Elsevier

Journal of Autoimmunity

Volume 23, Issue 4, December 2004, Pages 301-309
Journal of Autoimmunity

Cytokines activate caspase-3 in insulinoma cells of diabetes-prone NOD mice directly and via upregulation of Fas

https://doi.org/10.1016/j.jaut.2004.09.006Get rights and content

Abstract

In type 1 diabetes, autoimmune inflammation of pancreatic islets of Langerhans (‘insulitis’) results in destruction of insulin-producing β cells. Cytokines released from islet-infiltrating mononuclear cells are known to be cytotoxic both directly and by upregulating Fas for FasL-induced apoptosis. To investigate the role of caspase-3, a major effector of apoptosis in β-cell death, we asked whether cytokine- and/or FasL-induced apoptosis was associated with increased activity of caspase-3 in NIT-1 insulinoma cells and islets of autoimmune diabetes-prone NOD mice. Measurement of caspase-3 activity using a fluorogenic cleavage assay was validated in NOD mouse thymocytes undergoing dexamethasone (Dex)-induced apoptosis. For cytokine-induced apoptosis, NIT-1 cells or islets were exposed to IL-1β and IFN-γ for 24 h. Caspase-3-like activity was increased 2.1 ± 0.7 and 2.4 ± 0.9-fold in lysates of cytokine-treated NIT-1 cells and NOD mouse islets, respectively. However, NIT-1 cells exhibited 2.1% (4.7 pg active caspase-3/μg protein) and islets 0.8% (1.9 pg active caspase-3/μg protein) of the active caspase-3 content observed in Dex-treated thymocytes (225.1 pg active caspase-3/μg protein). After 24 h cytokine-exposure, the percentage of Fas-positive NIT-1 cells increased from 1.4 ± 1.1 to 29.7 ± 11.6%. Addition of FasL for a further 3 h increased caspase-3-like activity an additional 1.8-fold in cytokine-treated NIT-1 cells. In summary, exposure of NOD mouse insulinoma cells or islets to IL-1β and IFN-γ for 24 h induced caspase-3-like activity that, in the case of insulinoma cells at least, can be further enhanced by interaction of cytokine-induced Fas receptor with FasL. Compared to thymocytes, insulinoma cells and islets from NOD mice were characterised by low basal and cytokine-induced caspase-3 activity.

Introduction

Type 1 diabetes is an autoimmune disease in which mononuclear cell infiltration of the pancreatic islets of Langerhans (‘insulitis’) leads to destruction of the insulin-producing β cells [1]. Chromatin condensation, DNA fragmentation and Fas expression observed in β cells during insulitis implicate β-cell apoptosis as one mode of β-cell death [2], [3], [4]. The insulitis lesion is a rich source of pro-apoptotic cytokines such as IL-1β, IFN-γ and TNF-α, and of the radical NO produced by cytokine-induced iNOS [5]. The combination of IL-1β and IFN-γ provokes β-cell dysfunction, DNA-damage and apoptosis by activation of networks of transcription factors and genes [6].

Cytokine-induced expression of the Fas receptor sensitises β cells to FasL-induced apoptosis [7], [8]. Studies with well-established models of apoptosis demonstrate that triggering of Fas by FasL rapidly induces cell death by recruitment of the adaptor protein Fas-associated death domain (FADD), formation of the death-inducing signalling complex (DISC) and activation of the caspase cascade [9]. Caspases cleave proteins after an aspartate residue, specificity residing in a four-residue sequence upstream of the cleavage site which, for the main effector caspase, caspase-3, is DEVD [10], [11].

To investigate the role of caspase-3 in β-cell death, we asked whether cytokine- and/or FasL-induced apoptosis is associated with increased activity of caspase-3 in NIT-1 insulinoma cells and islets of autoimmune diabetes-prone NOD mice. Moreover, we compared the content of active caspase-3 in cytokine-treated islets and insulinoma cells with that of Dex-treated thymocytes.

Section snippets

Reagents

Super-FasL, general caspase inhibitor (ZVAD), caspase-3 substrate (DEVD-pNA) and caspase-3 inhibitor (DEVD-fmk) used for colorimetric detection of caspase-3-like activity were purchased from Alexis (San Diego, CA). Bioluminescent caspase-3 activity assay (CleavaLite™) was from Chemicon International (Temecula, CA). Active caspase-3 set containing fluorogenic substrate (AC-DEVD-AFC), recombinant caspase-3 and caspase-3 inhibitor (AC-DEVD-CHO) as well as polyclonal rabbit anti-caspase-3 antibody

Comparison of colorimetric, fluorogenic and bioluminescent cleavage assays for detection of caspase-3-like activity

To establish a sensitive method for detecting caspase-3-like activity in insulinoma cells and islets, we compared cleavage assays based on the peptide substrates DEVD-pNA, DEVD-AFC and CleavaLite™, a bioluminescent substrate containing the DEVD motif (Fig. 1). Active recombinant human caspase-3 was applied as source of enzyme activity and signals monitored at concentrations ranging in log2-steps from 200 to 0.78 ng/ml. The resulting curve was analysed to obtain assay parameters. All assays

Discussion

In autoimmune diabetes a variety of mechanisms contribute to β-cell death [1], [6]. Prominent are pro-inflammatory cytokines, that may be directly cytotoxic to β cells or may act indirectly to upregulate Fas receptor and sensitise for FasL–Fas-induced apoptosis [13]. To investigate the activation of caspase-3 by the cytokine combination IL-1β and IFN-γ alone or in presence of FasL, we first compared assays to detect caspase-3-like activity in NIT-1 insulinoma cells and NOD mouse islets. The

Acknowledgements

The advice of Dr. Birgit Finke for the fluorometic detection method is gratefully acknowledged. We thank Mrs. Christel Salzsieder for excellent technical assistance.

This work was supported by grants from the Deutsche Forschungsgemeinschaft AU 151/1-1, 1-2 and from the Ministerium für Bildung, Wissenschaft und Kultur Mecklenburg-Vorpommern IDK 97 007 80/SOM and IDK 97 007 80/HSP III.

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