Lysophosphatidic acid-induced oxidized low-density lipoprotein uptake is class A scavenger receptor-dependent in macrophages

Lysophosphatidic acid (LPA) is a low-molecular-weight lysophospholipid enriched in platelets and mildly oxidized low-density lipoprotein (OxLDL). It is suggested that LPA is involved in atherosclerosis, and our previous studies showed that LPA regulates inflammation in multiple cell types. The main aim of this study was to investigate the effects of LPA on the uptake of OxLDL by mouse J774A.1 macrophages. We observed that LPA upregulated fluorescence-labeled DiI-OxLDL uptake in J774A.1 cells. Meanwhile, expression of the class A scavenger receptor (SR-A), a receptor for modified LDL, was also enhanced. Furthermore, pertussis toxin (PTx) or Ki16425 significantly abolished LPA's effects, indicating that G_i and LPA₃ are involved in OxLDL uptake and SR-A expression. Of most importance, the LPA-induced OxLDL uptake could be inhibited when cells were incubated with a functional blocking antibody of SR-A. Our results suggest that LPA-enhanced OxLDL uptake is mediated via LPA₃-G_i activation and subsequent SR-A expression.     

Disparate regulation and function of the class A scavenger receptors SR-AI/II and MARCO

The macrophage class A scavenger receptors, macrophage receptor with a collagenous structure (MARCO) and type I/II class A scavenger receptor (SR-AI/II), share structural features and roles in host defense, but little is known about their regulation and signaling properties. Ligation of MARCO on mouse thioglycollate-elicited peritoneal macrophages (PEMs) with immobilized mAb costimulated IL-12 production, in contrast to previously reported inhibition by SR-AI/II. PEMs from MARCO-deficient mice exhibited 2.7 times lower IL-12 production in responses to stimulation with LPS and IFN-gamma and lack of significant IL-12 production on stimulation with LPS alone. Conversely, SR-AI/II-deficient PEMs produced 2.4 and 1.8 times more IL-12 than wild-type PEMs in response to LPS or LPS and IFN-gamma, respectively. Corresponding differences in regulation of SR-A and MARCO expression were also observed. Th1 adjuvants (LPS, a CpG motif-containing oligodeoxynucleotide (CpG-ODN), IL-12, and GM-CSF) increased, whereas Th2-polarizing factors (IL-4, M-CSF, and non-CpG ODN) decreased expression of MARCO on J774 macrophage-like cells. Expression of SR-A was regulated in the opposite manner to MARCO or not affected. Whereas MARCO was involved in opsonin-independent phagocytosis in CpG-ODN-pretreated but not in IL-4-pretreated J774 cells, anti-SR-A Abs inhibited particle uptake in untreated and IL-4-pretreated but not in CpG-ODN-pretreated cells. SR-A and MARCO are regulated differently and mediate distinct negative and positive effects on IL-12 production in macrophages. These differences may contribute to sustained Th1 or Th2 polarization of ongoing immune responses.     

Scavenger receptors for oxidized and glycated proteins

Summary. Our present knowledge on chemically modified proteins and their receptor systems is originated from a proposal by Goldstein and Brown in 1979 for the receptor for acetylated LDL which is involved in foam cell formation, one of critical steps in atherogenesis. Subsequent extensive studies using oxidized LDL (OxLDL) as a representative ligand disclosed at least 11 different scavenger receptors which are collectively categorized as scavenger receptor family. Advanced glycation endproducts (AGE) and their receptor systems have been studied independently until recent findings that AGE-proteins are also recognized as active ligands by scavenger receptors including class A scavenger receptor (SR-A), class B scavenger receptors such as CD36 and SR-BI, type D scavenger receptor (LOX-1) and FEEL-1/FEEL-2. Three messages can be summarized from these experiments; (i) endocytic uptake of OxLDL and AGE-proteins by macrophages or macrophage-derived cells is mainly mediated by SR-A and CD36, which is an important step for foam cell formation in the early stage of atherosclerosis, (ii) selective uptake of cholesteryl esters of high density lipoprotein (HDL) mediated by SR-BI is inhibited by AGE-proteins, suggesting a potential pathological role of AGE in a HDL-mediated reverse cholesterol transport system, (iii) a novel scavenger receptor is involved in hepatic clearance of plasma OxLDL and AGE-proteins.     

Minimally modified LDL binds to CD14, induces macrophage spreading via TLR4/MD-2, and inhibits phagocytosis of apoptotic cells

Minimally modified low density lipoprotein (mmLDL) is a pro-inflammatory and pro-atherogenic lipoprotein that, unlike profoundly oxidized LDL (OxLDL), is not recognized by scavenger receptors and thus does not have enhanced uptake by macrophages. However, here we demonstrate that mmLDL (as well as OxLDL) induces actin polymerization and spreading of macrophages, which results in such pro-atherogenic consequences as inhibition of phagocytosis of apoptotic cells but enhancement of OxLDL uptake. We also demonstrate for the first time that the lipopolysaccharide receptor, CD14, and toll-like receptor-4/MD-2 are involved in these mmLDL effects. Macrophages of the J774 cell line exhibited higher mmLDL binding and F-actin response than its CD14-deficient mutant, LR-9 cells. Similarly, Chinese hamster ovary cells transfected with human CD14 specifically bound mmLDL and responded with higher F-actin compared with control cells. Macrophages from C3H/HeJ mice, which have a point mutation in the Tlr4 gene, responded with lower F-actin to mmLDL and did not spread as well as macrophages from control animals. A significantly higher F-actin response was also observed in Chinese hamster ovary cells transfected with human toll-like receptor-4/MD-2 but not with TLR4 alone or TLR2. Thus, in addition to inhibition of phagocytosis, the recognition of mmLDL by macrophage lipopolysaccharide receptors results in convergence of cellular immune responses to products of microorganisms and to oxidation-specific self-antigens, which could both influence macrophage function and atherogenesis.     

Alpha-tocopherol decreases CD36 expression in human monocyte-derived macrophages

Cholesterol-laden macrophages are the hallmark of atherogenesis. The class B scavenger receptor, CD36, binds oxidized low density lipoprotein (OxLDL), is found in atherosclerotic lesions, and is upregulated by OxLDL. We tested the effects of alpha-tocopherol (AT) enrichment of human monocyte-derived macrophages on CD36 expression and cholesteryl ester accumulation. Monocytes isolated from normal volunteers were cultured into macrophages. Macrophages were enriched overnight with various doses of AT (25, 50, and 100 microM). LDL from normal volunteers was oxidized or acetylated (AcLDL) and incubated with macrophages for 48 h at a concentration of 50 or 100 microg/ml. CD36 expression was assessed by flow cytometry. Quantitative analysis of scavenger receptor class A (SR-A) activity was performed with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide perchlorate (DiI)-labeled LDL. CD36 expression was maximal after 8;-10 days of culture. AT (> or =50 microM) significantly decreased CD36 expression upregulated by OxLDL and AcLDL (P < 0.01). Other antioxidants (beta- or gamma-tocopherol) or protein kinase C inhibitors failed to decrease CD36 expression. Concomitantly, DiI-AcLDL and DiI-OxLDL uptake was significantly decreased after AT treatment (P < 0.001). Cholesteryl ester accumulation was significantly decreased after AT enrichment (AcLDL + AT, 77% inhibition; OxLDL + AT, 42% inhibition). In conclusion, AT decreases both CD36 and SR-A expression and cholesteryl ester accumulation in human macrophages. This provides additional scientific support for the antiatherogenic properties of AT.     

Expression of caveolin‐1 in hepatic cells increases oxidized LDL uptake and preserves the expression of lipoprotein receptors

Oxidized LDL (OxLDL) that are positively associated with the risk of developing cardiovascular diseases are ligands of scavenger receptor‐class B type I (SR‐BI) and cluster of differentiation‐36 (CD36) which can be found in caveolae. The contribution of these receptors in human hepatic cell is however unknown. The HepG2 cell, a human hepatic parenchymal cell model, expresses these receptors and is characterized by a very low level of caveolin‐1. Our aim was to define the contribution of human CD36, SR‐BI, and caveolin‐1 in the metabolism of OxLDL in HepG2 cells and conversely the effects of OxLDL on the levels/localization of these receptors. By comparing mildly (M)‐ and heavily (H)‐OxLDL metabolism between control HepG2 cells and HepG2 cells overexpressing CD36, SR‐BI, or caveolin‐1, we found that (1) CD36 increases M‐ and H‐OxLDL‐protein uptake; (2) SR‐BI drives M‐OxLDL through a degradation pathway at the expense of the cholesterol ester (CE) selective uptake pathway; (3) caveolin‐1 increases M‐ and H‐OxLDL‐protein uptake and decreases CE selective uptake from M‐OxLDL. Also, incubation with M‐ or H‐OxLDL decreases the levels of SR‐BI and LDL‐receptor in control HepG2 cells which can be overcome by caveolin‐1 expression. In addition, OxLDL move CD36 from low to high buoyant density membrane fractions, as well as caveolin‐1 in cells overexpressing this protein. Thus, hepatic caveolin‐1 expression has significant effects on OxLDL metabolism and on lipoprotein receptor levels. J. Cell. Biochem. 108: 906–915, 2009. © 2009 Wiley‐Liss, Inc.     

4-hydroxynonenal contributes to macrophage foam cell formation through increased expression of class A scavenger receptor at the level of translation

4-Hydroxynonenal (HNE) is known to be atherogenic, but its mechanism of action in atherogenesis is not clear. Therefore, this study investigated the role of HNE in macrophage foam cell formation and the underlying mechanism involved in HNE-induced expression of scavenger receptors (SRs). In the aortic sinus of ApoE-deficient mice fed a high-fat diet, multiple plaque lesions were accompanied by increased accumulation of HNE adducts in the enhanced Mac-2 stained area. In an in vitro study, HNE exposure to J774A.1 macrophages led to increased expression of class A SR (SR-A) and CD36 at the protein level with a concomitant increase in endocytic uptake of oxLDL. In contrast to CD36 protein expression, which was associated with an increase in mRNA expression, the HNE-enhanced SR-A protein expression was neither accompanied by its mRNA expression nor affected by actinomycin D. HNE enhanced the incorporation rates of ³⁵S-Met/Cys into SR-A, and HNE-induced SR-A protein expression was effectively attenuated by translation inhibitors such as cycloheximide and rapamycin. Taken together, these data suggest that HNE contributes to macrophage foam cell formation through increased synthesis of SR-A at the level of mRNA translation, consequently leading to the progression of atherosclerosis.     

Oxidized low density lipoprotein decreases macrophage expression of scavenger receptor B-I

Scavenger receptor class B type I (SR-BI) has recently been identified as a high density lipoprotein (HDL) receptor that mediates bidirectional flux of cholesterol across the plasma membrane. We have previously demonstrated that oxidized low density lipoprotein (OxLDL) will increase expression of another class B scavenger receptor, CD36 (Han, J., Hajjar, D. P., Febbraio, M., and Nicholson, A. C. (1997) J. Biol. Chem. 272, 21654-21659). In studies reported herein, we evaluated the effects of OxLDL on expression of SR-BI in macrophages to determine how exposure to this modified lipoprotein could alter SR-BI expression and cellular lipid flux. OxLDL decreased SR-BI expression in a dose- and time-dependent manner. Incubation with OxLDL had no effect on the membrane distribution of SB-BI, and it decreased expression of both cytosolic and membrane protein. Consistent with its effect on SR-BI protein expression, OxLDL decreased SR-BI mRNA in a dose-dependent manner. The ability of OxLDL to decrease SR-BI expression was dependent on the degree of LDL oxidation. OxLDL decreased both [(14)C]cholesteryl oleate/HDL uptake and efflux of [(14)C]cholesterol to HDL in a time-dependent manner. Incubation of macrophages with 7-ketocholesterol, but not free cholesterol, also inhibited expression of SR-BI. Finally, we demonstrate that the effect of OxLDL on SR-BI is dependent on the differentiation state of the monocyte/macrophage. These results imply that in addition to its effect in inducing foam cell formation in macrophages through increased uptake of oxidized lipids, OxLDL may also enhance foam cell formation by altering SR-BI-mediated lipid flux across the cell membrane.     

CD36 is not involved in scavenger receptor-mediated endocytic uptake of glycolaldehyde- and methylglyoxal-modified proteins by liver endothelial cells

Circulating proteins modified by advanced glycation end-products (AGE) are mainly taken up by liver endothelial cells (LECs) via scavenger receptor-mediated endocytosis. Endocytic uptake of chemically modified proteins by macrophages and macrophage-derived cells is mediated by class A scavenger receptor (SR-A) and CD36. In a previous study using SR-A knockout mice, we demonstrated that SR-A is not involved in endocytic uptake of AGE proteins by LECs [Matsumoto et al. (2000) Biochem. J. 352, 233-240]. The present study was conducted to determine the contribution of CD36 to this process. Glycolaldehyde-modified BSA (GA-BSA) and methylglyoxal-modified BSA (MG-BSA) were used as AGE proteins. 125I-GA-BSA and 125I-MG-BSA underwent endocytic degradation by these cells at 37 degrees C, and this process was inhibited by several ligands for the scavenger receptors. However, this endocytic uptake of 125I-GA-BSA by LECs was not inhibited by a neutralizing anti-CD36 antibody. Similarly, hepatic uptake of (111)In-GA-BSA after its intravenous injection was not significantly attenuated by co-administration of the anti-CD36 antibody. These results clarify that CD36 does not play a significant role in elimination of GA-BSA and MG-BSA from the circulation, suggesting that the receptor involved in endocytic uptake of circulating AGE proteins by LEC is not SR-A or CD36.     

Impaired ATP-binding cassette transporter A1-mediated sterol efflux from oxidized LDL-loaded macrophages

We investigated the interaction of oxidized low density lipoprotein (OxLDL) with the ATP-binding cassette A1 (ABCA1) pathway in J774 macrophages. Cellular efflux to apolipoprotein AI (apo-AI) of OxLDL-derived cholesterol was lower than efflux of cholesterol derived from acetylated low density lipoprotein (AcLDL). ABCA1 upregulation by 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (cpt-cAMP) or 22 (R)-hydroxycholesterol (22-OH) and 9-cis retinoic acid (9cRA) increased the efflux to apo-AI of cellular sterols derived from AcLDL, but not of those from OxLDL. AcLDL, but not OxLDL, induced ABCA1 protein content and activity in J774. However, OxLDL did not influence J774 ABCA1 upregulation by cpt-cAMP or 22-OH/9cRA. We conclude that sterols released to cells by OxLDL are available neither as substrate nor as modulator of ABCA1.     

Specific and efficient targeting of adenovirus vectors to macrophages: application of a fusion protein between an adenovirus-binding fragment and avidin, linked to a biotinylated oligonucleotide

BACKGROUND: The application of serotype 5 adenoviruses (Ad5) in macrophages is hampered by the absence of the endogenous coxsackie adenovirus receptor (CAR). METHODS: To overcome this limitation, we first generated a linker protein consisting of the virus-binding domain of CAR and the C-terminus of avidin. Second, to target macrophages, this linker protein was equipped with the biotinylated (bio) oligonucleotide dA6G10, which was previously shown to display a high affinity for the scavenger receptor A (SR-A). RESULTS: As compared to nontargeted virus, the linker protein equipped with bio-dA6G10 showed a 500-fold increased reporter gene expression in mouse macrophage RAW264.7 cells. A linker protein equipped with a bio-dA16 control oligonucleotide was inactive. Moreover, the bio-dA6G10-equipped linker showed a 390-fold increased luciferase expression in the macrophage cell line J774 and 276- and 150-fold increased reporter gene expression in primary peritoneal and bone marrow (BM)-derived macrophages, respectively. Using BM-derived macrophages from SR-A knockout mice, it was shown that the dA6G10-mediated uptake is predominantly SR-A-mediated. CONCLUSIONS: Thus, we have developed a novel tool to link biotinylated ligands to a virus-binding fragment of CAR and have exploited this linker protein to extend the applicability of Ad5 to infect transformed and primary macrophages.     

Regulation of acetylated low density lipoprotein uptake in macrophages by pertussis toxin-sensitive G proteins

Class A scavenger receptors (SR-A) mediate the uptake of modified low density lipoprotein (LDL) by macrophages. Although not typically associated with the activation of intracellular signaling cascades, results with peritoneal macrophages indicate that the SR-A ligand acetylated LDL (AcLDL) promotes activation of cytosolic kinases and phospholipases. These signaling responses were blocked by the treatment of cells with pertussis toxin (PTX) indicating that SR-A activates G(i/o)-linked signaling pathways. The functional significance of SR-A-mediated G(i/o) activation is not clear. In this study, we investigated the potential role of G(i/o) activation in regulating SR-A-mediated lipoprotein uptake. Treatment of mouse peritoneal macrophages with PTX decreased association of fluorescently labeled AcLDL with cells. This inhibition was dependent on the catalytic activity of the toxin confirming that the decrease in AcLDL uptake involved inhibiting G(i/o) activation. In contrast to the inhibitory effect on AcLDL uptake, PTX treatment did not alter beta-VLDL-induced cholesterol esterification or deposition of cholesterol. The ability of polyinosine to completely inhibit AcLDL uptake, and the lack of PTX effect on beta-VLDL uptake, demonstrated that the inhibitory effect is specific for SR-A and not the result of non-specific effects on lipoprotein metabolism. Despite having an effect on an SR-A-mediated lipoprotein uptake, there was no change in the relative abundance of SR-A protein after PTX treatment.These results demonstrate that activation of a PTX-sensitive G protein is involved in a feedback process that positively regulates SR-A function.     

Uptake of vitamin A in macrophages from physiologic transport proteins: role of retinol-binding protein and chylomicron remnants

Vitamin A plays an important role in reducing infectious disease morbidity and mortality by enhancing immunity, an effect that is partly mediated by macrophages. Thus, knowing how these cells take up vitamin A is important. The results in the present study demonstrate that J774 macrophages efficiently take up chylomicron remnant retinyl esters and retinol-binding protein (retinol-RBP) bound retinol by specific and saturable mechanisms. The binding of (125)I-RBP to plasma membrane vesicles demonstrated that the macrophage receptor had a similar binding affinity, as was discovered previously for other cells. The B(max) for the macrophages was smaller than the values reported for placenta, bone marrow, and kidney, but larger than that reported for liver. The J774 cells also bound and took up [(3)H]retinol-RBP. Approximately 50 to 60% of the uptake may compete with excess unlabeled retinol-RBP and approximately 30 to 40% with excess transtyrethin. Following the uptake of [(3)H]retinol-RBP, an extensive esterification occurred: After 5 hours of incubation, 77.8 +/- 3.9% (SD; n = 3) of the cellular radioactivity was recovered as retinyl esters. The J774 cells also demonstrated saturable binding of chylomicron remnant [(3)H]retinyl esters, and a continuous uptake at 37 degrees C followed by an extensive hydrolysis of the retinyl esters. Binding could be inhibited by approximately 50% by excess unlabeled low density lipoprotein (LDL). In addition, lipoprotein lipase increased the binding of chylomicron remnant [(3)H]retinyl esters by approximately 30% and the uptake of chylomicron remnant [(3)H]retinyl ester by more than 300%. Furthermore, because sodium chlorate reduced binding with 40% and uptake with 55%, the results suggest that proteoglycans are involved in the uptake. Thus, the results suggest that both LDL receptor and LDL-related protein are involved in the uptake of chylomicron remnant [(3)H]retinyl ester in macrophages.     

Scavenger receptor expressed by endothelial cells I (SREC-I) mediates the uptake of acetylated low density lipoproteins by macrophages stimulated with lipopolysaccharide

Scavenger receptor expressed by endothelial cells I (SREC-I) is a novel endocytic receptor for acetylated low density lipoprotein (LDL). Here we show that SREC-I is expressed in a wide variety of tissues, including macrophages and aortas. Lipopolysaccharide (LPS) robustly stimulated the expression of SREC-I in macrophages. In an initial attempt to clarify the role of SREC-I in the uptake of modified lipoproteins as well as in the development of atherosclerosis, we generated mice with a targeted disruption of the SREC-I gene by homologous recombination in embryonic stem cells. To exclude the overwhelming effect of the type A scavenger receptor (SR-A) on the uptake of Ac-LDL, we further generated mice lacking both SR-A and SREC-I (SR-A(-/-);SREC-I(-/-)) by cross-breeding and compared the uptake and degradation of Ac-LDL in the isolated macrophages. The contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 85 and 5%, respectively, in a non-stimulated condition. LPS increased the uptake and degradation of Ac-LDL by 1.8-fold. In this condition, the contribution of SR-A and SREC-I to the overall degradation of Ac-LDL was 90 and 6%, respectively. LPS increased the absolute contribution of SR-A and SREC-I by 1.9- and 2.3-fold, respectively. On the other hand, LPS decreased the absolute contribution of other pathways by 31%. Consistently, LPS did not increase the expression of other members of the scavenger receptor family such as CD36. In conclusion, SREC-I serves as a major endocytic receptor for Ac-LDL in LPS-stimulated macrophages lacking SR-A, suggesting that it has a key role in the development of atherosclerosis in concert with SR-A.     

A macrophage Fc receptor for IgG is also a receptor for oxidized low density lipoprotein.

The internalization of oxidized low density lipoprotein (OxLDL) by macrophages is hypothesized to contribute to foam cell formation and eventually to atherosclerotic lesion formation. OxLDL is a ligand for the acetylated low density lipoprotein (AcLDL) receptor, however, our data show that this receptor accounts for less than half of OxLDL uptake by mouse macrophages, suggesting additional receptors for OxLDL. We have developed a novel expression cloning strategy in order to isolate clones encoding OxLDL receptors. In addition to the AcLDL receptor, we isolated a molecular clone for a structurally unrelated receptor capable of mediating the high affinity uptake of OxLDL following transfection into cells. This receptor has been identified as the mouse Fc gamma RII-B2, a member of a family of receptors known to mediate immune complex uptake through recognition of the Fc region of IgG. The uptake of OxLDL by cells transfected with the Fc gamma RII-B2 clone is not blocked by AcLDL but is blocked by the anti-Fc gamma RII monoclonal antibody, 2.4G2.     

Mannose-binding lectin augments the uptake of lipid A, Staphylococcus aureus, and Escherichia coli by Kupffer cells through increased cell surface expression of scavenger receptor A

We investigated roles of scavenger receptor A (SR-A) and mannose-binding lectin (MBL) in the uptake of endotoxin and bacteria by Kupffer cells. When [3H]lipid A was injected into retro-orbital plexus of mice, significantly less accumulation of lipid A in the liver was observed in SR-A-deficient mice and wild-type mice coinjected with fucoidan or acetylated low-density lipoprotein, which are known ligands for SR-A. Isolated Kupffer cells were able to take up [3H]lipid A in a time-dependent manner. The amount of lipid A associated with nonadherent Kupffer cells derived from SR-A-deficient mice was reduced by approximately 80% when compared with wild-type cells, indicating an important role of SR-A in endotoxin uptake by Kupffer cells. The lipid A uptake by Kupffer cells was significantly enhanced in the presence of rMBL. Coincubation of fucoidan with [3H]lipid A significantly inhibited the basal and the MBL-stimulated uptake of lipid A by Kupffer cells. Preincubation of MBL with Kupffer cells also increased the uptake of lipid A. These results indicate that MBL augments the SR-A-mediated uptake of lipid A by Kupffer cells. Consistently, the exposure of MBL to Kupffer cells increased cell surface SR-A expression. The phagocytosis of Staphylococcus aureus and Escherichia coli by Kupffer cells was also enhanced by preincubation of MBL with the cells. In addition, MBL bound to lipid A, LPS, and S. aureus, and precipitated S. aureus. This study demonstrates important roles of SR-A and MBL in the uptake of endotoxin and bacteria by Kupffer cells.     

Lysophosphatidic acid-induced interleukin-1β expression is mediated through G<Subscript>i</Subscript>/Rho and the generation of reactive oxygen species in macrophages

Lysophophatidic acid (LPA), a low-molecular-weight lysophospholipid enriched in platelets and mildly oxidized low-density lipoproteins, is known to regulate inflammation and atherosclerosis by binding to its cognate receptors. In this study, we reported that LPA upregulated interleukin-1β (IL-1β) expression in mouse J774A.1 macrophages. By using pharmacological inhibitors, it was suggested that G_i/Rho activation and subsequent reactive oxygen species (ROS) production were involved in IL-1β induction. In addition, IL-1β induction by LPA was also observed in human primary macrophages. In summary, LPA is involved in the processes of inflammation by affecting macrophage behavior.