Cyclic hydrostatic pressure and particles increase synthesis of 1,25-dihydroxyvitamin D3 by human macrophages in vitro.

1,25-Dihydroxyvitamin D(3) has a pivotal role in bone resorption and osteoclast activity. As activated macrophages are known to synthesise 1,25-dihydroxyvitamin D(3), this study examined whether pressure modulated its synthesis. Pressure and particles have been shown to increase synthesis of pro-resorptive cytokines and other factors by cultured macrophages. Human peripheral blood macrophages were isolated, cultured and exposed to pressure (similar to that found in the human joint) and/or particles. Synthesis of 1,25-dihydroxyvitamin D(3) by macrophages was assayed using high pressure liquid chromatography and in situ hybridization. Synthesis of 1,25-dihydroxyvitamin D(3) but not 24,25-dihydroxyvitamin D(3) was increased in macrophages under pressure. In situ hybridization demonstrated an increase in 1alpha-hydroxylase expression in response to pressure or particles and simultaneous exposure to both stimuli generated higher expression of 1alpha-hydroxylase. In conclusion, this is the first study to demonstrate that mechanical loading, in the form of pressure, stimulates 1,25-dihydroxyvitamin D(3) synthesis in human macrophages. These findings have implications for the in vivo situation, as they suggest that 1,25-dihydroxyvitamin D(3) could be one factor stimulating osteoclastic bone resorption in pathologies, such as arthritis or implant loosening, where intra-articular or intra-osseous pressure is raised or where wear particles interact with macrophages.

Age of donor alters the effect of cyclic hydrostatic pressure on production by human macrophages and osteoblasts of sRANKL, OPG and RANK.

BACKGROUND: Cyclic hydrostatic pressure within bone has been proposed both as a stimulus of aseptic implant loosening and associated bone resorption and of bone formation. We showed previously that cyclical hydrostatic pressure influenced macrophage synthesis of several factors linked to osteoclastogenesis. The osteoprotegerin/soluble receptor activator of NF-kappa beta ligand /receptor activator of NF-kappa beta (OPG/ RANKL/ RANK) triumvirate has been implicated in control of bone resorption under various circumstances. We studied whether cyclical pressure might affect bone turnover via effects on OPG/ sRANKL/ RANK. METHODS: In this study, cultures of human osteoblasts or macrophages (supplemented with osteoclastogenic factors) or co-cultures of macrophages and osteoblasts (from the same donor), were subjected to cyclic hydrostatic pressure. Secretion of OPG and sRANKL was assayed in the culture media and the cells were stained for RANK and osteoclast markers. Data were analysed by nonparametric statistics. RESULTS: In co-cultures of macrophages and osteoblasts, pressure modulated secretion of sRANKL or OPG in a variable manner. Examination of the OPG:sRANKL ratio in co cultures without pressurisation showed that the ratio was greater in donors <70 years at the time of operation (p < 0.05 Mann Whitney U) than it was in patients >70 years. However, with pressure the difference in the OPG:sRANKL ratios between young and old donors was not significant. It was striking that in some patients the OPG:sRANKL ratio increased with pressure whereas in some it decreased. The tendency was for the ratio to decrease with pressure in patients younger than 70 years, and increase in patients > or = 70 years (Fishers exact p < 0.01). Cultures of osteoblasts alone showed a significant increase in both sRANKL and OPG with pressure, and again there was a decrease in the ratio of OPG:RANKL. Secretion of sRANKL by cultures of macrophages alone was not modulated by pressure. Only sRANKL was assayed in this study, but transmembrane RANKL may also be important in this system. Macrophages subjected to pressure (both alone and in co-culture) stained more strongly for RANK on immunohistochemstry than non-pressurized controls and 1,25-dihydroxyvitamin D3 (1,25 D3) further increased this. Immunocytochemical staining also demonstrated that more cells in pressurized co-cultures exhibited osteoclast markers (tartrate-resistant acid phosphatase, vitronectin receptor and multinuclearity) than did unpressurized controls. CONCLUSION: These data show that in co-cultures of osteoblasts and macrophages the ratio of OPG : sRANKL was decreased by pressure in younger patients but increased in older patients. As falls in this ratio promote bone resorption, this finding may be important in explaining the relatively high incidence of osteolysis around orthopaedic implants in young patients. The finding that secretion of OPG and sRANKL by osteoblasts in monoculture was sensitive to hydrostatic pressure, and that hydrostatic pressure stimulated the differentiation of macrophages into cells exhibiting osteoclast markers indicates that both osteoblasts and preosteoclasts are sensitive to cyclic pressure. However, the effects of pressure on cocultures were not simply additive and coculture appears useful to examine the interaction of these cell types. These findings have implications for future therapies for aseptic loosening and for the development of tests to predict the development of this condition.

Novel mutations in the 1alpha-hydroxylase (P450c1) gene in three families with pseudovitamin D-deficiency rickets resulting in loss of functional enzyme activity in blood-derived macrophages.

Pseudovitamin D-defiency rickets (PDDR) is an autosomal recessive disorder characterized by hypocalcemia, rickets (which are resistant to treatment with vitamin D), and low or undetectable serum levels of 1,25-dihydroxyvitamin D (1,25(OH)2D). The symptoms are corrected with 1,25(OH)2D treatment, and the disease is now believed to result from a defect in the cytochrome P450 component (P450c1; CYP27B1) of the renal 25-hydroxyvitamin D-1alpha-hydroxylase (1-OHase). We have studied genomic DNA from three families with PDDR and have identified the same homozygous mutation in the P450c1 gene in two of the index cases, causing a frameshift in exon 8, resulting in a premature stop codon in the heme-binding domain. The two cases in the third kindred were compound heterozygotes with missense mutations in exons 6 and 9. We have also identified a C/T polymorphism in intron 6 of the P450c1 genomic DNA. Interferon gamma-inducible 1-OHase activity in blood-derived macrophages was shown by 1,25(OH)2D synthesis in all control cells tested (37-184 fmol/h/106 cells) and those from the PDDR family parents (34-116 fmol/h/106 cells) but was totally absent from the patients' cells, indicating a defect in their macrophage 1-OHase, similar to the presumed renal defect. The assumption of similarity between the renal and macrophage P450c1 was supported by our ability to clone a 514 bp sequence, including the heme-binding region of the macrophage P450c1 cDNA from controls, which was identical to that published for both the renal and keratinocyte P450c1 cDNAs.