Cationic polypeptides are required for antibacterial activity of human airway fluid

In a search for direct evidence leading to the biological relevance of airway secretions in innate host defense, we characterized the antibacterial function of cationic polypeptides within minimally manipulated nasal fluid. In this study, we show that cationic antimicrobial polypeptides are responsible for most of the bactericidal activity of whole nasal fluid. The removal of cationic polypeptides using a cation-exchange resin ablated the activity of nasal fluid against Escherichia coli, Listeria monocytogenes, and Pseudomonas aeruginosa. By using a novel proteomic approach, we identified a dozen cationic peptides and proteins within nasal fluid, all of which either are known antimicrobial polypeptides or have other proposed roles in host defense. Of the three most abundant cationic polypeptides in nasal fluid, lysozyme was more effective than either lactoferrin or secretory leukoprotease inhibitor in restoring the antibacterial activity of the cationic polypeptide-depleted fluid against a mucoid cystic fibrosis isolate of P. aeruginosa.     

Properties of cholesteryl esters in pure and mixed monolayers

The surface properties of cholesteryl palmitate, stearate, linoleate, linolenate, arachidonate, and acetate were investigated. Long-chain esters were not surface-active and force-area (pi-A) isotherms were not obtained. Unsaturated cholesteryl esters were oxidized at the air-water interface and these oxidized lipids gave expanded pi-A isotherms. Cholesteryl acetate had an equilibrium spreading pressure of 14.0 dynes/cm and formed a stable monolayer indistinguishable from cholesterol below that surface pressure. Cholesteryl linoleate formed mixed monolayers with surface-active lipids, and the amount of cholesteryl linoleate in the monolayer depended both on its solubility in the other lipid and on the surface pressure. Even at moderate surface pressures cholesteryl linoleate was extruded from the monolayer into a bulk phase. Cholesteryl acetate exhibited the well-known condensing effect of cholesterol in mixed monolayers with egg lecithin.     

Specificity of lipid transfer protein for molecular species of cholesteryl ester

The capacity of the plasma-derived lipid transfer protein to facilitate the transfer of various cholesteryl ester species has been investigated. Four different molecular species of cholesteryl ester were incorporated into either reconstituted high density lipoproteins or phosphatidylcholine liposomes, and the resulting particles were used as donors in standardized lipid transfer assays. With reconstituted high density lipoproteins as substrate, the rate of transfer of cholesteryl esters was cholesteryl oleate greater than cholesteryl linoleate greater than cholesteryl arachidonate greater than cholesteryl palmitate. The transfer rate for cholesteryl oleate was 154% of that for cholesteryl palmitate. Liposome substrates gave similar results. It is concluded that lipid transfer protein transfers all major species of cholesteryl ester found in plasma; however, the relative rates of transfer were significantly affected by acyl chain composition. The transfer rates appeared to reflect substrate specificity rather than substrate availability within the donor particle.     

Macrophages can decrease the level of cholesteryl ester hydroperoxides in low density lipoprotein

Murine and human macrophages rapidly decreased the level of cholesteryl ester hydroperoxides in low density lipoprotein (LDL) when cultured in media non-permissive for LDL oxidation. This process was proportional to cell number but could not be attributed to the net lipoprotein uptake. Macrophage-mediated loss of lipid hydroperoxides in LDL appears to be metal ion-independent. Degradation of cholesteryl linoleate hydroperoxides was accompanied by accumulation of the corresponding hydroxide as the major product and cholesteryl keto-octadecadienoate as a minor product, although taken together these products could not completely account for the hydroperoxide consumption. Cell-conditioned medium possessed a similar capacity to remove lipid hydroperoxides as seen with cellular monolayers, suggesting that the activity is not an integral component of the cell but is secreted from it. The activity of cell-conditioned medium to lower the level of LDL lipid hydroperoxides is associated with its high molecular weight fraction and is modulated by the availability of free thiol groups. Cell-mediated loss of LDL cholesteryl ester hydroperoxides is facilitated by the presence of alpha-tocopherol in the lipoprotein. Together with our earlier reports on the ability of macrophages to remove peroxides rapidly from oxidized amino acids, peptides, and proteins as well as to clear selectively cholesterol 7-beta-hydroperoxide, results presented in this paper provide evidence of a potential protective activity of the cell against further LDL oxidation by removing reactive peroxide groups in the lipoprotein.     

Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins

Mouse peritoneal macrophages accumulate large amounts of cholesteryl ester when incubated with human low-density lipoprotein that has been modified by chemical acetylation (acetyl-LDL). This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of acetyl-LDL by adsorptive endocytosis and its delivery to lysosomes. The current studies demonstrate that the cholesteryl ester accumulation can be considered in terms of a two-compartment model: (a) the incoming cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and (b) the resultant free cholesterol is re-esterified in the cytosol where the newly formed esters are stored as lipid droplets. The following biochemical and morphologic evidence supports the hydrolysis-re-esterification mechanism: (a) Incubation of macrophages with acetyl-LDL markedly increased the rate of cholesteryl ester synthesis from [14C]oleate, and this was accompanied by an increase in the acyl-CoA:cholesteryl acyltransferase activity of cell-free extracts. (b) When macrophages were incubated with reconstituted acetyl-LDL in which the endogenous cholesterol was replaced with [3H]-cholesteryl linoleate, the [3H]cholesteryl linoleate was hydrolyzed, and at least one-half of the resultant [3H]cholesterol was re-esterified to form [3H]cholesteryl oleate, which accumulated within the cell. The lysosomal enzyme inhibitor chloroquine inhibited the hydrolysis of the [3H]cholesteryl linoleate, thus preventing the formation of [3H]cholesteryl oleate and leading to the accumulation of unhydrolyzed [3H]cholesteryl linoleate within the cells. (c) In the electron microscope, macrophages incubated with acetyl-LDL had numerous cytoplasmic lipid droplets that were not surrounded by a limiting membrane. The time course of droplet accumulation was similar to the time course of cholesteryl ester accumulation as measured biochemically. (d) When acetyl-LDL was removed from the incubation medium, biochemical and morphological studies showed that cytoplasmic cholesteryl esters were rapidly hydrolyzed and that the resultant free cholesterol was excreted from the cell.     

Microanalysis of antimicrobial properties of human fluids

Host defense responses of animals and plants to pathogenic microbes are mediated in part by the release of antimicrobial substances into tissue fluids. Exploration of the antimicrobial properties of tissue fluids is often limited by their small quantity. We have developed assays of antimicrobial activity that require only 1 microl of fluid. Using normal nasal secretions as a model mucosal fluid we demonstrated that the kinetics of the 1 microl colony-forming unit (CFU) assays were equivalent to the larger CFU assays. The handling of viscous mucin-containing fluids was facilitated by pretreatment with N-acetylcysteine (NAC), a treatment that did not alter the performance of the assay. This low-volume assay will facilitate studies of the antimicrobial properties of scarce biological fluids.     

Cholesteryl Esters Are Elevated in the Lipid Fraction of Bronchoalveolar Lavage Fluid Collected from Pediatric Cystic Fibrosis Patients

Background

Host-derived lipids including cholesteryl esters (CEs) such as cholesteryl linoleate have emerged as important antibacterial effectors of innate immunity in the airways and cholesteryl linoleate has been found elevated in the context of inflammation. Cystic fibrosis (CF) patients suffer from chronic infection and severe inflammation in the airways. Here, we identified and quantified CEs in bronchoalveolar lavage fluid (BALF) from CF patients and non-CF disease controls, and tested whether CE concentrations are linked to the disease.

Materials and Methods

CEs in BALF from 6 pediatric subjects with CF and 7 pediatric subjects with non-CF chronic lung disease were quantified by mass spectral analysis using liquid chromatography coupled with tandem mass spectrometry and multiple reaction monitoring. BALFs were also examined for total lipid, total protein, albumin, and, as a marker for inflammation, human neutrophil peptide (HNP) 1–3 concentrations. Statistical analysis was conducted after log 10 transformation of the data.

Results

Total lipid/protein ratio was reduced in CF BALF (p = 0.018) but the concentrations of CEs, including cholesteryl linoleate, were elevated in the total lipid fraction in CF BALF compared to non-CF disease controls (p < 0.050). In addition, the concentrations of CEs and HNP1-3 correlated with one another (p < 0.050).

Conclusions

The data suggests that the lipid composition of BALF is altered in CF with less total lipid relative to protein but with increased CE concentrations in the lipid fraction, likely contributed by inflammation. Future longitudinal studies may reveal the suitability of CEs as a novel biomarker for CF disease activity which may provide new information on the lipid mediated pathophysiology of the disease.     

Antimicrobial activity of PLUNC protects against Pseudomonas aeruginosa infection

Epithelial antimicrobial activity may protect the lung against inhaled pathogens. The bactericidal/permeability-increasing protein family has demonstrated antimicrobial activity in vitro. PLUNC (palate, lung, and nasal epithelium associated) is a 25-kDa secreted protein that shares homology with bactericidal/permeability-increasing proteins and is expressed in nasopharyngeal and respiratory epithelium. The objective of this study was to determine whether PLUNC can limit Pseudomonas aeruginosa infection in mice. Transgenic mice (Scgb1a1-hPLUNC) were generated in which human PLUNC (hPLUNC) was directed to the airway epithelium with the Scgb1a1 promoter. The hPLUNC protein (hPLUNC) was detected in the epithelium throughout the trachea and bronchial airways and in bronchoalveolar lavage fluid. Bronchoalveolar lavage fluid from transgenic mice exhibited higher antibacterial activity than that from wild type littermates in vitro. After in vivo P. aeruginosa challenge, Scgb1a1-hPLUNC transgenic mice displayed enhanced bacterial clearance. This was accompanied by a decrease in neutrophil infiltration and cytokine levels. More importantly, the overexpressed hPLUNC in Scgb1a1-hPLUNC transgenic mouse airway significantly enhanced mouse survival against P. aeruginosa-induced respiratory infection. These data indicate that PLUNC is a novel antibacterial protein that likely plays a critical role in airway epithelium-mediated innate immune response.     

Presence of regio-isomeric cholesteryl linoleate hydroperoxides and hydroxides in plasma from healthy humans as evidence of free radical-mediated lipid peroxidation in vivo

Abstract

Lipid hydroperoxides are the primary stable products of lipid peroxidation. We have developed an ultrasensitive method for the detection of lipid hydroperoxides¹ and found about 3 nM cholesteryl ester hydroperoxides (CE-OOH), mostly cholesteryl linoleate hydroperoxides (Ch18:2-OOH), in blood plasma obtained from healthy subjects.² Autoxidation of cholesteryl linoleate (Ch18:2) gives cholesteryl 13-hydroperoxy-9Z,11E-octadecadienoate (13ZE-Ch18:-OOH), cholesteryl 13-hydroperoxy-9E,11E-octadecadienoate (13EE-Ch18:2-OOH), cholesteryl 9-hydroperoxy-10E,12Z-octadecadienoate (9EZ-Ch18:2-OOH), and cholesteryl 9-hydroperoxy-10E,12E-octadecadienoate (9EE-Ch18:2-OOH). Enzymatic oxidation of Ch18:2 with 15-lipoxygenase gives predominantly only one product (13ZE-Ch18:2-OOH).³ To help elucidate the production mechanisms of cholesteryl linoleate hydroperoxides in vivo, we examined the distribution of Ch18:2-O(O)H regioisomers in human blood plasma.     

Isoprostane levels in lipids extracted from atherosclerotic arteries of nonhuman primates

Nonhuman primates used in these studies had been fed for 5 years diets enriched with cholesterol and one of three classes of fatty acids: saturated, monounsaturated, or polyunsaturated fatty acids. Atherosclerotic iliac artery lipid extracts were quantitatively analyzed for cholesterol, cholesteryl esters, fatty acid composition, and a marker of lipid oxidation, the F₂-isoprostanes. There was no significant difference in the mean accumulation of F₂-isoprostanes among the different diet groups. To account for the small, individual variation in the arachidonate concentration the F₂-isoprostane mass from each sample was normalized by dividing by arachidonate mass: F₂-isoprostane mass/(mass arachidonate). At lower levels of cholesterol accumulation, the F₂-isoprostane mass/(mass arachidonate) ratio was greater in lipids from POLY arteries compared to SAT arteries, but the reverse was true at high levels of cholesterol. F₂-isoprostane/(mass arachidonate) increased with mole fraction linoleate for the SAT group, but decreased for the POLY group. In summary, these studies demonstrated that there is no simple explanation of how F₂-isoprostane accumulation did not depend on the concentration of oxidizable lipids that promote free-radical lipid oxidation.     

Enrichment of endothelial cell arachidonate by lipid transfer from high density lipoproteins: relationship to prostaglandin I2 synthesis

We have previously shown that plasma high density lipoproteins (HDL) stimulate release of prostacyclin, measured as its stable metabolite, 6-keto-PGF1 alpha, by cultured porcine aortic endothelial cells. The present experiments were designed to elucidate the contribution of HDL lipids to endothelial cellular phospholipid pools and to prostacyclin synthesis. In experiments with reconstituted HDL, both the lipid and protein moieties were required to stimulate prostacyclin release in amounts equivalent to the native HDL particle. Endothelial cells incorporated label from reconstituted HDL containing cholesteryl [1-14C]arachidonate into the cellular neutral and phospholipid pools as well as into 6-keto-PGF1 alpha and PGE2. Labeled arachidonate incorporated into endothelial cell lipids from reconstituted HDL containing cholesteryl [1-14C]arachidonate was also metabolized to prostaglandins after the cells were exposed to the calcium ionophore, A-23187. Both rat and human HDL which stimulated 6-keto-PGF1 alpha release (rat greater than human) increased the weight percentage of arachidonate in endothelial cell phospholipids; phospholipid arachidonate in the enriched cells fell after exposure to the phospholipase activator, A-23187, with release of 6-keto-PGF1 alpha which was greater than in control cells. Rat HDL that was depleted of cholesteryl arachidonate (achieved by incubation with human low density lipoproteins (LDL) in the presence of cholesteryl ester transfer protein) stimulated 6-keto-PGF1 alpha release less than native rat HDL. LDL enriched in cholesteryl arachidonate stimulated 6-keto-PGF1 alpha release more than native LDL. ApoE-depleted HDL also stimulated 6-keto-PGF1 alpha release more than apoE-rich HDL suggesting the apoE receptor was not involved in the response.(ABSTRACT TRUNCATED AT 250 WORDS)     

CD1d-dependent macrophage-mediated clearance of Pseudomonas aeruginosa from lung

CD1d-restricted T cells are implicated as key players in host defense against various microbial infections. However, the mechanisms involved and the role they play, if any, at the mucosal surfaces where pathogenic infections are initiated is unknown. In a murine pneumonia model established by intranasal application of Pseudomonas aeruginosa, CD1d(-/-) mice showed markedly reduced pulmonary eradication of P. aeruginosa compared with wild-type mice; this was associated with significantly lower amounts of macrophage inflammatory protein-2 and reduced numbers of neutrophils within the bronchoalveolar lavage fluid. Corollarily, treatment of mice with alpha-galactosylceramide--a lipid that activates CD1d-restricted T cells--increased the amount of interferon-gamma; this was associated with rapid pulmonary clearance through enhanced phagocytosis of P. aeruginosa by alveolar macrophages. These results reveal a crucial role played by CD1d-restricted T cells in regulating the antimicrobial immune functions of macrophages at the lung mucosal surface.     

Fatty acid specificity of the lysosomal acid cholesterol esterase in intact human arterial smooth muscle cells

The fatty-acid specificity of the lysosomal cholesterol esterase was examined in cultured human arterial smooth muscle cells. The lysosomal compartment of cultured cells was enriched with cholesteryl esters by incubation of cells with 0.2 mg/ml low-density lipoprotein and 50 microM chloroquine for 24 h. The hydrolysis of cholesteryl esters was subsequently induced by incubating cells in a medium containing 5% lipoprotein-deficient serum without chloroquine. Cellular cholesteryl ester mass was markedly reduced after 23 h in the lipoprotein-deficient serum. Fatty-acid analysis of cholesteryl esters in cells before and after the 23 h incubation with lipoprotein-deficient serum revealed that polyunsaturated cholesteryl esters (linoleate and arachidonate) were preferentially hydrolyzed compared to cholesteryl oleate or saturated cholesteryl esters. An increase in the ratio of cholesteryl oleate to cholesteryl linoleate was observed even when the cellular activity of acyl-CoA:cholesterol acyltransferase was inhibited with Sandoz Compound 58-035. We conclude that, in human arterial smooth muscle cells, the lysosomal acid cholesterol esterase preferentially hydrolyzes polyunsaturated cholesteryl esters.     

Thin-layer chromatography blotting for the fluorescence detection of phospholipid hydroperoxides and cholesteryl ester hydroperoxides

A blotting technique was developed to specifically detect lipid hydroperoxides in thin-layer chromatography. Phosphatidylcholine hydroperoxides and cholesteryl linoleate hydroperoxides ranging from 0.1 to 0.5 nmol, which were prepared by reaction with soybean lipoxygenase, were visualized as fluorescent spots on the blotted membrane by immersing the plate into a blotting solvent containing 0.01% (w/v) diphenyl-1-pyrenylphosphine. This technique was applied successfully to monitor lipid peroxidation in human low-density lipoprotein in vitro.     

Effect of the stress of unilateral adrenalectomy on the depletion of individual cholesteryl esters in the rat adrenal

Normal rats were subjected to unilateral adrenalectomy and were killed 3 hr later. The concentration and composition of the cholesteryl esters in adrenals removed at operation and after death were compared. The esterified cholesterol concentration was lower in the adrenals obtained 3 hr after surgery. Cholesteryl arachidonate decreased in concentration significantly more than any other ester, followed in order of magnitude by linoleate and oleate. The cholesteryl ester concentration of adrenals removed from sham-operated rats 3 hr after surgery was also greatly reduced. On the basis of comparison with other work on the hydrolysis of cholesteryl esters by adrenal homogenates, it is concluded that the apparent selectivity in depletion of cholesteryl esters is due to differences in their rates of hydrolysis.     

Isolation and specificity of rat lecithin: cholesterol acyltransferase: comparison with the human enzyme using reassembled high-density lipoproteins containing ether analogs of phosphatidylcholine

Rat plasma lecithin: cholesterol acyltransferase, a 68 kDa glycoprotein, has been purified 14 000-fold by a modification of a procedure used for the human enzyme. The activity of lecithin: cholesteryl acyltransferase in human and rat plasma are the same, although activation of both enzymes by human apolipoprotein A-I is greater than that produced by rat apolipoprotein A-I. Using reassembled high-density lipoproteins composed of human apolipoprotein A-I, phosphatidylcholine ethers and a series of different phosphatidylcholines, the separate effects of molecular species specificity and microenvironment on the rate of cholesteryl ester formation was determined. Substitution of a fluid lipid, 1-palmityl-2-oleyl-sn-glycero-3-phosphorylcholine, for a solid lipid, 1,2-dipalmityl-sn-glycero-3-phosphorylcholine, produced an 8-fold increase in the activity of all molecular species of phosphatidylcholine. With either solid or fluid lipid environments, the activity decreased as a function of increasing chain length of saturated acyl groups. Addition of one or more double bonds greatly increased the activity of a given saturated homologue. One major difference between the molecular specificity of rat and human lecithin: cholesteryl acyltransferase was that the latter had a two-fold preference for phosphatidylcholines containing arachidonate at the sn-2-position.     

Functional interaction of human neutrophil peptide-1 with the cell wall precursor lipid II

Defensins constitute a major class of cationic antimicrobial peptides in mammals and vertebrates, acting as effectors of innate immunity against infectious microorganisms. It is generally accepted that defensins are bactericidal by disrupting the anionic microbial membrane. Here, we provide evidence that membrane activity of human α-defensins does not correlate with antibacterial killing. We further show that the α-defensin human neutrophil peptide-1 (HNP1) binds to the cell wall precursor lipid II and that reduction of lipid II levels in the bacterial membrane significantly reduces bacterial killing. The interaction between defensins and lipid II suggests the inhibition of cell wall synthesis as a novel antibacterial mechanism of this important class of host defense peptides.     

Functional characterisation of the 1-18 fragment of esculentin-1b, an antimicrobial peptide from Rana esculenta

Esculentin-1 is a 46-amino acid residue peptide isolated from skin secretions of Rana esculenta, displaying the most potent antimicrobial activity among the bioactive molecules found in the secretion, with negligible effects on eukaryotic cell membranes. From skin secretions, the 19-46 fragment of esculentin-1, devoid of antibacterial activity, was also isolated. We studied in detail the activity of the N-terminal fragment (1-18) of esculentin-1 using a synthetic amidated analogue. The results show that this fragment is highly active against most bacterial and fungal species, although at a lower extent than the full-length peptide, being four-fold more potent against Phytophthora nicotianae. It has a reduced activity against human erythrocytes with respect to the full-length peptide. The killing curves in liquid medium are similar for the two molecules and the shorter peptide is able to increase the bacterial outer and inner membrane permeability. Overall these data indicate that the antimicrobial properties of esculentin-1 are exerted by its N-terminal 1-18 region and that the positively charged residue distribution as well as peptide length represent important determinants for cell selectivity.     

The activity of cholesterol ester hydrolase in the enzymatic determination of cholesterol: comparison of five enzymes obtained commercially

The use of five cholesterol ester hydrolases (CEH), numbered 1 to 5, for the enzymatic determination of total cholesterol of human and rat serum are compared. All CEH gave approximately the same value (no statistical difference) for human serum. However, when rat serum cholesterol was determined, CEH-2 yielded a value significantly lower when compared to the four other CEH. The ability of each CEH to hydrolyze individual cholesterol esters was tested. During a 15-min incubation, all CEH were capable of hydrolyzing nearly 100% of cholesteryl oleate and linoleate. In contrast, the hydrolysis of cholesteryl arachidonate was only partial and varied from 20 to 80% depending on the CEH used. The highest hydrolysis was obtained by CEH-1 while the value given by CEH-2 was only 22% of that obtained by CEH-1. The rate of hydrolysis of cholesteryl arachidonate differed markedly among the CEH. The CEH-2-hydrolyzed the cholesteryl arachidonate at a rate seven times lower than the rate obtained with CEH-1. The data suggest that, Under our incubation conditions, CEH-2 did not properly hydrolyze the cholesteryl arachidonate. This phenomenon may be crucial whenever total cholesterol has to be determined enzymatically in the serum of species that contain large amount of cholesteryl arachidonate such as rat, mouse, or dog serum.     

Specificity of the ferrous oxidation of xylenol orange assay: analysis of autoxidation products of cholesteryl arachidonate

Autoxidation of polyunsaturated fatty acids and esters leads to a complex mixture containing hydroperoxides and cyclic peroxides. The oxidation mixture of cholesteryl arachidonate, which has been characterized by a variety of mass spectrometry techniques, was subject to analysis by conventional thiobarbituric acid-reactive substance (TBARS) and ferrous oxidation in xylenol orange (FOX) assays. Our results indicate that the FOX assay is not specific for hydroperoxides. Cyclic peroxides, such as monocyclic peroxides and serial-cyclic peroxides, give a positive FOX response even after triphenylphosphine reduction. We suggest that bicyclic endoperoxides are the major TBARS active compounds present in cholesteryl arachidonate oxidation mixtures. These compounds give a positive FOX assay before reaction with triphenylphosphine but negative TBARS and FOX assays after this reaction. Caution should be exercised when the FOX assay is used to analyze highly oxidized lipids, especially arachidonyl-containing lipids.