Eur. J. Immunol. 1990.20: 691-694

Induction of IL 6 by human and murine rIL 1 in mice

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Short paper Claude Libert+, Peter Brouckaert, Alan ShawO and Walter Fiers

Induction of interleukin 6 by human and murine recombinant interleukin 1 in mice*

Laboratory of Molecular Biology, State University, Gent and The Glaxo Institute for Molecular Biologyo, Geneva

Interleukin (IL) 6 is a pleistropic cytokine with activities, among others, on immune cells, hematopoietic precursor cells and hepatocytes. We have investigated the kinetics and amplitude of its in vivo induction in mice after injection of four different IL 1 species as well as murine (m) and human (h) tumor necrosis factor (TNF) and bacterial lipopolysaccharide(LPS) using a sensitive bioassay on 7TD1 cells to measure the IL6 concentrations. Recombinant mIL lp, administered as a single i.v. injection in mice, induced the appearance of IL 6 in the plasma with peak levels observed after 2 h. A dose-response correlation was found between serum IL6 levels and injected IL la concentrations at 3 and 8 h after the injection.We then compared the ability of W m I L l a , h/mIL lp, h/mTNF and LPS to induce IL6 in mice.We found: (a) LPS is the most potent inducer of IL6; (b) 3 h after injection, the four IL 1preparations had induced IL 6 levels comparable with the IL 6 levels observed after TNF injection; (c) high doses of mIL 1, a or p, but not hIL 1, resulted in a high IL 6 level persisting for over 8 h.We conclude that IL 1is a potent inducer of IL 6 in vivo and that no major differences are observed between the four IL1 preparations, as evaluated at 3 h after the injection. However, mILla and mILlp, in contrast to hILla and hILlp, induced a sustained IL6 level over a longer time period. This pattern of prolonged IL6 induction is even much more pronounced after mTNF injection, but not after hTNF injection.

1 Introduction IL6 is a cytokine which exerts a variety of activities, for example on immune cells, hematopoietic precursor cells and hepatocytes [1-31. The activities exerted by IL 6 cover the following range: helper function for T cell responsiveness to IL2; Tcell production of IL2 and differentiation of CTL; growth stimulation of plasmacytoma,hybridoma and myeloma cells, as well as some EBV-transformed cell lines; induction of MQ differentiation; growth inhibition of some cell lines; stimulation of hematopoietic progenitors, induction of neural differentiation and, last but not least, regulation of the production of acute-phase reactants. Known in vivo activities of IL6 are: induction of neutrophilia [l], fever [4], acute-phase proteins (APP) [5] and adrenocorticotropic hormone (ACTH) [6], as well as stimulation of myelopoiesis and erythropoiesis [7].

myxoma cells, have been shown to produce IL6 either constitutively or after stimulation. LPS,viral infection,TNF and especially IL 1 have been recognized as “natural” inducing agents for IL6 in vifro [3]. In animals, IL6 in circulation was detected, both by us and other groups, after injection of bacterial LPS or TNF [8,9]. Furthermore, IL6 has been found in some pathological situations, such as burns [lo], autoimmune disease, rheumatoid arthritis [ll], meningoencephalitis [12], septic shock [13] and cardiac myxoma [14].

IL 1is, like IL6 and TNF, a pleiotropic cytokine. In man as well as in mouse, two forms of IL 1have been described, i.e. ILla and ILlp, the chemical structure and PI values of which are quite different. Among the biological activities previously attributed to IL1 are: induction of fever, neutrophilia [15] and acute-phase reactants [16]. The So far, several cell types and lines, such as fibroblasts, induction of CSF [17] and the changes in BM population monocytes, endothelial cells, glioblastoma cells and cardiac [18] are currently under investigation for the protection and restoration of the BM in the context of radiotherapy and chemotherapy. The fact that IL1 acts as a very strong [I 81691 inducer of IL6 in several cell lines [19,20] is important because IL6 may be a potential mediator of at least some * Research supported by The Program on Interuniversity Attrac- IL 1 activities, or both cytokines may act in concert.

+

tion Poles (Belgian State, Science Policy Programming), the Program on Biosciences and the Fund for Scientific and Medical Research (FGWO). Research assistant with the NFWO.

Correspondence: Walter Fiers, RUG, Ledeganckstraat 35, B-9000 Gent, Belgium Abbreviations: APP: Acute-phase protein h: Human m: Murine 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

In this context, we investigated the kinetics of IL6 appearance and disappearance in plasma of mice after a bolus injection of IL 1, and compared these with the kinetics after TNF or LPS injection. We also compared the ability of murine (m)IL la and IL lp, and human (h)IL la and IL l p to induce IL 6 in mice. The four IL 1species were tested at concentrations corresponding with nonlethal and lethal concentrations of mTNF (2 pg and 20 pg, respectively). 0014-2980/90/0303-0691$02.50/0

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Eur. J. Immunol. 1990.20: 691-694

C. Libert, I? Brouckaert, A. Shaw and W. Fiers

2 Materials and methods 2.1 Laboratory animals and reagents Male C57BLlcnb mice (SCK, Mol, Belgium) were used at the age of 10 weeks.The animals were given food and water ad libitum.E. coli 0111 : B4 LPS was from Difco Laboratories, Detroit, MI. Purified hrTNF and mrTNF [21] were obtained from Dr. J. Tavernier (formerly Biogent, Gent, Belgium). hTNF had a sp. act. of 2.5x107U/mg and contained < 0.11 ng LPS/mg protein; mTNF had a sp. act. of 7.5 x lo7 U/mg and contained 1). Comparing the four IL 1 preparations, the 20 :2 pg ratio at 3 h was the highest for IL 1p (Table 1). For all groups treated with 2 pg, the I L 6 levels at 3 h were the highest after IL la injection, albeit still much below the level reached after LPS injec-

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tion.The values after 20 pg injection of hIL la or hIL l g did not differ much at 3 h; the difference between mIL la and mIL 1p was, however, sevenfold (Fig. 3). The 20 : 2 pg ratio recorded for IL 6 at 8 h was much higher after m I L l than after hIL1 injection; but the ratio was spectacularly high for mTNF (Table 1).For the 2-pg groups, all plasma I L 6 levels at this time point were lower than 1 ng/ml, except for m I L l a and LPS (3.3 ng/ml and 12.1 ng/ml). For the 20-pg groups, however, the IL 6 levels amounted to only a few ng/ml at 8 h following the injection of either human protein, whereas the IL 6 titers reached values up to several hundreds ng/ml following injection of the murine proteins, especially mTNF. It should be noted that the plasma of saline-injected animals never contained detectable IL6.

4 Discussion ni

LL 6 has been identified as a molecule with a wide range of activities both on white blood cells, such as lymphocytes and monocytes, and on other cells, such as hepatocytes. I L 6 can induce B lymphocytes to enter their final differentiation step and stimulates the production of antibodies [23]. IL 6 acts both on CD4+ and CD8+ T cells. In these respects, and even more because of its important role in the induction of the acute-phase response, I L 6 might be of crucial importance in the body's defense mechanisms. Due to its ability to enhance Ig production, IL 6 has recently also been recognized as playing a major role in some autoimmune diseases. Consequently, it is important to understand the mechanisms leading to circulatory IL 6 and the factors which regulate the plasma levels.

riL[ Bl

256.

Figure 3. Comparison of the ability of IL 1 , W a n d LPS to induce IL 6 in mice. The cytokines were i.v. injected either at 2 pg or 20 pg dose per mouse; LPS was i.v. injected as a 2-pg dose. Results are means If: SD of plasma IL 6 levels of five mice per group. IL 6 was determined in a 7TD1 bioassay. (A) Levels of plasma IL6 observed 3 h after injection. (B) Levels of plasma IL6 observed after 8 h. The percentage of IL6 concentration in plasma at 8 h compared with concentration at 3 h is shown above each column.

Table 1. Ratios of IL6 levels after a high-dose cytokine injection relative to a low dose

Timea)

Ratio 20 pg :2 pg of injected cytolciae (h) hILla hILlf3 mILla mILlf3 hTNF mTNF

3 8

1.2 5.9

13.2 3.9

3.4 45.3

4.3 105.7

22.8 3.3

5.4 907.9

a) After i.v. injection of either 2 pg or 20pg cytokine per mouse.

Several reasons led us to study the IL 6 induction by IL 1 in mice: (a) it is well documented that IL 1is the most potent physiological inducer of IL 6 in v i m ; (b) IL 1might play an important role in vivo, more particularly in septic shock and in protection against radiotherapy and chemotherapy; (c) a number of nonrecombinant IL 1preparations were apparently contaminated with IL 6 [4]; (d) IL 6 seems to mediate some in vivo and in vitro activities that were previously attributed to IL 1 [4]. Our results show that IL 1can indeed induce high circulating I L 6 levels in the plasma of mice. A bolus injection of 2 pg mIL l p gave rise to a transient IL 6 peak. These peak concentrations appeared between 2 and 4 h after the injection, which corresponds with results obtained with TNF and LPS [9]. The observation that IL 1strongly induces IL 6 in vivo has important implications: it means that I L 1 can, at least indirectly, exert all in vivo activities of IL 6. As the majority of these in vivo activities were formerly attributed to a direct action of IL 1, the question arises to what extent IL6 is acting as a mediator. Furthermore, it has recently been demonstrated that IL 1 and IL 6 share particular biological activities, and also that they are even synergistic in at least one system, i.e. thymocyte proliferation [4]. As far as the induction of APP in hepatocytes is concerned, it seems very likely that IL6 can be considered the main inducer [24]. However, depending on the in vitro systems, IL 1can induce the same APP [25] or a different APP [26] as IL 6. In other systems, IL 1 and IL 6 synergistically induce

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C. Libert, I? Brouckaert, A. Shaw and W. Fiers

APP [27], while sometimes IL 1 inhibits the induction of APP by IL 6 [28]. Injection of IL 6 does not, however, seem to change the concentration of acute-phase reactants to the same extent as an injection of IL 1[25].Takinginto account the rapid clearance of IL6 after an injection (data not shown), we suppose that the exposure time to IL6 is of crucial importance. After comparison of the ability of I L l a and ILlp, both murine and human, to induce IL6 in mice, we found that IL 18 was somewhat less effective than IL la at the early time point. Results from the samples taken at 8 h show that only high doses of mIL 1, either a or p, led to a sustained IL6 level. We also observed that mTNF is a far better inducer of IL6 than hTNE When assayed at 3 h, the ability of mTNF to induce IL6 is intermediate as compared with mIL la and mILlf3, whereas hTNF is a much weaker inducer than hILla or hIL.1p. However, the IL6 levels after mTNF injection were remarkably higher after 8 h than after 3 h. As we described elsewhere [9,29], a sustained IL6 level in the serum is obtained after lethal bolus injections of mTNF or LPS. A possible explanation could be that only lethal injections of mTNF or LPS induce appreciable concentrations of IL 1,which in its turn induces a second IL6 wave. There is, however, no reason to believe that this high, sustained IL 6 level is causally related to mortality, since 20 pg mIL1, which is not lethal at all, also induces a sustained IL6 level. Besides, injections of mIL 6 or hIL 6, even in very high concentration, never led to lethality and could not sensitize mice to the lethal effect of TNE The highest peak value of IL6 was found after LPS injection; however, this IL6 was rapidly cleared, which is what we would expect for a nonlethal dose of LPS. It seems that injection of the higher concentration of homologous (i. e. murine) IL 1or TNF in mice, the latter but not the former being lethal, exceeds a threshold regulatory mechanism, resulting in a sustained IL6 level, possibly triggered by induced, endogenous IL 1. We thank Dr. J. Van Snick for his gift of 7TDI cells, mrlL6 and mrlL6 mAb, as well as Dr. J. Tavernier and Dr. E! Wingfreld for providing the TNF and IL I preparations, respectively.

Received November 27, 1989.

5 References Le, J. and Vilkk, J., Lab. Invest. 1989. 61: 588. Wong, G. G. and Clark, S. C., Immunol. Today 1988. 9: 137. Fiers,W.,Brouckaert, F!,Content, J., Contreras, R., Everaerdt, B., Guisez,Y, Libert, C., Spriggs, D. ,Takahashi, N.,Tison, B., Vandenabeele, I? and Van Snick, J., Adv. Immunopharmacol. 1989. 4: 185.

Eur. J. Immunol. 1990.20: 691-694 4 Helle, M., Brakenhoff, J. I? J., De Groot, E. R. and Aarden, L. A., Eur. J. Imrnunol. 1988. 18: 957. 5 Geiger, T., Andus, T., Klapproth, J., Hirano,T., Kishimoto, T. and Heinrich, €? C., Eur. J. Immunol. 1988. 18: 717. 6 Naitoh,Y., Fukata, J. ,Tominaga,T., Nakai,Y ,Tamai, S., Mori, K. and Imura, H., Biochem. Biophys. Res. Commun. 1988. 155: 1459. 7 Ulich, T. R., Del Castillo, J. and Guo, K., Blood 1989. 73: 108. 8 Coulie, I? G., Cayphas, S.,Vink, A., Uyttenhove, C. and Van Snick, J., Eur. J. Immunol. 1987. 17: 1217. 9 Brouckaert, P. G., Libert, C., Everaerdt, B. ,Takahashi, N. and Fiers, W., Lymphokine Res. 1989. 8: 269. 10 Nijsten, M.W. N., De Groot, E. R.,Ten Duis, H. J., Klasen, H. J., Hack, C. E. and Aarden, L. A., Lancet 1987. ii: 921. 11 Waage, A., Kaufmann, C., Espevik, T. and Husby, G., Clin. Immunol. Immunopathol. 1989. 50: 394. 12 Frei, K., Leist, T. I?, Meager, A., Gallo, I?, Leppert, D., Zinkernagel, R. M. andFontana, A., J. Exp. Med. 1988.168: 449. 13 Waage, A., Brandtzaeg, I?, Halstensen, A., Kierulf, P. and Espevik,T., J. Exp. Med. 1989. 169: 333. 14 Hirano,T. ,Thga,T.,Yasukawa, K., Nakajima, K., Nakano, N., Thkatsuki, E , Shimizu, M., Murashima, A., Tsunasawa, S., Sakiyama, F. and Kishimoto, T., Proc. Natl. Acad. Sci. USA 1987. 84: 228. 15 Dinarello, C. A., Cannon, J. G., Mier, J.W., Bernheim, H. A., LoPreste, G., Lynn, D. L., Love, R. N.,Webb,A. C.,Auron,I? E., Reuben, R. C., Rich, A. and Wolff, S. M., J. Clin. Invest. 1986. 77: 1734. 16 Mortensen, R. F., Shapiro, J., Lin, B. F., Douches, S. and Neta, R., J. Immunol. 1988. 140: 2260. 17 Vogel, S. N., Douches, S. D., Kaufman, E. N. and Neta, R., J. Immunol. 1987. 138: 2143. 18 Neta, R., Oppenheim, J. J., Douches, S. D., Giclas, I? C., Imbra, R. J. and Karin, M., Prog. Immunol. 1986. 6: 900. 19 Defilippi, F!,Poupart, I? ,Tavernier,J., Fiers,W. and Content, J., Proc. Natl. Acad. Sci. USA 1987. 84: 4557. 20 Van Damme, J. and Van Snick, J., Dev. Biol. Stand. 1988. 69: 31. 21 Tavernier, J., Fransen, L., Marmenout, A. ,Van der Heyden, J., Miiller, R., Ruysschaert, M. R.,VanVliet, A., Bauden, R. and Fiers, W., Lymphokines 1987. 13: 181. 22 Brouckaert, P., Spriggs, D. R., Demetri, G., Kufe, D. W.and Fiers, W., J. Exp. Med. 1989. 169: 2257. 23 Muraguchi, A., Hirano, T., Tang, B., Matsuda, T., Horii, Y , Nakajima, K. and Kishimoto, T., J. Exp. Med. 1988. 167: 332. 24 Castell, J. V., GOmez-Lech611, M. J., David, M., Hirano, T., Kishimoto, T. and Heinrich, I? C., FEBS Lett. 1988. 232: 347. 25 Ramadori, G.,Van Damme, J., Rieder, H. and Meyer zum Biischenfelde, K. H., Eur. J. Immunol. 1988. 18: 1259. 26 Ritchie, D. G. and Zuckerman, S. H., Immunology 1987.61: 429. 27 Baumann, H., Richards, C. and Gauldie, J., J. Immunol. 1987. 139: 4122. 28 Andus,T., Geiger,T., Hirano,T., Kishimoto,T.,Tran-Thi,T.-A., Decker, K. and Heinrich, €? C., Eur. J. Biochem. 1988. 173: 287. 29 Brouckaert, I? G., Everaerdt, B., Libert, C.,Takahashi, N. and Fiers, W., Agents Actions 1989. 26: 196.

Induction of interleukin 6 by human and murine recombinant interleukin 1 in mice.

Interleukin (IL) 6 is a pleistropic cytokine with activities, among others, on immune cells, hematopoietic precursor cells and hepatocytes. We have in...
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