inhibition of lymphocyte proliferation by the anti-arthritic drug sinomenine

Pergamon Int. J. lmmunopharmac., Vol. 16, No. 8, pp. 685-691, 1994

Elsevier Science Ltd Copyright © 1994 International Society for lmmunopharmacology

Printed in Great Britain. All rights reserved 0192-0561/94 $7.00 + .00

0192 - 0561(94)E0029-M

SHORT C O M M U N I C A T I O N

INHIBITION OF L Y M P H O C Y T E P R O L I F E R A T I O N BY THE ANTI-ARTHRITIC D R U G SINOMENINE

LIANG LIU, *t KLAUS RESCH* and VOLKHARD KAEVER**

*Institute of Molecular Pharmacology, Medical School Hannover, D-30623 Hannover, Germany; and *Institute of Clinical Pharmacology, Guangzhou College of Traditional Chinese Medicine,

510407 Guangzhou, Republic of China

(Received 29 November 1993 and in f inal form 15 February 1994)

Abstract - - The effect on lymphocyte proliferation of sinomenine, a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum was investigated in vitro using mouse spleen cells and human peripheral blood mononuclear cells. It could be demonstrated that sinomenine markedly inhibited [3H]thymidine incorporation in mouse spleen cells activated with concanavalin A (1c50 = 400 laM) or by two-way mixed lymphocyte culture (Its0 = 60 taM) and also in human peripheral blood mononuclear cells activated with phytohemagglutinin, 12-O-tetradecanoylphorbol-13-acetate plus ionomycin, or mixed lymphocyte culture (tcs0 ranging from 34 to 129 ~M). Time kinetic experiments revealed that sinomenine was effective only when added within the first 48 h after the onset of mixed lymphocyte culture, which lasted for 5 days. Inhibition of lymphocyte proliferation by sinomenine was reversible. Accordingly, the drug showed no direct cytotoxicity in our cellular systems and had no inhibitory effect on the proliferation of the cytokine- independent growth of the human leukaemic T-cell lymphoblast cell line Jurkat. It can be considered that these anti-proliferative effects are part of the anti-inflammatory and anti-arthritic mechanisms of sinomenine obvious in clinical trials.

Keywords: sinomenine, lymphocyte proliferation, mixed lymphocyte culture, rheumatoid arthritis.

The Chinese medical plant S & o m e n i u m acu tum has been used in China since ancient times for the treatment o f rheumatic diseases (Shen & Shen, 1596). The alkaloid sinomenine (7,8-didehydro-4-hydroxy-3, 7-dimethoxy- 17 -methylmorphinane- 6-one) purified f rom this plant has been shown to inhibit significantly inf lammatory reactions caused by various phlogistic agents (Irino, 1958; Chang et al., 1964; Huo & Che, 1989). The therapeutic efficacy of sinomenine was confirmed in patients with rheumatoid arthritis (RA) (Ke & Xiu, 1986; Shi et al., 1986). Studies in our laboratory have revealed that the pure alkaloid has marked effects on mediator release f rom macrophages. Thus prosta- glandin E2 (PGE2) and leukotriene C4 (LTC4) synthesis, or nitric oxide product ion was significantly

inhibited by sinomenine (Liu et al., 1994), which may provide the basis for its acute ant i - inf lammatory action. However , in complex diseases such as RA sinomenine may exert its therapeutic effect by different pathways. Pathological processes involved in RA can be influenced by a variety of cellular humoral factors (Ziff, 1990). Activated lymphocytes, macrophages, fibroblasts, synovial cells, and chondrocytes are among the main cell populations implicated in inf lammatory events of RA. Moreover , one common hypothesis proposes that RA may represent a chronic immune-mediated disease, whose initiation and perpetuation is dependent on T-lymphocyte responses to unknown antigens (Firestein & Zvaifler, 1990; Panayi, 1993). There- fore, we investigated the effects of sinomenine on

*Author to whom correspondence should be addressed, at: Medizinische Hochschule Hannover, Institut fiir Klinische Molekularpharmakologie, D-30623 Hannover, Germany.

685

686 Short Communication

lymphocyte activation in order to obtain further insight into the relevant pharmacological mechanisms of this drug. In this study we have examined the actions of sinomenine on the proliferation of human peripheral blood mononuclear cells and mouse spleen cells activated with various mitogens or by two-way mixed lymphocyte culture (MLC).

EXPERIMENTAL PROCEDURES

Reagents

RPMI 1640 medium with L-glutamine, fetal calf serum (FCS), and non-essential amino acids (NEA) were obtained from Gibco (Eggenstein, Germany). F icol l -Hypaque medium was from Biochrom (Berlin, Germany). Phytohemagglutinin (PHA), concanavalin A (Con A), 12-O-tetradecanoylphorbol-13-acetate (TPA), and ionomycin were purchased from Sigma (Deisenhofen, Germany). [3H]Thymidine (spec. act. 25 Ci/mmol) was from Amersham (Braunschweig, Germany). Sinomenine (7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methylmorphinane-6-one) was kindly provided by L. D. Zhang (Shanxi Institute of Pharmaceutical Industry, Xi-an, China). The purity of the drug was checked by rp-HPLC as described elsewhere (Liu et al., 1994). A stock solution (0.1 M) of sinomenine was freshly prepared in RPMI 1640/1% dimethylsulf- oxide and further diluted in medium.

Animals

Female DBA/2 and C57/BL6 mice at the age of 8 - 1 0 weeks were purchased from the Zentrales Tierinstitut (Hannover, Germany).

Isolation o f mouse spleen cells and human peripheral blood mononuclear cells

Mouse spleen cells were isolated from whole spleens of DBA/2 and C57/BL6 mice, using a loosely fitting glass homogenizer. The cell suspen- sions were washed three times by centrifugation (1400 rpm, 10 min) using RPMI 1640 medium.

Human peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood drawn from healthy adults by density gradient centrifugation on Ficol l -Hypaque (whole blood : Ficoll = 1 . 5 : 1 , 1800rpm, 20min) and washed twice with RPMI 1640 medium (1600 rpm, 10 rain).

100

2 % 80

~ 60

(3

o 40

. E

E- 20

0 , , , , , , . i . . . . . . . . i . . . . . . . . J . . . . .

1 1 10 1@0 1000 Smemenane (~9/ml)

Fig. 1. Inhibitory effect of sinomenine on mouse spleen cell proliferation. Spleen cells from DBA/2 and C57/BL6 mice were activated in the absence or presence of sinomenine at the indicated concentrations by Con A (V1) or MLC (11) as described in Experimental Procedures. Proliferation was determined by [3H]thymidine incorporation after 2 (Con A) or 5 (MLC) days, respectively. Data are the mean ___ S.E.M. of three independent experiments. Mean control values

were 91029 cpm (Con A) or 7187 cpm (MLC).

Proliferation o f mouse spleen cells

For the measurement of lymphocyte activation [3H]thymidine incorporation was used as a marker. Briefly, 4 × 105 DBA/2 spleen cells were incubated in 96-well microtitre plates (Nunc, Wiesbaden, Germany) in a total volume of 0.2 ml in RPMI 1640 supplemented with L-glutamine (2 mM), Hepes (5 mM), /3-mercaptoethanol (50 CM), penicillin (100 U/ml), streptomycin (100~g/ml), and 10% FCS. The cells were activated with the mitogen Con A at a concentration of 5 ~g/ml. After a 48 h incubation at 37°C in a humidified atmosphere of 95°7o air/5% CO2 0.5 ~Ci [3H]thymidine was added and the cells further incubated for 4 h. The cells were harvested by an automatic cell harvester on glassfibre filters, and the radioactivity of dried filters was measured by liquid scintillation counting. In two-way MLC 2 x 105 cells from DBA/2 and C57/BL6 mice were mixed and incubated for 5 days. Then 0.5 ~Ci [3H]thymidine was added and the incubation was carried on for further 6 h. In these experiments sinomenine was added at the beginning of the cell culture at the indicated concentrations.

Proliferation o f PBMC

PBMC (4 x 105 ) were incubated in 96-well microtitre plates in a total volume of 0.2 ml in RPMI 1640 supplemented with L-glutamine (2 raM),

100

o

% 8o o

0 ~, U

o ~ 6o

• u

c ~ ~

o 40

2 ~

0 1 10 100 1000

Sinornenine (~g/ral)

Fig. 2. Inhibitory effect of sinomenine on human PBMC proliferation. Human PBMC were activated in the absence or presence of sinomenine at the indicated concentrations by PHA (O), TPA plus ionomycin (A), or MLC ( 0 ) as described in Experimental Procedures. Proliferation was determined by [3H]thymidine incorporation after 2 (PHA, TPA plus ionomycin) or 5 (MLC) days, respectively. Data are the mean of three independent experiments (activation by PHA or MLC) or one experiment with triplicate cell incubations (TPA plus ionomycin). Mean control values were 31665 cpm (PHA), 25079 cpm (TPA plus ionomycin),

or 16310 cpm (MLC).

N E A (1%), penicill in (100 U / m l ) , s t rep tomycin (100 tag/ml), and 10% FCS. The cells were act ivated with P H A at a concen t ra t ion of 10 tag/ml or with T P A ( 1 0 0 n g / m l ) plus ionomycin ( 5 0 0 n g / m l ) . Af ter incuba t ion of 48 h at 37°C in a humidi - fied a tmosphere of 95% a i r / 5 % CO2 0.5 ~Ci [3H]thymidine was added and the cul ture was carr ied on for fu r ther 4 h pr ior to cell harvest . In two-way M L C 2 x 105 P B M C f rom two dif ferent donors were mixed as described as for mouse MLC. In exper iments , in which the reversibil i ty of the drug ac t ion was tested, P B M C were act ivated by M L C in the presence (125 taM) or absence of s inomenine . Af ter 48 h the drug was washed of f with R P M I 1640/10% FCS for three t imes by centr i fuga- t ion. Then the cell cul ture was con t inued in the absence or af ter re -addi t ion of s inomenine (125/~M) and [3H]thymidine inco rpora t ion was de te rmined af ter fu r ther 3 days as described. In t ime kinetic exper iments h u m a n P B M C were act ivated by M L C as described above but s inomenine at a fixed concen t ra t ion of 100 taM was added at d i f ferent t ime points af ter the onset o f the MLC.

Proliferation of Jurkat cells

The h u m a n leukaemic T-cell l ymphob las t cell line Ju rka t was chosen to test any direct cytotoxic activity

Short Communication 687

Table 1. Reversibility of the anti-proliferative effect of sinomenine on human PBMC

cpm % of control

Control 8391 _ 853

Sinomenine (present for 5 days) 2470 -+ 367 49.4***

Sinomenine (washed off after 2 days) 7749 _+ 610 92.3 ....

Sinomenine (washed off after 2 days and re-added for 3 days)

3661 _+ 1003 56.5*

Proliferation of human PBMC activated by MLC for 5 days in the absense or presence of sinomenine at a fixed concentration (125/aM) was determined by [3H]thymidine incorporation. Sinomenine was either present for the whole incubation time or washed off after 2 days and then the culture was continued for 3 days in the absence or after re- addition of sinomenine (125/aM). Data are the mean _+ S.E.M. from one experiment with triplicate cell incubations. ***P<0.005; *P~<0.05; n.s., not significantly different from control.

of s inomenine on lymphocytes . Cells (5 x 104) were cul tured in 96-well microt i t re plates in a to ta l vo lume of 0.2 ml of R P M I 1640/5% FCS. S inomenine at the indicated concen t ra t ions was added at the beginning of the culture. Af ter incuba t ion for 48 h 0.5 taCi [3H]thymidine was added and the incuba t ion was con t inued for fur ther 4 h pr ior to cell harvest .

Statistics

Results are expressed as mean _+ S.E.M. Where appropr ia te , da ta were analysed for statistical s ignificance by S tuden t ' s t-test.

RESULTS

We first invest igated the effect of s inomenine on mi togen- induced prol i fera t ion of mouse spleen cells. The cells were act ivated either with Con A for 2 days or by M L C for 5 days in the absence or presence of s inomenine ( 0 . 1 6 - 329 tag/ml, cor responding to 0.5 t a M - 1 mM) added for the whole incuba t ion time. As can be seen in Fig. 1 s inomenine decreased [3H]thymidine inco rpora t ion of s t imulated lymphocytes in a dose-dependent manne r . A t the highest concen t ra t ion used (1 mM) no enhancemen t of spleen cell prol i fera t ion , compared to tha t of non-


Table 2. Effect of sinomenine on Jurkat cell proliferation c-

8,000 cpm % of control %

Control 8008 _+ 426 b EL

Sinomenine (/~M) ~ 6,000 3.9 7626 _+ 489 95.2 ~

E EL 7.8 7020 _+ 165 87.7 ~ u 4,000

15.6 8486 _+ 1163 106 - "0

31.2 7033 _+ 889 87.8 62.5 6523 _+ 439 81.5 .~

125 6909 _ 646 86.3 e- 2,000 250 9162 _+ 655 114 m 500 9046 _+ 214 113

1000 8972 _+ 741 112

Human leukaemic Jurkat cells were cultured in the absence or presence of sinomenine at the indicated concentrations for 2 days and proliferation was determined by [3H]thymidine incorporation. Data are the mean _+ S.E.M. from one experiment out of two similar ones with triplicate cell incubations.

act ivated cells, was seen. In the case of Con A- act ivated D B A / 2 spleen cells ha l f -maximal inhibi- t ion of pro l i fe ra t ion was achieved at a s inomenine concen t ra t ion of abou t 400 taM. Pro l i fe ra t ion induced by M L C was even more sensitive to inhib i t ion by this drug. Unde r this condi t ion an ICs0 value of abou t 60/aM was calculated (Fig. 1) but a very flat inh ib i t ion curve was ob ta ined .

In a similar way the prol i fera t ion of s t imulated h u m a n P B M C , p redominan t ly consist ing of lymphocytes, was also effectively inhibi ted by s inomenine (Fig. 2). C o m p a r a b l e lcs0 values regarding the anti- prol i ferat ive act ion of s inomenine were found under var ious ac t iva t ion condi t ions ( P H A : 34/aM; MLC: 100/aM; T P A / i o n o m y c i n : 129/aM).

A n addi t iona l exper imenta l design was chosen to test the reversibil i ty of the inh ib i to ry act ion of s inomenine on lymphocyte prol i fera t ion (Table 1). P B M C act ivated by M L C were t rea ted with sinomenine (125/aM), which led to abou t 50% reduct ion of cell pro l i fera t ion af ter 5 days. However , P B M C f rom which s inomenine had been washed of f af ter 48 h with R P M I 1640/10% FCS by cen t r i fuga t ion for three t imes showed a [3H]thymidine incorpora- t ion not d is t inguishable f rom tha t of cont ro l cul- tures. Re-addi t ion of s inomenine af ter washing again resulted in s ignif icant inh ib i t ion of cell p ro l i fe ra t ion (Table 1).

In a fu r ther exper iment the h u m a n leukaemic T-cell lymphoblas t cell line Ju rka t was used. In this au tonomous ly growing cellular system s inomenine did not show an inhib i tory inf luence on [3H]thymidine inco rpora t ion (Table 2).

0 c 0 1 2 4 8 24 48

Time aftzer NLC s t a r t (h rz )

Fig. 3. Time kinetics of the anti-proliferative effect of sinomenine on human PBMC. Human PBMC were activated by MLC for 5 days and proliferation was determined by [~H]thymidine incorporation as described in Experimental Procedures. Sinomenine (100/aM) was added at the indicated time points (0 -48 h) after the MLC start. Data are the mean _+ S.E.M. from one experiment out of two similar ones with triplicate cell incubations, c, Control incubation in the absence of sinomenine; ***P~<0.005;

*P~<0.05; n.s., not significantly different from control.

In all exper iments described so far s inomenine was added at the beginning of cell ac t ivat ion and remained in culture dur ing the whole incuba t ion t ime (2 or 5 days). In kinetic experiments s inomenine (100/aM) was added at dif ferent t ime points af ter the start of M L C (Fig. 3). A d d e d within 8 h af ter mixing of P B M C the drug markedly decreased cell prolife- ra t ion. W h e n given af ter 24 h a largely decreased but still s ignificant inh ib i tory effect became obvious. Inclus ion of s inomenine af ter 48 h but still for the last 3 days of M L C did not s ignif icantly change [3H]thymidine incorpora t ion compared to tha t of act ivated cells in the absence of the drug (Fig. 3).

DISCUSSION

Affec ted jo in ts of symptomat ic pat ients suffer ing f rom RA characterist ical ly exhibi t over lapping mani- festat ions of i n f l ammato ry processes, i.e. a b n o r m a l cellular and humora l i m m u n e responses, and synovial hyperplas ia (Gay et al., 1993). These recent results have suggested that par t icular ly T- lymphocytes have a central role in the pathogenesis of RA (Harr is , 1990). One of the main immunohis to log ica l characteris t ics of RA is the hyper t rophy of the synovial m e m b r a n e (SM). This is mainly due to the


o%

C19H23N04 (mol wt 329.38)

(7,8-didehydro-4-hydroxy-3,7-dirnethoxy- 17-methylmorphinane-6-one)

Fig. 4. Chemical structure and molecular weight of sinomenine.

entry of leukocytes from the circulation as well as the local proliferation of synoviai fibroblasts (Bergroth et aL, 1985). Numerous studies have established that synovial hypertrophy and hyperplasia of type A- and B-cells take place, in line with an increase in perivascular connective tissue accumulation of T-cells and monocytes/macrophages, and a marked new blood vessel formation (Gruber et al., 1988). Evidence pointing to the SM hypertrophy and the central role of T-cell activation in the pathogenesis of RA has fostered the development of several new therapeutic strategies (Kingsley et aL, 1990).

In studies in rats it was found that sinomenine, an alkaloid with anti-inflammatory and anti-rheumatic properties, could significantly inhibit granulation tissue growth induced by filter-paper disks soaked with formaldehyde (Saeki et al., 1975a, b). It has also been described that in mice sinomenine could inhibit both humoral and cellular immunological reactions (Wang & Xiao, 1992). After treatment of the animals with sinomenine (50-150 mg/kg/day, i.p.) for 5 days the weights of spleen and thymus were drastically reduced. Concomitantly, antibody production and delayed-type hypersensitivity stimulated by sheep red blood cells were also diminished by sinomenine. The same authors described that in vivo sinomenine suppressed spleen cell proliferation induced by LPS or Con A. It can be postulated that the above-mentioned pharmacological actions of sinomenine may contribute to its clinical benefit.

Here we show that sinomenine markedly inhibits & vitro proliferation of mouse spleen cells, activated either by the mitogen Con A or by two-way MLC.

Comparison of the inhibitory potencies of sinomenine in both systems revealed that the drug was more effective in the reduction of proliferation induced by MLC. We also examined the effect of sinomenine on human PBMC proliferation. In our hands sinomenine had a marked inhibitory effect on human lymphocyte proliferation stimulated either artificially by mitogens like PHA or TPA plus ionomycin, or in a more physiological way by MLC. Independent from the used activators a reduced PBMC proliferation was obvious even at low concentrations of the drug (1 - 10/aM).

In time kinetic experiments we obtained evidence that the anti-proliferative effect of sinomenine is based on the impairment of early T-cell activation steps. The drug had to be administered within the first 48 h after the onset of PBMC activation by MLC in order to achieve a significant reduction of [3H]thymidine incorporation after 5 days. After prolonged incubation times the addition and then continuous presence of sinomenine was without effect. From these results it can be concluded that the drug is not simply cytotoxic for PBMC. Accord- ingly, we could demonstrate that sinomenine acts in a reversible manner. When the drug was incubated with PBMC for 2 days in MLC and then washed off the cells could well proliferate again.

With regard to T-cell proliferation mitogenic or antigenic activation of quiescent lymphocytes induces an enhanced generation of inositol trisphosphate and diacylglycerol, which act as second messengers to liberate the intracellular calcium level and to activate protein kinase C (PKC), respectively


(Nishizuka, 1984; Berridge, 1987). PKC- or calcium- dependen t pa thways may be different ial ly involved in cellular processes induced by different mi togens (Planelles et al . , 1992). Our previous studies conf i rmed tha t s inomenine had a marked inhib i tory effect on e icosanoid and nitric oxide synthesis in macrophages , which might be due to an influence on the intracel lular calcium level and PKC activity of these cells (Liu et al . , 1994). The molecular mechanism by which s inomenine suppresses lymphocyte ac t iva t ion is far f rom being elucidated. However , it can be summar ized that the t ime kinetic exper iments suggest tha t an early ac t ivat ion step is impaired. Fu r the rmore , s inomenine inhibi ts with equal potency the pro l i fe ra t ion of h u m a n lymphocytes act ivated by mitogens , via the T-cell receptor (MLC), or by T P A plus ionomycin . This indicates tha t it affects processes distal to the act ivat ion of PKC.

Wi th respect to the role of lymphocytes in the pa thogenes is of RA it is clear tha t as a consequence of T-cell s t imula t ion lymphokines such as inter- feron-y are released, which then act ivate m o n o c y t e / macrophages to release a variety of cytokines as well as o ther i n f l a m m a t o r y media tors such as growth factors (Panayi , 1993). Signif icant levels of T-cell specific lymphokines could directly be detected in r h e u m a t o i d jo in ts (Firestein & Zvaif ler , 1990). This

also suppor ts the hypothesis tha t cytokines play an impor t an t role in lymphocyte prol i fera t ion and pathogenesis of RA. In our exper iments s inomenine could powerful ly inhibi t the pro l i fe ra t ion of h u m a n P B M C and mouse spleen cells caused by various act ivators but it had no inhib i tory influence on the prol i fera t ion of Ju rka t cells, which are lymphocyte- like cells whose pro l i fe ra t ion does not depend on lymphokine produc t ion . This implies tha t the reduct ion of T-cell p ro l i fe ra t ion by s inomenine is p robab ly related to its inf luence on cytokine synthesis. Exper iments address ing this aspect are in progress now.

In order to invest igate the in v ivo usefulness of s inomenine as an effective ant i -ar thr i t ic drug detailed studies on its pharmacokine t ics and organ cytotoxici ty as well as addi t iona l control led clinical studies have to be pe r fo rmed in the near future .

Acknowledgements - - We wish to thank H. Hartmann, A. Garbe, and C. Urban for their skilful technical assistance. We would also like to thank L. D. Zhang, Shanxi Institute of Pharmaceutical Industry, China, who has kindly provided us with pure sinomenine. This study was supported by a grant from the Deutsche Forschungsgemein- schaft (SFB 244). Liang Liu is a recipient of a scholarship from the Chinese Educational Committee.

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inhibition of lymphocyte proliferation by the anti-arthritic drug sinomenine

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