Macrophages and lymphocytes differentially ... - Semantic Scholar

Macrophages and lymphocytes differentially modulate the ability of RANTES to inhibit HIV-1 infection Eleanore Gross,* Carol A. Amella,* Lorena Pompucci,* Giovanni Franchin,† Barbara Sherry,* and Helena Schmidtmayerova*,1 *Immunology and Inflammation Center, North Shore-LIJ Research Institute, New York, and †Department of Medicine, Albert Einstein College of Medicine, New York

Abstract: The ␤-chemokines MIP-1␣, MIP-1␤, and RANTES inhibit HIV-1 infection of CD4ⴙ T cells by inhibiting interactions between the virus and CCR5 receptors. However, while ␤-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 ␤-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 ␤-chemokines through intracellular signals that result in increased production of MIP-1␣ and MIP-1␤, 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 ␤-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. J. Leukoc. Biol. 74: 781–790; 2003. Key words: CCR5 expression production 䡠 MIP-1␣ 䡠 MIP-1␤

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internalization

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␤-chemokine

INTRODUCTION ⫹

The CD4 T lymphocytes and cells of the monocyte/macrophage lineage are the major targets of HIV-1 in vivo. Infection

of these cells is initiated by interactions between the viral envelope proteins and specific cellular receptors. In addition to CD4 glycoprotein, which is a major HIV-1 receptor on target cells, several members of the chemokine receptor family have been identified as coreceptors for HIV-1. Two chemokine receptors, CCR5 and CXCR4, are the principal coreceptors used by HIV-1 to gain entry into the target cells. CCR5, a member of the ␤-chemokine receptor family, functions as a coreceptor for M-tropic strains, or R5 viruses [1– 4]. CXCR4, an ␣-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 –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 ␤-chemokines inhibit HIV-1 replication in lymphocytes by competing with the viral protein Env for binding [13–15] and by down-regulating the chemokine receptors from the cell surface [16 –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

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Correspondence: North Shore-LIJ Research Institute, 350 Community Drive, Manhasset, NY 11030. E-mail: [email protected] Received April 25, 2003; revised June 16, 2003; accepted June 17, 2003; doi: 10.1189/jlb.0403187.

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␤-chemokines [19 –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 ␤-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 GMCSF [26]. Furthermore, dichotomous effects of ␤-chemokines on HIV-1 replication in macrophages have been observed, depending on the time of treatment. Pretreatment with ␤-chemokines resulted in an increase in viral replication, while adding ␤-chemokines to macrophages during or immediately after infection resulted in the inhibition of virus replication [27]. The in vivo role of ␤-chemokines in the pathogenesis of HIV-1 infection is still not clearly defined. A number of studies have analyzed levels of circulating ␤-chemokines in the blood of HIV-1-infected patients. Several of those suggested a protective role for ␤-chemokines in HIV-1 infection [28, 29]. However, others showed an association between elevated serum levels of ␤-chemokines and HIV disease progression and low CD4 cell counts [30 –32]. Additionally, a clear correlation between high cervical HIV-1 RNA levels and increased genital fluid concentration of three ␤-chemokines, MIP-1␣, MIP-1␤, and RANTES has been shown [33]. Interestingly, in the same study, only a trend toward correlation existed between the ␤-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 ␤-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 ␤-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 ␤-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. 782

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MATERIALS AND METHODS Isolation and culture of lymphocytes and macrophages Peripheral blood mononuclear cells from healthy donors undergoing leukopheresis were separated on a Ficoll⫺Hypaque gradient. Cells were allowed to adhere to plastic for 2 h at 37°C in RPMI 1640 supplemented with 10% heat-inactivated human serum (BioWhittaker, Walkersville, MD). Afterward, nonadherent cells were resuspended in RPMI 1600 medium supplemented with 10% heat-inactivated fetal calf serum, stimulated with 5 ␮g/ml of phytohemagglutinin for three days, and then cultivated in medium containing 20 U/ml of IL-2 (Roche, Indianapolis, IN). Adherent cells were washed and incubated for additional 24 h before detachment and plating in either 24-well (1 ml) or 48-well plates (0.5 ml) at the concentration of 106 cells/ml. If not otherwise indicated, cells were allowed to differentiate for seven days in the presence of 2 ng/ml of human M-CSF (Sigma, St. Louis, MO).

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-␮m 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 ⫻ 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-␮l volume, containing 0.5 ␮g 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 ⫻ 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

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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-1␤ level in cell culture supernatants was determined by specific ELISA (Endogen, Woburn, MA), according to the manufacturer’s instruction.

RESULTS RANTES inhibits entry, but not replication of HIV1 in primary macrophages We have previously reported the failure of ␤-chemokines to inhibit HIV-1 replication in macrophages [24]. Similar results were published by others [3, 22]. Yet, potent inhibition of fusion by ␤-chemokines in a cell fusion assay employing macrophages as the fusion target have been reported [1, 20]. Because of this contradiction we thought to independently analyze the effect of RANTES on the entry step and on longterm replication of HIV-1 in primary macrophages and lymphocytes isolated from the same donors. We chose RANTES for these studies, since it has been shown previously that RANTES is the most potent inhibitor of HIV-1 infection in lymphocytes [40]. To determine virus entry, cell lysates prepared two hours after infection were analyzed for the presence of HIV-1 specific strong-stop DNA (LTR RU5), the early product of reverse transcription synthesized shortly after viral entry. We have found that RANTES inhibited entry of HIV-1 into both lymphocytes and macrophages (Fig. 1 A). However, while the virus production was inhibited in long-term, RANTES-treated cultures of lymphocytes, in macrophages maintained under the same conditions, virus replication was not suppressed, even though fresh RANTES was added to the cultures twice a week (Fig. 1 B). Relatively high donor-dependent variability in the infectability of macrophages is a wellknown phenomenon. Because we anticipated similar differences in the response of macrophages to RANTES treatment, we analyzed the effect of RANTES on virus replication in cells isolated from 10 additional blood donors. The results (data not shown) corroborated our assumption, as the kinetics of virus replication in RANTES-treated macrophages varied from donor to donor, while presence of M-CSF during the macrophage differentiation did not affect obtained results. Cells from most donors responded similarly to the donor shown in Fig. 1 B. In some donors virus replication was delayed or lowered in RANTES-treated cultures. However, it was never suppressed completely in macrophages as it was in lymphocyte cultures. Interestingly, the RANTES-mediated enhancement of HIV-1 replication that we observed in macrophage cultures infected with primary HIV-1 isolates 92US660 and 92US657 [24] was

Fig. 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.

rarely seen in cultures infected with HIV-1 ADA, the viral strain highly adapted to growth in macrophages.

RANTES treatment does not prevent HIV-1 spread during long-term cultivation of macrophages Next, we wanted to determine why the inhibition of HIV-1 entry is overcome in macrophages, but not in lymphocyte cultures. We first examined whether the inhibition of HIV-1 entry observed at the start point of the infection (e.g., two hours after infection, Fig. 1A) is maintained during long-term cultivation of RANTES-treated macrophages. We cultivated infected macrophages and lymphocytes in the presence or abGross et al. Inhibition of HIV-1 infection by RANTES

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sence of RANTES for three weeks. Cells cultivated in the presence of AZT from day three after infection were used as a control. Cell lysates were prepared once a week and analyzed for HIV-1 specific strong-stop DNA, the early product of reverse transcription, and 2LTR DNA circles, which provide a useful marker for successful nuclear translocation of the HIV-1 DNA. In principle, if RANTES-mediated inhibition of virus entry is maintained throughout long-term cultivation, no virus spread and/or reinfection will take place, and therefore, levels of strong-stop DNA and 2LTR circles will be maintained at steady levels or will decrease. Indeed, no increase in strongstop DNA or 2LTR circles was detected in either AZT or RANTES-treated lymphocytes (Fig. 2A). Although similar results were obtained in AZT-treated macrophages, HIV-1-specific DNA increased over the time in RANTES-treated macrophages. These results strongly indicate that the virus is spreading in RANTES-treated cultures of macrophages. To further validate this observation, we analyzed HIV-1-specific strongstop DNA in cytoplasmic fractions prepared from infected macrophages. As Fig. 2B shows, there is a higher rate of strong-stop DNA increase in RANTES-treated macrophages than in infected controls. Therefore, while RANTES inhibits entry and spread of the virus in lymphocyte cultures, in macrophages, RANTES treatment does not prevent virus spread and/or reinfection over long-term cultivation period. The in-

creased rate of strong-stop DNA amount in RANTES-treated macrophages as compared with controls also suggests that the amount of infectious virus produced per infected cell is higher in RANTES-treated cultures leading to accelerated spreading. One of the known mechanisms of antiviral activity of ␤-chemokines at the level of virus entry is down-regulation of chemokine receptors [17, 18]. Although decreased CCR5 expression in macrophages treated with RANTES for two hours has been observed, we analyzed CCR5 expression in RANTEStreated cells at later time intervals. As shown in Fig. 3, surface expression of CCR5 was markedly decreased in RANTEStreated uninfected and HIV-1 infected cells shortly after treatment (day 0), albeit less efficiently in macrophages than in lymphocytes. However, at day 3 and 10 after infection, a clear difference appeared between lymphocytes and macrophages. Although cultivation of lymphocytes in the presence of RANTES resulted in a continued suppression of CCR5 surface expression, surface expression of CCR5 in macrophages was restored even though fresh RANTES was added to the cultures twice a week. Thus, although in RANTES-treated macrophages, full surface expression of CCR5 might fully support viral spread, a sustained down-regulation of CCR5 in RANTES-treated lymphocytes can significantly dampen subsequent rounds of HIV-1infection.

Fig. 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 ␮M 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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Fig. 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.

RANTES is efficiently cleared in macrophage cultures We next addressed the question of whether RANTES might be inactivated in macrophage cultures. To determine the activity of RANTES we analyzed supernatants from RANTES-treated macrophages for their ability to downregulate CCR5. Supernatants from RANTES-treated macrophages and lymphocytes were collected three days after treatment and transferred to untreated cells isolated from the same donors. To rule out possible effects of lymphocyte media on CCR5 expression in macrophages, supernatants from untreated lymphocytes were used as a control. After incubation for two hours, CCR5 expression was analyzed and compared to that of untreated cells and those treated with fresh RANTES. As shown in Fig. 4, while supernatants from RANTES-treated lymphocytes decreased surface expression of CCR5 as efficiently as fresh RANTES, supernatants collected from RANTES-treated macrophages failed to down-regulate CCR5. Next, we asked whether the failure of macrophage supernatants to down-regulate CCR5 was due to either the inactivation of RANTES, or its degradation. To address this issue, we analyzed an aliquot of each of the supernatants collected above for RANTES by ELISA. Results of this analysis (Table 1) suggested that RANTES is either degraded or efficiently cleared over the period of three days in macrophage cultures. Macrophages can produce relatively high levels of active substances, such as matrix metalloproteinases (MMP) [41], which can selectively cleave and inactivate chemokines [42–

44]. In light of these observations, we evaluated the possibility that RANTES is cleaved and subsequently degraded by macrophage-secreted proteases. Aliquots of RANTES were resuspended in freshly collected supernatants from macrophage and lymphocyte cultures and incubated at 37°C for three days. Afterward, the activity of RANTES was analyzed. Surprisingly, supernatants from macrophage cultures did not affect the ability of RANTES to down-regulate CCR5 or inhibit HIV-1 entry into primary macrophages (Fig. 5A). Similar results were obtained when lymphocytes were used in these assays. To rule out the possibility that RANTES by itself could induce or activate proteases, we analyzed whether RANTES added again to the pretreated cultures of macrophages will still downregulate CCR5 receptors. After seven days cultivation of macrophages with or without RANTES, fresh RANTES was added to the treated and control cells either directly or after media change, and the surface expression of CCR5 was analyzed two hours later. RANTES added to the pretreated cultures was as efficient as in control cells (Fig. 5 B). Taken together, these results indicate that loss of RANTES activity in macrophage cultures is due to the degradation or clearance independent of substances secreted by macrophages.

RANTES internalization and turnover rate is significantly higher in macrophages than in lymphocytes We further examined whether the observed differences between macrophages and lymphocytes might be due to differences in the rate of RANTES internalization in these two cell Gross et al. Inhibition of HIV-1 infection by RANTES

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Fig. 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.

types. To determine the rate of internalization, we incubated untreated cells and cells pretreated with RANTES for three days with radio-labeled RANTES for the times indicated in Fig. 6A. The amount of intracellular radioactivity was determined after subtraction of background activity. At least 5 times more radioactivity was detected in macrophages than in lymphocytes after a 1-hour incubation period. In agreement with the sustained decrease in CCR5 expression in RANTEStreated lymphocytes (Fig. 3), lower intracellular activity was detected in lymphocyte cultures pretreated with RANTES for three days, whereas in macrophages, the levels were similar in pretreated and untreated samples. Higher levels of ligand internalization might be achieved in cells expressing significantly more receptors on cell surface that can bind and internalize a ligand. However, this is not a case with CCR5, as

TABLE 1. RANTES Is Efficiently Cleared in Macrophage Cultures

Untreated control RANTES treated

Macrophages (RANTES ng/ml)

Lymphocytes (RANTES ng/ml)

0.2 19.0

0.6 201.0

Cells were incubated with or without RANTES (400 ng/ml) for three days. Afterwards, supernatants were collected and RANTES levels were analyzed by ELISA.

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Fig. 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 (f), 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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Fig. 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.

CCR5 is expressed at similar levels in macrophages and lymphocytes at the time of assay (as detected by FACS analysis). RANTES may bind and be internalized also through CCR1 receptors. Except, surface expression of CCR1 decreases during macrophage cultivation, and at the time that we performed these internalization studies, the levels of CCR1 were relatively low as compared with CCR5 (our unpublished observation). Another explanation is that receptor turnover is faster in macrophages than in lymphocytes. To address this possibility, we analyzed turnover rates in macrophage and lymphocyte cultures. Cells were incubated with radio-labeled RANTES for 40 min, at which time intracellular activity was maximal (Fig. 6A). After elimination of noninternalized ligands, cells were returned to 37°C, and supernatants and cell lysates were collected as indicated in Fig. 6B. Equilibrium between intracellular (internalized) and extracellular radioactivity was achieved in 20 min in both untreated and RANTES pretreated macrophage cultures. In lymphocytes, the turnover rate was markedly slower. Equilibrium was achieved after more than 1 hour. A slight difference was observed between control and RANTES pretreated lymphocytes, which might be due to the much lower levels of internalization in pretreated samples resulting from significantly lower CCR5 expression. Together,

these results indicate that RANTES is cleared in macrophage cultures by continuous and efficient internalization and intracellular degradation.

RANTES induces production of MIP-1␣ and MIP-1␤ in primary lymphocytes, but not macrophages Chemokine binding to the appropriate receptors induces a cascade of intracellular signals [45– 47] that may lead to the synthesis of bioactive substances. In agreement with these reports, RANTES was recently shown to induce a chemokine cascade in dendritic cells [48]. Thus, we finally examined whether RANTES treatment might induce production of ␤-chemokines in the cell cultures used in our experiments. HIV-1-infected and control cells were cultivated in the presence or absence of RANTES for 21 days. Supernatants were collected twice a week, and production of CCR5 binding ␤-chemokines was analyzed in collected samples by ELISA. Following RANTES stimulation, lymphocytes, but not macrophages, continuously secreted MIP-1␣ and MIP-1␤ 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 Gross et al. Inhibition of HIV-1 infection by RANTES

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Fig. 7. RANTES induces ␤-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-1␤ (bottom panels) by ELISA. Results of one representative experiment out of two, each performed in duplicate. Standard deviations are indicated as vertical bars.

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.

DISCUSSION Many different experimental approaches analyzing distinct steps in the viral life cycle have been used to study the effects of ␤-chemokines on HIV-1 infection in primary macrophages. It is plausible that this might significantly contribute to the apparent discrepancies in published results that range from observations describing efficient inhibition of HIV infection of macrophages [19 –21] to observations reporting either failure [22, 23] or even enhancement of virus replication [24, 25] by ␤-chemokines. In an attempt to bring some clarification to this issue, we have independently analyzed the effects of RANTES on the entry steps and also on replication and spread of HIV-1 in parallel cultures of primary macrophages and lymphocytes. In agreement with studies demonstrating the inhibitory effects of ␤-chemokines on HIV-1 entry [1, 20], we show here that RANTES inhibits HIV-1 entry into both lymphocytes and macrophages at the moment of infection. However, the amount of virus progeny is suppressed by RANTES only in lymphocytes. In macrophages, the amount of virus produced by RANTES-treated and untreated cultures is similar. One expla788

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nation might be that HIV-1 infection by itself or together with RANTES treatment induced secretion of proinflammatory cytokines, such as TNF-␣, 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 ␤-chemokines was not detected in macrophages, lymphocytes selectively responded to RANTES treatment by continuous synthesis of MIP-1␣ and MIP-1␤. A slight increase in chemokine production was detected in RANTES-treated macrophages 2 hours after infection and at day 14 after infection in HIV-1infected 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 ␤-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– 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 RANTESmediated 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 http://www.jleukbio.org

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 ␤-chemokines in vivo. It has been suggested previously that elevated ␤-chemokine levels may play an important role in preventing HIV-1 infection in uninfected individuals with multiple highrisk sexual exposures [10, 29]. However, contradictory results have been reported when ␤-chemokine levels were analyzed during established HIV-1 infection [30 –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 ␤-chemokine activity are necessary to clarify the role of ␤-chemokines in HIV-1 pathogenesis.

ACKNOWLEDGMENTS This work was supported by NIH grants AI43743 (H.S.) and AI29110 (B.S.), and by The Picower Foundation. We thank Amanda Proudfoot from Serono Pharmaceutical for recombinant RANTES and Michael Bukrinsky for thoughtful discussion.

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