Figure 5. IL-6 production by scarring trachoma fibroblasts contributes to macrophage activation.

Fibroblasts alone (F) or fibroblasts with macrophages (F+M) at a 1:4 ratio were embedded in collagen gels and contraction was allowed to proceed for 7 days in the presence of 10% serum (a) or in serum free medium (b–d) with/without IL-6 inhibition or soluble recombinant IL-6. (a) Macrophages do not affect fibroblast-mediated contraction in the presence of serum. Shown is the mean+/− SEM for contraction at day 7 (n = 2–3 for 4 CFs and 5 STFs; **p = 0.0002, t-test). (b) Fibroblast and macrophage co-culture in the absence of serum led to a significant increase in contraction for both CFs and STFs (p = 0.0013 and p = 0.0002, 2-tailed t-test), and higher for STFs (*p = 0.030, non-parametric t-test; n = 2–3 for 5 CFs and 5 STFs). (c) IL-6 inhibition in co-culture, either with neutralizing antibody (nAb, 300 ng/ml) or siRNA treatment against IL-6, did not alter contraction (mean+/− SEM for 1–3 experiments for 4 CFs and 5 STFs). (d) Addition of IL-6 (20 ng/ml) did not alter contraction in co-cultures (mean+/− SEM for n = 1-3 for 4 CFs and 6 STFs). (e–g) Condition medium (CM) was generated from CFs and STFs cultures and IL-6 nAb or control IgG (200 ng/ml) was added to it prior to adding it to macrophages. After 24 h incubation the cells were lysed and pathway activation was tested by western blot. Macrophages treated with STF-derived CM exhibited an increased activation of STAT3 (e) and Akt pathways (f), which was significantly reduced upon IL-6 inhibition. Graphs show protein levels relative to GAPDH (**p = 0.0129, two-tailed t-test; n = 4 for 4 CFs and 5 STFs). (g) Representative corresponding western blots.