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Abstract
Among a myriad of pathogen-associated molecular pattern-sensing receptors, toll-like receptors (TLRs) are the principal core sensors of the host. Despite intensive studies for the expression of TLRs and their roles in the central nervous system, controversies remain regarding the expression and the function of TLR4 in human astrocytes. In order to clarify this issue, we attempted to verify functional expression of TLR4 in human astrocytes. Using Reverse transcription-polymerase chain reaction (RT-PCR), we confirmed that the human astrocytes express the TLR4 constitutively. To determine the function of TLR4, astrocytes were treated with TLR4 ligand or lipopolysaccharide (LPS), and then inflammatory cytokines expressions were checked using RT-PCR and enzyme-linked immunosorbent assay. Nuclear factor (NF)-κB activation was checked using electrophoretic mobility shift assay. Treatment of astrocytes with LPS increased tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 expression and induced NF-κB activation. Neutralizing anti-TLR4 antibody blocked the effect of LPS on cytokine production and NF-κB activation in astrocytes. The effect of LPS on cytokine production and NF-κB activation was shown in the presence of serum but not in the absence of serum. Therefore, we investigated the sensing mechanism of LPS in human astrocytes. Human astrocytes were treated with LPS following neutralizing anti-CD14 antibody treatment in the presence of serum. Neutralizing anti-CD14 antibody treatment abolished the effect of LPS on cytokine expression and NF-κB activation. Additionally, supplement of recombinant CD14 in serum-free media induced LPS effect on cytokine production and NF-κB activation. In these results, we showed that human astrocytes constitutively express functional TLR4 and require soluble CD14 to recognize LPS.
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Figure 1.
The expression of TLRs in human astrocytes (HA). Total RNA was isolated and RT-PCR for TLR1-9 was performed in the astrocytes and THP-1 cells (A). After LPS treatment for 7 h at variable dosages in HA, total RNA was isolated and RT-PCR for TLR2 was performed (B).
Figure 2.
The effect of LPS on cytokine production in astrocytes. The astrocytes were cultured in DMEM containing 10% serum. After LPS treatment for 6 h at variable dosages, total RNA was isolated and RT-PCR for TNF-α, IL-6 and GAPDH was per-formed (A). The astrocytes were incubated with LPS or 1 ng/ml of IL-1β for 24 h, and culture supernatants were then harvested. The supernatants were assayed for immunoreactive IL-6 (B) and IL-8 (C) using ELISA. ∗, p < 0.05
Figure 3.
The effect of LPS on cytokine expression in the absence of serum. The astrocytes were treated with LPS for 6 h in serum free DMEM. Total RNA was isolated and RT-PCR for TNF-α was performed (A). The cultures were incubated with LPS or 1 ng/ ml of IL-1β for 24 h in serum free media, and culture supernatants were then harvested. The supernatants were assayed for immunoreactive IL-6 (B) and IL-8 (C) using ELISA. ∗, p < 0.05
Figure 4.
The serum effect on NF-κB activation in LPS-treated astrocytes. Cells were incubated with LPS (100 ng/ml) in the presence or absence of serum. After incubation for the indicated amount of time, nuclear proteins were extracted and EMSA for NF-κB was performed.
Figure 5.
The effect of sCD14 supplementation on LPS-induced cytokine production in astrocytes. The cells were incubated with indicated doses of sCD14 and 10 ng/ml of LPS for 24 h in serum free media. The culture supernatants were harvested and assessed for IL-6 production using ELISA (A). The cells were treated with different concentrations of LPS or 10 ng/ml of IL-1β in the presence or absence of 0.1 μg/ml of sCD14. After incubation for 24 h, the culture supernatants were harvested and assessed for IL-6 production (B) or IL-8 production (C) using ELISA. ∗, p < 0.05
Figure 6.
The NF-κB activation induced by LPS treatment is dependent on sCD14. The cells were incubated with 10 ng/ml of LPS or 1 μg/ml of sCD14 or both for 30 min in serum free media. After nuclear extracts were harvested, EMSA for NF-κB was performed. (A). The cells were incubated with or without 5 μg/ml of neutralizing anti-CD14 antibodies in serum containing media for 1 h. Ten ng/ml of LPS was added. After incubation for 30 min, nuclear extracts were harvested, and EMSA for NF-κB was performed. (B).
Figure 7.
The effect of LPS-inducing cytokine expression was inhibited by anti-TLR4 antibody. Serum-free DMEM containing 0.1 μg/ml of sCD14 was used for the experiments. Cells were incubated with 0.1, 0.5, 2 and 10 μg/ml of anti-TLR4 antibody for 1 hr. Ten ng/ml of LPS was added. After incubation for 6 h, total RNAs were isolated and used for RT-PCR for TNF-α and IL-6 (A). After incubation for 24 h, culture supernatants were harvested and IL-6 production was assessed using ELISA (B). ∗, p < 0.05, ∗∗∗, p < 0.001
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