The effects of 1α,25-dihydroxyvitamin D₃ on matrix metalloproteinase and prostaglandin E₂ production by cells of the rheumatoid lesion

1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], the biologically active metabolite of vitamin D₃, acts through an intracellular vitamin D receptor (VDR) and has several immunostimulatory effects. Animal studies have shown that production of some matrix metalloproteinases (MMPs) may be upregulated in rat chondrocytes by administration of 1α,25(OH)₂D₃; and cell cultures have suggested that 1α,25(OH)₂D₃ may affect chondrocytic function. Discoordinate regulation by vitamin D of MMP-1 and MMP-9 in human mononuclear phagocytes has also been reported. These data suggest that vitamin D may regulate MMP expression in tissues where VDRs are expressed. Production of 1α,25(OH)₂D₃ within synovial fluids of arthritic joints has been shown and VDRs have been found in rheumatoid synovial tissues and at sites of cartilage erosion. The physiological function of 1α,25(OH)₂D₃ at these sites remains obscure. MMPs play a major role in cartilage breakdown in the rheumatoid joint and are produced locally by several cell types under strict control by regulatory factors. As 1α,25(OH)₂D₃ modulates the production of specific MMPs and is produced within the rheumatoid joint, the present study investigates its effects on MMP and prostaglandin E₂ (PGE₂) production in two cell types known to express chondrolytic enzymes.

To investigate VDR expression in rheumatoid tissues and to examine the effects of 1α,25-dihydroxyvitamin D₃ on cultured rheumatoid synovial fibroblasts (RSFs) and human articular chondrocytes (HACs) with respect to MMP and PGE₂ production.

Rheumatoid synovial tissues were obtained from arthroplasty procedures on patients with late-stage rheumatoid arthritis; normal articular cartilage was obtained from lower limb amputations. Samples were embedded in paraffin, and examined for presence of VDRs by immunolocalisation using a biotinylated antibody and alkaline-phosphatase-conjugated avidin-biotin complex system. Cultured synovial fibroblasts and chondrocytes were treated with either 1α,25(OH)₂D₃, or interleukin (IL)-1β or both. Conditioned medium was assayed for MMP and PGE₂ by enzyme-linked immunosorbent assay (ELISA), and the results were normalised relative to control values.

The rheumatoid synovial tissue specimens (n = 18) immunostained for VDRs showed positive staining but at variable distributions and in no observable pattern. VDR-positive cells were also observed in association with some cartilage-pannus junctions (the rheumatoid lesion). MMP production by RSFs in monolayer culture was not affected by treatment with 1α,25(OH)₂D₃ alone, but when added simultaneously with IL-1β the stimulation by IL-1β was reduced from expected levels by up to 50%. In contrast, 1α,25(OH)₂D₃ had a slight stimulatory effect on basal production of MMPs 1 and 3 by monolayer cultures of HACs, but stimulation of MMP-1 by IL-1β was not affected by the simultaneous addition of 1α,25(OH)₂D₃ whilst MMP-3 production was enhanced (Table 1). The production of PGE₂ by RSFs was unaffected by 1α,25(OH)₂D₃ addition, but when added concomitantly with IL-1β the expected IL-1 β-stimulated increase was reduced to almost basal levels. In contrast, IL-1β stimulation of PGE₂ in HACs was not affected by the simultaneous addition of 1α,25(OH)₂D₃ (Table 2). Pretreatment of RSFs with 1α,25(OH)₂D₃ for 1 h made no significant difference to IL-1β-induced stimulation of PGE₂, but incubation for 16 h suppressed the expected increase in PGE₂ to control values. This effect was also noted when 1α,25(OH)₂D₃ was removed after the 16h and the IL-1 added alone. Thus it appears that 1α,25(OH)₂D₃ does not interfere with the IL-1β receptor, but reduces the capacity of RSFs to elaborate PGE₂ after IL-1β induction.

Cells within the rheumatoid lesion which expressed VDR were fibroblasts, macrophages, lymphocytes and endothelial cells. These cells are thought to be involved in the degradative processes associated with rheumatoid arthritis (RA), thus providing evidence of a functional role of 1α,25(OH)₂D₃ in RA. MMPs may play important roles in the chondrolytic processes of the rheumatoid lesion and are known to be produced by both fibroblasts and chondrocytes. The 1α,25(OH)₂D₃ had little effect on basal MMP production by RSFs, although more pronounced differences were noted when IL-1β-stimulated cells were treated with 1α,25(OH)₂D₃, with the RSF and HAC showing quite disparate responses. These opposite effects may be relevant to the processes of joint destruction, especially cartilage loss, as the ability of 1α,25(OH)₂D₃ to potentiate MMP-1 and MMP-3 expression by 'activated' chondrocytes might facilitate intrinsic cartilage chondrolysis in vivo. By contrast, the MMP-suppressive effects observed for 1α,25(OH)₂D₃ treatment of 'activated' synovial fibroblasts might reduce extrinsic chondrolysis and also matrix degradation within the synovial tissue. Prostaglandins have a role in the immune response and inflammatory processes associated with RA. The 1α,25(OH)₂D₃ had little effect on basal PGE₂ production by RSF, but the enhanced PGE₂ production observed following IL-1β stimulation of these cells was markedly suppressed by the concomitant addition of 1α,25(OH)₂D₃. As with MMP production, there are disparate effects of 1α,25(OH)₂D₃ on IL-1β stimulated PGE₂ production by the two cell types; 1α,25(OH)₂D₃ added concomitantly with IL-1β had no effect on PGE₂ production by HACs. In summary, the presence of VDRs in the rheumatoid lesion demonstrates that 1α,25(OH)₂D₃ may have a functional role in the joint disease process. 1α,25(OH)₂D₃ does not appear to directly affect MMP or PGE₂ production but does modulate cytokine-induced production.

Samples of rheumatoid synovial tissue, cartilage and cartilage-pannus junction were obtained from arthroplasty procedures performed on patients with classic late-stage rheumatoid arthritis. Normal articular cartilage samples were obtained from lower limb amputations. Samples were fixed in Carnoy's fixative at 20°C for 2 h, embedded in paraffin wax and 5 μm sections cut. Tissue sections were dewaxed, rehydrated and examined for the presence of VDR.

Tissue sections were treated with 2N HCl at 37°C for 30 min, this being the antigen retrieval procedure recommended by the supplier of the primary antibody. Nonimmune rabbit serum at 10% (vol : vol) in TRIS-buffered saline was applied to the sections for 20 min at 20°C before incubation with the primary antibody. Rat monoclonal antibody to chick VDR (Biogenex, San Remo, USA), which is known to cross-react with human VDR, was applied to the sections for 2 h at 20°C after dilution 1 : 40 in TRIS-buffered saline. After 3 × 10 min washing in TRIS-buffered saline, biotinylated rabbit anti-rat immunoglobulin G (DAKO, Glostrup, Denmark) diluted 1 : 200 in TRIS-buffered saline was applied to the sections for 45 min at 20°C. After further washing in TRIS-buffered saline, alkaline phosphatase-conjugated ABC (Avidin-biotin complex system; DAKO) was applied to the sections for 45min at 20°C, diluted as instructed by the supplier. After further washing the alkaline phosphatase was developed using new fuchsin substrate to give a red colour. Sections were lightly counterstained using Harris's haematoxylin or toluidine blue. Non-immune rat immunoglobulin G was substituted for the primary antibody at similar concentrations on control tissue sections [10].

Rheumatoid synovial tissue and human articular cartilage were enzymically digested to provide synovial fibroblast and chondrocyte cultures as previously described [18,19]. Cells were grown in Dulbecco's Modified Eagle's Medium + 10% (vol : vol) foetal calf serum, harvested and seeded into 12-well culture dishes (Nunc, Gibco, UK) Triplicate wells of confluent cell cultures in Dulbecco's Modified Eagle's Medium + 2% foetal calf serum were treated with 1α,25(OH)₂D₃ (10^-8 mol/l), IL-1β (0.05 ng/ml), or IL-1β + 1α,25(OH)₂D₃ (0.05 ng/ml and 10^-8 mol/l, respectively) and incubated for 48 h at 37°C. The conditioned medium was collected and assayed for MMP-1, MMP-2, MMP-3 and MMP-9, and PGE₂ using enzyme-linked immunosorbent assay (ELISA) methodology. Cell numbers per well were counted at the end of each experiment after 70% ethanol fixation and toluidine blue staining.

ELISA methodology was used to determine protein levels of MMP-1 (collagenase 1), MMP-3 (stromelysin) and MMP-9 (gelatinase B) as previously described [20]. MMP-2 (Gelatinase A) was measured using ELISA kits purchased from The Binding Site (Birmingham, UK); and PGE₂ was measured using an ELISA assay kit purchased from R & D Systems Europe, Ltd (Abingdon,UK).

All ELISA results were initially calculated in ng or pg protein/ml culture medium/10⁶ cells per 48 h. Three different cultures of both RSFs and HACs were examined, but the capacities of each cell type to produce the MMPs and PGE₂ varied between the individual cultures. Therefore, the data from each culture was 'normalized' relative to control values, and the data sets from the three cultures of each cell type were subsequently pooled. This provided an evaluation that showed qualitative similarities for the RSFs and HACs, but demonstrated differences in 1α,25(OH)₂D₃ responses by each of these two cell types.

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).

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].

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.

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.