1. Volume 122, Issue 3, Pages 697-708 (March 2002)
Induction of anaphylatoxin C5a receptors in rat hepatocytes by lipopolysaccharide in vivo: Mediation by interleukin-6 from Kupffer cells 
Milena Koleva, Gerald Schlaf, Regine Landmann, Otto Götze, Kurt Jungermann, Henrike L. Schieferdecker 
Gastroenterology 
Volume 122, Issue 3, Pages (March 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
De novo expression of C5a receptor mRNA in hepatocytes isolated from LPS-treated rats. Animals were injected with 2.5 μg/g body weight LPS (LPS) or NaCl/RSA (control). cDNA was generated by reverse transcription of equal amounts of total RNA from hepatocytes isolated directly or 2, 4, 8, 10, or 30 hours after LPS injection and amplified in the presence of sequence-specific primers for the rat C5a receptor (511 bp) or rat β-actin (769 bp). PCR products were visualized after electrophoresis in agarose gels by ethidium bromide staining. Data represent 1 representative result of 3 independent experiments.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
De novo expression of C5a receptor protein in HCs isolated from LPS-treated rats. HCs isolated directly or 4, 8, 10, and 30 hours after injection of rats with LPS (LPS) or NaCl/RSA (controls), respectively, were centrifuged onto glass slides and (A) fixed with paraformaldehyde or (B) resuspended in FACS buffer. Cells were stained with MAb R63 (anti-C5aR) or MOPC-21 (IgG1-isotype control), followed by a secondary FITC-conjugated goat-anti-mouse IgG, and were then analyzed by (A) fluorescence microscopy or (B) flow cytometry. (A) Hepatocytes isolated 4 hours after LPS treatment weakly expressed C5aR protein; the expression was significantly stronger in hepatocytes isolated 8 and 10 hours after LPS treatment. C5aR protein expression was neither detectable in hepatocytes isolated 30 hours after LPS application nor in HCs isolated 4, 8, 10, and 30 hours after NaCl/RSA treatment (not shown). The Figure shows 1 representative result from 2 independent experiments. Control staining with the IgG-isotype control antibody MOPC-21 in HCs from control and LPS-treated rats was always negative (not shown). (B) Autofluorescence of cells from control and LPS-treated rats as seen after labeling with MOPC-21 is shown in white; fluorescence staining of the cells with R63 (anti-C5aR) is shown in grey. The Figure shows 1 representative result from 3 independent experiments.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Direct activation of glycogen phosphorylase by rrC5a in HCs isolated from LPS-treated but not in HCs from control rats. Isolated HCs from rats that had been treated with LPS or with NaCl/RSA (controls) for the time periods indicated were stimulated with rrC5a (final concentration 100 nmol/L) or, for comparison, with noradrenaline (NA; 1 μmol/L) in the presence of indomethacin (final concentration 20 μmol/L) as described in the Materials and Methods section. The reactions were stopped after 2 minutes, and glycogen phosphorylase (GPH) activity was determined in cell homogenates. Values represent increases in glycogen phosphorylase activity compared with basal unstimulated values (100%) and are means ± SEM of 3 independent experiments. *P ≤ 0.05, significant differences compared with basal unstimulated values (Student t test for unpaired samples).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Lack of inhibition of rrC5a-induced glucose output by the prostanoid synthesis inhibitor indomethacin and the thromboxane A2 receptor antagonist daltroban in perfused livers of LPS-treated but not of control rats. Isolated livers of LPS-treated or control rats were perfused via the portal vein in a nonrecirculating manner. After 15 minutes of preperfusion with Krebs-Henseleit bicarbonate buffer and an additional 15 minutes with the same buffer in the absence or presence of 20 μmol/L indomethacin (Indo) and 20 μmol/L daltroban (Dalt), rrC5a was infused for 30 seconds starting at minute 31 to a final concentration of 100 nmol/L. The perfusate was fractionated at 1-minute intervals for the determination of glucose concentrations. Glucose output (μmol × min−1 × g liver−1) was calculated as (posthepatic concentration − prehepatic concentration) [μmol × mL−1] × flow (mL × min−1 × g liver−1). Values are means ± SEM of 3 experiments. Data for controls were taken from Schieferdecker et al.26 AUC, areas under the curves.
Gastroenterology  , DOI: ( /gast )
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6. Fig. 5
Induction of C5a receptor mRNA by IL-6 and IL-1β but not TNFα or LPS in cultured HCs. Twenty-four–hour cultured HCs from normal rat livers were treated for 24 hours with 100 ng/mL rhIL-6, 10 ng/mL rhIL-1β, 100 ng/mL rhTNFα, or 1 μg/mL LPS. cDNA was generated, amplified with PCR, and detected as described in Figure 1. Data represent 1 exemplary result of 3 independent experiments.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Induction of C5a receptor protein by IL-6 and IL-1β but not TNFα or LPS in cultured HCs. Cells were cultured and treated as described in Figure 5. FACS analysis was performed as described in Figure 2. Autofluorescence of cells as detected with the isotype control antibody MOPC-21 is shown in white; the fluorescence caused by staining of the cells with the anti-C5aR MAb R63 is shown in grey. The Figure shows 1 representative result from 3 independent experiments.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Direct activation of glycogen phosphorylase by rrC5a in cultured HCs pretreated with IL-6 and IL-1β but not TNFα or LPS. Cells were cultured and treated as described in Figure 8. For activation of glycogen phosphorylase, cells were stimulated with rrC5a (final concentration 100 nmol/L) or, for comparison, with noradrenaline (NA; 1 μmol/L) in the presence of indomethacin (final concentration 20 μmol/L) as described in Figure 3. Values represent increases in glycogen phosphorylase (GPH) activity compared with basal unstimulated values (100%) and are means ± SEM of 3 independent experiments. *P ≤ 0.05, significant differences compared with basal unstimulated values (Student t test for unpaired samples).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

9. Fig. 8
IL-6–mediated induction of C5a receptor protein by LPS in HCs in KC/HC cocultures. HCs were seeded on top of 24-hour cultured KCs. The cells were cocultured for another 24 hours and then stimulated for 24 hours with 1 μg/mL LPS in the absence or presence of 1 μg/mL polyclonal antibody against rat IL-6. Cells were then fixed with paraformaldehyde, incubated with the anti-C5aR-MAb R63, followed by a biotinylated secondary goat-anti-mouse IgG antibody, and labeled by incubating with peroxidase-conjugated streptavidin. Although in untreated cocultures only the KCs (arrow) showed positive brownish staining, both KCs and HCs were stained in the LPS-treated cocultures. The LPS-dependent induction of C5aR expression in HCs was drastically reduced in the presence of an anti–IL-6 antibody (only KCs [arrow], but not HCs were stained); no staining was observed in monocultures of HCs.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions