Thorvaldson Lina, Johansson Sofia E, Höglund Petter, Sandler Stellan
Department of Medical Cell Biology, Biomedicum, Uppsala, Sweden.
J Immunol Methods. 2005 Dec 20;307(1-2):73-81. doi: 10.1016/j.jim.2005.09.008. Epub 2005 Oct 17.
Cytokines produced by Th1 or Th2 cells have been postulated to be important in the development of type 1 diabetes in humans and animal models, such as murine multiple low-dose streptozotocin (MLDSTZ)-induced diabetes. The aim of this study was to investigate cytokine production with or without in vitro depletion of plastic adherent cells from spleens isolated after MLDSTZ treatment. Spleen cells were prepared on day 14 from MLDSTZ- and saline-treated mice and divided into two fractions. One cell fraction was depleted of adherent cells by plastic adherence and the other was not. Both cell fractions were analysed by FACS for the distribution of immune cells. In other experiments, the cells were cultured for 48 h with concanavalin A stimulation. Supernatant samples were analysed by ELISA for TNFalpha, IFNgamma and IL-10 production. Either before or after the 48-h culture cytokine mRNA expression was determined by RT-PCR. Plastic adhesion decreased the macrophage numbers by approximately 30% and CD4(+)CD25(+) cells by about 60%. This was accompanied by increased medium levels of TNFalpha, IFNgamma and IL-10, which suggest that either CD4(+)CD25(+) cells, macrophages, or both, down-regulate production of both Th1 and certain Th2 cytokines. Depletion of adherent cells also decreased IL-4 mRNA amounts. MLDSTZ treatment increased the production of Th1 cytokines mainly at the protein level, and IL-10 mainly at the mRNA level. This indicates a sustained increase in Th1 production after MLDSTZ treatment and an increase in IL-10 that might reflect an attempt to counteract the MLDSTZ-induced immune damage. Plastic adhesion during cell preparation may affect the relative distribution of certain immune cells.
在人类和动物模型（如小鼠多次低剂量链脲佐菌素（MLDSTZ）诱导的糖尿病）中，Th1或Th2细胞产生的细胞因子被认为在1型糖尿病的发生发展中起重要作用。本研究的目的是调查在MLDSTZ治疗后分离的脾脏中，去除或不去除塑料贴壁细胞的情况下细胞因子的产生情况。在第14天从接受MLDSTZ和生理盐水处理的小鼠制备脾细胞，并分为两部分。一部分细胞通过塑料贴壁去除贴壁细胞，另一部分则不去除。通过流式细胞术分析两部分细胞中免疫细胞的分布。在其他实验中，细胞用伴刀豆球蛋白A刺激培养48小时。通过酶联免疫吸附测定法（ELISA）分析上清液样本中肿瘤坏死因子α（TNFalpha）、干扰素γ（IFNgamma）和白细胞介素10（IL-10）的产生。在48小时培养之前或之后，通过逆转录聚合酶链反应（RT-PCR）测定细胞因子mRNA的表达。塑料贴壁使巨噬细胞数量减少约30%，CD4(+)CD25(+)细胞减少约60%。这伴随着培养基中TNFalpha、IFNgamma和IL-10水平的升高，这表明CD4(+)CD25(+)细胞、巨噬细胞或两者都下调Th1和某些Th2细胞因子的产生。去除贴壁细胞也降低了IL-4 mRNA的量。MLDSTZ治疗主要在蛋白质水平增加Th1细胞因子的产生，而IL-10主要在mRNA水平增加。这表明MLDSTZ治疗后Th1产生持续增加，以及IL-10增加，这可能反映了对抗MLDSTZ诱导的免疫损伤的一种尝试。细胞制备过程中的塑料贴壁可能会影响某些免疫细胞的相对分布。
