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 4
Azathioprine induces a mitochondrial pathway of apoptosis. (a) Activity of caspase-3, -8, and -9 upon treatment of T cells with 6-MP. CD45RA and CD45RO T cell subsets were stimulated with antibodies to CD3 and CD28 and recombinant IL-2 for 5 days in the presence or absence of 6-MP, as indicated. There was a marked induction of caspase-9 activity upon azathioprine treatment. A second independent experiment showed similar results (data not shown). Data on caspase-9 activity from three independent healthy blood donors are shown in the right lower panel. (b) Specific blockade of caspase-9 by acetyl-LEHD-CHO (Ac-LEHD-CHO) suppresses 6-MP–induced apoptosis. CD4⁺ T lymphocytes from the peripheral blood of healthy volunteers were stimulated with antibodies to CD3 and CD28 in the presence or absence of 6-MP, 10 μM acetyl-LEHD-CHO, and 10 μm acetyl-IETD-CHO. Although acetyl-IETD-CHO had little effect, 6-MP–induced T cell apoptosis could be suppressed by acetyl-LEHD-CHO. (c) Measurement of ΔΨ_m in primary CD4⁺ T lymphocytes upon treatment with azathioprine, 6-MP, and FCCP (positive control). Peripheral blood CD4⁺ T cells from healthy volunteers were stimulated with antibodies to CD3 and CD28 and recombinant IL-2 and cultured in the presence or absence of azathioprine or 6-MP for 5 days as indicated. Cells were then loaded with JC-1 for 20 minutes followed by FACS analysis to determine ΔΨ_m. Both azathioprine and 6-MP as well as FCCP led to a marked reduction of ΔΨ_m as compared with untreated primary CD4⁺ T cells. One representative experiment of two is shown. Aza, azathioprine; UT, untreated.