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。2000 Nov；68（11）：6233-9。
doi:10.1128/IAI.68.1.6233-6239.2000。

出口出口出口出口出口出口和出口出口出口出口出口

R Uchikawa 1个单击功能区上S Matsuda单击功能区上N Arizono
疲劳，疲劳

出口出口出口出口出口出口和出口出口出口出口出口

R Uchikawaet al。 Infect Immun。 2000 Nov。

Abstract

Although certain helminth infections preferentially induce type2T-cell responses，the immunological mechanism responsible for type2T-cell polarization remain unclear。In the present study，we investigated the effects of excretory-secretory（ES）antigen from the nematode Nippostrongylus brasiliensis on cytokine production by mesenteric lymph node（MLN）cells isolated from naiverats。LN cells produced considerable levels of gamma interferon（IFN-gamma）during a72-h stimulation with concanavlin A（ConA）or with immobilized anti-CD3plus soluble anti-CD28antibodies（anti-CD3/CD28）。With either stimulation，10microg of ES antigen per ml significantly suppressed IFN-gamma and interleukin-2（IL-2）production without cytotoxic activity。The copresence of anti-IL-4，anti-IL-10，or transforming growth factor beta（TGF-beta）blocking antibodies did not alter the suppressive effect of ES antigen on IFN-gamma production。ES antigen did not affect IL-10 production。Kinetic studies of the effect of ES antigen indicated that the antigen suppressed even ongoing IFN-gamma production。Reverse transcription-PCR study showed that in the presence of ES antigen，IFN-gamma mRNA expression by MLN cells was suppressed6and12h after ConA or anti-CD3/CD28 stimulation。ES antigen also significantly suppressed IFN-gamma production by purified CD4（+）or CD8（+）T cells during anti-CD3/CD28 stimulation but did not affect IL-4 production by CD4（+）T cells。These findings suggested that the nematode antigen suppressed production of IFN-gamma and IL-2but not IL-4or IL-10 production。ES antigen-mediated suppression of IFN-gamma during the initiation of the immune response may provide a microenvironment that helps generation of type2T cells。

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Figures

FIG.1
FIG.1
Effect of ES antigen on spontaneous IFN-γproduction of MLN cells isolated from naive rats。Pooled MLN cells（5×106个/ml）were cultured with ES antigen at the indicated doses。After72h，supernatants were harvested and IFN-γlevels were determined by ELISA。The data are the means±standard deviations of triplicate cultures。The results are representative of three independent experiments。
FIG.2
FIG.2
Effect of ES antigen on IFN-γproduction by MLN cells from naive rats during polyclonal stimulation。Pooled MLN cells（106个/ml）were stimulated with ConA or anti-CD3/CD28 in culture medium containing various doses of ES antigen in the absence or presence of anti-IL-4 monoclonal antibody。After72h，supernatants were harvested and IFN-γlevels were determined by ELISA。The data are the means±standard deviations of triplicate cultures。The results are representative of five independent experiments。
FIG.3
FIG.3
Effect of anti-IL-10 or anti-TGF-βantibody on IFN-γproduction by MLN cells from naive rats during polyclonal stimulation。Pooled MLN cells（106个/ml）were stimulated with ConA（A）or anti-CD3/CD28（B）in culture medium containing various doses of ES antigen in the absence or presence of anti-IL-10 or anti-TGF-βneutralizing antibody。After72h，supernatants were harvested and IFN-γlevels were determined by ELISA。The data are the means±standard deviations of triplicate cultures。The results are representative of three independent experiments。
FIG.4
FIG.4
Effects of ES antigen on proliferation and viability of MLN cells。Pooled MLN cells（106个/ml）were cultured with ConA plus various doses of ES antigen for 72h，and proliferation（A）and cell viability（B）were determined by[3H]thymidine incorporation and trypan blue exclusion，respectively。（C）MLN cells were cultured for72h with various doses of ES antigen under stimulation with ConA（closed circles）or with anti-CD3/CD28（open circles），and cell numbers were estimated by MTS assay。The data are the means±standard deviations of triplicate cultures。These results are representative of three independent experiments。OD，optical density。
FIG.5
FIG.5
Effect of ES antigen on IL-10 production by MLN cells from naive rats。Pooled MLN cells（106个/ml）were incubated with or without ES antigen under stimulation with ConA（A）or with anti-CD3/CD28（B）。At the indicated time points，supernatants were harvested and IL-10 levels were determined by ELISA。The data are the means±standard deviations of triplicate cultures。These results are representative of two independent experiments。
FIG.6
FIG.6
Effect of ES antigen on IL-2 production by MLN cells from naive rats during polyclonal stimulation。Pooled MLN cells（106个/ml）were stimulated with ConA（A）or anti-CD3/CD28（B）in culture medium containing various doses of ES antigen。After72h，supernatants were harvested and IL-2levels were determined by ELISA。The data are the means±standard deviations of triplicate cultures。The results are representative of three independent experiments。
FIG.7
FIG.7
Kinetics of the effect of ES antigen on IFN-γproduction by MLN cells from naive rats。Pooled MLN cells（106个/ml）were cultured in medium containing ConA，and ES antigen（10μg/ml）was added to culture wells at the indicated time points after the start of cultures。Supernatants were harvested72h after the start of cultures，and IFN-γlevels were determined by ELISA。时间zero indicates the addition of ES antigen from the start of culture；at72h ES antigen was not added at all。The data are the means±standard deviations of triplicate cultures。The results are representative of five independent experiments。
FIG.8
FIG.8
ELN cells with ES antigen on ConA-stimulated IFN-γproduction。Pooled MLN cells（106个/ml）were cultured in medium containing ES antigen（10μg/ml）but not ConA for the indicated times，and after being washed，cells were cultured in medium containing ConA but not ES antigen。After72h，IFN-γlevels in culture supernatants were determined by ELISa。The data are the means±standard deviations of triplicate cultures。The results are representative of five independent experiments。
FIG.9
FIG.9
Effect of ES antigen on IFN-γmRNA expression by MLN cells isolated from naive rats。Pooled MLN cells were stimulated with ConA or anti-CD3/CD28 for 12 h in the presence or absence of ES antigen（10μg/ml）。高速IFN-γand，andβ-actin mRNA was analyzed by RT-PCR。The results are representative of three independent experiments。
FIG.10
FIG.10
Effect of ES antigen on cytokine production by CD4+and CD8+T cells separated from pooled naive rat MLNs。Purified CD4+and CD8+T cells（106个/ml）were cultured with or without ES antigen under stimulation with anti-CD3/CD28。After72h，supernatants were harvested and IFN-γand IL-4levels were determined by ELISA。The numbers of viable cells were evaluated by MTS assay（C）。The data are the means±standard deviations of triplicate cultures。The results are representative of three independent experiments。ND，not detectable。
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