Noriaki Kobayashi

IL-2-Induced IL-13 Production by Allergen-Specific Human Helper T Cell Clones

Background: Interleukin (IL)-5 and IL-13 are important cytokines in allergic diseases such as asthma and atopic dermatitis. We have reported that the production of IL-5 and IL-13 by mite-responsive T helper cells (Th) is controlled under similar signal requirements, but precise mechanisms are not yet well characterized. Methods: Allergen-specific Th clones were established from peripheral blood lymphocytes of atopic asthmatics, and cytokine synthesis in response to various stimuli was determined by specific ELISAs. IL-13 gene expression was enumerated by quantitative real-time PCR. Results: IL-13 production was clearly induced by IL-2. IL-13 mRNA expression was also induced. Conclusion: The IL-2 signal by itself causes IL-13 synthesis independent of T cell receptor stimulation.

IL-5 production by peripheral blood Th cells of adult asthma patients in response to Candida albicans allergen.

Cytokines produced by T cells are involved in chronic asthma. Relevant antigens for nonatopic asthma are still mostly unknown. Peripheral blood mononuclear cells (PBMCs) were obtained from adult asthmatic donors and incubated with Candida albicans extract. Proliferation response and cytokine production were analyzed. IL-2 and IL-5 were detectable in the cultures incubated with C. albicans extract, whereas IL-4 was undetectable. PBMCs obtained from some nonatopic asthma patients produced IL-5 upon stimulation of C. albicans extract. C. albicans allergen may be involved in the airway eosinophilic inflammation through the induction of IL-5 production by Th cells.

A contraction assay system using established human bronchial smooth muscle cells.

Background: To further understand the mechanisms of airway obstruction in asthma, it is crucial to investigate contractile responses of human airway smooth muscle. An in vitro assay system employing collagen gels embedded with well-established bronchial smooth muscle cells of human origin was explored in the present study. Methods: Commercially available cultured human bronchial smooth muscle cells were embedded into a collagen gel. Well-known constrictors, histamine and methacholine, were added to the gel. The gel images were captured by an image analyzer, and contractile responses were evaluated. Results: Histamine and methacholine induced contraction of the gels in a dose-dependent manner. Pyrilamine, an H1 receptor antagonist, inhibited gel contraction in an agonist-specific manner. Conclusion: Our contraction assay system, employing widely distributed cultured cells, was highly reproducible and precise. It may go a long way toward understanding mechanisms of asthmatic responses and evaluation of antiasthma drugs.

A solid-phase enzyme immunoassay for anti-acetylcholine receptor antibody in myasthenia gravis patients

A solid-phase enzyme immunoassay for the measurement of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis is reported. Sufficient amounts of acetylcholine receptor for the sensitive detection of anti-acetylcholine receptor antibody were directly fixed to Costar serocluster 96-well EIA plates coated with poly-L-lysine hydrobromide. The solid-phase enzyme immunoassay detected anti-acetylcholine receptor antibodies in 91% of the myasthenia gravis patients including 4 out of 4 ocular type myasthenia patients, anti-acetylcholine receptor antibodies of which were not detectable by the immunoprecipitation assay. Correlation between antibody titers measured by enzyme immunoassay and the immunoprecipitation assay was significant.

IL-9 Production by Peripheral Blood Mononuclear Cells of Atopic Asthmatics

Background: IL-9 might play a critical role in pathogenesis and development of atopic asthma, but there are few reports on allergen-specific IL-9 production by peripheral blood mononuclear cells (PBMCs) obtained from adult asthmatics. Methods: PBMCs were obtained from adult atopic asthmatics and incubated with Dermatophagoides farinae(Der f) extract for the designated time periods. The resulting supernatants were assayed for IL-9 by specific sandwich ELISA. Results: IL-9 production was detectable on day 2 and reached maximum on day 6 after stimulation of PBMCs with Der f extract. IL-9 production in response to Der f extract increased in a dose-dependent manner. CD2- or CD4-bearing cell depletion completely abolished IL-9 production by PBMCs, while CD8-bearing cell depletion did not affect it. Conclusion: CD4+ lymphocytes are the principal source of IL-9 produced by PBMCs of adult atopic asthmatics.

Selective Regulation of T Cell IL-5 Synthesis by OM-01, JTE-711 and p38 MAP Kinase Inhibitor: Independent Control of Th2 Cytokines, IL-4 and IL-5

Background: Helper T cells are involved in chronic eosinophilic inflammation. Control of cytokine production seems to be an effective management. Methods: The effect of nonactin, SB203580, a p38 MAP kinase inhibitor, and JTE-711 on the cytokine production of allergen-specific T cell clones was determined. The effect of nonactin on the airway eosinophilia was investigated using murine asthma model. Results: Nonactin suppressed IL-5 synthesis by human Th cells in a dose-dependent manner without affecting IL-2 or IL-4 synthesis, and still significantly suppressed murine airway eosinophilia induced by the antigen inhalation. SB203580 and JTE-711 also selectively inhibited IL-5 synthesis in vitro. Conclusion: Synthesis of IL-5 by human Th cells can be differentially regulated from that of other major T cell cytokines. The in vivo effects of selective IL-5 synthesis inhibitors suggest that IL-5 is the reasonable target for the regulation of allergic disorders accompanied by eosinophilic inflammation.

Regulatory mechanisms of human T cell IL-5 synthesis: differential roles of the proximal promoter-binding proteins in IL-5 gene transcription.

Background: IL–5 is crucially involved in eosinophilic inflammation. We have previously reported that IL–5 synthesis by atopic and nonatopic asthmatics is significantly enhanced compared to control subjects. T cell IL–5 synthesis is regulated at the transcriptional level. The proximal human IL–5 promoter (–62 to –46) homologous to the conserved lymphokine element 0 (CLE0) is essential for activation–induced gene transcription by Th cells. Methods: Luciferase reporter analysis and gel shift analysis were performed. Results: The CLE0 homologous element is the overlapping binding site for a constitutive and an inducible binding factor. Site–directed mutagenesis successfully differentiated the two bindings, and revealed that the transcriptional induction was ascribed to the inducible binding, while the constitutive binding was rather inhibitory. A mutant element which lost the constitutive binding, but retained the inducible binding, exerted 3 times more transcriptional activity compared to the wild–type element. In contrast, another mutant element which lost the inducible binding and retained the constitutive binding exhibited no transcriptional induction. Gel shift analysis was performed to clarify that the inducible binding was more prominent and the constitutive binding was less in IL–5 producing Th clones compared to IL–5–nonproducing clones. Conclusion: The ratio of the inducible/constitutive binding to the CLE0 homologous element may determine the capacity of human Th cells to transcribe the IL–5 gene.

Effect of formoterol on allergen-induced cytokine synthesis by atopic asthmatics.

Background: Effect of formoterol, long-acting β2 agonists, on T cell cytokine synthesis was examined. Methods: Human peripheral blood mononuclear cells were obtained from atopic asthmatics, and stimulated with Dermatophagoidesfarinae extract. Various concentrations of formoterol were added from the start of some cultures. Cytokine production and cell proliferation were analyzed. Results: Allergen-induced IL-5, IL-13, and IFN-γ production of PBMC were significantly suppressed by formoterol in a dose-dependent manner, whereas the proliferation response was not suppressed. Conclusion: Formoterol downregulates T cell functions of atopic asthmatics in vitro.

Synthesis of FF8181-A

Both enantiomers of FF8181-A were synthesized through optical resolution from the known Diels-Alder reaction product in 15 steps. The absolute configuration of the natural product was determined to be 1S,5S,5aS,9aS,9bS.

A solid-phase enzyme immunoassay for anti-insulin antibody in diabetes mellitus patients

A solid-phase enzyme immunoassay for the measurement of anti-insulin antibodies in the sera of patients with diabetes mellitus was developed. Porcine insulin conjugated with bovine serum albumin was used for coating microtiter plates. This assay was as sensitive as the conventional radioimmunoassay. Anti-insulin antibody titers measured by the enzyme immunoassay and conventional radioimmunoassay correlated well and the correlation coefficient was 0.920, which was statistically significant (P less than 0.001). The enzyme immunoassay detected anti-insulin antibody in 23 out of 35 sera of patients with insulin-dependent diabetes mellitus. The present enzyme immunoassay does not require radioactive materials, is less expensive and is concluded to be practically useful.