M0, M1, and M2 macrophage phenotypes were induced for the in vitro inflammation culture model. (A) Macrophage phenotypes were verified by examining expression of surface markers and expression of proteins one day after M1-priming, M2-priming, or no treatment (M0). Geometric mean fluorescent intensities of the M2 markers CD206 and CD301 were significantly increased in the M2 group compared to the M0 and M1 groups. (B) M1 macrophages expressed significantly higher levels of pro-inflammatory factors, IL-1β, TNFα, PGE2, and NO. There was a significant effect of macrophage type for all proteins. (C) Protein expression was determined by measuring the levels in the medium (that is, representing the cumulative expression from all cell types in a particular culture). M1 macrophages induced the secretion of inflammatory factors by TFs. Protein expression of IL-1β, TNFα, PGE2, and NO after one day of co-culture with M0, M1, or M2 macrophages in the presence and absence of TFs. There was a significant effect of TF for IL-1β, TNFα, and PGE2. Bars indicate significant differences (* P <0.05, N = 4 for flow cytometry, N = 5 for protein expression). Note that some data are repeated in (B) and (C) to highlight effects of macrophage type and TF, respectively. PGE2, prostaglandin E2; TF, tendon fibroblasts.