Antibodies and reagents
The mAbs TP1/55 (anti-CD69), HP2/6 (anti-CD4), Lia3/2 (anti-CD18), B942 (anti-CD8) and DR (anti-HLA-DR) have been described previously [15
]. The mAbs T3b (anti-CD3) and BU12 (anti-CD19) were generously donated by Dr J De Vries (DNAX, Palo Alto, CA, USA). The BAB281 (anti-NKp46), MA152 (anti-NKp80), z199 (anti CD94/NKG2A) and KD1 (anti-CD16) mAbs were kindly provided by Dr A Moretta (Universita degli Studi di Genova, Genova, Italy). Phycoerythrin-conjugated Leu-19 (anti-CD56), Leu-19 (anti-CD56 pure) and isotype-matched controls were purchased from Becton Dickinson (Mountain View, CA, USA). Anti-human NKG2D (MAB139), blocking anti-human IL-15 (MAB647), anti-human CD244 (2B4; MAB1039) and the negative control MAB002 mAb were obtained from R&D Systems (Abingdon, Oxon., UK). The anti-human CD244 (2-69) was from BD-Pharmigen (San Diego, CA, USA) and the anti-human CD48 (156-4H9) was from NeoMarkers (Freemont, CA, USA).
Recombinant human IL-15, IFN-γ, TNF and IL-1 were supplied by PeproTech EC, Ltd (London, UK). FCS was purchased from Boehringer Mannheim (Mannheim, Germany), RPMI 1640 medium, Dulbecco's modified Eagle's medium, penicillin and streptomycin were provided by BioWhittaker (Verviers, Belgium) and L-glutamine by Gibco BRL (Paisley, Renfrewshire, Scotland). Lipopolysaccharide was supplied by Sigma Diagnostics (St Louis, MO, USA).
Isolation of lymphocyte subsets
Peripheral blood lymphocytes (PBL) were isolated from healthy donors by Histopaque-1077 density-gradient centrifugation (Sigma Diagnostics). This was followed by the removal of monocytes by adhesion for 1 hour to Petri dishes (Costar, Cambridge, MA, USA) in RPMI 1640 medium supplemented with 10% FCS at 37°C. The lymphocyte-enriched fraction contained less than 1% CD14+ cells. CD4+ and CD8+ cells were then obtained by negative selection with Subset Enrichment Column kits (R&D Systems). Cell purity was determined by flow cytometry and was always greater than 90% for CD4+ and 95% for CD8+ T lymphocytes. NK cells were purified by negative selection with goat anti-mouse IgG Dynabeads (Dynal Biotech, Oslo, Norway) previously coupled to anti-CD3, anti-CD4 and anti-HLA-DR mAbs. After a second round of selection with beads coupled to CD3 and CD19 mAbs (Dynal Biotech) the cell population obtained was more than 95% CD56+ with less than 1% CD3+ cells.
In other experiments, PBL were depleted of T cells, B cells or NK cells, and the NK-depleted PBL were obtained by incubating the PBL with immunomagnetic beads coupled to BAB281 (anti-NKp46), KD1 (anti-CD16) and Leu-19 (anti-CD56) mAbs. This process was repeated and the cell population obtained was less than 1% CD56+. B cell-depleted PBL and T cell-depleted PBL populations were isolated by using the same procedure with the anti-CD19 and anti-HLA-DR mAbs, yielding a B cell-depleted PBL population that was less than 0.5% CD19+. When anti-CD3, anti-CD4 and anti-CD8 was used, the T cell-depleted PBL population was less than 1% CD3+.
Most experiments were performed with the human monocytic leukaemic cell line THP-1 obtained from ATCC/LGC Promochem (Barcelona, Spain). These cells were maintained in culture with RPMI 1640 medium supplemented with 10% heat-inactivated FCS, penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere consisting of 5% CO2 .
In experiments performed with human peripheral blood monocytes, these cells were obtained with the following purification procedure: peripheral blood mononuclear cells were obtained by Histopaque-1077 density-gradient centrifugation and resuspended in RPMI 1640 medium supplemented with 10% FCS. A sample of this cellular suspension was analysed through a Hitachi Coulter counter to determine the concentration of monocytes. A volume containing 105 monocytes was then added to each well in 24-well plates (Costar) to allow the attachment of monocytes and, after 1 hour at 37°C, wells were washed three timed with RPMI 1640 medium. The population attached to wells was more than 90% CD14+ after cell detachment and flow cytometry analysis. To perform co-culture assays, the autologous lymphocytes were treated as described above and co-cultured with the monocytes at a 10:1 ratio (106 lymphocytes or subpopulations per 105 monocytes attached at the wells of 24-well plates).
Patients and synovial fluid samples
Synovial fluid samples were obtained, with previous oral informed consent, from patients attending our out-patient clinic. Diagnoses included RA (n = 5), seronegative spondyloarthropathies (n = 6) and crystal-induced arthritis (n = 4). Unfractionated or NK-depleted synovial fluid lymphocytes (SFL) were purified as described above and this population was, on average, less than 5% CD56+.
This study was approved by the ethics committee for clinical research at Hospital Universitario de La Princesa.
Cell-cell contact assays
PBL or different lymphocyte subsets were incubated for 24 hours in the presence of medium alone or with IL-15 (1 to 100 ng/ml). After being washed, the cells were resuspended in medium and added to 24-well plates (Costar). Unless otherwise stated, then THP-1 cells were added in the proportion 10 lymphocytes to 1 THP-1. As a negative control, lymphocyte–THP-1 cell contact was prevented by using a 0.4 μm pore-size transwell insert (Costar). In some experiments, lymphocytes or THP-1 cells were fixed before cell co-culture (0.05% glutaraldehyde at 4°C for 30 to 45 seconds). After 24 hours the supernatants were harvested and stored at -80°C until the cytokines were quantified.
To investigate the involvement of different cell surface molecules in these experiments, the following purified mAbs were added: MAB002 (negative control), MAB647 (anti-IL15), BAB281 (anti-NKp46), MA152 (anti-NKp80), MAB139 (anti-NKG2D), z199 (anti-CD94/NKG2A), Lia3/2 (anti-CD18), 156-4H9 (anti-CD48) and 2-69 (anti-CD244). All mAbs were used at a final concentration of 10 to 20 μg/ml.
Induction of IL-15 expression on THP-1 cells
THP-1 cells were stimulated with different concentrations of IFN-γ (1 to 100 ng/ml), TNF (1 to 100 ng/ml), IL-1 (1 to 100 ng/ml) or medium alone for 24 hours; the expression of membrane-bound IL-15 was then analysed by flow cytometry.
Flow cytometry analysis
Cells were incubated with the specific mAbs at 4°C for 30 minutes. After being washed in PBS, the cells were labelled with fluorescein isothiocyanate-tagged goat anti-mouse Ig (Dako, Salstrup, Denmark) for 30 minutes at 4°C. For double staining, cells were additionally incubated for 15 minutes with mouse serum diluted 1:100 (ICN Biomedicals Inc, Aurora, OH, USA); they were washed and then incubated with a phycoerythrin-conjugated anti-CD56 mAb (Becton Dickinson) for 20 minutes. At least 5 × 103 cells were analysed with a FACScan flow cytometer (Becton Dickinson).
Quantification of cytokines in cell-free supernatant
Human TNF concentrations in supernatants were determined by an enzyme immunoassay (EIA). In brief, 96-well high-binding EIA plates (Costar) were coated overnight at 4°C with 50 μl of MAB610 (R&D Systems) per well at 8 μg/ml in PBS, pH 7.4. Subsequently, each well was washed twice with 200 μl of wash buffer (0.05% Tween 20 in PBS, pH 7.4) and blocked for 1 hour by adding 200 μl of PBS containing 2% BSA at 37°C. After each step, the wells were washed three times with 200 μl of wash buffer; 50 μl of dilution buffer (0.1% BSA, 0.05% Tween20, 20 mM Trizma base, 150 mM NaCl, pH 7.3) per well plus 50 μl of each sample or standard dilutions for recombinant human TNF (10,000 to 39 pg/ml; R&D Systems) were then added to the respective wells (in duplicate) and incubated at room temperature for 2 hours. Bound TNF was detected by incubation for 1 hour with, in each well, 50 μl of BAF210 (R&D Systems) diluted to 200 ng/ml in dilution buffer at room temperature. After washing, 100 μl streptavidin HRP (Calbiochem, San Diego, CA) diluted 1:5,000 in dilution buffer was added to each well for 20 minutes at room temperature; the reaction was then developed with 100 μl 3,3',5,5' -tetramethylbenzidine (Chemicon International Inc., Temecula, CA, USA) per well. The optical density of each well was determined with a SpectraII microtitre plate reader (Innogenetics Diagnóstica y Terapéutica, Barcelona, Spain) set to 450 nm, with wavelength correction set to 550 nm. Cytokine values were calculated from the standard curve. Samples that generated values higher than the highest standard were diluted (1:1) in dilution buffer and assayed again.
Because TNF production can vary depending on the lymphocyte donor, in the experiments in which cell–cell interactions were blocked with mAbs the results were normalized with the following equation: TNF production = 100 × TNFmAb /TNFmedium .
Human IFN-γ concentrations were measured with an EIA kit from R&D Systems.
Statistical analysis was performed with Stata 9.1 for Windows (StataCorp LP, College Station, TX, USA), by using one-way analysis-of-variance model with Bonferroni multiple-comparison correction for multiple sample experiments and the Mann–Whitney test for experiments with comparison between two groups.