Effect of liposome on gene expression of splenic macrophages

Myeloid-derived suppressor cells (MDSC) are induced in various situations. Generation of MDSC has been investigated extensively. Several cytokines are involved in regulating MDSC. Cellular microvesicles, which comprise cellular membrane (single lipid bilayer) are involved in the induction of MDSC. Especially, proteins or nucleic acids encapsulated in them or expressed on their surface are thought to play a role in the induction of MDSC. Nevertheless, the role of lipid components remains to be investigated. Our earlier study using WKAH rat revealed that MDSC-like cells can be induced in the rat spleen by intravenous injection of liposomal microparticles. They comprise a single lipid bilayer of approximately 250 nm diameter, with no cellular component. Therefore, they resemble cellular microvesicles, Cells capable of suppressing T cell proliferation were liposome-internalized cells. The cell surface phenotype was class II low/-, CD11b/c+. Nitric oxide was responsible for their suppression. In this study, we showed that they express B7-H3 molecule on their surface, removal of B7-H3 positive cells restored T cell proliferation. Western blot analysis confirmed that they are positive for iNOS and NFkB was activated 4 hr after liposomal injection. These results indicate that these cells in question belongs to a subset of MDSCs. Furthermore, we elucidated that vesicular form of lipid is important for the induction of T cell suppressive function. Based on these data, we concluded that liposomal microparticles can induce B7-H3 positive MDSC in vivo and the vesicular formation of lipid is essential for the induction of MDSC. Spleens were excised from liposome-loaded or control rats 24 hr after liposome or saline injection. Enrichment of MHC-class II-/CD11b/c+ cell was performed using magnetic beads according to the manufacturer’s protocol. Cells were applied to anti-MHC class II(OX-6) microbeads (Miltenyi Biotec) for negative selection of MHC-class II positive cells. Subsequently they were mixed with PE conjugated anti CD11b/c(OX-42), then stained with anti-PE microbeads and applied for the MACS column for positive selection of CD11b/c positive cells. Total RNA extracted from MHC-class II negative and CD11b/c positive cell enriched fractions of both liposome-loaded and saline-loaded rat were applied to a microarray system