Macrophages are present throughout the human body and play important roles in the regulation of oxidative stress and inflammation.1,2 Oxidative stress and inflammation act as cooperative and synergistic partners in the pathophysiology of numerous diseases such as cancer, diabetes, obesity and cardiovascular and neurological disorders.3,4 Acetylsalicylic acid (ASA, aspirin), a specific inhibitor of cyclooxygenase (COX) enzyme and a commonly used anti-inflammatory drug, has multiple pharmacological effects which may not be associated with its COX-inhibitory activity.5,6 Lipopolysaccharide (LPS) is a component of Gram-negative bacterial endotoxin, which penetrates cells and induces inflammatory and other toxic responses.7,8 LPS induces generation of reactive oxygen species (ROS), reactive nitrogen species (RNS), proinflammatory cytokines and prostaglandins (PGs) in macrophages and other cells.9,10
Our previous studies on murine macrophage J774.2 cells and human hepatoma HepG2 cells, treated with either acetaminophen or aspirin, have indicated increased oxidative stress and mitochondrial dysfunction, accompanied by increased apoptosis.11–13 Although aspirin is considered to be a specific inhibitor of PG synthesis, its effect on COX is still controversial in different cell lines,14,15 and its broad pharmacological effects may be associated with the regulation of redox (reduction–oxidation) metabolism, cell signalling and mitochondrial functions. We, therefore, have studied the effects of LPS stimulation using murine macrophage J774.2 and human hepatoma HepG2 cells in conjunction with ASA. Results from these studies suggest that toxicological responses towards LPS stimulation and ASA treatment are more pronounced in macrophages than in HepG2 cells.
Materials and methods
Aspirin, LPS, malondialdehyde, nicotinamide adenine dinucleotide phosphate (NADPH) and thiobarbituric acid were purchased from Sigma (St Louis, MO, USA). 2,7-Dichlorofluorescein diacetate (DCFDA) was purchased from Molecular Probes Inc. (Eugene, OR, USA). Kits for the nitric oxide assay were procured from R&D Systems (Minneapolis, MN, USA) and those for aconitase from Oxis International Inc. (Portland, OR, USA). The apoptosis detection kit for flow cytometry was from BD Pharmingen (BD Biosciences, San Jose, CA, USA). Murine macrophage J774.2 cells were purchased from the European Collection of Cell Cultures (Health Protection Agency Culture Collections, Salisbury, UK) and HepG2 cells from the American Type Culture Collection (Manassas, VA, USA). Polyclonal antibodies against beta-actin, nitric oxide synthase (iNOS), COX-2 and caspase-3 were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), aconitase-2 from Abcam (Cambridge, MA, USA) and caspase-8 from Cell Signaling Technology Inc. (Boston, MA, USA). Reagents for electrophoresis and Western blot analyses were purchased from Bio-Rad Laboratories (Richmond, CA, USA).
Cell culture and treatments
J774.2 cells were grown in poly-l-lysine-coated 75-cm2 flasks (≈2.0–2.5 × 106 cells/ml) in Dulbecco’s modified Eagle medium (DMEM) supplemented with 2 mM glutamine and 10% heat-inactivated fetal bovine serum in the presence of 5% CO2–95% air at 37°C. HepG2 cells were grown in poly-l-lysine-coated 75-cm2 flasks (≈2.0–2.5×106 cells/ml) in DMEM supplemented with 1% non-essential amino acids, 2 mM glutamine and 10% heat-inactivated fetal bovine serum in a humidified incubator in the presence of 5% CO2–95% air at 37°C. Cells were cultured to 80% confluence and treated with 1 µg/ml Escherichia coli LPS for 24 hours. In some cases, the cells were treated with 5 mM ASA for 24 hours with or without LPS. Cells were harvested after the treatments and cellular fractions, mitochondria and postmitochondrial supernatant (PMS) were used for further analyses, as described in our previous study.12
Measurement of reactive nitrogen species, reactive oxygen species and lipid peroxidation
J774.2 macrophages and HepG2 cells (1–5 × 105 cells/well) were cultured in six-well plates for 24 hours prior to LPS treatment. In some experiments, cells were also treated with 5 mM ASA (as described above). Nitric oxide (NO) production was determined by measuring the concentration of total nitrite in the culture supernatants with Griess reagent (R&D Systems Inc.) according to the vendor’s protocol.
The intracellular production of ROS was measured spectrofluorimetrically in the mitochondria and total homogenates from ASA/LPS-treated and untreated J774.2 and HepG2 cells by using the cell-permeable probe DCFDA, which preferentially measures peroxides.
NADPH-dependent membrane lipid peroxidation in the cell lysates from ASA/LPS-treated and untreated J774.2 and HepG2 cells was measured as thiobarbituric acid-reactive substances (TBARS) using malonedialdehyde as standard, as described in our previous research.12,13
Measurement of aconitase
Mitochondrial aconitase activity was measured by the NADPH-coupled conversion of citrate to isocitrate in the presence of isocitrate dehydrogenase using the Bioxytech Aconitase-340 assay kit (Oxis International Inc.) described in our previous study.13 Aconitase activity was expressed as the rate of formation of NADPH determined at 340 nm.
Measurement of apoptosis
The annexin V externalization assay for apoptosis was performed using flow cytometry after treating J774.2 and HepG2 cells with LPS/ASA, as described in the vendor’s protocol (BD Pharmingen). Briefly, untreated cells and cells treated with LPS/ASA from 60–70% confluent plates were trypsinized, washed in phosphate-buffered saline (PBS) and resuspended (1 × 106 cells/ml) in binding buffer [10 mM HEPES (hydroxyethyl piperazineethanesulfonic acid), pH 7.4, 140 mM NaCl, 2.5 mM CaCl2]. A fraction (100 µl/1 × 105 cells) of the cell suspension was incubated with 5 µl annexin V conjugated to fluorescein isothiocyanate (FITC) and 5 µl propidium iodide (PI) for 15 minutes at 25°C in the dark. Binding buffer (400 µl) was added to the suspension and apoptosis was measured immediately using a Becton Dickinson FACScan analyser as described in our previous research.13 The apoptotic cells were estimated as the percentage of cells that stained positive for annexin V(AV)-FITC while remaining impermeable to PI (AV+/PI−). This method also distinguished viable cells (AV−/PI−) and cells undergoing necrosis (AV+/PI+).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis
Proteins (50–100 µg) from the different subcellular fractions of control and treated J774.2 and HepG2 cells were separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)16 and electrophoretically transferred onto a nitrocellulose membrane by Western blotting.17 Transferred proteins were checked by reversible Ponceau S staining for equal loading and then probed with primary antibodies against aconitase, COX-2, iNOS, caspase-3 and caspase-8. Immunoreactive bands were visualized using the appropriate conjugated secondary antibodies. Equal loading of protein was confirmed using beta-actin as loading control. After development of the blots, the bands were visualized and further densitometric analysis was performed using the Typhoon FLA 9500 system (GE Healthcare, Uppsala, Sweden) and expressed as relative intensity (RI) compared with the untreated control.
Values shown are expressed as mean ± standard error of the mean (SEM) of three individual experiments. Statistical significance of the data was assessed using SPSS software Version 21 (SPSS Inc., Chicago, IL, USA) by analysis of variance followed by Dunnett’s post-hoc analysis. P-values ≤ 0.05 were considered statistically significant.
Effects of lipopolysaccharide and acetylsalicylic acid on oxidative and nitrosative stress
Figure 1 shows the differential effects of LPS alone or in combination with ASA on NO production in macrophages and HepG2 cells. In macrophages, a threefold increase in NO production was observed with LPS alone. Treatment with ASA alone caused approximately a twofold increase in NO production. A combination of LPS and ASA resulted in about a fourfold increase in NO production in J774.2 cells. However, a moderate increase (10–15%) in NO production was observed in HepG2 cells when treated with LPS and ASA alone, or a combination of both.
Reactive oxygen species production (Figure 2) in isolated mitochondria from macrophages and HepG2 cells was significantly increased after treatment with LPS and ASA alone, or a combination of both. This pattern was also observed when total ROS production was measured in a whole cell lysate.
Figure 3 shows an increased lipid peroxide production after treatment with LPS and ASA alone or a combination of both. The increase in lipid peroxidation (LPO) was more pronounced in macrophages than in HepG2 cells.
Mitochondrial matrix enzyme, aconitase is a sensitive marker of mitochondrial stress. Aconitase activity was inhibited by more than 70% in macrophages treated with LPS and ASA alone (Figure 4). However, only 20–30% inhibition was observed in HepG2 cells. This indicates that J774.2 cells are more sensitive to LPS and ASA treatment than HepG2 cells. A combination of LPS and ASA treatment also resulted in significant inhibition of aconitase activity in both cell systems.
Effects of lipopolysaccharide and acetylsalicylic acid on apoptosis
Figure 5 shows approximately 22% and 18% apoptotic cell death in macrophages after treatment with LPS and ASA respectively. A combination of LPS and ASA further increased the apoptotic cell death rate to 37%. However, no significant increase in apoptosis was observed in HepG2 cells treated with LPS alone (Figure 6) and only a moderate increase (15–19%) in apoptosis was observed in HepG2 cells treated with ASA alone or in combination with LPS. These results again indicate that macrophages are more sensitive to LPS treatment than hepatocytes.
Effects of lipopolysaccharide and acetylsalicylic acid on the expression of redox markers and apoptotic proteins
Figure 7 shows a marked decrease in the expression of aconitase enzyme protein after treatment with LPS in macrophages in comparison with HepG2 cells. Expression of COX-2 and iNOS was markedly increased in macrophages after treatment with LPS alone. Treatment with ASA alone, or in combination, also resulted in increased expression of these proteins in macrophages compared with HepG2 cells. Interestingly, an increased expression of the apoptotic marker protein caspase-3 was observed in macrophages after treatment with LPS while the expression in HepG2 cells was reduced. However, expression of caspase-8 was markedly increased in HepG2 cells after treatment with LPS but no significant increase was observed in macrophages. These results have confirmed an increased sensitivity of macrophages towards LPS treatment and also suggest that apoptosis in these cells is mediated by different mechanisms.
Circulating or resident macrophages are important immune-responsive cells and have multiple roles in attenuating or augmenting the pathophysiology of the host.18 Pathogen- and drug-induced cytotoxicity and tissue injury are the major challenges in clinical and experimental pharmacology. The endotoxin LPS is released on infection from the gastrointestinal fauna and enters the bloodstream and other tissues, such as the liver, which also regulate the inflammatory and toxic responses towards LPS exposure.19 Therefore, we have investigated the oxidative/nitrosative stress and associated complications of LPS and ASA alone or in combination on macrophages and HepG2 cells. Our previous study on acetaminophen showed increased mitochondrial and oxidative stress in J774.2 cells.11 Using HepG2 cells, our previous studies also provided evidence that ASA induces cell cycle arrest and mitochondrial dysfunction, accompanied by apoptosis.12,13 There is extensive evidence that inflammatory, pharmacological and toxicological properties of non-steroidal anti-inflammatory drugs (NSAIDs) are regulated by macrophage-produced cytokines and liver cells and that there are cooperative roles of inflammation and oxidative stress in the pathogenesis of disease.3,20,21 Acetaminophen has also been shown to act in synergy with LPS in the production of proinflammatory cytokines in murine macrophages.22 We have observed an increase in NO and ROS production by LPS in cells, but more so in macrophages, and have also shown that ASA has synergistic effects when applied in conjunction with LPS. Both mitochondrial and extramitochondrial ROS production appear to cooperatively increase after treatment with LPS. Lu et al.7 observed that enhanced oxidative and nitrosative stress by LPS are potentiated by alcohol and cytochrome P450 2E1 induction, which enhanced ROS production both in vitro and in vivo. LPS and ASA treatment alone or in combination has also resulted in increased LPO and inhibition of ROS-sensitive mitochondrial aconitase. Previously we reported the inhibition of aconitase activity by ASA treatment in HepG2.13 The result of increased apoptotic cell death by LPS alone, or more so in conjunction with ASA, especially in macrophages, has supported the evidence that increased mitochondrial oxidative and nitrosative stress trigger the apoptotic events in macrophages and HepG2 cells. Western blot analysis of the expression of COX-2, iNOS and aconitase enzyme expression has also confirmed this increased stress in macrophages. The expression of terminal caspase-3 enzyme appears to be more markedly induced by LPS in macrophages than in HepG2 cells. Increased expression of caspase-8 by LPS treatment in HepG2 cells, but not in macrophages, suggests the involvement of different pathways to induce apoptosis in macrophages and HepG2 cells.
Increased oxidative and nitrosative stress accompanied by increased lipid peroxidation and apoptosis were observed in cells treated with LPS. Macrophages appeared to be more sensitive to LPS treatment than HepG2 cells when treated with LPS either alone or in combination with ASA. Acetylsalicylic acid treatment appears to have synergistic effects for sensitizing macrophages in response to LPS treatment. Therefore, a substantial variability in responsiveness towards pathogen/toxicants and drugs in different cellular populations can be associated with specific physiological and pathological outcomes.