title: Immunohistochemical study of caspase-3-expressing cells within the pancreas of non-obese diabetic mice during cyclophosphamide-accelerated diabetes.
abstract: During insulin-dependent diabetes mellitus, immune cells infiltrate pancreatic islets progressively and mediate beta cell destruction over a prolonged asymptomatic prediabetic period. Apoptosis may be a major mechanism of beta cell loss during the disease. This process involves a proteolytic cascade in which upstream procaspases are activated which themselves activate downstream caspases, including caspase-3, a key enzyme involved in the terminal apoptotic cascade. Here dual-label immunohistochemistry was employed to examine the intra-islet expression, distribution and cellular sources of active caspase-3 in the non-obese diabetic (NOD) mouse given cyclophosphamide to accelerate diabetes. NOD mice were treated at day 95 and caspase-3 expression was studied at days 0, 4, 7, 11 and 14. Its expression was also correlated with advancing disease and compared with age-matched NOD mice treated with diluent alone. At day 0 (=day 95), caspase-3 immunolabelling was observed in several peri-islet and intra-islet macrophages, but not in CD4 and CD8 cells and only extremely rarely in beta cells. At day 4, only a few beta cells weakly expressed the enzyme, in the absence of significant insulitis. At day 7, caspase-3 expression was observed in a small proportion of intra-islet macrophages. At day 11, there was a marked increase in the number of intra-islet macrophages positive for caspase-3 while only a few CD4 cells expressed the enzyme. At day 14, caspase-3 labelling became prominent in a significant proportion of macrophages. Only a few CD4 and CD8 cells expressed the enzyme. Capase-3 labelling was also present in a proportion of macrophages in perivascular and exocrine regions. Surprisingly, beta cell labelling of caspase-3 at days 11 and 14 was rare. At this stage of heightened beta cell loss, a proportion of intra-islet interleukin-1beta-positive cells coexpressed the enzyme. Caspase-3 was also observed in numerous Fas-positive cells in heavily infiltrated islets. During this late stage, only a proportion of caspase-3-positive cells contained apoptotic nuclei, as judged by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). We conclude that during cyclophosphamide-accelerated diabetes in the NOD mouse, the predominant immunolabelling of caspase-3 in intra-islet macrophages suggests that apoptosis of macrophages may be an important mechanism for its elimination. The virtual absence of caspase-3 immunolabelling in most beta cells even during heightened beta cell loss supports their rapid clearance following their death during insulin-dependent diabetes mellitus.
authors.affiliation: School of Biological Sciences and the Department of Paediatrics, University of Auckland, Private Bag 92019, Auckland, New Zealand. s.reddy@auckland.ac.nz
authors.forename: Shiva
authors.initials: S
authors.lastname: Reddy
authors.forename: Joshua
authors.initials: J
authors.lastname: Bradley
authors.forename: Suyin
authors.initials: S
authors.lastname: Ginn
authors.forename: Praneeti
authors.initials: P
authors.lastname: Pathipati
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Ross
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D003520:Cyclophosphamide / Q000633:toxicity; D003921:Diabetes Mellitus, Experimental / Q000139:chemically induced / Q000201:enzymology* / Q000473:pathology; D003922:Diabetes Mellitus, Type 1 / Q000139:chemically induced / Q000201:enzymology* / Q000473:pathology; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D020287:In Situ Nick-End Labeling; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D051379:Mice; D016688:Mice, Inbred NOD; D018613:Microscopy, Confocal
chemical_list: D003520:Cyclophosphamide; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases