Macrophages induce the adhesion phenotype in normal peritoneal fibroblasts

Abstract

To determine whether macrophages, exposed to hypoxia, stimulate primary cultures of fibroblasts to acquire the adhesion phenotype. The adhesion phenotype has been previously characterized, in part, by increased fibroblast expression of transforming growth factor (TGF) β1, vascular endothelial growth factor (VEGF), and type I collagen. Media collected from human macrophages cultured under hypoxic conditions (2% O(2)) were used to treat human peritoneal fibroblasts. Additionally, human peritoneal fibroblasts were treated with varying concentrations of TGF-β1. Real-time reverse-transcription polymerase chain reaction and Western blot analysis were used to measure mRNA and protein levels, respectively, for select adhesion markers: TGF-β1, VEGF, and, type I collagen. We hypothesized that macrophage secretion, under hypoxic conditions, is responsible for inducing the adhesion phenotype in human peritoneal fibroblasts. University research laboratory. Human macrophages and peritoneal fibroblasts. Macrophage-fibroblast interaction. Ability of macrophages to induce the adhesion phenotype in human peritoneal fibroblasts. Hypoxia treatment resulted in a significant increase in TGF-β1 expression in human macrophages. Additionally, hypoxia treatment resulted in a significant increase in TGF-β1, VEGF, and type I collagen mRNA and protein levels in normal peritoneal fibroblasts compared with normoxic conditions. Similarly, normal peritoneal fibroblasts treated with media collected from macrophages cultured under hypoxic conditions resulted in a significant increase in TGF-β1, VEGF, and type I collagen mRNA and protein levels compared with normal peritoneal fibroblasts treated with media collected from macrophages cultured under normoxic conditions. Additionally, human peritoneal fibroblasts exposed to varying concentrations of TGF-β1 exhibited a dose-dependent response in the expression of TGF-β1, VEGF, and type I collagen. At a low TGF-β1 concentration (12.5 ng TGF-β1/mL medium), TGF-β1, VEGF, and type I collagen were significantly increased. In contrast, at higher TGF-β1 concentrations (25 and 50 ng TGF-β1/mL media), TGF-β1, VEGF, and type I collagen mRNA levels were significantly reduced compared with 12.5 ng TGF-β1/mL medium. Human macrophages, cultured under hypoxic conditions, release factors that induce the expression of the adhesion phenotype in normal peritoneal fibroblasts. Particularly, TGF-β1 reproduces this response by regulating the expression of TGF-β1, VEGF, and type I collagen in a dose-dependent manner. Therefore, these findings highlight an important role for human macrophages in peritoneal wound healing.