Anti-inflammatory effects of interleukin-4 on intervertebral disc cells

BACKGROUND CONTEXT: Inflammation has been associated with a number of pathological conditions including intervertebral disc (IVD) degeneration, increased risks of low back pain and other spinal diseases. Downregulating disc inflammation may be a strategy to reduce degeneration and more importantly back pain. Interleukin (IL)-4 was first discovered as a T-cell secreted factor that enhanced the proliferation of anti-IgM stimulated B cells and is now known as a cytokine that can stimulate cell proliferation and differentiation, tissue regeneration and neurological functions. IL-4 has been shown to be effective in inhibiting inflammatory pathways in chondrocytes. Immunohistochemical studies have shown that disc tissues are immunopositive for IL-4 receptor a (IL-4Ra) and IL-4. Yet, the roles of IL-4 and IL-4R in disc biology remain unknown. PURPOSE: The purpose of this study is to understand the roles of IL-4 and IL-4Ra in IVDs and to determine if IL-4 can function to inhibit inflammation in IVD cells. STUDY DESIGN/SETTING: In vitro experiment. METHODS: Deidentified patient IVD tissues were collected after surgery under the Orthopedic Information, Tissue and Implant Repository (ORA L00011021). IVD cells were isolated and cultured in monolayer. IL-4R protein expression was analyzed using immunocytochemistry. To test if the IL-4R was responsive to its ligand, signal transducer and activator of transcription 6 (STAT6) phosphorylation was analyzed on cell lysates of IVD cells treated with recombinant human IL-4 for 30 minutes using enzyme linked immunosorbent assay kit. Gene expression analysis of IL-4 up- and downregulated genes were analyzed using real-time RT-PCR. Anti-inflammatory effects of IL-4 were determined by cotreating disc cells with lipopolysaccharide (LPS) and IL-4 and measuring gene expression and protein release of inflammatory markers, IL-6 and IL-8. The significance of differences among means of data on gene expression and protein analyses were analyzed by one-way analysis of variance or student t test. Differences were considered significant when the p value was below 0.05. RESULTS: Immunocytochemistry staining for IL-4Ra in primary IVD cells (n=8) showed the majority of immunopositive staining was intracellular. After IVD cells (n=3-7) were treated with different concentrations of recombinant human IL-4 (0.1-100 ng/mL) for 30 minutes, phospho-STAT6 levels significantly increased by two-to four-fold at all concentrations tested compared with untreated cells. Gene expression of IL-4Ra and IL-6 increased significantly in cells undergoing IL-4 treatment for 24 hours compared with control treated IVD cells (n=5-10). LPS stimulated inflammatory gene expression of interferon (IFN)beta, IL-12, IL-6, and IL-8 were downregulated significantly in the presence of IL-4 (n=7). Lastly, protein release of IL-6 and IL-8 were reduced significantly in cells treated with IL-4 and LPS compared with those treated with LPS alone (n=7). CONCLUSIONS: This study was the first to explore the function of IL-4 and IL-4R in IVD cells. Immunocytochemistry studies confirmed that the majority of cells isolated from patient IVDs expressed IL-4Ra at the protein level. Also, IVD cells can respond to IL-4 by up-regulating IL-4Ra and IL-6 genes and inhibiting inflammatory genes and proteins induced by LPS. Further studies to test the anti-inflammatory effects of IL-4 in the IVD would be needed in animal models. (C) 2019 Elsevier Inc. All rights reserved.