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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Figure 2

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(a) Peripheral blood CD4+ T cells from healthy volunteers were stimulate...
(a) 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 6-MP and 6-TG for 4–5 days. T cell apoptosis was assessed by FACS analysis (upper panels). The average level of 6-MP– and 6-TG–specific apoptosis (induction of annexin-positive, propidium iodide–negative T cells compared with untreated cells) ± SEM from four independent experiments is shown in the lower panel. (b) Kinetics of azathioprine-induced apoptosis in primary CD4+ T lymphocytes. CD4+ T cells were cultured in the presence or absence of azathioprine for 2–5 days as indicated. T cell apoptosis was assessed by FACS analysis at the indicated time points. (c) 6-MP suppresses clonal expansion of activated primary CD4+ T lymphocytes in cell culture. CD4+ T cells were cultured in the presence (+) or absence (–) of 6-MP for 3–5 days. The clonal expansion of T cells during cell culture was calculated as specified in Methods. (d) CD4+ T cells were cultured in the absence of azathioprine or 6-MP for 5 days, followed by addition of azathioprine or 6-MP to the cell culture for an additional 5 days. The percentage of annexin-positive, propidium iodide–negative cells was then determined at day 10 by FACS analysis. The average percentage of azathioprine- and 6-MP–specific apoptosis (induction of annexin-positive, propidium iodide–negative cells compared with untreated cells indicated by black sections of bars, induction of annexin-positive, propidium iodide–positive cells compared with untreated cells indicated by white sections) ± SEM is shown in the lower panel.