Interleukin-6 upregulates extracellular matrix gene expression and transforming growth factor ß1 activity of tendon progenitor cells.

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-)1ß, 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), TGFß1 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 TGFß1 gene expression (6.3-fold; p = 0.01) in TPC, and significantly increased the TGFß1 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 TGFß1 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 TGFß1 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.

Equine peripheral blood CD14+ monocyte-derived macrophage in-vitro characteristics after GM-CSF pretreatment and LPS+IFN-γ or IL-4+IL-10 differentiation.

Macrophages are a heterogeneous population of immune cells that exhibit dynamic plasticity, polarize into inflammatory or regulatory/pro-resolving macrophages, and influence the healing tissue microenvironment. This study evaluated the in-vitro morphological, proliferative, cell surface marker expression and cytokine/soluble factor secretion characteristics of control, GM-CSF pretreated and inflammatory (LPS+IFN-γ) and regulatory (IL-4 + IL-10) differentiated equine CD14+ monocyte-derived macrophages. Phase contrast microscopy demonstrated that LPS+IFN-γ-primed macrophages exhibited a rounded, granular morphology, whereas IL-4 +IL-10-primed macrophages were elongated with a spindle-shaped morphology. GM-CSF enhanced the proliferation rate of monocytes/macrophages during adherent in-vitro culture. Flow cytometry analysis showed that GM-CSF alone and GM-CSF pretreatment with LPS+IFN-γ or IL-4 +IL-10 priming increased CD86 immunopositivity by 2-fold (p = 0.6); and CD206 immunopositivity remained unchanged. GM-CSF pretreatment and subsequent priming with LPS and IFN-γ yielded inflammatory macrophages that secrete significantly increased quantities of IL-1ß compared to control (p = 0.012) and IL-4 +IL-10-primed (p = 0.0047) macrophages. GM-CSF pretreatment followed by both LPS + IFN-γ and IL-4 + IL-10 priming significantly increased IL-1Ra secretion by 6-fold (p < 0.05). There were no differences in TGFß-1 secretion among control, LPS+IFN-γ or IL-4 + IL-10 primed macrophages (p = 0.85). All groups contained an average of 643 ± 51.5 pg/mL of TGFß1. Among the culture conditions evaluated, IL-4 +IL-10 priming for 24 h after 6 days of adherent culture yielded macrophages that were the least inflammatory compared to GM-CSF pretreated and LPS+IFN-γ or IL-4 +IL-10-primed macrophages. These results provide a basis for subsequent in-vitro and in-vivo studies that investigate macrophage-tissue cell interactions and related biological mechanisms relevant to the field of immunomodulatory approaches for enhancing tissue healing.

Macrophage phenotype impacts in vitro equine intrasynovial deep digital flexor tenocyte matrix metalloproteinase gene expression and secretion.

OBJECTIVE: To investigate matrix metalloproteinase (MMP) and their inhibitors tissue inhibitor matrix metalloproteinase (TIMP) gene expression and secretion during equine deep digital flexor tendon (DDFT) tenocyte and macrophage (undifferentiated, proinflammatory, and regulatory) co-culture. SAMPLE: Third passage DDF tenocytes and donor-matched macrophages differentiated from peripheral blood CD14+ monocytes from 5 healthy horses ages 9-11 years, euthanized for reasons unrelated to musculoskeletal conditions. METHODS: Passage 3 DDT tenocyte aggregate cultures were co-cultured with undifferentiated (control), proinflammatory (granulocyte-macrophage colony-stimulating factor; GM-CSF pretreated and lipopolysaccharide + interferon gamma-primed; LPS+IFN-γ) or regulatory (interleukin-4 and interleukin-10-primed; IL-4 + IL-10) macrophages in direct and transwell co-cultures for 72 hours. MMP-1, -2, -3, -9, -13, and TIMP -1, -2 mRNA were measured via real-time Polymerase Chain Reaction (rtPCR). Co-culture media MMP -3, -9, and TIMP -1, -2 concentrations were quantified via ELISA. RESULTS: Direct co-culture of DDF tenocytes with proinflammatory macrophages for 72 hours increased MMP-1, -3, and -13 mRNA levels whereas, MMP-9 mRNA levels decreased. Direct and transwell co-culture with proinflammatory and regulatory macrophages resulted in increased MMP-3 and decreased MMP-9 media concentrations. While direct co-culture with regulatory macrophages significantly increased TIMP-1 mRNA, overall, TIMP mRNA and culture media concentrations were largely unchanged. CLINICAL RELEVANCE: Cell-to-cell contact between DDF tenocytes and macrophages is not essential to induce MMP gene expression and secretion. Co-culture systems offer a viable in vitro platform to screen and evaluate immunomodulatory properties of therapies aimed at improving equine intrasynovial tendon healing.

Equine inflammatory response to abdominal surgery in the absence of gastrointestinal disease.

OBJECTIVE: To investigate the, equine inflammatory response to ventral midline celiotomy in the absence of gastrointestinal disease in horses of varying body condition scores primarily using serial measurements of serum amyloid A (SAA). DESIGN: Experimental clinical study. SETTING: University teaching hospital. ANIMALS: Ten adult light breed horses free of any clinical disease, 5 with body condition score (BCS) 3-4/9 and 5 with BCS 7-8/9. INTERVENTIONS: Horses had a ventral midline celiotomy performed under general anesthesia, including manual decompression of the small intestine. SAA, semiquantitative fibrinogen, plasma lactate, and WBC count were measured in the blood preoperatively and at 12, 24, 48, 72, 120, and 168 hours postoperatively. Complete serum biochemistry was performed preoperatively and 24 and 72 hours postoperatively. Serial abdominocentesis was also performed with peritoneal fluid analysis of SAA, total protein, lactate, WBC count, and cytology. MEASUREMENTS AND MAIN RESULTS: Significant (P < 0.05) increases in serum SAA were noted at 12, 24, and 48 hours postoperatively (124.6 ± 68.6, 390.8 ± 209.0, 568.6 ± 197.7 µg/mL), and most horses had values approaching normal at 168 hours postoperatively (174.4 ± 307.7 µg/mL). Other values such as fibrinogen also increased in response to surgery but did not return to normal within the measured time points. Horses with high BCS did not have significantly different serum SAA compared to horses with low BCS. Peritoneal fluid SAA did not increase significantly at 12 hours postoperatively. CONCLUSIONS: The information from this study can be used to help determine the effect of anesthesia and surgical intestinal manipulation resulting in increased SAA when a comparison to clinical or experimental cases is needed.