INTERLEUKIN-6 ENHANCES THE EXPRESSION OF TUMOR NECROSIS FACTOR RECEPTORS ON HEPATOMA CELLS AND HEPATOCYTES Sigrid Van Bladel,
Claude Libert,
Walter Fiers”
We have studied the effect of interleukin-6 (IL-6) on the binding of tumor necrosis factor (TNF) to various cell lines. A significant increase (up to 250%) in binding was observed on rat hepatocytes and on the human hepatoma cell line HepG2, while no changes in the number of cells or cell morphology could be observed. Scatchard plot analysis showed that IL-6 enhanced the number of TNF receptors without affecting the receptor affinity. The effect reached plateau levels after approximately 6 h and at IL-6 concentrations of 10 @ml. It could be completely eliminated by cotreatment of cells with anti-IL-6 antibodies, but not by treatment with anti-interferon-y (IFN-y), suggesting that IFN-y, which can enhance TNF receptor expression on a variety of cells, was not a mediator in this IL-6 effect. Treatment with inhibitors of protein or RNA synthesis completely abolished the IL&induced increase, suggesting that IL-6 caused an enhanced transcription of TNF receptor mRNA. IL-1 had no effect on TNF binding to HepG2. However, when cells were cotreated with IL-1 and IL-6, IL-1 could completely abrogate the IL-6 effect. Copyright o 1991 by W.B. Saunders Company
Tumor necrosis factor (TNF) is a cytokine which is produced primarily by activated macrophages and which exerts a wide variety of biological activities in vitro and in vivo. It is cytotoxic for tumor cells, stimulates the growth of fibroblasts, can modulate gene expression, and is believed to play a role in a number of human pathologies.’ TNF binds to high-affinity receptors (Kd = 10-l’ to lo-l1 M) and is subsequently internalized and degraded.2,3 TNF receptors are detectable on all cells, with the exception of erythrocytes, unstimulated T cells, and cells with autocrine TNF production. The number of receptors per cell varies from a few hundred to ten thousands, but has not proven, so far, to correlate with the responsiveness of cells to TNF. Interleukin-6 @L-6), like TNF, is a pleiotropic protein. It acts as a growth and/or differentiation factor on B and T lymphocyte subsets, leads to induction of adrenocorticotropic hormone synthesis in vivo, and is the major inducer of acute-phase proteins in hepatocytes.4 The liver seems to play a central role in the in vivo
Laboratory of Molecular Biology, State University, straat 35, B-9000 Gent, Belgium. *To whom reprint requests should be sent. Copyright o 1991 by W.B. Saunders Company 1043-4666/91/0302-0002$05.00/O KEY
WORDS:
CYTOKINE,
IL-6/TNF
K.L.
receptors/hepatocytes
Vol. 3, No. 2 (March),
1991: pp 149-154
Ledeganck-
action of both TNF and IL-6: it might be involved in alleviating the toxic effect of TNF,’ and responds to IL-6 by the production of acute-phase proteins.6 In this paper we present results regarding the effect of IL-6 on the expression of TNF receptors on various cell lines and more particularly on a human hepatoma cell line (HepG2).
RESULTS Initially, several cell lines were tested for a possible effect of IL-6 on the binding of TNF to its receptor (Table 1). Only in the case of HepG2 and of freshly prepared rat hepatocytes could a significant increase in binding be observed after treatment for 24 h with recombinant human (rh) IL-6. This IL-bcaused increase in TNF binding on HepG2 cells was observed repeatedly and varied within the range of 80 to 250%. For hepatocytes, an increase of 22 to 100% was observed. Interestingly, the omission of insulin from the hepatocyte culture medium caused a decrease in TNF binding. No changes in the number of cells or cell morphology could be observed after IL-6 treatment. In order to investigate whether the IL-6-induced increase in binding was caused by an alteration of receptor affinity or by an enhancement of receptor expression, binding was performed with varying amounts of ligand on both untreated and IL-6-treated HepG2 cells and the results analyzed in a Scatchard 149
150 1 Van Bladel,
Libert,
CYTOKINE,
Fiers
Vol. 3, No. 2 (March 1991: 149-1.54)
Table 1. Influence of IL-6 on the specific binding of TNF to various cell lines. Cells were incubated for 24 h with 1 &ml rhIL-6. Binding assays with ‘%rmTNF were then performed as described in Materials and Methods. Rat primary hepatocytes were isolated 48 h prior to addition of IL-6 and cultured either with (+) or without (-) 0.1 pM dexamethasone (DEX) or 5 ug/ml insulin (INS). Specificallybound ‘2SI-rmTNF Cell line
u937 WEHI L929 HepG2 Hepatocytes -DEX +DEX -DEX + INS +DEX +INS
(cpm)
Untreated
IL-6 treated
889 970 388 276
k f f +
147 72 29 10
970 950 468 777
f f + +-
148 81 33 44
183 162 349 388
It f It -+
43 34 49 91
253 324 478 475
+r f It
33 66 55 17
time Figure
plot (Fig. 1). From the results it can be concluded that IL-6 causes an increase in TNF receptor (TNF-R) expression without affecting the TNF-R affinity. In both cases, a Kd of 3.4 x 10-l’ M was calculated; untreated cells expressed 660 receptors/cell, while in this particular experiment the number of receptors on IL&treated cells increased to 1,23O/cell. A kinetic study of the increased expression of TNF-R by IL-6 revealed that the effect reached a maximum after 6 h of treatment, whereupon a plateau was maintained until at least 48 h of treatment (Fig. 2). Significant enhancement of TNF-R expression was already observed at concentrations of 1 rig/ml IL-6, and reached a plateau at approximately 10 rig/ml (Fig. 3). The effect showed a t,,* of about 16 h after IL-6 was B/FO.O07 0.006--
0.000 4 0
2.
Time
course
of TNF-R
20 (hours)
induction
on HepG2
by IL-6.
Cells were treated with 800 rig/ml rhIL-6 for varying periods of time, after which a binding assay was performed with 120 U/ml of ‘251-rmTNF (250,000 cpm). The increase in binding was calculated using an untreated control tested simultaneously as a reference. Each point is the mean of a triplicate determination and was corrected for unspecific binding.
removed from the cell culture (data not shown). As shown in Fig. 4, the TNF-R augmentation could be completely abolished by coadministration of anti-IL-6 polyclonal antibodies. Although our observations strongly resemble the effect of interferon-y (IFN-y) on the expression of TNF-R observed on a wide variety of cell lines: the effect described here is not IFN-y-mediated. This is supported by the following observations: (1) pretreatment with 0.5 &ml IFN-y caused only a slight (30%) increase in TNF-R expression on HepG2 cells (data not shown); (2) no IFN-y could be detected in the supernatant of IL6-treated cells (data not shown); (3) treatment with anti-IFN-y did not affect the observed enhancement of TNF-R expression with IL-6 (Fig. 4).
ountreated 011-6 treated
5
10 specific
15 20 25 30 binding of ~2%TNF
Figure 1. Scatchard analysis of competitive with ‘*%rmTNF on HepGZ cells treated (0) rhIL-6 (100 rig/ml for 18 h).
35 40 (10-13~)
45
50
binding experiments or not treated (0) with
Cells were incubated with 120 U/ml ‘=I-rmTNF (250,000 cpm) and increasing amounts of unlabeled rmTNF (in the range of a 1.5-fold to 500-fold excess) for 4 h at 4”C, and further treated as described in Materials and Methods. Each point represents the mean of a triplicate determination and was corrected for unspecific binding. B and F represent the concentrations of bound and free TNF.
ng/mlIL-6 Figure HepG2
3. Effect cells.
of IL-6
concentration
added on the binding
of TNF
to
Cells were treated with rhIL-6 for 18 h prior to binding with 120 U/ml of ‘“I-rmTNF (250,000 cpm). Each point is the mean of a triplicate determination and was corrected for unspecific binding.
Enhancement of TNF-R expression by IL-6 / 151 1000, 900 73 [I : D $
800
y-
500
7
600
E ff200 F 100 0i-;l
$-
-I-
-F
700
400 I 300
not treated
Figure 4. IL-&induced
riL-b
^
l-7 +IL-6 ana criL-6
Effect of anti-IL-6 and increase in TNF binding
+IL-
and a m-y
! 6
anti-IFN-y on HepG2
-I!-
II :
+IL-b
and preimmune serum
antibody cells.
on the
Cells were treated for 18 h with 20 rig/ml rhIL-6 (2 x lo4 U/ml) and 4 x lo4 neutralizing U/ml anti-rhll-6, 4 x lo4 neutralizing U/ml anti-rhIFN-y, or an equivalent amount of nonimmune rabbit serum. For binding, cells were incubated with 120 U/ml of lZI-rmTNF (250,000 cpm). Each measurement is the mean of a triplicate determination and was corrected for unspecific binding.
In order to obtain further insight into the mechanism of this IL-6-mediated induction of TNF-R, we treated HepG2 cells with IL-6 in the presence of cycloheximide (CHX) (an inhibitor of protein synthesis) or actinomycin D (ActD) (an inhibitor of RNA synthesis). As shown in Fig. 5, both inhibitors completely abolished the effect of IL-6, which strongly suggests that the IL-6 effect is transcriptionally mediated. We cannot explain the drop in TNF-R levels in CHX- plus IL-6-treated cells as compared to the CHX control. A similar effect, however, was observed in IFN-y-treated ME-180 cells.7 The IL-6-mediated increase in receptor expression did not affect the sensitivity of HepG2 cells to the cytotoxic action of TNF: no difference could be seen in
0 a3
an 18-h ActD assay between untreated cells and IL-6-treated cells (the cells were not sensitive to TNF in the absence of ActD; data not shown). The effect of IL-l on the expression of TNF-R was also investigated. IL-l has been shown to downregulate the expression of TNF-R on some cell lines,* while it has also proven to enhance the expression of IL-6 receptors on hepatocytes.’ As shown in Fig. 6, IL-1 on its own had no effect on TNF-R expression on HepG2 cells. However, when cells were cotreated with IL-l and IL-6, IL-l completely abrogated the induction by IL-6; this effect was weakened when the ratio of the concentration of added IL-6 to the concentration of added IL-l was raised (not shown), suggesting that both proteins, though they have different effects and use different mechanisms of actions, might share one or several common factors in their respective signaling pathways. This phenomenon strongly resembles the effect of IL-l on the induction of certain acute-phase proteins by IL-6 in rat hepat0cytes.l’
DISCUSSION Ever since TNF has been recognized as the major mediator of the antitumor and shock-inducing properties of endotoxin, it has become one of the most studied molecules in biomedical research. TNF is very pleiotropic in its activities, and can influence physiological processes in almost all cells and tissues. This includes the liver, which fulfills a key role in the defense of the body against toxic agents and seems to be responsible for clearing TNF from the bloodstream.” Hepatocytes, like most cells, express TNF-R, and several studies have shown that animals
untreated IL-6 treated
E zoo 8 100
i?l not treated
ActD CHX
Figure 5. Influence of ActD and CHX in TNF binding on HepG2 cells.
on the IL-Cinduced
increase
Cells were treated with 100 &ml rhIL-6 and 1 &ml ActD or 50 kg/ml CHX for 3 h at 37°C prior to binding with 120 U/ml of rZ51-TNF (250,000 cpm). Each measurement represents the mean of a triplicate determination and was corrected for unspecific binding.
Figure 6. Effect on the IL&induced
+IL-6
!---i-L +IL-1
of IL-1 on the binding of TNF increase in TNF binding.
+IL-6 +IL-1
to IIepG2
cells and
Cells were treated with 100 rig/ml rhlL-6, 500 ngfml rhIL-1, or both cytokines simultaneously for 18 h prior to binding with 120 U/ml of lZ51-rmTNF (250,000 cpm). Each point represents the specific binding of TNF and is the mean of a triplicate determination.
CYTOIUNE, Vol. 3, No. 2 (March 1991:149-154)
152 I Van Bladel, Libert, Fiers
with a defective liver function are much more vulnerable to the toxic effects of TNF.‘2-‘4 Since upregulation or downregulation of TNF-R has often proven to coincide with regulation of TNF activity (e.g., with IL-l or IFN-y; see above), we studied the regulation of TNF-R by IL-6, which is a cytokine with the liver as a main target.6 IL-6 is produced by a variety of cells (e.g., monocytes, fibroblasts, endothelial cells, and others), and can be induced by a variety of stimuli (TNF, IL-l, and LPS).4 Its most important role consists in the induction of the hepatic acute phase response. Here we describe another activity of IL-6 on hepatocytes. When hepatocytes or HepG2, which is a representative hepatic cell line, are incubated with IL-6, an increased specific binding of TNF to its receptors is observed. We could demonstrate that an incubation period of 2 to 48 h and a minimal dose of 1 to 10 rig/ml were required for a significant increase in TNF binding. This dose of IL-6 is comparable to the serum IL-6 levels found in mice after treatment with IL-l, TNF, or LPS (10 to 100 rig/ml).” The IL-6 effect on HepG2 cells could be completely neutralized by an anti-IL-6 antibody, indicating that the effect is specifically IL-6-mediated and not due to a possible contaminant in the IL-6 preparation. We could demonstrate that IFN-y, which is capable of increasing the number of TNF-R on a variety of cell lines, is not a mediator in this IL-6 effect. Furthermore, IL-6 was shown to be active at a transcriptional level, and did not alter the affinity of the TNF/TNF-R interaction. Reports suggest that TNF activity can be modulated by itself as well as by other cytokines. IFN-y, for example, causes an increased expression of TNF-R and sensitizes cells in vitro to the cytotoxic action of TNF.7,17 However, these two effects of IFN-y (receptor induction and synergistic cytotoxity) were shown not to be coupled.7 IL-l can cause a protein synthesisindependent decrease in TNF-R, and at the same time causes a desensitization to TNF cytotoxicity in vitro and to TNF lethality in viv0.~2~~ We have reported previously, however, that IL-ltreated animals do not show decreased TNF binding to hepatocytes.5 In this paper, we show that a dose of IL-l as high as 0.5 kg/ml does not decrease the number of TNF-R on HepG2 cells. Nevertheless, IL-l can completely abolish the enhanced expression of TNF-R caused by IL-6. A possible explanation for this phenomenon would be that IL-l downregulates the expression of IL-6R on HepG2 cells. However, this would contradict the findings of Bauer et a1.,9who showed that IL-l causes an increase in IL-6R expression on hepatocytes, as well as the results obtained in the laboratory of Dr. P. Heinrich (personal communication), which suggest that IL-l does not alter the expression of IL-6R on
HepG2 cells. Alternatively, IL-l and IL-6 might compete for a common factor in their respective action pathways. This inhibitory effect of IL-l on IL-6induced effects is not totally unexpected. It has been shown10~‘9that IL-l can inhibit the induction of CQmacroglobulin, cystein protease inhibitor, thiostatin, and fibrinogen by IL-6 in rat hepatocytes. The physiological relevance of the observed phenomena remains to be elucidated. IL-6 does not enhance TNF cytotoxicity on HepG2 cells in an 18-h TNF assay with ActD. Nevertheless, the possibility that other TNF effects on hepatocytes (such as the induction of acute-phase proteins) may be influenced, remains to be investigated. In vivo, the upregulation of TNF-R in the liver could cause a more efficient clearing of TNF, which could mean that IL-6 plays a beneficial role in disorders where TNF is the main mediator (e.g., septic shock). No clear protection or sensitization of animals to the toxic effect of TNF, IL-l, or LPS by IL-6 has been described so far. Nevertheless, several observations suggest a protective role for IL-6: IL-6 induces the synthesis of acute-phase reactants in the liver, some of which are thought to play a protective role in the body; IL-6 reduces TNF production in monocytes20~21 and leads, through the induction of adrenocorticotropic hormone,” to elevated corticosteroid levels, which not only inhibit the production of TNF and IL-1,23,24 but also protect against the toxic effects of TNF.
MATERIALS AND METHODS Actinomycin D (ActD) and cycloheximide (CHX) were obtained from Sigma Chemical Co., St. Louis, MO. Recombinant murine (rm) TNF was purified from E. coli and had a specific activity of 7.5 x lo7 Uimg. Recombinant human (rh) IL-6 was cloned in our laboratory, expressed in yeast, and purified to a specific activity of 1 x lo9 U/mg (Y. Guisez, J. Demolder, and R. Contreras, unpublished results). rhIL-1B was obtained from the Glaxo Institute for Molecular Biology, Geneva, Switzerland, through the courtesy of Dr. A. Shaw. Polyclonal anti-hIL-6 and anti-hIFN-y antibodies were prepared in rabbits, and had a neutralizing activity of 7 x 10’ U/ml and 8 x 10’ U/ml of stock solution, respectively.
Cell Culture U937, a human myeloma cell line (obtained from Biogen, Geneva, Switzerland), and WEHI 164~113, a murine fibrosarcoma line (obtained from Dr. T. Espevik, University of Trondheim, Norway), were grown in RPM1 medium (Gibco Bio-Cult, Paisley, UK), supplemented with 10% fetal calf serum (FCS) and antibiotics. HepG2, a human hepatoma cell line (obtained from Dr. H. Schaller, University of Heidelberg, Germany) and L929, a murine fibrosarcoma cell line (obtained from Dr. R. Konings, Rega Institute, Leuven, Belgium) were grown in DMEM
Enhancementof TNF-R expressionby IL-6 / 153 (Gibco Bio-Cult), supplemented with 10% FCS, antibiotics, and nonessential amino acids. HepG2 cells were kept in culture for maximum ten cell passages. Rat hepatocytes (a kind gift of Dr. C. Szpirer and S. Wasmine, Free University of Brussels, Belgium), were cultured in a mixture of 50% Ham’s F12 medium and 50% DMEM, supplemented with 10% FCS, antibiotics, 0.1 PM dexamethasone, and 5 pg/ml insulin.25 All cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO, and 95% air.
IL-4 Treatment and Binding Assays mTNF was radiolabeled with Na’*‘I (Amersham International, Amersham, UK) using Iodogen (Pierce Chemicals, Rockford, IL) as oxidant,26which led to a specific activity of 8 x lo7 cpm/pg without significant loss of biological activity. Cells were trypsinized and seeded into 6-well plates 48 h prior to the binding assay at a maximum density of lo6 cells/well in a volume of 2 ml. For standard IL-6 treatments, IL-6 was added to the cells 18 h prior to the binding assay without renewal of the medium. For the binding assay, the supernatants of the cells were removed and the cells were incubated with lZI-TNF in 1 ml of fresh culture medium, supplemented with 0.02% NaN, to prevent internalization of receptor-bound ligand, at 4°C for 4 h. After binding, the cells were washed twice with ice-cold medium, detached from the culture plates by trypsinization, and counted in a y counter (Pharmacia LKB Biotechnology, Uppsala, Sweden). All measurements were performed in triplicate, and corrected for nonspecific binding by parallel incubations with a 500-fold excess of cold rmTNF. Unspecific binding was rather high in these experiments and represented between 30% and 60% of total binding. The number of TNF-R/cell and the K, were determined by Scatchard plot analysis.16 Acknowledgments The authors thank Dr. C. Szpirer and S. Wasmine for their kind gift of rat primary hepatocytes, Dr. Y. Guisez for providing hIL-6 and hIFN-y, as well as
polyclonal anti-hIL-6 and anti-hIFN-y antisera, Dr. A. Shaw for his kind gift of IL-l, A. Raeymaekers for the preparation of mTNF, and W. Burm for technical assistance. Research was supported by the Belgian ASLK and FGWO. S. Van Blade1 and C. Libert are research assistants with the NFWO. Note Added in Proof
Recently, Starnes et a1.27 reported protection against TNF lethality in mice by pretreatment with monoclonal anti-IL-6 antibodies, which suggests that IL-6 might play a role in mediating, rather than in protecting against, TNF toxicity. So far, it is not clear how this correlates with the regulation of TNF receptors in the liver.
REFERENCES 1. Fiers W, Beyaert R, Brouckaert P, Everaerdt B, Haegeman G, Libert C, Suffys P, Takahashi N, Tavernier J, Van Blade1 S,
Vanhaesebroeck B, Van Ostade X, Van Roy F (1990) In vitro and in vivo action of tumor necrosis factor. In Bonavida B, Granger G (eds) Tumor Necrosis Factor: Structure, Mechanism of Action, Role in Disease and Therapy, Karger, Base], pp 77-81. 2. Watanabe N, Kuriyama H, Sone H, Neda H, Yamauchi N, Maeda M, Niitsu Y (1988) Continuous internalization of tumor necrosis factor receptors in a human myosarcoma cell line. J Biol Chem 263:10262-10266. 3. Mosselmans R, Hepburn A, Dumont JE, Fiers W, Galand P (1988) Endocytic pathway-of recombinant murine tumor necrosis factor in L-929 cells. J Immunol 141:3096-3100. 4. Van Snick J (1990) Interleukin-6: An overview. Annu Rev Immunol8:253-278. 5. Libert C, Van Blade1 S, Brouckaert P, Shaw A, Fiers W (in press) Involvement of the liver, but not of IL-6, in IL-l-induced desensitization to the lethal effects of tumor necrosis factor. J Immunol. 6. Heinrich PC, Caste11JV, Andus T (1990) Interleukin-6 and the acute phase response. Biochem J 265:621-636. 7. Aggarwal BB, Eessalu TE (1987) Induction of receptors for tumor necrosis factor-a by interferons is not a major mechanism for their synergistic cytotoxic response. J Biol Chem 262:10000-10007. 8. Holtmann H, Wallach D (1987) Downregulation of the receptors for tumor necrosis factor by interleukin 1 and 4@phorbol12-myristate-13-acetate. J Immunol139:1161-1167. 9. Bauer J, Bauer TM, Kalb T, Taga T, Lengyel G, Hirano T, Kishimoto T, Acs G, Mayer L, Gerok W (1989) Regulation of interleukin 6 receptor expression in human monocytes and monocytederived macrophages. Comparison with the expression in human hepatocytes. J Exp Med 170:1537-1549. 10. Andus T, Geiger T, Hirano T, Kishimoto T, Tran-Thi T, Decker K, Heinrich PC (1988) Regulation of synthesis and secretion of major rat acute-phase proteins by recombinant human interleukin-6 (BSF-2/IL-6) in hepatocyte primary cultures. Eur J Biochem 173:287-293. 11. Ferraiolo BL, Moore JA, Crase D, Gribling P, Wilking H, Baughman RA (1988) Pharmacokinetics and tissue distribution of recombinant human tumor necrosis factor-a in mice. Drug Metab Dispos 16:270-275. 12. Lehman V, Freudenberg MA, Galanos C (1987) Lethal toxicity of lipopolysaccharide and tumor necrosis factor in normal and D-galactosamine-treated mice. J Exp Med 165:657-663. 13. Libert C, Brouckaert P, Fiers W (submitted) The influence of modulating substances on tumor necrosis factor and interleukin 6 levels after injection of murine tumor necrosis factor or lipopolysaccharide in mice. 14. Fukushima H, Ikeuchi J, Tohkin M, Matsubara T, Harada M (1988) Lethal shock in partially hepatectomized rats administered tumor necrosis serum. Circ Shock 26:1-14. 15. Libert C, Brouckaert P, Shaw A, Fiers W (1990) Induction of interleukin 6 by human and murine recombinant interleukin 1 in mice. Eur J Immunol20:691-694. 16. Scatchard G (1949) The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660-672. 17. Fransen L, Van der Heyden J, Ruysschaert R, Fiers W (1986) Recombinant tumor necrosis factor: Its effect and its synergism with interferon-y on a variety of normal and transformed human cell lines. Eur J Cancer Clin Oncol22:419-426. 18. Wallach D, Holtmann H, Engelmann H, Nophar Y (1988) Sensitization and desensitization to lethal effects of timor n&rosi$ factor and IL-l. J Immunol 140:2994-2999. 19. Gauldie J, Richards C, Northemann W, Fey G, Baumann H (1989) IFN P2iBSF2/IL-6 is the monocyte-derived HSF that regulates receptor-specific acute phase gene regulation in hepatocytes. Ann NY Acad Sci 557:46-58. 20. Aderka D, Le J, VilEek J (1989) IL-6 inhibits lipopolysaccharide-induced tumor necrosis factor production in cultured human monocytes, U937 cells, and in mice. J Immunol 143:3517-3523.
154 I Van Blade& Libert,
Fiers
21. Schindler R, Mantilla J, Endres S, Ghorbani R, Clark SC, Dinarello CA (1990) Correlations and interactions in the production of interleukin-6 (IL-6), IL-l, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF. Blood 75:40-47. 22. Naitoh Y, Fukata J, Tominaga T, Nakai Y, Tamai S, Mori K, Imura H (1988) Interleukin-6 stimulates the secretion of adrenocorticotropic hormone in conscious, freely-moving rats. Biochem Biophys Res Commun 155:1459-1463. 23. Beutler B, Krochin N, Milsark IW, Luedke C, Cerami A (1986) Control of cachectin (tumor necrosis factor) synthesis: Mechanisms of endotoxin resistance. Science 232:977-980. 24. Lew W, Oppenheim JJ, Matsushima K (1988) Analysis of the suppression of IL-lo and IL-lp production in human peripheral
CYTOKINE,
Vol. 3, No. 2 (March 1991: 149-154)
blood mononuclear adherent cells by a glucocorticoid hormone. J Immunol140:1895-1902. 25. Wanson JC, Bernaert D, May C (1978) Morphology and functional properties of isolated and cultured hepatocytes. In Popper H, Schaffner F (eds) Progress in Liver Diseases, Grune & Stratton, New York, ~016, pp l-22. 26. Fraker PJ, Speck Jr JC (1978) Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro3a,6a-diphenylglycol uril. Biochem Biophys Res Commun 80:849857. 27. Starnes HF Jr, Pearce MK, Tewari A, Yim JH, Zou J-C, Abrams JS (1990) Anti-IL-6 monoclonal antibodies protect against lethal tumor necrosis factor-u challenge in mice. J Immunol145:41854191.
Claude Libert,
Walter Fiers”
We have studied the effect of interleukin-6 (IL-6) on the binding of tumor necrosis factor (TNF) to various cell lines. A significant increase (up to 250%) in binding was observed on rat hepatocytes and on the human hepatoma cell line HepG2, while no changes in the number of cells or cell morphology could be observed. Scatchard plot analysis showed that IL-6 enhanced the number of TNF receptors without affecting the receptor affinity. The effect reached plateau levels after approximately 6 h and at IL-6 concentrations of 10 @ml. It could be completely eliminated by cotreatment of cells with anti-IL-6 antibodies, but not by treatment with anti-interferon-y (IFN-y), suggesting that IFN-y, which can enhance TNF receptor expression on a variety of cells, was not a mediator in this IL-6 effect. Treatment with inhibitors of protein or RNA synthesis completely abolished the IL&induced increase, suggesting that IL-6 caused an enhanced transcription of TNF receptor mRNA. IL-1 had no effect on TNF binding to HepG2. However, when cells were cotreated with IL-1 and IL-6, IL-1 could completely abrogate the IL-6 effect. Copyright o 1991 by W.B. Saunders Company
Tumor necrosis factor (TNF) is a cytokine which is produced primarily by activated macrophages and which exerts a wide variety of biological activities in vitro and in vivo. It is cytotoxic for tumor cells, stimulates the growth of fibroblasts, can modulate gene expression, and is believed to play a role in a number of human pathologies.’ TNF binds to high-affinity receptors (Kd = 10-l’ to lo-l1 M) and is subsequently internalized and degraded.2,3 TNF receptors are detectable on all cells, with the exception of erythrocytes, unstimulated T cells, and cells with autocrine TNF production. The number of receptors per cell varies from a few hundred to ten thousands, but has not proven, so far, to correlate with the responsiveness of cells to TNF. Interleukin-6 @L-6), like TNF, is a pleiotropic protein. It acts as a growth and/or differentiation factor on B and T lymphocyte subsets, leads to induction of adrenocorticotropic hormone synthesis in vivo, and is the major inducer of acute-phase proteins in hepatocytes.4 The liver seems to play a central role in the in vivo
Laboratory of Molecular Biology, State University, straat 35, B-9000 Gent, Belgium. *To whom reprint requests should be sent. Copyright o 1991 by W.B. Saunders Company 1043-4666/91/0302-0002$05.00/O KEY
WORDS:
CYTOKINE,
IL-6/TNF
K.L.
receptors/hepatocytes
Vol. 3, No. 2 (March),
1991: pp 149-154
Ledeganck-
action of both TNF and IL-6: it might be involved in alleviating the toxic effect of TNF,’ and responds to IL-6 by the production of acute-phase proteins.6 In this paper we present results regarding the effect of IL-6 on the expression of TNF receptors on various cell lines and more particularly on a human hepatoma cell line (HepG2).
RESULTS Initially, several cell lines were tested for a possible effect of IL-6 on the binding of TNF to its receptor (Table 1). Only in the case of HepG2 and of freshly prepared rat hepatocytes could a significant increase in binding be observed after treatment for 24 h with recombinant human (rh) IL-6. This IL-bcaused increase in TNF binding on HepG2 cells was observed repeatedly and varied within the range of 80 to 250%. For hepatocytes, an increase of 22 to 100% was observed. Interestingly, the omission of insulin from the hepatocyte culture medium caused a decrease in TNF binding. No changes in the number of cells or cell morphology could be observed after IL-6 treatment. In order to investigate whether the IL-6-induced increase in binding was caused by an alteration of receptor affinity or by an enhancement of receptor expression, binding was performed with varying amounts of ligand on both untreated and IL-6-treated HepG2 cells and the results analyzed in a Scatchard 149
150 1 Van Bladel,
Libert,
CYTOKINE,
Fiers
Vol. 3, No. 2 (March 1991: 149-1.54)
Table 1. Influence of IL-6 on the specific binding of TNF to various cell lines. Cells were incubated for 24 h with 1 &ml rhIL-6. Binding assays with ‘%rmTNF were then performed as described in Materials and Methods. Rat primary hepatocytes were isolated 48 h prior to addition of IL-6 and cultured either with (+) or without (-) 0.1 pM dexamethasone (DEX) or 5 ug/ml insulin (INS). Specificallybound ‘2SI-rmTNF Cell line
u937 WEHI L929 HepG2 Hepatocytes -DEX +DEX -DEX + INS +DEX +INS
(cpm)
Untreated
IL-6 treated
889 970 388 276
k f f +
147 72 29 10
970 950 468 777
f f + +-
148 81 33 44
183 162 349 388
It f It -+
43 34 49 91
253 324 478 475
+r f It
33 66 55 17
time Figure
plot (Fig. 1). From the results it can be concluded that IL-6 causes an increase in TNF receptor (TNF-R) expression without affecting the TNF-R affinity. In both cases, a Kd of 3.4 x 10-l’ M was calculated; untreated cells expressed 660 receptors/cell, while in this particular experiment the number of receptors on IL&treated cells increased to 1,23O/cell. A kinetic study of the increased expression of TNF-R by IL-6 revealed that the effect reached a maximum after 6 h of treatment, whereupon a plateau was maintained until at least 48 h of treatment (Fig. 2). Significant enhancement of TNF-R expression was already observed at concentrations of 1 rig/ml IL-6, and reached a plateau at approximately 10 rig/ml (Fig. 3). The effect showed a t,,* of about 16 h after IL-6 was B/FO.O07 0.006--
0.000 4 0
2.
Time
course
of TNF-R
20 (hours)
induction
on HepG2
by IL-6.
Cells were treated with 800 rig/ml rhIL-6 for varying periods of time, after which a binding assay was performed with 120 U/ml of ‘251-rmTNF (250,000 cpm). The increase in binding was calculated using an untreated control tested simultaneously as a reference. Each point is the mean of a triplicate determination and was corrected for unspecific binding.
removed from the cell culture (data not shown). As shown in Fig. 4, the TNF-R augmentation could be completely abolished by coadministration of anti-IL-6 polyclonal antibodies. Although our observations strongly resemble the effect of interferon-y (IFN-y) on the expression of TNF-R observed on a wide variety of cell lines: the effect described here is not IFN-y-mediated. This is supported by the following observations: (1) pretreatment with 0.5 &ml IFN-y caused only a slight (30%) increase in TNF-R expression on HepG2 cells (data not shown); (2) no IFN-y could be detected in the supernatant of IL6-treated cells (data not shown); (3) treatment with anti-IFN-y did not affect the observed enhancement of TNF-R expression with IL-6 (Fig. 4).
ountreated 011-6 treated
5
10 specific
15 20 25 30 binding of ~2%TNF
Figure 1. Scatchard analysis of competitive with ‘*%rmTNF on HepGZ cells treated (0) rhIL-6 (100 rig/ml for 18 h).
35 40 (10-13~)
45
50
binding experiments or not treated (0) with
Cells were incubated with 120 U/ml ‘=I-rmTNF (250,000 cpm) and increasing amounts of unlabeled rmTNF (in the range of a 1.5-fold to 500-fold excess) for 4 h at 4”C, and further treated as described in Materials and Methods. Each point represents the mean of a triplicate determination and was corrected for unspecific binding. B and F represent the concentrations of bound and free TNF.
ng/mlIL-6 Figure HepG2
3. Effect cells.
of IL-6
concentration
added on the binding
of TNF
to
Cells were treated with rhIL-6 for 18 h prior to binding with 120 U/ml of ‘“I-rmTNF (250,000 cpm). Each point is the mean of a triplicate determination and was corrected for unspecific binding.
Enhancement of TNF-R expression by IL-6 / 151 1000, 900 73 [I : D $
800
y-
500
7
600
E ff200 F 100 0i-;l
$-
-I-
-F
700
400 I 300
not treated
Figure 4. IL-&induced
riL-b
^
l-7 +IL-6 ana criL-6
Effect of anti-IL-6 and increase in TNF binding
+IL-
and a m-y
! 6
anti-IFN-y on HepG2
-I!-
II :
+IL-b
and preimmune serum
antibody cells.
on the
Cells were treated for 18 h with 20 rig/ml rhIL-6 (2 x lo4 U/ml) and 4 x lo4 neutralizing U/ml anti-rhll-6, 4 x lo4 neutralizing U/ml anti-rhIFN-y, or an equivalent amount of nonimmune rabbit serum. For binding, cells were incubated with 120 U/ml of lZI-rmTNF (250,000 cpm). Each measurement is the mean of a triplicate determination and was corrected for unspecific binding.
In order to obtain further insight into the mechanism of this IL-6-mediated induction of TNF-R, we treated HepG2 cells with IL-6 in the presence of cycloheximide (CHX) (an inhibitor of protein synthesis) or actinomycin D (ActD) (an inhibitor of RNA synthesis). As shown in Fig. 5, both inhibitors completely abolished the effect of IL-6, which strongly suggests that the IL-6 effect is transcriptionally mediated. We cannot explain the drop in TNF-R levels in CHX- plus IL-6-treated cells as compared to the CHX control. A similar effect, however, was observed in IFN-y-treated ME-180 cells.7 The IL-6-mediated increase in receptor expression did not affect the sensitivity of HepG2 cells to the cytotoxic action of TNF: no difference could be seen in
0 a3
an 18-h ActD assay between untreated cells and IL-6-treated cells (the cells were not sensitive to TNF in the absence of ActD; data not shown). The effect of IL-l on the expression of TNF-R was also investigated. IL-l has been shown to downregulate the expression of TNF-R on some cell lines,* while it has also proven to enhance the expression of IL-6 receptors on hepatocytes.’ As shown in Fig. 6, IL-1 on its own had no effect on TNF-R expression on HepG2 cells. However, when cells were cotreated with IL-l and IL-6, IL-l completely abrogated the induction by IL-6; this effect was weakened when the ratio of the concentration of added IL-6 to the concentration of added IL-l was raised (not shown), suggesting that both proteins, though they have different effects and use different mechanisms of actions, might share one or several common factors in their respective signaling pathways. This phenomenon strongly resembles the effect of IL-l on the induction of certain acute-phase proteins by IL-6 in rat hepat0cytes.l’
DISCUSSION Ever since TNF has been recognized as the major mediator of the antitumor and shock-inducing properties of endotoxin, it has become one of the most studied molecules in biomedical research. TNF is very pleiotropic in its activities, and can influence physiological processes in almost all cells and tissues. This includes the liver, which fulfills a key role in the defense of the body against toxic agents and seems to be responsible for clearing TNF from the bloodstream.” Hepatocytes, like most cells, express TNF-R, and several studies have shown that animals
untreated IL-6 treated
E zoo 8 100
i?l not treated
ActD CHX
Figure 5. Influence of ActD and CHX in TNF binding on HepG2 cells.
on the IL-Cinduced
increase
Cells were treated with 100 &ml rhIL-6 and 1 &ml ActD or 50 kg/ml CHX for 3 h at 37°C prior to binding with 120 U/ml of rZ51-TNF (250,000 cpm). Each measurement represents the mean of a triplicate determination and was corrected for unspecific binding.
Figure 6. Effect on the IL&induced
+IL-6
!---i-L +IL-1
of IL-1 on the binding of TNF increase in TNF binding.
+IL-6 +IL-1
to IIepG2
cells and
Cells were treated with 100 rig/ml rhlL-6, 500 ngfml rhIL-1, or both cytokines simultaneously for 18 h prior to binding with 120 U/ml of lZ51-rmTNF (250,000 cpm). Each point represents the specific binding of TNF and is the mean of a triplicate determination.
CYTOIUNE, Vol. 3, No. 2 (March 1991:149-154)
152 I Van Bladel, Libert, Fiers
with a defective liver function are much more vulnerable to the toxic effects of TNF.‘2-‘4 Since upregulation or downregulation of TNF-R has often proven to coincide with regulation of TNF activity (e.g., with IL-l or IFN-y; see above), we studied the regulation of TNF-R by IL-6, which is a cytokine with the liver as a main target.6 IL-6 is produced by a variety of cells (e.g., monocytes, fibroblasts, endothelial cells, and others), and can be induced by a variety of stimuli (TNF, IL-l, and LPS).4 Its most important role consists in the induction of the hepatic acute phase response. Here we describe another activity of IL-6 on hepatocytes. When hepatocytes or HepG2, which is a representative hepatic cell line, are incubated with IL-6, an increased specific binding of TNF to its receptors is observed. We could demonstrate that an incubation period of 2 to 48 h and a minimal dose of 1 to 10 rig/ml were required for a significant increase in TNF binding. This dose of IL-6 is comparable to the serum IL-6 levels found in mice after treatment with IL-l, TNF, or LPS (10 to 100 rig/ml).” The IL-6 effect on HepG2 cells could be completely neutralized by an anti-IL-6 antibody, indicating that the effect is specifically IL-6-mediated and not due to a possible contaminant in the IL-6 preparation. We could demonstrate that IFN-y, which is capable of increasing the number of TNF-R on a variety of cell lines, is not a mediator in this IL-6 effect. Furthermore, IL-6 was shown to be active at a transcriptional level, and did not alter the affinity of the TNF/TNF-R interaction. Reports suggest that TNF activity can be modulated by itself as well as by other cytokines. IFN-y, for example, causes an increased expression of TNF-R and sensitizes cells in vitro to the cytotoxic action of TNF.7,17 However, these two effects of IFN-y (receptor induction and synergistic cytotoxity) were shown not to be coupled.7 IL-l can cause a protein synthesisindependent decrease in TNF-R, and at the same time causes a desensitization to TNF cytotoxicity in vitro and to TNF lethality in viv0.~2~~ We have reported previously, however, that IL-ltreated animals do not show decreased TNF binding to hepatocytes.5 In this paper, we show that a dose of IL-l as high as 0.5 kg/ml does not decrease the number of TNF-R on HepG2 cells. Nevertheless, IL-l can completely abolish the enhanced expression of TNF-R caused by IL-6. A possible explanation for this phenomenon would be that IL-l downregulates the expression of IL-6R on HepG2 cells. However, this would contradict the findings of Bauer et a1.,9who showed that IL-l causes an increase in IL-6R expression on hepatocytes, as well as the results obtained in the laboratory of Dr. P. Heinrich (personal communication), which suggest that IL-l does not alter the expression of IL-6R on
HepG2 cells. Alternatively, IL-l and IL-6 might compete for a common factor in their respective action pathways. This inhibitory effect of IL-l on IL-6induced effects is not totally unexpected. It has been shown10~‘9that IL-l can inhibit the induction of CQmacroglobulin, cystein protease inhibitor, thiostatin, and fibrinogen by IL-6 in rat hepatocytes. The physiological relevance of the observed phenomena remains to be elucidated. IL-6 does not enhance TNF cytotoxicity on HepG2 cells in an 18-h TNF assay with ActD. Nevertheless, the possibility that other TNF effects on hepatocytes (such as the induction of acute-phase proteins) may be influenced, remains to be investigated. In vivo, the upregulation of TNF-R in the liver could cause a more efficient clearing of TNF, which could mean that IL-6 plays a beneficial role in disorders where TNF is the main mediator (e.g., septic shock). No clear protection or sensitization of animals to the toxic effect of TNF, IL-l, or LPS by IL-6 has been described so far. Nevertheless, several observations suggest a protective role for IL-6: IL-6 induces the synthesis of acute-phase reactants in the liver, some of which are thought to play a protective role in the body; IL-6 reduces TNF production in monocytes20~21 and leads, through the induction of adrenocorticotropic hormone,” to elevated corticosteroid levels, which not only inhibit the production of TNF and IL-1,23,24 but also protect against the toxic effects of TNF.
MATERIALS AND METHODS Actinomycin D (ActD) and cycloheximide (CHX) were obtained from Sigma Chemical Co., St. Louis, MO. Recombinant murine (rm) TNF was purified from E. coli and had a specific activity of 7.5 x lo7 Uimg. Recombinant human (rh) IL-6 was cloned in our laboratory, expressed in yeast, and purified to a specific activity of 1 x lo9 U/mg (Y. Guisez, J. Demolder, and R. Contreras, unpublished results). rhIL-1B was obtained from the Glaxo Institute for Molecular Biology, Geneva, Switzerland, through the courtesy of Dr. A. Shaw. Polyclonal anti-hIL-6 and anti-hIFN-y antibodies were prepared in rabbits, and had a neutralizing activity of 7 x 10’ U/ml and 8 x 10’ U/ml of stock solution, respectively.
Cell Culture U937, a human myeloma cell line (obtained from Biogen, Geneva, Switzerland), and WEHI 164~113, a murine fibrosarcoma line (obtained from Dr. T. Espevik, University of Trondheim, Norway), were grown in RPM1 medium (Gibco Bio-Cult, Paisley, UK), supplemented with 10% fetal calf serum (FCS) and antibiotics. HepG2, a human hepatoma cell line (obtained from Dr. H. Schaller, University of Heidelberg, Germany) and L929, a murine fibrosarcoma cell line (obtained from Dr. R. Konings, Rega Institute, Leuven, Belgium) were grown in DMEM
Enhancementof TNF-R expressionby IL-6 / 153 (Gibco Bio-Cult), supplemented with 10% FCS, antibiotics, and nonessential amino acids. HepG2 cells were kept in culture for maximum ten cell passages. Rat hepatocytes (a kind gift of Dr. C. Szpirer and S. Wasmine, Free University of Brussels, Belgium), were cultured in a mixture of 50% Ham’s F12 medium and 50% DMEM, supplemented with 10% FCS, antibiotics, 0.1 PM dexamethasone, and 5 pg/ml insulin.25 All cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO, and 95% air.
IL-4 Treatment and Binding Assays mTNF was radiolabeled with Na’*‘I (Amersham International, Amersham, UK) using Iodogen (Pierce Chemicals, Rockford, IL) as oxidant,26which led to a specific activity of 8 x lo7 cpm/pg without significant loss of biological activity. Cells were trypsinized and seeded into 6-well plates 48 h prior to the binding assay at a maximum density of lo6 cells/well in a volume of 2 ml. For standard IL-6 treatments, IL-6 was added to the cells 18 h prior to the binding assay without renewal of the medium. For the binding assay, the supernatants of the cells were removed and the cells were incubated with lZI-TNF in 1 ml of fresh culture medium, supplemented with 0.02% NaN, to prevent internalization of receptor-bound ligand, at 4°C for 4 h. After binding, the cells were washed twice with ice-cold medium, detached from the culture plates by trypsinization, and counted in a y counter (Pharmacia LKB Biotechnology, Uppsala, Sweden). All measurements were performed in triplicate, and corrected for nonspecific binding by parallel incubations with a 500-fold excess of cold rmTNF. Unspecific binding was rather high in these experiments and represented between 30% and 60% of total binding. The number of TNF-R/cell and the K, were determined by Scatchard plot analysis.16 Acknowledgments The authors thank Dr. C. Szpirer and S. Wasmine for their kind gift of rat primary hepatocytes, Dr. Y. Guisez for providing hIL-6 and hIFN-y, as well as
polyclonal anti-hIL-6 and anti-hIFN-y antisera, Dr. A. Shaw for his kind gift of IL-l, A. Raeymaekers for the preparation of mTNF, and W. Burm for technical assistance. Research was supported by the Belgian ASLK and FGWO. S. Van Blade1 and C. Libert are research assistants with the NFWO. Note Added in Proof
Recently, Starnes et a1.27 reported protection against TNF lethality in mice by pretreatment with monoclonal anti-IL-6 antibodies, which suggests that IL-6 might play a role in mediating, rather than in protecting against, TNF toxicity. So far, it is not clear how this correlates with the regulation of TNF receptors in the liver.
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Vanhaesebroeck B, Van Ostade X, Van Roy F (1990) In vitro and in vivo action of tumor necrosis factor. In Bonavida B, Granger G (eds) Tumor Necrosis Factor: Structure, Mechanism of Action, Role in Disease and Therapy, Karger, Base], pp 77-81. 2. Watanabe N, Kuriyama H, Sone H, Neda H, Yamauchi N, Maeda M, Niitsu Y (1988) Continuous internalization of tumor necrosis factor receptors in a human myosarcoma cell line. J Biol Chem 263:10262-10266. 3. Mosselmans R, Hepburn A, Dumont JE, Fiers W, Galand P (1988) Endocytic pathway-of recombinant murine tumor necrosis factor in L-929 cells. J Immunol 141:3096-3100. 4. Van Snick J (1990) Interleukin-6: An overview. Annu Rev Immunol8:253-278. 5. Libert C, Van Blade1 S, Brouckaert P, Shaw A, Fiers W (in press) Involvement of the liver, but not of IL-6, in IL-l-induced desensitization to the lethal effects of tumor necrosis factor. J Immunol. 6. Heinrich PC, Caste11JV, Andus T (1990) Interleukin-6 and the acute phase response. Biochem J 265:621-636. 7. Aggarwal BB, Eessalu TE (1987) Induction of receptors for tumor necrosis factor-a by interferons is not a major mechanism for their synergistic cytotoxic response. J Biol Chem 262:10000-10007. 8. Holtmann H, Wallach D (1987) Downregulation of the receptors for tumor necrosis factor by interleukin 1 and 4@phorbol12-myristate-13-acetate. J Immunol139:1161-1167. 9. Bauer J, Bauer TM, Kalb T, Taga T, Lengyel G, Hirano T, Kishimoto T, Acs G, Mayer L, Gerok W (1989) Regulation of interleukin 6 receptor expression in human monocytes and monocytederived macrophages. Comparison with the expression in human hepatocytes. J Exp Med 170:1537-1549. 10. Andus T, Geiger T, Hirano T, Kishimoto T, Tran-Thi T, Decker K, Heinrich PC (1988) Regulation of synthesis and secretion of major rat acute-phase proteins by recombinant human interleukin-6 (BSF-2/IL-6) in hepatocyte primary cultures. Eur J Biochem 173:287-293. 11. Ferraiolo BL, Moore JA, Crase D, Gribling P, Wilking H, Baughman RA (1988) Pharmacokinetics and tissue distribution of recombinant human tumor necrosis factor-a in mice. Drug Metab Dispos 16:270-275. 12. Lehman V, Freudenberg MA, Galanos C (1987) Lethal toxicity of lipopolysaccharide and tumor necrosis factor in normal and D-galactosamine-treated mice. J Exp Med 165:657-663. 13. Libert C, Brouckaert P, Fiers W (submitted) The influence of modulating substances on tumor necrosis factor and interleukin 6 levels after injection of murine tumor necrosis factor or lipopolysaccharide in mice. 14. Fukushima H, Ikeuchi J, Tohkin M, Matsubara T, Harada M (1988) Lethal shock in partially hepatectomized rats administered tumor necrosis serum. Circ Shock 26:1-14. 15. Libert C, Brouckaert P, Shaw A, Fiers W (1990) Induction of interleukin 6 by human and murine recombinant interleukin 1 in mice. Eur J Immunol20:691-694. 16. Scatchard G (1949) The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660-672. 17. Fransen L, Van der Heyden J, Ruysschaert R, Fiers W (1986) Recombinant tumor necrosis factor: Its effect and its synergism with interferon-y on a variety of normal and transformed human cell lines. Eur J Cancer Clin Oncol22:419-426. 18. Wallach D, Holtmann H, Engelmann H, Nophar Y (1988) Sensitization and desensitization to lethal effects of timor n&rosi$ factor and IL-l. J Immunol 140:2994-2999. 19. Gauldie J, Richards C, Northemann W, Fey G, Baumann H (1989) IFN P2iBSF2/IL-6 is the monocyte-derived HSF that regulates receptor-specific acute phase gene regulation in hepatocytes. Ann NY Acad Sci 557:46-58. 20. Aderka D, Le J, VilEek J (1989) IL-6 inhibits lipopolysaccharide-induced tumor necrosis factor production in cultured human monocytes, U937 cells, and in mice. J Immunol 143:3517-3523.
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