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The Journal of Immunology, 2002, 168: 6388-6395.
Copyright © 2002 by The American Association of Immunologists

Inhibition of CXCR4-Tropic HIV-1 Infection by Lipopolysaccharide: Evidence of Different Mechanisms in Macrophages and T Lymphocytes1

Alessia Verani2,*, Francesca Sironi*, Antonio G. Siccardi{dagger}, Paolo Lusso* and Donata Vercelli{ddagger}

* Human Virology Unit, DIBIT, San Raffaele Scientific Institute, and {dagger} Department of Biology and Genetics, University of Milan, Milan, Italy; and {ddagger} Respiratory Sciences Center, College of Medicine, University of Arizona, Tucson, AZ 85724


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Bacterial LPS protects primary human macrophages from infection by CCR5-tropic HIV-1 isolates through the release of the CC chemokines RANTES and macrophage inflammatory protein-1{alpha} and -1{beta}. Here, we show that LPS also suppresses infection of macrophages by CXCR4-tropic HIV-1 isolates. A marked down-regulation of both CD4 and CXCR4 expression was associated with this effect. Furthermore, a soluble factor(s) released by macrophages upon LPS treatment inhibited infection with CXCR4-tropic HIV-1 isolate viruses in both macrophages and T lymphocytes. Infection of both cell types appeared to be blocked at the level of viral entry and was independent of stromal cell-derived factor-1, the only known natural ligand of CXCR4. Moreover, the suppressive effect of LPS was unrelated to the release of IFN-{alpha} and -{beta}, macrophage-derived chemokine, leukemia inhibitory factor, or TNF-{alpha}. These results suggest the existence of potent HIV-1 inhibitory factor(s), uncharacterized to date, released by activated cells of the mononuclear phagocytic system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Macrophages play an important role in HIV transmission and propagation in vivo. HIV-infected macrophages represent the predominant cell type infected by HIV in different tissues (1, 2, 3, 4) and may provide a reservoir for persistent infection and virus dissemination. In addition, macrophages modulate immune responses and tissue functions through the release of a large array of secretory molecules. Indeed, HIV-1 may prime macrophages for an enhanced production of cytokines such as IL-1{beta}, TNF-{alpha}, and IL-6 in response to different exogenous stimuli (5, 6). Furthermore, it has been reported that systemic release of TNF-{alpha} and IL-6 by HIV-infected macrophages may contribute to HIV spreading, increased serum Ig levels, and the development of B cell lymphomas, while local release of TNF-{alpha} may be a direct cause of tissue damage (7, 8, 9). The levels of TNF-{alpha} are increased in HIV-1-associated dementia and neuronal dysfunction (10, 11).

Besides the important role that macrophages play in the pathogenesis and natural history of HIV infection, these cells are critically involved in immune responses to bacterial infections. Indeed, LPS, the major constituent of the cell wall in Gram-negative bacteria, activates multiple macrophage effector functions that coordinate the host immune and inflammatory responses, mostly through induction of the secretion of inflammatory cytokines such as TNF-{alpha}, IL-6, and IL-1. The effects of LPS are primarily mediated by the engagement of CD14, a pattern recognition receptor for foreign lipoglycans expressed at high levels on monocytes and macrophages (12, 13, 14), as well as by members of the Toll-like receptor family, which act as signal transducers (15, 16, 17). These molecules belong to a group of nonclonal immune receptors that are highly conserved throughout evolution and play a critical role in nonadaptive (innate) immunity (14).

Patients with symptomatic HIV infection are immunosuppressed, often severely, and therefore become susceptible to superinfection with bacteria. Indeed, LPS may reach significant levels in the blood and liver of these patients and may thus affect HIV replication. We have previously shown that LPS blocks the replication of CCR5-tropic (R5)3 HIV-1 isolates in in vitro cultures (18). LPS affects HIV-1 infection in macrophages as well as, indirectly, in T lymphocytes through the release of soluble suppressive factors, most notably the CC chemokines RANTES, macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), and MIP-1{beta} (18). Because we and others have shown that CXCR4 is a functional coreceptor for HIV-1 infection of human macrophages (19, 20, 21, 22), in this study we investigated the effects of LPS on the replication of the second major biological variant of HIV-1, which uses CXCR4 as a primary coreceptor. In particular, we examined the ability of LPS to modulate HIV infection in monocyte-derived macrophages (MDM) isolated from normal donors. This issue is important because macrophages appear to be major producers of HIV in patients with opportunistic infections (23), and CXCR4-dependent viruses commonly emerge during the advanced stages of HIV infection, when bacterial superinfections most frequently occur. Our results show that LPS inhibits infection of MDM by CXCR4-tropic (X4) HIV-1 isolates through different mechanisms. A marked down-regulation of both CD4 and CXCR4, the essential receptor and coreceptor for HIV entry into target cells, was documented in LPS-treated MDM. Moreover, a soluble factor(s) released upon LPS treatment neutralized infection with CXCR4-dependent viruses not only in macrophages, but also in T lymphocytes, without affecting CD4 or CXCR4 expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

mAbs specific for human CXCR4 (12G5) and CCR5 (2D7) were provided by J. Hoxie and Leukosite (Cambridge, MA), respectively, through the AIDS Reagent Project, National Institute for Biological Standards and Control. mAbs to CD4 (Leu 3A), CD14 (P9), and FITC-conjugated goat anti-mouse IgG and isotype controls were purchased from BD Biosciences (Mountain View, CA). Recombinant IFN-{alpha} (Roferon-A; sp. act., 3.75 U/ng) was a gift from Roche Milano Ricerche (Milan, Italy). IFN-{alpha} concentrations in macrophage supernatants were assessed by ELISA (BioSource, Camarillo, CA). Recombinant TNF-{alpha}, neutralizing goat polyclonal Ab against human leukemia inhibitory factor (LIF; 50% neutralizing dose, 0.04–0.08 µg/ml) and human TNF-{alpha} (50% neutralizing dose, 0.003–0.01 µg/ml), and chicken polyclonal Ab against human MDC (50% neutralizing dose, 0.5–1.5 µg/ml) were obtained from R&D Systems (Minneapolis, MN). LPS from Salmonella minnesota was purchased from Sigma-Aldrich (St. Louis, MO). Polymyxin B sulfate (PMB) was purchased from Calbiochem (La Jolla, CA). The endotoxin content of cell culture reagents was assessed by the Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD) and was <0.125 EU/ml.

Isolation of MDM and HIV-1 infection

PBMC were isolated by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation from buffy coat preparations obtained from healthy donors. The cells were then resuspended in RPMI 1640 (BioWhittaker) supplemented with 10% AB+ serum (Sigma-Aldrich), 20% FCS (Life Technologies), 2 mM glutamine, 50 µg/ml streptomycin, and 100 U/ml penicillin and cultured at a concentration of 1 x 106 cells/cm2 for 5–7 days at 37°C in 12-well tissue culture plates (Nunc, Roskilde, Denmark) in a 1-ml volume. Nonadherent cells were then removed by extensive washing with medium. MDM preparations contained >=90% CD14+ cells, as assessed by immunofluorescence.

MDM were infected with primary HIV-1 isolates that use CCR5 alone (HIV-16088 and HIV-110005), CXCR4 alone (HIV-126, HIV-127, HIV-134, and HIV-1130), or CCR5, CXCR4, and CCR3 interchangeably (HIV-15233). All isolates were characterized for coreceptor usage as previously described (24). MDM were infected with DNase-treated virus (50% tissue culture infectious dose, 50/106 cells). HIV-1 p24 Ag concentrations in culture supernatants were determined by ELISA (18).

For infection, MDM in RPMI 1640/20% FCS were incubated with viral isolates in a total volume of 0.5 ml cell-free viral supernatant. After overnight incubation, unbound virus was removed by extensive washing, fresh medium (1 ml) was added, and cultures were further incubated at 37°C. Supernatants were harvested every 3–4 days for p24 Ag determination.

Isolation of lymphocytes and HIV-1 infection

Normal PBL depleted of monocytes by two cycles of adherence to plastic were activated by incubation with PHA (3 µg/ml; Sigma) for 3 days. The resulting PHA blasts were collected, resuspended at 2.0 x 106 cells/ml in medium containing 10% FCS, supplemented with IL-2 (10 U/ml; Amersham, Little Chalfont, U.K.), and incubated overnight with HIV-1 isolates. Subsequently, free virus was removed by washing twice in RPMI 1640, and cells (1.0 x 106/ml) were cultured in 12-well plates in the presence of IL-2. Culture supernatants were harvested every 3–4 days and tested for the presence of HIV-1 p24 Ag by ELISA.

Preparation of LPS-conditioned and monokine-depleted supernatants

LPS-conditioned supernatants were prepared by incubating cultures of normal uninfected MDM in the presence or the absence of LPS (1 µg/ml). One day later, supernatants were harvested, centrifuged, and stored at -20°C until used. Before use these supernatants were treated with polymyxin B sulfate to neutralize residual LPS activity. Crude supernatants from MDM, unstimulated or stimulated with LPS (1 µg/ml), were depleted of IFN-{alpha} and IFN-{beta} as follows: a mixture of sheep polyclonal Abs to human IFN-{alpha} and IFN-{beta} (at the concentration required to neutralize 1000 U/ml human IFN-{alpha}{beta}) or sheep control IgG (50 µg/ml) were incubated with protein G-Sepharose (Pharmacia Biotech) for 1 h at room temperature. LPS-conditioned supernatants or control supernatants were then incubated with the activated protein G-Sepharose overnight at 4°C, collected, centrifuged, and used immediately. IFN-{alpha} concentrations in macrophage supernatants before and after the subtraction experiment were assessed by ELISA (BioSource).

Immunofluorescence

Expression of CD4, CXCR4, CCR5, and CD14 was detected by indirect immunofluorescence as previously described (18). MDM and lymphocytes in staining buffer (RPMI 1640/10% AB+ serum, containing 0.01% sodium azide) were incubated with specific mAbs or isotype controls for 30 min at 4°C, followed by a 30-min incubation with FITC-conjugated goat anti-mouse IgG Ab. Cells were then extensively washed and fixed in 4% paraformaldehyde. Percentages of positive cells and mean fluorescence intensity were analyzed by FACScan (BD Biosciences) gating on the monocyte or lymphocyte population, as defined by forward and side light scatters.

Semiquantitative PCR for HIV-1 proviral DNA

DNA was extracted from MDM or lymphocytes 14 h postinfection by the salting-out method. PCR was performed using primers 1 and 2II (25) that amplify a 218-bp fragment from the HIV-1 gag gene. Samples were submitted to 50 cycles of amplification (95°C for 1 min, 63°C for 1 min, 72°C for 1 min). PCR products were separated on a 1.8% agarose gel, transferred to a nylon membrane and hybridized with a gag-specific 32P-labeled oligonucleotide (20). To normalize for the amount of DNA contained in each sample, a 441-bp region of the GAPDH gene was amplified using a set of primers previously described (20). Results were calculated as the ratio between the intensities of the HIV-1 and GAPDH bands, as assessed by scanning densitometry on duplicate or triplicate samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Infection of MDM by primary X4 HIV-1 isolates is potently inhibited by LPS treatment

We have previously shown that LPS and supernatants harvested from MDM cultures stimulated with LPS for 24 h (LPS-conditioned) block the replication of R5 HIV-1 isolates in MDM (18). To investigate whether LPS can affect the replication of HIV-1 isolates that use coreceptors other than CCR5 for entry into target cells, we evaluated the effects of LPS treatment on MDM infected with three different primary X4 HIV-1 isolates. Two R5 HIV-1 isolates were used as a control. A dramatic inhibition of both the R5 and the primary X4 HIV-1 isolates was observed in MDM after treatment with LPS (Table IGo). Moreover, infection with a promiscuous HIV isolate, which can use CCR5, CXCR4, and CCR3 interchangeably, was also inhibited. Suppression by LPS was specific because it was completely reversed by addition of PMB, an antibiotic that binds and neutralizes LPS. LPS affected the replication of X4 HIV-1 isolates in a dose-dependent fashion, showing a potent inhibitory effect (>80–95%) at concentrations from 10 ng/ml to 1 µg/ml; inhibition was still apparent (>30%) when LPS was added at 100 pg/ml. These results indicate that the LPS-dependent blockade of HIV-1 replication has a broad spectrum of action, affecting viruses with different coreceptor tropism.


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  		Table I. LPS inhibits the replication of different HIV-1 isolates in MDM regardless of coreceptor usage1

 
LPS inhibits the entry of X4 HIV-1 isolates into MDM

To determine which step in MDM infection with X4 HIV-1 isolates is inhibited by LPS, we used a semiquantitative PCR to assess the level of proviral DNA synthesized after infection. Samples were collected 14 h after exposure of MDM to the virus in the presence or the absence of LPS (1 µg/ml). As a control, HIV-1 p24 Ag secretion was measured 7 days after infection. Table IIGo shows that addition of LPS dramatically decreased the level of proviral DNA in MDM cultures infected with all the primary X4 isolates tested at 14 h postinfection, suggesting a block at the level of viral entry. Extracellular p24 Ag levels in the same cultures were reduced 7 days postinfection. As expected based on our previous findings (18), entry of an R5 viral isolate was also inhibited. Together these data indicate that LPS stimulation results in a blockade of HIV-1 entry into MDM regardless of the viral coreceptor used.


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  		Table II. LPS inhibits the entry of X4 HIV-1 isolates into MDM1

 
LPS down-regulates the expression of the HIV-1 receptor and coreceptors on MDM

Because PCR analysis provided evidence for an LPS-dependent inhibition of the entry of X4 HIV-1 isolates into MDM, we investigated the effects of LPS on the expression of CD4 and CXCR4, the receptor and coreceptor, respectively, used by these viral isolates. To this purpose, MDM were incubated with LPS (1 µg/ml) for 24 h and then assessed for CD4 and CXCR4 expression by indirect immunofluorescence followed by flow cytometric analysis. Expression of membrane CCR5, which was previously shown to be down-regulated by LPS (26, 27) and/or LPS-induced CC chemokines (28, 29), was tested in parallel. Fig. 1Go shows the results obtained in a representative experiment. LPS treatment induced a dramatic down-regulation of both CD4 and CXCR4 expression in MDM. Likewise, CCR5 became barely detectable on the surface of LPS-treated MDM, while CD14 expression was enhanced (data not shown), consistent with our previous report (18). These results suggest that down-regulation of both CD4 and CXCR4 may underlie the LPS-dependent blockade of X4 HIV-1 entry into MDM.



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  		FIGURE 1. LPS stimulation down-regulates cell surface expression of CD4 and CXCR4 in MDM. MDM from healthy donors were cultured in the presence or the absence of LPS (1 µg/ml) for 24 h. The expression of CD4 and CXCR4 was then assessed by indirect immunofluorescence using mAb Leu 3A and 12G5, respectively, and unrelated isotype controls. Leu 3A and 12G5 stainings are shown as heavy lines. Isotype stainings are shown as dotted lines. The data are representative of four independent experiments with similar outcomes. The percentage of positive cells is indicated for each histogram.

 
LPS-induced inhibition of X4 HIV-1 isolates is mediated by soluble factors active on macrophages, T lymphocytes, and neoplastic T cell lines

To investigate whether the inhibitory effects of LPS are mediated by soluble factors, MDM cultures were infected with X4 HIV-1 isolates in the presence or the absence of LPS-conditioned supernatants. Fig. 2GoA shows that LPS markedly decreased p24 Ag release in MDM cultures infected with three different primary X4 HIV-1 isolates. Inhibition by LPS was fully neutralized by addition of PMB. Strikingly, supernatants from polymyxin-treated LPS-stimulated MDM inhibited HIV-1 replication as efficiently as LPS itself.



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  		FIGURE 2. LPS-induced inhibition of X4 HIV-1 isolates replication is mediated by soluble factors active on macrophages, T lymphocytes, and tumor T cell lines. MDM (A) and PBL (B) from healthy donors were infected in vitro with different X4 primary isolates (HIV-126, HIV-127, HIV-1130 for MDM; HIV-126, HIV-127, HIV-134 for PBL) in the presence or the absence of LPS (1 µg/ml) or LPS-conditioned supernatants (1/3, v/v; in the presence of 15 µg/ml PMB). The tumor T cell lines PM1 and MT-2 (C and D, respectively) were infected with the T cell line-adapted strain HIV-1IIIB in the presence or the absence of LPS-conditioned supernatants.

 
Human T lymphocytes, which constitute the major target of HIV-1 infection in vivo together with macrophages, are known to be unresponsive to LPS. However, these cells closely interact with macrophages and are thus exposed to their secretory products, including those induced by LPS during bacterial infections. We therefore asked whether infection of T lymphocytes with X4 primary isolates could also be affected by the addition of LPS-conditioned supernatants. Fig. 2GoB shows that soluble MDM-derived factors strongly suppressed the infection of primary T cells with three different X4 isolates. By contrast, LPS had no direct inhibitory effect, confirming that T cells are unresponsive when directly challenged with this bacterial product. LPS-conditioned MDM supernatants also effectively suppressed the replication of X4 isolates in the tumor T cell lines PM1 and MT-2 (Fig. 2Go, C and D, respectively). These results demonstrate that soluble factors released by MDM upon stimulation with LPS potently suppress the replication of X4 isolates not only in macrophages, but also in primary or immortalized T cells.

IFN-{alpha} secreted by LPS-stimulated MDM inhibits infection by X4 HIV-1 isolates in both macrophages and T lymphocytes

Despite the finding that LPS-dependent inhibition of HIV-1 occurs at the level of viral entry into MDM, the possibility of a concomitant effect of LPS and/or LPS-released mediators at later stages in the viral life cycle cannot be ruled out. Addition of LPS is known to induce the release of several monokines, including IFN-{alpha}, a factor that potently inhibits HIV-1 replication by interfering with postentry events (30, 31). We therefore asked whether secretion of IFN-{alpha} by LPS-stimulated MDM could contribute to the LPS-dependent suppression of HIV-1 replication in macrophages and/or T cells. To this purpose, we first assessed the IFN-{alpha} concentration in the supernatants of MDM cultures treated with LPS for 24 h. In three independent experiments, IFN-{alpha} levels ranged between 0.2 and 3 ng/ml. Infection with HIV-1 did not result in a significant augmentation of IFN-{alpha} release (data not shown). To ascertain whether these doses of IFN could be responsible for the inhibitory effect of LPS-treated MDM supernatants, we tested the activity of recombinant IFN-{alpha}. Table IIIGo shows that high doses of recombinant IFN-{alpha} (1000 U/ml; i.e., ~270 ng/ml) strongly inhibited HIV replication in both cell types, while a more physiological concentration (100 U/ml; i.e., ~27 ng/ml) induced a less efficient and consistent suppression. No inhibitory activity was documented at a concentration of 10 U/ml or less (data not shown). By contrast, LPS-conditioned supernatants virtually abolished the replication of all the HIV-1 X4 isolates tested. These results suggest that secretion of IFN-{alpha} may be one of the mechanisms of HIV-1 blockade by LPS, but it does not seem to play a dominant role.


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  		Table III. IFN-{alpha} blocks the replication of X4 HIV-1 isolates in both macrophages and T lymphocytes1

 
IFN-{alpha} released by LPS-stimulated MDM is not the main mediator of HIV-1 suppression

To further elucidate the role of IFN-{alpha} in the LPS-induced inhibition of X4 HIV-1 isolate expression in macrophages and T lymphocytes, supernatants from LPS-stimulated MDM were first depleted of type I IFNs (i.e., IFN-{alpha} and IFN-{beta}) by adsorption on Sepharose-bound antisera and then tested for their residual HIV-suppressive capacity. Depletion of IFNs was successful (>95%) as determined by ELISA. Table IVGo shows that Ab-mediated depletion of type I IFNs only modestly neutralized the inhibitory activity of LPS-conditioned supernatants (from 98.5 to 89.3% on PBL; from >97 to 89.4% in MDM). These results suggest that type I IFNs are not the major factors mediating the suppression of X4 HIV-1 replication induced by LPS.


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  		Table IV. IFN-{alpha} released by LPS-stimulated MDM is not the principal mediator of the suppression of X4 HIV-1 replication1

 
Expression of CD4 and CXCR4 on T lymphocytes is not affected by LPS-conditioned supernatants or IFN-{alpha}

To investigate whether LPS suppresses HIV-1 through similar mechanisms in MDM and T lymphocytes, we tested the expression of CD4, CCR5, and CXCR4 in T cells after treatment with LPS-conditioned MDM supernatants or IFN-{alpha}. Fig. 3Go shows the results obtained in a representative experiment. Immunofluorescence analysis revealed that the expression of CD4, CXCR4, and CCR5 was readily detectable in unstimulated T cells. Neither IFN-{alpha} nor LPS-conditioned supernatants affected CD4 and CXCR4 expression. By contrast, the LPS supernatants, but not IFN-{alpha}, induced down-modulation of the CCR5 receptor, most likely through LPS-released CC chemokine ligands. Surface expression of CD4, CCR5, and CXCR4 was not modulated by treatment with LPS or polymyxin B alone (data not shown). These results showed that inhibition of the replication of X4 isolates in T lymphocytes by LPS-conditioned supernatants cannot be ascribed to a down-regulation of the HIV-1 receptor or coreceptors. Thus, different mechanisms seem to underlie inhibition of HIV replication in MDM and T cells.



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  		FIGURE 3. Effects of LPS-conditioned supernatants and IFN-{alpha} on the expression of CD4, CXCR4, and CCR5 in primary human T lymphocytes. PBL from healthy donors were stimulated with LPS-conditioned supernatants (1/3, v/v; in the presence of 15 µg/ml PMB) or IFN-{alpha} (1000 U/ml). After 1 day of culture, CD4, CCR5, and CXCR4 expression was assessed by indirect immunofluorescence, using mAb Leu 3A, 2D7, and 12G5, respectively. Leu 3A, 2D7, and 12G5 staining are shown as heavy lines; isotype controls are shown as dotted lines. The data are representative of three independent experiments.

 
A suppressive factor(s) released by LPS-stimulated MDM, but not IFN-{alpha}, blocks HIV-1 entry into T lymphocytes

The finding that treatment with LPS-conditioned supernatants failed to down-modulate the expression of CD4 and CXCR4 in T cells suggested that different inhibitory mechanisms may be involved. Therefore, we tested whether HIV-1 entry was affected by LPS-derived soluble suppressive factors. To this purpose, we assessed the levels of proviral DNA in T lymphocyte cultures incubated for 14 h with primary X4 isolates in the presence or the absence of LPS-conditioned supernatants or IFN-{alpha}. Table VGo shows that LPS-conditioned supernatants markedly reduced the levels of HIV-1 proviral DNA 14 h postinfection. By contrast, the level of viral entry was comparable for PBL-infected cultures treated with or without IFN-{alpha}. However, both treatments inhibited p24 Ag secretion on day 10 of infection. These results confirmed that IFN-{alpha} inhibits HIV-1 replication by interfering with postentry events, suggesting that LPS treatment interferes with HIV-1 entry in T lymphocytes despite the lack of effect on the expression of the receptor and coreceptor for X4 isolates.


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  		Table V. Entry of CXCR4-dependent isolates into primary T lymphocytes is inhibited by LPS-conditioned supernatants1

 
LPS-induced HIV-1 suppression is not mediated by secretion of macrophage-derived chemokine (MDC), LIF, or TNF-{alpha}

Several macrophage-derived cytokines have been reported to suppress HIV-1 replication, at least in some experimental systems, in both MDM and PBL, including MDC (32), LIF (33), and TNF-{alpha} (34, 35). We tested whether the release of these monokines could be involved in the LPS-induced inhibition of HIV-1 expression. To this purpose, neutralizing anti-MDC, anti-LIF (Fig. 4GoA) or anti-TNF-{alpha} (Fig. 4GoB) Abs were added to PBL cultures infected with X4-HIV-1 primary isolates in the presence of LPS-conditioned supernatants. Addition of neither Ab reversed the suppression of HIV-1 replication caused by LPS supernatants, thus ruling out a major role of MDC, LIF, or TNF-{alpha} in the observed HIV-1 suppression. Control Abs or rTNF-{alpha} (10 ng/ml) alone had no effect on p24 Ag secretion.



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  		FIGURE 4. LPS-induced HIV-1 suppression is not mediated by secretion of MDC, LIF, or TNF-{alpha}. PBL from healthy donors were infected in vitro with the X4 primary isolate HIV-126 in the presence or the absence of LPS-conditioned supernatants (1/3, v/v; in the presence of 15 µg/ml PMB) treated with or without neutralizing anti-MDC (A), anti-LIF (A) or anti-TNF-{alpha} (B) Abs (5 µg/ml). Culture supernatants were harvested on day 5 and tested for p24 Ag secretion by ELISA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Multiple lines of evidence highlight the central role played by macrophages in HIV-1 disease. Unlike HIV-infected T cells, macrophages appear to be relatively resistant to the cytopathic effects of the virus and thus have been suggested to serve as a reservoir for persistent infection and virus dissemination (36). HIV-infected macrophages are found in the brain, lungs, lymph nodes, and skin of seropositive patients and are likely to contribute directly to HIV-induced immunosuppression, CNS dysfunctions, and pulmonary complications. In addition, HIV-1 infection of human macrophages is associated with increased apoptosis of CD8+ T lymphocytes (37), cells that are known to play a major role in the control of infection of persisting viruses through their cytotoxic activity and the release of soluble HIV suppressive factors. The persistence in the body of infected macrophages represents a key challenge for therapeutic efforts aimed at eradicating HIV infection (38, 39). However, it is increasingly clear that macrophages may also play a defensive role against HIV, as demonstrated by the potent inhibitory effect of the CC chemokines RANTES, MIP-1{alpha}, and MIP-1{beta} on HIV replication (40), which are vigorously secreted not only by CD8+ T lymphocytes, the cells traditionally implicated in HIV suppression (40), but also by activated monocyte/macrophages (41).

We previously reported that CC chemokines released by LPS-stimulated human macrophages potently suppress infection by R5 HIV-1 isolates in both macrophages and T cells (18). In the present study we showed that a soluble suppressive factor(s) released by macrophages upon LPS treatment is also able to neutralize infection with primary X4 HIV-1 isolates, the second major biological variant of HIV-1, and that this inhibition occurs in both macrophages and T lymphocytes. Infection of both cell types appears to be blocked primarily at the level of viral entry. For this reason, an LPS-induced suppressive factor(s) appears to be distinct from the to date unidentified CD8+ T cell antiviral factor (42), which has been reported to be selectively produced by CD8+ T cells and to inhibit replication of X4 strains of HIV-1 at the level of viral transcription by suppressing long terminal repeat-driven viral expression (43). Interestingly, however, our data suggest that different inhibitory mechanisms operate in these two cell lineages. HIV-1 suppression in macrophages was associated with a loss of expression of both the HIV receptor (CD4) and coreceptor (CXCR4) for X4 HIV-1. By contrast, the surface expression of these molecules was not affected in T lymphocytes, while CCR5 was down-modulated. The mechanisms underlying the down-modulation of CD4 and CXCR4 remain to be clarified. In this regard it has been demonstrated that bacterial endotoxin induces a marked reduction in the cell surface levels of CCR2 in human monocytes, which is dependent upon tyrosine kinase activation and serine proteinase-mediated receptor degradation (44). Moreover, LPS was shown to mediate a direct and sustained down-regulation of CCR5 through inhibition of recycling and/or trafficking of the receptors to the cell membrane (27). In addition, CXCR4 has been recently identified, along with three additional cell surface proteins distinct from CD14 and the Toll-like receptors, as an LPS-binding molecule (45).

Because macrophages do not secrete stromal cell-derived factor-1 (46) (A. Verani, S. Polo, P. Lusso, and D. Vercelli, unpublished observations), the only known natural ligand for CXCR4, LPS-induced suppression of X4 HIV-1 isolates has to be ascribed to other soluble mediators. Among the possible candidates are type I IFNs. However, we found that inhibition of HIV-1 was unrelated to the release of IFN-{alpha} and -{beta}. Indeed, unlike LPS-conditioned supernatants, rIFN-{alpha} did not reduce proviral DNA levels at early times postinfection; moreover, depletion of IFN-{alpha} and -{beta} from LPS-conditioned supernatants did not significantly reduce their inhibitory potential in macrophages or T cells.

Additional factors that were reported to block the replication of both R5 and X4 HIV isolates in PBMC, at least in some experimental systems, are the CC chemokine MDC (32) and the cytokines LIF (33) and TNF-{alpha} (34, 35). An enhanced secretion of all these molecules by macrophages was reported upon LPS stimulation (47, 48, 49, 50). Nevertheless, in our hands, addition of TNF-{alpha} did not affect the replication of X4 primary HIV-1 isolates. Moreover, although the mechanism by which MDC may inhibit both R5 and X4 isolates remains elusive (51, 52, 53) (A. Verani and D. Vercelli, unpublished observations), MDC has been reported to affect postentry steps in the HIV-1 life cycle (53) unlike LPS-derived factors. Finally, supernatants from LPS-stimulated MDM cultures depleted of MDC, TNF-{alpha}, or LIF were not capable of neutralizing the inhibitory activity of the supernatants. Thus, it is unlikely that the release of these molecules may account for a significant proportion of the LPS-dependent HIV-1 inhibitory activity observed in our experiments.

In conclusion, our results clearly illustrate the dual role that macrophages play in HIV infection, as they sustain long term HIV persistence and replication, but at the same time can dramatically suppress the spread of HIV to both MDM and T cells following activation by bacterial pathogens. The observation that soluble factors produced by LPS-treated macrophages inhibit the entry of X4 HIV-1 isolates strongly suggests the existence of a novel soluble suppressive factor(s) uncharacterized to date. The identification of this putative HIV-suppressive molecule(s) will have significant implications for our understanding of the mechanisms of HIV control in vivo as well as for the development of novel therapeutic approaches.


   Acknowledgments
 
We thank Dr. Gabriella Scarlatti for kindly providing the primary HIV-1 isolates used in this paper.


   Footnotes
 
1 This work was supported by the AIDS Program, Istituto Superiore di Sanità, Rome, Grant 40C.91 (to A.V.). Back

2 Address correspondence and reprint requests to Dr. Alessia Verani, Human Virology Unit, DIBIT, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy. E-mail address: alessia.verani{at}hsr.it Back

3 Abbreviations used in this paper: R5, CCR5-tropic; LIF, leukemia inhibitory factor; MDC, macrophage-derived chemokine; MDM, monocyte-derived macrophage; MIP-1{alpha}, macrophage inflammatory protein-1{alpha}; PMB, polymyxin B sulfate; X4, CXCR4-tropic. Back

Received for publication November 16, 2001. Accepted for publication April 23, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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