Novel immunosuppressive effect of FK506 by augmentation of T cell apoptosis

Abstract

We have recently reported the accumulation of oligoclonal activated T cells in the spontaneously developed autoimmune pancreatitis in aly/aly mouse. In this study, we examined the effects of FK506 in this mouse model in preventing autoimmune pancreatitis and investigated its action on calcium signalling apoptosis of alymphoplasia (aly) lymphocytes in vitro. Mice were treated with FK506 from 8 to 25 weeks of age. At the age of 15 weeks, minimal mononuclear cell infiltration was observed in the pancreas in both the FK506 treated group and the control group. Furthermore, a marked cell infiltration associated with destruction of acini and partial fatty changes were observed in 25-week-old control mice. In contrast, FK506 treated mice showed almost no tissue destruction or mononuclear cell infiltration at the age of 25 weeks. Furthermore, at 15 weeks of age, most mononuclear cells in FK506-treated mice were TUNEL positive, whereas only a few were positive in control mice. This augmentation of T cell apoptosis by FK506 was confirmed using naive splenocytes activated by PMA and ionomycin in vitro. Finally, a suppressive effect of FK506 on Bcl-2 production but not on Bax production was confirmed by Western blotting. This unique effect of FK506 on the augmentation of T cell apoptosis is probably one of the mechanisms explaining its beneficial effect on aly autoimmune pancreatitis.

Fig. 1

Hematoxylin–eosin staining of pancreatic tissue samples from a representative 15-week-old mouse injected with (a) serine and (b) FK506, and a 25-week-old mouse injected with (c) serine and (d) FK506 (original magnification × 100).

Fig. 2

TUNEL staining of pancreatic tissue samples from a representative 15-week-old mouse injected with (a) serine and (b) FK506 (original magnification × 400), and a 25-week-old mouse injected with (c) serine and (d) FK506 (original magnification × 100).

Fig. 3

PCR products of pancreas tissue samples from 15-week-old aly/aly mice treated without FK506 (lanes 1 and 2) with FK506 (lanes 3 and 4). Left side: β-actin products (235 bp), right side: TCR constant region products (410 bp). B, blank; M, molecular marker.

Fig. 4

Serial changes (a) and dose-dependent (b) effect of FK506 on calcium-signalling apoptosis. Splenocytes were obtained from naive mice and cultured with complete medium supplemented with rIL-2. After 7 days of culture, the cells were stimulated by PMA and for 6–36 h with various concentration of FK506. Apoptosis was analysed by quantification of hypodiploid DNA. (a) Abscissa: incubation time (hours); ordinate: percentage of hypodiploid DNA. Triangles: with FK506, circles: without FK506, *P < 0·01 versus control (no FK506). (b) Abscissa: concentration of FK506 (nM); ordinate: percentage of hypodiploid DNA. Values are mean ±s.d. of five experiments. *P < 0·01 versus control (no FK506)

Fig. 5

Western blot analysis of Bcl-2 and Bax of aly splenocytes. Splenocytes were cultured with or without FK506 using the optimal dose and period. Cells were lysed and purified, and an identical amount of protein (20 µg/well) for each lysate was subjected to Western blot analysis. Upper panels: blots of Bcl-2 and Bax of each sample, lower panels: band size in arbitrary units.