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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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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(a) Azathioprine-induced apoptosis is critically dependent on costimulat...
(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-xL protein expression. Cellular proteins were isolated after 3 days of cell culture and assessed for bcl-xL or cellular NF-κB expression by Western blot analysis. (d) FACS analysis for intracellular bcl-xL 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-xL 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.