CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4⁺ T lymphocytes
J. Clin. Invest. Imke Tiede, et al. 111:1133 doi:10.1172/JCI16432 [Go to this article.]

Figure 5
(a) Azathioprine-induced apoptosis is critically dependent on costimulation with CD28. CD4⁺ T lymphocytes were stimulated in the presence or absence of azathioprine and 6-MP, as indicated. T cell apoptosis was assessed by FACS analysis using annexin V/propidium iodide staining at day 5 of cell culture. (b) Azathioprine-induced apoptosis is independent of the CD95/CD95L system. Primary CD4⁺ T lymphocytes were stimulated as above in the presence or absence of azathioprine and a neutralizing CD95L antibody. T cell apoptosis was assessed by FACS analysis at day 5 of cell culture. (c) The left panel shows a gene array for apoptosis-related genes in T lymphocytes. CD4⁺ T lymphocytes were stimulated as above in the presence or absence of azathioprine. The right panel shows that 6-MP suppresses bcl-x_L protein expression. Cellular proteins were isolated after 3 days of cell culture and assessed for bcl-x_L or cellular NF-κB expression by Western blot analysis. (d) FACS analysis for intracellular bcl-x_L expression in permeabilized lymphocytes upon 6-MP treatment. Purified CD4⁺ T lymphocytes were stimulated in the presence or absence of 6-MP. FACS analysis for bcl-x_L in permeabilized cells was performed after 5 days of cell culture. (e) 6-MP suppresses nuclear NF-κB activation. CD4⁺ T lymphocytes were stimulated in the presence or absence of 6-MP, as indicated. Nuclear proteins were isolated after 3 days and analyzed for NF-κB (upper panel) or SP-1 (middle panel) activity by gel retardation assays (EMSAs). Nuclear extracts from PMA-stimulated Jurkat T cells served as positive controls. The lower panel represents a supershift analysis of the upper complex using extracts from anti-CD3– plus anti-CD28–stimulated primary T cells. The addition of antibodies to p50 or p65 to the EMSA reaction is indicated. 6-MP treatment led to downregulation of the NF-κB p50/p65 complex.