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Am J Physiol Gastrointest Liver Physiol 287: G620-G626, 2004. First published April 15, 2004; doi:10.1152/ajpgi.00314.2003
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INFLAMMATION/IMMUNITY/MEDIATORS

Lipopolysaccharides induced increases in Fas ligand expression by Kupffer cells via mechanisms dependent on reactive oxygen species

Keiichiro Uchikura,1 Tatehiko Wada,1 Sumito Hoshino,1 Yuichi Nagakawa,1 Takashi Aiko,2 Gregory B. Bulkley,1 Andrew S. Klein,1 and Zhaoli Sun1

1Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; and 2First Department of Surgery, Faculty of Medicine, Kagoshima University, Kagoshima, Japan

Submitted 23 July 2003 ; accepted in final form 2 April 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fas-Fas ligand (FasL)-dependent pathways exert a suppressive effect on inflammatory responses in immune-privileged organs. FasL expression in hepatic Kupffer cells (KC) has been implicated in hepatic immunoregulation. In this study, modulation of FasL expression of KC by endogenous gut-derived bacterial LPS and the role of reactive oxygen species (ROS) as potential mediators of FasL expression in KC were investigated. LPS stimulation of KC resulted in upstream ROS generation and, subsequently, increased FasL expression and consequent Jurkat cell (Fas-positive) apoptosis. The NADPH oxidase and xanthine oxidase enzymatic pathways appear to be major sources of this upstream ROS generation. Increased FasL expression was blocked by antioxidants and by enzymatic blocking of ROS generation. Exogenous administration of H2O2 stimulated KC FasL expression and subsequent Jurkat cell apoptosis. Intracellular endogenous ROS generation may therefore represent an important signal transduction pathway for FasL expression in KC.

antioxidants; hepatic immunoregulation

ENDOGENOUS GUT-DERIVED BACTERIAL LPS have been implicated as important cofactors in the pathogenesis of liver injury (24, 25). Most of the toxicities of LPS, both intrahepatic and systemic, have been related to the release of these inflammatory cytokines and mediators (12, 37).

Kupffer cells (KC), the resident macrophage population in the liver, comprise one of major populations (20%) of the hepatic nonparenchymal cells. KC are found within the sinusoidal lumen, adhering to the liver sinusoidal endothelial cells. It has been reported that KC play a key role in clearing circulating LPS (5, 17, 19, 39), as demonstrated by experiments showing that the majority of injected 125I-labeled LPS can be localized to the KC within 30 min of injection (19). In addition to its ability to clear LPS, KC also respond to LPS with production of inflammatory cytokines (i.e., TNF-{alpha} and IL-1) (18) and a variety of mediators, including reactive oxygen species (ROS) (2, 3) and Fas ligand (FasL) (9). Moreover, KC can directly interact with passenger leukocytes and, thus, may play a role in immunomodulation (21), which includes antigen presentation (27) and induction of T cell apoptosis via Fas-FasL interactions (23).

Fas (Apo-1/CD95) belongs to the TNF receptor/nerve growth factor receptor family and transduces apoptotic death via its intracellular death domains on cross-linking by its cognate ligand (FasL), a 40-kDa membrane protein. Although the Fas-FasL system has been widely studied for its role in tissue growth (1) and elimination of malignant (16) and damaged cells (10), its role is perhaps best characterized in the immune system. Interactions between Fas and FasL are functionally involved in maintaining homeostasis and self-tolerance of the immune system via cytotoxic effector mechanisms; malfunctions of the Fas system cause lymphoproliferative disorders and lymphoadenopathy and facilitate autoimmune disorders (30). Induction of antigen-specific T cell tolerance by FasL-transfected antigen-presenting cells suggests a novel strategy for modulating T cell response (20, 38).

Knowledge of the regulation of FasL expression in KC is limited. ROS have been found to be important mediators of other inflammatory signaling pathways. We previously reported that ROS generated via the NADPH oxidase- and xanthine oxidase (XO)-dependent pathways are each important in the killing of circulating pathogens (Escherichia coli) and in antigen processing by KC (36, 26). This, together with the reported increased expression of FasL genes in LPS-stimulated KC associated with augmented ROS formation, prompted us to question whether formation of ROS in KC induced by LPS is capable of modifying FasL transcription. Consequently, we evaluated the potential role of ROS, and their generation, as upstream mediators of FasL expression in KC.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Male Lewis rats (225–250 g body wt; Harlan Sprague Dawley, Indianapolis, IN) were cared for according to National Institutes of Health guidelines and under a protocol approved by the Johns Hopkins University Animal Care Committee.

Reagents and target cells. Type IV collagenase, DNase I, pronase E, 2-mercaptoethanol, EDTA, and LPS from E. coli (0111:B4) were purchased from Sigma (St. Louis, MO). The antioxidants bovine erythrocyte superoxide dismutase (SOD) and human erythrocyte catalase were obtained from Calbiochem (La Jolla, CA). The reagents used to inhibit specific oxidant-generating enzymes, allopurinol, a specific inhibitor of XO, and diphenyleneiodonium (DPI), an inhibitor of the "neutrophil" NADPH oxidase, were obtained from Sigma. Anti-FasL antibodies MFL-4 and clone 33 were obtained from BD Pharmingen (San Diego, CA) and BD Transduction Laboratories (Lexington, KY), respectively. Jurkat cells, constitutively expressing Fas receptor and, thereby, sensitive to killing by FasL, were obtained from the American Type Culture Collection (Rockville, MD).

KC isolation and characterization. KC were isolated as described previously (7, 26). Briefly, the liver was perfused with 100 ml of 37°C Ca2+-free Hanks' balanced salt solution (HBSS; 20 ml/min) followed by 100 ml of 0.05% type IV collagenase in Ca2+-rich HBSS (20 ml/min). The liver was then removed and placed in a sterile bottle with 100 ml of HBSS containing 10 mg/ml DNase, 10 mM HEPES, 0.025% type IV collagenase, and 0.02% pronase E. After the liver was gently mashed, the digestion was allowed to proceed in this solution for 10 min at 37°C with stirring. The digest was then centrifuged at 300 g at 4°C for 5 min, and the pellet was washed three times with 40 ml of cold HBSS containing 10 mM HEPES and 10 mg/ml DNase (HBSS + HEPES-DNase). The final pellet was resuspended in 40 ml of HBSS + HEPES-DNase and centrifuged at 100 g at 4°C for 1 min. The resulting supernatant (containing most of the hepatic nonparenchymal cells) was layered on a sterile Percoll gradient (15 ml of 25% Percoll over 15 ml of 50% Percoll), which was then centrifuged at 900 g for 20 min. The lower zone, including the interface zone, was collected and resuspended in 40 ml of cold HBSS + HEPES-DNase and centrifuged at 900 g for 5 min. The pellet was resuspended, washed twice with HBSS + HEPES-DNase, and then resuspended in RPMI with 10 mM HEPES. The cell suspension was pipetted into 75-cm2 tissue culture flasks, which were previously coated with FBS. The flasks were incubated for 10 min at 37°C in a humidified incubator under 5% CO2 in air to allow the selective attachment of KC. Then the flasks were washed with HBSS + HEPES three times and placed on ice for 40 min. A shear force was applied by tapping the side of each flask until most of the cells were detached into suspension. The cell suspension was collected and centrifuged at 300 g for 5 min. This technique of cell isolation yielded, on average, 4–6 x 107 KC per liver, with 90–95% viability as determined by trypan blue exclusion. The cells showed typical macrophage morphological features and stained positively for nonspecific esterase and the antibodies ED1 and ED2 (Serotec) and phagocytosed 1-µm latex beads. Purity of the KC fraction was consistently >90% as determined by morphology (phagocytosed beads) and >90% as determined by ED2 staining (flow cytometry).

KC pretreatment. For most inhibition experiments, the inhibitor was added exogenously to the KC monolayers before each experiment. After the KC were washed with RPMI 1640 culture medium with 10 mM HEPES and 10% heat-inactivated FBS, they were incubated with antioxidants or enzyme inhibitors for 60 min. KC were washed three times and then stimulated with LPS or H2O2. Free radical scavengers, catalase and/or SOD, or selective inhibitors of XO (allopurinol) or NADPH oxidase (DPI) were used to inhibit enzymatic ROS production.

Cell viability assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) was used as an indicator of cell viability as determined by the mitochrondria-dependent reduction to formazan (22). KC were plated at a density of 105 cells/well into 96-well plates for 12 h and then treated with different concentrations of each compound for a further 24 h. Cells were washed with PBS three times, and MTT (50 mg/ml) was added to the medium for 4 h. The supernatant was removed, and the formazan crystals were dissolved using 0.04 N HCl in isopropanol. The absorbance was read at 600 nm with a microplate reader (Molecular Devices). The result was expressed as optical density.

Western blotting for FasL protein expression by KC. After 24 h of stimulation with H2O2 or LPS in 24-well plates, KC (1 x 106 cells/well) were homogenized in lysis buffer [60 mM Tris (pH 7.5), 4% SDS, and 10% glycerol]. KC proteins were extracted, and whole cell extracts were subjected to 12% SDS-PAGE. Resolved proteins were transferred to a nitrocellulose membrane and incubated with anti-FasL antibody (clone 33) and then with horseradish peroxidase-conjugated secondary antibody (1:1,000). After three 10-min washes with PBS-Tween 20, peroxidase activity was visualized with the enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's instruction, and FasL protein content was determined by densitometric scanning of the exposed X-ray film and compared with a control of {beta}-actin protein loading.

Real-time RT-PCR for FasL mRNA expression by KC. After 6 h of KC (3 x 106 cells/well) stimulation with LPS or H2O2 in six-well plates, total RNA was extracted using TRIzol (Life Technologies). To assess FasL mRNA expression, total RNA was subjected to RT-PCR using the following primers: 5'-AAGGCGGCCTTGTGATCA-3' and 5'-TTGCAAGACTGACCCCGG-3' and a fluorogenic probe (6FAM-TGAGGCTGGGTTGTACTTCGTATATTCCAAAGT-TAMRA),which were designed using Primer Express software (PE Biosystems, Foster City, CA). 18S ribosomal RNA (endogenous PCR control) primers were purchased from PE Biosystems and used as an endogenous PCR control. Standard curve preparation and quantification of mRNA in samples were performed by software provided with the ABI Prism 7700 sequence detector (PE Biosystems). Quantitative RT-PCR Thermoscript One-Step System (Life Technologies) was used as directed by the manufacturer.

Measurement of net, total H2O2 generation by KC. KC (2 x 105 cells/well) were cultured in medium with or without antioxidants in 96-well plates. At 60 min after incubation, the culture medium of each well was replaced with 100 µl of HBSS + HEPES buffer containing 20% FBS with or without LPS. Simultaneously, 100 µl of 50 µM Amplex red reagent (Molecular Probes, Eugene, OR) that had been mixed with 1 U/ml horseradish peroxidase were added to each well. After incubation for 60 min at 37°C in a 5% CO2 atmosphere, the fluorescence in each cell was measured in a fluorescence microplate reader (CytoFluor 2300, Millipore) using an excitation wavelength of 530 nm and an emission detection of 590 nm.

Detection of apoptosis of Jurkat target cells cocultured with KC. Apoptosis was assayed as follows: bromodeoxyuridine-labeled Fas antigen-positive Jurkat cells were used as target cells. The Jurkat cells (2 x 104 cells/well) were cocultured with KC (2 x 105 cells/well) as effector cells that had been stimulated with H2O2 or LPS for 24 h in 96-well plates. To confirm the mechanism of apoptosis, the ability of 10 µg/ml anti-FasL antibodies (MFL-4) to block target cell apoptosis was also assessed. KC were pretreated with anti-FasL antibodies for 1 h after LPS stimulation. Apoptotic cell death was measured with the cellular DNA fragmentation ELISA kit (Boehringer Mannheim, Indianapolis, IN). Each experiment was performed in triplicate.

Statistical analysis. Values are means ± SD, with n representing the number of cell preparations from different animals. Apparent differences between means were evaluated for significance using the two-tailed Student's t-test. Apparent differences between dose-response curves were evaluated for significance by single-factor ANOVA. P < 0.05 was considered statistically significant.


   RESULTS
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 MATERIALS AND METHODS
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DISCUSSION
 REFERENCES
 
FasL expression and H2O2 generation in KC stimulated by LPS. LPS stimulation of KC for 24 h resulted in dose-dependent increases of FasL protein expression over LPS concentrations of 0–3 µg/ml. KC stimulated by 1 µg/ml LPS increased FasL protein expression threefold (Fig. 1A). To quantify whether activation of KC stimulated by LPS is associated with the generation of ROS, we measured net, total H2O2 generation by KC. Significant amounts of H2O2 were detected from KC stimulated with LPS (1 µg/ml) for 60 min compared with nonstimulated KC. We quantified the contribution of NADPH oxidase and XO enzymatic pathways as sources of KC ROS generation. Allopurinol (300 µM) and DPI (300 nM) each decreased ROS generation significantly (Fig. 1B). Our results showed that LPS at high concentration shows no increase in FasL protein level (3 µg/ml); therefore, we determined whether LPS stimulation influences the viability of KC. The high concentration of LPS cocultured with KC resulted in ~50% death of KC as quantified by MTT assay (Fig. 1C). However, at low concentration of LPS (≤1 µg/ml), there was no effect on the viability of KC. To examine the inhibitory effects of antioxidants and inhibitors on LPS-induced ROS generation or FasL expression through their cytotoxic effect, MTT assay was performed. SOD (300 U/ml), catalase (300 U/ml), DPI (300 nM), and allopurinol (300 µM) showed no significant cytotoxic effect in our assay (Fig. 1C).



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  		Fig. 1. Effect of LPS and antioxidants on Kupffer cell (KC) Fas ligand (FasL) expression and reactive oxygen species (ROS) generation and viability. A: Western blot analysis of FasL protein expression by KC in response to LPS. KC (1 x 106 cells/well) were stimulated with LPS (0–3 µg/ml) in 24-well plates for 24 h. FasL expression was determined under control (unstimulated) and LPS-stimulated conditions. FasL expression was normalized to control {beta}-actin and expressed as a relative intensity ratio. Results are expressed as percentage of control values. Values are means ± SD from 8 experiments for each point. *Significantly different from control by single-factor ANOVA (P < 0.0001). B: H2O2 generation of LPS-pretreated KC and effects of antioxidants. KC (2 x 105 cells/well) were cultured with medium with or without LPS (0.1–1 µg/ml) and with or without antioxidants: 300 nM diphenyleneiodonium (DPI) or 300 µM allopurinol in 96-well plates. The presence of H2O2 was determined by horseradish peroxidase-dependent oxidation of Amplex red. Values are means ± SD from 6 experiments for each condition. *Significantly different from control by 2-tailed Student's t-test (P < 0.01). C: KC viability. KC were plated at a density of 105 cells/well into 96-well plates and then treated with LPS at 0–3 µg/ml, SOD at 300 U/ml, catalase at 300 U/ml, DPI at 300 nM, or allopurinol at 300 µM for 24 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Values [expressed as optical density (OD) at 600 nm] are means ± SD from 6 experiments for each condition. *Significantly different from control by single-factor ANOVA (P < 0.0001).

 
Increased FasL expression in KC was blocked by antioxidants and by enzymatic blocking of ROS generation. Increased FasL protein expression of KC stimulated with LPS was blocked by the antioxidants SOD (30–300 U/ml) and catalase (300 U/ml) and by the oxidant-generating enzyme inhibitors DPI (300 nM) and allopurinol (300 µM; Fig. 2 A–D). Even at low concentrations, DPI + allopurinol inhibited FasL protein expression of KC stimulated with LPS to a greater degree than DPI or allopurinol alone (Fig. 2E). The inhibitory effects of DPI and allopurinol were additive. Each of the antioxidants or oxidant enzyme inhibitors has no effect on FasL protein expression of KC cocultured without LPS compared with controls (Fig. 2F).



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  		Fig. 2. Effects of antioxidant pretreatment on FasL protein expression of LPS-activated KC. KC (1 x 106 cells/well) were incubated with antioxidants or enzyme inhibitors in 24-well plates for 60 min. KC were washed 3 times and then stimulated with LPS (1 µg/ml) for 24 h. SOD (30–300 U/ml; A), catalase (30–300 U/ml; B), DPI (30–300 nM; C), and allopurinol (30–300 µg; D) suppressed FasL expression levels dose dependently. E: DPI + allopurinol resulted in greater inhibition of FasL protein expression of KC stimulated with LPS than with DPI or allopurinol alone. F: effect of SOD (300 U/ml), catalase (300 U/ml), DPI (300 nM), and allopurinol (300 µM) on FasL protein expression of KC cocultured without LPS. FasL expression was determined by Western blot analysis. FasL expression was normalized to control {beta}-actin and expressed as a relative intensity ratio. Results are expressed as percentage of LPS-stimulated control values (no antioxidants or enzyme inhibitors). Values are means ± SD from 4 (A and B) or 3 (C–F) experiments for each point. Significant difference from control by single-factor ANOVA: *P < 0.03; **P < 0.01; ***P < 0.001.

 
H2O2 induces upregulation of FasL expression in KC. To estimate the direct role of ROS on FasL expression, unstimulated (i.e., no LPS) KC were exposed to H2O2 for 15 min. At 24 h, H2O2 stimulation produced dose-dependent increases of FasL protein expression. KC stimulated by 100 µM H2O2 increased FasL protein expression threefold (Fig. 3A). FasL protein expression was significantly inhibited by catalase at 30–300 U/ml (Fig. 3B).



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  		Fig. 3. Effect of H2O2 on KC FasL expression. A: FasL protein expression in response to H2O2 in KC. KC (1 x 106 cells/well) were exposed to 10–100 µM H2O2 for 15 min in 24-well plates and then cultured for 24 h. FasL expression was determined by Western blot analysis. Results are expressed as percentage of unstimulated control values. B: effects of antioxidant pretreatment on FasL protein expression of H2O2-activated KC. KC (1 x 106 cells/well) were incubated with catalase (30–300 U/ml) in 24-well plates for 60 min and then stimulated with 100 µM H2O2 for 15 min. FasL expression was determined by Western blot analysis after 24 h of culture. Results are expressed as percentage of maximally stimulated (100 µM H2O2), uninhibited KC. Values are means ± SD from 3 experiments for each point. *Significantly different from control by single-factor ANOVA (P < 0.0001).

 
Upregulation of FasL mRNA expression in KC in response to LPS or H2O2. LPS (1 µg/ml) stimulation of KC for 6 h resulted in a greater than threefold increase in FasL mRNA expression. FasL mRNA expression was blocked by the antioxidants SOD (300 U/ml) and catalase (300 U/ml; Fig. 4A). After 6 h of H2O2 (100 µM) stimulation (15 min), KC FasL mRNA expression increased more than threefold. Increased FasL mRNA expression was inhibited by catalase at 300 U/ml (Fig. 4B). However, SOD or catalase had no effect on FasL mRNA expression of KC in the absence of LPS or H2O2 stimulation, respectively.



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  		Fig. 4. KC FasL mRNA expression in response to LPS (A) or H2O2 (B) stimulation and effects of antioxidants. KC were pretreated with or without SOD (300 U/ml) or catalase (300 U/ml, Cat) for 60 min. After 6 h of KC (3 x 106 cells/well) stimulation with LPS (1 µg) or H2O2 (100 µM) in 6-well plates, total RNA was extracted using TRIzol. FasL mRNA expression was quantified by real-time RT-PCR. 18S ribosomal RNA was used as an endogenous PCR control. Results are expressed as percentage of control (no antioxidant, LPS, or H2O2) values. Values are means ± SD from 5 experiments for each condition. KC stimulated by LPS or H2O2 expressed >2-fold more FasL mRNA than nonstimulated KC. Significant difference from control by 2-tailed Student's t-test: *P < 0.03; **P < 0.01; ***P < 0.001.

 
Apoptosis of Fas-positive Jurkat cells induced by KC. To measure the functional capacity of KC to induce apoptosis via FasL, we quantified apoptosis of Fas-positive Jurkat target cells cocultured with KC after exposure to LPS or H2O2. With the use of the cellular DNA fragmentation ELISA kit, apoptosis of Jurkat cells was markedly increased in response to LPS (1 µg/ml) or H2O2 (100 µM) pretreatment of the KC. The apoptosis of Jurkat cells was significantly suppressed by the addition of anti-FasL antibody at 10 µg/ml (Fig. 5). The addition of catalase (300 U/ml) or SOD (300 U/ml) to LPS- or H2O2-stimulated KC reduced subsequent Jurkat cell apoptosis.



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  		Fig. 5. Apoptosis of Fas-positive Jurkat cells cocultured with KC. Bromodeoxyuridine-labeled Fas-positive Jurkat cells were used as target cells. Jurkat cells (2 x 104 cells/well) were cocultured with KC (2 x 105 cells/well), as effector cells, which had been stimulated with LPS (1 µg; A) or H2O2 (100 µM; B) for 24 h in 96-well plates. Apoptotic cell death was measured with the cellular DNA fragmentation ELISA kit. Ab, anti-FasL antibody. Values are means ± SD from 3 experiments for each condition. Significant difference from control values by 2-tailed Student's t-test: *P < 0.03; **P < 0.01; ***P < 0.001.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The highly efficient mediation of apoptosis of activated T lymphocytes by FasL-dependent mechanisms may, in some instances, create an environment of antigen-specific accommodation. Such "immune privilege" induced by FasL has been determined in the anterior chamber of the eye and in Sertoli cells of the testis (11). The immunologic repertoire of resident hepatic macrophages (KC) within the hepatic sinusoids may also endow the liver with similar immunologic privilege (21). Because KC can directly interact with circulating lymphocytes and also act as antigen-presenting cells for intrahepatic lymphocytes, they occupy a key position in the regulation of the hepatic immune response (21). It has been reported that IFN-{gamma} upregulates FasL expression in KC and, subsequently, induces lymphocyte death by a Fas/FasL-dependent mechanism (23). We recently reported that KC can regulate the T cell response via the Fas/FasL-dependent pathway and may play an important role in the induction of immune tolerance in liver allografts (33, 34). Expression of FasL in KC may represent an intrahepatic pathway of immunoregulation.

We have used LPS-stimulated KC FasL expression as a model to investigate the relation between KC ROS generation and FasL expression. Our results indicate that KC treated with LPS express functional FasL and induce apoptosis of Fas-positive target cells. The generation of intracellular endogenous ROS may represent an important signal transduction pathway for FasL expression in this system. To determine the pathways of ROS generation in KC stimulated with LPS, antioxidants and specific oxidant enzyme inhibitors were selected as blockers for ROS generation in our model. DPI is an inhibitor of NADPH oxidase, and allopurinol is a very specific inhibitor of XO (14), whereas SOD and catalase are enzymatic scavengers of O2– and H2O2, respectively. Our results show that antioxidants (SOD and catalase) significantly suppressed FasL expression in KC stimulated with LPS in a dose-dependent fashion. DPI or allopurinol partially blocked the FasL expression in KC stimulated with LPS. However, FasL expression was almost completely blocked when KC were pretreated with DPI + allopurinol at a relatively high dose (Fig. 2E). These findings suggest that NADPH and XO play a critical role in ROS-dependent FasL expression in KC stimulated with LPS. To confirm the role of ROS in this model of FasL expression, KC were stimulated directly with H2O2. H2O2 stimulation increased KC FasL expression, and this FasL expression was blocked by catalase, a specific enzymatic scavenger of H2O2. These results support the hypothesis that ROS may act as a signal to regulate FasL expression in KC.

We previously reported that ROS generated via the NADPH oxidase- and XO-dependent pathways are each important in the killing of circulating pathogens (E. coli) by KC (15, 36). Our data show that SOD is more effective in preventing the LPS-induced increase in FasL expression and suggest that O2– is more important than H2O2 in the observed LPS-induced increase in FasL expression. However, as a single agent, the XO inhibitor (allopurinol) or NADPH oxidase inhibitor (DPI) each prevents ~40% of the LPS-induced increase in FasL expression. Interestingly, the inhibitory effects of DPI and allopurinol were additive (Fig. 2E). Our group previously reported that DPI + allopurinol shows additive effects in reducing the generation of reactive oxygen metabolites by XO- and NADPH oxidase-dependent pathways (14). The additive effects of DPI and allopurinol in preventing an LPS-induced increase in FasL protein levels (~75%; Fig. 2) suggest that O2– and H2O2 are each important for the LPS-induced increase in FasL expression in KC.

It is not clear how extracellularly administered SOD was able to degrade intracellular O2–. Our previous studies (26, 36) showed that SOD inhibited phagocytic killing of bacteria in phagocytes (i.e., macrophages and KC) by suppressing ROS generation without influencing phagocytosis. SOD degradation of intracellular O2– may be explained, at least in part, by phagocytosis of SOD. Phagocytosis may transfer exogenous SOD into KC. In addition, SOD can directly degrade membrane-bound and released O2– from KC. Thus SOD may degrade intracellular and extracellular O2–. This may be one reason that SOD is more effective in preventing LPS-induced increase in FasL expression.

The correlation between the H2O2- and LPS-induced increase in FasL protein levels has been demonstrated by the observations that 1) LPS stimulation increases H2O2 generation in KC (Fig. 1), 2) H2O2 alone can increase FasL protein expression in KC (Fig. 3), and 3) antioxidants (SOD and catalase) and oxidant enzyme inhibitors (DPI and allopurinol) block the increase in FasL expression of KC stimulated with LPS (Figs. 2 and 4). A high concentration of exogenous H2O2 was required to increase FasL protein levels (Fig. 3A). Our data also show that LPS at 1 µg/ml produced <1 µM H2O2 within 60 min (Fig. 1B). In Western blotting assay for FasL protein expression, we used 1 x 106 cells/well in 24-well plates and stimulation with H2O2 or LPS for 24 h. However, for the H2O2 generation assay, we used 2 x 105 cells/well in 96-well plates and only quantified H2O2 generation of KC within 60 min after LPS stimulation. In addition, extracellular levels of H2O2 do not necessarily represent the intracellular levels of H2O2. That may explain why a relatively high concentration of H2O2 was required to increase FasL protein level, whereas LPS at 1 µg/ml produced only 1 µM H2O2 in our studies.

SOD or catalase completely prevents the LPS-induced increase in apoptosis (Fig. 5A), whereas SOD or catalase caused only partial inhibition of the LPS-induced increase in FasL mRNA (Fig. 4A) and FasL protein (Fig. 2, A and B). It should be recognized that KC constitutively express FasL, and, indeed, the coculture of untreated KC with Jurkat cells results in a baseline level of measurable apoptosis. In our study, coculture of Jurkat cells with untreated KC at a ratio of 1 to 10 did not significantly increase Jurkat cell apoptosis compared with coculture of Jurkat cells without KC (Fig. 5A). Thus the discrepancy between the effect of antioxidants on LPS-induced apoptosis and FasL expression could be explained by FasL dose-dependent induction of Jurkat cell apoptosis. The decreased FasL expression in KC by SOD or catalase insufficiently induces Fas-positive Jurkat T cell apoptosis. On the other hand, LPS-stimulated activation of KC not only induces increased FasL expression but also stimulates production of cytokines, such as TNF-{alpha} (31). These cytokines may also cause apoptosis of Jurkat T cells in our model. However, our observations that KC-induced T cell apoptosis can be blocked by addition of neutralizing anti-FasL antibody (34) further support the importance of FasL expression in LPS-induced apoptosis. We focused on FasL expression, although SOD or catalase may also inhibit cytokine production in LPS-stimulated KC. Catalase prevents up to 50% of H2O2-induced FasL expression in KC and similar levels of T cell apoptosis when cocultured with KC stimulated with H2O2 (Fig. 5B), suggesting a correlation between H2O2 and functional FasL expression in KC.

Although the mechanism of regulation of FasL expression in KC is unclear, it is likely that gut-derived LPS may act as a mediator to induce FasL expression of KC. LPS activates KC to release active substances such as ROS. Imbalance in oxidant production and elimination can lead to excessive exposure to ROS and subsequent oxidative damage of nucleic acids, proteins, and membrane lipids. A role for KC-dependent oxidative injury in hepatic ischemia-reperfusion is well supported (6). In contrast, the endogenous production of ROS may act as a signal to effect gene transcription, rather than cellular injury. ROS have been found to be important mediators of other inflammatory signaling pathways (35). Specifically, ROS have been shown to affect gene expression, including Fas and FasL gene expression in endothelial cells, microglia, and astrocytoma cells (8, 32). A number of signal transduction pathways has been implicated in FasL expression, including NF-{kappa}B, AP-1, p38 MAPK, and JNK (13, 28). LPS can activate the NF-{kappa}B signaling pathway mediated by ROS and MAPK signal transduction pathways such as JNK and p38 MAPK in KC (4, 29). The effect of "oxidant stress" on the activation of these transcription factors is cell-type specific, and the molecular mechanism by which ROS modulates these processes is unclear. However, these signals in KC might be mediated by the endogenous generation of ROS and, thereby, induce FasL expression. We observed that exogenous H2O2 induced upregulation of KC FasL and that the increased expression of FasL in LPS-stimulated KC was blocked by addition of antioxidants. These data suggest that LPS-induced upregulation of FasL expression in KC may be dependent, at least in part, on ROS generation. More extensive studies are needed to demonstrate which factors are key modulators of FasL expression via the generation of endogenous ROS in KC.

To our knowledge, this is the first study showing that LPS has the ability to upregulate FasL mRNA and protein expression in KC in vitro. Intracellular endogenous ROS generation may represent an important signal transduction pathway for FasL expression in KC. The NADPH oxidase and the XO enzymatic pathways appear to be major sources of this upstream ROS generation. We would propose that stimulation of KC FasL expression via ROS-dependent mechanisms may provide a novel strategy for modulating the T cell response.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. Sun, Dept. of Surgery, Johns Hopkins Univ. School of Medicine, 720 Rutland Ave., Ross 733, Baltimore, MD 21205 (E-mail: zlsun{at}jhmi.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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