Interleukins 4 and 13 modulate gene expression and promote proliferation of primary human tenocytes

Abstract

Background: Tendon disorders (tendinopathies) pose serious biomedical and socioeconomic problems. Despite diverse treatment approaches, the best treatment strategy remains unclear. Surgery remains the last resort because of the associated morbidity and inconsistent outcomes. We hypothesized that, similar to fibroblasts in various organs, tendon fibroblasts (tenocytes) might be responsive to stimulation with interleukins (ILs), particularly IL-4 and IL-13. These two cytokines share sequence homology, receptor chains and functional effects, including stimulation of fibrogenesis. It is unknown whether tenocytes are responsive to stimulation with IL-4 or IL-13. If true, local use of these cytokines might be used to facilitate tendon repair in patients with tendinopathies or used for tendon tissue-engineering approaches to facilitate tenocyte growth on scaffolds in culture.

Results: Tendon tissues that would normally be discarded were obtained during reconstructive surgery procedures performed for clinical indications. Primary tenocytes were derived from Achilles, posterior tibial, flexor digitorum longus and flexor hallucis longus tendon tissue samples. Reverse transcriptase quantitative PCR (RT-qPCR) experiments revealed that mRNAs for the receptor (R) chains IL-4Ralpha, IL-13Ralpha1 and IL-13Ralpha2, but not the common gamma-chain were present in all tested tendon tissues and in cultured tenocytes. Levels of IL-13R chain mRNAs were significantly higher than those of IL-4R mRNA. The cultures responded, in a dose-dependent fashion, to stimulation with recombinant human IL-4 or IL-13, by increasing proliferation rates 1.5 to 2.0-fold. The mRNA levels of 84 genes related to cell cycle regulation were measured by RT-qPCR after 6 h and 24 h of activation. The expression levels of several genes, notably CDK6 and CDKN2B changed more than twofold. In contrast to their effects on proliferation, stimulation with IL-4 or IL-13 had little if any effect on the levels of collagen mRNA or protein in cultured primary tenocytes. The mRNA levels of 84 other genes related to extracellular matrix and cell adhesion were also measured by RT-qPCR; expression of only five genes was consistently changed.

Conclusions: Stimulation with IL-4 or IL-13 could be used to facilitate tendon repair in vivo or to aid in tendon tissue engineering, through stimulation of tenocyte proliferation.

Figure 1

Reverse transcriptase quantitative (q)PCR analyses of interleukin (IL)-4 receptor (R)/IL-13R mRNA expression . (a) Ethidium bromide gels of PCR products after 35 cycles of qPCR with cDNAs from cultured indicated cells with indicated primers. Tenocytes (Tenoc) expressed scleraxis mRNA, confirming the phenotypic identity of these cells. Tenocytes and pulmonary fibroblasts but not T cells expressed mRNA for collagen chains. All tested cell types expressed IL-4Rα mRNA, and all cell types except T cells expressed IL-13Rα1 and IL-13Rα1 mRNAs. Only T cells expressed common gamma chain (γc). (b) Quantitative analyses reveal relative expression of receptor chains in cultured tenocytes from indicated tendons of five separate donors (SYBR Green-based quantification, normalized to 18S rRNA). The prime marks indicate separate cultures developed at a different time from the same tissue source and analyzed at a different passage. A549 = transformed pulmonary epithelial cell line; NHLF = normal human lung fibroblasts, PBMC = peripheral blood mononuclear cells from a healthy volunteer.

Figure 2

Effects of IL-13 and IL-4 on proliferation of primary tenocytes . Stimulation with (a) IL-13 or (b) IL-4 accelerated proliferation of primary tenocytes in a dose-dependent fashion (CellTiter Aqueous assays, day 7). Data show fold increase in proliferation rate ± SD, in tenocytes derived from indicated tendon types.