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 1

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Azathioprine and its metabolites induce T cell apoptosis. CD45RA (a) and...
Azathioprine and its metabolites induce T cell apoptosis. CD45RA (a) and CD45RO (b) T cell subsets were isolated from peripheral blood of healthy volunteers and stimulated with antibodies to CD3 and CD28 and recombinant IL-2 in the presence or absence of indicated concentrations of azathioprine and 6-MP. T cell apoptosis was assessed by FACS analysis after 5 days of cell culture. Azathioprine and 6-MP led to an induction of annexin-positive, propidium iodide–negative T cells, suggesting that they induced T cell apoptosis. The FACS data is representative of 6–10 independent experiments per group. The average induction of specific apoptosis from 6–10 patients per group (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) by azathioprine and 6-MP ± SEM is shown in the lower panels. Statistically significant changes are indicated (*P < 0.01). (c) Determination of apoptosis and cell cycle distribution by the Nicoletti technique. Peripheral blood CD4+ T cells from healthy volunteers were stimulated as above for 5 days, followed by analysis of DNA content by the Nicoletti technique. The percentage of apoptotic cells in the subdiploid peak is indicated by M1. (d) Azathioprine and 6-MP induce morphologic changes of CD45RA CD4+ T cells (upper panels) and CD45RO CD4+ T cells (lower panels) indicative of apoptosis, as assessed by transmission electron microscopy. Peripheral blood CD4+ T cells from healthy volunteers were stimulated as above for 5 days. Upon 6-MP treatment, T cells exhibited typical signs of apoptosis (arrowheads), with dense nuclear condensation and degeneration of organelles. Magnification, ×7200.