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 6

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Azathioprine blocks the Rac1/MEK kinase pathway. (a) Analysis of phospho...
Azathioprine blocks the Rac1/MEK kinase pathway. (a) Analysis of phosphorylation of MEK, a MAP kinase kinase that can be activated by MEKK (see Figure 8). Purified CD4+ T lymphocytes were stimulated in the presence or absence of 6-TG. Intracellular staining for phospho-MEK by FACS analysis was made after 3 days. (b) 6-MP suppresses CD28-induced MEK and IκB phosphorylation. Purified CD4+ T lymphocytes were stimulated in the presence or absence of 6-MP. Cellular proteins were isolated after 3 days and analyzed by Western blotting. The upper left panels show phospho-IκB (p-IκB) or phospho-MEK (p-MEK) activity upon 6-MP treatment. Band intensity was quantified by densitometry and normalized to actin levels. The lower left panels show IκB or MEK protein expression upon azathioprine and 6-MP treatment. Band intensity was normalized to ERK2 levels. The right panels show Rac1 and vav protein expression upon azathioprine and 6-MP treatment. Azathioprine and 6-MP treatment had little effect on Rac1 protein levels, whereas vav levels were increased in cellular extracts. Band intensity was quantified by densitometry and normalized to ERK2 levels. Den, densitometry. (c) 6-MP induces vav accumulation. CD4+ T lymphocytes were stimulated in the presence or absence of azathioprine, 6-MP, or 6-TG for 5 days. Cells were immunostained with Rac1-specific antibodies and vav-specific antibodies and Cy3-labeled secondary antibodies (red). Nuclei were counterstained with Hoechst blue. Confocal microscopy showed that the expression of the Rac1-associated guanosine exchange factor vav was increased upon treatment with azathioprine and its metabolites, whereas Rac1 levels were nearly unchanged.