We report that membrane CD14 (mCD14), a cell surface receptor found principally on leukocytes, can mediate the uptake and metabolism of extracellular phosphatidylinositol (PtdIns). mCD14 facilitates PtdIns internalization, targeting it to intracellular sites where, following stimulation with a calcium ionophore, it can be acted upon by cytosolic phospholipase A 2 . The [ 14 C]arachidonate released from mCD14-acquired [ 14 C]arachidonyl-PtdIns is either esterified to triacylglycerol and retained

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... in the cell or secreted as free arachidonate or leukotrienes. Although less than 10% of the arachidonate-derived lipids secreted from endogenous cellular stores are 5-lipoxygenase metabolites, over one-half of the secreted 14 C-lipids derived from mCD14-acquired PtdIns are hydroxyeicosatetraenoic acids or leukotriene B 4 . mCD14 may allow these highly active blood cells to acquire and use extracellular Pt-dIns as a source of arachidonate for leukotriene synthesis. Monocytes and neutrophils are highly differentiated, specialized cells that figure prominently in antimicrobial host defense. In addition to phagocytosing and killing bacterial and fungal cells, they play key roles in the innate immune process by which animals recognize invading microbes. They react to conserved microbial molecules, such as bacterial lipopolysaccharide, by releasing mediators that amplify and diversify the host inflammatory response. They are also major cellular sources of the leukotrienes, potent agonists for chemotaxis, smooth muscle contraction, and cell cycle stimulation (proliferation). Microbial recognition is mediated by a multistep process in which mCD14, 1 a 55-kDa glycosylphosphatidylinositol (GPI)anchored membrane protein, is thought to play an important role by binding bacterial lipopolysaccharide and other ligands to the plasma membrane. In addition to its key role in antimicrobial host defense, CD14 may be an important lipid transfer protein. Soluble CD14, which lacks the GPI anchor, can transfer several lipids to plasma lipoproteins or artificial lipid mem-branes (1, 2), and mCD14 is a receptor for phosphatidylinositol (PtdIns) and phosphatidylserine (3) . We recently reported that binding PtdIns and phosphatidylserine to mCD14 is facilitated by bacterial lipopolysaccharide-binding protein (LBP), a molecule that, like PtdIns, is found in plasma (3) . PtdIns is an important precursor of numerous signaling molecules (diacylglycerol, inositol 1,4,5-trisphosphate, and various phosphatidylinositides) and a potential source of arachidonic acid (the precursor of prostaglandins and leukotrienes). It is also incorporated into the glycosylphosphatidylinositol anchors used by many membrane proteins. We therefore asked whether extracellular, mCD14-bound PtdIns is utilized by the cells. We show here that mCD14-bound extracellular PtdIns is rapidly internalized to an intracellular compartment where, upon cell activation, it can be acted upon by cytosolic phospholipase A 2 . The arachidonate released from PtdIns then has several fates, including conversion to HETEs and leukotriene B 4 (LTB 4 ), but not to prostaglandins E 2 or F 2␣ , and secretion from the cell. EXPERIMENTAL PROCEDURES Reagents-1-Stearoyl-2-[ 14 C]arachidonyl-phosphatidylinositol (26.7 or 48 mCi/mmol), [ 3 H]arachidonic acid (212 Ci/mmol), and L-A-[myoinositol-2-3 H]phosphatidylinositol (6.6 Ci/mmol) were from NEN Life Science Products (Boston, MA). Arachidonic acid, prostaglandin E 2 , prostaglandin F 2␣ , 5-hydroperoxyeicosatetraenoic acid, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, diacylglycerol, triarachidonate, phorbol 12-myristate 13-acetate (PMA), and cholesteryl oleate were purchased from Sigma. 1,25-Dihydroxyvitamin D 3 (VD 3 ), leukotriene B 4, MK-886, ibuprofen, and MAFP were from Biomol Research Laboratories (Plymouth Meeting, PA). Phosphatidylinositol-specific phospholipase C (Bacillus cereus) was from Roche Molecular Biochemicals. Newborn calf serum (Sigma) was delipidated by ultracentrifugation at ϭ 1.3 g/ml (KBr) and then dialyzed against normal saline. After delipidation, it contained 38 mg/liter cholesterol, 27 mg/liter triglyceride, and 5.2 g/liter protein. Preparation of Phospholipids-Phospholipids were dried under argon and resuspended by sonication (400 watts, three pulses of 30 s each; Braunsonic 1510 sonicator; B. Braun, Melsungen AG) in serum-free RPMI 1640 medium. For most of the experiments, the resuspended lipids were then added to RPMI 1640 medium that contained 10% lipoprotein-poor newborn calf serum. Cells-Human monocytic THP-1 cells (4) were obtained, cultured in RPMI 1640 medium with 10% fetal calf serum, and transfected to express wild-type human CD14 (CD14-GPI) as described previously (5) . The human myeloid HL-60 cell line was obtained from the American Type Culture Collection and cultured in the same medium. Before the experiments, HL-60 cells were differentiated into monocyte-like cells by adding 100 nM VD 3 for 5 days. Human monocytes were kindly provided by David Wilkinson (The University of Texas Southwestern Medical Center, Dallas, TX). To label cellular lipids, [ 3 H]arachidonic acid was added to the culture medium (0.5 Ci/ml) for 24 h before each experiment. HL-60 cells were labeled on the fourth day of VD 3 treatment. Lipid Analysis-1 volume of cell suspension was added to 6 volumes of methanol/chloroform/acetic acid (2:1:0.01, v/v). After incubating with frequent mixing for 1 h at room temperature, 3 volumes of 0.05 M KCl were added. After vortexing, 2 volumes of chloroform were added. The sample was vortexed again and then centrifuged for 5 min at ϳ700 ϫ g.