Immune Cell Infiltration, Cytokine Expression, and β-Cell Apoptosis During the Development of Type 1 Diabetes in the Spontaneously Diabetic LEW.1AR1/Ztm-iddm Rat

Anne Jo rns 1 2 3 Armin Gu nther 1 3 Hans-J urgen Hedrich 0 1 Dirk Wedekind 0 1 Markus Tiedge 1 2 Sigurd Lenzen 1 2 0 Institute of Laboratory Animal Science, Hannover Medical School , Hannover , Germany. of Clinical Biochemistry, Hannover Medical School , D-30623 Hannover, Ger- many 1 RESEARCH DESIGN AND METHODS LEW.1AR1/Ztm-iddm rats were housed in the Institute of Laboratory Animal Science of Hannover Medical School as described previously (4). The animals originated from a breeding colony (4) that was maintained through mating of diabetic female and diabetic male IDDM rats (F7; seventh generation of the IDDM rat; see http://www.mh-hannover.de/institute/clinbiochemistry/iddmrat. htm for details). Homozygous pairing with both parents diabetic resulted in a diabetes incidence of 60%. All rats were maintained according to the FELASA (Federation of European Laboratory Animal Science Associations) guidelines. The animals were investigated at days 45 , 50, 55, 59, 60 , or 65 after birth. On the day the animals were killed, tissue samples of pancreas and spleen were collected for morphologic examination. All analyses were per- formed on the same set of animals. Blood glucose concentrations were determined using capillary blood samples from the tail vein by the glucose oxidase method (Glucometer Elite; Bayer , Leverkusen , Germany). Serum 2 Institute of Clinical Biochemistry, Hannover Medical School , Hannover, Germany; and the 3 Centre of Anatomy, Hannover Medical School , Hannover , Germany; the The IDDM (LEW.1AR1/Ztm-iddm) rat is a type 1 diabetic animal model characterized by a rapid apoptotic pancreatic -cell destruction. Here we have analyzed the time course of islet infiltration, changes in the cytokine expression pattern, and -cell apoptosis in the transition from the pre-diabetic to the diabetic state. Transition from normoglycemia to hyperglycemia occurred when -cell loss exceeded 60 -70%. At the early stages of islet infiltration, macrophages were the predominant immune cell type in the peripherally infiltrated islets. Progression of -cell loss was closely linked to a severe infiltration of the whole islet by CD8 T-cells. With progressive islet infiltration, interleukin-1 (IL-1) and tumor necrosis factor- (TNF-) were expressed in immune cells but not in -cells. This proinflammatory cytokine expression pattern coincided with the expression of inducible nitric oxide synthase (iNOS) and procaspase 3 in -cells and a peak apoptosis rate of 6.7%. Islet infiltration declined after manifestation of clinical diabetes, yielding end-stage islets devoid of -cells and immune cells without any sign of cytokine expression. The observed coincidence of IL-1 and TNF- expression in the immune cells and the induction of iNOS and procaspase 3 mRNA expression in the -cells depicts a sequence of pathological changes leading to apoptotic -cell death in the IDDM rat. This chain of events provides a mechanistic explanation for the development of the diabetic syndrome in this animal model of human type 1 diabetes. Diabetes 54:2041-2052, 2005 - Tto destruction of the pancreatic -cells (13). ype 1 diabetes is an autoimmune disease leading, after a pre-diabetic period, to overt diabetes due Despite many efforts, the mechanisms of interaction between the infiltrating immune cells in the pancreatic islets and pancreatic -cell death, which finally causes insulin-dependent diabetes, are not well understood (13). The pre-diabetic period is of special interest for understanding the mechanisms responsible for pancreatic -cell destruction. Animal models of human type 1 diabetes can provide an important contribution to a better understanding of the etiopathology underlying apoptotic -cell death in type 1 diabetes. The IDDM (LEW.1AR1/Ztm-iddm) rat is a new, particularly attractive autoimmune diabetes animal model (4) for the analysis of the different stages of the immune cell infiltration process because of the short normoglycemic pre-diabetic period with pancreatic islet infiltration of 1 week before ending in a state of overt hyperglycemia due to apoptotic -cell destruction (4,5). In contrast to the bio-breeding (BB) rat and the nonobese diabetic (NOD) mouse as the most studied rodent models, the IDDM rat closely resembles the diabetic syndrome in humans because the animals show a fully developed cellular immune system and no sex bias in diabetes incidence (4,5). In this study, the process of immune cell infiltration in the islets of the pancreas of littermates originating from a homozygous pairing of diabetic parents was investigated. The results allow us to delineate the sequence of pathological changes leading to apoptotic -cell death in this animal model of human type 1 diabetes. insulin concentrations were determined by a rat insulin microplate enzymelinked immunosorbent assay (Mercodia, Uppsala, Sweden). Tissue processing. Tissue specimens of pancreas and spleen were fixed in 4% paraformaldehyde, in 0.15 mol/l PBS, pH 7.3, or rapidly frozen in liquid nitrogen. Fixed tissue was embedded in paraffin, and frozen tissue was stored at 70C for cryostat histology. Additionally, small pancreatic tissue samples were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/l cacodylate buffer, pH 7.3, postfixed in 1% OsO4, and finally embedded in Epon for electron microscopical analysis (5). Immunohistochemistry. Serial paraffin and cryostat sections were stained by the avidin-biotin-complex (ABC) method (6). The slides were incubated overnight with the specific antibodies, followed by a 30-min incubation with biotinylated goat anti-rabbit IgG (1:100) or biotinylated rabbit anti-mouse IgG (1:100) (Santa Cruz Biotechnology, Santa Cruz, CA). The slides were then incubated with a mixture of streptavidin (1:100) and biotin-peroxidase (1: 1,000) (Jackson Immuno Research, West Grove, IL). The peroxidase reaction was visualized using 0.7 mmol/l diaminobenzidine and 0.002% hydrogen peroxidase in PBS, pH 7.3. The following primary antibodies were used: insulin (1:500) (polyclonal A565; DAKO, Hamburg, Germany), infiltrating macrophages (ED1), tissue macrophages (ED2), dendritic cells (Ox 62), B-cells (IgD), panT-cells (R 73), CD4 T-cells, CD8 T-cells (-chain), NK cells (CD 161), major histocompatibility complex class II (Ox 6), interleukin-1 (IL-1), tumor necrosis factor- (TNF-), interferon- (IFN-) (1:500) (all monoclonal; from Serotec, Oxford, U.K.), inducible nitric oxide synthase (iNOS) (1:50) (polyclonal; Santa Cruz Biotechnology), and activated caspase 3 (1:200) (polyclonal; Biosciences Pharmingen, Brussels, Belgium). All antibodies were certified for immunohistochemistry and showed specific immunostaining in tissue sections of the IDDM rat. In situ RT-PCR. Pancreatic sections were fixed on 3-Chamber SuperFrost Plus slides on a heating block at 100C for 2 min. Subsequently, the slides were treated with proteinase K (20 g/ml) for 20 min at 37C. Proteinase K was inactivated thereafter at 95C fo (...truncated)