In pancreatic lesions of non-obese diabetic (NOD) mice the expression of inducible nitric oxide synthase (iNOS) and of the cytokines interferon-gamma and interleukin-4 were studied. Strong iNOS expression as determined at the level of transcription, translation and of enzyme activity was associated with destructive insulitis as seen 8-10 days after cyclophosphamide treatment of 70- to 80-day-old female NOD mice. Immunohistochemistry showed iNOS associated with infiltrating macrophages but not in endocrine cells. The enhancement of iNOS after cyclophosphamide correlated with an increase of T-helper type 1 (Th1) associated interferon-gamma expression while T-helper type 2 (Th2) associated interleukin-4 was the dominant cytokine prior to cyclophosphamide and after diabetes onset. We conclude that insulitis in young NOD mice is carried by Th2 cells while cyclophosphamide enhanced insulitis is determined by Th1 cells. Macrophages show two different functional states in insulitis; strong iNOS expression in macrophages is associated with destructive insulitis.

In isologous islet transplantation in spontaneously diabetic nonobese (NOD) mice, destruction of the islet graft is caused by recurrence of T helper (Th)1-driven insulitis[fnc,1. We established a model of transplantation in which female NOD recipients were rendered diabetic by a single injection of cyclophosphamide (250 mg/kg). Under these conditions, 500 freshly isolated islets from young NOD mice transplanted under the kidney capsule did not lead to normoglycemia within 3 day after transplantation, but underwent immediate impairment of function. This primary nonfunction was seen in > 80% of the recipients. Treatment of the recipients with the Th2-associated cytokine interleukin (IL)-4 alone did not prevent primary nonfunction, whereas treatment of the recipients with a combination of IL-4 and IL-10 restored immediate function of the grafts. Cytokine treatment did not prevent later rejection of grafts. Histological analysis of the grafts revealed less severely infiltrated islets, with well preserved islet architecture, in only normoglycemic animals treated with IL-4 or with IL-4 and IL-10. Staining for lymphocytes, macrophages, and tumor necrosis factor (TNF)-alpha did not show differences between the groups, but IFN-gamma was markedly less expressed in IL-4- and IL-10-treated grafts. Concomitantly, analysis of animals treated for 8 days after injection of cyclophosphamide, with IL-4 and IL-10, revealed a reduction of IL-12 mRNA in the pancreas. We conclude from these data that primary nonfunction of islet grafts is prevented by treatment of the recipients with a combination of IL-4 and IL-10, via downregulation of Th1 cytokines.

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