Human osteoarthritic cartilage exhibits the 2B6 epitope without pretreatment with chondroitinase ABC

The monoclonal antibody, 2B6, recognizes the chondroitin-4-sulfate (C4S) stub of proteoglycans resulting from chondroitinase ABC digestion. Demonstration of C4S in normal control cartilage by immunohistochemistry using 2B6 requires pre-treatment by digestion via chondroitinase ABC. However, we found that osteoarthritic (OA) fibrillated cartilage reacted with 2B6 without enzymatic pre-digestion. The spontaneous immunoreactivity to 2B6 in OA cartilage was observed mainly intracellularly in superficial chondrocytes. To examine the possibility that this staining of C4S could have resulted from degradation in lysosomes, its co-localization with cathepsin B (CB) or D (CD), used as representative lysosomal proteinases, was analyzed by double immunostaining using confocal laser scanning microscopy. Immunoreactivity to CB and CD was not demonstrated in the chondrocyte granules immunostained by 2B6. Using double staining with 2B6 and propidium iodide, a nuclear marker, positive staining of 2B6 appeared both in the nucleus and cytoplasmic granules of chondrocytes. The results suggest that a native form of C4S recognized by 2B6 exists in the OA chondrocytes, both in the cytoplasmic granules and nuclei of the cells.