Background
Hepatic encephalopathy (HE) is a neurological disorder that presents in chronic and acute forms. Chronic HE is a neuropsychiatric disorder which commonly occurs in the setting of alcoholic cirrhosis and is associated with changes in personality, altered mood, decline in the intellectual capacity and abnormal muscle tone [
1]. Acute HE (acute liver failure, ALF) usually occurs following viral-mediated hepatitis, acetaminophen toxicity, and exposure to other hepatotoxins. ALF often presents with the abrupt onset of delirium, seizures and coma and has a high mortality rate (80-90%) [
2]. A major component of ALF is the development of brain edema leading to increased intracranial pressure and brain herniation, ultimately resulting in death [
3]. There is currently no satisfactory treatment for the edema in ALF except for an emergency liver transplantation [
4].
The preponderance of evidence indicates that the brain edema in ALF is "cytotoxic", i.e., due to astrocyte swelling [
5‐
9]. It has traditionally been considered that ammonia represents a major etiological factor in the development of neurological abnormalities associated with severe liver failure, including cytotoxic brain edema/astrocyte swelling [
6,
10]. Astrocytes primarily detoxify ammonia by converting glutamate into glutamine through a reaction mediated by glutamine synthetase, an enzyme predominantly present in astrocytes [
11]. Accordingly, several studies document that a pathophysiological concentration of ammonia results in astrocyte swelling in culture [
12‐
14], brain slices [
15,
16] and
in vivo models of hyperammonemia [
6,
17,
18]. The mechanism by which ammonia and the subsequent production of glutamine leads to astrocyte swelling/brain edema is incompletely understood, but appears to involve the development of oxidative/nitrosative stress, the mitochondrial permeability transition and activation of mitogen activated protein kinases. For review, see [
19].
In addition to ammonia, emerging evidence suggests that proinflammatory cytokines, likely derived from liver necrosis and/or sepsis (the latter a common complication in ALF) play an important role in brain edema formation in ALF. In support of this view, blood levels of TNF-α, IL-1β and IL-6 were found elevated in patients with ALF who had concurrent infections [
20‐
22]. Additionally, induction of endotoxemia was shown to exacerbate brain edema in an experimental model of hyperammonemia [
23].
While the above studies strongly suggest the involvement of inflammatory cytokines in the brain edema of ALF, the effect of various cytokines on astrocyte swelling has thus far not been investigated. The present study therefore examined the effect of various inflammatory cytokines on astrocyte swelling, as well as investigated the potential additive/synergistic interactions between ammonia and cytokines on cell swelling in cultured astrocytes.
We recently documented that exposure of cultured astrocytes to a pathophysiological concentration of ammonia results in the activation (nuclear translocation) of the transcriptional factor NF-κB and that inhibition of such activation caused a reduction in ammonia-induced astrocyte swelling [
24]. As cytokines are also well-known to activate NF-κB, we examined whether ammonia and cytokines exert additive/synergistic effects in the activation of NF-κB and whether such activation contributes to potential additive/synergistic effects on astrocyte swelling. Our studies show that a mixture of cytokines cause cell swelling in cultured astrocytes, and that sensitization of cultures with ammonia prior to treatment with cytokines caused a marked potentiation of both the activation of NF-κB and astrocyte swelling.
Discussion
This study demonstrates that a combination of inflammatory cytokines, including TNF-α, IL-1β, IL-6 and IFN-γ, as well as cytokines given individually, can induce astrocyte swelling. Simultaneous co-treatment of astrocytes with a pathophysiological concentration of ammonia and cytokines failed to elicit any additive/synergistic effect. However, treatment (24 h) of astrocyte cultures with ammonia prior to exposure to a mixture of cytokines for an additional 24 h resulted in a marked potentiation of astrocyte swelling. Treatment of cultures with a combination of cytokines, or cytokines given individually, resulted in the activation of NF-κB. Additionally, we observed that sensitization of cultures with ammonia before exposure to a mixture of cytokines resulted in a marked increase in the activation of NF-κB, similar to that observed with cell swelling. Treatment of cultures with BAY 11-7082, an inhibitor of NF-κB, completely blocked the potentiation effects of cell swelling of astrocytes sensitized by ammonia and subsequently treated with cytokines. Altogether, these data indicate that ammonia and cytokines independently cause the activation of NF-κB and astrocyte swelling, and that pretreatment of cultures with ammonia sensitizes these cells to the effect of cytokines, resulting in a marked increase in both NF-κB activation and cell swelling, indicating a critical role of NF-κB in the potentiation of astrocyte swelling.
While ammonia continues to play a prominent role in the mechanism of brain edema in ALF, the findings of this study on the role of cytokines in the mechanisms of astrocyte swelling are highly pertinent and add to a growing body of evidence suggesting that inflammation and inflammatory cytokines also contribute to the brain edema in ALF [
32‐
36]. The generation of cytokines is a consequence of extensive liver necrosis as well as infection/sepsis which frequently complicates ALF [
20,
37]. Additionally, lipopolysaccharide, a well-known inducer of inflammation [
38], was shown to exacerbate brain edema in an experimental model of hyperammonemia [
23]. Our observations on the marked potentiation of cell swelling by priming of cells with ammonia followed by treatment with cytokines in cultured astrocytes correspond well with the above clinical findings.
The present study also showed that TNF-α, IL-1β, or IL-6 given individually induces astrocyte swelling. Following treatment with these cytokines, astrocyte swelling was observed at 6 h and persisted for up to 12 h; however, by 24 h no swelling was detected. These findings suggest that cell swelling produced by cytokines is an early but transient event. Consistent with our results, one study documented that treatment of cultured astrocytes with TNF-α for 48 h did not affect astrocyte cell volume [
39]. Our findings are also consistent with a recent report showing that in transgenic mice deficient in TNF-α, IL-1β and IL-6 receptors, ALF results in a lesser degree of brain edema as compared to levels achieved in wild-type mice [
40]. To the best of our knowledge, this study documents for the first time that various inflammatory cytokines cause cell swelling in cultured astrocytes.
In contrast to TNF-α, IL-1β and IL-6, IFN-γ caused cell swelling which persisted for up to 24 h. The reason for the different pattern of swelling observed with IFN-γ is not known. The effect of IFN-γ, however, is at variance with a recent report noting that mice deficient in IFN-γ receptors do not show a reduction in brain edema following the induction of ALF [
40]. While the reason for the differences regarding the role of IFN-γ in cell swelling/brain edema is not known, studies by Bemeur et al [
40] noted that brain levels of IFN-γ were not elevated in wild-type mice after the administration of azoxymethane, a model of ALF that results in death within 16-18 h. By contrast, using a rat model of ALF induced by the hepatotoxin thioacetamide, which displays a more protracted clinical course (death occurring at 72-80 h), we found a modest increase (32%) in IFN-γ in brain, while the levels of TNF-α, IL-1β were elevated by 100-140% (unpublished observations). It is possible that differences in models of ALF, particularly in the rate of clinical progression and the degree of severity of liver failure, may be critical in the differential effects relative to the role of IFN-γ in the brain edema associated with ALF.
The present study additionally showed that a mixture of all four cytokines resulted in astrocyte swelling; however, the degree of swelling caused by a combination of cytokines did not differ from that exerted by individual cytokines. The reason for the absence of a potential additive effect on cell swelling when cytokines were added as a mixture is not clear. It is possible that negative interactions among cytokines may have precluded an additive effect. Consistent with this view, it has been shown that treatment of cultured astrocytes with TNF-α and IFN-γ showed no additive effects on the expression of complement (C3) genes, although individually these cytokines significantly enhanced C3 gene expression [
41]. Additionally, IFN- γ has been shown to antagonize the effect of TNF-α on the production of chemokines in human astrocytes [
42].
A notable finding of this study was that treatment of cultures with ammonia for 24 h prior to treatment with a mixture of cytokines for additional 24 h resulted in a marked potentiation of astrocyte swelling. By contrast, co-treatment with ammonia and cytokines did not result in such potentiation on astrocyte swelling. These data suggest that ammonia sensitizes astrocytes to the effect of cytokines, thereby resulting in a potentiation of cell swelling. This pattern parallels the clinical findings in ALF whereby the initial hyperammonemia is often followed by an inflammatory response as a consequence of liver necrosis and/or sepsis [
32,
33].
The mechanism by which prior sensitization of cultures with ammonia followed by treatment with a mixture of cytokines results in a marked increase in astrocyte swelling is not well understood. It is, however, reasonable to suggest that sensitization of cultures with ammonia facilitates a process by which cytokines potentiate their effects on astrocyte swelling. Pertinent to this view, it was recently demonstrated that exposure of cultured astrocytes to ammonia results in the activation of NF-κB as early as 12 h. Such activation appears to be mediated by oxidative/nitrosative stress (ONS), and activation of mitogen activated protein kinases (MAPKs) [
24]. It is therefore possible that the initial activation of NF-κB by ammonia (24 h ammonia sensitization) promotes a synergistic effect on NF-κB activation after the addition of cytokines. This sequence is plausible as cytokines are well-known to activate NF-κB [
31,
43‐
45].
A parallel pattern observed between cell swelling and NF-κB activation supports the proposed key role of NF-κB in such astrocyte swelling. Thus, the time-course of activation of NF-κB by TNF-α, IL-1β and IL-6, IFN-γ, correlate with the time-course of astrocyte swelling (Figures
1 and
6). Likewise, activation of NF-κB by a mixture of cytokines resulted in a commensurate increase in astrocyte swelling. Further, the observation that BAY 11-7082, an inhibitor of NF-κB, completely diminished the synergistic effect on astrocyte swelling produced by cytokines after prior-sensitization of cultures with ammonia, adds credence to the key role of NF-κB in synergism on cell swelling.
While the time-course of cell swelling and that of NF-κB activation by cytokines were similar, some inconsistencies were noted. Thus, while simultaneous co-treatment of cultures with ammonia and a mixture of cytokines continued to show astrocyte swelling, such treatment unexpectedly resulted in a significant reduction in NF-κB activation as compared to cultures treated with ammonia or with a mixture of cytokines (Figure
5). The reason for this discordance is not known. However, it is possible that the presence of ammonia during the course of cytokine treatment might have interfered with the ability of cytokines to activate NF-κB. It should be noted that this condition is very different from the ammonia pretreatment protocol, as by the time cytokines were added, all of the ammonia had been metabolized and was no longer present in the culture media (data not shown).
We also observed that exposure of cultures to ammonia prior to treatment with a mixture of cytokines caused a marked activation of NF-κB (428%), while the increase in astrocyte swelling was of lesser magnitude (129%). The disparity between these two events is probably due to the fact that the extent of swelling observed (129%) represents the maximal swelling capacity for astrocytes, as we previously documented that maximal increase in cell volume in cultured astrocytes incubated in hypoosmotic media was 120% ([
45] and references therein).
Precisely how NF-κB contributes to astrocyte swelling is not clear. NF-κB is known to activate various genes, including inducible nitric oxide synthase (iNOS) and NADPH oxidase (NOX) [
31], whose products nitric oxide, superoxide and peroxynitrite have been shown to cause astrocyte swelling [
46‐
48]. Additionally, NF-κB is known to activate phospholipase A2 (PLA2) as well as cycooxygenase-2 (COX-2) [
31], the products of which include arachidonic acid and prostaglandin E2, metabolites capable of inducing astrocyte swelling [
49]. Consistent with this view, recent studies have also shown that ammonia activates iNOS, NOX, PLA2 and COX-2 and that inhibition of these enzymes diminished ammonia-induced astrocyte swelling [
24,
50‐
52]. Nevertheless, the precise pathway(s) by which NF-κB activation contributes to astrocyte swelling remains to be established.
Competing interests
The authors declare that they have no conflicts of interest either financially or non-financially in this study.
Authors' contributions
KVR, ARJ, performed major parts of the experiments and XY and VMA assisted in some of the experiments. KVR, ARJ and MDN designed the experiments, analyzed the data and wrote the manuscript. All authors read and approved the final version of the manuscript.