Toll-like receptor expression on peripheral blood mononuclear cells in asthmatics; Implications for asthma management
SUPPLEMENTARY MATERIAL
Eunyoung Chun, So-Hee Lee, Soo-Yeon Lee, Eun-Jin Shim, Sang-Heon Cho, Kyung-Up Min, You-Young Kim, Heung-Woo Park
Detailed Methods
Isolation of PBMCs
Venous blood was diluted 1:1 with RPMI 1640 medium (WelGENE Inc.) and layered on Ficoll-PaqueTM Plus (Amersham Biosciences). After centrifugation (30 min at 2000g and room temperature), PBMCs were collected and washed twice with RPMI 1640. Cells were counted and stained with various fluorochrome-conjugated mAbs.
Flow cytometry analysis
FITC-conjugated anti-CD14, anti-CD8 mAb, PE-conjugated anti-CD4 mAb, and PerCP-conjugated anti-CD3 mAbs were purchased from BD Biosciences (San Jose, CA). PE-conjugated anti-TLR1, anti-TLR2, anti-TLR3, anti-TLR4, anti-TLR9, Biotin-conjugated anti-TLR6 mAbs, mouse IgG isotype control, rat IgG isotype control, and PE-streptavidin were purchased from eBioscience (San Diego, CA). For surface staining, cells were incubated with fluorochrome-conjugated mAbs for 30min at 4°C in the dark and washed twice with staining buffer (PBS containing 1% FBS (Invitrogen Corporation, Carlsbad) and 0.09% sodium azide (Sigma-Aldrich, St. Louis). Cells were also stained with isotype control for each of the specific antibodies. For TLR3 and TLR9 intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm kits (BD Biosciences, San Jose) and stained intracellularly with PE-conjugated anti-TLR3 and TLR9 or isotype controls. Stained cells were acquired on a FACSCalibur equipped with two lasers (BD Biosciences). Data analysis was performed with CellQuest software (BD Biosciences).
Cell culture and TLR-specific ligands stimulation
Isolated PBMCs (1x106/ml) were stimulated with 0.1 to 10 ㎍/mL LPS, 1 to 10 ㎍/mL PGN, 0.1 to 1 ㎍/mL Pam3Cys, 0.001 to 0.1 ㎍/mL Pam2Cys, and 0.01 to 1 ㎍/mL FSL-1 for 24 hours andcell-free supernatants were prepared from each experiment and stored at -80℃until required for cytokine determinations by ELISA. Our results demonstrate that 1 ㎍/mL LPS, 10 ㎍/mL PGN, 1㎍/mL Pam3Cys or 0.1 ㎍/mLFSL-1 resulted in the highest TNF-production. For intracellular cytokine assay, isolated PBMCs (1x106/mL) were stimulated for various times with1 ㎍/mL LPS, 10 ㎍/mL PGN, 1㎍/mL Pam3Cysand0.1㎍/mLFSL-1 in the presence of 10 ㎍/mL Brefeldin A (eBioscience, San Diego), an inhibitor of protein translocation from the endoplasmic reticulum to the Golgi apparatus. Phorbol myristate acetate (PMA) and ionomycin were used as non-specific controls. Cells were cultured in RPMI-1640 supplemented with 10% FCS (HyClone, Logan, UT). The intracellular cytokines TNF-α and IL-1β produced during 2-, 4-, or 6-hoursof stimulation with LPS, PGN, Pam3Cys, or FSL-1 were detected in CD14+ monocytes by adding BrefeldinA 2 or 4 hours before cell harvest.
Enzyme-linked immunosorbent Assay (ELISA)
Immulon microtiter plates (ThermoLabSystems, Franklin, MA) wereincubated overnight at 4°C with 100 Lof monoclonalanti-human TNF capture antibody (BD Biosciences) in coatingbuffer (0.1 M sodium carbonate, pH 9.5). Individual wells werewashed three times with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBST), at pH 7.4, and blocked with PBS containing 10%bovine serum albumin, at pH 7.0, for 1 h at room temperature. Individualwells were washed three times with PBST, and supernatantsof TLR ligand-stimulated PBMCs from individual wells were transferredto individual antibody-coated wells. Supernatants were then dilutedtwofold directly in the antibody-coated wells such that the amountof TNF-αwould fall within the range of TNF-αstandard curve range.To create the standard curve, serially diluted standard preparationof TNF-α(R&D Systems, Minneapolis, MN) was added to a separateset of individual antibody-coated wells in duplicate. Platescontaining samples were then incubated at room temperature for2 h, and washed by five times with PBST.To detect captured TNF-α, 100l of biotinylated anti-humanTNF-α sandwich monoclonal antibody (BD Biosciences) along withan avidin-horseradish peroxidase conjugate (BD Biosciences)was added to each well and the plate was incubated for 1 h atroom temperature. Individual wells were then washed five timeswith PBST and incubated with 100 Lof tetramethylbenzidineplus hydrogen peroxide substrate solution for 30 min at roomtemperature. The colorimetric reaction was stopped by adding 50 lof 1 M sulfuric acid, and absorbance was read at 450 nm usinga microtiter plate spectrophotometer (Biotek, Winooski, VT).To generate values for TNF-α production, the serially dilutedTNF-αstandard was used to create a curve within the spectrophotometersoftware environment to which experimental readings were mathematicallyapplied (KC junior; Biotek). TNF-αvalues obtained from duplicatesamples were averaged.
Detection of intracellular cytokine expression
For intracellular staining, cells harvested from stimulated cultures were washed twice with PBS/BSA, incubated in 0.5mL of lysing solution and 0.5 mL of permeabilizing solution (BD Biosciences) for 10 minutes at room temperature. They were then washed withPBS/BSA, and incubated for 30 minutes with appropriatelytitered PE-labeled anti–TNF-α, anti–IL-1β or IL-6 mAbs (BD Biosciences). The percentage of cytokine-producing cells on stimulation for the indicated period were calculated after subtracting background production. Mean fluorescence intensities (MFIs)were also used to assess levels of cytokine synthesis in the indicated populations.
Figure S1. Expression of TLRs on PBMCs according to the expression of CD14