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Immunolocalisation of Matrix Metalloproteinase 3 (Stromelysin) in Rheumatoid Synovioblasts (B Cells): Correlation with Rheumatoid Arthritis

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Immunolocalisation of Matrix Metalloproteinase 3 (Stromelysin) in Rheumatoid Synovioblasts (B Cells): Correlation with Rheumatoid Arthritis Ann Rheum Dis: first published as 10.1136/ard.48.8.645 on 1 August 1989. Downloaded from Annals of the Rheumatic Diseases 1989; 48: 645-653 Immunolocalisation of matrix metalloproteinase 3 (stromelysin) in rheumatoid synovioblasts (B cells): correlation with rheumatoid arthritis YASUNORI OKADA,' NAOTO TAKEUCHI,2 KATSURO TOMITA,2 ISAO NAKANISHI,' AND HIDEAKI NAGASE3 From the Departments of 'Pathology and 2Orthopedic Surgery, School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920, Japan; and the 3Department of Biochemistry, University of Kansas Medical Center, 39th and Rainbow Blvd, Kansas City, KS 66103, USA SUMMARY Metalloproteinases produced by connective tissue cells may play a key part in the destruction of joints in rheumatoid arthritis. Matrix metalloproteinase 3 (MMP-3; stromelysin) capable of degrading cartilage proteoglycans and type IX collagen and of activating procollagenase was immunolocalised in hyperplastic synovial lining cells in rheumatoid synovium, but not in the cells of normal synovium. Cells responsible for synthesis of MMP-3 have the phenotype of synovioblasts (B cells) by immunoelectron microscopy, but not of phagocytic synovial macrophages (A cells). Cultured monolayer of rheumatoid synovial cells synthesises MMP-3 only copyright. under treatment with macrophage conditioned medium. Immunolocalisation of MMP-3 in rheumatoid synovium and cultured synovial cells was possible when the specimens were treated with a monovalent ionophore, monensin. These results suggest that MMP-3 is synthesised and secreted continuously without storage from hyperplastic synovioblasts stimulated by factor(s) derived from activated macrophages present in the synovium. http://ard.bmj.com/ In rheumatoid arthritis the joints affected show lagen types I, II, III, and X,"5 MMP-2 (also called chronic proliferative synovitis that causes destruc- 'gelatinase')f' 7 and MMP-3 (stromelysin).8 9 Their tion of articular cartilage, subchondral bones, ten- synthesis and secretion are induced by factors such dons, and ligaments, resulting in deformity and as interleukin 1,10 tumour necrosis factor a,1" and disability of the joints. Although mechanical factors substance p.12 MMP-3 degrades a wide spectrum of or oxygen derived free radicals, or both, may extracellular matrix macromolecules, including car- on September 25, 2021 by guest. Protected contribute to progressive damage in inflamed joints, tilage proteoglycans, fibronectin, type IV collagen, the degradation of the major extracellular matrix and laminin. It also cleaves type IX collagen components of articular cartilage, proteoglycans, (manuscript in preparation), a recently charact- and collagens can be attributed to proteolytic erised collagen that stabilises cartilage by interacting enzymes.' All classes of proteinases have been with the triple helical domains of the type II collagen implicated in the degradation of connective tissue molecule.1314 In addition, an endogenous activator matrix components in various forms of arthritis.2 of procollagenase reported in various connective Among them, matrix metalloproteinases (MMPs) tissues in culture'57 has been proved to be MMP-3 derived from connective tissue cells are considered (stromelysin).'8 19 Direct evidence of the involve- to play an important part in joint destruction during ment of MMP-3 in rheumatoid arthritis has not been the long course of rheumatoid arthritis. They obtained, however, and identification of the cells include collagenase (EC 3.4.24.7) that digests col- leading to the synthesis and secretion of MMP-3 has not been investigated. Here we report that MMP-3 Accepted for publication 6 January 1989. Correspondence to Dr Yasunori Okada, Department of Pathology, is immunolocalised in hyperplastic synovial lining School of Medicine, Kanazawa University, 13-1 Takara-machi, cells and its synthesis is specific to synovioblasts (B Kanazawa, Ishikawa 920, Japan. cells) of rheumatoid synovium. 645 Ann Rheum Dis: first published as 10.1136/ard.48.8.645 on 1 August 1989. Downloaded from 646 Okada, Takeuchi, Tomita, Nakanishi, Nagase Materials and methods Nilsson.21 Immunoprecipitation of MMP-3 with the antiserum was carried out with a culture medium of PURIFICATION OF ACTIVE AND PRECURSOR [3H]leucine labelled . rheumatoid synovial cells FORMS OF MMP-3 stimulated with rabbit macrophage conditioned Active MMP-3 with relative molecular weight (Mr) medium22 according to the method of Nagase et al.23 28 000 and 45 000 and the precursor forms with Mr [3H]Leucine labelled MMP-3 was immunoprecipi- 57 000 and 59 000 (proMMP-3) were purified to tated before and after treatment with 1.5 mM 4- homogeneity from the culture medium of rheuma- aminophenyl mercuric acetate for two hours at toid synovial cells treated with macrophage con- 37°C. For electrophoretic immunoblotting analysis ditioned medium as described.8 20 Enzymic activities concentrated culture medium and two purified against [14C]collagen, [14C]gelatin, and [3H]car- forms of proMMP-3 with Mr 57 000 and 59 00020 boxymethylated transferrin were assayed as de- were electrophoresed in sodium dodecyl sulphate/ scribed.8 Purified proMMP-3 (17 1Ag) of Mr 57 000 polyacrylamide gel (10% total acrylamide) under was coupled to cyanogen bromide activated reduced conditions. Proteins separated in the gel Sepharose 4B (Pharmacia Fine Chemicals) accord- were electrotransferred to nitrocellulose paper. The ing to the manufacturer's directions and used for the paper was incubated for one hour at 22°C in the absorption of the specific antibody. antiserum diluted 1:1000 in PBS/1% bovine serum albumin/0005% Tween 20, washed, and incubated in PREPARATION AND CHARACTERISATION OF an alkaline phosphatase conjugated rabbit IgG to ANTIBODY TO MMP-3 sheep IgG (Cappel). Immunoreactive MMP-3 was A 50 1.g sample of purified MMP-3 with Mr 28 000 visualised with 165 jig/ml of 5-bromo-4-chloro-3- was emulsified with an equal volume of Freund's indolyl phosphate (Sigma Chemical Co) and 330 complete adjuvant (Difco Laboratories) and in- Rg/ml of nitroblue tetrazolium (Sigma Chemical Co) jected into an adult male sheep. Two further in 0-1 M trometamol-HCI pH 9-5 containing 0-1 M subcutaneous injections-30 [ig and 10 ,ug of enzyme NaCl and 5 mM MgC12. copyright. in incomplete adjuvant-were given after 14 and 24 days. A preimmune bleed and further bleeds were SYNOVIAL TISSUE AND CELL CULTURE taken on days 0, 14, 24, 35, 46, 49, 56, 60, and 67. Synovial tissue obtained at arthroplasty from nine The titre was checked by double immunodiffusion patients with classical (five cases) and definite (four on Ouchterlony plates. Immunoglobulin G was cases) rheumatoid arthritis was cut into pieces and prepared from the antiserum with the highest titre incubated in Dulbecco's modified Eagle's medium and preimmune serum by precipitation with ammo- containing 10% fetal calf serum in the presence or nium sulphate (30% saturation) and column chro- absence of 1 FiM monensin (Sigma Chemical Co) for http://ard.bmj.com/ matography on diethylaminoethyl cellulose equili- 10, 30 minutes, 1, 2, and 3 hours. The tissue brated with 0-1 M trometamol (TRIS)-HCl pH specimens freshly excised or incubated with or 8-0/0-02% NaN3. IgG F(ab')2 was purified by without monensin were embedded in Tissue-Tek application of the IgG digested with 2% (w/v) OCT compound (Miles Scientific) without fixation, pepsin for 49 hours at 37°C to a column of Ultrogel and frozen sections (4-6 ,um) were prepared for AcA 44 (15x 115 cm) equilibrated with phosphate immunofluorescent studies. Another set of samples buffered saline (PBS) containing 0-02% NaN3. was fixed in periodate-lysine-paraformaldehyde on September 25, 2021 by guest. Protected The ability of the antibody to inhibit MMP-3 was fixative24 for 15 hours at 4°C and embedded in Epon checked by using purified active MMP-3 of Mr 812 for immunohistochemistry of 1 tm sections by 45 O008 in [3H]carboxymethylated transferrin and the avidin-biotin-peroxidase complex method and [14C]gelatin assays. Purified IgG F(ab')2 treated immunoelectron microscopy. As controls, three with 2 mM di-isopropyl fluorophosphate was incu- samples of synovium with normal histology were bated with the enzyme solutions (110 ng) for one obtained from an ankle joint of an amputated leg of hour at 37°C and for an additional two hours at 22°C a patient with malignant giant cell tumour, from a before the enzyme assays. Inhibitory activities of the knee joint at meniscectomy, and from an elbow IgG F(ab')2 to partially purified collagenase and joint of a patient with osteochondritis dessicans. pure MMP-2 from the culture medium of rheuma- They were treated with or without monensin in toid synovial cells were also examined in Dulbecco's modified Eagle's medium containing [I4C]collagen and ['4C]gelatin assays respectively. 10% fetal calf serum for three hours and embedded Antibody specificity was studied by double im- in Tissue-Tek OCT compound. For ordinary histo- munodiffusion, immunoprecipitation, and electro- logical examination synovial tissue used in this study phoretic immunoblotting. Double immunodiffusion was also fixed in formaldehyde, and paraffin sec- was performed by the methods of Ouchterlony and tions were stained with haematoxylin and eosin. Ann Rheum Dis: first published as 10.1136/ard.48.8.645 on 1 August 1989. Downloaded from Immunolocalisation of matrix metalloproteinase 3 in rheumatoid synovium 647 Dissociated cells in the first generation of passage incubated with biotinated rabbit IgG to sheep IgG were prepared from the above mentioned rheuma- (Vector Laboratories, diluted 1:200) for 30 minutes toid synovial tissue according to the method of and with an avidin-biotin-peroxidase complex (Vec- Dayer et al5 and cultured on two-well Lab-Tek tor Laboratories) for 30 minutes at room tempera- slides (Miles Laboratories) in Dulbecco's modified ture. Colour was developed with 0-03% 3,3'- Eagle's medium with 10% fetal calf serum for five to diaminobenzidine tetrahydrochloride in 50 mM seven days.
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