Differential effects of tumor necrosis factor-α and interleukin-1β on cell death in human articular chondrocytes

Objective: The death of chondrocytes by apoptosis is characteristic of degenerative joint diseases, such as osteoarthritis (OA). Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) have been shown to play an important role in the development of OA. In this study we analyzed the effects of TNF-alpha and IL-1 beta on cell death in normal human chondrocytes. Methods: Normal human chondrocytes were isolated from knee cartilage obtained at autopsy from 30 adult cadaveric donors. The cells were stimulated with TNF-alpha (10 ng/ml) or IL-1 beta (5 ng/ml) in the presence or absence of Ro 31-8220 (Ro: a structurally related analog of bisindolylmaleimide that inhibits mitogen-activated protein kinase phosphatase 1 [MKP-1]) (Ro; 10 mu M), an MKP-1 inhibitor, which induces apoptosis in chondrocytes. Apoptosis was evaluated by flow cytometry (propidium iodide) and nuclear morphology was evaluated with 4',6'-dianidino-2-phenylindole dihydrochloride. The expressions of caspase-8, -7 and -3 and Bcl-2 were analyzed by Western blot and the activation of caspase-3 and -8 was measured by flow cytometry. Prostaglandin E2 (PGE2) was evaluated by enzyme-linked immunosorbent assay. Results: At 24 h the percentage of apoptotic (hypodiploid) nuclei induced by TNF-alpha + Ro was higher than the level induced by Ro alone. The combination of IL-1 beta (5 ng/ml) with Ro did not show a synergistic effect. A morphological analysis demonstrated that treatment with TNF-alpha + Ro resulted in a large number of cells with condensed nuclei and DNA fragmentation. Western blot studies indicated that IL-1 beta + Ro did not induce the time-dependent activation of caspase-8, -7 and -3 as seen with TNF-alpha + Ro. As quantified by flow cytometry, TNF-alpha + Ro induced a higher level of caspase-3 and -8 activation than that seen with IL-1 beta + Ro. Pre-incubation for 2 h with caspase inhibitors for caspase-3, -7, -8 and pan-caspase significantly decreased the hypodiploid DNA peak induced by treatment with TNF-alpha + Ro at 24 h. Indomethacin increased the cell death induced by IL-1 beta + Ro; however, apoptosis induced by TNF-alpha + Ro was not modified by indomethacin. Conclusions: These results confirm that TNF-alpha and IL-1 beta regulate apoptosis differently in this human chondrocyte model and that the differing effects of these cytokines are PGE2-independent. Indomethacin potentiates the effect of IL-1 on cell death and this may explain the reported effect of indomethacin on the progression of joint destruction. (C) 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.