Tenascin C upregulates interleukin-6 expression in human cardiac myofibroblasts via toll-like receptor 4

Figure 1 Tenascin C and toll-like receptor 4 staining in the murine left ventricle following infarction. A: Low power view of the infarcted mouse LV showing the proximity of TNC (brown) and TLR4 (pink) 3 d following the occlusion of the left anterior coronary artery; B: Diffuse TLR4 staining (open arrows) in myocytes and interstitial cells and intense TLR4 staining (solid arrows) in some myocytes. Interstitial TNC staining (brown) is evident around these cells; C: High powered view of TLR4 (pink) and alpha smooth muscle actin (brown) staining of cells in the infarcted LV. Intense TLR4 staining can be seen in some myocytes (solid arrow) with more diffuse staining seen in some cardiac myofibroblasts (labelled both pink and brown, open arrow); D: Low power view of the non-infarcted side of the mouse myocardium stained for TNC (brown) and TLR4 (pink). An absence of TNC staining and light diffuse TLR4 staining of the cells is observed. Inset image: a high powered view of cells in this area. In each image cell nuclei were stained with Mayer’s Haematoxylin. RV: Right ventricle. LV: Left ventricle; TNC: Tenascin C; TLR4: Toll-like receptor 4.

Figure 2 Changes in cytokine and matrix metalloproteinase mRNA expression in human cardiac myofibroblasts following incubation with Tenascin C. Effect of Tenascin C (TNC) (0.1 μmol/L, 24 h) on MMP1, MMP2, MMP3, MMP9, MMP10, MT1-MMP, IL-1β and IL-6 mRNA expression was assessed in human cardiac myofibroblasts (n = 4-6 donors). A significant increase in the expression of MMP3 and IL-6 was observed. Data are expressed as mean ± SEM. ^bP < 0.01 vs vehicle (student’s t-test). MMP: Matrix metalloproteinase; IL: Interleukin.

Figure 3 Toll-like receptor 4 expression in human cardiac myofibroblasts. A: Data from RT-PCR array showing abundance of TLR mRNA in a pooled sample of human CMF (n = 3 donors). Data expressed relative to 5 housekeeping genes; B: Taqman RT-PCR analysis of TLR2 and TLR4 mRNA levels in human CMF (n = 4 donors). Note log scale. ^bP < 0.001 (paired t-test); C: Western blot of CMF homogenates from 4 donors probed with anti-TLR4 antibody. Fifteen micrograms protein per lane. Molecular size (kDa) on left. TLR4 = 95 kDa; D1: Immunocytochemical localisation of TLR4 in human CMF using a primary antibody to human TLR4 and a Cy3-conjugated secondary antibody; D2: Effect of pre-absorption of primary antibody with TLR4 peptide (8 μg/mL) prior to immunostaining. Nuclei are labelled with DAPI. Loss of immunostaining confirms specificity of the antibody. Scale bar 50 μm; E: Effect of IL-1α (10 ng/mL, 6 and 24 h) on TLR4 mRNA expression in human CMF (n = 4 donors). Data are expressed as mean ± SEM. ^dP < 0.01 vs vehicle (ANOVA post-hoc). TLR: Toll-like receptor.

Figure 4 Tenascin C upregulates interleukin-6 expression in human cardiac myofibroblasts via toll-like receptor 4. A and B: Effect of TNC (0.1 μmol/L, 24 h) with and without 1 h pre-incubation in TLR4 antisera (25 μg/mL) on IL-6 mRNA (A) and IL-6 protein expression (B) in CMF (4-5 donors). Data expressed as mean ± SEM. ^bP < 0.01 vs vehicle (ANOVA post-hoc). TNC: Stimulation with TNC alone; TLR4-Ab: Pre-incubation with TLR4 neutralising antisera; TNC/TLR4-Ab: Stimulation with TNC following TLR4 pre-incubation; C: Effect of 1 h pre-incubation in TLR4 antisera (25 μg/mL) on IL-1α (10 ng/mL, 24 h)-stimulated IL-6 mRNA expression in CMF (n = 3 donors). The TLR4 neutralising antisera had no effect on IL-1α induced IL-6mRNA expression. Data expressed as mean ± SEM. ^dP < 0.0001 vs vehicle (ANOVA post-hoc). IL-1: Stimulation with IL-1α alone; TLR4-Ab: Pre-incubation with TLR4 neutralising antisera; IL-1/TLR4-Ab: Stimulation with IL-1α following TLR4 pre-incubation; TNC: Tenascin C; CMF: Cardiac myofibroblasts; IL: Interleukin; TLR: Toll-like receptor.

Figure 5 Fibrinogen-like globe domain of Tenascin C up-regulates interleukin-6 mRNA expression in human cardiac myofibroblasts via toll-like receptor 4. A and B: Effect of 1 μmol/L (24 h) recombinant protein, corresponding to either the FBG domain or the fibronectin type III domain (TNIII 5-7) of TNC, on IL-6 mRNA (A) and MMP3 mRNA (B) in human CMF (n = 3-5 donors). Data expressed as mean ± SEM. ^bP < 0.01 vs vehicle (ANOVA post-hoc); C: Effect of 1 h pre-incubation in TLR4 neutralising antisera (25 μg/mL) alone on IL-6 mRNA expression and on FBG (1 μmol/L, 24 h) stimulated IL-6 mRNA expression in CMF (n = 3 donors). Data expressed as mean ± SEM. ^bP < 0.01 vs vehicle (ANOVA post-hoc). FBG: Stimulation with FBG recombinant alone; TLR4-Ab: Pre-incubation with TLR4 neutralising antisera; FBG/TLR4-Ab: Stimulation with FBG following pre-incubation with TLR4 neutralising antisera. FBG: Fibrinogen-like globe; TNC: Tenascin C; IL: Interleukin; CMF: Cardiac myofibroblasts.