No 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.

Abstract-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.