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The Journal of Immunology, 2000, 164: 5453-5458.
Copyright © 2000 by The American Association of Immunologists

Induction of Functional Anaphylatoxin C5a Receptors on Hepatocytes by In Vivo Treatment of Rats with IL-6

Henrike L. Schieferdecker1,*, Gerald Schlaf{dagger}, Milena Koleva*, Otto Götze{dagger} and Kurt Jungermann*

* 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
 
In normal rat liver, anaphylatoxin C5a receptors (C5aR) are only expressed by nonparenchymal cells, mainly Kupffer cells and hepatic stellate cells, but not by parenchymal cells, i.e., hepatocytes (HC). Nevertheless, C5a stimulates glucose output by HC. This HC-specific defense reaction is induced indirectly via prostanoids secreted by the C5aR-expressing Kupffer cells and hepatic stellate cells. It is shown here that under inflammatory conditions simulated by in vivo treatment of rats with IL-6 C5aR mRNA and protein were induced in HC in a time-dependent manner. Maximal mRNA and protein expression were observed at 4–8 h and 8–10 h, respectively, after IL-6 injection. The newly expressed receptors were functional, because recombinant rat C5a significantly activated glycogen phosphorylase in HC isolated from IL-6-treated but not in HC from control rats. In perfused livers of IL-6-treated animals in contrast to control animals, recombinant rat C5a-induced glucose output was not impaired by inhibition of prostanoid synthesis and function with the cyclooxygenase inhibitor indomethacin and the thromboxane receptor antagonist daltroban. These results indicate that HC-specific defense reactions might be differently regulated under normal and inflammatory conditions as shown here for the indirect prostanoid-dependent or direct C5a-induced activation of hepatocellular glycogen phyosphorylase and glucose output in control or IL-6-treated rats, respectively.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Activation of the complement system via the classical, the alternative, or the mannan-binding lectin pathway leads to the generation of anaphylatoxins C3a and C5a. C5a, a glycopeptide of 77 and 74 aa in rat and human, respectively (1, 2, 3, 4), causes the degranulation of mast cells, the contraction of smooth muscle cells, an increase in vascular permeability, and the chemotaxis and activation of neutrophils with local release of reactive oxygen species, eicosanoids, and cytokines (5, 6, 7). C5a receptors (C5aR)2 have been cloned in various species including human (8, 9), mouse (10), dog (11), guinea pig (12), and rat (13, 14). They belong to the seven transmembrane domain receptor family, are coupled to the {alpha}-subunit of a pertussis toxin-sensitive G-protein (15, 16), and act via an increase in inositol-1,4,5-trisphosphate and intracellular Ca2+.

C5aR are known to be expressed on cells of the myeloid lineage including neutrophils, eosinophils, monocytes, and macrophages (17) but recently have also been found in nonmyeloid cells of human lung and liver (18) as well as in astrocyte-derived (19) and hepatocyte (HC)-derived (20, 21) cell lines. In contrast to these findings, C5aR mRNA (22) and protein (23) were expressed by Kupffer cells, hepatic stellate cells, and (weakly) sinusoidal endothelial cells but not by HC isolated from normal rat liver. This expression pattern was in line with functional studies demonstrating that recombinant rat C5a (rrC5a) activated glycogen phosphorylase (GPH) (24, 25) and induced glucose output (26, 27) in HC indirectly by stimulating PG and thromboxane release from Kupffer cells (24) and hepatic stellate cells (25). Analogously, C5a induced the synthesis of acute phase proteins in HC also indirectly by initiating proinflammatory cytokine formation by Kupffer cells (28). Thus, the lack of expression of C5aR on rat HC under normal conditions has been proven by molecular as well as functional evidence.

An up-regulation of C5aR expression in human skin and brain has been reported under various inflammatory conditions such as pyrogenic granuloma and lichen planus (29) or allergic encephalomyelitis (30) as well as in the inflamed CNS of different pathologies (31, 32). C5aR were induced on neurons and glia of transgenic mice with astrocytes constitutively expressing IL-3 (33), in rats with diffuse axonal injury (34), in mice with experimental Listeria meningoencephalitis (35), and in patients with Huntington’s disease (36).

Also, the i.p. injection of LPS, a major trigger of inflammation, increased C5aR mRNA expression in various tissues including the liver of mouse (18) and rat (14, 37). It was postulated that this enhanced expression, at least in part, was due to an induction of C5aR on HC. Because LPS does not act directly on HC (28), it was therefore investigated whether the proinflammatory cytokine IL-6, which is the major mediator of inflammation in the liver, as has been shown by its ability to induce the synthesis of acute phase proteins in HC (38, 39, 40, 41), might elicit the expression of C5aR mRNA and protein in rat HC. It was found that in vivo treatment of rats with IL-6 caused the expression of functional C5aR in HC as shown by RT-PCR, immunocytochemistry, flow cytometry, and C5a-induced direct activation of GPH in isolated HC as well as prostanoid-independent glucose output in the perfused liver.


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

All materials were of analytical grade and from commercial sources. Recombinant human IL-6 (rhIL-6) was purchased from Pharma Biotechnologie (Hannover, Germany), and recombinant rat IL-6 (rrIL-6) was obtained from R&D Systems (Wiesbaden, Germany). Percoll was obtained from Pharmacia (Freiburg, Germany); M199 was obtained from AppliChem (Darmstadt, Germany); newborn calf serum (NCS) was obtained from PAA Laboratories (Cölbe, Germany); dexamethasone and indomethacin was obtained from Sigma (Deisenhofen, Germany); and insulin, penicillin, streptomycin sulfate, and noradrenaline (NA) was obtained from Serva (Heidelberg, Germany). Tissue culture dishes were obtained from Nunc (Wiesbaden, Germany). Daltroban (BM 13.505, 4-[2-(4-chlorobenzenesulfonylamino)-ethyl] benzene-acetic acid) was kindly provided by Roche (Mannheim, Germany).

Abs

The mAb R63 against the rat C5aR was generated as described previously in detail (23). The IgG isotype control Ab MOPC-21 was obtained from Sigma, the FITC-labeled rabbit anti-mouse IgG was obtained from Dianova (Hamburg, Germany), and the biotinylated anti-mouse IgG was obtained from Amersham/Pharmacia (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, 24). 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. Endotoxin depletion of the C5a stock solution (100 µg/ml) was demonstrated by a negative Limulus amebocyte lysate assay (Sigma) that had a detection limit of 0.01 EU/ml or 1 pg/ml.

Animal treatment

Male Wistar rats (Winkelmann, Borchen, Germany), weighing 170–230 g for perfusion experiments and 130–200 g for the isolation of HC, 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 the experiments. The animals were injected with 0.02 µg rhIL-6 or rrIL-6/g body weight in 0.9% NaCl containing 0.1% rat serum albumin (RSA) ("IL-6-treated" animals) or only with 0.9% NaCl/0.1% RSA ("NaCl/RSA-treated" or "control" animals) directly or 2, 4, 8, 10, or 30 h before isolation of HC or start of the perfusion experiments as indicated. Due to the high costs of rrIL-6, experiments were performed with rhIL-6. The efficiency of rhIL-6 in the rodent system (42) was confirmed for each experimental series by one control experiment with rrIL-6 (see Results). Treatment of the animals followed the German Law on the Protection of Animals. The experimental protocol was approved by the local ethical committee for animal research.

HC preparation

HC were prepared according to Meredith by Ca2+-free liver perfusion without the use of collagenase (43). Briefly, the liver was perfused via the portal vein in a nonrecirculating manner with a Ca2+-free Krebs-Henseleit buffer containing 15 mM glucose, 2 mM lactate, 0.2 mM pyruvate, and 2 mM EDTA at 37°C and a flow rate of 10 ml/min. After 45 min, the liver was exised, the liver capsule was opened, and HC were suspended in Krebs-Henseleit buffer containing 1 mM CaCl2 and filtered through nylon gauze (mesh diameter, 60 µm). Viable HC were obtained by removing detritus in two subsequent washing steps with centrifugation at 50 x g and further purified through a gradient with 58% Percoll. Purity of HC as identified on the basis of their typical light microscopic appearance was >98%.

RNA isolation and RT-PCR

Total RNA from freshly isolated HC was isolated by the RNeasy Kit provided by Qiagen (Hilden, Germany), preincubated for 10 min at 68°C with 500 ng oligo-d(T)12–18, and transcribed into cDNA with reverse transcriptase (Superscript II; Life Technologies, Eggenstein, Germany). The cDNA thus generated was amplified with C5aR- or ß-actin-specific primers as described in detail elsewhere (22). The C5aR product yielded in HC from IL-6-treated rats was cloned with a SureClone ligation kit (Pharmacia) into pUC 18 and sequenced for identification using dyedeoxy terminator NTPs (Perkin-Elmer, Weiterstadt, Germany).

Cytospin immunofluorescence

A total of 5 x 104 HC in a volume of 150 µl PBS were centrifuged onto a glass slide using a Cytopro cytospin centrifuge (Berthold Schlag, Bergisch Gladbach, Germany) for 6 min at 1300 rpm. Cells on the slides were washed with PBS, and surface Ags were fixed with 0.75% paraformaldehyde at 4°C for 10 min. HC were then incubated with the primary mAbs R63 (anti-C5aR) or MOPC-21 (IgG1-isotype control) at concentrations of 5 µg/ml (R63) or 10 µg/ml (MOPC-21) in PBS for 50 min at 4°C, washed three times with PBS, and incubated with FITC-conjugated goat anti-mouse IgG at a dilution of 1:100 in PBS for another 50 min at 4°C. After three additional washing steps, immunofluorescence patterns were analyzed using a Zeiss Photomicroscope III (Zeiss, Oberkochen, Germany).

FACS analysis

For FACS analysis, HC were resuspended at 2 x 106 cells/ml in "FACS buffer" (PBS/1.5% FCS/0.1% NaN3). The subsequent incubation steps were performed on ice to prevent internalization of surface receptors. For each analysis, 2 x 105 HC were incubated for 45 min in 100 µl FACS buffer supplemented with 4 µg/ml R63 (anti-C5aR) or 10 µg/ml MOPC-21. After three washing steps, cells were incubated with a FITC-conjugated secondary rabbit anti-mouse IgG Ab (1:100) as above but in the dark. After three additional washing steps, cells were fixed by resuspension in 200 µl FACS buffer containing 1% formaldehyde, stored in the dark, and analyzed by flow cytometry with a Coulter EPICS XL-MCL (Beckmann-Coulter, Krefeld, Germany).

Determination of GPH activity

For the determination of GPH activity, HC isolated from IL-6-treated or control rats were suspended at 1 g wet weight/100 ml HEPES-buffered saline (20 mM HEPES, 120 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 5 mM glucose, 2 mM lactate, and 0.2 mM pyruvate, pH 7.4) containing 20 µM indomethacin in 0.1% DMSO. Next, 1-ml aliquots were sedimented by centrifugation for 2 min at 300 x g and resuspended in 300 µl HEPES-buffered saline containing indomethacin. After a preincubation period of 15 min at 37°C under gentle agitation, cells were stimulated with rrC5a (final concentration 100 nM) or NA (1 µM). After 2 min of incubation, the reaction was stopped by shock freezing in liquid nitrogen. GPH activity in cell lysates was determined by a standard assay (44).

Determination of glucose release and flow reduction in the perfused liver

Rat livers were perfused in situ via the portal vein in a nonrecirculating fashion with Krebs-Henseleit bicarbonate buffer, pH 7.4, containing 5 mM glucose, 2 mM lactate, and 0.2 mM pyruvate at a rate of 4 ml/min per gram of liver. The buffer was equilibrated with 95% O2 and 5% CO2 at 37°C. Livers were preperfused for 15 min with buffer and for an additional 15 min with buffer containing 0.1% DMSO and indomethacin and daltroban (20 µM each) where indicated. RrC5a was then infused to a final concentration of 100 nM for 30 s. After an additional 10 min, NA was infused for a second stimulation to a final concentration of 1 µM as a control. The effluate was fractionated in 1-min intervals to measure flow. Glucose was determined in these fractions using a commercial test kit from Merck (Darmstadt, Germany) (45).

Statistics

Values are given as means ± SEM of the number of experiments indicated. Two groups were compared by Student’s t test for unpaired samples. Values of p < 0.05 were regarded as significant differences.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of C5aR mRNA expression in HC by in vivo treatment of rats with IL-6

HC from normal rats do not express C5aR mRNA (22). Treatment of rats with 0.02 µg rhIL-6/g body weight caused a time-dependent expression of C5aR mRNA in HC (Fig. 1Go). HC isolated directly after IL-6 injection did not express C5aR mRNA. C5aR mRNA was first detected at 2 h and was maximally expressed between 4 and 8 h after IL-6 injection, depending on the experiment. Thereafter, C5aR mRNA expression declined until it was almost undetectable at 30 h after injection. Analogous results were obtained by treatment of the animals with 0.02 µg rrIL-6/g body weight (data not shown). RT-PCR amplification of total RNA with C5aR-specific primers yielded a single cDNA product corresponding to the expected 511 bp (Fig. 1GoB); its identity was verified by sequencing (data not shown). In HC from control animals, a very faint or no expression of C5aR mRNA could be detected at 4 and 8 h after NaCl/RSA injection, but the expression was always significantly lower than that induced by IL-6 treatment (Fig. 1GoA).



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  		FIGURE 1. C5aR mRNA expression in HC isolated from IL-6-treated but not in HC from control rats. Animals were injected with 0.02 µg/g body weight rhIL-6 (IL-6) or NaCl/RSA (control animals). cDNA was generated by reverse transcription of equal amounts of total RNA from HC isolated directly or 2, 4, 8, 10, or 30 h after injection and then was amplified in the presence of sequence-specific primers for the rat C5aR (511 bp) or rat ß-actin (769 bp). PCR products were visualized after electrophoresis in agarose gels by ethidium bromide staining (B) and semiquantified by video densitometry (A). A, Data represent means ± SEM of three (0, 2, 4, 8, and 10 h) or two (30 h) independent experiments. B, Data represent one exemplary result of the experiments shown in A.

 
Induction of C5aR protein expression in HC by in vivo treatment of rats with IL-6

HC from normal rats do not express C5aR protein (23). HC isolated after treatment of rats for 4 h with rhIL-6 did not express C5aR protein as revealed by immunofluorescence cytochemistry of cytospins (Fig. 2Go). In HC obtained 8 h after IL-6 injection, substantial C5aR protein expression was demonstrated; C5aR expression was similar or even slightly stronger in HC isolated 10 h after IL-6 treatment. Analogous results were obtained by treatment of the animals with 0.02 µg rrIL-6/g body weight (data not shown). In HC isolated from control animals, significant C5aR protein expression was neither detected 4, 8, nor 10 h after NaCl/RSA injection.



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  		FIGURE 2. C5aR protein expression in HC isolated from IL-6-treated but not in HC from control rats (immunocytochemistry). Cytospin preparations of HC isolated 4, 8, and 10 h after injection of rats with IL-6 or NaCl/RSA (controls), respectively, were fixed with paraformaldehyde and stained with mAb R63 (anti-C5aR) followed by FITC-conjugated goat anti-mouse IgG and photographed. C5aR protein expression was neither detectable in HC isolated 4, 8, and 10 h after NaCl/RSA treatment nor in HC isolated 4 h after IL-6 treatment. HC isolated 8 h after IL-6 treatment expressed C5aR protein; this expression was slightly stronger in HC isolated 10 h after IL-6 treatment. The figure shows one representative result from four independent experiments. Control staining with the IgG isotype control Ab MOPC-21 in HC from control and IL-6-treated rats was always negative (not shown).

 
For semiquantification, C5aR protein expression was also investigated by flow cytometry (Fig. 3Go). In HC isolated 4 h after IL-6 treatment of rats, no C5aR protein expression could be detected. In contrast, a substantial C5aR expression was observed in HC isolated 8 h after IL-6 injection. The expression remained stable or in some experiments slightly increased up to 10 h after IL-6 injection. Analogous results were obtained by treatment of the animals with 0.02 µg rrIL-6/g body weight (data not shown). In HC isolated from control animals, C5aR protein could not be detected 4, 8, or 10 h after NaCl/RSA injection.



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  		FIGURE 3. C5aR protein expression in HC isolated from IL-6-treated but not in HC from control rats (FACS analysis). HC isolated 4, 8, and 10 h after injection of rats with IL-6 or NaCl/RSA (controls), respectively, were stained with mAb R63 for C5aR followed by a FITC-conjugated secondary rabbit anti-mouse IgG Ab and analyzed by flow cytometry. Solid and dotted lines represent FITC-conjugated R63 (anti-)C5aR staining of control and IL-6-treated rats, respectively. The figure shows one representative result from three independent experiments. Control staining with the IgG isotype control Ab MOPC-21 in HC from control and IL-6-treated rats was always negative (not shown).

 
Induction of C5a reactivity in HC by in vivo treatment of rats with IL-6

Direct activation of hepatocellular GPH by rrC5a. To examine the functioning of the newly expressed C5aR, it was investigated whether rrC5a directly activated GPH in HC from IL-6-treated rats. RrC5a enhanced GPH activity in isolated HC slightly 4 h and strongly 8 and 10 h after IL-6 injection (Table IGo). These findings were in line with the expression of C5aR protein (cf. Fig. 2Go and 3Go). After 10 h, rrC5a activated hepatocellular GPH to a similar extent as NA, which is known to directly increase GPH activity in HC and therefore served as a positive control. Analogous results were obtained with HC isolated from rats treated with 0.02 µg rrIL-6/g body weight (data not shown). In HC isolated from control animals, rrC5a failed to activate GPH at any time point after NaCl/RSA injection. The lack of a direct rrC5a action on HC from normal rats has been shown previously using primary cultures (24, 25).


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  		Table I. Direct activation by rrC5a of GPH in HC isolated from IL-6-treated but not in HC from control rats1

 
Direct induction of glucose output in perfused livers by rrC5a. Basic glucose output in perfused livers of IL-6-treated rats was slightly higher than in livers of control animals (Fig. 4Go). RrC5a enhanced glucose output and reduced flow in perfused livers of rats 10 h after IL-6 treatment as it did in livers of control animals (Fig. 4Go). As shown previously (27), in perfused livers of control animals, these rrC5a-induced metabolic and hemodynamic effects were completely inhibited by the prostanoid synthesis inhibitor indomethacin and the thromboxane A2 receptor antagonist daltroban, indicating that rrC5a acted indirectly on HC via prostanoid release from nonparenchymal cells expressing C5aR (27). These results were independent of whether untreated animals (Fig. 4Go) or NaCl/RSA-treated animals (n = 2, data not shown) were used. In contrast, in livers from IL-6-treated animals, rrC5a-stimulated glucose output was not reduced by indomethacin and daltroban, demonstrating that C5a under these circumstances elicited glucose output from HC directly, i.e., independently of prostanoids released from Kupffer cells or hepatic stellate cells. Glucose output induced by NA, which directly acts on HC, was not affected by the prostanoid antagonists neither in IL-6-treated rats nor in control animals (data not shown).



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  		FIGURE 4. Lack of inhibiton of rrC5a-induced glucose output by the prostanoid synthesis inhibitor indomethacin and the thromboxane A2 receptor antagonist daltroban in perfused livers of IL-6-treated but not of control rats. Isolated livers of IL-6-treated or control rats were perfused via the portal vein in a nonrecirculating manner. After 15 min preperfusion with Krebs-Henseleit bicarbonate buffer and an additional 15 min with the same buffer in the absence or presence of 20 µM indomethacin (Indo) and 20 µM daltroban (Dalt), rrC5a was infused for 30 s starting at minute 31 to a final concentration of 100 nM. The perfusate was fractionated at 1-min intervals for the determination of glucose concentrations and flow rate. Glucose output (µmol/min per g liver) was calculated as (posthepatic concentration - prehepatic concentration; µmol/ml) x (flow; ml/min per g liver). Values are means ± SEM of three experiments. Data for controls were taken from Ref. 27 .

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is shown in this study that C5aR that are not normally expressed by HC (22, 23) were up-regulated in these cells both on the mRNA (Fig. 1Go) and the protein level (Fig. 2Go and 3Go) under inflammatory conditions as simulated by in vivo treatment of rats with the proinflammatory cytokine IL-6. It is demonstrated further that the up-regulated C5aR were functional both in isolated HC (Table IGo) and in perfused livers (Fig. 4Go).

C5aR expression in HC of IL-6-treated rats

In the early phase of inflammation, when the liver is still in its normal state and HC do not express C5aR (22, 23), the anaphylatoxin C5a generated systemically cannot directly elicit defense reactions in HC such as glucose release (26, 27) or acute phase protein synthesis (28); it can only act indirectly via the release of prostanoids or cytokines, respectively, from Kupffer cells (26, 28) and hepatic stellate cells (27), which bear the C5aR (22, 23). During prolonged systemic or local hepatic inflammation the pattern of receptor expression on liver cells may change. HC might express C5aR, so that they can be stimulated directly by C5a.

In this study, experimental evidence is provided that in vivo treatment of rats with the proinflammatory cytokine IL-6 indeed induced C5aR expression in HC ( Figs. 1–3GoGoGo). This induction was independent of whether rhIL-6 or rrIL-6 was used, confirming the activity of human IL-6 in the rodent system (42). In some cases, a slight increase in C5aR mRNA was also observed in HC isolated from NaCl/RSA-treated rats (Fig. 1GoA). Nevertheless this slight, presumably "stress-induced," C5aR mRNA expression was much weaker than that induced by IL-6 and did not lead to the surface expression of C5aR protein (Figs. 2Go and 3Go).

The finding of an inducible C5aR expression by the proinflammatory cytokine IL-6 is in line with previous observations in other tissues or cell systems having demonstrated an up-regulated C5aR expression during inflammatory processes. Keratinocytes expressed detectable levels of C5aR mRNA only in inflamed but not in normal skin (29), and reactive astrocytes and microglia and, to a lesser extent, also endothelial cells up-regulated C5aR expression in the inflamed human CNS (30).

Inducible C5aR expression under inflammatory conditions might explain contradictory results concerning C5aR expression by HC. While HC isolated from normal rats have been shown not to express C5aR mRNA and protein (22, 23), human HC (18, 20, 21) were reported to express C5aR. In these studies C5aR were detected either by in situ hybridization of human liver tissue (18), the source of which was not indicated, or on HepG2 cells (20, 21). The present investigation provides evidence that the findings with human liver tissue might not represent results obtained with HC in a normal state but most likely indicate a disease-induced up-regulation of C5aR mRNA in the livers of patients from whom the tissues were obtained. Similarly, the findings with HepG2 cells presumably are due to an up-regulation of C5aR during transformation of normal HC into hepatoma cells. These assumptions are confirmed by 1) a recent study demonstrating the absence of C5aR in human HC using normal liver tissue (46) and 2) the detection of C5aR mRNA in the rat hepatoma cell lines FAO and H4IIE but not in primary rat HC (23), presumably indicating a frequent or even general activation of the C5aR gene during malignant transformation.

An up-regulation of C5aR mRNA expression in the liver was observed previously after in vivo treatment of rats with LPS (14, 37). However, it was not investigated whether this enhanced expression occurred in nonparenchymal liver cells or in HC. Because LPS did not up-regulate C5aR mRNA expression directly in cultures neither of Kupffer cells, hepatic stellate cells, sinusoidal endothelial cells, nor of HC (C. Mäck, M. Koleva, H. L. Schieferdecker, and K. Jungermann, unpublished observations), it is likely that the enhanced C5aR mRNA expression in the liver following LPS treatment in vivo (14, 37) was induced by mediators released from responder cells in the periphery or locally within the liver. Among the most important mediators of inflammation is the proinflammatory cytokine IL-6, which can be released from Kupffer cells after stimulation with LPS (47). It has to be elucidated in further studies 1) whether the enhanced C5aR expression in the liver after LPS treatment in vivo occurs mainly in HC and 2) whether LPS acts indirectly via IL-6.

Functioning of C5aR in HC of IL-6-treated rats

Among the most important functions of HC is the release of glucose from glycogen stores. In normal livers in which the HC are devoid of C5aR, the anaphylatoxin cannot enhance glucose output from HC directly but only indirectly by stimulating the release of prostanoids from Kupffer cells and hepatic stellate cells (24, 25, 26, 27, 28). Thus, the C5a-dependent increase in glucose output from perfused livers of normal rats can be inhibited by the cyclooxygenase inhibitor indomethacin and the thromboxane receptor antagonist daltroban (27). Similarly, the C5a-dependent activation of GPH cannot be demonstrated in HC monocultures but only in cocultures of HC with prostanoid-secreting Kupffer cells (24) or hepatic stellate cells (25). Therefore, the findings of this study that C5a enhanced glucose output from perfused livers of IL-6-treated rats without impairment by indomethacin and daltroban (Fig. 4Go) and that it activated GPH in monocultures of HC from these animals (Table IGo) indicate that the newly expressed C5aR on HC were functional.

The data presented in this study demonstrate that hepatic defense reactions, as shown here for the short-term glucose output from HC, can be regulated by C5a via different mechanisms during the early and the later stages of inflammation. In the early stages, when HC are still devoid of C5aR, glucose output is stimulated by C5a indirectly via the release of prostanoids from Kupffer cells and hepatic stellate cells. In the later stages, when HC have been induced to express C5aR themselves, glucose output can be elicited by C5a directly without the intervention of nonparenchymal cells.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Henrike L. Schieferdecker, Institut für Biochemie und Molekulare Zellbiologie, Georg-August-Universität Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany. Back

2 Abbreviations used in this paper: C5aR, C5a receptor; GPH, glycogen phosphorylase; HC, hepatocytes; NA, noradrenaline; NCS, newborn calf serum; rhIL-6, recombinant human IL-6; rrC5a, recombinant rat C5a; rrIL-6, recombinant rat IL-6; RSA, rat serum albumin. Back

Received for publication November 24, 1999. Accepted for publication March 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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