Interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin production by human osteoblastic cells:: comparison of the effects of 17-β oestradiol and raloxifene

Oestrogen inhibits bone resorption, at least in part, by regulating the production of several cytokines, including interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor kappaB ligand (RANKL) and osteoprotegerin (OPG) by cells of the osteoblastic lineage. The selective oestrogen receptor modulator raloxifene (RAL) acts on bone in a similar manner to oestrogen, although the mechanisms of action of RAL on osteoblasts still remain unclear. We investigated and compared the effects of 17-beta oestradiol (E-2) and RAL on the regulation of IL-6, IL-1, RANKL and OPG in vitro in primary human osteoblastic (HOB) cells and in an immortalised clonal human bone marrow stromal cell line (HCC1) with osteoblastic characteristics. We tested E-2 and RAL at concentrations ranging from 10(-1)2 to 10(-6) M. IL-6, IL-1alpha and IL-1beta, OPG and RANKL were measured by ELISA. RANKL and OPG mRNA steady state level was assessed by quantitative PCR analysis. Both E-2 and RAL led to a significant reduction in IL-6 production in the HOB cells, although the effect was more marked with E-2 (P<0.05). IL-1alpha and IL-1beta also decreased significantly following treatment with E-2 and RAL in the HCC1 cells (E-2 (10(-8), 10(-7) and 10(-6) M), % reduction (means +/- S.E.M.) compared with vehicle-treated cells - IL-1alpha: 84 +/- 7.4, 70.8 +/- 2.9*, 78.2 +/- 4.8*; IL-1beta: 79 +/- 10, 72.8 +/- 8.2*, 66.6 +/- 2.8*; RAL (10(-8), 10(-7) and 10(-6) M) - IL-1alpha: 72.4 +/- 5*, 79 +/- 5.2*, 102 +/- 7.7; IL-1beta: 67.9 +/- 3.2*, 69 +/- 2.5*, 73.8 +/- 6.2*; *P<0.05). OPG protein concentration decreased significantly in a dose-dependent manner following treatment with E-2 and RAL (% reduction E-2 (10(-8), 10(-7) and 10(-6) M) - HOB: 72.5 +/- 8.4*, 80 +/- 6.7*, 62.8 +/- 8.9*; HCC1: 109 +/- 4, 98.8 +/- 6, 54.5 +/- 3.4*; RAL (10(-8), 10(-7) and 10.>6 M) - HOB: 81.5 +/- 5-5*, 62.7 +/- 7.4*, 55.2 +/- 10.9*; HCC1: 92.7 +/- 7.4, 67 +/- 12.2*, 39 +/- 4.5*; *P<0.05). In the HCC1 cells, RANKL protein did not change significantly following E2. In contrast, a significant reduction in RANKL was seen with RAL at 10-7 and 10(-6) M (66 +/- 6.4% and 74 +/- 3% respectively). There was no change in OPG mRNA expression following E2 or RAL in the HCC1 cells, although in the HOB cells we observed a significant reduction in OPG mRNA. RANKL mRNA decreased significantly in the HCC1 cells following RAL (10-8, 10-7 and 10-6 M) treatment (% change from controls: 52 +/- 2*, 62 +/- 1*, 53 +/- 5.8*; *P<0.05). Similar results were seen in the HOB cells with RAL at 10(-6) M (RANKL mRNA: 72 +/- 5.5, P<0.05). In addition, there was a significant decrease in the RANKL/ OPG ratio after RAL at 10(-6) M (HOB: 65.6 +/- 5*, HCC1: 56.9 +/- 20*; *P<0.05). RANKL/OPG ratio did not change significantly in the HCC1 cells following E-2. However, in contrast to RAL, we observed an increase in the RANKL/OPG ratio in the HOB cells following treatment with E-2. In conclusion, the study shows that RAL and E-2 have divergent cell-specific effects on the regulation of cytokines. The data also suggest that, in contrast to E-2, RAL may exert its anti-resorptive actions, at least in part, via the RANKL/OPG pathway. Further in vivo studies are required to confirm this.