Although considerable evidence implicates the cytokine interferon (IFN)-γ in atherogenesis, the proximal inducers and the range of sources of its expression remain unknown. This study tested the hypothesis that interleukin (IL)-18 regulates IFN-γ expression during atherogenesis. Indeed, human atheroma in situ expressed IL-18 and elevated levels of its receptor subunits, IL-18Rα/β, compared with nondiseased arterial tissue. IL-18 occurred predominantly as the mature, 18-kD form and colocalized with mononuclear phagocytes (MØ), while endothelial cells (ECs), smooth muscle cells (SMCs), and MØ all expressed IL-18Rα/β. Correspondingly in vitro, only MØ expressed IL-18, while all three cell types displayed the IL-18Rα/β complex constitutively, exhibiting enhanced expression upon stimulation with LPS, IL-1β, or tumor necrosis factor (TNF)-α. IL-18 signaling evoked effectors involved in atherogenesis, e.g., cytokines (IL-6), chemokines (IL-8), intracellular adhesion molecules (ICAM)-1, and matrix metalloproteinases (MMP-1/-9/-13), demonstrating functionality of the receptor on ECs, SMCs, and MØ. Finally, IL-18, particularly in combination with IL-12, induced the expression of IFN-γ in cultured MØ and, surprisingly, in SMCs (but not in ECs). The expression of functional IL-18 and IL-18 receptor on human atheroma-associated ECs, SMCs, and MØ, and its unexpected ability to induce IFN-γ expression in SMCs, suggests a novel paracrine proinflammatory pathway operating during atherogenesis.

Objective-Metformin may benefit the macrovascular complications of diabetes independently of its conventional hypoglycemic effects. Accumulating evidence suggests that inflammatory processes participate in type 2 diabetes and its atherothrombotic manifestations. Therefore, this study examined the potential action of metformin as an inhibitor of pro-inflammatory responses in human vascular smooth muscle cells (SMCs), macrophages (Ms), and endothelial cells (ECs). Methods and Results-Metformin

Thymic antigen-presenting cells (APCs) such as dendritic cells and medullary thymic epithelial cells (mTECs) use distinct strategies of self-antigen expression and presentation to mediate central tolerance. The thymus also harbors B cells; whether they also display unique tolerogenic features and how they genealogically relate to peripheral B cells is unclear. Here, we found that Aire is expressed in thymic but not peripheral B cells. Aire expression in thymic B cells coincided with major histocompatibility class II (MHCII) and CD80 upregulation and immunoglobulin class-switching. These features were recapitulated upon immigration of naive peripheral B cells into the thymus, whereby this intrathymic licensing required CD40 signaling in the context of cognate interactions with autoreactive CD4(+) thymocytes. Moreover, a licensing-dependent neo-antigen selectively upregulated in immigrating B cells mediated negative selection through direct presentation. Thus, autoreactivity within the nascent T cell repertoire fuels a feed forward loop that endows thymic B cells with tolerogenic features.

Atherosclerosis is a chronic inflammatory disease promoted by hyperlipidemia. Several studies support FOXP3-positive regulatory T cells (Tregs

Background-Strong evidence supports a role for CD40 ligand (CD40L) as marker and mediator of inflammatory diseases such as atherosclerosis. Despite extensive characterization of CD40, the classic receptor of CD40L, its role in immune defense against inflammatory diseases remains uncertain. The present study aimed to characterize the contribution of CD40 signaling to atherogenesis. Methods and Results-Surprisingly, mice deficient in both CD40 and the low-density lipoprotein receptor did not develop smaller lesions in the aortic arch, root, and thoracoabdominal aorta compared with mice deficient only in the low-density lipoprotein receptor that consumed an atherogenic diet for 8 and 16 weeks. By flow cytometry, radioactive binding assays, and immunoprecipitation, we demonstrate that CD40L interacts with the integrin Mac-1, which results in Mac-1-dependent adhesion and migration of inflammatory cells as well as myeloperoxidase release in vitro. Furthermore, mice deficient in CD40L show significantly reduced thioglycolate-elicited invasion of inflammatory cells into the peritoneal cavity compared with mice deficient in CD40 and wild-type controls. Inhibition of Mac-1 in low-density lipoprotein receptor-deficient mice attenuates lesion development and reduces lesional macrophage accumulation. Conclusions-These observations identify the interaction of CD40L and Mac-1 as an alternative pathway for CD40L-mediated inflammation. This novel mechanism expands understanding of inflammatory signaling during atherogenesis.

CD40 ligand (CD40L), identified as a costimulatory molecule expressed on T cells, is also expressed and functional on platelets. We investigated the thrombotic and inflammatory contributions of platelet CD40L in atherosclerosis. Although CD40L-deficient (Cd40l ؊/؊ ) platelets exhibited impaired platelet aggregation and thrombus stability, the effects of platelet CD40L on inflammatory processes in atherosclerosis were more remarkable. Repeated injections of activated Cd40l ؊/؊ platelets into Apoe ؊/؊ mice strongly decreased both platelet and leukocyte adhesion to the endothelium and decreased plasma CCL2 levels compared with wildtype platelets. Moreover, Cd40l ؊/؊ platelets failed to form proinflammatory plateletleukocyte aggregates. Expression of CD40L on platelets was required for plateletinduced atherosclerosis as injection of Cd40l ؊/؊ platelets in contrast to Cd40l ؉/؉ platelets did not promote lesion formation. Remarkably, injection of Cd40l ؉/؉ , but not Cd40l ؊/؊ , platelets transiently decreased the amount of regulatory T cells

Background-Loss of interstitial collagen, particularly type I collagen, the major load-bearing molecule of atherosclerotic plaques, renders atheroma prone to rupture. Initiation of collagen breakdown requires interstitial collagenases, a matrix metalloproteinase (MMP) subfamily consisting of MMP-1, MMP-8, and MMP-13. Previous work demonstrated the overexpression of MMP-1 and MMP-13 in human atheroma. However, no study has yet evaluated the expression of MMP-8, known as "neutrophil collagenase," the enzyme that preferentially degrades type I collagen, because granulocytes do not localize in plaques. Methods and Results-Transcriptional profiling and reverse transcription-polymerase chain reaction analysis revealed inducible expression of MMP-8 transcripts in CD40 ligand-stimulated mononuclear phagocytes. Western blot analysis demonstrated that 3 atheroma-associated cell types, namely, endothelial cells, smooth muscle cells, and mononuclear phagocytes, expressed MMP-8 in vitro upon stimulation with proinflammatory cytokines such as interleukin-1␤, tumor necrosis factor-␣, or CD40 ligand. MMP-8 protein elaborated from these atheroma-associated cell types migrated as 2 immunoreactive bands, corresponding to the molecular weights of the zymogen and the active molecule. Extracts from atherosclerotic, but not nondiseased arterial tissue, contained similar immunoreactive bands. Moreover, all 3 cell types expressed MMP-8 mRNA and protein in human atheroma in situ. Notably, MMP-8 colocalized with cleaved but not intact type I collagen within the shoulder region of the plaque, a frequent site of rupture. Conclusions-These data point to MMP-8 as a previously unsuspected participant in collagen breakdown, an important determinant of the vulnerability of human atheroma.

T lymphocytes localize within lesions of two diametrically opposed expressions of atherosclerosis: stenosis-producing plaques and ectasia-producing abdominal aortic aneurysm (AAA). T H 1 immune responses appear to predominate in human stenotic lesions. However, little information exists regarding the nature of the T-cell infiltrate in AAAs. We demonstrate here that AAAs predominantly express T H 2-associated cytokines and correspondingly lack mediators associated with the T H 1 response as determined by Western blot and immunohistochemical analysis. In the United States alone abdominal aortic aneurysms (AAA) affect 3% of individuals 60 years or older, necessitate 46,000 surgical interventions, and cause ϳ15,000 deaths annually.1 The prevalence of AAA will increase as the population ages and thus will continue to entail considerable morbidity, mortality, and medical expense. Despite considerable descriptive knowledge of the pathomorphology of AAA, 2,3 insufficient understanding of the molecular mechanisms underlying its pathogenesis currently limits the prevention and treatment of this human disease.No mechanistic model yet exists that explains why atherosclerosis can have diametrically opposed expressions: aneurysm versus occlusive disease. Previous work by us and other investigators, however, revealed certain salient morphological characteristics in the pathophysiology of aortic aneurysm, including the profound inflammation characterized by abundant infiltrates of leukocytes such as T lymphocytes. 4 -7 Studies demonstrating that stiffness of the aneurysmal aortic wall does not correlate with the risk for rupture further supported the hypothesis that the inflammatory composition, rather than morphology or mechanics alone, determines the risk for acute clinical complications.8 Moreover, inflammatory infiltrates and aneurysmal enlargement correlate temporally, suggesting that the inflammatory cells may participate in the destruction of the aneurysmal aortic wall.