Inflammatory signalling pathways involved in astroglial activation by unconjugated bilirubin

Abstract

During neonatal hyperbilirubinaemia, astrocytes activated by unconjugated bilirubin (UCB) may contibute to brain toxicity through the production of cytokines. As a first step in addressing the signal transduction cascades involved in the UCB-induced astroglial immunological response, we tested whether tumour necrosis factor (TNF)-a receptor 1 (TNFR1), mitogen-activated protein kinase (MAPK) and nuclear factor jB (NF-jB) would be activated in astrocytes exposed to UCB, and examined the profile of cytokine production. Astrocyte cultures stimulated with UCB showed a rapid rise in TNFR1 protein levels, followed by activation of the MAPKs p38, Jun N-terminal kinase1/2 and extracellular signal-regulated kinase1/2, and NF-jB. Interestingly, the induction of these signal effectors preceded the early up-regulation of TNF-a and interleukin (IL)-1b mRNAs, and later secretion of TNF-a, IL-1b and IL-6. Treatment of astrocytes with UCB also induced cell death, with levels comparable to those obtained after exposure of astrocytes to recombinant TNF-a and IL-1b. Moreover, loss of cell viability and cytokine secretion were reduced when the NF-jB signal transduction pathway was inhibited, suggesting a key role for NF-jB in the astroglial response to UCB. These results demonstrate the complexity of the molecular mechanisms involved in cell injury by UCB during hyperbilirubinaemia and provide a basis for the development of novel therapeutic strategies.

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“…In addition, pro-inflammatory cytokines could further promote apoptosis. For example, there are studies certifying that TNF-␣ or IL-1␤ activates the apoptosis pathway (10,26,27). The current study demonstrated that UCB might up-regulate or directly interact with the cell surface TNF receptor 1 and IL-1 receptor 1 as a ligand, similar to TNF-␣ and IL-1␤.…”

“…The current study demonstrated that UCB might up-regulate or directly interact with the cell surface TNF receptor 1 and IL-1 receptor 1 as a ligand, similar to TNF-␣ and IL-1␤. Intriguingly, this could induce NF-B activation and subsequently result in cell death and cytokine release; in turn, the increased secretion of TNF-␣ and IL-1␤ will again bind to TNF receptor 1 and IL-1 receptor 1, respectively, thus forming a vicious pathogenic cycle created by inflammation, which would exacerbate the inflammation and cell death (10,27,41). Therefore, many researchers have attempted to inhibit cytokine production as a therapeutic target for preventing bilirubin-mediated neurotoxicity.…”

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“…27 Incubation of astrocytes with 50 mM UCB and 100 mM HSA resulted in a biphasic activation of TNFR1 during the 24 h observation period. 25 The early phase was detected as early as 15 min, peaking at 1 h, and the late phase occurred at 12 h. Although the early-phase activation may result from a direct interaction of UCB with the TNFR1, we speculate that the secondary activation may be attributed to the action of IL-1b and IL-6 produced during astrocyte exposure to UCB (Figure 1), which are known to upregulate TNFR1. 28 Downstream from the UCB-induced expression of TNFR1, a rapid and transient activation of p38 (1-2 h), JNK1/2 (1-4 h) and ERK1/2 (2 h) was observed, with nuclear translocation of NF-kB occurring from 1 h onward with a maximal localization in the nuclei of astrocytes of B12% at 4 h, 25 which was also observed for 100 mM UCB (unpublished data).…”

“…24 In our earlier report 4 we have shown that UCB induces an immunological response in astrocytes, even after a short treatment of 4 h. For longer incubation periods, secretion of TNF-a and IL-1b reached a maximum at 12 h with a sustained elevation at 24 h after UCB addition. 25 The release of IL-6 was suppressed in the first 4 h of astrocyte incubation with UCB. However, for prolonged astroglial exposure to UCB, IL-6 showed a maximum production at 18 h. In these conditions, incubation of astrocytes with 100 mM UCB in the presence of the same amount of HSA for 24 h led to a marked increase in necrosis (B40%) and apoptosis (B16%).…”

“…26 Interestingly, TNF-a and IL-1b at the concentrations released by astrocytes exposed to UCB was shown to induce loss of cell viability. 25 Production and release of cytokines depend on inducible gene expression mediated by activation of cell signaling. Primary inflammatory stimulus may act through membrane receptors such as the TNF-a receptor 1 (TNFR1), which cause the activation of four major intracellular signaling pathways that are cascades of protein kinases, namely the three distinct mitogen-activated protein kinase (MAPK) pathways, p38, Jun-NH 2 -terminal kinase 1 and 2 (JNK1/2) and extracellular signal-regulated kinase 1 and 2 (ERK1/2), and the one leading to activation of the nuclear factor kB (NF-kB).…”