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. 2008 Nov;173(5):1440–1454. doi: 10.2353/ajpath.2008.080150

Figure 5.

Figure 5

Relationship between IL-2 and MAA intravenously induced tolerance. Total lymphocytes obtained from popliteal LNs of tolerized and nontolerized Lewis rats were cultured for 24 hours with or without MAA for RT-PCR (A) and ELISA (B). A: Expression of IL-2 transcript using RT-PCR. Low levels of IL-2 mRNA were observed in tolerized animals (lanes 1 and 3) compared to nontolerized rats (lanes 2 and 4). A strong band at 335 bp for β-actin indicated equal amounts of RNA in each lane. B: Levels of IL-2 protein were quantitated by ELISA. Compared with nontolerized rats the levels of IL-2 protein were significantly reduced in tolerized rats. Results shown are the mean ± SD of results in triplicate independent analyses. *P < 0.005. C and D: FACS analysis of intracellular IL-2. Total lymphocytes harvested from popliteal LNs at the onset of EAAU (day 14 after immunization) were stained with anti-IL-2 and anti-CD4. Dot plots show IL-2 content of gated CD4+ T cells in tolerized (C) and nontolerized (D) animals. R2 represents the CD4+ cells in total lymphocyte population and R3 represents the population of IL-2-expressing CD4+ cells. A decrease in IL-2-expressing CD4+ cells could be seen in tolerized animals compared to nontolerized animals. Recombinant IL-2 (rIL-2) reverses tolerance by reversing the suppressive effect of intravenous MAA. E: Lewis rats were divided into three groups. The first and second groups were tolerized with intravenous administration of MAA as described in the Materials and Methods. The rats in group 3 received the same volume of PBS intravenously. EAAU was induced by immunization with MAA (day 0). Lewis rats in group 2 were treated with rIL-2 intravenously on days 8, 10, and 12 after immunization. All animals in group 1 were tolerized and did not develop EAAU. All Lewis rats in group 2 that received rIL-2 failed to develop tolerance to intravenous MAA and developed disease. The clinical course and severity of EAAU in these animals was similar to that observed in intravenous PBS rats in group 3. All of the experiments were repeated three times with similar results.