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The present study demonstrates that liposomes coated with synthesized neoglycolipids constructed from mannotriose and dipalmitoylphosphatidylethanolamine (Man3-DPPE) activate peritoneal macrophages (PEMs) to up-regulate expression of co-stimulatory molecules and preferentially secrete IL-12. Injection of Man3-DPPE-coated liposomes (oligomannose-coated liposome, OMLs) into the peritoneal cavity of mice resulted in specific and rapid incorporation of OMLs into PEMs. Upon OML incorporation, expression of co-stimulatory molecules, CD40, CD80, and CD86, and of MHC class II molecules was clearly enhanced on PEMs. In addition, production of IL-12 from PEMs was clearly promoted in response to OML incorporation, while those of IL-1 and IL-6 were suppressed. In contrast, liposomes coated with other carbohydrates and those without a carbohydrate-coating did not induce production of these cytokines. The cytokine profile produced from PEMs in response to OML clearly differed from those in response to ligands for TLRs, in which productions of IL-1 and/or IL-6 were strongly enhanced. Taken together, the results indicate that OMLs activate PEMs through a particular type of signal pathway distinct from those activated with TLR ligands, leading to specific production of IL-12 and consequent maturation of PEMs. Thus, OMLs can be used as a novel adjuvant for efficient activation of specific cellular immunity.