European Journal of Clinical Investigation (1992) 22,27 1-276
Human recombinant C5a enhances Iipopolysaccharide-induced synthesis of interleukin-6 by human monocytes V. GROSS & T. ANDUS, Medizinische Universitatsklinik, Hugstetter Strasse 55, D-7800 Freiburg, Germany Received I5 May 1991 and in revised form 21 October 1991; accepted 22 October 1991
Abstract. The effect of human recombinant C5a (hrC5a) on the synthesis of interleukin-6 (IL-6) was studied in human monocytes. Monocytes incubated in the absence of hrC5a and of bacterial lipopolysaccharide (LPS) produced only low amounts ( < I 0 0 U/ 2 x 10' ce11/16 h) of IL-6 activity. LPS in concentrations from 10 pg m1-I to 10 ng ml-' greatly stimulated the synthesis of IL-6 to about 50.000 U/106 cells/l6 h. When hrC5a was added to the monocyte media maximal IL-6 synthesis was reached at lower LPS concentrations, i.e. at 0.1 ng ml-' LPS in the presence of 100 ng ml-l hrC5a. Maximal IL-6 production was not significantly enhanced by hrC5a. Metabolic labelling with [35S]-methioninefollowed by immunoprecipitation of IL-6 showed that the increased IL-6 activity in the medium of hrC5a treated monocytes was due to a stimulation of the de novo synthesis of IL-6. Increased amounts of IL-6 mRNA were found in monocytes treated with LPS and hrC5a compared with monocytes stimulated only with LPS. HrC5a prolonged the elevation of IL-6 mRNA levels after stimulation of monocytes with LPS. HrC5a thus enhanced the LPS-induced synthesis of IL-6 by human monocytes. Keywords. CSa, interleukin-6, lipopolysaccharide, monocytes. Introduction
Interleukin-6 plays a central role in the responses of the organism to disturbances of its homeostasis due to infection, tissue injury, neoplastic growth or immunological disorders [I]. It is essential for the terminal differentiation of B cells into immunoglobulin-secreting cells [2,3]. It is an important cofactor in T-cell activation and subsequent proliferation of activated T cells [4,5]. It enhances the proliferation of multipotential haematopoietic stem cells [6]. It is further an important inducer of the acute phase protein synthesis by hepatocytes [7,8]. In adult human hepatocytes Dedicated to Professor Dr W. Gerok on the occasion of his 651h birthday. Correspondence: Dr Volker Gross, Medizinische Universitatsklinik, Franz-Josef-Straws-Allee, D-8400 Regensburg, Germany.
interleukin-6 has been shown to be the most important inducer of acute phase protein synthesis whereas interleukin-I and tumour necrosis factor c1 play only a minor role [9,lO]. Interleukin-6 is synthesized by various cell types, e.g. fibroblasts [ 1 I , 121, endothelial cells [ 13,141, monocytes and macrophages [ 15,161, B and T lymphocytes [ 171, and various tumour cell lines (for a review see ref. 1). Different factors have been described to stimulate interleukin-6 synthesis. In monocytes/macrophages bacterial lipopolysaccharide [ 1 5,16,18], HIV virus [ 191, interleukin- 1 u//? [ 16,181, interferon-/? [20], granulocyte/macrophage colony-stimulating factor [20], tumour necrosis factor c1[ I81 and substances P and K [21] have been found to stimulate the synthesis of interleukin-6. The complement system plays an important role in host resistance to infections. Two types of ligands are generated during complement activation: those bound to the surface of infectious agents and those freely diffusible in the fluid phase (for a review see ref. 22). Among the diffusible ligands C5a plays an important role by promoting neutrophil chemotaxis and secretion. Specific receptors for C5a are also present on macrophages [23]. We therefore studied whether, in addition to the known bacterial and viral products and cytokines, C5a, which is generated as a diffusible ligand at the site of inflammation, regulates interleukin-6 synthesis by human monocytes. Materials and methods Materials
RPMI 1640 medium was purchased from Seromed (Berlin, Germany). PI/NaCI, L-glutamine, antibiotics, and vitamins were from Gibco (Karlsruhe, Germany). Ficoll-Paque and protein A-sepharose were obtained from Pharmacia (Freiburg, Germany). HrCSa, lipopolysaccharide and polymyxin B were from Sigma (Deisenhofen, Germany). ~[~~Sl-methionine ( > 1000 Ci mmol-' ) was purchased from Amersham International (Braunschweig, Germany). Human recombinant interleukin-6 was a generous gift of Dr W. Sebald (Wurzburg, Germany). The B9 cell line was kindly 27 1
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provided by Dr L. Aarden (Amsterdam, The Netherlands). Humun monocyte cultures
For the preparation of human monocytes buffy coats obtained from the blood of normal blood donors were used. The preparation of monocytes was carried out essentially as described by Andreesen et al. [24]. Mononuclear cells were separated from granulocytes and residual erythrocytes by centrifugation over Ficoll-Paque. Monocytes were then separated from lymphocytes by adherence to plastic at 37°C in RPMI 1640 medium containing 10% lipopolysaccharide-free human AB serum. RPMI 1640 was supplemented with L-glutamine,antibiotics and vitamins as described [24]. The non-adherent cells (lymphocytes) were removed by repeated gentle washing with RPMI 1640 medium. The adherent cells were then incubated further with RPMI 1640 medium containing 5% human AB serum and lipopolysaccharide and/or hrC5a in the concentrations indicated in the various experiments. For the determination of interleukin-6 activity the supernatants of 2 x lo6 monocytes were used after a 16-h incubation period. De novo synthesis of interleukin-6 and interleukin-6 mRNA was determined after a 16-h incubation period with lipopolysaccharide and/or hrC5a. Determinution of interleukin-6 activity
Interleukin-6 activity was determined by the growth of an interleukin-6 dependent hybridoma cell line (B9) by measuring [3H]-thymidine incorporation into DNA. One hundred microlitres of the supernatants were tested in triplicate in serial dilutions in the B9 cell assay according to the method described by Aarden et al. [25,26]. The dilution leading to half maximal stimulation of hybridoma cells was defined as 1 U ml-I. In all tests a standard interleukin-6 preparation was used as internal control. Starting dilution in all tests was 1 :4. Labelling of monocytes and immunoprecipitation of interleukin-6
Methionine free RPMI 1640 medium without serum was used for the radioactive labelling of monocytes obtained after 16 h incubation. A total of 2 x lo6cells/ dish were incubated with 150 pCi [%I- methionine for 2 h. After the labelling period the medium was separated from the cells. The radioactively labelled medium (1 ml/dish) was added to 5 ml of 20 mM Tris/ HCL (pH 7.6), 0.14 M NaCI, 5 mM EDTA, 1 YOTriton X-100. After addition of 10 p1 of a specific polyclonal rabbit anti-human interleukin-6 antiserum and incubation at 0°C overnight the antigen-antibody complexes were bound to 10 mg (dry weight) of protein A-sepharose and washed four times with the abovementioned buffer and twice with 50 mM sodium phosphate buffer, pH 7.5. Elution was performed by
incubation with 0.1 M Tris/HCI (pH 6.8) 5% 2mercaptoethanol, 0*5Y0sodium dodecylsulphate, 10% glycerol at 95°C for 5 min. The eluted proteins were analysed by electrophoresis in sodium dodecylsulphate/polyacrylamide ( 1 2%) gels [27] and fluorography [28l. Northern blots
For Northern blot analysis 8 x lo6 monocytes were lysed by guanidinium thiocyanate-phenol-chloroform, and RNA was isolated according to the protocol of Chomczynski & Sacchi [29]. Twenty pg of total RNA were separated on a 1 %, agarose, 6.6% formaldehyde gel prior to transfer to the gene screen filters [30]. Results Determination of interleukin-6 activity Human monocytes were incubated with increasing amounts of lipopolysaccharide to stimulate the synthesis of interleukin-6. As shown in Fig. I , increasing amounts of lipopolysaccharide enhanced the synthesis of interleukin-6, similar to previous studies [ 151. Maximal interleukin-6 activity was obtained at a lipopolysaccharide concentration of 10 ng ml- I. A further increase of the lipopolysaccharide concentration did not lead to a further stimulation of interleukin-6 synthesis. When hrC5a was added in a final concentration of 10 ng mi-l there was no induction of interleukin-6 synthesis in the absence of lipopolysaccharide; however, 10 ng mi-' hrc5a had a co-stimulatory effect on the lipopolysaccharide-induced interleukin-6 synthesis by monocytes. In the presence of 100 ng mi-' maximal interleukin-6 synthesis was obtained at a much lower lipopolysaccharide concentration (0.1 ng ml-I) than in the absence of hrC5a (10 ng ml-I). In the presence of 100 ng mi-' hrC5a there was a slight stimulation of interleukin-6 synthesis even in the absence of lipopolysaccharide. This stimulation of interleukin-6 by hrC5a was not inhibitable by preincubation of hrC5a with 5000 U ml-' Polymyxin B for 1 h at room temperature, a procedure which could neutralize the effect of 50 pg lipopolysaccharide (data not shown). HrC5a did not lead to a further significant increase in the maximal amount of interleukin-6 secreted by lipopolysaccharide-stimulated monocytes at lipopolysaccharide concentrations of 10 ng ml-' or 100 ng mi-'. De nouo synthesis of interleukin-6
To demonstrate that hrC5a affects the de nouo synthesis of interleukin-6, monocytes were metabolically labelled with [35S]-methionine after they had been incubated with lipopolysaccharide and hrC5a in increasing concentrations for 16 h. After a labelling period of 2 h interleukin-6 was immunoprecipitated by a specific antiserum. As shown in Fig. 2 no radioactively labelled newly synthesized interleukin-6 could be
C5a ENHANCES INTERLEUKIN-6 SYNTHESIS
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100000 ;
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Figure I . ElTect of hrC5a on interleukin-6 activity in lipopolysaccharide-treated monocyte cultures. Human monocytes (2 x lo6 cel1s)wereincubdted withoutorwith0.01 ngml-',0.1 ngml-'. 1 ngml-', IOngml-'and 100ngml 'oflipopolysaccharidein the absence or presence of 10 ng ml- I or 100 ng ml-' of hrC5a for 16 h. Interleukin-6 activity was determined by the B9-cell assay as described in Materials and methods. Mean values of 3 different experiments+SD are presented.
Figure 2. Du ~ ( J W synthesis of interleukin-6. Human monocytes (2 x lo6 cells) were incubated without (lanes I . 7, 13) or with lipopolysaccharide in concentrations of0.01 ng ml-' (lanes 2.8. 14). 0.1 ng ml (lanes 3,9. 15). I ng ml (lanes 4. 10. 16). 10 ng ml-'(lanes 5. I I . 17)or 100ngml-'(lanes6. 12, 18)eitherin theabsence(1anes 1-6)orpresenceof IOngml-'hrC5a(lanes7-12) or 100 ng ml-' hrC5a (13-18) for 16 h. The cells were radioactively labelled with [35S]-methionine(150 pCi/dish) for 2 h. Interleukin-6 was immunoprecipitated from the cell medium and subjected to sodium dodecylsulphate/polyacrylamide gel electrophoresis as described in Materials and methods. Molecular weight markers were glyceraldehyde 3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa) and soybean trypsin inhibitor (20 kDa).
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detected in the supernatants of monocytes incubated without lipopolysaccharide. Compared with monocytes stimulated only by lipopolysaccharide the costimulation of monocytes by lipopolysaccharide and by hrC5a led to the detection of radioactively labelled interleukin-6 at lower IiDoDolvsaccharide concentrations (0.1 ng ml- lipopolysaccharide in the presence of 10 ng ml-' or 100 ng ml-' of hrC5a vs. 1 ng ml-' lipopolysaccharide i n the absence of hrC5a). The various bands seen in Fig. 2 represent different molecular weight forms of interleukin-6, which are due to differences in 0- and N-glycosylation. Among others there are two predominant 0-glycosylated forms with apparent molecular weights of 24 kDa and L
~'
23 kDa and a form with an apparent molecular weight of 27.5 kDa, both 0- and N-glycosylated [31]. The labelling experiments demonstrate that hrC5a leads to a stimulation of the de nooo synthesis of interleukin-6 in the presence of lipopolysaccharide.
1
Determination of interleukin-6 mRNA
To study the effect of hrC5a on interleukin-6 mRNA, Northern-blots were done. Figure 3 shows that no detectable amounts of interleukin-6 mRNA could be found in monocytes incubated without lipopolysaccharide or with 10 pg ml-' lipopolysaccharide for 16 h (lanes 1, 3). Addition of 100 ng ml-' lipopolysacchar-
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Figure4. Effect of hrC5a on the kinetics of induction of IL-6 mRNA after LPS stimulation. Human monocytes were incubated without (lane I ) or with 100 ng ml-' of lipopolysaccharide for 2 h (lanes 2.3). 4 h (lanes 4, 5) or 8 h (lanes 6, 7) in the absence (lanes 2. 4, 6) or presence (lanes 3,5,7) of 100 ng ml I of hrC5a. RNA was extracted from 8 x 10' cells. An equal RNA load was assessed by probing of j-actin as house-keeping gene.
Discussion
Figure 3. Effect of hrC5a on IL-6 mRNA levels after stimulation with different concentrations of LPS. Human monocytes were incubated without (lanes I , 2) or with 0.01 ng ml-' (lanes 3, 4) or 100 ng m l ~(lanes 5,6) of lipopolysaccharide in the absence (lanes I , 3 . 5 ) or presence (lanes 2 , 4 , 6 ) of 100 ng ml- I hrC5a for 16 h. RNA was prepared from 8 x 10' cells. Northern blots were performed as described in Materials and methods.
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ide led to a clear increase in interleukin-6 mRNA (lane 5). HrC5a in a concentration of 100 ng ml-' did not enhance interleukin-6 mRNA in the absence of lipopolysaccharide (lane 2); however, it greatly enhanced the lipopolysaccharide-induced increase in interleukin-6 mRNA (lanes 4, 6). When the time-course of induction of interleukin-6 mRNA was studied (Fig. 4) it was found that 2 h or 4 h after addition of lipopolysacharide interleukin-6 mRNA levels were not different between cells incubated without or with hrC5a. In both treatment groups an interleukin-6 mRNA peak could be observed after 4 h. Eight hours after lipopolysaccharide stimulation, however, interleukin-6 mRNA levels were considerably higher in hrC5a treated monocytes than in untreated cells. This finding is in accordance with the mRNA data presented in Fig. 3 and indicates that hrC5a treatment of monocytes prolongs the elevation of interleukin-6 mRNA after stimulation with lipopolysaccharide.
C5a is an important diffusible inflammatory mediator generated during activation of the complement system. Binding of C5a to receptors on mast cells and basophils causes secretion of their granules and release of histamine. Binding of C5a or C5a desArg to receptors on neutrophils induces chemotaxis along a gradient of C5-derived glycopeptides, specific granule secretion, increased adhesive properties, increased oxygen consumption and superoxide generation, and enhanced expression of C3b receptors (for a review see ref. 22). In monocytes and macrophages C5a induces chemotaxis and the slow secretion of glycolytic and proteolytic enzymes. C5a thus has important pro-inflammatory properties. There are a number of systemic reactions of the body to maintain homeostasis during infection and inflammation. Many parts of this acute phase response, e.g. the synthesis of acute phase proteins in the liver, are mediated by interleukin-6 [I]. It was therefore of interest to study whether the pro-inflammatory peptide C5a is indirectly, i.e. via interleukin-6. involved in the regulation of this response. We found that hrC5a alone in a concentration of 10 ng ml- did not stimulate interleukin-6 synthesis by monocytes; however, it had a considerable co-stimulatory effect on the lipopolysaccharide-induced interleukin-6 synthesis. At a hrC5a concentration of 100 ng ml-' there was a slight stimulation of interleukin-6 synthesis even in the absence of lipopolysaccharide, but the most important effect of this high concentration of hrC5a was also seen in the presence of low concentrations (0.01 ng ml-' and 0.1 ng ml-I) of lipopolysaccharide. There are several lines of evidence to exclude the possibility that the observed effects of hrC5a on interleukin-6 synthesis might be due to
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C5a ENHANCES INTERLEUKIN-6 SYNTHESIS lipopolysaccharide contamination in the hrC5a preparation: 1. It had been shown that the hrC5a preparation used in our experiments had only a low lipopolysaccharide contamination of 1 pg pg-l hrC5a [32]. This amount is too low to cause any effects on interleukin-6 synthesis in our system. As shown in Figs 2 and 3, 10 pg of lipopolysaccharide did not lead to a detectable stimulation of the synthesis of interleukin-6. The slight stimulation of interleukin-6 synthesis by 100 ng C5a ml-' was not inhibitable by a preincubation with 5000 U ml- I of the lipopolysaccharide inhibitor Polymyxin B. Using this concentration we were able to inhibit the effect of up to 100 pg lipopolysaccharide mi-'. 2. In a recent report Scholz et af. [33] described that hrC5a-induced IL-6 synthesis by human peripheral blood-derived mononuclear cells was inhibited by heat treatment ( 1 00°C 15 min) suggesting that induction of interleukin-6 was not due to endotoxin contamination. 3. HrC5a alone had little effect on interleukin-6 synthesis by human monocytes in the absence of lipopolysaccharide. The effects of hrC5a were most pronounced in the presence of 0.01 and 0.1 ng mi-' of lipopolysaccharide. If the observed effect of hrC5a were only due to lipopolysaccharide contamination the most pronounced effects should have been found in the absence of lipopolysaccharide and addition of lipopolysaccharide should have abolished the effects.
The central finding of the present paper is that C5a alone only moderately enhances interleukin-6 production by human monocytes, but considerably increases the lipopolysaccharide stimulated interleukin-6 production. This finding adds new important aspects to the report of Scholz et al. [33] who described that C5a alone stimulates interleukin-6 synthesis by monocytes. Whereas the B9 assay showed no significant increase in interleukin-6 bioactivity by hrC5a at high concentrations of lipopolysaccharide, immunoprecipitation of interleukin-6 after labelling with [35S]-methionineor determination of mRNA levels by Northern blotting revealed an increase. This might be explained by the fact that immunoprecipitation and Northern blotting visualize differences at high interleukin-6 concentrations, which due to its logarithmic scale are not reflected by the B9 assay. Synergistic effects of C5a and lipopolysaccharide have also been observed for the synthesis of interleukin-l and tumour necrosis factor c1. In human peripheral blood monocytes, hrC5a had a co-stimulatory effect on the lipopolysaccharide-induced synthesis of both cytokines [34-371. It has been described that C5a induces the mRNA levels of interleukin-I and tumour necrosis factor c1 without inducing protein synthesis of these cytokines, and that the addition of lipopolysaccharide was needed for the translation and secretion of these mRNAs [37]. We did not find an induction of interleukin-6 mRNA synthesis by C5a alone (Fig. 3, lane 2), indicating that the synergistic effect of C5a and lipopolysaccharide on interleukin-6
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induction is not due to a selective action of C5a on interleukin-6 mRNA levels in human monocytes. The increased synthesis of interleukin-6 after lipopolysaccharide stimulation of monocytes in the presence of C5a might be due at least in part to a prolongation of the elevation of interleukin-6 mRNA (see Fig. 4). The mechanism by which hrC5a acts synergistically with lipopolysaccharide is not known. Several mechanisms, e.g. up-regulation of lipopolysaccharide receptors or secondary phenomena, e.g. increased interleukin-6 synthesis due to an increased interleukin- 1 synthesis, have to be considered. The findings presented in this paper show a mechanism by which the acute phase response of the body can be up-regulated. During bacterial infections lipopolysaccharides can directly stimulate the cytokine synthesis in monocytes. Since lipopolysaccharides also activate the complement system [38] the cytokine synthesis may be further enhanced by C5a. Thus, besides its potent pro-inflammatory properties C5a may indirectly stimulate protective mechanisms, such as acute phase protein synthesis in the liver.
Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (SFB 154 and a Heisenberg-Stipendium to V.G.). The skilful technical assistance of Mrs M. David and Mrs D. V. Berg and the help of Mrs G. Zahn with the preparation of the manuscript are greatly appreciated.
References Heinrich PC, Castell JV, Andus T. Interleukin-6 and the acute phase response. Biochem J 1990;265:621-36. Hirano T, Taga T, Nakano N, Yasukawa K, Kashiwamura S, Shimizu K, Nakajima K, Pyun KH, Kishimoto T. Purification to homogeneity and characterization of human B-cell differentiation factor (BCDF or BSFp-2). Proc Natl Acad Sci USA 1985;82:5490-4. Hirano T, Yasukawa K, Harada H et a/. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature (London) 1986;324:73-6. Garman RD, Jacobs KA, Clark SC, Raulet DH. B-cellstimulatory factor 2 (82 interferon) functions as a second signal for interleukin 2 production by mature murine T cells. Proc Natl Acad Sci USA 1987;84:7629-33. Habetswallner D, Pelosi E, Bulgarini D er a/. Activation and proliferation of normal resting human T lymphocytes in serumfree culture: role of IL-4 and IL-6. Immunology 1988;65:357-64. Leary AG, Ikebuchi K, Hirai Y el a/. Synergism between interleukin-6 and interleukin-3 in supporting proliferation of human hematopoetic stem cells: comparison with interleukin-I alpha. Blood 1988;71:1759-63. Gauldie J, Richards C, Harnish D, Lansdorp P, Baumann H. Interferon p2/BSF-2 shares identity with monocyte derived hepatocyte stimulating factor (HSF) and regulates the major acute phase protein response in liver cells. Proc Natl Acad Sci USA 1987;84:7251-5. Andus T, Geiger T, Hirano T el a/. Recombinant human B cell stimulatory factor 2 (BSF-Z/IFN-BZ)regulates 8-fibrinogen and albumin mRNA levels in Fao-9cells. FEBS Lett 1987;221:18-22. Castell JV, Gomez-Lechon MJ, David M, Hirano T, Kishimoto T, Heinrich PC. Recombinant human interleukin-6 (IL-6/BSF-
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2/HSF) regulates the synthesis of acute phase proteins in human hepatocytes. FEBS Lett 1988;232:347-50. 10 Castell JV, Gomez-Lechon MJ, David M et a/. Interleukin-6 is the major regulator of acute phase protein synthesis in human hepdtocytes. FEBS Lett 1989;242:237-9. I I Content J, DeWit L, Pierard D, Derynck R, DeClercq E, Fiers W. Secretory proteins induced in human fibroblasts under conditions used for the production of interferon B. Proc Natl Acad Sci USA 1982;79:2768-72. 12 Kohase M, Henriksen-DeStefano D, May LT, Vilcek J, Sehgal PB. Induction of B2-Interferon by tumor necrosis factor: A homeostatic mechanism in the control of cell proliferation. Cell I986;45:659-66. 13 Norioka K, Hara M, Harigai M er a/. Production of B cell stimulatory factor-2/interleukin-6 activity by human endothelial cells. Biochem Biophys Res Commun 1988;153:1045-50. 14 Jirik FR, Podor TJ, Hirano T et a/. Bacterial lipopolysaccharide and inflammatory mediators augment IL-6 secretion by human endothelial cells. J lmmunol 1989;142: 144-7. 15 Northoff H, Andus T, Tran-Thi T-A et al. The inflammation mediators interleukin-I and hepatocyte-stimulating factor are differently regulated in human monocytes. Eur J lmmunol 1987;17:707 I I . 16 Bauer J, Ganter V, Geiger T et a / . Regulation of Interleukin-6 expression in cultured human blood monocytes and monocytederived macrophages. Blood 1988;72:I 134-40. 17 Horii Y, Muraguchi A, Suematsu S et a/. Regulation of BSF-2/ IL-6 production by human mononuclear cells. Macrophagedependent synthesis of BSF-2/IL-6 by T cells. J Immunol 1988;141: 1529-35. 18 May LT, Ghrayeb J, Santhanam U era/. Synthesis and secretion of multiple forms of fi2-lnterferon/B-cell differentiation factor 2/ hepatocyte-stimulating factor by human fibroblasts and monocytes. J Biol Chem 1988;263:7760-6. 19 Nakajima K, Martinez-Maza 0, Hirano T et a/. Induction of IL-6 (B-cell stimulatory factor-2/IFN-/32) production by HIV. J lmmunol 1989;142:531- 6. 20 Navarro S, Debili N, Bernaudin J-F, Vainchenker W, Doly J. Regulation of the expression of 1L-6 in human monocytes. J Immunol 1989;142:4339-45. 21 Lotz M. Vaughan JH. Carson DA. Effect of neuropeptides on production of inflammatory cytokines by human monocytes. Science I988;24 I :I2 18-2 I , 22 Muller-Eberhard HJ. Molecular organization and function of the complement system. Ann Rev Biochem 1988;57:321 347. 23 Chenoweth DE, Goodman MG, Weigle WO. Demonstration of a specific receptor for human CSa anaphylatoxin on murine macrophages. J Exp Med 1982;156:68-78. 24 Andreesen R, Bross KJ, Osterholz J, Emmrich F. Human
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macrophage maturation and heterogeneity: Analysis with a newly generated set of monoclonal antibodies to differentiation antigens. Blood 1986;67:1257-64. Van Oers MHJ, Van der Heyden AAPAM, Aarden LA. Interleukin 6 (IL-6) in serum and urine of renal transplant recipients. Clin Exp Immunol 1988;71:314-19. Aarden LA, De Groot ER, Schaap OL, Lansdorp PM. Production of hybridoma growth factor by human monocytes. Eur J Immunol 1987;17:1411-16. King J, Laemmli U. Polypeptides of the tail fibres of bacteriophage T4. J Mol Biol 1971;62:465-77. Bonner WM, Laskey RA. A film detection method for tritiumlabelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 1974;46:83-8. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroformextraction. Anal Biochem 1987;162:156-9. Maniatis T, Fritsch EF, Sambrock J, eds. Molecular Cloning. Cold Spring Harbor, NY: Cold Spring Harbor. Laboratory Press, 1982. Gross V, Andus T, Castell J, vom Berg D, Heinrich PC, Gerok W. 0- and N-glycosylation lead to different molecular weight forms of human monocyte interleukin 6. FEBS Lett 1989; 247:323-6. Cavaillon J-M, Fitting C, Haeffner-Cavaillon N. Recombinant C5a enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide-stimulated monocytes and macrophages. Eur J Imrnunol 1990;20:253-7. Scholz W, Mcclurg MR, Cardenas GJ er al. C5a-mediated release of interleukin 6 by human monocytes. Clin Immunol Immunopathol 1990;57:297-307. Goodman MG, Chenoweth DE. Weigle WO. Induction of interleukin I secretion and enhancement of humoral immunity by binding of human C5a to macrophage surface C5a receptors. J Exp Med 1982; I 56:9 12- 17. Okusawa S, Dinarello C, Yancey KB er a/. CSa induction of human interleukin 1. Synergistic effect with endotoxin o r interferon-gamma. J Immunol 1987; I39:2635-40. Okusawa S, Yancey KB, Van Der Meer JWM et al. C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro. J Exp Med 1988;168:443-8. Schindler R, Lonnemann G , Shaldon S, Koch KM, Dinarello CA. Transcription, not synthesis, of interleukin-l and tumor necrosis factor by complement. Kidney Int 1990;37:85-93. Lkang-Takasaki VCJ, Saxen H, Makela PH. Leive L. Complement activation by polysaccharide of lipopolysaccharide: an important virulence determinant of salmonellae. Infect Immun 1983;41:563-9.
Human recombinant C5a enhances Iipopolysaccharide-induced synthesis of interleukin-6 by human monocytes V. GROSS & T. ANDUS, Medizinische Universitatsklinik, Hugstetter Strasse 55, D-7800 Freiburg, Germany Received I5 May 1991 and in revised form 21 October 1991; accepted 22 October 1991
Abstract. The effect of human recombinant C5a (hrC5a) on the synthesis of interleukin-6 (IL-6) was studied in human monocytes. Monocytes incubated in the absence of hrC5a and of bacterial lipopolysaccharide (LPS) produced only low amounts ( < I 0 0 U/ 2 x 10' ce11/16 h) of IL-6 activity. LPS in concentrations from 10 pg m1-I to 10 ng ml-' greatly stimulated the synthesis of IL-6 to about 50.000 U/106 cells/l6 h. When hrC5a was added to the monocyte media maximal IL-6 synthesis was reached at lower LPS concentrations, i.e. at 0.1 ng ml-' LPS in the presence of 100 ng ml-l hrC5a. Maximal IL-6 production was not significantly enhanced by hrC5a. Metabolic labelling with [35S]-methioninefollowed by immunoprecipitation of IL-6 showed that the increased IL-6 activity in the medium of hrC5a treated monocytes was due to a stimulation of the de novo synthesis of IL-6. Increased amounts of IL-6 mRNA were found in monocytes treated with LPS and hrC5a compared with monocytes stimulated only with LPS. HrC5a prolonged the elevation of IL-6 mRNA levels after stimulation of monocytes with LPS. HrC5a thus enhanced the LPS-induced synthesis of IL-6 by human monocytes. Keywords. CSa, interleukin-6, lipopolysaccharide, monocytes. Introduction
Interleukin-6 plays a central role in the responses of the organism to disturbances of its homeostasis due to infection, tissue injury, neoplastic growth or immunological disorders [I]. It is essential for the terminal differentiation of B cells into immunoglobulin-secreting cells [2,3]. It is an important cofactor in T-cell activation and subsequent proliferation of activated T cells [4,5]. It enhances the proliferation of multipotential haematopoietic stem cells [6]. It is further an important inducer of the acute phase protein synthesis by hepatocytes [7,8]. In adult human hepatocytes Dedicated to Professor Dr W. Gerok on the occasion of his 651h birthday. Correspondence: Dr Volker Gross, Medizinische Universitatsklinik, Franz-Josef-Straws-Allee, D-8400 Regensburg, Germany.
interleukin-6 has been shown to be the most important inducer of acute phase protein synthesis whereas interleukin-I and tumour necrosis factor c1 play only a minor role [9,lO]. Interleukin-6 is synthesized by various cell types, e.g. fibroblasts [ 1 I , 121, endothelial cells [ 13,141, monocytes and macrophages [ 15,161, B and T lymphocytes [ 171, and various tumour cell lines (for a review see ref. 1). Different factors have been described to stimulate interleukin-6 synthesis. In monocytes/macrophages bacterial lipopolysaccharide [ 1 5,16,18], HIV virus [ 191, interleukin- 1 u//? [ 16,181, interferon-/? [20], granulocyte/macrophage colony-stimulating factor [20], tumour necrosis factor c1[ I81 and substances P and K [21] have been found to stimulate the synthesis of interleukin-6. The complement system plays an important role in host resistance to infections. Two types of ligands are generated during complement activation: those bound to the surface of infectious agents and those freely diffusible in the fluid phase (for a review see ref. 22). Among the diffusible ligands C5a plays an important role by promoting neutrophil chemotaxis and secretion. Specific receptors for C5a are also present on macrophages [23]. We therefore studied whether, in addition to the known bacterial and viral products and cytokines, C5a, which is generated as a diffusible ligand at the site of inflammation, regulates interleukin-6 synthesis by human monocytes. Materials and methods Materials
RPMI 1640 medium was purchased from Seromed (Berlin, Germany). PI/NaCI, L-glutamine, antibiotics, and vitamins were from Gibco (Karlsruhe, Germany). Ficoll-Paque and protein A-sepharose were obtained from Pharmacia (Freiburg, Germany). HrCSa, lipopolysaccharide and polymyxin B were from Sigma (Deisenhofen, Germany). ~[~~Sl-methionine ( > 1000 Ci mmol-' ) was purchased from Amersham International (Braunschweig, Germany). Human recombinant interleukin-6 was a generous gift of Dr W. Sebald (Wurzburg, Germany). The B9 cell line was kindly 27 1
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provided by Dr L. Aarden (Amsterdam, The Netherlands). Humun monocyte cultures
For the preparation of human monocytes buffy coats obtained from the blood of normal blood donors were used. The preparation of monocytes was carried out essentially as described by Andreesen et al. [24]. Mononuclear cells were separated from granulocytes and residual erythrocytes by centrifugation over Ficoll-Paque. Monocytes were then separated from lymphocytes by adherence to plastic at 37°C in RPMI 1640 medium containing 10% lipopolysaccharide-free human AB serum. RPMI 1640 was supplemented with L-glutamine,antibiotics and vitamins as described [24]. The non-adherent cells (lymphocytes) were removed by repeated gentle washing with RPMI 1640 medium. The adherent cells were then incubated further with RPMI 1640 medium containing 5% human AB serum and lipopolysaccharide and/or hrC5a in the concentrations indicated in the various experiments. For the determination of interleukin-6 activity the supernatants of 2 x lo6 monocytes were used after a 16-h incubation period. De novo synthesis of interleukin-6 and interleukin-6 mRNA was determined after a 16-h incubation period with lipopolysaccharide and/or hrC5a. Determinution of interleukin-6 activity
Interleukin-6 activity was determined by the growth of an interleukin-6 dependent hybridoma cell line (B9) by measuring [3H]-thymidine incorporation into DNA. One hundred microlitres of the supernatants were tested in triplicate in serial dilutions in the B9 cell assay according to the method described by Aarden et al. [25,26]. The dilution leading to half maximal stimulation of hybridoma cells was defined as 1 U ml-I. In all tests a standard interleukin-6 preparation was used as internal control. Starting dilution in all tests was 1 :4. Labelling of monocytes and immunoprecipitation of interleukin-6
Methionine free RPMI 1640 medium without serum was used for the radioactive labelling of monocytes obtained after 16 h incubation. A total of 2 x lo6cells/ dish were incubated with 150 pCi [%I- methionine for 2 h. After the labelling period the medium was separated from the cells. The radioactively labelled medium (1 ml/dish) was added to 5 ml of 20 mM Tris/ HCL (pH 7.6), 0.14 M NaCI, 5 mM EDTA, 1 YOTriton X-100. After addition of 10 p1 of a specific polyclonal rabbit anti-human interleukin-6 antiserum and incubation at 0°C overnight the antigen-antibody complexes were bound to 10 mg (dry weight) of protein A-sepharose and washed four times with the abovementioned buffer and twice with 50 mM sodium phosphate buffer, pH 7.5. Elution was performed by
incubation with 0.1 M Tris/HCI (pH 6.8) 5% 2mercaptoethanol, 0*5Y0sodium dodecylsulphate, 10% glycerol at 95°C for 5 min. The eluted proteins were analysed by electrophoresis in sodium dodecylsulphate/polyacrylamide ( 1 2%) gels [27] and fluorography [28l. Northern blots
For Northern blot analysis 8 x lo6 monocytes were lysed by guanidinium thiocyanate-phenol-chloroform, and RNA was isolated according to the protocol of Chomczynski & Sacchi [29]. Twenty pg of total RNA were separated on a 1 %, agarose, 6.6% formaldehyde gel prior to transfer to the gene screen filters [30]. Results Determination of interleukin-6 activity Human monocytes were incubated with increasing amounts of lipopolysaccharide to stimulate the synthesis of interleukin-6. As shown in Fig. I , increasing amounts of lipopolysaccharide enhanced the synthesis of interleukin-6, similar to previous studies [ 151. Maximal interleukin-6 activity was obtained at a lipopolysaccharide concentration of 10 ng ml- I. A further increase of the lipopolysaccharide concentration did not lead to a further stimulation of interleukin-6 synthesis. When hrC5a was added in a final concentration of 10 ng mi-l there was no induction of interleukin-6 synthesis in the absence of lipopolysaccharide; however, 10 ng mi-' hrc5a had a co-stimulatory effect on the lipopolysaccharide-induced interleukin-6 synthesis by monocytes. In the presence of 100 ng mi-' maximal interleukin-6 synthesis was obtained at a much lower lipopolysaccharide concentration (0.1 ng ml-I) than in the absence of hrC5a (10 ng ml-I). In the presence of 100 ng mi-' hrC5a there was a slight stimulation of interleukin-6 synthesis even in the absence of lipopolysaccharide. This stimulation of interleukin-6 by hrC5a was not inhibitable by preincubation of hrC5a with 5000 U ml-' Polymyxin B for 1 h at room temperature, a procedure which could neutralize the effect of 50 pg lipopolysaccharide (data not shown). HrC5a did not lead to a further significant increase in the maximal amount of interleukin-6 secreted by lipopolysaccharide-stimulated monocytes at lipopolysaccharide concentrations of 10 ng ml-' or 100 ng mi-'. De nouo synthesis of interleukin-6
To demonstrate that hrC5a affects the de nouo synthesis of interleukin-6, monocytes were metabolically labelled with [35S]-methionine after they had been incubated with lipopolysaccharide and hrC5a in increasing concentrations for 16 h. After a labelling period of 2 h interleukin-6 was immunoprecipitated by a specific antiserum. As shown in Fig. 2 no radioactively labelled newly synthesized interleukin-6 could be
C5a ENHANCES INTERLEUKIN-6 SYNTHESIS
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100000 ;
100
10
1
co
0.01
1
0.1
10
100
LPS [ng/ml]
EBB
10 ng/ml
100 ng/ml
Figure I . ElTect of hrC5a on interleukin-6 activity in lipopolysaccharide-treated monocyte cultures. Human monocytes (2 x lo6 cel1s)wereincubdted withoutorwith0.01 ngml-',0.1 ngml-'. 1 ngml-', IOngml-'and 100ngml 'oflipopolysaccharidein the absence or presence of 10 ng ml- I or 100 ng ml-' of hrC5a for 16 h. Interleukin-6 activity was determined by the B9-cell assay as described in Materials and methods. Mean values of 3 different experiments+SD are presented.
Figure 2. Du ~ ( J W synthesis of interleukin-6. Human monocytes (2 x lo6 cells) were incubated without (lanes I . 7, 13) or with lipopolysaccharide in concentrations of0.01 ng ml-' (lanes 2.8. 14). 0.1 ng ml (lanes 3,9. 15). I ng ml (lanes 4. 10. 16). 10 ng ml-'(lanes 5. I I . 17)or 100ngml-'(lanes6. 12, 18)eitherin theabsence(1anes 1-6)orpresenceof IOngml-'hrC5a(lanes7-12) or 100 ng ml-' hrC5a (13-18) for 16 h. The cells were radioactively labelled with [35S]-methionine(150 pCi/dish) for 2 h. Interleukin-6 was immunoprecipitated from the cell medium and subjected to sodium dodecylsulphate/polyacrylamide gel electrophoresis as described in Materials and methods. Molecular weight markers were glyceraldehyde 3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa) and soybean trypsin inhibitor (20 kDa).
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detected in the supernatants of monocytes incubated without lipopolysaccharide. Compared with monocytes stimulated only by lipopolysaccharide the costimulation of monocytes by lipopolysaccharide and by hrC5a led to the detection of radioactively labelled interleukin-6 at lower IiDoDolvsaccharide concentrations (0.1 ng ml- lipopolysaccharide in the presence of 10 ng ml-' or 100 ng ml-' of hrC5a vs. 1 ng ml-' lipopolysaccharide i n the absence of hrC5a). The various bands seen in Fig. 2 represent different molecular weight forms of interleukin-6, which are due to differences in 0- and N-glycosylation. Among others there are two predominant 0-glycosylated forms with apparent molecular weights of 24 kDa and L
~'
23 kDa and a form with an apparent molecular weight of 27.5 kDa, both 0- and N-glycosylated [31]. The labelling experiments demonstrate that hrC5a leads to a stimulation of the de nooo synthesis of interleukin-6 in the presence of lipopolysaccharide.
1
Determination of interleukin-6 mRNA
To study the effect of hrC5a on interleukin-6 mRNA, Northern-blots were done. Figure 3 shows that no detectable amounts of interleukin-6 mRNA could be found in monocytes incubated without lipopolysaccharide or with 10 pg ml-' lipopolysaccharide for 16 h (lanes 1, 3). Addition of 100 ng ml-' lipopolysacchar-
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Figure4. Effect of hrC5a on the kinetics of induction of IL-6 mRNA after LPS stimulation. Human monocytes were incubated without (lane I ) or with 100 ng ml-' of lipopolysaccharide for 2 h (lanes 2.3). 4 h (lanes 4, 5) or 8 h (lanes 6, 7) in the absence (lanes 2. 4, 6) or presence (lanes 3,5,7) of 100 ng ml I of hrC5a. RNA was extracted from 8 x 10' cells. An equal RNA load was assessed by probing of j-actin as house-keeping gene.
Discussion
Figure 3. Effect of hrC5a on IL-6 mRNA levels after stimulation with different concentrations of LPS. Human monocytes were incubated without (lanes I , 2) or with 0.01 ng ml-' (lanes 3, 4) or 100 ng m l ~(lanes 5,6) of lipopolysaccharide in the absence (lanes I , 3 . 5 ) or presence (lanes 2 , 4 , 6 ) of 100 ng ml- I hrC5a for 16 h. RNA was prepared from 8 x 10' cells. Northern blots were performed as described in Materials and methods.
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ide led to a clear increase in interleukin-6 mRNA (lane 5). HrC5a in a concentration of 100 ng ml-' did not enhance interleukin-6 mRNA in the absence of lipopolysaccharide (lane 2); however, it greatly enhanced the lipopolysaccharide-induced increase in interleukin-6 mRNA (lanes 4, 6). When the time-course of induction of interleukin-6 mRNA was studied (Fig. 4) it was found that 2 h or 4 h after addition of lipopolysacharide interleukin-6 mRNA levels were not different between cells incubated without or with hrC5a. In both treatment groups an interleukin-6 mRNA peak could be observed after 4 h. Eight hours after lipopolysaccharide stimulation, however, interleukin-6 mRNA levels were considerably higher in hrC5a treated monocytes than in untreated cells. This finding is in accordance with the mRNA data presented in Fig. 3 and indicates that hrC5a treatment of monocytes prolongs the elevation of interleukin-6 mRNA after stimulation with lipopolysaccharide.
C5a is an important diffusible inflammatory mediator generated during activation of the complement system. Binding of C5a to receptors on mast cells and basophils causes secretion of their granules and release of histamine. Binding of C5a or C5a desArg to receptors on neutrophils induces chemotaxis along a gradient of C5-derived glycopeptides, specific granule secretion, increased adhesive properties, increased oxygen consumption and superoxide generation, and enhanced expression of C3b receptors (for a review see ref. 22). In monocytes and macrophages C5a induces chemotaxis and the slow secretion of glycolytic and proteolytic enzymes. C5a thus has important pro-inflammatory properties. There are a number of systemic reactions of the body to maintain homeostasis during infection and inflammation. Many parts of this acute phase response, e.g. the synthesis of acute phase proteins in the liver, are mediated by interleukin-6 [I]. It was therefore of interest to study whether the pro-inflammatory peptide C5a is indirectly, i.e. via interleukin-6. involved in the regulation of this response. We found that hrC5a alone in a concentration of 10 ng ml- did not stimulate interleukin-6 synthesis by monocytes; however, it had a considerable co-stimulatory effect on the lipopolysaccharide-induced interleukin-6 synthesis. At a hrC5a concentration of 100 ng ml-' there was a slight stimulation of interleukin-6 synthesis even in the absence of lipopolysaccharide, but the most important effect of this high concentration of hrC5a was also seen in the presence of low concentrations (0.01 ng ml-' and 0.1 ng ml-I) of lipopolysaccharide. There are several lines of evidence to exclude the possibility that the observed effects of hrC5a on interleukin-6 synthesis might be due to
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C5a ENHANCES INTERLEUKIN-6 SYNTHESIS lipopolysaccharide contamination in the hrC5a preparation: 1. It had been shown that the hrC5a preparation used in our experiments had only a low lipopolysaccharide contamination of 1 pg pg-l hrC5a [32]. This amount is too low to cause any effects on interleukin-6 synthesis in our system. As shown in Figs 2 and 3, 10 pg of lipopolysaccharide did not lead to a detectable stimulation of the synthesis of interleukin-6. The slight stimulation of interleukin-6 synthesis by 100 ng C5a ml-' was not inhibitable by a preincubation with 5000 U ml- I of the lipopolysaccharide inhibitor Polymyxin B. Using this concentration we were able to inhibit the effect of up to 100 pg lipopolysaccharide mi-'. 2. In a recent report Scholz et af. [33] described that hrC5a-induced IL-6 synthesis by human peripheral blood-derived mononuclear cells was inhibited by heat treatment ( 1 00°C 15 min) suggesting that induction of interleukin-6 was not due to endotoxin contamination. 3. HrC5a alone had little effect on interleukin-6 synthesis by human monocytes in the absence of lipopolysaccharide. The effects of hrC5a were most pronounced in the presence of 0.01 and 0.1 ng mi-' of lipopolysaccharide. If the observed effect of hrC5a were only due to lipopolysaccharide contamination the most pronounced effects should have been found in the absence of lipopolysaccharide and addition of lipopolysaccharide should have abolished the effects.
The central finding of the present paper is that C5a alone only moderately enhances interleukin-6 production by human monocytes, but considerably increases the lipopolysaccharide stimulated interleukin-6 production. This finding adds new important aspects to the report of Scholz et al. [33] who described that C5a alone stimulates interleukin-6 synthesis by monocytes. Whereas the B9 assay showed no significant increase in interleukin-6 bioactivity by hrC5a at high concentrations of lipopolysaccharide, immunoprecipitation of interleukin-6 after labelling with [35S]-methionineor determination of mRNA levels by Northern blotting revealed an increase. This might be explained by the fact that immunoprecipitation and Northern blotting visualize differences at high interleukin-6 concentrations, which due to its logarithmic scale are not reflected by the B9 assay. Synergistic effects of C5a and lipopolysaccharide have also been observed for the synthesis of interleukin-l and tumour necrosis factor c1. In human peripheral blood monocytes, hrC5a had a co-stimulatory effect on the lipopolysaccharide-induced synthesis of both cytokines [34-371. It has been described that C5a induces the mRNA levels of interleukin-I and tumour necrosis factor c1 without inducing protein synthesis of these cytokines, and that the addition of lipopolysaccharide was needed for the translation and secretion of these mRNAs [37]. We did not find an induction of interleukin-6 mRNA synthesis by C5a alone (Fig. 3, lane 2), indicating that the synergistic effect of C5a and lipopolysaccharide on interleukin-6
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induction is not due to a selective action of C5a on interleukin-6 mRNA levels in human monocytes. The increased synthesis of interleukin-6 after lipopolysaccharide stimulation of monocytes in the presence of C5a might be due at least in part to a prolongation of the elevation of interleukin-6 mRNA (see Fig. 4). The mechanism by which hrC5a acts synergistically with lipopolysaccharide is not known. Several mechanisms, e.g. up-regulation of lipopolysaccharide receptors or secondary phenomena, e.g. increased interleukin-6 synthesis due to an increased interleukin- 1 synthesis, have to be considered. The findings presented in this paper show a mechanism by which the acute phase response of the body can be up-regulated. During bacterial infections lipopolysaccharides can directly stimulate the cytokine synthesis in monocytes. Since lipopolysaccharides also activate the complement system [38] the cytokine synthesis may be further enhanced by C5a. Thus, besides its potent pro-inflammatory properties C5a may indirectly stimulate protective mechanisms, such as acute phase protein synthesis in the liver.
Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (SFB 154 and a Heisenberg-Stipendium to V.G.). The skilful technical assistance of Mrs M. David and Mrs D. V. Berg and the help of Mrs G. Zahn with the preparation of the manuscript are greatly appreciated.
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