All patients fulfilled the American College of Rheumatology classification criteria for RA, or for systemic lupus erythematosus, respectively. Healthy volunteers, matched for gender and age, were recruited at the hospitals or at the Blood Bank of the Swiss Red Cross, Basel. Inclusion criteria for healthy controls were fair general condition, age ≥28 and ≤70 years, and for blood donors fulfilling national criteria for blood donation. Exclusion criteria were current or previous systemic autoimmune disease, asthma and reconvalescence after major illness, surgery, current medication with corticosteroids, immunosuppressive agents, and malignant neoplasia or chemotherapy within 5 years before recruitment for the study. RA cases had a DAS ≥2.6, were from age ≥27 to ≤70 years, and had no other systemic autoimmune disease, including ankylosing spondylitis and psoriatic arthritis.
Exclusion criteria were corticosteroids ≥40 mg equivalent of prednisone daily, and those mentioned earlier for healthy controls. Informed, written consent was obtained from all subjects in the study, which was approved by the Cantonal Ethical Review Boards of Aargau-Solothurn and Basel/Basel-Land, Switzerland.
Preparation of plasma and serum
Plasma and serum were collected and processed as described previously
. Samples were studied immediately or stored at -80°C until analysis.
PMNs were isolated with Dextran-Ficoll density centrifugation
. Cell viability was 96% to 98%, with a purity of >95% PMNs. Neutrophils seeded in 24-well plates were allowed to settle for 1 hour at 37°C under 5% CO2 before further experimentation.
Cell-free DNA isolation and quantification
Cell-free DNA extracted from 850 μl plasma or serum by using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, CA, USA) was quantified by TaqMan real-time PCR (StepOne Plus Real-Time PCR System, Applied Biosystems, Foster City, CA, USA) specific for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene
Detection of neutrophil elastase (NE), myeloperoxidase (MPO), and cell-free nucleosomes
The concentrations of neutrophil elastase (NE) and myeloperoxidase (MPO) were measured with sandwich ELISA (Elastase/a1-PI Complex ELISA Kit, Calbiochem/EMD, Gibbstown, NJ, USA) and the human (MPO ELISA Kit, Hycult Biotech, Plymouth Meeting, PA, USA) respectively. Nucleosomes were measured by using the Human Cell Death Detection ELISAPLUS (Roche Diagnostics, Basel, Switzerland). Cell-culture supernatants were incubated with DNaseI (10 U for 5 minutes) (Roche Diagnostics) before analysis
MPO/DNA complex detection
MPO is present on extruded NETs. To detect such structures, NET-associated MPO/DNA complexes were quantified by using a modified capture ELISA
. In brief, NET-associated MPO in serum or culture supernatant was captured by using the coated 96-well plate of the human MPO ELISA Kit, (Hycult Biotech), after which the NET-associated DNA backbone was detected by using the detection antibody of the Human Cell Death Detection ELISAPLUS (Roche Diagnostics).
To detect the presence of PAD4 on extruded NETs in culture supernatants after spontaneous NETosis, cell-free PAD4/DNA complexes were quantified by using a modified capture ELISA, akin to that described for MPO earlier. In brief, cell-free PAD4 was captured by using the coated 96-well plate of a commercial human PAD4 ELISA (USCN Life Science, Inc., Wuhan, China), and associated DNA was detected by using Human Cell Death Detection ELISAPLUS kit (Roche Diagnostics).
ROS generation analysis
ROS was measured by using a 2′,7′-dichloro dihydrofluorescein diacetate (DCFH-DA) assay
. The 5 × 105 cells in a final volume of 500 μl were incubated for 30 minutes with 25 μM DCFH-DA (Sigma-Aldrich, St. Louis, MO, USA). Fluorescence was measured with flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA).
Fluorescence and scanning electron microscopy
The 5 × 104 cells isolated PMNs seeded on poly-L-lysine-coated coverslips (BD Biosciences) were stimulated with phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich) for 90 minutes and dehydrated with a graded ethanol series (30%, 50%, 70%, 100%)
, coated with 2-nm platinum, and analyzed with a Nova NanoSEM 230 scanning electron microscope (FEI Co., Hillsboro, OR, USA). PMNs were incubated for 10 minutes with 5 μM Sytox Green dye (Invitrogen Life Technologies, San Diego, CA, USA) for assessment of NETs with an Axiovert fluorescence microscope (Carl Zeiss) coupled to a Zeiss AxioCam color CCD camera (Carl Zeiss Microimaging, Oberkoch, Germany)
Immunohistochemical staining and quantification of NETs
The 5 × 104 isolated PMNs were seeded on poly-L-lysine-coated glass coverslips (BD Biosciences, San Jose, CA, USA) in tissue-culture wells and allowed to settle before stimulation, as described earlier. Coverslips were rinsed with ice-cold HBSS and the cells fixed with 4% paraformaldehyde and blocked overnight (HBSS with 10% goat serum, 1% BSA, 0.1% Tween20, and 2 mM EDTA) at 4°C. NETs were detected with rabbit anti-NE (Abcam, Cambridge, MA, USA), rabbit anti-MPO (Dako, Glostrup, Denmark), two different rabbit anti-PAD 4 (Abcam), mouse anti-PAD4 (Abcam), mouse anti-histone H1 + core proteins (EMD Millipore, Billerica, MA, USA), and rabbit anti-citrullinated histone H3 (citH3, Abcam). Secondary antibodies were goat anti-rabbit IgG AF555, goat anti-rabbit IgG AF488 (Invitrogen Life Technologies, San Diego, CA, USA), and goat anti-mouse IgG AF647. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), and NETs were visualized by using a Zeiss Axioplan 2 Imaging fluorescence microscope in conjunction with a Zeiss AxioCam MRm monochromatic CCD camera and analyzed with Axiovision 4.8.2 software (Carl Zeiss). A minimum of 20 fields (at least 1,000 PMNs) per case was evaluated for MPO/NE and DNA co-staining; nuclear phenotypes and NETs were counted and expressed as percentage of the total number of cells in the fields.
RA serum depletion, IgG purification, and quantification of NETs
After three washes with PBS, 200 μl protein G agarose (Pierce Biotechnology Inc, Rockford, IL, USA) was incubated with 200 μl ACPA + and ACPA- RA or control serum diluted in an equal volume of phenol red-free RPMI 1640 medium overnight at 4°C. The serum/protein G agarose mixture was centrifuged at 2,500 g for 5 minutes, and the supernatant (IgG-depleted serum) was carefully transferred into a new Eppendorf microcentrifuge tube. The protein G agarose pellet was gently washed 3 times with 500 μl ddH2O, and the bound antibody was released by the addition of 50 μl 0.1 M glycine pH 2–3, and immediately equilibrated with 10 μl of 1 M Tris pH 7.5-9. All protein concentrations were determined with the MN Protein Quantification Assay (Macherey-Nagel GmbH, Düren, Germany), and isolation of IgG was verified with Coomassie staining of SDS-PAGE.
Neutrophils from healthy donors (n = 3) were isolated and cultured for 2 hours in 96-well culture dishes (Thermo Fischer Scientific, Waltham, MA, USA), supplemented with: serum, depleted serum, and purified IgG from ACPA-positive RA patients (n = 3), ACPA-negative RA patients (n = 3), and healthy individuals (n = 3) to a final concentration of 100 μg/ml.
NETs were quantified after IHC staining with mouse anti-human MPO antibody (Abcam) and rabbit anti-human citH3 antibody (Abcam), or the respective isotype controls, followed by incubation with goat anti-mouse IgG AF555 and goat anti-rabbit IgG AF488 (Invitrogen Life Technologies). DNA was counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich). NETs were visualized by using an Olympus IX81 motorized epifluorescence microscope (Olympus America Inc., Center Valley, PA, USA) in conjunction with an Olympus XM10 monochromatic CCD camera (Olympus) and analyzed with the Olympus CellSens Dimension software (Olympus). A minimum of 20 fields at 10× magnification (at least 500 to 1,000 PMNs) per case was evaluated for MPO/citH3 and DNA co-staining through ImageJ analysis software (National Institutes of Health Image Processing, Bethesda, MD, USA); nuclear phenotypes and NETs were determined, counted, and expressed as percentage of the total area of cells in the fields
Protein isolation and Western blot analysis
Total protein was isolated with NucleoSpin TriPrep kit (Macherey-Nagel) from 3 × 106 PMNs. Proteins from the nuclear and cytoplasmic fractions were isolated by using the Nuclear and Cytoplasmic Protein Extraction Kit (Thermo Scientific). Western blotting was performed by using AnykD Mini-PROTEAN TGX Gels (Biorad, Hercules, CA, USA) and nylon/nitrocellulose membranes (Biorad). Primary and secondary antibodies used were: rabbit anti-PAD4 (Abcam), rabbit anti-MPO (Cell Signalling Technologies, Beverly, MA, USA), mouse anti-β-Actin (Sigma-Aldrich), goat anti-Mouse and/or anti-Rabbit, human anti-HRP (Southern Biotech). HRP activity was detected by using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Equal loading was verified by using beta-actin or histone H3, when appropriate. Western blots of citrullinated H3 (citH3) protein were performed according to Shechter et al.
. Densitometric analysis and protein quantification of the Western blots was performed by using the ImageJ software.
RNA isolation and quantitative real-time PCR
Total RNA was isolated by using RNeasy Mini Kit (Qiagen). TaqMan real-time quantitative RT-PCR was performed by using the Applied Biosystems StepOne Plus cycler (Applied Biosystems) and TaqMan Gene Expression Assay primer/probe sets (Applied Biosystems) for ELANE (HS00236952_m1), MPO (HS00924296_m1), PADI4 (HS00202612_m1), and β2-microglobulin B2M (HS99999907_m1). Data were normalized by using the housekeeping gene B2M, after a selection procedure involving six different endogenous reference genes, as suggested in the MIQE guidelines
. Relative values were calculated with 2-DDCt analysis
All data are presented as mean ± SEM. Descriptive statistics for continuous parameters consisted of median and range, and categoric variables were expressed as percentages. Comparisons between patients and healthy controls were by the Mann–Whitney U test with a Welch posttest correction. Statistical significance in multiple comparisons was by one-way analysis of variance (ANOVA) with a Dunn posttest correction. P < 0.05 was considered statistically significant.
Receiver operating characteristic (ROC) curves were calculated, and the area under the curve (AUC) with corresponding standard errors of means was calculated. Data were processed in GraphPad Prism version 5.0b for MacOSX (GraphPad Software Inc., San Diego, CA, USA). Analysis of covariance (ANCOVA) was conducted with SPSS version 21.0 statistical software (IBM SPSS Inc., Chicago, IL, USA). Additional professional statistical assistance was provided by A. Schoetzau, Basel.