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The terminal complement complex C5b-9 stimulates interleukin-6 production in human smooth muscle cells through activation of transcription factors NF-B and AP-1¹

CHRISTIANE VIEDT, GERTRUD-MARIA HÄNSCH^*, RALF P. BRANDES, WOLFGANG KÜBLER and JÖRG KREUZER²

Innere Medizin III,
^* Institut für Immunologie, Universität Heidelberg, and
Institut für Kardiovaskuläre Physiologie, Universität Frankfurt, Germany

²Correspondence: Innere Medizin III, Universität Heidelberg, Bergheimer Str. 58, 69115 Heidelberg, Germany. E-mail: Joerg_Kreuzer{at}med.uni-heidelberg.de

SPECIFIC AIMS

The aim of the study was to explore the functional link between complement activation and vascular inflammation. We investigated whether the terminal complement complex C5b-9 affects interleukin 6 (IL-6) production from vascular smooth muscle cells (VSMC) by measuring the IL-6 expression (Northern analysis, ELISA) and set out to determine the underlying signal transduction pathway.

PRINCIPAL FINDINGS

1. C5b-9 induces Gi-proteins, NF-B, and reactive oxygen species-dependent IL-6 secretion from VSMC
The proinflammatory activity of sublytic doses of the terminal complement complex C5b-9 on human VSMC was investigated by measuring the expression of IL-6 by ELISA. Stimulation of cells with purified C5b6, C7, C8, C9 added sequentially in order to form the terminal complement complex C5b-9 resulted in a time-dependent IL-6 secretion. During 24 h, untreated control cells produced 231 ± 132 pg/ml IL-6, whereas cells stimulated with C5b-9 or tumor necrosis factor (50 U/ml) accumulated 584 ± 187 pg/ml and 955 ± 265 pg/ml, respectively (mean ± SD, n=3 experiments, P<0.05). The necessity for an intact C5b-9 terminal complement complex was demonstrated using C5b6, C8, C9 in the absence of C7 or by using C7 alone, both of which failed to induce IL-6 secretion. To investigate the signal transduction pathways leading to C5b-9-mediated IL-6 release, VSMC were preincubated with the Gi-protein inhibitor pertussis toxin (PTX, 500 ng/ml, 18 h), the specific protein kinase C inhibitor GF109203X (2x10^-6 M, 60 min), or pyrrolidine dithiocarbamate (PDTC, 10 µM, 2 h), the pharmacological inhibitor of the IL-6 transcription regulator NF-B or the radical scavenger N-acetylcysteine (NAC, 10 mM, 60 min). Pretreatment with PTX, PDTC, and NAC completely inhibited IL-6 release after 24 h of stimulation; in contrast, GF109203X was without effect.

2. C5b-9-induced IL-6 release by VSMC is preceded by increased IL-6 mRNA
IL-6 generation was also investigated at the mRNA level by Northern blot analysis. C5b-9 increased steady-state IL-6 mRNA levels in VSMC, whereas C5b6, C8, C9 in the absence of C7 or C7 alone did not. mRNA expression peaked within 4 h, then gradually decreased and returned to baseline by 8 h. Similar to IL-6 protein, IL-6 mRNA expression was completely inhibited by preincubation with PTX or PDTC but not by GF109203X.

3. C5b-9 induces formation of reactive oxygen species (ROS), which along with IL-6 release is inhibited by the antioxidant N-acetylcysteine
ROS have been proposed to be important second messengers in atherogenesis and to be involved in the activation of the transcription factors NF-B and AP-1. As measured by dichlorofluorescein (DCF) fluorescence, C5b-9 induced, after a lag time of 5 min, a twofold increase in cellular ROS generation with maximal response within 20 min. The enhanced fluorescence could be completely attenuated by pretreating the cells with PTX (500 ng/ml, 18 h) and to a lesser extent by the radical scavenger NAC (10 mM). C5b-9-induced IL-6 expression also was significantly reduced (70%), but not completely abolished, by NAC and completely inhibited by PTX.

4. C5b-9 activates the transcription factors NF-B and AP-1
NF-B and AP-1, which are both activated by oxidative stress, are important transcriptional regulators of IL-6. Therefore, the effects of C5b-9 on NF-B and AP-1 as downstream effectors were examined by electrophoretic mobility shift assay. Active NF-B was already present after 30 min stimulation; maximal NF-B activation was found after 1 h, was still present after 2 h, and then gradually decreased and returned to base line level at 8 h. To determine which members of the NF-B family were involved in the C5b-9-induced complex, supershift experiments were performed with specific antibodies against the p65, p50 c-Rel, and p52 NF-B subunits. Only the p65 and p50 NF-B subunits are recruited in response to C5b-9 stimulation (schematic ). Excess unlabeled oligonucleotide reduced the signal intensity of the band associated with active NF-B, confirming specificity of the DNA–protein interaction.

View larger version (15K): [in this window] [in a new window]   Figure 3. Activation of IL-6 expression by the terminal complement complex C5b-9. Stimulation of VSMC with C5b-9 activates NF-B and AP-1 binding to their DNA consensus sequence (CS) resulting in IL-6 expression. The AP-1 complex mainly is comprised of c-Jun and the NF-B complex mainly of p65.

Accordingly, C5b-9-induced DNA binding activity of AP-1 was also investigated and found to increase after stimulation for 30 min, with a peak activation at 2 h, then gradually decreased and returned to the basal level at 8 h. Binding was determined to be specific as excess of unlabeled AP-1 consensus sequence reduced the signal. Addition of antibodies specific for c-Jun, c-Fos, and Fra-1 to the AP-1 binding reaction resulted in a shift of the binding complex and identified c-Jun as the prevailing protein in the AP-1 complexes (schematic ).

5. NF-B and AP-1 decoy ODN inhibits the C5b-9-induced IL-6 synthesis
The role of NF-B and AP-1 in C5b-9-mediated IL-6 gene regulation was elucidated further by the use of cis element double-stranded (decoy) oligonucleotides (ODN), which scavenge active transcription factors, thereby blocking their binding to the promoter regions in target genes. Gel mobility shift confirmed that decoy ODN against NF-B or AP-1 binding sites specifically competed, whereas control decoy ODN did not. Pretreatment with NF-B decoy ODN or AP-1 decoy ODN (10 µM) (Fig. 1 ), but not control ODN, specifically inhibited IL-6 secretion in response to C5b-9.

View larger version (25K): [in this window] [in a new window]   Figure 1. A) NF-B-dependent IL-6 release from VSMC. Cells were grown to confluence in 96-well plates and growth-arrested in serum-free medium for 24 h. For the experiment, VSMC were preincubated with the NF-B decoy ODN (10 µM) or control scrambled ODN (10 µM) for 6 h, followed by stimulation with complement components C5b6, C7, C8, and C9. The medium was collected after 24 h and assayed for IL-6 concentration by ELISA. B) AP-1-dependent IL-6 release from VSMC. Cells were preincubated with AP-1 decoy ODN (10 µM) or control scrambled ODN (10 µM) for 6 h, followed by stimulation with complement components C5b6, C7, C8, and C9. The medium was collected after 24 h and assayed for IL-6 concentration by ELISA. Results are representative of five independent experiments. Values are mean + SEM (n=5). *P < 0.05.

Taken together, these data show that the terminal complement complex C5b-9 induced a Gi-dependent expression of IL-6 that was mediated by ROS-dependent signal transduction and required activation of the downstream effectors NF-B and AP-1.

CONCLUSIONS

In this study we demonstrate for the first time that sublytic amounts of the terminal complement component, C5b-9 time-dependently increase expression of IL-6 in VSMC through the recruitment of the redox-sensitive transcription factors NF-B and AP-1. Using decoy oligonuclotides against these two transcription factors, we demonstrate the central role of NF-B and AP-1 for the C5b-9-induced IL-6 release. The effect of C5b-9 on IL-6 expression was demonstrated on a protein and mRNA level in VSMC.

Activation of the complement system plays an importent role in the pathogenesis of atherosclerosis. The exact mechanisms by which complement exerts its proatherosclerotic effects are not yet clear. Immunohistochemical studies demonstrated colocalisation of IL-6 and C5b-9 in atherosclerotic lesions. Although it is apparent that C5b-9 may modulate proinflammatory activation through several signal transduction pathways, the mechanisms through which it induces IL-6 producation in VSMC remain to be established. The excessive production of IL-6 seems to be related to the immunologic and inflammatory activities in the vessel wall. Therefore, it is essential to delineate the molecular and cellular mechanisms of IL-6 production by VSMC. A recent study investigated secretion of monocyte chemoattractant protein-1 (MCP-1) from VSMC after exposure to C5b-9. The rapid release of MCP-1 (within 10 min) infers a release of existing MCP-1 rather than the de novo induction of gene expression as seen in our studies with IL-6, suggesting different mechanisms to be at work.

To date, no specific receptor for activated complement has been identified. Upon binding of C7, C8, and C9 to the membrane, hydrophobic side chains are exposed allowing the insertion of the complex into the lipid bilayer. Through this mechanism, C5b-9 formed on nucleated cell membranes gains access to potential interactions with different intracellular second messengers. It has been described that C5b-9 can activate VSMC through Gi-proteins. In our study, IL-6 expression was also regulated through Gi. PTX not only inhibited IL-6 expression, but also C5b-9-dependent activation of transcription factors.

Other pathways might also be involved in the action of complement. In glomerular epithelial cells, it has been demonstrated that the components C5b-8 and C5b-9 increase intracellular levels of sn-1,2-diacylglycerol and affect protein kinase C activity. In the present study, however, the effects of C5b-9 were not mediated through PKC as demonstrated by the lack of effect of GF109203X, the specific PKC inhibitor used in the present study. This difference might be attributed to the different cell types used in the two studies.

Reactive oxygen species have been implicated as important mediators in inflammatory processes, such as atherosclerosis. In mesangial cells, it has also been reported that the terminal complement complex can elicit the production of ROS. Further, ROS formation can activate cell signal transduction pathways leading to the production of inflammatory mediators. Consistent with these findings, we observed that C5b-9 also induced ROS production in VSMC. Beyond that, IL-6 synthesis in VSMC was prevented by pretreatment with the antioxidant NAC. This indicates that C5b-9-induced reactive oxygen species influence intracellular redox status, which in turn modulates cell activation and proinflammatory cytokine gene expresssion.

ROS generation has been associated with the activation of NF-B, and NF-B and AP-1 are transcriptional regulators of IL-6. Accordingly, C5b-9-induced IL-6 protein and mRNA expression were inhibited by the pharmacological NF-B inhibitor, PDTC. The increase in the transcription rate of the IL-6 gene in our study suggest the activation of specific transcription factors that can bind to the regulatory regions of the IL-6 promoter. As different sets of transcription factors may regulate the IL-6 gene in a cell type-specific manner, it is still uncertain which element is functional in C5b-9-stimulated VSMC. In this study, we demonstrated that C5b-9 increased IL-6 expression and stimulated NF-B and AP-1 activation. Therefore, we reasoned that C5b-9 stimulates IL-6 gene expression through the NF-B and AP-1 complexes. To prove this hypothesis, we used the decoy approach against NF-B and AP-1 binding sites, respectively. Gel mobility shift assay showed that decoy against NF-B or AP-1 binding sites specifically competed, whereas control decoy ODN did not. Next, we examined the functional coupling between NF-B and AP-1 activation and C5b-9-induced IL-6 synthesis. Our results showed that NF-B and AP-1 decoy ODN effectively inhibited IL-6 production in response to C5b-9 (Fig. 1) . On the other hand, control decoy ODN had no effects. This clearly shows that AP-1 and NF-B play an important role in C5b-9-induced IL-6 secretion.

Our results demonstrate that C5b-9 stimulates the gene expression and production of the proinflammatory cytokine IL-6. C5b-9 mediates IL-6 production by a change in the cellular redox state. Activation of NF-B and AP-1 in VSMC by C5b-9 plays a pivotal role in this process. This may be one of the underlying mechanisms by which systemic inflammation can foster atherogenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0468fje To cite this article, use (October 6, 2000) FASEB J. 10.1096/fj.00-0468fje

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