© 1997 American Heart Association, Inc.
Articles |
Inhibition of Stimulus-Induced Endothelial Cell Intercellular Adhesion Molecule-1, E-Selectin, and Vascular Cellular Adhesion Molecule-1 Expression by Arachidonic Acid and Its Hydroxy and Hydroperoxy Derivatives
Department of Immunopathology and University of Adelaide Department of Paediatrics (Z.H.H., E.J.B., J.V.F., C.S.T.H., B.S.R, A.F.), and the Department of Chemical Pathology (A.P., B.S.R.), Women's and Children's Hospital, North Adelaide, South Australia.
Correspondence to Prof A. Ferrante, Department of Immunopathology, Women's and Children's Hospital, 72 King William Rd, North Adelaide, South Australia 5006.
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Abstract |
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Localized adhesion of peripheral blood leukocytes to the endothelial lining is essential for their exit from the blood under both physiological and pathological conditions. The establishment, development, and resolution of the inflammatory response is regulated by an array of mediators, many of which remain to be categorized. These include arachidonic acid (20:4n-6) and its hydroperoxy (HPETE) and hydroxy (HETE) derivatives, which are released during inflammation. The data show that human umbilical vein endothelial cells, pretreated with these fatty acids, have a reduced ability to be stimulated by tumor necrosis factor-
(TNF-) for enhanced neutrophil and monocyte adhesion; the order of inhibitory activity being 15-HPETE>15-HETE>20:4 (n-6). This fatty acid–induced inhibitory activity was reflected in the ability of the mediators to decrease the TNF-–induced expression of the following endothelial adhesion molecules: intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1), measured by both enzyme-linked immunosorbent assay and flow cytometric analysis. TNF-–induced increased expression of ICAM-1, E-selectin, and VCAM-1 mRNA was significantly depressed by 15-HPETE. Constitutively expressed ICAM-1 and ICAM-1 mRNAs were unchanged by the fatty acids. The saturated fatty acid 20:0 and the methyl ester of 20:4(n-6) had no inhibitory activity. The binding of TNF- to its receptors was not altered by these fatty acids. The fatty acids also inhibited the expression of ICAM-1 and E-selectin induced by phorbol 12-myristate 13-acetate, showing that inhibition occurred at a post–TNF- receptor binding level. The 15-HPETE was found to inhibit the TNF-–induced increase in adhesion molecule expression in the early stage of the incubation, but expression returned to normal after 18 hours. An effect of 15-HPETE on the early cell signaling system was demonstrated by the ability of this fatty acid to inhibit agonist-induced protein kinase C translocation.
Key Words: hydroperoxyeicosatetraenoic acid • monohydroxyeicosatetraenoic acid • 20:4(n-6) • adhesion molecule-1 • endothelial cell
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Localized adhesion of peripheral blood leukocytes to the endothelial lining is essential for their exit from the vascular space under both physiological and pathological conditions and is a critical event in the pathogenesis of certain vascular diseases. Adhesion of leukocytes to endothelial cells is a complex process involving adhesion molecules on both the leukocyte and the endothelial cell. At least four receptors on endothelial cells have been implicated in such interactions.1 Of these, ICAM-1 (CD54), E-selectin (CD62E), and VCAM-1 (CD106) play an important role in the tethering, activation, attachment, and transmigration of leukocytes into the extravascular space.2 3 4 5 6 7 8 9 ICAM-1, unlike E-selectin, is constitutively expressed by endothelial cells.10 11 The expression of both of these adhesion receptors is markedly upregulated by proinflammatory cytokines, such as interleukin-1ß and TNF-
,10 11 12 and by PMA, a direct stimulator of PKC.13 Expression is dependent on protein and mRNA synthesis11 14 and is characteristically evident by 4 hours and, for ICAM-1, remains high at 24 hours,10 whereas E-selectin expression peaks at 4 hours and rapidly declines to basal levels by 16 to 24 hours.11 VCAM-1 is not constitutively expressed but can be induced by cytokines, such as TNF-. Peak expressions after TNF- treatment occur after 6 to 8 hours of incubation, and expression persists for up to 72 hours.15 These adhesion molecules have been shown to be involved in vascular injury in animal models,16 17 acute and chronic airway inflammation,17 18 19 neutrophil-mediated damage associated with ischemic reperfusion injury in the heart,20 renal inflammation,21 and tumor cell adhesion to endothelium.22 23 ICAM-1 also serves as a receptor for two pathogens, namely rhinovirus, the causative agent of the common cold,24 and the malarial trophozoite Plasmodium falciparum.25 Many cell types, when stimulated, liberate arachidonic acid 20:4(n-6) from membrane phospholipids mainly through the action of phospholipase A2.26 20:4(n-6) is generally considered to be an inflammatory polyunsaturated fatty acid by virtue of the range of inflammatory eicosanoids to which it is metabolized.27 Recently, fatty acids such as 20:4(n-6) have also been suggested to act as second messengers, because they exert direct effects on components of signal transduction pathways.28 Within the blood system, under inflammatory and prothrombotic conditions, platelets and leukocytes interact with the endothelium and also between themselves,29 during which time they synthesize and release 20:4(n-6) together with a variety of its lipoxygenase and cyclooxygenase products.29 30 31 32 The first steps in the oxygenation of 20:4(n-6) by the lipoxygenase pathway generate a series of HPETEs. These HPETEs are the biosynthetic precursors of HETEs and are intermediates in the generation of leukotrienes.33 Metabolism of 20:4(n-6) through the cyclooxygenase pathway yields prostaglandins and thromboxanes. Recently 20:4(n-6) was found to inhibit neutrophil migration, with its HPETE and HETE derivatives having only moderate or negligible activity, respectively.34 In addition, 15-HETE, but not 5-HETE or 12-HETE, has been found to decrease neutrophil migration across endothelium in response to leukotriene B4 and other chemoattractants through a remodeling of neutrophil phospholipids and an associated reduction in the affinity of cell-surface receptors.35 These data demonstrate a potential anti-inflammatory function for such fatty acids. In the present study, we have assessed whether 20:4(n-6) together with 15-HPETE and 15-HETE can influence the expression of the adhesion receptors ICAM-1, E-selectin, and VCAM-1 on HUVECs.
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Materials and Methods |
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Unless indicated otherwise, general laboratory chemicals were from Sigma Chemical Co.
Isolation and Culture of Endothelial Cells
HUVECs were isolated from umbilical cords stored at 4°C after delivery as described previously36 but with 0.2% (wt/vol) gelatin (Cytosystems) to coat all tissue culture flasks and plates, 0.07% (wt/vol) collagenase (from Clostridium histolyticum, type II, Worthington) to digest the interior of the umbilical vein, and a culture medium consisting of RPMI 1640 (ICN-Flow) containing 40 mmol/L TES, 15 mmol/L D-glucose, 80 U/mL penicillin (Flow), 80 µg/mL streptomycin (Flow), and 3.2 mmol/L L-glutamine, which was brought to 260 to 300 mOsm/L before the addition of 20% (vol/vol) pooled heat-inactivated (56°C, 30 minutes) human group AB serum. Endothelial cells were identified by their characteristic contact-inhibited cobblestone morphology and positive staining for factor VIII–related antigen using peroxidase-conjugated anti-rabbit IgG to human von Willebrand factor (Dako) and 3,3'-diaminobenzidine. The cells from two cords were pooled for each experiment.
Confluent cultures were subcultured after a 2- to 5-minute exposure to trypsin (0.05% [wt/vol], Flow)–EDTA (0.02% [wt/vol]). For experimental use, second-passage cells were plated at 1 to 5x104 cells per well per 0.2 mL culture medium in 96-well culture plates (0.38-cm2 area per well, Linbro, Flow) for ELISA, 1x105 cells per well per 0.5 mL culture medium in 24-well plates (2.0-cm2 area per well, Linbro, Flow) for flow cytometry analyses, or 2x105 cells per well per 1.0 mL culture medium in six-well plates (9.4-cm2 area per well, Linbro, Flow) for mRNA studies and grown to confluence (2 to 7 days).
Solubilization of Fatty Acids
DPC, arachidonic acid 20:4(n-6), arachidic acid 20:0, and the methyl ester of 20:4(n-6) were obtained from Sigma Chemical Co. To overcome fatty acid insolubility in aqueous solution, we prepared mixed DPC (400 µg)–fatty acid (100 µg) micelles in HBSS by sonication, as previously described.37 Control incubations contained micelles of DPC alone appropriately diluted. Fatty acids were checked for purity by silica gel thin-layer chromatography in diethyl ether/hexane/glacial acetic acid (60:40:1 [vol/vol/vol]). The plates were visualized with iodine. There was no evidence of oxidation products of commercial fatty acids with storage.
Preparation of HPETE and HETE Derivatives of 20:4(n-6)
15-HPETE [15-(S)-monohydroperoxy 20:4(n-6)] was obtained by incubating 20:4(n-6) (50 mg in 1 mL ethanol) with soybean lipoxidase (7 mg, type 1B, Sigma) in 100 mL of 0.1 mol/L sodium borate, pH 9.0, for 20 minutes at room temperature.38 The reaction mixture was acidified with 5 mL of 1 mol/L citric acid, and fatty acid derivatives were extracted three times with 50 mL diethyl ether. The sample was evaporated under N2 and taken up in 3 mL hexane/diethyl ether (9:1 [vol/vol]) before application to a silicic acid column (4 g) equilibrated in the same solvent. Unoxidized 20:4(n-6) was eluted with 120 mL of this solvent, and 15-HPETE was eluted with 120 mL hexane/diethyl ether (1:1 [vol/vol]). A portion (5 mg) of 15-HPETE was reduced to 15-HETE with 2 mg sodium borohydride for 2 hours at 4°C and recovered by the addition of 2.5 mL water, acidification with formic acid, and extracted three times with 4 mL of diethyl ether. Fatty acids and their derivatives [20:4(n-6), 15-HPETE, and 15-HETE] were stored in chloroform at -20°C and purity-checked by thin-layer chromatography and, where possible, by gas-liquid chromatography. The HPETE and HETE derivatives consisted of at least 95% 15-HPETE and 15-HETE, respectively, with other isomers making up the rest. There was no evidence of 20:4(n-6) in the preparations.
Treatment of Endothelial Cells With Fatty Acids
Confluent monolayers (in 96-well plates) were washed once with RPMI 1640 (200 µL per well) before incubation at 37°C for 30 minutes in a total volume of 100 µL E-SFM (GIBCO). Incubations were in triplicate with the indicated concentrations of fatty acids. Control incubations contained vehicle alone. At the end of the incubation, 150 µL of E-SFM containing TNF- (125 U per well), PMA (final concentration, 10-7 mol/L), or diluent (DMSO) was added, and incubation continued for a further 4 hours at 37°C. Recombinant human TNF- (Genentech) was a gift from Dr G.R. Adolf, Ernst Boehringer Institut, Vienna, Austria.
Leukocyte/HUVEC Adhesion
For these studies, neutrophils and monocytes were prepared from the heparinized blood of healthy volunteers by a rapid single-step method39 in which blood was layered onto a Ficoll-Hypaque medium (density, 1.114g/L) and centrifuged at 600g for 30 to 40 minutes at room temperature. The leukocytes resolved into two distinct bands, the upper band, containing mononuclear leukocytes, and the lower band, containing neutrophils. Purified monocytes were obtained from the upper layer by adherence to cytodex 3 microcarriers as described previously.40 The lower neutrophil band was washed three times in HBSS before use.36
At the end of the incubation of HUVECs with fatty acids plus TNF-, the monolayers were washed once with 200 µL RPMI 1640 before incubation for 30 minutes at 37°C in the absence or presence of 5x105 neutrophils or monocytes in E-SFM (final volume, 100 µL). Nonadherent cells were removed by gentle aspiration, and the wells were washed twice with 200 µL HBSS containing 0.1% (wt/vol) BSA before staining with rose bengal.41 After release of the dye with 50% ethanol, the absorbance (570 nm) of each well was determined with an ELISA plate reader. Test and blank wells were performed in triplicate. Results were calculated after subtraction of the mean blank value (without leukocytes) from each test value (plus leukocytes).
ELISA for Adhesion Molecules on HUVECs
At the end of the incubation of HUVECs with fatty acids plus TNF- or PMA, the monolayers were washed once with 200 µL RPMI 1640 and fixed by incubation for 10 minutes at room temperature with 200 µL of a solution of 2% (wt/vol) paraformaldehyde, 0.075% (wt/vol) L-lysine monohydrochloride, and 2.1 mg/mL sodium periodate in HBSS.42 Fixation was stopped by aspiration of fixative, and the monolayers were washed twice with 200 µL of 0.1% (wt/vol) BSA in HBSS before the addition of 200 µL of 100 mmol/L glycine and 0.1% (wt/vol) BSA in HBSS to block reactive aldehyde groups. The plates were stored at 4°C for up to 3 days.
The ELISA was performed at room temperature with three washes of 200 µL of 0.1% (wt/vol) BSA in PBS between each step. HUVECs were incubated for 1 hour in turn with the primary monoclonal antibody (50 µL), secondary HRP-conjugated antibody (70 µL), and finally 100 µL of enzyme substrate consisting of 0.55 mg/mL 2,2'-azino-bis(3-ethylbenzthiazoline sulfonate) and 0.012% hydrogen peroxide in citrate-phosphate buffer, pH 4.2. Color was developed until TNF- alone wells gave an absorbance reading at 410 nm (optical density, 2.0 units) using an ELISA plate reader (Dynatech MR 7000).
Test and blank samples were performed in triplicate. Specific monoclonal antibody binding was calculated for each treatment by subtracting the mean blank value in the absence of TNF- or PMA from each test value. When constitutively expressed receptor was determined, the mean blank value in the absence of monoclonal antibody was subtracted from each test value. In each case, results were then expressed as percentage of DPC (ie, minus fatty acid) control for each treatment.
Primary monoclonal antibodies were mouse anti-human ICAM-1 (IgG1, B-C14, Serotec, diluted 1:1000), rat anti-human E-selectin (IgG2a, 4D10, Serotec, diluted 1:100), and mouse anti-human VCAM-1 (IgG1, E1/6, Becton Dickinson, diluted 1:1000). Corresponding secondary conjugated antibodies were sheep anti-mouse HRP conjugate (Silenus, diluted 1:2000) and goat anti-rat HRP conjugate (Sigma, diluted 1:1000). Dilutions were with 0.1% (wt/vol) BSA in PBS.
Flow Cytometry Analyses
After incubations with 15-HPETE plus TNF-, endothelial cell monolayers were treated with 0.05% trypsin/EDTA for 2 minutes to release the cells. These were immediately washed in HBSS and then treated with mouse anti-human ICAM-1 monoclonal antibody (IgG1, Serotec), mouse anti-human E-selectin (IgG2a, Becton Dickinson), or mouse anti-human VCAM-1 (IgG2a, Becton Dickinson). The cells were washed in Isoton II+1% BSA and then treated with FITC-conjugated goat anti-mouse IgG (Organon, Teknika) at 4°C for 30 minutes. After two washes in Isoton II+1% BSA, the cells were fixed with 1% paraformaldehyde. The fluorescence intensity of the cell population was analyzed by flow cytometry on a FACScan (Becton Dickinson). Approximately 10 000 cells were counted. Data were processed with a software Lysis II program (Becton Dickinson). Fluorescence values were corrected by subtraction of values for isotype-matched negative controls.
Measurement of ICAM-1, E-Selectin, and VCAM-1 mRNA
cDNA Probes
The human E-selectin c-DNA probe was a 3.8-kb insert cloned into the Xho-I site of a pB-SK vector, a gift from Dr J. Gamble, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, Australia. The ICAM-1 probe was kindly provided by Dr A. Boyd, The Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia, as a 1.4-kb insert between Xba-I and Hind III sites in a pGEM vector. The GAPDH probe cocktail was from Clontech. VCAM-1 cDNA was a 3080-bp fragment cloned into the Not I site of pCDM8 plasmid, obtained from Dr J. Gamble, a gift of Biogen Inc (Cambridge, Mass).
RNA Isolation and Hybridization
HUVECs were induced as described in the figure legends. RNA for the slot blots was isolated by the RNAzolB method (Cinna/Biotex) by direct addition and lysis on the tissue culture plate. Cells were lysed by the addition of 0.4 mL RNAzolB per 106 cells. To the lysate, 50 µL of chloroform was added, vigorously shaken for 15 seconds, and placed on ice for 5 minutes. The tubes were centrifuged at 12 000g for 15 minutes at 4°C. The upper phase was carefully removed, and the RNA was precipitated by the addition of an equal volume of isopropanol by incubation on ice for 15 minutes. The RNA was pelleted by centrifugation at 12 000g for 15 minutes at 4°C.
For preparing a series of threefold dilutions, the RNA samples were diluted in a diluent solution containing formaldehyde, SSC, SDS, and diethyl pyrocarbonate and heated at 65°C for 5 minutes. The samples were applied to a Gene Screen Plus nylon membrane using a slot-blot vacuum manifold (Hoeffer). The filters were baked for 2 hours at 80°C, and then prehybridization was performed for 16 to 24 hours in a solution containing formamide, dextran sulfate, SDS, SSC, Denhardt's solution, and denatured herring sperm DNA (NEN, Du Pont).
DNA probes were oligolabeled using [-32P]dCTP as described (Amersham) and used for hybridization. Autoradiographs were prepared by exposure with intensifying screens at -70°C to XAR films (Kodak). The extent of hybridization was quantified by scanning with a laser densitometer (LKB), and the relative intensities of the threefold dilution bands were calculated as the specific density of the band of interest (background-subtracted) divided by the specific density of the GAPDH reprobing of the same band (background-subtracted).
PKC Translocation
Endothelial cells were pretreated with 30 µmol/L of 15-HPETE or vehicle for 30 minutes and then stimulated with 100 nmol/L PMA for 3 minutes at 37°C. After incubation, cells were detached with a rubber policeman and sonicated in a buffer containing 20 mmol/L Tris, pH 7.5, 5 mmol/L EGTA, 2 mmol/L EDTA, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 10 µg/mL benzamidine, 10 µg/mL pepstatin A, 10 mmol/L phenylmethylsulfonyl fluoride, and 2 mmol/L mercaptoethanol, and the sonicate was centrifuged at 100 000g for 30 minutes. The pellet was resuspended in sonication buffer supplemented with 2% Triton X-100. After 30 minutes (4°C), the extract was centrifuged at 100 000g for 30 minutes. The supernatant was absorbed onto DE 52 (Bio-Rad), which was preequilibrated in 20 mmol/L HEPES (pH 7.5). After the beads were washed (twice with 0.5 mL HEPES buffer), PKC was eluted with 0.12 mol/L NaCl. PKC activity was assayed by using Histone type III-S (Sigma) as substrate according to the method of Hardy et al.43 The reactions were terminated by spotting aliquots onto P81 paper (Whatman Ltd). After the pieces of P 81 paper were extensively washed with 85 mmol/L orthophosphoric acid, the radioactivity was determined by liquid scintillation spectrometry.
HUVEC Viability
HUVECs were grown to confluence in 96-well plates as described above. During the last 16 hours of this culture, the medium was replaced with 200 µL of culture medium containing 1 µCi (37 kBq) of sodium [51Cr]chromate (10.4 MBq/µg chromium, Amersham). The monolayers were washed twice with 200 µL RPMI 1640 before treatment with fatty acids or vehicle alone in triplicate, followed by TNF-/PMA as described above. At the end of the incubation, the supernatants were removed to gamma-counting tubes, and the monolayers were washed gently with 200 µL RPMI. The wash and supernatant for each well were pooled before counting in a gamma counter (LKB 1282 Compugamma, Wallac). Monolayers were extracted overnight with 200 µL of 2 mol/L NaOH before gamma counting. Results were calculated by expressing counts per minute (supernatant+wash) as a percentage of the total counts per minute per well (supernatant+wash+NaOH extract).
TNF- Binding Assays
For these experiments, second-passage HUVECs were plated at 2x105 cells per well per 0.5-mL culture medium in 24-well culture plates (2.0-cm2 area per well, Linbro, Flow) and grown to confluence (2 days). The wells were washed once with 500 µL RPMI 1640 before incubation at 37°C for 4.5 hours with fatty acids or vehicle alone in a total volume of 500 µL E-SFM.
The plates were then placed on ice, the medium was removed, and the wells were washed with 500 µL cold (4°C) HBSS containing 5% (vol/vol) heat-inactivated human AB serum (binding medium). This medium was replaced with 0.3 mL cold binding medium containing 0.22 nmol/L [I125]human recombinant TNF- (105 dpm per well, 15 to 30 Tbq [terabecquerel]/mmol, Amersham). Selected wells also contained a 1000-fold excess of unlabeled TNF- (Pharma Biotechnologie). The plates were incubated on ice for 1 hour. Wells were washed three times with 1 mL binding medium at 4°C. Monolayers were extracted for 48 hours with 300 µL of 0.5 mol/L NaOH before gamma counting. For each fatty acid treatment, triplicate wells were used to calculate total binding, and duplicate wells were used to calculate nonspecific binding. Specific binding was defined as the difference between total counts in the absence of unlabeled TNF- and counts in the presence of unlabeled TNF- (nonspecific binding). Nonspecific binding ranged from 15% to 25% of total binding.
Expression of Results and Statistical Analysis of Data
Results are expressed as mean±SEM. Statistical significance was mainly evaluated by a two-tailed t test for paired data, except where indicated otherwise, in which case differences between groups were tested by ANOVA (pairwise comparisons were performed using Fisher's least significant difference test). The "raw" values generated for each experiment were used for the statistical analyses.
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Effect of 20:4(n-6) and Its 15-HETE and 15-HPETE Derivatives on the Adhesion of Neutrophils or Monocytes to TNF-
–Treated HUVECsOur initial experiments were designed to examine the adherence of leukocytes to cytokine-activated HUVECs pretreated with 20:4(n-6), 15-HETE, or 15-HPETE. Cytokine treatment maximizes the adhesive capacity of HUVECs, inducing the expression of adhesion molecules.8 9 10 HUVECs were therefore treated with fatty acids for 30 minutes, followed by TNF-
for 4 hours before incubation with either neutrophils (Fig 1A
) or monocytes (Fig 1B). The data shown in Fig 1 demonstrate that the binding of these cells to HUVECs was decreased with a potency of 20:4(n-6)<15-HETE<15-HPETE. Under these conditions, monocyte adhesion to TNF-–stimulated HUVECs was significantly inhibited by anti–ICAM-1 and anti–VCAM-1 antibodies. The optical density value due to TNF- treatment was 0.85± 0.04, which was reduced to 0.62±0.03 (P<.01) and 0.62±0.02 (P<.01) (after subtracting the basal level) in the presence of antibodies to ICAM-1 and VCAM-1, respectively. Neutrophil adhesion to TNF-–stimulated HUVECs was also significantly inhibited by antibodies to E-selectin and ICAM-1. The increase in optical density due to TNF- treatment was 0.78±0.10, which was reduced to 0.41±0.07 (P<.01) and 0.45±0.02 (P<.01), respectively.
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Effect of 20:4(n-6) and Its 15-HETE and 15-HPETE Derivatives on ICAM-1, E-Selectin, and VCAM-1 Expression
In order to investigate the mechanism whereby 20:4(n-6), 15-HETE, and 15-HPETE decreased the binding of leukocytes to HUVECs, we examined the expression of the endothelial adhesion molecules ICAM-1, E-selectin, and VCAM-1, which are known to support the binding of neutrophils and monocytes as well as a subpopulation of memory T lymphocytes, B cells, eosinophils, and basophils (reviewed in References 44 and 45).
ICAM-1 is constitutively expressed by endothelial cells,8 but its expression can be upregulated by TNF-.8 HUVECs were incubated with fatty acids for 30 minutes before the induction of ICAM-1 with TNF- (Fig 2). A significant inhibition of the expression of ICAM-1 induced with TNF- was evident with 10 µmol/L of 20:4(n-6), 15-HETE, or 15-HPETE (Fig 2). This inhibition increased slightly for 20:4(n-6) at 30 µmol/L but increased markedly for 15-HPETE at 30 µmol/L (Fig 2). The inhibition for 15-HETE was between that of 20:4(n-6) and 15-HPETE at 30 µmol/L (Fig 2).
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0.5 (ICAM-1, a twofold increase), 1.2 (E-selectin, a five-fold increase), and 0.5 (VCAM-1, a two-fold increase) over basal (control). *P<.05 and **P<.01.
E-Selectin is not present in unstimulated endothelium,11 which was confirmed in our experimental system, but is expressed on cytokine-activated endothelium.11 HUVECs were incubated with fatty acids for 30 minutes before the induction of E-selectin with TNF- (Fig 2
). A similar inhibition to that of ICAM-1 was found in the expression of E-selectin as the concentration of 20:4(n-6), 15-HETE, and 15-HPETE was increased to 30 µmol/L (Fig 2). As with ICAM-1, the order of effectiveness was 20:4(n-6)<15-HETE<15-HPETE.
VCAM-1 is also not constitutively expressed on HUVECs but can be induced by TNF-, usually requiring 12 hours of incubation for VCAM-1 protein to be expressed. Endothelial cells pretreated with 20 to 30 µmol/L of 15-HPETE showed significant inhibition of TNF-–induced VCAM-1 expression (Fig 2). Both 20:4(n-6) and 15-HETE were much less effective than 15-HPETE (Fig 2).
The expression of ICAM-1 and E-selectin can also be upregulated by PMA,13 which acts by directly activating PKC. HUVECs were incubated with fatty acids for 30 minutes before the induction of ICAM-1 or E-selectin with PMA (Fig 3). An inhibition of PMA-induced expression of adhesion molecules similar to that of TNF-–induced adhesion molecules was found as the concentration of fatty acids increased to 30 µmol/L. The order of effectiveness was 20:4(n-6)<15-HETE<15-HPETE.
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To exclude the possibility that the inhibition of ICAM-1, E-selectin, or VCAM-1 seen with 20:4(n-6), 15-HETE, and 15-HPETE might have resulted from an effect of these fatty acids on HUVEC viability, we examined the release of 51Cr from 51Cr-prelabeled HUVECs after treatment with fatty acids. The data (not presented) showed that the release of 51Cr was unchanged after fatty acid treatment in the absence or presence of either TNF- or PMA.
The time-related effects of 15-HPETE on the expression of ICAM-1, E-selectin, and VCAM-1 were examined. The results in Fig 4 show that ICAM-1, E-selectin, and VCAM-1 expression were significantly depressed by HPETE at 4 to 12 hours of culture but that by 18 hours these adhesion molecules were expressed at normal levels by the HUVECs.
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When HUVECs were incubated with 20:4(n-6), 15-HETE, or 15-HPETE was incubated under conditions identical to those used above but in the absence of TNF-, there was no significant inhibition of ICAM-1 expression, showing that the fatty acids had no effect on constitutively expressed ICAM-1 (data not shown).
When ethanol was used as an alternative method of solubilization of 15-HPETE, similar inhibition of ICAM-1 expression occurred with ethanol-solubilized 15-HPETE or DPC-micelle–incorporated 15-HPETE. The mean±SEM percent inhibition in six experiments was 69.8±10.2% for fatty acids in DPC and 68.5±10.2% for fatty acids in ethanol.
The fatty acid effects seen above could result from an inhibition of the binding of TNF- to its receptors on HUVECs. Experiments were conducted to establish if this were a mechanism for the fatty acid inhibitory actions. Fatty acids were incubated with HUVECs under conditions identical to those used for ICAM-1, E-selectin, and VCAM-1 determination, but TNF- was not added during incubation. No significant decrease in the binding of I125–TNF- to HUVECs was seen with 20:4(n-6), 15-HETE, or 15-HPETE (data not presented).
We were interested in establishing whether saturated fatty acids or esterified 20:4(n-6) had any inhibitory activity on the induction of ICAM-1, E-selectin, or VCAM-1. The results showed that the saturated fatty acid 20:0 had no inhibitory activity. Likewise, esterification of 20:4(n-6) [ie, 20:4(n-6) methyl ester] abolished any inhibitory activity of 20:4(n-6) (data not presented).
Flow Cytometry Analyses of ICAM-1, E-Selectin, and VCAM-1
The effects of 15-HPETE on the expression of ICAM-1, E-selectin, and VCAM-1 were confirmed using flow cytometric analyses (Fig 5 and Table). TNF- increased the expression of ICAM-1, E-selectin, and VCAM-1. HUVEC which had been pretreated with 15-HPETE showed significantly depressed expression of both of these adhesion molecules. However, the constitutively expressed ICAM-1 was not affected by 15-HPETE.
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Effect of 15-HPETE on Expression of ICAM-1, E-Selectin, and VCAM-1 mRNA Induced by TNF-
TNF- increased the expression of ICAM-1 mRNA and induced the expression of E-selectin and VCAM-1 mRNA as assayed by slot-blot analyses. Pretreatment of HUVECs with 15-HPETE significantly decreased the expression of ICAM-1, E-selectin, and VCAM-1 mRNA (Fig 6). ICAM-1 mRNA was constitutively expressed, and 15-HPETE had no effect on this expression of ICAM-1 mRNA.
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Effect of 15-HPETE on PMA-Induced PKC Translocation
The mechanisms by which TNF- induces/increases the expression of ICAM-1, E-selectin, and VCAM-1 still remain to be clearly defined. Since our data showed that 15-HPETE inhibited the PMA-induced expression of adhesion molecules and since PMA is known to initiate these events by stimulating translocation and activation of PKC, we used this system to examine the effects of 15-HPETE on early signals. PMA induced the translocation of PKC to a particulate fraction (Fig 7). However, HUVECs treated with 15-HPETE failed to show PMA-induced PKC translocation (Fig 7).
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Discussion |
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This is the first report to show that 15-hydroperoxy (15-HPETE) and 15-hydroxy (15-HETE) derivatives of 20:4(n-6) as well as 20:4(n-6) itself can significantly inhibit ICAM-1, E-selectin, and VCAM-1 expression induced in endothelial cells by TNF-
10 11 12 or PMA,11 as measured by ELISA. In this regard, the potency of inhibition was 15-HPETE>15-HETE>20:4(n-6), with there being little effect of 20:4(n-6) except at 30 µmol/L. Analysis of ICAM-1, E-selectin, and VCAM-1 expression by flow cytometric analysis similarly showed that 15-HPETE was very effective in inhibiting the TNF-–enhanced/induced expression of these adhesion molecules. These studies demonstrated that the suppression caused by the fatty acid was uniform in the whole population of cells. By both methods, it was evident that there was no effect on constitutively expressed ICAM-1, which suggests that these fatty acids are acting to suppress the synthesis of such adhesion molecules. This was supported by our studies, which showed that 15-HPETE inhibited the TNF-–enhanced/induced ICAM-1, E-selectin, and VCAM-1 mRNA expression but not the constitutively expressed ICAM-1 mRNA. This inhibition of ligand stimulation of ICAM-1, E-selectin, and VCAM-1 expression is likely to have in vivo significance, because fatty acid–treated HUVECs supported significantly less adhesion of neutrophils and monocytes than did untreated HUVECs. Localized adhesion of peripheral blood leukocytes to the endothelial lining is essential for their exit from the blood under both physiological and pathological conditions. Enhanced neutrophil margination occurs at sites of acute inflammation, whereas monocyte interactions with the microvasculature are seen more often in chronic inflammatory reactions. In vivo E-selectin is not found in the endothelium of normal tissues but is detected at sites of inflammation in postcapillary venules, the principal sites of leukocyte extravasation during inflammation.46 Both ICAM-1 and E-selectin have been shown to be involved in vascular injury,16 17 airway inflammation,17 18 19 reperfusion injury in the heart,20 renal inflammation,21 and tumor cell adhesion to endothelium.22 23 Attachment of monocytes to the endothelium also occurs in large arteries, as an early event in the development of atherosclerotic lesions, with ICAM-1 and VCAM-1 being found in atherosclerotic plaques.47 48
The effects of these fatty acids were not mediated by alterations in endothelial surface TNF- receptor expression, because inhibition occurred when ICAM-1 or E-selectin expression is induced either with TNF-, whose action is cell surface–mediated,49 or with PMA, which bypasses cell surface receptors, causing translocation and activation of PKC.50 This conclusion was supported by data showing that the specific binding of TNF- to its receptors was not altered by such fatty acid treatments. Recently, 15-HETE has been shown to reduce the affinity of neutrophil leukotriene B4 cell-surface receptors32 but to have no effect on receptors for the chemotactic peptide f-Met-Leu-Phe.51
When 15-HPETE was solubilized with ethanol, identical results to DPC-micellar 15-HPETE were obtained, demonstrating that the mode of administration of the fatty acid to HUVECs was not critical for the effect. The concentrations of fatty acids used in our studies are within the physiological range. Dietary polyunsaturated fatty acids can reach a free fatty acid concentration of 20 to 30 µmol/L.52 53 In addition, within the blood system, under inflammatory and prothrombotic conditions, platelets and leukocytes interact with the endothelium and also between themselves,29 during which time they synthesize and release 20:4(n-6) together with a variety of its lipoxygenase and cyclooxygenase products.29 30 31 32 Local high concentrations of fatty acids due to lipoprotein lipase activity on endothelial cells are also likely to occur. 15-HPETE and 15-HETE are known to be produced by leukocytes54 as well as endothelial cells.55 The finding that the HPETE effects are transient means that the continuous generation of this product is required to maintain inhibition of agonist-induced adherence molecule expression. It is most likely that under the conditions of an inflammatory reaction, a continuous supply of 15-HPETE would be present.
The present data demonstrate that the 20 carbon–saturated fatty acid had no inhibitory effect on ICAM-1 or E-selectin expression. In addition, esterification of 20:4(n-6) [ie, 20:4(n-6) methyl ester] abolished any inhibition due to 20:4(n-6), suggesting that a free carboxyl group on the polyunsaturated fatty acid is necessary for the effect. Similar results with saturated fatty acids and esterified polyunsaturated fatty acids have been found by us and by others in different experimental systems.32 33 35 36 37 40 56
The transient inhibitory effect of 15-HPETE on the expression of ICAM-1, E-selectin, and VCAM-1 may be due to the unstable nature of 15-HPETE, since it can be rapidly converted to 8,15-diHETE and other metabolites.55 Consequently, this may result in the waning of the suppression effect of 15-HPETE on agonist-induced expression of adhesion molecules.
Although the mechanisms by which 20:4(n-6), 15-HETE, and 15-HPETE decrease ICAM-1, E-selectin, and VCAM-1 expression in HUVECs remain to be clearly defined, it is known that HUVECs incorporate 20:4(n-6), 15-HETE, and 15-HPETE to varying extents into phospholipids and neutral lipids.57 58 59 60 This incorporation into phospholipids may perturb the packing of fatty acyl chains in certain regions of the lipid bilayer affecting membrane structure, resulting in physically reduced expression of ICAM-1, E-selectin, and VCAM-1. However, there was no effect of the fatty acids on constitutively expressed ICAM-1, which one would expect if physical alteration of receptor expression were occurring. In any case, the studies showed that the effects of 15-HPETE were evident at the level of ICAM-1, E-selectin, and VCAM-1 mRNA.
It is likely that interference with signaling via PKC could contribute, at least in part, to the inhibition of agonist-induced expression of adhesion molecules. Consistent with this suggestion, pretreatment of cells with 15-HPETE inhibited the PMA-stimulated translocation of PKC. Although it is generally accepted that PKC mediates most, if not all, of the activities of PMA, the role of PKC in TNF-induced expression of adhesion molecules has been less clear. Thus, both PKC-dependent and PKC-independent mechanisms for mediating the action of TNF have been proposed.12 60 61 Although oxidative stress may be a possible mechanism by which 15-HPETE exerts its action, it is unlikely that oxidative stress plays a major role, since oxidative stress has been reported to affect only the expression of VCAM-1, but not ICAM-1.62 63
Recently De Caterina et al64 and Weber et al65 have demonstrated that agonist-induced expression of adhesion molecules in cultured endothelial cells was inhibited by the n-3 fatty acid, docosahexaenoic acid [22:6(n-3)], but not the n-6 fatty acid, 20:4(n-6). This effect required prolonged exposure of the cells to 22:6(n-3) and demonstrates the protective antiatherogenic property of 22:6(n-3).
Our findings that 15-HPETE decreases TNF-–induced ICAM-1, E-selectin, and VCAM-1 expression demonstrate a novel action for this compound and extend previous findings involving changes induced by fatty acids on the endothelium.63 64 65 66 67 68 There are no reports, to our knowledge, of an anti-inflammatory action of this compound, but studies have indicated that it possesses vasoactive/inflammatory activity as does its metabolic product, 8,15-diHETE.69 70 In addition, Sultana et al71 have recently reported that nanomolar concentrations of 15-HPETE001/HETE stimulated the expression of adhesion molecules such as ICAM-1, E-selectin, and VCAM-1. The micromolar concentrations used in the present study showed only a minor but not significant increase in expression of these molecules and an associated depression in TNF--induced increase in expression of these molecules. The concentrations of HPETE/HETE used by Sultana et al were very small (<1 ng/mL), and the method of suspension (diluent/vehicle) is not outlined. In the present study, micelles mixed with DPC were used. Whether the observed differences in the direct fatty acid–induced effects on ICAM-1, E-selectin, or VCAM-1 are a function of variations in methods or a bimodial action of the 15-HPETE/HETE needs to be clarified. 15-HETE, when administered exogenously, decreases tissue injury in human psoriasis72 and carrageenin-induced experimental arthritis73 and is mildly anti-inflammatory in colitis.74 These findings may result, in part, from its ability to inhibit several enzymes involved in the synthesis of proinflammatory eicosanoids75 76 as well as in its ability to inhibit leukotriene B4–induced neutrophil migration across endothelium35 and receptor-triggered neutrophil function.76 In addition, 15-HETE and 15-HPETE do not stimulate neutrophil adherence and degranulation.76 We now demonstrate that 15-HETE also decreases the agonist-induced expression of the adhesion molecules ICAM-1, E-selectin, and VCAM-1 on HUVECs, a property that it shares with 15-HPETE. This is a mechanism that complements the inhibition of expression of adhesion molecules by 22:6(n-3).65 66 Strategies that reduce the expression of adhesion molecules on endothelial cells may provide new therapies for pathological conditions.
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Acknowledgments |
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This study was supported by the National Heart Foundation of Australia and the National Health and Medical Research Council of Australia. We thank the labor wards at Flinders Medical Centre, Ashford Community Hospital, and Calvary Hospital for the supply of umbilical cords, Madhuri Nandoskar for technical assistance, and Dr Debbie Rathjen for helpful discussions.
Received October 13, 1995; accepted November 7, 1996.
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M. Costabile, C. S. T. Hii, B. S. Robinson, D. A. Rathjen, M. Pitt, C. Easton, R. C. Miller, A. Poulos, A. W. Murray, and A. Ferrante A Novel Long Chain Polyunsaturated Fatty Acid, {beta}-Oxa 21:3n-3, Inhibits T Lymphocyte Proliferation, Cytokine Production, Delayed-Type Hypersensitivity, and Carrageenan-Induced Paw Reaction and Selectively Targets Intracellular Signals J. Immunol., October 1, 2001; 167(7): 3980 - 3987. [Abstract] [Full Text] [PDF]
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 R. Hattori, H. Otani, Y. Moriguchi, H. Matsubara, T. Yamamura, Y. Nakao, H. Omiya, M. Osako, and H. Imamura NHE and ICAM-1 expression in hypoxic/reoxygenated coronary microvascular endothelial cells Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2796 - H2803. [Abstract] [Full Text] [PDF]
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 H. P. A. Jersmann, C. S. T. Hii, J. V. Ferrante, and A. Ferrante Bacterial Lipopolysaccharide and Tumor Necrosis Factor Alpha Synergistically Increase Expression of Human Endothelial Adhesion Molecules through Activation of NF-{kappa}B and p38 Mitogen-Activated Protein Kinase Signaling Pathways Infect. Immun., March 1, 2001; 69(3): 1273 - 1279. [Abstract] [Full Text] [PDF]
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H. P. A. Jersmann, C. S. T. Hii, G. L. Hodge, and A. Ferrante Synthesis and Surface Expression of CD14 by Human Endothelial Cells Infect. Immun., January 1, 2001; 69(1): 479 - 485. [Abstract] [Full Text] [PDF]
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A. S. Orozco, X. Zhou, and S. G. Filler Mechanisms of the Proinflammatory Response of Endothelial Cells to Candida albicans Infection Infect. Immun., March 1, 2000; 68(3): 1134 - 1141. [Abstract] [Full Text] [PDF]
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 C. Kerkhoff, M. Klempt, V. Kaever, and C. Sorg The Two Calcium-binding Proteins, S100A8 and S100A9, Are Involved in the Metabolism of Arachidonic acid in Human Neutrophils J. Biol. Chem., November 12, 1999; 274(46): 32672 - 32679. [Abstract] [Full Text] [PDF]
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C. S. T. Hii, N. Moghadammi, A. Dunbar, and A. Ferrante Activation of the Phosphatidylinositol 3-Kinase-Akt/Protein Kinase B Signaling Pathway in Arachidonic Acid-stimulated Human Myeloid and Endothelial Cells. INVOLVEMENT OF THE ErbB RECEPTOR FAMILY J. Biol. Chem., July 13, 2001; 276(29): 27246 - 27255. [Abstract] [Full Text] [PDF]
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