Chronic Transplant Vasculopathy Microenvironment Present in the Renal Allograft Reprograms Macrophage Phenotype

Abstract

Macrophages and monocytes are important in chronic allograft dysfunction. Chronic transplant vasculopathy is an important cause of chronic renal allograft dysfunction. It is characterized by the presence of apoptotic endothelial cells, which generate an apocrine loop that leads to typical myointimal proliferation. However, the exact nature of the reprogramming induced by this microenvironment on recruited monocyte and macrophage phenotypes is ill defined. Human umbilical vein endothelial cells with a serum-starved condition (SSC) for 4 hours were used as a model of apoptotic endothelial cells. This conditioned medium was centrifuged to isolate the apoptotic bodies. Monocytes were harvested from healthy donors using Ficoll gradient and immunomagnetic selection. Blood monocyte–derived macrophages (5–7 days of maturation) were exposed to centrifuged apoptotic cell–conditioned media for 24 hours. Cells were harvested for immunoblotting, and supernates were retained for enzyme-linked immunosorbent assay to determine cytokine and chemokine production. Macrophages generated less IL-6 and IL-8 when cultured in SSC compared with control. Immunoblotting for STAT3 (signal transducer and activator of transcription 3) in macrophages revealed that SSC increased STAT3 levels, which persisted after lipopolysaccharide stimulation. These results suggest that SSC attenuates the pro-inflammatory phenotype in macrophages, through a STAT3-dependent mechanism.