Demonstration of the vitamin D receptor in rheumatoid tissues in vivo
Specimens of rheumatoid synovial tissue (n = 18) immunostained for VDR were shown to have variable distributions of the receptor. All specimens showed some positive staining, but this could be less than 5% or as much as 70% of the total cell population. Different cell types within the synovial specimens were shown to express the receptor, including macrophages, endothelial cells, lymphocytes and fibroblastic cells, but no regular pattern was observed. Cells with fibroblastic morphology immunostained for VDR are shown in Figure 1a. Chondrocytes within articular cartilage from rheumatoid joints also expressed the receptor in six out of 10 specimens (Fig. 1b), this being a much higher frequency compared with the one in 10 specimens of normal articular cartilage from nonarthritic joints (data not shown). VDR-positive cells were also observed in association with some cartilage–pannus junctions, described here as the rheumatoid lesion (Fig. 1c).
Effects of 1α,25-dihydroxyvitamin D3 on matrix metalloproteinase production by rheumatoid synovial fibroblasts
1α,25(OH)2D3 alone had no effect on basal MMP production by RSFs in monolayer culture, but the simultaneous addition of 1α,25(OH)2D3 with IL-1β reduced the expected stimulation of MMP-1, MMP-3 and MMP-9 by up to 50% (Fig. 2: P = 0.096, 0.009 and 0.01, for IL-1β versus IL-1β + 1α,25(OH)2D3 for MMP-1, MMP-3 and MMP-9, respectively, by Student's t-test). MMP-2 production was not affected by either IL-1β or IL-1β + 1α,25(OH)2D3 (data not shown), an observation that is in accord with the constitutive nature of MMP-2 expression [21].
Effects of 1α,25-dihydroxyvitamin D3 on matrix metalloproteinase production by human articular chondrocytes
In contrast to the data for RSFs, 1α,25(OH)2D3 had a slight stimulatory effect on basal production of MMP-1 and MMP-3 by monolayer cultures of HAC (Fig. 3: P = 0.098 and 0.002, for control versus 1α,25(OH)2D3, for MMP-1 and MMP-3, respectively, by Student's t-test). When stimulated with IL-1β MMP-1 and MMP-3 production was increased, and although simultaneous addition of 1α,25(OH)2D3 had no effect on the stimulation of the MMP-1 enzyme, MMP-3 production was further enhanced (Fig 3b: P = 0.008, by Students t-test). MMP-9 and MMP-2 were not produced in measurable quantities by these HAC cultures, either with or without IL-1β stimulation.
Effects of 1α,25-dihydroxyvitamin D3 on prostaglandin E2 production by rheumatoid synovial fibroblasts and human articular chondrocytes
PGE2 production by RSFs was unaffected by the addition of 1α,25(OH)2D3 alone. Treatment of RSFs with IL-1β upregulated the production of PGE2, but the addition of 1α,25(OH)2D3 together with IL-1β reduced the expected stimulation of PGE2 almost to control values (Fig. 4a:P = 0.014, for IL-1β versus IL-1β + 1α,25(OH)2D3, by Student's t-test).
Treatment of HACs with IL-1β also increased the production of PGE2, but in contrast to the effects noted for RSFs this IL-1-stimulation of PGE2 was not affected by the concomitant addition of 1α,25(OH)2D3 (Fig. 4b).
To examine the possibility that 1α,25(OH)2D3 might obscure or interact with the IL-1β receptor of RSFs the latter were pretreated with 1α,25(OH)2D3 before incubation with IL-1β. Figure 4c shows that a 1-h preincubation with 1α,25(OH)2D3 followed by IL-1β was not significantly different from the two factors added together, but preincubation with 1α,25(OH)2D3 for 16 h suppressed the expected increase in PGE2 production to control values. This effect was noted even when the 1α,25(OH)2D3 was removed after the 16 h and IL-1β then added alone (Fig.4c, data column F). Thus, rather than directly interfering with the IL-1β receptor, it appears that 1α,25(OH)2D3 reduces the capacity of the RSFs to elaborate PGE2 (and probably the MMPs shown in Fig. 2) after IL-1β induction.