Monocytes participate critically in atherosclerosis. There are 2 major subsets expressing different chemokine receptor patterns: CCR2 + CX3CR1 + Ly-6C hi and CCR2 -CX3CR1 ++ Ly-6C lo monocytes. Both C-C motif chemokine receptor 2 (CCR2) and C-X 3 -C motif chemokine receptor 1 (CX3CR1) are linked to progression of atherosclerotic plaques. Here, we analyzed mouse monocyte subsets in apoE-deficient mice and traced their differentiation and chemokine receptor usage as they accumulated within atherosclerotic plaques. Blood monocyte counts were elevated in apoE -/-mice and skewed toward an increased frequency of CCR2 + Ly-6C hi monocytes in apoE -/-mice fed a high-fat diet. CCR2 + Ly-6C hi monocytes efficiently accumulated in plaques, whereas CCR2 -Ly-6C lo monocytes entered less frequently but were more prone to developing into plaque cells expressing the dendritic cell-associated marker CD11c, indicating that phagocyte heterogeneity in plaques is linked to distinct types of entering monocytes. CCR2 -monocytes did not rely on CX3CR1 to enter plaques. Instead, they were partially dependent upon CCR5, which they selectively upregulated in apoE -/-mice. By comparison, CCR2 + Ly-6C hi monocytes unexpectedly required CX3CR1 in addition to CCR2 and CCR5 to accumulate within plaques. In many other inflammatory settings, these monocytes utilize CCR2, but not CX3CR1, for trafficking. Thus, antagonizing CX3CR1 may be effective therapeutically in ameliorating CCR2 + monocyte recruitment to plaques without impairing their CCR2-dependent responses to inflammation overall.

Blood of both humans and mice contains 2 main monocyte subsets. Here, we investigated the extent of their similarity using a microarray approach. Approximately 270 genes in humans and 550 genes in mice were differentially expressed between subsets by 2-fold or more. More than 130 of these gene expression differences were conserved between mouse and human monocyte subsets. We confirmed numerous of these differences at the cell surface protein level. Despite overall conservation, some molecules were conversely expressed between the 2 species' subsets, including CD36, CD9, and TREM-1. Other differences included a prominent peroxisome proliferatoractivated receptor ␥ (PPAR␥) signature in mouse monocytes, which is absent in humans, and strikingly opposed patterns of receptors involved in uptake of apoptotic cells and other phagocytic cargo between human and mouse monocyte subsets. Thus, whereas human and mouse monocyte subsets are far more broadly conserved than currently recognized, important differences between the species deserve consideration when models of human disease are studied in mice. (Blood. 2010;115:e10-e19) IntroductionSubpopulations of blood monocytes exist in humans, mice, and other species. 1-4 CD14 ϩϩ CD16 Ϫ and CD14 ϩ CD16 ϩ cell surface protein signatures 2 identify and distinguish the 2 major human monocyte subsets. In wild-type mice, monocytes can be identified as CD115 ϩ (c-fms/macrophage colony-stimulating factor [M-CSF] receptor), CD11b ϩ , F4/80 int blood cells, with monocyte subsets distinguished as Ly-6C ϩ and Ly-6C lo cells. [4][5][6][7] Ly-6C is frequently identified by flow cytometry using the Gr-1 antibody, which recognizes an epitope of both Ly-6C and Ly-6G. 8 It should be noted that monocytes express only Ly-6C. 4,9 It has been proposed that CD14 ϩϩ CD16 Ϫ human monocytes (here called CD16 Ϫ monocytes), which comprise approximately 95% of human blood monocytes, are counterparts to CD115 ϩ Ly-6C ϩ mouse monocytes (here called Ly-6C ϩ monocytes), which comprise approximately 50% of circulating mouse monocytes. 2,3,6,7,10 Moreover, CD14 ϩ CD16 ϩ human monocytes (here called CD16 ϩ monocytes) and CD115 ϩ Ly-6C lo mouse monocytes (here called Ly-6C lo monocytes) appear to bear similarity. 2,3,6,7,10 These proposed similarities arise from evidence that differential expression patterns of certain molecules between the 2 major subsets are shared in humans and mice. Namely, chemokine receptors CCR1 and CCR2 are more highly expressed on CD16 Ϫ human and Ly-6C ϩ mouse monocytes at the mRNA or protein level, 3,11,12 whereas CX 3 CR1 is elevated on CD16 ϩ human and Ly-6C lo mouse monocytes. 3 In addition, CD11a (␣ L integrin; Itgal), CD62L, and CD43 are conserved in their differential expression between monocyte subsets in humans and mice. [2][3][4]10,13 Further, CD16, the signature marker for distinguishing human monocyte subsets, was recently observed on the surface of mouse Ly-6C lo , but not Ly-6C ϩ , monocytes. 14 CD11c (␣ x integrin; Itgax) is more highly expressed on CD16 ϩ human monocytes...

Atherosclerosis involves a macrophage-rich inflammation in the aortic intima. It is increasingly recognized that this intimal inflammation is paralleled over time by a distinct inflammatory reaction in adjacent adventitia. Though cross talk between the coordinated inflammatory foci in the intima and the adventitia seems implicit, the mechanism(s) underlying their communication is unclear. Here, using detailed imaging analysis, microarray analyses, laser-capture microdissection, adoptive lymphocyte transfers, and functional blocking studies, we undertook to identify this mechanism. We show that in aged apoE−/− mice, medial smooth muscle cells (SMCs) beneath intimal plaques in abdominal aortae become activated through lymphotoxin β receptor (LTβR) to express the lymphorganogenic chemokines CXCL13 and CCL21. These signals in turn trigger the development of elaborate bona fide adventitial aortic tertiary lymphoid organs (ATLOs) containing functional conduit meshworks, germinal centers within B cell follicles, clusters of plasma cells, high endothelial venules (HEVs) in T cell areas, and a high proportion of T regulatory cells. Treatment of apoE−/− mice with LTβR-Ig to interrupt LTβR signaling in SMCs strongly reduced HEV abundance, CXCL13, and CCL21 expression, and disrupted the structure and maintenance of ATLOs. Thus, the LTβR pathway has a major role in shaping the immunological characteristics and overall integrity of the arterial wall.

Development of osteoporosis severely complicates long-term glucocorticoid (GC) therapy. Using a Cre-transgenic mouse line, we now demonstrate that GCs are unable to repress bone formation in the absence of glucocorticoid receptor (GR) expression in osteoblasts as they become refractory to hormone-induced apoptosis, inhibition of proliferation, and differentiation. In contrast, GC treatment still reduces bone formation in mice carrying a mutation that only disrupts GR dimerization, resulting in bone loss in vivo, enhanced apoptosis, and suppressed differentiation in vitro. The inhibitory GC effects on osteoblasts can be explained by a mechanism involving suppression of cytokines, such as interleukin 11, via interaction of the monomeric GR with AP-1, but not NF-kappaB. Thus, GCs inhibit cytokines independent of GR dimerization and thereby attenuate osteoblast differentiation, which accounts, in part, for bone loss during GC therapy.

Oxidation products of low-density lipoproteins have been suggested to promote inflammation during atherogenesis, and reticulocyte-type 15-lipoxygenase has been implicated to mediate this oxidation. In addition, the 5-lipoxygenase cascade leads to formation of leukotrienes, which exhibit strong proinflammatory activities in cardiovascular tissues. Here, we studied both lipoxygenase pathways in human atherosclerosis. The 5-lipoxygenase pathway was abundantly expressed in arterial walls of patients afflicted with various lesion stages of atherosclerosis of the aorta and of coronary and carotid arteries. 5-lipoxygenase localized to macrophages, dendritic cells, foam cells, mast cells, and neutrophilic granulocytes, and the number of 5-lipoxygenase expressing cells markedly increased in advanced lesions. By contrast, reticulocytetype 15-lipoxygenase was expressed at levels that were several orders of magnitude lower than 5-lipoxygenase in both normal and diseased arteries, and its expression could not be related to lesion pathology. Our data support a model of atherogenesis in which 5-lipoxygenase cascade-dependent inflammatory circuits consisting of several leukocyte lineages and arterial wall cells evolve within the blood vessel wall during critical stages of lesion development. They raise the possibility that antileukotriene drugs may be an effective treatment regimen in late-stage disease.arachidonic acid cascade ͉ coronary heart disease