Oxidized low density lipoprotein-induced LFA-1-dependent adhesion and transendothelial migration of monocytes via the protein kinase C pathway
Introduction
Atherosclerosis is both a disease entity and the principal process contributing to the pathogenesis of myocardial and cerebral infarction, gangrene and loss of function in the extremities [1]. The marked accumulation of T cells, monocytes and macrophages found in atherosclerotic plaques has reawakened interest in inflammatory and immune components in the genesis of atherosclerosis. T cells and monocytes/macrophages are features of the progressive growth of atherosclerotic plaques, but also contribute to the development of cell-mediated responses to lipoproteins, endothelial cells and smooth muscle cells in evolving lesions, where ‘chronic inflammation patterns’ are frequent features. Monocytes are particularly predominant in atherosclerotic plaques where they differentiate into lipid-filled ‘foam’ cells in the subendothelial space [2]. Recent interest in the pathogenesis of atherosclerosis has focused on recruitment and communication of monocytes in inflamed plaques.
We and others have reported that leukocyte transmigration is initiated by cellular interaction between circulating monocytes and the endothelium, a process resulting from a coordinated sequence of interactions between adhesion molecules on both the leukocytes and endothelial cells [3], [4], [5]. Integrin lymphocyte function- associated antigen-1 (LFA-1) and its endothelial ligand, intercellular adhesion molecule-1 (ICAM-1), are known to play a central role in the leukocyte-endothelial adhesion cascade. However, tethered leukocytes must be triggered efficiently to bind to the endothelium, since LFA-1 on circulating leukocytes cannot adhere to the ligand until activated [4], [5], [6], [7]. We have reported that the chemokines macrophage inflammatory protein-1β (MIP-1β) and MIP-1α trigger integrins and induce adhesion of T cell subsets to endothelial integrin ligands [5], [8], [9], [10]. Recent reports and reviews have supported the potential importance of chemokines in inflammatory responses: various chemokines other than MIP-1β produced in large amounts in inflamed tissues could trigger integrins on leukocytes and monocytes and induce accumulation of those cells in tissues [11], [12]. We have also reported that the chemoattractant, hepatocyte growth factor (HGF), induced integrin-mediated adhesion to endothelium and subsequent migration of T cells [13], [14]. Thus, multiple chemoattractants, including chemokines, HGF and certain lipoproteins are thought to be involved in integrin-triggering and chemotaxis of immune T cells, monocytes and neutrophils [3], [6].
A high plasma concentration of low-density lipoprotein (LDL) cholesterol is one of the principal risk factors for atherosclerosis. LDL, which may be modified by oxidation, glycation, aggregation, association with proteoglycans, or incorporation into immune complexes, is known to induce injury of the endothelium and underlying smooth muscles [15], [16], [17], [18], [19]. When LDL particles become trapped in an artery, they can undergo progressive oxidation and be internalized by macrophages by means of non-specific surface scavenger receptors [15], [17], [18], [19], [20]. Oxidized LDL produced by endothelial cells or macrophages, has been identified as a potent chemoattractant for monocytes and T cells, and is involved in infiltration of these cells into the subendothelial space during atherogenesis [21], [22], [23], [24]. Oxidized LDL has been reported to augment ICAM-1 expression on endothelial cells [25]. However, it remains unclear whether oxidized LDL induces any qualitative change in cell surface molecules such as monocyte-associated LFA-1, which is essential to adhesion of circulating monocytes to endothelial cells and subsequent recruitment into atherosclerotic plaques.
In this study, we used human resting monocytes prepared by counterflow centrifugal elutriation, to show that oxidized LDL triggered monocyte integrin LFA-1 activation and induced LFA-1-dependent monocyte adhesion to endothelial cells and transmigration through the endothelium in vitro. In particular, we investigated firstly whether oxidized LDL induced the activation-associated epitope of integrin LFA-1 and cytoskeletal rearrangement of monocytes, and secondly whether blocking agents such as protein kinase C (PKC) inhibitors or α-tocopherol were involved in oxidized LDL-induced adhesion of monocytes. Based on our results, we postulate that oxidized LDL may play an important role in integrin-mediated adhesion of resting monocytes to endothelial cells by efficiently activating monocyte LFA-1 through the PKC pathway, ultimately leading to the pathological processes of inflammation and atherosclerosis.
Section snippets
Preparation of lipoproteins
LDL (density=1.019–1.063 g/ml) was isolated by ultracentrifugation from pooled normal human plasma collected in EDTA (1 mg/ml) [21]. Protein was determined by the method of Lowry et al. [26] using bovine serum albumin (BSA) as a standard. Natural 2R, 4′R, 8′R-α-tocopherol and 2R, 4R′, 8′R-β-tocopherol were kindly supplied by Eisai Co. (Tokyo, Japan). Copper-oxidized LDL was prepared by incubating LDL, at a concentration of 100 μg protein/ml, with 10 μM Cu2+ in F10 medium at 37 °C for 24 h.
Preparation of human resting monocytes
Oxidized LDL induced LFA-1-mediated adhesion of resting monocytes to endothelial cells
Leukocyte integrins cannot mediate adhesion unless activated and therefore regulation of integrin-dependent adhesion is critical to the migration of virtually all hematopoietic cells [4], [30]. We and others have reported that chemokines such as MIP-1β and MIP-1α trigger T cell integrin functions [5], [8], [10], [12]. MCP-1 is known to be both a potent chemoattractant for monocytes and a trigger for integrin-mediated adhesion of monocytes. Resting monocytes prepared by counterflow centrifugal
Discussion
Circulating monocytes must adhere to endothelial cells via integrins LFA-1 and VLA-4 and be triggered by chemoattractants to facilitate their accumulation in atherosclerotic plaques. Recent reports have indicated that oxidized LDL, present in large amounts in such plaques, plays a pivotal role in the pathogenesis of atherosclerosis [41]. In this study, we used silent monocytes isolated by counterflow centrifugal elutriation and obtained the following sequence of results. Firstly, oxidized but
Acknowledgements
We thank Dr C. G. Figdor for providing NKI-L16 mAb, Dr K. Ades for providing cell line HMEC-1 and Ms T. Adachi for excellent technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and a Grant-in-Aid from Advances in Aging and Health Research of Japan.
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