Interleukin-4-induced IgG subclass and IgE secretion by mononuclear cells from atopic donors

To investigate the effect of interleukin-4 (IL-4) on the secretion of IgG subclasses and IgE in atopic individuals, peripheral blood mononuclear cells from 10 atopic and 10 normal donors were incubated with IL-4, and the production of IgE, IgG1, IgG2, IgG3, and IgG4 was determined after a 14-day culture period. Like normal peripheral blood mononuclear cells, atopic mononuclear cells were stimulated by IL-4 to secrete IgE and IgG4, but not the other IgG subclasses. This response was in the same quantitative ranges in both donor groups. Addition of interferon gamma to IL-4-containing cultures efficiently antagonized the IL-4-induced IgE and IgG4 secretion. These results do not support the hypothesis that an atopic condition is due to a discordant effect of IL-4 on IgE compared to IgG4 production or to an altered response to the potent antagonist interferon gamma.     

Differential requirements for induction of total immunoglobulin and physiological rheumatoid factor production by human peripheral blood B cells

Rheumatoid factors (RFs) are autoantibodies directed against the Fc part of IgG. Considerable evidence exists that there are two classes of RFs, pathological and physiological. Whereas pathological RFs are associated with disease, physiological RFs are considered to be a normal component of the immune response. RF(+) precursor B cells present as part of the B cell repertoire of healthy individuals are held responsible for the production of physiological RFs, which is a transient phenomenon with a clear correlation with an initiating stimulus such as immunization or exposure to an infection. Here we demonstrate a difference in the regulatory control of total Ig and RF production by peripheral blood (PB) B cells of both healthy controls (HC) and patients with rheumatoid arthritis (RA). Highly purified B cells from HC and patients with RA were cocultured with T cells stimulated with immobilized anti-CD3 mAb. Similar to IgM production, IgM-RF production was shown to be dependent on CD40 cross-linking. However, activation of PB B cells in the CD40 system in the presence of IL-2, IL-4, IL-10, combinations of these cytokines or supernatant of anti-CD3-stimulated T cells failed to induce detectable IgM-RF, whereas total IgM production was considerable. From these results we conclude that conditions to activate physiological RF(+) B cells require additional contact besides CD40--CD40L interactions between T and B cells. Since the requirements for RF production were similar using PB B cells from HC and patients with RA it is suggested that the regulatory properties of RF(+) precursors in the PB B cell compartment is equal among these groups. Together, these results indicate that conditions for the induction of total Ig and physiological RFs are different.     

Production of immunoglobulins by human sIgD+ and sIgD- human blood B lymphocytes in response to stimulation with activated T cells and agonistic antibodies; effect of IL-10, IL-2 and mode of activation of T cells.

Production of IgM, IgG and IgA was induced from human blood B lymphocytes by culturing with a CD40 MoAb and IL-2 for 9 days. Replacement of IL-2 by IL-10 markedly enhanced production of all three isotypes. High levels of immunoglobulin production also occurred when activated irradiated autologous T cells replaced the CD40 MoAb, and when IL-10 replaced IL-2 in these cultures a spectacular increase in IgG production occurred. The effectiveness of the T cell stimulus depended on the mode of purification of the T cells and the nature of the stimulant used to activate them. Differences in the kinetics and level of expression of CD40L on the various T cell preparations were observed, but did not account for variations in immunoglobulin-inducing efficiency. Immunoglobulin production from sIgD+ and sIgD- B cells was investigated. IgG and IgA were found in sIgD+ cultures, indicating that some isotype switching had occurred, but the major part of the IgG and IgA secreted was from cells already committed to these isotypes. Anti-IgD or anti-IgM MoAbs enhanced the proliferation of B cells induced by anti-CD40 antibody, but immunoglobulin production was not enhanced. Factors affecting the balance of proliferation and differentiation are discussed.     

A new multivalent B cell activation model--anti-IgD bound to Fc gamma RI: properties and comparison with CD40L-mediated activation

CHO cells permanently transfected with mouse Fc gamma RI alpha chain were prepared and used as a model to polyclonally activate murine B cells. The transfected CHO cells were treated with mitomycin C and placed into culture with varying quantities of anti-IgD. Using this model, murine splenic B cells (from BALB/c or C57Bl/6) were activated by mouse IgG2a-anti-IgD (10.4.22 or AF3.33) in a manner that is analogous to the activation of B cells seen with highly polyvalent anti- IgD (H delta(a)/1) prepared by chemical cross-linking to dextran. Efficient B cell activation was seen with nanogram quantities of anti- IgD. In the presence of IL-4 and IL-5, IgG1 production levels were equivalent to or better than seen when stimulation was with H delta(a)/1-dextran; however, IgE induction was not seen in either situation. The Ig production capacity was compared to that seen when B cells were activated with CD40L, using either CD40L-transfected CHO or a soluble CD40L construct. In the presence of IL-4 and IL-5, once a critical threshold of B cells was present, IgE and to a lesser extent IgG1 production was inversely proportional to B cell number when CD40L was the activating agent. In contrast, with Fc gamma RI-anti-IgD, IgM and IgG1 production was directly proportional to B cell number, while IgE production was never seen. Finally, when B cells were co-activated with immobilized anti-IgD and CD40L simultaneously, the IgE production from B cells induced by CD40L was strongly inhibited, while IgG1 and IgM production were not affected. Since B cell co-activation via sIg and CD40L would be a common scenario in secondary follicles, this inhibition of IgE production may be one of the reasons why serum IgE levels are much below IgG in normal immune situations.      

Phenotypic and functional analysis of human T lymphocytes in early second- and third-trimester fetuses

This study was undertaken to investigate the phenotypic and functional status of T lymphocytes of human fetuses from early second- to third-trimester. Cord blood samples were obtained from 19 healthy human fetuses (gestation weeks: 18-36), by cordocentesis, and 16 term newborns (gestation weeks 37-42). Maternal and unrelated male blood samples were also taken as controls. Percentage of lymphocytes in fetal white blood cells was 79.3%, reducing to 40% by term birth, much higher than that of adults. Cord blood mononuclear cells (CBMC), prepared by density gradient centrifugation followed by lysis of erythrocytes, were stained using PE- or FITC-labelled monoclonal Abs and analysed by flow cytometry. The frequencies of CD3+ T cells in fetal (40.1%) and neonatal (42.4%) CBMC were significantly lower than that of men (59.6%) and pregnant women (53.6%). Proportions of CD8+ T cells (9.5%), gammadelta-T cells (0.5%) and NK cells (4.8%) in fetal CBMC were also lower than that of neonates (except gammadelta-T cells) and adults. A negative linear correlation (r = -0.609) between the ratio of CD4+/CD8+ T cells in fetal blood and gestation age could also be established. Fetal CBMC showed vigorous spontaneous proliferation but failed to respond to mitogen (PHA) or allogeneic stimulation in vitro. The fetal mononuclear cells were unable to produce IL-2, IL-4 or IFN-gamma, but spontaneously secreted IL-10, IL-6 and TNF-alphain vitro. Stimulation with PHA up-regulated the production of IL-10, IL-6 and TNF-alpha substantially.     

The role of the interleukin-10 subfamily members in immunoglobulin production by human B cells

Interleukin (IL)‐10 has been shown to have various effects on B cells, including positively affecting the production of immunoglobulin A (IgA) and IgG. Several human IL‐10‐related molecules have been identified. These include IL‐19, IL‐20, IL‐22, IL‐24, IL‐26, IL‐28 and IL‐29. To determine the effects of the IL‐10 analogues on the class switch recombination in B cells, we analysed Ig production from naïve B cells stimulated with these cytokines in the presence of anti‐CD40. None of the cytokines were found to induce Ig production by themselves in the presence of anti‐CD40 Ab. However, all cytokines inhibited the production of IgA and IgG induced by anti‐CD40 Ab alone. In combination with anti‐CD40 Ab and IL‐4, IgG4 were inhibited in cultures stimulated with IL‐20, IL‐22, IL‐26, IL‐28 and IL‐29 compared with IL‐4 and anti‐CD40 Ab alone, whereas all IL‐10 analogues increased the production of total IgG. All analogues reduced anti‐CD40 Ab + transforming growth factor (TGF)‐β‐induced IgA production compared with cultures stimulated with anti‐CD40 Ab and TGF‐β alone. Together, these data show that the IL‐10‐related cytokines in combination with anti‐CD40 Ab are not by themselves directly involved in the Ig regulation in B cells. However, some of the analogues might have regulatory effects on CSR induced by CD40‐ligation in combination with IL‐4 or TGF‐β.     

Cytokine Responses of Human Blood Monocytes Stimulated with Igs

Using an in vitro system for stimulating human peripheral blood mononuclear cells (PBMC) with immobilized Ig, patterns of cytokine production as a function of different Ig classes and subclasses were elucidated. Wells were coated with IgA, IgG1, IgG2, IgG3 or IgG4. Equivalent protein content on surfaces of wells was demonstrated by a human kappa chain ELISA. Isolated human PBMC were added to Ig-coated wells and incubated for 24 hrs before supernatants were assayed for cytokines. The IgG subclasses showed differences in cytokine production stimulated from PBMC, with the relative stimulation for TNF being IgG2 IgG3 IgG1 > IgG4 and for IL-6 production, IgG2 IgG3 > IgG1 = IgG4. In contrast, the relative stimulation for IL-8 was IgG1 = IgG2 = IgG3 = IgG4. IgA caused less production of TNF when compared to IgG2, but similar levels of IL-8. Such differences may have important implications in the pathogenesis of immune complex mediated diseases.     

CD40-dependent and -independent activation of human tonsil B cells by CpG oligodeoxynucleotides

Immunostimulatory CpG oligodeoxynucleotide (CpG-ODN) sequences are known to directly activate B cells. We investigated the expression of the CpG receptor, Toll-like receptor 9 (TLR9), in human tonsil B cells, and determined functional responses following stimulation by a well-characterized stimulatory CpG-containing ODN sequence in the human immune system, ODN 2006. Tonsil B cells were found to express high amounts of TLR9 mRNA and protein, and exposure of B cells to CpG-ODN but not to an inactive control ODN induced a concentration- and time-dependent up-regulation of the activation markers CD23, CD25, CD40, CD54, CD80, CD86 and HLA-DR. However, significant induction of proliferation and the release of IL-6, IL-10, IgG and IgM were only noted when B cells were co-incubated with irradiated CD40L-expressing CHO cells. Endogenous IL-10 was identified as a critical mediator of Ig production, whereas all activating effects were independent of IL-6. Further, CpG-ODN counteracted IgE production induced by IL-4. Collectively, these findings suggest a synergistic role of the TLR9/CD40 system and a critical role for the immunomodulatory cytokine IL-10 in the orchestration of CpG-ODN-induced responses in B lymphocytes.     

IL-2和PWM对人淋巴细胞产生免疫球蛋白的影响

本文初步观察了IL-2和PWM 对人淋巴细胞产生免疫球蛋白的影响。将PWM、MLA-144细血培养上清(富含IL-2)分别加入人扁桃体和外周血淋巴细胞体外培养。用酶联免疫吸附试验双抗体夹心法,分别检测第七天培养上清液中IgG、IgM和IgA的含量。加入PWM(10μg/ml)或 MLA-144 细胞培养上清(1:5稀释)的刺激组,IgG、IgM、IgA 的含量均较对照组明显升高,三种免疫球蛋白比较,IgG、IgM 产量高于IgA。相同条件下,扁桃体淋巴细胞产生的IgA,高于外周血淋巴细胞所产生者。同时加入PWM和MLA-144细胞上清联合刺激组较加PWM 或MLA-144 细胞上清单独刺激组IgG、IgM、IgA的产量增高。结果说明:适当刺激浓度的 IL-2和PWM 对人淋巴细胞免疫球蛋白的产生有明显促进作用。一定条件下,PWM和 IL-2联合刺激诱生免疫球蛋白有相加作用。     

Immunoglobulin G subclasses secreted by human B cells in vitro in response to interleukin-2 and polyclonal activators

The IgG subclasses secreted by human B cells in vitro in response to IL-2 have been analysed. B cells were prepared from tonsil, blood and spleen, and cultured with recombinant IL-2 in the presence or absence of two polyclonal activators: Staphylococcus aureus Cowan 1 (SAC) and bacterial lipopolysaccharide (LPS). Secretion of all four subclasses and of IgM was stimulated by IL-2, but the relative amounts varied according to (i) the tissue source of the B cells, and (ii) which polyclonal activator was used. The amount of IgG1 tended to be higher and IgG2 tended to be lower when SAC was the polyclonal activator (compared to LPS). This difference was most marked for tonsil B cells, and it was found that SAC had a negative effect on secretion of IgM and IgG2 in these cultures, whilst synergizing with IL-2 to stimulate the production of IgG1, 3 and 4. When the degree of stimulation of different pairs of isotypes was analysed, several interesting positive correlations emerged. In tonsil B-cell cultures, stimulation of IgM and IgG2 was linked with each other, but not with IgG1, whilst in blood B-cell cultures all isotypes appeared to be stimulated co-ordinately. Stimulation of IgG1 and IgG3 were positively correlated in cultures of B cells from all tissues. The results emphasize that the effects of a single cytokine on immunoglobulin isotype production can be influenced by the source of the B cells, and by other signals delivered to the cells.     

Malaria-specific antibody subclasses in immune individuals: a key source of information for vaccine design

Immunity against the blood stage of Plasmodium falciparum malaria is associated with protective-type antibodies of certain classes and subclasses. Field studies have demonstrated the differential regulation of various IgG subclasses depending on the dynamics of parasite transmission and on the immune status of the individuals tested. The intrinsic properties of each IgG subclass has a crucial role in protection, both because immunoglobulin levels are dependent on their production and clearance from blood and because antibodies are actively used for parasite clearance. In vitro models using B cells obtained from P. falciparum-immune adults have enabled study of the production of various antibody subclasses depending on the individual and on the antigens used. Ex vivo and in vitro observations from immune donors have helped to extend our understanding of the development and regulation of the antibody response and to design more effective vaccine strategies.     

The effect of adherent cell-derived factors on immunoglobulin and anti-DNA synthesis in systemic lupus erythematosus

The role of adherent cells in the regulation of anti-DNA and immunoglobulin synthesis was investigated in patients with systemic lupus erythematosus (SLE). Peripheral blood mononuclear cells were cultured for 7 days, or were washed after 20 hr of incubation and recultured in fresh media. This washing resulted in a marked decrease in total IgG, IgM, and anti-DNA synthesis compared with unwashed cultures. Reculturing the washed cells in their original supernatant reconstituted Ig and anti-DNA synthesis. Hence it appeared that a supernatant factor, or factors, present in the first 20 hr of mononuclear cell cultures, was required for maximal Ig synthesis. Adherent cells were found to be the source of this Ig-stimulating activity. Moreover, adherent cell supernatants had no direct Ig-stimulating effect on B cells. Ig synthesis was stimulated, however, when T cells where present with the B cells at a 3:1 ratio. Autologous SLE mononuclear cell supernatants reconstituted Ig synthesis to a greater degree than did autologous normal supernatants. SLE adherent cell supernatants were fractionated on an HPLC sizing column. The fractions were tested for their ability to stimulate IgG synthesis by SLE mononuclear cells that had been washed after 20 hr of culture. A single peak of IgG-stimulating activity was found at approximately 14,000 Mr. A rabbit antiserum to interleukin-1 (IL-1) neutralized the Ig-stimulating activity in adherent cell supernatants. No correlations were found, however, between supernatant IL-1 levels assayed by C3H-HeJ mouse thymocyte proliferation and IgG stimulation in mononuclear cell cultures, suggesting that the effects of IL-1 on cell proliferation may not accurately reflect its effects on Ig synthesis. These observations suggest that in normal individuals and in patients with SLE in vitro polyclonal Ig and anti-DNA synthesis requires the presence of soluble adherent cell factors. The Ig-stimulating effect is facilitated by T cells and appears to be mediated at least in part by IL-1. This culture technique provides a new way of analyzing the role of soluble factors in autoantibody synthesis and suggests that IL-1 may be an important contributor to lupus B-cell hyperactivity.     

Differential effects of transforming growth factor-β1 on IgA vs. IgG2b production by lipopolysaccharide-stimulated lymph node B cells: a comparative study with spleen B cells

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that promotes IgA/IgG2b switching and secretion. Here, we show a differential effect of TGF-β1 on Ig production by lipopolysaccharide-stimulated spleen and lymph node (LN) B cells. Exogenous TGF-β1 increased IgA production in B cell cultures and IgG2b production by spleen B cells. In contrast, IgG2b was suppressed by TGF-β1 in cultures of LN B cells, although endogenous TGF-β was required for IgG2b production in LN B cell cultures. The suppressor properties of exogenous TGF-β1 (0.5 ng/ml) on IgG2b production by LN B cells were also seen when testing IgG1 or IgG2a induced by interleukin-4 or interferon-γ, respectively. These differences between B cells from each lymphoid tissue appeared to be related to a different TGF-β antiproliferative effect, since proliferation of LN B cells was extremely sensitive to TGF-β1 and IgG2b production was more sensitive than IgA to the TGF-β-mediated suppression. However, by counteracting the antiproliferative effect of TGF-β1 with a CD40 agonistic mAb (IC10), the IgG2b response by LN B cells was still lacking. IC10 was nevertheless inhibitory for IgG2b production in most cases, while increasing secretion of IgA in the very same cultures. Taken together, the results suggest that functional differences between spleen and LN B cells do exist, at least with regard to the immunomodulating properties of TGF-β on both proliferation and Ig production. Moreover, functional differences exist between cells committed for IgA and IgG2b regarding their sensitivity to the antiproliferative activity of TGF-β1 and the effect of CD40-derived signals on Ig secretion.     

Immunoglobulin isotype production by cycling human B lymphocytes in response to recombinant cytokines and anti-IgM

Actively cycling populations of purified human tonsilar B lymphocytes were examined for their capacity to secrete IgM, IgA, IgE and IgG of all four subclasses in direct response to recombinant cytokines; in some experiments, monoclonal antibody to IgM (anti-mu) was included in order to explore the influence of antigen receptor ligation on immunoglobulin (Ig) production. Enhanced IgM release was seen on culture of the cycling cells with either interleukin-2 (IL-2), IL-4 or interferon-alpha (IFN-alpha). IL-2 and IFN-alpha also augmented IgA production, whereas IL-4 had no effect on this isotype. IL-4 did, however, encourage the production of the IgG subclasses IgG1, IgG2 and IgG3, while IL-2 augmented IgG1 and IgG3 release and IFN-alpha increased IgG1 levels. IgG4 production, and that of IgE, failed to be perturbed by any of the cytokines assayed. Neither IL-1 alpha, IL-1 beta, IL-5 nor IFN-gamma significantly altered the profile of Ig isotype release. When confronted with anti-mu, cycling B cells demonstrated a marked suppression in IgM production. Suppression could not be overcome by the addition to culture of the normally IgM-promoting IL-4. Concomitant with the reduction in IgM levels, an increase in IgG release was observed. This was comprised of elevations in IgG1 and IgG3. Although not influencing IgA release directly, anti-mu was found to promote increased IgA production in co-culture with either IL-2 or IFN-alpha. The findings are discussed in the context of recent findings on Ig isotype control in both human and murine systems.     

低CD40L表达在儿童单纯性肾病综合征低IgG血症发生中的作用

为研究单纯性肾病综合征低IgG血症的发生机制。采用逆转录多聚酶链反应 (RT PCR ) ,流式细胞术 (FCM )等方法检测患儿外周血T淋巴细胞表面CD40配体 (CD40L )表达。结果在发作期CD40LmRNA及蛋白质表达降低 ,恢复期其表达可接近正常水平 ,且CD40L表达量与血清IgG水平呈显著正相关。体外实验中加入CD40单克隆抗体 (CD40mAb )可使患儿PBMC产生IgG及其亚类水平增高 ,但仍未达正常水平。单纯性肾病综合征低IgG血症的发生是综合因素作用的结果 ,CD40L信号减弱导致免疫球蛋白 (Ig )同种型转换障碍是因素之一。     

Disodium cromoglycate enhances ongoing immunoglobulin production in vitro in human B cells

The effect of disodium cromoglycate (DSCG) upon human immunoglobulin (Ig) isotypes and IgG subclasses production by purified B cells was studied. DSCG enhanced IgM, IgG1, IgG2, IgG3, IgG4 and IgA production in a dose-dependent fashion, while DSCG failed to induce IgE production at any concentrations tested by purified B cells. When B cells were separated into small resting and large activated B cells, DSCG failed to induce Ig production from small resting B cells in the presence or absence of Staphylococcus aureus Cowan strain I (SAC). In contrast, in large activated B cells DSCG significantly enhanced all types of Ig production (two-to threefold), especially IgG4 production (seven-to 11-fold), except IgE, which large B cells did not produce. The enhancement of IgG subclass production was not subclass switching, since DSCG failed to enhance IgG1 production in B cells depleted of surface IgG1+ cells (sIgG1+ cells). Similarly, DSCG did not enhance IgG2, IgG3 or IgG4 production from sIgG2-, sIgG3- or sIgG4- B cells, respectively, Interleukin-4 (IL-4) or interleukin-6 (IL-6) also enhanced Ig production except IgG4 from large activated B cells. The enhancing effect of DSCG was not mediated by IL-4 or IL-6 since anti-IL-4 or anti-IL-6 antibody failed to block the DSCG-induced enhancement. DSCG also enhanced IgG2 and IgM production from human B-cell lines GM-1500 and CBL, respectively. These results suggest that DSCG directly and preferentially stimulates activated B cells which are producing Ig and, in addition, enhances their Ig production.     

Influence of costimulatory molecules on immune response to Leishmania major by human cells in vitro

The importance of CD40, CD80, and CD86 costimulatory molecules in anti-Leishmania immune responses has been established in murine models. A role for these costimulatory molecules in human anti-Leishmania immune responses was investigated in this study. Autologous macrophages and peripheral blood leukocytes (PBL) were prepared from peripheral blood mononuclear cells of Leishmania-naive donors and cultured with or without Leishmania major in various combinations. After 7 days of culture, high levels of CD40 and CD86 were expressed on macrophages in the presence or absence of L. major. When macrophages were cultured for an additional 7 days with PBL, expression of all three costimulatory molecules was detected. When L. major was present in these cultures, the expression of CD80, and to a lesser extent CD40, on macrophages was enhanced. Blockade of CD80, CD86, or both molecules (in the order of greatest effect) in cultures containing macrophages, PBL, and L. major significantly inhibited the production of gamma interferon, interleukin-5 (IL-5), and IL-12. Blockade of CD40-CD154 interactions also significantly inhibited production of these cytokines in response to L. major. Production of IL-10 was unaltered by the blockade of these costimulatory molecules. Thus, these data suggest that CD40, CD80, and CD86 expression and regulation may significantly impact anti-Leishmania immune responses in humans.     

IgG subclass switch capacity is low in switched and in IgM-only, but high in IgD+IgM+, post-germinal center (CD27+) human B cells

Recent studies have shown that in humans the germinal center reactions produce three types of V(D)J mutated B cells in similar proportions, i.e. Ig-switched, IgD-IgM+ (IgM-only) and IgD+IgM+ cells, and that together they form the CD27+ compartment of recirculating B cells. We investigated the Ig isotype switch capacity of these cells. Peripheral blood B subsets were sorted and IgG subclass secretion in presence or absence of IL-4 was compared in B cell assays which lead to Ig secretion in all (coculture with EL-4 thymoma cells) or only in CD27+ (CD40L stimulation) B cells. Already switched IgG+ B cells showed no significant sequential switch and IgM-only cells also had a low switch capacity, but IgD+CD27+ switched as much as IgD+CD27- B cells to all IgG subclasses. Thus, in switched B cells some alterations compromising further switch options occur frequently; IgM-only cells may result from aborted switch. However, IgD+CD27+ human B cells, extensively V(D)J mutated and "naive" regarding switch, build up a repertoire of B cells combining (1) novel cross-reactive specificities, (2) increased differentiation capacity (including after T-independent stimulation by Staphylococcus aureus Cowan I) and (3) the capacity to produce appropriate isotypes when they respond to novel pathogens.