Thiol suppression of human immunodeficiency
virus type 1 replication in primary cord blood
monocyte-derived macrophages in vitro.
J Lioy, … , R A Polin, S D Douglas
J Clin Invest.
1993;
91(2)
:495-498.
https://doi.org/10.1172/JCI116227
.
We investigated the effects of glutathione (GSH), the major naturally occurring thiol, and a
pharmacologic thiol precursor of GSH, N-acetyl cysteine (NAC), on the expression of
human immunodeficiency type 1 (HIV-1) in primary cord blood and adult donor
monocyte-derived macrophages (MDM). HIV-1 infection of cord blood and adult MDM was
accomplished after incubating 10-15-d-old cultures for 4 h with a monocyte-tropic strain of
HIV-1 (Bal). After 1 wk in culture cell supernatants were tested for reverse transcriptase (RT)
activity. MDM were exposed to 5, 10 and 20 mM concentrations of both GSH and NAC
before infection, during infection, and after infection was established. GSH and NAC
suppressed the replication of HIV-1 in both primary cord blood and adult donor MDM in a
concentration dependent fashion. These suppressive effects were more pronounced in
cord-derived cells than in adult-derived cells. In cells treated with GSH or NAC before
infection, there was no significant rise in RT activity as compared with controls. Similarly,
when cells were treated with GSH and NAC and simultaneously infected, there was also no
significant rise in RT activity after 1 wk in culture. In cells treated after infection was
established, RT values were suppressed 80-90% that of untreated controls. This effect
persisted for 1-2 wk after exposure to GSH and NAC. Untreated controls demonstrated
syncytium formation and lost […]
Research Article
Thiol
Suppression
of Human
Immunodeficiency
Virus Type
1Replication
in
Primary
Cord Blood Monocyte-derived Macrophages
In
Vitro
JanetLioy,* Wen-ZheHo,*Joann R.Cutilli,t Richard A. Polin,*andSteven D. Douglast
Divisionsof *Neonatology and of tAllergy, Immunology andInfectiousDisease, TheChildren'sHospital ofPhiladelphia,
andDepartmentofPediatrics, UniversityofPennsylvania Medical School, Philadelphia, Pennsylvania19104
Abstract
Weinvestigated the effects of glutathione (GSH), the major naturallyoccurringthiol,and apharmacologic thiol precursor ofGSH, N-acetyl cysteine (NAC),ontheexpression ofhuman
immunodeficiency type I (HIV-1) in primary cord blood and adult donormonocyte-derived macrophages (MDM).HIV-1 infectionof cord blood and adult MDM was accomplished after incubating10-15-d-old cultures for 4 h with a monocyte-tropic strain of HIV-1(Bal). AfterIwk in culture cell supernatants weretestedfor reverse transcriptase (RT) activity. MDM were exposed to5, 10and20 mMconcentrationsof bothGSHand
NAC beforeinfection, during infection,andafterinfectionwas established.GSHandNACsuppressed thereplication of HIV-I in bothprimarycord blood and adult donor MDM in a con-centrationdependent fashion. Thesesuppressive effects were
more pronounced in cord-derived cells than in adult-derived cells. In cells treatedwith GSHorNAC before infection,there was nosignificantrise in RTactivity as compared with controls.
Similarly, when cellswere treated with GSH and NAC and
simultaneouslyinfected,therewasalsonosignificantrise in RT
activityafter1wkin culture. In cells treated after infectionwas
established, RT values were suppressed 80-90% that of
un-treated controls.Thiseffect persisted for1-2 wkafterexposure toGSHandNAC.Untreated controls demonstratedsyncytium formationandlostcharacteristics ofspreadingandelongation2 wk after HIV-1 infection, whereas most ofthe treated cells remained free of syncytiumand retained cytoplasmic spread-ing,adherence, andelongation.Thesedataareconsistent with
otherstudiesofthiolsuppression ofHIV-1replicationand
dem-onstrate asimilar observation for
primary
cultured cord MDM. These results mayoffernewapproaches toward cellular protec-tion afterinfectionwith HIV-1.(J. Clin.Invest. 1993.91:495-498.) Keywords:y-glutamyl
amino acids*'y-glutamylcysteine
synthetase * nuclear factor kB - phorbol
myristate
acetate-reversetranscriptase
Introduction
Overthe lastthreeyears there has beenan
significant
increasein the numberofpediatric AIDScasesreportedbytheCenters for Disease Control (1).
Although
asmall numberofchildren havebeeninfected through transfusions ofhumanimmunode-Addressreprintrequests to Dr.StevenD.Douglas, Sectionof Immunol-ogy, The Children's Hospital of Philadelphia, 34th Street & Civic CenterBlvd.,Philadelphia,PA 19104.
Receivedfor publication 21December1991andinrevisedform 3 September1992.
ficiencyvirus type 1 (HIV-1)-contaminated bloodproducts,
> 80% of infected children have acquired the virus perinatally. Viral transmission can occur prenatally via the placenta, at time ofdelivery bydirect inoculation or postnatally viabreast
milk. The estimated rates of perinatal transmission is- 30%.
Mean survival from the time of diagnosis is estimated at 38 months(1).
The monocyte-derived macrophage(MDM)' may play a central rolein the pathogenesis of HIV- 1 infection. In vitro the macrophage is believed to contribute to viral latency by acting asareservoirfor replication and dissemination (2-4). We have recentlydemonstratedthat cord MDMaresusceptibleto HIV-1 infection with the monocyte-tropic strains (Bal and Ada-M) andcapable ofaproductive infection (5).
Glutathione (GSH), the most abundant intracellular thiol (0.1-10 mM), playsakey role in intracellular defense against reactive oxygen intermediates and is one of the most active biological compounds available tothecell(6). Roederer et al. (7) have recentlydemonstratedthat N-acetylcysteine(NAC) inhibits tumor necrosis factor-a (TNF-a) and phorbol myris-tateacetate(PMA)stimulation of HIV- 1 replication in T cells andperipheral blood mononuclear cells. Kalebic et al. (8) have similarly demonstrated dose-dependent suppression of HIV-1 expression in achronically infected promonocytic U1 cell line with GSH and NAC. These cells were derived from infection of promonocytic U-937 cells carrying two copies of proviral DNA.Recentliteraturesuggeststhat agents thatincreaseGSH levels inhibit nuclear factor kB(NF-kB), which is a known enhancer of HIV- 1 transcription (9) and is a DNA binding
proteinresponsivetocellularactivation. This transcription
fac-torhas been shown to enhance HIV- 1 replication in T cells aftertreatmentwith PMAorTNF-a (10-13). The regulation of this nuclear factor may influence theexpression of certain genesresponsiblefor HIV- 1 expression.NAC has been shown
toblockPMA, TNF-a,andstimulation of HIVlongterminal
repeat-directed gene expression (lacZ gene expression) as
measuredby
f3-galactosidase
activity (7).Intheasymptomaticadultpatientwith HIV- 1 infection, peripheralblood
mononu-clearcells,serum, andlung epithelialfluidhave reduced levels of GSH and soluble thiols(14, 15).Thisdeficiencymay bea
markerforearlytransition fromlatencytothe active disease processin theasymptomaticindividuals.
This articlereportsthe effect ofGSH and NAConthe
infec-tivityof HIV- 1 forprimarycordblood MDM.
Methods
Heparinizedcordblood(20-50U/ml)wasobtained fromthe
umbili-calvein ofhealthytermnewborninfants afteruncomplicated
pregnan-ciesanddeliveries. All cord blood sampleswereidentifiedasHIV-l
1.Abbreviations used in thispaper: MDM,monocyte-derived macro-phage;NAC, N-acetylcysteine; NK-kB, nuclear factorkB.
J.Clin.Invest.
© TheAmerican Societyfor ClinicalInvestigation, Inc.
0021-9738/93/02/0495/04 $2.00
antibody negative by anonymoustestingwith the ELISA method
(Ab-bottLaboratories, NorthChicago, IL). Monocyteswerepurified
ac-cordingto ourpreviouslydescribedtechnique(16).Cord bloodwas
layered onaFicollgradientand monocyte fractionseparatedafter ad-herencetogelatincoated flasks.Cellswereplatedin24-well/cm2 cul-tureplates(Corning Glass, Inc., Corning, NY)at adensityof 3 xI05in RPMIcontaining30% fetal calfserum.Monocyteswerecultured for 10-15 d when theywereconsidered MDM, After the initial purifica-tion,>97% of the cellsweremonocytesasdeterminedby morphology
andfluorescence-activated cellsorting (FACS)analysis using monoclo-nalantibody (mAb) Leu-M3 andlow-densitylipoproteinspecificfor MDM(5).Inall cases,Limulus amebocyte lysateassay demonstrated thatmedia and reagents wereendotoxin-free. HIV- 1 strain Balwas
isolated from human infant lungtissue and provided by Dr. R.C. Gallo'slaboratorythroughthe AIDS Research and ReferenceReagent
Program, Division ofAIDS, National Institute ofAllergy and In-fectious Diseases, National Institutes ofHealth,Bethesda,MD. Infec-tionwasaccomplished by adding0.2 ml (24-wellplate)or0.05ml (48-wellplate) ofHIV-1(with5-8XI05 cpm/ ml ofreverse
transcrip-tase(RT)activity)toeach wellandincubatingthe cellsfor 3 hat370C. The inoculum wasremoved andcellswerewashedfour times with
RPMIto removeunabsorbed virus. The final washwastestedfor RT activity and showntobefree of residual inoculum.Every5-7d super-natantsweretestedforRTactivity.
LevelsofRTactivitypresent in HIV-l-infected cordMDM were
determined by the methods ofWilleyetal.( 17)withmodification.In
brief, 10ulof culture supernatantswasaddedto50
gl
ofacocktailcontaining poly (A), oligo (dt) (Pharmacia Inc., Piscataway, NJ),
MgCl2,Nonidetp-40 and [32P]dTTP (Amersham Corp.,Arlington Heights, IL) and incubated for 20 hat370C.A 30-ulportion of the reactionmixturewasthen spottedon DE81 paper (Whatman Int., Maidstone, England) and air-dried. The filterswerethenwashed in 2X standardsaline citrate(SSC0.3 MNaCl/0.03Msodiumcitrate,pH 7.0) and 100%ethanol,dried,cut, andplacedinascintillationcounter
(LS 7000, Beckman Instruments, Inc., Palo Alto, CA) for measure-mentof radioactivity.
NAC and GSH were purchased from Sigma Chemical Co. (St. Louis, MO). Cord blood MDM were incubated with NAC and GSH (5, 10, and 20 mM)either 24 hbefore addingHIV-l (Bal), simulta-neously with the addition ofHIV-1,orwithin 2 wk ofanalready estab-lishedproductiveinfection withHIV-l (asdeterminedbyRT activ-ity). The pretreated cells wereexposedtoGSHorNACfora24-h period before infection with HIV-1. Similarly, cells were simulta-neously exposedtoGSHorNACfor up to 3 h during infection with
HIV-l(Bal).Instudies of a third groupof cells in which infection was
alreadyestablished,fresh GSHorNAC were added at their respective concentrations every 3 dduringtheexperiment. Intheformer two groups, GSH andNAC were added only once. RT activity was assessed
at7-10 dpostinfectionandweekly thereafter for a total of three times.
Onlythose cells which demonstrated cytoplasmic spreading, adher-ence, andelongation were used for this study. NAC and GSH were dissolved in endotoxin-free media before use.
Results
InMDM that were infected and then treated, both cord blood-and adult blood-derived cells demonstrated suppression of RT
activity aftertreatmentwith GSH or NAC in a
concentration-dependent fashion (Fig. 1). Although both thiols suppressed
viralreplication, NAC had a slightly greater effect on RT values
than GSH. This suppressive effect was observed 3-5 d after
treatment with GSH or NAC, and persisted during 3 wk in culture. After6 wk,both treated and untreated cells demon-stratedlight microscopic features ofcelldeath (cell detachment loss ofspreading and elongation). Uninfected control cells,
however,
retained characteristics of livemacrophage culture,
-E
100
El
ADULTMDM* CORD MDM
80
60
40
20
5 10 20 0 5 10 20 mM GSH mM NAC
Figure 1. RT suppressionof HIV-l in adult and cord MDM. Con-centration-dependent suppression of RT activity with NAC and GSH incord and adult MDM is shown. RT activity was determined in culture supernatants from the average of two adult and seven cord cellexperiments.Allresults are shownas %controllevels and each experiment was done intriplicate. Variability between triplicate re-sultswas < 15%. RTlevels for infected control cells averaged 3,000 cpm/Mlcorresponding to 100% activity.
(adherence, spreading,andelongation). Syncytium formation wasa constantfeature of HIV- 1-infected cellsand was inhib-ited by treatment with GSH or NAC. Cord blood-derived MDM also demonstrated higherRT activity comparedwith
adult MDM after both were infected with the same HIV-l strain (Bal), and greater suppression after thiol treatment (Fig. 2).Cells that were pretreated for 24 h with 10 and 20 mM of GSH orNAC and then infected didnotdemonstrate signifi-cant RT activity after 10 d in culture. Similarly, cellswhich were treated and simultaneously infected demonstrated ab-sence ofRTactivity after 10 d. Treatment with 5 mM GSHor
NAC produced less RT inhibition than with 10 or 20 mM concentrations(Fig. 3,AandB).
Cell death was seen in all treated and untreated cells 30 d after infection. The influence ofGSH or NAC on cell deathwas
studied by using trypan blue exclusion assay, and there were no differences between the treated and untreated cells during the time course of the experiment.
Discussion
Glutathione is aubiquitousmoleculepresentinall mamma-lian cell systems. Itis synthesizedand degraded intracellularly,
occurring mainly in the reduced form (GSH). The cellular turnover of GSH is associated with its transport in and out of cells via atranspeptidase whichbreaks down GSH outside the cell membrane, transports its constituent
y-glutamyl
amino acids across into the cell, and resynthesizes GSH inside the cell. The transport ofGSHparticipates in reductive reactions that may occur on the cell membrane as well as the intracellularenvironment (6). In asymptomatic HIV-l-seropositive indi-viduals, low GSHlevels have beenfoundinplasma, peripheral blood mononuclear cells, and lung epithelial lining fluid
(14,
15).
-I
0
I~-z
0
0-CO)
-i w w
.4 I-cc
s0o
60
40-0
--
-0-CORD ADULT ADULT+NAC CORD+NAC
HIV-1 INFECTION
10 20 30
DAYS IN CULTURE
Figure 2. Duration ofsuppressiveeffects seen with 20 mM NAC in both cord and adult cells after infection with HIV- 1. Resultsarefrom the mean of two adult and seven cordexperimentsdone intriplicate.
Variability between triplicate results was<15%.RTlevelsfor in-fected control cellsaveraged3,000
cpm/,l
correspondingto 100%activity.Cell deathwas seenin both treated and untreated HIV- I-in-fected cells 30 d after infection.
15, 18).The transportof
cysteine
sulfurbetween cells andor-gansfor the reduction oftoxicoxygenradicalintermediates isa
pivotal function of GSH. Depleting GSHinhumanlymphoid cells markedly increases their sensitivitytoradiation(6, 18).It hasbeensuggested that this phenomenon is similartocellular damage duetofree radical formation and reactive oxygen
in-termediate damage. For
example,
tumorcellsalsodepletedofGSH by buthionine sulfoximine,a
y-glutamylcysteine
synthe-taseinhibitor,
werefoundtoexhibit increasedsusceptibilitytocytolysis
after exposureto oxygen radicals (6). Recently,we havedemonstrated depletion of GSH levels during superoxide formation afterPMAstimulation incord MDM.Early inthe course ofPMA stimulation, there is a dramatic decrease in.4
0
I-z
0
on 0 -A w ui -a
O-60
60
-40
intracellularGSHlevels with a slow recovery to baseline levels. (J.Lioyand M.Yudkoff, unpublished
data,
notshown). GSHis essential not only for its antioxidant function but is also importantintheearly stages of lymphocyte activation neces-saryfor hostdefense.Forexample,theaddition of glutathione
invivoenhances activation ofcytotoxic T-cells ( 19, 20).
Addi-tionally, intracellular glutathione depletionhasbeen foundto
inhibit mitogen induced lymphocyte activation and natural
killercellfunction(21 ).
Repleting intracellularGSH levels by suspending cells in
media containing GSH involves the enzymatic degradation,
transport across the cell membrane, andresynthesis ofGSH
inside thecell (6). In cord MDM infected with HIV-1, it is
likely that the intracellular GSH pool is increased either by
supplying NACdirectlytothemediaor by supplying enough
GSH to facilitate breakdown and transport of'y-glutamyl amino acids intothe cell.Increasingtheintracellular GSH pool mayinfluence
HIV-l
replication in primary cord MDM in anumberofways.Roederer et al. (7) have proposed that
oxida-tivestresscaused by PMA or TNF-a stimulation may enhance
thebinding ofthe DNA complexprotein, NF-kB, thus
regu-latingHIV-Itranscription through gene expression. In the
cy-toplasm, NF-kB exists ina complex with an inhibitor. After
stimulation withPMA,the I-kB componentofthecytoplasmic
NF-kB complex is phosphorylated allowing its dissociation from the complex, thereby activating HIV-l transcription
within the nucleus. Staal et al. (9) havedemonstratedNACto
inhibit the activation of NF-kB, possibly by interfering with phosphorylationoritstransport to the nucleus. Similarly,NAC
hasbeen shown to suppresstranscriptionof a lacZ gene under
control of theHIV promoter and enhancer regulated by NF-kB(9).
Our
findings
observed withprimary
cultureof cordblood MDMmayparalleleventsin vivo.NACandGSHsuppression ofHIV- 1in cells whichwerepretreatedsuggests thatintracellu-larGSHwasincreasedenoughpossibly limiting adsorption of
the virus through changes on the cell membrane. Similarly, suppression ofHIV- 1 incellstreated simultaneously while
in-A
0 5mM NAC
*
10 mMNACo
20 m NAc-a
0
I-z
0
C.)
o
-a
lu
I-Pretreated Treated & Infected Infected then
then Infected Simultaneously Treated thenPretreatedInfected Treated & Infected Infected thenSimutaneously Treated
Figure 3.SuppressionofRTlevelsbyNAC(A)and GSH(B)duringthreedifferentconditionsincordcells. Moresuppressionwasseenafter
treatmentwithhigherconcentrations(10and20mM) although 5 mMproducedsomeinhibition of RT values. All experiments were done in
triplicate.Variabilitybetweentriplicateresultswas< 15%.RT levels for infected control cellsaveraged 3,000 cpm/slcorresponding to100% activity.
processof viralentryintocells. The suppressionof cells treated 2 wk after infection was established may imply mechanisms similar to those involving downregulation oftranscription acti-vating factor NF-kB or asimilar transcription factor responsi-ble for viral replication once the virus has invaded the cell (7-9). Inpreviousworkusingstimulated and infected PBMC and a U 1 cell line, HIV- 1 infection was suppressed at
concen-trations of5 mMNAC(7). Ourstudydemonstratesahigher concentration dependent response to bothNAC and GSH(10
and 20 mM). The use of freshly isolated MDM which are
> 97% puritymay accountforthedecreased sensitivityof these cells to treatment with NAC and GSH.
The mechanismsresponsible for differences between cord andadultMDM responses to HIV- 1 infection and treatment with thiols remains unknown. We have previously reported
thatHIV-1infected cordMDM expresshigherP24antigenand RT levels than adult MDM (5). Thethiol suppressioneffect seen in this study is greater for cord than adult MDM (22)
infected withHIV- 1.Enhancedsuppressionof viral replication
incord MDM may representeitherincreasedintracellular
con-centrationofNACand GSH in MDMderived from neonates or greater sensitivity of NF-kB to the suppressive effects of NAC andGSH.Syncytiumformation inHIV-1 infectedadult
and cordMDMhasbeenreported (5, 23). Althoughthe
mecha-nism isnotknown,weobserved that NACandGSHinhibited
the formationof suchsyncytiaincord MDM.
In summary, this study demonstrates the suppressive
ef-fects of GSHand NACon HIV-1 replication in cord MDM. The use ofcord blood MDM may provide a close in vitro
modelin thestudy of thiolregulationof HIV- 1 expressionin
pediatric AIDS and in the effects of oxidant stress on viral
replicationand hostdefense.Finally, theuseof thiols in HIV-I
research mayprovide insight into mechanisms thatsuppress or prolong latencyofthevirus.
Acknowledgments
The authorsaregratefultoDr. MarcYudkofffor providing the authors with expertise in glutathione metabolism.Wealso thank Ms.Sharon Swink for assistance with monocyte isolation and to Mrs. Peggy McDonald forassistance inpreparation of this manuscript.
Thisinvestigationwassupported by W.W.Smith Charitable Trust
grant A4089, NIMH-MH grant 47422, NIH-NS grant 27405, and
grants500-185-11 PG and 500-270-13PGR, Pediatric AIDS Founda-tiongrantsfrom the American Foundation for AIDS Research.
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