Effects of inflammatory mediators on human osteoblasts

Abstract: Inflammatory mediators produced in or adjacent to bone have implications on bone and may induce both osteolysis and/or osteosclerosis. Inflammatory cytokines mediate these effects by acting either directly or indirectly on the bone cells. The present thesis focuses on the effects of pro- and anti-inflammatory mediators on primary cultures of human osteoblast-like cells.Three isolation techniques for primary cultures of human osteoblasts (hOB) were compared. The expression of the osteoblast phenotype was determined by use of biochemical markers. The potency of the cells to induce mineralisation of the extracellular matrix was investigated. Separate isolation techniques select for different sets of precursors cells, with different degrees of maturation. However, all cells form confluent primary cultures with the potential to differentiate into mature osteoblasts. The effects of thrombin and bradykinin, two inflammatory mediators, on the rate of hOB proliferation were studied. Thrombin stimulated proliferation of isolated human osteoblasts independently of prostaglandins by acting on the proteolytic activated thrombin receptor (PAR-1). No effect was seen with bradykinin. The effects of the pro-inflammatory cytokines interleukin-1α (IL-1α), tumour necrosis factor-α/β (TNF-α/β) and interleukin-6 (IL-6) on cell proliferation rate and the secretion of PGE2 in hOB cells were studied. IL-1α and TNF-β time- and dose-dependently enhanced the proliferation of osteoblasts. TNF-α stimulated proliferation at low doses, while it inhibited proliferation at doses at and above 100 pM. IL-6 did not affect the rate of proliferation.The effect of the anti-inflammatory cytokine interleukin-13 (IL-13) on proliferation and IL-6 transcription and secretion in primary isolated human osteoblasts was studied and compared with the related cytokine interleukin-4 (IL-4). The expression of receptor subunits was investigated by use of RT-PCR. mRNA was expressed for the subunits IL-4Rα, IL-13R and IL-13Rα. IL-4 and IL-13 dose-dependently inhibited [3H]thymidine incorporation and stimulated the IL-6 secretion. Using a blocking antibody to the receptor the latter effect was abolished. There was no effect on the reduction of [3H]thymidine incorporation.Conclusions: Primary cultures of human osteoblasts are useful tools for studying proliferation and differentiation. Pro- and anti-inflammatory cytokines have regulatory effects on osteoblast proliferation, differentiation and activation, implicating effects on bone formation and osteoclast resorption.