Expression of Glutamine Synthetase in Macrophages

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-γ-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). α-[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 collagenase-pronase perfusion and separated by a single Nycodenz gradient and centrifugal elutriation (Eyhorn et al. 1988). Kupffer cells (KCs) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and 1% gentamycin for up to 48 hr.

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 1-48 hr after seeding by their typical light microscopic appearance, by immunological staining for ED2 (specific macrophages marker protein) (Shi et al. 1996), and by their ability to phagocytose fluorescent 1.1-μm latex particles. Purity of SECs was ≥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 (PB-MCs) were isolated from blood by centrifugation on Ficoll-Hypaque as described (Böyum 1968). Cells were washed twice in PBS without Ca²⁺ and Mg²⁺. Cells were resuspended in medium, layered on top of a continuous Percoll gradient, and centrifuged for 5 min at 400 × g. Afterwards, the supernatant with contaminating platelets was removed. Monocytes were isolated by adherence on polystyrol for 60 min, followed by three washes with PBS to remove nonadherent cells (Wahl and Smith 1995). Purity was controlled by unspecific esterase staining as described (Li et al. 1973).

Isolation and Cultivation of Parenchymal Cells from Rat Liver

Isolated parenchymal cells (PCs) were prepared from livers of 5-8-week-old male Wistar rats by a collagenase perfusion technique as described (Meijer et al. 1975). Cells were plated on collagen-coated culture dishes and maintained in Krebs-Henseleit medium supplemented with 6 mmol/liter glucose in a humidified atmosphere of 5% CO₂ and 95% air at 37C. After 2 hr, medium was removed and the culture was continued for 24 hr in Dulbecco's modified Eagle's medium containing 5% FCS, penicillin/streptomycin, 0.1 mg/ ml, insulin 100 nmol/liter, dexamethasone 100 nmol/liter, Na-selenite 30 nmol/liter, and aprotinin 1 μg/ml.

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 Bradford (1976), using a commercially available reagent from BioRad. The cell lysate was mixed 2:1 with gel loading buffer containing 300 mmol/liter dithiothreitol (DTT, pH 6.8). After boiling for 5 min, the proteins were subjected to SDS gel electrophoresis (30 μg of protein per lane, 7.5% gel). After electrophoresis, gels were equilibrated with transfer buffer [39 mmol/liter glycine, 48 mmol/liter Tris base, 0.03% SDS, 20% methanol (v/v)]. Proteins were transferred to nitrocellulose membranes using a semidry transfer apparatus (Pharmacia; Freiburg, Germany) according to the manufacturer's instructions. Blots were blocked in 5% BSA containing 20 mmol/liter Tris, pH 7.5, 150 mmol/liter NaCI, 0.1% Tween-20 (TBST) and then incubated at 4C overnight with mouse anti-glutamine synthetase antibody (1:1000). After washing with TBST and incubation with horseradish peroxidase-coupled anti-mouse IgG antibody (dilution 1:10,000) at room temperature (RT) for 2 hr, the blot was washed extensively and developed using enhanced chemiluminescent detection (Amersham). Blots were exposed to Kodak X-OMAT AR-5 film (Eastman Kodak; Rochester, NY) for 1-10 min.

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 Ward and Bradford (1979) but without Triton. Aliquots of 60 μl of homogenate were incubated with 50 μl of reaction mixture at 37C. The final concentrations used were as follows: 100 mmol/liter imidazole buffer (pH 7.2), 12.5 mmol/liter MgCl₂, 10 mmol/liter sodium ATP, 50 mmol/liter potassium glutamate, 20 mmol/liter β-mercaptoethanol, 1 mmol/liter ouabain, 13 mmol/liter phosphoenolpyruvate, 100 mmol/ liter hydroxylamine (freshly neutralized to pH 7.2 with KOH), and 5 U/ml pyruvate kinase. Reaction was stopped after the time period indicated by adding 250 μl of a fresh mixture containing 500 mmol/liter FeCl₃, 400 mmol/liter perchloric acid, and 400 mmol/liter HCl. After 20-30-min incubation on ice, the protein precipitate was sedimented by centrifugation at 20,000 × g and 4C for 10 min. The optical density of the supernatant was measured at 492 nm. Standard curves were established using L-γ-glutamyl-hydroxamate. Values are expressed as μmol L-γ-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 × 10⁶ cells/coverslip. At the end of culture time (0-48 hr), cells were fixed with methanol for 10 min at 4C. Cryosectioning and staining of cryosections for immunofluorescence were performed as previously described by Kubitz et al. (1997). Cryosections of normal human liver were obtained from explants after surgery on benign or malignant liver tumors. This procedure is in accordance with German ethical regulations. Cryosections of rat liver, rat pancreas, or human liver were fixed with methanol for 10 min at 4C.

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 (argon-krypton 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 × 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 [α-³²P]-dCTP-labeled random-primed glutamine synthetase and GAPDH cDNA probes. Membranes were washed three times in 2 × 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=3-6). 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.

Glutamine Synthetase in Liver Macrophages

As shown in Figures 1A and 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 (Gebhardt et al. 1996), the GS mRNA levels in KCs and in GS-positive pericentral PCs may be similar.

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 (Figures 2A and 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 (Figure 2D), suggestive of a localization of GS in macrophages. In line with previous results (Grinko et al. 1995), KCs (ED2 positivity) were enriched in the periportal area (Figure 2E). In sections from normal human liver (Figure 2F), glutamine synthetase was also detectable in liver macrophages.

OPEN IN VIEWER

Figure 1

Glutamine synthetase mRNA (A) and protein (B) in various cell types. (A) Cells were harvested for RNA after the time period indicated and subjected to Northern blot analysis for glutamine synthetase (GS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The position of the 28S (∼4.7 kb) and 18S (∼1.9 kb) ribosomal RNA band is depicted at right. (B) For Western blot analysis, an antibody specifically raised against glutamine synthetase was used. KC, rat Kupffer cells; MC, rat blood monocytes; PC, rat parenchymal cells; SEC, rat sinusoidal endothelial cells; RAW: RAW 264.7 mouse macrophages.

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 (Figure 1). However, immunohistochemistry of rat pancreas revealed the presence of GS in pancreas macrophages (Figure 2G).

Only small amounts of GS protein and mRNA were found in isolated SECs (Figure 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 (Figure 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 15-20% KCs, our findings suggest that SECs do not contain GS protein or mRNA and that the signals found in Figure 1 are due to contaminating KCs.