Elevated oxidized lipids, anti-lipid autoantibodies and oxidized lipid immune complexes in active SLE.

BACKGROUND: Here, we explore the serum levels of anti-oxidized lipid autoantibodies as well as immune complexes in patients with SLE and determine their correlation with disease. METHODS: Serum levels of oxidized-LDL immune complexes, autoantibodies to dsDNA, ox-LDL, MDA-LDL, 9-HODE, 13-HODE and POVPC were detected by ELISA in 64 SLE patients and 9 healthy controls. RESULTS: Active SLE patients exhibited increased serum levels of autoantibodies compared to healthy controls, including anti-MDA-LDL-IgG (p = .003), anti-ox-LDL-IgG (p = .004), anti-9-HODE-IgG (p = .001), anti-13-HODE-IgG (p = .0003), anti-POVPC-IgG (p = .001) and ox-LDL-IC (p = .003). Serum anti-ox-LDL-IgG was positively correlated with SLEDAI (r = 0.34; p = .01), and negatively with C3 (r = -0.40; p = .01). Anti-9-HODE-IgG and anti-POVPC-IgG were positively correlated with SLEDAI and negatively with C4. CONCLUSIONS: Active SLE patients exhibit significantly increased serum levels of IgG anti-oxidized-lipid autoantibodies. Coordinated elevation of oxidized lipids, autoantibodies to these lipids, and immune complexes of these lipid-antibody components could potentially serve as pathogenic drivers and serum markers of SLE disease activity.

12/15-lipoxygenase expressed in non-epithelial cells causes airway epithelial injury in asthma.

The mechanisms underlying asthmatic airway epithelial injury are not clear. 12/15-lipoxygenase (an ortholog of human 15-LOX-1), which is induced by IL-13, is associated with mitochondrial degradation in reticulocytes at physiological conditions. In this study, we showed that 12/15-LOX expressed in nonepithelial cells caused epithelial injury in asthma pathogenesis. While 12/15-LOX overexpression or IL-13 administration to naïve mice showed airway epithelial injury, 12/15-LOX knockout/knockdown in allergic mice reduced airway epithelial injury. The constitutive expression of 15-LOX-1 in bronchial epithelia of normal human lungs further indicated that epithelial 15-LOX-1 may not cause epithelial injury. 12/15-LOX expression is increased in various inflammatory cells in allergic mice. Though non-epithelial cells such as macrophages or fibroblasts released 12/15-LOX metabolites upon IL-13 induction, bronchial epithelia didn't release. Further 12-S-HETE, arachidonic acid metabolite of 12/15-LOX leads to epithelial injury. These findings suggested 12/15-LOX expressed in non-epithelial cells such as macrophages and fibroblasts leads to bronchial epithelial injury.

The cytochrome P450 inhibitor, ketoconazole, inhibits oxidized linoleic acid metabolite-mediated peripheral inflammatory pain.

BACKGROUND: Oxidized linoleic acid metabolites (OLAMs) are a class of endogenous agonists to the transient receptor potential V1 (TRPV1) receptor. Although TRPV1 mediates inflammatory heat hyperalgesia, it is not known if the OLAMs contribute to the peripheral activation of this receptor during tissue inflammation. In the present study, we evaluated whether the OLAM system is activated during inflammation and whether cytochrome P450 enzymes mediate OLAM contributions to heat hyperalgesia using the complete Freund's adjuvant (CFA) model of inflammation. RESULTS: Our results demonstrate that the intraplantar (ipl) injection of anti-OLAM antibodies significantly reversed CFA-induced heat hyperalgesia. Moreover, application of lipid extracts from inflamed rat skin to cultured sensory neurons triggered a significant release of iCGRP that is blocked by co-treatment with I-RTX, a TRPV1 antagonist. To determine the role of CYP enzymes in mediating OLAM effects, we used a broad spectrum CYP inhibitor, ketoconazole. Pretreatment with ketoconazole inhibited the release of TRPV1 agonists in lipid extracts from inflamed skin and significantly reversed CFA-induced heat hyperalgesia by a peripheral mechanism of action. Moreover, the ipl injection of linoleic acid to rats 24 hr after CFA evoked spontaneous nocifensive behaviors that were significantly reduced by capsazepine, by knockout of the TRPV1 gene, or by pretreatment with either anti-OLAM antibodies or ketoconazole. CONCLUSIONS: Taken together, our data suggests that OLAMs contribute to inflammatory nociception in the periphery and that cytochrome P450 enzymes play a crucial role in mediating OLAM contributions to inflammatory heat hyperalgesia.

Structure-activity relationships in toll-like receptor 2-agonists leading to simplified monoacyl lipopeptides.

Toll-like receptor 2-agonistic lipopeptides typified by S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (PAM(2)CS) compounds are potential vaccine adjuvants. In continuation of previously reported structure-activity relationships on this chemotype, we have determined that at least one acyl group of optimal length (C(16)) and an appropriately oriented ester carbonyl group is essential for TLR2-agonistic activity. The spacing between one of the palmitoyl ester carbonyl and the thioether is crucial to allow for an important H-bond, which observed in the crystal structure of the lipopeptide:TLR2 complex; consequently, activity is lost in homologated compounds. Penicillamine-derived analogues are also inactive, likely due to unfavorable steric interactions with the carbonyl of Ser 12 in TLR2. The thioether in this chemotype can be replaced with a selenoether. Importantly, the thioglycerol motif can be dispensed with altogether and can be replaced with a thioethanol bridge. These results have led to a structurally simpler, synthetically more accessible, and water-soluble analogue possessing strong TLR2-agonistic activities in human blood.

Immunochemical detection of a novel lysine adduct using an antibody to linoleic acid hydroperoxide-modified protein.

We have previously prepared the polyclonal antibody to the 13-hydroperoxyoctadecadienoic acid-modified protein (13Ab) (Kato et al. 1997. J. Lipid Res. 38: 1334-1346), however, the epitopes have not yet been structurally identified. In this study, we identified a novel amide-type adduct as one of the major epitopes of 13Ab and characterized the endogenous formation. Upon incubation of the lysine derivative with peroxidized linoleic acid, the formation of N epsilon -(azelayl)lysine (AZL) was confirmed using liquid chromatography-mass spectrometry. The chemically synthesized azelayl protein was significantly recognized by 13Ab. The peroxidation products of different polyunsaturated fatty acids also generated several analogous carboxyalkylamide-type adducts to AZL by the reaction with the lysine derivative, whereas 13Ab specifically recognized AZL, suggesting that the AZL moiety may be one of the major epitopes of 13Ab. The immunoreactive materials of 13Ab were immunohistochemically detected in atherosclerotic lesions from hypercholesterolemic rabbits. More strikingly, the immunoreactivity was significantly enhanced when the sections were treated with alkali or phospholipase A2 for hydrolyzing the ester bonds prior to the staining. These results suggest that the lipid hydroperoxide-derived carboxylic adducts, such as AZL, and their esters linked with phospholipids may be generated in vivo and involved in the pathogenesis of atherosclerosis associated with oxidative stress.

Effects of conjugated linoleic acid (CLA) on immune responses, body composition and stearoyl-CoA desaturase.

Conjugated linoleic acid (CLA) has shown a wide range of biologically beneficial effects; reduction of incidence and severity of animal carcinogenesis, reduction of the adverse effects of immune stimulation, reduction of severity of atherosclerosis, growth promotion in young rats, and modulation of stearoyl-CoA desaturase (SCD). One of the most interesting aspects of CLA is its ability to reduce body fat while enhancing lean body mass which is associated with the trans-10,cis-12 isomer of CLA. The effects of CLA are unique characteristics that have not been observed with other polyunsaturated fatty acids. In this review, we will focus on the effects of CLA on immune responses, body compositional changes and stearoyl-CoA desaturase.

Influence of dietary conjugated linoleic acid isomers on early inflammatory responses in male broiler chickens.

1. The influence of dietary conjugated linoleic acid isomer (CLA, 0 and 10 g/kg) on the metabolic and physiological responses to immune stimulation induced by a single injection of Salmonella enteritidis lipopolysaccharide (LPS) or repeated injections of LPS and Sephadex G-50 was determined in male broiler chicks. 2. In experiment 1, 10-d-old chicks were fed on experimental diets for 14 d and half of the birds fed on each diet were injected intraperitoneally with LPS (1.5 mg/kg body weight). In experiment 2,7-d-old chicks were fed on experimental diets for 18 d. Immune stimulation was started at 19 d old and continued for 5 d. Half of the birds fed on each diet were injected intraperitoneally with 0.25 mg/kg body weight of LPS at 19, 21 and 23 d of age, and with 250 mg/kg body weight of Sephadex at 20 and 22 d of age to stimulate the immune system. 3. In experiment 1, giving CLA prevented an increase in blood heterophil to lymphocyte ratio 7 h after a single injection of LPS, and increases in plasma ceruloplasmin and alpha 1 acid glycoprotein (AGP) 24 h after the injection, but not 7 h after the injection. CLA also prevented a decrease in food intake for 24 h after LPS injection. 4. In experiment 2, the CLA diet partially prevented reductions in body weight gain and weight gain to feed intake ratio caused by repeated injections of LPS and Sephadex. Feeding CLA prevented increases in plasma ceruloplasmin and AGP at 24 d of age caused by repeated injections of LPS and Sephadex, but not at 20 d of age. 5. These results suggest that feeding CLA alleviates some undesirable metabolic and physiological changes induced by immunological stimulation in male broiler chicks.

Detection of oxidized phospholipid-protein adducts using anti-15-hydroperoxyeicosatetraenoic acid-modified protein antibody: contribution of esterified fatty acid-protein adduct to oxidative modification of LDL.

The reaction of lipid hydroperoxide with protein was investigated using an antibody, which was prepared using 15-hydroperoxyeicosatetraenoic acid (15-HPETE)-modified keyhole limpet hemocyanin as an immunogen. The obtained antibody recognized not only 15-HPETE-modified bovine serum albumin (BSA) but also 13-hydroperoxyoctadecadienoic acid (13-HPODE)-modified BSA. Glutaroyl-BSA adduct, which was prepared by the reaction of glutaric anhydride with protein, was also recognized by the antibody. The results revealed that the carboxyl terminus of lipid moiety in adducts was required for an appearance of the antigenicity. The cross-reactivity of phosphatidylcholine hydroperoxide-modified BSA (PCAOOH-BSA) with the antibody was examined. The antibody could not recognize the intact PCAOOH-BSA, whereas alkaline-treated modified BSA revealed the antigenicity. Furthermore, stearic acid at the 1 position in the phospholipid was liberated from the PCAOOH-BSA following treatment with 0.25 N NaOH. The result showed that the phospholipid moiety could be covalently bound to the protein molecule. The formation of esterified fatty acid-protein adduct during oxidation was confirmed using low-density lipoprotein (LDL). During oxidation of LDL by copper ion or 2,2'-azo-bis(2-amidinopropane)dihydrochloride, the formation of antigenic materials was observed in a time- or dose-dependent fashion. The antigenicity was significantly enhanced by the alkaline treatment on the oxidized LDL, suggesting that considerable amounts of oxidized esterified fatty acids can covalently react with apoprotein B-100 in oxidatively modified LDL.

Characterization of a specific polyclonal antibody against 13-hydroperoxyoctadecadienoic acid-modified protein: formation of lipid hydroperoxide-modified apoB-100 in oxidized LDL.

Lipid hydroperoxide may react with protein or amino phospholipid without secondary decomposition. We prepared a polyclonal antibody to lipid hydroperoxide-modified proteins using 13S-hydroperoxy-9Z, 11E-octadecadienoic acid-modified keyhole limpet hemocyanin (13-HPODE-KLH) as immunogen. The antibody recognized 13-HPODE-modified bovine serum albumin (BSA), but not aldehyde-modified proteins, such as malondialdehyde-modified BSA. The antibody also recognized adducts derived from 13-HPODE and 13S-hydroperoxy-9Z, 11E, 15Z-octadecatrienoic acid (13-HPOTRE(alpha)). The oxidized alpha-linolenic acid- and linoleate-protein adducts were recognized by the antibody. Oxidized phospholipid-protein adducts were scarcely recognized by the antibody. However, when ester bonds of phospholipids containing linoleic acid were hydrolyzed by alkaline treatment, the cross-reactivities appeared. The result suggests that a phospholipid hydroperoxide can react with a protein directly or indirectly, and a carboxyl terminal (COOH) of the lipid in an adduct was needed as an epitope. Oxidized LDL (ox-LDL) was prepared by the incubation of LDL with copper ion or 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and the formation of lipid hydroperoxide-modified apolipoprotein was confirmed using the antibody. A slight immunoreactivity was observed in ox-LDL without alkaline treatment. When the ox-LDL was treated with alkali to hydrolyze the ester bonds of the lipid, enhanced antigenicity appeared with time-dependency. The results suggest that lipid hydroperoxide-modified apolipoprotein was formed during the oxidation of LDL.

Production and characterization of antibodies reactive with leukotoxin.

A sensitive radioimmunoassay was developed for the determination of leukotoxin (9: 10-epoxy-12-octadecenoic acid), which was reported to exist in human burned skin and neutrophils, and was regarded as a toxic and/or defensive substance in living beings. The leukotoxin was conjugated with bovine serum albumin by means of the mixed anhydride technique as immunogen and rabbits were immunized over 4 months. According to a titration test of antiserum, forty-fold diluted antiserum was found to bind approximately 50% of about 4,000 dpm of methylated leukotoxin labeled with [14C] carbon. The detection limit of leukotoxin was at least as low as 5 ng in this radioimmunoassay by use of a polyetylene glycol precipitation method. This antiserum had a strong specificity to leukotoxin and no cross-reactivity with the other analogs tested.

Quantitation of 13-hydroxyoctadecadienoic acid (13-HODE) by radioimmunoassay.

Antibodies against 13-hydroxyoctadecadienoic acid (13-HODE) were produced in rabbits by immunizing the animal with 13-HODE-thyroglobulin conjugate. The antibodies appeared to be rather specific for 13-HODE since other hydroxy fatty acids showed minimal crossreaction. The radioimmunoassay was capable of detecting 50 pg per assay tube and was applied to the study of the biosynthesis of 13-HODE in platelets and leukocytes. In contrast to reported findings from endothelial cells, A-23187, thrombin and collagen stimulated synthesis and release of 13-HODE from platelets. However, insignificant synthesis of 13-HODE was found in leukocytes following A-23187 stimulation. Exogenous addition of linoleic acid stimulated the synthesis of 13-HODE from both platelets and leukocytes. The majority of 13-HODE synthesized was found in the medium. These studies suggest that both types of blood cells possess active (omega-6) lipoxygenase. Platelets may use endogenously released linoleic acid to synthesize 13-HODE, whereas leukocytes may utilize linoleic acid released from other cell types for 13-HODE synthesis.

Immunogenicity of fatty acid anilides in rabbits and the pathogenesis of the Spanish toxic oil syndrome.

Fatty acid anilides, the major xenobiotic found in the cooking oils responsible for the Spanish toxic oil syndrome, are immunogenic for rabbits as ascertained by a skin test reaction, the characterization of specific antibodies against anilides and the immunofluorescent detection of 'anilide dependent antigens' in tissue slices from treated animals.

Immunodiagnostic of multiple sclerosis by estimation of lymphokines in vitro and in vivo.

The basis of our investigations is a hypothesis about immunomechanisms responsible for aetiopathogenesis of multiple sclerosis including immungenetic connection (predisposition) and autoimmunity (pathogenesis). Some new results about cellular immunity using cell electrophoresis mobility technique, especially sensitization against normal brain tissue antigen (NTA) and investigations of lymphokines in cerebrospinal fluid are represented. The intention of this paper is to demonstrate a combined diagnostic regime for early detection of multiple sclerosis as well as for follow-up studies in course of treatment.

Immunostimulation or immunodepression?

Our experimental studies and extensive literature survey established that the overall response of an experimentally modified host defense system is strongly dose-dependent, irrespective of either the modifying agent or the test model system. If a sufficiently broad dose range is used, an irregular, nonlinear, nonmonotonic response curve is formed, generally W- or M-shaped, depending on the parameters selected for the representation, with two peaks of relative maximal effect. Accordingly, a modifying agent may exhibit, in some cases, dual effect--stimulation and depression, each at different dose levels. This typical response is defined as characteristic of the entire host defense system and therefore implies a biologically integrated system. The described dose-response relationship mandates the use of multiple doses in evaluation experiments, to establish efficacy and especially to design optimal dose schedules for experimental and clinical application of any agent modifying the host defense system activity.

Importance of the spleen for the immuno-inhibitory aciton of linoleic acid in mice.

Certain immuno-inhibitory effects of a polyunsaturated fatty acid, linoleic acid (C18:2), no longer occur after splenectomy of young adult CBA mice. This observation suggest that the spleen is a major intermediary in the action of C18:2 on the lymphoreticular system. Pathways of this action remain to be elucidated. Possibilities include C18:2-stimulated suppressor cell generation in the spleen, or excess biosynthesis of immuno-inhibitory prostaglandins by splenic macrophages.

Receptor-dependent hydrolysis of cholesteryl esters contained in plasma low density lipoprotein.

Selective radioactive labeling of the cholesteryl esters contained within human plasma low density lipoprotein has allowed the study of the metabolism of these molecules in monolayers and extracts of cultured human fibroblasts. In monolayers of normal cells, binding of low density lipoprotein to its cell surface receptor was followed by rapid hydrolysis of the [3H]cholesteryl linoleate contained within the lipoprotein and accumulation of the liberated [3H]cholesterol within the cell. The stoichiometry of the degradative process suggested that the protein and cholesteryl ester components of the lipoprotein were hydrolyzed in parallel. Incubation of intact fibroblasts with chloroquine, a known inhibitor of lysosomal degradative processes, inhibited the hydrolysis of the lipoprotein-bound cholesteryl esters. Fibroblasts from subjects with the homozygous form of familial hypercholesterolemia, which lack functional low density lipoprotein receptors and thus are unable to take up this lipoprotein when it is present in the culture medium at low concentrations, were therefore unable to hydrolyze the lipoprotein-bound [3H]cholesteryl linoleate. However, cell-free extracts from these mutant cells were capable of hydrolyzing the lipoprotein-bound [3H]cholesteryl linoleate at the same rapid rate as normal cells when incubated at acid pH. These data illustrate the essential role of the low density lipoprotein receptor and of lysosomal acid hydrolases in the metabolic utilization of low density lipoproteins by cultured human fibroblasts.