Yonsei Med J.  2005 Dec;46(6):818-826.

Differential Expression, Shedding, Cytokine Regulation and Function of TNFR1 and TNFR2 in Human Fetal Astrocytes

• Choi SJ
• Lee KH
• Park HS
• Kim SK
• Koh CM
• Park JY

Abstract

Tumor necrosis factor (TNF) -alpha induces pleiotropic cellular effects through a 55kDa, type 1 receptor (TNFR1) and a 75kDa type 2 receptor (TNFR2). Moreover, it participates in the pathogenesis of several CNS diseases, including demyelinating diseases. TNF- receptors are differentially expressed and are regulated in many cell types. However, data regarding the TNF-alpha receptor expression and regulation in human astrocytes is limited to date. We investigated TNF-alpha receptor expression, its regulation by cytokines, and its functional role in primary cultured human fetal astrocytes, which are the most abundant cellular population in the central nervous system and are known to be immunologically active. In this study, astrocytes were found to constitutively and predominantly transcribe, translate and shed TNFR1 rather than TNFR2, but TNFR2 expression was increased by adding TNF-alpha, IL-1, and IFN-gamma, but not by adding LPS. To determine the functional roles of TNFR1 and TNFR2 on TNF induction, we investigated NF-kappaB activation and TNF-alpha induction after neutralizing TNFR1 and TNFR2 by an antibody treatment. We found that NF-kappaB activation and TNF-alpha induction are blocked by TNFR1 neutralizing antibody treatments.

Keyword

Astrocytes; cytokine; TNFR1; TNFR2

MeSH Terms

Receptors, Tumor Necrosis Factor, Type II/genetics/*metabolism/physiology
Receptors, Tumor Necrosis Factor, Type I/genetics/*metabolism/physiology
RNA, Messenger/metabolism
NF-kappa B/metabolism
Humans
Gene Expression Regulation
Fetus/cytology
Cytokines/*pharmacology
Cells, Cultured
Astrocytes/drug effects/*metabolism

Figure

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  Fig. 1 Quantitation of TNFR1 and TNFR2 mRNA expressions in human fetal astrocytes. Total RNA was isolated from astrocytes and THP-1 cells using a Trizol reagent. TNFR1 and TNFR2 mRNA levels were analyzed by competitive RT-PCR and calculated using densitometry. Different copies (10-104) of competitors were used in competitive RT-PCR. Experiments were performed using at least three different batches of human fetal brain samples.

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  Fig. 2 Effects of TNF-α, IL-1β, IFN-γ or LPS on TNFR1 and TNFR2 mRNA expression in human astrocytes. Human astrocytes were cultured in the presence or absence of TNF-α (A), IL-1β (B), IFN-γ (C) or LPS (D). At different time intervals, the total RNA was isolated. Then, TNFR1 and TNFR2 mRNA levels were analyzed by competitive RT-PCR. One thousand copies of TNFR1 competitor and 10 copies of TNFR2 competitor were used in these experiments. This photograph was obtained from at least three repetitive experiments.

• 

  Fig. 3 Soluble TNFRs secretion in human astrocytes. Human astrocytes were cultured with DMEM, and the culture supernatants were harvested at 6, 12, 24, 36, 48, 60, and 72 h. The levels of soluble TNFRs in the culture supernatants were measured using ELISA. Values represent the means of at least three experiments.

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  Fig. 4 TNFR1 blocking antibody inhibits TNF-α dependent NF-κB activation in human astrocytes. Human astrocytes were preincubated for 1 h with TNFR1 (5 µg/mL or 0.5 µg/mL) or TNFR2 blocking antibodies (5 µg/mL), and then treated with TNF-α (100 U/mL) for 30 min at 37℃. The cells were then examined for NF-κB activation by using an electrophoretic mobility shift assay.

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  Fig. 5 TNFR1 blocking antibody inhibits TNF-α dependent TNF-α mRNA induction in human astrocytes. Human astrocytes were preincubated for 1 h with TNFR1 (5 µg/mL) or TNFR2 (5 µg/mL) blocking antibodies and treated with TNF-α (100 U/mL) for 2 h at 37℃. RT-PCR was done using TNF-α primer.

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