INDUCTION OF SWELLING, SYNOVIAL HYPERPLASIA AND CARTILAGE PROTEOGLYCAN LOSS UPON INTRA-ARTICULAR INJECTION OF TRANSFORMING GROWTH FACTOR p-2 IN THE RABBIT Peter R. Elford, Michael Graeber, Hiroshi Ohtsu,l Markus Aeberhard, Beatrice Legendre,l William L. Wishart,l Andrew R. MacKenzie Transforming growth factor p (TGF-P) is a multifunctional homodimeric polypeptide with potent actions upon many target cells, including those of mesenchymal and haemopoietic lineage. The recent reports of high levels of the cytokine in rheumatoid synovium and synovial fluid, prompted this study into the effect of intra-articular injection of TGFP-2 into rabbit knee-joints. Four daily injections of 1 pg caused swelling, probably as a consequence of prostaglandin E, production, synovial fibroblastic hyperplasia and a striking loss of femoral condyle proteoglycan. Using the polymerase chain reaction, no evidence could be obtained for the induction of interleukin-la gene expression in either synovial tissue or synovial fluid cells. These findings suggest that the TGF-P present in the rheumatoid joint may contribute directly to the pathogenesis of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a multi-faceted, chronic inflammatory process, involving reaction of both the immune system and the connective tissues of the joint. Prominent in the disease are profound hypertrophic and proliferative changes in the synovium, which lead to the formation of a pannus that invades articular cartilage and subchondral bone.’ A number of inflammatory mediators, such as interleukins 1,6 and 824 and granulocyte-macrophage colony-stimulating factor5 have been identified in

From

the Department of Bone and Joint Disease, Sandoz Research Institute, Berne, Switzerland and ‘Department of Biotechnology, Sandoz Preclinical Research, Basel, Switzerland. Address correspondence and reprint requests to: Dr Peter R. Elford, Department of Bone and Joint Disease, Sandoz Research Institute, Berne, P.O. Box 2173, CH-3001 Berne, Switzerland. Received 1 November 1991; accepted for publication 12 January 1992 0 1992 Academic Press Limited 1043-4666/92/030232+07 $05.00/O KEY

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WORDS: arthritis/cartilage/interleukin transforming growth factor-p

Usynovium

rheumatoid tissue or synovial fluid, although the complex interplay between these cytokines makes their relative importance in contributing to the disease process difficult to establish. Recently, transforming growth factor p (TGF-l3) has also been detected in rheumatoid synovial fluid6s7 and found to be produced by rheumatoid synovial explant9 and synovial fibroblasts.9 TGF-l3 is a multifunctional polypeptide, the cellular responses to which appear pertinent to the development and maintenance of a chronic synovitis. Cellular proliferation, extracellular matrix protein production and pericellular proteolysis are all influenced by TGF-l3. lo Furthermore, the molecule also has notable effects upon the function of cells involved in the inflammatory response.” The object of this study was to ascertain the consequence of injecting active TGF-6 into a normal rabbit joint. We found that four daily injections of 1 pg recombinant human TGF+2 induced joint swelling, fibroblastic proliferation of the synovial membrane and profound loss of articular cartilage proteoglycan. The latter occurred without the apparent induction of synthesis of interleukin-la (ILla), a cytokine known to induce cartilage breakdown in vivo.12

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RESULTS Injection of TGF-B into rabbit knee-joints resulted in a marked swelling not apparent with vehicle-injected knees (Fig. 1). Upon opening knees injected with TGF-B, it was found that the swelling was due in part to oedema of the soft tissues, and also the deposition of granulation tissue in the synovial membrane. No macroscopic pathological changes to cartilage or bone were observed. Prostaglandin E, (PGE,) levels increased in the synovial fluid lo-fold (Fig. 2); prostaglandin-induced hyperaemia, working in concert with additional vasoactive substances, probably contributed to the observed oedema. Microscopic examination of synovial membrane taken from joints injected with TGF-/3 revealed a fibroblastic hyperplasia with a diffuse monocytic infiltrate, but with a notable absence of neutrophils (Fig. 3A, B). Femoral condyle cartilage from these joints showed extensive loss of metachromasia, compared with vehicle-injected joints (Fig. 4A, B respectively), indicating loss of proteoglycan. Biochemical analysis of articular cartilage confirmed the loss of proteoglycan seen microscopically, a striking reduction in the glycosaminoglycan content of the femoral icondyle being observed (Fig. 5A). In contrast, the collagen (hydroxyproline) content remained unchanged (Fig. 5B). Since IL-1 causes cartilage proteoglycan breakdown both in vitroI and in vivo,12 and as TGF-B is reported to induce IL-l expression in resting monocytes14~15 synovial tissue and synovial fluid cells from four TGF-B-injected joints were examined for the presence of mRNA for the cytokine. This was carried out by the polymerase chain reaction (PCR), using specific primers to amplify cDNA for IL-la (or y-actin as positive control). Whereas a clear signal for y-actin was present, that for IL-la was absent from both synovial tissue (Fig. 6A) and synovial fluid cells (Fig. 6B). Although we cannot exclude a level of IL-1 expression below that detectable by our PCR system, it does appear likely that the pathological changes induced by TGF-B occurred in the absence of IL production.

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Figure 1. Swelling daily intra-articular

of rabbit injections

knee-joints 24 hours of 1 pg TGF-P.

Vehicle open bars, TGF-P striped. Bars are means per group, those with TGF-P were significantly vehicle, P

Induction of swelling, synovial hyperplasia and cartilage proteoglycan loss upon intra-articular injection of transforming growth factor beta-2 in the rabbit.

Transforming growth factor beta (TGF-beta) is a multifunctional homodimeric polypeptide with potent actions upon many target cells, including those of...
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