To determine the capacity of human subchondral osteoarthritic osteoblasts (Ob) to produce interleukin (IL)-1beta, IL-6, transforming growth factor-beta (TGF-beta) and prostaglandin E(2) (PGE(2)), and determine if a relationship exists between IL-1beta, TGF-beta, PGE(2) and IL-6 production.
We measured the abundance of IL-1beta, IL-6, TGF-beta and PGE(2) using very sensitive ELISA in conditioned-media of human primary subchondral Ob from normal individuals and osteoarthritic patients. Selective inhibition of IL-6 or IL-6 receptor signaling was performed to determine its effect on PGE(2) production whereas the inhibiton of PGE(2) production was performed to determine its effect on IL-6 production. The expression of bone cell markers and urokinase plasminogen activator (uPA) activity was also determined.
Osteoarthritic Ob produced all these factors with greater variability than normal cells. Interestingly, the production of IL-6 and PGE(2) by osteoarthritic Ob separated patients into two subgroups, those whose Ob produced levels comparable to normal (low producers) and those whose Ob produced higher levels (high producers). In those cells classified as high osteoarthritic Ob, PGE(2) and IL-6 levels were increased two- to three-fold and five- to six-fold, respectively, compared with normal. In contrast, while using their IL-6 and PGE(2) production to separate osteoarthritic Ob into low and high producers, we found that IL-1beta levels were similar in normal and all osteoarthritic Ob. Using the same criteria, TGF-beta levels were increased in all osteoarthritic Ob compared with normal. Reducing PGE(2) synthesis by Indomethacin [a cyclo-oxygenase (COX) -1 and -2 inhibitor] reduced IL-6 levels in all osteoarthritic Ob, whereas Naproxen (a more selective COX-2 inhbitor) reduced PGE(2) and IL-6 levels only in the high osteoarthritic group. Conversely, PGE(2) addition to osteoarthritic Ob enhanced IL-6 production in both groups. Moreover, the addition of parathyroid hormone also stimulated IL-6 production to similar normal levels in both osteoarthritic groups. In contrast, using an antibody against IL-6 or IL-6 receptors did not reduce PGE(2) levels in either group. The evaluation of alkaline phosphatase activity, osteocalcin release, collagen type I and uPA activity in osteoarthritic Ob failed to show any differences between these cells regardless to which subgroup they were assigned.
These results indicate that IL-6 and PGE(2) production by subchondral Ob can discriminate two subgroups of osteoarthritic patients that cannot otherwise be separated by their expression of cell markers, and that endogenous PGE(2) levels influence IL-6 synthesis in osteoarthritic Ob.