Homocysteine Elicits an Inflammatory Profile in Murine Macrophages Through an EMMPRIN Mediated Pathway

Abstract

IntroductionMacrophages are known to differentiate between two distinct phenotypic subsets: M1 and M2. The M1 phenotype elicits a pro‐inflammatory profile while M2 macrophages are categorized as being anti‐inflammatory. Hyperhomocysteinemia (HHcy) is associated with many inflammatory diseases such as obesity and atherosclerosis. It is known that HHcy increases specific macrophage secretory factors, such as MCP‐1 and MMP‐9. However, the direct effect of HHcy on macrophage phenotype is largely unexplored. We hypothesize that HHcy causes macrophages to shift into a more inflammatory phenotype or M1.MethodsWe utilized 2 murine macrophage cell lines: Raw 264.7 and J774A.1 cells. These cells were treated with 500 and 100 µM homocysteine, respectively, for 24 hours. Methods employed are Western Blot, ICC, FACS and PCR.ResultsIn Raw 264.7 cells, 24 hour treatment with 500 μM Hcy resulted in a 2.25 fold increase in CD40 protein expression: a cell surface marker on M1 macrophages. ICC confirms this increase in CD40. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) was also increased by 1.62 fold, indicating that MMP‐9 induction with Hcy may be through an EMMPRIN mediated pathway. J774A.1 cells were more responsive to Hcy treatment, demonstrating a 1.4 fold induction of CD40 with 24 hours of 100 μM treatment. ICC demonstrates increased MMP9 production in this cell line. We suggest that Hcy may alter IL‐1β, iNOS, TIMPs 1 and 4, and MMP‐2, as well as EMMPRIN in MMP9 production.ConclusionsOur current data suggests that Hcy treatment shifts macrophage phenotype towards the pro‐inflammatory, M1 subset. We believe that HHcy increases IL‐1 β, iNOS and MMP‐2, while demonstrating decreases in TIMPs 1 and 4.