TNF-induced structural joint damage is mediated by IL-1

Zwerina et al. 10.1073/pnas.0610812104.

Supporting Information

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SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10
SI Figure 11
SI Materials and Methods

SI Figure 6

Fig. 6. Spleen and bone marrow cells from 12-week-old wild-type, IL-1^-/-, hTNFtg, and IL-1^-/-hTNFtg mice were investigated for CD4, CD8, CD45R, and CD11b expression by FACS analysis (A). Cellular distribution of synovial inflammatory tissue in hTNFtg and IL-1^-/-hTNFtg mice was assessed by immunohistochemical analysis of markers for neutrophil granulocytes (7-4), B-cells (CD45R), macrophages (F4/80), and T-cells (CD3) (B).

SI Figure 7

Fig. 7. Quantification of cathepsin K levels in the culture supernatants of osteoclasts derived from monocytic precursor cells from wild-type, IL-1^-/-, hTNFtg, and IL-1^-/-hTNFtg mice and differentiated by MCSF and RANKL for 5 days (A). Quantification of serum levels of tartrate-resistant acid phosphatase 5b (TRAP5b) and collagen type I cross-laps (CTX) in wild-type, IL-1^-/-, hTNFtg, and IL-1^-/-hTNFtg mice. * and ** indicate significant (P < 0.05) difference compared to wild-type and hTNFtg cells, respectively.

SI Figure 8

Fig. 8. Inflamed synovial tissue of radiation chimeras mice was stained for IL-1 by immunohistochemistry.

SI Figure 9

Fig. 9. Synovial fibroblasts from knee joints of wild-type, IL-1^-/- hTNFtg, and IL-1^-/-hTNFtg mice were cultured and cell numbers, spontaneous as well as Fas- and staurosporin-induced apoptosis and MMP-3 production, were assessed (A and B). MMP-3 levels in culture supernatants of synovial fibroblasts from hTNFtg and IL-1^-/-hTNFtg mice were determined by ELISA (C).

SI Figure 10

Fig. 10. Quantitative mRNA analysis for MMP-3, MMP-13, ADAMTS-4, ADAMTS-5, and TIMP-1 from chondrocytes that were cocultured in a transwell system with TNF-stimulated wild-type or IL-1^-/- monocytes for indicated time periods. **, P [lt] 0.05 (difference between groups).

SI Figure 11

Fig. 11. Quantitative mRNA expression of MMP-3, MMP-13, and ADAMTS-5 (A) and AP-1 transcription factor members (B; c-jun, junD, c-fos, Fra-1, and Fra-2) in bone marrow macrophages from wild-type and IL-1^-/- mice. * indicates significant (P < 0.05) difference between wild-type and IL-1^-/- cells.

SI Materials and Methods

Animals

Heterozygous hTNFtg mice (strain Tg197; genetic background C57BL/6) and IL-1^-/- mice (genetic background C57BL/6) were interbred to receive IL-1^-/-hTNFtg mice. All four genotypes (wild-type, IL-1^-/-, hTNFtg, and IL-1^-/-hTNFtg mice) of the F2 generation were comparatively analyzed in this study, and for all of the experiments only littermates were used. In the present study, 100 animals were included in clinical, histological, and cellular analyses Animals were killed at ages 8, 12, and 16 weeks by cervical dislocation, and blood was withdrawn by cardiac puncture. The local ethics committee approved all animal procedures.

Primers

The following primers were used: hTNF transgene: 5'-TACCCCCTCCTTCAGACACC-3' and 5'-GCCCTTCATAATATCCCCCA-3'; IL-1a/b primers were as follows: IL-1aF, 5'-CTTGGCCATACTGCAAAGGTCATG-3'; IL-1aR, 5'-CAGGTCATTTAACCAAGTGGTGCTG-3'; IL-1a/b KO, 5'-GAGGTGCTGTTTCTGGTCTTCACC-3'; IL-1bF, 5'-GCGAATGTGTCACTATCTGCCACC-3'; IL-1bR, 5'-CCATGCCACAGTCCCTCCAC-3'; MMP-3: F, 5'-TGGAGATGCTCACTTTGACG-3'; R, 5'-GCCTTGGCTGAGTGGTAGAG-3'; MMP-9: F, 5'-CATTCGCGTGGATAAGGAGT-3'; R, 5'-ATTTTGGAAACTCACACGCC-3'; MMP-13: F, 5'-AGTTGACAGGCTCCGAGAAA-3'; R, 5'-GGCACTCCACATCTTGGTTT-3'; ADAMTS4: F, 5'-TGCAGGTAGAGGGTTTGACT-3'; R, 5'-GACTCCGGATCTCCATTGAT-3'; ADAMTS5: F, 5'-CCAGCAGTGCAACTTGACAT-3'; R, 5'-AGACACACCATTTGGCCTTG-3'. TIMP-1: F, 5'-GCATCTGGCATCCTCTTGTT-3'; R, 5'-CCTTATGACCAGGTCCGAGT-3'; Cathepsin K: F, 5'-GGAAGAAGACTCACCAGAAGC-3'; R, 5'GTCATATAGCCGCCTCCA CAG-3'; MMP-9: F, 5'CCTGTGTGTTCCCGTTCATCT-3'; R, 5'CGCTGGAATGATCTAAG CCCA-3'; TRAP: F, 5'ACAGCCCCCACTCCCACCCT-3'; R, 5'TCAGGGTCTGGGTCTCCTTGG-3'; Calcitonin receptor: F, 5'CATTCCTGTACTTGGTTGGC-3'; R, 5'AGCAATCGACAAGGAGTGAC-3'; c-fms: F, 5'-CTGTGAATGGCTCTGATGTCCTGTTCTG-3'; R, 5'-CTCCCACTT CTCATTGTAGGGCAACTGA-3'; RANK: F, 5'-CTTCGACTGGTTCACTGCTCCTAAT-3'; R, 5'-TTACTGTTTCCAGTCACGTTCCCAGAGG-3'; b-actin: F, 5'TGTGATGGTGGGAATGGGTCAG-3'; R, 5'TTTGATGTCACGCACGATTTCC-3'; porphobilinogen deaminase: F, 5'TCCCTGAAGGATGTGCCTAC-3'; R, 5'GCACTTTTCTCTGGCAAGGT-3'; c-jun: F, 5'-ACTCGGACCTTCTCACGTC-3'; R, 5'-CGGTGTAGTGGTGATGTGCC-3'; junD: F, 5'-CATCGACATGGACACGCAAG-3'; R, 5'-CGGTGTTCTGGCTTTTGAGG-3'; c-fos: F, 5'-CGGGTTTCAACGCCGACTAC-3'; R, 5'-CAGGTCTGGGCTGGTGGAGA-3'; Fra-1: F, 5'-GAGACGCGAGCGGAACAAG-3'; R, 5'-CTTCCAGCACCAGCTCAAGG-3'; Fra-2: F, 5'-AGCCTCCCGAAGAGGACAG-3'; R, 5'-AGGACATTGGGGTAGGTGAAG-3';

RT-PCR

RNA was isolated from cell cultures by Trizol reagent (Invitrogen, Carlsbad, CA) and reverse transcribed with murine leukemia virus reverse transcriptase using the Gene Amp RNA PCR kit (Applied Biosystems, Foster City, CA) and oligo(dT) primers. Quantitative real-time RT-PCR was performed using LightCycler technology (Roche Diagnostics, Indianapolis, IN) and the Fast Start SYBR Green I kit for amplification and detection. In all assays, cDNA was amplified using a standardized program (10-min denaturing step; 55 cycles of 5 sec at 95°C, 15 sec at 65°C, and 15 sec at 72°C; melting point analysis in 0.1°C steps; final cooling step). Each LightCycler capillary was loaded with 1.5 ml of DNA Master Mix, 1.8 ml of MgCl₂ (25 mM), 10.1 ml of H₂O, 0.4 ml of 10 mM stock of each primer. The final amount of cDNA per reaction corresponded to 2.5 ng of total RNA used for reverse transcription. Quantification of target gene expression was performed using a mathematical model by Pfaffl. The expression of the target molecule was normalized to the expression of b-actin.

Clinical Assessment

Scoring of clinical signs of arthritis and measurement of body weight was done in all mice once weekly starting from 5 weeks of age. Arthritis was evaluated in a blinded manner as described previously (14). Briefly, joint swelling was examined in all four paws, and a clinical score of 0 to 3 was assigned (0, no swelling; 1, mild; 2, moderate; 3, severe swelling of the toes and ankle). In addition, grip strength was examined in each paw using a 3-mm-diameter wire and was scored on a scale from 0 to -4 (0, normal grip strength; -1, mildly reduced; -2, moderately reduced; -3, severely reduced; -4, no grip strength).

FACS Analysis

For immunophenotyping cells were isolated from the spleen and the tibial and femoral bone (n = 4-5 per group); stained with appropriate antibodies against T cells (CD4, CD8), B cells (CD45R), and monocytes (CD11b) (BD Biosciences, Heidelberg, Germany); and analyzed by FACS using standard settings.

MicroCT Imaging

MicroCT images were acquired on a vivaCT40 (Scanco Medical, Bassersdorf, Switzerland). The scanner generates a cone beam at 5-mm spot size and operates at 50 keV. Images of knees and hind paws were generated from wild-type, IL-1^-/-, hTNFtg, and IL-1^-/-hTNFtg mice.

Joint Histology

Hind paws were fixed overnight in 4.0% formalin and then decalcified in 14% EDTA (Sigma-Aldrich, St. Louis, MO) until the bones were pliable. Serial paraffin sections (2 mm) of hind paws were stained with H&E for assessment of synovial inflammation and bone erosion, toluidine blue for proteoglycan loss of articular cartilage, and tartrate-resistant acid phosphatase (TRAP) for detection of osteoclasts. TRAP staining was performed using a leukocyte acid phosphatase staining kit (Sigma-Aldrich, Munich, Germany). Synovial inflammation, bone erosions, osteoclast numbers, and cartilage destruction were quantified with the use of a Zeiss Axioskop 2 microscope (Carl Zeiss, Marburg, Germany) equipped with a digital camera and image analysis system (Osteomeasure, OsteoMetrics, Decatur, Georgia). The area of inflammation was quantified on H&E-stained sections. Total scores were calculated as the sum of the areas of inflammation in all tarsal joints in each mouse. Erosions were quantified in the same H&E-stained sections. Osteoclasts (three or more nuclei, TRAP-positive) were assessed in TRAP-stained serial sections. For proteoglycan content analysis, toluidine blue sections were used. Negatively stained cartilage appears as light blue as opposed to proteoglycan-rich, dark blue-stained cartilage. The area of negatively stained and total cartilage was measured, calculated, and given in percent cartilage that contains no proteoglycan. In addition, the number of cartilage lacunae containing no chondrocytes ("empty lacunae") was counted.

Immunohistochemistry

For immunohistochemistry, deparaffinized, ethanol-dehydrated tissue sections were stained with monoclonal antibodies against surface markers of neutrophil granulocytes (MCA771G; clone 7-4; Serotec, Dusseldorf, Germany), macrophages (anti-F4/80; clone Cl:A3-1; Serotec), T-lymphocytes (anti-CD3; CD3-12; Novocastra, Newcastle, UK) and B-lymphocytes (anti-B220; clone RA3-6B2; PharMingen, San Jose, CA), as well as polyclonal antibody against IL-1 (R&D, Minneapolis, MN). Sections were pretreated with proteinase K (Roche, Mannheim, Germany; 0.05% for 5 min at 37°C) for detection of neutrophils, macrophages and T-lymphocytes, or heat (95°C, 20 min) for IL-1 or were left without pretreatment for staining of B-lymphocytes. All above mentioned procedures were followed by blocking of endogenous peroxidase and subsequent incubation with a biotinylated species-specific anti-IgG secondary antibody. Sections were incubated with the appropriate ABC-complex using 3,3-diaminobenzidine (Sigma-Aldrich) as chromogen, resulting in brown staining of antigen-expressing cells.

Cartilage Neoepitope formation

Irreversible proteoglycan damage by MMP activity was assessed by immunohistochemistry staining for the VDIPEN neoepitope. Joint sections were deparaffinized, rehydrated, and digested for 1 h at 37°C in 0.25 units/ml of chondroitinase ABC in 0.1 M Tris·HCl (pH 8.0) (Sigma-Aldrich) to remove chondroitin sulfate from the proteoglycans. Sections were treated with 1% H₂O₂ in methanol, followed by treatment with 0.1% Triton X-100 in PBS. After incubation with 1.5% normal goat serum, sections were incubated with affinity-purified rabbit anti-VDIPEN IgG or with normal rabbit IgG, followed by biotinylated goat anti-rabbit IgG and avidin- peroxidase (Vector, Burlingame, CA). Peroxidase development was performed using nickel enhancement to increase sensitivity. Sections were counterstained with 2% orange G. Scoring was performed as follows: Total cell numbers and numbers of positively stained cells were counted in at least three different sites of the tarsal joint pannus in each mouse for determination of cellular distribution. Semiquantitative scoring using a scale from 0 to 3 (0, no staining; 1, 1-20% positive cells; 2, 20-50% positive cells; 3, >50% positive cells) was applied. Eight mice from the hTNFtg and IL-1^-/-hTNFtg mice (n = 4 per group) were evaluated.

Generation of Radiation Chimera

Recipient 4-week-old hTNFtg and IL-1^-/-hTNFtg mice received a lethal whole-body irradiation of 11 Gy. Bone marrow cells were isolated from long bones of age-matched donor hTNFtg or IL-1^-/-hTNFtg mice, washed in M199 medium (Sigma-Aldrich) supplemented with 10 mM Hepes buffer (ICN Biomedicals), 10 ng/ml DNase and 4 ng/ml gentamycin (both from Sigma-Aldrich), and 5 ´ 10⁶ bone marrow cells were injected into the tail vein of recipient mice. Chimeras were weekly assessed for clinical signs of arthritis as describe above and killed after 7 weeks for histological analyses of the tarsal joints.

Efficacy of bone marrow transplanation was ascertained by color in situ hybridization (CISH) analysis of the number of Y chromosome-positive male donor cells in the spleens of female recipients. Briefly, spleen cytospin preparations were denaturated and then dehydrated in a series of ethanol solutions and air-dried. The mouse Y chromosome probe (a kind gift from Dr. Diane Krause, Yale University, New Haven, CT) was labeled with digoxygenin by nick translation (Boehringer-Ingelheim, Germany). Detection was performed with an alkaline phosphatase labeled anti-digoxygenin antibody. For the color reaction, nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate was applied, and counterstaining with Kernechtrot was performed. Engraftment was complete as depicted in SI Fig. 8; 100% positive cells are not reached even in male positive controls because the cell nucleus is not fully sectioned and visible in all cells of cytospin preparations.

Isolation of Bone Marrow Macrophages

Bone marrow macrophages were prepared by flushing long bones of mice using a 25G needle and syringe filled with 5 ml RPMI 1640 medium. After washing with PBS, bone marrow cells were plated at 5 ´ 10⁶ cells per 10-cm dish with 6 ml of RPMI 1640 medium containing 10% FBS and 25 ng/ml murine M-CSF. After a 1-day culture, 3 ml of the above medium was added to the culture. After 3 or 4 days, floating cells were discarded and adherent cells used for assays. For immunoblotting analysis, protein extracts from RANKL-stimulated (50 ng/ml) cells were separated on SDS/12% PAGE and stained with antibodies against phosphorylated and total p38 MAPK, JNK, and ERK (Cell Signaling, Beverly, MA).

Generation of Osteoclasts and Osteoclast Analyses

Murine osteoclasts were generated as previously described (34). In brief, spleen cells from 6-week-old mice of all four genotypes were cultured overnight in aMEM containing 10% FBS (FBS) and 30 ng/ml M-CSF. After 24 h, nonadherent cells were harvested and subjected to gradient purification, seeded in 48-well plates (1 ´ 10⁶ cells/ml), and supplemented with 30 ng/ml M-CSF and 50 ng/ml RANKL (both R&D). For some experiments, human TNF (100 ng/ml) was additionally supplied. TRAP staining was performed after 5 days, and osteoclasts were identified by the presence of three or more nuclei and purple color. Bone resorption was performed on 0.4-mm-thick bovine cortical slices. Areas of bone resorption were quantitatively analyzed using an Axioskop 2 microscope (Carl Zeiss) equipped with a digital camera and Osteomeasure image analysis system (OsteoMetrics).

Enzyme-Linked Immunosorbent Assays (ELISAs). Supernatant levels of cathepsin K (Biomedica, Vienna, Austria) from osteoclast cultures as well as serum levels of collagen type I fragments (RatLaps, Nordic Bioscience) and TRAP5b (IDS, Frankfurt, Germany) were measured by ELISAs according to the manufacturer's recommendations. The lower limits of detection were 1.1 pmol/liter for cathepsin K, 2 ng/ml for collagen type I fragments, and 0.1units/liter for TRAP5b. Analyses were performed on 32 mice from the four treatment groups (n = 8 per group) and three different osteoclast cultures.

Synovial Fibroblast Analyses

Synovial tissue from 10--eek old wild-type, IL-1^-/-, hTNFtg, and IL-1^-/-hTNFtg mice (two mice per genotype) was minced, digested by 1.5 mg/ml dispase II (Roche Diagnostics, Mannheim, Germany), and cultivated in DMEM supplemented with 10% FCS (Invitrogen). Fibroblasts were passaged two to three times before the analyses when displaying typical synovial fibroblast morphology. Cell numbers were counted after various periods of time to show proliferative potential of cells. Apoptosis was determined by Apo-One Homogenous Caspase 3/7 Assay (Promega). Analyses were performed in unstimulated cells as well as cells treated with 100 ng/ml FasL or staurosporin (1 mg/ml). MMP-3 production was measured in supernatants by ELISA (Quantikine mouse MMP-3 ELISA kit, R&D Systems, Wiesbaden, Germany) after 24 h of culture.

Cartilage Explant Cultures and Macromolecule Biosynthesis

Knee joints of 10-week-old wild-type, IL-1^-/-, TNFtg, and IL-1^-/-TNFtg (n = 3-5 per group) mice were opened after sacrifice, and cartilage was obtained aseptically. Cartilage tissue was then cultivated in 24-well cell culture plates (Costar, Cambridge, MA) and labeled with 20mCi/ml of [³⁵S]sulfate (carrier-free; Amersham Pharmacia, Buckinghamshire, U.K.) for 3 h at 37°C. Explants were washed in ice-cold buffer [10 nM EDTA, 0.1 M sodium phosphate (pH 6.5)] and digested overnight by 1 mg/ml Proteinase K at 80°C. Unincorporated isotope was removed by using Sephadex G-25 (PD-10 columns; Pharmacia Biotech, Piscataway, NJ) gel chromatography. Values were obtained by liquid scintillation counting (1410 liquid scintillation counter; Wallac Oy, Turku, Finland) of aliquots from void volume fractions and normalized to DNA content. DNA content was assessed by use of bisbenzimide (Hoechst 33258; Sigma-Aldrich).

Primary Chondrocytes and Cellular Cocultures

Primary mouse chondrocytes were prepared from ventral rib cages of 1- to 3-day-old wild-type mice by collagenase digestion [0.1% collagenase D (Roche Applied Science) in F12/DMEM containing 5% FCS] after adhering connective tissue, and muscle was completely removed by trypsin and collagenase pretreatment. Cells were cultured in 12-well plates until confluency in F12/DMEM containing 10% FCS.

For coculture experiments, primary monocytes were isolated from the bone marrow of wild-type and IL-1^-/- mice by selection of CD11b using MACS technique (Miltenyi Biotec, Germany); 1 ´ 10⁶ monocytes per ml were placed in transwell cell culture inserts, incubated with TNF (10 ng/ml) for 1 h, and then thoroughly washed. Transwells with adherent monocytes were then placed in each well of 12-well plates with confluent primary chondrocytes, thus forming transwell chambers. Cells were cultured in F12/DMEM containing 10% FCS for indicated time periods. Total RNA from chondrocytes was isolated from cell cultures by Trizol reagent as described above.

Statistical Analysis

Data are shown as means ± SEM. Group mean values of histological data were compared by paired Student´s t test. For comparison of clinical assessments, a nonparametric Wilcoxon signed-rank test was used.