Department of Pharmacology and Toxicology, Center for Toxicology, The University of Arizona, Tucson, Arizona 85721
Received October 5, 1999; accepted December 15, 1999
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ABSTRACT |
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Key Words: diethyldithiocarbamate (DDC); rat Kupffer cells; tumor necrosis factor- (TNF-
); nitric oxide; oxidative stress; NF-
B; protein kinase C.
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INTRODUCTION |
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It has been reported that low doses of DDC protect against liver injury induced by many hepatotoxic agents via its inhibition of drug metabolizing enzymes and antioxidant effects in rats (Stott et al., 1997; Yamano and Morita, 1992
; Younes and Siegers, 1980
). Previous studies have shown that large doses of DDC cause liver injury in rats (Ishiyama et al., 1990
). The pathogenesis of this injury involves bioactivation as well as dysfunction of antioxidant systems in parenchymal cells (Ishiyama et al., 1990
). Importantly, this hepatotoxicity can be prevented if rats are pretreated with gadolinium chloride, an inhibitor of Kupffer cells (Ishiyama et al., 1995
). DDC has been shown to stimulate the release of reactive oxygen species by Kupffer cells. Thus, activation of Kupffer cells appears to be a critical factor in DDC hepatotoxicity.
The activation of Kupffer cells plays a critical role in the pathogenesis of chemical-induced liver injury in animals (Edwards et al., 1993; El Sisiet al., 1993
; Gunawardhana et al., 1993
; Hoglen et al., 1998
; Laskin, 1990
; Sipes et al., 1989
), and has been associated with human liver disease (see review in Winwood and Arthur, 1993). Kupffer cells release a variety of cytotoxic factors such as reactive oxygen species, including superoxide anion, nitric oxide (NO), TNF-
, interleukin-1, and interleukin-6 (Decker, 1990
). Thus, the purpose of this study was to investigate if DDC stimulated the production of toxic secretory products (TNF-
and NO) in isolated Kupffer cells. Although DDC alone did not stimulate the release of NO and TNF-
by Kupffer cells, DDC enhanced the production of both when Kupffer cells were stimulated by lipopolysaccharide (LPS), a potent activator of Kupffer cells. Therefore, we further investigated DDC as modulating agent of LPS-induced intracellular signaling transduction pathways. A part of this study has been presented at the 1998 SOT meeting (Ishiyama et al., 1998
).
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MATERIALS AND METHODS |
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Chemicals
2', 7'-Dichlorofluorescin diacetate was obtained from Acros Organics (Pittsburgh, PA). Lipopolysaccharide (LPS, E.coli; 0111:B4) was obtained from Difco Laboratories (Detroit, MI). Collagenase was purchased from Worthington Biochemcal Co. (Lakewood, NJ). Antibody to inducible nitric oxide synthase (iNOS) was from Transduction Laboratories Inc. (Lexington, KY). Tumor necrosis factor (TNF)- antibody was from Genzyme (Cambridge, MA). NF-
Bp65 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Diethyldithiocarbamate (DDC), N-acetyl-L-cysteine (NAC), L-N6-(1-iminoethyl)lysine (NIL), phorbol 12-myristate 13-acetate (PMA), 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), N-(2-[methylamino]ethyl)-5-isoquinoline sulfonamide (H-8) and all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).
Isolation of Kupffer cells
Rats (250300 g) were anesthetized with pentobarbital (50 mg/kg, ip). Livers were digested with a collagenase/protease solution and Kupffer cells were purified from other cells by centrifugal elutriation (Knook and Sleyster 1976; Gunawardhana et al. 1995
). The viability of cells was greater than 98% as determined by trypan blue exclusion. Phagocytosis of opsonized 3-µm latex beads and endogenous peroxidase staining indicated the purity was > 90%. Cells were cultured at a concentration of 1 x 106 cells/well in 6- or 24-well plates in RPMI-1640 medium supplemented with 10% fetal calf serum, 25 mM NaHCO3, 10 mM HEPES and 50 µg/ml gentamicin at 37°C in a humidified 5% CO2/95% air.
Experimental Design
Following 24 h of culture, the effect of DDC and/or LPS on Kupffer cell-derived nitric oxide (NO) or TNF- was determined. DDC (030 µM) and/or LPS in fresh culture medium was added to wells containing 1 x 106 cells/well. DDC (> 100 µM) showed cytotoxicity over 4-h incubation (not shown). In certain incubations, the various inhibitors (NIL, NAC, H-7 and H-8) were added 3 h before DDC and/or LPS. Cells were then maintained in culture medium for 20 h. The culture medium was collected and measured for NO and TNF-
. The medium for TNF-
determination was frozen at 20°C until measurement. For immunocytochemistry determination and detection of oxidative stress, the isolated cells (1 x 106 cells/well) were plated on sterile cover slips in a 6-well plate. The cells were treated either with DDC and/or LPS for the time indicated for the various determinations: 20 h (iNOS); 3 h (TNF-
); 1 h (NF-
Bp65 or oxidative stress).
Assays
NO release.
Nitrite, a stable metabolite of NO, was determined by the Griess reaction (Green et al., 1981). Culture medium was reacted with 1% sulfanilamide in 5% H3PO4 and 0.1% N-(1-naphthyl) ethylenediamine solutions. The optical density was measured using a microplate reader (Dynatech Labolatories) at 570 nm. Additional evidence for the release of NO was estimated by the oxidation of 2', 7'-dichlorofluorescin free base (DCFH) (Le Bel et al., 1992
; Gunasekar et al., 1995
). This assay is based on conversion of nonfluorescent DCFH to a fluorescent product, DCF, by NO. DCFH was prepared from 2', 7'-dichlorofluorescin diacetate (DCFH-DA) by the method of Cathcart et al. (1983). One mM DCFH-DA and 0.01 N NaOH (1:4) were mixed at room temperature for 30 min, and the solution was neutralized with phosphate-buffered saline (PBS, pH 7.4). DCFH (50 µM, final concentration) was added to the culture medium with or without test compounds. After 20 h of incubation, the intensities of DCF fluorescence in the media were measured by a Hitachi F-2000 spectrofluorometer at an excitation wavelength of 488 nm and emission wavelength of 525 nm.
TNF- release.
TNF- released into the culture medium was determined by the L929 bioassay method (Stadler et al., 1993
). L929 fibroblasts were cultured at a concentration of 2 x 105 cells/well in 96-well cultured plates. Media obtained from the incubations of Kupffer cells and actinomycin D (1 µg/ml, final concentration) were added to the cultures of L929 cells. After 18 h, the cytotoxicity was determined by staining the cells with 0.5% crystal violet solution. The optical density was read at 570 nm using the microplate reader.
Immunocytochemistry.
iNOS, TNF- and NF-
Bp65 proteins were determined by an immunofluorescence analysis. The cultured cells were washed with phosphate-buffered saline (PBS, pH7.4) and fixed with 2% paraformaldehyde for 15 min at 4°C. Preparations were stored in a freezer at 70°C until further processing. After being hydrated with PBS for 5 min at 4°C, the preparations were incubated in PBS containing 0.1% Triton X-100 for 1 h. After washing with PBS/1% bovine serum albumin (BSA), normal goat serum was applied to the preparations for 1 h when staining for TNF-
and NF-
Bp65. The preparations were then incubated for 1 h with primary antibody (mouse monoclonal antibody directed against iNOS, rabbit polyclonal anti-mouse TNF-
, or rabbit polyclonal antibody specific NF-
Bp65). After washing with PBS containing 1% BSA, the preparations were incubated with an FITC-conjugated goat anti-murine IgG for 1 h in the dark. Cells were washed 3 times with PBS containing 1% BSA, and the cover slips were mounted on the slides in p-phenylene diamine mounting solution. The preparations were observed with an Olympus BH-2 fluorescent microscope at the excitation wavelength of 495 nm and emission wavelength of 520 nm. No labeling was seen on the sections when the secondary antibody alone was used.
Determination of oxidative stress.
Oxidative stress was determined by the loading of Kupffer cells with DCFH-DA (Cathcart et al., 1983). DCFH-DA is hydrolyzed by intracellular esterases to DCFH. DCFH is oxidized to fluorescent DCF by reactive oxygen species. Cultured Kupffer cells on cover slips were incubated with RPMI-1640 medium without serum containing 50 µM DCFH-DA for 2 h at 37°C in the CO2 incubator. The cells were washed with fresh incubation medium and then were treated with test compounds in RPMI-1640 with serum for 1 h. The treated cells were fixed with methanol for 10 min at 20°C and the fluorescence was observed with the fluorescent microscope (excitation wavelength: 495 nm, emission wavelength: 520 nm).
Statistics
Statistical analysis of results was performed by analysis of variance (ANOVA). Between-group variance was determined using the Student-Newman-Keuls post hoc test. Data were expressed as mean ± SD and were considered significant at a p value of < 0.05.
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RESULTS |
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NF-Bp65 protein.
Untreated Kupffer cells revealed NF-Bp65 immunoreactivity, indicated by green fluorescence, in cytoplasm rather than in the nuclear area (Fig. 5G
). In contrast, the nuclei of cells were strongly immunopositive 1 h after LPS exposure. This nuclear fluorescence implies the translocation of NF-
Bp65 protein from cytosol to nucleus. The degree of nuclear immunoreactivity in DDC + LPS-treated cells was stronger than that in LPS alone (Figs. 5H and 5I
).
Effect on Oxidative Stress
When Kupffer cells were incubated for 1 h with PMA or H2O2, two agents known to induce intracellular oxidative stress, DCF associated green fluorescence was observed in Kupffer cells. Thus, these agents caused an intracellular oxidative stress (Fig. 6). In control Kupffer cells or Kupffer cells incubated with DDC, LPS or DDC + LPS, only yellow auto-fluorescence was observed (Fig. 6
). Therefore, under the condition of these experiments, these treatments did not cause detectable formation of DCF reactive species.
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DISCUSSION |
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DDC alone did not affect the detectable release of NO and TNF- by Kupffer cells. However, DDC, in a concentration-dependent manner, enhanced the release of both NO and TNF-
by Kupffer cells when cells were stimulated with LPS. These results suggest that DDC sensitizes the Kupffer cell to LPS-stimulated TNF-
and to NO production.
It is well known that NO is synthesized by the enzyme NOS. NOS is inducible in Kupffer cells as well as other mammalian cells when exposed to LPS (Nathan, 1992; Nussler and Billiar, 1993
). To confirm whether DDC modulates inducible NOS (iNOS), iNOS protein was detected immunocytochemically. Our results showed that DDC enhanced the expression of iNOS protein when Kupffer cells were stimulated with LPS. Mülsch et al. (1993) reported that higher concentrations of DDC (> 100 µM) inhibited LPS-induced NO production and iNOS mRNA expression by bone marrow cells. On the other hand, Togashi et al. (1997) reported that pyrrolidine dithiocarbamate, a DDC derivative, enhanced NO production, iNOS mRNA expression, and NF-
B activation after LPS stimulation in glial cells. Thus, it is considered from these findings that the different action of DDC depends on cell types and concentration of DDC.
TNF- release from Kupffer cells after LPS stimulation is thought to be the result of protein biosynthesis (Lichtman et al., 1996
). In the studies reported here, DDC enhanced the increase in the immunoreactivity of cytosolic TNF-
protein by LPS. Interestingly, the staining intensities for TNF-
protein of both LPS- and LPS + DDC-treated Kupffer cells were most strongly increased in the perinuclear area. It is in these perinuclear vesicles where TNF-
accumulates before secretion by LPS-stimulated human monocytes (Hofsli et al., 1989
). Schmalbach et al. (1992) reported that DDC stimulates the expression of TNF-
mRNA in HL-60 cells. More recently, Bulger et al. (1998) reported that DDC and its derivative enhanced the production of TNF-
by LPS in rabbit alveolar macrophages. Taken together, these results suggest that DDC enhances the production and secretion of TNF-
by Kupffer cells and similar cell types upon stimulation.
The transcription of TNF- and iNOS genes by LPS is regulated by transcription factor NF-
B, which is composed of a heterodimer of 2 DNA binding subunits, p65 and p50 (Baeuerle and Henkel, 1994
). In Kupffer cells as well as other macrophages, activation of NF-
B is a critical step after LPS exposure (Tran-Thi et al., 1995
). In an inactivated form, NF-
B localizes in the cytoplasm, complexed with the inhibitor protein
B (I
B). After activation of NF-
B by LPS, the activated NF-
B translocates into the nucleus and stimulates the transcription of target genes encoding immunologically relevant proteins. To determine if DDC promotes the translocation of activated NF-
B into the nucleus at the individual cell level, it was detected using a specific antibody for NF-
Bp65. The translocation of NF-
Bp65 into the nucleus was detected 1 h after exposure to LPS. Interestingly, DDC enhanced the intensity of immunoreactivity in the nucleus of LPS-stimulated Kupffer cells. The enhancement effects of DDC on LPS-induced NO and TNF-
release were inhibited by NAC. Since NAC can inhibit the activation of NF-
B by LPS in rat Kupffer cells (Fox and Leingang, 1998
), these findings suggest that DDC enhances the activation of NF-
B by LPS. Contrary to our findings, previous studies have shown that DDC (> 100 µM) inhibited TNF-
and NO production through NF-
B inactivation (Mülsch et al., 1993
; Schreck et al., 1992
). However, we used lower concentrations of DDC than the concentrations used in those experiments in which DDC was reported to inactivate NF-
B. Thus, DDC may affect NF-
B by multiple mechanisms that are dose-dependent.
Reactive oxygen species stimulate the activation of NF-B by LPS and other cytotoxic factors in many cell types (Baeuerle and Henkel, 1994
). There have been reports regarding a pro-oxidant effect of DDC (> 100 µM) and its derivatives, resulting in the generation of intracellular oxidative stress (Nobel et al., 1995
). Therefore, we investigated whether oxidative stress in Kupffer cells might occur after lower concentration of DDC and/or LPS exposure. As expected, PMA and H2O2 increased the intensity of DCFH fluorescence in cytoplasm, but neither LPS alone nor in combination with DDC caused such an increase in fluorescence intensity. Therefore, it is concluded that an intracellular oxidative stress does not mediate the effect of DDC in this system. Fox and Leingang (1998) demonstrated that the inactivation of NF-
B by NAC in LPS-stimulated Kupffer cells might involve a regulatory protein synthesis rather than the inhibition of oxidative stress. Thus, there may be alternative factors which activate NF-
B.
Glutathione disulfide status plays a critical role in the regulation of NF-B activation (Dröge et al., 1994
). DDC is known to interact with thiols and to deplete intracellular GSH (Kelner and Alexander, 1986
). However, this action seems to occur at concentrations greater than 1 mM. In preliminary experiments, DDC (< 30 µM) had no significant effect on the concentration of GSH in isolated Kupffer cells (unpublished data).
Recently, there have been reports regarding the early sequence of intracellular signaling events following stimulation with LPS. These are upstream to the activation of gene transcription. It has been reported that in Kupffer cells the production of TNF- and NO by LPS might involve the activation of protein kinase C (PKC) (Bankey et al., 1990
; Spitzer, 1994
). More recently, Lichtman et al. (1998) reported that the production of TNF-
by LPS-stimulated Kupffer cells might be mediated by 2 parallel pathways, one consisting of endocytosis, endocytic processing, and a rise of free intracellular calcium, and the other, as an independent tyrosine kinase pathway. In addition, these investigators mentioned that activation of PKC by LPS was a third independent pathway. Izumi et al. (1994) demonstrated that the action of DDC on superoxide production might be through PKC. Moreover, PKC pathways may link to NF-
B activation via inactivation of I
B (Baeuerle and Henkel, 1994
). In our study, the PKC inhibitor H-7 prevented NO and TNF-
production by both LPS alone and LPS + DDC. On the other hand, the cAMP-dependent protein kinase inhibitor, H-8, had no significant effect on the actions of LPS or DDC + LPS. PMA, a direct PKC activator, also enhances LPS stimulation of Kupffer cells (Bankey et al., 1990
). Thus, as described by Lichtman et al. (1998) multiple cellular mechanisms exist that link LPS to the production of NO and TNF-
. One of these, the PKC signaling pathway, may be a critical target for DDC. Additional studies are needed to examine the effect on signaling events upstream of PKC.
In conclusion, DDC enhanced the production of NO and TNF- by LPS-stimulated Kupffer cells. NAC and H-7 inhibited the enhancement caused by DDC. DDC caused an increase in the translocation of activated NF-
B to the nucleus of LPS-stimulated Kupffer cells. Intracellular oxidative stress associated with activation of NF-
B was not detected by treatments with either LPS or DDC. These findings demonstrate that DDC may affect a PKC signaling pathway and may trigger other key signaling events for LPS-stimulated NO and TNF-
production.
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ACKNOWLEDGMENTS |
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NOTES |
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2 Present address: Idun Pharmaceuticals, Inc., La Jolla, CA 92037.
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