ARTICLE |
Correspondence to: Dieter Häussinger, Medizinische Universitätsklinik, Klinik für Gastroenterologie, Hepatologie und Infektiologie, Heinrich-Heine Universität, Moorenstr. 5, D-40225 Düsseldorf, Germany.
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Summary |
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We studied the expression of glutamine synthetase in liver macrophages (Kupffer cells, KCs) in situ and in culture. Glutamine synthetase was detectable at the mRNA and protein level in freshly isolated and short-term-cultured rat liver macrophages. Enzyme activity and protein content were about 9% of that in liver parenchymal cells. In contrast, glutamine synthetase mRNA levels in liver macrophages apparently exceeded those in parenchymal liver cells (PCs). By use of confocal laser scanning microscopy and specific macrophage markers, immunoreactive glutamine synthetase was localized to macrophages in normal rat liver and normal human liver in situ. All liver macrophages stained positive for glutamine synthetase. In addition, macrophages in rat pancreas contained immunoreactive glutamine synthetase, whereas glutamine synthetase was not detectable at the mRNA and protein level in blood monocytes and RAW 264.7 mouse macrophages. No significant amounts of glutamine synthetase were found in isolated rat liver sinusoidal endothelial cells (SECs). The data suggest a constitutive expression of glutamine synthetase not only, as previously believed, in perivenous liver parenchymal cells but also in resident liver macrophages. (J Histochem Cytochem 48:415421, 2000)
Key Words: glutamine metabolism, sinusoidal endothelial cells, monocytes
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Introduction |
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The liver plays an important role in glutamine metabolism, with implications for ammonia and bicarbonate homeostasis (for review see -actin-expressing myofibroblast-like cells (
Recent studies suggested that glutamine is utilized at a high rate by peritoneal macrophages and that the activity of glutaminase is high in these cells (
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Materials and Methods |
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Animals
Male Wistar rats were used in all experiments. All rats were raised in the local institute for laboratory animals and held, fed ad libitum on stock diet, according to the local ethical guidelines. GS immunohistochemistry was performed on cryosections from normal liver and pancreas of four different animals.
Materials
RPMI 1640 and DMEM medium, glutamine, and fetal calf serum were from Biochrom (Berlin, Germany). Nycodenz was from Nycomed (Oslo, Norway). Pronase was from Merck (Darmstadt, Germany). DNase I and collagenase were from Boehringer Mannheim (Mannheim, Germany). FITC-conjugated anti-mouse IgG, chicken serum albumin, and L--glutamyl-hydroxamate were obtained from Sigma (Deisenhofen, Germany). Anti-glutamine synthetase antibody from mouse was from Chemicon International (Harrow, UK). Anti-glutamine synthetase antibody from goat was from Santa Cruz Biotechnology (Heidelberg, Germany). Cy3-conjugated anti-goat IgG (Fab fragment) was obtained from Dianova (Hamburg, Germany). Anti-ED2 antibody and anti-human mature macrophage antibody (MCA1122) from mouse were from Serotec (Raleigh, NC). Horseradish peroxidase-conjugated anti-mouse IgG antibody was from BioRad (Hercules, CA). RNeasy total RNA kit was obtained from Qiagen (Hilden, Germany). The glutamine synthetase cDNA fragment used for detection of glutamine synthetase mRNA was kindly provided by Dr. W.H. Lamers (University of Amsterdam, Netherlands). The 1.0-kb cDNA fragment for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used for standardization, was from Clontech (Heidelberg, Germany). Nitrocellulose membranes were purchased from Schleicher & Schuell (Dassel, Germany).
-[32P]-dCTP was from Amersham (Braunschweig, Germany). Polybead fluorescent microspheres (2.5% solid latex, 1.1 µm diameter) were obtained from Polysciences (St. Goar, Germany). All other chemicals were from Merck.
Isolation and Cultivation of Kupffer Cells and Sinusoidal Endothelial Cells
Cells were isolated from 1-year-old male Wistar rats by collagenasepronase perfusion and separated by a single Nycodenz gradient and centrifugal elutriation (
Sinusoidal endothelial cells (SECs) were plated on collagen 1-coated culture dishes and maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FCS and 1% gentamycin for 24 hr.
Culture medium was changed after 24 hr. Culture was performed in a humidified atmosphere of 5% CO2 and 95% air at 37C. Purity of KCs was 98% as assessed 148 hr after seeding by their typical light microscopic appearance, by immunological staining for ED2 (specific macrophages marker protein) (
80% as assessed 24 hr after seeding by their typical light microscopic appearance and by their inability to phagocytose fluorescent 1.1-µm latex particles.
Isolation of Blood Monocytes
Monocytes were isolated from the blood of 1-year-old male Wistar rats. The peripheral blood mononuclear cells (PBMCs) were isolated from blood by centrifugation on FicollHypaque as described (
Isolation and Cultivation of Parenchymal Cells from Rat Liver
Isolated parenchymal cells (PCs) were prepared from livers of 58-week-old male Wistar rats by a collagenase perfusion technique as described (
Culture of RAW 264.7 Cells
The murine peritoneal macrophage cell line RAW 264.7 was maintained in DME medium at 37C in 5% CO2, supplemented with 10% FCS, and 1% gentamycin, and were grown to near confluency before experiments were performed.
Determination of Glutamine Synthetase Expression
Cells were harvested in a buffer containing 63 mmol/liter Tris-HCl (pH 6.8) and 1% SDS. Protein content of the lysate was determined according to
Determination of Glutamine Synthetase Activity
Cells were cultured for the time period indicated, harvested by scraping in distilled water, and homogenized by sonication for 4 sec at 4C. Glutamine synthetase activity was measured according to the method described in -glutamyl-hydroxamate. Values are expressed as µmol L-
-glutamyl-hydroxamate formed per hour per mg of protein at 37C. The reaction was tested for linearity and dependence on the protein concentration employed.
Fluorescence Staining of Glutamine Synthetase
For immunocytochemistry, KCs and ECs were cultured on glass coverslips with a diameter of 10 mm at a density of 0.25 x 106 cells/coverslip. At the end of culture time (048 hr), cells were fixed with methanol for 10 min at 4C. Cryosectioning and staining of cryosections for immunofluorescence were performed as previously described by
After rinsing twice with PBS, the methanol-fixed samples were incubated in PBS containing 0.1% Triton for 10 min at RT. The cells were washed again and incubated for at least 3 hr at RT with goat anti-glutamine synthetase antibody (1:25) plus mouse anti-ED2 (for rat) antibody (1:50) or mouse antibody against human mature macrophages (1:250) in a wet chamber. Then the cells were washed with PBS and incubation was continued for 2 hr with Cy3-conjugated anti-goat immunoglobulin (1:300) plus FITC-conjugated anti-mouse immunoglobulin (1:100). To rule out false positivity, negative controls were done by incubation of samples with either the combination of secondary antibodies alone or with each single first antibody together with both secondary antibodies. Microscopy was performed using a confocal laser scanning system (argonkrypton laser; Leica, Bensheim, Germany).
Northern Blot Analysis
Total RNA from cultured cells was isolated by using the RNeasy total RNA Kit (Qiagen). RNA samples (2 µg per lane) were electrophoresed in 0.8% agarose containing 3% formaldehyde and then blotted onto Hybond-N nylon membranes (Amersham) with 20 x SSC (3 mol/liter NaCl/0.3 mol/liter Na-citrate). After brief rinsing with water and UV crosslinking (Hoefer UV-crosslinker 500), the membranes were observed under UV illumination to determine RNA integrity and location of the 28 S and 18 S rRNA bands. The blots were then subjected to prehybridization for 3 hr at 43C in 50% deionized formamide in sodium phosphate buffer (0.25 mol/liter; pH 7.2) containing 0.25 mol/liter NaCl, 1 mmol/liter EDTA, 100 mg/ml salmon sperm DNA, and 7% SDS. Hybridization was carried out in the same solution with approximately 106 cpm/ml [-32P]-dCTP-labeled random-primed glutamine synthetase and GAPDH cDNA probes. Membranes were washed three times in 2 x SSC/0.1% SDS for 10 min, twice in sodium phosphate buffer (25 mmol/liter; pH 7.2)/EDTA (1 mmol/liter)/0.1% SDS, and twice in sodium phosphate buffer (25 mmol/liter; pH 7.2)/ EDTA (1 mmol/liter)/1% SDS. Blots were then exposed to Kodak X-OMAT AR-5 film at -70C with intensifying screens.
Suitably exposed autoradiographs were then analyzed by densitometry scanning (PDI; New York, NY). Relative glutamine synthetase mRNA levels were determined by standardization to the absorption of GAPDH mRNA.
Analysis of Results
Values are expressed as means ± SEM (n=36). Results of at least three independent experiments are given. For statistical analysis the one-way analysis of variance was performed using the one-way ANOVA test. p<0.05 was considered statistically significant.
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Results |
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Glutamine Synthetase in Liver Macrophages
As shown in Fig 1A and Fig 1B, glutamine synthetase mRNA and protein were detectable in isolated rat liver parenchymal cells and liver macrophages. The amount of GS protein in KCs was only about 9% of the amount found in isolated PCs. In line with this, specific GS activity in isolated PCs and KCs was 0.56 ± 0.03 (n=4) and 0.06 ± 0.01 µmol/hr/mg protein (n=4), respectively. In contrast, GS mRNA levels in KCs apparently exceeded those in PCs. Culture of KCs for 2 days had no effect on GS protein and mRNA levels. However, the PC preparation used includes GS-positive (pericentral) as well as GS-negative (periportal) cells. Because the GS mRNA content in pericentral PCs is about 20-fold higher than in periportal PCs (
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Immunocytochemical studies on freshly isolated KCs or on KCs kept for 2 days in culture revealed co-localization of GS and ED2, a specific macrophage marker protein (Fig 2A and Fig 2B). GS was present in all KCs and not only in subpopulations thereof. GS was also detectable in KCs in situ. In sections from rat liver tissue, intense staining for GS was found in the perivenous hepatocytes surrounding the terminal hepatic venules, but punctate staining was also found throughout the parenchyma, with some enrichment in the periportal areas. Counterstaining with the macrophage-specific antibody against ED2 revealed that the punctate GS-positive staining co-localized with ED2 (Fig 2D), suggestive of a localization of GS in macrophages. In line with previous results (
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GS in Other Macrophages and Sinusoidal Endothelial Cells
In mouse RAW 264.7 macrophages and blood monocytes, neither GS protein nor GS mRNA was detectable (Fig 1). However, immunohistochemistry of rat pancreas revealed the presence of GS in pancreas macrophages (Fig 2G).
Only small amounts of GS protein and mRNA were found in isolated SECs (Fig 1), and no GS activity was detectable. At the immunocytochemical level, which allowed the differentiation of SECs from contamination of KCs by simultaneous use of the ED2 antibody, no staining for GS was detectable in SECs (Fig 2C). The amounts of GS protein and mRNA found in SEC preparations were only about 17% of the amounts found in isolated KCs. Because our SEC preparation contained 1520% KCs, our findings suggest that SECs do not contain GS protein or mRNA and that the signals found in Fig 1 are due to contaminating KCs.
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Discussion |
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This study demonstrates a constitutive expression of glutamine synthetase in liver macrophages. This was shown at the level of mRNA and of protein and enzyme activity as well as by immunohistochemistry and confocal laser scanning microscopy. Kupffer cell-associated GS showed some enrichment in the periportal area of the liver lobule due to the known higher amount of KCs in periportal compared to perivenous areas (
Our data further indicate that there are differences in the ratio of GS mRNA to protein and specific activity between KCs and PCs (Fig 1). This might be explained by differences in the regulatory mechanisms for expression or degradation of GS and GS mRNA in these cell types. With respect to the enhanced oxidative metabolism resulting from the activation of macrophages (
Expression of GS does not appear to be a feature of macrophages in general, as blood monocytes and RAW 264.7 peritoneal mouse macrophages contain neither GS protein nor GS mRNA. Because not only liver macrophages but also pancreas macrophages contain glutamine synthetase, it is attractive to speculate that GS expression is a feature of sessile tissue macrophages only. However, GS is inducible by corticosteroids (
GS expression in KCs may relate to the involvement of glutamine in proliferation, phagocytosis and cytokine release by the cells of the immune system (, or NO synthesis and the macrophage-mediated lysis of a ß-cell line has been reported for macrophages stimulated either by lipopolysaccharide (
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Footnotes |
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1 These authors contributed equally to this work.
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Acknowledgments |
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Supported by the Deutsche Forschungsgemeinschaft and the Gottfried-Wilhelm-Leibniz-Prize.
The GS cDNA fragment used for detection of GS mRNA was kindly provided by Dr W. H. Lamers (University of Amsterdam, The Netherlands).
Received for publication July 14, 1999; accepted October 13, 1999.
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