Generation of nondividing high rate Ig-secreting plasma cells in cultures of human B cells stimulated with anti-CD3-activated T cells.

The capacity of human B cells to differentiate into high rate nondividing antibody-secreting plasma cells was investigated. Highly purified human peripheral blood B cells were stimulated with polyclonal B cell activators in the presence of a variety of recombinant cytokines (IL-2, IL-4, IL-6). Maximal production of Ig of all isotypes was observed when B cells were stimulated with intact T cells that were activated with mAb to the CD3 molecular complex. In these cultures, Ig production continued for more than 16 days. Moreover, differentiation to nondividing high rate Ig-producing cells was induced, as evidenced by a ninefold increase in the amount of Ig produced per Ig-secreting cell and the acquisition of resistance of ongoing Ig secretion to the inhibitor of DNA synthesis, hydroxyurea. To determine whether intact T cells were required for the entire culture period to achieve maximal Ig production, B cells were cultured with activated T cells for various lengths of time, reisolated and cultured with fresh activated T cells or various cytokines, then analyzed for Ig secretion. B cells preactivated for 6 days with anti-CD3-stimulated T cells required contact with intact T cells for continued Ig secretion. However, after 9 days of preactivation, dividing B cells responded maximally to anti-CD3-stimulated T cells, whereas cytokines were able to drive continued IgG secretion by nondividing B cells in the absence of intact T cells. IL-6 alone, or in combination with either IL-2 or IL-4, was the major cytokine driving ongoing Ig secreting by nondividing preactivated B cells. These results suggest that continued clonal expansion of Ig-secreting B cell blasts requires intact anti-CD3-activated T cells, whereas terminal differentiation of B cells into plasma cells after extensive clonal expansion is driven by cytokines, most notably IL-6.     

Syntaxin-4 is essential for IgE secretion by plasma cells

The humoral immune system provides a crucial first defense against the invasion of microbial pathogens via the secretion of antigen specific immunoglobulins (Ig). The secretion of Ig is carried out by terminally differentiated B-lymphocytes called plasma cells. Despite the key role of plasma cells in the immune response, the mechanisms by which they constitutively traffic large volumes of Ig out of the cell is poorly understood. The involvement of Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins in the regulation of protein trafficking from cells has been well documented. Syntaxin-4, a member of the Qa SNARE syntaxin family has been implicated in fusion events at the plasma membrane in a number of cells in the immune system. In this work we show that knock-down of syntaxin-4 in the multiple myeloma U266 human plasma cell line results in a loss of IgE secretion and accumulation of IgE within the cells. Furthermore, we show that IgE co-localises with syntaxin-4 in U266 plasma cells suggesting direct involvement in secretion at the plasma membrane. This study demonstrates that syntaxin-4 plays a critical role in the secretion of IgE from plasma cells and sheds some light on the mechanisms by which these cells constitutively traffic vesicles to the surface for secretion. An understanding of this machinery may be beneficial in identifying potential therapeutic targets in multiple myeloma and autoimmune disease where over-production of Ig leads to severe pathology in patients.     

Cytokine network regulating terminal maturation of human bone marrow B cells capable of spontaneous and high rate Ig secretion in vitro.

Human bone marrow (BM) B cells capable of spontaneous and high rate Ig secretion for 14 days in vitro have been described previously. We have shown recently that Ig secretion by these BM cells depends on stromal adherent BM cell-derived factors identified as IL-6 and fibronectin. Our report shows that the endogenous generation of IL-1 beta and TNF-alpha in serum-containing cultures of BM mononuclear cells (BMMC) is also involved in the control of Ig-secreting cells, because their blockade with specific antibodies markedly reduced Ig production. Further experiments revealed that IL-1 beta and TNF-alpha acted by regulating IL-6 production, as can be deduced from the following findings: 1) the inhibition of Ig secretion caused by either anti-IL-1 beta or anti-TNF-alpha antibodies could be reversed by exogenous IL-6; 2) the addition of either of these antibodies inhibited endogenous IL-6 production in BMMC cultures; 3) IL-1 beta plus TNF-alpha, but neither one alone, restored complete IL-6 and Ig production by BMMC in serum-free cultures. Moreover, adherent, but not nonadherent, BM cells were responsible for endogenous IL-1 beta and TNF-alpha secretion. Finally, IL-1 beta plus TNF-alpha induced the production of IL-6, but not of Ig, by adherent BM cells. Neither IL-6 nor Ig production was induced by adding this cytokine combination to nonadherent BM cell cultures, despite the fact that this fraction contained all the Ig-secreting cells. However, the addition of IL-6 restored Ig secretion in this cell fraction. These results suggest that IL-1 beta and TNF-alpha produced by adherent BM cells synergistically induce early IL-6 generation, which, in turn, drives BM B cell producers into the high rate Ig-secreting state.     

Genetic control of B- and T-Lymphocyte abnormalities of NZB mice in crosses with B10.D2 mice

Parental NZB and B10.D2, F₁ and F₁ × B10.D2 mice were studied to determine the genetic control of (1) altered B-cell IgD expression, (2) plasma cell frequency, (3) IgM secretion per plasma cell, (4) primary in vitro cytotoxic T-cell responses to H-2-compatible cells, (5) production of thymocyte-binding antibodies, and (6) production of red-cell-specific antibodies. The results demonstrate that, in this cross, IgD abnormalities and production of red-cell-specific antibodies were recessive traits. There was a common genetic influence on plasma cell frequency, IgM secretion per plasma cell and production of thymocyte-binding antibodies which was distinct from the genes governing the ability to generate a cytotoxic T lymphocyte response to H-2-compatible cells.Abbreviations used in this paper CTL cytotoxic T lymphocyte - F1 anti-Fab fluorescein-labeled antimouse Fab - FMF flow microfluorometry - Ig immunoglobulin - IgM/PC IgM secretion per PC - PC plasma cell - sIg surface immunoglobulin - TBA thymocyte-binding antibody     

Platelet-activating factor enhances Ig production in B lymphoblastoid cell lines

Platelet-activating factor (PAF) is a highly potent phospholipid mediator known to be active in many biologic systems. To date, little is known of the effect of PAF on B lymphocytes. Using two Ig-secreting B lymphoblastoid cell lines, we have demonstrated that PAF can enhance Ig production by these cells in a dose-dependent fashion. PAF also causes significant alteration of the kinetics of Ig secretion in these lymphoblastoid cell lines. The effect of PAF is rapid, with detection of 6- to 12-fold increases in Ig production in the first 24 h of cell culture, followed by a plateau during the next 24 to 48 h. The specificity of the PAF effect on Ig secretion is emphasized by lyso-PAF having no Ig-enhancing properties and by the inhibition of Ig enhancement in the presence of the structural analogue PAF antagonist CV3988 and the soluble nonstructural analogue PAF receptor antagonist Web 2086. PAF does not cause an increase in the kinetics of cell proliferation or an increase in cell numbers at any time during a 72-h culture period. In an attempt to explain the increase in Ig secretion in the absence of changes in growth parameters, an ELISA spot assay for enumeration of Ig-secreting cells was developed. This assay demonstrated that the increase in Ig production is likely due to enhancement of single cell Ig secretion rather than an increase in cell number. These data indicate that PAF may have an important immunomodulatory role in the production of Ig by B lymphocytes.     

Plasmablast and plasma cell production and distribution in trout immune tissues

These studies describe the in vitro and ex vivo generation of plasmablasts and plasma cells in trout (Oncorhynchus mykiss) peripheral blood and splenic and anterior kidney tissues. Cells were derived either from naive trout and cultured with the polyclonal activator, Escherichia coli LPS, or from trout that had been immunized with trinitrophenyl-keyhole limpet hemocyanin. Hydroxyurea was used to resolve populations of replicating (plasmablast) and nonreplicating (plasma cell) Ab-secreting cells (ASC). Complete inhibition of Ig secretion was only observed within the PBL. Both anterior kidney and splenic lymphocytes possessed a subset of ASCs that were hydroxyurea resistant. Thus, in vitro production of plasma cells appears to be restricted to the latter two tissues, whereas peripheral blood is exclusively restricted to the production of plasmablasts. After immunization with trinitrophenyl-keyhole limpet hemocyanin, specific ASC could be isolated from all immune organs; however, the anterior kidney contained 98% of all ASC. Late in the response (>10 wk), anterior kidney ASC secreted specific Ab for at least 15 days in culture, indicating that they were long-lived plasma cells. Cells from spleen and peripheral blood lost all capacity to secrete specific Ab in the absence of Ag. Late in the Ab response, high serum titer levels are solely the result of Ig secretion from anterior kidney plasma cells.     

CD5+ peritoneal B cells express high levels of membrane, but not secretory, C mu mRNA.

We used in situ hybridization to study Ig mRNA levels in murine peritoneal and splenic B cells. Ig mRNA production fell into three distinct groups: low, intermediate, and high. Splenic B cells primarily exhibited low levels characteristic of resting B cells or high Ig mRNA levels characteristic of plasma cells. In contrast, a significant fraction of peritoneal B cells exhibited intermediate Ig mRNA levels. Intermediate Ig mRNA was T cell dependent in that congenic nu/nu mice had far fewer peritoneal cells expressing the intermediate Ig message than their wild type counterparts. CD5+ CD11b+ IgMbright+ peritoneal B cells were found to be mainly responsible for the production of intermediate Ig mRNA levels. The peritoneal CD5- CD11b+ IgMbright+ "sister" B cell subpopulation contained a lower percentage of intermediate Ig mRNA-producing B cells. CD5-CD11b-IgMdull+ "conventional" B cells produced negligible levels of Ig mRNA, comparable to those of unfractionated splenic B cells. Northern analysis showed that the majority of Ig mRNA expressed in the peritoneum is of the membrane rather than the secreted form. Consistent with that result, in short-term culture, peritoneal cells showed markedly less Ig secretion than did spleen cells. These studies describe novel Ig mRNA expression by peritoneal B cells and emphasize that within the peritoneal cavity, B cells do not tend to become antibody-secreting cells.     

Basophils support the survival of plasma cells in mice

We have previously shown that basophils support humoral memory immune responses by increasing B cell proliferation and Ig production as well as inducing a Th2 and B helper phenotype in T cells. Based on the high frequency of basophils in spleen and bone marrow, in this study we investigated whether basophils also support plasma cell survival and Ig production. In the absence of basophils, plasma cells of naive or immunized mice rapidly undergo apoptosis in vitro and produce only low amounts of Igs. In contrast, in the presence of basophils and even more in the presence of activated basophils, the survival of plasma cells is markedly increased and continuous production of Igs enabled. This effect is partially dependent on IL-4 and IL-6 released from basophils. Similar results were obtained when total bone marrow cells or bone marrow cells depleted of basophils were cultured in the presence or absence of substances activating basophils. When basophils were depleted in vivo 6 mo after immunization with an Ag, specific Ig production in subsequent bone marrow cultures was significantly reduced. In addition, depletion of basophils for 18 d in naive mice significantly reduced the number of plasma cells in the spleen. These data indicate that basophils are important for survival of plasma cells in vitro and in vivo.     

B cell activation. VIII. Membrane immunoglobulins transduce signals via activation of phosphatidylinositol hydrolysis

Considerable evidence indicates that cross-linking of B cell surface Ig results in a "first signal" in B cell activation. We have shown that transduction of this signal is manifest by changes in plasma membrane potential leading to increased expression of surface I-A antigen. In previous studies, we have provided evidence that suggests that this signal is transduced via phosphatidylinositol (PI) hydrolysis liberating diacylglycerol (DAG), which subsequently activates protein kinase C. These biochemical events are aspects of a transmembrane signal transduction mechanism that is common in nature and utilizes the PI metabolic cycle for generation of "second messenger" diacylglycerol. Here we report direct evidence that treatment of B cells with various antibodies to surface Ig results in activation of the PI cycle. Results suggest that the increased phospholipid metabolism that occurs in B cells in response to anti-Ig involves only those phospholipids in the PI cycle and is a consequence of turnover of existing lipid rather than de novo synthesis. Furthermore, we show that PI cycle activation requires cross-linking of membrane Ig and is inhibitable by increased intracellular cyclic AMP. These findings are particularly important in view of previous studies that have shown identical requirements for and inhibitability of induction of B cell membrane depolarization and increased I-A expression. Thus, these results are consistent with our previous hypothesis that early B cell activation events initiated by receptor Ig occupancy are mediated via PI hydrolysis, diacylglycerol generation, and protein kinase C activation.     

Inhibition of N-linked oligosaccharide trimming mannosidases blocks human B cell development.

Deoxymannojirimycin (dMM) or swainsonine (SW), which block conversion of high-mannose to complex-type N-linked glycans, strongly inhibited the production of immunoglobulin (Ig) when added to cultures of human lymphocytes together with the polyclonal B cell activators pokeweed mitogen (PWM) and Staphylococcus aureus (SAC). To obtain the inhibitory effect, inhibitor had to be present during the first 36 h of culture. Addition at later timepoints was less effective and showed that neither inhibitor interfered with rate of production or secretion of Ig as such. Viability and proliferation of the lymphocytes, as defined by cell number and rate of DNA synthesis, were not influenced by the presence of dMM or SW, and no changes in the relative number of helper (T4+) or suppressor (T8+) cells were observed. Thus, for normal differentiation of human B lymphocytes into Ig secreting (plasma) cells in response to PWM and SAC, conversion of high-mannose to complex N-linked glycans is essential.     

Lymphocyte function in human bone marrow. I. Characterization of two T cell populations regulating immunoglobulin secretion

We analyzed the regulation of immunoglobulin (Ig) production in short-term cultures of human (rib) bone marrow cells. In contrast to blood or tonsil cell cultures, large quantities of IgG and IgA, but not IgM, were secreted by unstimulated marrow cells. The addition of pokeweed mitogen or phytohemagglutinin resulted in the suppression of this Ig secretion. Both mitogens induced the production of high levels of interleukin 2 (IL 2) in marrow cultures, and the addition of IL 2 alone mimicked the suppressive effect of mitogens. Incubation of marrow cells with Epstein Barr virus resulted in enhanced Ig secretion, primarily of the IgM isotype. The addition of mitogen or IL 2 suppressed Ig production in these cultures as well. The mitogen-induced suppression of Ig secretion in stimulated or unstimulated marrow cultures was inhibited by the monoclonal anti-TAC (IL 2 receptor) antibody. Cell separation experiments indicated that the induction of suppressor activity in marrow cultures involved two distinct populations of marrow-resident T lineage cells. The first population responds to activation by mitogens with the production of IL 2. This population has a surface phenotype appropriate for helper T cells. The second T cell population expresses T8 and TAC determinants. These cells acquire suppressor cell activity after exposure to IL 2. The expression of suppressor function does not require additional (e.g., mitogenic) activation signals. The IL 2-dependent marrow suppressor T cells represent a newly recognized T lymphocyte subset. The regulatory pathway delineated may be important for the regulation of antibody formation in bone marrow, the major site of Ig production in man.     

Promotion of activated human B cell apoptosis and inhibition of Ig production by soluble CD95 ligand: CD95-based downregulation of Ig production need not culminate in activated B cell death

CD95/CD95L interactions are vital to normal lymphoid homeostasis and in the protection against autoimmunity. To directly assess the effects of CD95L on activated B cell survival and Ig responses, purified human peripheral blood B cells, activated in vitro with SAC + rIL2, were incubated with a soluble CD95L fusion protein (fp) and assayed for apoptosis and IgG/IgM production. CD95L fp reproducibly increased apoptosis of these activated B cells and inhibited their Ig production. However, CD95L fp-mediated effects on activated B cell survival could be uncoupled from those on Ig production in that a soluble CD40L fp was incapable of reversing CD95L fp-mediated downregulation of Ig responses despite inhibiting CD95L fp-mediated apoptosis. Moreover, despite the specific caspase-8 inhibitor z-IETD-fmk substantially protecting transformed CL-01 B cells from CD95L fp-mediated apoptosis and permitting their ongoing proliferation, caspase-8 inhibition had no protective effects on CD95L fp-mediated inhibition of constitutive IgM production by CL-01 B cells. Collectively, these results point to a CD95-based downregulatory pathway in activated B cells that need not necessarily culminate in their death.     

Mesenchymal stem cells infected with Mycoplasma arginini secrete complement C3 to regulate immunoglobulin production in b lymphocytes

Mesenchymal stem cells (MSCs) have immunomodulatory functions such as the suppression of T and B cells. MSCs suppress immunoglobulin (Ig) production by B cells via cell–cell contact as well as via secretion of soluble factors. Our study showed that the conditioned medium (CM) of MSCs infected with a mycoplasma strain, Mycoplasma arginini, has marked inhibitory effects on Ig production by lipopolysaccharide/interleukin-4-induced B cells compared with mycoplasma-free MSC-CM. We analyzed mycoplasma-infected MSC-CM by fast protein liquid chromatography and liquid chromatography to screen the molecules responsible for Ig inhibition. Complement C3 (C3) was the most critical molecule among the candidates identified. C3 was shown to be involved in the suppression of the Ig production of B cells. C3 was secreted by mycoplasma-infected MSCs, but not by mycoplasma-free MSCs or B cells. It was able to directly inhibit Ig production by B cells. In the presence of a C3 inhibitor, Ig inhibition by MSC-CM was abrogated. This inhibitory effect was concomitant with the downregulation of B-cell-induced maturation protein-1, which is a regulator of the differentiation of antibody-secreting plasma cells. These results suggest that C3 secreted from mycoplasma-infected MSCs has an important role in the immunomodulatory functions of MSCs. However, its role in vivo needs to be explored.     

Immunomodulatory role of IL-4 on the secretion of Ig by human B cells

The effect of IL-4 on the production of Ig by human B cells was examined. Highly purified B cells were stimulated with Staphylococcus aureus (SA) and IL-4 alone or in combination with various other cytokines and the supernatants assayed for Ig by isotype-specific ELISA. IL-4 (10 to 100 U/ml) did not support Ig secretion by SA-stimulated blood, spleen, or lymph node B cells, whereas IL-2 supported the production of all isotypes including IgE. Moreover, IL-4 suppressed the production of all isotypes of Ig by B cells stimulated with SA and IL-2 including IgG1, IgG2, and IgE. IL-4-mediated suppression was partially reversed by IFN-gamma or -alpha and low m.w. B cell growth factor. TNF-alpha and IL-6 did not reverse the IL-4-induced suppression of Ig production. The inhibitory action of IL-4 on Ig production appeared to depend on the polyclonal activator used to stimulate the B cells. Thus, Ig secretion by B cells activated by LPS and supported by IL-2 was not inhibited by IL-4. Whereas IL-4 alone supported minimal Ig production by LPS-activated B cells, it augmented production of all Ig isotypes in cultures stimulated with LPS and supported by IL-2. IFN-gamma further enhanced production of Ig in these cultures. When the effect of IL-4 on the responsiveness of B cells preactivated with SA and IL-2 was examined, it was found not to inhibit but rather to promote Ig production modestly. A direct effect of IL-4 on the terminal differentiation of B cells was demonstrated using B lymphoblastoid cell lines. IL-4 was able to enhance the Ig secreted by an IgA-secreting hybridoma, 219 and by SKW6-CL-4, an IL-6-responsive IgM-secreting EBV transformed B cell line. These results indicate that IL-4 exerts a number of immunoregulatory actions on human B cell differentiation. It interferes with the activation of B cells by SA and IL-2, but promotes the differentiation of preactivated B cells, B cell lines, and B cells activated by LPS without apparent isotype specificity.