Interleukin-1alpha and 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 proteinase 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 D3 (1,25(OH)2D3; 1,25) and 24,25(OH)2D3 (24,25), regulate the level of IL-1 found in this tissue. Sprague-Dawley rats were placed on normal (Normal rats) or rachitogenic diet (-VDP rats). The -VDP rats were either left untreated, 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 adding authentic cytokine or growth plate extracts to cultures of lapine articular cartilage and assaying release of glycosaminoglycans (GAGs) and changes in collagenase and neutral metalloproteinase activity. Neutralization of activity in the extracts was performed using polyclonal antisera to IL-1alpha or IL-1beta. An ELISA was used to determine levels of IL-1alpha and beta in the extracts. All extracts contained IL-1alpha and beta, as determined by ELISA. Levels of IL-1beta, but not IL-1alpha, were affected by the vitamin D status of the animal. Extracts from -VDP+24,25 animals contained significantly more IL-1beta than any of the other treatment groups, with the level found in these animals being 3-fold higher than normal and 2-fold higher than -VDP. Extracts were also tested in the bioassay to determine the level of active cytokine present. All growth plate extracts contained activity which altered GAG and proteinase release by lapine articular cartilage. Extracts 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 stimulated a 300% increase in glycosaminoglycan release. Both collagenase and neutral metalloproteinase activity of lapine cartilage were increased after incubation with the growth plate extracts. Collagenase activity was significantly increased 8- to 13-fold by the addition of extracts from -VDP-, -VDP+24,25-, or -VDP+1,25-treated animals. Neutral metalloproteinase activity was similarly increased by 4- to 10-fold. To characterize this activity further, growth plate extracts were incubated with neutralizing antibody to IL-1alpha or beta prior to addition to the lapine articular cartilage cultures. When antibodies were used separately, only partial inhibition was observed; incubation with both antibodies blocked 25% of the glycosaminoglycan release observed without antibody and greater than 80% of the enzyme activity released by the articular cartilage cultures. The results of this study show that growth plate cartilage contains both IL-1alpha and beta and indicate that vitamin D regulates the level of IL-1 in this tissue.
Dean
D D
DD
Department of Orthopaedics, University of Health Science Center at San Antonio, Texas 78284-7774, U.S.A.
Schwartz
Z
Z
Muniz
O E
OE
Arsenis
C H
CH
Boyan
B D
BD
Howell
D S
DS
D015650:24,25-Dihydroxyvitamin D 3 / Q000494:pharmacology*; D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D017364:Collagenases / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004872:Ergocalciferols / Q000494:pharmacology*; D006025:Glycosaminoglycans / Q000378:metabolism; D006132:Growth Plate / Q000187:drug effects* / Q000378:metabolism; D007375:Interleukin-1 / Q000378:metabolism*; D008297:Male; D008666:Metalloendopeptidases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014020:Tissue Extracts / Q000032:analysis; D014808:Vitamin D Deficiency / Q000503:physiopathology
D004872:Ergocalciferols; D006025:Glycosaminoglycans; D007375:Interleukin-1; D014020:Tissue Extracts; D015650:24,25-Dihydroxyvitamin D 3; D017364:Collagenases; D008666:Metalloendopeptidases; D002117:Calcitriol