Macrophages regulate lung ILC2 activation via Pla2g5-dependent mechanisms.

Group V phospholipase A2 (Pla2g5) is a lipid-generating enzyme necessary for macrophage effector functions in pulmonary inflammation. However, the lipid mediators involved and their cellular targets have not been identified. Mice lacking Pla2g5 showed markedly reduced lung ILC2 activation and eosinophilia following repetitive Alternaria Alternata inhalation. While Pla2g5-null mice had Wt levels of immediate IL-33 release after one Alternaria dose, they failed to upregulate IL-33 in macrophages following repeated Alternaria administration. Unexpectedly, while adoptive transfer of bone marrow-derived (BM)-macrophages restored ILC2 activation and eosinophilia in Alternaria-exposed Pla2g5-null mice, exogenous IL-33 did not. Conversely, transfers of Pla2g5-null BM-macrophages reduced inflammation in Alternaria-exposed Wt mice. Mass spectrometry analysis of free fatty acids (FFAs) demonstrated significantly reduced FFAs (including linoleic acid (LA) and oleic acid (OA)) in lung and BM-macrophages lacking Pla2g5. Exogenous administration of LA or LA+OA to Wt mice sharply potentiated IL-33-induced lung eosinophilia and ILC2 expansion in vitro and in vivo. In contrast, OA potentiated IL-33-induced inflammation and ILC2 expansion in Pla2g5-null mice, but LA was inactive both in vivo and in vitro. Notably, Pla2g5-null ILC2s showed significantly reduced expression of the FFA-receptor-1 compared to Wt ILC2s. Thus, macrophage-associated Pla2g5 contributes significantly to type-2 immunity through regulation of IL-33 induction and FFA-driven ILC2 activation.

Dysregulation of soluble epoxide hydrolase and lipidomic profiles in anorexia nervosa.

Individuals with anorexia nervosa (AN) restrict eating and become emaciated. They tend to have an aversion to foods rich in fat. Because epoxide hydrolase 2 (EPHX2) was identified as a novel AN susceptibility gene, and because its protein product, soluble epoxide hydrolase (sEH), converts bioactive epoxides of polyunsaturated fatty acid (PUFA) to the corresponding diols, lipidomic and metabolomic targets of EPHX2 were assessed to evaluate the biological functions of EPHX2 and their role in AN. Epoxide substrates of sEH and associated oxylipins were measured in ill AN, recovered AN and gender- and race-matched controls. PUFA and oxylipin markers were tested as potential biomarkers for AN. Oxylipin ratios were calculated as proxy markers of in vivo sEH activity. Several free- and total PUFAs were associated with AN diagnosis and with AN recovery. AN displayed elevated n-3 PUFAs and may differ from controls in PUFA elongation and desaturation processes. Cytochrome P450 pathway oxylipins from arachidonic acid, linoleic acid, alpha-linolenic acid and docosahexaenoic acid PUFAs are associated with AN diagnosis. The diol:epoxide ratios suggest the sEH activity is higher in AN compared with controls. Multivariate analysis illustrates normalization of lipidomic profiles in recovered ANs. EPHX2 influences AN risk through in vivo interaction with dietary PUFAs. PUFA composition and concentrations as well as sEH activity may contribute to the pathogenesis and prognosis of AN. Our data support the involvement of EPHX2-associated lipidomic and oxylipin dysregulations in AN, and reveal their potential as biomarkers to assess responsiveness to future intervention or treatment.

Leukocyte transglutaminase 2 expression limits atherosclerotic lesion size.

OBJECTIVE: Transglutaminase 2 (TG2), a broadly expressed regulator of protein cross-linking, wound healing, and tissue fibrosis, mediates apoptotic cell ingestion and transforming growth factor-beta release by macrophages and thereby can limit leukocyte-mediated inflammation. In atherosclerosis, oxidative stress and accumulation of unesterified cholesterol stimulate atherosclerotic lesion cell apoptosis. Cell death in advanced atherosclerotic lesions promotes lesion expansion and vulnerable plaques prone to rupture. Hence, we tested the hypothesis that leukocyte TG2 expression limits atherosclerosis. METHODS AND RESULTS: We transplanted TG2-/- or TG2+/+ bone marrow into lethally irradiated low-density lipoprotein receptor (LDLR)-/- mice and evaluated diet-induced atherosclerosis after 16 weeks. We subsequently studied cultured TG2-/- and congenic TG2+/+ mouse macrophages for selected atherogenesis regulatory functions. Atherosclerotic aortic valve lesions in LDLR-/- recipients of TG2-/- bone marrow were larger and more subintimal lesional macrophage penetration than in TG2+/+ marrow recipients. Lesion intimal TG2 expression appeared robust in TG2+/+ but not TG2-/- marrow recipients. Cultured TG2-/- macrophages demonstrated diminished phagocytosis of apoptotic leukocytes, unaltered endocytosis, and degradation of oxidized LDL but decreased retinoic acid induction of the reverse cholesterol transport and apoptotic cell uptake mediator ABCA1. CONCLUSIONS: We conclude that macrophage TG2 expression promotes both apoptotic cell clearance and ABCA1 expression in vitro and limits atherosclerotic lesion size in vivo.

Scavenger receptor class B type I as a receptor for oxidized low density lipoprotein.

Scavenger receptor class B type I (SR-BI) has been established as the primary mediator of the selective transfer of lipids from HDL to mammalian cells. In addition to its role in cholesterol metabolism, SR-BI has been shown to bind apoptotic cells and thus could in theory also function as a scavenger receptor. We now show that SR-BI binds oxidized LDL (OxLDL) with high affinity (K(d) of 4.0 +/- 0.5 microg/ml) and mediates internalization and degradation to an extent comparable to that of other scavenger receptors, when normalized to binding activity. The best competitors for OxLDL binding to SR-BI were oxidized lipoproteins, whereas native or acetylated lipoproteins only competed for a small fraction of OxLDL binding. Both the isolated lipids and the isolated protein from OxLDL bound with high affinity to SR-BI and showed partial reciprocal competition. Monoclonal antibody EO6, an antibody against oxidized phospholipids, and 1-palmitoyl-2-(5-oxovaleroyl) phosphatidylcholine (POVPC) both competed effectively with intact OxLDL and with isolated lipids from OxLDL for SR-BI binding.Together, these results demonstrate a potential function of SR-BI, in addition to its role in selective uptake of lipids, to mediate internalization of OxLDL by macrophages and suggest a central role for oxidized phospholipids in this process.

Scavenger receptors, oxidized LDL, and atherosclerosis.

Oxidized LDL (OxLDL) competes with oxidatively damaged and apoptotic cells for binding to mouse peritoneal macrophages, implying the presence of one or more common domains. However, the nature of the ligands involved has not been determined. Studies in this laboratory over the last several years provide evidence that oxidized phospholipids, present in OxLDL and also in the membrane of apoptotic cells, represent one such ligand. These oxidized phospholipids, either in the lipid phase of OxLDL or becoming attached covalently to apoprotein B during LDL oxidation, have been shown to play a major role in the binding of OxLDL to CD36 and to SR-B1 expressed in transfected cells. The lipid and protein moieties compete with each other to some extent, indicating that they are binding to at least one common site. A monoclonal antibody selected because of its reactivity with OxLDL proved to be an antibody against oxidized phospholipids (but not native phospholipids). This antibody (EO6) blocked the uptake of OxLDL by CD36 and by SR-B1 in transfected cells by as much as 80%; it also inhibited macrophage phagocytosis of apoptotic cells by about 40%. Thus, the persistence of receptors for OxLDL during evolution is probably accounted for by their role in recognition of ligands on the surfaces of oxidatively damaged or apoptotic cells. This has important implications in biology generally and specifically in atherogenesis, because apoptosis is a prominent feature of late lesions.

Mildly oxidized low density lipoprotein activates multiple apoptotic signaling pathways in human coronary cells.

Apoptosis of arterial cells induced by oxidized low density lipoproteins (OxLDL) is thought to contribute to the progression of atherosclerosis. However, most data on apoptotic effects and mechanisms of OxLDL were obtained with extensively oxidized LDL unlikely to occur in early stages of atherosclerotic lesions. We now demonstrate that mildly oxidized LDL generated by incubation with oxygen radical-producing xanthine/xanthine oxidase (X/XO) induces apoptosis in primary cultures of human coronary endothelial and SMC, as determined by TUNEL technique, DNA laddering, and FACS analysis. Apoptosis was markedly reduced when X/XO-LDL was generated in the presence of different oxygen radical scavengers. Apoptotic signals were mediated by intramembrane domains of both Fas and tumor necrosis factor (TNF) receptors I and II. Blocking of Fas ligand (FasL) reduced apoptosis by 50% and simultaneous blocking of FasL and TNF receptors by 70%. Activation of apoptotic receptors was accompanied by an increase of proapoptotic and a decrease in antiapoptotic proteins of the Bcl-2 family and resulted in marked activation of class I and II caspases. Mildly oxidized LDL also activated MAP and Jun kinases and increased p53 and other transcription factors (ATF-2, ELK-1, CREB, AP-1). Inhibitors of Map and Jun kinase significantly reduced apoptosis. Our results provide the first evidence that OxLDL-induced apoptosis involves TNF receptors and Jun activation. More important, they demonstrate that even mildly oxidized LDL formed in atherosclerotic lesions may activate a broad cascade of oxygen radical-sensitive signaling pathways affecting apoptosis and other processes influencing the evolution of plaques. Thus, we suggest that extensive oxidative modifications of LDL are not necessary to influence signal transduction and transcription in vivo.

Oxidized LDL reduces monocyte CCR2 expression through pathways involving peroxisome proliferator-activated receptor gamma.

The CCR2-mediated recruitment of monocytes into the vessel wall plays an important role in all stages of atherosclerosis. In recent studies, we have shown that lipoproteins can modulate CCR2 expression and have identified native LDL as a positive regulator. In contrast, oxidized LDL (OxLDL), which is mainly formed in the aortic intima, reduces CCR2 expression, promotes monocyte retention, and may cause pathological accumulation of monocytes in the vessel wall. We now provide evidence that OxLDL reduces monocyte CCR2 expression by activating intracellular signaling pathways that may involve peroxisome proliferator-activated receptor gamma (PPARgamma). Receptor-mediated uptake of the lipoprotein particle was required and allows for delivery of the exogenous ligand to the nuclear receptor. The suppression of CCR2 expression by OxLDL was mediated by lipid components of OxLDL, such as the oxidized linoleic acid metabolites 9-HODE and 13-HODE, known activators of PPARgamma. Modified apoB had no such effect. Consistent with a participation of the PPARgamma signaling pathway, BRL49653 reduced CCR2 expression in freshly isolated human monocytes ex vivo and in circulating mouse monocytes in vivo. These results implicate PPARgamma in the inhibition of CCR2 gene expression by oxidized lipids, which may help retain monocytes at sites of inflammation, such as the atherosclerotic lesion.

Enrichment of acyl coenzyme A:cholesterol O-acyltransferase near trans-golgi network and endocytic recycling compartment.

Acyl coenzyme A:cholesterol O-acyltransferase (ACAT) is the enzyme responsible for cholesterol esterification in macrophages leading to foam cell formation. The determination of its localization is a critical step in understanding its regulation by cholesterol. Using immunofluorescence and confocal microscopy, we previously showed that the enzyme colocalized with markers of the endoplasmic reticulum, but in addition, ACAT was found in an unidentified paranuclear site. In the present study, we further define the localization of paranuclear ACAT. First, we found that ACAT does not colocalize with sorting endosomes or late endosomes labeled with fluorescent alpha(2)-macroglobulin. The paranuclear ACAT is close to the endocytic recycling compartment labeled with fluorescent transferrin. We also show that the paranuclear structure containing ACAT is very close to TGN38, a membrane protein of the trans-Golgi network (TGN), but farther from Gos28, a marker of cis, medial, and trans Golgi. After treatment with nocodazole, the central localization of ACAT did not colocalize with markers of the TGN. These data indicate that a significant fraction of ACAT resides in membranes that may be a subcompartment of the endoplasmic reticulum in proximity to the TGN and the endocytic recycling compartment. Because the TGN and the endocytic recycling compartment are engaged in extensive membrane traffic with the plasma membrane, esterification of cholesterol in these membranes may play an important role in macrophage foam cell formation during atherogenesis.

Human scavenger receptor B1 is involved in recognition of apoptotic thymocytes by thymic nurse cells.

Recognition and uptake of apoptotic cells by neighboring phagocytes is essential for the clearance of dying cells without accompanying inflammation or tissue damage. In the thymus, many apoptotic cells are generated in the process of negative selection, and both thymic macrophages (professional phagocytes) and nursing thymic epithelial cells (nursing TEC; nonprofessional phagocytes) recognize and ingest them. However the receptors responsible for this recognition and uptake have not been identified. In the present study, we have established a human nursing TEC line and examined the expression of several genes of the scavenger receptor family considered to be potential receptors for apoptotic cells. Human scavenger receptor-B1 (hSR-B1)/CLA-1, previously shown to recognize apoptotic cells, was strongly expressed in nursing TEC, whereas there was little or no expression of the other scavenger receptors tested: scavenger receptor class A, CD36, or CD68. Suppression of hSR-B1/CLA-1 expression using antisense oligonucleotides decreased the binding of apoptotic thymocytes to nursing TEC by more than 40%. These results indicate that hSR-B1/CLA-1 may play a major role in the clearance of apoptotic cells in the thymus, mediating the recognition and ingestion of apoptotic thymocytes by nursing TEC.

The binding of oxidized low density lipoprotein to mouse CD36 is mediated in part by oxidized phospholipids that are associated with both the lipid and protein moieties of the lipoprotein.

There is growing evidence that CD36 has an important physiological function in the uptake of oxidized low density lipoprotein (OxLDL) by macrophages. However, the ligand specificity and the nature of the ligands on OxLDL that mediate the binding to CD36 remain ill defined. Results from recent studies suggested that some of the macrophage scavenger receptors involved in the uptake of OxLDL recognized both the lipid and the protein moieties of OxLDL, but there was no conclusive direct evidence for this. The present studies were undertaken to test whether a single, well characterized OxLDL receptor, CD36, could bind both the lipid and protein moieties of OxLDL. COS-7 cells transiently transfected with mouse CD36 cDNA bound intact OxLDL with high affinity. This binding was very effectively inhibited ( approximately 50%) both by the reconstituted apoB from OxLDL and by microemulsions prepared from OxLDL lipids. The specific binding of both moieties to CD36 was further confirmed by direct ligand binding analysis and by demonstrating reciprocal inhibition, i.e. apoB from OxLDL inhibited the binding of the OxLDL lipids and vice versa. Furthermore, a monoclonal mouse antibody that recognizes oxidation-specific epitopes in OxLDL inhibited the binding of intact OxLDL and also that of its purified protein and lipid moieties to CD36. This antibody recognizes the phospholipid 1-palmitoyl 2-(5'-oxovaleroyl) phosphatidylcholine. This model of an oxidized phospholipid was also an effective competitor for the CD36 binding of both the resolubilized apoB and the lipid microemulsions from OxLDL. Our results demonstrate that oxidized phospholipids in the lipid phase or covalently attached to apoB serve as ligands for recognition by CD36 and, at least in part, mediate the high affinity binding of OxLDL to macrophages.

Surface expression and rapid internalization of macrosialin (mouse CD68) on elicited mouse peritoneal macrophages.

Macrosialin, the mouse homolog of human CD68, is a heavily glycosylated transmembrane protein found almost exclusively in macrophages. Its function remains uncertain. It has a high affinity for oxidized low-density lipoprotein (LDL) in ligand blots and antibodies against the human homolog, CD68, inhibit the binding of oxidized LDL to a human monocyte-derived cell line (THP-1). However, there is still controversy as to whether macrosialin, found predominantly in late endosomes, is expressed at all on the plasma membrane. The present studies, done in thioglycollate-elicited peritoneal macrophages, confirm that macrosialin is predominantly intracellular but show clearly that 10-15% of it is expressed on the cell surface. Exchange with intracellular pools occurs at an extremely high rate. The results are compatible with a surface function, including internalization of bound ligands or adhesion to surfaces.

Ligands for peroxisome proliferator-activated receptor inhibit monocyte CCR2 expression stimulated by plasma lipoproteins.

Substantial evidence supports a causal role for monocyte chemoattractant protein-1 (MCP-1) and its receptor, CCR2, in the recruitment of monocytes from the circulation into atherosclerotic lesions. MCP-1 is produced and secreted by virtually every cellular component of the vessel wall. It generally is assumed that the magnitude of the monocyte chemotactic activity, which is initiated by the functional activation of CCR2 by MCP-1, is directly proportional to the concentration of the chemoattractant. However, we recently demonstrated that an inflammatory response of monocytes is finely regulated and also depends on the expression levels of CCR2. We identified plasma low-density lipoprotein (LDL) as a positive regulator and showed that it greatly increased monocyte CCR2 gene expression. In contrast, activation of peroxisome proliferator-activated receptor by synthetic ligands or components of oxidized LDL reduces monocyte CCR2 expression and blocks chemotaxis mediated by MCP-1. We hypothesized that the excessive monocyte accumulation in the vessel wall during atherogenesis may result in part from an enhanced chemotactic response. These findings suggest CCR2 gene expression in circulating monocytes as a potential additional target for intervention and prevention of atherosclerosis.

Role of the first extracellular loop in the functional activation of CCR2. The first extracellular loop contains distinct domains necessary for both agonist binding and transmembrane signaling.

The physiological cellular responses to monocyte chemoattractant protein-1 (MCP-1), a potent chemotactic and activating factor for mononuclear leukocytes, are mediated by specific binding to CCR2. The aim of this investigation is to identify receptor microdomains that are involved in high affinity agonist binding and receptor activation. The results from our functional studies in which we utilized neutralizing antisera against CCR2 are consistent with a multidomain binding model, previously proposed by others. The first extracellular loop was of particular interest, because in addition to a ligand-binding domain it contained also information for receptor activation, crucial for transmembrane signaling. Replacement of the first extracellular loop of CCR2 with the corresponding region of CCR1 decreased the MCP-1 binding affinity about 10-fold and prevented transmembrane signaling. A more detailed analysis by site-directed mutagenesis revealed that this receptor segment contains two distinct microdomains. The amino acid residues Asn(104) and Glu(105) are essential for high affinity agonist binding but are not involved in receptor activation. In contrast, the charged amino acid residue His(100) does not contribute to ligand binding but is vital for receptor activation and initiation of transmembrane signaling. We hypothesize that the interaction of agonist with this residue initiates the conformational switch that allows the formation of the functional CCR2-G protein complex.

Expression of the monocyte chemoattractant protein-1 receptor CCR2 is increased in hypercholesterolemia. Differential effects of plasma lipoproteins on monocyte function.

Monocytes are recruited from the circulation into the subendothelial space where they differentiate into mature macrophages and internalize modified lipoproteins to become lipid-laden foam cells. The accumulation of monocytes is mediated by the interaction of locally produced chemoattractant protein-1 (MCP-1) with its receptor CCR2. The objective of the present study is to demonstrate the differential effects of plasma lipoproteins on monocyte CCR2 expression. The CCR2 expression was increased about 2.4-fold in monocytes isolated from hypercholesterolemic patients, compared to monocytes from normal controls. There was a significant correlation between CCR2 expression and plasma low density lipoprotein (LDL). Elevated levels of high density lipoprotein (HDL) blunted and even reverted the effects of LDL on CCR2 expression, both in vivo and in vitro. The causal relationship between plasma lipoproteins and CCR2 expression was further confirmed by modulating the lipoprotein profile. Estrogen supplement therapy decreased plasma LDL cholesterol, increased plasma HDL cholesterol, and reduced CCR2 expression in hypercholesterolemic postmenopausal women, but had no effect on the plasma lipid profile or CCR2 expression in normocholesterolemic subjects. The physiological significance of altered CCR2 expression was tested by chemotaxis assay, and our results demonstrated that treatment of THP-1 monocytes with LDL induced CCR2 expression and substantially enhanced the chemotaxis elicited by MCP-1. Our findings suggest that plasma lipoproteins differentially control monocyte function and that monocytes from hypercholesterolemic subjects are hyperresponsive to chemotactic stimuli. This may increase their accumulation in the vessel wall and accelerate the pathogenic events of atherogenesis.

Chemokine receptor CCR2 expression and monocyte chemoattractant protein-1-mediated chemotaxis in human monocytes. A regulatory role for plasma LDL.

The subendothelial accumulation of macrophage-derived foam cells is one of the hallmarks of atherosclerosis. The recruitment of monocytes to the intima requires the interaction of locally produced chemokines with specific cell surface receptors, including the receptor (CCR2) for monocyte chemoattractant protein-1 (MCP-1). We have previously reported that monocyte CCR2 gene expression and function are effectively downregulated by proinflammatory cytokines. In this study we identified low density lipoprotein (LDL) as a positive regulator of CCR2 expression. Monocyte CCR2 expression was dramatically increased in hypercholesterolemic patients compared with normocholesterolemic controls. Similarly, incubation of human THP-1 monocytes with LDL induced a rapid increase in CCR2 mRNA and protein. By 24 hours the number of cell surface receptors was doubled, causing a 3-fold increase in the chemotactic response to MCP-1. The increase in CCR2 expression and chemotaxis was promoted by native LDL but not by oxidized LDL. Oxidized LDL rapidly downregulated CCR2 expression, whereas reductively methylated LDL, which does not bind to the LDL receptor, had only modest effects on CCR2 expression. A neutralizing anti-LDL receptor antibody prevented the effect of LDL, suggesting that binding and internalization of LDL were essential for CCR2 upregulation. The induction of CCR2 expression appeared to be mediated by LDL-derived cholesterol, because cells treated with free cholesterol also showed increased CCR2 expression. These data suggest that elevated plasma LDL levels in conditions such as hypercholesterolemia enhance monocyte CCR2 expression and chemotactic response and potentially contribute to increased monocyte recruitment to the vessel wall in chronic inflammation and atherogenesis.

Identification of the lectin-like receptor for oxidized low-density lipoprotein in human macrophages and its potential role as a scavenger receptor.

A new receptor for oxidized low-density lipoprotein (LDL), lectin-like oxidized LDL receptor-1 (LOX-1), has recently been cloned from bovine endothelial cells and human lung. A limited tissue-distribution study suggested that the protein was mainly produced by the vascular endothelium. In the present study we demonstrate that LOX-1 is also expressed in macrophages, where it may function as a scavenger receptor. LOX-1 was not detected in undifferentiated THP-1 cells or in freshly isolated human blood monocytes. However, mature human monocyte-derived macrophages and differentiated THP-1 cells showed high levels of LOX-1 transcripts. Consistent with these results, immunofluorescence staining and FACS analysis demonstrated that LOX-1 protein is expressed on the plasma membrane of macrophages. Western-blot analysis of membranes from macrophages (but not those from monocytes) identified a single band, with an apparent molecular mass of about 40 kDa, that displayed oxidized LDL-binding activity. These results suggest that differentiation induces the expression of LOX-1 in macrophages, where it may play a role as a scavenger receptor and/or a receptor for oxidized LDL.

Minimally oxidized low-density lipoprotein increases expression of scavenger receptor A, CD36, and macrosialin in resident mouse peritoneal macrophages.

Fully oxidized LDL (OxLDL) is believed to contribute to atherogenesis in part by virtue of uptake into macrophages via specific scavenger receptors. This phenomenon results in the formation of cholesterol-loaded foam cells, a major component of atherosclerotic lesions. The present study is directed at examining the effects of OxLDL and minimally oxidized LDL (MM-LDL) on scavenger receptor expression and activity in mouse peritoneal resident macrophages. Macrophages were preincubated with MM-LDL or OxLDL at concentrations of 25 or 50 microg/mL for 24 to 48 hours, after which their ability to bind and take up 125I-OxLDL or 125I-acetylated LDL (AcLDL) was determined. MM-LDL pretreatment induced a clear increase of cell association and degradation of 125I-OxLDL and 125I-AcLDL. Pretreatment with OxLDL also enhanced scavenger receptor activity, but to a lesser degree. Neither native LDL nor AcLDL had any effect. Scatchard analysis showed that preincubation with 50 microg/mL MM-LDL for 48 hours increased the Bmax of 125I-OxLDL and 125I-AcLDL by 139% and 154%, respectively, without significantly changing their affinity. Lipids extracted from MM-LDL also significantly induced scavenger receptor activity, but to a lesser extent than did intact MM-LDL. MM-LDL pretreatment increased both mRNA levels and protein levels of scavenger receptor A, CD36, and macrosialin. On the other hand, OxLDL pretreatment increased expression of macrosialin only. These results, showing that MM-LDL can upregulate scavenger receptor expression in mouse resident peritoneal macrophages, suggest that clearance of OxLDL by macrophages in lesions is more effective, in part because the OxLDL precursor, MM-LDL, primes the macrophage for foam cell generation.

Identification of an N-formyl peptide receptor ligand binding domain by a gain-of-function approach.

Replacement of N-formyl peptide receptor (FPR) domains with those from a homologous receptor, FPR2, resulted in chimeric receptors displaying low binding affinity to fMet-Leu-Phe (fMLF). To characterize fMLF binding domain, we adopted a "gain-of-function" approach by selective replacement of non-conserved residues in the FPR2 portion of the chimeric receptors with those from the FPR. This led to the identification of 3 clusters of residues required for high-affinity fMLF binding. Introduction of 2 positively charged amino acids, Arg84 and Lys85, dramatically improved binding affinity of one chimeric receptor (Kd from 105 nM to 1.6 nM). Similarly, restoration of either Gly89/His90 or Phe102/Thr103 improved the binding affinity of another chimeric receptor from a Kd of 275 nM to a 2.3 Kd and 3.3 nM, respectively. Increased ligand binding affinity was accompanied by a gain in calcium mobilization capability, suggesting functional coupling to G proteins. These results demonstrate the presence of structural determinants in the first extracellular loop and its adjacent transmembrane domains that are essential for high affinity fMLF binding.

Characterization of CLA-1, a human homologue of rodent scavenger receptor BI, as a receptor for high density lipoprotein and apoptotic thymocytes.

Recently, a murine scavenger receptor type B class I (SR-BI) was identified that binds high density lipoprotein (HDL) and mediates the selective uptake of cholesterol esters. The human CD36 and LIMPII analogous-1 (CLA-1) receptor shows high sequence homology with SR-BI, but their functional relationship has not been determined. Transfected cells expressing CLA-1 bound HDL with a Kd of about 35 microg/ml, similar to the Kd for HDL binding to rodent SR-BI. This binding resulted in an intracellular accumulation of HDL-derived [3H]cholesterol esters without internalization or degradation of 125I-apolipoprotein. CLA-1 was strongly expressed in the adrenal gland and was also abundant in liver and testis, suggesting that CLA-1, like SR-BI, could play a role in the metabolism of HDL. However, CLA-1 was also expressed in monocytes and, like SR-BI, recognized modified forms of low density lipoproteins as well as native LDL and anionic phospholipids. These findings suggest that CLA-1 might have additional physiological functions. We found that CLA-1 recognizes apoptotic thymocytes. Our results demonstrate that CLA-1, a close structural homologue of SR-BI, is also functionally related to SR-BI and may play an important role as a "docking receptor" for HDL in connection with selective uptake of cholesterol esters. An additional role in recognition of damaged cells is suggested by these studies.