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

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 α (IL-4Rα) and IL-4. Yet, the roles of IL-4 and IL-4R in disc biology remain unknown.

The purpose of this study is to understand the roles of IL-4 and IL-4Rα in IVDs and to determine if IL-4 can function to inhibit inflammation in IVD cells.

In vitro experiment.

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.

Immunocytochemistry staining for IL-4Rα 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-4Rα 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)β, 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).

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-4Rα at the protein level. Also, IVD cells can respond to IL-4 by up-regulating IL-4Rα 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.

Biological therapies which include intradiscal delivery of cells, anti-inflammatories or growth factors are being investigated to treat disc degeneration and back pain in animal models and in the clinic. Based on our findings that IL-4 has anti-inflammatory effects on IVD cells, the results of this study suggest including recombinant IL-4 delivery into the intervertebral disc may be a beneficial therapeutic strategy to treat patients with back pain by reducing disc inflammation.