Identification of IgG1 isotype phosphorylcholine antibodies for the treatment of inflammatory cardiovascular diseases.

BACKGROUND: Phosphorylcholine (PC) is an important pro-inflammatory damage-associated molecular pattern. Previous data have shown that natural IgM anti-PC protects against cardiovascular disease. We aimed to develop a monoclonal PC IgG antibody with anti-inflammatory and anti-atherosclerotic properties. METHODS: Using various techniques PC antibodies were validated and optimized. In vivo testing was performed in a femoral artery cuff model in ApoE3*Leiden mice. Safety studies are performed in rats and cynomolgus monkeys. RESULTS: A chimeric anti-PC (PC-mAb(T15), consisting of a human IgG1 Fc and a mouse T15/E06 Fab) was produced, and this was shown to bind specifically to epitopes in human atherosclerotic tissues. The cuff model results in rapid induction of inflammatory genes and altered expression of genes associated with ER stress and choline metabolism in the lesions. Treatment with PC-mAb(T15) reduced accelerated atherosclerosis via reduced expression of endoplasmic reticulum stress markers and CCL2 production. Recombinant anti-PC Fab fragments were identified by phage display and cloned into fully human IgG1 backbones creating a human monoclonal IgG1 anti-PC (PC-mAbs) that specifically bind PC, apoptotic cells and oxLDL. Based on preventing macrophage oxLDL uptake and CCL2 production, four monoclonal PC-mAbs were selected, which to various extent reduced vascular inflammation and lesion development. Additional optimization and validation of two PC-mAb antibodies resulted in selection of PC-mAb X19-A05, which inhibited accelerated atherosclerosis. Clinical grade production of this antibody (ATH3G10) significantly attenuated vascular inflammation and accelerated atherosclerosis and was tolerated in safety studies in rats and cynomolgus monkeys. CONCLUSIONS: Chimeric anti-PCs can prevent accelerated atherosclerosis by inhibiting vascular inflammation directly and through reduced macrophage oxLDL uptake resulting in decreased lesions. PC-mAb represents a novel strategy for cardiovascular disease prevention.

Arg123-Tyr166 domain of human ApoA-I is critical for HDL-mediated inhibition of macrophage homing and early atherosclerosis in mice.

Atherosclerosis was studied in apolipoprotein E (apoE) knockout mice expressing human apolipoprotein A-I (apoA-I) or an apoA-I/apolipoprotein A-II (apoA-II) chimera in which the Arg123-Tyr166 central domain of apoA-I was substituted with the Ser12-Ala75 segment of apoA-II. High density lipoprotein (HDL) cholesterol levels were identical in apoA-I and apoA-I/apoA-II mice, but at 4 months, plaques were 2.7-fold larger in the aortic root of the apoA-I/apoA-II mice (P<0.01). The macrophage-to-smooth muscle cell ratio of lesions was 2.1-fold higher in apo-I/apoA-II mice than in apoA-I mice (P<0.01). This was due to a 2.7-fold higher (P<0.001) in vivo macrophage homing in the aortic root of apoA-I/apoA-II mice. Plasma platelet-activating factor acetyl hydrolase activity was lower (P<0.01) in apoA-I/apoA-II mice, resulting in increased oxidative stress, as evidenced by the higher titer of antibodies against oxidized low density lipoprotein (P<0.01). Increased oxidative stress resulted in increased stimulation of ex vivo macrophage adhesion by apoA-I/apoA-II beta-very low density lipoprotein and decreased inhibition of beta-very low density lipoprotein-induced adhesion by HDL from apoA-I/apoA-II mice. The cellular cholesterol efflux capacity of HDL from apoA-I/apoA-II mice was very similar to that of apoA-I mice. Thus, the Arg123-Tyr166 central domain of apoA-I is critical for reducing oxidative stress, macrophage homing, and early atherosclerosis in apoE knockout mice independent of its role in HDL production and cholesterol efflux.

N-linked glycosylation of macrophage-derived PAF-AH is a major determinant of enzyme association with plasma HDL.

Human plasma PAF-AH (platelet-activating factor-acetylhydrolase) is a Ca(2)+-independent phospholipase A2 of hematopoietic origin associated with LDL and HDL; it degrades PAF and oxidizes phospholipids. We show that human macrophages synthesize PAF-AH as a premedial Golgi precursor containing high mannose N-linked glycans. Secreted PAF-AH possesses a molecular mass of approximately 55 kDa and contains mature N-linked glycans. Secreted PAF-AH activity (90 +/- 4% of the total) bound to a wheat germ lectin column and could be eluted with N-acetylglucosamine, whereas digestion with N-acetylneuraminidase II completely abolished enzyme absorption. Tunicamycin significantly reduced cell-associated PAF-AH activity and inhibited enzyme secretion; but it did not alter the ratio of secreted to cell-associated enzyme (1.8 at 6 h and 3.1 at 24 h), suggesting that glycosylation is not essential for PAF-AH secretion. Digestion of cell-associated PAF-AH or secreted PAF-AH with peptide N-glycosidase F affected neither catalytic activity nor its resistance to proteolysis with trypsin or proteinase K; in addition, it did not affect PAF-AH association with LDL, but significantly increased its association with HDL. We suggest that macrophage-derived PAF-AH contains heterogeneous asparagine-conjugated sugar chain(s) involving sialic acid, which hinders its association with HDL but does not influence the secretion, catalytic activity, or resistance of PAF-AH to proteases.

Oxidized low density lipoproteins downregulate LPS-induced platelet-activating factor receptor expression in human monocyte-derived macrophages: implications for LPS-induced nuclear factor-kappaB binding activity.

Monocytes/macrophages play a key role in atherogenesis due to their inflammatory properties including formation of lipid mediators such as platelet-activating-factor (PAF). We investigated the effect of oxidized low-density lipoprotein (oxLDL) on lipopolysaccharide (LPS)-induced PAF receptor (PAF-R) expression in human macrophages and the implication of the nuclear factor (NF)-kappaB in this regulation. LPS-treatment (1 microg.mL(-1)) of macrophages increased PAF binding and PAF-R mRNA expression by 56% (P < 0.05) and twofold (P < 0.01), respectively. In contrast, highly oxidized low-density lipoprotein [ox24hLDL; 100 microg.mL(-1); thiobarbituric acid reacting substances: 31 +/- 3 nmol equiv. malondialdehyde (MDA).mg protein LDL-1] diminished PAF-R expression (-69%; P < 0.05) and mRNA level (- 45%; P < 0.01). LPS pretreatment induced the activated form of p65 in the nuclear compartment of macrophages (detected by Western blotting) and NF-kappaB binding activity (by electrophoretic mobility shift assay). Treatment of macrophages with ox24hLDL suppressed the LPS-induced binding of NF-kappaB to DNA. In addition, treatment of macrophages with lysophosphatidylcholine (2 and 10 microM), a major component of oxLDL, inhibited the LPS-induced NF-kappaB binding to DNA and reduced PAF binding by 30 and 70%, respectively. In conclusion, oxLDL may downregulate PAF-R expression in human macrophages by inhibiting LPS-induced NF-kappaB binding to DNA.

Adenovirus-mediated gene transfer of human platelet-activating factor-acetylhydrolase prevents injury-induced neointima formation and reduces spontaneous atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Atherosclerosis is characterized by an early inflammatory response involving proinflammatory mediators such as platelet-activating factor (PAF)-like phospholipids, which are inactivated by PAF-acetylhydrolase (PAF-AH). The effect of adenovirus-mediated expression of PAF-AH on injury-induced neointima formation and spontaneous atherosclerosis was studied in apolipoprotein E-deficient mice. METHODS AND RESULTS: Intravenous administration of an adenovirus (5 x 10(8) plaque-forming units) directing liver-specific expression of human PAF-AH resulted in a 3.5-fold increase of plasma PAF-AH activity at day 7 (P<0.001); this was associated with a 2.4- and 2.3-fold decrease in malondialdehyde-modified LDL autoantibodies and the lysophosphatidylcholine/phosphatidylcholine ratio, respectively (P<0.001 for both). Non-HDL and HDL cholesterol levels in PAF-AH-treated mice were similar to those of control virus-treated mice. Seven days after virus injection, endothelial denudation of the common left carotid artery was induced with a guidewire. Neointima formation was assessed 18 days later. PAF-AH gene transfer reduced oxidized lipoproteins by 82% (P<0.001), macrophages by 69% (P=0.006), and smooth muscle cells by 84% (P=0.002) in the arterial wall. This resulted in a 77% reduction (P<0.001) of neointimal area. Six weeks after adenovirus-mediated gene transfer, spontaneous atherosclerotic lesions in the aortic root were analyzed. PAF-AH gene transfer reduced atherosclerotic lesions by 42% (P=0.02) in male mice, whereas a nonsignificant 14% reduction was observed in female mice. Basal and PAF-AH activity after gene transfer were higher in male mice than in female mice (P=0.01 and P=0.04, respectively). CONCLUSIONS: Gene transfer of PAF-AH inhibited injury-induced neointima formation and spontaneous atherosclerosis in apolipoprotein E-deficient mice. Our data indicate that PAF-AH, by reducing oxidized lipoprotein accumulation, is a potent protective enzyme against atherosclerosis.

Oxidized low-density lipoprotein and peroxisome-proliferator-activated receptor alpha down-regulate platelet-activating-factor receptor expression in human macrophages.

Regulation of the expression of platelet-activating factor (PAF) receptor by atherogenic lipoproteins might contribute to atherogenesis. We show that progressive oxidation of low-density lipoprotein (LDL) gradually inhibits PAF receptor expression on the macrophage cell surface. We tested the effect of oxidized LDL (oxLDL) on PAF receptor expression in human monocytes that do not contain peroxisome-proliferator-activated receptor gamma (PPARgamma), a nuclear receptor activated by oxLDL. OxLDL decreased by 50% (P < or = 0.001) and by 29% (P < or = 0.05) the binding of PAF and the expression of PAF receptor mRNA respectively. Next we demonstrated that progressive oxidation of LDLs significantly activated PPARalpha-dependent transcription in transfected mouse aortic endothelial cells. Finally we demonstrated, in mature macrophages, that fenofibrate (20 microM), a specific PPARalpha agonist, but not the specific PPARgamma agonist BRL49653 (20 nM), significantly decreased both PAF binding and PAF receptor mRNA expression, by 65% and 40% (P < or = 0.001) respectively. Additionally, another PPARalpha agonist, Wy14,643, decreased PAF receptor promoter activity by 70% (P < or = 0.05) in transfected THP-1 cells, suggesting the involvement of the proximal promoter region (-980 to -500) containing a series of four nuclear factor (NF)-kappaB motifs. Thus PPARalpha might be involved in the down-regulation of PAF receptor gene expression by oxLDLs in human monocytes/macrophages. The oxidation of one or more lipid components of LDLs might result in the formation of natural activators of PPARalpha. It is hypothesized that such activators might modulate inflammation and apoptosis upon atherogenesis by decreasing the expression of PAF receptor.

Expression of platelet-activating factor receptor in human carotid atherosclerotic plaques: relevance to progression of atherosclerosis.

BACKGROUND: Human monocyte-derived macrophages synthesize numerous proinflammatory and prothrombotic substances, including lipid mediators, such as platelet-activating factor (PAF), which may play a major role in the onset and perpetuation of atherosclerotic lesions. In addition, both monocytes and macrophages express PAF receptors (PAF-R). The expression of PAF-R is transcriptionally downregulated by oxidized LDL in in vitro primary cultures of monocyte/macrophages. In this study, we evaluated the expression of PAF-R in human carotid plaque tissue, in foam cells isolated from human carotid plaques, and in primary cultures of umbilical smooth muscle cells (SMCs). METHODS AND RESULTS: We show that PAF-R was expressed at low levels in foam cells compared with monocyte/macrophages in plaques, as assessed by immunohistochemical staining and in situ hybridization. In addition, low levels of mRNA were also detected by RT-PCR in isolated human carotid foam cells. A prominent finding of our study was the demonstration that contractile SMCs were positive for PAF-R, and its mRNA was extracted from primary cultures of umbilical SMCs. CONCLUSIONS: As macrophages loose their inflammatory phenotype on transformation into foam cells, they may equally loose their capacity of defense against aggression. We postulate that the diminished expression of PAF-R may be deleterious in the context of plaque formation and progression. The observation that arterial SMCs of contractile phenotype express PAF-R opens new avenues concerning the migration of these cells from media to intima and atherosclerotic plaque formation.

HDL-associated PAF-AH reduces endothelial adhesiveness in apoE-/- mice.

Macrophage infiltration into the subendothelial space at lesion prone sites is the primary event in atherogenesis. Inhibition of macrophage homing might therefore prevent atherosclerosis. Since HDL levels are inversely correlated with cardiovascular risk, their effect on macrophage homing was assessed in apoE-deficient (apoE-/-) mice. Overexpression of human apolipoprotein AI in apoE-/- mice increased HDL levels 3-fold and reduced macrophage accumulation in an established assay of leukocyte homing to aortic root endothelium 3.2-fold (P<0.005). This was due to reduced in vivo betaVLDL oxidation, reduced betaVLDL triggered endothelial cytosolic Ca2+ signaling through PAF-like bioactivity, lower ICAM-1 and VCAM-1 expression, and diminished ex vivo leukocyte adhesion. Adenoviral gene transfer of human PAF-acetylhydrolase (PAF-AH) in apoE-/- mice increased PAF-AH activity 1.5-fold (P<0.001), reduced betaVLDL-induced ex vivo macrophage adhesion 3.5-fold (P<0.01), and reduced in vivo macrophage homing 2.6-fold (P<0.02). These inhibitory effects were observed in the absence of increased HDL cholesterol levels. In conclusion, HDL reduces macrophage homing to endothelium by reducing oxidative stress via its associated PAF-AH activity. This protective mechanism is independent of the function of HDL as cholesterol acceptor. Modulation of lipoprotein oxidation by PAF-AH may prevent leukocyte recruitment to the vessel wall, a key feature in atherogenesis.

Effect of overexpression of human apo A-I in C57BL/6 and C57BL/6 apo E-deficient mice on 2 lipoprotein-associated enzymes, platelet-activating factor acetylhydrolase and paraoxonase. Comparison of adenovirus-mediated human apo A-I gene transfer and human apo A-I transgenesis.

Various mechanisms may contribute to the antiatherogenic potential of apolipoprotein A-I (apo A-I) and high density lipoproteins (HDLs). Therefore, the effect of adenovirus-mediated human apo A-I gene transfer or human apo A-I transgenesis on platelet-activating factor acetylhydrolase (PAF-AH) and arylesterase/paraoxonase (PON1) was studied in C57BL/6 and C57BL/6 apo E(-/-) mice. Human apo A-I transgenesis in C57BL/6 mice resulted in a 4.2-fold (P<0.0001) increase of PAF-AH and a 1.7-fold (P=0.0012) increase of PON1 activity. The apo E deficiency was associated with a 1.6-fold (P=0.008) lower PAF-AH and a 2.0-fold (P=0.012) lower PON1 activity. Human apo A-I transgenesis in C57BL/6 apo E(-/-)mice increased PAF-AH and PON1 activity by 2.1-fold (P=0.01) and 2.5-fold (P=0.029), respectively. After adenovirus-mediated gene transfer of human apo A-I into C57BL/6 apo E(-/-)mice, a strong correlation between human apo A-I plasma levels and PAF-AH activity was observed at day 6 (r=0.92, P<0.0001). However, PON1 activity failed to increase, probably as a result of cytokine-mediated inhibition of PON 1 expression. In conclusion, this study indicates that overexpression of human apo A-I increases HDL-associated PAF-AH activity. PON1 activity was also increased in human apo A-I transgenic mice, but not after human apo A-I gene transfer, a result that was probably related to cytokine production induced in the liver by the adenoviral vectors. Increased levels of these HDL-associated enzymes may contribute to the anti-inflammatory and antioxidative potential of HDL and thereby to the protection conferred by HDL against atherothrombosis.

Mildly oxidized LDL induces expression of group IIa secretory phospholipase A(2) in human monocyte-derived macrophages.

Phospholipase A(2)s (PLA(2)s) constitute a family of enzymes that hydrolyze fatty acids of membrane phospholipids, thus initiating the synthesis of proinflammatory mediators. Various PLA(2)s have been detected in human atherosclerotic arteries (advanced lesions); however, only the secretory group of PLA(2) has been shown to specifically hydrolyze low density lipoprotein (LDL)-associated phospholipids and, as such, may play a potential role in atherogenesis. In the present study, we investigated the expression pattern of group IIa, IV, and V PLA(2)s in human macrophages, which are the key cells involved in the onset and perpetuation of atherosclerosis. Immunohistochemical staining by double labeling showed that the secretory nonpancreatic PLA(2) (snpPLA(2)) is detectable in macrophages in the intima of early atherosclerotic lesions. Reverse transcription-polymerase chain reaction analysis of RNA extracted from human monocytes clearly showed that expression of group IV PLA(2) was enhanced during differentiation into macrophages, with an onset of induction at days 2 to 3 of differentiation. Group V snpPLA(2) was constitutively expressed on differentiation, whereas the detection of group IIa snpPLA(2) was dependent on both differentiation and subsequent stimulation of macrophages. Indeed, the transcription of group IIa snpPLA(2) in macrophages was induced by treatment with minimally modified or mildly oxidized LDL, whereas native, extensively oxidized, or acetylated LDL had no effect. To our knowledge, this is the first report describing induction of group IIa snpPLA(2) expression in human monocyte-derived macrophages. The mRNA levels of cytosolic PLA(2) group IV and snpPLA(2) group V remained unchanged on LDL treatment. Thus, our results show that the expression of distinct PLA(2) enzymes is regulated not only during differentiation of monocytes into macrophages but also on exposure of macrophages to distinct LDL species. Consequently, our results indicate a potential role for both cytosolic and secretory PLA(2) enzymes in inflammation and in macrophage functions related to atherosclerosis, with a specific role for group IIa snpPLA2 in LDL scavenging.

Interleukin-8 mediates downregulation of tissue inhibitor of metalloproteinase-1 expression in cholesterol-loaded human macrophages: relevance to stability of atherosclerotic plaque.

BACKGROUND: The accumulation of macrophage-derived foam cells in atherosclerotic lesions correlates with increased local release of matrix-degrading metalloproteinases (MMPs) and a thin fibrous cap. The activity of these enzymes is controlled by specific tissue inhibitors of metalloproteinases (TIMPs). METHODS AND RESULTS: Because oxidized low-density lipoprotein (OxLDL) modulates gene expression, we investigated the effect of these particles on the levels of MMP-1, MMP-3, MMP-9, TIMP-1, and TIMP-2 in the culture media of human monocyte-derived macrophages. OxLDL but not native LDL or high-density lipoprotein reduced the level of TIMP-1 in a dose-dependent manner with maximal effect (60% of control) at approximately 100 microg protein/mL. In addition, Northern blotting revealed marked reduction in the abundance of TIMP-1 mRNA in OxLDL-treated cells. Evaluation of the effect of oxysterol components of OxLDL on TIMP-1 production revealed that 25-hydroxycholesterol (1 microg/mL) was the most potent inhibitor ( approximately 30% of control). Such inhibition was partially mediated by interleukin (IL)-8. Indeed, IL-8 (2.5 ng/mL) induced maximal inhibition of TIMP-1 accumulation (30% of control) in 4 of 6 cell preparations. In addition, the inhibitory effect of OxLDL-treated cells in the presence of an anti-IL-8 neutralizing antibody was partially reversed. CONCLUSIONS: Immunohistochemical analyses of human atherosclerotic plaques revealed the expression of TIMP-1 in some but not all macrophage-rich and IL-8-rich areas. Therefore, IL-8 may play a potential atherogenic role by inhibiting local TIMP-1 expression, thereby leading to an imbalance between MMPs and TIMPs at focal sites in the atherosclerotic plaque.

Inhibition of LPL expression in human monocyte-derived macrophages is dependent on LDL oxidation state: a key role for lysophosphatidylcholine.

The regulation of macrophage lipoprotein lipase (LPL) secretion and mRNA expression by atherogenic lipoproteins is of critical relevance to foam cell formation. LPL is present in arterial lesions and constitutes a bridging ligand between lipoproteins, proteoglycans, and cell receptors, thus favoring macrophage lipoprotein uptake and lipid accumulation. We investigated the effects of native and of oxidized lipoproteins on the expression of LPL in an in vitro human monocyte-macrophage system. Exposure of mature macrophages (day 12) to highly copper-oxidized human low density lipoprotein (LDL) (100 microg protein per milliliter) led to marked reduction in the expression of LPL activity (-62%, P<0.01) and mRNA level (-47%, P<0.05); native LDL, acetylated LDL, and LDL oxidized for <6 hours were without effect. The reduction in LPL activity became significant at a threshold of 6 hours of LDL oxidation (-31%, P<0.05). Among the biologically active sterols formed during LDL oxidation, only 7beta-hydroxycholesterol (5 microg/mL) induced a minor reduction in macrophage LPL activity, whereas 25-hydroxycholesterol was without effect. By contrast, lysophosphatidylcholine, whose LDL content increased in parallel with the degree of oxidation, induced significant reductions in LPL activity and mRNA levels at concentrations of 2 to 20 micromol/L (-34% to -53%, P<0.01). Our results demonstrate that highly oxidized LDL (>6-hour oxidation) exerts negative feedback on LPL secretion in human monocytes-macrophages via a reduction in mRNA levels. By contrast, native LDL and mildly oxidized LDL (<6-hour oxidation) did not exert a feedback effect on LPL expression. We speculate that the content of lysophosphatidylcholine and, to a lesser degree, of 7beta-hydroxycholesterol in oxidized LDLs is responsible for the downregulation of LPL activity and mRNA abundance in human monocyte-derived macrophages and may therefore modulate LPL-mediated pathways of lipoprotein uptake during conversion of macrophages to foam cells.

Expression of the PAF receptor in human monocyte-derived macrophages is downregulated by oxidized LDL: relevance to the inflammatory phase of atherogenesis.

Human monocyte-derived macrophages play a major role in the initiation and progression of atherosclerotic lesions as a result of the production of a wide spectrum of proinflammatory and prothrombotic factors. Among such factors is a potent inflammatory phospholipid, platelet-activating factor (PAF), which is produced after macrophage activation. Because the cells involved in PAF biosynthesis are typically targets for the bioactions of PAF via specific cell surface receptors, we evaluated the expression of the PAF receptor in human monocyte-derived macrophages. Oxidized LDL (oxLDL) exerts multiple cellular effects that enhance lesion progression; we therefore investigated the potential modulation of expression of the macrophage PAF receptor by oxLDL. [3H]PAF bound to adherent human macrophages with a K(d) of 2.1 nmol/L and a B(max) of 19 fmol/10(6) cells; approximately 5300 binding sites per cell were detected. OxLDL (100 microg protein per milliliter) induced a twofold decrease in cellular PAF binding after 3 hours at 37 degrees C. Analysis of macrophage mRNA by reverse transcription-polymerase chain reaction (RT-PCR) revealed two forms corresponding to the PAF receptor, of which the leukocyte type (type 1 promoter) predominated. Expression of PAF receptor mRNA, evaluated by quantitative RT-PCR using an actin or a GAPDH mimic, was progressively reduced (up to 70%) by oxLDL up to 6 hours and remained low for at least 24 hours. Such downregulation was reversible after incubation of the cells for 24 hours in oxLDL-free medium. Addition of forskolin (3 micromol/L) or dibutyryl cAMP (1 mmol/L) to macrophage cultures reproduced the oxLDL-mediated inhibition of PAF receptor expression; carbamyl PAF reduced PAF binding and PAF mRNA to a similar degree (approximately 50%). These data demonstrate that atherogenic oxLDL downregulates the expression of both cellular PAF receptors and PAF receptor mRNA in macrophages, consistent with both a diminished bioresponse to PAF and decreased cell motility. Such diminished bioresponse to a powerful antacoid reflects the suppression of an acute inflammatory reaction, thereby leading to chronic, low-level inflammation, such as that characteristic of fatty streaks and more advanced atherosclerotic plaques.

Modulation of platelet-activating-factor production by incorporation of naturally occurring 1-O-alkylglycerols in phospholipids of human leukemic monocyte-like THP-1 cells.

1-O-Alkylglycerols (alkyl-Gro), naturally occurring compounds abundant in shark liver oil, protect patients from radiotherapy side-effects. However, the protection mechanism is not well understood. It might be mediated by alkyl-Gro incorporation into pools of platelet-activating factor (PAF) precursor and subsequent modification of PAF biosynthesis. Using a 3H-labelled or unlabelled natural alkyl-Gro mixture, in which prominent alkyl chains were C18:1(9) (54-65%), C16:1(7) (5-15.5%), and C16:0 (5-10%), we investigated the incorporation of alkyl-Gro into phospholipids of human leukemic monocyte-like THP-1 cells. Incubation of cells for 24 h with [3H]alkyl-Gro (10 microM) resulted in their incorporation into 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (1097+/-25.1 pmol/2x10(6) cells) and into 1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine (640.4+/-12.5 pmol/2x10(6) cells) with a total yield of 6.5%. Such incorporation induced production of 1-O-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine ([3H]PAF), which was increased after stimulation by the calcium ionophore A23187. HPLC analysis of the [3H]PAF molecular species indicated that the three major [3H]alkyl-Gro were used for [3H]PAF synthesis in ratios similar to that of the mixture. Total production of biologically active PAF, as measured by the platelet-aggregation bioassay, was also increased by alkyl-Gro incorporation in resting (+20%) and in A23187-stimulated (+59%) THP-1 cells. HPLC analysis of the [3H]PAF produced in the presence of [3H]acetate, confirmed that levels of PAF, but not of its 1-acyl analog, were increased by alkyl-Gro incorporation in resting and stimulated cells. However, the rise in [3H]acetyl-PAF, which resulted mainly from C16:0 PAF, was reduced by about 50% in the presence of the PAF-receptor antagonist SR 27417, providing evidence that stimulation of total PAF synthesis was caused by the increase in the precursor pool and autocrine amplification of PAF-induced PAF production. Thus, the supplementation of THP-1 cells in culture with naturally occurring alkyl-Gro led to the incorporation of alkyl-Gro into ether-containing phospholipids, which were subsequently used for PAF synthesis. Furthermore, alkyl-Gro incorporation resulted in a significant rise in PAF production by THP-1 cells under resting and stimulated conditions. These results may be of importance for modulating PAF production in several pathophysiological conditions, such as peroxysome deficiencies, that are associated with a lack of ether lipid synthesis.

Identification on human CD36 of a domain (155-183) implicated in binding oxidized low-density lipoproteins (Ox-LDL).

Uptake of oxidized LDL (oxLDL) by macrophages is one of the key events implicated in the initiation and perpetuation of atherosclerotic lesions. One of the major scavenging receptors, which binds modified LDL, on macrophages is CD36. The domain on CD36 implicated in the binding of oxLDL remains to be elucidated. In this study, COS cells transfected with human CD36 cDNA bound FITC-oxidized human LDL in a dose-dependent, saturable manner. This binding was inhibited by an excess of oxLDL but not by native LDL. Anti-CD36 monoclonal antibodies (mAbs) 10/5, FA6-152, and 8A6 (directed against domain 155-183), but not mAb 13/10 (directed against domain 30-76), completely inhibited oxLDL binding to human CD36-transfected COS cells. Cells transfected with a chimeric human CD36 construct (hmh 155-183), resulting from the swapping of human domain 155-183 with its murine counterpart, resulted in low binding of oxLDL. In contrast, cells transfected with a chimeric murine CD36 construct (mhm 155-183), resulting from the swapping of murine domain 155-183 with its human counterpart, resulted in high binding of oxidized human LDL. Binding of oxLDL to cells transfected by chimeric construct mhm 155-183 were only partially blocked by mAbs 10/5, FA6-152, and 8A6. In the present study we have identified, for the first time, an important functional domain (encompassing amino acids 155-183) on CD36 involved in the binding of oxLDL. In addition, the binding site for oxidized human LDL on murine CD36 seems to differ from its human counterpart.

Phagocytic activation induces formation of platelet-activating factor in human monocyte-derived macrophages and in macrophage-derived foam cells. Relevance to the inflammatory reaction in atherogenesis.

Monocyte-derived macrophages and macrophage-derived foam cells in arterial tissue may undergo phagocytic activation and thereby contribute to an inflammatory reaction. We have investigated the effect of phagocytic activation on the formation of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF-acether, PAF), a proinflammatory phospholipid, in human monocyte-derived macrophages (macrophages) and in cholesterol-loaded macrophage foam cells (foam cells). Adherent human monocyte-derived macrophages were transformed into foam cells upon incubation with acetylated low-density lipoproteins (Ac-LDL). Such foam cells characteristically displayed a markedly increased content of cholesteryl esters compared with macrophages (4.3 +/- 1.3 microgram/microgram DNA and 0.2 +/- 0.3 microgram/microgram DNA, n = 5, respectively). After phagocytic stimulation with serum-opsonized zymosan (OPZ), both macrophages and foam cells synthesized PAF transiently with maximal production (0.5-1.1 pmol PAF/microgram DNA, n = 5, corresponding to 4.0-8.8 pmol PAF/10(6) cells, as assessed by bioassay) occurring approximately 15 min after stimulation. A major fraction of the synthesized PAF remained cell-associated; such PAF was composed mainly of the hexadecyl (16:0 PAF, approximately 75%) and the octadecenyl (18:1 PAF) species and of trace amounts of octadecyl (18:0 PAF), as assessed by reverse-phase liquid chromatography. Addition of exogenous 16:0 lyso-PAF alone triggered PAF formation (0.9-1.7 pmol PAF/microgram DNA, after 15 min of cellular stimulation); simultaneous cellular stimulation with OPZ and 16:0 lyso-PAF increased PAF formation in an additive manner. Acetyltransferase, the enzyme which acetylates the precursor lyso-PAF and transforms it into PAF, displayed elevated activity both in macrophages and in foam cells, attaining 83-240 pmol PAF formed per min per mg DNA (n = 4); such elevated activity was not increased by OPZ-stimulation. The activity of acetylhydrolase, the PAF-degrading enzyme, was similar in macrophages and in foam cells, and varied between 120 pmol and 320 pmol PAF degraded per min per mg DNA (n = 5). Cell-associated acetylhydrolase activity was increased significantly by 40+/-15 % (P < 0.003, n = 5) after 15 - 30 min of activation with OPZ compared with non-stimulated cells and may account for the rapid decrease in cellular PAF content observed approximately 30 min after stimulation. These studies have established that metabolism of PAF in foam cells closely resembles that in macrophages, and thus PAF metabolism is largely independent of cellular cholesterol content. Moreover our data are consistent with the hypothesis that both macrophages and macrophage-derived foam cells upon phagocytic-activation constitute a significant transient source of PAF at inflammatory sites in the arterial intima where this phospholipidic mediator may exert potent proatherogenic and prothrombotic effects.

Effects of organometals on cellular signaling. II. Inhibition of reincorporation of free arachidonic acid and influence on paf-acether synthesis by triethyllead.

Organometal compounds affect many enzymes, especially those containing SH-groups as acyl- and acetyltransferases involved in lysophospholipid reacylation. In HL-60 cells, organotin and -lead compounds stimulate phospholipase A2 activity, contributing thus to increase the level of lysophospholipids. In the present study, we have tested whether paf-acether (paf) biosynthesis was affected by treatment with triethyllead (Et3PbCl) in HL-60 cells. Et3PbCl inhibits the incorporation of exogenous arachidonic acid in the presence of high (> or = 50 microM) but not low concentrations (< or = 1 microM). High concentrations of the lead compound are unable to induce paf formation by itself, however, lower concentrations (< or = 10 microM) acted synergistically with TPA or fMLP to stimulate paf formation. Whereas unstimulated cells produced 0.4 pmole paf/2 x 10(6) cells, the stimulation with low fMLP (0.1 microM) resulted in the synthesis of 1.7 pmole and with low TPA (2 ng/ml) in 0.5 pmole paf. Preincubation of the cells with 10 microM Et3PbCl for 20 to 30 min increased the amount of paf formed by these cells to 3.3 pmole after treatment with 0.1 microM fMLP and 1.5 pmole after TPA. Furthermore, the results showed an inhibition of acetyltransferase (the key enzyme of paf synthesis) by the high and not by low concentrations of the lead compound. We conclude that low concentrations of Et3PbCl (< or = 10 microM) may act as a synergistic inducer of paf synthesis initiated via a receptor-coupled stimulation.

Biosynthesis of paf-acether in cultured-mouse mast cells: the role of calcium and G proteins.

We examined the potential role of a guanine nucleotide-binding protein in the biosynthesis of paf-acether (paf) and the release of beta-hexosaminidase during antigenic stimulation of cultured mouse bone marrow-derived mast cells. Unlike pertussis toxin, cholera toxin treatment enhanced the antigen-stimulated production of paf and calcium mobilisation without affecting acetyltransferase activation and cell degranulation. The level of intracellular cAMP doubled in cholera toxin-treated cells. Our data suggest that a cholera toxin-sensitive guanine nucleotide-binding protein is involved in the IgE receptor-mediated signal transduction leading to paf production most probably at the level of Ca2+ influx.

Heterogeneity of arachidonate and paf-acether precursor pools in mast cells.

In mammalian cells, arachidonate release and paf-acether formation are frequently associated. The alkyl-acyl-GPC has been proposed as an important source for released arachidonic acid and arachidonate-containing alkylacyl-GPC species as unique precursor for paf-acether. However, the specificity of precursor pools either concerning arachidonic acid or paf-acether is still a matter of controversy. We studied the relationship between the precursor pools for both autacoids in antigenically-stimulated cultured mast cells. We took advantage of the particular arachidonate turnover rate in each phospholipid to investigate the role of alkyl-arachidonyl-GPC in the supply of arachidonic acid by using newly and previously [14C]arachidonate-labeled cells. The specific activity of the released arachidonate was reduced 2-fold following overnight cell incubation, whereas labeling in alkyl-arachidonoyl-GPC was only slightly modified and never corresponded to that of released arachidonate when newly or previously labeled cells were triggered with the antigen. These results are not in favor of a major role for alkyl-arachidonoyl-GPC in supplying arachidonate. In contrast, by using previously labeled cells, we demonstrated that all arachidonate-containing phospholipids were involved in the release of arachidonic acid. The pattern of alkyl chains in alkyl-arachidonoyl-GPC, as well as in total alkylacyl-GPC, is unique since it consists mainly of 18:1 (more than 55%), whereas the 16:0 represents only about 30% of total alkyl chains. Therefore, we analyzed paf-acether molecular composition in order to compare it to the alkyl composition of the precursor pools. The content in 18:1 species of paf-acether, as measured by bioassay (aggregation of rabbit platelets), was always lower than that of 16:0 species and then did not correspond to the alkyl composition of the precursor. These data suggest that the enzymes involved in paf synthesis might be specific for 16:0 alkyl chains of precursor pool.

Biosynthesis of paf-acether. Activators of protein kinase C stimulate cultured mast cell acetyltransferase without stimulating paf-acether synthesis.

Antigen stimulation of cultured bone-marrow-derived mast cells sensitized with specific monoclonal IgE induced cell degranulation and paf-acether (paf; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) biosynthesis via the deacylation/acetylation (remodelling) pathway. Phorbol myristate acetate (PMA; 20-100 ng/ml) triggered only acetyltransferase activation, without concomitant lyso-paf (1-O-alkyl-sn-glycero-3-phosphocholine) and paf formation. A low concentration of PMA (5 ng/ml) potentiated antigen-induced degranulation, acetyltransferase activation and paf formation by about 30% but did not change the level of lyso-paf formation. Stimulation of mast cells with antigen increased intracellular Ca2+ from 61 to 269 nM, whereas no modification of Ca2+ influx was observed when cells were pretreated with PMA (5 ng/ml) before antigen challenge. Gas chromatography coupled to electron capture detection revealed that the composition of paf formed by cells stimulated by antigen alone was similar to that of paf formed by PMA-primed antigen-stimulated cells; 84 +/- 8% and 79 +/- 2% (means +/- S.E.M., n = 3) of molecules respectively bore the C16:0 alkyl chain moiety, with the remainder bearing essentially C18:0 molecules. Overnight treatment of mast cells with PMA (200 ng/ml) caused disappearance of protein kinase C (PKC) from both cytosol and membranes. When such cells were stimulated further with antigen, they failed to degranulate, and acetyltransferase activation, paf production and lyso-paf production were decreased by 33 +/- 11%, 57 +/- 4% and 96 +/- 3% respectively (n = 3 or 5). The PKC inhibitors chlorpromazine and staurosporine inhibited to a significant extent both cell degranulation and all steps leading to paf biosynthesis. Our data suggest that PKC-dependent mechanisms are operational during cell degranulation and contribute only in part to paf biosynthesis. The PKC-dependent signal directly generated by PMA or diacylglycerol is not sufficient to trigger the full cell response, which is obtained only through receptor-operated antigen challenge.