CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes
Imke Tiede, … , Mohammad Reza Ahmadian, Markus F. Neurath
Imke Tiede, … , Mohammad Reza Ahmadian, Markus F. Neurath
Published April 15, 2003
Citation Information: J Clin Invest. 2003;111(8):1133-1145. https://doi.org/10.1172/JCI16432.
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Article Immunology

CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

Abstract

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are immunosuppressive drugs that are used in organ transplantation and autoimmune and chronic inflammatory diseases such as Crohn disease. However, their molecular mechanism of action is unknown. In the present study, we have identified a unique and unexpected role for azathioprine and its metabolites in the control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation. We found that azathioprine and its metabolites induced apoptosis of T cells from patients with Crohn disease and control patients. Apoptosis induction required costimulation with CD28 and was mediated by specific blockade of Rac1 activation through binding of azathioprine-generated 6-thioguanine triphosphate (6-Thio-GTP) to Rac1 instead of GTP. The activation of Rac1 target genes such as mitogen-activated protein kinase kinase (MEK), NF-κB, and bcl-xL was suppressed by azathioprine, leading to a mitochondrial pathway of apoptosis. Azathioprine thus converts a costimulatory signal into an apoptotic signal by modulating Rac1 activity. These findings explain the immunosuppressive effects of azathioprine and suggest that 6-Thio-GTP derivates may be useful as potent immunosuppressive agents in autoimmune diseases and organ transplantation.

Authors

Imke Tiede, Gerhard Fritz, Susanne Strand, Daniela Poppe, Radovan Dvorsky, Dennis Strand, Hans Anton Lehr, Stefan Wirtz, Christoph Becker, Raja Atreya, Jonas Mudter, Kai Hildner, Brigitte Bartsch, Martin Holtmann, Richard Blumberg, Henning Walczak, Heiko Iven, Peter R. Galle, Mohammad Reza Ahmadian, Markus F. Neurath

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Azathioprine induces a mitochondrial pathway of apoptosis. (a) Activity ...
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 ΔΨm 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 ΔΨm. Both azathioprine and 6-MP as well as FCCP led to a marked reduction of ΔΨm as compared with untreated primary CD4+ T cells. One representative experiment of two is shown. Aza, azathioprine; UT, untreated.