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.

Introduction

Apoptosis is an active process invoked in many steps of development and maintenance of the immune system [1,2]. Stimulation of TCR on immature T cells induces apoptosis rather than proliferation, as occurs in mature T cells. Moreover, even mature T cells undergo apoptosis when they are persistently exposed to TCR stimulation [3,4]. In addition to TCR stimulation, calcium signalling, which causes elevation of intracellular calcium, also leads to apoptosis of T cells through certain pathways [2,5]. Recent studies by Shibaski and coworkers [6] demonstrated that the ability of Bcl-2 to block calcium-induced apoptosis through the formation of tight Bcl-2/calcineurin complexes results in targeting calcineurin to Bcl-2 sites on cytoplasmic membranes.

The immunosuppressant agent FK506 is known to inhibit the activation of calmodulin-dependent protein phosphatase by binding to FK506-binding protein 12 and cyclophilin [7], resulting in inhibition TCR/CD3-mediated induction of apoptosis in T cell hybridomas [8]. In contrast to these findings, we have demonstrated that FK506 augmented apoptosis of only activated thymocytes [9].

In autoimmune diseases affecting humans and animals with disease-prone genetic backgrounds there is convincing evidence for accumulation of T cells with activated phenotypes expressing specific TCR gene segments [10–13]. Furthermore, studies examining the repertoire of TCR of infiltrates suggest that only oligoclonal T cells expand and contribute to tissue destruction, even in the late stages of the disease process [14,15]. Using the alymphoplasia/alymphoplasia (aly/aly) mouse, we have demonstrated previously the accumulation of oligoclonal activated T cells in spontaneously developed autoimmune pancreatitis and sieloadenitis [13]. In the present study, we treated these mice with FK506 to prevent autoimmune disease. We also examined the mechanisms of action of FK506 with a special focus on the mechanisms regulating apoptosis.

Materials and methods

Animals

Aly/aly mice were purchased from Nippon Shinyaku (Tokyo, Japan). They were housed in a stable temperature environment and provided with water and food (chow 5015, Purina) ad libitum. The experimental protocol was approved by the Animal Care Ethics Review Committee of our institution. Homozygosity was confirmed by serum IgA level (homozygotes, < 0·01 mg/dl; heterozygotes, 0·3 ± 0·03 mg/dl). Fk506 (2 mg/kg, Fujisawa Pharmaceutical Co., Tokyo) or serine were administrated to the animals intracutaneously every 2 days from the age of 8 weeks. Animals were sacrificed between 15 and 25 weeks of age and the pancreas, salivary glands and spleen were removed. In some experiments, 50% of the pancreatic tissue was fixed in 4% paraformaldehyde (Waco Co., Japan) for histological examination while the remaining 50% was frozen in liquid nitrogen and stored at −70°C.

Immunohistochemistry

Fresh pancreas and salivary glands sections fixed in 4% paraformaldehyde were embedded in OCT compound. The 6-µm-thick sections were cut and stained with haematoxylin and eosin. In addition, other sections were treated with antimouse CD4, antimouse CD8, antimouse CD20, antimouse Thy1 or antimouse Thy2 using the labelled streptavidin–biotin method (Histfine staining kit, Nichirei Co., Tokyo). All antibodies were purchased from Coulter Immunology (Hialeah, FL, USA). The fixed sections were also stained by hamster antimouse Bcl-2 MoAb and anti-Bax MoAb (Immunoteck). TUNEL was used to examine DNA fragmentation in histological sections. For this purpose, nuclei of tissue sections were stripped from proteins by incubation with 0·1 µg/ml proteinase K for 7·5 min at 37°C, and the slides were then washed in PBS three times (each for 5 min). The sections were rinsed once with distilled deionized water and covered with terminal deozynucleotidyl transferase (TdT, Boehringer Mannheim, Germany) buffer alone. Then, TdT buffer containing 0·3 U/µl TdT and 3 µm biotinylated 16-dUTP (Boehringer Mannheim) was added to the sections, and the slides were incubated in a humidified atmosphere at 37°C for 1 h. The reaction was terminated by immersing the slides in 50 mm Tris-HCl, pH 7·5 four times (at 10 min each) at room temperature, followed by rinsing with PBS. Endogenous peroxidase was inactivated by immersing the sections in 0·3% H₂O₂ in methanol for 15 min at room temperature and washed three times with PBS (each for 5 min). After incubation with 5% BSA in PBS for 1 h at room temperature to block non-specific binding, the sections were incubated with HRP goat antibiotin (1:100, Boehringer Mannheim) diluted with 5% BSA in PBS for 2 h at room temperature, followed by washing with PBS. HRP was visualized by H₂O₂ and DBA for 5 min. As a control, some sections were reacted without TdT.

TCR Vβ gene polymerase chain reaction (PCR)

The PCR reaction was based on the RACE protocol [16]. Briefly, total cellular RNA was extracted from the frozen pancreas, spleen and salivary glands using guanidium thiocyanate and phenol (RNAzol B, Cinna/Biotek Labs International, Friendswood, TX, USA) and stored at −70°C in sterile diethylpyrocarbonate (DEPC, Sigma, St Louis, MO, USA)-treated water. Complementary DNA (cDNA) transcripts were prepared from 1 µg of cellular RNA using oligo-dT priming (1 µg/20 µl vol) and avian reverse transcriptase (30 U/20 µl) (Life Science Inc.) in the presence of RNAsin (40 U/20 µl) (Promega Inc, Madison, WI, USA). The reactions were diluted to 200 µl with sterile water and stored at −20°C. For PCR reactions, 5 µl of denatured cDNA was amplified in a 20-µl final volume with 1 U Taq DNA polymerase (Promega), 0·3 µg of both primers, Taq polymerase buffer containing 1·5 mm MgCl₂ and with 1·5 mm of each dNTP (Promega Inc). A 35-cycle step programme (95°C for 1 min, 56°C for 2 min, and 72°C for 3 min) was followed by a 10-min extension at 72°C (Model PJ2000 DNA Thermal cycler, Perkin Elmer, Norwalk, CN, USA). The amplified products were subjected to electrophoresis on 1·5% agarose gels.

TCR constant region primers:

5′-ATG TGA CTC CAC CCA AGG TCT CCT TGT TTG-3′

5′-TTG CAG ACA GAA CCC CCT GAT GAT AGG ATG-3′

β-actin primers:

5′-ATG TAC CGA CCC CAC AAC TT-3′

5′-AAG AGA GGC ATC CTC ACC CT-3′

Cell culture

Splenocytes were obtained by simple needle perfusion of the intact organ, as described previously [17]. Spleen cells were cultured with RPMI-1640 culture medium (Gibco, Paisley, UK) containing 10% FBS supplement with penicillin (100 U/ml, Meiji Seika, Tokyo), streptomycin sulphate (120 mg/l, Gibco), l-glutamic acid (300 mg/l, Sigma), pyruvic acid (110 mg/l, Sigma) and rIL-2 (10 IU, Gibco) for 7 days at 37°C in 10% CO₂ in 24 wells culture plate (Falcon). The cells were re-stimulated by PMA (10 ng/ml, Sigma) and ionomycin (500 ng/ml, Sigma) for the next 6–36 h with or without various concentrations of FK506.

In vitro detection of DNA fragmentation

After re-stimulation by PMA and ionomycin, splenocytes were fixed with 70% ethanol and treated with RNAase (100 µg/ml, Sigma) and then stained with propidium iodide (100 µg/ml, Sigma) for 30 min on ice. The stained cells were analysed using a flow cytometer (Epics Profile-II, Coulter). Apoptosis was quantified by flow cytometric determination of the proportion of cells with hypodiploid DNA.

Bcl-2 and Bax quantification by Western blotting

Splenocytes were also cultured with or without FK506 using the optimal dose and period. Cells were collected and lysed by the addition of a lysis buffer (1% NP-40, 50 mm Tris at pH 7·5, 100 nm NaCl, 5 mm EDTA, 1 mm polymethylsulphonylfluoride) for 20 min at 4°C, and insoluble material was removed by centrifugation at 6 000 × g for 10 min at 4°C. The supernatants were collected and protein concentration was determined by the DC protein assay kit (Bio-Rad, Melville, NY, USA). An identical amount of protein (20 µg/well) for each lysate was subjected to 10% sodium dodecyl sulphide polyacrylamide gel electrophoresis. Proteins were transferred to a nitrocellulose filter and the filter was blocked for 1·5 h using 5% non-fat dried milk in TBS (50 mm Tris, 0·15 m NaCl, pH 7·5) containing 0·1% Tween 20 (PBS-T), washed with PBS-T and incubated at room temperature for 2 h in a 1:1000 dilution of hamster antimouse Bcl-2 MoAb (Biomol Research Laboratories, Plymouth Meeting, PA, USA) or rabbit anti-Bax polyclonal antibody (Santa Cruz Biotechnology, Delaware Avenue, CA, USA). The filter was washed with PBS-T and incubated at room temperature with 1 : 1000 dilution of rabbit antihamster IgG (Rockland, Gilbertsville, PA, USA) or donkey antirabbit IgG (Amersham, Arlington Height, IL, USA), coupled with horseradish peroxidase (Immunoteck). The enhanced chemiluminescence (ECL) system (Amersham, Arlington Heights, IL, USA) was used for detection.

Results

In vivo effect of FK506 on autoimmune pancreatitis

First, we examined the in vivo effect of FK506 by examining H-E sections. As reported previously, mice treated with serine (control group) showed minimal mononuclear cell infiltration in the pancreas at the age of 15 weeks (Fig. 1a and Table 1) and a marked cell infiltration with destruction of acini and partial fatty changes at the age of 25 weeks (Fig. 1b and Table 1). In mice treated with FK506 (FK506 group), mononuclear cell infiltration at the age of 15 weeks was almost similar to that of the control (Fig. 1c and Table 1). However, FK506 resulted in the almost complete disappearance of mononuclear cells at the age of 20 weeks, and prevented tissue destruction as well as mononuclear cell infiltration at the age of 25 weeks (Fig. 1d and Table 1).

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).

Table 1.

Degree of mononuclear cell infiltration in the pancreas

	Control	FK506
15w	15 ± 8	18 ± 8
25w	480 ± 25	10 ± 8*

Degree of mononuclear cell infiltration was expressed by the average number of mononuclear cells counted per power field (E) (100 power fields were examined per sample). Values are mean ± s.d. of 30 animals.

^*P < 0·01 versus control.

To investigate the mechanisms of action of FK506 on mononuclear cell infiltration between 15 and 20 weeks of age, apoptosis in fixed sections was then examined using TUNEL staining. H&E staining showed no difference in the degree of cell infiltration between the two groups at the age of 15 weeks. Surprisingly, most mononuclear cells in mice injected with FK506 were TUNEL positive at the age of 15 weeks (Fig. 2a and Table 2), whereas TUNEL-positive cells were rare in control mice of the same age (Fig. 2b and Table 2). In mice aged 25 weeks, pancreatic ducts, acini and mononuclear cells were TUNEL-positive in the control group (Fig. 2c and Table 2), but no such positive cells were present in pancreatic ducts and acini in the FK506 group (Fig. 2d and Table 2).

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).

Table 2.

TUNEL positive infiltrated mononuclear cells in the pancreas

	Control	FK506
15w	3 ± 1	16 ± 6*
25w	58 ± 10	8 ± 6*

The number of TUNEL positive mononuclear cells represents the average number of mononuclear cells counted per power field (E) (100 power fields were examined per sample). Values are mean ± s.d. of 30 mice.

^*P < 0·01 versus control

Effect of FK506 on TCR expression

We have studied previously the phenotype and TCR repertoire of infiltrated T cells in the aly autoimmune pancreatitis and sieloadenitis, and demonstrated their oligoclonality [13]. In the present study, we compared TCR repertoire in the control and FK506 groups. In control mice TCR repertoire showed oligoclonal T cells (data not shown), while in the FK506 group even TCR constant region messages, that are usually stronger than each Vβ gene messages, could not be detected (Fig. 3).

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.

In vitro effect of FK506 on calcium signalling apoptosis

Based on the results of TUNEL staining, apoptosis of mononuclear cells that infiltrated the pancreas seemed to be augmented by FK506. To confirm this conclusion, we examined the effect of FK506 on apoptosis using cultured naive splenocytes. Splenocytes were obtained from naive mice by the simple perfusion method [17] and cultured with the complete medium supplemented by rIL-2. After 7 days of culture, cells were stimulated by PMA and ionomycin for 6–36 h without or with FK506 at various concentrations, to induce calcium signalling apoptosis, as described previously [8]. Apoptosis was analysed by quantification of hypodiploid DNA. Our results showed induction of apoptosis 6 h after the addition of PMA and ionomycin and reached a peak level after 24–36 h (Fig. 4a). FK506 clearly augmented the level of apoptosis (Fig. 4a). In the next series of experiments, we examined the augmentation effect 24 h after administration of PMA and ionomycin. FK506 enhanced apoptosis in a dose-dependent manner and the maximum effect was observed using 500 nm of FK506 (Fig. 4b).

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)

Effect of FK506 on Bcl-2 and Bax production

The ability of Bcl-2 to block most cases of calcium signalling apoptosis has been reported previously [2]. We therefore fixed the tissue sections and examined the production of Bcl-2 and Bax in these tissues. Most mononuclear cells were Bcl-2 and Bax positive in both untreated and FK506-treated groups (data not shown), and therefore it was difficult to compare their quantities in these two groups. We then examined the effect of FK506 on Bcl-2 production using naive splenocytes. According to the results of the previous section, cells were cultured with or without 500 nm of FK506 for 24 h. Cells were then lysed, purified and subjected to Western blotting using a mouse Bcl-2 specific MoAb. The quantity of Bcl-2 protein in splenocytes cultured with FK506 was almost half that of cells cultured without FK506 (Fig. 5, Table 3). Examination of Bax production using the same method showed that FK506 did not influence production (Fig. 5, Table 3).

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.

Table 3.

Quantity of Bcl-2 protein examined by Western blotting

	Control	FK506
Bcl-2	100	53 ± 8*
Bax	100	90 ± 12

Band density in arbitrary units (control: 100). Values are mean ± s.d. of five independent experiments.

^*P < 0·01 versus control (no FK506).

Discussion

In our previous study, we demonstrated the accumulation of oligoclonal activated T cells in spontaneously developed autoimmune pancreatitis [13]. In this study, we examined the effects of the immunosuppressant FK506 on this mouse model. Based on examination of H&E-stained tissue sections, infiltrated T cells seemed to be killed by FK506 before proliferation. Subsequent studies using TUNEL staining showed that at the age of 15 weeks most accumulated T cells were TUNEL positive, whereas only a very small proportion of these cells were TUNEL positive in the control group. The results of these histological studies suggested that FK506 enhanced apoptosis of T cells. Further studies of the effect of FK506 on calcium-signalling apoptosis in vitro using naive aly splenocytes showed that culture of these cells with rIL-2 (10 IU) for 7 days followed by stimulation by PMA and ionomycin resulted in apoptosis 6 h after the addition of PMA and ionomycin. Furthermore, our studies demonstrated that FK506 clearly augmented the apoptotic effect of PMA and ionomycin in a dose-dependent manner.

Previous studies have shown that Bcl-2 blocks the majority of calcium signalling apoptosis [2]. In the present study, we also examined the effect of FK506 on Bcl-2. Most T cells showed positive staining for Bcl-2 in the pancreas of the control and pancreatitis groups and that the positive ratio of Bcl-2 by immunohistochemistory was similar in both groups. The effect of FK506 on Bcl-2 was examined by Western blotting using splenocytes treated as above, as well as by a mouse Bcl-2 specific MoAb. FK506 produced a marked decrease in the amount of Bcl-2 protein of cultured splenocytes relative to the control. Bcl-2 inhibits apoptosis by heterodimerization with Bax [18], although Bax production was not influenced by FK506.

FK506 and cyclosporin A are known to inhibit cytokine production of T cells via inhibition of calmodulin-dependent protein phosphatase through binding to FK506-binding protein 12 and cyclophilin [7]. Calcineurin is a calcium-dependent protein phosphate that induces T cell activation. Calcineurin has also been demonstrated to induce calcium-triggered apoptosis in mammalian cells deprived of growth factor and co-expression of Bcl-2 efficiently blocks calcineurin-induced cell death [19]. Furthermore, calcium-independent calcineurin mutant induces apoptosis in the absence calcium, and this apoptotic response is a direct consequence of calcineurin's phosphatase activity, suggesting that calcineurin influences the upstream events in calcium-activated cell death [19]. Cyclosporin A and FK506 inhibit activation-induced cell death caused by phorbol ester in the murine B cell line [20], and also abolish the inhibition of glucocorticoid-induced apoptosis by ionomycin in T cell hybridomas [8]. Furthermore, FK506 significantly augments thymic apoptosis induced by administration of anti-CD3 antibodies in mice [21]. Cross-linking of TCR-CD3 together with CD4, CD8 or LFA-1 markedly inhibits glucocorticoid-induced death of murine thymocytes in vitro, and such inhibition is abrogated by FK506 [21]. These results suggest that FK506 may augment activation-induced apoptosis independent of the interference of Fas–FasL interaction. In this study, we have demonstrated that FK506 reduced the production of Bcl-2 protein. Since Bcl-2 blocks the majority of calcium signalling apoptosis, this action may be one of the mechanisms of FK506 augmentation of activation-induced apoptosis.

Accumulation of activated self-reactive T cells at the site of inflammation has been reported in a variety of autoimmune diseases [10–15]. In addition, a persistent increase of oligoclonal T cells (these cells are supposed to be pathogenic), has been demonstrated in these diseases [14,15,22]. Thus, it is possible that these pathogenic T cells undergo apoptosis before clonal expansion. FK506 augments apoptosis of activated T cells but not resting ones. We suggest that FK506 may act selectively on activated T cells at the site of inflammation and prevent the host from systemic immune deficiency.

In summary, we have shown that the immunosuppressant agent FK506 was effective in the suppression of the development of aly autoimmune pancreatitis. This action was mediated by augmentation of apoptosis of infiltrating T cells. FK506 also suppressed Bcl-2 production by activated T cells in vitro, and this effect is probably involved in the augmentation of apoptosis. Our results suggest that FK506 is potentially useful in various autoimmune diseases, including autoimmune pancreatitis.