LPS receptor CD14 participates in release of TNF-alpha in RAW 264.7 and peritoneal cells but not in Kupffer cells

Steven N. Lichtman1, Jian Wang1, and John J. Lemasters2

Departments of 1 Pediatrics and 2 Cell Biology and Anatomy, University of North Carolina, Chapel Hill, North Carolina 27599-7220

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Lipopolysaccharide (LPS) is a bacterial polymer that stimulates macrophages to release tumor necrosis factor-alpha (TNF-alpha ). In macrophages (RAW 264.7 and peritoneal cells), LPS binds to the CD14 surface receptor as the first step toward signaling. Liver macrophages, Kupffer cells, are the most numerous fixed-tissue macrophage in the body. The presence of CD14 on Kupffer cells and its role in LPS stimulation of TNF-alpha were examined. TNF-alpha release by Kupffer cells after LPS stimulation was the same in the presence and absence of serum. RAW 264.7 and peritoneal cells, which utilize the CD14 receptor, released significantly less TNF-alpha after LPS stimulation in the absence of serum because of the absence of LPS-binding protein. Phosphatidylinositol-phospholipase C treatment, which cleaves the CD14 receptor, decreased LPS-stimulated TNF-alpha release by RAW 264.7 cells but not by Kupffer cells. Deacylated LPS (dLPS) competes with LPS at the CD14 receptor when incubated in a ratio of 100:1 (dLPS/LPS). Such competition blocked LPS-stimulated TNF-alpha release from RAW 264.7 cells but not from Kupffer cells. Western and fluorescence-activated cell sorter analysis directly demonstrated the presence of CD14 on RAW 264.7 cells and murine peritoneal cells but showed only minimal amounts of CD14 in murine Kupffer cells. LPS stimulation did not increase the amount of CD14 detectable on mouse Kupffer cells. CD14 expression is very low in Kupffer cells, and LPS-stimulated TNF-alpha release is independent of CD14 in these cells.

macrophages; tumor necrosis factor-alpha ; endotoxin

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

ENDOTOXIN (lipopolysaccharide; LPS) is a cell wall polymer present in gram-negative bacteria that stimulates macrophages to release proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha ), interleukin-1, interleukin-6, and others. As the first step toward stimulation of TNF-alpha synthesis, LPS must bind to a receptor on the macrophage cell surface. Several LPS receptors have been identified in different macrophages (2, 8, 10, 26, 28). The most thoroughly studied receptor is CD14, which has been identified in several types of macrophages and macrophage cell lines, including the mouse peritoneal macrophage cell line, RAW 264.7 (11). In cells that possess the CD14 receptor, the presence of serum increases the sensitivity to LPS by 100- to 1,000-fold (23, 29). Increased sensitivity to LPS is mediated by LPS-binding protein (LBP), a protein found in abundance in serum. The LPS-LBP complex interacts with the CD14 receptor in a manner that stimulates TNF-alpha production much more than does LPS alone (23, 29).

The Kupffer cell is the resident macrophage of the liver and is the most abundant fixed-tissue macrophage in the body. The Kupffer cell receives blood from the portal vein, which drains the intestinal tract, and portal blood contains LPS absorbed from the large number of gram-negative organisms in the intestinal lumen. Because Kupffer cells can release large amounts of proinflammatory cytokines in response to LPS, understanding of the mechanisms by which LPS stimulates TNF-alpha release by Kupffer cells is important. The first step of LPS stimulation of Kupffer cells is by interaction with the LPS receptor. Tracy and Fox (25) suggested that rat Kupffer cells in nonstimulated livers may not possess the CD14 receptor but, after bile duct ligation, CD14 expression increased, which may have been the result of an influx of activated monocytes and macrophages. In addition, Bellezzo et al. (1) showed that LPS stimulation of nuclear factor (NF)-kappa B in isolated Kupffer cells was not serum dependent, implying that it may not be CD14 dependent. Similarly, Matsuura and colleagues (17) showed that nonstimulated livers had very low CD14 expression, which increased after LPS injection in mice. They also showed that CD14 expression increased in peritonal macrophages after LPS stimulation in cell culture (17). In the present studies, we investigated whether isolated rat and mouse Kupffer cells possess the CD14 receptor, comparing them with RAW 264.7 and peritoneal cells, which are known to possess the CD14 receptor. Our results indicate that CD14 expression is very low in isolated mouse and rat Kupffer cells and that LPS-stimulated TNF-alpha release may not depend on CD14.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. Monoclonal anti-vinculin antibody, deacylated LPS (dLPS), Escherichia coli LPS (serotype 0111:B4), and Pronase were purchased from Sigma Chemical (St. Louis, MO). Collagenase and phosphatidylinositol-phospholipase C (PI-PLC) were purchased from Calbiochem (La Jolla, CA).

Experimental design. Sprague-Dawley rats (300 g) and C57b6 mice were used (Charles River, Raleigh, NC). After isolation of Kupffer cells and culture for 48 h, cells were stimulated with LPS, and culture supernatant was collected after 4 h for measurement of TNF-alpha by ELISA. In most studies, LPS was added in the presence of 10% fetal calf serum (FCS) unless the conditions involved serum-free medium.

In some studies, cells were preincubated with 0.5 U/ml of the enzyme PI-PLC, and 10 ng/ml LPS were added in the presence of PI-PLC for 4 h. CD14 is a glucosyl-phosphatidylinositol (GPI)-linked protein that is cleaved by the enzyme PI-PLC (10).

In other studies, cells were preincubated with dLPS competitively, which inhibits LPS-stimulated activation when used in 100-fold excess, presumably by competitively blocking the CD14 receptor (11, 22).

Isolation and culture of rat Kupffer cells. Kupffer cells were isolated from 300-g Sprague-Dawley rats by modification of the procedure of Knook and Sleyster (12), as previously reported (15). Kupffer cells have been characterized by positive peroxide staining, characteristic electron micrographic appearance, and by labeling with an anti-Kupffer cell antibody (KU-1 monoclonal antibody provided by Dr. J. Reichner, Rhode Island Hospital, Providence, RI). All animals received humane care in compliance with the regulations of the University of North Carolina and with the guidelines set forth in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and endorsed by the National Institutes of Health (NIH publication no. 86-23, revised 1985). Briefly, each liver was perfused first with Gey's balanced salt solution (GBSS) for 5 min, second with calcium- and magnesium-free GBSS for another 5 min, and third with GBSS containing 0.25% collagenase and 0.02% Pronase for 20 min. Perfusion rates were 30 ml/min. The liver was then raked with a steel comb to disperse the cells, which were centrifuged three times at 50 g for 2 min each to sediment hepatocytes. The supernatant, composed of nonparenchymal cells, was centrifuged at 250 g for 5 min and resuspended in 15 ml of GBSS and then loaded into a Beckman J2/21ME centrifugal elutriator (Palo Alto, CA) at 4°C. At a rotor speed of 2,500 rpm (Beckman JE-6B), cells were elutriated at 11, 18, 21, and 36 ml/min with GBSS and 1% albumin. Purified Kupffer cells were collected at 36 ml/min.

Kupffer cells were cultured in 96-well microtiter plates (Falcon Plastics, Becton-Dickinson Labware, Lincoln Park, NJ) in RPMI 1640 medium supplemented with 10% endotoxin-free FCS and 10 mM HEPES, using 400,000 cells in 200 l of culture medium per well at 37°C in 5% CO2-air. Culture medium was changed at 2 h and then every 24 h. Cells were used for experiments 48 h after initial plating.

Isolation of Kupffer cells from mice. A similar method of cell isolation of rat Kupffer cells was used for isolation of mouse Kupffer cells. The portal vein was cannulated and flushed with GBSS for 3 min at 15 ml/min. Next, livers were perfused with calcium-free, magnesium-free GBSS for 5 min at 15 ml/min. Livers were then perfused with 0.125% collagenase and 0.02% Pronase for ~10 min at 7 ml/min. As for rat cells, the liver was then raked with a steel comb, and cells were centrifuged three times at 50 g for 2 min each to sediment hepatocytes. Three livers were pooled before centrifugal elutriation to obtain 15-30 × 106 Kupffer cells, and elutriation was the same as for rat Kupffer cells. More than 95% of these cells stained positive for peroxidase, had characteristic electron microscopic appearance, and phagocytosed fluorescent latex beads.

RAW 264.7 cells. RAW 264.7 cells (American Tissue Culture Center TIB 71) were propagated in T-75 culture flasks (Costar, Cambridge, MA) in Dulbecco's minimal essential medium supplemented with 25 mM glucose (DMEM, high glucose), 10% FCS, and gentamicin. Before use, cells were detached at 37°C with trypsin-EDTA (GIBCO, Grand Island, NY) and plated in 96-well microtiter plates at a density of 200,000 cells/200-µl well or in 35-mm petri dishes at a density of 107 cells/ml in 1 ml. Medium was changed every 24 h, and cells were used after 48-72 h of incubation.

Isolation of murine peritoneal cells. Mice were killed by CO2 narcosis. The peritoneal cavity was washed two times with 5 ml of cold heparinized Hanks' balanced salt solution (HBSS). The HBSS was pooled and centrifuged for 10 min at 1,500 rpm at 4°C. The pelleted cells were resuspended in 2 ml of cold red blood cell lysis buffer for 1 min and 35 ml RPMI were added, and this was then centrifuged for 10 min at 1,500 rpm. This pellet was resuspended in 1 ml RPMI with 5% FCS, and cells were cultured at a final concentration of 106 cells/ml in 35-mm culture dishes.

Measurement of TNF-alpha . TNF-alpha was measured in aliquots of culture supernatants using a commercial ELISA kit (Genzyme, Cambridge, MA) with mouse recombinant TNF-alpha as a standard. Color changes at 450 nm were measured using a Dynetec ELISA reader (Chantilly, VA). The standard curve measured TNF-alpha between 50 and 1,000 pg/ml, the range that our studies covered. The anti-mouse TNF-alpha antibody employed cross-reacted with rat TNF-alpha , permitting accurate measurements of the rat protein.

Western blotting for CD14. Cells were cultured in 30-mm dishes, washed, and collected with a rubber policeman. The cells were sonicated, and total protein was measured by the bicinchoninic acid protein assay method (Pierce, Rockford, IL). Aliquots of 20 µg protein were used for Western analysis.

Cells were prepared by sonication, and 20 µg were loaded onto a 10% polyacrylamide gel for electrophoresis. Protein was transferred to nitrocellulose membranes. Blots were blocked with 5% Carnation evaporated milk and then incubated with rat anti-mouse CD14 at 1:300 dilution (Pharmingen, San Diego, CA) for 1 h at room temperature. After washing, blots were incubated with goat anti-rat horseradish peroxidase at a dilution of 1:1,000 (Pharmingen) for 1 h. The blot was then washed and incubated with the enhanced chemiluminescence system for 1 min (Amersham). Blots were subsequently exposed to X-ray film (Eastman Kodak, Rochester, NY).

To confirm that blots were loaded with equal amounts of protein, we reprobed each blot with an anti-vinculin antibody (Sigma Chemical) diluted 1:100 in PBS-Carnation evaporated milk. Vinculin is a cell surface protein (12) that does not change after LPS stimulation (personal communication, Dr. M. Schaller, Dept. of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, NC).

Flow cytometry. Isolated Kupffer cells, mouse peritoneal cells, and RAW 264.7 cells were washed with fluorescence-activated cell sorter (FACS) buffer (HBSS containing 1% FCS and 0.1% sodium azide). Cells were incubated with mouse IgG (50 µg/106 cells) on ice for 30 min. Next, cells were incubated with a 1:100 dilution of FITC-anti-CD14 antibody (IgG1-kappa isotype; Pharmingen) or FITC-control IgG1-kappa antibody (Pharmingen) for 30 min at 4°C. The cells were washed three times and fixed with 100 µl of 1% paraformaldehyde. Samples were wrapped in foil and stored at 4°C until studied by FACS analysis (FACScan; Becton-Dickinson, Mountain View, CA). As a positive control, a 1:100 dilution of anti-MAC-1-FITC (Pharmingen) was used to label Kupffer cells.

Treatment of RAW 264.7 and peritoneal cells using centrifugal elutriation. The isolation technique for Kupffer cells involves proteolytic digestion with collagenase and Pronase. These conditions could remove the CD14 receptor from the cell surface of Kupffer cells. Therefore, we treated RAW 264.7 cells and peritoneal cells using similar proteolytic conditions and examined these cells for CD14 receptor using Western blotting.

Cells were suspended in 50-ml culture tubes in RPMI with 10% FCS. Cells were pelleted at 250 g for 20 min, washed, and then incubated with GBSS for 5 min, pelleted, and washed. Next, the cells were suspended in a calcium- and magnesium-free GBSS for another 5 min, pelleted, washed, and subsequently suspended with GBSS containing 0.25% collagenase and 0.02% Pronase for 20 min. After another wash, cells were resuspended in 15 ml of GBSS, which was then loaded into a Beckman J2/21ME centrifugal elutriator at 4°C. At a rotor speed of 2,500 rpm (Beckman JE-6B), cells were elutriated at 11, 18, 21, and 36 ml/min with GBSS and 1% albumin. RAW 264.7 and peritoneal cells were collected at 36 ml/min. Subsequently, cells were cultured for 48 h in 35-mm culture dishes in RPMI without serum, and cell lysates were obtained for Western analysis as described above.

Statistical analysis. All values are expressed as means ± SD. Groups were compared by Student's t-test. P values less than 0.05 were considered statistically significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effect of serum on LPS-stimulated TNF-alpha release. Serum contains high concentrations of LBP, which binds to LPS to produce a complex that binds to membrane-associated CD14 (23, 29). Previously, Wright and co-workers (23, 29) showed that LPS stimulation of RAW 264.7 cells was markedly enhanced in the presence of 10% FCS and that serum-free conditions produced 100- to 1,000-fold less activation when LPS was used in concentrations below 10 ng/ml. When greater LPS concentrations were used, the effect of serum was abrogated. We replicated these results by incubating RAW 264.7 cells and peritoneal cells with 0.1 and 1.0 ng/ml LPS in the presence or absence of serum (Fig. 1, A and B). By contrast, when Kupffer cells derived from either mouse (Fig. 1C) or rat livers (Fig. 1D) were tested in the presence or absence of serum, LPS-stimulated TNF-alpha release was not significantly different.


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Fig. 1.   Lipopolysaccharide (LPS)-stimulated tumor necrosis factor-alpha (TNF-alpha ) release from cells in the presence and absence of serum. All cells were cultured in RPMI without FCS for 48 h and then stimulated with increasing doses of LPS in the presence of 10% FCS or absence of FCS. LPS was added for 4 h, and supernatants were assayed for TNF-alpha by ELISA. Each value was derived from 4-10 experiments, and values are means ± SD. A: RAW 264.7 cells. B: peritoneal cells. C: mouse Kupffer cells. D: rat Kupffer cells.

Dose-response studies (Fig. 1) demonstrated that maximum TNF-alpha release occurred in Kupffer cells when 1 ng/ml LPS or greater was used, both in the presence and absence of serum in the medium. With serum-containing medium, maximum TNF-alpha responses were also seen in RAW 264.7 and peritoneal cells using 1 ng/ml LPS. However, when RAW 264.7 and peritoneal cells were cultured in medium without serum, there was no significant TNF-alpha release above background levels when doses of 0.1-10 ng/ml LPS were used. TNF-alpha release did increase when 100 ng/ml or higher doses (data not shown) of LPS were used. Therefore, the dependency of the response to serum was overcome in RAW 264.7 and peritoneal cells at high concentrations of LPS (Fig. 1).

Effect of dLPS on LPS-stimulated TNF-alpha release. It has been shown that dLPS inhibited LPS stimulation of prostaglandins and plasminogen activator inhibitor-1 release by enothelial cells (22) and dLPS inhibited LPS-stimulated interleukin-8 release by THP-1 cells (11). In RAW 264.7 cells, 100-fold excess dLPS (i.e., 100 ng dLPS mixed with 1 ng LPS) inhibited LPS-stimulated TNF-alpha release by >90%. However, dLPS failed to inhibit LPS-stimulated TNF-alpha release from mouse and rat Kupffer cells (Fig. 2).


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Fig. 2.   Effect of deacylated LPS (dLPS) and phosphatidylinositol-phospholipase C (PI-PLC) on LPS-stimulated TNF-alpha release by RAW 264.7 cells and Kupffer cells (KC). Kupffer cells were isolated from rats or mice as described in METHODS and were cultured in microtiter wells for 48 h before study. RAW 264.7 cells were also studied 48 h after plating. Cells were preincubated with PI-PLC for 1 h, and then LPS was added for 4 h in the presence of PI-PLC. The following doses were used: 1 ng/ml LPS, 100 ng/ml dLPS, 0.5 U PI-PLC. Medium used was RPMI with 10% FCS. TNF-alpha was measured in culture supernatants by ELISA. * P < 0.001 compared with mouse or rat Kupffer cells.

Effect of PI-PLC on LPS-stimulated TNF-alpha release. CD14 is a GPI-linked protein. Thus CD14 is not a transmembrane receptor and does not interact directly with cytosolic proteins. The enzyme PI-PLC cleaves GPI-linked proteins from the cell surface (10). In the presence of PI-PLC, TNF-alpha release from RAW 264.7 cells was markedly diminished after exposure to 1 ng/ml LPS (Fig. 2). However, PI-PLC did not inhibit LPS-stimulated TNF-alpha from mouse or rat Kupffer cells (Fig. 2).

Western analysis of CD14. We used rat anti-mouse CD14 antibody to identify CD14 in cell extracts by Western analysis. Twenty micrograms of protein were loaded per lane. RAW 264.7 cells contained large amounts of CD14 by Western analysis (Fig. 3A). Mouse peritoneal cells also possessed CD14 when tested using Western analysis (Fig. 3A). By contrast, Kupffer cells derived from the same mouse strain as the peritoneal cells did not possess CD14 when examined by Western analysis (Fig. 3A). In separate experiments, PI-PLC treatment greatly decreased CD14 content in RAW 264.7 cells and peritoneal cells (Fig. 3B). In all gels, equal loading of protein was demonstrated by the presence of equal amounts of the cell surface protein, vinculin.


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Fig. 3.   CD14 expression in RAW 264.7 cells, mouse peritoneal macrophages, and Kupffer cells. Mouse Kupffer cells and peritoneal cells were isolated as described in METHODS. All cells were grown on 35-mm dishes for 48 h, culture medium was removed, and cells were washed and lysed. Twenty micrograms of cell lysate were loaded onto each lane for SDS-PAGE. Gels were probed with rat anti-mouse CD14 antibody as described in METHODS. The same gels were reprobed with an anti-vinculin antibody as described in METHODS. A: RAW 264.7 cells (lane 1), murine Kupffer cells (Mur KC; lane 2), and mouse peritoneal cells (Mur PC; lane 3). B: lane 1, RAW 264.7 cell lysate without LPS stimulation; lane 2, murine Kupffer cell lysate without LPS stimulation; lane 3, cell lysate from RAW 264.7 cells treated with 0.5 U/ml PI-PLC; lane 4, cell lysate from peritoneal cells treated with 0.5 U/ml PI-PLC.

Flow cytometry. To confirm the results of Western analysis, RAW 264.7 cells, peritoneal cells, and murine Kupffer cells were incubated with anti-CD14-FITC or control isotype antibody. Cells were sorted using FACScan and showed that 100% of RAW 264.7 cells and peritoneal cells were labeled by the anti-CD14 antibody (Fig. 4, A and B). However, <15% of freshly isolated mouse Kupffer cells were labeled with anti-CD14 (Fig. 4C), and <2% stained positive after 48 h of culture (Fig. 4D). Figure 4 displays representative results from three experiments each. The other two experiments using freshly isolated Kupffer cells showed 12 and 3% of cells stained with CD14, and all Kupffer cells cultured for 48 h showed <2% staining. Figure 4E shows that mouse Kupffer cells are labeled with anti-MAC-1 antibody.


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Fig. 4.   Fluorescence-activated cell sorter (FACS) analysis of RAW 264.7, peritoneal, and Kupffer cells. Using FITC-labeled antibodies and isotype controls, we examined cells for surface marker using FACS. A: RAW 264.7 cells labeled with anti-CD14 FITC. B: mouse peritoneal cells labeled with anti-CD14 FITC. C: freshly isolated mouse Kupffer cells labeled with anti-CD14 FITC. D: mouse Kupffer cells cultured for 48 h labeled with anti-CD14 FITC. E: mouse Kupffer cells cultured for 48 h labeled with anti-MAC-1 FITC. Vertical axis, number of cells; horizontal axis, log of fluorescence intensity.

Effect of centrifugal elutriation on CD14 expression. RAW 264.7 and peritoneal cells were treated in a manner similar to isolated Kupffer cells. Cells were therefore exposed to collagenase, Pronase, and centrifugal elutriation under the same conditions used for Kupffer cell isolation. This treatment did not significantly diminish CD14 expression in these cells (Fig. 5).


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Fig. 5.   Effect of centrifugal elutriation and enzyme digestion on CD14 expression. Cell lysates from RAW 264.7 and peritoneal cells were collected after appropriate treatment. Twenty micrograms of protein were loaded per lane for SDS-PAGE. Gels were probed with rat anti-mouse CD14, and gels were reprobed using an anti-vinculin antibody as described in METHODS. Lane 1, RAW 264.7 cells cultured on plastic dishes; lane 2, RAW 264.7 cells treated with collagenase, Pronase, and centrifugal elutriation (Elut); lane 3, mouse peritoneal cells cultured on plastic dishes; lane 4, mouse peritoneal cells treated with collagenase, Pronase, and centrifugal elutriation.

Effect of LPS stimulation on CD14 expression. When RAW 264.7 and peritoneal cells were stimulated with LPS for 2 h, CD14 expression was increased, but LPS stimulation did not increase CD14 expression in purified Kupffer cells (Fig. 6).


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Fig. 6.   Effect of LPS stimulation on CD14 expression. Cell lysates from RAW 264.7, Kupffer, and peritoneal cells were collected after appropriate treatment. Twenty micrograms of protein were loaded per lane for SDS-PAGE. Gels were probed with rat anti-mouse CD14, and gels were reprobed using an anti-vinculin antibody as described in METHODS. Lane 1, RAW 264.7 cells cultured on plastic dishes without LPS treatment; lane 2, RAW 264.7 cells cultured on plastic dishes stimulated with 10 ng/ml LPS for 2 h; lane 3, mouse Kupffer cells cultured on plastic dishes without LPS treatment; lane 4, mouse Kupffer cells cultured on plastic dishes stimulated with 10 ng/ml LPS for 2 h; lane 5, murine peritoneal cells cultured on plastic dishes without LPS treatment; lane 6, murine peritoneal cells stimulated with 10 ng/ml LPS for 2 h.

TNF-alpha release from Kupffer cells cultured in serum-free medium. It is possible that serum contains small amounts of soluble CD14 (sCD14) (6) and that sCD14 may bind to Kupffer cells while in culture for 48 h after elutriation. Therefore, Kupffer cells were isolated and cultured in serum-free medium for 48 h before LPS stimulation, and these results were compared with results from cells incubated in medium with serum. Kupffer cells cultured without serum vs. those cultured for 48 h with serum showed similar TNF-alpha release after stimulation with 1, 10, and 100 ng/ml LPS (Table 1).

                              
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Table 1.   LPS stimulation of isolated Kupffer cells in the presence and absence of serum during culture

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Kupffer cells are the most abundant fixed-tissue macrophage in the body, and they are capable of releasing large quantities of proinflammatory cytokines such as TNF-alpha and interleukin-1 after stimulation with LPS. The first step of Kupffer cell stimulation is the binding of LPS to a cell surface receptor. The most widely studied receptor is CD14, but other LPS receptors have also been described (2, 8, 10, 26, 28).

Our studies indicate that Kupffer cells derived from mice and rats express very little or no CD14. Western analysis directly demonstrated the presence of CD14 on RAW 264.7 cells and murine peritoneal cells. However, the same technique detected either no CD14 in cultured murine Kupffer cells or small amounts in freshly isolated Kupffer cells. The anti-CD14 antibody used in this study detected CD14 on murine tissue but did not functionally inhibit the LPS-stimulated TNF-alpha response (data not shown). This is not unusual since Viriyayakosol and Kirkland (27) listed several anti-human CD14 antibodies that all labeled CD14, but most did not inhibit LPS-stimulated responses. FACS analysis confirmed the findings of Western blots by showing that <15% of fresh Kupffer cells were labeled by the anti-CD14 FITC antibody, whereas 100% of RAW 264.7 and peritoneal cells were labeled. The 15% of Kupffer cells labeled in freshly isolated cells may represent contamination from other cells (5) that was diminished to <2% by incubating the cells for 48 h. Other evidence that Kupffer cells express little CD14 is inferred by the experiments using the enzyme PI-PLC, which cleaves the GPI-linked CD14 receptor. PI-PLC causes cells that utilize the CD14 receptor to release less TNF-alpha after LPS stimulation. As expected, PI-PLC treatment decreased LPS-stimulated TNF-alpha release by RAW 264.7 cells. In contrast, PI-PLC treatment did not decrease TNF-alpha release by Kupffer cells.

Functionally, the CD14 receptor seemed to play little role in LPS stimulation of Kupffer cells. TNF-alpha release by Kupffer cells after LPS stimulation (with doses of 100 ng/ml or less) is the same in the presence and absence of serum. Serum contains LBP, which complexes with LPS and enhances signaling through the CD14 receptor (23, 29). RAW 264.7 cells, which utilize the CD14 receptor, released substantially less TNF-alpha after LPS stimulation in the absence of serum. Our data are in agreement with Bellezzo et al. (1) who showed that stimulation of NF-kappa B after LPS exposure was not serum dependent in isolated Kupffer cells but was serum dependent in peritoneal macrophages. In addition, dLPS competes with LPS at the CD14 receptor and inhibits the effect of LPS when they are incubated together in a ratio of 100:1 (dLPS-LPS; see Ref. 22). Such competition blocked LPS-stimulated TNF-alpha release from RAW 264.7 cells but did not inhibit LPS-stimulated TNF-alpha release from Kupffer cells. The low expression of CD14 and the lack of typical function attributed to the CD14 receptor in Kupffer cells together strongly support the conclusion that LPS stimulates Kupffer cells to release TNF-alpha by a non-CD14 pathway.

RAW 264.7 cells are a transformed murine macrophage cell line. We used these cells since they have been shown to possess the CD14 receptor and to be sensitive to PI-PLC treatment (9), and they are sensitive to the absence of serum (which contains LBP) in the medium (23, 29). The method of isolation was not responsible for the loss of CD14 expression in Kupffer cells since RAW 264.7 and peritoneal cells did not lose CD14 after similar proteolytic treatment and centrifugal elutriation (Fig. 5).

Our results appear to differ from those reported by Matsuura and colleagues (17). They found that nonstimulated mouse livers showed evidence of CD14 using immunohistochemistry. However, only a few cells stained positively. In addition, nonstimulated livers had no detectable levels of CD14 mRNA. After LPS stimulation, CD14-positive cells increased sharply in their study. These cells may represent newly recruited monocytes that infiltrate the liver after LPS injection (20). Tracy and Fox (25) showed that nonstimulated livers had low CD14 expression but that, after bile duct ligation, CD14 expression increased. Fearns and colleagues (4) also showed that CD14 mRNA was not detectable in nonstimulated mouse livers but increased 4 h after LPS exposure. Before LPS stimulation, immunohistochemistry demonstrated a few CD14-positive cells in the liver that may have been Kupffer cells (4) and nearly undetectable amounts of CD14 mRNA in the liver before LPS stimulation (5). In addition, they showed that bile duct epithelia and hepatocytes had some CD14 mRNA, which could account for at least some of the low levels detected in unstimulated livers. Takakuwa and colleagues (24) found no detectable CD14 mRNA in mouse livers before LPS stimulation. Leicester et al. (14) showed that there was virtually no detectable CD14 in control human livers, but expression increased in livers from patients with primary biliary cirrhosis. Therefore, five research groups (4, 14, 17, 24, 25) showed that nonstimulated livers demonstrated little or no CD14 expression, but, after stimulation with LPS, TNF-alpha , or bile duct ligation, CD14 expression increased. In view of our findings, it is possible that the small number of CD14-positive cells identified in nonstimulated livers were actually not Kupffer cells but monocytes, cells moving through the hepatic vasculature, or even bile duct epithelial cells (5). Another interpretation is that a small subset of Kupffer cells do contain CD14. If there is a small subset of CD14-positive staining Kupffer cells, then functionally this does not seem to be relevant since the absence of serum, cleavage of CD14 with PI-PLC, and competition with dLPS did not decrease LPS-stimulated TNF-alpha release from mouse or rat Kupffer cells.

Recently, Haziot et al. (9) and Perera et al. (19) used CD14 knockout mice to study CD14-dependent and -independent pathways. These mice are resistant to death after injections of LPS and produce only a very small elevation of serum TNF-alpha . In these mice, peritoneal exudate cells had no CD14 and no response to LPS with respect to TNF-alpha release. Kupffer cells were not examined in that study. If Kupffer cells do not require CD14 for TNF-alpha release, then this may indicate that Kupffer cells do not play as much of a role in endotoxic shock as do other macrophages, such as lung and splenic macrophages. Direct study of Kupffer cells isolated from these CD14 knockout mice is planned for future studies.

The significance of a lack of CD14 on Kupffer cells is not known. A variety of LPS receptors have been described (2, 8, 10, 26, 28). It is possible that constant exposure of Kupffer cells to LPS in the portal system downregulates CD14 expression in vivo. Kupffer cells are phagocytes, and we have postulated that internalization and endosomal acidification are required for LPS stimulation of Kupffer cells (16). Pollack and co-workers (21) also demonstrated that inhibition of uptake of LPS with monoclonal antibodies inhibited TNF-alpha release by human phagocytes. In addition, Detmers and co-workers (3) demonstrated that endocytosis was necessary for LPS-stimulated adhesion by neutrophils. Perhaps the specific LPS receptor is simply a means of enhancing internalization of LPS into macrophages, and subsequently similar intracellular signaling events occur whether or not CD14 is initially present.

LPS stimulation of RAW 264.7 cells for 2 h caused a significant increase in CD14 expression, but this did not occur in isolated Kupffer cells that were cultured for 48 h and then stimulated with LPS. Fearns and Loskutoff (5) noted that myeloid-derived cells, which include Kupffer cells, responded quickly (within 2-4 h of LPS stimulation) to increase surface CD14 expression, whereas epithelial-derived cells (hepatocytes, bile duct epithelial cells, and kidney epithelial cells) required 8 h to increase CD14 expression (5).

If Kupffer cells do not possess surface CD14 receptors, then how does LPS bind to these cells? Fox et al. (6) demonstrated that Kupffer cells showed no saturation kinetics for LPS, and they postulated that LPS could intercalate into the cell membrane based in part on the lipid content of LPS. It is also possible that one of the other LPS receptors mediates LPS interaction of Kupffer cells (2, 8, 10, 26, 28). There is no role for soluble CD14 in LPS-stimulated TNF-alpha from Kupffer cells (Table 1) as has been found in endothelial cells (7).

In summary, this study directly examined isolated Kupffer cells for CD14 using Western analysis and FACS and found little or no CD14. LPS-stimulated TNF-alpha release from Kupffer cells occurred under conditions that were CD14 independent. Together, these data show that CD14 does not play an important role in LPS-stimulated TNF-alpha release from nonstimulated Kupffer cells.

    ACKNOWLEDGEMENTS

This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-44233 (to S. N. Lichtman) and DK-37034 (to J. J. Lemasters).

    FOOTNOTES

Address for reprint requests: S. N. Lichtman, 310 Burnett Womack Bldg., Univ. of North Carolina, Chapel Hill, North Carolina 27599-7220.

Received 19 September 1997; accepted in final form 7 April 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 275(1):G39-G46
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