Ethics Statement and Patients
Written informed consent was obtained from healthy volunteers and patients with RA and osteoarthritis. All RA patients fulfilled the 2010 ACR/EULAR classification criteria for RA [20]. Serum was collected from 40 RA patients and 40 healthy volunteers. SF was collected from 32 RA patients and 32 patients with osteoarthritis of knee joints. All SF samples were obtained through therapeutic arthrocentesis before steroid injection into the swollen joints. SF mononuclear cells (SFMC) were obtained from 10 RA patients and 10 patients with knee osteoarthritis. All protocols were performed in accordance with relevant guidelines and regulations and were approved by the Institutional Review Board for Human Research in Konkuk University Hospital (KUH1010960).
Reagents
Recombinant IL-33, RANKL, and M-CSF were purchased from R&D systems (Minneapolis, MN).
Human Mast Cell Line Culture And Stimulation
The human mast cell line was purchased from Kerafast (Kerafast, MA, USA) and maintained in StemPro-34 SFM at a concentration of 5 × 105 cells/ml. The cells were grown at 37 °C with 5% CO2, and the medium was exchanged weekly with 50% fresh medium without exogenous cytokines. The mast cells were seeded in 6-well plates at a density of 5 × 104 cells/ml. After one day, the medium was replaced with StemPro-34 SFM culture medium with or without cytokines. The mast cells were then stimulated with 100 ng/mL human recombinant IL-33 (R&D Systems, Inc.) for 24 and 72 h. The supernatants and RNA were harvested and stored at − 80 °C until analysis.
Enzyme-linked Immunosorbent Assay (ELISA)
SF and serum samples obtained from healthy volunteers and patients with RA and osteoarthritis were subjected to tryptase, chymase, and histamine analyses using tryptase (Boster Biological Technology, CA), chymase (Aviva Systems Biology, CA), and histamine (Enzo Life Sciences Inc., NY) ELISA kits, according to the manufacturers’ instructions. The levels of cytokines such as TNF-α, IL-1, IL-6, IL-17, RANKL, MMP-9, and MMP-13 in the culture supernatants from human mast cells were measured using sandwich ELISA (R&D Systems), according to the manufacturer's instructions. Absorbance at 405 nm was also measured using an ELISA microplate reader (Molecular Devices, CA).
Flow Cytometry
In the samples used for in vitro experiments, flow cytometry was performed after collecting the mast cells. The cells were first stained with monoclonal antibodies. For surface staining, the cells were stained with mAbs against CD117 (c-kit)-APC (YB5.B8, IgG1κ) (eBioscience, CA). The cells were then washed, fixed, permeabilized, and stained with mAbs against FcεRI alpha-PE-Cy7 (AER-37-CRA1, IgG2b) (eBioscience), tryptase-FITC (G3, IgG1κ) (Santa Cruz Biotechnology, Inc, Texas), and chymase-PE (CC1, IgG1κ) (Santa Cruz Biotechnology, Inc) to detect intracellular cytokines. Appropriate isotype controls were used for gate setting. Cells were analyzed using a FACSCalibur flow cytometer and FlowJo software.
Immunocytologic Staining And Confocal Microscopy
To measure changes in protein expression, 4 µm-thick formalin-fixed and paraffin-embedded (FFPE) synovial sections were deparaffinized in xylene and rehydrated in ethanol and deionized water. Antigen retrieval was performed by heating these sections in a buffer solution (citrate buffer pH 6) (DAKO, Glostrup, Denmark) at 120 °C for 15 min. Slides were washed thrice (5 min each) with phosphate-buffered saline (PBS). The slides were then removed from PBS and each section was covered with 3% H₂O₂ solution (Sigma Aldrich, Saint Louis, Missouri (MO)) for 15 min at room temperature to block endogenous peroxidase activity. After washing, non-specific binding was blocked by incubation in 10% normal goat serum in PBS for 30 min at room temperature. The blocking buffer was then removed and the sections were incubated overnight with c-kit (CD117) polyclonal antibody (DAKO), FcεR1 monoclonal antibody (Abcam, Cambridge, MA), FcεR1 polyclonal antibody (LSBio, Seattle, WA), FITC-conjugated tryptase antibody (Santa Cruz, California), and chymase monoclonal antibody (MBL, Nagoya, Japan) at 4 °C. The sections were then washed and incubated with the secondary antibodies, anti-rabbit IgG Alexa Fluor 594 and anti-mouse IgG-APC (Invitrogen, Carlsbad, California), for 2 h at room temperature. The nuclei were also stained with DAPI (Invitrogen). The slides were then covered with a fluorescent mounting medium (DAKO) and the stained sections were visualized under a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany) at 200 × and 400 × magnifications.
Real-time PCR For mRNA Quantitation
mRNA was extracted from the samples using RNAzol B, according to the instructions of the manufacturer (Biotex, Friendswood, TX). The total mRNA (2 µg) was reverse transcribed at 42 °C using a SuperScript RT system (Takara). PCR was performed at a final volume of 20 µl in capillary tubes in a LightCycler (Roche Diagnostics). The reaction mixture contained 2 µl LightCycler FastStart DNAMaster Mix for SYBR Green I (Roche Diagnostics), 0.5 µM of each primer, 4 mM MgCl2, and 2 µl of the template DNA. All the capillaries were amplified in a LightCycler with the following program: activation of polymerase at 95 °C for 10 min, followed by 45 cycles of 10 s at 95 °C and 10 s at 60 °C (beta-actin, IL-17, MMP-13) or 57 °C (TNF, IL-1β, IL-6, RANKL, MMP-9), and a final 10 s at 72 °C. The temperature transition rate was 20 °C/s for all steps. Melting curve analyses were performed immediately after amplification, using the following program: 0 s (hold time) at 95 °C, 15 s at 71 °C, and 0 s (hold time) at 95 °C. The rate of temperature change was 20 °C/s for all steps except the final step, during which it was 0.1 °C/s. The melting peaks generated represented the quantity of each amplified product. The crossing point was defined as the maximum of the second derivative from the fluorescence curve. Negative controls, which contained all elements of the reaction mixture except for the template DNA, were also included. All samples were processed in duplicate.
Isolation Of Peripheral Blood (PB) Monocytes And Osteoclast Differentiation
PB mononuclear cells were separated, washed thrice with sterile PBS, and resuspended in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin-streptomycin (henceforth called complete medium). Freshly isolated PBMCs were incubated at 37 °C in the complete medium and allowed to adhere for 45 min. The non-adherent cells were removed, and the adherent cells were washed with sterile PBS, harvested with a rubber policeman, and stained with monocyte-specific anti-CD14 monoclonal antibodies to assess the purity of the preparation. Ninety percent of the isolated cells were monocytes expressing CD14. Osteoclast precursors were prepared using the monocyte-enriched fraction from PB. The cells were co-cultured for 3 weeks in minimal essential medium (MEM)-α with 10% heat-inactivated FBS in the presence of 25 ng/ml rhM-CSF and IL-33/IL-33-stimulated mast cells. The medium was changed on day 3 and then on alternate days thereafter. On day 21, tartrate-resistant acid phosphatase (TRAP)-positive cells were identified using a leukocyte acid phosphatase kit, according to the manufacturer’s instructions (Sigma-Aldrich). The osteoclast precursors and mast cells/IL-33-stimulated mast cells were co-cultured in 24-well transwell plates separated by the membrane in lower and upper chambers, respectively (Costar, New York, NY). The medium was changed on day 3 and then every other day thereafter. On day 21, TRAP-positive cells were identified using a leukocyte acid phosphatase kit, according to the manufacturer’s protocol (Sigma-Aldrich).
Western Blotting
PB monocytes were incubated with or without IL-33 in the presence of RANKL. After incubation for 1 h, whole-cell lysates were prepared from approximately 1⋅107 cells by homogenization in the lysis buffer and then centrifuged at 14,000 rpm for 15 min. Protein concentration in the supernatant was determined using the Bradford method (Bio-Rad, Hercules, CA). Protein samples were separated using 10% SDS–PAGE and transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). For western blotting, the membrane was pre-incubated with 0.5% skim milk in 0.1% Tween-20 and Tris-buffered saline (TTBS) at room temperature for 2 h. Primary antibodies against TRAF6, phospho-Src, Src, phospho-JNK, JNK, phospho-ERK, ERK, phospho-p38, p38, phospho-Akt, Akt, phospho-IκBα, IκBα, phospho-c-Jun, and c-Jun (Cell Signaling Technology Inc., Danvers, MA), diluted 1:1000 in 5% BSA-0.1% Tween-20/TBS, were added and incubated overnight at 4 °C. After washing the membrane 4 times with TTBS, horseradish peroxidase-conjugated secondary antibody was added and incubated for 1 h at room temperature. After TTBS washing, hybridized bands were detected using the ECL detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia).
Statistical analysis
All data are expressed as mean ± standard error of the mean (SEM). Statistical differences between two groups were assessed using Mann-Whitney U test and those between more than three groups were assessed using one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison post-hoc test. Differences with p < 0.05 were considered statistically significant.