Oxidative metabolism and release of myeloperoxidase from polymorphonuclear leukocytes obtained from blood sedimentation in a ficoll‐hypaque gradient

CELL BIOCHEMISTRY AND FUNCTION

Cell Biochem[ Funct[ 07\ 016Ð021 "1999#

Received 16 September 0888Copyright Þ 1999 John Wiley + Sons\ Ltd[ Accepted 10 December 0888

Oxidative Metabolism and Release of Myeloperoxidase fromPolymorphonuclear Leukocytes Obtained from BloodSedimentation in a Ficoll!Hypaque Gradient

IVANISE M[ M[ REBECCHI\0 NEIL FERREIRA NOVO\1 YARA JULIANO1 AND ANA CAMPA0�

0 Departmento de Ana�lises Cl(�nicas e Toxicolo�`icas\ Faculdade de Cie¼ncias Farmace¼uticas\ Universidade de Sa½o Paulo"USP#\ Brazil1 Departamento de Bioestat(�stica da Universidade Federal de Sa½o Paulo "UNIFESP#\ Brazil

Polymorphonuclear neutrophils "PMN# have an important role in the host defence response to infection[ These cellsproduce large amounts of reactive oxygen species "O=−

1 \ H1O1 and ONOO−# with microbicidal activity[ PMN arecommonly isolated from peripheral blood by sedimentation through a gradient of density "Ficoll!Hypaque gradientand dextran#\ yielding a highly homogeneous cellular population[ However\ some cellular activation due to membraneperturbation is also expected[ We studied how the production of reactive oxygen species and release of myelo!peroxidase "MPO# from blood PMN are a}ected by the use of the Ficoll!Hypaque density gradient[ PMN isolatedby spontaneous sedimentation and total blood were used for comparisons[ Lucigenin! and luminol!enhanced chemi!luminescence was used to estimate the production of reactive oxygen from intact cells and shown to be higher forcells isolated by density gradient both in the absence and presence of added stimuli[ The release of MPO\ estimatedby the chemiluminescence of the luminol:H1O1 reaction in the supernatant of PMN incubated in the absence andpresence of stimuli and absence and presence of cytochalasin B\ was also higher for PMN isolated by a densitygradient[ In conclusion\ it was shown that the PMN isolation procedure a}ects reactive oxygen species productionand MPO release and in some cases may cause a misinterpretation of results[ Copyright Þ 1999 John Wiley + Sons\Ltd[

KEY WORDS * oxidative metabolism^ myeloperoxidase^ polymorphonuclear leukocytes^ Ficoll!Hypaque^ spontaneous sedi!mentation^ blood cells^ chemiluminescence

ABBREVIATIONS * C\ control^ FMLP\ N!formyl!methionyl!leucyl!phenylalanine^ MPO\ myeloperoxidase^ PMA\ phorbol miristateacetate^ PMN\ polymorphonuclear leukocytes^ ZY\ opsonized zymosan\ SG\ sedimentation in gradient^ SS\spontaneous sedimentation^ TB\ total blood^ HRP\ horseradish peroxidase

INTRODUCTION

Leukocyte separation through spontaneous sedi!mentation "SS# of heparinized blood was _rstdescribed by Hirschhorn and Weissmann0 and wasfurther improved by the use of gradients of density"SG#[1Ð3 Besides the advantages of yielding more

� Correspondence to Dr Ana Campa\ Faculdade de Cie¼nciasFarmace¼uticas\ Universidade de Sa½o Paulo\ Sa½o Paulo\ CEP94497!899\ SP\ Brazil[ Tel] "44!00# 7072526[ Fax] "44!00#7021086[ E!mail] anacampaÝusp[br[

Contract grant sponsor] Fundacža½o de Amparo a� Pesquisa doEstado de Sa½o Paulo "FAPESP\ Sa½o Paulo#[Contract grant sponsor] Conselho Nacional de Desenvolvi!mento Gient(�f(�co e Tecnolo�gico "CNPq\ Bras(�lia#[

puri_ed cell preparations\ these procedures addedsome disadvantages[ It is known that chemicalagents or osmotic shock "heparin\ EDTA\ NH3CLor dextran# can change the size\ shape\ number andintensity of azurophil granules of blood neutro!phils[4\5 Granule exocytosis or the release ofenzymes can occur in response to mechanical shock\hypotonic or acidic medias or to the presence ofcalcium[6 Concern about modi_cations in metab!olism and functionality has also been pointed out[7\8

Neutrophils are key cells in the immune response[The whole cell or subcellular structures are widelyused in cellular biochemistry or immunology stud!ies[ The activation state of this cell has been cor!related with the production of reactive oxygen

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species and is easily estimated by chemi!luminescence in the presence of luminol and luci!genin[ The utilization of total blood "TB# orheterogeneous preparations for the monitoring ofthe neutrophil oxidative burst\ although avoidingextensive manipulation\ might add to the dis!advantages of low sensitivity and of high inter!ference from other blood components[

In this work we evaluate the in~uence of SG onthe neutrophil activation state and the MPOrelease[ Cells obtained from SG were comparedwith cells obtained from SS or TB[ Luminol!and lucigenin!enhanced chemiluminescence09 wasmonitored in the absence and presence of phorbolmyristate acetate "PMA#\ zymosan "ZY# or N!formyl!methionyl!leucyl!phenylalanine "FMLP#[Azurophil degranulation was estimated in thesupernatant of PMN incubated in the absence andpresence of stimuli and in the presence and absenceof cytochalasin B "cyt B#\ by the chemiluminescenceof luminol:H1O1[

MATERIAL AND METHODS

Zymosan A\ PMA\ FMLP\ luminol\ lucigenin\HistopaqueR\ dextran\ horseradish peroxidase"HRP#\ myeloperoxidase from human leukocytes"MPO# and cytochalasin B were purchased fromSigma Chemical Co[ and PerhidrolR was obtainedfrom Merck[ All other reagents were high puritycommercial samples from Sigma\ Merck andAldrich[

The ZY was opsonized by incubation withhuman serum\ at 26>C for 0 h[ PMA was solubilizedin dimethyl sulfoxide and FMLP was dissolved inaqueous solution[ All these stimulants were frozenat −09>C in small aliquots[ Luminol and lucigeninwere dissolved in water[ Dextran was prepared in9=8 per cent NaCl[ Cytochalasin B\ MPO and HRPwere dissolved in PBS Dulbecco 09 mM at pH 6=3[

PMN was obtained from the blood of healthyvolunteers after 01 h of fasting and collected intoheparin "49 UI ml−0 blood#[

PMN Isolation by SG

PMN were isolated from blood essentially by themethod of Bo�yum[2 Blood "4 ml# was diluted withan equal volume of PBS Dulbecco 09 mM at pH 6=3containing 099 mM CaCl1\ 49 mM MgCl1 and glu!cose 0 mg ml−0 and carefully layered on 4 ml ofa commercial gradient of Ficoll!Hypaque "Histo!paqueR\ d�0=966#[ The tube was centrifuged at

1499 r[p[m[ at room temperature for 19 min[ Thesupernatant\ rich in mononuclear cells wasdiscarded\ and 09 ml of dextran 4 per cent wasadded to the pellet[ The tube was homogenized andmaintained for 29 min at room temperature to allowerythrocyte sedimentation[ The resulting super!natant\ rich in granulocytes\ was recovered\ washedwith PBS Dulbecco and the pellet submitted tohypotonic treatment with 09 ml of distilled water topromote lysis of contaminated erythrocytes[ After 0min\ the isotonicity was restored by the addition of4 ml of 1=6 per cent NaCl and PBS Dulbecco[

PMN Isolation by SS

PMN were isolated from blood essentially by themethod of Hirschhorn and Weissmann[0 Leu!kocyte!rich plasma was obtained by spontaneouserythrocyte sedimentation from 09 ml of blood keptat 3>C for 89 min[ The supernatant was then gentlyremoved and centrifuged at 0499 r[p[m[ for 4 minat room temperature[ The pellet was submitted tohypotonic treatment with 09 ml of distilled waterto promote lysis of contaminating erythrocytes andafter 0 min the isotonicity was restored by theaddition of 4 ml of 1=6 per cent NaCl and PBSDulbecco[

Cells obtained from both procedures of sep!aration were counted in a Neubauer chamber andwere morphologically evaluated by MayÐGru�n!waldÐGiemsa staining[ The viability was deter!minate by Trypan blue exclusion[

Chemiluminescence Assays

Chemiluminescence was followed in Packard0899TR liquid scintillation analyser in the mode outof coincidence at room temperature[ The luminol"0×09−4 M# or lucigenin "1=4×09−4 M#!enhancedchemiluminescence of 0×094 PMN was monitoredin phosphate bu}ered Dulbecco "without glucose#in a _nal volume of 1 ml[ PMA "49 ng ml−0#\ ZY"1=4 mg ml−0# or FMLP "3×09−5 M# were used tostimulate the cells[ Integrated values of light emis!sion were taken after 093 min of reaction[

A chemiluminescence assay for MPO using theluminol "0×09−4 M#:H1O1 "0×09−4 M# reactionwas standardized with commercial HRP or MPOin phosphate bu}ered Dulbecco "without glucose#[The release of MPO was estimated in the super!natant "049 ml# of PMN "0×094# incubated in theabsence and presence of stimuli and cytochalasin B"4 mg ml−0# in the same concentrations mentioned

PMN AND FICOLL!HYPAQUE GRADIENTS

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above for 29 min at 26>C[ Integrated values forlight emission were taken after 177 min[

Statistical Analysis

Comparisons among SS\ SG and TB and amongnon!stimulated and PMA!\ ZY!\ FMLP!stimulatedcells were made using the Friedman two!way analy!sis of variance[ When di}erences were observed\ themultiple comparisons test was used[ For the MPOassays\ comparisons between SS and SG were doneusing the Wilcoxon test for two non!independentsamples[

RESULTS AND DISCUSSION

Isolated PMN can be primed and activated in vitroby several classes of natural and syntheticcompounds[ Simultaneous action of some of thesecompounds triggers a complex event resulting fromsynergistic and antagonistic e}ects among them[It has sometimes been di.cult to give the correctinterpretation to the action of isolated or combinedimmunomodulators\ serum compounds and knownstimuli on PMN responses[ Undoubtedly\ one ofthe most studied PMN function is the oxidativeburst[ How di}erent compounds modify the oxi!dative burst and how to distinguish intra! fromextracellular release of reactive oxygen species"ROS# are additional di.culties[ The complexity ofthis picture can be even greater when chemicals orprocedures used to isolate PMN cells can a}ect theoxidative burst[ The use of TB as a source of PMNis more likely to minimize the contribution of theprocedure of cell isolation itself and consequentlyproduce conditions closer to those found in vivo[However serum compounds and blood cells causelight scattering and absorption\ hindering the useof spectrophotometric assays[ In this work we com!pare the ROS production and MPO release fromPMN isolated through SG and SS with PMN pre!sent in TB[

The lucigenin! and luminol!enhanced chemi!luminescence were used here to estimate the sum ofintra! and extra!cellular formation of ROS[ Despitenot being speci_c\ it was considered that at leastpart of the luminol!enhanced chemiluminescence isrelated to the MPO:H1O1:halide system[00\01

As expected\ the cellular population obtainedthrough SG was highly enriched with neutrophils"over 89 per cent# when compared to SS "around59 per cent#[ Among leukocyte cells\ the oxidativeburst\ and consequently the luminol! and lucigenin!

enhanced light emission\ is mainly related to PMN"neutrophils plus eosinophils#[ For comparisonsamong the procedures of cell isolation\ PMN acti!vation status was estimated with the same numberof PMN cells\ independent of the presence of othercell types[ For SS and TB\ it is expected that thepresence of cellular types other than PMN con!tribute to increased light scattering and non!speci_clight absorption[ The status of PMN activation fol!lowing its isolation by SG or SS or in TB is shownin Figure 0[ Di}erences among SS\ SG and TB areonly observed for the luminol!enhanced chemi!luminescence[ PMN obtained through SG gives riseto a chemiluminescent intensity around one orderof magnitude higher than SS and TB[ The fact thatdi}erences could only be observed in the presenceof luminol and not with lucigenin indicates that] "i#light absorption deriving from cells other thanPMN was not su.cient to explain the lower chemi!luminescence seen for the SS preparation and\ "ii#activation of cells occurred during SG preparationand that this activation may be in some way relatedto the MPO system[

It was further investigated how cells obtainedthrough SS\ SG and TB respond to a stimulus[The integrated light emissions observed after theaddition of PMA\ ZY and FMLP are shown inFigures 1Ð3\ for PMN obtained through SS\ SGand TB\ respectively[ The pro_le of response ofthese cells to the di}erent stimuli was essentiallythe same\ independent of the procedure of PMNisolation[ For the conditions used "number of cells#\FMLP was not able to trigger chemiluminescencethat was statistically di}erent from the control"without stimulus#[ Di}erences between stimulatedand non!stimulated cells were always seen for ZY[Statistical di}erences in relation to the control werenot seen for all the reactions with PMA[ For allstimuli\ the luminol!enhanced chemiluminescencewas much higher for SG than for SS and TB[

In sequence\ the release of MPO from PMNobtained through SG and SS was evaluated[ Inorder to enhance degranulation\ this study was alsodone in the presence of cytochalasin B[02\03 Com!mercial and puri_ed HRP and MPO samples wereused to standardize the conditions of a chem!iluminescent determination of MPO using luminol:H1O1[ The values of integrated light emission werelinear with respect to HRP "r�9=88# and MPO"r�9=86# concentration in the range of 0Ð19×09−8

M[ The supernatant resulting from the incubationof PMN isolated through SS or SG with PMA\ ZYor FMLP\ in the absence or presence of cyto!

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Figure 0[ Luminol! "A# and lucigenin! "B# enhanced chemiluminescence of PMN obtained through SS\ SG and TB[ The numberof experiments was four and _ve for the luminol! and lucingenin!enhanced chemiluminescence\ respectively[ � P ³ 9=91 for di}erencesin relation to TB[

Figure 1[ Luminol! "A# and lucigenin! "B# enhanced chemiluminescence of PMN obtained through SS stimulated with PMA\ ZY\FMLP or without stimulation "C#[ The number of experiments was four and _ve for the luminol! and lucigenin!enhancedchemiluminescence\ respectively[ � P ³ 9=90^ è P ³ 9=94 for di}erences in relation to the control[

chalasin B\ were added to the luminol H1O1 reactionand the chemiluminescence was monitored[ Thevalues of integrated light emission are shown inTable 0[ Comparing the procedures\ higher valuesof chemiluminescence were found when PMNobtained through SG were stimulated with ZY andFMLP[ This e}ect was much stronger in the pres!ence of cytochalasin B[ Although cytochalasin B byitself is not able to activate the superoxide!gen!erating oxidase\ its inhibitory e}ect on FMLP!induced actin polymerization can enhance the oxi!dase activity and degranulation[03

Our data show that the presence of cytochalasinB does not signi_cantly a}ect the release of MPO

by PMN isolated by SS exposed to PMA\ ZY andFMLP[ However\ when cells were isolated by SG\a marked e}ect of cytochalasin B was obtained\especially for cells stimulated with FMLP[ It is wellknown that although cytochalasin B has only aweak e}ect on FMLP!mediated H1O1 production\it enhances the FMLP stimulatory e}ect on MPOrelease[04 The e}ect of cytochalasin B found herewas in some way similar to that reported by Elferinket al[05 where polyarginine\ polylysine and poly!ethyleneamine were able to express an activatinge}ect of cytochalasin B[ It was suggested that poly!cation!elicited membrane perturbation can provideactivation of some steps in the superoxide!gen!

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Figure 2[ Luminol! "A# and lucigenin! "B# enhanced chemiluminescence of PMN obtained through SG stimulated with PMA\ZY\ FMLP or without stimulation "C#[ The number of experiments was four and _ve for the luminol! and lucigenin!enhancedchemiluminescence\ respectively[ � P ³ 9=91^ è P ³ 9=94 for di}erences in relation to the control[

Figure 3[ Luminol! "A# and lucigenin! "B# enhanced chemiluminescence of PMN obtained through TB stimulated with PMA\ ZY\FMLP or without stimulation "C#[ The number of experiments was four and _ve for the luminol! and lucigenin!enhancedchemiluminescence\ respectively[ � P ³ 9=91^ èP ³ 9=94 for di}erences in relation to the control[

Table 0[ Integrated light emission values originated from luminol:H1O1 plus supernatants obtained after incubation of PMN withPMA\ ZY or FMLP in the presence or not of cytochalasin B[ In parentheses are the range of _ve determinations[

Means and range of integrated light emission of the supernatant of stimulated PMN

Without cytochalasin B With cytochalasin B

Stimulus SS SG� SS SG$

PMA 9=11 "9=93Ð9=56# 9=41 "9=10Ð0=14# 9=24 "9=02Ð9=44# 9=42 "9=02Ð0=12#ZY 0=53 "9=07Ð4=09# 3=29 "9=70Ð8=56# 9=13 "9=05Ð9=26# 7=16 "0=55Ð02=05#FMLP 9=00 "9=97Ð9=05# 9=41 "9=17Ð0=96# 9=25 "9=09Ð9=49# 49=38 "9=40Ð89=85#Control 9=26 "9=95Ð9=87# 0=04 "9=09Ð1=15# 9=03 "9=93Ð9=17# 9=22 "9=17Ð9=24#

� P ³ 9=994 in relation to SS\ independent of stimulus^ $ P ³ 9=991 in relation to SS\ independent of stimulus[

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erating oxidase system that were not activatedsolely by cytochalasin B[

The _ndings here described\ associated with thefact that FMLP and ZY lead to degranulation ofthe primary granule\06\07 while PMA mainly stimu!lates the degranulation of non!speci_c granules\07

support our initial suspicion that the SG proceduresomehow a}ects the MPO system\ i[e[ degranu!lation[ The e}ect of SG on MPO release can beclearly observed when ZY or FMLP were used asa stimulus and is even more marked in the presenceof cytochalasin "Table 0#[ It is important to notethat in a previous study the e}ect of a gradient ofdensity on MPO release was not observed\ probablydue to the use of PMA as stimulus[08

The fact that a frequently utilized cell isolationprocedure "Ficoll!Hypaque# interferes with MPOrelease can explain some contradictory resultsfound in the literature[ For instance\ while Pemberand Kinkade19 reported an enhancement in MPOactivity in di}erent populations of PMN\ the samewas not observed by Miyasaki et al[10 Curiously\while the former used Ficoll!Hypaque\ the latterused Percoll gradients to isolate human bloodPMN[

In conclusion\ it was observed that\ although SSintroduces the inconvenience of a heterogeneouspreparation\ it could o}er a source of PMN in anactivation state more similar to that found in vivo[Together\ these _ndings have to be considered instudies where the activation of PMN is sought[

ACKNOWLEDGEMENTS

The authors thank Fundacža½o de Amparo a� Pes!quisa do Estado de Sa½o Paulo "FAPESP\ Sa½oPaulo# and Conselho Nacional de Desen!volvimento Cientif(�f(�co e Tecnolo�gico "CNPq\Bras(�lia# for grant support[

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