C57BL/6 mice, 6-8 wk of age, were purchased from Hyo Chang Bioscience (Korea). Peritoneal macrophages were isolated by peritoneal larvage with 1x PBS (WelGENE, Korea). Mixture containing macrophages and RBCs was incubated in RBC lysis buffer for 5 min on ice and washed with 1x PBS three times. After that, cells were counted using hemocytometer and cultured in DMEM (WelGENE) containing 1% FBS (WelGENE). The following Abs were used for immunoblotting: Abs to p-p38, p38, p-Erk, Erk (all from Cell signaling Technology, Danvers, MA), IkBα (Santa Cruz Biotechnology), GAPDH (Millipore), and His (Santa Cruz Biotechnology, Santa Cruz, CA). Erk inhibitor (PD98059), p38 inhibitor (SB203580), and Jnk inhibitor (SP600125) were purchased from Merk (Germany).

Total RNA isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) from mouse spleens was subjected to cDNA synthesis using Olig dT and AMV reverse transcriptase (Promega, Madison, WI). The cytoplasmic domain of mouse CD137L gene was amplified by PCR using sense (5'-GAATTCATGG ACCAGCACACA-3') and antisense primers (5'-CTCGAGTCAT GGGTGGCGGGA-3'). CD137Lct DNA fragments were inserted in pET21a-HisTAT vector (TAT-CD137Lct). To construct pET21a-HisTAT vector, double-stranded oligonucleotide including his-tag and TAT sequences was prepared by annealing (heating at 95℃ for 5 min and chilling at room temperature for 60 min) of single-strand oligonucleotides (Forward, 5'-CAT ATG CAC CAC CAC CAC CAC CAC TAT GGC AGG AAG-3'; Reverse, 5'-GGA TCC TCG TCG TCG CTG TCT CCG CTT CTT CCT GCC-3') and then inserted in pET21a vector. EGFP sequence was obtained from pEGFPN1 vector (Clonetech Laboratories, Mountain View, CA) by PCR amplification using the following primers: sense, 5'-GAA TTC ATG AGT AAA GGA GAA-3'; antisense, 5'-CTC GAG TTT GTA TAG TTC ATC-3'.

BL12 star E. coli (Merk, Germany) were transformed with each DNA construct, cultured overnight, and added with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for protein induction at 37℃ for 6 h. Harvested cells were sonicated in lyisis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole). Lysates were clarified by centrifugation at 13,000 rpm for 20 min at 4℃. Clarified lysates were bound with Ni-NTA agarose bead (QIAGEN, Valencia, CA) for 1 h. Bound proteins were washed 5 times with washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, 50 mM imidazole) and eluted with elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, 250 mM imidazole). Purified proteins were dialysed using 1x PSB (pH 8.0) and LPS was removed using ToxinEsaer TM Endotoxin Removal kit (GeneScript, Piscataway, NY). Protein concentration was measured using bicinchoninic acid (BCA) kit (Thermo Scientific, Rockford, IL).

Peritoneal macrophages (1×10⁶) were incubated with 10 µM TAT-EGFP, CD137Lct, TAT-CD137Lct and TAT-CD137Lct-EGFP for 3 min. Cells were lysed in lysis buffer (50 mM Tris-HCl [pH7.4], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM PMSF, protease inhibitors, 1 mM NaF, and 0.1 mM Na₃VO₄) on ice for 30 min. Proteins were resolved in 17% SDS-PAGE and transferred onto nitrocellulose membranes. Immunoblot procedure was described in the next section. Transduced proteins were detected by anti-his Abs. Intracellular localization of TAT-EGFP and TAT-CD137Lct-EGFP protein was visualized by confocal microscopy. Cells were cytospinned at 1,000 rpm for 15 min on slide glass. Cytospinned cells were mounted with Fluromount-G solution (Southern Biotech).

Protein lysates were prepared from peritoneal macrophages and their protein concentrations were determined using the Bio-Rad assay kit (Hercules, CA). Lysates (5 µg of protein per sample) were mixed with gel loading buffer (60 mM Tris/HCl, pH 6.8, 25% glycerol, 2% SDS, 14.4 mM β-mercaptoethanol, 0.1% bromphenol blue) and boiled for 3 min. Proteins were resolved in 15% or 17% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Bedford, MA). Membranes were blocked for 1 h in blocking buffer (1X TBST [50 mM Tris-HCl, pH 7.5, 150 mM NaCl] including 5% dried milk), followed by overnight incubation at 4℃ with first Abs. Membranes were washed 3 times with 1X TBST and incubated with secondary Abs conjugated with alkaline phosphatase (Santa Cruz Biotechnology) for 1 h at room temperature and developed by ECL-plus substrate kit (Amersham Biosciences, Piscataway, NJ).

Peritoneal macrophages (1×10⁵) were treated with 20 ng TAT-EGFP, CD137Lct, TAT-CD137Lct, and TAT-CD137Lct-EGFP for 4 h. Culture supernatants were collected and the amount of IL-6 and TNF-α was quantified using a CBA kit (BD Biosciences, San Diego, CA) with a FACS-Caliber cytometer equipped with Cell-QUEST-PRO and CBA software according to the manufacturer's instructions.

Total RNA isolated using TRIzol reagent (Invitrogen) was subjected to cDNA synthesis using Olig dT and AMV reverse transcriptase (Promega). Dilutions (5- or 10-fold) of cDNA reaction mixtures were subjected to PCR amplification using the following primers: IL-6 sense, 5'-GATGCTACCAAACTGGATA TAATC-3'; IL-6 antisense, 5'-GGTCCTTAGCCACTCCTTCTGTG-3'; TNF-α sense, 5'- AGGGGCCACCACGCTCTT CT-3'; TNF-α antisense, 5'- CGGGGCAGCCTTGTCCCTTG-3'; GAPDH sense, 5'-TGAAGGTCGGTGTGAACGGATTTG-3'; GAPDH antisense, 5'-CATGTAGGCCATGAGG TCCACCAC-3'.

Nuclear extracts were prepared from 1×10⁶ of peritoneal macrophages stimulated with 50 ng TAT-EGFP, CD137Lct and TAT-CD137Lct proteins. Cells were washed with ice-cold PBS, resuspended in buffer A (20 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride), and left on ice for 10 min. Nuclei were pelleted by centrifugation at 5,000 rpm for 10 min at 4℃ and resuspended in buffer B (20 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 420 mM NaCl, 0.2 mM EDTA, 20% glycerol, and 1 mM dithiothreitol). After incubation for 30 min at 4℃, the mixture was centrifuged at 13,000 rpm for 15 min at 4℃. The supernatant as nuclear extract was collected and stored at -70℃ until use. The C/EBP (sc-2525) and CREB (sc-2504) probe was purchased from Santa Cruz. Each probe was end-labeled with [γ-³²P]dATP (Amersham Biosciences) by T4 polynucleotide kinase (Promega). The nuclear extracts and probe were incubated for 30 min at room temperature in reaction buffer containing 10 mM HEPES, pH 7.9, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, and 5% glycerol. Each reaction contained 0.02 unit of poly[dI-dC] (Sigma, St. Louis, MO). Reactions were electrophoresed on a 4% non-denaturing polyacrylamide gel in 0.5X Tris borate-EDTA buffer at 150 V for 2 h, and then analyzed by autoradiography.

We generated DNA constructs to produce polyhistidine-tagged TAT-CD137Lct, CD137Lct, TAT-CD137Lct-EGFP, and TAT-EGFP proteins in E. coli (Fig. 1A). Recombinant proteins purified using Ni-NTA agarose beads had expected molecular weights when separated in 13% SDS-PAGE gel (Fig. 1B). Western blot analysis showed that all TAT fusion proteins were able to be permeabilized into peritoneal macrophages (Fig. 1C). Transduced TAT-CD137Lct-EGFP was localized beneath the cell membrane of peritoneal macrophages and TAT-EGFP was found throughout the cytoplasm (Fig. 1D). Our results suggest that TAT fusion proteins could easily penetrate into the cell.

CD137L reverse signaling induces IL-6 and TNF-α production in monocytes (4). We investigated whether transduction of TAT-CD137Lct would result in the production of IL-6 and TNF-α in peritoneal macrophages. Peritoneal macrophages produced high levels of IL-6 and TNF-α in response to TAT-CD137Lct and TAT-CD137Lct-EGFP in a dose-dependent manner (Fig. 2A and B). A high concentration of CD137Lct slightly induced the production of IL-6 and TNF-α. This might be due to the ability of macrophages to uptake extracellular proteins, since TAT-EGFP had no effect on the cytokine production of macrophages. Heat-inactivated TAT-CD137Lct did not stimulate macrophages to produce IL-6 and TNF-α, suggesting that there was no LPS contamination. As expected, LPS and CD137-Fc (100 ng/ml) was potent in inducing the production of IL-6 and TNF-α in peritoneal macrophages. RT-PCR analysis showed that TAT-CD137Lct also significantly increased mRNA levels of IL-6 and TNF-α in peritoneal macrophages (Fig. 2C and D).

To further define the mechanism by which TAT-CD137Lct controls macrophage production of IL-6 and TNF-α, we investigated what intracellular signaling molecules are involved in this process. TAT-CD137Lct increased phosphorylation of the kinases Erk, p38 and JNK but had no visible effect on the degradation rate of the inhibitor of NF-κB (IκBα) (Fig. 3A). Consistent with this result, the production of IL-6 and TNF-α in peritoneal macrophages was inhibited specifically by the Erk (PD98059) and p38 (SB203580) inhibitors (Fig. 3B and C). Interestingly, the JNK inhibitor (SP600125) blocked the production of TNF-α but not IL-6 in TAT-CD137Lct- stimulated macrophages. We also found that TAT-CD137Lct strongly activated CREB and C/EBP in macrophages at 12 h after its treatment (Fig. 3D). Our data are consistent with previous studies showing that CD137L is required for activation of CREB and C/EBP but not NF-κB in macrophages (19).

It has been shown that interactions of cell surface CD137L with TLR4 increase LPS-induced cytokine production in macrophages (19). Since TAT-CD137Lct seemed to evoke the CD137L signaling pathway regardless of CD137L on the cell surface, we predicted that TAT-CD137Lct would have an additive effect with LPS on cytokine production in macrophages. Indeed, co-treatment of TAT-CD137Lct and LPS showed an additive effect on IL-6 and TNF production in peritoneal macrophages (Fig. 4).