Anti-Fas-induced apoptosis in chondrocytes reduced by hyaluronan - Evidence for CD44 and CD54 (intercellular adhesion molecule 1) involvement

Objective. To investigate the in vitro effect of therapeutic hyaluronan (HA ) of 500-730 kd on anti-Fas-induced apoptosis of chondrocytes from osteoart hritis (OA) patients, and to assess its mechanism of action by analyzing th e role of the 2 HA receptors, CD44 and CD54 (intercellular adhesion molecul e 1 [ICAM-1]). Methods. Chondrocytes isolated from human OA knee cartilage were cultured a nd the effect of HA on both spontaneous and anti-Fas-induced apoptosis was evaluated. Apoptosis was analyzed by JAM test (for quantitative analysis of fragmented DNA), cell death detection immunoassay (for quantitative analys is of oligonucleosome), TUNEL assay, and electron microscopy. Blocking expe riments with anti-CD44 and anti-CD54 alone or in combination were performed to investigate the HA mechanism of action. Results. Both quantitative tests demonstrated that anti-Fas significantly i nduced apoptosis of isolated OA chondrocytes. HA at 1,000 mug/ml significan tly reduced the anti-Fas-induced apoptosis of chondrocytes but did not affe ct spontaneous chondrocyte apoptosis. These data were also confirmed by TUN EL staining and by electron microscopy morphologic evaluation. The antiapop totic effects of HA on anti-FAS-induced chondrocyte apoptosis were signific antly decreased by both anti-CD44 (mean +/- SD 57 +/- 12% inhibition) and a nti-ICAM-1 (31 +/- 22% inhibition). The mixture of the 2 antibodies had an additive effect, since the rate of inhibition increased to 87 +/- 13%. Conclusion. These data demonstrate that 500-730-kd HA exerts an antiapoptot ic effect on anti-FAS-induced chondrocyte apoptosis by binding its specific receptors (CD44 and ICAM-1). Furthermore, this HA fraction may be able to slow down chondrocyte apoptosis in OA by regulating the processes of cartil age matrix degradation.