Differential induction of metallothionein synthesis by interleukin-6 and tumor necrosis factor-α in rat tissues

lnt. J. Immunopharmac., Vol. 16, No. 2, pp. 187-195, 1994 Elsevier Science Ltd Copyright © 1994 International Society for lmmunopharmacology Printed in Great Britain. All rights reserved 0192-0561/94 $6.00 + .00

~ Pergamon

D I F F E R E N T I A L I N D U C T I O N OF M E T A L L O T H I O N E I N SYNTHESIS BY I N T E R L E U K I N - 6 A N D T U M O R NECROSIS F A C T O R - a IN RAT TISSUES* M A S A O S A T O , t* M I S A O SASAKI t a n d

HIROSHI HOJO §

*Environmental Pollution Research Laboratory, Research Laboratory, Fukushima Medical College, 1 Hikarigaoka, Fukushima, 960- 12 Japan; and ~Department of Hygienic Chemistry, Pharmaceutical Institute, Tohoku University, Aoba-ku, Sendai, 980 Japan

(Received 26 May 1993 and in final form 10 August 1993)

Abstract - - Metallothionein (MT) synthesis induced by the inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-a (TNF), was studied in vivo. Administration of recombinant human IL-6 or TNF to rats caused the acute phase responses including rapid decreases in plasma zinc (Zn), and increases in plasma copper (Cu) and ceruloplasmin. Hepatic concentration of MT-1, one of MT isoforms, began to increase within 3 h after the injection of IL-6 or TNF. In IL-6-treated rats, MT-I concentration in liver reached a maximum level at 12 h and decreased with a transient rebound, whereas, in TNF-treated rats, a high level of MT-1 lasted for about 48 h. MT-II, the other MT isoform, was induced more than MT-I in liver by both cytokines. MT-I was also induced in lung and heart by TNF, but little by IL-6. The data suggest that IL-6 may be responsible for MT synthesis in liver, whereas TNF may be responsible not only in liver but also in lung and heart. Furthermore plasma concentration of MT did not always reflect the enhanced concentration of MT by TNF and IL-6 in liver, suggesting involvement of many factors influencing plasma MT levels. The interrelation between IL-6 and TNF for MT synthesis has also been discussed.

Disturbances of the physiological homeostasis lead to a complex series of local and systemic reactions (Geiger, Andus, Klapproth, Hirano, Kishimoto & Heinrich, 1988). An increase in synthesis of acute phase proteins such as fibrinogen, C-reactive protein, aracid glycoprotein in liver is a major feature of the systemic reactions (Heinrich, Castell & Andus, 1990). Recently, metallothionein (MT), which is a low molecular, ubiquitous, and metal binding protein, has also been referred to as one of the acute phase proteins (Sato, Sasaki & Hojo, 1993). It is widely believed that MT has a protective role against toxicity of heavy metals and regulates the metabolism of the essential metals such as zinc (Zn) and copper (Cu) (Kagi, Hunziker & Vasak, 1990). Moreover, it has been proposed that MT may play a protective role as a radical scavenger (Thornalley & Vasak, 1985). MT synthesis is induced not only by metals such as Zn, Cu and cadmium (Cd) but also by

a variety of compounds including cytokines, hormones such as glucocorticoid and glucagon, drugs and poisons, though the significance of the induction is uncertain (Bremner, 1987). Among cytokines, interleukin-1 (IL-1) (DiSilvestro & Cousins, 1984) and interleukin-6 (IL-6) (Schroeder & Cousins, 1990), tumor necrosis factor-a (TNF) (Grimble & Bremner, 1 9 8 9 ) and interferon (Friedman & Stark, 1985) have the ability to induce synthesis of MT in tissues and cells. It has been reported that induction of MT synthesis by bacterial endotoxin is mediated by IL-1, IL-6 and TNF (De, McMaster & Andrews, 1990; Liu, Liu, Sendelbach & Klaassen, 1991). Exposure to oxidative stresses increased the plasma and hepatic concentrations of MT in rats (Sato, 1991). Pretreatment with dexamethasone, which is an inhibitor of the secretion of cytokines from macrophages, prevented the increase in MT synthesis by oxidative stress, suggesting that MT synthesis induced by oxidative stress is mediated

*This work was presented in part at the 3rd International Meeting on Metallothionein, Tsukuba, Japan, 9 December 1992. *Author to whom correspondence should be addressed. 1~7

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through cytokines (Min, Mukai, Ohta, Onosaka & Tanaka, 1992). The mechanisms of MT induction by cytokines and the role of MT during the acute phase response are not yet known. IL-6 is the major regulator of the acute phase protein synthesis (Geiger et al., 1988). Administration of IL-1 into rats induces tissue specific synthesis of MT (Cousins & Leinart, 1988), but lately, IL-6 rather IL-1 has been ascribed as a mediator of hepatic MT production and/or Zn metabolism (Schroeder & Cousins, 1990). We previously reported tissue specific synthesis of MT induced by TNF (Sato, Sasaki & Hojo, 1992). However, the relation between IL-6 and TNF in the regulation of MT induction is uncertain. Although IL-6 induces expression of MT mRNA in rat liver (De et al., 1990), the effect of IL-6 on MT levels in other organs than liver has not yet been examined. To better understand the significance of the MT induction by cytokines, the present study was designed to evaluate the effects of recombinant human (rh) IL-6 and rh TNF on MT synthesis and Zn metabolism in vivo. Tissue specificity, time course, and isoforms of MT induced by IL-6 or TNF, were studied. EXPERIMENTAL PROCEDURES

R eagen ts

B o l t o n - H u n t e r reagent was obtained from ICN Biochemicals Inc. (Irvine, CA, U.S.A.). Donkey anti-sheep-goat IgG serum was obtained from Scottish Antibody Production Unit (Carluke, Lanarkshire, U.K.). Sheep anti-rat liver MT-I serum was kindly provided from Dr Ian Bremner (Rowett Research Institute, Aberdeen, U.K.). Recombinant human TNF-a (2.2 x 106 units/mg) or IL-6 (4 / 106 units/mg) was given by Asahikasei Co. Ltd (Tokyo) or by Ajinomoto Co. Ltd (Yokohama, Japan), respectively. TNF was assayed by the cytotoxicity test using L-M cells and the activity in units/ml is defined as the reciprocal of the dilution resulting in 50°7o cell survival. The specific activity of IL-6 was estimated by an E p s t e i n - B a r r virustransformed SKW6-CL-4 cell line (Hirano et al., 1986). Animals

Wistar male rats (Funabashi Farm Co., Funabashi, Japan), weighing approximately 83 + 1 g in IL-6-treated group or 87 _+ 1 g in TNF-treated group were used. Rats were housed in groups of four per cage in an environmentally controlled room (light on

0 7 : 0 0 - 19:00 h; temperature 23.0 _+ 1.5°C; humidity 55 + 5%). Animals were allowed free access to water and a commercial laboratory chow diet (Clea Japan Inc., Tokyo). Dose-dependent increases in MT-I were observed in tissues of both the TNFtreated rats (Sato et al., 1992) and the IL-6-treated rats (data not shown). The concentration of the TNF or IL-6 which induces hepatic synthesis of MT to a similar extent was chosen. Rats were injected subcutaneously with TNF (105 units/rat), or IL-6 (5 x 104 units/rat). Saline was injected as the control (5 ml/kg). Heparinized blood was collected by cardiac puncture under pentobarbital anesthesia. Rats were killed 0, 3, 6, 12, 24, 36 and 48 h after the injection. Tissues were quickly removed and stored at - 4 0 ° C before use. Determination o f Zn and M T

Tissues were homogenized in nine volumes of cold 50 mM T r i s - H C 1 , pH 8.0. For analyzing Zn a portion of homogenates was digested with acid mixture (HNO3/HC104/H2SO4, 5 : 2 : 1, by vol. and HNO~/HC104, 5 : 1 , v/v). After digestion the inorganic residues were dissolved in ultrapure water (Japan Millipore Ltd, Tokyo) and metal analysis was carried out by atomic absorption by a Hitachi spectrometer Z-6100. Two forms of MT have been identified in the rat and are referred to as metallothionein-I (MT-I) and metallothionein-II (MT-II) (Kagi et al., 1990). The homogenates were centrifuged at 1500g for 15 rain, and the supernatants were used for the estimation of total MT by the Cd-heme method (Onosaka, Tanaka, Doi & Okahara, 1978; Onosaka & Cherian, 1981) or MT-I in the radioimmunoassay which was developed by Mehra and Bremner (1983). This radioimmunoassay is suitable for measurement of 100-25,000 pg of MT-I in all tissues and fluids. MT-I and MT-II contents in the liver were determined by the FPLC method (Sato et al., 1992). Statistical analysis

Data were analyzed by analysis of variance, followed by Duncan's multiple range test, or, where applicable, by Student's t-test. The acceptable level of significance was set at P<0.05. RESULTS

Effects o f T N F and IL-6 administration on the plasma concentrations o f Zn, Cu, and ceruloplasmin

The plasma concentration of Zn decreased at 3 h after injection of TNF or IL-6, and showed the

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lowest level at 6 h, t h e n r e t u r n e d to n o r m a l levels at 24 h (Fig. 1). T h e extent o f decrease was a little larger in T N F - t r e a t e d rats t h a n in IL-6-treated rats at the dose used in the experiment. In c o n t r a s t to Z n , the p l a s m a c o n c e n t r a t i o n s o f Cu a n d c e r u l o p l a s m i n increased after i n j e c t i o n o f T N F or IL-6 (Fig. 1). The kinetics s u p p o r t s the fact t h a t m o s t o f the Cu is b o u n d to c e r u l o p l a s m i n in the plasma. T h e m a x i m a l increase o f Cu a n d c e r u l o p l a s m i n was greater in TNF-treated rats t h a n in IL-6-treated rats. A d m i n i s t r a t i o n o f T N F to rats resulted in decreases in b o d y weight at 6, 12, 24 a n d 36 h postinjection whereas IL-6 did n o t show a n y changes c o m p a r e d with the controls.

Hepatic concentrations of Z n , Cu and MT-I after injection of TNF and IL-6 T h e a d m i n i s t r a t i o n o f either T N F or IL-6 to rats increased hepatic c o n c e n t r a t i o n s o f Z n , but did n o t affect the Cu levels d u r i n g the experiments (Fig. 2). Hepatic M T - I levels were r e m a r k a b l y increased after T N F or IL-6 a d m i n i s t r a t i o n (Fig. 3). M T - I levels reached the m a x i m u m at 12 h after injection of IL-6 a n d rapidly decreased with a transient r e b o u n d , whereas in the T N F - t r e a t e d rats the high levels of M T - I lasted for a long period (48 h). Elution profiles o f F P L C c o l u m n c h r o m a t o g r a p h y

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E f f e c t o f T N F a n d I L - 6 administration on M T - I concentration in other tissues T N F or IL-6 a d m i n i s t r a t i o n significantly increased MT-I c o n c e n t r a t i o n s in the lung a n d heart (Fig. 5), however, the extent o f the m a x i m a l increase was m u c h greater in T N F - t r e a t e d rats (3 - 4-fold) t h a n in IL-6-treated rats (1.4-fold). T h e c o n c e n t r a t i o n of MT-I in the lung a n d h e a r t of T N F - t r e a t e d rats r e m a i n e d elevated over the d u r a t i o n of the study. The M T - I levels in the kidney a n d t h y m u s

were not affected by either cytokines (Fig. 6), and MT-I c o n c e n t r a t i o n s in the spleen were decreased by injection of T N F (Fig. 7), a l t h o u g h the m e c h a n i s m is u n k n o w n . IL-6 h a d little effect on M T - I levels in the spleen. The p l a s m a MT-I levels after injection o f T N F b e g a n to increase at 3 h a n d r e m a i n e d elevated at 48 h with a peak at 12 h (Fig. 8). IL-6 also increased the MT-I levels, but the increase was very small c o m p a r e d with that caused by TNF. DISCUSSION The present study d e m o n s t r a t e d t h a t M T synthesis by T N F or IL-6 was induced in tissues a n d the tissue specificity was different between the two cytokines.

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(Fig. 6) and expression of MT m R N A , but induced expression of manganese superoxide dismutase m R N A (data not shown). These data indicate that receptor for T N F exists in the kidney cells, but MT induction does not occur in this organ postbinding of TNF. T N F and IL-6 produced a transient depression of the plasma Zn levels (Fig. 1) and concomitant increases in hepatic concentrations of Zn (Fig. 2) and M T (Fig. 3). The increased M T was found as Znbound form (data not shown), and contained both MT-I and MT-II (Fig. 4). Concentrations of MT-I began to increase soon after injections of T N F or IL-6 in the liver. MT-I levels remained elevated in the liver, lung and heart of TNF-treated rats over the duration of the experiment. This persistence of high levels may be due to the concerted effect of several kinds of cytokines released from a variety of cells upon a stimulation with TNF. T N F has been reported to effectively induce hepatic expression of

Metallothionein Synthesis by IL-6 and TNF mRNA of IL-1 (De et al., 1990). IL-1 administration gradually increases hepatic concentrations of MT in rats, which peak at 36 h postinjection (Cousins & Leinart, 1988). TNF administration also induces circulating IL-6 (Mclntosh et al., 1989), whereas IL-6 does not induce expression of mRNA of any cytokines in mouse liver (De et al., 1990). It is possible that the injected TNF induces IL-6 secretion at local sites and indirectly induces MT synthesis. However, MT-1 concentrations in the lung were obviously greater in TNF-treated rats than in IL-6-treated rats (Fig. 5), suggesting that TNF directly induces MT synthesis in the tissues. The fact that TNF induces gene expression of manganese superoxide dismutase mRNA, in rat pulmonary epithelial as well as endothelial cells (Shiki, Meyrick, Briglram & Burr, 1987), suggests that TNF may act directly to induce MT synthesis in the lung. Even though the MT-I level observed in the liver of TNF-treated rats was comparable to that of IL-6treated rats (Fig. 3), the plasma MT-I level was higher in TNF-treated rats than in the IL-6-treated ones (Fig. 8). Similar results were obtained in fasted rats, that is, restriction of food intake resulted in increases in the hepatic concentration of MT, but has little effect on plasma MT levels (Sato & Sasaki, 1991). Although secretion of MT into blood is thought to be a normal physiological process, the data suggest that plasma concentration of MT does not always reflect hepatic concentrations of MT. In addition, the maximal increase of MT in tissues by TNF was observed at 24 h or later (Figs 3 and 5), whereas the peak in plasma was at 12 h (Fig. 8). Although the mechanism by which MT secretes from tissues into the blood is still unknown, TNF itself may affect secretion process of MT early times after the injection, increasing the plasma levels of MT. Increases in plasma concentration of MT have also been reported in animals exposed to endotoxin (Sato, Mehra & Bremner, 1984), paraquat (Sato & Sasaki, 1991; Sato, Ohtake, Takeda, Mizunuma & Nagai, 1989), carbon tetrachloride (Sato & Sasaki, 1991) and in animals subjected to the restraint stress (Hidalgo, Campany, Borras, Garvey & Armario, 1989). The source of the plasma MT has not yet been

193

established, however, factors affecting plasma MT levels have been discussed (Bremner, Mehra & Sato, 1985). Increased plasma MT concentrations accompany an increase in the degree of liver damage (Sato et al., 1984), but TNF administration did not injure liver tissues, judging from the absence of increase in aspartate aminotransferase activities of plasma (data not shown). Further studies are required to establish factors influencing excretion of MT into the blood. Whether TNF plays a role in secreting MT from tissues to the blood or not should also be investigated. Physiological roles of MT induced by cytokines are still unknown. In tissue injury, infection or inflammation, cytokines such as IL-I, IL-6 and TNF are released from macrophages, fibroblasts and endothelial cells to serum, and mediate acute phase protein synthesis (Geiger et al., 1988). Induction of MT synthesis was observed in the liver, lung and heart of TNF-treated rats, whereas, in IL-6-treated rats MT induction seemed to be restricted in the liver (Figs 3 and 5). In addition, plasma MT levels increased only slightly in IL-6-treated rats (Fig. 8). These data suggest that the target organ of IL-6 is the liver and that IL-6 exerts an important role there. Hepatic MT induced by IL-6 or TNF may act as a radical scavenger, since pretreatment of hepatocytes with IL-6 or TNF prevents cell death caused by carbon tetrachloride in liver (Schroeder & Cousins, 1990), and MT has a strong ability to scavenge free radicals (Thornalley & Vasak, 1985). Inflammation is known to cause oxidative damage to tissues and cells. The physiological role of MT induced by inflammatory cytokines may be the protection of the tissues from free radicals generated during inflammation. Further studies to establish roles of MT induced by cytokines are under investigation in our laboratory. Acknowledgements - - The authors are grateful to Ms

Emiko Yoshioka for secretarial work and the staff of the Experimental Animal Laboratory of the Fukushima Medical College for the care of the animals. The authors express appreciation to Asahikasei Corp. Ltd, and Ajinomoto Corp. Ltd for supplying the recombinant human TNF and IL-6, respectively.

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