Immune Netw.  2011 Aug;11(4):216-222. 10.4110/in.2011.11.4.216.

Recombinant TAT-CD137 Ligand Cytoplasmic Domain Fusion Protein Induces the Production of IL-6 and TNF-alpha in Peritoneal Macrophages

• Kim JD
• Lee EA
• Quang NN
• Cho HR
• Kwon B

• KMID: 1449813
• DOI: http://doi.org/10.4110/in.2011.11.4.216

Abstract

BACKGROUND
The ligand for CD137 (CD137L; also called 4-1BBL) is mainly expressed on activated APCs such as dendritic cells, B cells and macrophages. Even though CD137L functions as a trigger of the CD137 signaling pathway for T cell activation and expansion, engagement of CD137L can deliver a signal leading to the production of proinflammatory cytokines in macrophages.
METHODS
We generated cell-permeable TAT-CD137L cytoplasmic domain fusion protein (TAT-CD137Lct) and examined its ability to initiate the CD137L reverse signaling pathway.
RESULTS
Treatment of TAT-CD137Lct induced the production of high levels of IL-6 and TNF-alpha mRNAs and proteins in peritoneal macrophages. TAT-CD137Lct increased phosphorylation of Erk, p38 MAPK and Jnk, and activated transcription factors C/EBP and CREB. However, TAT-CD137Lct did not visibly affect the degradation of the inhibitor of NF-kB (IkBalpha). We further demonstrated that JNK activation was required for TAT-CD137Lct-induced production of TNF-alpha, while activation of Erk and p38 MAPK were involved in IL-6 and TNF-alpha production.
CONCLUSION
Our results suggest that TAT-CD137Lct is an effective activator for the CD137L reverse signaling pathway.

Keyword

CD137 ligand; CD137; Reverse signaling; TAT; Peritoneal macrophages

MeSH Terms

4-1BB Ligand
B-Lymphocytes
Cytokines
Cytoplasm
Dendritic Cells
Interleukin-6
Macrophages
Macrophages, Peritoneal
NF-kappa B
p38 Mitogen-Activated Protein Kinases
Phosphorylation
Proteins
RNA, Messenger
Transcription Factors
Tumor Necrosis Factor-alpha
4-1BB Ligand
Cytokines
Interleukin-6
NF-kappa B
Proteins
RNA, Messenger
Transcription Factors
Tumor Necrosis Factor-alpha
p38 Mitogen-Activated Protein Kinases

Figure

• 

  Figure 1 Production and transduction of TAT fusion proteins. (A) Plasmid constructs of the TAT fusion proteins. (B) Purified proteins were separated on 13% SDS-PAGE and then stained with Commasie blue. (C, D) Transduction activity of purified proteins was analyzed using immunoblot (C) and confocal microscope (D). Peritoneal macrophages (1×106) were incubated with 10 µM of TAT-EGFP, CD137Lct, TAT-CD137Lct, and TAT-CD137Lct-EGFP for 30 min. Transduced proteins were detected using anti-his Abs (C). Intracellular localization of TAT-EGFP and TAT-CD137Lct-EGFP were observed using confocal microscopy (D). Scale bar is 10 µm.

• 

  Figure 2 TAT-CD137Lct induces IL-6 and TNF production in peritoneal macrophages. (A, B) Peritoneal macrophages (1×105) were treated with indicated concentrations of TAT- EGFP, CD137Lct, TAT-CD137Lct, and TAT-CD137Lct-EGFP and cultured for 4 h. Culture medium was collected and levels of IL-6 (A) and TNF-α (B) were measured using a CBA kit. Heat-inactivated (HI) TAT-CD137Lct was used as a negative control and LPS (10 ng/ml) and CD137-Fc (100 ng/ml) as a positive control. Data are the mean±SD of triplicates. (C) Total RNA was extracted from peritoneal macrophages (1×106) that were treated with 20 ng of protein. Total RNA was subjected to cDNA synthesis. The TNF-α and IL-6 transcripts were amplified using specific primer sets and the PCR products were analyzed using 1.5% agarose gel electrophoresis. GAPDH mRNA expression was used as an internal control. (D) Density of amplified PCR product bands was analyzed using Image J program and normalized against the density of the GAPDH band.

• 

  Figure 3 TAT-CD137Lct induced phosphorylation of Erk, p38, and Jnk and activaton of C/EBP and CREB. (A) Peritoneal macrophages (1×106) were treated with 50 ng of indicated proteins and harvested at 0, 15, 30, 60, and 120 min after their treatment. Lysates (10 µg) were used for immunoblot. LPS (50 ng/ml) is used as a positive control. p-, phosphorylated. (B, C) Peritoneal macrophages (1×105) were treated with TAT-CD137Lct (20 ng) in the presence of PD98059, SB203580, or SP600125 (10 µM each) and cultured for 4 h. Levels of IL-6 (B) and TNF (C) in culture medium were measured using a CBA kit. Data are the mean±SD of triplicates. (D) EMSA for nuclear extracts from peritoneal macrophages stimulated with 50 ng of indicated proteins was performed with radiolabeled C/EBP and CREB probes. LPS (50 ng/ml) is used as a positive control.

• 

  Figure 4 An additive effect of TAT-CD137Lct and LPS. Peritoneal macrophages (1×105) were treated with indicated concentrations of TAT-CD137Lct and LPS and cultured for 4 h. Culture medium was collected and levels of IL-6 (A) and TNF-α (B) was measured using a CBA kit.

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