Elsevier

Cytokine

Volume 40, Issue 3, December 2007, Pages 241-250
Cytokine

Preferential production of IL-12 by peritoneal macrophages activated by liposomes prepared from neoglycolipids containing oligomannose residues

https://doi.org/10.1016/j.cyto.2007.10.005Get rights and content

Abstract

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.

Introduction

Antigen-presenting cells (APCs)1, especially macrophages and dendritic cells (DCs), play a central role in the innate immune response to a wide range of bacterial components [1], [2]. On activation by these components, APCs produce pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-6 and IL-1, express high levels of MHC class II and co-stimulatory molecules such as CD80 and CD86 on their cell surface, and mature to produce IL-12, which greatly potentiates IFN-γ-producing Th1-type T cell differentiation [3], [4]. Thus, the maturation and cytokine production profiles of APCs are critical in the link between innate and adaptive immunity.

APCs interact with pathogens using pattern recognition receptors (PRRs), which can recognize highly conserved molecular signatures: so-called pathogen-associated molecular patterns (PAMPs) [5]. Toll-like receptors (TLRs) are a major pattern recognition receptor family. TLRs respond to PAMPs, and the subsequent signaling events can lead to detection of microbial infections, activation of innate immune responses, subsequent activation and modulation of adaptive immunity, and consequent triggering of the antibacterial host defense response [1], [2]. In addition to TLRs, carbohydrate-binding receptors are another part of the larger PRR family and are expressed on a wide variety of cell types, including APCs. Carbohydrate-binding receptors participate in recognition of carbohydrate structures associated with a wide variety of microbes and microbial-derived products [6], [7], [8]. The macrophage mannose receptor (CD206) expressed on macrophages and DCs recognizes mannose, fucose and N-acetylglucosamine residues of glycoconjugates, and is a well-characterized endocytic receptor in uptake of bacteria, yeast, and other pathogenic microorganisms [9]. The β-glucan receptor, dectin-1, mediates phagocytosis of nonopsonic yeast by macrophages and acts in synergy with TLR-2 to augment pro-inflammatory cytokine responses [10], [11]. DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN), a new dendritic cell-specific membrane lectin, binds to high-mannose oligosaccharides and is involved in uptake of mannose-exposed particles and microbes by DCs [12], [13], [14], [15], [16], and DC-SIGN-related proteins, SIGNR1 and SIGNR3, expressed on macrophages also recognize polysaccharides and microbes [17]. Thus, carbohydrate-binding receptors may play important roles in recognition of PAMPs in innate immunity.

Recently, we have reported that liposomes coated with synthesized neoglycolipids constructed from mannotriose and dipalmitoylphosphatidylethanolamine (Man3-DPPE) are preferentially and rapidly taken up by peritoneal macrophages, and that liposome-incorporating macrophages accumulate in extranodal lymphoid tissues following injection of Man3-DPPE-coated liposomes (hereafter referred to as oligomannose-coated liposomes, OMLs) into the peritoneal cavity [18], [19]. It has also been shown that intraperitoneal immunization of OMLs entrapped with soluble leishmanial antigen induces an antigen-specific Th1 immune response and protects against subsequent infection with Leishmania major in BALB/c mice [20], [21]. These findings indicate that specific incorporation of OMLs into peritoneal macrophages triggers the induction of Th1 immune response, but the underlying mechanism is unclear. Up-regulation of co-stimulatory molecules and MHC class II on APCs is crucial in determining the nature and extent of the immune response [22], [23], [24], and IL-12 secretion from APCs is critical for development of Th1 cells and initiation of a cell-mediated immune response [3]. Thus, the present study was conducted to investigate the activation of peritoneal macrophages by OMLs and the subsequent profile of pro-inflammatory cytokines. Our results indicate that OMLs exhibit a novel adjuvant activity that results in increased expression of MHC class II and co-stimulatory molecules and leads to preferential induction of IL-12 production by peritoneal macrophages.

Section snippets

Antibodies and reagents

Fluoroscein isothiocyanate (FITC)-, phycoerythrin (PE)-, or peridinin chlorophyll protein-cyanin 5.5 (PerCP-Cy5.5)-labeled antibodies directed against mouse CD3ε, CD11b, CD19, CD40, Gr.-1 and I-A/I-E class II molecules and an Fc-block (anti-mouse CD16/32) were purchased from BD PharMingen (San Diego, CA, USA). PE-labeled anti-F4/80 antibody was purchased from Caltag Laboratories (Burlingame, CA, USA). FITC-conjugated antibodies directed against mouse CD11c, mouse CD80, and CD86 were purchased

Intraperitoneal immunization of OMLs induces a Th1 response against encased antigen

Liposomes coated with Man3-DPPE were used as oligomannose-coated liposomes (OMLs) in all experiments, unless otherwise indicated. We used OMLs consisting of DPPC:cholesterol:Man3-DPPE (10:10:1) and with a particle size of 1 μm, since it has been shown that OMLs of this composition and size are preferentially taken up by peritoneal macrophages (PEMs), with subsequent induction of a Th1 immune response specific for an antigen entrapped in the OML in mice [21]. To confirm preferential uptake of

Discussion

Our results show that production of IL-12 in response to OMLs (Man3-DPPE- or Man5-DPPE-coated liposomes) is a characteristic of peritoneal macrophages, with subsequent maturation to antigen-presenting cells with enhanced expression of co-stimulatory molecules and MHC class II molecules, and induction of an antigen-specific Th1 immune response. Thus, OMLs possess novel adjuvant properties for induction of cell-mediated immunity. These responses are characteristic to OMLs, since liposomes coated

Acknowledgments

This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), and in part by a grant for Hi-Tech Research from Tokai University, and by the Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization (NEDO) of Japan.

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