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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Figure 3

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Azathioprine treatment induces T cell apoptosis in IBD. (a) LPMCs were i...
Azathioprine treatment induces T cell apoptosis in IBD. (a) LPMCs were isolated and stimulated with anti-CD2/CD28 plus IL-2 in the presence or absence of 6-MP for 5 days followed by FACS analysis. (b) Peripheral blood CD4+ T cells were isolated from patients with IBD receiving azathioprine/6-MP and patients with IBD before therapy and stimulated with anti-CD3/CD28 plus IL-2 in the presence or absence of 6-MP for 5 days. The number of apoptotic cells (annexin-positive, propidium iodide–negative cells) was determined by FACS analysis after 5 days and stratified according to clinical data on azathioprine therapy. Data represent mean values ± SEM from eight patients with IBD per group. (c–h) TUNEL assays for the detection of apoptotic lamina propria cells in patients with IBD under azathioprine therapy. Successful azathioprine treatment associated with low-level gut inflammation was characterized by a high number of apoptotic cells (shown at low [d] and high [g] magnification) as compared with control patients with low-level gut inflammation not receiving azathioprine (c and f). In contrast, unsuccessful azathioprine treatment associated with high-level gut inflammation was characterized by a very low percentage of apoptotic cells (e and h). Data are representative of 5–13 patients per group. Magnification, ×20 (bar = 100 μm) (c–e) and ×40 (bar = 50 μm) (f–h). (i) Quantification of the percentage of apoptotic, TUNEL-positive LPMCs in patients with IBD receiving 6-MP as compared with untreated control patients. Clinical 6-MP responsiveness was associated with a marked, significant (P < 0.01) increase in the percentage of apoptotic LPMCs as compared with control patients with IBD not receiving 6-MP. Data are mean values ± SEM of 5–13 patients per group.