The susceptibility of macrophages to human immunodeficiency virus type 1 X4 isolates dependson their activation state

Biomed Pharmacother 2001 ; 55 : 32-8 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0753332200000159/FLA

Dossier: AIDS

The susceptibility of macrophages to human immunodeficiency virus type 1 X4 isolates depends on their activation state Y. Bakri*, S. Amzazi, A. Mannioui, A. Benjouad Laboratoire de Biochimie-Immunologie, JER 3012 associée à l’Agence Universitaire Francophone, Faculté des Sciences, Avenue Ibn Batouta, 11400, Rabat, Morocco; Institut National de la Santé et de la Recherche Médicale E0013, faculté de Médecine Saint-Antoine, Faculté de Médecine Saint-Antoine, 27 rue Chaligny, 75571 Paris cedex 12, France (Received 30 October 2000; accepted 3 November 2000)

Summary – The demonstration that macrophages express CXCR4 has led to a reexamination of their susceptibility to human immunodeficiency (HIV)-1 X4 strains. Here, we examined the susceptibility to X4 HIV-1Lai of two previously characterized macrophage populations, obtained either as 1) adherent cells of five-day cultures of blood mononuclear cells (PBMC), followed by two days without nonadherent PBMC nor added cytokines (MDM-5d); or 2) as adherent cells recovered from one-hour incubation of PBMC, which were cultured for seven days with macrophage colony-stimulating factor (MDM-MCSF). Exposing MDM-5d or MDM-MCSF to HIV-1Lai did not lead to productive infection, as indicated by a lack of (MDM-MCSF) or low (MDM-5d) viral p24 levels in culture supernatants. However, MDM-5d vigorously transmitted HIV-1Lai to autologous T lymphocytes, which was not the case of HIV-1Lai-exposed MDM-MCSF. PCR analysis of the LTR RU5 region showed that X4 HIV-1Lai entered into both types of macrophages in the same manner as R5 HIV-1BaL. However, in contrast to MDM-5d, there was a block of HIV-1Lai retrotransciption in MDM-MCSF. Cytokine profile analysis of the two types of macrophages showed that TNF-α, IL-6 and RANTES levels were higher in MDM-5d than in MDM-MSCF, while the IL10 level was higher in MDM-MCSF, both producing similar IL16 levels. Altogether, these data indicate that HIV-1 X4 strains enter into macrophages but that their replication is blocked thereafter in a different manner according to the activation status of the cells. © 2001 Éditions scientifiques et médicales Elsevier SAS activation status / cytokines / macrophages / X4 HIV-1

Human immunodeficiency virus (HIV)-1 strains isolated from recently infected individuals are predominantly macrophage-tropic (M-tropic) and use CCR5 as a coreceptor in combination with CD4 [20]. Later in the course of HIV-1 disease, the virus frequently expands its coreceptor use to include CXCR4 and other coreceptors in addition to CCR5, resulting in a selective advantage for the virus [8]. The emergence of CXCR4-dependent (X4) HIV-1 strains precedes a more rapid decline in CD4+ T cell counts and pro*Correspondence and reprints.

gression to AIDS, suggesting that X4 strains contribute to AIDS pathogenesis [4]. The high levels of viremia at advanced stages of HIV-1 disease when CD4+ T cells are markedly depleted suggests that other cells may be responsible for viral replication in vivo. Macrophages play an important role in the natural history of HIV-1 infection. They are assumed to be among the first targets of viral infection in the genital or rectal mucosae [2, 5] and serve as long-term reservoirs in chronically HIV-1-infected patients [13]. Tissue macrophages represent a highly productive source of HIV during opportunistic infections at

X4 HIV-1 isolate and macrophages

advanced stages of HIV disease [16]. Macrophages are readily infected by R5 strains of HIV-1, the entry of which is mediated by CCR5 [7, 9, 30], but they have been considered to be refractory to X4 HIV-1 isolates [7, 27] even though they express functional CXCR4 in addition to CCR5 [1, 9, 15, 23, 27]. Some studies reported that X4 strains entered efficiently into macrophages but were blocked post-entry [21], whereas others have shown that some primary isolates using CXCR4 but not CCR5 replicated in macrophages [27], and that there was no post-entry block for X4 isolates in macrophages [24]. These inconsistencies may be ascribed at least to variation in culture conditions, which may generate macrophages at different stages of activation. In this respect, cytokines are important regulators of cell activation as well as modulators of HIV infection [17, 18, 22, 26]. In this study, we investigated whether macrophage culture conditions and, hence, their activation state may affect their susceptibility to HIV-1 X4 strains. Actually, we found that the capacity of macrophages to replicate X4 strains depended largely on how these cells were cultured and that these conditions correlated with the cytokine and chemokine environment. MATERIALS AND METHODS Reagents Recombinant human macrophage colony-stimulating factor (M-CSF) was from R&D Systems (Minneapolis, MN). Monoclonal antibodies (mAbs) CD4 (phycoerythrin [PE]-Leu3a), CD14 (fluorescein isothiocyanate [FITC]-LeuM3) were from BectonDickinson (Mountain View, CA). Anti-CCR5, -CCR3 and -CXCR4 mAbs were from the NIH AIDS Reagent Program (Bethesda, MD) or from R&D Systems.

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glutamine (all from GIBCO BRL, Paisley, UK), 20% fetal calf serum ([FCS]; Boehringer Mannheim, Mannheim, Germany), and 10% heat-inactivated normal human AB serum (Pitié-Salpêtrière Blood Bank), without added exogenous growth factor, after which nonadherent cells were removed and adherent cells were cultured for a further two days; nonadherent cells were then removed by an extensive washing in Ca2+/Mg2+-free phosphate-buffered saline (PBS), and macrophages were cultured in medium with only 20% FCS for another two days before use. 2) MDMMCSF: monocytes from the same donors were separated by one-hour adherence, and cultured for seven days in the same culture medium, but with only 20% FCS, and with 50 U/mL M-CSF. The macrophages obtained were characterized by flow cytometry analysis (FACScan, Becton Dickinson) as described [1, 28]. They were 87 to 99% CD14+, without detectable contaminating T and B lymphocytes; they expressed CD4, CCR5 and CXCR4 [1]. HIV-1 infection of cells Macrophages (5 × 105) were exposed for 18 hours to 500 TCID50 HIV-1BaL or 500 TCID50 HIV-1Lai in 1 mL culture medium as described [1, 29]. Excess virus was washed away, and cells were cultured further for four weeks. Cocultures were developed at day 7 post-infection: macrophages were extensively washed and cocultured with PHA/IL-2-activated autologous T lymphocytes. Virus production was assessed by measuring p24 by a highly sensitive enzyme-linked immunosorbent assay (ELISA) (Coulter/Immunotech, S.A., Marseilles, France). Accurate quantification of samples was made by using the HIV-1 p24 antigen kinetic standard and kinetic reading of the assay plate according to the manufacturer’s instructions. Semi-quantitative detection of viral DNA

Cells Buffy-coat peripheral blood mononuclear cells (PBMC) from healthy donors were isolated by FicollPaque (Pharmacia, Uppsala, Sweden) centrifugation. Monocytes were obtained by one-hour adherence of PBMC, and macrophages were obtained according to two procedures [1]. 1) MDM-5d: PBMC were cultured for five days in RPMI 1640, penicillinstreptomycin-neomycin (50 µg/mL), 2 mM

HIV-1 DNA was detected by nested PCR as described [3]. Briefly, macrophages were exposed for three hours at 37° C to 500 TCID50 of virus. HIV-1 R-U5 and LTR-gag DNA were analyzed 48 hours later: cells were extensively washed with cold PBS and scarped with a rubber policeman, washed again and lysed (1 × 106/mL) in 10 mM TRIS HCl, pH 8.3, 50 nM KCl, 0.5% Tween 20 (Biorad, Hercules, CA), 0.5% Nonidet (Sigma, St. Louis, MO). Proteinase K

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(4 µg/mL; Boehringer, Mannheim, Germany) was added, lysates were incubated at 56° C for one hour, and proteinase K was inactivated at 95° C for 10 minutes. Infected lysates were then serially diluted (1 to 1:10,000) in lysates of HIV-negative A301 cells (1 x 106/mL). Samples (30 µl) were then subjected to 35 rounds of PCR amplification with primers designed to detect early (R-U5) or late (LTR-gag) reverse transcription products. For nested PCR, 2 µl of amplified products were submitted to another 30-cycle amplification under the same conditions, using internal primers. Amplifications were performed in an automated DNA Thermal Cycler (Crocodile III, Appligene, Strasbourg, France). The following primers were used for the first amplification (numbering positions correspond to the HXB2 DNA sequence): LTR R-U5 sens primer 5’-CTAACTAGGGAACCCACTG-3’(nucleotides 498–516), antisens primer 5’-CTGCTAGA GATTTTCCACAC-3’(nucleotides 616–635); LTR-gag sens primer 5’-CAGATATCCACTG ACCTTTGG3’(nucleotides 110–130), antisens 5’-GCTTAATACTGACGCTCTCGCA-3’(nucleotides 795–816). Primers for the nested PCR were as follows: LTR R-U5 sens primer 5’-ACTAGGGAACCCACTGCT3’(nucleotides 501–518), antisens primer 5’-GGTCTGAG GGATCTCTAG-3’(nucleotides 588–605); LTR-gag sens primer 5’-CTAACTAGGGAACCC ACTG-3’(nucleotides 498–516), antisens 5’-TCCTGCGTCGAGAGAGCTC-3’(nucleotides 678–696). Amplified fragments (15 µl) of the correct size (R-U5: 105 bp; LTR-gag: 199 bp) were electrophoresed onto 2% agarose and stained with ethidium bromide for UV visualization. The PCR sensitivity (1 HIV copy/3 × 104 cells) was determined relative to serial dilutions of 8E5/LAV cells (1 copy/cell) in HIV-negative A301 parental cells. Detection of cytokines in culture supernatants Macrophages obtained as above were washed extensively at day 7, which corresponds to the day of exposure to virus in the above experiments, and then cultured for 48 hours before collecting supernatants that were kept at –70° C until use. Cytokines and chemokines levels (TNF-α, IL-6, IL-10, IL-16 and RANTES) were measured by ELISA according to the manufacturer’s instructions (R&D Systems).

Figure 1. Effect of culture conditions on the infection of macrophages by R5 or X4 HIV-1 strains. Culture day 7 MDM-5d (■, [) or MDM-MCSF ( , ·) were infected by HIV-1BaL (black symbols) or HIV-1Lai (open symbols) and cultured up to day 21 post-infection. Virus production was assessed by measuring p24 levels in culture supernatants. Data are from one representative experiment out of three.

RESULTS Susceptiblity of monocyte-derived macrophages to HIV-1Lai/X4 isolate As reported, macrophages generated under either condition, i.e., MDM-5d or MDM-MCSF, expressed comparable CXCR4 and CCR5 levels [1]. Here, we analysed the susceptibilty of these two macrophage populations to X4 HIV-1Lai infection. Exposure of MDM-MCSF to 500 TCID50 HIV-1Lai did not result in production of detectable p24 in supernatants (figure 1), even over three to four weeks of culture (data not shown). HIV-1Lai-exposed MDM-5d produced p24 that did not exceed 200 pg/mL (i.e., 500- to 1000fold lower than levels currently observed in HIV1BaL-infected macrophages or in HIV-1Lai-infected T lymphocytes), in keeping with another report [21]. The fact that weak p24 production persisted at the same level through four weeks, a period that did not allow survival of T lymphocytes that should also already have been killed by the virus, indicates that p24 was actually produced by infected macrophages. To ascertain whether macrophages actually produced virus, day-7 HIV-1Lai-infected macrophages were extensively washed to eliminate residual viral particles as well as hypothetical contaminating T lymphocytes, before being cocultured with autologous PHA-activated T lymphocytes. Cocultures with MDM-5d produced then high p24 levels in supernatants, but only very low p24 appeared later on in co-

X4 HIV-1 isolate and macrophages

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MSCF, MDM-5d transmitted vigorously X4 virus to lymphocytes. Analysis of HIV-1 Lai/X4 entry and retrotranscription in macrophages

Figure 2. Virus transmission from macrophages to activated T lymphocytes. MDM-5d (■, [) or MDM-MCSF ( , ·) were infected by HIV-1BaL (black symbols) or HIV-1Lai (open symbols). At day 7 post-infection they were extensively washed to remove all free virions and then cocultured with autologous PHA/IL-2 activated T lymphocytes. Virus production was assessed by measuring p24 levels in culture supernatants. Data are from one experiment of two.

cultures with MDM-MCSF (figure 2). The appearance of p24 was delayed because of the time necessary to its amplification by several rounds of infection in lymphocytes, attesting again to the low replication rate of HIV-1Lai in macrophages. Alternatively, we cannot exclude that lymphocytes stimulate MDM-5d, but not MDM-MCSF, to replicate X4 virus, which may again attest to the replicative capacity of X4 isolates in such macrophages when stimulated. These results indicate that, in contrast to MDM-

To understand the differences between MDM-5d and MDM-MSCF with respect to susceptibility to HIV1Lai, we analyzed the first steps of the HIV replicative cycle described as limiting X4 virus replication in macrophages [7, 21, 27]. Both type of macrophages were exposed to HIV-1Lai or HIV-1BaL for 48 hours and then extensively washed, after which virus entry was evaluated by semi-quantitative endpoint dilution nested PCR as described [3]. As indicated, (figure 3a), HIV-1Lai efficiently entered into both types of macrophages, although 1 Log less efficiently than R5 HIV-1BaL. That HIV-1Lai enters efficiently and to similar levels into both types of macrophages indicates that CXCR4 is functional on both. We then assessed the efficiency of reverse transcription, reported as one limiting step of X4 virus replication in macrophages [21], by comparing the difference between the Log endpoint titers (i.e., the inverse of the last dilution yielding a positive signal) obtained by LTR-gag and R-U5 PCR. The efficacy of HIV-1Lai retrotranscription in macrophages was comparable to that observed with HIV-1BaL, but there was a block of HIV-1Lai retrotranscription in MDM-MCSF, which was 2 Log lower than in MDM-5d (figure 3b).

Figure 3. Nested PCR detection of total HIV DNA in MDM-5d and MDM-MCSF. Culture day-7 macrophages were exposed to HIV-1Ba-L or HIV-1LAI, and cultured for 48 hours. PCR lysates were then prepared, and HIV-1 DNA was amplified by using LTR R-U5 primers to detect early retrotranscripts (a), or LTR-gag primers to detect late retrotranscripts (b). Control amplification was performed in lysates from uninfected macrophages and heat-inactivated virus (data not shown). Endpoint dilution analysis was performed as indicated. Results of one experiment out of three.

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Figure 4. Cytokine production profile of macrophages. Culture day-7 MDM-5d or MDM-MCSF were washed extensively, and cultured further for 48 hours. Cytokines were assessed by ELISA in supernatants. The results are the means of four independent experiments, with values expressed as mean (pg/mL) ± SD. Statistical analysis was performed with the paired Student’s t-test (N = 4); (*) significant P-value.

The cytokine profile of macrophages correlates with susceptibility to HIV-1Lai The above differences between MDM-5d and MDMMCSF may be ascribed at least to variation in culture conditions, which may generate macrophages at different stages of activation. Since cytokines are important regulators of cell activation as well as modulators of HIV infection [17, 18, 22, 26], we analyzed the cytokine profile of the cells, in particular those known to affect HIV-1 infection. We observed that TNF-α, IL-6 and RANTES production was fourto fivefold increased in MDM-5d relative to MDMMSCF (figure 4). In contrast, IL-10 level was sixfold higher in MDM-MCSF than in MDM-5d. IL-16 was produced to similar low levels in both macrophages. The differences in cytokine production together with differences in proteoglycan profiles [1] underline the activation status of the cells, which may condition their susceptibility to HIV-1 X4 isolates. DISCUSSION Macrophages are important targets of HIV-1 in vivo and serve as reservoirs for viral persistence. In fact, macrophages express CD4, CCR5, and CXCR4 [1, 7, 27], but the ability of macrophage chemokine receptors to support HIV-1 entry and infection is both cell- and strain-specific. Actually, it has been described that macrophages are permissive for R5 and dual-tropic R5X4 viruses but not for X4 strains [7, 23, 27]. Recent data showed that this restricted

susceptibility of macrophages to such strains could not be explained by the absence or nonfunctional expression of CXCR4 [1, 7, 24, 25]. In line with these reports our data show that the capacity of X4 strains to infect macrophages depends on the conditions used to obtain and culture macrophages: whereas R5 HIV1Ba-L strongly replicated in MDM-MCSF, X4 HIV1Lai retrotranscription was reduced and no virus production occurred, as already reported [21]. Such a block was confirmed by the absence or very low transmission of virus from X4 HIV-1-exposed MDMMCSF to autologous T lymphocytes, indicating that MDM-MCSF not only do not replicate but also cannot transmit this virus to activated T lymphocytes. These observations are in line with the fact that M-CSF strongly reduced viral reverse transcriptase activity in infected macrophages [12]. In contrast, when macrophages were obtained as MDM-5d, they supported low replication of HIV-1Lai, in line with another report [21]; in this case viral retrotranscription was not dramatically reduced, and we noted efficient viral transmission to T lymphocytes, which may attest for viral replication even at low rates. Of note, we did not observe a real difference of either R5 or X4 virus entry into MDM-5d or MDM-MCSF, which suggests that both CCR5 and CXCR4 are similarly functional on these cells. The activation state of macrophages obtained under either condition may account for the differences observed. We have reported [1] that MDM-5d and MDM-MCSF differed as to proteoglycan cell surface expression, but other factors such as cytokines may also vary with the activation status of the cells. That the capacity of macrophages to support efficient replication of X4 strains seems to depend on postentry signals, which should be necessary for productive replication of these viruses, may be due to the effect of some proinflammatory cytokines or chemokines [6, 17] that modulate HIV replication post-entry. Here we found that MDM-5d produced higher TNF-α amounts than MDM-MCSF. Though TNF-α is considered as the most potent HIV-inducing cytokine that activates NFkB, which is a strong inducer of HIV LTR-mediated transcription [10, 19], one cannot exclude its effect on replication of X4 viruses in MDM-5d. The latter cells also produced more IL-6 than their M-CSF-stimulated counterparts; once again, it is well known that IL-6 alone increases HIV expression primarily by a post-transcriptional

X4 HIV-1 isolate and macrophages

mechanism, but it can also synergize with NFkBinducing cytokines (i.e., TNF-α) to enhance HIV transcription [18]. In contrast, we found that MDMMCSF secreted higher amounts of IL-10 than MDM5d. IL10 is known to exert suppressive activity on the virus cycle in HIV-infected macrophages [22]. These reports support our finding that MDM-MCSF secreted low TNF-α and IL-6 levels. Of note, IL-16, which is known to inhibit virus replication in macrophages [14], was secreted at the same low levels in both types of macrophages. Also, higher levels of RANTES, which is reported to increase infectivity of X4 isolates [11], were detected in cultures of MDM-5d than in those of MDM-MCSF, whereas MIP-1α and MIP-1β were at the same low levels (data not shown). In summary, these data indicate that the capacity of macrophages to replicate X4 strains depended largely on how these cells were cultured and that these conditions correlated with the cytokine and chemokine environment. ACKNOWLEDGEMENTS We thank Birgitta Äsjo (Bergen, Norway) for the gift of HIV-1BaL, and the NIH AIDS Reagent Program (Bethesda, MD) for providing anti-CCR5, -CCR3 and -CXCR4 mAbs. This work was supported by the Agence Nationale de Recherche sur le SIDA (ANRS), Ensemble contre le SIDA - Sidaction, and the Moroccan Programme d’Appui à la Recherche Scientifique (PARS). YB is the recipient of a predoctoral fellowship from ANRS. REFERENCES 1 Amzazi SD, Lylisastigui L, Bakri Y, Rabehi L, Gattegno L, Parmentier M, et al. The inhibitory effect of RANTES on the infection of primary macrophages by R5 human immunodeficiency virus type-1 depends on the macrophage activation state. Virology 1998 ; 252 : 96-105. 2 Balter M. HIV’s other immune-system targets: macrophages. Science 1996 ; 274 : 1464-5. 3 Canque B, Bakri Y, Camus S, Yagello M, Benjouad A, Gluckman JC. The susceptibility to X4 and R5 human immunodeficiency virus-1 strains of dendritic cells derived in vitro from CD34+ hematopoietic progenitor cells is primarily determined by their maturation stage. Blood 1999 ; 93 : 3866-75. 4 Chen JD, Bai X, Yang AG, Cong Y, Chen SY. Inactivation of HIV-1 chemokine co-receptor CXCR-4 by a novel intrakine strategy. Nat Med 1997 ; 3 : 1110-6. 5 Chun TW, Stuyver L, Mizell SB, Ehler LA, Mican JA, Baseler M, et al. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc Natl Acad Sci USA 1997 ; 94 : 13193-7.

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