Bisphenol A (BPA), Superoxide Dismutase from bovine erythrocytes, Cytochrome C from equine heart, Griess reagent, nitroblue tetrazolium (NBT), N-Formyl-Met-Leu-Phe (fMLP) and Latex were from Merck Millipore, Burlington, USA. May-Grunwald and Giemsa were from Aqua-med; Polimorphoprep™ (Axis Shield), PBS with or without ions of CaCl2 and MgCl2 (Thermo Fisher Scientific, Waltham, USA). Wortmannin was from Calbiochem (San Diego, CA); ICI 182.780 (ICI/Fulvestrant, 98% pure) and 17-β-estradiol (E2) were from SigmaAldrich.
The study involved a group of 15 healthy voluntary blood donors [5 women (in the follicular phase of the menstrual cycle) and 10 men] aged 22–30 years, from Regional Centre for Transfusion Medicine, Bialystok, Poland. Blood donors were healthy people, who did not smoke and did not drink alcohol for 48 hours before blood collection. All donors gave written informed consent before to blood donations. Material to study was whole blood taken from a vein in the arm into the test-tube with anticoagulant - Heparin (10 U/ml).
All of the experiments were performed in accordance with good laboratory practice.
Polymorphonuclear leukocytes (PMNs) were isolated by centrifugation in density gradient Polymorphoprep™. This method enables to obtain 91% pure fraction of PMNs .
To purify neutrophils (up to 99,9%), cells positive selection using Midi-MACS magnetic separation system (Miltenyi Biotec, Bergisch Gladbach, Germany) that employed MicroBeads conjugated to monoclonal anti-human CD16 antibodies were performed .
The neutrophils survival was evaluated with use of trypan blue and light microscope and was equal 97%.
Assessment Of Cell Capacity To Chemotaxis By Boyden Chamber
Previously isolated neutrophils were suspended in PBS with CaCl2 and MgCl2 ions and incubated for 30 minutes in an incubator with 5% CO2 flow and 37 °C without stimulants, or in the presence of BPA (16 nM or 1.6 µM) or E2 (100 pM). Using the Boyden chamber (Neuro Probe, Gaithersburg, USA) enables the quantitative assessment of chemotaxis-capable cells using cell movement towards a chemotactic agent. Boyden chamber consists of two compartments separated by a filter with a pore diameter of 5 µm. The lower part of the chamber was filled with a chemoattractant solution – N-Formyl-Met-Leu-Phe (fMLP). Upper part of the chamber was filled out with suspension of 50,000 cells incubated with or without tested compounds. After an hour incubation in an incubator with a 5% CO2 flow and a temperature of 37 °C, the filter was removed by placing it on a slide. The preparation was stained using the May-Grunwald-Giemza method and evaluated under a light microscope using immersion oil by counting all the cells in the filter. Result were presents as percentage of cells which presents ability to chemotaxis.
Assessment of cell capacity to phagocytosis by Park’s method with latex
Blood collected on heparin was centrifuged at 2000 rpm/5 minutes. Then, neutrophils collected from the "leukocyte coat" fraction were incubated (5% CO2, 37 °C) for 30 min in the presence of BPA (16 nM or 1.6 µM) or E2 (100 pM). Latex (an exogenous beads) was added to the cells and absorbed into the cytoplasm via cells capable of phagocytosis. After incubation (55 minutes; (5% CO2, 37 °C)), smears were made, which were fixed and stained using the May-Grunwald-Giemza method. The preparations were evaluated under immersion in a light microscope by differentiating neutrophils in terms of the amount of absorbed latex beads into 4 classes: 0 class - neutrophils without absorbed latex deads, I class - neutrophils containing 1–10 latex beads, II class - neutrophils containing 11–30 latex beads, and III class - neutrophils containing more than 30 latex beads in cytoplasm. The application of this method allows to quantitative (percentage of cells capable of phagocytosis) and qualitative (social cohesion and reconciliation (SCORE) index)) assessment of phagocytosis. SCORE index is a result of multiplying the group’s name and number of neutrophils in that group.
Assessment of nicotinamide adenine dinucleotide phosphate hydrogen oxidase (NADPH) activity by Park’s test with nitroblue tetrazolium (NBT)
The use of the by Park’s test with nitroblue tetrazolium (NBT) allows to assess the effect of BPA and E2 on NADPH oxidase activity in human neutrophils. The method is based on the phenomenon of reduction of the absorbed into cells cytoplasm NBT into insoluble blue formazan crystals.
Whole blood samples were incubated for 30 minutes with BPA (16 nM or 1.6 µM) or E2 (100 pM). Then, NBT was added; moreover, latex beads which stimulate cells to absorption were added into NBT stimulated samples. The samples were then incubated for 15 minutes at 37 °C with a constant flow of 5% CO2 and 15 minutes at room temperature. Then, smears were made on slides, which were fixed and stained using the May-Grunwald-Giemza method. The preparations were evaluated using immersion oil using a light microscope. The results were expressed as percent of cells with formazan in spontaneous test or as percent of cells with formazan and latex in stimulated test.
Neutrophil Extracellular Traps
Isolated neutrophils were suspended in culture medium containing Rosewell Park Memorial Institute (RPMI) 1640, antibiotic, and serum (4%). Neutrophils were stimulated to form NETs on 96-well culture plates. Cells were introduced in the amount of 5 × 104 per well. Neutrophils were preincubated for 30 min at 37°C, 5% CO2 in the absence or presence of BPA (16 nM or 1.6 µM) or E2 (100 pM). Then, LPS was added to part of the samples to initiate NET formation by neutrophils. Neutrophil stimulation was performed for 1 h at 37°C, 5% CO2. Hoechst 3342 dye (Invitrogen) (at a concentration of 1 µg/ml prepared in phosphate-buffered saline (PBS), providing DNA detection) and antihuman myeloperoxidase (MPO) antibody (Life Technologies), which allowed detection of the main NET component, were added to the samples. The analysis of NET formation in wells was carried out under the confocal microscope–IN Cell Analyzer 2200 (GE Healthcare Life Sciences) using the IN Cell Analyzer Workstation program.
Cluster of Differentiation
The expression of cluster of differentiation (CD) antigens on cells suspended in PBS was assessed by the direct fluorescence method using Canto II flow cytometer (Becton Dickinson) and monoclonal antibodies (Becton Dickinson). 20 µl/5 µl of monoclonal antiCD14/antiCD284 antibodies, respectively, were added to 50 µl of the mixture. After 30 min of incubation in the dark, the samples were suspended in PBS and analyzed for 30 min using the FASCSDiva software.
Some of the cells were previously preincubated for 60 min with a wortmannin inhibitor (0.1 µM), Calbiochem (San Diego, CA), or an estrogen-receptor antagonist—ICI 182.780 (1 µM, ICI/Fulvestrant, Sigma Aldrich, 98% pure).
Assessment of total NO concentration in PMN supernatants by Griess reaction
Isolated neutrophils were suspended in Hank’s Salt Solution 1X (HBSS) culture medium (5 × 105 cells/ml) in the presence of donor serum, antibiotics: 100 U penicillin/ml and 50 ng streptomycin/ml. The microplates (Microtest III-Falcon) was incubated for 2 hours (temperature – 37 °C, with a constant flow of 5% CO2 (Nuarie™ US Autoflow, Plymouth, MN) in presence of BPA (16 nM and 1.6 µM) and E2 (100 pM).Some cells were previously preincubated 60 min with a wortmannin inhibitor (0.1 µM) Calbiochem (San Diego, CA) or ICI 182.780 (1 µM, ICI/Fulvestrant, SigmaAldrich, 98% pure). Then, the plate was centrifuged and the culture supernatants obtained were used to assess total nitric oxide (NO) concentration. Cells precipitate was collected and used to assess protein expression by Western Blot.
The assessment of NO production by neutrophils was performed using an indirect method based on measuring the concentration of NO2− ions in culture supernatants according to the Griess reaction. The total NO concentration is equal to the sum of the nitrate (III) and nitrate (V) concentrations. The Griess reaction detects only nitrates (III) and consists of a two-stage reaction. At first stage, neutrophils supernatants were incubated for 30 minutes with cadmium to reduce nitrates (V) to nitrates (III). Then, Griess reagent was added, and absorbance was read at 540 nm in spectrophotometer. Nitric oxide products were expressed as mM (5 × 105 cells in 270 ml supernatant).
The cytoplasmic fraction of neutrophils was isolated by the NucBuster™ Protein Extraction Kit (Merck). The isolated cytoplasmic protein was suspended in 2x Laemmli Sample Buffer (Bio-Rad) with βME (Bio-Rad). Protein in the amount of 10 µg/well was applied to polyacrylamide gel and electrophoresis under denaturing conditions (SDS-PAGE) was performed in Mini-PROTEAN® Tetra Cell (Bio-Rad). The Transfer Buffer solution was used to transfer the electrophoretically separated proteins from gel to nitrocellulose membranes on a 0.45 m roll (Bio-Rad) of the Mini-PROTEAN ® Tetra Cell apparatus. The next stages of the procedure were carried out in SNAP i.d.® 2.0 (Millipore) apparatus. The membrane was blocked in 1 × TBS 1% Casein Blocker (Bio-Rad). Nitrocellulose was incubated for 10 min at room temperature with primary antibodies: anti-p-PI3K goat polyclonal, anti-p-Akt1/2/3 (B-5) mouse monoclonal, anti-p-Akt1/2/3 (Ser473) rabbit polyclonal, anti-iNOS rabbit polyclonal (1:100, Santa Cruz Biotechnology). The membrane was rinsed three times in Tris Buffered Saline (Bio-Rad) with Tween® 20 (Sigma). Nitrocellulose was incubated for 10 min at room temperature with appropriate secondary antibodies labeled with alkaline phosphatase (1:5000, Santa Cruz Biotechnology). Again, the membrane was rinsed three times. Immunoreactive strips were obtained by adding BCIP®/NBT Liquid Substrate System (Sigma). The intensity of the stained strips was evaluated using ImageJ software (NIMH, Bethesda, MD). The obtained results are presented in arbitrary units.
All data are reported as the mean ± standard deviation (SD). Untransformed variables were statistically analysed if two criteria were met: data were normally distributed (assumption: SD was less than one-half of the mean of non-negative variables) and the Shapiro-Wilk W-test was insignificant. For the comparison between groups, the t-Student test was used. A significance level of p < 0.05 was considered statistically significant. STATISTICA version 13.3 program (StatSoft, Inc., Tulsa, OK) was used for all analyses.