Background Alkaptonuria (AKU) is a rare autosomal recessive disorder of tyrosine metabolism which causes rapidly progressing, early onset osteoarthritis (OA). The disease results from absence of the enzyme homogentisate 1,2 dioxygenase which breaks down homogentisic acid (HGA). Over time HGA undergoes polymerisation, a process called ochronosis, which occurs predominantly in weight bearing cartilages in hips, knees and intervertebral discs. This rapidly progresses into ochronotic osteoarthropathy. Research into AKU has demonstrated that focal change within cartilage is required for ochronosis to occur, before progression into ochronotic osteoarthropathy.
Objectives We examine the effect of interleukins (IL's); IL-1β, -6 and -10, on pigmentation (ochronosis) and cell viability in an in vitro model of ochronotic osteoarthropathy in AKU.
Methods C20 chondrosarcoma cells were cultured for 21 days in under the following conditions: control, HGA, IL-1β or IL-6 or IL-10, HGA+IL. HGA was added to medium at 0.33M and 1ng/ml for IL's. Cultures (n=6) for histological examination were stained with schmorl's reagent and nuclear fast red to highlight and quantify pigmentation before fixation. Cell viability was assessed by trypan blue assay. Statistical analysis consisted of ANOVA, differences between groups was determined by Newman-Keuls post-test.
Results C20 cultures treated with HGA (solely or in combination with IL's) demonstrated significant increases in pigment deposition compared to control and IL cultures (p<0.001). Comparing HGA solely against HGA+IL-1, -6 or -10 there were no significant differences. Cell viability analysis with IL-1 showed a significant decrease in cell numbers of all cultures compared to control (p<0.001). There was no significance between control and IL-1. HGA alone cultures showed a significant decrease in cell number compared to HGA+IL-1. Analysis of cultures with IL-6 showed HGA and HGA+IL-6 had a significant decrease compared to control (p<0.001). HGA+IL-6 compared to IL-6 alone showed a significant decrease in cell numbers (p<0.001) but no difference to HGA alone. Cells cultured in IL-6 alone showed no difference compared to control. C20 cultures with HGA and HGA+IL-10 showed a significant decrease in cell numbers compared to control, as did HGA+IL-10 compared to IL-10 alone (p<0.001), but no difference when compared to HGA alone. There was no difference between HGA and HGA+IL-10.
Conclusions We demonstrate that addition of IL's alongside HGA does not appear to have an effect on pigment deposition compared to HGA alone. This suggests that the presence of IL's alongside HGA does not produce more pigmentation. However the IL's do impact on cell viability. Interestingly HGA+IL-1 Does not appear to be as detrimental to cell number as HGA alone. This may explain the early and rapid onset of the arthropathy of AKU compared to OA. Furthermore, anti-inflammatory IL's do not appear to affect the decrease in cell number when used in combination with HGA. These results are the first to explore the combination of HGA and IL's in ochronotic osteoarthropathy. They suggest that the presence of IL's does not cause more pigmentation but does have a detrimental effect on the number of cells present and as such may be a factor in altering the extracellular milieu and promoting the degeneration process.
Disclosure of Interest None declared