Yukiyoshi Yanagihara

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.

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.

Cord–Blood–Derived Human Cultured Mast Cells Produce Interleukin 13 in the Presence of Stem Cell Factor

Background: Mast cells have been regarded as a potential source of cytokines. Although the human mast cell line HMC–1 and human lung mast cells have been shown to produce interleukin (IL) 13, it still remains uncertain whether cord–blood–derived human cultured mast cells produce IL–13. Methods: Human cultured mast cells were raised from cord blood cells in the presence of stem cell factor (SCF) and IL–6. Levels of IL–13 mRNA were examined by a semiquantitative reverse transcriptase–polymerase chain reaction. IL–13 levels in the supernatants were measured with an enzyme–linked immunosorbent assay. Results: When the IgE–sensitized cultured mast cells were activated with anti–IgE, mRNA for IL–13 was amplified with a peak at 3 h after the stimulation. IL–13 was not detected in the supernatants of the activated mast cells in the absence of SCF, whereas the mast cells secreted significant amounts of IL–13 after the stimulation in the presence of SCF. Calcium ionophore A23187 also stimulated the mast cells to release IL–13 into the supernatant in the presence of SCF. Conclusions: These observations suggest that human mast cells can produce IL–13 under the condition with SCF. The cord–blood–derived human cultured mast cells will help in studying the functional properties of human mast cells in allergic diseases.

Ligation of Toll-like receptor 3 differentially regulates M2 and M3 muscarinic receptor expression and function in human airway smooth muscle cells.

Background: Viral infection causes asthma exacerbations and airway hyperreactivity. Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA) of viral or synthetic origin in a fashion different from protein kinase R (PKR). The aim of this study was to examine the expression and function of TLR3 in human airway smooth muscle (ASM) cells. Methods: Expression of TLR3 and muscarinic receptor (MR), histamine receptor (HR), and cysteinyl leukotriene receptor (CysLTR) subtypes was analyzed by quantitative real-time PCR, flow cytometry, or Western blotting. It was assessed whether ASM cells respond to polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of dsRNA, with alterations in M2R, M3R, H1R, and CysLT1R expression. The function of these subtypes was evaluated by cholinergic regulation of forskolin-stimulated cyclic AMP accumulation or by mobilization of intracellular calcium upon stimulation. Results: ASM cells expressed TLR3 and PKR, and intracellular TLR3 expression was demonstrated. Poly I:C caused decreased M2R and increased M3R expression, without affecting H1R and CysLT1R expression. Poly I:C-treated cells showed decreased cholinergic inhibition of forskolin-stimulated cyclic AMP accumulation and enhanced calcium flux in response to acetylcholine, but not to histamine and LTD4. These modulating effects of poly I:C were reversed by chloroquine, but not by 2-aminopurine. Conclusions: The data indicate that poly I:C internalized by ASM cells differentially regulates M2R and M3R expression and function by interacting with TLR3 rather than with PKR, suggesting that these changes may contribute to airway hyperreactivity.

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.

Immunopharmacological actions of an extract isolated from inflamed skin of rabbits inoculated with vaccinia virus (neurotropin); enhancing effect on delayed type hypersensitivity response through the induction of Lyt-1+2− T cells

To clarify the immunopharmacological action of an extract isolated from inflamed skin of rabbits inoculated with vaccinia virus (Neurotropin), its effect on delayed type hypersensitivity (DTH) response to sheep red blood cells (SRBC) in mice was examined. Neurotropin enhanced the DTH response in C57BL/6 mice which were low responders to SRBC, but not in either BALB/c or C3H/He mice (high responders) when administered i.p. for 4 consecutive days prior to sensitization. However, Neurotropin did not affect the formation of plaque-forming cells to SRBC in C57BL/6 mice under the condition where it enhanced the DTH response. We further examined the mechanism by which Neurotropin enhanced the DTH response in C57BL/6 mice by means of cell transfer experiments. Spleen cells from mice administered Neurotropin i.p. for 4 days, but not saline, could enhance the DTH response when transferred i.v. into normal syngeneic mice just before sensitization. However, the treatment of the spleen cells with anti-Thy-1.2 + complement (C) or with anti-Lyt-1.2 + C, but not with anti-Lyt-2.2 + C, abrogated its enhancing effect. The depletion of macrophages from the cells had no effect. On the other hand, the spleen cells from mice administered Neurotropin had no enhancing effect in the effector phase of DTH response, and they showed a helper T cell activity in a DTH helper T cell assay system in which cyclophosphamide-treated mice were used as recipients. These results suggest that Neurotropin enhances the DTH response in low responder mice through the induction of Lyt-1+2- DTH helper T cells.

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.

Enhancement of IgE synthesis and histamine release by T cell factors derived from atopic patients with bronchial asthma

Culture supernatants of unstimulated T cells (TCS) derived from normal donors or from atopic patients with bronchial asthma were tested for their ability to regulate the spontaneous IgE synthesis by B cells of normal and atopic subjects. The same TCS were also tested for their influence on the histamine release from leukocytes of house dust mites-sensitive patients. Addition of TCS to B cell cultures from allergic donors induced a dose-dependent increase of the spontaneous IgE production without affecting the synthesis of IgG, IgM, and IgA. The potentiating activity of TCS was observed only in B cell cultures spontaneously producing IgE; TCS were still active on irradiated B cells. The maximal IgE-enhancing activity was observed when TCS were added at the onset of B cell cultures. The supernatants of T cells lysed at day 0 did not contain IgE-potentiating factors. The antigen-induced but not the spontaneous histamine release from leukocytes of house dust mite-sensitive patients was enhanced by pretreatment with TCS from allergic donors. The enhancing activities of TCS on IgE synthesis and on histamine release could be removed by absorption with IgE-Sepharose and subsequently recovered by elution with glycine buffer. The results indicate that T cells of patients with asthma spontaneously release IgE-binding factors capable of increasing both the spontaneous IgE synthesis by B cells and the antigen-induced histamine release.

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.

Platelet-activating-factor-induced augmentation of production of eosinophil-lineage cells in hematopoietic precursor cells obtained from human umbilical cord blood.

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 (p < 0.01) inhibited by the addition of the anti-PAF agent WEB 2086. This enhancing effect of PAF was significantly (p < 0.01) 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.