Interleukin-6 upregulates extracellular matrix gene expression and transforming growth factor β1 activity of tendon progenitor cells

Abstract

Background: Prolonged inflammation during tendon healing and poor intrinsic healing capacity of tendon are causal factors associated with tendon structural and functional degeneration. Tendon cells, consisting of mature tenocytes and tendon progenitor cells (TPC) function to maintain tendon structure via extracellular matrix (ECM) synthesis. Tendon cells can succumb to tissue cytokine/chemokine alterations during healing and consequently contribute to tendon degeneration. Interleukin-(IL-)1β, IL-6 and TNFα are key cytokines upregulated in injured tendons; the specific effects of IL-6 on flexor tendon-derived TPC have not been discerned.

Methods: Passage 3 equine superficial digital flexor tendon (SDFT)-derived TPC were isolated from 6 horses. IL-6 impact on the viability (MMT assay with 0, 1, 5 and 10 ng/mL concentrations), migration (scratch motility assay at 0, 10ng/mL concentration) of TPC in monolayer culture were assessed. IL-6 effect on tendon ECM and chondrogenic gene expression (qRT-PCR), TGFβ1 gene expression and activity (ELISA), and MMP-1, -3 and - 13 gene expression of TPC was evaluated.

Results: IL-6 decreased TPC viability and migration. IL-6 treatment at 10 ng/mL significantly up-regulated TGFβ1 gene expression (6.3-fold; p = 0.01) in TPC, and significantly increased the TGFβ1 concentration in cell culture supernates. IL-6 (at 10 ng/mL) significantly up-regulated both tendon ECM (COL1A1:5.3-fold, COL3A1:5.4-fold, COMP 5.5-fold) and chondrogenic (COL2A1:3.9-fold, ACAN:6.2-fold, SOX9:4.8-fold) mRNA expression in TPC. Addition of SB431542, a TGFβ1 receptor inhibitor, to TPC in the presence of IL-6, attenuated the up-regulated tendon ECM and chondrogenic genes.

Conclusion: IL-6 alters TPC phenotype during in vitro monolayer culture. Pro- and anti-inflammatory roles of IL-6 have been implicated on tendon healing. Our findings demonstrate that IL-6 induces TGFβ1 activity in TPC and affects the basal TPC phenotype (as evidenced via increased tendon ECM and chondrogenic gene expressions). Further investigation of this biological link may serve as a foundation for therapeutic strategies that modulate IL-6 to enhance tendon healing.

Keywords: ECM mRNA gene expression; Interleukin-6; Matrix metalloproteinases; Superficial digital flexor tendon; TGFβ1.

Fig. 1

TPC viability measured via MTT assay after 24 h of monolayer culture in basal medium supplemented with 0, 1, 5 and 10 ng/mL IL-6. The y-axis denotes absorbance readings measured at 490 nm. Bars and errors represent mean ± SD

Fig. 2

TPC migration assessed via ‘scratch assay’ measured as the width of the defect remaining after 24 h of monolayer culture in basal medium supplemented with 0, 1, 5 and 10 ng/mL IL-6. Bars and errors represent mean ± SD of “scratch” defect width after 24 h. Supporting 5X photomicrographs of TPC monolayers at baseline, and at 24-hour timepoint with 0 and 10 ng/mL IL-6. Scale bar represents 200 microns

Fig. 3

(A) TGFβ1 mRNA and (B) ELISA quantification of TGFβ1 concentration measured in TPC monolayer culture and culture media, respectively, (mean ± SD) after 72 h of culture with 0, 1, 5 and 10 ng/mL IL-6. TGFβ1 mRNA and concentration were also measured in TPC in basal medium and 10 ng/mL IL-6 supplemented with 2 μm TGFβ type I receptor (ALK-4, − 5, −7) inhibitor SB431542. Bars and errors represent mean ± SD of TGFβ1 mRNA and concentration (pg/mL)

Fig. 4

(A) Tendon ECM and (B) Chondrogenic gene expression of TPC monolayers cultured in basal medium with 10 ng/mL IL-6, basal medium with 2 μm TGFβ type I receptor (ALK-4, − 5, −7) inhibitor SB431542, and 10 ng/mL IL-6 + SB431542 for 72 h. Data represents mean ± SD of fold change (normalized to housekeeping gene, EF1α) from respective values obtained from TPC monolayer cultures in basal medium alone (represented via dotted line in all graphs)