Preparation of RrTCTP, truncated TCTPs, and mutated TCTPs
The sequences coding rat TCTP and deleted rat TCTPs (RrTCTP, residue 1–172; Del-N11TCTP, residues 11–172; Del-N35TCTP, residues 35–172; Del-N39C110TCTP, residues 39–110) were amplified using the oligonucleotide primers for cloning into the bacterial expression vector, pRSET A (Invitrogen). The residues 1–172 was amplified using forward-CG GGATCC (BamHI) ATG ATC ATC TAC CGG GAC- and reverse primer-CCC AAGCTT (HindIII) ACA TTT TTC CAT CTC TAA GCC, the residues 11–172 was amplified using forward-CG GGATCC (BamHI) GAC GAG CTG TCC TCC GAC AT- and reverse primer-CCC AAGCTT (HindIII) ACA TTT TTC CAT CTC TAA, the residues 35–172 was amplified using forward-CG GGATCC (BamHI) AGT GTC AGT AGA ACA GAG- and reverse primer-CCC AAGCTT (HindIII) ACA TTT TTC CAT CTC TAA, and the residues 39–110 was amplified using forward-CG GGATCC (BamHI) ACA GAG GGT GCC ATC GA and reverse primer-G GAATTC (EcoRI) CCT TTC TGG TTT CTG TT.
Three mutations were introduced into the Del-N11TCTP (Del-N11TCTP-C28S and Del-N11TCTP-C172S) and Del-N35TCTP (Del-N35TCTP-C172S) in order to eliminate the cysteine residues using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The pRSET A/Del-N11TCTP and pRSET A/Del-N35TCTP were amplified with complimentary oligonucleotide primers containing the mutation; for Del-N11TCTP-C28S, CG GAC GGG CTG TCT CTG GAG GTG GA and TC CAC CTC CAG AGA CAG CCC GTC CG, for Del-N11TCTP-C172S and Del-N35TCTP-C172S, GAG ATG GAA AAA TCT AAG CTT GAT CCG and CGG ATC AAG CTT AGA TTT TTC CAT CTC.
BL21(DE3)pLysS cells transformed with the pRSET A/TCTPs or pRSET A/mutated TCTPs were overexpressed and purified using a Ni2+
-charged His-Bind column according to manufacturer's protocol (Novagen). The NH2
-terminal fusion proteins were separated by fast protein liquid chromatography (FPLC) on a Mono Q HR 5/5 column (Amersham Pharmacia Biotech) using a NaCl gradient. Buffer A was 20 mM Tris-HCl, 1 mM EDTA, 50 mM NaCl, pH 7.4, and Buffer B was 20 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, pH 7.4. A linear gradient from 0% to 15% Buffer B was run over 5 column volumes and it was followed by a linear gradient from 15% to 50% Buffer B over 25 column volumes to elute the TCTPs. Each eluted fraction was desalted and transferred into PBS using PD-10 column (Amersham Pharmacia Biotech). Final proteins were detected LPS contaminations by the Limulus Amebocyte lysate (LAL) assay (Cambrex). The LAL assay showed that LPS contaminations ranged from 1 to 100 pg/µg protein. Because it had been known that BEAS-2B cells were relatively insensitive to LPS 
, proteins purified using FPLC were used in cytokine assays without further purification. The purity of the proteins was 85–98%. For T cells and sensitization and challenge of mice airway, proteins were additionally purified with EndoTrap (Cambrex).
Measurement of IL-8 and GM-CSF
Human bronchial epithelial cells, BEAS-2B (ATCC), were maintained in bronchial epithelial growth medium (BEGM, Clonetics). When BEAS-2B cells became 80% confluent, they were passaged in 48-well culture plates, incubated for 20–24 h in BEGM, washing twice with 1% penicillin-streptomycin/bronchial epithelial basal medium (BEBM), and then incubated for the indicated time with 1–10 µm/ml of proteins or without protein (negative control, NC) in 1% penicillin-streptomycin/BEBM. IL-8 and GM-CSF secreted into media were measured by ELISA using a commercial kit (PIERCE).
Measurement of IL-2
Human jurkat T cells (ATCC) were maintained in RPMI 1640 containing, 10 mM HEPES, 2 mM L-Glutamine, 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10% heat-inactivated FBS, 1% penicillin-streptomycin. Cells were preincubated at 106 cells/well for 4 h with TCTPs in growth medium or with medium alone (NC), and then stimulated with 10 µm/ml PMA and 25–500 µm/ml ionomycin for 20 h. IL-2 in supernatant was measured using ELISA kit (PIERCE).
In vitro activation and differentiation of TH cells
CD4+ TH cells were isolated from the draining lymph node of the C57BL6 mice using mouse CD4 microbeads according to the manufacturer's instruction (Miltenyi Biotec). Cells were stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml) antibodies (BD Pharmingen) for 48 h, or restimulated with PMA (10 µm/ml) and ionomycin (1 µM) at 72 h. For TH2 cell differentiation, cells were co-stimulated with IL-4 (10 µm/ml) and anti-IFN-γ Ab (5 µg/ml) at day 1. Cells were induced to differentiate for additional 5 days and re-stimulated with anti-CD3 for 24 h. CD4+ TH cells were incubated with RrTCTP or Del-N11TCTP accord with TCR stimulation. Supernatants were harvested at 48 h or at 24 h after secondary PMA/ionomycin or anti-CD3 stimulation. Supernatants were incubated with capturing antibodies specific for IL-2, IL-4, or IFN-γ, and subsequently biotinylated anti-cytokine antibodies as instructed (BD Pharmingen).
Intracellular cytokine staining
CD4+ TH cells were stimulated with anti-CD3 and anti-CD28 antibodies in the presence of RrTCTP or Del-N11TCTP for 72 h and additionally stimulated with PMA and ionomycin for 4 h. Cells were treated with Monensin (Sigma-Aldrich) for 2 h before harvest and subsequently incubated with phycoerythrin (PE)-tagged anti-IL-2 Ab or allophycocyanin (APC)-conjugated IL-4 Ab. After washing the cells with PBS, cells were analyzed by FACS Calibur and CellQuest program (BD Biosciences).
Development of airway inflammation and lung analyses
BALB/c mice were sensitized at days 0 and 14 (i.p.) with OVA, RrTCTP or Del-N11TCTP. At day 28, the sensitized mice were challenged with the same allergen (300 µg of OVA, 10 µg of RrTCTP or 10 µg of Del-N11TCTP) by intranasal administration six times. At day 40, the mice were sacrificed and lungs and bronchoalveolar lavage (BAL) fluids were collected and immune cells were counted from the cytocentifuged cells. The supernatant was used for IL-5 measurement by ELISA (PIERCE) and TCTP detection by Western blotting. Lungs and rhinal tissues were fixed in 4% paraformaldehyde. After dehydration in alcohol, tissues were embedded in paraffin and cut into 5 µm thick sections. Tissue sections were stained with PAS (Periodic Acid Schiff) solution (Sigma-Aldrich). All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by Ewha Womans University's institutional animal care and use committee.
Detection of the dimerized TCTP in sera of atopic and atopic/asthmatic patients
All the subjects enrolled in this study gave written informed consent, and the study protocol was approved by the Institutional review board of Seoul National University Hospital (No. 0706-040-211). The serum was precipitated with acetone/TCA, denatured in 8 M urea/2% CHAPS, and analyzed on non-reducing SDS-PAGE.
BEAS-2B cells seeded on confocal dish (SPL) were blocked with 1% BSA/BEBM at RT for 10 min prior to 30 min incubation with 5 µM of RrTCTP or Del-N11TCTP at 4°C. NC (without protein), RrTCTP, Del-N11TCTP treated cells were washed and labeled with rabbit anti-TCTP and rhodamine-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch) at 4°C. The cells washed and then fixed in 3.7% formaldehyde/PBS for 15 min. Confocal microscopy was performed using a Carl Zeiss Laser Scanning Systems LSM 510. Z-stack images were taken at 0.1-µm intervals, and the profile was analyzed.
Chemical Cross-linking of RrTCTPs
RrTCTP and Del-N35TCTP were irreversibly cross-linked by a crosslinker for primary amines, DMS (dimethyl suberimidate· 2HCl, PIERCE), and two crosslinkers for sulfhydryl groups having different lengths of spacer arms, BMOE (bis-maleimidoethane, PIERCE), and BM(PEO)3 (1, 11-bismaleimidotriethylene glycol, PIERCE). FPLC purified proteins were incubated with thirty-fold molar excess of DMS for 30 min, five-fold molar excess of BMOE for 2 h, or five-fold molar excess of BM(PEO)3 for 1 h at room temperature. The reaction of DMS was stopped by adding Tris, pH 7.5 at 50 mM final concentration; in all reactions, the excess nonreacted crosslinkers was removed by vivaspin ultrafiltration spin column (Vivascience). The resulting products were analyzed on SDS-PAGE, and assessed the IL-8 secretion activity in BEAS-2B cells.
Overexpression and purification of HrTCTP and its variants
pCEP4 expression vector (Invitrogen) was modified to facilitate the construction of rabbit Fc fusion proteins by fusing sequences encoding HrTCTP and its variants, Del-N11HrTCTP and Del-N35HrTCTP, to the Fc region of rabbit IgG heavy chain. Human IgG kappa chain leader sequence and the rabbit Fc were linked by overlap extension PCR and the product was cloned in between the HindIII and BamHI sites of the pCEP4 vector. Three cysteines in the hinge region of the rabbit Fc region were converted to serine. The TCTP and its variants were cloned in SfiI site between the leader sequence and the rabbit Fc region. Full length human TCTP gene was amplified by using a sense primer with SfiI site (GGCCCAGGCGGCCATG ATT ATC TAC CGG GAC CTC ATC AGC C) and an anti-sense primer with SfiI site (GGCCGGCCTGGCC ACA TTT TTC CAT TTC TAA ACC ATC CTT AAA GAA AAT CAT ATA TGG GGT CAC ACC ATC C). This anti-sense primer was also used for amplifying Del-N11HrTCTP and Del-N35HrTCTP genes. A sense primer with SfiI site for Del-N11HRF gene was GGCCCAGGCGGCC GAT GAG ATG TTC TCC GAC ATC TAC AAG ATC CGG G and for Del-N35HRF gene was GGCCCAGGCGGCC ATG GTC AGT AGG ACA GAA GGT AAC ATT G.
The constructs were transfected into HEK293T cells, and the culture supernatant was harvested and clarified by centrifugation at 4,000 rpm for 30 min and passed through a bottle-top filter (Millipore Corporation) before application to a protein A-agarose column (Repligen). After washing with binding buffer (PIERCE), the binding proteins were eluted with 0.1 M glycine-HCl (pH 2.2). The eluate was neutralized with 1 M Tris (pH 9.5). Protein concentration was measured by BCA assay kit (PIERCE).
Results are expressed as means±SD. We evaluated the results with a one-way ANOVA followed by multiple-comparison tests. Differences were considered significant if p values were<0.05. The Tukey's HSD test is applied to all pairwise differences between means, and Dunnett's test was performed to compare each group with the control.