Published online before print July 15, 2003
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* Immunology and Inflammation Center, North Shore-LIJ Research Institute, New York, and
Department of Medicine, Albert Einstein College of Medicine, New York
1Correspondence: North Shore-LIJ Research Institute, 350 Community Drive, Manhasset, NY 11030. E-mail: HSchmidt{at}nshs.edu
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, MIP-1ß, and RANTES inhibit HIV-1 infection of CD4+ T cells by inhibiting interactions between the virus and CCR5 receptors. However, while ß-chemokine-mediated inhibition of HIV-1 infection of primary lymphocytes is well documented, conflicting results have been obtained using primary macrophages as the virus target. Here, we show that the ß-chemokine RANTES inhibits virus entry into both cellular targets of the virus, lymphocytes and macrophages. However, while virus entry is inhibited at the moment of infection in both cell types, the amount of virus progeny is lowered only in lymphocytes. In macrophages, early-entry restriction is lost during long-term cultivation, and the amount of virus produced by RANTES-treated macrophages is similar to the untreated cultures, suggesting an enhanced virus replication. We further show that at least two distinct cellular responses to RANTES treatment in primary lymphocytes and macrophages contribute to this phenomenon. In lymphocytes, exposure to RANTES significantly increases the pool of inhibitory ß-chemokines through intracellular signals that result in increased production of MIP-1 and MIP-1ß, thereby amplifying the antiviral effects of RANTES. In macrophages this amplification step does not occur. In fact, RANTES added to the macrophages is efficiently cleared from the culture, without inducing synthesis of ß-chemokines. Our results demonstrate dichotomous effects of RANTES on HIV-1 entry at the moment of infection, and on production and spread of virus progeny in primary macrophages. Since macrophages serve as a reservoir of HIV-1, this may contribute to the failure of endogenous chemokines to successfully eradicate the virus.
Key Words: CCR5 expression • internalization • ß-chemokine production • MIP-1 • MIP-1ß
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-chemokine receptor, acts as a coreceptor for T-tropic strains, or X4 viruses [5
].
The identification of viral coreceptors revealed a molecular feature, which explains in part one of the important phenomena of HIV-1 disease, the evolution of viral tropism during the course of infection. M-tropic, or R5 strains of HIV-1, predominate during the initial viremia after HIV-1 transmission, while T-tropic, or X4 strains are detected only at later disease stages [6
7
8
9
], indicating that macrophages are probably one of the first cell types infected during HIV-1 transmission. Identification of individuals with a homozygous deletion in the CCR5 gene (
ccr5) further demonstrated the important role of macrophages in HIV pathogenesis. These individuals are highly resistant to HIV-1 infection despite multiple high-risk sexual exposures to the virus [10
, 11
]. In vitro, macrophages isolated from ccr5 individuals are refractory to HIV-1 infection by R5 viruses, although lymphocytes isolated from the same donors can be productively infected by X4 viruses [12
]
Since chemokine receptors play such a crucial role in HIV-1 infection, their natural ligands have been considered important players in HIV pathogenesis, and numerous studies have been directed toward characterization of their effects on HIV-1 infection. It is now well documented that ß-chemokines inhibit HIV-1 replication in lymphocytes by competing with the viral protein Env for binding [13 14 15 ] and by down-regulating the chemokine receptors from the cell surface [16 17 18 ], thus inhibiting virus entry into the target cells. However, their effect on virus replication in primary macrophages is still in question. The results of in vitro studies range from observations describing efficient inhibition of HIV infection of macrophages by ß-chemokines [19 20 21 ] to observations reporting either failure [22 , 23 ] or even enhancement of virus replication [24 , 25 ]. Variable methods of isolation and cultivation of macrophages may affect the antiviral activity of ß-chemokines, as suggested in a study comparing the effect of RANTES on HIV replication in macrophages prepared by two different methods. The HIV-1 infection was inhibited by RANTES when macrophages were cultivated without exogenous growth factors, but not when cultured with a physiological concentration of M-CSF or GM-CSF [26 ]. Furthermore, dichotomous effects of ß-chemokines on HIV-1 replication in macrophages have been observed, depending on the time of treatment. Pretreatment with ß-chemokines resulted in an increase in viral replication, while adding ß-chemokines to macrophages during or immediately after infection resulted in the inhibition of virus replication [27 ].
The in vivo role of ß-chemokines in the pathogenesis of HIV-1 infection is still not clearly defined. A number of studies have analyzed levels of circulating ß-chemokines in the blood of HIV-1-infected patients. Several of those suggested a protective role for ß-chemokines in HIV-1 infection [28
, 29
]. However, others showed an association between elevated serum levels of ß-chemokines and HIV disease progression and low CD4 cell counts [30
31
32
]. Additionally, a clear correlation between high cervical HIV-1 RNA levels and increased genital fluid concentration of three ß-chemokines, MIP-1, MIP-1ß, and RANTES has been shown [33
]. Interestingly, in the same study, only a trend toward correlation existed between the ß-chemokine concentration and the virus load in plasma. Assuming a role for macrophages in natural transmission of HIV-1 infection, the latter results raise the possibility that ß-chemokines affect virus replication in macrophages and blood lymphocytes differently.
Macrophages represent a unique target for HIV-1. Virus may persist in macrophages for a prolonged period of time within intracellular vacuoles [34 , 35 ], hidden from the host immune surveillance. Cells harboring the virus may serve as a reservoir for continued infection and dissemination of HIV through the body over an extended period of time. However, a number of macrophage functions could be impaired following HIV-1 infection, including phagocytosis, intracellular killing, chemotaxis, and cytokine production [36 ], which, in turn, may contribute to the pathogenesis of AIDS. Thus, understanding the effects of ß-chemokines on HIV-1 infection in primary macrophages may contribute to a better understanding of their role in HIV-1 pathogenesis. In this study, we have characterized and compared the effects of ß-chemokines on HIV-1 infection in primary macrophages and lymphocytes isolated from the same donors. Furthermore, we have examined cellular responses to RANTES treatment and their possible involvement in the modulation of the ability of RANTES to inhibit HIV-1 infection. Our results demonstrate that while virus entry is inhibited at the moment of infection in both cell types, the amount of virus progeny is lowered only in lymphocytes, which suggest a loss of entry restriction and an enhanced virus replication. Further, our results suggest that the antiviral activity of RANTES is controlled by the target cells.
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Virus, infection, and treatment
A macrophage-tropic strain HIV-1 ADA [37
] was used in this study. Viral stock was prepared as follows; cell supernatants from infected macrophages were pooled, filtered through 0.45-µm filter, aliquoted, and stored at -80°C. Immediately before infection, an aliquot of the viral stock was treated with 200 U/ml of RNase-free DNase (Roche Molecular Biochemicals, Indianapolis, IN) to eliminate contamination with viral DNA. Cells were infected for 2 h at 37°C with an amount of virus corresponding to 5 x 104 cpm of reverse transcriptase activity per million cells. Virus replication was measured in culture supernatants using a standard reverse transcriptase (RT) assay. To analyze the effects of RANTES on HIV infection, cells were treated with 200 ng/ml (if not indicated otherwise) of RANTES obtained from Serono Pharmaceutical, Geneva, Switzerland (a kind gift from Amanda Proudfoot).
Detection of HIV-1-specific DNA by polymerase chain reaction (PCR)
Samples from infected cultures were prepared and subjected to PCR analysis using HIV-1-specific primers, amplifying LTR RU5 transcripts and 2LTR circles as described previously [38
]. Cytoplasmic fractions were prepared as described elsewhere [39
]. Briefly, cells were resuspended in buffer containing 10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.3 M sucrose, and incubated on ice for 5 min. An equal volume of buffer containing 0.2% of Nonidet P40 (NP40) was added, and the cells were incubated for additional 5 min on ice. After centrifugation at 1000 rpm, supernatants containing cytoplasmic fractions were collected and subjected to PCR analysis. Amplified DNA was analyzed by Southern blot hybridization using 32P-labeled probes and quantified on an Instant Imager (Packard, Meriden, CT). Results are expressed as counts per minute. Amplification of the -tubulin gene or the mitochondrial DNA gene was used to control for the amount of DNA in each sample. Serial dilutions of 8E5/LAI cells, containing one HIV-1 genome per cell, were included in each amplification reaction to standardize obtained results.
Flow cytometric analysis
Macrophages were stained directly in 48-well plates. Macrophages and lymphocytes were preincubated with 20% normal human serum in PBS, containing 0.1% sodium azide for 20 min at room temperature. After washing, cells were stained with anti-CCR5 monoclonal antibody directly labeled with either fluorescein isothyocyanate (2D7-FITC, BD PharMingen, San Diego, CA) or phycoerythrin (2D7-PE, BD PharMingen, San Diego, CA) for 30 min at room temperature in a 100-µl volume, containing 0.5 µg of antibody. As a control, mouse isotype antibody IgG2b-FITC or IgG2b-PE (BD PharMingen, San Diego, CA) was used. After washing, cells were resuspended in 0.8% formaldehyde in PBS. Macrophages were then detached and transferred to tubes. Staining was analyzed on FACS Calibur (Becton Dickinson, San Jose, CA).
Radio-labeled RANTES internalization studies
For internalization experiments, 0.5 x 106 cells were incubated with 0.5 nM of [125I]-RANTES (Perkin Elmer Life Sciences, Boston, MA; specific activity 2200 Ci/mmol) in culture media for 1, 2, 5, 10, 15, 20, 30, 40, 50, and 60 min at 37°C. Afterward, noninternalized [125I]-RANTES molecules were removed by washing cells with an acid solution (0.05 M glycine HCl buffer, pH 3.0, containing 0.1 M NaCl) for 1 min, followed by washing four times with PBS. Cells were then lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% deoxycholic acid, and 1% SDS) and counted in a gamma counter (Gamma Track 1193). Nonspecific uptake was determined by counting radioactivity internalized in the presence of a 100-fold excess of unlabeled RANTES. To analyze turnover rate, cells were incubated with 0.25 nM of [125I]-RANTES for 40 min at 37°C. Afterward, noninternalized [125I]-RANTES was removed by acid wash, and cells were incubated again at 37°C. At the sequential time points, media and cell lysates were collected and counted in a gamma counter.
Chemokine assays
Each human MIP-1 and MIP-1ß level in cell culture supernatants was determined by specific ELISA (Endogen, Woburn, MA), according to the manufacturer’s instruction.
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Figure 1. RANTES inhibits the entry, but not the replication of HIV-1 in primary macrophages. Macrophage and lymphocyte cultures, isolated from the same donor, were treated with 200 ng/ml of RANTES for 1 hour before infection with HIV-1 ADA. (A) Two hours after infection, half of the cultures were lysed and subjected to 30 cycles of polymerase chain reaction amplification using LTR RU5 primers amplifying HIV-1-specific strong-stop DNA. Amplified products were hybridized with a specific 32P-labeled probe and quantified on an Instant Imager (Packard). Results are expressed as counts per minute (cpm). Amplification of the -tubulin gene was used to control the amount of DNA in each sample. (B) Remaining cells were used to analyze virus replication in long-term follow-up. Medium was changed twice a week and assayed for RT activity. After each medium change, cultures were supplemented with fresh RANTES. Data (A and B) show results of one representative experiment out of five, each performed in duplicate.
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Figure 2. HIV-1-specific strong-stop DNA increases in RANTES-treated macrophages during long-term cultivation of infected cultures. (A) Macrophages (left panels) and lymphocytes (right panels) were pretreated with 200 ng/ml of RANTES before infection with HIV-1 ADA. Two hours after infection, an aliquot from each sample was lysed for PCR analysis. Remaining cells were cultivated in the presence or absence of RANTES. Three days later, an aliquot of infected cells was treated with 10 µM AZT. Cells were maintained in culture for 3 weeks. Fresh RANTES and AZT were added twice a week after media change. At indicated time points, an aliquot from each sample was lysed and analyzed by PCR using primers from HIV-1 LTR RU5 region (upper panels) and primers amplifying HIV-1-specific 2LTR circles (bottom panels). Quantification of the results obtained after hybridization of PCR product with radio-labeled probe is shown. Each bar represents mean ± standard deviation of HIV-specific transcript levels from one representative experiment out of three, each performed in duplicate. (B) Macrophages were treated with RANTES, infected, and cultured as described in part (A). Cytoplasmic fractions were prepared at indicated intervals and analyzed by PCR using primers specific for HIV-1 LTR RU5. Amplification of a mitochondrial DNA gene was used to control the amount of DNA in each sample. Primers specific for the -tubulin gene were used to rule out possible contamination of cytoplasmic fraction with nuclei. Each bar represents mean ± standard deviation of HIV-specific transcript levels from one representative experiment out of two, each performed in duplicate.
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Figure 3. Differential cell surface expression of CCR5 in RANTES-treated macrophages and lymphocytes. Macrophages (left panels) and lymphocytes (right panels) were treated with RANTES, infected, and cultured as described in the legend to Fig. 2
. An aliquot of cells from each sample was taken for immunofluorescence staining at day 0 (2 hours after infection), day 3, and day 10. Cells were stained with directly labeled 2D7-FITC (anti-CCR5) antibody. Parallel samples were stained with mouse isotype antibody IgG2a-FITC. Staining was analyzed by flow cytometry. CCR5 expression is shown as mean fluorescence intensity (MFI) after subtraction of mean fluorescence of matched isotype control. Data show results of one representative experiment out of four, each performed in duplicate. Standard deviations are indicated as vertical bars.
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Figure 4. Culture supernatants from RANTES-treated lymphocytes, but not macrophages, down-regulate CCR5. Macrophages and lymphocytes were incubated with RANTES (200 ng/ml) for three days. Afterward, cell supernatants were transferred to the untreated macrophages isolated from the same donors. Parallel samples were treated with 200 ng/ml of RANTES. After 2 hours incubation, surface expression of CCR5 was analyzed by immunofluorescence staining as described in the legend to Fig. 3
. Data show results of one representative experiment out of three, each performed in duplicate. Standard deviations are indicated as vertical bars.
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Table 1. RANTES Is Efficiently Cleared in Macrophage Cultures
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Figure 5. Supernatants from macrophage cultures do not alter RANTES activity. (A) Aliquots of RANTES were resuspended in supernatants collected from macrophage and lymphocyte cultures, or in media supplemented with human serum and incubated at 37°C. After 3 days, macrophage cultures were treated with appropriate RANTES sample for 2 hours. Afterward, cells were either stained for CCR5 expression as described in the legend to Fig. 3
(upper panel) or were infected with HIV-1 ADA (lower panel). Lysates prepared from infected cells were subjected to PCR amplification using LTR RU5 primers. Amplification of the -tubulin gene was used to control the amount of DNA in each sample. Data show results of one representative experiment out of three, each performed in duplicate. (B) Macrophages were cultivated in the presence or absence of RANTES (200 ng/ml) for seven days. Media were changed and fresh RANTES was added to the cultures 3 days after treatment. At day 7, fresh RANTES was added to the pretreated and control untreated cells either directly ( ), or after media change (
). After 2 hours incubation, cells were stained with anti-CCR5 antibody (2D7-PE). Data show results of one representative experiment out of two, each performed in duplicate.
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Figure 6. Levels of RANTES internalization and turnover rate are significantly higher in macrophages than in lymphocyte cultures. Macrophages and lymphocytes were cultivated for 3 days in the absence (labeled as control) or presence of 200 ng/ml of RANTES (labeled as RANTES). Afterward, cells were washed and analyzed as follows. (A) Cells were incubated with [125I]-RANTES for the indicated time at 37°C. Afterward, noninternalized ligand was removed by acid wash, cells were lysed and intracellular radioactivity was counted. Each time point indicates internal radioactivity after subtraction of the background activity measured in parallel samples incubated with 100 times excess of cold RANTES. Each point represents average of at least two experimental values from one representative experiment out of three. (B) Macrophages (upper panel) and lymphocytes (bottom panel) were incubated with 125I-RANTES for 40 min at 37°C. Afterward, noninternalized ligand was removed by acid wash, and cells were returned to 37°C. At the indicated time points, culture supernatants and cell lysates were collected independently and counted in a gamma counter. Each point represents an average of at least two experimental values from one representative experiment out of two.
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and MIP-1ß in primary lymphocytes, but not macrophages and MIP-1ß into the culture medium (Fig. 7
). A slight increase in chemokine production was detected in RANTES-treated macrophages 2 hours (day 0) after infection. At day 14 after infection, when virus replication reached a peak, a slight increase in chemokines was detected in HIV-1-infected and RANTES-treated infected cultures. However, chemokine levels in these samples were far below those produced by RANTES-treated lymphocytes.
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Figure 7. RANTES induces ß-chemokine production in primary lymphocytes, but not macrophages. Macrophages (left panels) and lymphocytes (right panels) were treated with 200 ng/ml of RANTES and infected with HIV-1 ADA. Cells were cultivated with or without RANTES for 21 days. Twice a week, the medium was changed, and fresh RANTES was added to the appropriate cultures. Supernatants, collected at the indicated times were analyzed for production of MIP-1 (upper panels) and MIP-1ß (bottom panels) by ELISA. Results of one representative experiment out of two, each performed in duplicate. Standard deviations are indicated as vertical bars.
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, which could enhance HIV-1 replication and counterbalance the inhibitory effect of RANTES at the level of entry [49
]. Although several lines of evidence presented here suggest that cellular responses modulate the antiviral activity of RANTES, we did not detect a sustained increase in TNF levels in supernatants from HIV-1-infected and RANTES-treated macrophages (data not shown). In fact, although production of inflammatory cytokines and ß-chemokines was not detected in macrophages, lymphocytes selectively responded to RANTES treatment by continuous synthesis of MIP-1 and MIP-1ß. A slight increase in chemokine production was detected in RANTES-treated macrophages 2 hours after infection and at day 14 after infection in HIV-1-infected and RANTES-treated infected cultures. In experiments presented here, we did not see substantial chemokine production in HIV-1-infected cultures of macrophages. At the first glance, this seems to contradict what we have shown previously, for example, that HIV-1 infection induces ß-chemokines in primary macrophages [50
]. The induction that we observed depended on productive viral infection. In our present experiments, virus replication was lower and peaked only late after infection, in average from 17 to 20 days. We assume that viral levels were not high enough to induce high levels of chemokine synthesis. A similar effect of RANTES on the induction and amplification of proinflammatory chemokines, as we observed in lymphocytes, has recently been described using dendritic cells [48
, 51
]. In light of these reports, our results are surprising, yet the RANTES-induced signal transduction pathways controlling this response may be different in lymphocytes and dendritic cells than in macrophages. It has been demonstrated previously that chemokine receptors may couple to different G proteins and that coupling is cell type-specific [52
]. Our results further revealed that RANTES is efficiently cleared from the macrophage cultures over a 3-day incubation period. We identified rapid internalization and turnover as one of the possible mechanisms that may contribute to RANTES clearance in macrophage cultures. It has been shown that differentiation and activation of macrophages leads to the expression of matrix metalloproteinases (MMPs), endoproteinases with extracellular matrix-degrading activity [41 , 53 ]. Several chemokines serve as substrates for secreted MMPs, and proteolysis by MMPs may result in selective potentiating [43 ] or degradation [42 43 44 ] of chemokines. We did not detect an alteration of RANTES activity either by supernatants collected from macrophages that might contain secreted proteases, or in RANTES-treated cultures. Although, we cannot exclude completely involvement of additional mechanisms in RANTES depletion, our results strongly suggest that RANTES is not degraded by proteases secreted by macrophages themselves or after RANTES treatment.
It has been reported recently that binding of RANTES to CCR5 triggers intracellular signaling that may enhance viral replication at the postentry levels [25 , 54 ]. The RANTES-mediated inhibition of HIV-1entry is not complete in either of the cellular targets of the virus (Fig. 1A) . Removal of RANTES from infected PM1 cells leads to the rapid burst of viral replication [55 ], further confirming that RANTES suppresses but does not completely eliminate HIV-1entry. In light of these observations, it is likely that before being cleared from macrophage cultures, RANTES may induce a signaling cascade that enhances virus replication. The increased rate of LTR RU5 amount in cytoplasm of RANTES-treated macrophages supports this hypothesis. Our results also show that RANTES clearance in macrophage cultures is followed by full, or in some donors even increased (data not shown), re-expression of CCR5 on the cell surface. Thus, even if only a limited amount of virus is produced in RANTES-treated cultures during early rounds of viral replication, simultaneous activation of enhancing signaling pathways might reinforce the more efficient propagation of the virus in the treated cultures. Further investigation is necessary to confirm this hypothesis as well as to determine why RANTES-induced signaling does not enhance virus replication in lymphocytes.
It has been reported that differentiation of macrophages in the presence of M-CSF or GM-CSF abrogates the inhibitory effect of RANTES on virus infection [26 ]. The effect has been attributed to the differences in proteoglycan surface expression on growth factors stimulated vs. unstimulated cells. In contrast, chemokine-mediated inhibition of HIV-1 Env-mediated fusion with GM-CSF-treated macrophages was slightly more efficient in another report [20 ]. In our hands, the presence or absence of M-CSF during macrophage differentiation did not significantly affect RANTES’ effects on HIV-1 replication in long-term cultures. However, in agreement with a previous report [40 ], we have observed donor-dependent variation in RANTES’ effects on virus replication in macrophages.
In conclusion, our data suggest that under in vitro conditions, the antiviral activity of RANTES at the moment of infection is the same in lymphocytes and macrophages. However, during virus replication in long-term cultures, lymphocytes and macrophages differentially modulate the ability of RANTES to inhibit HIV-1 infection. These observations explain the apparent contradictions reported previously and contribute toward better understanding of the role of ß-chemokines in vivo. It has been suggested previously that elevated ß-chemokine levels may play an important role in preventing HIV-1 infection in uninfected individuals with multiple high-risk sexual exposures [10 , 29 ]. However, contradictory results have been reported when ß-chemokine levels were analyzed during established HIV-1 infection [30 31 32 33 ], when other factors may counterbalance the inhibitory effects at the level of virus entry. Further studies directed toward the characterization of possible endogenous and exogenous factors that are involved in HIV-1 replication and may alter ß-chemokine activity are necessary to clarify the role of ß-chemokines in HIV-1 pathogenesis.
Received April 25, 2003; revised June 16, 2003; accepted June 17, 2003.
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