Microparticles from patients with systemic lupus erythematosus induce production of reactive oxygen species and degranulation of polymorphonuclear leukocytes

SLE patients were included from our inpatient and outpatient rheumatology clinic and fulfilled internationally accepted classification criteria [24, 25]. Disease activity was described using the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI)-2 K 30 days [26] and other available clinical information. The healthy controls included were anonymous blood donors at the Blood Bank of Copenhagen University Hospital, Rigshospitalet. The study was approved by the regional scientific ethics committee (protocol no. H-1-2013-046). For experiments including only SLE and healthy control sera or MPs as variables, frozen samples from this study and an earlier study [19] were used. Patient characteristics are presented in Table 1.

MPs were purified as described previously [19]. In brief, 6 ml of blood was collected in a K₂EDTA tube (Vacuette; Greiner Bio-one GmbH, Kremsmünster, Austria) by venous puncture. After transfer of 600 μl of the blood to other tubes for the purification of leukocytes, as described later, the blood was centrifuged at 1800 × g for 10 min at 37 °C and supernatants were transferred to a new tube and centrifuged at 3000 × g for 10 min at 37 °C for removal of platelets. The platelet-poor plasma was then filtered through a 1.2-μm syringe filter (Minisart; Sartorius, Göttingen, Germany) and divided into aliquots of 460 μl. Each aliquot was added 10% RPMI-1640 (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and ultracentrifuged twice at 19,000 × g for 30 min at 21 °C; the first step was followed by washing in RPMI, and the last step was followed by resuspension in RPMI at a total volume of 100 μl.

Aliquots of 200 μl K₂EDTA blood were stored at 4 °C for approximately 1 hour, until the last step of the MP purification. Afterwards, the red blood cells were removed by 15 min of lysis in a 1:10 dilution of BD Pharm Lyse Lysing Buffer (BD Biosciences, San Jose, CA, USA), according to the manufacturer’s instructions. After lysis, the solution was centrifuged twice at 300 × g for 5 min at room temperature; the first step was followed by washing in Dulbecco’s Phosphate Buffered Saline (Thermo Fisher Scientific), and the final volume was adjusted to 200 μl. Aliquots of 36.6 μl of the cell solution were incubated for 30 min at 37 °C in RPMI-1640 containing 25% (v/v) serum at a total volume of 200 μl with no stimulation, 10 μg/ml E. Coli LPS (Sigma, Merck, Kenilworth, NJ, USA), 20 μl of purified MP solution, 10 μg/ml LPS plus 20 μl of purified MP solution or 60 nM PMA (Sigma). All samples contained 0.66 mM dihydrorhodamine 123 (DHR) (Molecular Probes, Thermo Fisher Scientific), which is oxidized intracellularly by H₂O₂ to fluorescent rhodamine 123. An additional aliquot was incubated without DHR to measure background fluorescence. After incubation, the samples were washed in cold PBS and centrifuged at 740 × g for 10 min at 4 °C. Supernatants were removed for analysis of granule content (see later) and cells were incubated with anti-CD15 APC (BD Biosciences) for 30 min at 4 °C. The LIVE/DEAD Fixable Near-IR Dead Cell Stain (Molecular Probes) was used to discriminate between live and dead PMNs. After a similar wash, cells were analysed by flow cytometry using a BD FACSCanto II (BD Biosciences). Additional file 1 shows the gating strategy.

In another series of experiments, leukocytes from SLE patients and healthy controls were stimulated with the previously described stimuli in the presence of either autologous serum or normal human serum (NHS) from of a pool of sera from blood group AB-positive donors (Sigma Aldrich, Merck). In a third series, leukocytes from a single healthy donor were stimulated in the presence of various sera from SLE patients or healthy controls. In a fourth series of experiments, leukocytes from healthy controls were stimulated in RPMI-1640 containing autologous serum supplemented with IgG purified from sera of five SLE patients, as already mentioned, or with normal IgG for intravenous use (IVIg; CSL Behring, King of Prussia, PA, USA). In a fifth series of experiments, leukocytes from a healthy control, suspended in RPMI containing autologous serum, were stimulated with MPs from various SLE patients or healthy controls. Table 2 presents an overview of the different experimental conditions applied.

Supernatants were kept frozen at –80 °C until analysis of granule content using R&D Magnetic Luminex Screening Assays (R&D Systems, Minneapolis, MN, USA) on a Luminex Bio-Plex® 200 system (Bio-Rad Laboratories, Hercules, CA, USA). The content of MPO from primary granules was analysed with the aid of a one-plex MPO, and the content of NGAL and gelatinase (MMP-9) from secondary and tertiary granules, respectively, was analysed using a two-plex MMP-9 and Lipocalin-2/NGAL. Analysis of the fluorescence data was done using Bio-Plex Manager 6.0 Software (Bio-Rad Laboratories). The experiments were carried out according to the manufacturer’s instructions.

Because the study data were not normally distributed, non-parametric statistical methods were applied. The effects of different stimuli were investigated using the Wilcoxon matched-pairs signed rank test. Comparisons between groups were done using the Mann–Whitney U test, and correlations were determined using Spearman’s correlation coefficient (r _s). The differences between ratios were calculated using a Wilcoxon signed rank test where the difference from a theoretical value (=1) was tested.