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.

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Free radical-mediated oxidation of cholesterol-rich LDL plays a key role in atherogenesis and involves the formation of oxidized phospholipids with proinflammatory biological activity. We evaluated the production of platelet-activating factor (PAF), a potent inflammatory mediator, in human LDL subspecies on copper-initiated oxidation (4 mumol/L CuCl2, 80 micrograms/mL for hours at 37 degrees C). PAF formation was determined by biological assay of HPLC-purified lipid extracts of copper-oxidized lipoproteins; chemical identity was confirmed by gas chromatographic and mass spectrometric analyses. PAF, characterized as the C16:0 molecular species, was preferentially produced in intermediate LDL (d = 1.029 to 1.039 g/mL) (8.6 +/- 5.7 pmol PAF/3 h per mg LDL protein) and light LDL (d = 1.019 to 1.029 g/mL), but was absent from dense LDL particles (d = 1.050 to 1.063 g/mL). As PAF:acetylhydrolase inactivates PAF and oxidized forms of phosphatidylcholine, we evaluated the relationship of lipoprotein-associated PAF:acetylhydrolase to PAF formation. We confirmed that PAF:acetylhydrolase activity was elevated in native, dense LDL (41.5 +/- 9.5 nmol/min per mg protein) but low in LDL subspecies of light and intermediate density (d 1.020 to 1.039 g/mL) (3.5 +/- 1.6 nmol/min per mg protein) [Tselepis et al, Arterioscler Thromb Vasc Biol. 1995;15:1764-1773]. On copper-mediated oxidation for 3 hours at 37 degrees C, dense LDL particles conserved 20 +/- 14% of their initial enzymatic activity; in contrast, PAF:acetylhydrolase activity was abolished in light and intermediate LDL subspecies. Clearly, the elevated PAF:acetylhydrolase activity of dense LDL efficiently diminishes the potential inflammatory role of endogenously formed PAF; nonetheless, formation of proatherogenic lysophospholipids results. In contrast, LDL particles of the light and intermediate subclasses can accumulate PAF on oxidative modification.