Cell Culture and Chemicals
The characteristics of the three MCL cell lines, Jeko-1, Mino, and SP53, have been previously described [43
]. Briefly, all of these three cell lines have the mature B-cell immunophenotype, carry the t(11;14)(q13;q32)
cytogenetic abnormality, and overexpress cyclin D1. All three cell lines are negative for the Epstein-Barr virus nuclear antigen. MCL cells were treated with 20 ng/ml of human recombinant IL-22 protein (rIL-22; R&D Systems, Minneapolis, MN) for 0 and 30 minutes and harvested for Western blot analysis. To obtain highly purified peripheral blood B cells from healthy donors, we first collected peripheral blood mononuclear cells by centrifugation over Ficoll-Hypaque. CD19-positive B cells were isolated by positive selection using specific monoclonal antibody-coated magnetic beads and a preparative magnetic cell sorter (Miltenyi, Bergisch Gladbach, Germany) in accordance with the manufacturer's recommended protocol. The purity of the isolated B-cell population was analyzed by flow cytometry (FACScan; Becton Dickinson, San Jose, CA) and confirmed to be greater than 98%. NF-κB activation inhibitor 6-amino-4-(4-phenoxyphenylethylamino quinazoline (catalog no. EI-352) was purchased from Enzo Life Sciences International (Farmingdale, NY) and E
-2-fluoro-4′-methoxystilbene (catalog no. 481412) was purchased from EMD (Gibbstown, NJ).
Western Blot Analysis and Antibodies
Western blot analysis was performed using standard techniques. Briefly, cells were lysed in a buffer containing 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 40 µg/ml leupeptin, 1 µM pepstatin, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride and centrifuged at 15,000g for 15 minutes at 4°C. The supernatant was removed, and 50 to 100 µg of protein was run on an SDS-polyacrylamide gel. After the proteins were transferred to nitrocellulose membranes, the membranes were blocked with 5% milk in TBS buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl) and then incubated with primary antibodies overnight followed by 1 hour of incubation with horseradish peroxidase-conjugated secondary antibody ( Jackson Immunoresearch Laboratories, Inc, West Grove, PA). Membranes were washed in PBS with 0.05% Tween-20 for 30 minutes between steps. Proteins were detected using the enhanced chemiluminescence detection kit (Amersham Life Sciences, Arlington Heights, IL). Antibodies used were anti-STAT3 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), antipSTAT3 (1:500; Santa Cruz Biotechnology), anti-IL-22RA1 (1:1000; Sigma-Aldrich, Oakville, Ontario, Canada), and anti-β-actin (1:3000; Sigma-Aldrich).
Immunofluorescence Staining and Confocal Microscopy
Immunofluorescence was performed using standard techniques. Briefly, cells grown on coverslip in a six-well plate were fixed with 4% paraformaldehyde in PBS. Cells were rinsed three times with 1x PBS, incubated with 30 µl of anti-IL-22RA1 (1:50; Sigma-Aldrich) antibody overnight followed by rinsing three times with 1x PBS. After incubating with 200 µl of Alexa Fluor 488 secondary antibody (1:250; Invitrogen, Burlington, Ontario, Canada) for 1 hour at room temperature, cells were rinsed with PBS, and the procedure was completed with a mounting medium (Dako, Mississauga, Ontario, Canada) added to the slides. Cells were visualized with a Zeiss LSM 510 (Carl Zeiss, Toronto, Canada) confocal microscope at the Core Cell Imaging Facility, Cross Cancer institute.
Flow Cytometric Detection of IL-22RA1 MCL Cell Lines
MCL cells were fixed in the CytoFix Buffer from Becton Dickinson Biosciences (Franklin Lakes, NJ) washed in cold PBS, centrifuged, and resuspended in the FACS staining buffer purchased from Becton Dickinson. Cells were incubated with primary antibodies for 60 minutes at 4°C in the dark and washed twice using cold buffer between incubations. The following antibodies were used: unconjugated mouse IgG1 as the isotype control (10 µg/ml; Santa Cruz Biotechnology), unconjugated mouse anti-human IL-22RA1 (IgG1, 10 µg/ml; R&D Systems), and phycoerythrin-conjugated rat anti-mouse antibody (IgG1, 5 µg/ml; Becton Dickinson). Flow cytometry was performed using the FACScan (Becton Dickinson, Toronto, Canada), and data were analyzed using the accompanying CELLQuest software as per manufacturer's guidelines.
Reverse Transcription-Polymerase Chain Reaction
Total cellular RNA was extracted from 1 x 106 cells from Mino, SP53, Jeko-1, purified human B cells, and HepG2 cell lines using the TRIzol extraction method (Invitrogen). Reverse transcription (RT) was performed using 500 ng of total RNA in a first-strand complementary DNA synthesis reaction with SuperScript Reverse Transcriptase as recommended by the manufacturer (Invitrogen). Primer pairs were designed to detect IL-22, IL-22RA1, and IL-10R2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Polymerase chain reaction (PCR) was performed by adding 5 µl of RT product into a 25-µl volume reaction containing 1x buffer, 200 µM of each dNTPs, oligonucleotide primer, and 0.2 U of AmpliTaq polymerase. The primer sequences and PCR cycles are shown in . For DNA amplification, complementary DNA was denatured at 94°C for 1 minute, subjected to primer annealing at 60°C for 1 minute, and then subjected to DNA extension at 72°C for 1minute for 35 cycles in a thermal cycler (Applied Biosystems, Foster City, CA). Amplified products were analyzed by DNA gel electrophoresis in 1% agarose and visualized by the Alpha Imager 3400 (Alpha Innotech, San Leandro, CA).
Cell Growth Assay
For the MTS assay (Promega, Madison, WI), 5000 cells were seeded in 96-well culture plates and treated daily with recombinant IL-22 up to 6 days. Cell proliferation was then measured colorimetrically at 450 nm using a Microplate reader (BioRad, Hercules, CA), and absorbance values were normalized using the Microplate Manager 5.2.1 (BioRad).
MCL Tumors and Immunohistochemistry
All MCL primary tumors were diagnosed at the Cross Cancer Institute, and the diagnostic criteria were based on those described in the World Health Organization Classification Scheme [1
]. All cases were confirmed to express cyclin D1 by immunohistochemistry. The use of these tissues has been approved by our institutional ethics committee. Immunohistochemical staining was performed using standard techniques. Briefly, formalin-fixed, paraffin-embedded tissue sections of 4-µm thickness were deparaffinized and hydrated. Heat-induced epitope retrieval was performed using Tris buffer (pH 9.9; Dako) and a rapid microwave histoprocessor (RHS; Milestone, Bergamo, Italy). After incubating at 100°C for 10 minutes, slides were washed in running tap water for 5 minutes, and endogenous peroxidase was blocked using 10% H2
and methanol followed by washing in running tap water. Tissue sections were then incubated with anti-IL-22RA1 (1:500; Capralogics, Hardwick, MA) overnight in a humidified chamber at 4°C. After three washes with PBS, tissue sections were incubated with anti-rabbit IgG (EnVision, Dako) for 30 minutes at room temperature. The tissue sections were incubated with 3,3′-diaminobenzidine/H2
(Dako) for color development, using hematoxylin as a counterstain. For IL-22RA1, liver tissues were used as a positive control, and benign tonsils were used as a negative control.
All the experiments were performed in triplicate. Student's t test was used. P < .05 was considered to be statistically significant.