Hybridoma production and monoclonal antibody purification
Two BALB/c mice were immunized subcutaneously with 100 μL of complete Freund’s adjuvant (CFA) (Difco Laboratories, Detroit, MI, USA) containing 100 μg of recombinant human adiponectin expressed in E. coli (ProSpec, Rehovot, Israel). After 2 weeks, the mice were injected with incomplete Freund’s adjuvant. The mice were boosted with antigen only (that is, 50 μg of adiponectin intravenously) 2 weeks later. Two days after the last boost, sera were tested for reactivity to recombinant adiponectin using enzyme-linked immunosorbent assay (ELISA). Splenic lymphocytes were fused to FO myeloma cells (ATCC, Manassas, VA, USA) and plated on 96-well plates in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum (FBS) (Invitrogen, Waltham, MA, USA) and HAT component (Sigma-Aldrich Korea, Yongin, Korea) as described previously [28]. The culture supernatants were tested by Western blot and ELISA for reactivity to recombinant human adiponectin. mAbs were purified from culture supernatants of the screened clones by using Protein G-Sepharose column chromatography (GenScript, Piscataway, NJ, USA) in accordance with the protocol of the manufacturer. Studies were conducted in accordance with the National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee of Kyung Hee University.
Collagen-induced arthritis mouse model
Male DBA/1 J mice (4 weeks old) were purchased from Central Lab. Animal Inc. (Seoul, Korea). The animals were kept in a rodent facility and adapted for at least 1 week before CIA induction as previously described [29]. Briefly, the mice (6 weeks old) were immunized at the base of the tail with a 100-μL mixture of chicken collagen type II (CII) 100 μg (Sigma-Aldrich Korea) and CFA. After 14 days, the mice were given a booster injection of 100 μg CII and incomplete Freund’s adjuvant. The mice were divided into five groups (n = 8) containing normal, CIA (saline, control), CIA + KH4–8 mAb (6 mg/kg, intraperitoneal injection, three times a week for 6 weeks), CIA + KH7–33 mAb (6 mg/kg, intraperitoneal injection, three times a week for 6 weeks), and CIA + prednisolone (10 mg/kg, intragastric administration, twice a week for 6 weeks). Normal mice and CIA control mice were given an equal volume of normal saline. The treatment antibody concentration was decided on the basis of the treatment dose for therapeutic antibodies in mice [30, 31]. To evaluate the therapeutic effect of mAbs KH4–8 and KH7–33 on the progression of arthritis in CIA mice, body weight, paw volume, squeaking score, and arthritic score were measured [29]. All methods were approved by the Animal Care and Use Committee of Kyung-Hee University (KHUASP[SE]-15–115). All procedures were executed in accordance with the guide for the Care and Use of Laboratory Animals by the Korea National Institute of Health.
Cell culture for in vitro functional testing of monoclonal antibodies
Human umbilical vein endothelial cells (HUVECs) and human osteoblasts were obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea) and Cell Applications, Inc. (San Diego, CA, USA), respectively. Endothelial cells and osteoblasts were cultured in T-75 flasks (Nunc, Thermo Fisher Scientific, Waltham, MA, USA) containing EGM-2 (Lonza, Alpharetta, GA, USA) and osteoblast growth medium (Cell Applications, Inc. San Diego, CA, USA), respectively. After the cells had grown to confluence, they were split at a 1:4 ratio. Cell passages 5–6 were used for all experiments. HUVECs (2 × 105 cells per six-well plate in 2 mL of medium) and osteoblasts (1 × 105 cells per six-well plate in 2 mL of medium) were cultured overnight and treated with human recombinant adiponectin, which was produced by using E. coli (ProSpec, Rehovot, Israel ). To test the ability of the mAb to block adiponectin function, mAb (~120 μg/mL) and adiponectin (2.5 or 5 μg/mL) were mixed and incubated for 1 h before being used to treat cells. After 24-h treatment, the culture supernatants were collected and frozen, and IL-6 and IL-8 were measured by ELISA.
Epitope mapping of monoclonal antibody
Epitope mapping of the KH4–8 mAb was performed by using PEPperMAP® technology (PEPperPRINT GmbH, Heidelberg, Germany) [32]. PEPperMAP® Linear Epitope Mapping of mouse mAb KH4–8 was performed against human adiponectin translated into linear 15–amino acid peptides with a peptide-peptide overlap of 14 amino acids. Human adiponectin peptide microarrays were incubated with the mouse mAb at concentrations of 1 μg/mL, 10 μg/mL, and 100 μg/mL in incubation buffer followed by staining with secondary goat anti-mouse IgG (H + L) DyLight680 antibody. Samples were processed by using an Odyssey Imaging System (LI-COR, Lincoln, NE, USA). Quantification of spot intensities and peptide annotation were performed using a PepSlide® Analyzer (PEPperPRINT GmbH).
Western blotting
To screen hybridomas for production of mAbs against recombinant human adiponectin, adiponectin (100 ng/well) was resolved via 12% SDS-PAGE and transferred to Hybond-ECL membranes (Amersham, Arlington Heights, IL, USA). The membranes were blocked with 6% non-fat milk dissolved in TBST buffer (10 mM Tris-Cl [pH 8.0], 150 mM NaCl, 0.05% Tween 20). The blots were probed with hybridoma culture supernatants at 4 °C overnight and incubated with a 1:1,000 dilution of horseradish peroxidase–conjugated goat anti-mouse IgG secondary antibody (Sigma-Aldrich Korea). The blots were developed by using the ECL method (Amersham). To detect adiponectin in human, mouse, and rat serum samples, serum was subjected to gradient SDS-PAGE (4–12%) (NuPAGE® Bis-Tris Mini Gels, Invitrogen), blotted, and probed with purified mAb (5 μg/mL) as the primary antibody, as described above. Mouse and rat serum were obtained via heart puncture of male BALB/c mice and Sprague Dawley (SD) rats (8 weeks old), respectively. Human serum was obtained from a male volunteer (55 years old).
Enzyme-linked immunosorbent assay
Culture supernatants from cells treated with mAb plus adiponectin were analyzed with IL-6 and IL-8 ELISA kits (BD Bioscience Korea, Seoul, Korea) in accordance with the protocol of the manufacturer. To screen hybridoma clones for mAbs against adiponectin, 96-well plates were coated with adiponectin (200 ng/well), hybridoma culture supernatants were added after blocking, wells were incubated with a 1:1,000 dilution of horseradish peroxidase–conjugated goat anti-mouse IgG secondary antibody (Sigma-Aldrich Korea), and the ELISA procedure was completed. Sera from CIA were obtained from heart puncture and analyzed for adiponectin, IL-6, TNF-α, and RANKL by using the Luminex® 200™ Total System (Luminex Corporation, Austin, TX, USA) as previously described [33].
Histological assessment of inflammation
Mice were killed after 56 days of CII + CFA treatments. Histochemical staining was performed to determine the degree of immune cell infiltration into the joints. Mice knee joints were dissected, fixed for 3 days in 10% formalin, dehydrated through a graded ethanol series, cleared in xylene, and processed for embedding in paraffin wax with routine protocols. Coronal sections 8 mm thick were cut through the knee joint by using a manual rotary microtome (Finesse 325, Thermo Shandon Inc., Pittsburgh, PA, USA) and stained with hematoxylin and eosin for routine histological evaluation. Paraffin tissue sections obtained from rat knees were deparaffinized in xylene. The tissue samples were hydrated with ethanol and washed in distilled water, followed by antigen retrieval via heating with 100 mM citrate buffer (pH 6.0) at 65 °C for 1–2 h. The samples were examined with a confocal laser scanning microscope (Olympus BX53, Olympus Corporation, Tokyo, Japan). The degree of inflammation was evaluated on a scale from 0 to 4 by three different experts who were blinded to treatment information. The scale was defined as follows: 0 = no inflammation, 1 = minimal inflammation, 2 = mild inflammation, 3 = moderate inflammation, and 4 = severe inflammation.
Immunohistochemistry
Normal human tissues were obtained from archived paraffin collections at the Department of Pathology, Inje University Sanggye Paik Hospital (Seoul, Korea). Adipose, lung, kidney, and pancreas tissues were obtained from different donors (a 59-year-old woman, a 72-year-old man, a 62-year-old woman, and a 67-year-old man, respectively). Sections (4 μm) of the paraffin blocks were cut and immunohistochemically stained with mAbs (50 μg/mL) by using an automated system (Vision BioSystems Ltd., Mount Waverley, Australia), as described below. Antigen was retrieved with epitope retrieval solution 1 (Leica Microsystems, Newcastle, UK). Slides were incubated with antibody at room temperature for 20 min and then with a biotinylated secondary antibody for 8 min. The resulting complexes were detected with avidin-peroxidase-conjugated polymer. Color was developed by using 3,3′-diaminobenzidine (DAB) (ScyTek, Logan, UT, USA). Mayer’s hematoxylin was used as a counterstain. Positive and negative control stains were used in each run. The study protocol was reviewed and approved by the institutional review board at Inje University Sanggye Paik Hospital. For immunohistochemical staining of adiponectin in the joints of mice using anti-adiponectin antibody (Abcam, Cambridge, MA, USA), moderate nuclear or cell membranous staining was determined as a percentage and scored as follows: 0 = staining in less than 10% of cells, 1 = staining in 10–50% of cells, and 2 = staining in more than 50% of cells. Cases with a score of 1 or 2 were classified as positive.
Statistical analysis
In vitro data are expressed as the mean ± standard error of the mean of quadruplicate samples. The expression levels of the factors were compared between groups by using the Mann–Whitney test. Prism 5.02 software (GraphPad Software, San Diego, CA, USA) was used for statistical analysis and graphing. Statistical differences between CIA mouse groups were identified by using t tests, one-way analysis of variance (ANOVA) with Dunn’s Multiple Comparison Test, and two-way ANOVA followed by Bonferroni post-test correction (for multiple comparisons of body weight, squeaking score, paw volume, and arthritis index score). Differences were considered significant at a P value of less than 0.05.