The role of scavenger receptor class A in the adhesion of cells is dependent on cell type and cellular activation state.

Scavenger receptor class A (SR-A) facilitates the development of atherosclerosis, which might be due to its role in the uptake of modified low-density lipoproteins. However, the receptor is also suggested to be important for cell adhesion, thereby potentially influencing the residence time of cells in vivo. Using SR-A-deficient mice, we investigated the role of SR-A in the adhesion of peritoneal macrophages (PM) and tissue macrophages (Kupffer cells). In resident PM no effect of the absence or presence of SR-A on cell adhesion was observed, either in the presence or in the absence of serum. However, in thioglycollate-induced PM, SR-A is important for adhesion both in the presence and in the absence of serum and more than 85% of the divalent-cation-independent adhesion in the presence of serum is mediated by SR-A. In unactivated Kupffer cells, like in resident PM, adhesion is not influenced by the absence or presence of SR-A. In vivo administration of phorbol 12-myristate 13-acetate leads to the activation of Kupffer cells, and it appears that under these conditions SR-A does contribute to adhesion, since both in the absence and in the presence of serum SR-A is responsible for about 35% of cell adhesion. It is concluded that SR-A is important for the divalent-cation-independent adhesion of activated PM and Kupffer cells, suggesting that SR-A may influence the residence time of cells at sites of cellular activation, e.g., in atherosclerotic plaques and during liver infection.     

Scavenger Receptors SR-AI/II and MARCO limit pulmonary dendritic cell migration and allergic airway inflammation

The class A scavenger receptors (SR-A) MARCO and SR-AI/II are expressed on lung macrophages (MPhis) and dendritic cells (DCs) and function in innate defenses against inhaled pathogens and particles. Increased expression of SR-As in the lungs of mice in an OVA-asthma model suggested an additional role in modulating responses to an inhaled allergen. After OVA sensitization and aerosol challenge, SR-AI/II and MARCO-deficient mice exhibited greater eosinophilic airway inflammation and airway hyperresponsiveness compared with wild-type mice. A role for simple SR-A-mediated Ag clearance ("scavenging") by lung MPhis was excluded by the observation of a comparable uptake of fluorescent OVA by wild-type and SR-A-deficient lung MPhis and DCs. In contrast, airway instillation of fluorescent Ag revealed a significantly higher traffic of labeled DCs to thoracic lymph nodes in SR-A-deficient mice than in controls. The increased migration of SR-A-deficient DCs was accompanied by the enhanced proliferation in thoracic lymph nodes of adoptively transferred OVA-specific T cells after airway OVA challenge. The data identify a novel role for SR-As expressed on lung DCs in the down-regulation of specific immune responses to aeroallergens by the reduction of DC migration from the site of Ag uptake to the draining lymph nodes.     

The uptake and metabolism of chylomicron-remnant lipids by rat liver parenchymal and non-parenchymal cells in vitro.

The uptake and metabolism of chylomicron-remnant lipids by individual liver cell types was examined by incubating remnants with monolayer cultures of hepatocytes, Kupffer cells, and endothelial cells from rat liver. Remnants were prepared in vitro from radiolabelled mesenteric-lymph chylomicra, utilizing either purified lipoprotein lipase from bovine milk, or plasma isolated from heparinized rats. The resulting particles contained [3H]phosphatidylcholine and cholesterol, and [14C]oleate in the acylglycerol, phospholipid, fatty-acid and cholesterol-ester fractions. The capacities of the three cell types for uptake of both [3H]lipids and [14C]lipids were determined to be, on a per-cell basis, in the order: Kupffer greater than hepatocytes greater than endothelial. The relative proportions of [3H]phospholipid and total [3H]cholesterol taken up by hepatocytes and non-parenchymal cells remained constant with time. The uptake of [14C]oleoyl lipids by all three cell types was slightly greater than that of the total [3H]cholesterol and [3H]phospholipid components. There was evidence of cholesterol-ester hydrolysis and turnover of [14C]oleate in the phospholipid fraction in hepatocytes and Kupffer cells, but not endothelial cells, over the first 2 h. With both remnant preparations, these observations indicate that significant differences exist between the three major liver cell types with respect to the uptake and metabolism of remnant lipid components.     

Blastogenesis and polyclonal immunoglobulin synthesis in murine spleen cells stimulated with lipopolysaccharide, lipid A and acid-degraded polysaccharide from Fusobacterium nucleatum

Lipopolysaccharide of Fusobacterium nucleatum strain Fevl was split by acid hydrolysis. The split products, i.e. lipid A and degraded polysaccharide were mitogenic for murine spleen cells as measured by uptake of [3H]thymidine. The uptake of [3H]thymidine was dose-dependent. Incubation of spleen cells with stimulants for 3 days resulted in a polyclonal activation of immunoglobulin synthesis. Higher mitogenic response and immunoglobulin production were found in spleen cells of athymic mice compared to those of thymic mice. The activity of lipid A in stimulating immunoglobulin synthesis was comparable with the parent lipopolysaccharide-Fevl, the degraded polysaccharide being the less potent stimulator.     

Surfactant protein A (SP-A)-mediated clearance of Staphylococcus aureus involves binding of SP-A to the staphylococcal adhesin eap and the macrophage receptors SP-A receptor 210 and scavenger receptor class A

Staphylococcus aureus causes life-threatening pneumonia in hospitals and deadly superinfection during viral influenza. The current study investigated the role of surfactant protein A (SP-A) in opsonization and clearance of S. aureus. Previous studies showed that SP-A mediates phagocytosis via the SP-A receptor 210 (SP-R210). Here, we show that SP-R210 mediates binding and control of SP-A-opsonized S. aureus by macrophages. We determined that SP-A binds S. aureus through the extracellular adhesin Eap. Consequently, SP-A enhanced macrophage uptake of Eap-expressing (Eap(+)) but not Eap-deficient (Eap(-)) S. aureus. In a reciprocal fashion, SP-A failed to enhance uptake of Eap(+) S. aureus in peritoneal Raw264.7 macrophages with a dominant negative mutation (SP-R210(DN)) blocking surface expression of SP-R210. Accordingly, WT mice cleared infection with Eap(+) but succumbed to sublethal infection with Eap- S. aureus. However, SP-R210(DN) cells compensated by increasing non-opsonic phagocytosis of Eap(+) S. aureus via the scavenger receptor scavenger receptor class A (SR-A), while non-opsonic uptake of Eap(-) S. aureus was impaired. Macrophages express two isoforms: SP-R210(L) and SP-R210(S). The results show that WT alveolar macrophages are distinguished by expression of SP-R210(L), whereas SR-A(-/-) alveolar macrophages are deficient in SP-R210(L) expressing only SP-R210(S). Accordingly, SR-A(-/-) mice were highly susceptible to both Eap(+) and Eap(-) S. aureus. The lungs of susceptible mice generated abnormal inflammatory responses that were associated with impaired killing and persistence of S. aureus infection in the lung. In conclusion, alveolar macrophage SP-R210(L) mediates recognition and killing of SP-A-opsonized S. aureus in vivo, coordinating inflammatory responses and resolution of S. aureus pneumonia through interaction with SR-A.     

Effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of liposomes by Kupffer cells in culture

We investigated the effects of (dihydro)cytochalasin B, colchicine, monensin and trifluoperazine on uptake and processing of large unilamellar liposomes by rat Kupffer cells in maintenance culture. The phospholipid vesicles were labeled in the lipid moiety with phosphatidyl[14C]choline and contained [3H]inulin or [125I]iodoalbumin as nondegradable and degradable markers of the aqueous vesicle content, respectively. Cytochalasin B and dihydrocytochalasin B, inhibitors of microfilament function, reduced inert inulin label uptake by 75% maximally, but residual uptake was not followed by release of lipid degradation products from the cells. By contrast, colchicine, an inhibitor of microtubule assembly, reduced uptake of liposomal inulin by maximally 55% but could not inhibit release of lipid degradation products from the cells. It is concluded that the cytochalasins partly inhibit uptake but fully prevent the arrival of internalized liposomes in the lysosomal compartment, while the action of colchicine is to slow down the overall process of uptake and subsequent transportation to the lysosomes. Monensin reduced inulin uptake to an extent similar to that found with colchicine, but reversibly blocked degradation of liposomal lipid and encapsulated protein. The kinetics of degradation of liposomal constituents suggests that residual uptake in the presence of monensin represents accumulation in an intracellular compartment. Trifluoperazine did not affect binding, internalization or degradation of encapsulated protein at low concentration (6 microM), but completely inhibited release of liposomal lipid degradation products under these conditions. At intermediate concentration (14 microM), the drug also reduced the internalization, while a high concentration (22 microM) was required to inhibit protein degradation as well. We conclude that trifluoperazine has multiple sites of action in the uptake and processing of liposomal constituents by Kupffer cells.     

Intrahepatic uptake and processing of intravenously injected small unilamellar phospholipid vesicles in rats

Small unilamellar vesicles consisting of sphingomyelin, cholesterol and phosphatidylserine in a molar ratio of 4:5:1 containing [³H]inulin as a marker of the aqueous space or [Me-¹⁴C]choline-labeled sphingomyelin as a marker of the lipid phase were injected intravenously into rats. After separation of the non-parenchymal cells into a Kupffer cell fraction and an endothelial cell fraction by elutriation centrifugation analysis of the radioactivity contents demonstrated that Kupffer cells were actively involved in the uptake of the vesicles whereas endothelial cells did not contribute at all. Uptake by total parenchymal cells was also substantial but, on a per cell base, significantly lower than that by the Kupffer cells. By comparising the fate of the [³H]inulin label and the [¹⁴C]sphingomyelin label it was concluded that release of liposomal lipid degradation products especially occurred from Kupffer cells rather than from parenchymal cells. In both cell types, however, substantial proportions of the ¹⁴C-label accumulated in the phosphatidylcholine fraction, indicating intracellular degradation of sphingomyelin and subsequent phosphatidylcholine synthesis. Treatment of the animals with the lysosomotropic agent chloroquine prior to liposome injection effectively blocked the conversion of the choline-labeled sphingomyelin into phosphatidylcholine in both cell types. This observation indicates that uptake of the vesicles occurred by way of an endocytic mechanism.     

Kinetics of lipopolysaccharide clearance by Kupffer and parenchyma cells in perfused rat liver

We studied the kinetics of [3H]lipopolysaccharide ([3H]LPS) (endotoxin) binding to Kupffer cells and hepatocytes at the level of the microtubular system after treatment with gadolinium chloride (GdCl(3)) and colchicine. Liver perfusion in Sprague-Dawley rats involves both portal vein and thoracic inferior vena cava cannulations as inlet and outlet, respectively. The subhepatic inferior vena cava is ligated to prevent perfusate leakage. Buffer containing 2% serum and [3H]LPS is administered at 1 ml/min and collected for 50 min. Rate constants for hepatocellular clearance of [3H]LPS in controls, colchicine-treated rats, GdCl(3)-treated rats, and colchicine plus GdCl(3)-treated rats are assessed using a simplified mathematical model. Forward-binding, reversal-binding, residency time, and influx rate constants are estimated. Results show that in GdCl(3)-treated rats, the hepatocytes effectively clear endotoxin from the circulation, and its ultimate binding affinity at the hepatocyte site is somewhat reduced compared to the Kupffer cells. In colchicine-treated rats, the disruption of the microtubule network altered [3H]LPS binding with Kupffer cells, suggesting that the microfilament-microtubular network also affects Kupffer cell function. Simultaneous treatments with colchicine and GdCl(3) increased the influx rate constant, suggesting that the compiled morphological alterations up-regulated endotoxin clearance by the liver, as indicated by a drastic increase in cellular vacuolation. In conclusion, the kinetics of the trafficking process of [3H]LPS clearance are regulated by apical-sinusoidal endocytotic and canalicular routes.     

Effects of recombinant human macrophage colony-stimulating factor on proliferation, differentiation and survival of Kupffer cells in the liver of adult mice

OBJECTIVE: To investigate the effects of macrophage colony-stimulating factor (M-CSF) on the proliferation, differentiation and survival of Kupffer cells in the liver of adult mice. STUDY DESIGN: By the combined method of autoradiography with [3H]thymidine and immunohistochemistry using a monoclonal antibody against mouse macrophages (F4/80 or BM8), the labeling rate of [3H]thymidine in macrophages within the liver sinusoids was examined at various intervals after single flash labeling with [3H]thymidine in adult mice with or without daily administration of recombinant human M-CSF. RESULTS: A minor population of Kupffer cells (about 2%) possessed proliferative capacity under a normal steady state condition. With time after flash labeling, the influx of monocytes and their differentiation into macrophages were demonstrated in the liver, and their labeling rate returned to the baseline level one week later. Afterward, the labeling rate of Kupffer cells was maintained at the baseline level until the end of five weeks. Administration of M-CSF enhanced the proliferative capacity of Kupffer cells, increased the number of macrophages and delayed the time of peaking. However, it did not prolong the survival of Kupffer cells. CONCLUSION: In normal mice, Kupffer cells can survive for at least five weeks. Daily M-CSF administration induces the increased number and proliferative capacity of Kupffer cells.     

Suppression of TLR4-mediated inflammatory response by macrophage class A scavenger receptor (CD204)

The class A scavenger receptor (SR-A, CD204), one of the principal receptors expressed on macrophages, has been found to regulate inflammatory response and attenuate septic endotoxemia. However, the detailed mechanism of this process has not yet been well characterized. To clarify the regulative mechanisms of lipopolysaccharide (LPS)-induced macrophage activation by SR-A, we evaluated the activation of Toll-like receptor 4 (TLR4)-mediated signaling molecules in SR-A-deficient (SR-A^−/−) macrophages. In a septic shock model, the blood levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and interferon (IFN)-β were significantly increased in SR-A^−/− mice compared to wild-type mice, and elevated nuclear factor kappa B (NFκB) activation was detected in SR-A^−/− macrophages. SR-A deletion increased the production of pro-inflammatory cytokines, and the phosphorylation of mitogen-activated protein kinase (MAPK) and NFκB in vitro. SR-A deletion also promoted the nuclear translocation of NFκB and IFN regulatory factor (IRF)-3. In addition, a competitive binding assay with acetylated low-density lipoprotein, an SR-A-specific ligand, and anti-SR-A antibody induced significant activation of TLR4-mediated signaling molecules in wild-type macrophages but not in SR-A^−/− macrophages. These results suggest that SR-A suppresses the macrophage activation by inhibiting the binding of LPS to TLR4 in a competitive manner and it plays a pivotal role in the regulation of the LPS-induced inflammatory response.     

GM-CSF regulates protein and lipid catabolism by alveolar macrophages

Metabolism of surfactant protein (SP) A and dipalmitoylphosphatidylcholine (DPPC) was assessed in alveolar macrophages isolated from granulocyte-macrophage colony-stimulated factor (GM-CSF) gene-targeted [GM(-/-)] mice, wild-type mice, and GM(-/-) mice expressing GM-CSF under control of the SP-C promoter element (SP-C-GM). Although binding and uptake of (125)I-SP-A were significantly increased in alveolar macrophages from GM(-/-) compared with wild type or SP-C-GM mice, catabolism of (125)I-SP-A was markedly decreased in GM(-/-) mice. Association of [(3)H]DPPC with alveolar macrophages from GM(-/-), wild-type, and SP-C-GM mice was similar; however, catabolism of DPPC was markedly reduced in cells from GM(-/-) mice. Fluorescence-activated cell sorter analysis demonstrated decreased catabolism of rhodamine-labeled dipalmitoylphosphatidylethanolamine by alveolar macrophages from GM(-/-) mice. GM-CSF deficiency was associated with increased SP-A uptake by alveolar macrophages but with impaired surfactant lipid and SP-A degradation. These findings demonstrate the important role of GM-CSF in the regulation of alveolar macrophage lipid and SP-A catabolism.     

Activation of signaling pathways by putative scavenger receptor class A (SR-A) ligands requires CD14 but not SR-A

Macrophage scavenger class A type I and type II receptors (SR-A) are trimeric, integral membrane glycoproteins that bind an unusually broad array of macromolecular ligands. These ligands include modified proteins and lipoproteins, nucleic acids, and a variety of plant and microbial cell wall constituents, such as fucoidan and lipoteichoic acid. Early studies of SR-A functions indicated that the receptors bound, internalized, and degraded their ligands without provoking any macrophage activating signaling events. More recent studies have provided evidence that several SR-A ligands can activate macrophage gene expression via utilization of a receptor-linked, PI3-kinase pathway. To investigate the role of SR-A in engaging signal transduction events, we employed macrophages taken from mice lacking these receptors. Using either fucoidan or lipoteichoic acid, we confirm that both ligands stimulate tyrosine phosphorylation of PI3-kinase and production of modest levels of the cytokine, TNFalpha. However, macrophages taken from SR-A null mice did not differ from wild type macrophages in these responses, indicating that these signaling events arise independently of SR-A activity. Employing mice lacking CD14, a GPI anchored receptor that binds bacterial lipopolysaccharide and signals via activation of Toll-like receptors, we show that the fucoidan and lipoteichoic acid responses are largely abrogated when CD14 is absent. These data do not provide support for direct SR-A involvement in signal transduction events and suggest that the early characterization of these receptors as initiators of a non-phlogistic, pathogen clearance pathway was correct.     

Uptake and processing of immunoglobulin-coated liposomes by subpopulations of rat liver macrophages

In vivo uptake and processing by liver macrophages (Kupffer cells) of liposomes, covalently coated with rabbit immunoglobulin (Ig liposomes) was studied following intravenous injection in rats. Rabbit Ig liposomes were labeled with trace amounts of cholesteryl[14C]oleate and [3H]cholesteryl hexadecyl ether. 1 h after injection of the liposomes, the non-parenchymal cells were isolated and subjected to centrifugal elutriation with stepwise-increasing flow rates; thus, five sub-fractions of Kupffer cells were obtained ranging in size from 9 to 14 micron in diameter. The cells were assayed for peroxidase activity and protein content. Rabbit Ig liposomes were taken up preferentially by Kupffer cells with diameters larger than 11 micron, which constitute less than 25% of the total Kupffer cell population. The intralysosomal degradation of the ingested liposomes was monitored by measuring the 3H/14C ratio of the cells. Due to the rapid release from the cells of the [14C]oleate formed from the cholesteryl[14C]oleate and the virtually complete retention of the non-metabolizable [3H]cholesteryl hexadecyl ether the 3H/14C ratio of the cells increases with proceeding hydrolysis of the liposomes. Thus, we were able to show that, in vivo, the Kupffer cells of the larger size classes, are not only more active in liposome uptake, but are also substantially more active in liposome degradation than smaller cells. The maintenance of the observed heterogeneity of rat liver Kupffer cells, with respect to liposome uptake under in vitro culture conditions, was examined. Subfractions were maintained in monolayer culture for 2 days and incubated with rabbit Ig liposomes. Binding and uptake of liposomes by the cells was monitored by measuring cell-associated radioactivity at 4 degrees C and 37 degrees C, respectively. In contrast to our in vivo results, we observed maximal in vitro liposome binding and uptake in those subfractions containing small cells (10-11 micron diameter), while the fractions containing cells larger than 12 micron, which were more active in vivo, were substantially less active than the smaller cells. The maximum we observed was even more pronounced when the liposome concentration was increased. We conclude that liver macrophage subfractions that barely participate in liposome uptake from the bloodstream in vivo, possess the potential to develop the capacity in vitro to phagocytose rabbit Ig-coated liposomes to extents equal to or even higher than the cells belonging to those subfractions containing the phagocytically most active cells under in vivo conditions.     

Blastogenesis and polyclonal immunoglobulin synthesis in murine spleen cells stimulated with lipoolysaccharide, lipid A and acid-degrade polysaccharide from Fusobacterium nucleatum

Abstract Lipopolysaccharide of Fusobacterium nucleatum strain Fevl was split by acid hydrolysis. The split products, i.e. lipid A and degraded polysaccharide were mitogenic for murine spleen cells as measured by uptake of [³H]thymidine. The uptake of [³H]thymidine was dose-dependent. Incubation of spleen cells with stimulants for 3 days resulted in a polyclonal activation of immunoglobulin synthesis. Higher mitogenic response and immunoglobulin production were found in spleen cells of athymic mice compared to those of thymic mice. The activity of lipid A in stimulating immunoglobulin synthesis was comparable with the parent lipopolysaccharide-Fevl, the degraded polysaccharide being the less potent stimulator.     

Hepatic processing of the cholesteryl ester from low density lipoprotein in the rat

Human low density lipoprotein (LDL), radiolabeled in the cholesteryl ester moiety, was injected into estrogen-treated and -untreated rats. The hepatic and extrahepatic distribution and biliary secretion of [3H]cholesteryl esters were determined at various times after injection. In order to follow the intrahepatic metabolism of the cholesteryl esters of LDL in vivo, the liver was subfractioned into parenchymal and Kupffer cells by a low temperature cell isolation procedure. In control rats, the LDL cholesteryl esters were mainly taken up by the Kupffer cells. After uptake, the [3H]cholesteryl esters are rapidly hydrolyzed, followed by release of [3H]cholesterol from the cells to other sites in the body. Up to 24 h after injection of LDL, only 9% of the radioactivity appeared in the bile, whereas after 72 h, this value was 30%. Hepatic and especially the parenchymal cell uptake of [3H]cholesteryl esters from LDL was strongly increased upon 17 alpha-ethinylestradiol treatment (3 days, 5 mg/kg). After rapid hydrolysis of the esters, [3H]cholesterol was both secreted into bile (28% of the injected dose in the first 24 h) as well as stored inside the cells as re-esterified cholesterol ester. It is concluded that uptake of human LDL by the liver in untreated rats is not efficiently coupled to biliary secretion of cholesterol (derivatives), which might be due to the anatomical localization of the principal uptake site, the Kupffer cells. In contrast, uptake of LDL cholesterol ester by liver hepatocytes is tightly coupled to bile excretion. The Kupffer cell uptake of LDL might be necessary in order to convert LDL cholesterol (esters) into a less toxic form. This activity can be functional in animals with low receptor activity on hepatocytes, as observed in untreated rats, or after diet-induced down-regulation of hepatocyte LDL receptors in other animals.     

Scavenger receptor A (SR-A) is required for LPS-induced TLR4 mediated NF-κB activation in macrophages

Recent evidence suggests that the macrophage scavenger receptor class A (SR-A, aka, CD204) plays a role in the induction of innate immune and inflammatory responses. We investigated whether SR-A will cooperate with Toll-like receptors (TLRs) in response to TLR ligand stimulation. Macrophages (J774/a) were treated with Pam2CSK4, (TLR2 ligand), Polyinosinic:polycytidylic acid (Poly I:C) (TLR3 ligand), and Lipopolysaccharides (LPS) (TLR4 ligand) for 15 min in the presence or absence of fucoidan (the SR-A ligand). The levels of phosphorylated IκBα (p-IκBα) were examined by Western blot. We observed that Poly I:C and LPS alone, but not Pam2CSK4 or fucoidan increased the levels of p-IκBα. However, LPS-induced increases in p-IκBα levels were further enhanced when presence of the fucoidan. Immunoprecipitation and double fluorescent staining showed that LPS stimulation promotes SR-A association with TLR4 in the presence of fucoidan. To further confirm our observation, we isolated peritoneal macrophages from SR-A deficient (SR-A(-/-)), TLR4(-/-) and wild type (WT) mice, respectively. The peritoneal macrophages were treated with LPS for 15min in the presence and absence of fucoidan. We observed that LPS-stimulated TNFα and IL-1β production was further enhanced in the WT macrophages, but did not in either TLR4(-/-) or SR-A(-/-) macrophages, when fucoidan was present. Similarly, in the presence of fucoidan, LPS-induced IκBα phosphorylation, NF-κB binding activity, and association between TLR4 and SR-A were significantly enhanced in WT macrophages compared with LPS stimulation alone. The data suggests that SR-A is needed for LPS-induced inflammatory responses in macrophages.     

FDG-PET imaging of pulmonary inflammation in healthy volunteers after airway instillation of endotoxin.

Recent studies indicate that a focal, limited, inflammatory response can be safely elicited after direct bronchial instillation of small doses of endotoxin into a single lung segment. Because the radiotracer [18F]fluorodeoxyglucose ([18F]FDG) is taken up at accelerated rates within inflamed tissues, we hypothesized that we could detect and quantify this regional inflammatory response with positron emission tomography (PET). We imaged 18 normal volunteers in a dose-escalation study with 3 endotoxin dosing groups (n = 6 in each group): 1 ng/kg, 2 ng/kg, and 4 ng/kg. Endotoxin was instilled by bronchoscopy into a segment of the right middle lobe, with imaging performed approximately 24 h later, followed by bronchoalveolar lavage (BAL). A "subtraction imaging analysis" was performed in the highest dose cohort to identify the area of inflammation, using the preendotoxin scan as a baseline. BAL neutrophil counts were significantly higher in the highest dose group compared with the other two groups (1,413 +/- 625 vs. 511 +/- 396 and 395 +/- 400 cells/mm3; P < 0.05). Autoradiography performed on cells harvested by BAL showed specific [3H]deoxyglucose ([3H]DG) uptake limited to neutrophils. In vitro [3H]DG uptake in BAL neutrophils in the 4 ng/kg dose group (but not in the 2 ng/kg group) was statistically greater than in peripheral blood neutrophils obtained before endotoxin instillation. The rate of [18F]FDG uptake was greatest in the 4 ng/kg group, with a consistent, statistically significant increase in the rate of uptake after endotoxin instillation compared with baseline. We conclude that the inflammatory response to low-dose endotoxin in a single lung segment can be visualized and quantified by imaging with FDG-PET.     

Major Vault Protein Regulates Class A Scavenger Receptor-mediated Tumor Necrosis Factor-α Synthesis and Apoptosis in Macrophages

Atherosclerosis is considered a disease of chronic inflammation largely initiated and perpetuated by macrophage-dependent synthesis and release of pro-inflammatory mediators. Class A scavenger receptor (SR-A) expressed on macrophages plays a key role in this process. However, how SR-A-mediated pro-inflammatory response is modulated in macrophages remains ill defined. Here through immunoprecipitation coupled with mass spectrometry, we reported major vault protein (MVP) as a novel binding partner for SR-A. The interaction between SR-A and MVP was confirmed by immunofluorescence staining and chemical cross-linking assay. Treatment of macrophages with fucoidan, a SR-A ligand, led to a marked increase in TNF-α production, which was attenuated by MVP depletion. Further analysis revealed that SR-A stimulated TNF-α synthesis in macrophages via the caveolin- instead of clathrin-mediated endocytic pathway linked to p38 and JNK, but not ERK, signaling pathways. Importantly, fucoidan invoked an enrichment of MVP in lipid raft, a caveolin-reliant membrane structure, and enhanced the interaction among SR-A, caveolin, and MVP. Finally, we demonstrated that MVP elimination ameliorated SR-A-mediated apoptosis in macrophages. As such, MVP may fine-tune SR-A activity in macrophages which contributes to the development of atherosclerosis.     

A PEST deletion mutant of ABCA1 shows impaired internalization and defective cholesterol efflux from late endosomes

ATP-binding cassette transporter A1 (ABCA1) promotes the efflux of cellular cholesterol and phospholipids to apoA-I. We described previously a cytoplasmic PEST sequence in ABCA1 and showed that deletion of the PEST sequence results in a prominent increase in the cell surface concentration of ABCA1. In the current study we evaluated the hypothesis that the PEST sequence-deleted ABCA1 might display defective internalization and trafficking to the late endosomes/lysosomes. As assessed by monensin treatment and cell surface biotinylation, the internalization rate of PEST sequence-deleted ABCA1 (ABCA1-dPEST) was markedly decreased compared with wild-type ABCA1 (ABCA1-wt). Immunofluorescence confocal microscopy of ABCA1-wt showed both plasma membrane localization and substantial co-localization with LAMP2 in late endosomes. In contrast, ABCA1-dPEST showed more prominent plasma membrane localization but little co-localization with LAMP2. To assess cholesterol efflux from late endosomes, HEK293 cells were transiently co-transfected with scavenger receptor A (SR-A) and incubated with [3H]cholesterol/acetyl low density lipoprotein (acLDL). Although ABCA1-dPEST showed higher cholesterol efflux than did ABCA1-wt following cell surface labeling ([3H]cholesterol/acLDL in the absence of SR-A co-transfection), it showed impaired cholesterol efflux after late endosomal labeling ([3H]cholesterol/acLDL in the presence of SR-A). Thus, deletion of the PEST sequence leads to a decrease in the internalization of ABCA1 and decreased cholesterol efflux from late endosomal cholesterol pools, providing evidence that the internalization and trafficking of ABCA1 is functionally important in mediating cholesterol efflux from intracellular cholesterol pools.