| Staphylococcal enterotoxin B (SEB) is not only a toxic substance but also a mostimportant superantigen (SAg) which is more investigated in nine serotypes ofstaphylococcal enterotoxin. SEB as a SAg can cross-link major histocompatibility complexclass II (MHC II) molecules with a variable portion of the β chain (Vβ) of the T-cellreceptor, binding beyond the antigen-specific site, which does not require classicalantigen-processing and presentation and can polyclonally stimulate T cells. A line ofstudies have shown that the immune response to SAgs is biphasic: an initial activationphase characterized by T-cell proliferation is followed by a period of anergy and/ortolerance owing to depletion of the appropriate Vβ-expressing T cells by apoptosis.Althrough a lot of literatures report the influence of SEB administration during adulthoodor neonatal period on immune organs, T cells and cytokines, it is little known about effectof maternal SEB administration during pregnancy on cellular immunity of neonataloffspring rats. Therefore, pregnant rats at gestational day (GD)16were injectedintravenously by15μg SEB(as SEB group) or PBS (as PBS group). Both CD4/CD8T cellsand TCR Vβ8~+T cells subpopulation were determined in the thymus, spleen or peripheralblood of neonatal offspring rats from day0to5after birth. It was also investigated theinfluence of either ConA or SEB on thymocytes development by cultured thymus organ oron splenocytes proliferation response by in vitro cultured splenocytes in neonatal offspringrats.Methods:1. Pregnant rats at GD16were randomly divided two group: rats at SEB group wereinjected intravenously by15μgSEB and at PBS group byPBS at the same volume.2. At GD21, pregnant rats were anesthesized by4%chloral hydrate and their abdominalcavity and uterus were opened. The body, thymus and spleen of fetal rats and placenta wereweighed. 3. The suspensions of thymocytes, splenocytes and peripheral blood cells of neonataloffspring rats from day0to5after birth were stained by CD3-FITC,CD4-APC andCD8-PE or by Vβ8.2-FITC,CD4-APC and CD8-PE, then T cells subpopulations weredetermined by flow cytometry.4. Thymuses of neonatal rats in day0after birth were harvested and co-cultured in vitrowith either ConA or SEB for three days. At the end of culture, thymocytes suspension wasstained by above antibody and determined for T cells subpopulation by flow cytometry.5. Spleens of neonatal rats in day3after birth were harvested. Then lymphocytes in spleenswere acquired by gradient centrifugation method and co-cultured in vitro with either ConAor SEB for three days. At the end of day1,2,3, splenocytes suspension was harvested andstained by above antibody and determined for T cells subpopulation by flow cytometry.When proliferation activity of lymophocytes was detected by3H incorporation,3H-TdRwas added into the cultured system5hours before the end of the timed points.Results:1. Effect of maternal SEB administration during pregnancy on weight of fetal rats.The weights and net weights of both thymus and spleen of fetal rats in SEB group weresignificantly decreased than that in PBS group. But weights of both placenta and fetal bodyin two groups had no difference.2. Effect of maternal SEB administration during pregnancy on CD4/CD8T cells ofneonatal rats.2.1Effect of maternal SEB administration during pregnancy on CD4/CD8T cells in thethymus of neonatal rats.From day0to5after birth, percentage of thymic CD4~+T cells in SEB group wassignificantly higher than that in PBS group, while percentage of thymic CD8~+T cells inSEB group was significantly lower than that in PBS group.2.2Effect of maternal SEB administration during pregnancy on CD4/CD8T cells in thespleen of neonatal rats.From day0to5after birth, percentage of splenic CD4~+T cells in SEB group wassignificantly higher than that in PBS group, but percentage of thymic CD8~+T cells in twogroups had no difference. 2.3Effect of maternal SEB administration during pregnancy on CD4/CD8T cells in theperipheral blood of neonatal rats.From day0to5after birth, the change of percentage of either CD4~+or CD8~+T cells inthe peripheral blood of neonatal rats was similar to that in spleen. The ratio of CD4~+andCD8~+T cells in two groups was less than1from day0to1after birth, then increased tomore than1from day2to5, but the ratio in SEB group was significantly increased thanthat in PBS group.3. Effect of maternal SEB administration during pregnancy on TCR Vβ8.2~+T cells ofneonatal rats.Percentage of thymic CD4~+TCR Vβ8.2~+T cells in SEB group was significantly lowerthan that in PBS group from day0to3after birth, but from day4to5in two groups hadno difference. While Percentages of thymic CD8~+TCR Vβ8.2~+T, both CD4~+and CD8~+TCR Vβ8.2~+T in the peripheral blood of SEB group were lower than that in PBS groupfrom day0to5after birth.4. Effect of maternal SEB administration during pregnancy on in vitro cultured thymusdevelopment of neonatal rats.4.1Effect of maternal SEB administration during pregnancy on CD4/CD8T cells in invitro cultured thymus of neonatal rats.Percentages of both CD4~+and CD8~+T cells in SEB group were significantly decreasedthan that in PBS group in thymus in vitro co-cultured with ConA or SEB for three days, butpercentage of CD4~+CD8~+T cells was sighificantly increased, while percentage ofCD4-CD8-T cells had no difference.4.2Effect of maternal SEB administration during pregnancy on TCR Vβ8.2~+T cells in invitro cultured thymus of neonatal rats.After in vitro co-cultured with ConA or SEB for three days, percentages of both CD4~+and CD8~+TCR Vβ8.2~+T cells in SEB group were significantly decreased than that in PBSgroup.5. Effect of maternal SEB administration during pregnancy on in vitro lymphocytesproliferation in the spleen of neonatal rats.5.1Effect of maternal SEB administration during pregnancy on in vitro T cells subpopulation proliferation.After in vitro co-cultured with ConA or SEB, percentage of CD4~+T cells in SEB groupwas significantly increased at day1and decreased at day2and3than that in PBS group,while percentage of CD8~+T cells in two groups had no difference. After in vitro co-culturedwith ConA or SEB, percentages of CD4~+TCR Vβ8.2~+T cells at day1and CD8~+TCRVβ8.2~+T cells at the three days in SEB group were significantly increased than that in PBSgroup, but percentages of CD4~+TCR Vβ8.2~+T cells at day2and3in two groups had nodifference.5.2Effect of maternal SEB administration during pregnancy on proliferation index oflymphocytesAfter in vitro co-cultured with ConA or SEB, proliferation activity of lymphocytes inSEB group was significantly increased at day1and decreased at day2and3than that inPBS group.Conclusions：1. Maternal SEB administration during pregnancy can affect the development of boththymus and spleen in fetal rats.2. Maternal SEB administration during pregnancycan cause decreased TCR Vβ8.2~+T cellsby intrathymic and peripheral (post-thymic) clonal deletion. SEB could affect thedevelopment of other T cells besides TCR Vβ8.2~+T cells.3. In the neonatal thymus in vitro co-cultured with ConA or SEB, maternal SEBadministration during pregnancy could not change the development of thymocytes fromDN to DP, but significantly reduce the mature of thymocytes from DP to SP.4. CD4~+T cells are the majar subpopulation to response ConA or SEB in in vitro culturedsplenocytes of neonatal rats born to mother injected by SEB during pregnancy.5. Maternal SEB administration during pregnancy imprints CD4~+, CD8~+and TCR Vβ8.2~+Tcells in neonatal rats, which may be the fetal origin of adult disease. |
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