News and Publications

Division of Arthritis Research

Martin Lotz, M.D., Division Head

Chondrocyte Apoptosis in Human and Experimentally Induced Osteoarthritis

S. Hashimoto, K. Kühn, R.L. Ochs, L. Creighton-Achermann, M. Lotz

Age-related changes in human articular cartilage include a reduction in tissue cellularity, an increase in the number of empty lacunae, and abnormal calcification of the extracellular matrix. The age-related decreases in cell numbers correlate with increased frequencies of fibrillation.

We studied the occurrence of apoptosis and the relationship between chondrocyte apoptosis and cartilage degradation in human osteoarthritis. Knee cartilage was obtained at autopsy, from tissue banks, and from patients undergoing total joint replacement surgery. Approximately 22% of chondrocytes from subjects with osteoarthritis and 5% of normal chondrocytes were undergoing apoptosis. Areas of cartilage that contained apoptotic cells showed proteoglycan depletion, and the number of apoptotic cells correlated significantly with the severity of osteoarthritis.

To determine the relationship between synthesis of nitric oxide, chondrocyte apoptosis, and the development of cartilage degradation, we used a model of osteoarthritis in rabbits. Osteoarthritis was induced in experimental animals by transection of the anterior cruciate ligament, and samples of cartilage from the treated joints were examined. Controls consisted of cartilage from untreated joints in rabbits that had transection of the ligament in a joint and cartilage from rabbits that had no surgery in any limb. Conditioned media from organ cultures of cartilage obtained from treated joints contained higher levels of nitric oxide than did the media of organ cultures of cartilage obtained from control joints. Approximately 29% of chondrocytes isolated from cartilage from surgically treated joints and 7% of chondrocytes from cartilage from untreated joints in the same animals were undergoing apoptosis. The prevalence of apoptotic cells correlated with the levels of nitrite production and the grade of the osteoarthritis.

These observations indicate an increase in chondrocyte apoptosis in cartilage in humans with osteoarthritis and in animals with experimentally osteoarthritis. Chondrocyte apoptosis and proteoglycan depletion are anatomically linked and may be mechanistically related. Thus, inhibitors of the production of nitric oxide synthesis or chondrocyte apoptosis may be of therapeutic value after cartilage injury and in osteoarthritis.

Articular cartilage has several features that affect the fate of apoptotic bodies. This tissue is not vascularized and does not contain mononuclear phagocytes. Furthermore, chondrocytes are anchored in the extracellular matrix, are surrounded by a pericellular matrix, and are not known to migrate within cartilage. Thus, no apparent mechanism exists for the clearance of apoptotic bodies. Electron microscopy of apoptotic chondrocytes suggested a similarity between apoptotic bodies and matrix vesicles, which are essential in cartilage calcification during the formation of endochondral bone. We examined structural and functional properties of chondrocyte-derived apoptotic bodies.

In cartilage treated with nitric oxide, the dense pericellular matrix that normally surrounds the cells was degraded, and apoptotic bodies accumulated within and in the vicinity of the chondrocyte lacunae. Functional analysis indicated that apoptotic bodies isolated from treated chondrocytes or cartilage produced pyrophosphate. Apoptotic bodies contained alkaline phosphatase and nucleoside triphosphate pyrophosphohydrolase activities and precipitated calcium. These results suggest that chondrocyte-derived apoptotic bodies express functional properties that may contribute to the pathologic calcification of cartilage observed in aging and osteoarthritis.

Signal Transduction in Cartilage Degradation

Y. Geng, T. Shalom-Barak, J. Valbracht, M. Lotz

In human articular chondrocytes, IL-1 induces the expression of a large number of genes associated with joint inflammation and cartilage degradation. We used pharmacologic inhibitors to analyze the role of different protein kinases in the expression of chondrocyte genes.

Inhibition of protein tyrosine kinases with herbimycin A or genistein prevented the expression of most IL-1­induced genes, including those for inducible cyclooxygenase, inducible nitric oxide synthase, cytokines, and collagenase. In contrast, protein kinase C and protein kinase A inhibitors had a selective effect on the expression of these genes. The effects of herbimycin were selective; it did not inhibit the IL-1--induced expression of the immediate-early gene product c-Fos. Analysis of known IL-1--activated intracellular signaling events showed that herbimycin only partially reduced the activities of the MAP kinases Erk and JNK and had almost no effect on p38 kinase. IL-1 also increased the DNA-binding activities of the transcription factors NF-B and activator protein-1, and this increase was only marginally reduced by herbimycin A.

These results with kinase inhibitors were supported by the discovery of tyrosine phosphorylation of 32-, 34-, 60-, 80-, and 150-kD proteins after IL-1 stimulation of chondrocytes. Tyrosine-phosphorylated proteins were coprecipitated with the Grb2 adaptor molecule, and the amino acid sequence was determined. The results indicated that histone H1 was one of the tyrosine-phosphorylated proteins and suggest that the central role of tyrosine kinases in IL-1--induced expression of chondrocyte genes may be related to changes in chromatin structure.

Cytokine Networks in Synovial Inflammation and Cartilage Degradation

T. Olee, M. Setareh, J. Quach, T. Shalom-Barak, M. Lotz

Synovial inflammation, hyperplasia of the synovial membrane, and degradation of articular cartilage are major components in the pathogenesis of arthritis. These events are, in part, the consequence of activation of synoviocytes and chondrocytes by cytokines and growth factors. The cytokines IL-1 and TNF- are principal mediators in the pathogenesis of arthritis.

IL-17 was originally identified as a antigen associated with cytotoxic T lymphocytes. The receptor for IL-17 is not related to previously identified cytokine receptor families. Stimulation of human articular chondrocytes with IL-17 induced expression of several genes associated with inflammation and cartilage degradation, including those for IL-1ß, IL-6, cyclooxygenase 2, inducible nitric oxide synthase, and stromelysin. MAP kinases and the nuclear transcription factor NF-B were activated in response to IL-17. Dexamethasone reduced IL-17--induced release of nitric oxide, inhibited expression of inducible nitric oxide synthase and cyclooxygenase 2, and reduced IL-17--induced gene expression. The p38 kinase--specific inhibitor SB203580 had qualitatively similar effects but was less effective. These results suggest that IL-17 activation of chondrocytes is associated with and depends at least in part on the activation of MAP kinases and NF-B.

IL-18, also known as IFN---inducing factor, has structural similarities with the IL-1 family of proteins. IL-18 mRNA was induced by cytokines, including IL-1ß, TNF-, and IL-17, in chondrocytes and synovial fibroblasts. Chondrocytes produced the IL-18 precursor and in response to IL-1 stimulation secreted the mature form of IL-18. This event was prevented by an inhibitor of caspase I.

Studies of the effects of IL-18 on chondrocytes showed that it inhibits proliferation and induces production of nitric oxide. IL-18 stimulated the expression of several genes in normal human articular chondrocytes, including those for inducible nitric oxide synthase, inducible cyclooxygenase, IL-6, and stromelysin. Gene expression was associated with the synthesis of the corresponding proteins. Treatment of normal human articular cartilage with IL-18 increased the release of glycosaminoglycans. These findings indicate that IL-18 is a cytokine that promotes synovial inflammation and cartilage degradation.

PUBLICATIONS

Hashimoto, S., Ochs, R.L., Lotz, M. The Linkage of chondrocyte apoptosis and cartilage degradation in human osteoarthritis. Arthritis Rheum., in press.

Hashimoto, S., Ochs, R.L., Rosen, F., Quach, J., McCabe, G., Solan, J., Seegmiller, J.E., Terkeltaub, R., Lotz, M. Chondrocyte-derived apoptotic bodies and calcification of articular cartilage. Proc. Natl. Acad. Sci. U.S.A. 95:3094, 1998.

Hashimoto, S., Takahashi, K., Amiel, D., Coutts, R.D., Lotz, M. Chondrocyte apoptosis and nitric oxide production during experimentally induced osteoarthritis. Arthritis Rheum. 41:1266, 1998.

Lotz, M. Mechanisms of pain in arthritis. In: Anesthesia: Biologic Foundations. Biebuyck, J.F., et al. (Eds.). Lippincott-Raven, Philadelphia, 1998, p. 819.

Lotz, M. Mechanisms of tissue destruction in rheumatoid arthritis. In: Rheumatology, 2nd ed. Klippel, J.H., Dieppe, P.A. (Eds.). Mosby, St. Louis, 1998, p. 11.1.

Lotz, M. Nitric oxide and chondrocyte survival. Osteoarthritis Cartilage, in press.

Lotz, M. The role of nitric oxide in articular cartilage damage. Rheum. Dis. Clin. North Am., in press.

Lotz, M., Hashimoto, S., Ochs, R.L., Kühn, K. Chondrocyte apoptosis. In: Apoptosis and Inflammation. Winkler, J.D. (Ed.). Birkhaüser, Basel, in press.

Lotz, M., Shalom-Barak, T. IL-17 signaling. In: Cytokine signal transduction mechanisms. Lewis, A.J., Manning, A.M. (Eds.). Birkhäuser, Basel, Switzerland, in press.

Schwarz, H., Arden, A., Lotz, M. CD137, a member of the tumor necrosis factor receptor family, is located on chromosome 1p36, in a cluster of related genes, and colocalizes with several malignancies. Biochem. Biophys. Res. Commun. 235:699, 1997.

Terkeltaub, R., Lotz, M., Johnson, K., Deng, D., Hashimoto, S., Goldring, M.B., Burton, D., Deftos, L.J. Parathyroid hormone related protein (PTHrP) 1-173 expression is increased in osteoarthritic cartilage and is selectively induced by TGF-ß in articular chondrocytes: PTHrP 1-173 inhibits inorganic pyrophosphate (PPi) release. Arthritis Rheum., in press.

von Kempis, J., Schwarz, H., Lotz, M. Differentiation-dependent and stimulus-specific expression of ILA, the human 4-1BB-homologue, in cells of mesenchymal origin. Osteoarthritis Cartilage 5:394, 1997.