Prevention of autoimmune diabetes by FTY720 in nonobese... : Transplantation (original) (raw)
Oral administration of a novel immunomodulatory agent FTY720 prolongs allograft survival in experimental transplantation with remarkable potency without producing any noticeable side effects (1). FTY720 is also effective in preventing experimental autoimmune diseases such as adjuvant-induced or collagen-induced arthritis (2), myocarditis (3), uveoretinitis (4), and thyroiditis (5). FTY720 mediates immunomodulation by decreasing circulating blood lymphocytes because of their chemokine-dependent homing to secondary lymphoid tissues (lymph nodes and Peyer’s patches) (6) and by reducing the recruitment of effector lymphocytes into grafted organs and peripheral lesions (7). The decrease in blood lymphocytes caused by oral administration of FTY720 is also a result of apoptosis (8).
Nonobese diabetic (NOD) mice spontaneously develop diabetes that has features similar to those of human type 1 diabetes mellitus (9). The onset of diabetes is preceded by a long period of insulitis, which progresses from lymphoid cell infiltration in the periphery of the islets (peri-insulitis) to cellular infiltration invading the islets (insulitis) (10). The result of autoimmune insult is the destruction of beta cells followed by loss of intrinsic insulin secretion, hyperglycemia, polydipsia, polyuria, loss of body weight, and ketosis, as seen in human type 1 diabetes mellitus. Because FTY720 accelerates lymphocyte homing to secondary lymphoid tissues and reduces the recruitment of effector lymphocytes into the peripheral lesions, we hypothesized that treatment of NOD mice with FTY720 starting at an early age would prevent infiltration of effector lymphocytes into islets and prevent the onset of overt diabetes.
Young (3 weeks of age) female NOD mice were purchased from Taconic (Germantown, NY). Beginning from 4 weeks of age, a group of NOD mice (n=16) was given 0.5 mg/kg FTY720 (a generous gift from Novartis Pharmaceuticals Corporation, East Hanover, NJ) by gavage needle once per day, five times per week (Monday through Friday). An untreated group (n=33) of age-matched NOD mice served as controls. As shown in Figure 1, 15 of 16 mice treated with FTY720 remained normoglycemic until 35 weeks of age, when treatment was stopped. The treated mice showed no side effect such as infection, diarrhea, or weight loss. In contrast, 70% of untreated NOD mice became diabetic by 35 weeks of age. Withdrawal of FTY720 was associated with development of diabetes within 2 weeks in five additional mice. Nine of 16 treated mice (one normoglycemic mouse was killed at 37 weeks of age for histologic examination) remained free of diabetes at 44 weeks of age despite withdrawal of treatment. The difference in diabetes incidence between the two groups is statistically significant (P =0.005 by Mantel’s log-rank test using Kaplan-Meier estimates). Light microscopic examination of the pancreases removed from the FTY720-treated normoglycemic mice at 37 or 44 weeks of age revealed islets that were either without lymphocytic infiltrates (Fig. 2A) or with varying degrees of peripheral lymphocytic infiltrates (Fig. 2B). Invading insulitis was never seen. These islets contained intracellular insulin granules by insulin staining (Fig. 2C). In contrast, the pancreases of mice that became diabetic had either no islets or heavy cellular infiltration without identifiable insulin staining (Fig. 2D).
Effect of FTY720 treatment on development of spontaneous diabetes in NOD mice. FTY720 was given orally once per day five times per week at 0.5 mg/kg starting at 4 weeks of age and ending at 35 weeks of age. Diabetes was defined as nonfasting morning blood glucose levels of more than 300 mg/dL in two consecutive measurements.
(A and B) Representative histologic specimens of pancreas removed at 37 or 44 weeks of age after successful treatment with FTY720. Hematoxylin and eosin staining (magnification, ×200). (C) Representative insulin staining of the pancreas in FTY720-treated normoglycemic mice. Counter staining by hematoxylin (magnification, ×400). (D) Insulin staining of the pancreas removed from untreated spontaneously diabetic mice (magnification, ×400).
Administration of cyclophosphamide (CY) in nondiabetic NOD mice triggers accelerated development of diabetes approximately 2 weeks after injection (11). Therefore, we investigated whether administration of FTY720 prevents CY-induced diabetes. Two groups of nondiabetic NOD mice were given 200 mg/kg CY intraperitoneally at 40 weeks of age. One group (n=6) received daily (5 days per week) oral administration of 0.5 mg/kg FTY720 starting on the day of CY treatment, whereas the control group (n=6) was treated with CY alone (no FTY720). As shown in Figure 3, CY injection caused rapid development of diabetes in five of six control mice, whereas FTY720 prevented the development of diabetes in six of six treated mice. Withdrawal of FTY720 after 6 weeks resulted in onset of diabetes 2 weeks later in two of six mice.
Effect of FTY720 treatment on CY-induced diabetes in NOD mice. CY was given at 200 mg/kg to normoglycemic mice at 40 weeks of age. FTY720 was given orally five times per week starting from the day of CY injection for 6 weeks.
These results demonstrate that FTY720 is effective in preventing autoimmune diabetes. It has been reported that NOD islet isografts transplanted into overtly diabetic NOD mice survived long term (>100 days) when the recipient mice were continuously treated with 3 mg/kg FTY720, whereas the long-term islet grafts were destroyed within 2 weeks after withdrawal of FTY720 (12), indicating persistence of autoimmunity in FTY720-treated mice. Thus, we expected that beta cells that were spared from autoimmune destruction during FTY720 treatment would be destroyed after the cessation of treatment. To our surprise, the majority of FTY720-treated NOD mice maintained the diabetes-free condition even after the withdrawal of FTY720, suggesting that FTY720 is also capable of abrogating autoimmunity. This was also true with FTY720-mediated prevention of CY-induced diabetes; four of six treated mice remained diabetes free after withdrawal of FTY720. Because FTY720 is capable of inducing apoptosis (8), it is conceivable that continuous treatment with FTY720 might lead to deletion of beta-cell–reactive T cells, hence abrogation of autoimmunity. Studies examining this possibility are currently underway.
Although polyclonal antilymphocyte serum and anti-CD4 monoclonal antibody are capable of preventing the development of autoimmune diabetes in NOD mice (13), they induce generalized immunosuppression. In contrast, FTY720 does not impair either humoral immune responses or immunologic memory at either the T or B cell levels and does not suppress the generation of primary virus-specific cytotoxic T lymphocytes in lymph nodes (14). No generalized immunosuppression was associated with FTY720. Together with good tolerability in phase I and II clinical trials of FTY720, our present results suggest that FTY720 may be a safe and benign therapeutic agent that could be used chronically in prediabetic individuals to prevent type 1 diabetes mellitus.
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© 2002 Lippincott Williams & Wilkins, Inc.