Rapid Publications Distinct Macrophage Subpopulations in Pancreas of Prediabetic BB/E Rats Possible Role for Macrophages in Pathogenesis of IDDM ROBERT WALKER, ADRIAN J. BONE, ANNE COOKE, AND JOYCE D. BAIRD Use of monoclonal antibodies directed against rat macrophages and serial pancreatic biopsy in the prediabetic period have enabled us to document the involvement of macrophages in the pancreatic events leading to onset of diabetes in the spontaneously diabetic BB/E rat. A few weeks before onset of disease, there is marked recruitment and accumulation of ED1 + macrophages at periductal and perivascular locations adjacent to noninfiltrated islets. These recruited cells, distinct from the resident ED2 + tissue macrophages, then infiltrate the islets. Infiltration of the pancreas by ED1 + macrophages is therefore a very early event in the prediabetic period and suggests a possible role for macrophages in the pathogenesis of insulin-dependent diabetes mellitus (IDDM) in this animal model. Diabetes 37:1301-304,1988 T he BB rat develops a diabetic syndrome closely resembling human insulin-dependent diabetes mellitus (IDDM) (1). In a prospective longitudinal cohort study in which batches of prediabetic BB/ Edinburgh (BB/E) rats were killed at intervals in the predi- abetic period, we have previously reported the presence of 0X19 + (T-) and W3/25 + (helper T-) lymphocytes in the early stages of pancreatic infiltration (2). However, because W3/ 25 also recognizes certain macrophage populations (3), it is important to use a different marker to allow discrimination between T- lymphocytes and macrophages. Recently, sev- eral monoclonal antibodies (MoAbs) identifying distinct rat macrophage subpopulations have been described (4). We have used these along with serial pancreatic biopsy in in- From the Metabolic Unit, University Department of Medicine, Western General Hospital, Edinburgh, Scotland; and the Department of Immunology, University College and Middlesex School of Medicine, London, England, United King- dom. Address correspondence and reprint requests to Dr. J. D. Baird, Metabolic Unit, University Department of Medicine, Western General Hospital, Edinburgh EH4 2XU, UK. Received for publication 16 May 1988 and accepted 20 May 1988. dividual animals to investigate a possible role for macro- phages in the development of diabetes in BB/E rats. MATERIALS AND METHODS The animals used in this study were from the Edinburgh colony of BB rats, the nucleus of which was kindly donated in 1982 by P. Thibert (Animal Resources Division of Canada, Ottawa). The BB/E colony consists of two sublines of animals created by selective breeding. The diabetes-prone main line has a 60-70% incidence of diabetes, with the mean age of onset —96 days. The diabetes-resistant subline has an in- cidence of diabetes of <1% at 120 days. Animals (20 male, 20 female) were selected at random from age-matched di- abetes-prone litters. All animals were monitored daily in re- lation to body weight and glycosuria. Animals developing overt diabetes were maintained on a single daily subcuta- neous injection of Ultratard insulin (Novo, Copenhagen). An- imals from the diabetes-resistant subline and normal Wistar rats were used as nondiabetic and non-BB control rats, re- spectively. Protocol. Pancreatic biopsy was performed essentially as described by Logothetopoulos et al.(5). Eight groups (n = 5) of diabetes-prone animals were biopsied twice (with a 10- day interval) between 30 and 110 days of age. Animals de- veloping diabetes received a third biopsy at onset of dis- ease. Biopsies were also performed on diabetes-resistant animals (n = 12) and on normal Wistar rats (n = 6) between 100 and 120 days of age. Biopsies (50-75 mg of tissue) were immediately snap frozen in isopentane at -70°C for subsequent immunohistochemical examination. One hun- dred twenty serial cryostat sections (4 fxm) were cut from each biopsy. Scan slides with 8 sections/slide, each section taken at a different level through 80 fim of tissue, were used as an initial screen to detect areas of pancreatic infiltration with class II MHC-positive cells. Detailed analysis of areas with infiltration via the antisera listed below were performed by referring back to subsequent serial sections coincident with the areas represented by the scan slides. Antisera. The following MoAbs were generously provided as clone supernatants (D. Mason, Sir William Dunn School DIABETES, VOL. 37, SEPTEMBER 1988 1301