Modulation of Epstein-Barr virus-induced B-cell activation by concanavalin A

Direct addition of the T-cell mitogen, concanavalin A (Con A), to cultures of Epstein-Barr virus (EBV)-stimulated peripheral blood mononuclear cells (PBMC) resulted in a dose-dependent inhibition of immunoglobulin M (IgM) secreted in the supernatant, as measured by an enzyme-linked immunosorbent assay. Furthermore, Con A inhibited IgM secretion of isolated T-depleted cells stimulated with EBV, and both the proliferation and IgM secretion of EBV-driven lymphoblastoid cell lines. T-Enriched cells, precultured for 48 hr with Con A, were also able to suppress the IgM response of fresh autologous PBMC stimulated with EBV. This suppression was radiation sensitive (2000 rad), a procedure which resulted in enhancement of the IgM secretion of the responder cells in two out of three experiments. Studies on the long-term effects of Con A showed that the early suppression of IgM secretion was transient and that the mitogen prevented the development of the cytotoxic T-cell response normally seen with lymphocytes from EBV-seropositive donors after 5 weeks of culture. Thus, Con A appears to modulate human lymphocyte responses to EBV by multiple mechanisms.     

Molecular mechanisms of B-lymphocyte transformation by Epstein-Barr virus

The B-lymphotropic virus Epstein-Barr virus (EBV) has been implicated in the pathogenesis of B-cell malignancies, particularly in immunodeficient individuals. This review provides a brief overview of the EBV-encoded proteins involved in B-cell transformation, and the current state of knowledge about their roles in this process.     

CD19 signalling improves the Epstein-Barr virus-induced immortalization of human B cell

Epstein-Barr virus (EBV) infection in vitro immortalizes primary B cells and generates B lymphoblastoid cell lines (LCLs). These EBV-LCLs have been used for several purposes in immunological and genetic studies, but some trials involving these transformations fail for unknown reasons, and several EBV-LCLs do not grow in normal culture. In this study, we improved the immortalization method by CD19 and B-cell receptor (BCR) co-ligation. This method shortens the time required for the immortalization and generation of EBV-LCLs but does not alter the cell phenotype of the LCLs nor the expression of the EBV genes. In particular, the CD19 and BCR co-ligation method was found to be the most effective method examined. EBV-infected B cells induced by CD19 and/or BCR ligation expressed the intracellular latent membrane protein LMP-1 earlier than EBV-infected B cells, and the expression of intracellular LMP-1 was found to be closely related to the time of immortalization. These results suggest that the modified method, using CD19 and/or BCR ligation, may efficiently generate EBV-LCLs, by expressing intracellular LMP-1 at an early stage.     

Suppressive effect of human natural killer cells on Epstein-Barr virus-induced immunoglobulin synthesis

The suppressive effect of human natural killer (NK) cells on Epstein-Barr virus (EBV)-induced immunoglobulin (Ig) synthesis by autologous B cells was investigated. By Percoll discontinuous density gradient centrifugation, low-density fractions enriched for NK cells were isolated from human peripheral blood lymphocytes. These NK-enriched fractions were added to purified autologous B cells in the presence of EBV, were cultivated for 8 days, and were examined for their suppressive effect on Ig synthesis by an enzyme-linked immunosorbent assay. The fractions markedly suppressed both IgM and IgG synthesis induced by EBV. It was possible to reduce the suppressive effect of NK-enriched cells by complement-dependent lysis of NK cells and Leu-11, but not by OKT3 monoclonal antibody, indicating that NK cells may be responsible for the suppression of Ig synthesis. Upon close examination of interferon (IFN) activity, it was revealed that the co-cultures of NK-enriched cells and EBV-infected B cells generated production of IFN-alpha, which might be produced by NK cells in response to EBV-stimulated B cells. Addition of anti-IFN-alpha but not anti-IFN-gamma serum almost completely abrogated the suppressive effect of NK-enriched cells on Ig synthesis, indicating that IFN-alpha produced are required for the NK cell-mediated suppression of Ig synthesis. However, addition of IFN-alpha into purified B cells showed no direct suppressive effect on EBV-induced Ig synthesis by B cells in the absence of NK cells. Nevertheless, NK cells when previously incubated with IFN-alpha and added to B cells showed a suppressor activity on Ig synthesis to a level higher than that of untreated NK controls. These results strongly suggest the possibility that NK cells display an interaction with EBV-infected B cells and produce IFN-alpha, which in turn activates NK cells. These activated NK cells suppress the Ig synthesis by B cells, which undergo transformation induced by EBV.     

Severe thrombocytopenia in Epstein-Barr virus-induced mononucleosis.

Severe thrombocytopenia is a rare complication of Epstein-Barr virus-induced infectious mononucleosis. We evaluated the clinical and laboratory data from seven patients seen between 1976 and 1985 whose lowest platelet counts varied from 3 to 25 x 10(9) per liter. Five of the seven patients were initially thought to have either acute leukemia or idiopathic thrombocytopenic purpura; eventually, however, primary Epstein-Barr virus infections were confirmed in all patients. Two of six patients tested had antiplatelet antibodies during the acute phase of their illnesses. Eight additional patients with acute disease who had only mild thrombocytopenia (94 to 144 x 10(9) per liter) were also tested for platelet antibodies with negative results. Steroid therapy was administered to three patients and platelet transfusions to one. All seven patients recovered with no serious hemorrhagic sequelae.     

Limiting dilution analysis of Epstein-Barr virus-induced immunoglobulin production

The major goal of this work is to establish a culture system for the growth of human B lymphocytes at the single-cell level so that the immunoglobulin secreted by the clonal progeny of that cell can be analyzed. A method which involves culturing small numbers (1–1000) of lymphocytes, which have been infected with Epstein-Barr virus (EBV) prior to plating, in round-bottom microtiter plates is described. A feeder layer of irradiated (2500 R) umbilical cord blood lymphocytes to which phytohemagglutinin has been added was found to be optimal. Culture supernatants collected from 3 to 6 weeks postinfection are assayed for the production of IgG and IgM by radioimmunoassay in order to determine the overall cloning efficiency of the system. We have shown that up to 33% of surface Ig-positive cells produce detectable clones in this system. Umbilical cord blood cells are superior to T-cell and macrophage cell lines as feeder layers. Furthermore, culture supernatants from phytohemagglutinin-stimulated umbilical cord lymphocytes do not adequately replace these cells. We also observed that while most IgM-secreting clones continued to produce immunoglobulin during the 7-week time period analyzed, the majority of IgG-secreting clones had a relatively short half-life in vitro. This culture system allows us to examine a significant proportion of the human B-cell population and carry out studies on the frequency of specific antibody- and isotype-producing clones.     

Characterization of an Epstein-Barr virus-induced thymidine kinase.

Previous work from our laboratory suggested that the selective inhibition of Epstein-Barr virus (EBV) replication by 1-beta-D-arabinofuranosylthymine in human lymphoid cell lines involved the induction of a new thymidine kinase (TK) able to phosphorylate the thymidine analog. We further characterized this enzyme induced in various EBV-positive cell lines after viral genome activation with a combination of sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate. The following results confirmed the existence of an EBV-specific deoxypyrimidine kinase: induction of EBV-related TK was connected with the appearance of viral early antigens in EBV-carrying cells; unexpected behaviors of the enzyme activity upon different fractionating treatments led to the conclusion that EBV-induced TK was extracted as a complex molecular form, larger than other known cellular or viral isozymes; enzymatic properties distinguished EBV-induced TK from host lymphoid cell isozymes but made it resemble other herpesvirus-specific deoxypyrimidine kinases, i.e., by partial inhibition by dTTP or ammonium sulfate, insensitiveness to dCTP, and nonstringent specificity for normal TK substrates. Genetic evidence is required to definitively ensure that EBV-specific TK actually is virus coded in EBV-transformed human lymphoid cells.     

Distinct regulation of dengue virus-induced inflammasome activation in human macrophage subsets

Macrophages (Mϕ) are the major source of inflammatory cytokines and are target cells for dengue virus (DV) replication. However, Mϕ are heterogeneous and their phenotypic and functional diversities are influenced by cytokines that regulate their differentiation, tissue distribution, and defense against invading pathogens. In vitro, human primary macrophages are derived from peripheral blood CD14⁺ monocytes in the presence of macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF). These are essential for developing tissue/resting macrophages (M-Mϕ) and inflammatory macrophages (GM-Mϕ), respectively. While IFN production is similar between M-Mϕ and GM-Mϕ, M-Mϕ cannot produce IL-1β after DV infection. In contrast, GM-Mϕ is more susceptible to DV infection and DV triggers CLEC5A in GM-Mϕ to activate NLRP3 inflammasomes, which in turn release IL-18 and IL-1β that are critical for Th17 activation and contribute to disease severity. Thus, GM-Mϕ is more representative than M-Mϕ for investigating inflammasome activation in dengue infection, and is invaluable for revealing the molecular mechanism of pathogen-induced inflammatory reaction. Distinct phenotypes of macrophage subsets under the influence of M-CSF and GM-CSF raise the question of optimal conditions for culturing primary macrophages to study host-pathogen interaction.     

Papain solubilization of the Epstein-Barr virus-induced membrane antigen.

The Epstein-Barr virus (EBV)-induced membrane antigen (MA) was successfully solubilized from the membranes of viable EBV-infected Raji cells by treatment with papain (5 to 6 U per 1 X 10(7) to 2 X 10(7) cells). The loss of MA from viable cells was monitored by membrane immunofluorescence and antibody-dependent cellular cytotoxicity. Soluble MA was demonstrated in papain digests through inhibition of antibody-dependent cellular cytotoxicity and by inhibition of the binding of anti-MA antibodies to cells as detected by use of 125I-labeled staphylococcal protein A. Approximately 75% of the MA activity in the extracts was not sedimentable at 100,000 X g,, indicating that the majority of EBV MA activity that was released by this procedure was associated with small-molecular-weight material. Antiserum prepared from an owl monkey immunized with these papain extracts contained antibody to MA and neutralizing antibodies, but lacked detectable antibodies against viral capsid antigens and EBV-induced early antigens.     

Mechanism of human immunodeficiency virus-induced complement expression in astrocytes and neurons

The cerebral complement system is hypothesized to contribute to neurodegeneration in the pathogenesis of AIDS-associated neurological disorders. Our former results have shown that the human immunodeficiency virus (HIV) strongly induces the synthesis of complement factor C3 in astrocytes. This upregulation explains in vivo data showing elevated complement levels in the cerebrospinal fluid of patients with AIDS-associated neurological symptoms. Since inhibition of complement synthesis and activation in the brain may represent a putative therapeutic goal to prevent virus-induced damage, we analyzed in detail the mechanisms of HIV-induced modulation of C3 expression. HIV-1 increased the C3 levels in astrocyte culture supernatants from 30 to up to 400 ng/ml; signal transduction studies revealed that adenylate cyclase activation with upregulation of cyclic AMP is the central signaling pathway to mediate that increase. Furthermore, activity of protein kinase C is necessary for HIV induction of C3, since inhibition of protein kinase C by prolonged exposure to the phorbol ester tetradecanoyl phorbol acetate partly abolished the HIV effect. The cytokines tumor necrosis factor alpha and gamma interferon were not involved in mediating the HIV-induced C3 upregulation, since neutralizing antibodies had no effect. Besides whole HIV virions, the purified viral proteins Nef and gp41 are biologically active in upregulating C3, whereas Tat, gp120, and gp160 were not able to modulate C3 synthesis. Further experiments revealed that neurons were also able to respond on incubation with HIV with increased C3 synthesis, although the precise pattern was slightly different from that in astrocytes. This strengthens the hypothesis that HIV-induced complement synthesis represents an important mechanism for the pathogenesis of AIDS in the brain.     

Transferrin receptor induction is required for human B-lymphocyte activation but not for immunoglobulin secretion

Transferrin receptors are expressed on proliferating cells and are required for their growth. Transferrin receptors can be detected after, but not before, mitogenic stimulation of normal peripheral blood T and B cells. In the experiments reported here we have examined the regulation of transferrin receptor expression on activated human B cells and whether or not these receptors are necessary for activation to occur. Activation was assessed by studying both proliferation and immunoglobulin secretion. We have determined that transferrin receptor expression on B cells is regulated by a factor contained in supernatants of mitogen-stimulated T cells (probably B-cell growth factor). This expression is required for proliferation to occur, since antibody to transferrin receptor (42/6) blocks B-cell proliferation. Induction of immunoglobulin secretion, however, although dependent on PHA-treated T-cell supernatant, is not dependent on transferrin receptor expression and can occur in mitogen-stimulated cells whose proliferation has been blocked by antitransferrin receptor antibody. In addition, we have demonstrated that IgM messenger RNA induction following mitogen stimulation is unaffected by antitransferrin receptor antibody. These findings support a model for B-cell activation in which mitogen (or antigen) delivers two concurrent but distinct signals to B cells: one, dependent on B-cell growth factor and transferrin receptor expression, for proliferation, and a second, dependent on T cell-derived factors and not requiring transferrin receptors, which leads to immunoglobulin secretion.     

Surface antigen changes during B-lymphocyte activation in primates

It is shown that B-cell-specific surface antigens are conserved on lymphocytes from phylogenetically distant primate species. Characterization of the expression of those antigens on the surface of simian B lymphocytes has led to two observations with important implications for human B-cell physiology. First, lectin stimulation in vitro or antigen stimulation in situ in lymph nodes drives a population of human B lymphocytes to express the B2 but not the B1 antigen on its surface. Second, under pathologic circumstances, this activated B cell can be found in the peripheral blood of monkeys. Thus, the “B2 only” cell defines an activated B lymphocyte whose presence may provide useful diagnostic information concerning pathologic processes.     

CD4+ T-cell effectors inhibit Epstein-Barr virus-induced B-cell proliferation

In immunodeficient hosts, Epstein-Barr virus (EBV) often induces extensive B-cell lymphoproliferative disease and lymphoma. Without effective in vitro immune surveillance, B cells infected by the virus readily form immortalized cell lines. In the regression assay, memory T cells inhibit the formation of foci of EBV-transformed B cells that follows recent in vitro infection by EBV. No one has yet addressed which T cell regulates the early proliferative phase of B cells newly infected by EBV. Using new quantitative methods, we analyzed T-cell surveillance of EBV-mediated B-cell proliferation. We found that CD4+ T cells play a significant role in limiting proliferation of newly infected, activated CD23+ B cells. In the absence of T cells, EBV-infected CD23+ B cells divided rapidly during the first 3 weeks after infection. Removal of CD4+ but not CD8+ T cells also abrogated immune control. Purified CD4+ T cells eliminated outgrowth when added to EBV-infected B cells. Thus, unlike the killing of EBV-infected lymphoblastoid cell lines, in which CD8+ cytolytic T cells play an essential role, prevention of early-phase EBV-induced B-cell proliferation requires CD4+ effector T cells.     

Mechanism of collagen activation in human platelets

The mechanism of collagen-induced human platelet activation was examined using Ca2+, Na+, and the pH-sensitive fluorescent dyes calcium green/fura red, sodium-binding benzofuran isophthalate, and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Administration of a moderate dose of collagen (10 microg/ml) to human platelets resulted in an increase in [Ca2+](i) and platelet aggregation. The majority of this increase in [Ca2+](i) resulted from the influx of calcium from the extracellular milieu via the Na+/Ca2+ exchanger (NCX) functioning in the reverse mode and was reduced in a dose-dependent manner by the NCX inhibitors 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (KD(50) = 4.7 +/- 1.1 microm) and KB-R7943 (KD(50) = 35.1 +/- 4.8 microm). Collagen-induced platelet aggregation was dependent on an increase in [Ca2+](i) and could be inhibited by chelation of intra- and extracellular calcium through the administration of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) and EGTA, respectively, or via the administration of BAPTA-AM to platelets suspended in no-Na+/HEPES buffer. Collagen induced an increase in [Ca2+](i) (23.2 +/- 7.6 mm) via the actions of thromboxane A(2) and, to a lesser extent, of the Na+/H+ exchanger. This study demonstrates that the collagen-induced increase in [Ca2+](i) is dependent on the concentration of Na+ in the extracellular milieu, indicating that the collagen-induced increase in [Ca2+](i) causes the reversal of the NCX, ultimately resulting in an increase in [Ca2+](i) and platelet aggregation.