Abstract
Inflammation plays an important role in the development and resolution of tendon diseases, but underlying mechanisms are poorly understood. We therefore aimed to analyze the response of human tenocytes to inflammatory stimuli and to uncover their interplay with macrophages in vitro. Tenocytes from human ruptured supraspinatus tendons (n = 10) were treated for three days with a stimulation mixture derived from activated mononuclear cells isolated from healthy human peripheral blood. Significantly increased expression levels of selected adhesion- and human leukocyte antigen (HLA)-molecules, and enhanced interleukin (IL)-6 release were detected by flow cytometry. Tenocyte stimulation with the pro-inflammatory cytokines interferon gamma, tumor necrosis factor alpha and IL-1ß triggered similar changes in surface markers and enhanced the release of IL-6, IL-8 and monocyte chemoattractant protein 1 (MCP-1). In co-cultures of macrophages with pre-stimulated tenocytes, macrophages significantly increased CD80 expression, but simultaneously decreased HLA-DR-expression, which are both typical pro-inflammatory polarization markers. Co-cultures also released more IL-6, IL-8, MCP-1 than tenocyte-cultures alone. We demonstrate that tenocytes respond to inflammatory environments in vitro with altered surface marker and cytokine profiles and influence macrophage polarization. Importantly, all changes detected in direct co-cultures were also present in a transwell setting, implicating that communication between the cells involves soluble factors.
Highlights
Tendon ruptures can result from either acute traumatic injury or follow chronic overuse leading to degeneration
To mimic the clinically relevant inflammatory conditions that might influence tenocytes during tissue injury, a complex stimulation media from αCD3αCD28 stimulated peripheral blood mononuclear cells (PBMCs) cultures containing a set of human cytokines, predominantly IL-6, IL-8, monocyte chemoattractant protein 1 (MCP-1), TNFα, and IFNγ was generated (Supplemental Fig. 1)
Macrophages were generated by a 6 day M-CSF stimulation of blood-derived CD14+ monocytes, and added to tenocytes which were seeded at day-1, with or without pre-stimulation for 3 days using the stimulation media derived from αCD3αCD28 stimulated PBMCs
Summary
Tendon ruptures can result from either acute traumatic injury or follow chronic overuse leading to degeneration. In the later stages of acute tendon healing, tissue repair macrophages dominate, and release anti-inflammatory cytokines to dampen inflammation and promote tendon remodeling[10,11,12]. The balance between pro- and anti-inflammatory cells and soluble factors within the tendon healing process will have an important impact on the successful resolution of inflammation. The role of monocyte derived macrophages for tissue remodeling and repair is known from many other tissues and diseases[15,16,17,18], and is likely important in tendon injury as infiltration of CD14+CD68+ myeloid cells was already demonstrated, mostly by immunohistochemistry, flow cytometry or mRNA expression analyses[7, 13, 19, 20]
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