Objective-Atherosclerosis is a chronic inflammatory disease in which the immune system plays an important role.Neutrophils have not been thoroughly studied in the context of atherogenesis. Here, we investigated neutrophils in the development of murine atherosclerotic lesions. Methods and Results-LDLRϪ/Ϫ mice were given a high-fat diet for different time periods and subsequently atherosclerotic lesions were studied by immunohistochemistry. Staining with anti-Ly-6G monoclonal antibody, a specific marker for neutrophils, revealed a marked accumulation of neutrophils during atherosclerosis development. Neutrophils were observed in the lesion, attached to the cap, and in the arterial adventitia. In addition, at some sites, neutrophil accumulation colocalized with endothelial E-selectin expression. Immunofluorescence double staining with anti-myeloperoxidase and anti-Ly-6G antibodies demonstrated the presence of myeloperoxidase in atherosclerotic lesions and its colocalization with neutrophils. After introducing the high-fat diet, levels of circulating myeloperoxidase in plasma strongly increased, with a peak at 6 weeks and a subsequent decrease to almost normal levels after 16 weeks of diet.Conclusions-We here demonstrate for the first time the presence of neutrophils and myeloperoxidase in murine atherosclerotic lesions. As a major cell type in inflammatory responses the neutrophil may also be an important mediator in the development of atherosclerosis. Key Words:therosclerosis is a multifactorial chronic inflammatory disease with prominent involvement of the immune system. Although incompletely understood, atherogenesis is most likely the result of a complex interplay between various immune and nonimmune cell types. The most abundant immune cells in atherosclerotic lesions are monocyte-derived macrophages and T cells. 1,2 These cells are therefore considered the immunologic key players in atherogenesis and have been extensively studied. In contrast, neutrophils, the principal cellular component of the innate immune system, have received little attention, and their histological presence in atherosclerotic lesions has not been described in detail before.Neutrophils are professional phagocytes, whose main function is to sense and destroy pathogenic organisms. In addition, they are the most prominent leukocytes in acute inflammatory reactions and contribute to host tissue injury in a number of inflammatory conditions, including ischemiareperfusion injury, sepsis, and vasculitis. 3 Importantly, being the key cellular component of the acute inflammatory response, neutrophils are thought to contribute to the initiation and shaping of the immune response. 4 For example, neutrophils generate chemokines that recruit monocytes and dendritic cells and can determine whether macrophages differentiate to a predominantly pro-or antiinflammatory phenotype.Although not extensively investigated, indirect evidence exists implicating a role for neutrophils in the process of atherogenesis. First, atherosclerotic mice deficient in P-sele...

Abstract. Tissue-selective lymphocyte homing is directed in part by specialized vessels that define sites of lymphocyte exit from the blood. These vessels, the post capillary high endothelial venules (HEV), are found in organized lymphoid tissues, and at sites of chronic inflammation . Lymphocytes bearing specific receptors, called homing receptors, recognize and adhere to their putative ligands on high endothelial cells, the vascular addressins . After adhesion, lymphocytes enter organized lymphoid tissues by migrating through the endothelial cell wall . Cells and/or soluble factors arriving in lymph nodes by way of the afferent lymph supply have been implicated in the maintenance of HEV morphology and efficient lymphocyte homing . In the study reported here, we assessed the influence of afferent lymphatic vessel interruption on lymph node composition, organization of cellular elements; and on expression of vascular addressins. At 1 wk after occlusion of afferent lymphatic vessels, HEV became L PHOCYTES migrate continuously between various lymphoid and extra-lymphoid tissues of the body by way of the blood and lymph vascular systems. Lymphocyte entry into peripheral lymph nodes, mucosal lymphoid tissues, and sites of chronic inflammation is directed in part by tissue-selective interactions between blood-borne lymphocytes and specialized cells lining postcapillary high endothelial venules (HEV) . I Functional studies have revealed that lymphocyte adhesion to HEV is governed at the lymphocyte level by homing receptors (6,12,13,18), and at the endothelial cell level by vascular addressins (21,22). In the mouse system, two structurally and functionally distinct vascular addressins have been described. The peripheral lymph node addressin mediates the interaction of lymphocytes with HEV in peripheral lymphoid organs (22), and the mucosal addressin directs equivalent cellular interactions in mucosal lymphoid tissues (21). The peripheral lymph node and mucosal addressins are defined, and the adhesive interactions directed by these molecules are functionally flat walled and expression of the peripheral lymph node addressin disappeared from the luminal aspect of most vessels, while being retained on the abluminal side . In addition, an HEVspecific differentiation marker, defined by mAb MECA 325, was undetectable at 7-d postocclusion. In vivo homing studies revealed that these modified vessels support minimal lymphocyte traffic from the blood. After occlusion, we observed dramatic changes in lymphocyte populations and at 7-d postsurgery, lymph nodes were populated predominantly by cells lacking the peripheral lymph node homing receptor LECAM-1. In addition, effects on nonlymphoid cells were observed : subcapsular sinus macrophages, defined by mAb MOMA-1, disappeared ; and interdigitating dendritic cells, defined by mAb NLDC-145, were dramatically reduced. These data reveal that functioning afferent lymphatics are centrally involved in maintaining normal lymph node homeostasis.inhibited by the anti-addressin mAb and , res...

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Immune cell accumulation in adipose tissue (AT) is associated with the development of AT inflammation, resulting in metabolic dysfunction. Circulating immune cell patterns may reflect immune cell accumulation in expanding AT. However, data linking human leukocytes in blood and AT is lacking. We investigated whether blood immune cell populations are associated with their counterparts in subcutaneous (scAT) or visceral AT (vAT). Flow cytometry was performed on blood, scAT and vAT from 16 lean and 29 obese men. Circulating natural killer (NK)-cells, classical monocytes and nonclassical monocytes were higher in obese individuals. vAT, but not scAT, of obese individuals contained more inflammatory CD11c+ “M1” macrophages and NK cells compared to lean individuals. Blood classical monocytes were associated with CD11c+ macrophages in vAT but not scAT. This association was unrelated to expression of the adhesion molecules CD11b and CD11c or of the chemokine receptor CX3CR1 on these monocytes. Other AT immune cells were not associated with their respective counterparts in blood. Finally, CD11c+ macrophages and CD4+ T-cells in vAT were associated with their counterparts in scAT. In conclusion, blood classical monocytes reflect CD11c+ macrophages in vAT.

Objective-Previously, the peptide sequence cNGR has been shown to home specifically to CD13/APN (aminopeptidase N) on tumor endothelium. Here, we investigated the feasibility of selective imaging of cardiac angiogenesis using the cNGR-CD13/APN system. Methods and Results-CD13/APN induction and cNGR homing were studied in the murine myocardial infarction (MI) model. By real-time polymerase chain reaction (PCR) at 7 days after MI, CD13/APN expression was 10-to 20-fold higher in the angiogenic infarct border zone and the MI area than in non-MI areas. In vivo fluorescence microscopy confirmed specific homing of fluorophore-tagged cNGR to the border zone and MI territory at 4 and 7 days after MI with a local advantage of 2.3, but not at 1 or 14 days after MI. Tissue residence half-life was 9.1Ϯ0.3 hours, whereas the half-life in plasma was 15.4Ϯ3.4 minutes. Pulse chase experiments confirmed reversible binding of cNGR in the infarct area. Fluorescent labeled cNGR conjugates or antibodies were injected in vivo, and their distribution was studied ex vivo by 2-photon laser scanning microscopy (TPLSM). cNGR co-localized exclusively with CD13/APN and the endothelial marker CD31 on vessels. Conclusions-In cardiac angiogenesis endothelial CD13/APN is upregulated. It can be targeted specifically with cNGR conjugates. In the heart cNGR binds its endothelial target only in angiogenic areas.