Synovial fluid was obtained via knee aspiration from 27 arthritis patients (26 rheumatoid arthritis and 1 juvenile idiopathic arthritis patient) visiting the rheumatology outpatient clinic of the LUMC as part of standard clinical care. Written informed consent was obtained from all these donors. Anonymized leftover synovial fluid of two osteoarthritis patients was collected using a syringe before knee-replacement surgery performed at the departments of orthopedic surgery at the LUMC and the Alrijne hospital in Leiden. The study was approved by the local ethical committee.
Processing of synovial fluid for molecular measurements
On the same day of knee aspiration, the fluid was divided in two sets of 3–5 aliquots ranging between 300 μL and 1 mL to be able to fit the fluid in a heat block with vortex modus. 1 mg/mL hyaluronidase from bovine testis (Sigma-Aldrich) was added in a 1:11 ratio. Hyaluronidase was suspended just before use in phosphate-buffered saline (PBS, B.Braun). SF was vortexed for 5 min and subsequently incubated for 25 min at 37°C. Next, the first set of aliquots (set 1) was directly centrifuged for 10 min at 931xg, while the second set (set 2) was first pooled together before centrifugation. After centrifugation, the pooled set was divided again in 35 individual aliquots. Of each of the aliquots in set 1 and set 2, 100 μL supernatant was transferred to a glass vial (Agilent Technologies) together with 294 μL methanol (Fluka LC-MS CHROMASOLV grade, Sigma-Aldrich) and 6 μL internal standards (50 ng/mL leukotriene B4-d4, prostaglandin E2-d4, 15-HETE-d8, and 500 ng/mL of docosahexaenoic acid-d5, all from Cayman Chemicals). Argon gas was added and the samples were stored at −80°C. Similarly, 200 μL supernatant was also stored at −20°C for immunoglobulin and IL-8 analysis. For IL-6, IL-10, CXCL1, CXCL5, and TNFα analysis, SF was centrifuged for 10 min at 931xg on the day of knee aspiration and supernatant was stored directly at −80°C. At the day of analysis, SF was thawed to room temperature and treated as described above for the fresh SF.
Interleukin 8 (IL-8) and IgG ELISA were performed according to manufacturer’s protocol (Invitrogen, BD Biosciences, and Bethyl Laboratories, respectively).
Bio-Plex pro reagent kit III and Bio-Plex Pro Human IL-6 set (171BK29MR2), IL-10 set (171BK32MR2), TNF-α (171BK55MR2), CXCL1 (171BK22MR2), and CXCL5 (171BK14MR2) were purchased from Bio-rad. The cytokines and chemokines were determined according to the manufacturer’s protocol. SF samples were measured on the Bio-Plex 200 system (Bio-Rad), and analysis was done using Bio-plex Manager 6.2 Software (Bio-Rad).
Lipidomic analysis of synovial fluid
LC-MS/MS-based lipid mediator and oxylipin profiling were carried out as described elsewhere . A QTrap 6500 mass spectrometer (Sciex) was used, coupled to a Shimadzu Nexera LC30-system including auto-sampler and column oven (Shimadzu). The column was a Kinetex C18 50 × 2.1 mm, 1.7 μm, protected with a C8 pre-column (Phenomenex). LC-MS/MS peaks were integrated with manual supervision, and the areas were corrected to corresponding IS using MultiQuant™ 2.1 (Sciex). For quantitation, the multiple reaction monitoring (MRM) transitions and collision energies (CE) were used together with calibration lines for quantification. Calibration lines were constructed using 15-HETE, 17R-HDHA, leukotriene B4, prostaglandin E2, arachidonic acid, docosahexaenoic acid, and eicosapentaenoic (Cayman Chemicals)
Flow cytometry analyses of synovial fluid cells
For general analysis, the fluid was centrifuged for 10 min at 931xg and cells were collected and analyzed. The supernatant was treated with hyaluronidase as stated above and subsequently centrifuged for 10 min at 931xg to isolate remaining cells. For the effect of hyaluronidase treatment on cell marker expression, the SF was diluted 20x in PBS and divided in two. One sample was treated with hyaluronidase (treated), and the other sample was treated similarly, but without the addition of hyaluronidase (untreated). Isolated cells were resuspended in PBS and filtered through a 70-μm cell strainer before flow cytometry. For general cell characterization, cells were stained with anti-CD3 (AF700, clone UCHT1), anti-CD14 (FITC, clone M5E2), anti-CD15 (APC, clone HI98), anti-CD16 (PE, clone B73.1), anti-CD19 (PerCp/Cy5.5, clone SJ25C1), and anti-CD45(APC/Cy7, clone 2D1). The gating strategy is depicted in supplementary figure 1. For additional characterization into CD4+ T cells and CD8+ T cells, cells were stained with anti-CD3 (PE, clone SK7), anti-CD4 (APC, clone SK3), and anti-CD8 (FITC, clone SK1). For the effect of hyaluronidase treatment on cell marker expression, cells were stained with anti-CD3 (Pacific Blue, clone SK7), anti-CD4 (APC, clone SK3), anti-CD8 (FITC, clone SK1), anti-CD19 (APC/Cy7, clone SJ25C1), anti-CD44 (PE/Cy7, clone G44-26), and anti-CD69 (PE/CF594, clone FN50). The positivity for CD44 and CD69 was determined using isotype controls IgG2bk (PE/Cy7, clone 27-35) and IgG1 (PE/CF594, clone X40). For additional characterization into naïve B cells, memory B cells and plasmablasts/cells, cells were stained with anti-CD3 (Pacific Blue, clone SK7), anti-CD14 (Pacific Blue, clone M5E2), anti-CD19 (APC/Cy7, clone SJ25C1), anti-CD20 (AF 700, clone 2H7), and anti-CD27 (PE/Cy7, clone M-T271). For gating strategy for the T cell and B cell subsets, see supplementary figure 2.
All antibodies were from BD except for the anti-CD20 which was from Sony Biotechnology (USA, CA, San Jose). For all analysis, dead cells were excluded using DAPI (Molecular Probes) and cells were quantified using Flow count Fluorospheres (Beckman Coulter). Cells were measured on a LSR Fortessa (BD) and were analyzed with FACSDiva Software (BD).
Statistical analyses and calculations
Wilcoxon signed rank tests were performed to evaluate significance between groups in all figures. Coefficient of variation (CV) was calculated by dividing the standard deviation of a group of measurements by the mean and expressing this as a percentage.