Koichi Ikizawa

Suppression of IgE production by IPD-1151T (suplatast tosilate), a new dimethylsulfonium agent: (1). Regulation of murine IgE response.

The effect of IPD-1151T, a new dimethylsulfonium compound, on the IgE response was investigated in the mouse system. The oral administration of IPD-1151T to immunized BALB/c mice suppressed the primary IgE antibody response and depressed the elevation of serum IgE levels, whereas the same treatment did not affect the IgG antibody response. The enhanced expression of low-affinity IgE receptor (Fc epsilon RII/CD23) on the spleen cells of immunized mice was also inhibited by IPD-1151T administration. It was further demonstrated from the adoptive transfer experiment that IPD-1151T, administered to hapten-primed B cell donors, but not to carrier-primed T cell donors, exerted its suppressive influence on the hapten-specific secondary IgE antibody response in irradiated syngeneic recipients. Interestingly, IPD-1151T concentration-dependently inhibited the production of interleukin 4 (IL-4) by D10G4.1, known to be a typical Th2 clone. However, IPD-1151T did not suppress the production of IgE and IgG1 by normal splenic B cells stimulated with lipopolysaccharide and IL-4. Moreover, IL-4-induced expression of Fc epsilon RII on normal spleen cells was not inhibited by the agent. These results strongly suggest that the IgE-suppressive activity of IPD-1151T is most likely due to the inhibition of IL-4 production at the T cell level.

The expression of murine cutaneous late phase reaction requires both IgE antibodies and CD4 T cells

Background Exposure of atopic patients to a specific allergen evokes an immediate response which is followed, in many cases, by a late phase reaction (LPR) some hours later. Here we have examined the immunological mechanisms required for the expression of cutaneous LPR in mice.

Functional significance of IL-4 receptor on B cells in IL-4-induced human IgE production.

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.

Species-specific differences in the glucocorticoid receptor transactivation function upon binding with betamethasone-esters.

Glucocorticoids (GCs) are the most effective drugs for anti-inflammatory diseases. A number of adverse side effects, however, limit chronic treatment with GCs. To improve their therapeutic usefulness, attempts have been made to dissociate the two main actions of the glucocorticoid receptor (GR), transactivation and transrepression, which are believed to be responsible for the side effects and anti-inflammatory effects, respectively. We report here species-specific differences in the transactivation response mediated by GR. Dexamethasone (DEX), betamethasone (BM), and their esterified-derivatives had full transrepression agonistic activity in a reporter assay using CV-1 cells transfected with either human or rat GR. These GCs also had full transactivation agonistic activity in CV-1 cells transfected with human GR. The esterified-BM, however, had only partial transactivation agonistic activity in cells transfected with rat GR, whereas BM and esterified-DEX had full transactivation agonistic activity. Moreover, in rat hepatoma H4-II-E cells, the esterified-BM failed to induce tyrosine aminotransferase, which is regulated by GR-mediated transactivation activity. There were no significant differences between the binding affinity of these GCs to human and rat GR. Consistent with the weak transactivation activity of esterified-BM mediated by rat GR, there were few side effects, evaluated by thymus involution and body weight loss, in an antigen-induced asthmatic model in rats. These results suggest that the potency of esterified-BM to induce transactivation activity is different between species and that this difference is not due to differences in receptor binding.

A thiol antioxidant regulates IgE isotype switching by inhibiting activation of nuclear factor-κB

The binding site for nuclear factor-kappaB (NF-kappaB) is present at the promoter region of the germline Cepsilon gene, but there is little information on whether this factor is involved in regulating IgE synthesis by human B cells. Accordingly, we studied the role of NF-kappaB in germline Cepsilon transcription by using two human Burkitts lymphoma B cell lines, DND39 and DG75. In both cell lines, n-acetyl-L-cysteine (NAC), a potent thiol antioxidant, inhibited the triggering of the nuclear expression of NF-kappaB by IL-4 and by anti-CD40 monoclonal antibody. Although IL-4 activated signal transducers and activators of transcription (STAT) 6 in addition to NF-kappaB, NAC treatment or the transfection of decoy oligodeoxynucleotides for NF-kappaB or STAT6 only partly blocked IL-4-induced germline Cepsilon transcription. However, these two decoy oligodeoxynucleotides together almost completely abrogated IL-4-induced germline Cepsilon transcription. Of note, CD40-mediated enhancement of IL-4-driven germline Cepsilon transcription was markedly decreased by NAC or by a decoy oligodeoxynucleotide for NF-kappaB. The effect of NAC was also examined on deletional switch recombination underlying the isotype switch to IgE. NAC inhibited the generation of Smu/Sepsilon switch fragments in normal human B cells costimulated with IL-4 and anti-CD40 monoclonal antibody. It also abolished IL-4-induced upregulation of CD40 but promoted upregulation of CD23. These results suggest that coordination of NF-kappaB and STAT6 may be required for induction of germline Cepsilon transcription by IL-4, and that CD40-mediated NF-kappaB activation may be important in regulating both enhancement of germline Cepsilon transcription and class switching to IgE.

Possible role of tyrosine kinase activity in interleukin 4–induced expression of germ-line Cϵ transcripts in a human Burkitt lymphoma B-cell line, DND39

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.

CD40-mediated tumor necrosis factor receptor-associated factor 3 signaling upregulates IL-4-induced germline Cε transcription in a human B cell line

Induction of germline C epsilon transcription in B cells by IL-4, which is a critical initiating step for IgE class switching, is enhanced by CD40 engagement. Although signaling by CD40 is initiated by the binding of tumor necrosis factor receptor-associated factor (TRAF) family members to its cytoplasmic domain, whether those TRAF family proteins mediate enhancement of germline Cepsilon transcription is not evident. We report here that CD40-induced TRAF3-dependent activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) is involved in the upregulation of IL-4-driven germline C epsilon transcription in a human Burkitts lymphoma B cell line, DG75. Among the six known TRAF proteins, TRAF2, 3, 5, and 6 associated with CD40 in an unstimulated state, and the levels of these four proteins were unaffected by anti-CD40 stimulation. Antisense oligodeoxynucleotide (ODN) for TRAF3 inhibited CD40-induced activation of MEK1-ERK pathway by decreasing expression of TRAF3 protein, but antisense ODNs for TRAF2, 5, and 6 were ineffective. Furthermore, CD40-mediated enhancement of IL-4-driven germline C epsilon transcription was inhibited by antisense ODN for TRAF3 and by a MEK1 inhibitor, PD98059. These results suggest that in DG75 cells, TRAF3-induced MEK1 activation may be involved in CD40-mediated upregulation of IL-4-driven germline C epsilon transcription.

Possible Role of CD5+ B Cells Expressing CD23 in Mediating the Elevation of Serum-Soluble CD23 in Patients with Rheumatoid Arthritis

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.

Involvement of nuclear factor-κB activation in IgE synthesis in human B cells☆☆☆★★★

Abstract Nuclear factor-κB (NF-κB) is a transcription factor that binds to the consensus DNA sequence in the cis-acting elements of various genes. Although NF-κB activates the expression of many genes involved in immune and inflammatory responses, little is known about the role of NF-κB activation in the induction of IgE synthesis in human B cells. Therefore we first examined the participation of NF-κB in germline Cϵ 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-κB, which was identified by electrophoretic mobility shift assays. n -Acetyl-L-cysteine (NAC), a potent antioxidant, blocked NF-κB activation caused by IL-4 and by anti-CD40 mAb. Although inhibition of IL-4–driven germline Cϵ transcription by NAC was not sufficient, the agent remarkably diminished anti-CD40 mAb–mediated up-regulation of germline Cϵ 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 Cϵ 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-κB activity is commonly inducible in DND39 cells by IL-4 and anti-CD40 mAb and suggest that NF-κB sensitive to NAC may play a role in regulating IgE synthesis in B cells. (J Allergy Clin Immunol 1996;98:S224-9.)

Allergen-Specific Human IgE Helper T Cell Lines Derived from Patients Allergic to Japanese Cedar Pollen

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 Japanese 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-gamma (IFN-gamma) 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-gamma 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.(ABSTRACT TRUNCATED AT 250 WORDS)