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Chondrocytes stimulated with IL-1 produce high levels of nitric oxide (NO), which inhibits proliferation induced by transforming growth factor-beta or serum. This study analyzes the role of NO and IL-1 in the induction of chondrocyte cell death. NO generated from sodium nitroprusside induced apoptosis in cultured chondrocytes as demonstrated by electron microscopy, 4',6-dianidino-2-phenylindole dihydrochloride staining, FACS analysis, and histochemical detection of DNA fragmentation. Similar results were obtained with two other NO donors, 3-morpholinosynonimide-hydrochloride and s-nitroso-N-acetyl-D-L-penicillamine. In contrast, oxygen radicals generated by hypoxanthine/xanthine oxidase caused necrosis but did not induce chondrocyte apoptosis. To analyze whether endogenously generated NO induces apoptosis, chondrocytes were stimulated with IL-1, but there was no evidence for apoptotic changes. Combinations of NO inducers such as IL-1, lipopolysaccharide, tumor necrosis factor, and interferon-gamma also failed to trigger apoptosis. IL-1-stimulated chondrocytes are known to produce oxygen radicals that react with NO to form products that can induce cell death in other systems. We thus tested IL-1 in combination with the oxygen radical scavengers N-acetyl cysteine, dimethyl sulfoxide, or 5,5'-dimetylpyrroline 1-oxide. Under these conditions IL-1 was able to induce apoptosis, which was inhibited in a dose-dependent manner by the NO synthase inhibitor N-monomethyl L-arginine. Conversely, endogenous oxygen radicals induced by inflammatory mediators caused necrosis under conditions in which the simultaneous production of NO was reduced. These results suggest that NO, but not oxygen radicals, is the primary inducer of apoptosis in human articular chondrocytes.

By means of immunofluorescence and immunoelectron microscopy we have studied the fate of different nucleolar components during the apoptotic process in camptothecin-treated HL60 cells. We have found that RNA polymerase I disappeared while UBF was associated with previously described fibrogranular threaded bodies. In contrast, fibrillarin, C23/nucleolin, and B23/nucleophosmin remained detectable in granular material present amid micronuclei of late apoptotic cells. Double immunolabeling experiments showed colocalization of both C23 and B23 with fibrillarin. Immunoblotting analysis showed that UBF was proteolytically degraded, whereas fibrillarin, C23/nucleolin, and B23/nucleophosmin were not. These results may help explain the presence of anti-nucleolar antibodies seen in various pathological disorders. J. Cell. Biochem. 78:264–277, 2000. © 2000 Wiley-Liss, Inc.