation of CD40 can stimulate B cell proliferation in the presence of interleukin-4. A recently identified counterstructure for CD40 is found on T helper cells and is believed to represent the cognate ligand for B cell activation. This study investigates the role of CD40 as an accessory molecule in sIg-dependent B cell activation. Simultaneous ligation of sIg and CD40 by monoclonal antibodies (mAb) in the presence of mouse L cells which express human Fc gamma receptor type II (Fc gamma RII-L cells) results in potent stimulation of small resting B cells. When CD40 is co-ligated, picomolar concentrations of mouse IgG1 anti-mu, and anti-delta mAb can stimulate B cell proliferation. This requires interaction of the anti-Ig mAb with the Fc gamma RII-L cells: a mouse IgG2a anti-mu mAb which is not recognized by Fc gamma RII, was > or = 1000-fold less effective. These findings suggest a mechanism for B cell activation whereby engagement of T cells via CD40 and its cognate ligand provides potent enhancement of signals delivered through sIg. Based on these observations, models for the activation of B cells by T-dependent antigens are presented.

Coregulation of the APO-1 Antigen With Intercellular Adhesion Molecule-1 (CD54) in Tonsillar B Cells and Coordinate Expression in Follicular Center B Cells and in Follicle Center and Mediastinal B-Cell Lymphomas

APO-1 is a 48-Kd transmembrane glycoprotein identical to the Fas antigen and belongs to the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor family of surface molecules. Cross-linking of APO-1 induces apoptotic cell death in sensitive cells. We show here that APO-1 is an activation molecule on B cells. It was induced/enhanced on dense and buoyant tonsillar B cells, respectively, through surface Ig cross-linking in combination with interleukin-2 or by interferon-gamma together with tumor necrosis factor-alpha. These conditions also increased the amount of intercellular adhesion molecule-1 (CD54) on these cells. Epstein-Barr virus transformants of peripheral B cells coexpressed APO-1 and CD54 at very high levels. Immunohistologically, Apo-1 was detectable at low levels in a subpopulation of follicle center B blasts and, at higher levels, in sinusoidal B cells. APO-1 was undetectable in follicular mantle B cells and plasma cells. In isolated tonsillar B cells, APO-1 was expressed in CD10+ follicle center cells. In acute B lymphoblastic leukemia, chronic B lymphocytic leukemia, and Burkitt's lymphomas, APO-1 and CD54 molecules were immunohistochemically undetectable. Coordinate expression of these antigens was found in mediastinal B-cell lymphomas. The mode of APO-1 and CD54 expression was correlated in follicle center cell lymphomas (P < .0019), but less stringently in hairy cell leukemia. No association was found in plasmacytomas. This was in line with the differential expression of these molecules found in reactive plasma cells. Expression of APO-1 in B cells of different stages of differentiation and, correspondingly, in certain B-cell neoplasias might suggest a role of this molecule in the induction of B-cell apoptosis. This function might be influenced by CD54 and CD54-mediated signals.

Coregulation of the APO-1 antigen with intercellular adhesion molecule- 1 (CD54) in tonsillar B cells and coordinate expression in follicular center B cells and in follicle center and mediastinal B-cell lymphomas

APO-1 is a 48-Kd transmembrane glycoprotein identical to the Fas antigen and belongs to the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor family of surface molecules. Cross-linking of APO- 1 induces apoptotic cell death in sensitive cells. We show here that APO-1 is an activation molecule on B cells. It was induced/enhanced on dense and buoyant tonsillar B cells, respectively, through surface Ig cross-linking in combination with interleukin-2 or by interferon-gamma together with tumor necrosis factor-alpha. These conditions also increased the amount of intercellular adhesion molecule-1 (CD54) on these cells. Epstein-Barr virus transformants of peripheral B cells coexpressed APO-1 and CD54 at very high levels. Immunohistologically, Apo-1 was detectable at low levels in a subpopulation of follicle center B blasts and, at higher levels, in sinusoidal B cells. APO-1 was undetectable in follicular mantle B cells and plasma cells. In isolated tonsillar B cells, APO-1 was expressed in CD10+ follicle center cells. In acute B lymphoblastic leukemia, chronic B lymphocytic leukemia, and Burkitt's lymphomas, APO-1 and CD54 molecules were immunohistochemically undetectable. Coordinate expression of these antigens was found in mediastinal B-cell lymphomas. The mode of APO-1 and CD54 expression was correlated in follicle center cell lymphomas (P &lt; .0019), but less stringently in hairy cell leukemia. No association was found in plasmacytomas. This was in line with the differential expression of these molecules found in reactive plasma cells. Expression of APO-1 in B cells of different stages of differentiation and, correspondingly, in certain B-cell neoplasias might suggest a role of this molecule in the induction of B-cell apoptosis. This function might be influenced by CD54 and CD54-mediated signals.

Protein kinase C regulates secretion of lymphotoxin (LT/TNFβ) and TNFα by human EBV-transformed B cells

Epstein-Barr virus (EBV)-transformed cell lines constitutively secrete lymphotoxin (LT/TNF beta) and not tumor necrosis factor-alpha (TNF alpha). To analyze the cellular processes that regulate LT and TNF alpha secretion by lymphoblastoid cell lines, we studied the role of two signal transduction pathways leading to either protein kinase C (PK-C) or PK-A activation. We demonstrate that PK-C activation, either after cross-linking of surface Ig or by direct activation with phorbolester, leads to increased production of both LT and TNF alpha, whereas no prominent role for PK-A was found. Interleukin (Il)-4 was found to synergize with PK-C activation in raising levels of secreted LT and TNF alpha. Increased levels of LT and TNF alpha did not correlate with augmented levels of immunoglobulin secreted by the cell lines nor with improved proliferation. These observations demonstrate that EBV B cells respond to B cell activation signals leading to PK-C activation with increased production of both LT and TNF alpha. It is, however, unlikely that these molecules serve as autostimulatory factors for EBV B cells, but in contrast might play a role in downregulation of biological functions in these cells.

The CD19/CD21 signal transducing complex of human B lymphocytes includes the target of antiproliferative antibody-1 and Leu-13 molecules.

CD19 is a member of the Ig superfamily expressed on the surface of B lymphocytes that may be involved in the regulation of B cell function. Immunoprecipitation studies with B cell lines solubilized by digitonin have shown CD19 to be part of a multimolecular complex that includes CD21 (CR2) and other unidentified proteins. In this study, two of the CD19-associated proteins were identified as TAPA-1, which is expressed on most cell types, and Leu-13, which is expressed on subsets of lymphoid cells. TAPA-1 and Leu-13 are physically associated in many cell lineages. CD19 and CD21 mAb each specifically coprecipitated proteins of the same size as those precipitated by TAPA-1 and Leu-13 mAb from B cell lines and cDNA-transfected K562 cell lines. Western blot analysis with a TAPA-1 mAb verified the identity of TAPA-1 in CD19 and CD21 immunoprecipitated materials. In addition, when TAPA-1 or Leu-13 were crosslinked and patched on the cell surface, all of the CD19 comigrated with TAPA-1 and some of the CD19 comigrated with Leu-13. Furthermore, mAb binding to CD19, CD21, TAPA-1, and Leu-13 on B cell lines induced similar biologic responses, including the induction of homotypic adhesion, inhibition of proliferation, and an augmentation of the increase in intracellular [Ca2+] induced by suboptimal cross-linking of surface Ig on B cell lines. Together, these data suggest that TAPA-1 and Leu-13 are broadly expressed members of a signal transduction complex in which lineage-specific proteins, such as CD19 and CD21, provide cell-specific functions.

IL-4 induces IL-2 receptor p75 beta-chain gene expression and IL-2-dependent proliferation in mouse T lymphocytes.

T and B cells exhibit complex responses to the combination of IL-2 and IL-4, each of which can act as a growth or differentiation factor for lymphocytes under certain circumstances. To characterize better the mechanism by which these cytokines interact, mRNA levels of the signal-transducing p75 beta-chain of the IL-2R were analyzed. These studies show that IL-4 increases expression of the IL-2R beta-chain in mouse splenic B and T cells, and the response of B cells was potentiated by concurrent cross-linking of surface Ig. Kinetic analysis of the IL-2R beta response showed a slow onset but maintenance of peak levels of expression between 10 and 24 h. These data indicate that the pathways involved in the lymphocyte response to IL-4 differ for IL-2R and IL-4R, and that the induction of IL-4R precedes the increase in IL-2R. The effect of IL-4 on IL-2R beta mRNA levels was mediated in part by an increase in the rate of gene transcription, and was associated with increased IL-2 binding in the absence of any change in IL-2R alpha levels. In addition, IL-4 increased the level of IL-2R beta expression in thymocytes. Proliferation assays demonstrated that pretreatment of splenic T cells with IL-4 led to a substantial increase in IL-2-dependent proliferation. These results are consistent with a mechanism by which IL-4 can prime T cells and certain thymocytes for responsiveness to IL-2 by increasing IL-2R p75 chain gene expression, independent of general T cell activation.

Tolerance induction in resting memory B cells specific for a protein antigen.

Resting memory B lymphocytes specific for the model protein Ag cytochrome c have been shown to be susceptible to tolerance induction in in vitro splenic fragment cultures. This induction of nonresponsiveness is dependent upon the strength of the interaction between surface Ig and specific Ag, where concentration, valency, affinity, and time of exposure all appear to be important factors, as is the case for tolerance induction in immature or primary B cells. The induction of nonresponsivenes in greater than 80% of Ag-specific memory B cells was achieved by incubation with 1 microM cytochrome polymer for 24 h in the absence of T cell help. Not only were memory B cells unresponsive to specific Ag, they were also unable to become activated through nonspecific uptake and presentation of an Ag to which T cells have been primed, demonstrating that the induction of nonresponsiveness involves more than a modulation or blockade of surface Ig receptors. Although soluble factors collected from activated T cells failed to prevent memory B cells from becoming nonresponsive after surface Ig cross-linking, the direct activation of T cells within splenic fragment cultures did partially inhibit tolerance induction in splenic fragment memory B cells. In addition, the induction of tolerance was partially blocked by protein tyrosine kinase inhibitors, suggesting a physiologic change within the B cells associated with the state of nonresponsiveness and resulting from tyrosine-specific phosphorylation.

Crosslinking of the cell surface immunoglobulin (mu-surrogate light chains complex) on pre-B cells induces activation of V gene rearrangements at the immunoglobulin kappa locus.

We constructed an expression vector encoding a truncated Ig mu chain that lacks both VH and CH1 domains (mu delta m chain) and introduced the mu delta m vector into the Ig negative Abelson pre-B cell line P17-27. The transfectants expressed a large amount of the mu delta m chain on their surface, which was not complexed with the lambda 5 and VpreB surrogate light chain molecules. While P17-27 transfected with a vector for the intact micron chain (P17-27 micron) shows V kappa rearrangements in culture, V kappa rearrangements were not detected in P17-27 mu delta m cells. When the mu delta m chains on the cell surface were crosslinked by anti-mu antibodies, V kappa gene rearrangements were induced in P17-27 mu delta m. These results strongly suggest that crosslinking of the micron-lambda 5-VpreB complex on the pre-B cell surface generates a signal that activates V kappa gene rearrangement, and that the lambda 5 and VpreB molecules are necessary for the spontaneous crosslinking of surface Ig on pre-B cells.

A polyclonal model for B-cell tolerance: II. Linkage between signaling of B-cell egress from G 0, class II upregulation and unresponsiveness

Overnight exposure of adult splenic B cells to anti-Ig, a surrogate for antigen/tolerogen, can result in a hyporesponsive state in terms of antibody synthesis. Since B cells treated with either intact of F(ab′) 2 fragments of anti-Ig will exit the G 0 phase of the cell cycle and enter G 1 or S, respectively, we examined which steps in B-cell activation were required for this form of hyporesponsiveness. We found that B-cell hyporesponsiveness could be induced under conditions leading to either abortive or productive B-cell cycle progression, depending on the immunogenic challenge employed. Thus, PMA + ionomycin, concanavalin A, PMA alone, or ionomycin alone induced hyporesponsiveness. Each of these reagents is able to drive B-cell exit from G 0 into G 1 and cause class II hyperexpression. We next examined the effect of cyclosporin A (CSA), a reagent that blocks anti-Ig but not by PMA-induced class II hyperexpression. Interestingly, CSA only interfered with the induction of B-cell hyporesponsiveness with anti-Ig. These results suggest that upregulation of MHC class II may be coincident with a CSA-sensitive tolerance pathway in B cells stimulated by anti-Ig. Finally, IL-4 pretreatment was found to ablate hyporesponsiveness induced by either intact anti-Ig or PMA. These results parallel the Fc-dependent induction of hyporesponsiveness reported earlier (G. Warner and D. W. Scott, J. Immunol., 146, 2185, 1991) . We propose that crosslinking of surface Ig, leading to cell cycle progression out of G 0 as well as class II hyperexpression, in the absence of a cognate T cell signal, leads to B-cell hyporesponsiveness.

Induction of IL-5 receptors on normal B cells by cross-linking surface Ig with anti-Ig-dextran.

Regulation of IL-5R expression in normal, non-Ly-1 (CD5) B cells was evaluated. Freshly isolated unfractionated spleen B cells express little or no detectable IL-5R. In contrast, B cells stimulated with anti-Ig-dextran conjugates express substantial numbers of IL-5R. Phenotypic analysis of the B cells responding to anti-Ig-dextran, and expressing IL-5R, demonstrates that these cells do not express Ly-1 or Mac-1. Scatchard analysis of B cells stimulated with anti-IgD-dextran reveals two classes of IL-5R: a high affinity receptor with a Kd of 17 pM and approximately 300 receptors/cell, and a low affinity receptor with a Kd of 0.6 nM and approximately 1000 receptors/cell. Peak receptor expression in response to anti-IgD-dextran is seen 72 h after stimulation and with a dose of 10 ng/ml. The induced receptors are functional, because both proliferation and Ig secretion by B cells treated with anti-IgD-dextran are enhanced by IL-5. Other B cell mitogens such as LPS, soluble anti-Ig/IL-4, or phorbol esters/ionomycin are poor inducers of the IL-5R. Finally, not only does LPS fail to induce significant IL-5R expression on spleen B cells, it suppresses both high and low affinity IL-5R expression induced by anti-IgD-dextran. These data indicate that normal, non-Ly-1 B cells are capable of expressing both high and low affinity IL-5R but that receptor expression is critically dependent on the type of stimulus provided to the B cell. A stimulus that produces extensive cross-linking of surface Ig on B cells, i.e., anti-Ig-dextran, is very effective in inducing IL-5R whereas a variety of other B cell mitogens are ineffective.

Cross-linking of Fc gamma receptors and surface antibodies. Theory and application.

B lymphocyte responses to the cross-linking of surface Ig (sIg) are known to be inhibited, when IgG is the cross-linking agent, by the concurrent binding of the Fc portion of the IgG to Fc gamma R. We present a mathematical framework for designing and analyzing experiments aimed at uncovering the inhibition mechanism(s). From our model, we calculate concentrations of receptors and ligands in the different cell surface states, at equilibrium or as a function of time. IgG can cross-link surface receptors in three ways, i.e., by bridging two sIg molecules without Fc binding, by bridging two sIg while binding as well to an Fc gamma R, and by binding to an Fc gamma R and only one sIg. We show how the concentrations or fractions of these distinct cross-linked states depend on experimentally manipulable variables, including the concentrations of intact IgG, bivalent and monovalent IgG fragments, and agents that block Fc binding. Then, using simple signal/response relationships, reflecting active and passive mechanisms of Fc-mediated inhibition, we simulate the results of a variety of experiments. In cases where published experimental results are available, we find that the qualitative predictions of our general model are consistent with the data and that comparisons of simulations with available data provide some quantitative information about the parameters governing the cell surface signaling events. In particular, comparison of model predictions with published experiments on the kinetics of IgG-induced inositol trisphosphate production indicate that sIg cross-links form more rapidly than sIg-Fc gamma R "co-cross-links." Further, IgG-sIg bonds stabilize Fc attachments, i.e., the dissociation of IgG from Fc gamma R is slowed significantly when the IgG is also cross-linked to sIg. Predictions of the model suggest other experiments and ways of presenting the data that will help to identify relationships between the molecular signaling events occurring on the cell surface and the various cellular responses.

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

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 x 10(-4) micrograms/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 microgram/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.

Altered signal transduction secondary to surface IgM cross-linking on B-chronic lymphocytic leukemia cells. Differential activation of the phosphatidylinositol-specific phospholipase C.

To further study the mechanisms by which surface Ig triggering activates the inositol phospholipid signaling pathway, we have used B cells from chronic lymphocytic leukemia patients which, as previously described, display two patterns of response upon sIg cross-linking: in one group this cross-linking induces an inositol phosphate release, an intracellular free Ca2+ concentration elevation and a subsequent cell proliferation; in a second group none of these events occur although there is an increased class II Ag expression following anti-mu stimulation as in the first group. We have been able to demonstrate that the phosphatidyl inositol specific phospholipase C (PI-PLC) can be activated in permeabilized B cells from the first group by direct stimulation, with GPT gamma S, of a guanine nucleotide binding (G) protein. In addition, since anti-mu + GTP gamma S stimulate an increased inositol phosphate production in these cells, this suggests that surface Ig cross-linking activates PI-PLC via a G protein. However, in cells from the second group no inositol phosphate is released after GTP gamma S stimulation although PI-PLC can be directly activated by high Ca2+ concentrations. This reflects in these cells, an interruption of the signaling cascade sIg/G protein/PI-PLC at the level of the G protein or at the G protein/PI-PLC coupling. In cells from both groups PMA treatment, which is known to alter phosphatidyl inositol metabolism in B cells, completely inhibits PI-PLC activation even by high Ca2+ concentrations. These studies show that the phosphatidyl inositol-dependent signaling cascade after surface Ig triggering can be altered at different levels in B cells.

Augmentation by Cytochalasin B of Antigen Receptor‐Mediated Activation of Normal and Malignant Human B Lymphocytes

In the present study, the effects of cytochalasin B on the proliferation of normal and malignant human B lymphocytes activated by monoclonal antibodies (MoAb) reactive with IgM heavy chain determinants were evaluated. The following data indicate that heterogeneous populations of normal peripheral blood and tonsillar B cells and certain monoclonal populations of leukaemic B cells proliferate after exposure to monoclonal anti-IgM antibodies. The relationship of these findings to B-cell activation caused by surface Ig cross-linking is discussed

Cholera toxin-sensitive and insensitive signaling via surface Ig.

We have observed that a 2-h pretreatment of murine B cells with cholera toxin (CT) renders the B cell incapable of receiving an activation signal via surface Ig as measured by cell volume increase and entry into the S phase of the cell cycle. In contrast, CT pretreatment does not inhibit the delivery of a signal by IL-4, as measured by increase in cell volume. In fact, CT pretreated B cells are able to respond to anti-Ig in the presence of IL-4, as measured by both an increase in cell size and entry into S suggesting that IL-4 overcomes the effects of CT on normal B cell activation. Despite blocking the anti-Ig-mediated entry into the cell cycle, CT was not able to interfere with the induction of nonresponsiveness by anti-Ig in normal B cells or with the delivery of growth-inhibitory signal to the B cell lymphoma WEHI-231. These results suggest that there are two signaling pathways mediated by cross-linking of surface Ig: one pathway sensitive and the other insensitive to modulation by CT.

Superoxide-dependent nitroblue tetrazolium reduction and expression of cytochrome b-245 components by human tonsillar B lymphocytes and B cell lines.

EBV-transformed B lymphocyte cell lines can generate superoxide, using an electron transport chain homologous, or even identical, to phagocytic NADPH-oxidase. We searched for normal, not virally transformed, B lymphocytes with analogous properties, using tonsils as the source of B cells. Unseparated tonsillar leukocytes contained cells capable of PMA-triggered superoxide dismutase-inhibitable reduction of nitroblue tetrazolium (NBT+ cells) well in excess of phagocytes (18.9 +/- 6.4% NBT+ cells with 1.3 +/- 0.9% granulocytes and 1.9 +/- 2.3% monocytes/macrophages, n = 8). NBT reduction was also inhibited by diphenylene iodonium, a selective inhibitor of phagocytic NADPH-oxidase. Cross-linking of surface Ig was equally effective as PMA in inducing NBT reduction among tonsillar leukocytes. NBT+ cells co-distributed with B cells on Percoll density gradients and were enriched among purified B cells obtained by SRBC rosetting twice and Sephadex G10 adherence (47.8 +/- 15.2% NBT+ cells among 90.5 +/- 5.5% B cells, 4.8 +/- 5.1% T cells, 1.2 +/- 0.77% monocytes/macrophages, and 0.73 +/- 0.6% granulocytes, n = 10). Further, mAb 7D5, directed against an extracellularly located epitope of the small subunit of cytochrome b-245 of phagocytes, stained the majority of tonsillar B cells (85 +/- 9.2% 7D5+ cells and 91.6 +/- 4.04% B cells, n = 3). Superoxide production, staining with 7D5 antibody, and expression of mRNA for the beta chain of cytochrome b-245 were further analyzed in cell lines. The EBV-BLCL F1 and the Burkitt lymphoma P3HR-1 both carried 7D5-detectable cytochrome b-245 Ag and expressed mRNA for the beta chain of the cytochrome b, both in similar amounts. However, only F1, not P3HR-1, was capable of PMA-triggered superoxide production. These data indicate that also normal nontransformed B lymphocytes possess the capacity to generate superoxide by a system apparently similar to phagocytic NADPH-oxidase, provisionally termed "B cell oxidase." Discrepancies observed in certain B cells and lines between expression of cytochrome b components and stimulus-induced superoxide production may be related to an absence or low level of other oxidase components or of the signal transduction mechanism. Conceivably, production of superoxide and derived reactive oxygen species by B cells may have cytotoxic, immunomodulatory, or mutagenic effects on the B cells themselves or on cells in their immediate vicinity.

Prostaglandin E2 modulation of rheumatoid factor synthesis.

We examined the influence of prostaglandin E2 (PGE2) on the in vitro synthesis of rheumatoid factor (RF) by purified human B and T lymphocytes stimulated with Staphylococcus aureus Cowan 1 or pokeweed mitogen (PWM). Supernatants were assayed for total IgM and RF. PGE2 at concentrations of 10(-7) M to 10(-9) M significantly inhibited RF and IgM secretion stimulated by S aureus Cowan 1, a cross-linker of B cell surface Ig. The magnitude of inhibition of RF production was significantly greater than that of total IgM at low PGE2 concentrations (P less than 0.05). In contrast, PWM-stimulated cultures were only minimally inhibited by PGE2 at all concentrations tested. Since cross-linking of surface Ig renders B cells more susceptible to inhibition by PGE2, heat-aggregated IgG (HAIgG) was added to the PWM-stimulated cultures in an attempt to increase the sensitivity of precursors of RF-secreting cells to the inhibitory effects of PGE2. Addition of HAIgG markedly increased PGE2-mediated inhibition of RF synthesis without significantly affecting IgM production. Inhibition could not be overcome by the addition of soluble T helper cell factors, indicating that PGE2-mediated suppression was not the result of an inhibitory action of T helper cells. When lymphocytes from patients with rheumatoid arthritis were examined, HAIgG was found to be unable to induce sensitivity to PGE2-mediated inhibition of responsiveness. These results suggest that down-regulation of RF synthesis requires both cross-linking of surface Ig and the influence of PGE2. Abnormalities in this immunoregulatory mechanism may explain the ongoing production of RF in patients with rheumatoid arthritis.

Cross-linking of membrane IgM on B CLL cells: dissociation between intracellular free Ca2+ mobilization and cell proliferation.

It has been shown previously that chronic lymphocytic leukemia (CLL) B cells are frozen at different stages of activation with unique requirements for proliferation. Although most B CLL cells express surface IgM, anti-mu antibodies are able to trigger only some of them to proliferate and/or respond to B cell growth factor (BCGF) or interleukin 2 (IL2), as normal B cells. In this report we extend these observations using three different monoclonal antibodies (mAb) to human mu chain (one mitogenic in soluble form for normal B cells, the two others mitogenic only when coupled on Sepharose 4B beads). Cells from only 3 out of 11 B CLL patients proliferated in the presence of either mitogenic anti-mu. When the early events following surface Ig cross-linking, such as calcium mobilization (by flow cytometry on indo-1-labeled cells), were studied all three mAb in soluble form were able to induce a similar increase in the intracellular free calcium concentration ([Ca2+]i); analogous to [Ca2+]i rise after the mitogenic F(ab')2 anti-mu stimulation. This response was seen only in 8 out of the 12 CLL B cells studied. All B CLL cells, however, proliferate in response to a combination of phorbol ester 12,13-dibutyrate (PBt2) and ionomycin. Therefore, three patterns of response to sIg cross-linking by anti-mu could be distinguished: cells from 4 out of 12 cases proliferate and mobilize Ca2+ upon anti-mu triggering (behaving like resting B lymphocytes); in 4 other cases anti-mu lead to Ca2+ mobilization without cell proliferation; in the last 4 cases neither Ca2+ mobilization, IP3 generation (in the one case studied) nor cell proliferation are observed although these cells do proliferate directly in response to growth factors. Moreover, anti-mu stimulation in this group leads to increased proliferation in response to BCGF and IL2 suggesting an anti-Ig signaling independent of inositol phosphate metabolism. These results are interpreted in terms of differential anti-mu signaling on B cells at different stages of activation.

The superoxide generating system of B cell lines. Structural homology with the phagocytic oxidase and triggering via surface Ig.

EBV-transformed B lymphocyte cell lines (EBV-BLCL) produce superoxide after stimulation with phorbol ester, a capacity unique among nonmyeloid cells. The superoxide producing system of EBV-BLCL (B cell oxidase) was compared with the phagocytic NADPH-oxidase and the relationship of the capacity to produce superoxide to the presence of the EBV-genome was analyzed. The two EBV-transformed B cell lines F1 and HELL generated superoxide in response to PMA (2.3 nmol/10(6) F1 cells x 1 h and 6.27 nmol/10(6) HELL cells x 1 h with 1 microgram/ml of PMA), whereas no superoxide release was detected with the EBV-positive Burkitt lymphoma line WIL-2 and the EBV-negative plasmocytoma line U-266. Also, F1 and HELL showed lucigenin-dependent chemiluminescence (CL) after PMA-treatment, whereas no CL responses were detected from WIL-2 or U-266. Further, F1 and HELL cells contained a low potential cytochrome b-245 (10.9 and 61.0 pmol/mg protein, respectively) and also a 45 kDa diphenylene-iodonium (DPI)-binding peptide, both components of the phagocytic NADPH-oxidase. In contrast, neither the cytochrome b-245 nor the 45 kDa DPI-binding peptide were detected in WIL-2 and U-266. In addition, DPI inhibited O2- production by PMA-stimulated EBV-BLCL and polymorphonuclear granulocytes. Further, F1 line cells showed superoxide dismutase-inhibitable lucigenin-dependent CL when triggered by protein A-bearing staphylococci (Cowan strain I) or by a mAb directed against human IgG in the presence of solid-phase goat anti-mouse-Ig antibody. From a panel of eight EBV-BLCL, only five responded with CL when exposed to protein A-bearing staphylococci, whereas all showed CL when treated with phorbol ester. Inasmuch as all eight EBV-BLCL possessed surface Ig and a "functional" oxidase, their differential response to cross-linking of surface Ig may be determined by differences in signal transduction. Superoxide production by EBV-BLCL appears thus related to expression of an electron transport chain structurally homologous, if not identical, with the "phagocytic" NADPH-oxidase. Apparently, the presence of EBV-genome in B cell lines does not per se lead to expression of this oxidase. This suggests that nontransformed B cells may, at a certain differentiation stage, also express a superoxide-generating chain. From the finding of stimulation of superoxide production of EBV-BLCL via surface Ig it appears possible that also Ag may be able to trigger such B cells to production of superoxide which might have an important role in the physiology of B cells.

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.