MICA/B expression in macrophage foam cells infiltrating atherosclerotic plaques

https://doi.org/10.1016/j.yexmp.2014.07.002Get rights and content

Abstract

Infiltrating macrophages accumulate in fatty streak lesions and transform into foam cells, leading to the formation of atherosclerotic plaques. Inflammatory mechanisms underlying the plaque formation mediated by NKG2D-positive lymphocytes such as CD8+ T cells, natural killer cells and natural killer T cells have been extensively investigated. Yet, the involvement of the NKG2D system itself remains poorly understood. Recent work in mouse models has shown that blockade of an NKG2D receptor–ligand interaction reduces plaque formation and suppresses inflammation in aortae. In this study, we conducted immunohistochemical analysis of NKG2D ligand expression in autopsy-derived aortic specimens. Foam cells expressing NKG2D ligands MICA/B were found in advanced atherosclerotic lesions accompanied by a large necrotic core or hemorrhage. Human monocyte-derived macrophages treated in vitro with acetylated low-density lipoproteins enhanced expression of MICA/B and scavenger receptor A, thus accounting for NKG2D ligand expression in foam cells infiltrating atherosclerotic plaques. Our results suggest that, as in mice, the NKG2D system might be involved in the development of atherosclerosis in humans.

Introduction

Infiltration of monocyte-derived macrophages (MDMs) into the arterial wall is assumed to be the initial step toward the development of atherosclerosis (Woollard and Geissmann, 2010). Infiltrating macrophages accumulates in fatty streak lesions and transforms into foam cells, which then undergo necrotic or apoptotic cell death in loco, leading to the formation of atherosclerotic plaques (Sakakura et al., 2013). The class A scavenger receptor (SR-A) is a major macrophage receptor responsible for the uptake of modified low-density lipoprotein (LDL) such as oxidized LDL and acetylated LDL (AcLDL) (Goldstein et al., 1979, Kunjathoor et al., 2002) that converts macrophages into foam cells. Thus, SR-A expression is frequently observed in atherosclerotic lesions (Naito et al., 1992). Recent work has shown that exposure to high glucose in vitro enhances SR-A expression in human MDMs, accounting for the high incidence of atherosclerosis in diabetic patients (Fukuhara-Takaki et al., 2005). SR-A expression is also up-regulated by interferon-α, providing a possible explanation for the high risk of atherosclerosis in patients with systemic lupus erythematosus (Li et al., 2011).

Activation of lymphocytes, such as CD8+ T cells, natural killer (NK) cells and natural killer T (NKT) cells, plays a crucial role in the development of atherosclerosis (Kyaw et al., 2013, Tse et al., 2013, Whitman and Ramsamy, 2006). NK cells are atherogenic, and their production of perforin and granzyme B contributes to atherosclerosis and the expansion of necrotic cores (Selathurai et al., 2014). Thioglycolate-elicited macrophages pulsed with oxidized LDL express elevated levels of CD1d and induce interferon-γ production by NKT cells (Nakai et al., 2004). CD8+ T cells also promote the development of atherosclerotic plaques by perforin- and granzyme B-mediated apoptosis of macrophages and tumor necrosis factor α-mediated inflammation (Kyaw et al., 2013).

NKG2D (natural killer group 2, member D) is a major activating receptor (Raulet, 2003) expressed constitutively on NK cells, NKT cells, CD8+ T cells and γδ T cells (Eagle and Trowsdale, 2007, Kasahara and Yoshida, 2012). In humans, six ligands for NKG2D have been identified: MHC class I-related chains A and B (MICA and B) and four ULBP molecules (Bauer et al., 1999, Groh et al., 2001). NKG2D ligands are barely detectable on the surface of healthy cells and tissues, but are frequently expressed by tumor cells or infected cells (Chitadze et al., 2013, Nausch and Cerwenka, 2008). Thus, NKG2D receptor–ligand interactions play an important role in eliminating transformed or infected cells (Jonjić et al., 2008, Nausch and Cerwenka, 2008). Recent work has shown that various cellular stresses such as reactive oxygen species and DNA damage up-regulate MICA/B expression (Peraldi et al., 2009) and that dysregulated expression of NKG2D ligands is involved in the development of inflammatory and autoimmune diseases such as rheumatoid arthritis, celiac disease and type 1 diabetes (Groh et al., 2003, Meresse et al., 2004, Ogasawara et al., 2004).

NK, NKT and CD8+ T cells implicated in the development of atherosclerosis express NKG2D; however, the involvement of the NKG2D system itself is poorly understood. Recently, the inhibition of NKG2D functions was shown to suppress inflammation by immune cells and reduce aortic plaque formation in mice (Xia et al., 2011). The same work also showed that endothelial cells and macrophages infiltrating atherosclerotic plaques express MICA/B in patients with type 2 diabetes (Lin et al., 2012, Xia et al., 2011).

In this study, we conducted immunohistochemical analysis of NKG2D ligand expression in autopsy-derived aortic specimens and confirmed that foam cells infiltrating atherosclerotic plaques can express MICA/B. We found that the incubation with AcLDL increases SR-A expression in MDMs in vitro and transforms them into MICA/B + foam cells, thus accounting for NKG2D ligand expression in foam cells infiltrating atherosclerotic plaques.

Section snippets

Antibodies (Abs) and reagents

Mouse monoclonal Abs (mAbs) for human MICA/B (6D4; eBioscience, Inc., San Diego, CA), human CD68 (KP1; Dako, Glostrup, Denmark) and human macrophage SR-A (SRA-C6; TransGenic Inc., Kumamoto, Japan) were used for immunofluorescent and immunohistochemical staining. Mouse mAbs for human CD3 (F7.2.38; Dako) and CD8 (C8/144B; Nichirei Biosciences Inc., Tokyo, Japan) were used for immunohistochemical staining. Mouse mAbs for human CD80 (2D10; BioLegend, Inc., San Diego, CA) and human CD163 (RM3/1;

MICA/B expression in foam cells infiltrating atherosclerotic plaques

Previous work showed that MICA/B is expressed in endothelial cells and foam cells in atherosclerotic lesions of type 2 diabetic patients (Xia et al., 2011). To examine whether MICA/B is expressed in atherosclerotic lesions in general, we performed immunohistochemical staining of MICA/B in 10 autopsy-derived aortic samples. Positive staining for MICA/B was observed in two samples with advanced atherosclerotic lesions accompanied by large necrotic cores (Fig. 1A). Staining was observed in foam

Discussion

It was shown previously that MICA/B is expressed in endothelial cells and foam cells in atherosclerotic lesions in patients with type 2 diabetes (Xia et al., 2011). In the present study, we observed positive staining for MICA/B only in foam cells infiltrating advanced atherosclerotic lesions (Fig. 1). Normal human MDMs treated with AcLDL in vitro transformed into foam cells and enhanced MICA/B expression (Fig. 2). These results indicate that the uptake of modified LDL induces MICA/B expression

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by Grants-in-Aid for Scientific Research Grant Number 24390091 from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT).

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