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...

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Objective : We assessed the effects of an intervention aimed at increasing the consumption of fruits and vegetables on plasma folate and homocysteine concentrations. Methods: Seventy-one healthy non-smoking women (mean Ϯ SD 41 Ϯ 4 y of age) were randomized to an intervention or a control group. Participants in the intervention group (n ϭ 36) received weekly packets containing fruits and vegetables free of charge and were asked to consume a daily amount of Ն200 g of vegetables and two pieces of fruit (the Dutch recommended intake level) over a period of 1 mo. Control subjects did not receive any intervention. Results: Compared with the control group, reported fruit and vegetable intakes in the intervention group increased by 133 g/d (95% confidence interval [CI] 87-179, P Ͻ 0.001) for fruits and juice and 64 g/d (95% CI 37-91, P Ͻ 0.001) for vegetables and estimated folate intake from fruits and vegetables increased by 40 g/d (95% CI 22-58, P Ͻ 0.001). However, no effect was observed on plasma folate concentrations (intervention effect 0.3 nmol/L, 95% CI Ϫ1.8 to 2.8, P ϭ 0.77) or homocysteine concentrations (intervention effect 0.26 mol/L, 95% CI Ϫ0.34 to 0.87, P ϭ 0.39). Conclusion:The results suggest that 4 wk of increased fruit and vegetable consumption to the recommended amounts may be insufficient to change plasma folate and homocysteine concentrations.

SummaryIn humans and animal models of atherosclerosis, antibodies against oxidized LDL have been associated with atherosclerotic lesion development. It has been suggested that IgM anti-oxLDL antibodies are anti-atherogenic, whereas IgG anti-oxLDL antibodies are pro-atherogenic. In this study, we examined the relation between IgM and IgG antibody levels and atherosclerosis severity in APOE -/-CD40L -/-mice, which are deficient for IgG and develop moderate advanced atherosclerosis, and compared results with mice developing severe (APOE -/-) or no atherosclerosis (C57Bl/6). Mice were followed in time for anti-oxLDL antibodies while on high-fat diet or normal chow. Anti-oxLDL antibody levels were determined by ELISA. Results revealed that 24-week-old APOE -/-CD40L -/-mice had enhanced IgM anti-oxLDL antibody levels when compared with wild-type mice, but similar levels to those of APOE -/-mice. As expected, IgG anti-oxLDL antibody levels were almost absent in APOE -/-CD40L -/-mice. The transition from early to advanced lesions in APOE -/-mice was reflected by elevated IgM anti-oxLDL antibody levels. IgM anti-oxLDL levels did not further increase during progression to more advanced lesions. No relation was found between IgG anti-oxLDL levels and atherosclerosis severity. In conclusion, the severity of advanced atherosclerosis in mice is not reflected by IgM and/or IgG anti-oxLDL antibody levels. Furthermore, less advanced atherosclerotic lesion development in APOE -/-CD40L -/-mice does not seem to be the result of higher levels of protective IgM anti-oxLDL antibodies. Therefore, our study does not support the idea that the previously observed inconsistency in the relation between anti-oxLDL and atherosclerosis severity is due to differences in antibody isotypes.

AimsNew strategies to overcome complications of cardiovascular diseases are needed. Since it has been demonstrated that atherosclerosis is an inflammatory disease, modulation of the immune system may be a promising approach. Previously, it was suggested that antibodies may confer protective effects on the development of atherosclerosis. In this study, we hypothesised that passive immunization with anti-oxLDL IgM antibodies specific for hypochlorite (HOCl) may be athero-protective in mice.Methods and ResultsMonoclonal mouse IgM antibodies were produced and the antibody with specificity for hypochlorite-oxLDL (HOCl-oxLDL) (Moab A7S8) was selected. VH sequence determination revealed that Moab A7S8 is a natural IgM antibody. Atherosclerosis in LDLr−/− mice was induced by a perivascular collar placement around the right carotid artery in combination with feeding a high-fat diet. Subsequently, the mice were treated every six days with 500 µg Moab A7S8, non-relevant IgM or with PBS and the carotid arteries and aortic roots were studied for atherosclerosis. Passive immunization with this Moab A7S8 resulted in a significant reduced plaque volume formation in LDLr−/− mice when compared with PBS treatment (P = 0.002 and P = 0.035). Cholesterol levels decreased by 20% when mice were treated with Moab A7S8 compared to PBS. Furthermore, anti-oxLDL specific IgM and IgG antibody production increased significantly in the Moab A7S8 treated mice in comparison with PBS treated mice.ConclusionOur data show that passive immunization with a natural IgM antibody, directed to HOCl-oxLDL, can reduce atherosclerotic plaque development. We postulate that specific antibody therapy may be developed for use in human cardiovascular diseases.

Our results show that during atherogenesis there is a strong induction of IgM antibodies to HOCl-modified LDL particles. Whether these induced IgM antibodies are pro- or anti-atherogenic remains to be established.