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HIV Replication in CD4+ T Cells of HIV-Infected Individuals is Regulated by a Balance between the Viral Suppressive Effects of Endogenous beta -chemokines and the Viral Inductive Effects of other Endogenous Cytokines

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This study demonstrates that the β -chemokines macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β ) and, RANTES (regulated on activation, normally T-cell expressed and secreted) inhibit human immunodeficiency virus (HIV) replication in
   Proc. Natl. Acad. Sci. USA Vol. 93, pp. 14076–14081, November 1996Medical Sciences HIV replication in CD4  T cells of HIV-infected individuals isregulated by a balance between the viral suppressive effectsof endogenous   -chemokines and the viral inductiveeffects of other endogenous cytokines (proinflammatory  regulation)  A  UDREY  L. K  INTER *, M  ARIO  O STROWSKI , D ELIA   G OLETTI , A  LESSANDRA   O LIVA  , D REW  W EISSMAN , K  IRA   G  ANTT ,E LENA   H  ARDY , R OBERT  J  ACKSON , L  INDA   E HLER ,  AND  A  NTHONY  S. F  AUCI Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 6A33, 10 Center Drive,MSC-1576, Bethesda, MD 20892-1576 Contributed by Anthony S. Fauci, September 19, 1996  ABSTRACT This study demonstrates that the   -chemo-kines macrophage inflammatory proteins 1  and 1  (MIP-1  and MIP-1  ) and, RANTES (regulated on activation, nor-mally T-cell expressed and secreted) inhibit human immuno-deficiency virus (HIV) replication in anti-CD3 or recall an-tigen-stimulated peripheral blood mononuclear cells (PB-MCs) of asymptomatic HIV-infected subjects. Significantlevels of    -chemokines were produced by both CD4  andCD8  PBMC subsets from HIV-infected individuals. Neutral-ization of endogenous MIP-1  , MIP-1  , and RANTES did notrescue HIV replication in cultures to which greater than 10%CD8  T cells had been added, indicating that the HIV suppressor activity of CD8  T cells cannot be explainedentirely by the   -chemokines. However, significant enhance-ment of viral replication was observed upon neutralization of endogenous   -chemokines in CD8-depleted or CD4  PBMCsfrom most donors, particularly in cultures with low induciblelevels of HIV production. In contrast, certain endogenousproinflammatory cytokines induced HIV replication in thesesame cells. These data suggest that the levels of HIV replica-tion in CD4  PBMC reflect the balance of the opposing effectsof endogenous suppressive factors, such as the  -chemokines,and HIV-inducing cytokines, such as tumor necrosis factor   and interleukin 1  . The regulation of human immunodeficiency virus (HIV)replication by the network of endogenous cytokines is enor-mously complex (reviewed in ref. 1). Certain cytokines, suchas interferon    (2) and interleukin (IL)-10 (3, 4), primarilydown-regulate virus replication, whereas others, particularlythe proinflammatory cytokines tumor necrosis factor (TNF)-  (5–7) and IL-1   (7–10), have been found to enhance HIVproduction  in vitro . Cytokines may act synergistically (9, 11) orantagonistically (3, 10, 12) to regulate HIV expression  replication; the overall effect on HIV production being de-pendent upon the cytokines present in a particular microen- vironment. Recent studies (13–17) demonstrate that the che-mokines, a superfamily of chemotactic factors involved in therecruitment and activation of leukocytes during inflammation(reviewed in ref. 18), can now be included in the group of factorsthatregulateHIVreplicationandspread.Thesestudiesdemonstrated that certain members of the   -chemokine fam-ily, namely macrophage inflammatory protein-1   and 1  (MIP-1  andMIP-1  ),andRANTES(regulatedonactivation,normally T-cell expressed and secreted), suppress the replica-tion of macrophage-tropic, but not T-cell-tropic, HIV strainsin  in vitro  infected mitogen-activated primary T cells fromuninfected donors or T-cell lines. The mechanism for thisinhibition is now known to be related to the downregulation orblocking by these chemokines of the C-C chemokine recep-tor-5 (CCR-5), which has recently been identified as a neces-sary coreceptor used by macrophage tropic HIV strains forfusion with the cell membrane (15–17). However, the role that  -chemokines may play in the regulation of virus replicationand spread in  in vivo  infected cells from HIV-infected subjectshas yet to be determined.This study demonstrates that   -chemokines exert an inhib-itory effect on HIV replication in primary CD8-depleted andCD4  peripheral blood mononuclear cells (PBMCs) fromHIV-infected individuals stimulated  in vitro  with recall antigenor anti-CD3 antibody. CD8-depleted and CD4  PBMCs of most asymptomatic HIV-infected subjects tested produce sub-stantial levels of   -chemokines, which play a significant role incontrolling HIV replication and spread  in vitro , as determinedby neutralization assays. Furthermore, the levels of HIVreplication in CD4  PBMC cultures were found to reflect abalance of the effects of endogenous HIV-suppressive factors,such as the   -chemokines, and those of endogenous HIV-inducing cytokines, such as TNF-   and IL-1. Finally, the  -chemokines cannot fully account for the CD8  T-cell-mediated suppression of HIV replication in PBMC fromHIV-infected subjects. MATERIALS AND METHODS Cellular Populations.  PBMCs were obtained from apheresisof 20 HIV-infected individuals (CD4  T-cell count: range159–885   l; mean 495   l) after separation over FicollHypaque density gradients. PBMCs were separated into CD4-depleted or CD8-depleted subsets (  96% depleted as deter-mined by FACS analysis) using immunomagnetic beads (Dy-nal, Great Neck, NY); CD4  and CD8  T-cell subsets (  96%pure as determined by FACS analysis) were obtained bydepletion of either CD4  or CD8  cells from E-rosette (  ) Tcells, unless otherwise indicated. Monocyte  macrophages were obtained by adherence for 45 min of CD8-depletedPBMCs onto flasks followed by five vigorous washes in PBSand gentle scraping. The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked ‘‘  advertisement ’’ inaccordance with 18 U.S.C. §1734 solely to indicate this fact.  Abbreviations: CCR-5, chemokine receptor 5; PBMC, peripheralbloodmononuclearcells;MIP-1  ,MIP-1  ,macrophageinflammatoryproteins 1  and 1  ; RANTES, regulated on activation normally T-cellexpressed and secreted; IL, interleukin; TNF, tumor necrosis factor;rh, recombinant human; TT, tetanus toxoid; PHA, phytohemaggluti-nin.*To whom reprint requests should be addressed. 14076  Effect of Exogenous   -Chemokines.  CD8-depleted PBMCs were cultured at 1.5–2  10 6 per well in 48-well plates in RPMImedium with 10% fetal calf serum (endotoxin   10 pg  ml),supplemented with 1 mM antibiotics, glutamine, and Hepesbuffer. Cultures were treated with various concentrations(0.5–100 ng  ml) of    -chemokines [recombinant human (rh)MIP-1  , rhMIP-1  , rhRANTES, rhMCP-1, or rhIL-8; R & DSystems], added individually or in combination and stimulated with either anti-CD3 (mouse ascites, 1:4000 dilution) plus IL-2(10 units  ml; Boehringer Mannheim) or with tetanus toxoid(12.5   g  ml; Wyeth Ayerst Laboratories, Marietta, PA). Cul-tures were refed with   -chemokines alone (recall antigencultures) or with the addition of IL-2 (10 units  ml) (anti-CD3-stimulated cultures) twice weekly. Endogenous   -Chemokine Assays.  CD8-depleted PBMCs,cultured alone as described above or with various proportionsof autologous CD8  T cells, were cultured in the absence orpresence of IgG isotype control mAb antibodies (R & DSystems) or neutralizing antibodies directed against   -chemo-kines, individually or in combination [unless otherwise indi-cated: polyclonal anti-MIP-1   (50   g  ml), polyclonal anti-MIP-1   (50   g  ml), and monoclonal anti-RANTES (10   g  ml); R & D Systems] immediately prior to stimulation of cultures with either anti-CD3 plus IL-2 or recall antigen asdescribed above. Cultures were refed twice weekly with anti-bodies and, in anti-CD3-stimulated cultures, with IL-2. CD4  T cells were supplemented with an additional 5% monocyte  macrophages and cultured in anti-CD3 plus IL-2-stimulatedconditions in the presence of either isotype control antibodiesor a combination of the anti-  -chemokine antibodies (asdescribed above), a combination of antagonists of proinflam-matorycytokines[IL-1ra(200ng  ml),sTNFR(10  g  ml),andgoat anti-IL-6 (5   g  ml); R & D Systems] or both. Cultures were refed with antibodies and maintained as described above.  Analysis of    -Chemokine Production.  Unfractionated,CD8-depleted, CD4-depleted, and negatively or positivelyselected CD4  and CD8  T-cell subsets were cultured at 2  10 6  ml and left untreated or stimulated with phytohemagglu-tinin (PHA) (4   g  ml). Culture supernatants were harvest at6, 18, 24, 48, and 72 h and 5 and 7 days after stimulation andfrozen at  80  C for later analysis of    -chemokine productionby ELISA for MIP-1  , MIP-1  , and RANTES (R & DSystems). Quantitation of HIV.  Culture supernatants were analyzedfor levels of HIV either by reverse transcriptase assay, aspreviously described (19), or by HIV p24 ELISA (DuPont). RESULTS The Effect of Exogenous MIP-1  , MIP-1  , and RANTES onHIV Replication in CD8-Depleted or CD4  PBMCs of HIV-Infected Subjects.  The effect of exogenous   -chemokines onHIV replication was assessed in CD8-depleted PBMCs from15 HIV-infected subjects (CD4  T cells 154–885   l; mean  495   l), five of whom had been recently boosted with tetanustoxoid (TT). Various concentrations of MIP-1  , MIP-1  , orRANTES were added with either recall antigen (TT) oranti-CD3 plus IL-2. MIP-1  , MIP-1  , and RANTES dramat-ically suppressed HIV replication and this effect was oftenobtained,particularlyinTT-stimulatedconditions(Fig.1  A ),atchemokine concentrations (5 ng  ml) 100-fold less than thosepreviously shown to effectively inhibit HIV replication in  in vitro -infected, mitogen-stimulated PBMCs or T-cell lines (13,17). Addition of    -chemokines (10–100 ng  ml) inhibited HIVreplication induced by either recall antigen (TT) or anti-CD3by 45–95% in the majority (12 of 15) of donor PBMCs (Fig.1  B ); the   -chemokine macrophage chemotactic protein-1 andthe  -chemokine IL-8 either had no effect or slightly enhancedHIV replication (data not shown). While the degree of inhi-bition of HIV replication exerted by a particular chemokine varied among PBMCs from different HIV-infected donors,RANTES consistently produced the most dramatic inhibition(Fig. 1  B ). Of interest,   -chemokine-mediated inhibition of HIV replication frequently did not exhibit linear dose depen-dence, particularly in recall antigen-stimulated conditions(data not shown).   -chemokine-mediated inhibition of HIVreplication was not related to suppression of CD4  T-cellactivation as determined by the induction of IL-2 production,expression of CD25 (IL-2R  ) on CD4  T cells, and cellularproliferation in recall antigen-stimulated cultures (data notshown). Numerous PBMC Subsets Produce   -Chemokines.  To de-termine which PBMC subsets were the primary source of endogenous   -chemokine production, unfractionated, CD4-depleted, CD8-depleted or CD4  and CD8  PBMC subsetsfrom HIV-infected subjects were assayed for the secretion of MIP-1  , MIP-1  , and RANTES under various stimulatoryconditions. Unfractionated, CD8-depleted and CD4-depletedPBMC produced comparable levels of    -chemokines undermost conditions (data not shown). Significant upregulation of   -chemokine production in response to PHA was observed inboth CD4  and CD8  T cells (Table 1). Of interest,   -che-mokine production by negatively selected CD4  T cells wasequal to or often greater than levels produced by parallelcultures of autologous CD8  T cells (Table 1). Levels of   -chemokines produced in PHA-stimulated conditions by F IG . 1. Exogenous   -chemokines inhibit HIV replication in CD8-depleted PBMCs from HIV-infected subjects. (  A ) Supernatant-associated reverse transcriptase activity present in cultures of CD8-depleted PBMC stimulated with tetanus toxoid in the absence orpresence of 5 ng  ml of either rhMIP-1  , rhMIP-1  , or RANTES. (  B )Summary of the reduction of peak levels of   in vitro  HIV replicationupon treatment of CD8-depleted PBMCs from 12 HIV-infectedindividuals with MIP-1   (x ¯  23% of control), MIP-1   (x ¯  28% of control), or RANTES (x ¯    16% of control) (  -chemokines used at0.5–100 ng  ml). Medical Sciences: Kinter  et al .  Proc. Natl. Acad. Sci. USA 93 (1996)  14077  either CD4  or CD8  PBMC subsets of asymptomatic HIV-infected subjects did not differ significantly from levels pro-duced by those of HIV-uninfected donors (data not shown).Recall antigen stimulation of CD8-depleted PBMC fromHIV-infected donors resulted in significantly lower levels of   -chemokine production (2- to 10-fold), and the peak produc-tion of    -chemokines was delayed compared with that ob-served with more potent cellular activators such as PHA oranti-CD3 (data not shown). Modulation of HIV Replication in CD4  and CD8-DepletedPBMC by Endogenous   -Chemokines and Other Proinflam-matory Cytokines.  To determine whether endogenous   -che-mokines regulate HIV replication in an autocrine  paracrinemanner in PBMC of HIV-infected subjects, neutralizing anti-  -chemokine antibodies were added in combination to CD8-depleted PBMC cultured in the absence or the presence of  various proportions of CD8  T cells. Concomitant neutraliza-tion of all three  -chemokines failed to rescue HIV replicationin CD8-depleted PBMC to which 10% (recall antigen-stimulated cultures; Fig. 2  A  and  B ) or 30% (anti-CD3-stimulated cultures; Fig. 2  C  and  D ) CD8  T cells had beenadded. The addition of    -chemokine antibodies to CD8-depleted cultures to which low proportions of CD8  T cells(  10%) had been added resulted in enhanced HIV replica-tion; however, the degree of enhancement was comparable tothat observed in the absence of CD8  T cells and thus couldnot be attributed specifically to inhibition of CD8  T-cellactivity (data not shown). Tetanus toxoid is a considerably lesspotent activator of T cells than is the polyclonal activatoranti-CD3, particularly in HIV-infected subjects with reducedcapacity to respond to recall antigens. Therefore, the levels of HIV replication and the number of CD8  cells required toinhibit viral production (Fig. 2  A  and  B ) are lower in tetanustoxoid stimulated conditions as compared with anti-CD3-stimulated cultures (Fig. 2  C  and  D ). Althoughneutralizationofendogenous  -chemokinesfailedto rescue HIV replication in the presence of CD8  T cells, asignificant increase in the levels of HIV replication by CD8-depleted PBMC cultured in the absence of CD8  T cells wasobserved upon neutralization of endogenous   -chemokines ineither recall antigen-stimulated (Fig. 2  A  and  B ) or anti-CD3plus IL-2-stimulated (Fig. 2  C  and  D  and Fig. 3) conditions inthe majority (12, 16) of donors tested. Of interest, the enhanc-ing effect of    -chemokine neutralization on HIV replication was most dramatic in CD8-depleted PBMC cultures fromdonors in which control levels of   in vitro  viral production werelowascomparedwithCD8-depletedculturesfromdonorswithhigh control levels of   in vitro  HIV replication (Fig. 3); a similarcorrelation was observed in CD8-depleted PBMC culturesfrom most of the 16 HIV-infected donors tested. Furthermore,although the numbers of individuals were small (2 of 5 shownin Fig. 3), those with high levels of   in vitro  viral replication inisotype control cultures had lower CD4  T-cell counts. Thedegree of enhancement of HIV replication in CD8-depletedPBMC observed upon neutralization of a particular   -chemo-kine varied among different donors (Fig. 4) and occasionallyenhancement of HIV replication was observed upon neutral-ization of only one of the   -chemokines (Fig. 4 C ).Several studies have demonstrated that  in vitro  HIV repli-cation can be inhibited by neutralization of endogenous proin-flammatory cytokines, such as TNF-  and IL-1  (7, 16). It wastherefore of interest to determine whether the net levels of HIV replication in CD4  T cells were determined by a balancebetween the effects of endogenous HIV-inducing proinflam-matory cytokines and the HIV-inhibitory   -chemokines.CD4  PBMC (T cells plus 5% monocytes) from HIV-infectedindividuals were stimulated with anti-CD3 plus IL-2 in thepresenceorabsenceofanti-  -chemokineantibodies,acocktailof antagonists of proinflammatory cytokines [sTNF receptor(R) plus IL-1 receptor antagonist (ra) plus anti-IL-6] or thetwo treatments in combination. In CD4  PBMC of certainHIV-infected donors the levels of HIV replication clearlyreflected the balance of positive and negative effects of endogenous proinflammatory cytokines and   -chemokines,respectively(Fig.5).Cellularproliferationwasnotsignificantlyaltered by any of the treatments despite the dramatic differ-ence in the effects of each treatment on HIV replication (datanot shown). DISCUSSION This study demonstrates that the  -chemokines MIP-1  , MIP-1  , and RANTES inhibit HIV replication in CD8-depletedPBMC of most asymptomatic HIV-infected subjects. It hasbeen well established that   -chemokines are secreted by a variety of cell types including monocytes and a number of lymphocyte subsets (reviewed in ref. 18). We demonstrate thatCD4  T cells from HIV-infected subjects produce   -chemo-kines at levels comparable to those produced by autologousCD8  T cells. Of particular interest, neutralization of endog-enously produced   -chemokines, individually or in combina-tion, by anti-  -chemokine antibodies, resulted in a significantenhancement of HIV replication in CD8-depleted and CD4  PBMC from most donors tested; however, these same anti-bodies failed to eliminate the HIV suppressive effects of CD8  T cells, when such cells were added in coculture to CD8-depleted PBMCs. Of note is the fact that the level of HIVreplication in CD4  PBMC was found to reflect the netbalance of positive and negative regulatory effects of endog-enous proinflammatory cytokines (TNF-  , IL-1  , and IL-6)and endogenous   -chemokines, respectively. These data sug-gest that the   -chemokines, MIP-1  , MIP-1  , and RANTESmay play a role in controlling the levels of viral replication  in vivo  and may counteract or antagonize the effects of HIV-inducing cytokines.Previous studies from several laboratories have shown thatexogenous   -chemokines inhibit the ability of macrophage-tropic HIV strains or cells expressing macrophage-tropic envproteins to acutely infect or fuse with CD4  T cells and PBMCof normal donors or T-cell lines (15–17). Our study furthersthese observations by demonstrating that the   -chemokinessuppress HIV replication in CD8-depleted PBMCs from mostasymptomaticHIV-infectedsubjectsstimulatedwithanti-CD3plus IL-2 or with recall antigen. Of note is the fact that underthe conditions of   in vitro  antigen stimulation of   in vivo  infectedPBMC, inhibition of HIV replication was often observed at Table 1. Production of MIP-1  , MIP-1  , and RANTES (pg  ml)by unfractionated, CD8  , and CD4  PBMC from threeHIV-infected subjectsUnfractionated CD8  CD4  Unstim-ulated PHA Unstim-ulated PHA Unstim-ulated PHA MIP-1  1 40 7,418 10 2,693 10 3,8352* 13 1,531 10 4,396 10 7333 10 8,138 224 3,201 1729 6,580MIP-1  1 838 19,305 59 20,975 46 20,3082* 104 24,928 123 28,357 19 4,0943 10 29,773 171 23,759 1738 28,557RANTES1 10 4,759 10 1,914 10 1,7422* 10 2,739 10 2,383 10 1853 10 4,019 10 825 10 1,825  -chemokine production in culture supernatants was assessed onday 5 post-stimulation.*CD4  and CD8  cells were obtained by positive selection usingimmunomagnetic beads rather than by negative selection. 14078 Medical Sciences: Kinter  et al .  Proc. Natl. Acad. Sci. USA 93 (1996)   -chemokine concentrations up to 100-fold less than thosepreviously reported to be required to inhibit HIV replicationin acutely infected PHA blasts or T-cell lines (13–17). It isunclear whether the greater sensitivity to the   -chemokine- F IG . 4. The effect of neutralization of individual endogenous  -chemokines on HIV replication in tetanus toxoid-stimulated CD8-depleted PBMCs from HIV-infected individuals. CD8-depleted PB-MCs from three HIV-infected subjects were stimulated with tetanustoxoid in the presence of isotype control antibody (50   g  ml) oranti-MIP-1   (50   g  ml), anti-MIP-1   (50   g  ml), or anti-RANTES(10  g  ml).Culturesupernatantswereassayedforthelevelsofreversetranscriptase activity or p24 antigen at various time points during theculture period.F IG . 2. The role of endogenous   -chemokines vs. CD8  T cells inthe regulation of HIV replication in CD8-depleted PBMCs fromHIV-infected individuals. CD8-depleted PBMC of four HIV-infectedsubjects were stimulated with (  A  and  B ) tetanus toxoid in the absenceor the presence of 10% CD8  T cells or ( C  and  D ) anti-CD3 plus IL-2in the absence or the presence of 30% CD8  T cells and treated witheither isotype control antibody (110   g  ml) or a cocktail of anti-  -chemokine antibodies (anti-MIP-1  , 50   g  ml; anti-MIP-1  , 50   g  ml; anti-RANTES, 10   g  ml).F IG . 3. Variable sensitivity of CD8-depleted PBMC of HIV-infected individuals to the enhancing effect on HIV replication byneutralization of   -chemokines. CD8-depleted PBMC from five HIV-infected donors (154–885 CD4  T cells   l) were stimulated withanti-CD3 plus IL-2 and cultured in the presence of isotype controlantibody or a combination of neutralizing anti-  -chemokine antibod-ies. Data represent peak HIV replication as measured by reversetranscriptase assay. Medical Sciences: Kinter  et al .  Proc. Natl. Acad. Sci. USA 93 (1996)  14079  mediated HIV inhibition that we observe in our endogenousinfection system is due to increased sensitivity of the donor’sHIV quasi-species to inhibition of   env  binding to CCR-5 by  -chemokines as compared with viruses used in acute  in vitro infection systems (15–17); such strain variability has beennoted in previous studies (17). Alternatively, the greatersensitivitycouldbeduetolowerlevelsofCCR-5onCD4  cellsof the HIV-infected subjects tested or to differences in meth-odology of this and previous studies (13–17).Our observation that simultaneous neutralization of MIP-1  , MIP-1  , and RANTES did not abrogate CD8  T-cell-mediated HIV suppression is of interest and strongly suggeststhat the   -chemokines cannot completely explain the HIVsuppressor effects of CD8  T cells, at least in our system of endogenous HIV replication in cells from HIV-infected indi- viduals. However, the assay of suppression of HIV replicationconducted in this study on PBMC from HIV-infected subjectsused CD8  T-cell cocultures and not supernatants from CD8  TcellsorhumanT-lymphocytevirustypeI-transformedCD8  T-cell lines as did the previous study srcinally describing theHIV-suppressor effects of the   -chemokines (13). Our at-tempts to conduct these experiments with culture supernatantsof primary CD8  T cells or with transwell coculture systemsfailed to yield consistent results. In this regard, it is possiblethat other labile soluble factors or cell contact-mediatedfactors play a dominant role in suppressing HIV replication indirect coculture systems. However, the observation that neg-atively selected CD8  and CD4  T cells from asymptomaticHIV-infected subjects produce comparable levels of    -che-mokines argues against these   -chemokines as the sole medi-ators of HIV-suppressor activity that is specific for CD8  Tcells. Of particular interest, crosslinking of CD4 molecules bypositive selection of CD4  cells using anti-CD4-coated mag-netic beads appeared to reduce the capacity of these cells toproduce   -chemokines upon stimulation with PHA (Table 1).This observation suggests that antibody crosslinking of CD4 onthe surface of T cells, as seen here, or by glycoprotein 120  160 in vivo , may deliver a negative signal with regard to theproduction of    -chemokines.The role of endogenous  -chemokines produced by PBMCsubsets other than CD8  T cells in the regulation of HIVreplication in CD4  T cells from HIV-infected subjects hasnot been previously demonstrated. Previous studies (14)suggested that elevated   -chemokine production by CD4  cells from exposed uninfected individuals may be responsiblefor the lack of susceptibility to acute infection with a primaryisolate of HIV, but not a variant of this strain modified toexpress a T-cell tropic-like  env  gene product; this observa-tion was later found to be due to the lack of CCR-5expression in these exposed uninfected individuals (20).Data from our   -chemokine neutralization studies suggestthat high levels of the natural ligands of CCR-5, MIP-1  ,MIP-1  , and RANTES, in HIV-infected subjects, can play asignificant role in limiting the spread of HIV infection inmost asymptomatic HIV-infected individuals. We have pre-sumed that the individuals in this study were harboringpredominantly macrophage-tropic strains because, for themost part, they were in the early stages of HIV disease (21).The variability in the capacity of anti-  -chemokine antibod-ies to enhance  in vitro  HIV replication in CD8-depleted orCD4  PBMC from HIV-infected individuals may be areflection of the relative representation of T-cell-tropic vs.macrophage-tropic viruses in the PBMCs of the subjectsunder study. In this regard, neutralization of endogenous  -chemokines appeared to have a more consistent anddramatic enhancing effect on viral replication in thoseCD8-depleted PBMCs from donors with higher numbers of CD4  T cells   l and in whose cultures the control levels of  in vitro  HIV production were moderate-to-low (Fig. 3). Although the reasons for these observations are unclear atpresent they are consistent with the hypothesis that thepredominant virus(es) in individuals with early-to-intermediate stage disease are slow  low, macrophage tropicstrains (21). Correlations of the type of virus obtained fromPBMC cultures with the suppressive capabilities of theendogenous   -chemokines in these cultures are currentlyunder investigation in our laboratory. Alternatively, theinability of anti-  -chemokine antibodies to enhance HIVreplication in CD8-depleted PBMC cultures from certainindividuals could be due to lack of or a great reduction of   -chemokine production; however, based on our measure-ments of    -chemokine levels, this does not appear to be thecase.The fact that endogenous proinflammatory cytokines areimportant modulators of HIV replication in primary PBMCshasbeenpreviouslydemonstrated(reviewedinrefs.1,3,7,and22). The enhancing effect of proinflammatory cytokines hasbeen demonstrated in culture systems employing both T-cell-tropic (7, 16) and macrophage-tropic (3, 7, 16) strains of HIV.It is interesting to consider the selective pressures exerted byproinflammatory cytokines and   -chemokines on the emer-gence of predominantly T-cell-tropic, rapid  high virus strainsin HIV-infected individuals during disease progression. In thisregard, both the   -chemokines and the HIV-inducing cyto-kines TNF-  , IL-1  , and IL-6 are involved in primary proin-flammatory immune responses (23) and have been reported tobe produced at elevated levels in HIV-infected individuals, asdetermined either by  in situ  tissue analysis or plasma levels(24–29). Taken together, these data strongly suggest that thesteady state of virus replication in HIV-infected individualsreflects, at least in part, a delicate balance between cytokines F IG . 5. Opposing effects of endogenous   -chemokines and proin-flammatory cytokines on HIV replication in CD4  PBMC of twoHIV-infected individuals. CD4  PBMC (T cells plus 5% monocytes)from HIV-infected subjects were stimulated with anti-CD3 plus IL-2in the presence of either a cocktail of anti-  -chemokine antibodies (asdescribed in Fig. 3), a cocktail of proinflammatory cytokine antago-nists [sTNFR (10   g  ml), IL-1ra (200 ng  ml), and anti-IL-6 (5  g  ml)], a combination of both treatments, or isotype control anti-bodies (100   g  ml). 14080 Medical Sciences: Kinter  et al .  Proc. Natl. Acad. Sci. USA 93 (1996)
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