M.J. Howard, M.G. Chambers, R.M. Mason, C.M. Isacke

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Distribution of Endo180 receptor and ligand in developing articular cartilage M.J. Howard, M.G. Chambers, R.M. Mason, C.M. Isacke Osteoarthritis and Cartilage Volume 12, Issue 1, Pages 74-82 (January 2004) DOI: 10.1016/j.joca.2003.07.001

Fig. 1 Characterization of anti-Endo180 antibodies and soluble Endo180 constructs. (A) Diagram showing the domain structure of Endo180, the soluble Endo180 (sEndo180) construct, and the soluble Endo180-Fc chimera (sEndo180-Fc). CR, cysteine-rich domain; FNII, fibronectin type II domain; CTLD, C-type lectin-like domain. The signal sequence is not shown. COS-1 cells were transfected with pcDNA3 vector (-), pcDNA3ΔHindII-sEndo180 (sEndo180) or pIgplus-sEndo180-Fc (sEndo180-Fc), and the media harvested 5 days later. 20μl of medium was resolved by 10% non-reducing SDS-PAGE and blotted onto a nitrocellulose membrane. In parallel, 10μg of lysate from MG63 cells which express endogenous receptor (Endo180) was run and blotted. Membranes were incubated with anti-human Endo180mAb A5/158 followed by an HRP-conjugated anti-mouse Ig antibody, developed using the ECL reagent and exposed to film for 15s. The increased molecular size of the Fc construct results from dimerization via the Fc tail. (B) Serial cryosections of tibial articular cartilage from a 14-week STR/ort mouse were probed with anti-Endo180 antibody that had been incubated with untreated nitrocellulose (- sEndo180) or preabsorbed with sEndo180 protein bound to nitrocellulose (+ sEndo180), followed by FITC-anti-rabbit Ig. Sections were viewed by fluorescence microscopy and exposure settings were locked for qualitative analysis. AC, articular cartilage. Original magnification ×50. Osteoarthritis and Cartilage 2004 12, 74-82DOI: (10.1016/j.joca.2003.07.001)

Fig. 2 Endo180 expression in the medial tibial articular cartilage of CBA and STR/ort murine knee joint. Cryosections of tibial articular cartilage of (A) 5-week, (B) 9-week, (C) 14-week, (D) 35-week CBA mice and (F) 6-week, (G) 9-week, (H) 14-week, (I) 35-week STR/ort mice were probed with anti-Endo180 antibody followed by FITC-anti-rabbit Ig. (E, J) 5-week CBA and 6-week STR/ort mice probed with FITC-anti-rabbit Ig alone, respectively. Sections were viewed by fluorescence microscopy and exposure settings were locked for qualitative analysis. AC=articular cartilage, CT, calcified tissue. Original magnification ×50. Comparable results were obtained from a minimum of three different animals at each age examined on at least two separate occasions. Osteoarthritis and Cartilage 2004 12, 74-82DOI: (10.1016/j.joca.2003.07.001)

Fig. 3 Loss of Endo180 expression in the articular cartilage from older animals. Cryosections through the tibial lateral articular cartilage of (A, B) 14-week or (C, D) 35-week CBA mouse knee joints were probed with anti-Endo180 antibody followed by FITC-anti-rabbit Ig and counterstained with propidium iodide. Sections were photographed independently in the FITC (A, C) and TRITC (B, D) channels. Sections through the tibial lateral (E) and medial (F) articular cartilage from the same knee joint of a 35-week STR/ort mouse were probed with anti-Endo180 antibody followed by FITC-anti-rabbit Ig. Arrows in panel F shows a grade 3 osteoarthritic lesion.17AC, articular cartilage; CT, calcified tissue; FC, femoral cartilage. Original magnification ×50. Osteoarthritis and Cartilage 2004 12, 74-82DOI: (10.1016/j.joca.2003.07.001)

Fig. 4 Endo180 ligand distribution in articular cartilage. Cryostat sections were cut through the knee joint of a 6-week STR/ort mouse and probed with (A) purified sEndo180-Fc followed by FITC-anti-human Fc Ig, (B) purified ICAM-Fc followed by FITC-anti-human Fc Ig or (C) anti-Endo180 antibody followed by FITC-anti-rabbit Ig. AC, articular cartilage; CT, calcified tissue. Original magnification x50. Osteoarthritis and Cartilage 2004 12, 74-82DOI: (10.1016/j.joca.2003.07.001)

Fig. 5 Distribution of Endo180 receptor and Endo180 ligand in the growth plate. For Endo180 receptor staining, cryostat sections through the knee joints of (A) 5-week male CBA and (B) 6-week old male STR/ort mice were probed with anti-Endo180 antibody followed by FITC-anti-rabbit Ig. For Endo180 ligand staining, cryostat sections through the knee joint of a (C) 5-week male CBA or (D) 6-week STR/ort mouse were probed with sEndo180-Fc and detected by FITC-anti-human Fc Ig. Panel E shows a control section of a 6-week STR/ort mouse probed with FITC-anti-human Fc Ig alone. NHZ, non-hypertrophic zone. HZ, hypertrophic zone. Original magnification ×50. Osteoarthritis and Cartilage 2004 12, 74-82DOI: (10.1016/j.joca.2003.07.001)

Fig. 6 Expression of Endo180 by chondrocytes in vitro. (A) MC615 or HT1080 cells were stained with anti-Endo180 antibody followed by Alexa 488 anti-rabbit Ig, fixed in 1% paraformaldehyde and analysed by flow cytometry (solid profiles). Open profiles show cells stained with Alexa 488 anti-rabbit Ig alone. (B) MC615 chondrocyte or HT1080 fibrosarcoma cells cultured on coverslips were fixed in 3% paraformaldehyde, permeabilized with 0.2% saponin and stained with anti-Endo180 antibody followed by Alexa 488 anti-rabbit Ig antibody. Nuclei were counterstained with TO-PRO-3 and cells subject to confocal microscopy. Horizontal (xy) sections through the cells are shown in the upper panels, vertical (xz) sections are shown in the lower panels. Scale bar=20μm. Osteoarthritis and Cartilage 2004 12, 74-82DOI: (10.1016/j.joca.2003.07.001)