Induction of the chemokine IL-8/Kc by the articular cartilage: Possible influence on osteoarthritis

Abstract

Purpose

IL-8 and its murine equivalent keratinocyte chemoattractant (Kc), chemokines produced by chondrocytes, contribute to the pathophysiology of osteoarthritis. However, the mechanisms leading to their production are poorly known. We aimed to investigate whether mechanical (compression), inflammatory (IL-1β) and metabolic (visfatin) stresses may induce the release of Kc when applied on murine cartilage.

Methods

Mouse cartilage explants were subjected to intermittent compression for 4, 6 and 24 h. Primary cultures of immature murine articular chondrocytes were obtained by enzymatic digestion of articular cartilage from 6-days-old newborns mice. The effect of compression, IL-1β (10, 50, 100 pg/mL) and of visfatin (5 μg/mL) on the release of Kc was assessed by ELISA. IL-8 levels in conditioned media from human OA joint tissues (cartilage or synovium) were also assessed.

Results

In comparison with non-compressed explants, loading increased Kc release of 3.2-, 1.9- and 2.0-fold at 4, 6 and 24 h respectively (P < 0.004, n = 9). IL-1β triggered an increase of Kc release by primary cultured chondrocytes of 4.1-, 15.5- and 35.2-fold at 10, 50 and 100 pg/mL of IL-1β respectively (P < 0.05, n = 4). Likewise, visfatin (5 μg/mL) induced an increase in Kc release of 56.5 ± 25.2 fold (P = 0.002, n = 6). IL-8 was released in conditioned media by synovium as well as by cartilage.

Conclusion

We show for the first time that IL-8/Kc is highly responsive to mechanical, inflammatory and metabolic stresses, strengthening the hypothesis that IL-8/Kc could be added to the cytokines which may have a deleterious impact in osteoarthritis.

Introduction

Osteoarthritis (OA) is the most common form of arthritis characterized by a progressive disruption of the cartilage associated with synovial inflammation and subchondral bone remodeling. OA is characterized by quantitative and qualitative modifications of cartilage matrix related to an altered imbalance between biosynthesis and degradation of its components [1]. The OA chondrocyte acquires a pro-degradative status characterized by the release of proteolytic enzymes and of proinflammatory mediators such as prostaglandin E₂ (PGE₂) as well as a pre-apoptotic hypertrophic differentiation. Pro-inflammatory mediators such as cytokines produced locally into the OA joint are critical in these pathological processes: interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) which promote joint inflammation and subsequently cartilage degradation [2]. A better understanding of the cytokine network involved in the pathophysiology of OA represents a major objective to delineate the underlying disease mechanisms.

Taking into account their well-known roles in many inflammatory processes, chemokines represent additional key mediators in OA [2]. Among them, the CXCR2 ligands including interleukin-8 (IL-8 also called CXCL-8), its murine equivalent called keratinocyte chemoattractant (Kc), and growth-related oncogene alpha (GROα or CXCL-1) are synthesized in OA chondrocytes [3] and are able to induce hypertrophic differentiation on chondrocytes in culture [4]. Moreover, IL-8 promotes several pathogenic processes such as homing and activation of mononuclear cell leukocytes into the synovium, neutrophil degranulation, release of oxidative products, production of matrix metalloproteinase-13 (MMP-13) by articular chondrocytes, chondrocyte apoptosis and loss of proteoglycans [5], [6], [7], [8]. These effects are consecutive to the binding of these chemokines to their receptor CXCR2, expressed on OA or normal chondrocytes [3], [4], [9]. While the effects and the quantification of IL-8/Kc and GROα have been deeply studied in OA, the mechanisms leading to the IL-8 release by chondrocytes in OA remain poorly known. However, several pathogenic signals occurring in OA represent potential inducers of IL-8/Kc in OA, such as:

• mechanical stress, modeling the effect of overweight or injury on cartilage of lower limb joints [10]. The intermittent compression of cartilage explants induces the pro-degradative phenotype of osteoarthritic chondrocytes characterized by the release of PGE₂, nitrite oxide (NO) as well as matrix metalloproteinases all participating to the cartilage degradation [11], [12], [13], [14];

• inflammatory stress, represented by pro-inflammatory cytokines including IL-1β markedly elevated in the OA synovial fluid and considered as the most potent catabolic factor in OA stimulating the release of PGE₂, NO, IL-6 and IL-17 [2], [15], [16], [17], [18], [19];

• metabolic stress, since there is a systemic link between the obesity and OA [20] and involving soluble mediators, adipokines, acting at distance of fat tissue. These adipokines are also synthesized within the joint and may be important links between the immune response and metabolism [21]. Among them, visfatin, also called nicotinamide phosphoribosyltransferase (NAMPT) or pre-B cell colony-enhancing factor (PBEF), is detectable in all tissues of the OA joint and induces pro-inflammatory mediators (PGE₂) and pro-degradative enzymes (A Disintegrin And Metalloproteinase with Thrombospondin Motifs-4 [ADAMTS-4] [15], ADAMTS-5, MMP-3 and MMP-13) by chondrocytes [22], [23], [24], [25].

In the present study, we investigated whether some pathogenic stimuli observed in OA corresponding to inflammatory, mechanical or metabolic stresses were able to induce the release of the murine equivalent of IL-8/Kc by murine cartilage.