Expression of the PAF Receptor in Human Monocyte–Derived Macrophages Is Downregulated by Oxidized LDL

Abstract

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.

Inhibition of LPL Expression in Human Monocyte–Derived Macrophages Is Dependent on LDL Oxidation State

“…In addition, cell protein content was not affected, and the expression of actin and scavenger receptor I and II mRNAs remained unaffected or slightly increased after oxLDL treatment, as reported earlier. 41 Moreover, in the same experiments, inhibition of platelet-activating factor receptor gene expression by oxLDL was reversible, as shown by an additional 24-hour incubation in medium devoid of oxLDL. If cytotoxicity has been demonstrated in vitro for mouse macrophages incubated with LDL oxidized for 24 hours with 5 mol/L CuSO 4 43 , oxLDL prepared under our conditions (48 hours with 2.5 mol/L CuCl 2 , followed by extensive dialysis) had no detectable effect on macrophage morphology, as evaluated by scanning electron microscopy (G. Le Naour et al, unpublished findings, 1997).…”

Mildly Oxidized LDL Induces Expression of Group IIa Secretory Phospholipase A 2 in Human Monocyte–Derived Macrophages

“…Native and moderately modified LDLs have been shown to exhibit both stimulatory and inhibitory effects on gene transcription, eg, on macrophage scavenger receptors, 38 platelet-activating factor receptor, 26 granulocyte macrophage colony stimulating factor, 39 monocyte chemotactic protein-1, 40,41 and cellular adhesion molecules. 42,43 To examine whether LDL modulated the expression of PLA 2 genes, human monocyte-derived macrophages were incubated with various LDL preparations displaying different degrees of oxidation, and PLA 2 mRNA levels were assessed by RT-PCR.…”

“…26 In brief, buffy coats diluted with PBS (1:1, vol/vol) were carefully loaded onto a Ficoll gradient under sterile conditions. After an initial centrifugation step (20 minutes, 2700g), monocytes were collected at the interface and then washed 3 times at room temperature in PBS containing 0.1% EDTA (successively at 1000g, 340g, and 160g for 10 minutes) and then once in PBS alone at 160g for 10 minutes.…”

Down-Regulation of Platelet-Activating Factor Receptor Gene Expression During Focal Reversible Cerebral Ischemia in Rats

“…The PAF receptor promoter contains three nuclear transcription factor NF-jB binding sites. The transcription activity of the PAF receptor is regulated by cyclic adenosine monophosphate (cAMP) and protein kinase C (PKC), which are upstream messengers of NF-jB [31][32][33]. In many cells, low concentrations of PKC up-regulate the PAF receptor, while high concentrations demonstrate the opposite effect [21].…”