Fig. 1

Effect of the PM exposure of monocytes on: (I) Antigen-driven T cell proliferation. Antigen -driven T cell proliferation in the presence of monocytes exposed to NIST or LAP as APC was evaluated by [³H]-thymidine incorporation assay after 7 days stimulation with recall antigen PPD (purified protein derivative of tuberculin). Data are presented as counts per minute (cpm; median ± interquartile range from 9 independent experiments). (II) Cell morphology. Images show monocytes without exposition to PM (a) vs. exposed to NIST 100 µg/mL (b) or LAP 100 µg/mL (c). Arrows indicate alterations in cell morphology after 15 min exposure to PM. Data are presented from one representative experiment (May-Grünwald-Giemsa staining; magnification × 1000, scale 10 µm). (III) Annexin V binding. The viability of monocytes was evaluated by Annexin V binding assay and flow cytometry analysis. Data are presented as a percentage of Annexin V positive cells (median ± interquartile range from 5 independent experiments). Cells were cultured with a single dose of NIST, or LAP used in three different concentrations (1, 10 or 100 µg/mL) for 15 min (a), 2 h (b) and 4 h (c). In some experiments, monocytes were cultured with PM for 2 h and then the second dose of NIST or LAP was added to cell cultures for the next 15 min (d) or 2 h (e). (IV) ROS production. ROS formation was analyzed by luminol-dependent chemiluminescence measurement recorded at 37 °C. Data are presented in relative chemiluminescence units (RCU) as mean of the results of each cycle (from 1 to 60 cycles) (a, b) and results are expressed as cumulative counts (cc) of the response (c, d) recorded from 60 cycles during 100 min. of measurement. Data are presented as median ± interquartile range from 11 independent experiments. Additionally, detection of ROS in live monocytes were performed with CellROX Green reagent by flow cytometry after 15 min of the cell exposure to NIST or LAP (e). Data are expressed as the percentage of CellROX Green positive cells (median ± interquartile range from 6 independent experiments). Dot plots (CellROX Green—FITC vs. PE) show CellROX Green positive cells in unstimulated control and cells stimulated with NIST or LAP in the highest concentration of 100 µg/mL, for 15 min (f). As a positive control, 200 µM TBHP was used. As a negative control, monocytes were pre-treated with 1.5 mM Mito-TEMPO (c) or 50 µM Ac-yvad-cmk (d) for 1 h prior to the PM exposure. (V) Inhibitors of ROS production and Caspase-1 activity. Additionally, the viability of cells was evaluated by Annexin V binding assay and flow cytometry analysis after monocyte pre-incubation (1 h) with 1.5 mM Mito-TEMPO or 50 µM Ac-yvad-cmk prior to PM exposure (a, b). Dot plots (Annexin V—FITC vs. PE) show Annexin V positive cells in unstimulated control, cells stimulated with NIST (100 µg/mL) for 15 min and cells treated with 1.5 mM Mito-TEMPO or 50 µM Ac-yvad-cmk for 1 h prior to the NIST (100 µg/mL) exposure (c). (VI) Mitochondrial membrane potential (ΨMMP). Alteration of mitochondrial membrane potential (ΔΨMMP) was appointed by flow cytometry using MitoScreen JC-1 dye after 15 min the exposure of cells to NIST or LAP. ΔΨMMP of monocyte was expressed as a ratio of the percentages of cells with high fluorescence intensity (aggregates with high ΨMMP) to cells with low fluorescence intensity (monomers with low ΨMMP) (a). As a positive control, 10 µM CCCP was used. Dot plots (JC-1—FL-1 vs. JC-1 – FL-2) show JC-1 positive cells for aggregates with high ΨMMP; high fluorescence intensity and monomers with low ΨMMP; low fluorescence intensity in unstimulated control and cells stimulated with NIST (100 µg/mL) for 15 min (b). Data are presented as median ± interquartile range from 6 independent experiments. Additionally, the viability of cells was evaluated by Annexin V binding assay and flow cytometry analysis after cell incubation with 10 µM CCCP for 15 min (c). Data are presented as a percentage of Annexin V positive cells (median ± interquartile range from 5 independent experiments). Statistically significant differences were estimated at p < 0.05, p < 0.01, p < 0.001, p < 0.0001, ns – not significant. (d) Changes in mitochondrial membrane potential of cells