In this study, we showed that when the response to proinflammatory cytokines is inhibited, hepatocellular responses to hypoxia, oxidative stress, heat shock, and apoptosis remain intact after pretreatment with CM-LPS. The similar responses to stressors in pretreated and unpretreated hepatocytes were seen both in primary rat hepatocytes and McArdle cells.
McArdle cells (RH-7777) produced a uniform and reliable cell culture model for primary rat hepatocytes. McArdle (RH-7777) is a transformed rat hepatoma cell line that retains intact membrane receptors and lipoprotein metabolism, which makes these cells ideal for studying hepatic lipoprotein metabolism (20
). Studies in our laboratory have shown that McArdle cells can be grown into spheroid aggregates in the same manner as primary rat hepatocytes, and that after pretreatment with CM-LPS complexes, these cells exhibit cytokine tolerance in exactly the same time- and dose-dependency pattern as primary hepatocytes (unpublished data).
Oxidative stress is one of the most challenging conditions to cell survival. Although many other compounds are capable of inducing oxidative stress, oxygen is the most ubiquitous oxidant that cells are exposed to (22
). Many of the cellular enzymes, structural and membrane proteins, simple and complex sugars, intracellular signaling pathways, and DNA or RNA complexes are susceptible to oxidative damage (23
). Antioxidant compounds such as vitamins C and E, ubiquinone, and uric acid, are the first line of defense against oxidative stress. However, when the stress exceeds certain levels, cells adapt in a way to reduce the extent of the damage. Hydrogen peroxide at a concentration of 120 to 150 μM induces a transient adaptation to oxidative stress in many cells (24
). Similarly in this study, we found that H2
induces a transient but significant reduction in the mitochondrial activity of McArdle hepatocytes at 2 and 4 h after the exposure, and that mitochondrial activity returns to normal levels at 8 h. Cells pretreated with CM-LPS showed a pattern and magnitude of reduced mitochondrial activity that was similar to that of unpretreated cells, indicating that pretreatment with CM-LPS had no effect on the cellular response to oxidative stress induced by H2
In this study, we used mitochondrial activity as a good indicator of hepatocellular response to oxidative stress, instead of measuring the ratio of the reduced to the oxidized form of glutathione (GSH/GSSG). Measurement of GSH and GSSG levels as indicators of oxidative injury in hepatocytes is complicated by the fact that there is shuttling of glutathione, particularly GSSG, out of the cells, resulting in fluctuations in this ratio (25
). We have similarly found that oxidative injury incurred by H2
stimulation did not elevate GSSG levels or change the GSH/GSSG ratio in hepatocytes (unpublished data).
Oxygen homeostasis is critically important for all mammalian cells, which should maintain the oxygen concentration within the tight constraints essential for life. Induction of HIF-1 has been shown to be a central mechanism of oxygen-mediated gene expression in most cells, including hepatocytes (26
). As a member of the PAS superfamily of transcription factors, HIF-1 is a heterodimer of α and β subunits (27
). The β subunits, also known as hydrocarbon receptor nuclear translocators, are constitutively expressed in the nucleus. When oxygen tension is lowered, HIF-1 α subunits stabilize and translocate to the nucleus, where they dimerize with the β subunit. The HIF-1 complex then binds to the hypoxia response elements and activates transcription of a range of genes. In this study, we have clearly shown that 200 μM of CoCl2
induced the nuclear translocation of the HIF-1α subunits (). Although we have previously shown that NF-κB activation is inhibited following pretreatment with CM-LPS (9
), nuclear translocation of HIF-1 α subunits remains similar in pretreated and unpretreated cells, showing that pretreatment with CM-LPS has no effect on this hepatocellular response to hypoxia.
Exposure of cells to a sublethal increase in temperature activates a cellular stress response that renders the cells more resistant to subsequent lethal insults, a process called thermotolerance (28
). Thermotolerance is associated with the synthesis and accumulation of a family of highly conserved proteins referred to as heat-shock proteins (HSP), which are grouped according to their molecular weight. Within the family of heat-shock proteins, we chose three different ones: HSP 25, HSP 32, and HSP 72, to study the hepatocellular response to heat shock following pretreatment with CM-LPS. One of the major functions of heat-shock proteins is acting as a molecular chaperone for protein maturation and integrity (29
). However, they can also play a more specific role, such as maintaining the redox state of the cell (31
). We studied the induction of these heat-shock proteins to get an overall illustration of the heat stress and shock response in hepatocytes. Subjecting both unpretreated and CM-LPS pretreated hepatocytes to sublethal heat shock resulted in an identical level of expression of all three heat-shock proteins examined. Similarly, cells from both treatment groups were equally capable of adopting a thermotolerant phenotype. These findings indicate that pretreating hepatocytes with CM-LPS does not affect their capacity to initiate a heat shock response or to adopt a thermotolerant phenotype.
Apoptosis, or programmed cell death, is a genetically regulated mechanism. It has been shown to be a highly controlled mechanism involving death factors and death receptors, and is similar to mechanisms that determine cell growth and proliferation (32
). TNF-α is a potent inducer of apoptosis, signaling through the TNFR1 receptor (33
). TNF-α mediated apoptotic signaling leads to cleavage and activation of the apoptotic enzyme caspase 8, which, through a cascade of events, in turn cleaves and activates caspase 3. Active caspase 3 subunits break down the intracellular proteins that are mainly responsible for cellular proliferation and regulation of the cell cycle, leading to the fragmentation of DNA that is characteristic of apoptosis. In our study, pretreating hepatocytes with CM-LPS complexes did not result in any changes in the activity of either caspase 3 or caspase 8 when compared to unpretreated cells. These findings indicate that although CM-LPS pretreated cells are less responsive to the stimulatory effects of proinflammatory cytokines, the cellular response to other TNF-mediated processes remains unaltered.
Hepatocytes are hard to stimulate. Multiple proinflammatory cytokines are required to induce an inflammatory response in hepatocytes through a synergistic activity (34
). Although TNF-α and IL-1β have different cell membrane receptors and intracellular pathways, they eventually merge on phosphorylation and activation of the IKK complex (35
). Activation of the IKK complex, and specifically the IKK-β subunit of this complex, will lead to activation and intranuclear localization of the NF-κB heterodimeric complex. In the nucleus, this transcriptional protein promotes the gene activity of many proinflammatory responses, including NO production. In our study, a combination of proinflammatory cytokines (cytomix, TNF-α, IL-1β, and IFN-γ) could induce a detectable inflammatory response in hepatocytes in terms of NO production. Pretreating these cells with CM-LPS resulted in significant reduction in the amount of NO production. Although the amount of overall NO production was lower in hepatocytes stimulated with a combination of TNF-α and IL-1β, the inhibitory effect of CM-LPS was still evident in pretreated cells. Stimulating the hepatocytes with IL-1β or IFN-γ individually also resulted in a much lower amount of NO production, and no tolerance was observed in the pretreated cells. These two findings suggest that pretreatment has no effect on the proinflammatory activity of these two cytokines. However, TNF-α alone could not induce any detectable NO production in the primary rat hepatocytes.
To have a more direct study on the effect of TNF-α on hepatocytes and a better understanding of the possible mechanism of cytokine tolerance, we created a stable cell line of McArdle cells containing a NF-κB reporter plasmid. Even a small stimulation of the NF-κB pathway by proinflammatory cytokines could be amplified by the plasmid and generate an appropriate light luciferase response. The transformed McArdle cells were pretreated with CM-LPS and were stimulated with TNF-α and IL-1β individually or in combination. We found that when TNF-α was used alone or in combination, inhibition of NF-κB activity was observed in pretreated cells. This finding suggest that pretreatment with CM-LPS inhibits the intracellular signaling pathway of TNF-α and blocks the synergistic activity of this cytokine. Interestingly, while the proinflammatory response of the TNF-α is blocked there is no change in the proapoptotic activity in pretreated cells. This observation further indicates that the effect of CM-LPS on cytokine mediated intracellular signaling is independent of TNF-induced apoptotic signaling.
Throughout this study, we examined the cellular response to a variety of strong stressors in an attempt to understand how CM-LPS complexes affect hepatocytes. Our choice of stimulants was based on a variety of different stressors that could produce a strong and reliable cellular response through different mechanisms. Although, each of these stressors can be analyzed by different techniques or groups of assays, in this study, by carefully selecting key proteins and assays, we tried to use the most reliable and acceptable indicators of the hepatocellular response to these stressors.
In conclusion, increased circulating triglyceride levels may provide a protective role in sepsis, and cytokine tolerance is likely a means of negatively regulating the cytokine-mediated hepatic response to infection. As this study shows, induction of cytokine tolerance is a selective process, and this phenomenon is due to blocking of the TNF-α signaling pathway and disruption of the synergistic activity of the proinflammatory cytokines on hepatocytes. Although the hepatocellular response to proinflammatory cytokines is inhibited after pretreatment with CM-LPS complexes, hepatocytes are still capable of responding to many other cellular stressors in a normal manner. In addition, this study shows that McArdle cells are a suitable cell culture model for the study of cytokine tolerance in hepatocytes.