Effect of recombinant human interleukin 4 on spontaneous in vitro human IgE synthesis

Recombinant human interleukin 4 (IL 4) alone enhanced the spontaneous IgE synthesis in cultures of peripheral blood leukocytes (PBL) from atopic patients as well as from nonatopic individuals, suggesting the existence of preactivated PBL sensitive for IL 4. Preactivated cells were also obtained by stimulation with Staphylococcus aureus strain Cowan I (SAC). However, co-stimulation of PBL by IL 4 with SAC or anti-IgM antibody and pokeweed mitogen did not result in an enhanced IgE synthesis. Optimal IL 4 concentrations for the induction of IgE synthesis coincided with optimal proliferative responses in PBL. The effect of IL 4 was not isotype specific, and in terms of protein even more IgG and IgM antibodies were formed. The effect of IL 4 on IgE synthesis was counteracted by very low concentrations of interferon-gamma (IFN-gamma), suggesting that both IL 4 and IFN-gamma might be decisive cytokines for the human in vitro IgE synthesis.     

Immunostimulatory DNA inhibits IL-4-dependent IgE synthesis by human B cells.

BACKGROUND: Immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) is a potent antiallergic immunomodulating agent in mice. However, few studies have addressed its antiallergic potential in human subjects. OBJECTIVE: We sought to determine whether a phosphoro-thioate ISS-ODN could inhibit IL-4-dependent IgE synthesis by human B cells. METHODS: Initially, nonatopic- and atopic-donor PBMCs were incubated with ISS-ODN or mutated oligodeoxynucleotide, and cytokine production and B-cell expression of IFN-gamma receptor and IL-4 receptor were measured by using ELISA and flow cytometry, respectively. In subsequent studies atopic-donor PBMCs were incubated with IL-4 alone or with ISS-ODN or mutated oligodeoxynucleotide. After 14 days, IgE production and IgM, IgG, and IgA production were determined by using ELISA. In select IgE studies cytokines were neutralized with mAbs. RESULTS: ISS-ODN induced IL-12, IFN-alpha, IFN-gamma, IL-10, and IL-6 production from both nonatopic- and atopic-donor PBMCs. ISS-ODN also increased IFN-gamma receptor and inhibited IL-4 receptor expression on B cells from both donor populations. Furthermore, ISS-ODN inhibited IL-4-dependent IgE production by atopic-donor PBMCs. Neutralization of IL-12, IFN-alpha, IFN-gamma, and IL-10, but not IL-6, attenuated the inhibitory activity of ISS-ODN on IgE production. In contrast to its inhibition of IgE synthesis, ISS-ODN stimulated the production of IgM, IgG, and IgA. CONCLUSION: These in vitro studies demonstrate that phos-phorothioate ISS-ODN elicits an innate immune response by PBMCs, which inhibits IL-4-dependent IgE synthesis. In addition, these results provide further support for consideration of ISS-ODN therapy for the treatment of allergic disease in clinical practice.     

Serum levels of interleukin 4, interleukin 6 and interferon-gamma following in vivo isotype-specific activation of IgE synthesis in humans.

It is now generally accepted that interleukin 4 (IL4), interleukin 6 (IL6) and interferon-gamma (IFN gamma) play main roles in the regulation of human IgE synthesis. This concept is based mainly on in vitro data. To obtain corresponding in vivo data, we determined IL4, IL6 and IFN gamma by immunoassays in sera collected from 4 atopic patients following a clinical trial of selective IgE apheresis (plasmaimmunoadsorption). This treatment removes several milligrams of IgE from patient's blood and is suggested to induce strong and isotype-specific activation of the IgE system. Serum IgE levels restored rapidly within 3-5 days after IgE apheresis. However, very low and constant levels of IL4 (from less than 50 to 130 pg/ml) and IL6 (from less than 300 to 920 pg/ml) were detected in the sera of the treated patients. Serum IFN gamma was absent before treatment (concentrations less than 0.5 U/ml) and increased to low but detectable levels (0.90 and 8.05 U/ml) on the day following the last IgE apheresis in 2 of 4 patients. In our opinion, the data presented argue against in vivo participation of IL4 and IL6 in the activation of the human IgE system, at least in atopic patients under constant allergen exposure.     

Role for T cells, IL-2 and IL-6 in the IL-4-dependent in vitro human IgE synthesis.

The role of T cells and monocytes, as well as that of cytokines, such as IL-1, IL-2 and IL-6, on the IL-4-dependent in vitro human IgE synthesis was investigated. Recombinant IL-4, IL-4-containing T-cell clone supernatants and different combinations of recombinant cytokines failed to induce highly purified B cells to synthesize IgE. IL-4-dependent IgE synthesis was restored by addition to purified B cells of either untreated or mitomycin C-treated autologous T lymphocytes. Addition to purified B cells of autologous monocytes did not restore the IgE response, but usually it exerted a potentiating effect on the synthesis of IgE induced by IL-4 in the presence of suboptimal concentrations of T cells. The activity of T cells apparently preceded that of IL-4 and required a physical contact with B cells. The presence in culture of IL-2 also appeared to be necessary for the T-cell and IL-4-dependent IgE synthesis. Even though not essential, IL-6 was able to potentiate IgE synthesis in most experiments, whereas IL-1 did not display any modulatory effect.     

T cell clones providing helper function for IgE synthesis release soluble factor(s) that induce IgE production in human B cells: possible role for interleukin 4 (IL-4).

We have previously demonstrated that a proportion of human T cell clones (TCC) derived from tonsil or peripheral blood (PB) of non-allergic donors, upon triggering with phytohaemagglutinin (PHA) or anti-CD3 monoclonal antibody (MoAb), were able to provide help for IgE synthesis in B cells from both allergic and non-allergic individuals. In this study we show that, upon PHA stimulation, culture supernatants from 10 selected TCC active on IgE synthesis also provided helper activity for IgE, whereas supernatants from unstimulated cultures of the same TCC were ineffective. In contrast, culture supernatants derived from five PHA-stimulated TCC, unable to provide helper function for IgE synthesis, consistently failed to elicit production of IgE. While the induction of IgE synthesis by TCC occurred in B cells from virtually all allergic and non-allergic donors, their soluble factor(s) were found to be able to provide substantial help for IgE production only in B cells from a proportion of donors tested. In addition, B cells from non-atopic donors usually appeared to be less responsive than atopic B cells to the activity of such factor(s). In contrast, synthesis of both IgG and IgM was induced in every B cell donor by both TCC and their supernatants. Partial characterization of the factor(s) providing helper function for IgE synthesis in B cells showed that it apparently had a mol. wt between 10 and 50 kD and did not bind to immobilized IgE. Such an activity appeared to be associated with the presence of interleukin 4 (IL-4) in supernatants and it was inhibited by adding both gamma-interferon and anti-human IL-4 antibody in culture.     

IFN-gamma plays a dominant role in upregulation of Candida-specific IgE synthesis in patients with atopic dermatitis

BACKGROUND: Although Candida albicans (CA) is known to induce Th1 clones that suppress IgE synthesis, serum IgE antibody against CA is often increased in atopic patients. This study aims to elucidate the mechanism of IgE synthesis against CA in atopic patients. METHODS: We measured the production of IL-4 and IFN-gamma by peripheral blood mononuclear cells (PBMCs) from atopic patients upon stimulation with CA and examined the correlation with the level of serum IgE antibody against CA. Results: The level of serum CA-specific IgE antibody (CA-IgE) was significantly higher in patients with atopic dermatitis (AD) than in patients with bronchial asthma (BA) (geometric mean = 3.6 vs. 0.27 U(A)/ml, p < 0.02) (U(A) = unit allergen), while there was no difference in the level of house dust mite-specific IgE antibody between them (67.6 vs. 87.1 U(A)/ml). Although IL-4 production by PBMCs upon stimulation with CA in patients with AD was not significantly different from that in patients with BA (mean = 359.1 vs. 515.3 fg/ml), IFN-gamma production was significantly lower in the former than in the latter group (8.1 vs. 56.2 pg/ml, p < 0.001). Consequently, the ratio of IL-4/IFN-gamma production was apparently higher in patients with AD than in those with BA, which corresponds to the difference between them in the level of serum CA-IgE. A significant negative correlation was seen in patients with AD between IFN-gamma production by CA-stimulated PBMCs and the level of serum CA-IgE (p < 0.05). CONCLUSIONS: IgE synthesis against CA in atopic patients may be precipitated not by enhancing IL-4 production, but by reducing IFN-gamma secretion.     

Regulation of IgE synthesis by macrophages expressing FcE-receptors: role of interleukin 1.

Triggering rat macrophages with IgE complexes induced the production of interleukin 1-like activity (IL-1). The signal is delivered through the macrophage FcE receptor since stimulating macrophages with IgE bound to spleen cells (to avoid endocytosis) or with an anti-FcE receptor antibody linked to nonphagocytizable cells also led to IL-1 production. The molecular weight of IL-1 produced after IgE triggering is in the same range (30 kD) as previously described for rat IL-1. A positive feed-back effect of IL-1 on IgE response was suggested, as purified IL-1 was able to enhance IgE synthesis in vitro by lymphocytes from immunized animals.     

Role of interleukin 4 and gamma interferon in the regulation of human IgE synthesis: possible alterations in atopic patients

The IgE helper function of human T cell clones or their phytohemagglutinin-induced supernatants was positively correlated with their ability to produce or their content in interleukin 4 (IL-4), whereas it was inversely correlated with production of or content in gamma interferon. The addition to B cell cultures of anti-IL-4 antibody abolished not only the IgE synthesis induced by recombinant human IL-4, but also that induced by IL-4-producing T cell clones or their phytohemagglutinin-induced supernatants. A clonal analysis in nonatopic donors and patients with common atopy showed that atopics possess in their peripheral blood significantly higher numbers of T cells able to secrete IL-4 and to provide helper function for IgE.     

Interleukin 3 mediates interleukin 6 production in murine interleukin 3-dependent hemopoietic cells

A series of murine interleukin 3 (IL-3)-dependent hemopoietic cell lines was studied for the capacity to produce interleukin 6 (IL-6) in vitro. These included a bone marrow-derived mast cell line (L138.8A) and several early myeloid cell lines described in the literature (DA-1, DA-3, NFS-60, NFS-78, FDC-P1, FDC-P2, FDC-PmixA4, and 32Dcl.23). All of these cell lines produced growth factor activity for IL-6-dependent hybridoma cells (7TD1), which was completely neutralized by the monoclonal anti-IL-6-antibody 6B4. IL-6 expression was also evident at the mRNA level using a murine IL-6-specific cDNA probe. In 32Dcl.23 cells (2 x 10(5)/ml) stimulated for 24 hr with serial dilutions of purified murine IL-3, a positive correlation was found between the IL-3 dose and the amount of IL-6 measured in the conditioned media. At 24 hr this correlation was not evident at the mRNA level. However, prolonged exposure of 32Dcl.23 cells (up to 72 hr) to either a high (60 U/ml) or a low IL-3 concentration (1 U/ml) revealed a time-dependent increase and decrease, respectively, of IL-6 mRNA levels. At both IL-3 concentrations 32Dcl.23 cells remained in a fully viable and proliferative state. The influence of IL-3 on IL-6 release could be specifically counteracted by anti-IL-3-antiserum. IL-6 added alone or in concert with IL-3 did not stimulate 32Dcl.23 proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of interleukin 4 on an antigen-specific IgE response in vitro in murine lymphocytes

The present study shows that interleukin 4 (IL4) can exert either stimulatory or inhibitory effects on an antigen-specific IgE response in vitro. Spleen cells from BALB/c mice that had been primed with trinitrophenyl keyhole limpet hemocyanin (TNP-KLH) were cultured with the same antigen for 2 days, washed, transferred to an antigen-free culture medium, and then cultured for 4 days. Anti-TNP IgE antibodies secreted into the medium were determined by an enzyme immunoassay developed in our laboratory. Anti-TNP IgE response was elicited by TNP-KLH in cultures of the spleen cells from mice that had been primed twice with the antigen at an interval of 3 weeks, but not in cells from animals that had received only a single injection of the antigen. When the latter cells were cultured with the antigen in the presence of mouse recombinant IL4, a considerable level of the antigen-specific IgE response was induced. In contrast, the IgE response elicited in the cells of mice that had been primed twice with the antigen was markedly down-regulated by added IL4. These observations suggest a novel function of IL4 in the regulation of IgE antibody responses.     

Interleukin 4 induces synthesis of IgE and IgG4 in human B cells

Interleukin (IL)4 has been shown to regulate the IgG subclasses and induce IgE production in splenic mouse B cells. Here we show that IL4 and phorbol 12-myristate 13-acetate (PMA) induce, on a per cell basis, very high IgE secretion in purified human B cells by using a mouse thymoma (EL4) co-culture method. In addition, a marked increase in the number of IgG4-producing cells was also observed. Furthermore, IL2 could synergize with IL4 and PMA in the production of IgE. By using limiting dilution analysis, a considerable increase in the precursor frequency for IgE was found when IL4 and PMA were added to cultures as compared to cultures with PMA only. This indicates that IL4 induces an isotype switch in human B cells.     

Dysregulation of JAM-A plays an important role in human tumor progression

Junctional adhesion molecule A (JAM-A) is a transmembrane protein that belongs to the immunoglobulin (Ig) superfamily. Evidence determines that JAM-A plays a role in numerous cellular processes, including tight junction assembly, leukocyte migration, platelet activation, angiogenesis and virus binding. Recent research suggests that JAM-A is dysregulated in various cancers and is vital for tumor progression. JAM-A is implicated in carcinogenesis via different signal pathways such as TGF-β1 signaling. Furthermore, JAM-A expression in cancers is usually associated with certain outcome of patients and might be a prognostic indicator. In this review, the correlation between JAM-A expression and human cancers will be described.     

Inhibition of human interleukin 4-induced IgE synthesis by a subset of anti-CD23/Fc epsilon RII monoclonal antibodies.

Specific monoclonal antibodies (mAb) directed against the CD23 antigen were used to study human interleukin 4 (hIL4)-induced IgE production by blood and tonsillar mononuclear cells. Both peripheral blood and tonsillar mononuclear cells stimulated by hIL4 expressed membrane CD23 as detected by the binding of all anti-CD23 mAb. Nevertheless, two sets of anti-CD23 mAb could be distinguished. The first set, including mAb 25, was able to decrease significantly hIL4-induced IgE synthesis by mononuclear cells. The second set, including EBVCS#1, did not affect hIL4-induced IgE synthesis. All the anti-CD23 mAb were able to bind specifically to a human B cell line expressing recombinant CD23. Inhibition experiments revealed that the two sets of anti-CD23 mAb did not recognize the same epitope on the CD23 antigen. In fact, all the anti-CD23 mAb, except EBVCS#1, were able to inhibit IgE binding to CD23 on RPMI 8866 cells. Moreover, the first set of antibodies, which decreased IgE production, was able to up-regulate membrane CD23 expression on hIL4-stimulated tonsillar mononuclear cells. Conversely, EBVCS#1, which had no effect on IgE production, did not affect hIL4-induced CD23 expression. These results indicate that CD23 plays a key role in human IgE synthesis.     

Interleukin 5 enhances interleukin 4-induced IgE production by normal human B cells. The role of soluble CD23 antigen

Interleukin 4 (IL 4)-induced IgE production by peripheral blood lymphocytes and tonsil cells from normal donors was enhanced in a dose-dependent fashion by IL 5. IL 5 tested alone was not effective. The synergistic effects of IL 5 were most pronounced at suboptimal IL 4 concentrations, whereas at saturating IL 4 concentrations (200-300 U/ml), IL 5 had no effect. Interferon-gamma (IFN-gamma) and F(ab')2 fragments of monoclonal antibody 25 directed against the CD23 antigen, that blocked IL 4-induced IgE synthesis, also inhibited the production of IgE in the presence of combinations of IL 4 and IL 5, indicating that IL 5 potentiates the activation pathway through which IL 4 induces IgE production. In contrast, IL 4 (50 U/ml) blocked IL 5-induced IgA synthesis. IL 5 was ineffective in inducing the release of soluble CD23 (sCD23), but in the presence of IL 4 an enhanced release of sCD23 was observed, provided IL 4 was present at suboptimal concentrations. IFN-gamma completely blocked sCD23 release induced by IL 4 and IL 5. These results demonstrate that there is a strong quantitative correlation between sCD23 release and induction of IgE synthesis. sCD23 fraction-correlation between sCD23 release and induction of IgE synthesis. sCD23 fractionated from the Epstein-Barr virus-transformed B cell line RPMI 8866 was ineffective in inducing IgE production. However, sCD23 acted synergistically with suboptimal concentrations of IL 4. sCD23 did not modulate the IgE response at saturating concentrations of IL 4. Collectively, these data indicate that sCD23 plays an important regulatory role in the modulation of IL 4-induced IgE synthesis mediated by IFN-gamma and IL 5.     

The polysaccharide portion plays an indispensable role in Salmonella lipopolysaccharide-induced activation of NF-kappaB through human toll-like receptor 4

The lipid A portion has been identified as the active center responsible for lipopolysaccharide (LPS)-induced macrophage activation. However, we found that Salmonella (Salmonella enterica serovars Abortusequi, Minnesota, and Typhimurium) lipid A is inactive in human macrophages, despite its LPS being highly active. Thus we investigated the critical role of polysaccharide in Salmonella LPS-induced activation of NF-kappaB. In human monocytic cell line THP-1, Salmonella lipid A and synthetic Salmonella-type lipid A (516) did not induce NF-kappaB-dependent reporter activity up to 1 micro g/ml, whereas strong activation was observed in response to Salmonella LPS. The difference in activity between this lipid A and LPS was further examined by using 293 cells expressing human CD14/Toll-like receptor 4 (TLR4)/MD-2, and similar results were obtained in these cells as well. A polysaccharide preparation obtained from Salmonella LPS was inactive in 293 cells expressing human CD14/TLR4/MD-2 even in combination with 516. Salmonella enterica serovar Minnesota Re LPS, whose structure consists of lipid A and two molecules of 2-keto-3-deoxyoctonic acid, but not its lipid A exhibited strong activity in THP-1 cells and 293 cells expressing human CD14/TLR4/MD-2. These results indicate that the polysaccharide portion covalently bound to lipid A plays the principal role in Salmonella LPS-induced activation of NF-kappaB through human CD14/TLR4/MD-2.