Cross-linking Of Surface Immunoglobulin Research Articles

Major histocompatibility complex class II compartments in human B lymphoblastoid cells are distinct from early endosomes.

In human B lymphoblastoid cell lines, the majority of major histocompatibility complex (MHC) class II heterodimers are located on the cell surface and in endocytic compartments, while invariant chain (Ii)-associated class II molecules represent biosynthetic intermediates which are present mostly in the endoplasmic reticulum and Golgi complex. To investigate the origin of the MHC class II-positive compartments and their relation to early endosomes, the intracellular distribution of MHC class II molecules and Ii in relation to endocytic tracers was studied in human lymphoblastoid B cells by immunoelectronmicroscopy on ultrathin cryosections. Cross-linking of surface immunoglobulins, followed by a brief period of internalization of the immune complexes, did not alter the intracellular distribution of MHC class II molecules. While early endosomes were abundantly labeled for the cross-linked immunoglobulins, < 1% of total MHC class II molecules were detectable in early endosomes. MHC class II- and Ii-positive structures associated with the trans-Golgi network can be reached by endocytosed bovine serum albumin (BSA)-gold conjugates after 30 min of internalization. Prolonged exposure to BSA-gold allowed visualization of later endocytic compartments, in which a progressive loss of Ii was observed: first the lumenal portion, and then the cytoplasmic portion of Ii escaped detection, culminating in the formation of MHC class II-positive compartments (MIIC) devoid of Ii. The loss of Ii also correlated with a transition from a multivesicular to a multilaminar, electron-dense MIIC. The intracellular compartments in which class II molecules reside (MIIC) are therefore a heterogeneous set of structures, part of the later aspects of the endocytic pathway.

The Zif268 cellular transcription factor activates expression of the Epstein-Barr virus immediate-early BRLF1 promoter.

The Epstein-Barr virus immediate-early protein BZLF1 mediates the switch from latent to lytic infection. BZLF1 transcription can be derived from either the BZLF1 promoter or the BRLF1 promoter (Rp). Productive viral infection of EBV-infected B cells can be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, as well as cross-linking of surface immunoglobulin with antiimmunoglobulin antibody. Both TPA and antiimmunoglobulin antibody are known to activate expression of the cellular transcription factor Zif268 in B cells. In this study, we have examined the regulation of BZLF1 transcription by Zif268. We show that Rp (but not the BZLF1 promoter) is activated by Zif268. Bacterially synthesized Zif268 binds strongly to an upstream sequence in the Rp promoter (located from -131 to -123 relative to the start site) and more weakly to a proximal sequence (-49 to -40). Zif268 activation of Rp requires these two Zif268 binding sites. TPA treatment of B cells induces the expression of Zif268 protein, which binds to Rp. Furthermore, TPA activation of Rp requires the upstream Zif268 site. These findings indicate that Zif268 can activate a critical Epstein-Barr virus immediate-early promoter and, therefore, may play a key role in the regulation of viral latency.

IgM-mediated B cell apoptosis.

Cross-linking of surface immunoglobulin M (sIgM) on normal mature B cells induces different signaling consequences, including DNA synthesis (positive signaling) and cell cycle arrest and/or death by apoptosis (negative signaling). Presumably, the difference depends on the intensity of sIgM cross-linking: relatively weak cross-linking induces DNA synthesis, moderate cross-linking induces DNA synthesis with cell cycle arrest at the G2/M interphase, and intense cross-linking induces apoptosis. In vivo experiments with transgenic mice have shown that relatively weak cross-linking of sIgM by soluble antigens induces anergy in autoreactive B cells, whereas intense sIgM cross-linking by membrane-bound forms of antigens induces deletion of them. However, it is still unknown whether the different intensities of sIgM cross-linking generate qualitatively different signals responsible for DNA synthesis or cell death or whether they generate qualitatively the same but quantitatively different signals, and the quantitative difference is responsible for the induction of positive or negative signaling. The sIgM-mediated negative signaling presumably plays an important role in the induction and maintenance of B cell tolerance, and sIgD and sIgG also possess the machinery necessary for negative signaling. Negative signaling through sIgM is dependent on tyrosine kinase(s) and Ca2+ influx and is sensitive to cyclosporin A in certain types of B cells but not in all B cells. It has been suggested that there are different intracellular signaling pathways that transduce negative signaling via sIgM, and that activation-induced B cell death by sIgM cross-linking does not necessarily show DNA fragmentation and the morphology of apoptosis. On the other hand, sIgM-mediated B cell death may be inhibited in the presence of appropriate co-stimulators such as IL-4, alpha-, and beta-interferons and CD40-mediated signaling. The CD40-mediated signaling effectively inhibits sIgM-mediated B cell apoptosis in many but not all experimental systems. Although homotypic cell adhesion through the LFA-1/ICAM-1 dependent pathway was shown to be involved in certain types of CD40-mediated inhibition of sIgM-mediated negative signaling, it is still not known how the cytokines and CD40-mediated signaling inhibit sIgM-mediated B cell death. The molecular mechanisms responsible for sIgM-mediated negative signaling and for the inhibitory signaling against sIgM-mediated negative signaling need further elucidation.

Activation of AP-1 in Primary B Lymphocytes by Surface Immunoglobulin Requires de Novo Jun-B Synthesis

We demonstrate herein that resting primary B lymphocytes do not contain detectable levels of AP-1 (TRE)-binding activity. Upon cross-linking of surface immunoglobulin (sIg) receptors, TRE-binding activity is induced within 2 hr and its appearance requires de novo protein synthesis. Antisera to Jun-B inhibits the vast majority of TRE-binding activity, indicating that Jun-B is a primary component of B cell TRE-binding complexes. In keeping with this, Jun-B protein is not detectable in cytosol or nuclear extracts from resting B lymphocytes, as determined by immunoblotting with Jun-B antisera. However, the nuclear expression of Jun-B is induced within 2 hr following sIg cross-linking and is completely blocked by cycloheximide. 35S-labeling studies suggest that the increase in Jun-B expression results from de novo protein synthesis. Moreover, Jun-B migrates in SDS-polyacrylamide gels as two distinct electrophoretic proteins that correspond to a 41-kDa species and a phosphorylated 47-kDa form. These results suggest that the induction of AP-1-binding activity in primary B lymphocytes following sIg cross-linking does not result from post-translational phosphorylation of a preexisting cellular pool of Jun-B protein, but rather is coupled to the stimulation of de novo Jun-B synthesis. Thus Jun-B synthesis represents an integral event in the production of receptor-mediated AP-1 in B cells. The significance of these results with respect to the role of Jun-B in controlling gene expression during the activation of primary B cells is discussed.

Cyclosporin-a sensitive induction of NF-AT in murine B cells

Primary B cells are induced to proliferate by crosslinking surface immunoglobulin or by its pharmacological equivalent, phorbol ester and calcium ionophore. However, nuclear responses that have been studied in activated B cells are typically inducible with phorbol esters alone. We show that a factor, indistinguishable from the nuclear factor of activated T cells (NF-AT), is induced in B cells in response to anti-immunoglobulin signals or the combined action of phorbol ester and ionomycin, but not in response to either reagent alone. The signals necessary for NF-AT induction in B cells, therefore, closely parallel those required to induce B cell proliferation. Transfection analysis shows that B cell NF-AT is a transcriptional activator. Furthermore, NF-AT induction in spienic cells is suppressed by cyclosporin A, suggesting a mechanism by which immunosuppressive agents act on the B cell compartment. We propose that NF-AT should be considered more generally as a nuclear factor of activated lymphoid cells.

Molecular cloning of human Syk. A B cell protein-tyrosine kinase associated with the surface immunoglobulin M-B cell receptor complex.

B cell antigen receptors are multicomponent complexes consisting of the surface immunoglobulin and accessory molecules with associating protein-tyrosine kinases. A spleen tyrosine kinase, Syk, in porcine B cells and a 72-kDa protein-tyrosine kinase, PTK72, in murine B cells associate with the B cell antigen receptor. Herein, we report the isolation of a full-length cDNA encoding the human homologue of Syk. This cDNA predicted a polypeptide consisting of two NH2-terminal SH2 domains and a COOH-terminal tyrosine kinase domain. Syk is highly conserved between human and swine and is homologous to the T cell-associated protein-tyrosine kinase ZAP-70. Both Syk mRNA and protein were detected in cells derived from multiple hematopoietic lineages. Within the B cell compartment, Syk was expressed from pro-B cells to plasma cells. In vitro kinase assays conducted on the human Syk protein isolated from B cells revealed the presence of autophosphorylation activity on Syk tyrosine residues. Tyrosine phosphorylation of Syk associating with the B cell receptor complex in human was augmented rapidly after surface immunoglobulin cross-linking. The human SYK locus was mapped to chromosome 9 at band q22.

Plastic‐immobilized anti‐μ or anti‐δ antibodies induce apoptosis in mature murine B lymphocytes

It is widely accepted that extensive cross-linking of surface immunoglobulin (sIg) receptors on mature B cells promotes their activation and progression through the cell cycle. A commonly employed method to maximize receptor cross-linking via anti-receptor antibodies is to immobilize them on tissue culture plastic. We show here that immobilizing monoclonal anti-mu or anti-delta antibodies, which are mitogenic in solution, on plastic abrogates their capacity to induce DNA synthesis in mature murine B cells, even in the presence of interleukin-4 (IL-4). The cells do become abortively activated, as evidenced by up-regulation of major histocompatibility complex class II antigen levels, but subsequently virtually all of them die, manifesting DNA fragmentation characteristic of apoptosis. The induction of apoptosis is abrogated by the inclusion of either IL-4 or anti-CD40 antibodies in the cultures, with the two stimuli acting in concert. We believe that the system represents a polyclonal model of clonal deletion tolerance in mature B cells, such as may be induced under physiological conditions by antigens with repeating epitopes.

A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1.

CaM kinase-Gr is a multifunctional Ca2+/calmodulin-dependent protein kinase which is enriched in neurons and T lymphocytes. The kinase is absent from primary human B lymphocytes but is expressed in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines, suggesting that expression of the kinase can be upregulated by an EBV gene product(s). We investigated the basis of CaM kinase-Gr expression in EBV-transformed cells and the mechanisms that regulate its activity therein by using an EBV-negative Burkitt lymphoma cell line, BJAB, and BJAB cells converted to expression of individual EBV proteins by single-gene transfer. CaM kinase-Gr expression was upregulated in BJAB cells by EBV latent-infection membrane protein 1 (LMP1) but not by LMP2A or by nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C. In LMP1-converted BJAB cells, the kinase was functional and was dramatically activated upon cross-linking of surface immunoglobulin M. Overlapping cDNA clones that encode human CaM kinase-Gr were sequenced, revealing 81% amino acid identity between the rat and human proteins. Transfection of BJAB cells with an expression construct for the human enzyme resulted in a functional kinase which was shown by epitope tagging to localize primarily to cytoplasmic and perinuclear structures. Induction of CaM kinase-Gr expression by LMP1 provides the first example of a Ca2+/calmodulin-dependent protein kinase upregulated by a viral protein. In view of the key role played by LMP1 in B-lymphocyte immortalization by EBV, these findings implicate CaM kinase-Gr as a potential mediator of B-lymphocyte growth transformation.

PMA-activation of peripheral blood and tonsillar B lymphocytes induces large adhesive cells reminiscent of large extrafollicular (monocytoid) B cells.

Extrafollicular (EF) B lymphocytes differ in size and morphology depending on the lymphatic organ involved and the kind of inflammatory reaction. On re-evaluating EF B cells in various sites and conditions we discriminated three forms: a small (lymphoid) and intermediate (centrocytoid), and a large (monocytoid) variant. Immunohistochemically, these variants could be discriminated by their differential expression of adhesion molecules CD62L (L-selectin) and CD11c: small EF B cells were strongly L-selectin+ and CD11c-; intermediate cells were moderately CD62L+ and CD11c-; large cells were faintly CD62L+ or - but expressed CD11c. In 72 h cultures of normal peripheral and tonsillar B cells, cross-linking surface immunoglobulin in the presence of interleukin-2 or interleukin-4 led to formation of clusters in vitro together with an increase in cell size and a slight up-regulation of CD11c, as determined by flow cytometry. Stimulation with phorbol 12-myristate 13-acetate (PMA), however, gave rise to large, plastic adherent cells which also showed strong homotypic adhesion, expressed CD62L at minimal levels and CD11c at comparably highest levels and altogether mimicked the large cell variant of EF B cells. We conclude that EF B cells are subjected to cytokine-induced metamorphosis and that differences in cell size and morphology reflect their state of activation and activation-associated adhesion properties. Our data suggest that EF B cells in all anatomical sites are functionally closely related cells which--possibly mediated by CD11c/CD18--may become sessile and proliferate locally once activated by appropriate signals.

An integral membrane protein (LMP2) blocks reactivation of Epstein-Barr virus from latency following surface immunoglobulin crosslinking.

The role of latent membrane protein 2 (LMP2) in Epstein-Barr virus (EBV) infection was evaluated by using latently infected primary B lymphocytes that had been growth transformed by wild-type or specifically mutated EBV recombinants. LMP2 null mutant recombinant EBV-infected cells were similar to normal B lymphocytes in their rapid increase in intracellular free calcium after surface immunoglobulin crosslinking. These cells also became more permissive for lytic EBV replication. In sharp contrast, wild-type control infected cells had little or no increase in intracellular free calcium or in permissivity for EBV replication. The block to surface immunoglobulin crosslinking-induced permissivity in cells expressing wild-type LMP2 could be bypassed by raising intracellular free calcium levels with an ionophore and by activating protein kinase C with phorbol 12-myristate 13-acetate. LMP2A, not LMP2B, mediates this effect on calcium mobilization. Genetic and biochemical data are consistent with these effects being due to the interaction of the LMP2A N-terminal cytoplasmic domain with B lymphocyte src family tyrosine kinases.

Antigen-receptor cross-linking induces peritoneal B-cell apoptosis in normal but not autoimmunity-prone mice

Background: Programmed cell death (apoptosis) is an essential process in the development of various tissues and seems to be involved in the elimination of self-reactive immature T and B lymphocytes when they interact with self antigens. Indeed, signaling through the antigen receptor of immature T cells induces their apoptotic cell death. Immature B cells have also been shown to be eliminated when they interact with antigens, although the involvement of apoptosis has yet to be demonstrated. In contrast, little is known about the elimination of mature lymphocytes upon interaction with antigens. We have previously demonstrated that Ly1 B cells in the peritoneal cavity of transgenic mice undergo apoptotic cell death upon interaction with antigens. As Ly1 B cells constitute a B-cell lineage distinct from conventional B cells, it is important to know whether conventional B cells also undergo apoptosis upon antigen-receptor cross-linking.Results Our experiments show that, in vivo, strong cross-linking of cell-surface immunoglobulins induced apoptotic death of normal, mature B cells in the peritoneal cavity, regardless of whether they were conventional or Ly1 B cells. The same treatment did not kill, but rather activated, B cells in bcl-2-transgenic, apoptosis-resistant mice. Peritoneal B cells from autoimmune-disease-prone New Zealand mouse strains were also found to be resistant to cell death induced by surface immunoglobulin cross-linking.Conclusion Self-reactive B cells are eliminated by the binding of antigen at both mature and immature stages. B-cell activation appears to require, in addition to antigen binding, a second signal that induces expression of rescue molecules such as the bcl-2 gene product. Resistance to B-cell apoptosis induced by antigen receptor cross-linking may play a crucial role in the production of autoantibodies and in the pathogenesis of the autoimmune diseases found in the strains of mice used here.

Restitution of superoxide generation in autosomal cytochrome-negative chronic granulomatous disease (A22(0) CGD)-derived B lymphocyte cell lines by transfection with p22phax cDNA.

The respiratory burst oxidase of phagocytes and B lymphocytes is a multicomponent enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2-production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or physiologic stimuli, such as phagocytosis in phagocytes or cross-linking of surface immunoglobulin in B lymphocytes. The activity of this enzyme is greatly diminished or absent in patients with chronic granulomatous disease (CGD), an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every CGD patient studied so far, an abnormality has been found in a gene encoding one of the four components of the respiratory burst oxidase: the membrane proteins p22phox or gp91phox which together form the cytochrome b558 protein, or the cytosolic proteins p47phox or p67phox. Autosomal recessive cytochrome-negative CGD (A22(0) CGD) is associated with mutations in the gene coding for p22phox. We report here that the capacity for O2- production and cytochrome b558 protein expression were restored to Epstein-Barr virus-transformed B lymphocytes from two A22(0) CGD patients by transfection with an expression plasmid containing a p22phox cDNA. No detectable O2- was generated by untransfected p22phox-deficient lymphocytes. The genetic reconstitution of the respiratory burst in A22(0) CGD B lymphocytes by transfer of the wild-type p22phox cDNA represents a further step towards somatic gene therapy for this subgroup of A22(0) CGD. This system will also be useful for expression of genetically engineered mutant p22phox proteins in intact cells, facilitating the structure-function analysis of cytochrome b558.

Inhibition of superoxide production in B lymphocytes by rac antisense oligonucleotides.

Rac1 and Rac2 gene products are small GTP-binding proteins showing 92% homology to each other. According to recent studies performed in cell-free systems, Rac1 and Rac2 proteins may be involved in the activation of NADPH-oxidase, the superoxide-generating enzymatic complex active in phagocytes. Epstein-Barr virus (EBV) transformed B lymphocytes, which express rac1 and rac2 genes, also efficiently release superoxide anions when triggered by various cell surface stimuli. To investigate the regulatory role of Rac proteins in living cells, we analyzed superoxide production in response to cross-linking of surface immunoglobulins or phorbol ester treatment in human EBV-transformed B lymphocytes pretreated with Rac sense and antisense oligonucleotides. We report here that (i) the rac protein content estimated by immunoblotting can be decreased by 60% in Rac antisense pretreated cells and (ii) a strong (50-60%), dose-dependent inhibition of superoxide production is observed in antisense pretreated cells whereas cells pretreated with sense oligonucleotide are unaffected. The data presented show, for the first time in whole cells, that superoxide production is modulated by the Rac protein content, thus demonstrating the physiological role of Rac proteins in the regulation of NADPH-oxidase.

Cross-linking of surface immunoglobulin activates src-related tyrosine kinases in WEHI 231 cells

Crosslinking of sIgM on the B cell line WEHI 231 with anti-sIgM antibody induces protein tyrosine phosphorylation, implicating protein tyrosine kinases (PTKs) in sIg-mediated signal transduction. We have analyzed this cell line for members of the src family of PTKs and have evaluated whether these PTKs might be involved in the process of sIgM-mediated signaling. Our results show that Blk, Lyn, Lck, and Hck are detectable in WEHI 231 cells. Addition of antibodies to sIgM were found to variably stimulate the activities of Blk, Lyn, Lck, and Hck as measured by immune-complex protein kinase assays. Autophosphorylation of these src PTKs, as assessed by reaction with anti-phosphotyrosine antibodies, increased over the time course of sIgM-mediated activation. Co-immunoprecipitation studies to investigate the potential physical interaction of src PTKs with the sIgM receptor complex revealed that, under digitonin and Brij 96 lysis conditions Lyn, Lck, Hck, but not Blk associated with sIgM.

Relationships between the degree of cross-linking of surface immunoglobulin and the associated inositol 1,4,5-trisphosphate and Ca2+ signals in human B cells.

Cross-linking of surface immunoglobulin (Ig) receptors on human B cells leads to the activation of a tyrosine kinase. The activated tyrosine kinase subsequently phosphorylates a number of substrates, including phospholipase C-gamma. This enzyme breaks down phosphoinositol bisphosphate to form two intracellular messengers, diacylglycerol and inositol 1,4,5-trisphosphate, leading to the activation of protein kinase C and the release of intracellular Ca2+ respectively. We have used h.p.l.c. and flow cytometry to measure accurately the inositol phosphate turnover and Ca2+ release in anti-Ig-stimulated human B cells. In particular, we have examined the effect of dose of the cross-linking antibody on the two responses. The identity of putative messenger inositol phosphates has been verified by structural analysis, and the amounts of both inositol phosphates and Ca2+ present have been quantified. In the Ramos Burkitt lymphoma, which is very sensitive to stimulus through its Ig receptors, both inositol phosphate production and Ca2+ release were found to be related to the dose of anti-Ig antibody applied. This suggests that phospholipase C-mediated signal transduction in human B cells converts the degree of cross-linking of the immunoglobulin receptor quantitatively into intracellular signals.

Intracellular localization of tyrosine kinase substrates beneath crosslinked surface immunoglobulins in B cells.

Crosslinking of surface immunoglobulins (sIg) in B cells led to the accumulation of submembranal phosphotyrosine, which was followed morphologically with the PY20 antiphosphotyrosine monoclonal antibody. Phosphotyrosine was not detected before sIg crosslinking. After sIg crosslinking, phosphotyrosine-containing proteins were redistributed from scattered small clusters near the plasma membrane to a juxtanuclear region, where immunofluorescent staining decreased with time. Double immunofluorescent staining of individual cells showed accumulation of phosphotyrosine beneath crosslinked sIg molecules at the cell surface. The sIg molecules were subsequently internalized more rapidly than the phosphotyrosine-containing molecules were redistributed. Genistein, a protein tyrosine kinase (PTK) inhibitor, blocked intracellular tyrosine phosphorylations but not cell surface patching of crosslinked sIg. When polyacrylamide beads coated with anti-Ig antibodies were added to the cells, intracellular tyrosine phosphorylation occurred beneath the regions of contact with the beads. This study provides an independent line of evidence confirming recent biochemical experiments that show that crosslinking of the antigen receptor induces PTK activity in B cells, and that components of the newly described sIg complex are among the PTK substrates. The surprising finding that the bulk of the induced phosphotyrosine remains associated with crosslinked sIg for many minutes suggests a role for complex local protein interactions in phosphotyrosine-mediated signal transduction through the antigen receptor of B cells.

The Protein Kinase C-Independent Human B Cell Proliferation Induced via Surface Immunoglobulins is Unaffected by CD45 Monoclonal Antibodies

In the present study the effect of 72-5D3 monoclonal antibody (CD45) on the proliferation induced by cross-linking of surface immunoglobulins on untreated and 4β-phorbol 12-myristate 13-acetate-treated human dense B cells was studied. The 72-5D3 mAb inhibited the proliferation induced via surface immunoglobulins alone or plus soluble T cell factors without affecting the release of inositol phosphate metabolites. However, after prolonged incubation (24 h) with high doses (100 ng/ml) of 4β-phorbol 12-myristate 13-acetate or mezerein, but not with 4β-phorbol, the same inhibitory effect did not take place. Therefore, our data support the hypothesis that protein kinase C is necessary for the antiproliferative effect of the 72-5D3 monoclonal antibodies.

Surface immunoglobulin crosslinking activates a tyrosine kinase pathway in B cells that is independent of protein kinase C.

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 of diacylglycerol 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 signaling 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 of anti-immunoglobulin-stimulated phosphatidylinositol bisphosphate hydrolysis does not inhibit activation of this tyrosine kinase-dependent pathway. These findings suggest that occupancy of the membrane immunoglobulin receptor may induce multiple pathways of activation.

Signaling through CD19, Fc receptors or transforming growth factor‐β: each inhibits the activation of resting human B cells differently

To understand further the roles that negative regulatory signals may play in B cell immune responses, we compared three inhibitors of B cell proliferation: cross-linking CD19 with monoclonal antibody (mAb), signaling through Fc receptors by intact anti-mu mAb, and transforming growth factor-beta (TGF-beta). Each agent was tested for its ability to block proliferation and specific activation events induced in human tonsilar B cells activated by either cross-linking surface immunoglobulin, signaling through CD20, or direct activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate. We found that each inhibitor was functionally distinct. Both anti-CD19 mAb and anti-mu mAb inhibited anti-immunoglobulin activated cells and anti-CD20-activated cells, but neither inhibited cells activated by phorbol 12-myristate 13-acetate. TGF-beta, on the other hand, inhibited equally profoundly cells activated by each of the three regimens. These results suggest that TGF-beta blocks B cell activation at a step following the activation of PKC, whereas both signaling through CD19 and Fc receptor block early steps in the PKC activation pathway. Signaling through anti-CD19 mAb was unique in that proliferation of anti-immunoglobulin-activated cells was reduced on day 3 and then augmented subsequently. With all other inhibitory combinations the block was permanent. We conclude that each of these three inhibitors has unique important functions and therefore suggest that the effectiveness of negative signaling in B cell immune regulation will depend on the combinations of specific inhibitors modulating a specific activation program.

Mitogen-induced phosphorylation of human B-lymphocyte proteins. Relationship to protein kinase C activation.

We have investigated the rapid phosphorylation of proteins in B-lymphocytes incubated with the tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), anti-Ig and combinations of TPA and the Ca2+ ionophore ionomycin. Two-dimensional electrophoretic analysis was used to identify the proteins phosphorylated in cells preincubated with [32P]Pi. TPA induced a characteristic pattern of labelled proteins, four of which (pp85, pp76, pp66 and pp63) showed a dose-dependent incorporation of 32P on serine residues. The phosphorylation of pp63 and pp66, in particular, correlated with the mitogenic dose-response curve. Addition of the Ca2+ ionophore ionomycin to B-cells also stimulated a characteristic incorporation of 32P into proteins, which included pp63 and pp66. With combined doses of TPA and ionomycin, these two proteins show an enhanced phosphorylation, which correlated well with the synergistic enhancement of proliferation shown by this combination of agents. Protein kinase C (PKC) was partially purified from B-cells and separated into alpha and beta subtypes. The activation of both PKCs was assessed with increasing doses of TPA and concentrations of Ca2+ of 0.1 microM and 2 microM. For both forms of PKC, in particular the beta form, higher concentrations of Ca2+ shifted the dose-response curve for TPA to the left and increased the maximum activation. Anti-Ig, which stimulated B-cells by cross-linking surface immunoglobulin and causing hydrolysis of PtdIns(4,5)P2, also caused increased phosphorylation of several proteins, which again included pp63 and pp66. These data suggest that PKC, particularly the beta form, is involved in the early part of the proliferation cascade for human B-lymphocytes. It is most probably activated in a synergistic manner by the increased Ca2+ and diacylglycerol levels which result from the earlier hydrolysis of PtdIns(4,5)P2.