Niemann-Pick C1 Like 1 (NPC1L1) is a protein localized in jejunal enterocytes that is critical for intestinal cholesterol absorption. The uptake of intestinal phytosterols and cholesterol into absorptive enterocytes in the intestine is not fully defined on a molecular level, and the role of NPC1L1 in maintaining whole body cholesterol homeostasis is not known. NPC1L1 null mice had substantially reduced intestinal uptake of cholesterol and sitosterol, with dramatically reduced plasma phytosterol levels. The NPC1L1 null mice were completely resistant to diet-induced hypercholesterolemia, with plasma lipoprotein and hepatic cholesterol profiles similar to those of wild type mice treated with the cholesterol absorption inhibitor ezetimibe. Cholesterol/cholate feeding resulted in down-regulation of intestinal NPC1L1 mRNA expression in wild type mice. NPC1L1 deficiency resulted in up-regulation of intestinal hydroxymethylglutaryl-CoA synthase mRNA and an increase in intestinal cholesterol synthesis, down-regulation of ABCA1 mRNA, and no change in ABCG5 and ABCG8 mRNA expression. NPC1L1 is required for intestinal uptake of both cholesterol and phytosterols and plays a major role in cholesterol homeostasis. Thus, NPC1L1 may be a useful drug target for the treatment of hypercholesterolemia and sitosterolemia.Cholesterol absorption of both dietary cholesterol and cholesterol cleared from the liver through biliary secretion contributes along with regulation of cholesterol biosynthesis to maintain a tight control of cholesterol homeostasis. The mechanism by which cholesterol moves from the intestinal lumen into the absorptive enterocytes lining the proximal small intestine is poorly understood. The identification of ezetimibe as a potent selective inhibitor of intestinal cholesterol uptake and absorption confirmed this mechanism as a key point of therapeutic intervention for lowering plasma cholesterol levels and indicated that this process is mediated by a specific transporter (1-4). Based on the properties of ezetimibe in animal models of cholesterol uptake, it was predicted that such a transporter would be expressed in jejunal enterocytes and localized to the brush border membrane, which forms the interface between the intestinal lumen and the intracellular compartments responsible for cholesterol esterification and packaging into chylomicrons.Through studies designed to understand the mechanism by which ezetimibe inhibits cholesterol absorption, we recently identified Niemann-Pick C1 Like 1 (NPC1L1) 1 as a critical protein for the intestinal absorption of dietary and biliary cholesterol (5). NPC1L1 was identified through a genomics-bioinformatics approach by sequencing an expression sequence tags library from rat jejunum, annotating the sequences, and searching databases for intestinal proteins with features of a cholesterol transporter (5). NPC1L1 was found to be highly expressed in the jejunum and localized on the surface of the absorptive enterocytes. Mice deficient in NPC1L1 exhibited a significant reduction in chol...

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia, but its molecular target has been elusive. Using a genetic approach, we recently identified Niemann-Pick C1-Like 1 (NPC1L1) as a critical mediator of cholesterol absorption and an essential component of the ezetimibe-sensitive pathway. To determine whether NPC1L1 is the direct molecular target of ezetimibe, we have developed a binding assay and shown that labeled ezetimibe glucuronide binds specifically to a single site in brush border membranes and to human embryonic kidney 293 cells expressing NPC1L1. Moreover, the binding affinities of ezetimibe and several key analogs to recombinant NPC1L1 are virtually identical to those observed for native enterocyte membranes. KD values of ezetimibe glucuronide for mouse, rat, rhesus monkey, and human NPC1L1 are 12,000, 540, 40, and 220 nM, respectively. Last, ezetimibe no longer binds to membranes from NPC1L1 knockout mice. These results unequivocally establish NPC1L1 as the direct target of ezetimibe and should facilitate efforts to identify the molecular mechanism of cholesterol transport.cholesterol ͉ intestinal brush border membranes

Abstract-Inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, such as simvastatin, lower circulating cholesterol levels and prevent myocardial infarction. Several studies have shown an unexpected effect of HMG-CoA reductase inhibitors on inflammation. Here, we confirm that simvastatin is anti-inflammatory by using a classic model of inflammation: carrageenan-induced foot pad edema. Simvastatin administered orally to mice 1 hour before carrageenan injection significantly reduced the extent of edema. Simvastatin was comparable to indomethacin in this model. To determine whether the anti-inflammatory activity of simvastatin might affect atherogenesis, simvastatin was tested in mice deficient in apoE. Mice were dosed daily for 6 weeks with simvastatin (100 mg/kg body wt). Simvastatin did not alter plasma lipids. Atherosclerosis was quantified through the measurement of aortic cholesterol content. Aortas from control mice (nϭ20) contained 56Ϯ4 nmol total cholesterol/mg wet wt tissue, 38Ϯ2 nmol free cholesterol/mg, and 17Ϯ2 nmol cholesteryl ester/mg. Simvastatin (nϭ22) significantly (PϽ0.02) decreased these 3 parameters by 23%, 19%, and 34%, respectively. Histology of the atherosclerotic lesions showed that simvastatin did not dramatically alter lesion morphology. These data support the hypothesis that simvastatin has antiatherosclerotic activity beyond its plasma cholesterol-lowering activity.

Neutrophil-activating protein 1/interleukin 8 (NAP-1/IL-8)' was first identified as a 72 amino acid peptide secreted by monocytes in response to bacterial LPS, with the properties of activating and attracting polymorphonuclear leukocytes (PMN) in vitro (1). Subsequently several different types ofcells, including macrophages and endothelial cells, were found to synthesize NAP-1/IL-8 upon stimulation with TNF or IL-1. Sequence data indicate that NAP-1/IL-8 is homologous to a group of peptides including platelet basic protein, platelet factor 4, and macrophage inflammatory protein 2 . In vivo accumulation of PMN at sites of injection of NAP-1/IL-8 suggests that it may participate in the recruitment of PMN into inflamed tissue, and NAP-1/IL-8 has been identified in skin lesions of patients with psoriasis (1).Several lines of evidence indicate that the CD11/CD18 complex ofleukocyte adhesion receptors may participate in the adhesion of PMN to the vascular endothelium which is necessary for extravasation . mAbs specific for CD18 are effective in blocking the adherence of stimulated PMN to endothelium in vivo (2) and in vitro (3, 4), and patients genetically deficient in the CDII/CD18 complex exhibit poor recruitment of PMN to Rebuck skin windows (5). Here we describe the response of CD11/CD18 on PMN to NAP-1/IL-8, with particular emphasis on the numerically dominant member, CD11b/CD18 . This receptor not only recognizes ligands on endothelium but also binds to complement protein C3bi (6), fibrinogen (7), and LPS (8).

SummaryTumor necrosis factor a, granulocyte colony-stimulating factor, granulocyte/macrophage colonystimulating factor, and formyl peptide were each found to cause a twofold increase in expression of CD14 on the surface of polymorphonuclear leukocytes (PMN). Upregulation of CD14 was complete by 20 min and thus appeared to result from expression of preformed stores ofprotein . The CD14 on the surface ofPMN was shown to serve two biological functions. It bound particles coated with complexes oflipopolysaccharide (LPS) and LPS binding protein (LBP). This binding activity was enhanced by agonists that upregulated CD14 expression and may serve in the clearance of Gram-negative bacteria opsonized with LBP. Interaction of CD14 with LPS in the presence of LBP or serum also caused a dramatic, transient increase in the adhesive activity of CR3 (CD11b/CD18) on PMN . Enhanced activity of CR3 and other members of the CD11/CD18 family underlies many of the known physiological responses ofPMN to LPS and may be a central feature of the in vivo responses of PMN to endotoxin. BacterialLPS(endotoxin) is known to have profound physiological effects on PMN both in vivo and in vitro. Animals respond to intravenous LPS with a rapid fall in the number of circulating PMN and a concomitant accumulation ofPMN in the lungs (1) . Isolated PMN respond to LPS with increased adhesion to protein-coated surfaces (2), enhanced ability to mount an oxidative burst (3), and enhanced microbicidal powers (4) . Many of these responses to LPS appear to depend at least in part on enhanced function of the three members of the CD11/CD18 family of adhesion-promoting receptors (LFA1, CR3, and p150,95, also known as the 02 or leukocyte integrins) on the surface of PMN . Adhesion of PMN to endothelium requires the participation ofCDII/CD18 molecules (5-7), and blockade ofCD18 with mAbs prevents accumulation ofPMN in the lungs ofendotoxin-treated animals (8) . Adhesion ofPMN to protein-coated glass (5), enhanced oxidative burst in response to soluble agonists (9), and microbicidal activity (5) are also dependent on increased CD11/CD18 function. Here, we directly measure the effect of LPS on the adhesive function ofCR3 (CD11b/CD18) and describe a dramatic enhancement of its adhesive activity by complexes of LPS with proteins from the serum.Recent studies have described a novel mechanism by which mononuclear cells may respond to LPS (10, 11) . LPS is first bound by the serum protein LPS binding protein (LBP)l, and the resulting LPS-LBP complex is then recognized by CD14, a 55-kD glycoprotein that is strongly expressed on monocytes and macrophages. LBP and CD14 serve two physiological roles. These proteins act as opsonin and opsonic receptor, respectively, to promote the phagocytic uptake of bacteria or LPS-coated particles by macrophages (12). They also dramatically enhance the ability of mononuclear cells to synthesize TNF in response to endotoxin . Addition of LBP speeds the synthesis of TNF and enables a response to doses of LPS 100-fold lower than are otherwi...