Astilbin selectively facilitates the apoptosis of interleukin-2-dependent phytohemagglutinin-activated jurkat cells

Pharmacological Research, Vol. 44, No. 2, 2001 doi:10.1006/phrs.2001.0838, available online at http://www.idealibrary.com on

ASTILBIN SELECTIVELY FACILITATES THE APOPTOSIS OF INTERLEUKIN-2-DEPENDENT PHYTOHEMAGGLUTININ-ACTIVATED JURKAT CELLS RU YANa and QIANG XUa,b,∗ a Department

of Pharmacology for Chinese Materia Medica, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, China, b State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, 22 Han Kou Road, Nanjing 210093, China Accepted May 3 2001

The present study examined the relationship between the activation of T cells and the apoptosisfacilitating effect of astilbin on them. By the stimulation of PHA, a remarkable IL-2 production was detected in the supernatant of Jurkat cells after 120 h among 72–144 h incubation. This kinetics was quite in accordance with that of astilbin-induced apoptosis of Jurkat cells, where 1 h-exposure of the PHA-activated cells to astilbin caused a significantly increased apoptosis in a dose-dependent manner. To the Jurkat cells that had been cultivated for 72–144 h without PHA, however, astilbin did not show any facilitation of the cell apoptosis. Pre-treatment by cyclosporine A simultaneously with PHA dose-dependently lowered the IL-2 production and susceptibility of the cells to astilbin, while the treatment after 120 h of PHA-activation did not. The exogenous IL-2 treatment after 72 h of PHA-activation significantly and dose-dependently raised the susceptibility of the Jurkat cells to astilbin. These results indicated the dependency of the apoptosis-facilitating effect of astilbin on appropriate status of activated T lymphocytes with a relation to IL-2 production. This characteristic of astilbin may be of great significance for the treatment of a variety of c 2001 Academic Press immunologically related diseases.

K EY WORDS : astilbin, apoptosis, IL-2, selective immunosuppression, Jurkat cells.

INTRODUCTION The immunologically related diseases, such as multiple sclerosis, insulin-dependent diabetes mellitus and rheumatoid arthritis, are common and frequently occurring diseases which are difficult to cure. To date, the most useful strategy for treating these diseases is still to suppress the immune response by using potent immunosuppressants, such as cyclosporin A, FK506 and rapamycin [1, 2]. However, the immunosuppressive therapy is limited by the lack of antigen specificity, which usually results in general suppression of whole immune system with various side effects [3]. For this reason, emphasis should be laid on developing specific treatments that down-regulate deleterious immune reactions without affecting normal immune surveillance. To search for the specific immunosuppressants, we have previously screened a variety of drugs from traditional Chinese medicines by investigating their selective efficacy on the T cell-mediated immune responses including delayed-type hypersensitivity (DTH). As ∗ Corresponding author. E-mail: [email protected]

1043–6618/01/080135–05/$35.00/0

expected, several extracts from Chinese herbs have been found to show an interesting activity, selectively inhibiting either the induction or effector phase of the DTH reaction without affecting immune organs [4–7]. Such characteristics are quite different from those of the immunosuppressants in present use, and may provide a new possibility for the selective treatment of various immune diseases. One of the extracts, that from Rhizoma Smilacis Glabrae (RSG), selectively inhibited the effector phase of DTH without reducing the antibody formation to sheep red blood cells [7]. RSG extract also improved adjuvant-induced arthritis by inhibiting the DTH reaction as well as by a direct antiinflammatory activity via reducing PGE2 production [8]. To the DTH-induced liver injury model established by us, the extract showed a significant improvement by a novel mechanism, causing the apoptosis of liverinfiltrating lymphocytes [9, 10], mainly CD4+ and CD8+ T lymphocytes [11]. Furthermore, we isolated an effective principle, astilbin, from RSG. A similar immunosuppressive feature to the RSG extract was evidenced in astilbin, where the administration during c 2001 Academic Press

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MATERIALS AND METHODS

Cell line The human leukemia Jurkat cell line is maintained in RPMI-1640 medium containing 10% fetal calf serum (FCS).

Drugs and reagents Astilbin, 3, 30 , 40 , 5, 7-pentahydroxyflavanone 3-(6deoxy- (L-mannopyranoside)), was isolated from the rhizome of Smilax glabra (RSG), a Liliaceae plant. Phytohemagglutinin (PHA) was the product of Sigma. Cyclosporine A (CsA) was from Sandoz Ltd Basel, Switzerland. Recombinant human interleukin-2 (rhIL-2) was purchased from Changsheng Gene Pharm Co Ltd (Changchun, China). The ELISA kits for human IL-2 was obtained from Jingmei Biotech Co Ltd (Shenzhen, China).

Activation of Jurkat cells in vitro Jurkat cells (1×105 cells ml−1 ) were cultured in RPMI1640 medium containing 1 µg ml−1 of PHA or not for indicated times in flasks. After being centrifuged at 200g for 10 min, the supernatants were used for assaying IL-2 production. After washing the mitogen, the cells were resuspended and used for examining the effects of astilbin on DNA fragmentation.

Apoptosis measurements DNA fragmentation was quantified by the diphenylamine method with a minor modification [13]. Briefly, cells were lysed by 0.4 ml lysis buffer (10 mM Tris, pH 7.5, 1 mM EDTA, pH 8.0, 0.5% Triton X-100) at room temperature for 30 min. The lysate was centrifuged at 13 000g for 10 min to separate intact from fragmented chromatin. The supernatant, containing fragmented DNA, was removed to a separate tube. Both sediment and supernatant were brought to 12.5% trichloroacetic acid (TCA) and left overnight at 4 ◦ C. After being centrifuged at 20 000g at 4 ◦ C, the DNA in the precipitates was hydrolyzed by heating to 90 ◦ C for 10 min in 80 µl 5% trichloroacetic acid, and then 160 µl of diphenylamine reagent (0.15 g diphenylamine, 0.15 ml sulfuric acid, 2.5 µl 40% acetaidehyde/10 ml glacial acetic acid) was added to each tube. After the color development overnight at room temperature, the absorbance was read at 595 nm. The ratio of DNA fragmentation was recorded as the percentage of the

150 120 IL-2 (U/ml)

the effector but not induction phase significantly inhibited the DTH-induced liver injury. This activity of astilbin was associated with the selective dysfunction of liver-infiltrating lymphocytes [12]. The aim of the present study, therefore, is to examine the requirement of an appropriate activation of T cells for the selective inhibition of astilbin by using the human leukemia cell line, Jurkat cells.

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Fig. 1. IL-2 production in supernatants of Jurkat cells activated by PHA. Jurkat cells (1 × 106 ) were stimulated with PHA (1 µg ml−1 ) for 24, 48, 72, 96, 120 and 144 h, respectively. Then, supernatants were collected for IL-2 assay by ELISA. For other details, see the text. Each point represents the mean ± SD of three independent experiments and each experiment included triplicate sets. ∗ P< 0.05; ∗∗ P< 0.01 vs without PHA.

fragmented DNA to the total DNA. DNA electrophoresis was measured as described by Yamada et al. [14]. Briefly, portions of 1 × 106 cells with or without drug treatment were washed and spun down. Pellets were re-suspended in 0.6 ml of lysis buffer and incubated for 10 min on ice. After centrifugation at 20 000g, supernatants were mixed gently for 2–3 min with an equal volume of TE-saturated phenol (Wako Pure Chemical Industries Ltd, Osaka, Japan) (Wako), followed by centrifuging and mixing supernatants with chloroform: isoamylalcohol (24 : 1). Then, the supernatants containing DNA after centrifugation at 20 000g were precipitated overnight at −20 ◦ C in 0.3 M NaCl and 70% ethanol. The loading buffer (Wako) containing 0.02% bromophenol blue, 0.02% xylene cyanol FF, 50% glycerol and 0.1% sodium dodecyl sulfate was then added to the samples at a 1:10 (v/v) ratio. Electrophoresis was carried out in 2% agarose for 120 min at 50 V and DNA was visualized with ethidium bromide.

Statistics Data were expressed as mean ± evaluated using student’s t-test.

SD

and statistically

RESULTS

IL-2 production in supernatants of Jurkat cells activated by PHA for 120 h Jurkat cells were stimulated with PHA (1 µg ml−1 ) for 24, 48, 72, 96, 120 and 144 h. Then, supernatants were collected for IL-2 assay. As shown in Fig. 1, IL-2 production was observed in the supernatant of Jurkat cells significantly 96–144 h after co-cultured with PHA. The peak production was seen at 120 h. In the cases of PHA absence, the IL-2 production was not detected in any incubated times.

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Fig. 3. Concentration-dependent facilitation of apoptosis in PHAactivated Jurkat cells by astilbin. Jurkat cells (1 × 106 ) were incubated with PHA (1 µg ml−1 ) for 120 h. After washing, they were treated with astilbin for 1 h. Then, cells were collected for diphenylamine assay. Each column represents the mean ± SD of three separate experiments and each experiment included triplicate sets. ∗ P< 0.05 vs control.

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Fig. 2. Effect of astilbin on DNA fragmentation of Jurkat cells using the diphenylamine method. Jurkat cells (1 × 106 ) were incubated with (a) or without (b) PHA (1 µg ml−1 ) for 72, 96, 120, or 144 h respectively. After cultivation in the absence (control) or presence of 1 × 10−6 g ml−1 of astilbin for 1h, DNA was extracted and used for diphenylamine assay. Each point represents the mean ± SD of three separate experiments and each experiment included triplicate sets. ∗ P< 0.05 vs control.

Facilitated apoptosis by astilbin of PHA-activated Jurkat cells for 120 h Jurkat cells were incubated with PHA (1 µg ml−1 ) for 72, 96, 120, and 144 h respectively. After incubation, the cells were treated with 1 × 10−6 g ml−1 of astilbin for 1 h. As shown in Fig.2(a), the apoptosis of Jurkat cells began to increase at 96 h, reached a peak at 120 h, and then reduced at 144 h. However, astilbin did not show any facilitating activity on the apoptosis of Jurkat cells without PHA activation [Fig. 2(b)]. To those cells that had been treated for 120 h with PHA, astilbin at concentrations of 1 × 10−8 to 1 × 10−6 g ml−1 showed a dose-dependent facilitation of apoptosis (Fig. 3). Such dose-dependent activity was also demonstrated with a typical 180-bp ladder band in agarose electrophoresis (Fig. 4).

Decrease by CsA in IL-2 production and susceptibility of PHA-activated Jurkat cells to astilbin Jurkat cells were cultured for 120 h in the copresence of PHA (1 µg ml−1 ) and CsA (1 × 10−8 to 1 × 10−6 g ml−1 ). After washing, the cells were incubated with astilbin for 1 h. As shown in Fig. 5(a), the co-presence of CsA mildly increased the apoptosis of PHA-activated Jurkat cells (open column) and dosedependently inhibited IL-2 production (dotted column). To the cells without CsA treatment, astilbin significantly facilitated the apoptosis. Such facilitation was reduced in

Fig. 4. Gel agarose electrophoresis of DNA extracted from PHAactivated Jurkat cells and the effect of astilbin on it. Jurkat cells (1 × 106 ) activated with PHA (1 µg ml−1 ) for 120 h were cultivated in the absence (control) or presence of astilbin for 1 h. The results are representative of two separate experiments. 1: control, 2–4: 1 × 10−8 , 1 × 10−7 , 1 × 10−6 g ml−1 of astilbin, M: DNA marker. For other details, see the text.

CsA and PHA-co-treated cells in a CsA-dose-dependent manner (hatched column). At the concentration of 1 × 10−6 g ml−1 of CsA, the astilbin-induced facilitation was completely abolished. However, when CsA was cultured after 120 h of PHA stimulation for 30 min and then washed, no influence was observed on the astilbin-induced apoptosis [Fig. 5(b)].

Increase by IL-2 in the susceptibility of PHA-activated Jurkat cells to astilbin Considering the IL-2 production in supernatant of Jurkat cells activated by PHA shown in Fig. 1, Jurkat cells were cultured with 50 or 200 U ml−1 of IL-2 at 72 h after removing PHA for a further 12, 24, 48 and 72 h, respectively. Then, they were harvested for exposure to 1 × 10−6 g ml−1 of astilbin for 1 h. As

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Fig. 5. Effects of CsA on IL-2 production and susceptibility of PHAactivated Jurkat cells to astilbin-induced apoptosis. (a) Jurkat cells (1×106 ) were cultured for 120 h in the co-presence of PHA (1 µg ml−1 ) and CsA (1 × 10−8 to 1 × 10−6 g ml−1 ). Then, the supernatants were collected for IL-2 assay. (b) Jurkat cells (1 × 106 ) were cultured for 120 h with PHA (1 µg ml−1 ). After washing, they were incubated with CsA (1 × 10−6 g ml−1 ) for 30 min. After the treatment with PHA and CsA, the cells (in both (a) and (b)) were washed and incubated with astilbin (1 × 10−6 g ml−1 ) for 1 h followed by determining the DNA fragmentation by diphenylamine assay. Each column represents the mean ± SD of three separate experiments and each experiment included triplicate sets. ∗ : P< 0.05, ∗∗ : P< 0.01 vs control; # : P< 0.05, ## : P< 0.01 vs PHA alone.

shown in Fig. 6, the incubation of IL-2 dose-dependently increased the susceptibility of the Jurkat cells to astilbin. A greatly increased apoptosis was observed in the cells after treatment at the dose of 200 U ml−1 IL-2 for 24– 72 h. However, IL-2 itself did not influence the apoptosis of the cells (open circles).

DISCUSSION To investigate the appropriate status of T cell activation for examining the effect of astilbin, the present study first measured the time course of IL-2 production in Jurkat cells by the stimulation of PHA. IL-2, known as a hallmark of T lymphocyte activation, could stimulate the proliferation of T cells and regulate the magnitude and duration of the T cell immune response following antigen encounter [15]. As a result, we detected a peak IL-2 production in the supernatant of Jurkat cells after 120 h of PHA stimulation [Fig. 1(a)], suggesting a good status of activated Jurkat cells at this time. The kinetics of IL-2 production was in fair accordance

Control IL-2 50 U/ml IL-2 200 U/ml

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Astilbin Astilbin + IL-2 50 U/ml Astilbin + IL-2 200 U/ml

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Fig. 6. Effect of exogenous IL-2 on the susceptibility of PHAactivated Jurkat cells to astilbin-induced apoptosis. Jurkat cells (1×106 ) were cultured with PHA (1 µg ml−1 ) for 72 h and then incubated with 50 or 200 U ml−1 of IL-2 after removing PHA for further 12, 24, 48 and 72 h, respectively. Then, they were harvested for exposure to 1 × 10−6 g ml−1 of astilbin for 1 h. DNA fragmentation was assayed by diphenylamine method. Each point represents the mean ± SD of three separate experiments and each experiment included triplicate sets. ∗ P< 0.05; ∗∗ P< 0.01 vs astilbin alone.

with that of astilbin-induced apoptosis of Jurkat cells. When the cells, after various periods of PHA activation, were exposed to astilbin for 1 h, those cells which experienced 96 h-activation began to show susceptibility to the flavanoid. Such susceptibility reached a maximum after 120 h and then declined after 144 h [Fig. 2(a)]. These results indicated that PHA activation for 120 h is the most suitable timing for the apoptosis-facilitating effect of astilbin. It should be noted that for the Jurkat cells cultivated for 72–144 h without PHA, astilbin did not show any effect on the cell apoptosis [Fig. 2(b)]. This selective action of astilbin against the cells in some special status is quite different from other potent immunosuppressants, such as glucocorticoids and cyclophosphamide that inhibit the whole immune system through suppressing immune cells at any stage of their differentiations. This characteristic of astilbin may ensure selectively eliminating specific lymphocyte populations participating in certain diseases without lowering the whole immunocompetence of the body. Based on these findings, the Jurkat cells after 120 h of PHA activation were used for the exposure to astilbin to confirm its selective effect. A significant facilitation of the apoptosis was observed in a dose-dependent manner in both quantitative analysis (Fig. 3) and agarose electrophoresis (Fig. 4) of DNA fragmentation. The consistency between IL-2 content and the effects of astilbin hint that astilbin could selectively inhibit T cells at a certain stage in which the existence of IL-2 is needed. To ascertain the involvement of IL-2, CsA, a famous inhibitor of IL-2 transcription [1] was applied to the PHA activation system for Jurkat cells. The co-culture of CsA at the same time as PHA dose-dependently lowered the production of IL-2 and the susceptibility of the cells

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to astilbin. Namely, the apoptosis-facilitating effect of astilbin on the cells was weakened by the pretreatment of CsA accompanying with the decrease in IL-2 production. CsA at a dose of 1 × 10−6 g ml−1 completely blocked the effect of astilbin [Fig. 5(a)]. However, when CsA was added after 120 h of PHA-activation, the Jurkat cells remained susceptible to astilbin and induction of apoptosis was not influenced [Fig. 5(b)]. In order to further elucidate the role of IL-2 in the effects of astilbin, exogenous IL-2 was added to the culture system of Jurkat cells after 72 h of PHA-activation for a further 12–72 h. The treatment of IL-2 significantly and dose-dependently raised the susceptibility of the cells to astilbin. In the case of the IL-2 doses used, the higher the amount of IL-2 added, we note that the effect of astilbin on apoptosis appeared more quickly and was increased in strength (Fig. 6). These results suggest that the apoptosisfacilitating effect of astilbin on PHA-activated Jurkat cells is at least well correlated to the IL-2 production. Previously, we have found that astilbin could improve a DTH-induced liver injury when administered during the effector but not induction phase of the DTH reaction. This activity was associated with the dysfunction of liverinfiltrating nonparenchymal cells including apoptosis induction [12]. Considering the main population of T lymphocytes in the nonparenchymal cells, the dysfunction-inducing effect of astilbin on activated T lymphocytes could be supposed to include the apoptosisfacilitating effect. This supposition was supported by the present study on PHA-activated Jurkat cells. On the other hand, Th1 cells are known as the main effector cells in some autoimmune diseases [16–19]. The production of Th1-like cytokines includes IL-2 (Fig. 1) and IFN-γ (data not shown) in PHA-activated Jurkat cells indicated a possible involvement of Th1 cells as the main target of astilbin. Overall, the present study revealed the dependency of astilbin on a certain stage of activated T lymphocytes. Activation with PHA for 120 h in the presence of Jurkat cells may mimic the activation status for the effect of astilbin on inflammatory T lymphocytes in some immunoinflammatory models as previously reported by us [12]. This selective activity of astilbin may be of great significance to pave a selective treatment for a variety of immunologically related diseases. Further investigation is in progress on the detailed mechanism of the selective role of astilbin.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No. 39925041 and No. 30070876).

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