Induction of apoptosis in chondrocytes by tumor necrosis factor-alpha

Tumor necrosis factor alpha (TNF-alpha) induces apoptosis in a number of ce ll types and plays an essential role in bone remodeling, both stimulating t he proliferation of osteoblasts and activating osteoclasts. During endochon dral ossification, apoptosis of chondrocytes occurs concurrently with new b one formation and the resorption and replacement of mineralized cartilage w ith woven bone. In the present study, the role of TNF-alpha in promoting ch ondrocyte apoptosis was examined. Chondrocyte cell populations, enriched in either hypertrophic or non-hypertrophic cells, were isolated from the ceph alic and caudal portions of 17-day chick embryo sterna, respectively, and t reated in vitro with 0.1-10 nM recombinant human TNF-alpha. As a positive c ontrol, apoptosis was also induced by Fas receptor antibody binding. Dye ex clusion assays of the live/dead ratios of cells showed that TNF-alpha cause d a dose-dependent 1.5- and 2.0-fold increase in the number of dead cells i n both hypertrophic and non-hypertrophic chondrocytes. Induction of apoptos is was independently assayed by measurement of interleukin-1 beta -converti ng enzyme (ICE) activity, and analyzed by a semi-quantitative determination of DNA fragmentation. When compared to untreated cells, these analyses als o showed dose-dependent increases in TNF-alpha induced apoptosis in both ch ondrocyte populations, with increases in the levels of ICE activity for all doses of TNF-alpha (from similar to5 to similar to 20 fold). Osteoblasts, however, were not affected by treatment with TNF-alpha or by Fas antibody/p rotein G induction. Immunostaining of chondrocytes for Fas receptor and cas pase-2 protein expression showed that most of the chondrocytes expressed th ese two markers of apoptosis after treatment with TNF-alpha. Although cell killing and ICE induction were higher in the more hypertrophic cells, TNF-a lpha induced apoptosis in both hypertrophic and non-hypertrophic chondrocyt e populations, These results demonstrate that apoptosis may be induced in b oth hypertrophic and non-hypertrophic chondrocytes through both Fas and TNF -alpha receptor mediated signaling, and suggest that chondrocytes are more sensitive to apoptotic effects of TNF-alpha within the skeletal lineage tha n are osteoblasts. (C) 2001 Orthopaedic Research Society. Published by Else vier Science Ltd. All rights reserved.