Macrophages generate reactive oxygen species in response to minimally oxidized low-density lipoprotein: toll-like receptor 4- and spleen tyrosine kinase-dependent activation of NADPH oxidase 2.
Oxidative modification of low-density lipoprotein (LDL) plays a causative role in the development of atherosclerosis. In this study, we demonstrate that minimally oxidized LDL (mmLDL) stimulates intracellular reactive oxygen species (ROS) generation in macrophages through NADPH oxidase 2 (gp91phox/Nox2), which, in turn, induces production of RANTES and migration of smooth muscle cells. Peritoneal macrophages from gp91phox/Nox2(-/-) mice or J774 macrophages in which Nox2 was knocked down by small interfering RNA failed to generate ROS in response to mmLDL. Because mmLDL-induced cytoskeletal changes were dependent on Toll-like receptor (TLR)4, we analyzed ROS generation in peritoneal macrophages from wild-type, TLR4(-/-), or MyD88(-/-) mice and found that mmLDL-mediated ROS was generated in a TLR4-dependent, but MyD88-independent, manner. Furthermore, we found that ROS generation required the recruitment and activation of spleen tyrosine kinase (Syk) and that mmLDL also induced phospholipase PLCgamma1 phosphorylation and protein kinase C membrane translocation. Importantly, the phospholipase Cgamma1 phosphorylation was reduced in J774 cells expressing Syk-specific short hairpin RNA. Nox2 modulated mmLDL activation of macrophages by regulating the expression of proinflammatory cytokines interleukin-1beta, interleukin-6, and RANTES. We showed that purified RANTES was able to stimulate migration of mouse aortic smooth muscle cells and addition of neutralizing antibody against RANTES abolished the migration of mouse aortic smooth muscle cells stimulated by mmLDL-stimulated macrophages. These results suggest that mmLDL induces generation of ROS through sequential activation of TLR4, Syk, phospholipase Cgamma1, protein kinase C, and gp91phox/Nox2 and thereby stimulates expression of proinflammatory cytokines. These data help explain mechanisms by which endogenous ligands, such as mmLDL, can induce TLR4-dependent, proatherogenic activation of macrophages.
Bae
Yun Soo
YS
Department of Life Sciences, Ewha Womans University, Seoul, Korea. baeys@ewha.ac.kr
Lee
Jee Hyun
JH
Choi
Soo Ho
SH
Kim
Sunah
S
Almazan
Felicidad
F
Witztum
Joseph L
JL
Miller
Yury I
YI
D000818:Animals; D050197:Atherosclerosis / Q000201:enzymology* / Q000276:immunology; D002460:Cell Line; D002465:Cell Movement; D018946:Chemokine CCL5 / Q000378:metabolism; D004789:Enzyme Activation; D055785:Gene Knockdown Techniques; D018836:Inflammation Mediators / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000201:enzymology* / Q000276:immunology; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D000072377:Syk Kinase; D013997:Time Factors; D051197:Toll-Like Receptor 4 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*
C503460:Ccl5 protein, mouse; D018946:Chemokine CCL5; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D015850:Interleukin-6; D047908:Intracellular Signaling Peptides and Proteins; D008077:Lipoproteins, LDL; D008562:Membrane Glycoproteins; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D017382:Reactive Oxygen Species; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; C099252:oxidized low density lipoprotein; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606897:Syk protein, mouse; D011493:Protein Kinase C; D051966:Phospholipase C gamma; C117399:Plcg1 protein, mouse