IL-40 is up-regulated in the synovial fluid and cartilage of osteoarthritis patients and contributes to the alteration of chondrocytes phenotype in vitro

Abstract

Introduction: IL-40 is a novel cytokine associated with autoimmune connective tissue disorders such as rheumatoid arthritis (RA) or Sjögren syndrome. We have previously shown an accumulation of IL-40 in the RA joint and its expression by immune cells and fibroblasts. Therefore, we aimed to assess the role of IL-40 in association with hyaline cartilage and chondrocyte activity.

Methods: Immunohistochemistry was employed to detect IL-40 in paired samples of loaded and unloaded regions of osteoarthritis (OA) cartilage (n=5). Synovial fluid IL-40 was analysed by ELISA in OA (n=31) and control individuals after knee injury (n=34). The impact of IL-40 on chondrocytes was tested in vitro.

Results: IL-40 was found in chondrocytes of the superficial zone of the OA cartilage, both in loaded and unloaded explants. Additionally, only biopsies from loaded explants showed significant IL-40 positivity in transitional zone chondrocytes. Levels of IL-40 were significantly elevated in the synovial fluid from OA patients compared to controls (p<0.0009) and correlated with synovial fluid leukocyte counts in OA (r=0.444, p=0.014). Chondrocytes exposed to IL-40 dose dependently increased in the secretion of pro-inflammatory cytokines IL-6 (p<0.0001) and IL-8 (p=0.004). Moreover, a dose dependent up-regulation of matrix degrading metalloproteinases MMP-1 (p=0.004), MMP-3 (p=0.031) and MMP-13 (p=0.0002) upon IL-40 treatment was observed in contrast to untreated chondrocytes.

Conclusion: This study is the first to demonstrate the accumulation of IL-40 in OA cartilage and its up-regulation in the synovial fluid of OA patients compared to controls. In addition, extracellular IL-40 appears to play a role in promoting inflammation and cartilage destruction by driving chondrocyte behaviour towards a more aggressive phenotype.

Keywords: Cartilage; Chondrocytes; Interleukin 40; Osteoarthritis.

Fig. 1

IL-40 was highly expressed in the superficial and transitional zone chondrocytes in the loaded areas of OA cartilage. In the radial zone of loaded explants, IL-40 positive chondrocytes were found only in some patients (A, C). A. In the unloaded areas, superficial zone chondrocytes tested positive for IL-40, whereas only random IL-40 staining was found in transitional and radial zones (B, D). Semi‐quantitative analysis was performed to evaluate the percentage of the IL-40 positive cells in all analysed samples of loaded (C) and unloaded (D) cartilage explants (n=5). Rabbit IgG was used as isotype control (A, B). Safranin O – Fast Green and haematoxylin were used to assess the cartilage morphology (A, B). Representative images are shown at x40 and x200 magnification. Levels of IL-40 in the synovial fluid were significantly up-regulated in osteoarthritis (OA) patients compared to controls (E). No relation between synovial fluid IL-40 levels and radiographic severity (KL grade) was observed in OA patients (F). Synovial fluid IL-40 levels were positively associated with synovial fluid leukocyte counts in OA patients (G) The data are represented as the median; the error bars show 1^st and 3^rd quartiles. ***, p<0.001

Fig. 2

Normal human articular chondrocytes (n=7-9) significantly increased the secretion of cytokines IL-6, IL-8 a matrix degrading metalloproteinases (MMPs) but not IL-1β upon the exposure to recombinant IL-40 (5-200 ng/ml) for 24h in in vitro conditions (A-F). The data are represented as the median; the error bars show 1^st and 3^rd quartiles. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001