Different responses of healthy, relative healthy and OA chondrocytes with IL1β treatment under hypoxia and normoxia

Abstract

Purpose: Due to its avascular nature, articular cartilage is relatively hypoxic. Depending on the zone in the cartilage, the residing chondrocytes are exposed to a concentration of approximately 6% O2 in the superficial layer, and to a concentration as low as less than 1% O2 in the calcified layer. In contrast most experiments with cultured chondrocytes are performed at an atmospheric oxygen concentration, which can be considered as a non-physiological, hyperoxic condition for chondrocytes. We hypothesized that oxygen tension critically influences the responses of articular chondrocytes to extracellular stimuli like pro-inflammatory cytokines. The aim of this study is to determine how oxygen tension influences the response of healthy, relatively healthy and osteoarthritic chondrocytes to IL1β. Methods: Cartilage specimens were isolated from healthy patients undergoing ankle replacement surgery and OA patients undergoing total knee replacement surgery and graded by the OARSI grading method, based on Alcian blue and Safranin O staining. Healthy (HL), relative healthy (RH) and osteoarthritic (OA) chondrocytes were isolated from cartilage with grade 0, grade 1 and grade 4 respectively. Cells were cultured in monolayer with or without IL-1β stimulation under 2.5% (hypoxia) or 21% O2 (normoxia) in proliferation medium. Cell morphology, proliferation, gene expression, protein expression, apoptosis were measured. Results: In hypoxia chondrocytes showed a higher proliferation rate irrespective of their origin. Gene expression analysis demonstrated higher levels of SOX9 and ACAN mRNA expression in hypoxia compared to normoxia. Remarkably in normoxia the expression of both genes decreased in the order of HL > RH > OA, while this difference was not present when cells were cultured under hypoxia. MMP1, 3 and 13 mRNA were expressed at higher levels in normoxia with no apparent difference between cells from different origins. Treatment with IL1β induced MMP1 expression with highest responses in OA chondrocytes followed by RH and HL. Absolute levels of MMP mRNAs were lower in hypoxia in control conditions and after stimulation with IL1β (Figure 1A) but fold stimulation was higher. Interestingly, in RH and OA chondrocytes IL1β increased AXIN2, an established WNT signaling target gene, mRNA expression in normoxia. This response was blunted in hypoxia in RH or even decreased in OA chondrocytes (Figure 1B). Conclusions: A supraphysiological oxygen concentration, but not an hypoxic condition, negatively affected the expression of SOX9 and ACAN mRNA particularly in chondrocytes from macroscopically diseased cartilage. In addition, high oxygen increases the expression of MMPs mRNA in chondrocytes under basal conditions compared to hypoxia. This suggests that exposure of chondrocytes to a supraphysiological oxygen concentration may induce a mild inflammatory response. While absolute levels of MMPs mRNA after treatment with IL1β were lower in hypoxia compared to normoxia, fold increase was increased suggesting that the chondrocytes become more sensitive to IL1β treatment in hypoxic conditions.