Suppression of polyclonal human B cell activation by IgG binding factors: interference with the maturation of Ig-containing cells into Ig-secreting cells

Human IgG binding factors (IgG BF) were prepared by immunopurification on IgG immunosorbents from cell-free supernatants of unstimulated peripheral blood mononuclear cells (PB MNC). The suppressive effects of IgG BF was studied using PB MNC stimulated by pokeweed mitogen or by nocardia delipidated cell mitogen. At the end of the culture three parameters of B cell activation were measured: (1) the numbers of IgM-, IgG-, or IgA-containing cells (CC) using direct immunofluorescence, (2) the numbers of IgM, IgG, or IgA plaque-forming cells (PFC) using a Protein A hemolytic plaque assay, and (3) the concentrations of IgM, IgG, or IgA in culture supernatants using an enzyme-linked immunosorbent assay. Addition of IgG BF at the third day of culture resulted in a selective decrease of IgG CC, while IgM CC and IgA CC were increased or unchanged. Conversely, IgG BF induced a nonselective diminution of the number of PFC and of the amount of secreted Ig of the three major Ig classes. Therefore the results demonstrate two distinct effects of IgG BF: (1) an isotype-specific suppression of cells producing IgG, demonstrated by the parallel decrease of IgG CC and IgG PFC, and (2) a blocking of the late stages of B cell maturation evidenced by the discrepancy between normal or elevated Ig CC and decreased Ig PFC of the IgM and IgA classes.     

B cell response to T helper cell subsets. II. Both the stage of T cell differentiation and the cytokines secreted determine the extent and nature of helper activity.

Helper activity of several murine CD4+ T cell subsets was examined. Effector Th, derived from naive cells after 4 days of in vitro stimulation with alloantigen, when generated in the presence of IL-4, secreted high levels of IL-4, IL-5, and IL-6, and low levels of IL-2 and IFN-gamma, and induced the secretion of all Ig isotypes particularly IgM, IgG1, IgA, and IgE from resting allogeneic B cells. Effectors generated with IL-6 secreted IL-2, IL-4, IL-5, IL-6, and IFN-gamma, and induced similar levels of total Ig, 25 to 35 micrograms/ml, but with IgM, IgG3, IgG1, and IgG2a isotypes predominating. Helper activity of these Th was significantly greater than that of effectors generated with IL-2 (10-15 micrograms/ml Ig) and of 24-h-activated naive and memory cells (2-4 micrograms/ml), both of which induced mainly IgM. Unlike other isotypes, IgE was induced only by effector Th generated with IL-4. Blocking studies showed that secretion of all isotypes in response to IL-6-primed effectors was dependent on IL-2, IL-5, and IL-6. IL-4 was required for optimal IgM, IgG1, and IgA secretion, but limited secretion of IgG2a, whereas IFN-gamma was required for optimal IgG2a secretion, and limited IgM, IgG1, and IgA. In contrast, secretion of all isotypes in response to IL-4-primed effectors was dependent on IL-5, although IL-4 and IFN-gamma were also essential for IgE and IgG2a, respectively. Addition of exogenous IL-5 to B cell cultures driven by IL-6-primed effectors did not obviate the requirement for IL-2, IL-4, and IL-6, suggesting that interaction of IL-4-primed effectors with B cells was qualitatively different from that of IL-6-primed effectors, driving B cells to a stage requiring only IL-5 for differentiation. Addition of exogenous factors to IL-2-primed effector Th, particularly IL-4 in the presence of anti-IFN-gamma, resulted in levels of Ig, including IgE, comparable to those induced with other effectors. These results show that functionally distinct Th cell subsets can be generated rapidly in vitro, under the influence of distinct cytokines, which vary dramatically in their levels of help for resting B cells. The cytokines involved in responses to distinct Th cells differ depending on the quality of interaction with the B cell, and the extent of help is strongly determined by the quantity and nature of cytokines secreted by the T cells.     

Sequential synergistic effect of interleukin 2 and interferon-gamma on the differentiation of a Tac-antigen-positive B cell line

The presence of Tac-antigen (Tac-Ag) on human B lymphocytes and its functional significance with regard to the ability of interleukin 2 (IL 2) to modulate B cell differentiation is currently an area of high interest. An Epstein-Barr virus-transformed B cell line (CB) that secretes IgG was 30 to 40% Tac-Ag+ and was used as a model for examining the role of Tac-Ag and IL 2 in B cell differentiation. Recombinant IL 2 alone was found to have a modest but significant effect on CB in enhancing IgG secretion, increasing the plaque-forming cell response from 637 to 1734 at high concentrations (1000 U/ml IL 2) and to 888 at lower concentrations (100 U/ml). In contrast, recombinant interferon-gamma (IFN-gamma) alone had no effect on the differentiation of CB. However, both factors together showed marked synergy in increasing the number of plaque-forming cells to over 3000 by using only 10 U/ml of IFN-gamma and 100 U/ml of IL 2. These two factors were shown to act sequentially in that IL 2 was needed initially, while IFN-gamma was required for the next differentiation step into IgG-secreting cells. The effect of IL 2 on stimulating differentiation was blocked by anti-Tac, indicating that the action of IL 2 is mediated through its Tac-Ag receptor. CB cells were also sorted into Tac+ and Tac- populations and were cultured separately. In 2 wk, both populations reverted to the pattern of the original cell line. Moreover, cell cycle analysis when using double staining procedures indicated that Tac-Ag on the cell surface of CB appears and disappears according to the stage of the cell cycle, and that Tac is most strongly expressed in the S and G2 + M phases. Thus, the present study suggests that certain B cells are capable of responding to sequential stimulation by IL 2 and IFN-gamma with terminal differentiation into Ig-secreting cells, and that the amount of Tac-Ag expression is cell cycle dependent.     

Ophiopogonin D attenuates the progression of murine systemic lupus erythematosus by reducing B cell numbers

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by immune abnormalities leading to multi-organ damage. The activation of autoreactive B cell differentiation will lead to the production of pathogenic autoantibodies, contributing to the development of SLE. However, the effects of Ophiopogonin D (OP-D) on B cell activation and autoantibody production as well as renal injury in the pathogenesis of SLE remain unclear. MRL/lpr mice, one of the most commonly used animal models of SLE, were intragastrically administered with 5 mg/kg/d OP-D at 17 weeks of age for 3 weeks. The survival rates of mice in each group were monitored for 6 weeks until 23 weeks of age. Proteinuria and serum creatinine levels were measured. Serum levels of immunoglobulin (Ig)G, IgM, and anti-dsDNA autoantibodies were detected by enzyme-linked immunosorbent assay. Numbers of CD19⁺ B cells in the blood, spleen and bone marrow and numbers of splenic germinal center (GC) B cells were calculated by using flow cytometry. OP-D treatment prolonged survival in MRL/lpr mice. OP-D treatment reduced proteinuria and serum creatinine levels as well as mitigated renal pathological alternation in MRL/lpr mice. Furthermore, serum levels of IgG, IgM, and anti-dsDNA autoantibodies were reduced by OP-D treatment. OP-D lessened not only CD19⁺ B cells in the spleen and bone marrow but also plasma cells that secreted anti-dsDNA autoantibodies, IgG and IgM in the spleen and bone marrow. OP-D ameliorated the progression of SLE by inhibiting the secretion of autoantibodies though reducing B cell numbers.     

Effect of recombinant IL 2 and gamma-IFN on proliferation and differentiation of human B cells

The effects of IL 2 and gamma-IFN on the activation of human B cells was studied with recombinant IL 2 and gamma-IFN. BCDF-responsive B lymphoblastoid cell lines and highly purified human B cells were employed as target B cells. IL 2 or gamma-IFN did not induce any IgG or IgM secretion in the B cell lines CESS and SKW6-CL4, in which IgG and IgM were inducible with conventional T cell factor(s). IL 2 alone did not induce the optimum production of Ig, but did induce proliferation in the SAC-stimulated B cell population. No Leu-1-, Leu-4-, or Leu-7-positive cells were detected in B cell populations that had been stimulated with SAC for 3 days. FACS analysis showed that a portion of the SAC-stimulated B cells (30%) were in the G2 or M stages by IL 2 stimulation. The addition of gamma-IFN together with IL 2 induced IgM and IgG secretion in SAC-stimulated B cells that was comparable with that induced by a conventional T cell factor(s). IL 2 induced proliferation not only in SAC-stimulated B cells but also in an anti-mu-stimulated B cell population. Stimulation of T cell populations with anti-mu and IL 2 did not induce significant proliferation, suggesting the direct effect of IL 2 on B cells. Double staining of anti-mu-stimulated B cells with anti-Ig and anti-Tac antibodies demonstrated that anti-mu stimulation induced an increased expression of Tac antigen on surface Ig-positive B cells. All of these results strongly supported the notion that IL 2 was one of the growth factors for B cells, and gamma-IFN was one of the differentiation factors for B cells.     

Functional studies on B cell hybridomas with B cell surface antigens. IV. Direct effects of cytochalasin B on differentiation

We previously established B cell hybridomas between M12.4.1 B lymphoma of BALB/c mice and normal B cell of C57BL/6 (B6) mice. These hybridomas express Iab, Iad, and IgM molecules on the cell membrane, and can induce the generation of IgM secretion when treated with purified goat anti-mouse-mu antibody (anti-mu) without T cell factors. In this study, TH2.54, a subclone of a B cell hybridoma, was treated with cytochalasin B (CB), a fungal product that disrupts microfilaments, and the direct effect of CB on the proliferation and differentiation of TH2.54 was examined. CB considerably suppressed the spontaneous proliferation of hybrid cells. This product, however, did not inhibit the generation of IgM secretion by TH2.54 treated with anti-mu. Surprisingly, CB could directly induce the development of IgM-secreting cells by TH2.54 at a relatively high frequency. Among cytochalasins, dihydrocytochalasin B (H2CB), cytochalasin C (CC), and cytochalasin D (CD) showed marked effects on the induction of IgM secretion as well as CB. In addition, the differentiative effect of CB was greatly inhibited by N6, O2-dibutyryladenosine 3':5'-cyclic monophosphate (dbc-AMP), but not by N2, O2-dibutyrylguanosine 3':5'-cyclic monophosphate (dbc-GMP). Analysis by flow microfluorometry (FMF), cytotoxicity assays, and quantitative absorption tests demonstrated that CB treatment of TH2.54 resulted in a significant decrease in the expression of Iab, Iad, and IgM molecules on the cell membrane. In contrast, parental M12.4.1 neither generated any IgM secretion nor changed Iad expression on the cell membrane under the same conditions. The present study suggests very strongly that microfilament-microtubule systems are not involved in the differentiative process of TH2.54 induced by anti-mu. The results also indicate that CB can provide the initiative signal for differentiation of TH2.54 into the maturation lineage; this is followed by a significant change in the expression of Ia and IgM molecules on the cell membrane.     

Regulation of mucosal B cell immunoglobulin secretion by intestinal epithelial cell-derived cytokines

Intestinal epithelial cells (IEC) secrete a variety of cytokines and, because of their close proximity to B cells in the lamina propria, may affect local antibody production via these cytokines. However, studies have not yet addressed which and to what extent these IEC-derived cytokines may affect B cell antibody production. In this study, rat mesenteric lymph node B cells were cultured with culture supernatants from the rat IEC-6 intestinal epithelial cell line to determine their effect on immunoglobulin (Ig) secretion. Unstimulated IEC-6 cells were found to secrete sufficient levels of IL-6 to enhance IgA, IgG and IgM secretion by unstimulated B cells. However, culture of lipopolysaccharide (LPS)-stimulated B cells with the unstimulated IEC-6 supernatant resulted in an enhancement of IgA secretion while IgM secretion was significantly suppressed. Depletion of the IEC-6 supernatant using cytokine specific antibodies revealed that both interleukin 6 (IL-6) and transforming growth factor beta (TGF-beta) were responsible for the enhanced IgA secretion while TGF-beta suppressed IgM secretion. More importantly, culture supernatants from LPS stimulated IEC-6 cells contained enhanced levels of IL-6 which enhanced both IgG and IgA production and partially overcame the suppressive effect of TGF-beta on IgM secretion. These results suggest that intestinal epithelial cells may secrete IL-6 and TGF-beta to regulate local B cell antibody secretion and their effect may be highly dependent upon the activation state of the epithelial cells.     

Rapid loss of IgM expression by normal murine B cells undergoing IgG1 and IgE class switching after in vivo immunization

Injection of mice with polyclonal goat anti-mouse IgD antibody (G alpha M delta) stimulates a potent T cell-dependent immune response characterized by large increases in serum IgG1 and IgE concentrations and by the generation of substantial numbers of membrane (m)IgG1+ B cells. The onset of this response occurs 6 days after G alpha M delta injection and peaks by day 7 to 8. Utilizing two color fluorescence analysis and cell sorting we demonstrate that most mIgG1-expressing B cells lack mIgM during the period of onset of Ig isotype switching (day 6). Both IgG1 and IgE are produced predominantly by mIgM- cells. On day 6, IgG1 and IgE are secreted predominantly by cells expressing mIgG1 and mIgE, respectively. By day 8, a majority of the IgG1 secretion occurs among the mIgG1- cells but virtually all IgE secretion continues to come from the mIgE+ population. B cells that strongly express mIgG1 secrete little IgM or IgE. Freshly harvested B cells expressing mIgG1, 6 days after G alpha M delta injection, have undergone substantial deletion of CH mu-specific DNA in contrast to their mIgG1- counterparts. Hence, the great majority of B cells that switch to the IgG1 or IgE isotypes in vivo rapidly lose their expression of IgM.     

Analysis of T cell and B cell function in Peyer's patch and lamina propria of New Zealand Black and DBA/2 mice

We have analyzed gastrointestinal immune function in both DBA/2 and spontaneously autoimmune New Zealand Black (NZB) mice. We have studied both in vitro proliferation and differentiation of Peyer's patch cells and have measured immunoglobulin (Ig) secretion by cultured jejunal segments. Peyer's patch B cells and T cells from both DBA/2 and NZB mice showed similar proliferative responses to Con A and lipopolysaccharide (LPS), respectively. Unlike NZB splenic B cells, isolated Peyer's patch B cells from NZB mice did not spontaneously secrete Ig of any isotype. Seven-day cultures of equal numbers of Peyer's patch T cells and B cells resulted in similar patterns of secretion of IgA, IgG, and IgM in both strains. The addition of Con A to cultures of DBA/2 Peyer's patch cells consistently resulted in a onefold to threefold increase in IgA secretion after 7 days. Con A stimulation of NZB Peyer's patch cells did not produce any increment in IgA secretion. LPS stimulation of Peyer's patch cells from either strain resulted in a similar increase in IgG secretion with little effect on IgA secretion. The in vivo correlate of this finding was seen in the IgA to IgG ratio of Ig secreted by cultured jejunal fragments. In DBA/2 mice the rates of IgA/IgG varied from 2.36 to 4.85, whereas in NZB mice the ratio never exceeded 0.5. These experiments show that defects on the T cell compartment of NZB mice encompass gut-associated lymphoid tissue. The possible relationship of these findings and previously observed defects in oral tolerance is discussed.     

Kinetics of interleukin-4 induction and interferon-gamma inhibition of IgE secretion by Epstein-Barr virus-infected human peripheral blood B cells.

Interleukin-4 (IL-4) acts directly on purified human peripheral blood B cells cultured in the presence of Epstein-Barr virus (EBV) to induce IgE secretion and to enhance the secretion of IgG and IgM. Interferon-gamma (IFN-gamma) inhibits IgE secretion in this system, without affecting the secretion of the other Ig isotypes. To identify the time period during which EBV-infected B cells can be induced by IL-4 to secrete IgE, we have studied the effects of delayed addition of IL-4, or the termination of IL-4 stimulation by wash out or by neutralization with anti-IL-4 antibodies, on the induction of an IgE response. To induce a maximal IgE response, IL-4 had to be added to cultures of B cells plus EBV no later than 2 days after the initiation of culture, and had to remain present through the tenth day of culture. These two time points correspond to the initiation of detectable DNA synthesis (Days 3 to 4) and the earliest detectable Ig secretion (Days 10 to 12) by EBV-stimulated B cells. No IgE response was induced if the period during which EBV-stimulated B cells were cultured with IL-4 was less than 4 days, or if IL-4 were added later than the tenth day of culture, regardless of how long the culture was continued beyond that time. In contrast, IL-4 considerably enhanced IgG and IgM secretion and B cell CD23 expression, even if it was added after the tenth day of culture. IFN-gamma strongly inhibited the IgE response of B cells cultured with IL-4 plus EBV if added within 6 days of the initiation of culture, but had little effect on the generation of IgM or IgG responses made by these cells, regardless of the time of addition. Neither IL-4 nor IFN-gamma affected ongoing IgE secretion by an established, IgE-secreting, EBV-transformed cell line. These observations suggest that: (i) IL-4 first becomes able to induce EBV-activated B cells to secrete IgE as these cells begin to synthesize DNA, must stimulate B cells for at least 4 days to induce IgE secretion, and loses its ability to induce IgE secretion as these cells differentiate into Ig-secreting cells; (ii) the ability of IFN-gamma to suppress an IgE response is limited to this same time period; and (iii) IL-4 enhancement of CD23 expression and IgM and IgG secretion are independent of IL-4 induction of an IgE response.     

Double immunocytochemical staining in the study of antibody-producing cells in vivo. Combined detection of antigen specificity (anti-TNP) and (sub)class of intracellular antibodies

Mice and rabbits were immunized with trinitrophenyl (TNP)-conjugated keyhole limpet hemocyanin (KLH). Cells producing specific antibodies against the hapten TNP were detected in vivo in spleen and lymph nodes using a TNP--alkaline phosphatase (AP) conjugate. Using horseradish peroxidase (HRP)-conjugated anti-mouse (sub)class (IgG2A, IgG2B, IgM) antibodies and anti-rabbit class (IgG, IgM) antibodies and a double immunocytochemical staining technique for simultaneous demonstration of the enzymes AP and HRP, we were able to determine both the antigen specificity (anti-TNP) and the (sub)class of intracellular antibodies produced by individual antibody-forming cells in vivo.     

Role of DNA synthesis in secretion of immunoglobulin from murine B cells stimulated by T cell derived lymphokines

We have investigated whether cell division is required for induction of Ig secretion from three types of B cells, which represent distinct activation states: normal splenic B cells, anti-Ig-treated B cells, and a monoclonal murine B cell tumor, BCL1. Polyclonal Ig secretion was stimulated in vitro by LPS or by lymphokines produced by EL-4 cells (EL-4 SN), which includes B cell growth factor II (BCGF II). LPS and EL-4 SN were mitogenic for all three cell populations and stimulated substantial IgM secretion from both B cells and anti-Ig blasts. Aphidicolin, a reversible inhibitor of DNA synthesis, abolished IgM secretion from B cells and anti-Ig blasts induced by either mitogen, indicating that Ig-secreting cells in these cultures are part of a cycling population. BCL1 tumor cells respond to BCGF II (but not to interleukin 2 or B cell stimulatory factor 1) with IgM secretion and cell division, allowing a direct assessment of the influence of BCGF II-stimulated cell division on secretion of IgM. Secretion by these cells during the first 24 hr of culture was not substantially affected by aphidicolin, but secretion at 48 or 72 hr was markedly inhibited. Culture of BCL1 cells for 48 hr with aphidicolin alone had no effect on cell viability or on subsequent responsiveness if the drug was removed, eliminating non-specific toxicity as an explanation of the drug's effect. Addition of aphidicolin during the last 24 hr of culture to either normal B cells or BCL1 cells was much less effective at inhibiting IgM secretion. These results indicate that the cells that secrete IgM in response to BCGF II also synthesize DNA when exposed to this factor. Thus, induction of high-rate Ig secretion from murine B cells by some stimuli, including BCGF II, may require at least one round of cell division.     

B cell response to fresh and effector T helper cells. Role of cognate T-B interaction and the cytokines IL-2, IL-4, and IL-6

The helper activity of resting T cells and in vitro generated effector T cells and the relative roles of cognate interaction, diffusible cytokines, and non-cognate T-B contact in B cell antibody responses were evaluated in a model in which normal murine CD4+ T cells (Th), activated with alloantigen-bearing APC, were used to support the growth and differentiation of unstimulated allogeneic B cells. Both "fresh" T cells, consisting of memory and naive cells, stimulated for 24 h, and "effector" T cells, derived from naive cells after 4 days of in vitro stimulation, induced the secretion of IgM, IgG3, IgG1, IgG2a, and IgA. Effector T cells were significantly better helpers of the response of small dense B cells, inducing Ig at lower numbers and inducing at optimal numbers 2- to 3-fold more Ig production than fresh T cells. The predominant isotype secreted was IgM. Supernatants derived from fresh T cell cultures contained moderate levels of IL-2, whereas those from effector cultures contained significant levels of IL-6 and IFN-gamma in addition to IL-2. The involvement of soluble factors in the B cell response was demonstrated by the ability of antibodies to the cytokines IL-2, IL-4, and IL-6 to each block Ig secretion. Antibodies to IL-5 and IFN-gamma had no effect on the T cell-induced response. Kinetic studies suggested that IL-4 acted during the initial stages of the response, whereas the inability of anti-IL-6 to block B cell proliferation suggested that IL-6 was involved in part in promoting differentiation of the B cells. The relative contributions of cognate (MHC-restricted) and bystander (MHC-unrestricted) T-B cell contact vs cytokine (non-contact)-mediated responses were assessed in a transwell culture system. The majority of the IgM, IgG3, IgG1, and IgG2a response induced by both fresh and effector T cells was dependent on cognate interaction with small, high density B cells. In contrast, a small proportion of these isotypes and most of the IgA secreted resulted from the action of IL-6 on large, presumably preactivated, B cells. The IgA response did not require cell contact or vary when fresh and effector cells were the helpers. The contribution of bystander contact in the overall antibody response to both T cell populations was minimal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Patterns of isotype commitment in human B cells: limiting dilution analysis of Epstein Barr virus-infected cells

The frequency of Epstein Barr virus- (EBV) inducible IgM, IgG, and IgA-secreting cells in human peripheral blood and tonsil was determined by performing limiting dilution experiments in suspension culture. We devised a method of propagating small numbers of EBV-infected B cells with irradiated umbilical cord blood cells as a feeder layer. Precursor cell frequencies can be derived from these experiments; we have shown by statistical analysis that they conform to the single hit model of the Poisson distribution. By using this technique, a significant percentage of surface immunoglobulin-bearing lymphocytes are activated to secrete immunoglobulin in vitro. On the average, 8 to 38% of B cells in peripheral blood and tonsil can be propagated and the secreted immunoglobulin from the clonal progeny can be analyzed. Neither the EBV immune status of the donor nor the source of the umbilical cord blood feeder layer could account for the variations in cloning efficiency observed among donors. A surprisingly high frequency of B cells secreted IgA in vitro and we have shown that a small proportion of B cell clones in tonsil and peripheral blood secrete both IgM and IgA during the 4-wk culture period. Other B cells, including all those that produce IgG, appear to be committed to the secretion of a single isotype. Thus, these studies establish methodology for the analysis of the secreted products of human B cells at the single cell level and demonstrate that the progeny of at least some clones can secrete more than one isotype in vitro.     

Immunoglobulin M and immunoglobulin G secretion by human B cells exposed to RU 41.740, a glycoprotein extract from Klebsiella pneumoniae

RU 41.740, a glycoprotein extract from Klebsiella pneumoniae, was seen to activate human B cells to immunoglobulin secretion in vitro. The effects of RU 41.740 on human B cells were compared to those induced by pokeweed mitogen, a T-cell-dependent polyclonal B-cell activator, and Epstein-Barr virus, a T-cell-independent polyclonal B-cell activator. Exposure of human B cells to all of these agents resulted in increased immunoglobulin M (IgM) and immunoglobulin G (IgG) secretion. IgM and IgG secretion induced by RU 41.740 appeared to be T cell dependent when B cells were isolated from human peripheral blood. However, this activity may have been T cell independent when B cells were isolated from human spleen. RU 41.740-induced IgM secretion by peripheral blood B cells was seen to peak after 6 days in culture; IgG secretion peaked after 7 days in culture. The optimal concentration of RU 41.740 for the induction of IgM and IgG secretion by human B cells in vitro was seen to be 200 micrograms/ml.     

IL-4 induces co-expression of intrinsic membrane IgG1 and IgE by murine B cells stimulated with lipopolysaccharide

IL-4 promotes IgG1 and IgE secretion by murine B cells stimulated with bacterial LPS. We show that stimulation of unprimed resting splenic B cells with LPS and 10(4) U/ml rIL-4 results in the expression of membrane (m) IgG1 and mIgE on 40 to 50% and 15 to 25% of the total B cell population, respectively, on day 4 of culture. The possibility of a significant contribution to cell surface staining by cytophilic, secreted Ig isotypes was eliminated by either the addition of anti-Fc gamma or anti-Fc epsilon R mAb during the culture or by acid treatment before staining. A similar proportion of IgE-expressing B cells are also found, after stimulation with LPS and 10(4) U/ml IL-4, by cytoplasmic staining using fluorescence microscopy. Cell sorting analysis further indicates that B cell populations that express mIgG1 and mIgE secrete these respective Ig isotypes. In addition, such cells show striking diminution in IgM secretion compared to mIgG1- or mIgE- sorted B cells. Stimulation with LPS and IL-4 (10(4) U/ml) induces co-expression of mIgG1 and mIgE on LPS-stimulated B cells; up to 75% of mIgE+ B cells co-express mIgG1 and up to 19% of mIgG1+ B cells express mIgE. This striking co-expression of mIgG1 and mIgE is mirrored by the co-expression of mIgG1 with mIgG3 and mIgG2b by B cells stimulated with LPS and 200 U/ml IL-4. Cell sorting analysis demonstrates that the B cell population that co-expresses mIgG1 and mIgE secretes both IgG1 and IgE. However, "two-color" cytoplasmic staining fails to demonstrate any B cells that simultaneously secrete both IgG1 and IgE.     

Jacalin, an IgA-binding lectin, inhibits differentiation of human B cells by both a direct effect and by activating T-suppressor cells

Jacalin, a lectin extracted from the seeds of Artocarpus intergifolia (jackfruit), has been reported to bind specifically to IgA while inducing B-cell polyclonal immunoglobulin secretion. We confirmed that jacalin only binds to IgA and not to IgG or IgM and extended these findings by showing that it does not bind to IgE. Addition of jacalin to either unfractioned peripheral blood lymphocytes or purified B cells failed to induce immunoglobulin synthesis; indeed immunoglobulin production was diminished in the presence of jacalin. We found that jacalin directly inhibited the induction of immunoglobulin synthesis from B cells in the presence of T-cell replacing factor. Cell lines making IgG, IgM, and IgA were inhibited by jacalin. Furthermore, T cells incubated with jacalin also inhibited immunoglobulin production by stimulated B cells. Under these conditions jacalin was found to be a potent mitogen for T cells but to induce little or no activation of B cells. Jacalin appears to be a potent T-cell mitogen which can induce suppressor T cells for Ig production. It also has a direct inhibitory effect on B-cell Ig production.     

Regulation of B cell maturation and differentiation. I. Suppression of pokeweed mitogen-induced B cell differentiation by tumor necrosis factor (TNF)

Growth and differentiation of B cells into Ig-secreting plasma cells is regulated by both T cells and macrophages and/or their secreted factors. Although the regulatory role of various cell-derived factors has been examined, the involvement of the macrophage-derived factor, TNF, in human B cell growth and differentiation has not yet been investigated. In the present study we examine the role of rTNF in polyclonal B cell response of human PBL induced by PWM. The addition of rTNF at the initiation of the culture resulted in the dose-dependent inhibition of the generation of both IgG and IgM PFC. Inhibition of PFC development followed the same dose response as rTNF-mediated cytotoxicity against a TNF-sensitive tumor target. The mechanism of rTNF-mediated suppression was examined in different experimental systems. Recombinant TNF did not affect the viability or proliferation of either the T cell or B cell subpopulations, suggesting that TNF does not mediate its suppressive effect by cytotoxic mechanisms. Kinetic studies in which rTNF was added at different times after initiation of culture indicated that inhibition can be observed as late as 4 days of culture and suggested that TNF acts at a late phase of the growth and differentiation pathway of B cells. In further studies we examined the cellular level of TNF-mediated suppression. The addition of rTNF to supernatants containing helper factors and enriched B cells resulted in no inhibition, suggesting that TNF does not act at the B cell level. This was confirmed by demonstrating that rTNF does not inhibit spontaneous PFC development by the CESS B cell line. The effect of TNF on T cell subpopulations was examined by using normal or irradiated T cells, which inactivate suppressor cells. Addition of rTNF to B cells combined with either T cell population suppressed both IgG and IgM PFC development, indicating that the target cell for suppression is the T helper cell but not ruling out an effect on macrophages or the T suppressor cells. Combined, the observed results demonstrate that rTNF suppresses PWM-induced B cell differentiation without affecting B cell proliferation. TNF appears to mediate the suppression by acting directly on T helper cells or else by regulating the production of factors controlling T cell activation and lymphokine secretion.     

IL-4-dependent IgE switch in membrane IgA-positive human B cells

IgE responses by human B cells, separated according to membrane Ig classes, were analyzed in a clonal assay using EL-4 thymoma cells as helper cells, T cell supernatant, and rIL-4. In cultures seeded by means of the autoclone apparatus of the FACS, IgE responses were generated frequently by either IgM (mu+/gamma-alpha-) or IgA (alpha +/mu-)-positive B cells (16 and 14% of the Ig producing wells, respectively), but rarely by IgG (gamma +/mu-)-positive B cells (1.3% of Ig producing wells). The total amounts of Ig secreted by IgM-, IgG-, or IgA-positive cells and the total proportions of responding autoclone wells (23-27%) were comparable. All IgE secretion was IL-4 dependent. When the Ig secretion patterns from alpha +/mu- vs alpha +/mu-epsilon- B cells were compared, most autoclone wells from both types of cells produced IgA only, and similar proportions of IgA producing wells (6.2 and 6.0%) also secreted IgE. In addition, IgE restricted responses occurred 6 times more frequently with alpha +/mu- than with alpha +/mu-epsilon- cells, which suggests that membrane IgA+E double-positive, IgE committed B cells occur in vivo. The isotype pattern generated by alpha +/mu-epsilon- B cells cannot be explained by a chance assortment of separate IgA and IgE precursors or by cytophilic antibody. Thus, IL-4 dependent switch to IgE occurred frequently in IgM- or IgA-positive, but rarely among total IgG-positive, B cells. This could be relevant to IgE production in mucosal tissues rich in IgA expressing B cells.