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Association of interleukin-4 receptor (IL-4R) with phosphatidylinositol 3-kinase (PI3-kinase) has been demonstrated as the proximal event of IL-4 signaling. We investigated the role of this enzyme in the IL-4 signaling pathway in a human Burkitt lymphoma B cell line, DND39, that expresses germline C epsilon transcripts in response to IL-4. Stimulation of DND39 cells with IL-4 resulted in an accumulation of PI-3-monophosphate as well as a decrease of PI-4,5-bisphosphate, which were abrogated by wortmannin, a potent inhibitor of PI3-kinase. Activation of PI3-kinase was further confirmed by the finding that IL-4 caused an increase in PI3-kinase activity coimmunoprecipitated with anti-IL-4R and with anti-JAK3 kinase antibodies. As a possible downstream event of PI3-kinase activation, the translocation of a zeta isoform of protein kinase C (PKC) from the cytosol to the membrane fraction was observed after IL-4 stimulation, and wortmannin also suppressed this translocation. Moreover, IL-4-induced expression of germline C epsilon transcription was inhibited not only by wortmannin, but also by a PKC inhibitor, K252a. These results suggest that the signaling pathway involving PI3-kinase and PKC zeta plays an important role in induction of germline C epsilon transcription in DND39 cells by IL-4.

Despite the recent advances in knowledge of the molecular mechanism by which interleukin-4 (IL-4) induces IgE production, little is known about the signal transduction pathway that leads to this event. This study investigated the signal transduction mechanism responsible for IL-4-induced expression of germ-line C epsilon transcripts with use of a human Burkitt lymphoma B-cell line, DND39, which is known to express germ-line C epsilon transcripts in response to IL-4. On stimulation with IL-4, the generation of inositol triphosphate was observed in the cells. In addition, this generation was associated with activation of phospholipase C-gamma 1 (PLC-gamma 1). Although herbimycin A, a potent inhibitor of tryosine kinase, inhibited IL-4-induced activation of PLC-gamma 1 and generation of inositol triphosphate, direct phosphorylation of PCL-gamma 1 was not determined. Nevertheless, IL-4 stimulation could induce activation of FYN but not LYN kinase, suggesting that additional molecule(s) might link FYN kinase to PLC-gamma 1. Interestingly, herbimycin A almost completely inhibited IL-4-induced expression of germ-line C epsilon transcripts when present during the entire culture period. These results indicate that the induction of germ-line C epsilon transcripts in IL-4-stimulated DND39 cells is essentially dependent on the activation of tyrosine kinase, possibly FYN kinase.

Since increased levels of serum soluble CD23/Fc epsilon RII (sCD23) were evidently demonstrated in patients with autoimmune diseases such as rheumatoid arthritis (RA), the possible mechanisms responsible for the elevation of serum sCD23 were investigated in RA patients. In keeping with increased serum sCD23, high proportion of CD23+ B cells was detected in the patients; this was associated with the enhanced expression of only Fc epsilon RIIa mRNA. Upon incubation at 37 degrees C, peripheral blood mononuclear cells of the patients spontaneously released high levels of sCD23 into the culture supernatant, while the CD23 expression on their B cells was considerably maintained even after the culture. Dot blot analysis further revealed that in contrast to normal subjects, RA patients showed no complete disappearance of Fc epsilon RIIa mRNA after the spontaneous culture. In addition, sCD23 release was significantly reduced in the patients by the addition of cycloheximide. It was also found that cycloheximide exerted the inhibitory influence on the spontaneous culture-mediated expression of CD23 on CD5+ but not CD5- B cells of the patients. However, the disappearance of CD23 from CD5+ as well as CD5- B cells of cord blood samples was unaffected by the agent. These results strongly suggest that CD5+ B cells of RA patients may be specifically activated by some mechanisms responsible for the persistent expression of Fc epsilon RIIa mRNA leading to the accelerated turnover of CD23 and in turn the increased release of sCD23.

Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis in humans. EAE can be passively transferred into naive syngeneic animals by administration of MOG-specific T cell clones. Lymphocytes isolated from green fluorescent protein (GFP)-transgenic (Tg) mice can light up by emitting green fluorescence, thus making it feasible to use such animals in a passive transfer model for EAE. When MOG-sensitized splenic lymphocytes from GFP-Tg mice were adoptively transferred to irradiated, syngeneic C57BL/6 and RAG-1(-/-)mice, typical symptoms of EAE developed. Analysis of the reconstituted mice with EAE revealed prominent infiltration of fluorescing (GFP+), CD4+ T cells into the central nervous system (CNS). Real-time confocal imaging revealed these cells in the spinal cords and brains of recipient mice. This infiltration was also confirmed by anti-GFP monoclonal antibodies. Furthermore, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) evaluation indicated that the infiltrating GFP+, CD4+ T cells exclusively produced T helper type 1 (Th1) cytokines, especially interferon-gamma (IFN-gamma). These results clearly show that MOG-specific CD4+ T cells preferentially invade into the CNS and mediate the development of EAE by producing Th1-biased cytokines.

Background: The role of CD8 + T cells in IgE synthesis remains unclear. Objective: The aim of this study was to investigate IL-4 production and CD40 ligand expression by human CD8 + T cells. Methods: We conducted functional and phenotypic analyses of human T cells in peritoneal washings from severe combined immunodeficiency mice reconstituted with PBMCs from normal and atopic human donors. We also examined the expression of IL-4 and CD40 ligand by CD8 + T cells from a patient with adenosine deaminase deficiency who received autologous T cell–directed gene therapy. Results: Transfer of atopic cells into the mice caused production of IgE and IgG with increased expression of IL-4 and CD40 ligand mRNA. In addition, both intracellular IL-4 and cell surface CD40 ligand were detected in CD8 + and in CD4 + T cells. CD8 + T-cell lines generated from the patient's T cells carrying the adenosine deaminase gene expressed not only IL-4 mRNA and protein but also CD40 ligand mRNA and protein after being stimulated with an anti-CD3 mAb. After anti-CD3 stimulation and paraformaldehyde fixation, CD8 + T cells induced IgE synthesis by normal human B cells in the presence of recombinant IL-4. Conclusion: Taken together, these results demonstrate that IL-4–producing and CD40 ligand–expressing CD8 + cells are detectable among human T cells and suggest that such cells may promote IgE production by B cells under some conditions. (J Allergy Clin Immunol 1999;103:S405-11.)

Nuclear factor-kappa B (NF-kappa B) is a transcription factor that binds to the consensus DNA sequence in the cis-acting elements of various genes. Although NF-kappa B activates the expression of many genes involved in immune and inflammatory responses, little is known about the role of NF-kappa B activation in the induction of IgE synthesis in human B cells. Therefore we first examined the participation of NF-kappa B in germline C epsilon transcription in a human Burkitt lymphoma B cell line, DND39. Stimulation of DND39 cells with IL-4 or anti-CD40 monoclonal antibody (mAb) activated phosphatidylinositol 3-kinase and subsequently induced nuclear expression of NF-kappa B, which was identified by electrophoretic mobility shift assays. n-Acetyl-L-cysteine (NAC), a potent antioxidant, blocked NF-kappa B activation caused by IL-4 and by anti-CD40 mAb. Although inhibition of IL-4-driven germline C epsilon transcription by NAC was not sufficient, the agent remarkably diminished anti-CD40 mAb-mediated up-regulation of germline C epsilon transcription. Second, we studied the effect of NAC on IgE synthesis in human normal B cells costimulated with IL-4 and anti-CD40 mAb. NAC was effective in inhibiting mature C epsilon transcription and IgE synthesis in the T cell-independent culture system. However, NAC did not significantly affect the spontaneous production of IgE by atopic B cells. These results indicate that NF-kappa B activity is commonly inducible in DND39 cells by IL-4 and anti-CD40 mAb and suggest that NF-kappa B sensitive to NAC may play a role in regulating IgE synthesis in B cells.

IL-4 with the IgE-inducing activity is shown to upregulate the expression of IL-4 receptor (IL-4R) on lymphocytes. Antisense strategy was used that aimed at investigating the significance of IL-4-induced upregulation of IL-4R on B cells in human IgE production. When an antisense phosphorothioate oligodeoxynucleotide to IL-4R (S-oligo 1) was added to B cells together with IL-4, the agent selectively abrogated the upregulation of IL-4R without affecting its constitutive level expression. Moreover, S-oligo 1 had a suppressive effect on the T-cell-independent synthesis of IgE by B cells costimulated with IL-4 and anti-CD40 antibody. This suppression was accompanied by inhibition of mature but not germline C epsilon transcription. These findings indicate that constitutively expressed IL-4R provides a signal or signals responsible for the induction of germline C epsilon transcription and suggest that IL-4R upregulation may be required for the subsequent class switch recombination that leads to mature C epsilon transcription and IgE synthesis. The IL-4R signal transduction mechanism underlying germline C epsilon transcription was also analyzed in a human Burkitt lymphoma B-cell line, DND39. Induction of germline C epsilon transcripts in DND39 cells by IL-4 required at least two distinct signaling cascades. One was mediated by enhancement of tyrosine phosphorylation of a 57 kd protein associated with phospholipase C-gamma 1 (PLC-gamma 1) that resulted in PLC-gamma 1 activation, inositol lipid hydrolysis, and protein kinase C delta translocation. The other was dependent on phosphatidylinositol 3-kinase, whose activation induced protein kinase C zeta translocation. In fact, kinase inhibitors such as herbimycin A, K-252a, and wortmannin were effective in inhibiting IL-4-induced germline C epsilon transcription. Therefore, in addition to activation of protein tyrosine kinases, coordinated actions of PLC-gamma 1 and phosphatidylinositol 3-kinase may be involved in IL-4-driven germline C epsilon transcription in DND39 cells.

To study the regulatory mechanism of allergen-dependent human IgE synthesis, Cry j I-specific and interleukin 4 (IL-4)-producing CD4+ T cell lines (SN-4 and SS-12) were established from 2 patients allergic to Japenese cedar pollen who highly expressed IL-4 mRNA in T cells in response to Cry j I stimulation. Upon stimulation of SN-4 and SS-12 cells with Cry j I, IL-4 production, which was observed at the protein and the mRNA levels, was induced in an HLA-DR-restricted manner, using autologous and allogeneic antigen-presenting cells. In addition to IL-4, not only considerable amounts of IL-5 and IL-6 but also very small amounts of IL-2 and interferon-γ (IFN-γ) were secreted by SN-4 and SS-12 cells, indicating that they fit into the Th2-like phenotype. The culture supernatant from Cry j I-activated SN-4 cells had the ability to induce IL-4-dependent IgE synthesis, CD23 expression and soluble CD23 release. Moreover, Cry j I-dependent IgE synthesis medated by SN-4 cells derived from 1 patient expressing HLA-DRw8, w9 could be specifically induced in both autologous and HLA-DRw9-matched allogeneic B cell cultures. This IgE induction was inhibited by neutralizing antibodies to IL-4, IL-5 and IL-6, but was not enhanced by anti-IFN-γ antibody. On the other hand, neither IL-4 production nor IgE synthesis was induced when SN-4 cells were cocultured in the presence of Cry j I with HLA-DRw8-matched or histoincompatible allogeneic cells. A similar result was obtained with HLA-DR4-restricted SS-12 cells generated from another patient with HLA-DR4, w13. These data demonstrate that allergen-reactive and HLA-DR-restricted CD4+ T cells with the Th2 phenotype such as SN-4 and SS-12 cells specific for Cry j I are responsible for the induction and enhancement of IgE synthesis through the absolute production of Th2-specific cytokines.

Platelet-activating factor (PAF)-induced augmentation of spontaneous differentiation into eosinophil-lineage cells from hematopoietic precursor cells was found among human umbilical cord blood mononuclear cells (CBMCs). This ability of PAF to augment eosinophil production was significantly (p0.01) inhibited by the addition of the anti-PAF agent WEB 2086. This enhancing effect of PAF was significantly (p0.01) diminished by the presence of antibodies to interleukin (IL)-1 alpha, -beta and IL-3, whereas antibodies to IL-5 or granulocyte-macrophage colony-stimulating factor (GM-CSF) did not diminish it. IL-1 beta, IL-3 or IL-5 gene expression was detected in cellular RNA isolated from both medium- and PAF-stimulated CBMCs using the reverse transcription polymerase chain reaction (RT-PCR) method; however, no expression of GM-CSF was detected. Moreover, 5 times as much IL-3 was detected in the PAF-stimulated CBMC culture supernatant than in that of cells treated with the medium. In the case of IL-1 beta, there was no difference between the 2 cell preparations. On the other hand, no IL-5 or GM-CSF was detected in the supernatant after stimulation with medium or PAF. Depletion of CD2+, CD16+ or CD19+ cells, but not CD14+ cells, from CBMCs resulted in a marked decrease in PAF-induced augmentation of eosinophil production. These results suggest that PAF induces the augmentation of IL-3 production in CBMCs which in turn enhances differentiation into eosinophil-lineage cells.

Using severe combined immunodeficiency mice engrafted with peripheral blood mononuclear cells from atopic patients, we analyzed the regulatory effects of anti-CD154 antibody on the in vivo induction of human interleukin (IL)-4 and IgE expression. Although anti-CD154 treatment of engrafted mice abrogated mature Ce transcription and IgE production, IL-4 mRNA levels were enhanced by the treatment. In addition, anti-CD154-induced enhancement of intracellular IL-4 synthesis was detectable in both CD4+ and CD8+ T cell subsets. Furthermore, upregulation of germline Ce transcription could be seen in anti-CD154-treated mice. These results demonstrate that blocking the CD154-CD40 interaction with anti-CD154 differentially regulates IL-4 synthesis in T cells and IgE production in B cells. Our data also indicate that antibody ligation of CD154 on T cells causes enhanced synthesis of IL-4, thereby contributing to upregulation of germline Ce transcription in B cells.