IL-4 gene therapy for collagen arthritis suppresses synovial IL-17 and osteoprotegerin ligand and prevents bone erosion (original) (raw)

Animals. Male DBA-1/BOM mice were purchased from Bomholdgärd (Ry, Denmark). The mice were housed in filter-top cages. The mice were immunized 10–12 weeks of age. Female C57bl/6 and balb/c mice were obtained at our university breeding facilities in Nijmegen. Balb/c IL-4 gene knockout mice were kindly provided by NV Organon (Oss, The Netherlands). Water and food were provided ad libitum.

Adenoviral vectors. The recombinant replication-deficient adenovirus Ad5E1mIL-4 was generated by homologous recombination after cotransfecting 293 cells with PACCMVmIL-4 and a virus-rescuing vector pAdBHG10 as described elsewhere (37). The empty recombinant replication-deficient adenovirus Ad5del70-3 was used as a control vector throughout the study. High titers of recombinant adenoviruses were amplified, purified, titered, and stored as described previously (38). Biologic activity of IL-4 produced by Ad5E1mIL-4 was verified by blocking experiments with anti–IL-4 on AdE1mIL-4–induced inflammation and synovial cell mass.

Intra-articular gene transfer with Ad5E1mIL-4. Naive mice were intra-articularly injected in the right knee joint with 108, 107, or 106 pfu/6 μL of either Ad5E1mIL-4 or Ad5del70-3. At different time points, mice were bled and sacrificed by cervical dislocation. Patella with adjacent synovium was dissected in a standardized manner (25) from the right and the contralateral knee. The levels of IL-4 in sera and washouts of joint tissue were measured by ELISA as described later here.

Induction of collagen-induced arthritis. Bovine type II collagen was prepared as described (24) and diluted in 0.05 M acetic acid to a concentration of 2 mg/mL and was emulsified in equal volumes of CFA (2 mg/mL of Mycobacterium tuberculosis; strain H37Ra; Difco Laboratories, Detroit, Michigan, USA). The mice were immunized intradermally at the base of the tail with 100 μL of emulsion (100 μg of collagen). On day 21, mice were given an intraperitoneal booster injection of 100 μg of type II collagen dissolved in PBS, and normally arthritis onset will then occur around days 25–28.

Study protocol. The collagen arthritis model (CIA) was induced in male DBA-1 mice as already described here. Just before expected onset of CIA, mice were scored visually for the appearance of arthritis. Mice without macroscopic signs of arthritis in the paws were selected. Mice were anesthetized with ether, and a small aperture in the skin of the knee was performed for the intra-articular injection procedure. When absence of arthritis was confirmed in the knee joint, intra-articular injections were performed with 107 pfu/6 μL of either an IL-4–expressing (Ad5E1mIL-4) or an empty control (Ad5del70-3) recombinant human type 5 adenovirus vector or with saline (26). At days 1, 3, 5, and 7 after the intra-articular injection of the viral vector, mice were sacrificed by cervical dislocation, and the skin of the knee joint was removed. The appearance of arthritis in the injected joints was assessed and severity score was recorded as described previously (24). Thereafter, knee joints were isolated and processed for light microscopy.

Assessment of arthritis. Mice were considered to have arthritis when significant changes in redness and/or swelling were noted in the digits or in other parts of the paws. Knee joint inflammation was scored visually after skin dissection, using the following scale: 0, noninflamed; 1, mild inflammation; 1.5, marked inflammation; 2, severe inflammation. Scoring was done by two independent observers, without knowledge of the experimental groups.

Assessment of IL-4, IL-6, and IL-12 protein in 1-hour patella washouts. To determine the levels of IL-4, IL-6, and IL-12 in patella washouts, patellae were isolated in a standardized manner from knee joints as described previously (25). Patella were incubated in RPMI 1640 medium (GIBCO BRL, Breda, The Netherlands) with 0.1% BSA, gentamicin (50 μg/mL), and L-glutamine (2 mM) (200 μL/patella) for 1 hour at room temperature. After supernatant was harvested the IL-4, IL-6, and IL-12 levels were measured by ELISA (25, 26). Anti-murine IL-4 antibodies (Ab’s) were purchased from PharMingen (San Diego, California, USA; capture Ab: rat anti-mouse IL-4 mAb [clone: BVD4-1D11]; detection Ab: rat anti-mouse IL-4 mAb biotin labeled [clone: BVD6-24G2]). The detection range of the IL-4 ELISA is 1,280–1.2 pg/mL. The sensitivity of the IL-4 ELISA is 5 pg/mL. No cross-reactivity was found with the cytokines IL-1, IL-6, or IL-10. Anti-murine IL-6 Ab’s were from Biosource International (Camarillo, California, USA; capture Ab: rat anti-mouse IL-6 mAb [clone: MP5-20F3]; detection Ab: rat anti-mouse IL-6 mAb biotin labeled [clone MP5-32CK]). The detection range of the IL-6 ELISA is 2,560–40 pg/mL, with a sensitivity of 78 pg/mL. No cross-reactivity was found with the cytokines IL-1β, IL-4, and IL-10. Anti-murine IL-12 Ab’s were obtained from Genzyme (Cambridge, Massachusetts, USA; capture Ab: monoclonal rat anti-mouse IL-12 [clone C15.6]; detection Ab: monoclonal rat anti-mouse IL-12 biotin labeled [clone C17.8]). The detection range of the IL-12 ELISA is 1,280–1.2 pg/mL, with a sensitivity of 10 pg/mL. No cross-reactivity was found with IL-4, IL-6, TNF-α, and IL-1.

Briefly, ELISA plates (Maxisorb; Nunc, Copenhagen, Denmark) were coated with the capture Ab (3 μg/mL) by overnight incubation at 4°C in 0.1 M carbonate buffer (pH 9.6). Nonspecific binding sites were blocked by 1-hour incubation at 37°C with 1% BSA in PBS/Tween-20. The supernatants were tested by 3-hour incubation at 37°C. The plates were then incubated at 37°C with the biotin-labeled second Ab (0.25 μg/mL) diluted in PBS/0.5% BSA (pH 7.5), followed by a 30-minute incubation at 37°C with streptavidin conjugated to poly-horseradish peroxidase (0.25 μg/mL) (source: Streptomyces avidinii; Central Laboratory of Blood Transfusion, Amsterdam, The Netherlands) diluted in 1% casein colloid/PBS buffer (pH 7.5) (Central Laboratory of Blood Transfusion). Bound complexes were detected by reaction with 0.08% orthophenylenediamine (OPD) diluted in 50 mM phosphate buffer (pH 6.0) and 0.03% H2O2. Absorbance was measured at 492 nm using an ELISA plate reader (Titertek Multiscan MCC/340; Labsystems, Helsinki, Finland). The cytokine concentration in the samples was calculated as picograms per milliliter using recombinant murine IL-4 (a kind gift of S. Smith [Schering-Plough, Kenilworth, New Jersey, USA]), IL-6 (Biosource International) and IL-12 (kindly provided by S. Wolf, Genetic Institute Inc., Cambridge, Massachusetts, USA) as a standard.

Isolation of RNA. Mice were sacrificed by cervical dislocation, and the patella and adjacent synovium were immediately dissected (39). Synovium biopsy tissue was taken from six patella specimens. Two biopsy specimens with a diameter of 3 mm were punched out, using a biopsy punch (Stifle, Wachtersbach, Germany): one from the lateral side and one from the medial side. Three lateral and three medial biopsy samples were pooled to yield two samples per group. The synovium samples were immediately frozen in liquid nitrogen. Synovium biopsy samples were ground to powder using a microdismembrator II (Braun Inc., Melsungen, Germany). Total RNA was extracted in 1 mL of Trizol reagent (GIBCO BRL), a monophasic solution of phenol and guanidine isothiocyanate, which is an improved single-step RNA isolation method based on the method described by Chomczynski and Sacchi (40).

PCR amplification One microgram of synovial RNA was used for RT-PCR. Messenger RNA was reverse transcribed to complementary DNA (cDNA) using oligo-dT primers, and one twentieth of the cDNA was used in one PCR amplification. PCR was performed at a final concentration of 200 μM dNTPs, 0.1 μM of each primer, and 1 unit of Tag polymerase (GIBCO BRL) in standard PCR buffer (20 mM Tris-HCL [pH 8.4] and 50 mM KCl) (GIBCO BRL). The mixture was overlaid with mineral oil and amplified in a thermocycler (Omnigene, Hybaid, United Kingdom). Message for GAPDH was amplified using the primers described elsewhere (24). Primers for cathepsin K, IL-17, IL-12, OPGL and osteoprotegerin (OPG) were designed using Oligo 4.0 and Primer Software (Molecular Biology Insights Inc., Cascade, Colorado, USA). For every mediator that is tested in the PCR reaction, GAPDH expression was also measured in the same reaction mix. Samples (5 μL) were taken from the reaction tubes after a certain number of cycles. PCR products were separated on 1.6% agarose and stained with ethidium bromide. The expression for GAPDH is normalized between the control vector group and the IL-4 group before differences in mRNA expression for a particular mediator were determined.

Radiology. At the end of the experiment, knee joints were isolated and used for x-ray analysis as a marker for joint destruction. X-ray films were carefully examined using a stereo microscope, and joint destruction was scored on a scale of 0–5, ranging from no damage to complete destruction of the joint.

Tartrate-resistant acid phosphatase staining. At the end of the experiment, whole knee joints were fixed for 2 days in 10% formalin, followed by decalcification in 10% EDTA (Titriplex III; Merck, Darmsadt, Germany) in 1 mM Tris-HCl (pH 7.4) for up to 2 weeks at 4°C (10). Decalcified specimens were processed for paraffin embedding (41). Staining of tissue sections (7 μm) for tartrate-resistant acid phosphatase (TRAP) was performed by a leukocyte acid phosphatase kit, a cell-staining kit for the detection of tartrate resistant acid phosphatase from Sigma Chemical Co. (St. Louis, Missouri, USA).

Histology. Whole knee joints were removed and fixed for 4 days in 10% formalin. After decalcification in 5% formic acid, the specimens were processed for paraffin embedding (41). Tissue sections (7 μm) were stained with hematoxylin and eosin (H&E) or Safranin O. Histopathological changes were scored using the following parameters. Infiltration of cells was scored on a scale of 0–3, depending on the amount of inflammatory cells in the synovial cavity (exudate) and synovial tissue (infiltrate). A characteristic parameter in CIA is the progressive loss of bone. This destruction was graded on a scale of 0–3, ranging from no damage to complete loss of the bone structure.

Histopathological changes in the knee joints were scored in the patella and femur/tibia regions on five semiserial sections of the joint, spaced 70 μm apart. Immunohistochemistry was quantified using an automated image analysis system (Leica Q500/N; Leica Imaging Systems Ltd., Cambridge, United Kingdom). Microscopic images were recorded by a CDD video camera (Victor Company of Japan Ltd., Tokyo, Japan) and processed by a personal computer. Optical density was measured in the cartilage or synovium. For cartilage, the whole area of noncalcified cartilage was scanned, and staining was expressed per unit of tissue (μm2). For synovium, a defined area along the cortical bone was scanned, using a standardized template. Five sections per joint were scanned. Staining values were corrected for background staining, as measured in the control vector group. Scoring was performed by two observers without knowledge of the experimental group, as described earlier (24).

Immunohistochemistry of OPGL. Whole knee joints were fixed, decalcified and paraffin embedded as already describe here. Tissue sections (7 μm) were treated with 1% H2O2 for 10 minutes at room temperature. Sections were incubated for 1 hour with the primary Ab RANKL (goat polyclonal Ab raised against a peptide mapping at the NH2-terminus of RANKL (RANK ligand) of mouse origin (N-19; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) or a control goat IgG Ab (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, USA). After rinsing, sections were blocked with 4% normal mouse serum for 20 minutes at room temperature. Thereafter, sections were incubated for 30 minutes with biotinylated mouse anti-goat IgG (Jackson ImmunoResearch Laboratories) and detected using biotin-streptavidin/peroxidase staining (Elite kit; Vector Laboratories, Burlingame, California, USA). Development of the peroxidase staining was done with 3′,3′diaminobenzidine (Sigma Chemical Co.). Counterstaining was done with Mayer’s hematoxylin.

Immunohistochemical staining of type II collagen neoepitopes. Whole knee joints were fixed for 2 days in 10% formalin, followed by decalcification in 10% EDTA (Titriplex III), 7.5% polyvinylpyrrolidone (PVP; Mr 29,000; Serva, Amsterdam, The Netherlands) in 0.1M phosphate buffer (pH 7.4) for 2 weeks at 4°C. After extensive rinsing with 7.5% PVP in 0.1M phosphate buffer, tissue blocks were rapidly frozen in liquid nitrogen and stored at –70°C. Whole knee joint sections (7 μm) were cut at 22°C on a micron cryostat and mounted on glass microscope slides precoated with 3-aminopropyltriethoxysilan (Sigma Chemical Co.). Sections were dried for 1 hour and stored at –70°C until further use. After thawing, the sections were fixed in freshly prepared 4% formaldehyde (5 minutes) and washed extensively in 0.1 M PBS (pH 7.4) for 15 minutes. Sections were incubated with 1% hyaluronidase (type I-s; Sigma Chemical Co.) for 30 minutes at 37°C, to remove proteoglycans. After treatment with 1% H2O2 for 30 minutes, nonspecific staining was blocked by incubation with 10% normal goat serum with 1% BSA. Sections were incubated overnight with the primary rabbit Ab Col2-3/4Cshort (kindly provided by A.R. Poole [McGill University, Montreal, Canada]) directed against the COOH-terminal neoepitope generated by cleavage of native human type II collagen by collagenases, which has been described and characterized previously (42). The Col2-3/4Cshort antiserum detects the COOH-terminal neoepitope that can be generated by matrix metalloprotease-1 (MMP-1), MMP-8, MMP-13, and probably also by MMP-2 (43). After extensive rinsing, sections were incubated with biotinylated goat anti-rabbit IgG and detected using biotin-streptavidin/peroxidase staining (Elite kit). Development of the peroxidase staining was done with 3′,3′diaminobenzidine (Sigma Chemical Co.). Counterstaining was done with Mayer’s hematoxylin.

Preparation of bone fragments. Rheumatoid bone samples were obtained from patients with osteoarthritis (OA) and RA, according to the revised criteria of the American College of Rheumatology (44), who were undergoing knee or wrist synovectomy, or joint replacement. Bone fragments were prepared as described previously (30). Samples were cut into small pieces of approximately 2 mm3 and incubated in triplicate in complete medium consisting of MEM medium (GIBCO BRL), 2 mM L-glutamin, 100 U/mL penicillin, 50 mg/mL gentamicin, 20 mM HEPES buffer, and 10% FCS. Cultures were performed at 37°C in a 5% CO2/95% humidified air. Bone fragments were cultured in 24-well plates (Falcon, Oxnard, CA) in a final volume of 2 mL. The cytokines to be tested were added at the beginning of the culture.

Measurement of collagen degradation. Type I collagen C-telopeptide breakdown products (CTX) were measured in synovium piece culture supernatants by a two-site ELISA (Serum Crosslaps One-Step, Osteometer Biotech, Ballerup, Denmark) using two mAb’s raised against a synthetic peptide with an amino acid sequence specific for a part of the C-telopeptide of α1-chain of type I collagen (45). Intra- and interassay CVs are lower than 5% and 8%, respectively, and the sensitivity is 154 pmol/L.

Determination of type I collagen production. The production of type I collagen was estimated in 48-hour culture media by measuring the concentration of the C-propeptide of type I collagen (PICP) using a two-site ELISA that uses an mAb and a polyclonal Ab raised against human PICP purified from skin fibroblast cultures (Procollagen-C, Metra Biosystem Inc., Palo Alto, California, USA) (46, 47). The sensitivity of the assay is 1 ng/mL, and the intra- and interassay CV were below 7%.

Statistical analysis. Means ± SD of the various groups were determined and potential differences between experimental groups were tested using the Mann-Whitney rank sum test, unless stated otherwise.