Marked potentiation of cell swelling by cytokines in ammonia-sensitized cultured astrocytes

Abstract

Background: Brain edema leading to high intracranial pressure is a lethal complication of acute liver failure (ALF), which is believed to be cytotoxic due to swelling of astrocytes. In addition to the traditional view that elevated levels of blood and brain ammonia are involved in the mechanism of brain edema in ALF, emerging evidence suggests that inflammatory cytokines also contribute to this process. We earlier reported that treatment of astrocyte cultures with a pathophysiological concentration of ammonia (5 mM NH4Cl) resulted in the activation of nuclear factor-kappaB (NF-κB) and that inhibition of such activation diminished astrocyte swelling, suggesting a key role of NF-κB in the mechanism of ammonia-induced astrocyte swelling. Since cytokines are also well-known to activate NF-κB, this study examined for additive/synergistic effects of ammonia and cytokines in the activation of NF-κB and their role in astrocyte swelling.

Methods: Primary cultures of astrocytes were treated with ammonia and cytokines (TNF-α, IL-1, IL-6, IFN-γ, each at 10 ng/ml), individually or in combination, and cell volume was determined by the [3H]-O-methylglucose equilibration method. The effect of ammonia and cytokines on the activation of NF-κB was determined by immunoblots.

Results: Cell swelling was increased by ammonia (43%) and by cytokines (37%) at 24 h. Simultaneous co-treatment with cytokines and ammonia showed no additional swelling. By contrast, cultures pretreated with ammonia for 24 h and then exposed to cytokines for an additional 24 h, showed a marked increase in astrocyte swelling (129%). Treatment of cultures with ammonia or cytokines alone also activated NF-κB (80-130%), while co-treatment had no additive effect. However, in cultures pre-treated with ammonia for 24 h, cytokines induced a marked activation of NF-κB (428%). BAY 11-7082, an inhibitor of NF-κB, completely blocked the astrocyte swelling in cultures pre-treated with ammonia and followed by the addition of a mixture of cytokines.

Conclusion: Our results indicate that ammonia and a mixture of cytokines each cause astrocyte swelling but when these agents are added simultaneously, no additive effects were found. On the other hand, when cells were initially treated with ammonia and 24 h later given a mixture of cytokines, a marked potentiation in cell swelling and NF-κB activation occurred. These data suggest that the potentiation in cell swelling is a consequence of the initial activation of NF-κB by ammonia. These findings provide a likely mechanism for the exacerbation of brain edema in patients with ALF in the setting of sepsis/inflammation.

Figure 1

Time course of astrocyte cell volume following treatment with individual cytokines (TNF-α, IL-1β, IL-6 and IFN-γ). Cultures were treated with 10 ng/ml concentration of each cytokine. * vs. Control, p < 0.05. Values in each group are mean ± S.E.M of 5 individual culture plates taken from 3 separate seedings (n = 15).

Figure 2

Time course of astrocyte cell volume following treatment with a mixture of cytokines (TNF-α, IL-1β, IL-6 and IFN-γ, 10 ng/ml each). * vs. Control, p < 0.05. CKs, cytokines. Values in each group are mean ± S.E.M of 5 individual culture plates taken from 3 separate seedings (n = 15).

Figure 3

Effect of a co-treatment of ammonia and a mixture of cytokines on cell volume (24 h) in cultured astrocytes. * vs. Control, p < 0.05. Values in each group are mean ± S.E.M of 4 individual culture plates taken from 3 separate seedings (n = 12).

Figure 4

Synergistic effects on astrocyte cell volume by a pre-treatment of cultures (sensitization) with ammonia followed by treatment with a mixture of cytokines on astrocyte cell volume (24 h). * vs. Control, p < 0.05; ** vs. CKs and NH₄, p < 0.05. Values in each group are mean ± S.E.M of 5 individual culture plates taken from 3 separate seedings (n = 15).

Figure 5

Effect of a co-treatment of ammonia and a mixture of cytokines on the activation (nuclear translocation) of NF-κB in cultured astrocytes. * vs. Control, p < 0.05; ** vs. CKs and NH₄, p < 0.01. Values in each group are mean ± S.E.M of 3 individual culture plates taken from 3 separate seedings (n = 9).

Figure 6

Time course of NF-κB activation following treatment with individual cytokines (TNF-α, IL-1β, IL-6 and IFN-γ, 10 ng/ml each). * vs. Control, p < 0.05. Values in each group are mean ± S.E.M of 3 individual culture plates taken from 3 separate seedings (n = 9).

Figure 7

Synergistic effects on NF-κB activation by a 24 h treatment of cultures with ammonia followed by treatment with mixture of cytokines on astrocyte cell volume (24 h). * vs. Control, p < 0.05; ** vs. CKs and NH₄, p < 0.01. Values in each group are mean ± S.E.M of 3 individual culture plates taken from 3 separate seedings (n = 9).

Figure 8

Effect of inhibition of NF-κB activation by BAY 11-7082 on astrocyte cell volume (24 h) induced by a mixture of cytokines and ammonia. * vs. Control, p < 0.05; ** vs. CKs; NH₄ and NH₄ + CKs, p < 0.05. Values in each group are mean ± S.E.M of 5 individual culture plates taken from 3 separate seedings (n = 15).