Hong-Wei Yao, Jun Li, Yong Jin, Yun-Fang Zhang, Chang-Yu Li, Shu-Yun Xu, Institute of Clinical Pharmacology, Anhui Medical University, Heifei 230032, Anhui Province, China; School of Pharmacy, Anhui Medical University, Heifei 230032, Anhui Province, China
Supported by Natural Science Foundations of Anhui Province, No. 98446733
Correspondence to: Prof. Jun Li, Institute of Clinical Pharmacology, Anhui Medical University; School of Pharmacy, Anhui Medical University, Heifei 230032, Anhui Province China. amuicplj@mail.hf.ah.cn
Telephone: +86-551-5161040 Fax: +86-551-5161040
Received: 2002-07-31 Accepted: 2002-09-12

Abstract
AIM: To study the effect of leflunomide on immunological liver injury (ILI) in mice.

METHODS: ILI was induced by tail vein injection of 2.5 mg Bacillus Calmette-Guerin (BCG), and 10 d later with 10 mg lipopolysaccharide (LPS) in 0.2 mL saline (BCG+LPS). The alanine aminotransferase (ALT), aspartate aminotransferase (AST), nitric oxide (NO) level in plasma and molondiadehyde (MDA), glutathione peroxidase (GSHpx) in liver homogenate were assayed by spectroscopy. The serum content of tumor necrosis factors-a (TNF-a) was determined by ELISA. Interleukin-1 (IL-1), interleukin-2 (IL-2) and Concanavalin A (ConA)-induced splenocyte proliferation response were determined by methods of 3H-infiltrated cell proliferation.

RESULTS: Leflunomide (4, 12, 36 mg.kg^-1) was found to significantly decrease the serum transaminase (ALT, AST) activity and MDA content in liver homogenate, and improve reduced GSHpx level of liver homogenate. Leflunomide (4, 12, 36 mg.kg^-1) significantly lowered TNF-a and NO level in serum, and IL-1 produced by intraperitoneal macrophages(PMF). Moreover, the decreased IL-2 production and ConA-induced splenocyte proliferation response were further inhibited.

CONCLUSION: These findings suggested that leflunomide had significant protective action on ILI in mice.

Yao HW, Li J, Jin Y, Zhang YF, Li CY, Xu SY. Effect of leflunomide on immunological liver injury in mice. World J Gastroenterol 2003; 9(2): 320-323
http://www.wjgnet.com/1007-9327/9/320.htm


INTRODUCTION
Earlier studies have identified leflunomide, an isoxazole derivative, as a unique immunomodulatory agent capable of treating rheumatoid arthritis, allograft and xenograft rejection, systemic lupus erythematosus, prostate carcinoma, and neuronal-glial tumours, etc^[1-12]. Our studies indicated that leflunomide had significantly therapeutic effects on the secondary inflammation response of adjuvant arthritis (AA) in rats. Recent evidence suggested the anti-inflammatory and immunoregulatory effects of leflunomide were related to its ability to suppress IL-1 and TNF-a selectively over their inhibitors in T lymphocyte/monocyte activation, and the activation of nuclear factor kappa B, a potent mediator of inflammation when stimulated by inflammatory egents^[13-16]. Jankovic reported that A₇₇₁₇₂₆, leflunomide's active metabolite, also had inhibitory effect on NO production and iNOS mRNA expression in IFN-g+LPS-activated murine and rat primary fibroblast^{[17, 18]}.
   As we known, the activity of cytokines such as TNF-a, IL-1, IL-6, NO and T cell mediated immunity were closely related to the degree of liver injury caused by virus, endotoxin, ConA, and GalN^[19-21]. Thus, inhibition of proinflammatory cytokines and regulation of host immunity would be beneficial to alleviating liver injury.
   Based on the immunological dysfunction in liver injury and leflunomide's immunomodulatory feature with high effication and low toxicity, we assumed that leflunomide might have therapeutic effect on ILI. To the best of our knowledge, however, there has been no report so far concerning the effect of leflunomide on ILI. In this study, therefore, we have clarified the therapeutical effect of leflunomide on ILI in mice.

MATERIALS AND METHODS
Animals and reagents
Male Kunming strain mice weighing 18-22 g were purchased from Animal Center of Anhui Medical University. Mice were allowed to take food and tap water ad libitum. Leflunomide was kindly donated by Cinkate Co., USA. ConA and LPS from Escherichia coli were purchased from Sigma Co., St. Louis, M, USA. 1, 1, 3, 1-tetraethoxypropane (TEP) and 5, 5?dithibis-(2-nitrobenzicacid) (DTNB) were purchased from FLUKA Co., Switzerland. BCG was purchased from Institute of Shanghai Biological Products.

Preparation of ILI ^[22]
   Each mouse was injected with 2.5 mg BCG (viable bacilli) in 0.2 mL saline via tail vein, and 10 d later with 10 mg LPS in 0.2 mL saline. At 0, 4, 8, and 12 h post-injection of LPS, animals received either leflunomide (4, 12, and 36 mg/kg, ig) or appropriate volume (25 mL/kg, ig) of vehicle (3 % prednisone). The mice were anesthetized with ether, then sacrificed by cervical dislocation 16 h after LPS injection and trunk blood was collected into heparinised tubes (50 U/mL) and centrifuged (1 500×g, 10 min, room temperature). Plasma was aspirated and stored at -70 ℃ until assayed as described below. The liver was also removed and stored at -70 ℃ until required.

Measurement of plasma ALT, AST, NO and TNF-a
Plasma ALT, and AST were determined using commercial kits produced by Institute of Shanghai Biological Products affiliated to the Ministry of Health. These activities are expressed as an international unit (U/L). Serum TNF-a and NO were measured using commercial kits produced by Sigma Co. and Beijing Biotinge-Tech., Co.Ltd, and their levels were expressed as pg.L^-1 and mmol.l^-1 respectively.

Measurement of MDA and GSHpx in liver homogenate
Livers were thawed, weighed and homogenized with Tris-Hcl buffer (5 mM containing 2 mM EDTA, pH 7.4). Homogenates were centrifuged (1 000×g, 10 min, room temperature) and the supernatant was used immediately for the assays of MDA and GSHpx. MDA was measured by the thiobarbituric acid method according to standard techniques (Gavino VG., 1981). The content of MDA was expressed as nmol per gram liver tissue. GSHpx was measured by the DTNB method, and its content was expressed as U per milligram protein.

Measurement of ConA induced splenocyte proliferation, IL-1 and IL-2
ConA induced splenocyte proliferation was determined according to the report by Yamamoto I in 1982. IL-1 and IL-2 were measured according to the reference (Liang JS, 1989; Ding GF, 1988).

Statistical analysis
Results were expressed by x±s. Statistical significance of differences between groups were determined by ANOVA followed by Student's t test. P value of less than 0.05 was considered statistically significance.

RESULTS
Therapeutic effects of leflunomide on ILI induced by BCG+LPS in mice
Results are shown in Tables 1 and 2. ALT, AST, and NO in plasma and MDA content in liver homogenate were significantly increased after the interval injection of BCG and LPS. Meanwhile, the GSHpx level in liver homogenate was sharply decreased. Both leflunomide (12, 36 mg/kg) and prednisone (3 mg/kg) could not only significantly decrease ALT, AST, NO and MDA level, but evidently increase GSHpx in mice with ILI.

Table 1 Effects of leflunomide on serum ALT and AST activities induced by BCG+LPS in mice (n=10, x±s)

^aP<0.05, ^bP<0.01 vs model group; ^dP<0.01 vs normal group.

Effect of leflunomide on IL-2 generation and ConA induced splenocyte proliferation
IL-2 and ConA induced splenocyte proliferation were significantly inhibited in the model group (Table 4). Leflunomide (4, 12, and 36 mg/kg) further inhibited IL-2 production and ConA induced splenocyte proliferation response.

DISCUSSION
It has been demonstrated that severe hepatitis could be induced by injecting a small dose of bacterial LPS into BCG-pretreated mice^[22]. In this article, ILI was successfully induced by BCG+LPS. On this basis, leflunomide (4, 12, and 36 mg/kg) could significantly lower the increased plasma transaminase level and MDA content in liver homogenate, meanwhile, GSHpx level rose significantly. All these indicated that leflunomide markedly protected ILI. Leflunomide significantly inhibited the generation of NO, TNF-a and IL-1 excreted by PMF, moreover, IL-2 production and ConA induced splenocyte proliferation was further inhibited by leflunomide. Therefore, the protective effects of leflunomide on ILI might be related with its function of balancing cytokine generation and modulating immune.
   As it is known, TNF-a is one of the important mediators in liver injury. It has been demonstrated that liver injury induced by endotoxin was conducted by TNF-a, and the activity of TNF-a was positively related with the extent of liver necrosis^[22-24]. However, TNF-a itself could not directly result in liver injury. The damaging degree of TNF-a on liver might be involved with infection, activity of Kupffer cell, and endogenous serine type protease, etc^[25-30]. TNF-a could act as the first factor of liver injury, its elevation would stimulate a number of proinflammatory mediators including NO, IL-1, IL-6, IL-8 and SIL-2R^[31-36], which further deteriorated the liver injury intoxicated by TNF. Therefore, although the TNF lever was low, liver was damaged significantly.
   Leflunomide, an immunomodulatory reagent, is mainly aimed to inhibit the activity of dihydroorotate dehydrogenase (DHODH) involved in de novo pyrimidine biosynthesis. But at a higher concentration, it mainly inhibited protein tyrosine kinases initiating signaling^{[1,13,14,37,38]}, and therefore could reduce the cell response to mitogen and cytokine. In the model of ILI induced by BCG+LPS, leflunomide could significantly lower the increased TNF-a level in serum, which agreed with the results of Smith's experiment that leflunomide significantly lowered the increased TNF level in joints from AA rats^[15,16,39]. As it is known, TNF mainly come from Kupffer cell in liver. In this article, leflunomide significantly inhibited TNF-a level in serum of ILI. It deserved further investigation on about whether it is related to leflunomide's effect of regulating the immunological dysfunction through inhibiting the growth and differentiation of Kupffer cell and production of TNF, thus, alleviating liver injury.
   As reported in documents, the synthesis of NO was regulated by many immunological factors including TNF-a, IL-1, and IFN-g, which is composed of a complicated web system, could act on hepatocytes, Kupffer cells and Ito in endotoxemia mice to increase the generation of NO^{[31,32,35,40]}. Likewise, LPS could also induce Ito cells to express iNOS and synthesis of a large amount of NO^[41,42]. According to our investigation, the effects of leflunomide to inhibit ILI might well be related with its function of decreasing the degeneration of NO.
   Although IL-1 itself has no damage on liver, its elevation could stimulate many kinds of immunological and inflammatory cells to excrete cytokine including TNF-a, IFN-g, IL-6, and IL-8, which mediate the inflammatory and immunological injury. Apart from these, IL-1,TNF-a, IFN-g and LPS could act on hepatocyte to enhance the expression of iNOS mRNA in synergetic manner, and to increase the generation of NO, thus deteriorating the liver injury. Leflunomide significantly regulated abnormal IL-1 level excreted by PMF in ILI mice in vivo, which agrees with Deage's investigation^[43] in effect of leflunomide on AA rats.
   Suzuki found that splenectomy could modulate the excretion of inflammatory mediators, which prevented liver injury intoxicated by LPS after hepatectomy. In this study, we discovered that IL-2 production and ConA induced splenocyte proliferation were reduced in ILI induced by BCG+LPS. However, leflunomide further inhibited the production of IL-2 and ConA induced splenocyte proliferation response. Hoskin et al^[44] reported that leflunomide inhibited the T lymphatic cell growth and response to IL-2 and production of IL-2. Further studies are needed to elucidate the relationship between the protective effect of leflunomide on ILI and its inhibitory action on cellular immune function.

REFERENCES
1    Sanders S, Harisdangkul V. Leflunomide for the treatment of rheumatoid arthritis and autoimmunity. Am J Med
      Sci 2002; 323: 190-193
2    Wendling D. Leflunomide in the treatment of rheumatoid arthritis. Ann Med Interne 2002; 153: 21-24
3    Alldred A, Emery P. Leflunomide: a novel DMARD for the treatment of rheumatoid arthritis. Expert Opin
      Pharmacother 2001; 2: 125-137
4    Jakez-Ocampo J, Richaud-Patin Y, Simon JA, Llorente L. Weekly dose of leflunomide for the treatment of
      refractory rheumatoid arthritis: an open pilot comparative study. Joint Bone Spine 2002; 69: 307-311
5    Williams JW, Mital D, Chong A, Kottayil A, Millis M, Longstreth J, Huang W, Brady L, Jensik S, Anita C, Anita K, Michael
      M, James L, Wanyun H, Lynda B, Stephen J. Experiences with leflunomide in solid organ transplantation.
      Transplantation 2002; 73: 358-366
6    Jin MB, Nakayama M, Ogata T, Fujita M, Mino K, Taniguchi M, Suzuki T, Shimamura T, Furukawa H, Todo S. A
      novel leflunomide derivative, FK778, for immunosuppression after kidney transplantation in dogs. Surgery 2002; 132: 72-79
7    Barthel HR. Leflunomide for the treatment of systemic lupus erythematosus: comment on the article by McMurray.
      Arthritis Rheum 2001; 45: 472
8    Kessel A, Toubi E. Leflunomide in systemic lupus erythematosus. Harefuah 2002; 141: 355-357
9    Remer CF, Weisman MH, Wallace DJ. Benefits of leflunomide in systemic lupus erythematosus: a pilot observational
      study. Lupus 2001; 10: 480-483
10  Shawver LK, Schwartz DP, Mann E, Chen H, Tsai J, Chu L, Taylorson L, Longhi M, Meredith S, Germain L, Jacobs JS, Tang
      C, Ullrich A, Berens ME, Hersh E, McMahon G, Hirth KP, Powell TJ. Inhibition of platelet-derived growth factor-mediated
      signal transduction and tumor growth by N-[4-(trifluoromethyl)-phenyl]5-methylisoxazole-4-carboxamide. Clin Cancer
      Res1997; 3: 1167-1177
11  Xu X, Shen J, Mall JW, Myers JA, Huang W, Blinder L, Saclarides TJ, Williams JW, Chong ASF. In vitro and in vivo
      antitumor activity of a novel immunomodulatory drug, leflunomide: mechanisms of action. Biochem
      Pharmacol 1999; 58: 1405-1413
12  Huang M, Wang Y, Collins M, Mitchell BS, Graves LM. A77 1726 induces differentiation of human myeloid leukemia K562
      cells by depletion of intracellular CTP pools. Mol Pharmacol 2002; 62: 463-472
13  Herrmann ML, Schleyerbach R, Kirschbaum BJ. Leflunomide: an immunomodulatory drug for the treatment of
      rheumatoid arthritis and other autoimmune diseases. Immunopharmacology 2000; 47: 273-289
14  Breedveld FC, Dayer JM. Leflunomide: mode of action in the treatment of rheumatoid arthritis. Ann Rheum
      Dis 2000; 59: 841-849
15  Li WD, Ran GX, Teng HL, Lin ZB. Dynamic effects of leflunomide on IL-1, IL-6, and TNF-a activity produced from
      peritoneal macrophages in adjuvant arthritis rats. Acta Pharmacol Sin 2002; 23: 752-756
16  Manna SK, Mukhopadhyay A, Aggarwal BB. Leflunomide Suppresses TNF-Induced Cellular Responses: Effects on
      NF-kB, Activator Protein-1, c-Jun N-Terminal Protein Kinase, and Apoptosis. J Immunol 2000; 165: 5962-5969
17  Jankovic V, Samardzic T, Stosic-Grujicic S, Popadic D, Trajkovic V. Cell-specific inhibition of inducible nitric oxide
      synthase activation by leflunomide. Cell Immunol 2000; 199: 73-80
18  Trajkovic V. Modulation of inducible nitric oxide synthase activation by immunosuppressive drugs. Curr Drug
      Metab 2001; 2: 315-329
19  Hoek JB, Pastorino JG. Ethanol, oxidative stress, and cytokine-induced liver cell injury. Alcohol 2002; 27: 63-68
20  Sass G, Koerber K, Tiegs G. TNF tolerance and cytotoxicity in the liver: the role of interleukin-1beta, inducible
      nitric oxide-synthase and heme oxygenase-1 in D-galactosamine-sensitized mice. Inflamm Res 2002; 51: 229-235
21  Trautwein C, Rakemann T, Malek NP, Piumpe J, Tiegs G, Manns MP. Concanavalin A-induced liver injury triggers
      hepatocyte proliferation. J Clin Invest 1998; 101: 1960-1969
22  Wang GS, Liu GT. Influences of Kupffer cell stimulation and suppression on immunological liver injury in mice.
      Acta Pharmaceutica Sinica 1997; 18: 173-176
23  Zhang XL, Quan QZ, Sun ZQ, Wang YJ, Jiang XL, Wang D, LI WB. Protective effects of cyclosporine A on T-cell
      dependent ConA-induced liver injury in Kunming mice. World J Gastroenterol 2001; 7: 569-571
24  Zang GQ, Zhou XQ, Yu H, Xie Q, Zhao GM, Wang B, Guo Q, Xiang YQ, Liao D. Effect of hepatocyte apoptosis induced by
      TNF-a on acute severe hepatitis in mouse models. World J Gastroenterol 2000; 6: 688-692
25  Enomoto N, Ikejima K, Yamashina S, Hirose M, Shimizu H, Kitamura T, Takei Y, Sato And N, Thurman RG. Kupffer
      cell sensitization by alcohol involves increased permeability to gut-derived endotoxin. Alcohol Clin Exp
      Res 2001; 25 (Suppl): 51S-54S
26  Enomoto N, Yamashina S, Kono H, Schemmer P, Rivera CA, Enomoto A, Nishiura T, Nishimura T, Brenner DA, Thurman
      RG. Development of a new, simple rat model of early alcohol-induced liver injury based on sensitization of Kupffer
      cells. Hepatology 1999; 29: 1680-1689
27  Nagaki M, Muto Y, Ohnishi H, Moriwaki H. Significance of tumor necrosis factor (TNF) and interleukin-1 (IL-1) in
      the pathogenesis of fulminant hepatitis: possible involvement of serine protease in TNF-mediated liver injury.
      Gastroenterologia Japonica 1991; 26: 448-455
28  Murr MM, Yang J, Fier A, Kaylor P, Mastorides S, Norman JG. Pancreatic elastase induces liver injury by activating
      cytokine production within Kupffer cells via nuclear factor-Kappa B. J Gastrointest Surg 2002; 6: 474-480
29  McClain CJ, Hill DB, Song Z, Deaciuc I, Barve S. Monocyte activation in alcoholic liver disease. Alcohol 2002; 27: 53-61
30  Hoebe KHN, Witkamp RF, Fink-Gremmels J, Miert ASJPAM, Monshouwer M. Direct cell-to-cell contact between Kupffer
      cells and hepatocytes augments endotoxin-induced hepatic injury. Am J Physiol Gastrointest Liver
      Physiol 2001; 280: G720-G728
31  Muntane J, Rodriguez FJ, Segado O, Quintero A, Lozano JM, Siendones E, Pedraza CA, Delgado M, O'alle F,
      Garcia R, Montero JL, De La Mata M, Mino G. TNF-alpha dependent production of inducible nitric oxide is involved in
      PGE(1) protection against acute liver injury. Gut 2000; 47: 553-562
32  Nadler EP, Dickinson EC, Beerstolz D, Alber SM, Watkins SC, Pratt DW, Ford HR. Scavenging nitric oxide reduces
      hepatocellular injury after endotoxin challenge. Am J Physiol Gastrointest Liver Physiol 2001; 281: G173-G181
33  Sopena B, Fernandez-Rodriguez CM, Martinez Vazquez C, Mendez MX, de la Fuente J, Freire M, Arnillas E, Outon A.
      Serum levels of soluble interleukin-2 receptor in alcoholic patients. An Med Interna 1998; 15: 189-193
34  Tulek N, Saglam SK, Saglam M, Turkyilmaz R, Yildiz M. Soluble interleukin-2 receptor and interleukin-10 levels in patients
      with chronic hepatitis B infection. Hepatogastroenterology 2000; 47: 828-831
35  Shiratori Y, Ohmura K, Hikiba Y, Matsumura M, Nagura T, Okano K, Kamii K, Omata M. Hepatocyte nitric oxide production
      is induced by Kupffer cells. Dig Dis Sci 1998; 43: 1737-1746
36  Simeonova PP, Gallucci RM, Hulderman T, Wilson R, Kommineni C, Rao M, Luster MI. The role of tumor necrosis factor-
      alpha in liver toxicity, inflammation, and fibrosis induced by carbon tetrachloride. Toxicol Appl
      Pharmacol 2001; 177: 112-120
37  Xu X, Gong H, Blinder L, Shen J, Williams JW, Chong AS. Control of lymphoproliferative and autoimmune disease in MRL-
      lpr/lpr mice by brequinar sodium: mechanisms of action. J Pharmacol Exp Ther 1997; 283: 869-875
38  Elder RT, Xu X, Williams JW, Gong H, Finnegan A, Chong AS. The immunosuppressive metabolite of leflunomide,
      A₇₇₁₇₂₆, affects murine T cells through two biochemical mechanisms. J Immunol 1997; 159: 22-27
39  Smith-Oliver T, Noel LS, Stimpson SS, Yarnall DP. Connolly KM. Elevated levels of TNF in the joints of adjuvant arthritic
      rats. Cytokine 1993; 5: 298-304
40  Billiar TR. The delicate balance of nitric oxide and superoxide in liver pathology. Gastroenterology 1995; 108: 603-605
41  Saibara T, Ono M, Iwasaki S, Maeda T, Onishi S, Hayashi And Y, Enzan H. Effects of ethanol on L-arginine transport in rat
      Ito cells in relation to nitric oxide production. Alcohol Clin Exp Res 2001; 25( Suppl): 39S-45S
42  Rockey DC, Chung JJ. Inducible nitric oxide synthase in rat hepatic lipocytes and the effect of nitric oxide on
      lipocyte contractility. J Clin Invest 1995; 95: 1199-1206
43  Deage V, Burger D, Dayer JM. Exposure of T lymphocytes to leflunomide but not to dexamethasone favors the production
      by monocytic cells of interleukin-1 receptor antagonist and the tissue-inhibitor of metalloproteinases-1 over that of
      interleukin-1beta and metalloproteinases. Eur Cytokine Netw 1998; 9: 663-668
44  Hoskin DW, Taylor RM, Makrigiannis AP, James H, Lee TD. Dose-dependent enhancing and inhibitory effects of
      A₇₇₁₇₂₆ (leflunomide) on cytotoxic T lymphocyte induction. Int J Immunopharmacol 1998; 20: 505-513