Cytokines activate caspase-3 in insulinoma cells of diabetes-prone NOD mice directly and via upregulation of Fas.
In type 1 diabetes, autoimmune inflammation of pancreatic islets of Langerhans ('insulitis') results in destruction of insulin-producing beta 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 beta-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-1 beta and IFN-gamma 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/microg protein) and islets 0.8% (1.9 pg active caspase-3/microg protein) of the active caspase-3 content observed in Dex-treated thymocytes (225.1 pg active caspase-3/microg 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-1 beta and IFN-gamma 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.
Augstein
Petra
P
Institute of Diabetes Gerhardt Katsch Karlsburg e.V., Greifswalder Strasse 11e, 17495 Karlsburg, Germany. augstein@uni-greifswald.de
Bahr
Jeanette
J
Wachlin
Gerhild
G
Heinke
Peter
P
Berg
Sabine
S
Salzsieder
Eckhard
E
Harrison
Leonard C
LC
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001681:Biological Assay; D053148:Caspase 3; D020169:Caspases / Q000032:analysis / Q000378:metabolism*; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology / Q000502:physiology*; D003907:Dexamethasone / Q000633:toxicity; D003922:Diabetes Mellitus, Type 1 / Q000201:enzymology* / Q000276:immunology; D053222:Fas Ligand Protein; D007340:Insulinoma; D007371:Interferon-gamma / Q000494:pharmacology / Q000502:physiology; D007375:Interleukin-1 / Q000494:pharmacology / Q000502:physiology; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D008562:Membrane Glycoproteins / Q000032:analysis / Q000502:physiology*; D051379:Mice; D016688:Mice, Inbred NOD; D010190:Pancreatic Neoplasms; D013950:Thymus Gland / Q000166:cytology / Q000187:drug effects; D015854:Up-Regulation
D016207:Cytokines; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D007375:Interleukin-1; D008562:Membrane Glycoproteins; D003907:Dexamethasone; D007371:Interferon-gamma; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases