Easy-blue total RNA extraction kit for total RNA isolation was purchased from iNtRON Biotechnology (Seoul, Korea). Dulbecco’s phosphate-buffered saline, penicillin-streptomycin, and fetal bovine serum (FBS) were purchased from Gibco/BRL (Life Technologies, Gaithersburg, MD, USA). Ovalbumin (OVA), phytohemagglutinin (PHA), and lipopolysaccharides (LPS) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Interleukin (IL)-1β and monocyte chemotactic protein-1 (MCP-1) were obtained from R&D Systems (Minneapolis, MN, USA). The Qiagen Mini kit was purchased from Qiagen Sciences (Germantown, MD, USA). LightCycler FastStart DNA SYBR Green I Mix was obtained from Roche (Mannheim, Germany). Rabbit anti-human Sulf-1 polyclonal antibody and rabbit anti-human Sulf-2 were purchased from Santa Cruz Biotechnology (CA, USA). Antibodies against CD68 were purchased from Abcam Inc. (Cambridge, MA, USA); goat anti-mouse immunoglobulin G (IgG) Alexa Fluor 488, goat anti-rabbit IgG Alexa Fluor 546, rabbit anti-goat IgG Alexa Fluor 546, normal goat serum, and normal rabbit serum from Invitrogen Corp. (Carlsbad, CA, USA). Primer sequences for Sulf-1, Sulf-2, and β-actin were synthesized by Bionics (Daejeon, Korea). Sulf-1 and Sulf-2 small interfering RNA (siRNA) sequences were purchased from Santa Cruz Biotechnology. Negative control siRNA was purchased from Invitrogen (Carlsbad). pcDNA3.1(–) vector for transfection was obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). pcDNA3.1/myc-His(–) Sulf-1 and pcDNA3.1/myc-His(–) Sulf-2 from Addgene Inc. (Cambridge, MA, USA).

With informed consent, synovial tissues were obtained from patients with rheumatoid arthritis (RA) who underwent total knee replacement surgery at Yeungnam University Medical Center. A CD68 primary antibody specific to macrophages and anti-Sulf-1, Sulf-2 primary antibodies were applied in parallel for immunofluorescence after pretreatment for paraffin sections and blocking. After overnight incubation with the primary antibodies at 4°C, the sections were treated with the respective secondary antibodies labeled with fluorescence for 30 minutes avoiding light at room temperature. Cell nuclei were counterstained with Hoechst 33258 diluted at 1:5,000 in distilled water for 2 minutes protected from light at room temperature. Expression of Sulf-1, Sulf-2, and CD68 was observed using a fluorescence microscope, and the images were merged for double staining using Photoshop 7.0 (Adobe Systems Inc., San Jose, CA, USA).

Peripheral blood mononuclear cells (PBMCs) were isolated by endotoxin-free Ficoll-Paque PLUS centrifugation (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) from buffy coats obtained from healthy adult blood donors at Yeungnam University Medical Center. PBMCs were cultured at a cell concentration of 10⁶ cells/mL in RPMI 1640 medium (Lonza, Basel, Switzerland) supplemented with 10% FBS and 1% penicillin-streptomycin. Prior to stimulation, plates were incubated for 1.5 hours (37°C, 5% CO₂, 100% humidity).

Specific pathogen-free, 7-week-old inbred BALB/c mice (six mice, three experiments/mouse) were purchased from Central Lab Animal Inc. (Seoul, Korea). The utmost precautions were taken to ensure that the mice remained free from infection by environmental pathogens. The mice were cared for in accordance with the principles of the Guide to the Care and Use of Experimental Animals of Yeungnam Medical Center. Peritoneal macrophages were obtained by lavage using Hanks’ balanced salt solution (HBSS) 4 days after injection with 2 mL of 4% thioglycollate medium (BBL-Becton Dickinson, Cockeysville, MD, USA). Macrophages in complete medium (RPMI 1640 supplemented with 1% penicillin-streptomycin and 10% FBS) were plated in six well tissue culture plates, incubated for 2 hours at 37°C in an atmosphere of 5% CO₂, and then washed three times with HBSS to remove any non-adherent cells. Macrophages were cultured overnight in a complete medium at 37°C in 5% CO₂. The medium was then replaced with serum-free RPMI 1640, and the cells were cultured in the presence or absence of stimuli for the indicated times.

Macrophages were transfected with Sulf-1 and Sulf-2 siRNA oligomers (50 nmol/L) using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s instructions. After 24 hours of incubation, the macrophages were placed in a growth medium for 24 hours before the experiments.

pcDNA3.1, pcDNA3.1/Sulf-1, and pcDNA3.1/Sulf-2 plasmids were amplified after transformation into MAX Efficiency DH5αCompetent cells on LB medium with 100 µg/mL ampicillin. The amplified plasmids were purified from cell lysates using the Qiagen Mini kit, according to the manufacturer’s instructions. One microgram of each complementary DNA (cDNA) was transfected into macrophages using 2.5 µL of Lipofectamine 2000 reagent diluted in Opti-MEM (Thermo Fisher Scientific) separately.

Macrophages were cultured with OVA (2 mg/mL) for 24 hours. T cell hybridomas (DO-11-10, 10⁵ cells/well) were added, and the cultures were incubated for 48 hours. The cultures were then centrifuged at 1,000×g, followed by the removal of 100 µL of the supernatant. The cell supernatant fluids were assayed for IL-2 activity immediately or frozen at −70°C until use. The supernatant fluids were assayed for the presence of IL-2 using the IL-2 dependent CTLL-2 cell line. CTLL-2 cells (10⁴ cells/well) were added to 100 µL of culture supernatant and incubated for 40 hours. The cultures were then pulsed overnight with [³H]-thymidine (1 µCi/well) (Perkin Elmer, Boston, MA, USA). [³H]-Thymidine-labeled cells were collected, and radioactivity was measured using a Packard scintillation counter (Packard Instrument Co., Meriden, CT, USA).

Mouse peritoneal macrophages were transfected with siRNA oligomers or control plasmid DNA. After 24 hours of incubation, a phagocytosis assay using a Cytoselect 96-well phagocytosis assay kit was performed according to the manufacturer’s instructions (Cell Biolabs, San Diego, CA, USA). Images of phagocytic macrophages were taken with a Leica DFC 495 camera (Leica Microsystems, Cambridge, UK) mounted on a Nikon microscope (Eclipse TE300; Nikon, Tokyo, Japan) at 200× magnification.

Total RNA was extracted using the easy-BLUE Total RNA Extraction Kit (iNtRON Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. One microgram of total RNA per sample was reverse-transcribed using a Maxime RT premix kit (iNtRON Biotechnology, Daejeon, Korea) according to the manufacturer’s instructions. cDNA synthesis was performed at 45°C for 60 minutes, followed by real-time (RT) inactivation at 95°C for 5 minutes. Sulf-1 and Sulf-2 were amplified by RT polymerase chain reaction (PCR) using a LightCycler (Roche). The total PCR volume was 20 µL, and each PCR reaction consisted of LightCycler FastStart DNA SYBR Green I mix (Roche), primer, and 2 µL of cDNA. Prior to PCR amplification, the mixture was incubated at 9°C for 10 minutes. The amplification step consisted of 45 cycles of denaturation (10 seconds at 95°C), annealing (5 seconds at the primer-appropriate temperature), and extension (10 seconds at 72°C) with fluorescence detection at 72°C after each cycle. After the final cycle, melting point analyses of all samples were performed over a temperature range of 65°C–95°C with continuous fluorescence detection. β-Actin was used as a reference gene for the normalization of sample expression levels. The primers used for PCR were as follows: Sulf-1 (195 bp) sense, 5′-tgctgaacagtcaccctgatccaa-3′; antisense, 5′-tcagatgcagggtttggaggttga-3′; Sulf-2 (195 bp) sense, 5′-tcaaagtgacccatcggtgctaca-3′, antisense, 5′-agtcacattcttccggtcgcttct-3′; and β-actin (148 bp) sense, 5′-agagggaaatcgtgcgtgac-3′, antisense, 5′-caatagtgatgacctggccgt-3′’. The mRNA levels of Sulf-1 and Sulf-2 were determined by comparing experimental levels to standard curves and were expressed as relative fold expression levels.

Total lysates were prepared in PRO-PREP buffer (iNtRON Biotechnology, Seoul, Korea). Protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as a standard. Twenty micrograms of protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. Membranes were soaked in 5% non-fat dried milk in TBST (10 mmol/L Tris/HCl pH 7.5, 150 mmol NaCl, and 0.05% Tween-20) for 1 hour, followed by incubation for 16 to 18 hours with primary antibodies against Sulf-1, Sulf-2, and β-actin at 4°C. Membranes were then washed three times with TBST for 10 minutes, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. Finally, membranes were rinsed three times with TBST for 10 minutes, and antigen-antibody complexes were detected using an enhanced chemiluminescence detection system (LAS-3000; Fujifilm, Tokyo, Japan).

Statistical analysis was performed using SPSS version 12.0 (SPSS Inc., Chicago, IL, USA). Independent sample t-test was used for parametric analysis, and Mann-Whitney test was used for nonparametric analysis with a confidence interval of 95% and p-value of <0.05.