BLAST-2 [EBVCS], an early cell surface marker of human B cell activation, is superinduced by Epstein Barr virus

Activation of human B cells by pokeweed mitogen (PWM), protein A, anti-IgM, or EBV infection results in the expression of a new surface antigen, termed BLAST-2 [EBVCS]. This marker appears before the cells undergo blast transformation as assessed by the initiation of DNA synthesis and expression of the BLAST-1 antigen. Thus, the BLAST-2 [EBVCS] antigen is expressed on both activated and lymphoblastoid cells. The antigen is, in addition, restricted to B cells, as it is not found on cells of T or myeloid lineage derived from peripheral blood, cell lines, or neoplastic cells. However, it is readily detected on chronic lymphocytic leukemia cells of B cell origin and in the germinal centers of tonsils and lymph nodes. Like the BLAST-1 antigen, BLAST-2 [EBVCS] is expressed at a high level only on EBV-transformed B lymphoblasts and has a m.w. close to 45,000. Immunoprecipitation experiments show, however, that the two antigens are expressed on distinct populations of molecules.     

Growth inhibition of Epstein-Barr virus-transformed B cells by anti-HLA-DR antibody L243: possible relationship to L243-induced down-regulation of CD23 antigen expression

Among five anti-HLA class II monoclonal antibodies (mab's) tested, the anti-HLA-DR mab L243 selectively inhibited the in vitro proliferation of Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL). Saturating amounts of L243 mab induced 50% suppression of LCL growth as revealed by measuring [3H]thymidine incorporation or counting cell numbers. Preincubation for 20 hr at 37 degrees C in the presence of L243 mab dramatically reduced the expression of certain B cell-specific antigens on LCL, notably CD23 (BLAST-2, low affinity Fc epsilon receptor). In view of the known function of the CD23 antigen as a B cell growth factor receptor, we discuss the possibility that the suppressive effect of L243 mab on LCL proliferation is due to down-regulation of CD23 antigen expression.     

Direct human T helper cell-induced B cell activation is not mediated by inositol lipid hydrolysis

The Ag-specific interaction between cloned allospecific human Th cells and class II MHC determinants on the surface of allogeneic B cells induces a significant fraction of resting B cells to express a B cell specific activation Ag BLAST-2 (CD23). On the other hand, cross-linking of B cell surface Ig R by Ag analogues does not lead to BLAST-2 expression. By utilizing the BLAST-2 induction assay as a positive control for efficient Th-B cell interaction, we have investigated the biochemical basis of human B cell activation mediated by Ag and Th cells. Our data demonstrate that ligands for sIg R, including F(ab')2 goat anti-human IgM and Staphylococcus aureus protein A, stimulate the metabolism of B cell membrane inositol lipids as assessed by: 1) increased [3H]inositol phosphates formation in myo-[3H]inositol-labeled B cells; 2) selective incorporation of [32P]orthophosphate into phosphatidic acid and phosphatidylinositol, but not into phosphatidylethanolamine or phosphatidylcholine; and 3) rapid increase in B cell cytoplasmic ionized Ca2+ concentration ([Ca2+]i). In contrast, direct Th-B cell interaction leads to high intensity BLAST-2 expression on the B cell surface but this response is not mediated by changes in inositol lipid metabolism or [Ca2+]i. Further, Th-B cell interaction does not affect the changes in B cell inositol lipid metabolism or [Ca2+]i triggered by sIg cross-linking. Taken together, our results suggest that Ag and Th cells induce different functional B cell responses by activating distinct second messenger systems within the B cell.     

Biochemical analysis suggests distinct functional roles for the BLAST-1 and BLAST-2 antigens

The biochemical processing of the BLAST-1 and BLAST-2 activation antigens has been studied. Both are glycoproteins that derive from different precursors of the same apparent m.w. on SDS-PAGE. BLAST-1 is synthesized as a 43,000 m.w. light chain in association with a second heavier chain of 55,000 m.w. The light chain acquires sialylated O-linked glycans and is stably expressed at the cell surface with a half-life of 14 hr. BLAST-2 is also synthesized as a 43,000 m.w. precursor, but it acquires only unsialylated N-linked glycans. The mature glycoprotein is only expressed briefly at the cell surface (half-life of 1 to 2 hr), and is then shed into the culture supernatant as a soluble 33,000 m.w. derivative. The different fates of these molecules, one stably expressed at the cell surface and one shed, suggest disparate roles for these two antigens in B cell activation.     

Purification and biochemical characterization of a human autocrine growth factor produced by Epstein-Barr virus-transformed B cells

Autocrine growth factor for Epstein-Barr virus-transformed human B cells (aBGF), a protein that is constitutively produced by the human EBV-transformed B cell line 5/2, has been purified from serum-free conditioned medium. The purification involved sequential ammonium sulfate precipitation, ion exchange chromatography, gel filtration, and reversed-phase high performance liquid chromatography. The purified protein has a m.w. of 16,000 in NaDodSO4/polyacrylamide gel electrophoresis and an isoelectric point between 7.0 and 8.0. The relative molecular mass 16,000 form exists in equilibrium with dimeric and tetrameric forms. aBGF supports the growth of EBV-transformed B cells, which have been deprived of their own conditioned medium. The purified aBGF is fully effective at 0.5 ng/ml and has no interleukin 1 activity in the lymphocyte activation factor assay. Because several randomly selected lines of EBV-transformed cells and one EBV-negative lymphoma cell line both produce aBGF activity and show growth dependency on aBGF and because stimulation of normal B cells with anti-immunoglobulin M is increased by aBGF, we propose that aBGF has general significance for growth control of human B cells.     

Modulation of cardiac rhythm by allatostatins in the cockroach Blattella germanica (L.) (Dictyoptera,Blattellidae)

Cardiac rhythm was measured in Blattella germanica females during the reproductive cycle. The rate increased from day 0 to 1, remained constant during the vitellogenic period and fell by about 20% during the period of oothecal transport. The effects of allatostatins, allatostatin analogues and corazonin were tested on semi-isolated heart preparations. Allatostatins showed a rapid, reversible and dose-dependent cardioinhibitory activity. Blattella allatostatin 1 (BLAST-1: LYDFGL-NH(2)), was the most active, eliciting 76% inhibition at 10(-7) M and even 19% inhibition at 10(-9) M. BLAST-2 (DRLYSFGL-NH(2)), BLAST-3 (AGSDGRLYSFGL-NH(2)) and BLAST-4 (APSSAQRLYGFGL-NH(2)) were less active. An analogue of BLAST-2 with C-terminus in acid form and a pseudopeptide analogue of BLAST-2 with a methyleneamino Psi[CH(2)NH] peptide bond surrogate between residues L(3) and Y(4) were inactive. Corazonin elicited rapid, reversible and dose-dependent cardioacceleratory activity. When tested together with BLAST-1, corazonin overrode the cardioinhibitory effect of allatostatin. Our previous results had shown that high levels of allatostatin were maintained during the period of oothecal transport. This and the fact that physiological concentrations of allatostatins produce physiological levels of inhibition, suggest that allatostatins are involved in the modulation of cardiac rhythm in this cockroach.     

Epstein-Barr virus nuclear protein 2A forms oligomers in vitro and in vivo through a region required for B-cell transformation.

Epstein-Barr virus nuclear antigen 2 (EBNA-2) has been shown to be indispensable for immortalization of latently infected B lymphocytes, and it has been shown that EBNA-2 exists in a high-molecular-weight complex in these cells. In order to study the components of this protein machinery, we have purified baculovirus-expressed EBNA-2 from insect cells to greater than 95% homogeneity. We have shown by both gel filtration and sucrose gradient analysis that the purified material corresponds to a multimer containing eight EBNA-2 subunits. This multimeric complex is stable in 1.0 M NaCl, suggesting that the self-association is quite strong in vitro. By expressing portions of the EBNA-2 open reading frame to generate fusion proteins in yeast cells, we have used the two-hybrid system to demonstrate that this self-association occurs in vivo and is mediated at least in part by a domain of EBNA-2 encompassing amino acids 122 to 344. Mutational analysis of the self-association function suggests that two subdomains that flank amino acid 232 may each play a role in EBNA-2 protein-protein interaction.     

Binding of C3 and C3dg to the CR2 complement receptor induces growth of an Epstein-Barr virus-positive human B cell line

The effect of ligand interactions with the C3d/C3dg complement receptor (CR2) on proliferation of human B lymphoblastoid cells was investigated by using cell cultures performed at low density (1 to 1.5 x 10(3) cells/ml) in a serum-free defined medium to which only transferrin had been added. This medium does not allow proliferation of Raji cells which die within 48 hr with formation of polykaryons. Addition of purified human C3 to the cultures resulted in a dose-dependent proliferation of the cells. A steady growth of Raji cells with a doubling time of 36 hr was observed in cultures containing 10 micrograms/ml of C3. A growth rate similar to that observed in the presence of native C3 was found in the presence of equimolar concentrations of purified C3dg but not of C3c. F(ab')2 anti-C3d but not F(ab')2 anti-C3c antibodies inhibited the mitogenic effect of C3. Preincubation of Raji cells with monoclonal antibody OKB7 which directly inhibits the binding of C3dg to CR2, totally suppressed C3-induced growth of the cells. C3 did not enhance growth of the T lymphoma-derived cell line JM and monocytic cell line U937 which do not express CR2. These results provide direct evidence that the interaction between CR2 and C3 fragments stimulates proliferation of human cells of the B lineage. Because CR2 also acts as a receptor for Epstein-Barr virus on B cells, our results may pertain to the B cell mitogenic properties of the virus.     

Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death.

Epstein-Barr virus (EBV) not only induces growth transformation in human B lymphocytes, but has more recently been shown to enhance B cell survival under suboptimal conditions where growth is inhibited; both effects are mediated through the coordinate action of eight virus-coded latent proteins. The effect upon cell survival is best recognized in EBV-positive Burkitt's lymphoma cell lines where activation of full virus latent gene expression protects the cells from programmed cell death (apoptosis). Here we show by DNA transfection into human B cells that protection from apoptosis is conferred through expression of a single EBV latent protein, the latent membrane protein LMP 1. Furthermore, we demonstrate that LMP 1 mediates this effect by up-regulating expression of the cellular oncogene bcl-2. The interplay between EBV infection and expression of this cellular oncogene has important implications for virus persistence and for the pathogenesis of virus-associated malignant disease.     

Human peripheral blood T helper cell-induced B cell activation results in B cell surface expression of the CD23 (BLAST-2) antigen

We have developed an in vitro system to assess the early stages of B cell activation induced by peripheral blood T helper cells. Peripheral blood mononuclear cells are cultured for 16 hr with anti-CD3 monoclonal antibody (mAb), T lymphocytes are then removed by sheep red blood cell rosette depletion, and expression of the B cell surface activation antigen CD23 (BLAST-2) is assessed by indirect immunofluorescence. Anti-CD3 mAb, but not a control anti-CD5 mAb, stimulates the expression of CD23 on 20-50% of peripheral blood B cells cultured with autologous T cells. T cell subset depletion studies show that the CD4+ T cell subset is responsible for anti-CD3-mediated induction of CD23 on autologous B cells. Anti-CD3-induced, T helper cell-dependent CD23 expression is not MHC-restricted, as allogeneic combinations of T and non-T cells, cultured in the presence of anti-CD3 antibody, also result in the expression of B cell CD23. Individuals whose monocyte Fc receptors bind murine IgG1 mAb poorly fail to trigger T cell proliferation in response to murine IgG1 anti-CD3 mAb and also fail to express B cell CD23 following culture of PBMC with IgG1 anti-CD3 mAb, while the usual expression of CD23 is seen after culture with IgG2a anti-CD3 mAb. The mechanism of anti-CD3-induced B cell activation was addressed in experiments using a two-chamber culture system. While little IL-4 activity was detected in anti-CD3-stimulated culture supernatants, optimal induction of CD23 was observed when T and B cells were cultured together in a single chamber. This suggests that under physiologic conditions, in which quantities of lymphokine may be limiting, close physical contact between the anti-CD3-activated Th cell and B cell may be required for CD23 expression. The anti-CD3-induced BLAST-2 assay will facilitate the analysis of Th cell-mediated B cell activation in any individual and should permit us to separately evaluate the roles of Th cells and B cells in the impaired immunoregulation characteristic of autoimmune disorders.     

Epstein-Barr virus nuclear antigen 2 induces expression of the virus-encoded latent membrane protein.

Infection of Epstein-Barr virus-negative human B-lymphoma cell lines with the fully transforming B95.8 Epstein-Barr virus strain was associated with complete virus latent gene expression and a change in the cell surface and growth phenotype toward that of in vitro-transformed lymphoblastoid cell lines. In contrast, the cells infected with the P3HR1 Epstein-Barr virus strain, a deletion mutant that cannot encode Epstein-Barr nuclear antigen 2 (EBNA2) or a full-length EBNA-LP, expressed EBNAs1, 3a, 3b, and 3c but were negative for the latent membrane protein (LMP) and showed no change in cellular phenotype. This suggests that EBNA2 and/or EBNA-LP may be required for subsequent expression of LMP in Epstein-Barr virus-infected B cells. Recombinant vectors capable of expressing the B95.8 EBNA2A protein were introduced by electroporation into two P3HR1-converted B-lymphoma cell lines, BL30/P3 and BL41/P3. In both cases, stable expression of EBNA2A was accompanied by activation of LMP expression from the resident P3HR1 genome; control transfectants that did not express the EBNA2A protein never showed induction of LMP. In further experiments, a recombinant vector capable of expressing the full-length B95.8 EBNA-LP was introduced into the same target lines. Strong EBNA-LP expression was consistently observed in the transfected clones but was never accompanied by induction of LMP. The EBNA2A gene transfectants expressing EBNA2A and LMP showed a dramatic change in cell surface and growth phenotype toward a pattern like that of lymphoblastoid cell lines; some but not all of these changes could be reproduced in the absence of EBNA2A by transfection of P3HR1-converted cell lines with a recombinant vector expressing LMP. These studies suggest that EBNA2 plays an important dual role in the process of B-cell activation to the lymphoblastoid phenotype; the protein can have a direct effect upon cellular gene expression and is also involved in activating the expression of a second virus-encoded effector protein, LMP.     

Molecular mechanisms of TNFR-associated factor 6 (TRAF6) utilization by the oncogenic viral mimic of CD40, latent membrane protein 1 (LMP1)

Latent membrane protein 1 (LMP1), encoded by Epstein-Barr virus, is required for EBV-mediated B cell transformation and plays a significant role in the development of posttransplant B cell lymphomas. LMP1 has also been implicated in exacerbation of autoimmune diseases such as systemic lupus erythematosus. LMP1 is a constitutively active functional mimic of the tumor necrosis factor receptor superfamily member CD40, utilizing tumor necrosis factor receptor-associated factor (TRAF) adaptor proteins to induce signaling. However, LMP1-mediated B cell activation is amplified and sustained compared with CD40. We have previously shown that LMP1 and CD40 use TRAFs 1, 2, 3, and 5 differently. TRAF6 is important for CD40 signaling, but the role of TRAF6 in LMP1 signaling in B cells is not clear. Although TRAF6 binds directly to CD40, TRAF6 interaction with LMP1 in B cells has not been characterized. Here we tested the hypothesis that TRAF6 is a critical regulator of LMP1 signaling in B cells, either as part of a receptor-associated complex and/or as a cytoplasmic adaptor protein. Using TRAF6-deficient B cells, we determined that TRAF6 was critical for LMP1-mediated B cell activation. Although CD40-mediated TRAF6-dependent signaling does not require the TRAF6 receptor-binding domain, we found that LMP1 signaling required the presence of this domain. Furthermore, TRAF6 was recruited to the LMP1 signaling complex via the TRAF1/2/3/5 binding site within the cytoplasmic domain of LMP1.     

Epstein-Barr virus (EBV) antigens processed and presented by B cells, B blasts, and macrophages trigger T-cell-mediated inhibition of EBV-induced B-cell transformation.

The ability of B cells, B blasts, and macrophages to present Epstein-Barr virion antigens to autologous T cells and trigger their capacity to inhibit Epstein-Barr virus-induced B-cell transformation was tested. Macrophages were as efficient as B cells and B blasts in presenting the virus to T lymphocytes. This function required antigen processing, because it was inhibited by chloroquine treatment and by fixation of the antigen-presenting cells immediately after viral exposure but not 18 h later. T cells exposed to the purified Epstein-Barr virus envelope antigen gp350 coupled to immunostimulating complexes also showed inhibitory function. These results suggest that recognition of processed virion antigens elicits the generation of T-cell-mediated inhibition of Epstein-Barr virus-induced B-cell transformation.     

T cell-derived B cell growth factor(s) can induce stimulation of both resting and activated B cells

The effects of a preparation containing partially purified, EL4-derived B cell growth factor(s) (BCGF) on B cell growth and proliferation have been examined by using B lymphocyte subpopulations separated on the basis of size. BCGF was found to maintain and enhance proliferation of a significant proportion of large activated B cells. In contrast, small resting B cells required the presence of BCGF and a second stimulus such as anti-IgM antibody (anti-mu) to be induced to proliferate. This disparity was not due to a lack of an effect of BCGF on small resting B cells. A factor contained within the partially purified EL4 supernatant produced time-dependent increases in cell size and RNA content in all subpopulations. These effects were independent of possible effects due to contaminating lymphokines such as interleukin 2 (IL 2), concanavalin A (Con A), and phorbol myristate acetate (PMA). Nonmitogenic doses of lipopolysaccharide (LPS) failed to show similar effects. Our data suggest that B cells at all levels of in vivo activation are responsive to stimulation by a growth factor present in EL4 supernatant, as manifested by cell growth and RNA synthesis. This activity has not previously been described for BCGF preparations. However, because the partially purified, EL4-derived supernatant used as BCGF in these studies has not been purified to homogeneity, we cannot conclude whether the factors that induce resting B cells to increase in size are the same as the growth factors that synergize with anti-mu to induce B cell proliferation or that maintain the proliferation of activated B cells.     

Soluble Epstein-Barr virus glycoproteins gH, gL, and gp42 form a 1:1:1 stable complex that acts like soluble gp42 in B-cell fusion but not in epithelial cell fusion

Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cell membrane. The fusion process requires the actions of viral glycoproteins gH, gL, and gB for entry into epithelial cells and additionally requires gp42 for entry into B cells. To further study the roles of these membrane-associated glycoproteins, purified soluble forms of gp42, gH, and gL were expressed that lack the membrane-spanning regions. The soluble gH/gL protein complex binds to soluble gp42 with high affinity, forming a stable heterotrimer with 1:1:1 stoichiometry, and this complex is not formed by an N-terminally truncated variant of gp42. The effects of adding soluble gp42, gH/gL, and gH/gL/gp42 were examined with a virus-free cell-cell fusion assay. The results demonstrate that, in contrast to gp42, membrane fusion does not proceed with secreted gH/gL. The addition of soluble gH/gL does not inhibit or enhance B-cell or epithelial cell fusion when membrane-bound gH/gL, gB, and gp42 are present. However, the soluble gH/gL/gp42 complex does activate membrane fusion with B cells, similarly to soluble gp42, but it does not inhibit fusion with epithelial cells, as observed for gp42 alone. A gp42 peptide, derived from an N-terminal segment involved in gH/gL interactions, binds to soluble gH/gL and inhibits EBV-mediated epithelial cell fusion, mimicking gp42. These observations reveal distinct functional requirements for gH/gL and gp42 complexes in EBV-mediated membrane fusion.     

The BCL1 B lymphoma responds to IL-4, IL-5, and GM-CSF

Proliferation in vitro of the in vivo passaged murine B cell tumor line BCL1 has been used as a standard assay for mouse interleukin-5 (IL-5) for a number of years. We demonstrate that this line will also respond to human IL-5. The response to murine IL-5 is abrogated by transforming growth factor-beta and to a lesser extent by interferon-gamma. This suggests a possible regulatory role for these lymphokines in the proliferation of B cells induced by IL-5. Other purified recombinant lymphokines were also tested for their ability to induce BCL1 proliferation. The lymphokines IL-1, IL-2, IL-3, and IL-6 had no effect on the growth of BCL1. In contrast, IL-4 and more surprisingly granulocyte-macrophage colony-stimulating factor (GM-CSF) also induced proliferation of this cell. These effects could be inhibited by specific antibodies directed against the respective lymphokines. These data suggest that GM-CSF, as well as IL-4 and IL-5, may be yet another regulator of neoplastic and possibly even normal B-cell growth and differentiation.