Cells of macrophage lineage can be infected with macrophage-tropic (M-tropic) or CCR5-using strains of HIV-1 in vitro and in vivo[1–5], via the CD4 molecule and β-chemokine co-receptors [6,7]. Although freshly isolated peripheral blood monocytes (PBMC) are relatively refractory to HIV-1 infection in vitro, susceptibility to infection is dramatically improved by culturing monocytes prior to infection [8,9]. It is thought that the levels of maturation of monocytes/macrophages affects their susceptibility to HIV-1 infection, as tissue macrophages can be readily infected on the day of their isolation . HIV-1 replication in monocyte-derived macrophages (MDM) infected with M-tropic/CCR5 HIV-1 strains may be influenced by a variety of cytokines and growth factors such as various interleukins, interferons, chemokines, tumour necrosis factor alpha, colony-stimulating factor-1 (CSF-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) [10–17].
GM-CSF, produced by a variety of cell types, including activated T cells, monocytes, macrophages and fibroblasts, is required for the survival, proliferation and differentiation of granulocyte–macrophage precursor cells and for the function of their mature progeny (reviewed by Metcalf  and Armitage ). Early studies investigating the effect of GM-CSF on HIV-1 replication reported that this factor exerted an upregulatory effect on viral production in both MDM [14,20–23] and chronically infected promonocytic lines U937 and U1 [24,25]. However, other investigators have reported inconsistent [13,26] or negative effects of GM-CSF on HIV-1 entry or replication in MDM [27,28] associated with reduced β-chemokine receptor expression.
GM-CSF mediates its activities through binding to a heterodimer receptor comprising a ligand-specific α-chain and a β-chain that is shared with interleukin (IL) 3 and 5 (reviewed by Armitage  and Crowe and Lopez ). Amino acid 21 within the first α-helix of GM-CSF has been found to be critical for the biological function of GM-CSF and for the interactions between GM-CSF and the β-chain of the GM-CSF receptor that are necessary for high-affinity binding . In order to clarify the effects of GM-CSF on HIV-1 replication in MDM in vitro, the activity of this growth factor was examined by several different criteria. A mutant form of GM-CSF (E21R) that binds only to the α-chain of the receptor [30,31] and this form was used to examine the effect of GM-CSF on HIV-1 replication.
Isolation and culture of monocytes
Human monocytes were isolated from buffy coats of HIV-seronegative blood donors (supplied by the Red Cross Blood Bank, Melbourne, Australia) by Ficoll-Paque density gradient centrifugation and plastic adherence as previously described . Immediately after isolation, cell viability was greater than 95% as assessed by Trypan blue exclusion; the purity of monocytes was greater than 90% as determined by immunofluorescent staining with anti-CD14 monoclonal antibody (MAb) (Becton Dickinson, Mountain View, California, USA) and flow cytometric analysis. Cells were cultured in Iscove's modified Dulbecco medium (Cytosystem, Castle Hill, Australia) supplemented with 10% heat-inactivated human AB+ serum, 2 mmol/l l-glutamine and 24 μg/ml gentamicin (supplemented Iscove's medium). Monocytes were cultured adherent to plastic in 24-well plates (Costar, Cambridge, Massachusetts, USA) or cultured in suspension in polytetrafluorethylene (Teflon) pots (Savillex, Minnetonka, Minnesota, USA) at a concentration of 1 × 106 cells/ml.
Freshly isolated monocytes were treated with recombinant human GM-CSF (Genzyme, Cambridge, Massachusetts, USA or Genetics Institute, Cambridge, Massachusetts, USA) at varying concentrations (1–100 ng/ml). In selected experiments, monocytes were cultured for 5 days prior to the addition of GM-CSF. Control cells from the same donors were cultured in the absence of GM-CSF. MDM cultured in suspension in the presence of GM-CSF (0.01–100 ng/ml) for 21 days showed no evidence of toxicity as assessed by Trypan blue exclusion. To determine the specificity of the effect of GM-CSF on HIV-1 replication in MDM, GM-CSF was incubated with a neutralizing anti-human GM-CSF MAb (0.1–100 μg/ml; 4D4) or a non-neutralizing anti-GM-CSF control MAb (0.1–100 μg/ml; 4A12) for 30 min at 4°C prior to its addition to MDM.
HIV-1 infection of monocyte-derived macrophages
The M-tropic strain of HIV-1Ba−L (the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Disease, NIH, Bethesda, Maryland, USA) or an M-tropic primary isolate of HIV-1 obtained from an HIV-infected patient, HIV-1D36, was amplified in PBMC. Briefly, PBMC stimulated with phytohaemagglutinin (10 μg/ml, Murex Diagnostics, Dartford, UK) for 3 days were infected with HIV-1Ba−L or primary isolate and cultured in Iscove's medium containing 10% fetal calf serum (PA Biologicals, New South Wales, Australia) and IL-2 10 U/ml (Boehringer-Mannheim, Mannheim, Germany). The culture supernatants collected 14 days later were clarified using 0.2 μm filters (Schleicher and Schuller, Dassel, Germany), stored at −70°C in small volumes and thawed immediately before use.
Following their isolation, monocytes were cultured under three sets of conditions to determine whether alteration of HIV replication depended upon the timing of addition of GM-CSF with respect to cellular maturation or addition of HIV. Cells were (i) cultured from time of isolation in the presence or absence of GM-CSF, infected with HIV-1 on day 5 (day 0 GM-CSF, day 5 infection), as described earlier [20,21]; (ii) treated with GM-CSF on the day of isolation and infected the following day (day 0 GM-CSF, day 1 infection) ; or (iii) exposed to GM-CSF and HIV-1 on day 5 after isolation (day 5 GM-CSF, day 5 infection) [13,14]. For infections, HIV-1Ba−L was pretreated with 10 U RNase-free DNase (Boehringer Mannheim Australia, Castle Hill, New South Wales, Australia) for 20 min at room temperature in the presence of 10 mmol/l MgCl2to remove contaminating viral DNA and used at a concentration of > 50 000 pg/ml HIV antigen (HIV-1 p24 antigen immunoassay, Abbott Laboratories, Abbott Park, Illinois, USA) for 1 × 106 cells for 2 h. Cells were then washed with phosphate-buffered saline (PBS; Trace Biosciences, New South Wales, Australia) and resuspended in fresh Iscove's supplemented medium in the presence or absence of GM-CSF. MDM were then cultured for 10 days following infection either in suspension in Teflon pots or adherent to plastic in 24-well plates. Uninfected MDM from the same donors were used as controls for each experiment. Since endotoxin contamination has been shown to alter HIV-1 replication in MDM, culture supernatants and GM-CSF stocks were tested for LPS levels using the Limulus Amebocyte Lysate Assay (Biowhitaker, Walkersville, Maryland, USA).
Quantification of HIV-1 replication
HIV-1 replication in MDM cultured in 24-well plates was measured as p24 antigen production (HIV-1 p24 antigen immunoassay, Abbott Laboratories, according to manufacturer's instructions) using serial dilutions of culture supernatant obtained 10 days after infection or by monitoring reverse transcriptase (RT) activity using a micro-RT assay. Briefly, 10 μl culture supernatant was added to 10 μl 0.3% NP40 in a 96-well plate. Thereafter, 40 μl RT mixture [50 mmol/l Tris pH 7.8, 7.5 mmol/l KCl, 5 mmol/l MgCl2, 2 mmol/l dithiothreitol (Sigma, St Louis, Montana, USA), distilled H2O up to 4 ml per plate], 5 μg/ml template-primer p.An.dT12-18 (Pharmacia-Biotec, Buckinghamshire, UK) and 3 μCi 33P-dTTP (Amersham Co., Amersham, UK) was added and the mixture was incubated for 2 h at 37°C. The reaction products were spotted on a DE81 chromatography paper (Whatman, Maidstone, UK) and air-dried. Dry filters were washed six times with 2× SSC buffer (0.3 mol/l sodium chloride and 34 mmol/l sodium citrate) to remove free radioactive dNTP, rinsed once in 95% ethanol and air-dried. Meltilex scintillant (Wallax, Turku, Finland) was spotted onto the filters and bound radioactivity was counted in the LKB micro betacounter (Wallex).
Infection in suspension-cultured macrophages was determined in pre-fixed and permeabilized cells using a previously described method , by staining for intracellular p24 antigen using a MAb directed against p24 (2 μg/ml; IgG1, Olympus, Lake Success, New York, USA) or isotype-matched control (MOPC 21; 2 μg/ml; IgG1, Bionetics, Charlestone, South Carolina, USA) followed by goat anti-mouse IgG conjugated to FITC (FITC-GAM; Tago, Burlingame, California, USA). The proportion of cells containing intracellular p24 antigen was quantified by flow cytometric analysis (FACStarPlus, Becton Dickinson).
Expression of GM-CSF, CCR5 and CD4 receptors
Monocytes from HIV-1 seronegative donors were analysed for expression of the α- and β-chain of the GM-CSF receptor on the day of isolation and at times up to 14 days in culture in Teflon pots. Cells were stained with MAb directed against the α-chain (8G6; 5 μg/ml), β-chain (4F3; 5 μg/ml)  or isotype-matched control followed by FITC conjugate. The proportion of monocytes expressing α- or β-chain was quantified by flow cytometric analysis.
To determine regulation of CCR5 and CD4 surface expression by GM-CSF, monocytes were exposed to GM-CSF on the day of isolation and infected with HIV-1Ba−L7 days after isolation. CCR5 and CD4 surface levels were assessed on the day of isolation, the day of HIV-1 infection and 2 and 5 days after infection. CCR5 expression was quantified using MAb against CCR5 (5C7; IgG2a, 0.5 μg/ml, LeukoSite Cambridge, Massachusetts, USA) or isotype-matched control (RPC5; IgG2a) on ice for 30 min. After two washes with cold PBS, cells were incubated with anti-mouse immunoglobulin conjugated with biotin (Silenus, Melbourne, Australia). After two further washes with cold PBS, MDM were incubated with fluorescein-conjugated streptavidin (Calbiochem, La Jolla, California, USA) for 30 min, washed with cold PBS and analysed by flow cytometry. The expression of CD4 receptor was assessed using anti-CD4 MAb conjugated to FITC (Leu-3; 1 μg; Becton Dickinson) on ice for 30 min, followed by a wash with cold PBS and flow cytometric analysis.
The effect of mutant granulocyte-macrophage colony-stimulating factor on HIV-1 replication
In selected experiments, MDM were also exposed to a mutant form of GM-CSF (E21R; BresaGen, Adelaide, South Australia) with a site mutation within the first α-helix, resulting in a substitution of arginine for glutamic acid [30,31]. This mutant GM-CSF only binds with a low affinity to the α-chain of the GM-CSF receptor and was used at a concentration of 100 ng/ml. The significance of the effect of treatment with GM-CSF or the E21R mutant GM-CSF on HIV replication was assessed using the Student's t-test (paired, 2-tailed).
HIV-1 DNA and RNA extraction and amplification
DNA was extracted as previously described . Briefly, 1 × 106 MDM cells infected with HIV-1 were cultured for 7 days in the presence or absence of GM-CSF or mutant GM-CSF (E21R). Cells were lysed in 0.5 ml lysis buffer [50 mmol/l KCl, 10 mmol/l Tris (pH 8.3), 2.5 mmol/l MgCl2, 0.5% Tween 20 (BDH Chemicals, Kilsyth, Australia), 0.5% NP40 (Sigma)]. Following the addition of 5 μl proteinase K (Boehringer Mannheim), cell lysates were heated at 60°C for 1 h and 95°C for 30 min. Sample lysates and lysates from controls (8E5 cells, containing a single HIV-1 provirus per cell)  were used in the polymerase chain reaction (PCR) reaction after making serial threefold dilutions in lysis buffer. Amplification of gag using SK38 and SK39 primers and hybridization using a 32P-labelled probe SK19 was according to published methods in use in our laboratory . HLA-DQ alpha was amplified in the same reaction using primers GH-26 and -27 to standardize the amount of DNA in the lysates.
Using oligo (dT)25 beads (Dynabeads, Dynal, Australia), mRNA was extracted from MDM lysates according to the manufacturer's protocol. Briefly, 1 × 106 MDM that had been cultured in the presence or absence of GM-CSF for 7 days after infection were lysed in lysis/binding buffer [100 mmol/l Tris-HCl (pH 8), 500 mmol/l LiCl, 10 mmol/l ethylenediamine tetraacetic acid (EDTA) (pH 8), 0.1% LiDS (ICN Pharmaceuticals Mesa, California, USA), 5 mmol/l dithiothreitol]. After beads were preconditioned in lysis/binding buffer, MDM lysates were hybridized with beads (1 × 106 MDM/20 μl beads) and incubated at room temperature for 10 min to form a Dynabeads oligo(dT)25/mRNA complex. Beads were washed twice with 100 μl washing buffer [10 mmol/l Tris-HCl (pH 8), 0.15 mol/l LiCl, 1 mmol/l EDTA (pH 8)] (BDH Chemicals) with 0.1% LiDS (ICN Pharmaceuticals Mesa, California, USA), and then three times with 100 ml washing buffer. After beads were washed four times with RT buffer [10 mmol/l Tris-HCl (pH 8.3), 75 mmol/l KCl] the Dynabeads oligo(dT)25/mRNA complex was resuspended in 25 μl AMV-RT mix (1 mmol/l each of dGTP, dATP, dTTP, dCTP, 4 mmol/l sodium pyrophosphate, 25 U RNasin, 18 U AMV/RT, 5 μl 5× RT-buffer) and incubated for 1 h at 42°C to synthesize cDNA. After removing the RT mix, beads were resuspended in 100 μl elution solution (2 mmol/l EDTA) and heated at 95°C to remove mRNA. Beads were resuspended in 100 μl TE buffer (pH 8) and stored at 4°C in 10-fold dilutions in lysis buffer until used for PCR using gag- specific primers and detection by hybridization with a 32P-labelled probe SK19. Levels of cDNA were standardized according to β-actin levels  as assessed by laser densitometry. To check for viral DNA contamination, samples prepared without AMV-RT were included within each experiment.
GM-CSF inhibited HIV-1Ba−L replication in MDM in a dose-dependent manner (Table 1). Results of six experiments using MDM from different donors confirmed that 0.1 ng/ml or greater GM- CSF significantly inhibited HIV-1Ba−L replication by a mean of 67.7 ± 4.9% (standard error of the mean, SEM) compared with untreated controls. GM-CSF inhibition was blocked by a neutralizing MAb (4D4) against GM-CSF in a concentration-dependent fashion but not by a non-neutralizing anti-GM-CSF MAb (4A12), suggesting the specificity of the observed effect (Table 2).
MDM from a total of 30 donors were cultured in the absence of GM-CSF for the first 5 days after isolation then infected with HIV-1Ba−L on day 5 and simultaneously treated with GM-CSF (day 5 onwards). In 26 of 30 experiments, GM-CSF pre-treatment of MDM resulted in a two- to tenfold (mean 54.6 ± 5.5%, n = 30) decrease in the level of HIV p24 antigen in culture supernatants harvested 10 days after infection compared with untreated cells. In the remaining four experiments, there was no or insignificant (< 10%) inhibition (data not shown). Replication of a primary isolate HIV-1D36 was similarly inhibited. There was no difference observed in results from experiments using GM-CSF from Genzyme (n = 8) and Genetics Institute (n = 10) with a mean inhibition of 41.0 ± 8.4% (SEM) and 51.0 ± 11.3%, respectively, compared with p24 antigen concentrations in control supernatants (data not shown).
To determine whether the level of maturation of the monocyte at the time of exposure to GM-CSF, and the timing of exposure to GM-CSF prior to infection influenced the effect of GM-CSF on HIV-1 replication, monocytes were cultured under one of three conditions. The addition of GM-CSF resulted in downregulation of HIV-1 replication under all conditions (P < 0.01) compared with controls. As anticipated from results of previous studies in this laboratory [9,35], infection on day 1 resulted in lower levels of viral replication than in MDM infected on day 5, regardless of length of time of exposure to GM-CSF prior to infection (data not shown).
Intracellular p24 antigen was quantified in MDM cultured and infected in suspension; GM-CSF dramatically decreased intracellular p24 antigen concentrations as assessed by flow cytometry, whereas the mutant GM-CSF E21R did not significantly alter intracellular p24 antigen levels from those in MDM not exposed to GM-CSF. The MESF (molecules of equivalent soluble fluorochrome) values were 7.2 × 105, 1.8 × 105 and 5.2 × 105 for intracellular p24 fluorescence of MDM cultured in the absence of GM-CSF, in presence of GM-CSF and in presence of mutant E21R GM-CSF, respectively. GM-CSF also inhibited supernatant RT production (82 ± 5.2% inhibition; n = 7;P < 0.01), whereas E21R GM-CSF did not significantly suppress RT (13.6 ± 18% inhibition; n = 7;P = 0.5).
To establish whether the surface expression of α- and β-chains of GM-CSF receptor on freshly isolated monocytes and differentiated MDM could affect their susceptibility to GM-CSF and potentially contribute to the effect of GM-CSF on HIV-1 replication, the level of both chains were assessed during 14 days of culture. The α-chain of the GM-CSF receptor was present in abundance on monocytes on the day of isolation, declined during the first 3 days of culture and then subsequently increased to the same level as on the day of isolation (Fig. 1). Levels of β-chain expression on monocytes were low on the day of isolation and increased during the 14-day culture period (Fig. 1).
To investigate the effect of GM-CSF at the viral entry level, surface expression of both CD4 receptor and CCR5 chemokine co-receptor was assessed by flow cytometry. As previously reported by our group , surface CD4 was readily detected on freshly isolated monocytes and increased more than fivefold after 7 days of culture. GM-CSF significantly downregulated surface expression of CD4 by more than 50% on days 7, 9 or 12 after monocyte isolation and GM-CSF treatment (Fig. 2a). GM-CSF also decreased expression of CD4 in HIV-infected MDM on days 2 and 5 after HIV-infection (corresponding to days 9 and 12 post-isolation) (Fig. 2a). Surface expression of CCR5 was undetectable or barely detectable on freshly isolated monocytes and significantly increased with in vitro differentiation (as previously demonstrated by a number of studies [28,36,37]). Exposure of fresh monocytes and MDM to GM-CSF resulted in an increase in CCR5 surface expression in both mock- and HIV-infected cultures compared with cells unexposed to GM-CSF (Fig. 2b).
The effect of GM-CSF on HIV-1 DNA and mRNA levels was examined to establish whether the decrease in HIV replication occurs before or after transcription. There was a minimal one- to threefold decrease in the level of HIV gag DNA in infected cells cultured in the presence of GM-CSF (n = 3) compared with cells not exposed to GM-CSF or treated with E21R GM-CSF (Fig. 3a). As assessed by laser densitometry, the ratio of gag to DQ signal was 0.47, 0.18 and 0.47 (mean of the first three dilutions, results for a representative experiment) for cells cultured in the absence of GM-CSF, presence of GM-CSF and presence of E21R GM-CSF, respectively. Analysis of cell lysates from three donors showed a three- to tenfold decrease in HIV-1 gag mRNA expression in MDM infected with HIV-1Ba−L that were exposed to GM-CSF and an approximately twofold inhibition in those exposed to GM-CSF E21R compared with that in untreated MDM, suggesting that HIV-1 replication is inhibited before or at transcription. Densitometry units for gag signal standardized to equivalent levels of β-actin levels were 1956, 732 and 957 for MDM cultured in the absence of GM-CSF, presence of GM-CSF and presence of E21R GM-CSF, respectively (Fig. 3b).
This study shows that GM-CSF consistently suppresses HIV-1 replication in human MDM in a dose-dependent manner. The inhibitory effect is specific since it is totally reversed by addition of neutralizing MAb 4D4 but not by addition of non-neutralizing anti-GM-CSF control MAb, 4A12. The inhibitory effect of GM-CSF is unrelated to the level of maturation of MDM at the time of GM-CSF stimulation or HIV infection. E21R GM-CSF, binding only to the α-chain of the GM-CSF receptor , does not affect HIV-1 replication. We conclude, therefore, that the inhibition of HIV-1 replication by GM-CSF results from signaling through the β-chain of its receptor.
Our report addresses a longstanding controversy in the literature, with most of the early studies reporting augmentation [14,20–25] or no change [13,26] in viral production and two recent studies suggesting inhibition of HIV-1 replication in MDM by GM-CSF [27,28]. A number of laboratory variables could potentially contribute to such variation. We have extensively examined potential confounders to determine why our results differ to some previous studies. We have reproduced the assay conditions used by other investigators, including strain of HIV-1, source/concentration of GM-CSF, timing of incubation with cytokine in relation to cell maturity and HIV-1 infection. Regardless of the experimental conditions, we observed reduced replication of HIV-1.
The mechanism by which GM-CSF alters HIV replication in MDM and in promonocytic cell lines is also controversial. Wang et al. reported that the GM-CSF-induced increase in HIV-1 replication in MDM was attributable to upregulation of CCR5 expression. These data are in direct contrast to those of Di Marzio et al., who demonstrated that GM-CSF suppressed CCR5 and CD4 expression on MDM and reduced HIV-1 entry into these cells.
Stimulation of monocytes with GM-CSF for 7 days prior to HIV-1 infection resulted in modest downregulation of CD4 surface expression and augmentation of CCR5 levels on MDM, coincident with inhibition of HIV-1 replication in those cells. It is unlikely that these opposing effects on the expression of CD4 and CCR5 will affect viral entry of M-tropic strains of HIV-1. We have previously demonstrated that the susceptibility of human monocytes/macrophages to HIV-1 infection is not dependent on the level of CD4 expression . Recent studies by Fear et al. and Kozak et al. demonstrate that expression of CD4 is not a rate-determining factor for viral entry in the presence of adequate CCR5 levels, since M-tropic strains of HIV-1 have been able to infect CCR5-expressing cells by utilizing very low densities of CD4.
In support of our findings, other cytokines have also been shown to have opposing effects on the expression of CD4 and CCR5. Interferon-gamma (IFNγ), a cytokine that has bidirectional effects on HIV-1 replication in MDM, significantly upregulated CCR5 surface expression and inhibited CD4 surface levels, coincident with suppression of HIV-1 replication . These results are not surprising since both GM-CSF and IFNγ are known to transduce signals via common pathways, e.g., JAK/STAT. The relationship between JAK2 activation and HIV-1 replication in MDM is currently being investigated.
GM-CSF mediates its activities through binding to its receptor, a heterodimer comprising a ligand-specific α-chain with low-affinity binding  and a non-ligand-binding β-chain that increases binding affinity . Although there is high expression of the α-chain and low expression of the β-chain on the surface of monocytes , to our knowledge the kinetics of expression over time in culture has not previously been documented. It appears that monocyte differentiation is associated with an increase in α/β dimers available for GM-CSF binding and transduction of the signal to the cell.
Our data suggest minimal inhibition at gag DNA level and a three- to tenfold decrease in gag mRNA within MDM treated with GM-CSF compared with untreated cells, suggesting that the block to HIV-1 replication occurs at or prior to transcription. The difference in the inhibitory effect for DNA and mRNA levels could be a consequence of the effect of GM-CSF at multiple points, donor variation or the semiquantitative nature of the assays. Our results are in agreement with Matsuda et al., who has also reported inhibition of HIV-1 replication by GM-CSF with a decrease in pro-viral DNA.
Currently, GM-CSF is used rarely for the treatment of HIV-infected patients because of concerns regarding potential activation of HIV replication. Early studies showed that GM-CSF treatment of HIV-infected patients increased serum p24 antigen and plasma HIV RNA titres [44,45]. However, when used in combination with effective antiretroviral therapy, GM-CSF has been safely administered to patients [46–49]. Data from two studies of HIV-infected patients receiving antiretroviral therapy and GM-CSF have shown that patients experienced a decrease in viral load and an increase in CD4 counts [50,51]. Clinical improvement and augmented macrophage function without an increase in viral load has been also reported in patients with advanced HIV infection and drug-resistant opportunistic infections when treated with GM-CSF [46,52]. Our in vitro data, together with that contained in another recent report  and the clinical studies mentioned above, suggest that clinical utility of GM-CSF in the setting of HIV infection (especially with M-tropic strains of the virus) may be cautiously revisited.
The authors would like to thank John Mills for his critical review of the manuscript, Kathy Tolli for her secretarial assistance, Geza Paukovics for assistance with flow cytometric analysis and Amanda Handley, Antoniette Violo and Helen Mutimer for their technical assistance. We thank LeukoSite Inc. for kindly providing us with CCR5 antibody.
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