INTERLEUKIN-1-ALPHA AND INTERLEUKIN-1-BETA IN GROWTH-PLATE CARTILAGE ARE REGULATED BY VITAMIN-D METABOLITES IN-VIVO

Matrix remodeling plays a prominent role in growth plate calcification . Since interleukin-1 (IL-1) has been implicated in stimulating protei nase production and inhibiting matrix synthesis in articular cartilage , we examined whether IL-1 was present in growth plate and whether the vitamin D metabolites, 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3; 1,2 5) and 24,25(OH)(2)D-3 (24,25), regulate the level of IL-1 found in th is tissue. Sprague-Dawley rats were placed on normal (Normal rats) or rachitogenic diet (-VDP rats). The -VDP rats were either left untreate d, injected 24 h prior to euthanasia with 24,25 (-VDP+24,25 rats) or 1 ,25 (-VDP+1,25 rats), or were given ergocalciferol (Ergo rats) orally, 48 h prior to euthanasia. Growth plates were harvested and extracted in buffer containing 1 M guanidine. IL-1 activity was measured by addi ng authentic cytokine or growth plate extracts to cultures of lapine a rticular cartilage and assaying release of glycosaminoglycans (GAGs) a nd changes in collagenase and neutral metalloproteinase activity. Neut ralization of activity in the extracts was performed using polyclonal antisera to IL-1 alpha or IL-1 beta. An ELISA was used to determine le vels of IL-1 alpha and beta in the extracts. All extracts contained IL -1 alpha and beta, as determined by ELISA. Levels of IL-1 beta, but no t IL-1 alpha, were affected by the vitamin D status of the animal. Ext racts from -VDP+24,25 animals contained significantly more IL-1 beta t han any of the other treatment groups, with the level found in these a nimals being 3-fold higher than normal and 2-fold higher than -VDP. Ex tracts were also tested in the bioassay to determine the level of acti ve cytokine present. All growth plate extracts contained activity whic h altered GAG and proteinase release by lapine articular cartilage. Ex tracts from -VDP-, -VDP+1,25-, and -VDP+Ergo-treated rats stimulated a 40% increase in glycosaminoglycan release compared with extracts from normal rats. In contrast, extracts from -VDP+24,25-treated rats stimu lated a 300% increase in glycosaminoglycan release. Both collagenase a nd neutral metalloproteinase activity of lapine cartilage were increas ed after incubation with the growth plate extracts. Collagenase activi ty was significantly increased 8- to 13-fold by the addition of extrac ts from -VDP-, -VDP+24,25-, or -VDP+1,25-treated animals. Neutral meta lloproteinase activity was similarly increased by 4- to 10-fold. To ch aracterize this activity further, growth plate extracts were incubated with neutralizing antibody to IL-1 alpha or beta prior to addition to the lapine articular cartilage cultures. When antibodies were used se parately, only partial inhibition was observed; incubation with both a ntibodies blocked 25% of the glycosaminoglycan release observed withou t antibody and greater than 80% of the enzyme activity released by the articular cartilage cultures. The results of this study show that gro wth plate cartilage contains both IL-1 alpha and beta and indicate tha t vitamin D regulates the level of IL-1 in this tissue.