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Temporal Relationship Between Immune Cell Influx and the Expression of Inducible Nitric Oxide Synthase, Interleukin-4 and Interferon-γ in Pancreatic Islets of NOD Mice Following Adoptive Transfer of Diabetic Spleen Cells

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Abstract

Beta cell destruction in NOD mice can be accelerated by adoptive transfer of diabetic spleen cells into irradiated adult NOD mice. Here mice receiving diabetic spleen cells were examined at days 0, 7, 14, 21 and at onset of diabetes for the resulting insulitis and the number of intra-islet CD4 and CD8 cells and macrophages. The progression of insulitis and the number of intra-islet CD4 and CD8 cells and macrophages were correlated with the expression and co-localization of inducible nitric oxide synthase, interferon-γ and interleukin-4 by dual-label light and confocal immunofluorescence microscopy. Diabetes developed in 7/8 mice by 27 days following cell transfer. The insulitis score increased slightly by day 7 but rose sharply at day 14 (p=0.001) and was maintained until diabetes. The mean number of intra-islet CD4 and CD8 cells and macrophages showed a similar trend to the insulitis scores and were present in almost equal numbers within the islets. Immunolabelling for inducible nitric oxide synthase was observed at day 7 in only some cells of a few islets but increased sharply from day 14. It was restricted to islets with insulitis and was co-localized in selective macrophages. Weak intra-islet interleukin-4 labelling was observed at days 7 and 14 but became more pronounced at day 21 and at onset of diabetes, being present in selective CD4 cells. Intra-islet labelling for interferon-γ was first observed at day 21, but became more intense at onset of diabetes and was co-localized in a proportion of macrophages. Both cytokines were expressed in islets with advanced insulitis. Interferon-γ staining was also observed within endothelial cells located in the exocrine pancreas. We conclude that transfer of diabetic spleen cells results in a rapid influx of CD4 and CD8 cells and macrophages within the pancreas of recipient mice. During the period of heightened insulitis, selective immune cells begin to express inducible nitric oxide synthase and the opposing cytokines, interferon-γ and interleukin-4. Expression of these molecules becomes more pronounced immediately prior to and during the onset of diabetes.

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References cited

  • Bach JF (1994) Insulin-dependent diabetes mellitus as an autoimmune disease. Endocr Rev 15: 516-542.

    Google Scholar 

  • Bendelac A, Carnaud C, Boitard C, Bach JF (1987) Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates: requirement for both L3T4+ and Lyt-2+ cells. J Exp Med 166: 823-832.

    Google Scholar 

  • Cetkovic-Cvrlje M, Eizirik DL (1994) TNF-α and IFN-γ potentiate the deleterious effects of IL-1β on mouse pancreatic islets mainly via generation of nitric oxide. Cytokine 6: 399-406.

    Google Scholar 

  • Charlton B, Bacelj A, Mandel TE (1988) Administration of silica particles or anti-Lyt 2 antibody prevents β-cell destruction in NOD mice given cyclophosphamide. Diabetes 37: 930-935.

    Google Scholar 

  • Corbett JA, Mikhael A, Shimizu J, Frederick K, Misko TP, McDaniel ML, Kanagawa O, Unanue ER (1993) Nitric oxide production in islets from nonobese diabetic mice: aminoguanidine-sensitive and-resistant stages in the immunological diabetic process. Proc Natl Acad Sci USA 90: 8992-8995.

    Google Scholar 

  • Eisenbarth GS, Connelly J, Soeldner JS (1987) The ‘natural’ history of type 1 diabetes. Diabetes Metab Rev 3: 873-891.

    Google Scholar 

  • Faulkner-Jones BE, Dempsey-Collier M, Mandel TE, Harrison LC (1996) Both TH1 and TH2 cytokinem RNAs are expressed in the NOD mouse pancreas in vivo. Autoimmunity 23: 99-110.

    Google Scholar 

  • Foulis AK, Farquharson MA, Meager A (1987) Immunoreactive α-interferon in insulin-secreting β cells in type 1 diabetes mellitus. Lancet 2: 1423-1427.

    Google Scholar 

  • Foulis AK, Mcgill M, Farquharson MA (1991) Insulitis in type 1 (insulin-dependent) diabetes mellitus in man-macrophages, lymphocytes, and interferon-γ containing cells. J Histopathol 165: 97-103.

    Google Scholar 

  • Fultz MJ, Barber SA, Dieffenbach CW, Vogel SN (1993) Induction of IFN-γ in macrophages by lipopolysaccharide. Int Immunol 5: 1383-1392.

    Google Scholar 

  • Griffiths GM, Mueller C (1991) Expression of perforin and granzymes in vivo: Potential diagnostic markers for activated cytotoxic cells. Immunology Today 12: 415-419.

    Google Scholar 

  • Harada M, Makino S (1984) Promotion of spontaneous diabetes in non-obese diabetic-prone mice by cyclophosphamide. Diabetolgia 27: 604-606.

    Google Scholar 

  • Harrison LC, Dempsey-Collier M, Kramer DR, Takahashi K (1996) Aerosol insulin induces regulatory CD8 γδ T cells that prevent murine insulin-dependent diabetes. J Exp Med 184: 2167-2174.

    Google Scholar 

  • Held W, MacDonald HR, Weissman IL, Hess MW, Mueller C (1990) Genes encoding tumour necrosis factor alpha and granzyme A are expressed during development of autoimmune diabetes. Proc Natl Acad Sci USA 87: 2239-2243.

    Google Scholar 

  • Huang X, Hultgren B, Dybdal N, Stewart TA (1994) Islet expression of interferon-α precedes diabetes in both the BB rat and streptozotocin-treated mice. Immunity 1: 469-478.

    Google Scholar 

  • Huang X, Yuan J, Goddard A, Foulis A, James RFL, Lernmark A, Pujol-Borrell R, Rabinovitch A, Somoza N, Stewart TA (1995) Interferon expression in the pancreases of patients with type 1 diabetes. Diabetes 44: 658-664.

    Google Scholar 

  • Hultgren B, Huang X, Dybdal N, Stewart TA (1996) Genetic absence of γ-inteferon delays but does not prevent diabetes in NOD mice. Diabetes 45: 812-817.

    Google Scholar 

  • Hutchings P, Rosen H, O'Reilly L, Simpson E, Gordon S, Cooke A (1990) Transfer of diabetes in mice prevented by blockade of adhesion-promoting receptor on macrophages. Nature 348: 639-642.

    Google Scholar 

  • Jansen A, Homo-Delarche F, Hooijkaas H, Leenen PJ, Dardenne M, Drexhage HA (1994) Immunohistochemical characterization of monocytes-macrophages and dendritic cells involved in the initiation of the insulitis and β-cell destruction in NOD mice. Diabetes 43: 667-675.

    Google Scholar 

  • Kay TWH, Campbell IL, Harrison LC (1991) Characterization of pancreatic T lymphocytes associated with beta cell destruction in the non-obese diabetic (NOD) mouse. J Autoimmun 4: 263-276.

    Google Scholar 

  • Kikutani H, Makino S (1992) The murine autoimmune diabetes model: NOD and related strains. Adv Immunol 51: 285-322.

    Google Scholar 

  • Kilgore KS, Schmid E, Shanley TP, Flory CM, Maheshwari V, Tramontini NL, Cohen H, Ward PA, Friedl HP, Warren JS (1997) Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nuclear factor-κB activation. Am J Pathol 150: 2019-2031.

    Google Scholar 

  • Kleemann R, Rothe H, Kolb-Bachofen V, Xie Q-W, Nathan C, Martin S, Kolb H (1993) Transcription and translation of inducible nitric oxide synthase in the pancreas of prediabetic BB rats. FEBS Lett 328: 9-12.

    Google Scholar 

  • Kolb-Bachofen V, Epstein S, Kiesel U, Kolb H (1988) Low dose streptozotocin-induced diabetes in mice. Electron microscopy reveals single-cell insulitis before diabetes onset. Diabetes 37: 21-27.

    Google Scholar 

  • Lindsten T, June CH, Ledbetter JA, Stella G, Thompson CB (1989) Regulation of lymphokine mRNA stability by a surface mediated T cell activation pathway. Science 244: 339-343.

    Google Scholar 

  • Mandrup-Poulsen T, Helqvist S, Molvig J, Wogensen LD, Nerup J (1989) Cytokines as immune effector molecules in autoimmune endocrine diseases with special reference to insulin-dependent diabetes mellitus. Autoimmunity 4: 191-218.

    Google Scholar 

  • Mathieu C, Waer M, Casteels K, Laureys J, Bouillon R (1995) Prevention of type 1 diabetes in NOD mice by nonhypercalcemic doses of a new structural analog of 1,25-dihydroxyvitamin D3, KH1060. Endocrinology 136: 866-872.

    Google Scholar 

  • Mossmann TR, Coffman RL (1989) TH1 and TH2 cells: Different patterns of lymphokine secretion lead to different functional properties. Ann Rev Immunol 7: 145-173.

    Google Scholar 

  • Mueller C, Held W, Imboden MA, Carnaud C (1995) Accelerated β-cell destruction in adoptively transferred autoimmune diabetes correlates with an increased expression of the genes coding for TNF-α and granzyme A in the intra-islet infiltrates. Diabetes 44: 112-117.

    Google Scholar 

  • Munder M, Mallo M, Eichmann K, Modolell M (1998) Murine macrophages secrete interferon γ upon combined stimulation with interleukin (IL)-12 and IL-18: A novel pathway of autocrine macrophage activation. J Exp Med 187: 2103-2108.

    Google Scholar 

  • Nicoletti F, Zaccone P, Di Marco R, Di Mauro M, Magro G, Grasso S, Mughini L, Meroni P, Garotta G (1996) The effects of a nonimmunogenic form of murine soluble interferon-γ receptor on the development of autoimmune diabetes in the NOD mouse. Endocrinology 137: 5567-5575.

    Google Scholar 

  • O'Reilly LA, Hutchings PR, Crocker PR, Simpson E, Lund T, Kioussis D, Takei F, Baird J, Cooke A (1991) Characterization of pancreatic islet cell infiltrates in NOD mice: Effect of cell transfer and transgene expression. Eur J Immunol 21: 1171-1180.

    Google Scholar 

  • Pilstrom B, Bjork L, Bohme J (1997) Monokine-producing cells predominate in the recruitment phase of NOD insulitis while cells producing Th1-type cytokines characterize the effector phase. J Autoimmun 10: 147-155.

    Google Scholar 

  • Rabinovitch A (1993) Roles of cytokines in IDDM pathogenesis and islet β-cell destruction. Diabetes Rev 1: 215-240.

    Google Scholar 

  • Rabinovitch A (1998) An update on cytokines in the pathogenesis of insulin-dependent diabetes mellitus. Diabetes/Metab Rev 14: 129-151.

    Google Scholar 

  • Rabinovitch A, Suarez-Pinson WL, Sorensen O, Bleackley RC, Power RF (1995) IFN-γ gene expression in pancreatic islet-infiltrating mononuclear cells correlates with autoimmune diabetes in non-obese diabetic mice. J Immunol 154: 4874-4882.

    Google Scholar 

  • Rabinovitch A, Suarez-Pinson WL, Sorensen O (1996) Interleukin 12 mRNA expression in islets correlates with β-cell destruction in NOD mice. J Autoimmun 9: 645-651.

    Google Scholar 

  • Reddy S, Bibby NJ, Elliott RB (1988a) Ontogeny of islet cell antibodies, insulin autoantibodies and insulitis in the non-obese diabetic mouse. Diabetologia 31: 322-328.

    Google Scholar 

  • Reddy S, Bibby NJ, Elliott RB (1988b) An immunofluorescent study of insulin-, glucagon-, pancreatic polypeptide-and somatostatin-containing cells in the early ovine fetal pancreas. Quart J Exp Physiol 73: 225-232.

    Google Scholar 

  • Reddy S, Bibby NJ, Elliott RB (1991) Time course of immune markers in the NOD mouse: Comparison between sexes and animals on an anti-diabetogenic diet. Diabetes Nutr & Metab Clin and Exp 4: 259-265.

    Google Scholar 

  • Reddy S, Wu D, Swinney C, Elliott RB (1995) Immunohistochemical analyses of pancreatic macrophages and CD4 and CD8 T cell subsets prior to and following diabetes in the NOD mouse. Pancreas 11: 16-25.

    Google Scholar 

  • Reddy S, Kaill S, Poole CA, Ross J (1997) Inducible nitric oxide synthase in pancreatic islets of the non-obese diabetic mouse: A light and confocal microscopical study of its ontogeny, co-localization and up-regulation following cytokine administration. Histochemical J 29: 53-64.

    Google Scholar 

  • Reddy S, Yip S, Karanam M, Poole CA, Ross JM (1999) An immunohistochemical study of macrophage influx and the co-localization of inducible nitric oxide synthase in the pancreas of non-obese diabetic (NOD) mice during disease acceleration with cyclophosphamide. Histochemical J 31: 303-314.

    Google Scholar 

  • Rothe H, Faust A, Schade, U, Kleemann R, Bosse G, Hibino T, Martin S, Kolb H (1994) Cyclophosphamide treatment of female non-obese diabetic mice causes enhanced expression of inducible nitric oxide synthase and interferon-gamma, but not of interleukin-4. Diabetologia 37: 1154-1158.

    Google Scholar 

  • Shimada, A, Charlton B, Taylor-Edwards C, Fathman CG (1996) β-cell destruction may be a late consequence of the autoimmune process in non-obese diabetic mice. Diabetes 45: 1063-1067.

    Google Scholar 

  • Signore A, Pozzilli P, Gale EAM, Andreani D, Beverley PCL (1989) The natural history of lymphocyte subsets infiltrating the pancreas of NOD mice. Diabetologia 32: 282-289.

    Google Scholar 

  • Sobel DO, Ahvazi B (1998) α-Interferon inhibits the development of diabetes in NOD mice. Diabetes 47: 1867-1872.

    Google Scholar 

  • Suarez-Pinson W, Rajotte RV, Mosmann TR, Rabinovitch A (1996) Both CD4+ and CD8+T-cells in syngeneic islet grafts in NOD mice produce interferon-γ during β-cell destruction. Diabetes 45: 1350-1357.

    Google Scholar 

  • Tisch R, Yang XD, Liblau RS, McDevitt HO (1994) Administering glutamic acid decarboxylase to NOD mice prevents diabetes. J Autoimmun 7: 845-850.

    Google Scholar 

  • Vecchi A, Garlanda C, Lampugnani MG, Resnati M, Matteuccci C, Stoppacciaro A, Schnurch H, Risau W, Ruco L, Mantovani A, Dejana E (1994) Monoclonal antibodies specific for endothelial cells of mouse blood vessels. Their application in the identification of adult and embryonic epithelium. Eur J Cell Biol 63: 247-254.

    Google Scholar 

  • Wang J, Wakeham J, Harkness R, Xing Z (1999) Macrophages are a significant source of type 1 cytokines during mycobacterial infection. J Clin Invest 103: 1023-1029.

    Google Scholar 

  • Welsh M, Welsh N, Bendtzen K, Mares J, Strandell E, Oberg C, Sandler S (1995) Comparison of mRNA contents of interleukin-1β and nitric oxide synthase in pancreatic islets isolated from female and male nonobese diabetic mice. Diabetologia 38: 153-160.

    Google Scholar 

  • Wicker LS, Miller BJ, Mullen Y (1986) Transfer of autoimmune diabetes mellitus with splenocytes from nonobese diabetic (NOD) mice. Diabetes 35: 855-860.

    Google Scholar 

  • Young LHY, Peterson LB, Wicker LS, Persechini PM, Young JD (1989) In vivo expression of perforin by CD8 lymphocytes in autoimmune disease: Studies on spontaneous and adoptively transferred diabetes in nonobese diabetic mice. J Immunol 143: 3994-3999.

    Google Scholar 

  • Xie Q-W, Cho HJ, Calacay J, Mumford RA, Swiderek KM, Lee TD, Ding A, Troso T, Nathan C (1992) Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 256: 225-228.

    Google Scholar 

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Authors and Affiliations

  1. Department of Paediatrics, University of Auckland School of Medicine, Private Bag 92019, Auckland, New Zealand

    S. Reddy, M. Karanam, K. Nitschke & J. Neve

  2. Department of Molecular Medicine, University of Auckland School of Medicine, Private Bag 92019, Auckland, New Zealand

    G. Krissansen

  3. Department of Anatomy, University of Auckland School of Medicine, Private Bag 92019, Auckland, New Zealand

    C.A. Poole & J.M. Ross

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  1. S. Reddy
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Reddy, S., Karanam, M., Krissansen, G. et al. Temporal Relationship Between Immune Cell Influx and the Expression of Inducible Nitric Oxide Synthase, Interleukin-4 and Interferon-γ in Pancreatic Islets of NOD Mice Following Adoptive Transfer of Diabetic Spleen Cells. Histochem J 32, 195–206 (2000). https://doi.org/10.1023/A:1004084232446

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