After a second incubation with secondary antibodies, according to the manufacturer’s instructions, immune complexes were then detected with ECL reagent
After a second incubation with secondary antibodies, according to the manufacturer’s instructions, immune complexes were then detected with ECL reagent. Statistical analysis Results were expressed as means SEM. KLA were markedly attenuated. Moreover, KLA increased the expression of mRNA and protein of 5-LO, suggesting a pivotal role of 5-LO on these processes. In studies, KLA increased monocyte adhesion to aortic endothelium of wild-type (WT) mice, which was attenuated in WT mice treated with anti-Mac-1 antibody as well as in TLR4-deficient mice. Taken together, TLR4-mediated expression of Mac-1 in monocytes plays a pivotal role on monocyte adhesion to vascular endothelium, leading to increased foam cell formation in the development of atherosclerosis. Introduction Monocyte adhesion to the endothelium in blood vessels is usually a key early event in the development and progression of atherosclerosis , , . Monocytes, an important class of white blood cells, are known to contribute significantly to the development of atherosclerosis , SPDB . They are actively recruited to atherosclerotic lesions, and promote plaque development by sustaining a chronic inflammatory reaction . Recent evidence clearly demonstrates the role of toll-like receptors (TLRs) are known to mediate monocyte adhesion to EC via an increased expression of adhesion molecules on endothelial cells , , , however, its role around the expression of monocyte adhesion molecules is usually remain unknown. TLR4 is usually expressed in monocytes , and TLR4 SPDB signaling appears to be a critical for the activation of inflammatory reaction in the monocytes , . Recent studies have recognized specific adhesion molecules in monocytes, such as, members of the -2 integrin family, LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), CD11c/CD18, and -1 integrin, VLA-4 (CD49d/29), SPDB that interact with endothelial counter-ligands, such as, SPDB ICAM-1 or VCAM-1 , , , . Furthermore, monocytes adhesion molecules, which are required for adhesion to endothelium, play important functions in the pathogenesis and progression of atherosclerosis , . In our previous studies, we found that 5-LO is usually implicated in the development and progression of atherosclerosis , , . KLA, a glycolipid component of the gram-negative bacterial cell wall, binds to the TLR4 on the surface of a variety of cells, including monocytes , , stimulates monocytes, and affects the productions of a number of inflammatory mediators, such as, 5-LO , . Furthermore, the role played by TLR4 around the modulation of 5-LO suggests an important conversation between 5-LO-mediated inflammation and the development of atherosclerosis. In this study, we determined functional role of TLR4 in endothelial adhesion of monocytes, and recognized the involved mechanisms in studies. In studies, we investigated the potential role of TLR4 and Mac-1 on monocytes in endothelial adhesion of monocytes using wild-type control mice treated with an anti-Mac-1 antibody as well as TLR4-deficient mice. In addition, we also investigated the role of TLR4 signaling in 5-LO expression in monocytes. Methods Chemicals and antibodies KLA (Kdo-Lipid A) was purchased from Adipogen (San Diego, CA, USA). Purified anti-human TLR4 antibody, anti-human Mac-1 antibody and anti-mouse IgG isotype control antibody were purchased from eBioscience (San Diego, CA). R-phycoerythrin (PE)-conjugated mouse anti-human Mac-1 (clone ICRF44; BD) antibody and PE-conjugated mouse IgG isotype control (clone MOPC-21) antibody were obtained from BD (San Diego, CA). Rabbit anti-mouse Mac-2 antibody was purchased from Abcam (Cambridge UK). MK-886 and various transmission pathway inhibitors were purchased from EMD Serono, Inc. (Rockland, MA, USA) and Sigma-Aldrich Co. (Saint Louis, MO). Cell culture THP-1 cells (a human monocytic leukemia cell collection) were purchased from your ATCC (Manassas, VA, USA). Cells were produced in RPMI 1640 medium (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS), antibiotic-antimycotic, and L-glutamine (Life Technologies). Human Umbilical Vein Endothelial Cells (HUVEC) were obtained from Lonza Walkersville, Inc. (Walkersville, MD) and cultured in endothelial growth MGC20461 medium-2 (EGM-2 MV, Lonza). Cells were managed at 37C in a humidified 5% CO2/95% air flow atmosphere. Animals TLR4 deficient mice (TLR4(-/-)) in the C57BL/6 background were kindly provided Dr. G. Y. Koh (KAIST, Korea) and wild-type (WT) control mice (C57BL/6) were purchased from Jackson Laboratories (Harlan Nossan, Italy). All the animals procedures were confirmed in accordance with the Guideline for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85C23, revised 1996), and experimental protocols were approved by the Pusan National University or college Institutional Animal Care and Use Committee. Isolation of peripheral blood mononuclear cell (PBMC) Peripheral blood.