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The Journal of Immunology, 2001, 167: 3972-3979.
Copyright © 2001 by The American Association of Immunologists

Anaphylatoxin C5a Actions in Rat Liver: Synergistic Enhancement by C5a of Lipopolysaccharide-Dependent {alpha}2-Macroglobulin Gene Expression in Hepatocytes Via IL-6 Release from Kupffer Cells1

Claudia Mäck*, Kurt Jungermann*, Otto Götze{dagger} and Henrike L. Schieferdecker2,*

* Institut für Biochemie und Molekulare Zellbiologie and {dagger} Abteilung für Immunologie, Georg-August-Universität Göttingen, Göttingen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The effects of the anaphylatoxins C5a and C3a on the liver are only poorly characterized in contrast to their well known systemic actions. Recently, it has been demonstrated that the anaphylatoxin C5a enhanced glucose output from hepatocytes (HC) indirectly via prostanoid release from Kupffer cells (KC). In the present study, it is shown that recombinant rat C5a (rrC5a), together with LPS, activated the gene of the acute phase protein {alpha}2-macroglobulin ({alpha}2MG) in HC also indirectly via IL-6 release from KC. RrC5a alone increased neither IL-6 mRNA in nor IL-6 release from KC, whereas LPS alone did so. However, rrC5a synergistically enhanced the LPS-dependent increase in IL-6 mRNA and IL-6 release. Only rIL-6, but not TNF-{alpha} or IL-1{beta}, enhanced {alpha}2MG mRNA in HC. In line with the actions of rrC5a and LPS on KC, conditioned medium of KC stimulated only with rrC5a did not increase {alpha}2MG mRNA in HC. However, medium of KC stimulated with rrC5a plus LPS induced {alpha}2MG mRNA expression in HC more strongly than medium from cells stimulated only with LPS; thus, C5a acted synergistically with LPS. The stimulatory effects of KC-conditioned medium could partially be inhibited by a neutralizing anti-IL-6 Ab, indicating that KC-derived IL-6 was a major mediator in C5a- plus LPS-elicited {alpha}2MG gene expression. These results suggest that C5a, besides enhancing glucose output via prostanoids, is involved in the initiation of the acute phase response in HC via proinflammatory cytokines from KC. This provides evidence for another important function of C5a in the regulation of hepatocellular defense reactions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The anaphylatoxins C3a and C5a are generated during activation of the complement system via the classical, the alternative, or the mannan-binding lectin pathway. C5a, a glycopeptide of 77 (rat) and 74 (human) amino acids, respectively (1, 2, 3, 4), is the N-terminal cleavage product of the {alpha}-chain of the precursor protein C5. C5a is involved in systemic defense reactions by causing contraction of smooth muscle cells, an increased vascular permeability, degranulation of mast cells, and activation, chemotaxis, and margination of neutrophils (5, 6, 7). In the case of disorders of the protective barrier function of the gastrointestinal tract as with inflammatory bowel disease or liver cirrhosis, LPS as a component of the outer cell wall of various Gram-negative gut bacteria can leak via the mesenteric veins into the portal vein and thus can reach the liver. Because LPS is a major trigger of complement activation via the alternative pathway (8), the encounter of LPS with complement proteins circulating in the mesenteric/portal bed and being newly synthesized and secreted by hepatocytes (HC)3 (9, 10) can lead locally to the generation of anaphylatoxins and thus make the liver their primary target organ. Therefore, anaphylatoxins, alone or together with their trigger LPS, might play a key role in the initiation of liver-specific defense reactions.

In normal rat liver C5aR are only expressed by nonparenchymal cells, i.e., strongly by Kupffer cells (KC), the resident macrophages, by fat-storing hepatic stellate cells, and weakly by the fenestrated sinusoidal endothelial cells, but not by HC (11, 12). Nevertheless, C5a was shown to influence HC-specific effector functions. In perfused rat livers, complement-activated serum (13) and the anaphylatoxin C5a (14) enhanced glucose output, thereby providing energy substrates as well as electron donors for the generation of reactive oxygen species by KC. In accordance with the finding that HC do not express C5aR, the C5a-dependent glucose output was shown to be mediated indirectly by prostanoids (15) released from KC (16) and hepatic stellate cells (17).

Besides maintaining energy supply by glucose output, HC support intra- as well as extrahepatic defense reactions by an altered synthesis and secretion of positive and negative acute phase proteins (APP) in the course of the acute phase response (APR). Whereas the short-term increase in glucose release during inflammatory processes is elicited by prostanoids from KC (15, 16), the long-term enhancement of hepatocellular APP synthesis is mainly mediated by cytokines such as TNF-{alpha}, IL-1{beta}, and especially IL-6 (18, 19, 20, 21). One major source of these proinflammatory cytokines is macrophages like KC (22), which represent the largest population of macrophages in the organism. Therefore, it was investigated whether C5a, besides enhancing hepatic glucose release via prostanoids, might also initiate APP synthesis via cytokines. In detail, it was examined whether C5a might induce the synthesis of IL-6, TNF-{alpha}, and IL-1{beta} in KC and whether it might thus (alone or together with LPS, which triggers C5a formation) elicit the APR. It was found that C5a alone failed to induce IL-6 mRNA in and IL-6 protein secretion from KC but synergistically enhanced LPS-induced IL-6 mRNA expression and protein release. Accordingly, conditioned medium of KC stimulated with C5a together with LPS increased gene expression of the APP {alpha}2-macroglobulin ({alpha}2MG) in HC clearly more strongly than conditioned medium of cells stimulated solely with LPS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Male Wistar rats (270–370 g for the isolation of KC, 200–250 g for HC; Winkelmann, Borchen, Germany) were kept on a 12-h day/night rhythm (light 7 a.m. to 7 p.m.) with free access to water and a standard rat diet (Ssniff, Soest, Germany) for at least 2 wk before starting the experiments. Animal care followed the German Law on the Protection of Animals and was performed with permission of the state animal welfare committee.

Chemicals

All materials were of analytical grade and obtained from commercial sources. Pronase was obtained from Merck (Darmstadt, Germany), collagenase H and DNase from Roche Diagnostics (Mannheim, Germany), Nycodenz from Life Technologies (Eggenstein, Germany), and Percoll from Pharmacia (Freiburg, Germany). RPMI 1640 was purchased from Biochrom (Berlin, Germany), and M199 from AppliChem (Darmstadt, Germany). Newborn calf serum (NCS) was obtained from PAA Laboratories (Cölbe, Germany); insulin, penicillin, and streptomycin sulfate from Serva (Heidelberg, Germany). Tissue culture dishes were obtained from Nunc (Wiesbaden, Germany); dexamethasone, actinomycin D, and cycloheximide (CHX) from Sigma (Deisenhofen, Germany). Recombinant rat IL-6 (rrIL-6), recombinant human TNF-{alpha} (rhTNF-{alpha}), and rhIL-1{beta} were purchased from Strathmann Biotech (Hannover, Germany); LPS (Escherichia coli 026:B6) was obtained from Sigma. Polyclonal Abs against rat IL-6, rat TNF-{alpha}, and rat IL-1{beta} were obtained from R&D Systems (Wiesbaden, Germany). Oligonucleotide primers were custom synthesized by NAPS (Göttingen, Germany), and plasmids pBlueScript, pUC18, and pUC57 were obtained from MBI Fermentas (St. Leon-Rot, Germany). ELISA kits for IL-6 were purchased from BioSource International (Ratingen, Germany) and for INF-{alpha} and IL-1{beta} were from Amersham Pharmacia Biotech (Freiburg, Germany).

Preparation of rrC5a

RrC5a was prepared by synthesis of a cDNA from rat liver RNA and a subsequent PCR using degenerate 5' and 3' primers that were designed according to sequence data published in the EMBL GenBank (accession no. X91892, ID: RNC5AARPT) (4) as described previously (4, 16). RrC5a contained, in addition to the original sequence of amino acids 1–77, the N-terminal sequence MRGSHHHHHHGS used for its purification from bacterial lysates by Ni2+-chelate chromatography and was depleted of endotoxins by affinity chromatography on polymyxin B agarose (Sigma). Endotoxin depletion of the C5a stock solution (100 µg/ml) was demonstrated by a negative Limulus amebocyte lysate assay (Sigma), which had a detection limit of 0.01 EU/ml or 1 pg/ml. Thus, the LPS content of the amount of rrC5a used for stimulation was below 10 fg/ml.

KC preparation and culture

KC were isolated by combined collagenase/Pronase perfusion of rat liver and purified by density gradient centrifugation and subsequent counterflow elutriation using a Beckman JE-6 elutriation rotor in a J-21 Beckman centrifuge (Beckman Instruments, München, Germany) (23). KC were plated at 4 x 106 cells/dish on 3.5-cm-diameter tissue culture dishes in RPMI 1640 supplemented with 30% NCS and 1% penicillin/streptomycin (KC culture medium). After 24 h the NCS content of the medium was reduced to 10% and, after an additional 24 h, to 0% (serum-free KC culture medium). Seventy-two hours after the preparation, KC were stimulated with rrC5a and/or LPS in concentrations given in Results. When indicated, KC were pretreated for 30 min with actinomycin D (Act D, 1 µg/ml) or CHX (1 µg/ml) before stimulation with rrC5a and LPS. At the time points indicated, media were taken for the measurement of cytokine release and for the stimulation of HC, or cells were scraped off the culture dishes with lysis buffer (RNeasy kit; Qiagen, Hilden, Germany) for the isolation of total RNA.

HC preparation and culture

HC were prepared by collagenase digestion or according to Meredith (24) without the use of collagenase as described previously (25). Purity of HC was >99% as identified on the basis of their typical light microscopic appearance. They were plated at 1 x 106 cells/ml on 3.5-cm-diameter tissue culture dishes in M199 supplemented with 0.5 nM insulin, 100 nM dexamethasone, 1% penicillin/streptomycin, and additional 4% NCS for the first 4 h. Cells were then cultured in 1 ml of serum-free medium with one medium change after 24 h. After 48 h, 500 µl of medium was replaced by conditioned media of stimulated or unstimulated KC or by serum-free KC culture medium containing rrC5a, LPS, rrIL-6, rhTNF-{alpha}, or rhIL-1{beta}, as indicated. In some experiments KC-conditioned media were preincubated with polyclonal Abs against rat IL-6, TNF-{alpha}, or IL-1{beta} (0.09, 2, and 1 µg/ml, respectively) for 1 h at 37°C. Twenty-four hours after stimulation, HC were scraped off the culture dishes with lysis buffer (RNeasy kit; Qiagen) for the detection of {alpha}2MG mRNA expression.

RT-PCR

Total RNA from cultured KC was isolated by the RNeasy kit provided by Qiagen, preincubated for 10 min at 68°C with 500 ng of oligo(dT)12–18, and transcribed into cDNA with reverse transcriptase (Superscript II; Life Technologies, Eggenstein, Germany). The cDNA thus generated was amplified in a 50-µl reaction mixture containing 1.5 mM MgCl2, 0.6 µM forward and reverse oligonucleotide primers for rat {beta}-actin ({beta}-actin-f and -r), IL-6 (IL-6-f and -r), TNF-{alpha} (TNF-{alpha}-f and -r), or IL-1{beta} (IL-1{beta}-f and -r) (Table IGo), 0.2 mM dNTPs, 6% DMSO, and 0.5 U of thermostable DNA polymerase (Goldstar Red; Eurogentec, Seraing, Belgium). For PCR master mixtures were prepared before adding the respective cDNAs. The cDNA was denatured for 3 min at 94°C and then subjected to 35 cycles of 1 min at 94°C, 1 min at 61°C ({beta}-actin), 54°C (IL-6), 52°C (TNF-{alpha}), or 56°C (IL-1{beta}), and 2 min at 72°C with a final elongation step of 10 min at 72°C. After amplification, PCR products were separated on 2% agarose gels and visualized by ethidium bromide staining. PCR products were cloned with a SureClone ligation kit (Amersham Pharmacia Biotech) into pUC 18 (IL-6) or pBlueScript (TNF-{alpha}, IL-1{beta}) and sequenced for identification using the Ready Reaction BigDye Terminator kit (PerkinElmer, Weiterstadt, Germany).


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  		Table I. Oligonucleotide primers

 
LightCycler-PCR (LC-PCR) and melting curve analysis

For PCR analysis with the LightCycler (Roche Diagnostics, Mannheim, Germany), cDNA generated as described above was amplified in a 10-µl reaction mixture containing 3 mM MgCl2, 0.5 µM forward and reverse oligonucleotide primers for rat {beta}-actin (LC-{beta}-actin-f and -r); IL-6-f and -r; TNF-{alpha}-f and -r; and IL-1{beta}-f and -r (Table IGo), and 10x LC Master SYBR Green I (Roche Diagnostics). After initial denaturation for 30 s at 95°C, cDNA was subjected to 45 cycles of denaturation, annealing, elongation, and fluorescence reading with the following settings: LC-{beta}-actin: 0 s 95°C, 5 s 60°C, 12 s 72°C, 0 s 86°C; IL-6: 0 s 95°C, 5 s 54°C, 20 s 72°C, 0 s 79°C; TNF-{alpha}: 0 s 95°C, 5 s 52°C, 28 s 72°C, 0 s 87°C; IL-1{beta}: 0 s 95°C, 5 s 56°C, 20 s 72°C, 0 s 82°C. cDNA fragments cloned in pUC57 ({beta}-actin), pUC18 (IL-6), or pBlueScript (TNF-{alpha}, IL-1{beta}) served as standards. For melting curve analysis, samples were heated to 95°C at a transition rate of 0.2°C/s with continuous fluorescence readings. Fluorescence signals were quantified using LC Data Analysis software combined with melting peak correction.

Northern blot analysis

Total RNA from cultured HC was isolated by the RNeasy kit provided by Qiagen and separated on denaturing formaldehyde gels. Equal lane-to-lane loading of RNA was assured by ethidium bromide staining of the gels before blotting. RNA was transferred to nylon membranes according to standard procedures (26). {alpha}2MG mRNA was detected by hybridization with a digoxigenin (DIG)-labeled complementary RNA probe, provided by Dr. Bruno Christ (Institut für Biochemie und Molekulare Zellbiologie, Göttingen, Germany) and generated as follows: a PCR product was amplified from a 0.6-kb insert of rat {alpha}2MG cDNA (27) cloned in pBR322 (pBR-{alpha}2MG). Because this plasmid could not be used for in vitro transcription, a PCR product containing a promoter for T7 RNA polymerase was generated by amplification of pBR-{alpha}2MG with a sense primer complementary to {alpha}2MG ({alpha}2MG-f) and an antisense primer complementary to {alpha}2MG coupled to the sequence of the T7 RNA polymerase promoter ({alpha}2MG-r-T7) (Table IGo). After purification with the QIA Quick PCR Purification kit (Qiagen) this PCR product was used for the generation of a DIG-labeled cRNA probe by in vitro transcription. The reaction was conducted with T7 RNA polymerase (MBI Fermentas) according to the standard protocol of the DIG RNA Labeling Mix (Roche Diagnostics) in the presence of DIG-UTP. Subsequently, template DNA was digested with 1 U of RNase-free DNase I (Roche Diagnostics) for 15 min at 37°C. The DIG-labeled antisense {beta}-actin probe was generated in pBlueScript by in vitro transcription of a 550-bp {beta}-actin cDNA fragment (GenBank accession no. HSA1007, positions 69–618) using T3 RNA polymerase (28). Hybridization at 68°C and detection were performed according to the manufacturer’s application notes of the DIG-nucleic acid detection kit (Roche Diagnostics).

Determination of cytokine concentrations

IL-6, TNF-{alpha}, and IL-1{beta} protein concentrations in the culture media of KC were determined by commercial ELISA kits without further purification, according to the manufacturer’s instructions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction by rrC5a of TNF-{alpha} mRNA and IL-1{beta} mRNA but not of IL-6 mRNA in cultured KC

In a first series of experiments the dose and time dependence of the induction by rrC5a of IL-6-, TNF-{alpha}-, and IL-1{beta}-mRNA expression was characterized and compared with that of LPS. Untreated KC did not express IL-6 mRNA or TNF-{alpha} mRNA, but clearly expressed basal levels of IL-1{beta} mRNA (Fig. 1Go). RrC5a dose-dependently increased TNF-{alpha} mRNA and IL-1{beta} mRNA expression. The levels of TNF-{alpha} mRNA and IL-1{beta} mRNA were faint after stimulation with 1 nM (10 ng/ml) rrC5a, increased with 10 nM (100 ng/ml), and were highest with 100 nM (1 µg/ml) rrC5a (Fig. 1Go), consistent with the binding affinity of rrC5a for its receptor (29). At 100 nm rrC5a, i.e., at a concentration that was reached in human peripheral blood after complete activation of the complement system (30), rrC5a was a weaker inducer of TNF-{alpha} mRNA and IL-1{beta} mRNA than LPS at 1 ng/ml (Fig. 1Go). This LPS concentration was found in peripheral blood of patients with Gram-negative sepsis (31), and therefore might represent the maximal pathophysiologically relevant LPS level. Although rrC5a elicited TNF-{alpha} and IL-1{beta} mRNA expression, it induced IL-6 mRNA neither at the lower concentrations of 1 and 10 nM nor (in most experiments) at the higher concentration of 100 nM; if 100 nM rrC5a induced IL-6 mRNA in some experiments, it did so very weakly (Fig. 1Go). In contrast to rrC5a, LPS effectively induced IL-6 mRNA at the pathophysiologically relevant concentration of 1 ng/ml. At this concentration LPS was still submaximally effective, because 10 and 100 ng/ml LPS induced TNF-{alpha}, IL-1{beta}, and IL-6 mRNA more strongly than 1 ng/ml (data not shown). The stimulating effects of rrC5a on TNF-{alpha} and IL-1{beta} mRNA expression could not be ascribed to contaminating LPS, neither in the serum-free medium nor in the rrC5a used for stimulation, because 1) unstimulated in contrast to LPS-treated cells did not express IL-6 mRNA and TNF-{alpha} mRNA and 2) rrC5a in contrast to LPS failed to induce IL-6 mRNA.



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  		FIGURE 1. Dose dependence of the induction of IL-6 mRNA, TNF-{alpha} mRNA, and IL-1{beta} mRNA by rrC5a in cultured KC. KC were isolated by collagenase/Pronase perfusion of rat liver followed by Nycodenz gradient centrifugation and centrifugal elutriation. KC were cultured for 72 h and then stimulated with rrC5a at the given concentrations or for comparison with LPS (1 ng/ml). After 3 h cells were scraped off the tissue culture dishes for isolation of RNA. After reverse transcription with oligo(dT) as primers, cytokine cDNA was detected by conventional PCR with cytokine-specific primers (Table IGo). PCR products were visualized after electrophoresis in agarose gels by ethidium bromide staining. One representative of three different experiments is shown.

 
The finding that rrC5a did not induce IL-6 mRNA expression could not be explained by different kinetics of rrC5a- compared with LPS-caused IL-6 mRNA induction. At pathophysiological concentrations, 100 nM rrC5a failed to significantly induce IL-6 mRNA over a time period of up to 5 h (Fig. 2Go), whereas 1 ng/ml LPS strongly increased IL-6 gene expression to a maximum within 3 h, which remained stable up to 5 h (Fig. 2Go). For comparison, rrC5a induced the expression of TNF-{alpha} mRNA and IL-1{beta} mRNA in KC primary cultures with time courses similar to those after stimulation with LPS (Fig. 2Go). Both rrC5a and LPS induced TNF-{alpha} mRNA expression within 30 min. TNF-{alpha} mRNA expression was maximal at 60 min after rrC5a and at 90 min after LPS; it then declined. IL-1{beta} mRNA expression after rrC5a reached a maximum between 1 and 2 h and then slowly declined, whereas it was maximal after LPS at 90 min and remained stable for up to 5 h.



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  		FIGURE 2. Time dependence of the induction of IL-6 mRNA, TNF-{alpha} mRNA, and IL-1{beta} mRNA by rrC5a in cultured KC. KC were isolated and cultured as described in Fig. 1Go and then stimulated with rrC5a (100 nM) or for comparison with LPS (1 ng/ml) for the indicated times. Cytokine mRNA expression was detected by RT-PCR as described in Fig. 1Go and quantified by videodensitometry. The unstimulated control value was set equal to 1 (arbitrary unit). Data represent differences between stimulated and unstimulated cells and are means ± SEM of three to six experiments.

 
Enhancement by rrC5a of LPS-induced IL-6 mRNA and protein but not of TNF-{alpha} or IL-1{beta} mRNA and protein in cultured KC

To investigate whether rrC5a and LPS had any costimulatory effects on the synthesis of proinflammatory cytokines in cultured KC, both stimuli were given simultaneously. In these experiments demanding precise quantification, mRNA expression was analyzed by real-time PCR using the LC. Although 100 nM rrC5a by itself failed to induce IL-6 mRNA expression significantly (cf Figs. 1Go and 2Go), it clearly enhanced IL-6 mRNA expression elicited by 1 ng/ml LPS (Fig. 3Go), which induced IL-6 mRNA submaximally (data not shown). RrC5a enhanced LPS-dependent IL-6 mRNA expression slightly at 1 h after stimulation and clearly in a synergistic manner at 2 and 3 h after stimulation. Thus, the kinetics of rrC5a+LPS-induced IL-6 mRNA expression corresponded to that after stimulation with LPS. RrC5a, even though it weakly induced TNF-{alpha} mRNA as well as IL-1{beta} mRNA, did not influence LPS-caused TNF-{alpha} or IL-1{beta} gene expression at 1, 2, or 3 h after stimulation (Fig. 3Go).



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  		FIGURE 3. Time dependence of IL-6 mRNA, TNF-{alpha} mRNA, and IL-1{beta} mRNA induction by costimulation with rrC5a and LPS in cultured KC. KC were isolated and cultured as described in Fig. 1Go and then stimulated with rrC5a (100 nM) and/or LPS (1 ng/ml) for the time points indicated. Cytokine mRNA expression was detected and quantified by real-time PCR using the LC and correlated to the value of {beta}-actin mRNA expression (in arbitrary units). Data represent differences between stimulated and unstimulated cells and are means ± SEM of three to six experiments.

 
Also at a LPS concentration of 0.1 ng/ml, which is 10- to 30-fold higher than the normal LPS concentration found in plasma (31), 100 nM rrC5a clearly enhanced the LPS-dependent induction of IL-6 mRNA, but again had no costimulatory effect on the LPS-elicited TNF-{alpha} mRNA and IL-1{beta} mRNA induction (Fig. 4Go A). At a subthreshold LPS concentration of 0.01 ng/ml, which did not induce IL-6 mRNA, 100 nM rrC5a did not exert any additional effect (Fig. 4GoA). Over the whole range of LPS concentrations tested from 0.01 to 1 ng/ml rrC5a applied at the lower pathophysiological level of 10 nM had no significant effect (data not shown).



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  		FIGURE 4. Costimulatory effect of rrC5a on LPS-induced IL-6, but not TNF-{alpha} or IL-1{beta}, mRNA expression and protein release in cultured KC. KC were isolated and cultured as described in Fig. 1Go. After 72 h of culture, cells were stimulated with rrC5a and/or LPS at the given concentrations. A, After 2 h, cytokine mRNA expression was detected and quantified by real-time PCR with the LC as in Fig. 3Go. Data represent differences between stimulated and unstimulated cells and are means ± SEM of three to six experiments. *, p <= 0.05, significant differences between LPS- and rrC5a + LPS-stimulated KC (Student’s t test for paired samples). B, Protein concentrations were determined in the culture media by ELISA after 24 h (IL-6) or 6 h (TNF-{alpha}, IL-1{beta}). Data represent differences between stimulated and unstimulated cells and are means ± SEM of three experiments. *, p <= 0.05, significant differences between LPS- and rrC5a+LPS-stimulated KC (Student’s t test for paired samples).

 
On the protein level, 100 nM rrC5a did not induce the release of IL-6, TNF-{alpha}, or IL-1{beta} from KC between 90 min and 24 h, whereas 1 ng/ml LPS enhanced the release of IL-6 and TNF-{alpha}, but not of IL-1{beta} (Fig. 4GoB and data not shown). Again, paralleling the mRNA results, rrC5a synergistically enhanced the LPS-induced IL-6 release. This effect was first observed weakly 2 h after stimulation and strongly between 12 h (data not shown) and 24 h (Fig. 4GoB). As found on mRNA level, rrC5a and LPS had no costimulatory effect on TNF-{alpha} release or the very weak IL-1{beta} release at 90 min, 6 h, 12 h (data not shown), and 24 h (Fig. 4GoB).

Pretreatment of KC with the transcriptional inhibitor Act D completely inhibited C5a+LPS-dependent IL-6 mRNA expression (Fig. 5Go), demonstrating that IL-6 gene activation was regulated at the transcriptional level. C5a+LPS-dependent IL-6 mRNA expression was not significantly influenced by the protein synthesis inhibitor CHX (Fig. 5Go), indicating that the transcriptional activation by rrC5a and LPS was independent from de novo protein synthesis.



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  		FIGURE 5. Inhibition of rrC5a+LPS-dependent IL-6 mRNA induction by Act D, but not by CHX, in cultured KC. KC were isolated and cultured as described in Fig. 1Go. KC were then treated for 30 min with Act D (1 µg/ml) or CHX (1 µg/ml) before stimulation with rrC5a (100 nM) and LPS (1 ng/ml) for 2 h. IL-6 mRNA expression was detected by conventional PCR and quantified by real-time PCR with the LC as in Fig. 3Go. Data represent differences between treated and untreated cells and are means ± SEM of three experiments.

 
Induction of {alpha}2MG mRNA in cultured HCs by conditioned media of KC stimulated with rrC5a and LPS

{alpha}2MG is known to be a major IL-6-dependent type 2 APP in rat HC (18, 19). Therefore, the influence of conditioned media from rrC5a- and LPS-stimulated KC on the expression of {alpha}2MG mRNA was investigated. For these experiments, cultured KC were incubated with 100 nM rrC5a and 1 ng/ml LPS for 24 h, when IL-6 levels in the medium were still maximal (see above; cf. Fig. 4GoB). HC were then stimulated with these KC-conditioned media for another 24 h, because preliminary results had shown that rrIL-6 induced {alpha}2MG mRNA expression maximally 24 h after stimulation (data not shown). Conditioned medium of KC stimulated with rrC5a failed to induce {alpha}2MG mRNA significantly, whereas medium of KC stimulated with LPS slightly did so (Fig. 6Go, middle). Conditioned medium of KC treated with rrC5a together with LPS induced {alpha}2MG mRNA expression clearly more strongly than conditioned medium of cells treated only with LPS. In a first series of controls, the direct stimulation of HC with rrC5a and/or LPS did not affect {alpha}2MG gene expression (Fig. 6Go, right). In a second series of controls, as expected, rrIL-6 (10 ng/ml), but not rhTNF-{alpha} (100 ng/ml) or rhIL-1{beta} (100 ng/ml) strongly induced the expression of mRNA for the type 2 APP {alpha}2MG in HC (Fig. 6Go, left).



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  		FIGURE 6. Enhancement of {alpha}2MG mRNA expression in cultured HC by conditioned media of KC stimulated with rrC5a and/or LPS. HC were isolated and cultured as described in Materials and Methods. After 48 h they were stimulated for another 24 h with 1) rrIL-6 (10 ng/ml), rhTNF-{alpha} (100 ng/ml), or rhIL-1{beta} (100 ng/ml), 2) conditioned media of unstimulated KC or KC stimulated with rrC5a (100 nM) and/or LPS (1 ng/ml), which replaced 0.5 ml of the original 1 ml of medium, or 3) serum-free KC culture medium containing rrC5a (100 nM) and/or LPS (1 ng/ml), which again replaced 0.5 ml of the original 1 ml of medium. Total RNA was isolated and separated electrophoretically in denaturating agarose gels and analyzed by Northern blot hybridization with DIG-labeled {alpha}2MG and {beta}-actin probes. Chemiluminescence signals were quantified by videodensitometry. Data represent the ratios of {alpha}2MG mRNA in treated compared with untreated controls (fold increase) and are means ± SEM of three to four experiments. *, p <= 0.05; **, p <= 0.01, significant differences (Student’s t test for paired samples).

 
To investigate whether induction of {alpha}2MG mRNA expression in HC by KC-conditioned media was indeed mediated by IL-6, media of stimulated KC were pretreated with neutralizing polyclonal Abs against the proinflammatory cytokines IL-6, TNF-{alpha}, and IL-1{beta}. Induction of {alpha}2MG mRNA expression in HC by conditioned medium of LPS-stimulated KC was inhibited significantly by anti-IL-6 to ~55% of untreated controls but not by anti-TNF-{alpha} or anti-IL-1{beta} (Fig. 7Go, left). The stronger induction of {alpha}2MG expression induced by medium of KC stimulated with LPS together with rrC5a was again reduced by anti-IL-6 to ~60%, but it was also lowered slightly by anti-IL-1{beta} to ~80% while it remained essentially unaffected by anti-TNF-{alpha} (Fig. 7Go, right). The inhibitory effect of anti-IL-6 was significantly stronger than that of anti-IL-1{beta}, but it was not enhanced by coincubation with anti-IL-6 and anti-IL-1{beta} (data not shown). Nevertheless, {alpha}2MG expression was not completely inhibited by anti-IL-6. In control experiments, {alpha}2MG mRNA expression induced directly by 10 ng/ml rrIL-6 was inhibited by anti-IL-6 to ~40%, whereas the effect of 1 ng/ml rrIL-6, which is still higher than the concentration of native IL-6 measured in the KC-conditioned media (cf Fig. 4GoB) was abrogated (data not shown). This might be explained by a higher affinity of anti-IL-6 to the unglycosylated rrIL-6, against which it had been generated, than to the native glycosylated protein or by the involvement of other factors (cf Discussion). The inhibitory effect of anti-IL-1{beta} on {alpha}2MG expression induced by the conditioned media was most likely due to unspecific interactions of the Ab with rrIL-6, because it inhibited also the direct actions of rrIL-6 on HC to ~70 and 80%, respectively (n = 2, data not shown). Thus, the present data indicate that the induction of {alpha}2MG mRNA in HC by C5a plus LPS-conditioned medium from KC was mainly, but probably not exclusively, due to IL-6.



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  		FIGURE 7. Inhibition by a neutralizing anti-IL-6 Ab of {alpha}2MG mRNA expression in cultured HC induced by conditioned media of C5a- and LPS-stimulated KC. Experiments were performed as described in Fig. 6Go but additionally KC-conditioned media were pretreated for 1 h with polyclonal Abs against rat IL-6, TNF-{alpha}, and IL-1{beta} (3, 5, and 2 µg/ml, respectively) as indicated. Total RNA was analyzed for specific mRNA expression by Northern blot hybridization as described in Fig. 6Go. Data represent mRNA expression as a percentage of LPS-stimulated controls (these values were set equal to 100%) and are means ± SEM of three to four experiments. *, p <= 0.05, significant differences (Student’s t test for paired samples).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It was shown in the present investigation that C5a, which alone had no effect on IL-6 synthesis, synergistically enhanced LPS-elicited IL-6 but not TNF-{alpha} release from cultured KC (Figs. 3Go and 4Go). Moreover, it was found that conditioned medium of KC treated with C5a and LPS stimulated {alpha}2MG mRNA expression in HC more strongly than media of cells treated solely with LPS (Fig. 6Go). These stimulatory actions of the KC-conditioned medium were mainly mediated by IL-6 as verified by anti-IL-6 Abs (Fig. 7Go).

Synergistic enhancement by C5a of LPS-elicited IL-6 release from KC

The possible actions of C5a in the absence or presence of LPS on the production of proinflammatory cytokines by KC have not been studied before. Some investigations have been conducted with human PBMC (hPBMC). In line with the present results, two investigations reported that C5a alone had no effect on IL-6 release, but that it enhanced LPS-induced IL-6 formation in hPBMC (32, 33); yet, at variance with the present findings, one study found that C5a alone stimulated IL-6 release from hPBMC (34). Controversial observations have also been made on the action of C5a on TNF-{alpha} and IL-1{beta} release from hPBMC. In one examination, C5a alone had a stimulatory effect (35); in another, it had not (36), but in both it enhanced LPS-induced TNF-{alpha} and IL-1{beta} release from hPBMC. However, this latter costimulatory action of C5a and LPS on TNF-{alpha} and IL-1{beta} formation was not observed in the present study with KC (Fig. 4Go). The reasons for the discrepancies between different studies with hPBMC are not clear; the differences between studies with hPBMC and rat KC may be ascribed to cell type- and species-specific signaling pathways. C5a and LPS did not induce IL-1{beta} release from KC, probably due to their inability to activate caspase-1-dependent (37) or -independent (38) pathways of IL-1{beta} processing. Indeed, preliminary studies have shown that C5a as well as LPS induced the synthesis of the 35-kDa pro-IL-1{beta} in isolated KC, but failed to promote posttranslational processing and release into the medium under the experimental conditions used (39).

Mechanism of the synergistic action of C5a and LPS on IL-6 release from KC

Knowledge of the C5a-triggered and the LPS-triggered signaling chains is a prerequisite for understanding the possible interactions of the two regulatory pathways and thus of their synergistic action. However, controversial findings on both signaling pathways have been reported.

C5a stimulates responsive cells via the C5aR, which has a seven-transmembrane domain structure (40, 41). Coupling of the C5aR to G proteins was found to be pertussis toxin-resistant (42, 43, 44) as well as pertussis toxin-sensitive (43, 45, 46, 47) in PBMC (44, 45, 46) or in transfected cells (42, 43, 47); in KC the mode of coupling has not been elucidated so far. The short-term C5a actions appear to be mediated by an increase in intracellular Ca2+ (12, 42, 45, 47), the long-term effects by an activation of the ras/raf/mitogen-activated protein kinase pathway (41) finally involving among other events the activation of NF-{kappa}B (42).

LPS activates responsive cells mainly after binding to the circulating LPS binding protein via the CD14R (48, 49) with Toll-like receptor 4 as a coreceptor (50). CD14 is a GPI-anchored surface glycoprotein that cannot transmit a signal to the cell interior; signal transduction into the cell is achieved by the Toll-like receptor 4 initiating multiple intracellular signaling events, including the activation of NF-{kappa}B. At LPS concentrations up to 5 ng/ml, the CD14 pathway is dependent on serum containing the LPS binding protein (47, 48). However, in the present study, LPS at a concentration of 1 ng/ml was able to induce IL-6, TNF-{alpha}, and IL-1{beta} mRNA and stimulate the release of IL-6 and TNF-{alpha} protein in the absence of serum (Fig. 3Go). These findings corroborate the hypothesis that KC are activated by LPS independently of CD14 via an alternative and more sensitive pathway (51).

As long as the signaling pathways of C5a and of LPS in general and in particular in KC are not known in more detail, it is impossible to even speculate on a possible mechanism for the synergistic action of C5a and LPS on IL-6 release from KC.

Synergistic enhancement by C5a of LPS-elicited {alpha}2MG mRNA expression in HC via IL-6 release from KC

The present study demonstrates for the first time a synergistic action of C5a and LPS on the expression of {alpha}2MG in HC. This cooperative effect was mediated indirectly via IL-6 from KC. APP like {alpha}2MG function primarily as protease inhibitors to limit tissue damage caused by microorganisms or other infectious agents (20, 21, 22, 23). They are newly synthesized and released by HC in the course of the APR. IL-6, which appears to be the most important mediator of the hepatic APR (52), stimulates the formation of type 2 APP, e.g., {alpha}2MG in the rat, whereas IL-1{beta} and TNF-{alpha} stimulate that of type 1 APP (20, 21, 22, 23, 53). IL-6, after binding to the heterotrimeric IL-6R, activates {alpha}2MG expression presumably via the gp130/Janus kinase/STAT pathway (53) and via acute phase responsive elements in the 5'-flanking region of the {alpha}2MG gene (54) (Fig. 8Go).



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  		FIGURE 8. Cell-to-cell communication within the liver via C5a- and LPS-stimulated release of IL-6 from KC to HC. APRE, Acute phase responsive element.ñ

 
Even though IL-6 was identified as the main mediator of C5a+LPS-mediated {alpha}2MG expression in HC, additional factors might be involved. This is reflected by the incomplete inhibition of the effects of the conditioned KC media by anti-IL-6 (Fig. 7Go). It is known that besides IL-6, other IL-6-type cytokines such as leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, cardiotropin 1, and IL-11, as well as glucocorticoids and growth factors like HC growth factor, can be involved in the induction of APP by modulating the effects of IL-6 (55). Of these factors only HC growth factor, on the one hand, is known to be released from KC (56) and, on the other hand, has been reported to induce {alpha}2MG expression by enhancing the IL-6 effect (57).

Besides these soluble factors, other mechanisms could possibly mediate the effect of the KC conditioned media on {alpha}2MG expression. In a recent study, it was shown that the proinflammatory cytokines IL-6 and IL-1{beta} induced functional C5aRs in HC (our unpublished data), which under normal conditions do not express these receptors. Therefore, cytokines and possibly further mediators contained in the conditioned medium of C5a+LPS-stimulated KC might enhance APP synthesis as well as induce de novo expression of C5aRs in HC. In this case C5a used for stimulation and still present in the conditioned medium would mediate {alpha}2MG expression via two mechanisms: first, by enhancing IL-6 release from KC, and second, by acting directly on C5aRs in HC, which had been de novo induced by KC-derived mediators.

Conclusion

The present study provides a new example for the regulatory role of anaphylatoxin C5a in defense reactions of C5aR-free HC. C5a acted indirectly via the release of soluble mediators from C5aR-expressing nonparenchymal cells: C5a not only increased hepatocellular glucose output via prostanoids from KC (15, 16) but it enhanced synergistically with LPS {alpha}2MG gene expression in HC via IL-6 from KC. Thus C5a, by intrahepatic cell-to-cell signaling, was indirectly involved in the short-term regulation of energy metabolism and also in the long-term regulation of the hepatic APR ( Figs. 6–8GoGoGo). The enhancement by C5a of LPS-triggered processes probably reflects a necessary amplification step in infections in which the extent of LPS-elicited defense reactions may not be sufficient. Because LPS is a major activator of C5a formation, the synergistic actions of C5a and LPS would represent an autoamplification.


   Footnotes
 
1 This work was supported by a grant from the Fonds der Chemischen Industrie, Frankfurt (to K.J.), and by the Sonderforschungsbereich 402 Molecular and Cellular Hepato-Gastroenterology, Göttingen. Back

2 Address correspondence and reprint requests to Dr. Henrike Schieferdecker, Institut für Biochemie und Molekulare Zellbiologie, Georg-August-Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany. E-mail address: hschief{at}gwdg.de Back

3 Abbreviations used in this paper: HC, hepatocyte; APP, acute phase protein; KC, Kupffer cell; LC, LightCycler; NCS, newborn calf serum; {alpha}2MG, {alpha}2-macroglobulin; rh, recombinant human; rr, recombinant rat; hPBMC, human PBMC; DIG, digoxigenin; Act D, actinomycin D; CHX, cycloheximide; APR, acute phase response. Back

Received for publication December 7, 2000. Accepted for publication July 18, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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