effects of human low-density lipoproteins on superoxide

Eur J Clin Chem Clin Biochem 1997; 35(2):73-80 © 1997 by Walter de Gruyter · Berlin · New York

Effects of Human Low-Density Lipoproteins on Superoxide Production byFormyl-Methionyl-Leucyl-Phenylalanine Activated PolymorphonuclearLeukocytes

Christine Bonneau1, Remy Couderc1, Michele Tissot1, Anne Athias2, Monique Roch-Arveiller1 andJean Paul Giroud1

1 Laboratoire de Pharmacologie, Unite CNRS 15-35, Hopital Cochin, Paris, France2 Laboratoire de Biochimie des Lipoproteines, Höpital du Bocage, Dijon, France

Summary: Neutrophils play a major role in the host defence by producing reactive oxygen species. These productsare liberated by activated cells and are known to cause endothelial cell injury and damage. The present study showsthat low-density lipoproteins increase Superoxide anion production by twofold in polymorphonuclear leukocytesstimulated by formyl-Met-Leu-Phe in vitro. Moreover, LDL induced a large increase in phosphoinositides andcytosolic-free calcium. Data from experiments performed on neutrophils treated with pertussis toxin, staurosporine,propranolol or nifiumic acid suggest that modulation of phospholipase D and A2 activities could be involved in themodification by LDL of leukocyte response to formyl-Met-Leu-Phe. LDL lipid moiety could play a key role intheir action on polymorphonuclear functions because cholesterol was exchanged between lipoproteins and cells thatcan modify membrane fluidity and interact with the formyl-Met-Leu-Phe receptor.

IntroductionDyslipoproteinaemia, demonstrating increased blood li-poprotein concentrations, hypertension and tabagism, isoften associated with accelerated atherogenesis mecha-nisms (1, 2). Increased lipoprotein concentrations, andespecially high low-density lipoprotein (LDL) concen-trations, could modify polymorphonuclear leukocyte(PMN) functions. Some authors have reported the en-hanced oxidative metabolism of polymorphonuclearneutrophils isolated from hyperlipoproteinaemic sub-jects and an inhibition of polymorphonuclear neutrophilphagocytosis following their treatment with LDL in vi-tro (3, 4).

In a previous study, we demonstrated that LDL stimu-lates Superoxide (O^") generation by human polymor-phonuclear neutrophil in vitro (5). This effect is concen-tration dependent and might mediate venous endothelialstructure injury by arachidonic acid metabolites, proteo-lytic enzymes and reactive oxygen species released byactivated polymorphonuclear neutrophils (6—10). More-over, they might enhance LDL oxidation and contributeto the formation of foam cells mainly implicated in theatherogenic process (11 — 13).

The oxidative burst results from the assembly and acti-vation of the NADPH oxidase, a transmembraneelectron transport chain which reduces oxygen to super-oxide (14, 15). This (Oi~ generation is initiated in neu-trophils by a variety of agonists (16). Among them isthe formyl-Methionyl-Leucyl-Phenylalanine, a tripep-

tide analogous to bacterial wall constituents, and cur-rently being used for in vitro experiments. Polymorpho-nuclear neutrophil activation by formyl-Met-Leu-Phetriggers a cascade of tightly controlled biochemicalevents leading to an oxidative burst (17—19). The firststep consists of the ligand binding to specific cell sur-face receptors. Among them is the formyl-Met-Leu-Phereceptor which is well-characterized and coupled to cel-lular responses through a pertussis toxin-inhibitable Gprotein (20). Three phospholipases are mainly involvedat different steps of neutrophil activation; phospholipaseC, phospholipase A2 and phospholipase D. Hydrolysisof membrane phospholipids leads to the formation ofnumerous bioactive lipids. These pathways were impli-cated in the signal transduction of polymorphonuclearneutrophil activated by formyl-Met-Leu-Phe (18, 21).

In order to clarify LDL effects of formyl-Met-Leu-Phe-induced polymorphonuclear neutrophil oxidative burst,we investigated in vitro the action of pharmacologicaltools on some steps of transduction leading to the oxida-tive burst of polymorphonuclear neutrophil.

Materials and MethodsReagentsPertussis toxin (lot 72H-0641-1), staurosporine, propranolol,nifiumic acid, and formyl-Met-Leu-Phe were purchased fromSigma Chemical Co. (Saint-Louis MO, USA).Stock solutions of formyl-Met-Leu-Phe (1 mmol/l) and stauro-sporine (1 mmol/l) were prepared in dimethyl sulphoxide andstored at -20 °C.

74 Bonneau et al.: Eifects of human LDL on Superoxide production by formyl-Met-Leu-Phe activated granulocytes

Stock solution of propranolol (50 mmol/1) was prepared in phos-phate buffered saline without Ca2+ and Mg2"1" and stored at-20 °C.

Dilutions of stock solutions were made in phosphate buffered sa-line for each experiment.

Lipoprotein isolation

LDL (1.030 < d < 1.050 kg/1) was prepared from the serum ofnormolipaemic subjects (3.5 mmol/1 < total cholesterol < 6.5mmol/1 and triacylglyerols < 1.0 mmol/1) in the presence of ethyl-enediaminetetraacetic acid (2.69 μπιοΐ/ΐ) and phenylmethylsulpho-nyl fluoride (0.1 mmol/1) to prevent both action of lipolytic en-zymes and endogenous proteases and to avoid oxidation. Isolationof LDL was achieved through two successive ultracentrifugationsteps at 100 000 g for 18 hours at 4 °C. It was then dialyzed ex-haustively against the phosphate buffer and stored for less than fivedays under nitrogen at 4 °C after sterilization by filtration (0.22urn Millipore). The purity of the LDL fraction was assayed by SDSpolyacrylamide gel electrophoresis. Since the main constituent ofLDL is cholesterol, LDL concentration was expressed as LDL cho-lesterol in mmol/1.

Cell preparation

The cells were isolated from heparinized (10000 U/l) peripheralblood of healthy volunteers from the laboratory population, by suc-cessive use of Ficoll-Hypaque and polyvinylalcohol (22). After iso-lation, the cells were put in a phosphate buffered solution for deter-mination of Superoxide generation and viability was assessedthrough a trypan blue exclusion test.

Superoxide production

Superoxide production was measured in terms of ferricytochrome creduction as previously described (5, 23). The stimulating agentwas formyl-Methionyl-Leucyl-Phenylalanine 100 nmol/1. The re-sults were expressed as O^ release in mmol/106 cells.

Phospholipid turnover analysis

Polymorphonuclear neutrophil labelling

After isolation, polymorphonuclear neutrophil cell count was ad-justed to 1010/1 in Hank's balanced salt solution containing 20mmol/1 Hepes, pH = 7.4, supplemented with 250 mg/1 bovine se-rum albumin as previously described (24). Cells were labelled with7.4 GBq/1 (200 mCi/1) of Hiyo-[2-3H]inositol and incubated for16-20 h at 37 °C with gentle shaking.

At the end of incubation, cells were washed three times, countedand put in the same Hank's solution, supplemented with 1 g/1 bo-vine serum albumin and 10 mmol/1 LiCl (for inhibiting the en-zymes of inositol phosphate dephosphorylation). Cells were ad-justed to a concentration of 107 cells per 600 μΐ aliquots and incu-bated for 10 min at 37 °C before the stimulation.

Polymorphonuclear neutrophil stimulation

Stimulating agents were applied to aliquots tested in duplicate, asfollows:

— phosphate buffered saline for 15 s (controls)

- formyl-Met-Leu-Phe from 10~9 to 10~6 mol/1 for 15 s

- LDL (2.5 mmol/1) for 5 min

— LDL (2.5 mmol/1) for 5 min before the addition of formyl-Met-Leu-Phe for 15 s.

The reaction was stopped by addition of perchloric acid, followedby three cycles of freezing-thawing.

After centrifugation, the hydrosoluble perchloric acid precipitationsupernatants containing inositol phosphates were diluted and neu-tralized. The inositol lipids were extracted from the perchloric acid

insoluble pellets, dried and deacylated by alkaline hydrolysis asdescribed by Creba et al. (25) and Poggioli et al. (26). The[3H]inositol phosphates and [3H]glycerophosphoryl esters werethen separated by anion-exchange chromatography on DowexAG1-X8 columns using the buffer system described by Downes etal. (27) and Berridge et al. (28).

Cytosolic-free calcium concentrations

Changes in cytosolic free calcium concentration were measured inpolymorphonuclear neutrophil loaded with 1 umol/l Fura 2/AM at37 °C for 45 min (29, 30). Cells were then washed and suspendedin 10 mmol/1 HEPES/Hank's balanced salt solution. Fura 2 fluores-cence assays were performed with aliquots of 5 Χ 106 polymor-phonuclear neutrophil in 2 ml Hank's balanced salt solution, usinga fluorimeter (Jobin Yvon 3D, France) equipped with a thermallycontrolled cuvette holder and a magnetic stirrer. Various concentra-tions of LDL were tested: 0.01 to 5 mmol/1 cholesterol. Excitationand emission wavelengths for Fura 2 fluorescence assays were 340and 510 nm, respectively. Cytosolic calcium concentrations werecalculated as described (30).

Intracellular level of polymorphonuclear neutrophilcholesterol

Polymorphonuclear neutrophils were preincubated in duplicate at37 °C for 5 min and placed in contact with increasing concentra-tions of LDL for 15 min. Superoxide generation was measured inhalf of the tubes and cellular cholesterol content was measured inthe other. In these tubes, ferricytochrome c was omitted. After stop-ping the reaction in an ice-water bath, the cells were washed threetimes in phosphate buffered saline before freezing.

After lipid extraction from neutrophils, cellular cholesterol mea-surement was performed by gas chromatography coupled withmass spectrometry. This measurement was performed in the Labor-atoire de Biochimie des Lipoproteines de Dijon (Service du PrGamberf).

Statistical analysis

The Mann-Whitney's test and the Wilcoxon's test were used for thecomparison of unpaired and paired quantitative variables.

Results

After polymorphonuclear neutrophil preincubation for5 min at 37 °C with buffer or LDL, Superoxide pro-duction was assayed over the next 5 min in the presenceof formyl-Met-Leu-Phe 100 nmol/1.

In the presence of LDL, formyl-Met-Leu-Phe-inducedstimulation was increased twofold (Wilcoxon's test:p < 0.01) (control: Oi~ 6.72 ±1.65 nmol/106 cells;with LDL at 2.5 mmol/1: Ο^~ 14.92 ± 2.19 nmol/106

cells; with LDL at 5 mmol/1: ΟΓ 16.59 ±2.18 nmol/106 cells; means ± SEM, n = 9).

Pertussis toxin action

Preincubation with pertussis toxin (1 μg for 2 X 107

cells) drastically impaired polymorphonuclear neutro-phil response by ADP-ribosylation of G-protein (31).There was a variability in the activity of pertussis toxindepending on the batch purchased, but the maximal ef-fect was observed with polymorphonuclear neutrophiltreated for 2 h in Hank's solution under shaking, as pre-viously described by Gabig et al. (32) and Omann et al.

Bonneau et al.: Effects of human LDL on Superoxide production by formyl-Met-Leu-Phe activated granulocytes 75

(33). After centrifugation, cells were washed before theirstimulation by formyl-Met-Leu-Phe with or withoutLDL. The cells' response to formyl-Met-Leu-Phe wasinhibited after pertussis toxin treatment, in the absence(polymorphonuclear neutrophils + formyl-Met-Leu-Phe: O2~ 14.37 ± 0.83 nmol/106 cells; polymorphonu-clear neutrophils + pertussis toxin + formyl-Met-Leu-Phe: O2~ 3.28 ± 1.38 nmol/106 cells; means ± SEM,n = 3) or in the presence of LDL (polymorphonuclearneutrophils + LDL + formyl-Met-Leu-Phe: O2~ 18.45± 0.67 nmol/106 cells; polymorphonuclear neutrophils+ pertussis toxin + LDL + formyl-Met-Leu-Phe: O2~3.72 ± 1.44 nmol/106 cells).

Effects of LDL on phosphatidyl turnover

Polymorphonuclear neutrophil incubation with LDL ata concentration of 2.5 mmol/1 induced a large increasein phosphoinositides, and particularly in phosphatidyli-nositol 4,5-bisphosphate along with inositol 1,4,5-tris-phosphate (fig. 1). Polymorphonuclear neutrophil incu-bation with formyl-Met-Leu-Phe induced, as expected,an activation of phosphatidylinositol 4,5-bisphosphatebreakdown and formation of inositol 1,4,5-trisphos-phate.

Stimulation of polymorphonuclear neutrophils by for-myl-Met-Leu-Phe in the presence of LDL also led to anincrease of phosphatidylinositol 4,5-bisphosphate in allthe LDL or formyl-Met-Leu-Phe concentrations used(tab. 1).

Effect of LDL on calcium mobilization

The intracellular Ca2+ mobilization, measured as Fura 2fluorescence intensity, increased with LDL concentra-tions (fig. 2). At the lowest LDL concentration, thekinetics were similar to those obtained by formyl-Met-Leu-Phe stimulation. At LDL concentrations above 0.5mmol/1 cholesterol, kinetics were the same as those ob-tained after polymorphonuclear neutrophil stimulationby a particular agent, such as opsonized zymosan. Bo-vine albumin at concentrations from 0.1 to 1.0 mg/1 in-duced less than 1/10 of the increase in Fura 2 fluores-cence intensity induced by LDL at respective proteinconcentrations.

Staurosporine effect

At a concentration of 100 nmol/1, Staurosporine showeda potentiation of formyl-Met-Leu-Phe-induced polymer-

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Fig. 1 Variation of phosphoinositides and inositol phosphates of polymorphonuclear neutrophilsstimulated with formyl-Met-Leu-Phe or LDL.Mean ± SEM * significantly different from control p ^ 0.05Wilcoxon's test for a paired series (n = 10) ** significantly different from control p s 0.01

76 Bonneau et al.: Effects of human LDL on Superoxide production by formyl-Met-Leu-Phe activated granulocytes

Tab. 1 Effects of LDL on polymorphonuclear neutrophil stimulation by formyl-Met-Leu-Phe.Radioactivity was expressed in counts per minute

LDL(mmol/1)

2.52.52.55.05.02.55.0

Formyl-Met-Leu-Phe(mmol/1)

io-6

io-6

10~6

ΙΟ"6

io-6

io-7

io-9

Phosphatidylinositol 4,5-bisphosphate

Formyl-Met-Leu-Phe(counts/min)

291677033

16377

159

LDL + formyl-Met-Leu-Phe(counts/min)

65292263136344200340

Inositol 1,4,5-trisphosphate

Formyl-Met-Leu-Phe(counts/min)

124164251168167241137

LDL + formyl-Met-Leu-Phe(counts/min)

70170202136154220110

phonuclear neutrophil stimulation (polymorphonuclearneutrophils + formyl-Met-Leu-Phe: ΟΓ 2.58 ± 0.92nmol/106 cells; polymorphonuclear neutrophils + staur-osporine + formyl-Met-Leu-Phe: O^ 6.37 ± 2.06nmol/106 cells; means ± SEM, n = 3).

Staurosporine did not significantly modify the LDL ef-fect on the polymorphonuclear neutrophil response toformyl-Met-Leu-Phe (polymorphonuclear neutrophils +LDL + formyl-Met-Leu-Phe: Oi~ 8.12 ± 1.91 nmol/106

cells; polymorphonuclear neutrophils + Staurosporine +LDL + formyl-Met-Leu-Phe: Oi~ 8.87 ± 2.29 nmol/IO6 cells).

Propranolol effect

Polymorphonuclear neutrophil preincubation for 5 minat 37 °C with phosphate buffer or with various concen-

trations of propranolol, ranging from 0.05 mmol/1 to5 mmol/1, was performed before the polymorphonuclearneutrophil stimulation with LDL, formyl-Met-Leu-Pheor LDL + formyl-Met-Leu-Phe.

Propanolol exerted a potent stimulating effect at a con-centration of 0.5 mmol/1 but an inhibiting effect at thehighest concentration (5 mmol/1).

LDL added their proper effect to that exerted by formyl-Met-Leu-Phe (fig. 3a).

Niflumic acid effect

Niflumic acid had no effect on polymorphonuclear neu-trophil stimulation by LDL. When the cells were prein-cubated for 5 min at 37 °C with various concentrationsof niflumic acid (88.6 μηιοΐ/l, 443 μηιοΙ/1, 886 μηιοΙ/1,

0.46

Formyl-Met-Leu-Phe, ιl(T9mol/l

0.1 0.5 l

LDL cholesterol [mmol/1]

2.5

Fig. 2 Effect of LDL on cytosolic-free calcium concentration inpolymorphonuclear neutrophils. Calcium concentration statedabove the peaks was calculated from Fura 2 fluorescence intensityusing the formula: [Ca2+] nmol/1 = 224 (F - Fmin)/(Fmax - F)

where Fmin and Fmax were the fluorescence intensity measured onunstimulated and on Triton X-100 treated polymorphonuclear neu-trophils respectively. A representative experiment out of three isshown.


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LDL +formyl-Met-Leu-Phe

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Fig. 3 Effect of propranolol (a) and niflumic acid (b) on polymor-phonuclear neutrophil stimulation by formyl-Met-Leu-Phe andLDL.Concentrations of propranolol used:ffl control, E2 0.05 mmol/1, B 0.5 mmol/1, W 5 mmol/1Concentrations of niflumic acid used:• control, 0 88.6 μτηοΐ/l, Β 0.5 mmol/1, W 886 μτηοΐ/l,m 1772 μηιοΙ/1Result of one representative experiment

1772 μπιοΙ/1) before the addition of formyl-Met-Leu-Phe, an inhibiting effect was observed for concentrationshigher than 88.6 μηιοΐ/ΐ. LDL potentiated the formyl-Met-Leu-Phe stimulating effect and added their properstimulating effect to the response of control cells incu-bated in phosphate buffered saline (fig. 3b).

Intracellular cholesterol content

In the presence of LDL (0.25-1 mmol/1 cholesterol) aconcentration-dependent increase of polymorphonuclearneutrophil cholesterol content was observed (Mann-Whitney test: ρ < 0.05) (fig. 4).

DiscussionThis study has shown that LDL, at physiological con-centrations, interacts with polymorphonuclear neutrophil

stimulation either directly by its own stimulating effector indirectly by modification of the response to formyl-Met-Leu-Phe.

In previous work (5), a slight enhancement of the bind-ing of formyl-Met-Leu-Phe to its specific membrane re-ceptors was observed in the presence of LDL which mayinduce an increase in Oi~ released in response to for-myl-Met-Leu-Phe. Because LDL did not interfere withpertussis toxin inhibitable G proteins, they also mighthave a "priming" effect which can potentiate theNADPH oxidase stimulation by formyl-Met-Leu-Phe(34) rather than increase the number or affinity of theformyl-Met-Leu-Phe receptors.

Phospholipase C activity was investigated by means ofinositol phosphate measurement and a phosphoinositidemetabolism study. We observed a highly significantincrease of phosphatidylinositol 4,5-bisphosphatecontent which could not be due to an accelerated inositolturnover because dephosphorylating enzymes were in-hibited by the lithium added to the incubation buffer.However, it is possible that tritiated inositol, in excessin the cell medium, conjugated to CMP-activated phos-phatidic acid was able to replenish membrane stocks ofphosphatidyl inositol (phosphates). This phenomenonmight be due to phospholipase D activation by LDL,leading to an increased generation of phosphatidic acid.This activation may be greater than that of phospholi-pase C inducing a relatively weak enhancement of inosi-tol 1,4,5-trisphosphate release. Inositol 1,4,5-trisphos-.phate is released into the cytosol, binds to specific re-ceptors on intracellular Ca2+ storage organelles and in-duces the release of calcium. In our study, theintracellular calcium mobilization, measured as Fura 2fluorescence intensity, increased with LDL concentra-tions. However at LDL cholesterol concentrations above

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Fig. 4 Cellular cholesterol content of polymorphonuclear neutro-phils after contact of 15 min with LDL at different concentrations.Mean ± SEM; one experiment in triplicateMann-Whitney's test for non-paired values* significantly different from control (p ̂ 0.05)


0.5 mmo/1, Fura 2 fluorescence was sustained even inan incubation medium without calcium and in the pres-ence of EGTA (data not shown). These data suggest thatthe calcium efflux could be decreased by LDL. To thebest of our knowledge, there is no previous study on theeffect of LDL on polymorphonuclear neutrophil phos-phoinositide metabolism. However Block et al. (35) havealready described a stimulation of the phosphatidylinosi-tol cycle by LDL in human platelets, lymphocytes andfibroblasts.Protein kinase C, a phospholipid/Ca2+-dependent ki-nase, is a key enzyme in the regulation of intracellularsignal transduction. Staurosporine, an alkaloid extractedfrom Streptomyces, was described by Tamaoki et al. (36)as a protein kinase C inhibitor. These authors have sug-gested that Staurosporine interacts directly with proteinkinase C and this hypothesis has been corroborated byNakadate et al. (37) who have shown that Staurosporineinteracts at the active site of the catalytic fragment ofprotein kinase C. Staurosporine is one of the most potentinhibitors, active at nanomolar concentrations, but de-monstrates similar affinities for protein kinase C, tyro-sine kinases and c-AMP-dependent protein kinase (38,39). In our previous experiments, polymorphonuclearneutrophil stimulation by phorbol myristate acetate wasinhibited 90% by Staurosporine, 100 nmol/1. However,the effect of LDL on polymorphonuclear neutrophil doesnot seem to be directly and exclusively protein kinaseC-dependent because in the present study it was not in-hibited by staurosporine.Nevertheless, staurosporine exerts several effects onpolymorphonuclear neutrophil activation. This sub-stance at a concentration of 100 nmol/1 potentiates poly-morphonuclear neutrophil stimulation by formyl-Met-Leu-Phe (40) and inhibits Superoxide generation athigher concentrations. These effects might depend onthe length of polymorphonuclear neutrophil treatmentwith staurosporine. Combadiere et al. reported 125%O2~ production compared to the control (41) while wefound a 200% increase in O2~ production by polymor-phonuclear neutrophil incubation 10 min with stauro-sporine before the addition of formyl-Met-Leu-Phe.Likewise, an increase of phosphatidic acid release wasshown by Reinhold et al. (42) and Perianin et al. (43).Recently, Mori et al. (44) have shown that enhancementof Superoxide production by staurosporine 100 nmol/1via phospholipase D activation in formyl-Met-Leu-Phe-stimulated polymorphonuclear neutrophils was the resultof an increase in diacylglycerol production throughphosphatidic acid.Considering the phopsholipase D activation already de-scribed with staurosporine, we hypothesize that the ef-fects of LDL treatment on polymorphonuclear neutro-phil phosphoinositide metabolism could be explained bya similar mechanism.

In formyl-Met-Leu-Phe-activated polymorphonuclearneutrophils, phosphatidylcholine is the phospholipase Dsubstrate generating phosphatidic acid which in turn leadsto a diacylglycerol release after its dephosphorylation byphosphatidate phosphohydrolase. Rossi et al. have shownthat this enzyme was inhibited by propranolol (45). In thesame experimental conditions as those used by these au-thors, we showed an enhancement of O2~ generationwhich is for English et al. (46) correlated to phosphatidicacid release. However, Agwu et al. have shown that poly-morphonuclear neutrophil stimulation by formyl-Met-Leu-Phe was counteracted by high a propranolol concen-tration (5 mmol/1) which inhibited phospholipase D (47).Under the same experimental conditions, we observedthat LDL did not modify the propranolol effect.

In formyl-Met-Leu-Phe-stimulated human neutrophils,protein kinase C-independent activation mechanismshave been suggested and M ller & Nigam (48) haveshown that staurosporine enhances arachidonic acid andplatelet activation, and leukotriene B4 release. We havethus tested the activity of niflumic acid, a non-steroidalanti-inflammatory drug, on polymorphonuclear neutro-phil activation. According to Abramson et al. (49) vari-ous concentrations of niflumic acid inhibited formyl-Met-Leu-Phe-induced O2~ generation in a concentration-dependent manner. Nevertheless, niflumic acid did notinhibit the LDL-induced polymorphonuclear neutrophiloxidative metabolism and even the lowest concentrationused (88.5 μπιοΙ/1) potentiated an LDL effect, probablyby stimulating phospholipase A2, which leads to arachi-donic acid accumulation.

LDL is able to modify in vitro the polymorphonuclearneutrophil oxidative metabolism either by a direct stimu-lating effect or by alteration of the polymorphonuclearneutrophil response to formyl-Met-Leu-Phe by an actionwhich could involve phospholipase D and phospholipaseA2 activations.

The LDL structure plays a main role in its action ofpolymorphonuclear neutrophil functions. LDL modifiescell lipid content, since a concentration-dependentincrease in cholesterol level was observed after incuba-tion of polymorphonuclear neutrophil with increasingLDL concentrations. We have previously demonstrated(5) that LDL from hypertriglyceridaemic subjects stim-ulated the polymorphonuclear neutrophil oxidative me-tabolism and inhibited polymorphonuclear neutrophilchemotaxis less than LDL from normolipaemic subjectswhose composition is different. Moreover, LDL trypsi-nation experiments showed that the lipid moiety ismainly responsible for the described effects of LDL onpolymorphonuclear neutrophil migration (5). These ob-servations, added to the fact that the polymorphonuclearneutrophil cholesterol amount increases after the incuba-tion of these cells with increasing LDL concentrations,


suggest that lipid exchanges between lipoproteins andcell membranes might appear and modify the membranefluidity. These modifications could alter formyl-Met-Leu-Phe receptor expression and affinity. This hypothe-sis was corroborated upon viewing the alteration of for-myl-Met-Leu-Phe binding on polymorphonuclear neu-trophils extracted from hyperlipidaemic type lib pa-tients (50).

In spite of their short half-life, polymorphonuclearneutrophils could be involved in atherogenic statesbecause they display, in their excited state, a greatsource of free radicals involved in endothelial injuries.LDL are transported into the intimal layer through theendothelium (51) and their concentration in the arterial

intima is three times greater than in plasma (52). Thepolymorphonuclear neutrophils present in the intimamay be activated by LDL. Activated neutrophils mightproduce oxygen free radicals and therefore modifyLDL but they are found in small numbers in theatherosclerotic intima (53). However it has beenshown in rabbits fed a high cholesterol diet that poly-morphonuclear neutrophils were the most numerouscells at the beginning of the atheromateous process,and were soon replaced by monocytes and lympho-cytes (54). Therefore it seems particularly interestingto study the interaction between polymorphonuclearneutrophils and lipoproteins during the early stages ofatherogenesis in animal models.

References1. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum

JL. Beyond cholesterol. Modifications of low-density lipopro-teins that increase its atherogenicity. N Engl J Med 1989;320:915-24.

2. Slyper AH. Low-density lipoprotein density and atherosclero-sis. J Am Med Ass 1994; 272:305-8.

3. Terkeltaub R, Curtiss LK, Tenner AJ, Ginsberg MH. Lipopro-teins containing apoprotein B are a major regulator of neutro-phil responses to monosodium urate crystals. J Clin Invest1984; 73:1719-30.

4. Ludwig P, Hunninghake D, Hoidal J. Increased leukocyte oxi-dative metabolism in hyperlipoproteinaemia. Lancet 1982;14:348-50.

5. Bonneau C, Couderc R, Roch-Arveiller M, Giroud JP, Raich-varg D. Effects of low-density lipoproteins on polymorphonu-clear leukocyte functions in vitro. J Lipid Medial Cell Signal1994; 10:203-12.

6. Ricevuti G, Mazzone A, Pasotti D, De Servi S, Speccia G.Role of granulocytes in endothelial injury in coronary heartdisease in humans. Atherosclerosis 1991; 91:1 — 14.

7. Harats D, Mulkins MA, Sigal E. A possible role for 15-lipoxy-genase in atherogenesis. Trends Cardiovasc Med 1995;5:29-36.

8. Consigny PM. Pathogenesis of atherosclerosis. Am J Roent-genol 1995; 164:553-8.

9. Ward PA. Mechanisms of endothelial cell injury. J Lab ClinMed 1991; 188:421-6.

10. Natarajan V. Oxidants and signal transduction in vascular en-dothelium. J Lab Clin Med 1995; 125:26-37.

11. Wieland E, Brandes A, Armstrong VW, Oellerich M. Oxida-tive modification of low-density lipoproteins by human poly-morphonuclear leukocytes. Eur J Clin Chem Clin Biochem1993; 31:725-31.

12. Jackson RL, Ku G, Thomas CE. Antioxydants: a biologicaldefense mechanism for the prevention of atherosclerosis. MedRes Rev 1993; 13:161-82.

13. Kilgore KS, Lucchesi BR. Reperfusion injury after myocardialinfarction: the role of free radicals and the inflammatory re-sponse. Clin Biochem 1993; 26:359-70.

14. Babior BM. The respiratory burst of phagocytes. J Clin Invest1984; 73:599-601.

15. Chanock SJ, El Benna J, Smith RM, Babior BM. The respira-tory burst oxidase. J Biol Chem 1994; 269:24519-22.

16. Lambeth JD. Activation of the respiratory burst oxidase in neu-trophils: on the role of membrane-derived second messengers,Ca++, and protein kinase C. J Bioenerg Biomembr 1988;20:709-33.

17. Snyderman R, Uhig RJ. Phagocytic cells: stimulus-responsecoupling mechanisms. In: Gallin JI, Goldstein IM, SnydermanR, editors. Inflammation: basic principles and clinical corre-lates. New York: Raven Press, 1988:309-23.

18. Cockcroft S. G-Protein-regulated phospholipases C, D and A2-mediated signalling in neutrophils. Biochim Biophys Acta1992; 1113:135-60.

19. Baggiolini M, Boulay F, Badwey JA, Curnutte JT. Activationof neutrophil leukocytes: chemoattractant receptors and respi-ratory burst. FASEB J 1993; 7:1004-10.

20. Weingarten R, Bokoch GM. GTP binding proteins and signaltransduction in the human neutrophil. Immunol Lett 1990;26:1-6.

21. Thelen M, Dewald B, Baggiolini M. Neutrophil signal trans-duction and activation of the respiratory burst. Physiol Rev1993; 73:797-821.

22. Zenou M, Raichvarg D, Agneray J. Unmodified phagocytosisafter periodate treatment of human granulocytes separated by atwo-step method using Ficoll-Hypaque and polyvinylic alcoholmedium. Biomedicine 1981; 35:166—9.

23. Johnston RB, Keel BB, Misra HP, Lehmeyer JE, Webb LS,Baehner RL, et al. The role of Superoxide anion generation inphagocytic bactericidal activity. J Clin Invest 1975;55:1357-72.

24. Tissot M, Mathieu J, Mirossay L, Thuret A, Giroud JP. Poly-phosphoinositide metabolism in polymorphonuclear cells fromhealthy and thermally injured rats. Effect of the immunomodu-lator RU 41740. J Leukoc Biol 1991; 50:607-14.

25. Creba JA, Downes CP, Hawkins PT, Brewster G, Michell RH,Kirk CJ. Rapid breakdown of phosphatidylinositol 4-phos-phate and phosphatidylinositol 4,5-bis-phosphate in rat hepato-cytes stimulated by vasopressin and other Ca2+-mobilizinghormones. Biochem J 1983; 212:733-47.

26. Poggioli J, Mauger JP, Claret M. Effect of cyclic AMP-depen-dent hormones and Ca++ mobilizing hormones on the Ca++

influx and polyphosphoinositide metabolism in isolated rat he-patocytes. Biochem J 1986; 235:663-9.

27. Downes CP, Michell RH. The polyphosphoinositide phospho-diesterase of erythrocyte membranes. Biochem J 1981;198:133-40.

28. Berridge MJ, Dawson RMC, Downes CP, Heslop JP, IrvineRF. Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. Bio-chem J 1983; 212:473-82.

29. Periani A, Snyderman R. Analysis of calcium homeostasis inactivated human polymorphonuclear leucocytes: evidence fortwo distinct mechanisms for cytosolic calcium. J Biol Chem1989; 264:1005-9.

30. Grynkiewicz G, Poenie M, Tsien RY. A new generation ofCa2+ indicators with greatly improved fluorescence properties.J Biol Chem 1985; 260:3440-50.

31. Kopf GS, Woolkalis MJ. ADP-ribosylation of G proteins withpertussis toxin. Methods Enzymol 1991; 195:257-66.

32. Gabig TG, English D, Akard LP, Schell MJ. Regulation ofneutrophil NADPH oxidase activation in a cell-free system by


guanine nucleotides and fluoride. J Biol Chem 1987;262:1685-90.

33. Omann GM, Porasik-Lowes MM. Graded G-protein uncoupl-ing by pertussis toxin treatment of human polymorphonuclearleukocytes. J Immunol 1991; 146:1303-8.

34. Hallet MB, Lloyds D. Neutrophil priming: the cellular signalsthat say 'amber' but not 'green'. Immunol Today 1995;16:264-8.

35. Block LH, Knorr M, Vogt E, Locher R, Vetter W, GroscurthP, et al. Low density lipoprotein causes general cellular activa-tion with increased phosphatidylinositol turnover and lipopro-tein catabolism. Proc Natl Acad Sei USA 1988; 85:885-9.

36. Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M,Tomita F. Straurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase. Biochem Biophys Res Com-mun 1986; 135:397-402.

37. Nakadate T, Yeng AY, Blumberg PM. Comparison of proteinkinase C functional assays to clarify mechanisms of inhibitoraction. Biochem Pharmac 1988; 37:1541-5.

38. Blumberg PM. Complexities of the protein kinase C pathway.Mol Carcinog 1991; 4:339-44.

39. Badwey JA, Erickson RW, Curnutte JT. Staurosporine inhibitsthe soluble and membrane-bound protein tyrosine kinases ofhuman neutrophils. Biochem Biophys Res Commun 1991;178:423-9.

40. Combadiere C, Hakim J, Giroud JP, Perianin A. Staurosporine,a protein kinase inhibitor, up-regulates the stimulation of hu-man neutrophil respiratory burst by N-formyl peptides andplatelet activating factor. Biochem Biophys Res Commun1990; 168: 65-70.

41. Combadiere C, El Benna J, Pedruzzi E, Hakim J, Perianin A.Stimulation of the human neutrophil respiratory burst by for-myl peptides is primed by a protein kinase inhibitor, Stauro-sporine. Blood 1993; 82:2890-8.

42. Reinhold SL, Prescott SM, Zimmerman GA, Me Intyre TM.Activation of human neutrophil phospholipase D by three sep-arable mechanisms. FASEB J 1990; 4:208-14.

43. Perianin A, Combadiere C, Pedruzzi E, Djerdjouri B, Hakim J.Staurosporine stimulates phospholipase D activation in humanpolymorphonuclear leukocytes. FEBS Lett 1993; 315:33-7.

44. Mori T, Ando M, Takagi K. Staurosporine and its derivativesenhance f-Met-Phe-induced Superoxide production via phos-pholipase D activation in human polymorphonuclear leuko-cytes. Int J Clin Pharmacol Ther 1994; 32:422-8.

45. Rossi F, Grzeskowiak M, Delia Bianca V, Calzetti F, GandiniG. Phosphatidic acid and not diacylglycerol generated by phos-

pholipase D is functionally linked to the activation of theNADPH oxidase by fMLP in human neutrophils. BiochemBiophys Res Commun 1990; 168:320-7.

46. English D, Taylor GS. Divergent effects of propranolol on neu-trophil Superoxide release: involvement of phosphatidic acidand diacylglycerol as second messengers. Biochem BiophysRes Commun 1991; 175:423-9.

47. Agwu DE, Me Phail LC, Sozzani S, Bass DA, Me Call CE.Phosphatidic acid as a second messenger in human polymor-phonuclear leukocytes. Effects on activation of NADPH oxi-dase. J Clin Invest 1991; 88:531-9.

48. Müller S, Nigam S. Arachidonic acid release and platelet-acti-vating factor formation by Staurosporine in human neutrophilschallenged with n-formyl peptide. Eur J Pharmcol 1992;218:251-8.

49. Abramson SB, Leszczynska-Piziak J, Weissman G. Arachi-donic acid as a second messenger. Interactions with a GTP-binding protein of human neutrophils. J Immunol 1991;147:231-6.

50. Bonneau C, Couderc R, Chanu B, Bailleul S, Roch-ArveillerM, Rouffy J, et al. Fonctions des polynucleaires neutrophilesde sujets hypercholesterolemiques. In: Galteau MM, Siest G,Henny J, editors. Biologie Prospective. Comptes rendus duSeme colloque de Pont-a-Mousson. Paris: John Libbey Euro-text, 1993:549-52.

51. Muller WA. Migration of leukocytes across the vascular in-tima. Molecules and mechanisms. Trends Cardiovas Med1995; 5:15-20.

52. Kovanen PT. Atheroma formation: defective control in the inti-mal round-trip of cholesterol. Eur Heart J 1990; 11 (SupplE):238-46.

53. Jonasson L, Holm J, Skalli O, Bondjers G, Hansson GK. Re-gional accumulation of T cells, macrophages, and smooth mus-cle cells in the human atherosclerotic plaque. Arteriosclerosis1986; 6:131-8.

54. Kling D, Holzschuh T, Betz E. Recruitment and dynamics ofleukocytes in the formation of arterial thickening — a compar-ative study with normo- and hypercholesterolemic rabbits. Ath-erosclerosis 1993; 101:79-96.

Received November 6, 1996Corresponding author: Dr. R. Couderc, Service de Biochimie,Höpital Tenon, 4 rue de la Chine, F-75020 Paris, France

effects of human low-density lipoproteins on superoxide

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