Abstract
There is extensive evidence that pro-inflammatory cytokines produced by macrophages are involved in toxicity induced by drugs such as acetaminophen (APAP). We investigated the effect of subtoxic concentrations of acetaminophen in conjunction with bacterial lipopolysaccharide (LPS) on the expression of the pro-inflammatory cytokines TNFα and IL-1β using the mouse macrophage cell line RAW264.7 as a model. APAP alone induced in a dose-dependent manner the production of TNFα and IL-1β in this cell line. When LPS was added to APAP-treated cells, the increase in TNFα and IL-1β production observed was higher than the sum of cytokine amounts produced with each agent alone, suggesting a synergistic mechanism. Moreover, we found that p38MAPK, JNK, and ERK were activated by APAP or LPS alone or in association. In our model, the NFκB signaling pathway was not involved in cytokine production induced by APAP. When inhibiting MAPKs using pharmacological inhibitors, we showed that p38MAPK inhibition abrogated the synergistic effect of APAP and LPS found for TNFα production but not for IL-1β production. JNK and ERK have comparable roles in the production of the cytokines. Furafylline, a CYP1A inhibitor, and indomethacin, a PGHS inhibitor, exhibited a significant inhibitory effect on TNFα and IL-1β production induced by the APAP and LPS combination. This work suggests that in macrophages, APAP and LPS can synergistically provoke the induction of pro-inflammatory cytokines, an effect involving the MAPK pathway and APAP bioactivation by CYP and PGHS.
| Abbreviations: | ||
| APAP: | = | acetaminophen |
| LPS: | = | Lipopolysaccharide |
| TNFα: | = | Tumor Necrosis Factor-α |
| IL-1β: | = | Interleukin-1β |
| MAPK: | = | Mitogen-Activated Protein Kinase |
| JNK: | = | c-Jun N-terminal Kinase |
| ERK: | = | Extracellular signal-Regulated Kinase |
| NF-κB: | = | Nuclear Factor-κB |
| DON: | = | deoxynivalenol |
| PGHS: | = | Prostaglandin H synthase |
Introduction
Acetaminophen (APAP) is a widely used analgesic and antipyretic agent. It is also a dose-related toxin that can cause fulminant liver failure when taken in massive overdose (James et al., Citation2003). APAP-induced liver injury is not due to the drug itself but to the formation of N-acetyl-p-benzoquinone imine (NAPQI), a toxic metabolite generated through the cytochrome P-450 (CYP) drug-metabolizing system (James et al., Citation2003). Human CYP3A4 is the major CYP isoenzyme responsible for APAP bioactivation at therapeutic doses, whereas CYP2E1 and CYP1A2 are significantly involved at higher plasma levels (Bessems and Vermeulen, Citation2001). In mice, CYP2E1 is probably the most important CYP isoenzyme involved in the bioactivation of APAP at low doses, whereas CYP1A2 contributes to the bioactivation and toxicity of APAP at high doses (Hu et al., Citation1993; Snawder et al., Citation1994). APAP can also be metabolized by prostaglandin H synthase (PGHS) in tissues with low CYP activity, such as the kidney (Pirmohamed et al., Citation1996); for review (Bessems and Vermeulen, Citation2001).
Several authors have reported that APAP toxicity is associated with the release of several pro-inflammatory cytokines, such as tumor necrosis factor (TNF, -α) and interleukin (IL, -1β) in mice liver or plasma (Blazka et al., Citation1995, Citation1996; Bourdi et al., Citation2002; Gardner et al., Citation2002, Citation2003; James et al., Citation2003). Moreover, the use of neutralizing antibodies directed against TNFα has been shown to delay the hepatic necrosis and inflammation caused by APAP in animal models (Blazka et al., Citation1995, Citation1996). Resident macrophages in the liver (Kupffer cells) are known to have a central role in the production of inflammatory cytokines in response to APAP treatment. Indeed, inactivation or depletion of these cells causes a considerable reduction in the hepatic damage induced by agents such as APAP in animal models.
Lipopolysaccharide (LPS) is a major constituent of the outer cell wall of Gram-negative bacteria and certainly one of the most potent microbial inducers of inflammation. In macrophages, interaction of LPS with Toll-like receptor–4 (TLR-4) triggers cell activation, leading to production of several inflammatory mediators such as the pro-inflammatory cytokines TNFα and IL-1β. TLR-4 engagement results in activation of a multitude of signalling events, including the activation of both the Mitogen-Activated Protein Kinases (MAPK) and the Nuclear Factor-κB (NF-κB) pathways. The MAPK signalling pathways consist of a series of kinases that are sequentially activated and consequently phosphorylate downstream kinases and transduce extracellular stimuli into intracellular responses. MAPK family includes p38MAPK, c-Jun N-terminal Kinase (JNK), and Extracellular signal-Regulated Kinase (ERK). One of the major functions of MAPK is the activation of transcription factors, several of which bind to the promoters of pro-inflammatory cytokines (Baldassare et al., Citation1999; Zhu et al., Citation2000). LPS has been shown to act synergistically with APAP in the production of pro-inflammatory cytokines in co-cultures of rat hepatocytes and resident liver macrophages (Tukov et al, Citation2006).
The RAW264.7 murine macrophage cell line is widely used as a macrophage cell model to study inflammatory processes (Wong et al., Citation1998; Jung and Sung, Citation2004; Schildknecht et al., Citation2006). Pestka et al. used RAW264.7 as a model to study the effect of deoxynivalenol (DON), a trichothecene mycotoxin also called vomitoxin, on mRNA expression and pro-inflammatory cytokines production, and on the potential interactive effect with LPS. DON was shown to up-regulate inflammatory mediators such as TNFα, IL1-β, and IL-6; these effects were potentiated by LPS (Chung et al., Citation2003). These data suggested that LPS and DON might induce MAPK pathways leading to pro-inflammatory cytokine expression.
The objective of this work was to evaluate the interaction of APAP and LPS on TNFα and IL-1β production using the murine macrophage cell line RAW264.7 as a model. The effect of APAP alone or in combination with LPS on the production of the pro-inflammatory cytokines TNFα and IL-1β was evaluated at both the protein and mRNA levels. The activation of MAPK and NF-κB signal transduction pathways were also investigated.
Materials and methods
Chemicals and reagents
LPS derived from E. coli serotype 026:B6 (1.5 × 106 EU/mg), acetaminophen (APAP), furafylline, and indomethacin were obtained from Sigma-Aldrich (St. Quentin Fallavier, France). The p38 MAPK inhibitor SB203580, the MEK1 inhibitor PD98059, (which is up-stream of ERK 1/2), and the JNK inhibitor SP600125 were obtained from Calbiochem (VWR Internationals, Fontenay-sous-Bois, France).
Cells and treatment
The murine macrophage RAW264.7 cell line was obtained from the American Tissue Culture Collection (ATCC, Rockville, MD). Cells were maintained at 37°C in a 5 % CO2 humidified incubator in Dulbecco’s Modified Eagle’s Medium (DMEM, Biowhittaker Co., Fontenay sous Bois, France) supplemented with 10 % (v/v) heat-inactivated fetal calf serum (GibcoBRL, Life Technologies, Cergy Pontoise, France), and 1% (v/v) penicillin and streptomycin antibiotic solution (all from GibcoBRL).
For mRNA induction studies, cells (2.5 × 105 cells/ml) were incubated in 6-well flat-bottomed plates (Fisher Bioblock Scientific Co, Illkirch, France) in a volume of 2 ml for 24 hr. Supernatant was replaced with medium containing LPS (E. coli, 0.1 or 1 ng/ml), APAP (1 mM), or LPS (0.1 or 1 ng/ml) + APAP (1 mM). Cells were incubated for indicated times and total RNA was extracted and analyzed by Real-Time Polymerase Chain Reaction.
For TNFα and IL-1β production studies, cells (2.5 × 105 cells/ml) were cultured for 24 hr in 12-well tissue culture plates (Fisher Scientific Co.) with each well containing 1 ml of cell suspension. Supernatant was replaced with medium containing LPS (E. coli, 0.1 or 1 ng/ml), APAP (1 mM), or LPS (0.1 or 1 ng/ml) + APAP (1 mM). For studies using inhibitors, fresh medium containing inhibitors, e.g., MAPK inhibitors [SB 203580 (1 μM), SP600125 (20 μM), PD 98059 (30 μM)], CYP inhibitor [furafylline (30 μM)], or PGHS inhibitor [indomethacin (50 μM)], was added 1 hr before treatment with LPS (E. coli, 0.1 ng/ml), APAP (1 mM), or LPS (0.1 ng/ml) + APAP (1 mM). After 8 or 24 hr incubation, supernatants were collected and analyzed for cytokine production by ELISA.
For MAPK studies, cells (2.5 × 105 cells/ml) were incubated in 6-well dishes (Fisher Scientific Co.) in a volume of 2 ml for 24 hr. Supernatant was replaced with medium containing LPS (E. coli, 0.1 or 1 ng/ml), APAP (1 mM), or LPS (0.1 or 1 ng/ml) + APAP (1 mM). Cells were incubated for indicated times and then lysed for assay for MAPK phosphorylation.
Real-time reverse transcription-polymerase chain reaction
After treatment, cells were washed in cold phosphate- buffered Saline (PBS) before RNA extraction with Qiagen Rneasy Mini Kit according to the manufacter’s protocol (Qiagen, Courtaboeuf, France). The amount of RNA recovered was measured by spectrophotometry. cDNA was synthesized by reverse a transcriptase from 5 μg total RNA using a High-capacity cDNA archive Kit (Applied Biosystems, Foster City, CA). Murine TNFα and IL-1β and Cytochrome P450 CYP 2E1, CYP 1A2, USA and CYP 3A11 (CYP3A4 for human) mRNAs were tested relative to eukaryotic 18S rRNA. Primers were purchased as Assays-on-Demand (Applied Biosystems, Mm00443258, Mm00434228, Mm00491127, Mm00487227, Mm00731567 and Hs99999901). Real-Time PCR was performed using the ABI 7700 Sequence Detection System (Applied Biosystems) according to the manufacturer’s instructions. The Taqman Universal PCR Master Mix was used during PCR amplification (Applied Biosystems) in a 20 μL reaction volume. Amplification reactions were carried out using the following temperature profiles: 50°C, 2 min; 95°C, 10 min; 95 °C, 15 sec; and 60°C, 1 min for 40 cycles. Fluorescence emission was detected for each PCR cycle, and the threshold cycle (CT) values were determined.
The CT value was defined as the actual PCR cycle when the fluorescence signal increased above the background threshold. Results were expressed as 2−ΔCt. ΔCt corresponds to (Ct studied gene – Ct reference gene).
ELISA assays
Supernatants were collected at 8 or 24 hr, stored at -80°C, and then assayed for TNFα and IL-1β by ELISA using a Quantikine mouse set (R&D systems, Lille, France) according to the manufacter’s instructions. Cells were lysed and protein concentration was determined. Cell proteins were dosed using the Pierce method. Limits of quantification announced by the manufacturers were less than 5 and 3 pg/ml for TNFα and IL-1β, respectively. Results were expressed as pg/ml/μg protein. At least two different wells were analyzed on the same day for each experimental condition. The data presented correspond to the mean of these replicates. The variability of cytokine levels measured in separate wells for the same experimental conditions on the same day was below 20%. At least two independent experiments performed on different days were conducted for each experimental condition.
Western blotting analyses
RAW264.7 cells (2.5 × 105/ml) were exposed to LPS alone (0.1 or 1 ng/ml), APAP alone (1 mM) or a combination of LPS and APAP for the indicated periods of time. Cells were washed in cold PBS before lysis in 50 μl of lysis buffer (20 mM Tris [pH 7.4], 137 mM NaCl, 2 mM EDTA [pH 7.4], 1% Triton, 25 mM β-glycerophosphate, 1 mM Na3VO4, 2 mM sodium pyrophosphate, 10% glycerol, 1 mM PMSF, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). The homogenates were centrifuged at 15,000 rpm for 20 min at 4°C. Equal amounts of denatured proteins were separated over 12% SDS-PAGE gels and transferred on to PVDF membranes (Amersham Biosciences, Les Ulis, France). Membranes were then incubated with antiobodies raised against the phosphorylated forms of p38 MAPK, JNK 1/2, or ERK1/2 for MAPK pathway (all from Cell Signaling Technology, Ozyme, St-Quentin en Yvelines, France), or with antibody directed against IκBα for the NF-κB pathway (IκBα C-21, Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands were detected by chemiluminescence (ECL solution, Amersham Biosciences). Stripping of membranes was performed to analyze total p38 MAPK expression as a loading control with an antibody raised against p38 MAPK (p38 N20, Santa Cruz Biotechnology). The ratio (P-protein/p38 MAPK)treated / (P-protein/p38 MAPK)control was calculated using the imagequant software.
Statistical analysis
All data are represented as mean ± standard deviation of the mean. Differences between the chemical treated cells were evaluated by an unpaired Student’s t-test. *p < 0.05 was considered to be statistically significant. All tests were performed using the software GraphPad Instat 3.
Results
Effect of APAP on pro-inflammatory cytokine production
The production of the pro-inflammatory cytokines TNFα and IL-1β was assessed in the murine macrophages cell line RAW264.7 treated with APAP using ELISA assays. Results showed that when RAW264.7 cells were treated for 24 hr with APAP, TNFα and IL-1β were secreted in the medium in a concentration-dependent fashion (), without cell toxicity (data not shown). No significant increase in pro- inflammatory cytokines in the medium was detected after 8 hr with concentrations up to 1 mM APAP. TNFα was increased with 24 hr of treatment at 0.5 mM and 1 mM APAP (7.5X and 10.7X average fold-induction for 0.5 mM and 1 mM, respectively) (). IL-1β production was detected after 24 hr of treatment at 0.5 mM or 1 mM APAP (). The dose of 1 mM, which induced the stronger production of each cytokines at 24 hr, was then use for the continuation of the present work.
Figure 1. APAP treatment of the murine macrophage cell line RAW264.7 induced the production of TNFα and IL-1β. RAW264.7 cells were treated with APAP (0.02 - 1.00 mM) for 8 or 24 hr. Supernatants were collected and analysed for cytokine production by ELISA. Values are expressed here as pg/ml/μg protein. Data represent two independent experiments (□ experiment 1, ▪ experiment 2). NT: not treated.
Effect of the APAP and LPS combination on pro-inflammatory cytokine production
RAW264.7 cells were incubated with APAP or LPS alone, or with a combination of APAP and LPS, for 8 or 24 hr. LPS alone induced TNFα and IL-1β secretions after 8 hr and 24 hr of treatment in a dose-dependent fashion (). For TNFα, the maximum of production was observed after 8 hr of treatment with LPS and after 24 hr with APAP, whereas IL-1β secretion reached a maximum after 24 hr of LPS or APAP treatment. The combination of APAP and LPS enhanced pro-inflammatory cytokine production at both 8 hr and 24 hr. IL-1β secretion was the most markedly affected as compared to TNFα, with a clear synergistic effect of APAP and LPS. shows that the production of cytokines was almost always higher with the combination APAP and LPS, as compared to the sum of cytokines production obtained with APAP or LPS alone, suggesting that APAP and LPS acted in synergy on TNFα and IL-1β production. Although the relative effects of various treatments with respect to control were consistently reproducible, it was noted that the secretion of a cytokine under a given condition may vary by several-fold over repeated experiments. For example, IL-1β production in response to APAP 1 mM for 24 hr was approximately 50–60 pg/ml/μg protein in the APAP experiment shown in whereas the IL-1β response to APAP was 15 pg/ml/μg protein in another experiment (see ).
Figure 2. Effect of LPS on TNFα and IL-1β production induced by APAP in RAW264.7 cells. RAW264.7 cells were treated with APAP alone (1 mM), LPS (0.1 or 1.0 ng/ml) alone, or with APAP and LPS for 8 or 24 hr. Supernatants were collected and analysed for cytokine production by ELISA. Values (expressed as pg/ml/μg protein) are mean ± SD of the three independent experiments shown in .
Table 1. Effect of LPS on TNFα and IL-1β production induced by APAP.
Effect of APAP and LPS combination on pro-inflammatory cytokine mRNA expression
The expression of TNFα and IL-1β mRNA was measured in RAW 264.7 cells, treated with APAP or LPS, alone or in combination, by Real-Time RT-PCR. Results showed that a basal level of TNFα mRNA was detectable in these cells whereas this was not the case for IL-β (). LPS dose- dependently induced both TNFα and IL-1β mRNA expression with a maximum reached after 2 hr of treatment. APAP alone induced a very weak increase in close up TNFα and IL-1β mRNA expression at all the timepoints measured. When RAW264.7 cells were incubated with APAP in association with LPS, TNFα and IL-1β mRNA levels were not different compared to that induced by LPS treatment alone.
Figure 3. LPS and APAP alone or in combination modulate TNFα and IL-1β mRNA expression in RAW264.7 cells. Treatment of RAW264.7 cells with APAP alone (1 mM), LPS (0.1 or 1.0 ng/ml) alone or with APAP and LPS was performed for 2, 4, or 6 hr. mRNAs were extracted and measured by Real-Time RT-PCR using specific primers. Data are expressed as 2−ΔCt and are representative of two independent experiments. * Untreated; □ LPS; ○ APAP; ▪ APAP and LPS.
Activation of MAP kinases by APAP and LPS
To investigate the implication of MAPK in TNFα and IL-1β synthesis induced after APAP treatment (alone or in association with LPS) the phosphorylations of p38MAPK, ERK1/2, and JNK1/2 were measured in RAW 264.7 cells by Western blotting. LPS-Induced phosphorylation of p38MAPK was maximal at 30 min and was markedly reduced at 1 and 2 hr (). In the same way, LPS activation of ERK1/2 and JNK1/2 peaked at 30 min and then diminished at 1 and 2 hr after treatment. Cell treatment with APAP induced the phosphorylation of p38MAPK, JNK1/2 and ERK1/2 after 30 min of treatment that was still present up to 8 hr after the initiation of APAP treatment (). P38MAPK phosphorylation was more markedly affected by APAP compared to JNK and ERK. When RAW264.7 cells were treated with APAP and LPS, the level of p38MAPK phosphorylation observed was higher than the level of phosphorylation observed for APAP or LPS treatment alone, whereas this was not obvious for JNK and ERK. The effect on p38MAPK phosphorylation was more evident following 1 and 2 hr of treatment. At later treatment time, MAPK induced-phosphorylation by LPS + APAP was equivalent to the one observed for APAP alone.
Figure 4. Effect of LPS on APAP-induced phosphorylation of MAPK in RAW264.7 cells. (A) RAW264.7 cells were treated with APAP (1 mM) alone, LPS (0.1 ng/ml) alone, or an APAP and LPS combination for up to 8 hr and lysed for assay of MAPK phosphorylation. (B) RAW264.7 cells were treated with APAP (1 mM) alone, LPS (1 ng/ml) alone, or an APAP and LPS combination for up to 8 hr and lysed for assay of MAPK phosphorylation. Whole cell lysates were subjected to Western blotting using antibodies specific for the phosphorylated forms of p38MAPK, ERK1/2, JNK1/2. An anti-p38 MAPK antibody was used as a loading control. Numbers under each image correspond to the ratio (P-protein/p38 MAPK) treated/(P-protein/p38 MAPK) control. Results are representative of two independent experiments.
Effect of the APAP and LPS combination on the NFκB signaling pathway
To investigate the implication of the NFκB pathway in TNFα and IL-1β expression induced after APAP treatment (alone or in association with LPS) in the RAW264.7 cell line, the activation of NFκB was monitored through the degradation of IκBα by Western blotting. No degradation of IκBα was observed after APAP treatment, showing that in our conditions, the NFκB pathway was not involved in APAP-related effects on cytokine production at any timepoint studied (). When RAW264.7 cells were treated with LPS alone, degradation of IκBα was observed at 30 min of treatment. At later times of LPS incubation, IκBα was not degraded, showing evidence for a rapid and transitional activation of the NFκB pathway. The same result was observed when cells were incubated with the APAP and LPS combination.
Figure 5. Effect of the APAP and LPS combination on the NF-κB signaling pathway in RAW264.7 cells. A. RAW264.7 cells were treated with APAP (1 mM) alone, LPS (0.1 ng/ml) alone, or an APAP and LPS combination for up to 8 hr and lysed for assay of IκBα degradation by Western blotting. B. RAW264.7 cells were treated with APAP (1 mM) alone, LPS (1 ng/ml) alone, or an APAP and LPS combination for up to 8 hr and lysed for assay of IκBα degradation. Whole cell lysates were subjected to Western Blotting using antibodies specific for IκBα. An anti-p38 MAPK antibody was used as a loading control. Numbers under each image correspond to the ratio (IκBα/p38 MAPK) treated/(IκBα/p38 MAPK) control. Results are representative of two independent experiments.
Effect of MAPK inhibitors on cytokine production
To evaluate the role of MAPK in APAP and LPS-induced cytokine production, RAW 264.7 cells were pre-treated for 1 hr with the p38MAPK inhibitor SB203580 (1 μM), or the JNK inhibitor SP600125 (20 μM), or the ERK inhibitor PD98059 (30 μM) and then treated with LPS, APAP, or the combination of APAP and LPS for 24 hr. Results showed that TNFα produced by APAP treatment was strongly dependent on ERK and JNK, whereas p38MAPK inhibition had no effect (). By contrast, p38MAPK played a major role in IL-1β production after APAP treatment; JNK or ERK inhibition also affected IL-1β production but to a lesser extent. When both APAP and LPS were added, all the inhibitors decreased TNFα production at a level similar with LPS alone in the presence of inhibitors. Same results were obtained for IL-1β. Interestingly, inhibition of p38MAPK abolished the synergy between APAP and LPS for TNFα production, whereas inhibition of p38MAPK did not diminish TNFα production after APAP or LPS treatment alone.
Figure 6. MAPK inhibitors modulated TNFα and IL-1β production induced by APAP, LPS, or APAP and LPS in RAW264.7 cells. After 1 hr-pre-treatment with SB 203580 at 1 μM (
) or PD98059 at 30 μM (▪), APAP (1 mM) or LPS (0.1 ng/ml) or APAP and LPS were added to culture medium for a supplementary 24 hr. Supernatants were analyzed for cytokine production by ELISA. Values (expressed as pg/ml/μg protein) are mean ± SD of three independent experiments.
Effect of the APAP and LPS combination on CYP mRNA expression
The expression of CYP 2E1, CYP 1A2, and CYP 3A11 mRNAs was measured in RAW 264.7 cells treated for 2, 4, and 6 hr with APAP or LPS (alone or in combination) by Real Time RT-PCR. Results showed that no basal levels of CYP2E1 and CYP3A11 mRNA were detectable in RAW264.7 cells (data not shown). When RAW264.7 cells were incubated with APAP in association with LPS, CYP1A2 mRNA levels were increased compared to LPS or APAP treatment alone ().
Figure 7. Murine CYP1A2 mRNA expression (A) and CYP1A2 inhibitor modulation of cytokine production (B) by APAP, LPS, or APAP and LPS in RAW264.7 cells. (A) CYP1A2 mRNAs were measured in cells treated with APAP alone (1 mM), LPS (0.1 ng/ml) alone, or with APAP and LPS for 2, 4, or 6 hr. mRNAs were extracted and measured by Real-Time RT-PCR using specific primers. Data are expressed as 2−ΔCt and are representative of two independent experiments. * Untreated; □ LPS; ○ APAP; ▪ APAP and LPS. (B) After a 1 hr-pre-treatment with furafylline (□) at 30 μM or medium (▪), APAP (1 mM), LPS (0.1 ng/ml), or APAP and LPS were added to culture medium for a supplementary 24 hr. Supernatants were analyzed for cytokine production by ELISA. Values (expressed as pg/ml/μg protein) are mean ± SD of three independent experiments. *p < 0.05 vs fura value.
Effect of CYP and PGHS inhibitors on cytokine production
To evaluate the role of CYP-dependent APAP-bioactivation in APAP, and LPS-induced cytokine production, RAW 264.7 cells were pre-treated for 1 hr with the CYP1A inhibitor furafylline (30 μM) and then treated with LPS, APAP or the combination of APAP and LPS for 24 hr. Results showed that TNFα and IL-1β produced by APAP treatment were significantly inhibited by furafylline (). By contrast, furafylline had no inhibitory effect on cytokine production induced by LPS. When both APAP and LPS were added, furafylline decreased TNFα and IL-1β production to reach the level obtained when cells were treated with APAP alone. (ie. fura value.)
Other experiments were performed in order to evaluate the role of PGHS, another APAP bioactivation enzyme (). RAW264.7 were pre-treated 1 hr with indomethacin (50 μM) and then treated with LPS, APAP or the combination of APAP and LPS for 24 hr. Results showed that indomethacin reduced significantly TNFα and IL-1β production induced by the APAP and LPS combination, whereas it did not significantly modify cytokine production by APAP alone.
Figure 8. PGHS inhibitor modulated TNFα and IL-1β production induced by APAP and LPS in RAW264.7 cells. After a 1 hr-pre-treatment with indomethacine (□) at 50 μM or medium (▪), APAP (1 mM) or LPS (0.1 ng/ml) or APAP and LPS were added to culture medium for a supplementary 24 hr. Supernatants were analyzed for cytokine production by ELISA. Values (expressed as pg/ml/μg protein) are mean ± SD of three independent experiments. *p < 0.05 vs indo value.
Discussion
The present study was initiated by the observation that treatment of RAW 264.7 macrophages with APAP at concentrations ranging from 0.5 to 1 mM induced the production of the pro-inflammatory cytokines TNFα and IL-1β in the culture medium. Other authors have previously shown that treatment of co-culture systems using rat hepatocytes and resident liver macrophage cells incubated with APAP alone (1–10 mM) did not induce the secretion of TNFα (Milosevic et al., Citation1999; Tukov et al., Citation2006). Possible explanation for this discrepancy could include not only difference in cell types but also species differences between rat and mouses models. Our observation with APAP prompted us to further investigate the mechanism of action of APAP treatment alone and in association with LPS, mimicking signals provided by an infectious environment, on pro-inflammatory cytokine production in the RAW 264.7 macrophage cell line.
Our results showed that LPS co-treatment potentiated in a synergistic manner-pro-inflammatory cytokines production induced by APAP through MAPK activation but independently of the NFκB pathway, in RAW 264.7 cells. The effect on TNFα was observed as soon as 8 hr after treatment, whereas IL-1β modulation was observed 24 hr after treatment with APAP and LPS. However, when measuring TNFα and IL-1β mRNA expressions, our results did not evidence a synergy between APAP and LPS, suggesting that this effect did not occur at the transcriptional level. A synergistic effect of LPS on TNFα production was observed by other authors with the mycotoxin deoxynivalenol (DON) in RAW 264.7 cells (Wong et al., Citation1998). In PMA-differentiated U937 cells, the production of TNFα, IL-6, and IL-8 induced by DON and other 8-ketotrichothecenes was also superinduced by LPS at low doses (Sugita-Konishi and Pestka, Citation2001). Co-culture of rat hepatocytes and resident liver macrophage cells incubated for 6 hr with APAP and LPS also showed a synergy for TNFα production (Tukov et al., Citation2006).
Mitogen-activated protein kinases (MAPK) are proteins which transduce extracellular signals to intracellular responses in numbers of cells, through cascades of phosphorylations, and contribute to both activation of transcription and mRNA stability for cytokines and other pro-inflammatory genes (Johnson and Lapadat, Citation2002). In our model, APAP alone clearly induced MAPK activation as reflected in increased levels of P-p38MAPK, P-JNK and P-ERK observed in Western blots. Interestingly, MAPK phosphorylation occurred as soon as 30 min after APAP treatment and lasted for 8 hr. When cells were treated with both APAP and LPS, an increase in p38MAPK phosphorylation was found at 30 min compared to APAP or LPS alone. Up to now, few experiments have been performed to study MAPK activation after APAP treatment. Different authors have shown that JNK was activated after APAP treatment in vitro in different cellular model such as human hepatoma cells (Macanas-Pirard et al., Citation2005), murine hepatocytes (Matsumaru et al., Citation2003), and also rat glioma cells (Bae et al., Citation2001). Gunawan et al. showed that treatment of murine cultured hepatocytes with APAP induced a sustained activation of JNK as reflected by increased phospho-c-jun levels (Gunawan et al., Citation2006). Several mechanisms have been proposed for APAP-induced JNK phosphorylation including glutathione depletion resulting in alteration of thiol-disulfide redox status of the cell (Matsumaru et al., Citation2003; Gunawan et al., Citation2006). Oxidative stress with production of reactive oxygen species is also a possible mechanism since APAP metabolisation has been shown to generate reactive oxygen species (Jaeschke et al., Citation2003; James et al., Citation2003).
Our experiments with MAPK inhibitors indicated that TNFα production induced by APAP was strongly dependent on the JNK and ERK pathways, whereas IL-1β secretion was mainly dependent on p38MAPK and to a lesser extent on the JNK and ERK pathways. All inhibitors affected TNFα secretion after APAP and LPS co-treatment, with TNFα concentrations reaching the level measured in the presence of LPS and MAPK inhibitors. Moreover, p38MAPK inhibition had no effect on TNFα production induced by APAP or LPS, but reduced almost completely the synergistic effect of the APAP and LPS combination. So, p38MAPK in this model could play a major role in the synergy observed between APAP and LPS. As no clear increase of TNFα and IL-1β expression was observed at the mRNA level with the combination APAP and LPS as compared to APAP or LPS alone, it could be hypothesized that the synergistic effect for the production of pro-inflammatory cytokines following MAPK activation did not occur at the transcriptional level but at the translational/post-translational level.
APAP is known to be bioactivated in mice into reactive metabolites by several CYP isoenzymes (CYP2E1, CYP1A2, and CYP3A11) and by PGHS. We tested the hypothesis that APAP bioactivation mediated by CYP and/or PGHS could play a role in the synergistic cytokine production induced by APAP and LPS by incubating RAW 264.7 cells with specific inhibitors of CYP1A2 (furafylline) and PGHS (indomethacin). Only the CYP1A2 inhibitors were tested since only CYP1A2 mRNAs were detected in RAW264.7 cells and a synergistic increase of CYP1A2 mRNAs was observed when APAP was incubated in the presence of LPS. The inducible form of PGHS was found to be expressed in LPS stimulated RAW264.7 macrophages (Schildknecht et al., Citation2006). Our results showed that the CYP1A2 inhibitor furafylline and the PGHS inhibitor indomethacin significantly inhibited the production of TNFα and IL-1β upon treatment of RAW264.7 cells with APAP in the presence of the LPS. This suggests that potential APAP bioactivation mediated by CYP1A2 and by PGHS could play a role in the synergistic cytokine production induced by APAP and LPS. APAP bioactivation into a reactive metabolite has been shown to deplete glutathione and could thus result in JNK phosphorylation (Matsumaru et al., Citation2003; Gunawan et al., Citation2006) which is implicated in cytokine expression modulation (Johnson and Lapadat, Citation2002). The same mechanism seems to be involved in RAW264.7 cells; however, the precise mechanism by which the metabolic activation of APAP by CYP1A2 and PGHS mediates a synergistic effect on cytokine production remains unclear.
In conclusion, our results show for the first time that APAP can induce the production of the pro-inflammatory cytokines TNFα and IL-1β in the murine macrophage-like RAW264.7 cell line. Moreover, APAP and LPS act in synergy to enhance cytokine levels involving the MAPK, but not the NF-κB, signaling pathway. Further work is needed to better understand the mechanism of the synergistic effect observed with the combination of APAP and LPS in RAW264.7 cells, and involving the metabolic activation of APAP by CYP1A2 and PGHS.
Acknowledgements
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- Bae, M. A., Pie, J. E., and Song, B. J. 2001. Acetaminophen induces apoptosis of C6 glioma cells by activating the c-Jun NH(2)-terminal protein kinase-related cell death pathway. Mol. Pharmacol. 60:847–856.
- Baldassare, J. J., Bi, Y., and Bellone, C. J. 1999. The role of p38 mitogen-activated protein kinase in IL-1β transcription. J. Immunol. 162:5367–5373.
- Bessems, J. G., and Vermeulen, N. P. 2001. Paracetamol (acetaminophen)-induced toxicity: Molecular and biochemical mechanisms, analogues and protective approaches. Crit. Rev. Toxicol. 31:55–138.
- Blazka, M. E., Elwell, M. R., Holladay, S. D., Wilson, R. E., and Luster, M. I. 1996. Histopathology of acetaminophen-induced liver changes: Role of interleukin 1α and tumor necrosis factor-α. Toxicol. Pathol. 24:181–189.
- Blazka, M. E., Wilmer, J. L., Holladay, S. D., Wilson, R. E., and Luster, M. I. 1995. Role of pro-inflammatory cytokines in acetaminophen hepatotoxicity. Toxicol. Appl. Pharmacol. 133:43–52.
- Bourdi, M., Masubuchi, Y., Reilly, T. P., Amouzadeh, H. R., Martin, J. L., George, J. W., Shah, A. G., Pohl, L. R. 2002. Protection against acetaminophen-induced liver injury and lethality by interleukin 10: Role of inducible nitric oxide synthase. Hepatology 35:289–298.
- Chung, Y. J., Zhou, H. R., and Pestka, J. J. 2003. Transcriptional and posttranscriptional roles for p38 mitogen-activated protein kinase in up-regulation of TNFα expression by deoxynivalenol (vomitoxin). Toxicol. Appl. Pharmacol. 193:188–201.
- Gardner, C. R., Laskin, J. D., Dambach, D. M., Chiu, H., Durham, S. K., Zhou, P., Bruno, M., Gerecke, D. R., Gordon, M. K., and Laskin, D. L. 2003. Exaggerated hepatotoxicity of acetaminophen in mice lacking tumor necrosis factor receptor-1. Potential role of inflammatory mediators. Toxicol. Appl. Pharmacol. 192:119–130.
- Gardner, C. R., Laskin, J. D., Dambach, D. M., Sacco, M., Durham, S. K., Bruno, M. K., Cohen, S. D., Gordon, M. K., Gerecke, D. R., Zhou, P., and Laskin, D. L. 2002. Reduced hepatotoxicity of acetaminophen in mice lacking inducible nitric oxide synthase: potential role of tumor necrosis factor-α and interleukin-10. Toxicol. Appl. Pharmacol. 184:27–36.
- Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. 2006. c-Jun N-terminal kinase plays a major role in murine acetaminophen hepatotoxicity. Gastroenterology 131:165–178.
- Hu, J. J., Lee, M. J., Vapiwala, M., Reuhl, K., Thomas, P. E., and Yang, C. S. 1993. Sex-related differences in mouse renal metabolism and toxicity of acetaminophen. Toxicol. Appl. Pharmacol. 122:16–26.
- Jaeschke, H., Knight, T. R., and Bajt, M. L. 2003. The role of oxidant stress and reactive nitrogen species in acetaminophen hepatotoxicity. Toxicol. Lett. 144:279–288.
- James, L. P., Mayeux, P. R., and Hinson, J. A. 2003. Acetaminophen-induced hepatotoxicity. Drug Metab. Dispos. 31:1499–1506.
- Johnson, G. L., and Lapadat, R. 2002. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298:1911–1912.
- Jung, W. J., and Sung, M. K. 2004. Effects of major dietary antioxidants on inflammatory markers of RAW 264.7 macrophages. Biofactors 21:113–117.
- Macanas-Pirard, P., Yaacob, N. S., Lee, P. C., Holder, J. C., Hinton, R. H., and Kass, G. E. 2005. Glycogen synthase kinase-3 mediates acetaminophen-induced apoptosis in human hepatoma cells. J. Pharmacol. Exp. Ther. 313:780–789.
- Matsumaru, K., Ji, C., and Kaplowitz, N. 2003. Mechanisms for sensitization to TNF-induced apoptosis by acute glutathione depletion in murine hepatocytes. Hepatology 37:1425–1434.
- Milosevic, N., Schawalder, H., and Maier, P. 1999. Kupffer cell-mediated differential down-regulation of cytochrome P450 metabolism in rat hepatocytes. Eur. J. Pharmacol. 368:75–87.
- Pirmohamed, M., Madden, S., and Park, B. K. 1996. Idiosyncratic drug reactions. Metabolic bioactivation as a pathogenic mechanism. Clin. Pharmacokinet. 31:215–230.
- Schildknecht, S., Heinz, K., Daiber, A., Hamacher, J., Kavakli, C., Ullrich, V., and Bachschmid, M. 2006. Autocatalytic tyrosine nitration of prostaglandin endoperoxide synthase-2 in LPS-stimulated RAW 264.7 macrophages. Biochem. Biophys. Res. Commun. 340:318–325.
- Snawder, J. E., Roe, A. L., Benson, R. W., and Roberts, D. W. 1994. Loss of CYP2E1 CYP1A2 activity as a function of acetaminophen dose: Relation to toxicity. Biochem. Biophys. Res. Commun. 203:532–539.
- Sugita-Konishi, Y., and Pestka, J. J. 2001. Differential upregulation of TNFα, IL-6, and IL-8 production by deoxynivalenol (vomitoxin) and other 8-ketotrichothecenes in a human macrophage model. J. Toxicol. Environ. Health 64:619–636.
- Tukov, F. F., Maddox, J. F., Amacher, D. E., Bobrowski, W. F., Roth, R. A., and Ganey, P. E. 2006. Modeling inflammation-drug interactions in vitro: A rat Kupffer cell-hepatocyte coculture system. Toxicol. In Vitro 20:1488–1499.
- Wong, S. S., Zhou, H. R., Marin-Martinez, M. L., Brooks, K., and Pestka, J. J. 1998. Modulation of IL-1β, IL-6 and TNFα secretion and mRNA expression by the trichothecene vomitoxin in the RAW 264.7 murine macrophage cell line. Food Chem. Toxicol. 36:409–419.
- Zhu, W., Downey, J. S., Gu, J., Di Padova, F., Gram, H., and Han, J. 2000. Regulation of TNF expression by multiple mitogen-activated protein kinase pathways. J. Immunol. 164:6349–6358.