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Journal of Virology, November 2001, p. 10738-10745, Vol. 75, No. 22
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.22.10738-10745.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Induction of Rapid and Extensive beta -Chemokine Synthesis in Macrophages by Human Immunodeficiency Virus Type 1 and gp120, Independently of Their Coreceptor Phenotype

Wonkyu Choe, David J. Volsky, and Mary Jane Potash*

Division of Molecular Virology, St. Luke's-Roosevelt Hospital Center, Columbia University, New York, New York 10019

Received 12 May 2001/Accepted 17 August 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Human immunodeficiency virus type 1 (HIV-1) interacts with its target cells through CD4 and a coreceptor, generally CCR5 or CXCR4. Macrophages display CD4, CCR5, and CXCR4 that are competent for binding and entry of virus. Virus binding also induces several responses by lymphocytes and macrophages that can be dissociated from productive infection. We investigated the responses of macrophages to exposure to a series of HIV-1 species, R5 species that productively infect and X4 species that do not infect macrophages. We chose to monitor production of several physiologically relevant factors within hours of treatment to resolve virally induced effects that may be unlinked to HIV-1 production. Our novel findings indicate that independently of their coreceptor phenotype and independently of virus replication, exposure to certain R5 and X4 HIV-1 species induced secretion of high levels of macrophage inflammatory protein 1alpha (MIP-1alpha ), MIP-1beta , RANTES, and tumor necrosis factor alpha. However two of the six R5 species tested, despite efficient infection, were unable to induce rapid chemokine production. The acute effects of virus on macrophages could be mimicked by exposure to purified R5 or the X4 HIV-1 envelope glycoprotein gp120. Depletion of intracellular Ca2+ or inhibition of protein synthesis blocked the chemokine induction, implicating Ca2+-mediated signal transduction and new protein synthesis in the response. The group of viruses able to induce this chemokine response was not consistent with coreceptor usage. We conclude that human macrophages respond rapidly to R5 and X4 envelope binding by production of high levels of physiologically active proteins that are implicated in HIV-1 pathogenesis.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The complex interactions of human cells with human immunodeficiency virus type 1 (HIV-1) include effects restricted to productive infection and other responses that extend beyond active viral replication. Among the events following viral exposure that may be unrelated to infection, the greatest effects have been attributed to the envelope glycoprotein, gp120. In early studies, gp120 was shown to kill rodent neurons through a Ca2+-dependent pathway (10). By binding CD4, gp120 was found to activate protein kinase p56lck and thus induce translocation of NF-kappa B into the nucleus (34). Recent studies revisiting cytopathogenicity have demonstrated that gp120 can initiate apoptosis in multiple cell types (48). In particular, primary macrophages exposed to gp120 display membrane tumor necrosis factor alpha (TNF-alpha ) and trigger gp120-dependent apoptosis in bystander cells through TNF receptors (18). The latter studies reveal an apparent paradox regarding macrophage-gp120 interactions. Macrophages display both major HIV-1 coreceptors CCR5, a beta -chemokine receptor, and CXCR4, an alpha -chemokine receptor (45, 47). They are highly susceptible to viruses that utilize CCR5 for entry, but they are generally resistant to productive infection by laboratory-adapted virus species that are restricted to CXCR4 (32, 39). They respond to laboratory-adapted X4 HIV-1 and their envelope glycoproteins by Ca2+ uptake (27), by secretion of unidentified neurotoxins (16), by apoptosis (48), and by induction of apoptosis in neighboring cells (18). We showed that X4 viruses that do not replicate in macrophages still enter cells and undergo the early phases of virus replication (19, 36). More recent studies demonstrated that macrophage CXCR4 is competent to mediate virus entry and that some primary X4 HIV-1 species productively infect macrophages (40). R5 HIV-1 or envelope can also induce signal transduction, secretion of neurotoxins, and activation of ion channels in macrophages (3, 20, 48). These findings suggest that ligation of CD4 and CCR5 or CXCR4 on macrophages by HIV-1 envelope is not sufficient to predict subsequent completion of the viral life cycle or activation of cellular responses.

In the present work we focused upon acute effects of virus exposure to investigate potentially protective responses of macrophages to HIV-1 that can be dissociated from productive infection. Discrimination of effects unlinked to virus production was achieved by four approaches. First, we tested the effects of exposure of macrophages to six X4 HIV-1 species that do not productively infect macrophages (1, 11, 35, 41, 44), as well as six R5 species and one R5/X4 HIV-1 species that productively infect (8, 14, 15, 24, 26, 44). Second, we tested responses to isolated gp120 of both coreceptor phenotypes. Third, we monitored responses 6 to 24 h after virus exposure, which is well before the peak of infection of macrophages, about 2 weeks later. Finally, we evaluated responses in the presence and absence of inhibitors of HIV-1 infection. We measured production of a set of secreted proteins implicated in several phases of HIV-1 disease. Among these are certain beta -chemokines that block HIV-1 infection in vitro (27) and are elevated in some exposed but uninfected individuals (46). However, these factors also have been shown to stimulate HIV-1-infected T cells to enhanced viral replication in culture (22). In addition, we tested production of TNF-alpha , one of the primary inflammatory cytokines produced by HIV-1-infected cells, which also can be elevated in the brains of some HIV-1-infected persons (17, 43). We have compared the abilities of multiple species of HIV-1 to induce primary human macrophages to produce macrophage inflammatory protein 1alpha (MIP-1alpha ), MIP-1beta , macrophage chemotactic protein (MCP-1), RANTES, and TNF-alpha . In novel findings we report that within hours of exposure to HIV-1 or viral gp120, macrophages secreted very high levels of several chemokines and TNF-alpha . Two of six R5 viruses and three of six X4 viruses tested failed to induce this response, and neutralizing antibodies or soluble CD4 failed to block this response, indicating that binding CD4 and either CCR5 or CXCR4 was insufficient for induction. By contrast, all the R5 HIV-1 species induced beta -chemokine synthesis at the peak of viral infection, as previously reported (37, 42). We conclude that a major immediate response of macrophages to either R5 or X4 HIV-1 exposure is secretion of high levels of beta -chemokines and TNF-alpha . Such secretion may be seen as part of the innate immune response eliciting lymphocyte migration (13) to a viral source to establish an antigen-specific response prior to major viral spread.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cells and viruses. Human monocytes were prepared from peripheral blood mononuclear cells of HIV-1- and hepatitis B virus-negative donors by countercurrent centrifugal elutriation. Monocytes were >98% pure by Ham56 and CD68 staining. Monocytes were allowed to adhere and differentiate to macrophages (MDM) at a concentration of 2.5 × 105 cells/well in Dulbecco's Modified Eagle Medium (Sigma, St. Louis, Mo.) with 10% endotoxin-free, heat-inactivated human serum, 10% giant cell tumor conditioned medium (Sigma), 2 mM glutamine, and antibiotics. Cells were cultured for 7 days prior to infection or stimulation. The following HIV-1 species were used in this study: ADA (R5 [15]), BaL (R5 [14]), JR-CSF (R5 [24]), JR-FL (R5 [24]), Lai (X4 [33]), NDK (X4 [11]), NL4-3 (X4 [1]), NLHXADA-GP (R5 [44]), NLHXDADA-PG (X4 [44]), Yu2 (R5 [26]), Z6 (X4 [41]), IIIB (X4 [35]), and 89.6 (R5/X4 [8]). Viral stocks were prepared by either proviral DNA transfection of 293T cell (pYu2, pGP, pNDK, pNL4-3, pZ6, p89.6, pLai.2, and pPG), by cell-free virus infection of macrophages (ADA, BaL, JR-FL, and JR-CSF) or by culturing of chronically infected H9-HTLV-IIIB cells (35). ADA, JR-FL, and BaL were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program; ADA and BaL were also obtained from H. Gendelman. Viral stocks were concentrated by high-speed centrifugation (12,000 × g, 2 h, 4°C) and resuspended in phosphate-buffered saline to rule out medium effect for future experiments and were frozen at -80°C until use. Viral stocks were quantified for p24 antigen level using an HIV Ag kit (Coulter, Hialeah, Fla.) according to the manufacturer's instructions.

HIV-1 and gp120 exposure. MDM cells were differentiated for 7 days in differentiation medium prior to infection, and then medium was replaced with maintenance medium (DMEM with 10% endotoxin-free fetal bovine serum, 2 mM glutamine, and antibiotics). Cells were infected overnight with HIV-1 at a dose of 0.2 pg of p24 per cell or were cultured in the presence of purified HIV-1 envelope gp120 at a 20 nM concentration, and supernatants were sampled for assay of chemokines and TNF-alpha . gp120s used for this experiment include BaL, CM235 (R5 [28]), IIIB (X4 [38]), MN (X4 [38]), and SF2 [R5/X4 [25]), which were obtained from the AIDS Research and Reference Reagent Program. Virus stocks and gp120 preparations were screened for endotoxin contamination using the E-TOXATE kit (Limulus Amebocyte Lysate; Sigma) and found to be negative (<0.06 EU per ml).

ELISA. beta -chemokines MIP-1alpha , MIP-1beta , MCP-1, and RANTES and cytokine TNF-alpha were measured by using the Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, Minn.) according to the manufacturer's instructions. Macrophage cultures in triplicate in 24-well plates were exposed to either infectious species of HIV-1 or purified HIV-1 gp120, and culture supernatants were sampled for the ELISA assay. Experiments were repeated two to five times with cells derived from different donors. Macrophages were stimulated with 1 µg of lipopolysaccharide (LPS) (Sigma)/ml to mimic the maximum immune stimulation.

Treatment with inhibitors of HIV-1 replication. Fully differentiated macrophages were pretreated with 1 µM 2',3'-dideoxycytidine (ddC) (Sigma) for 12 h followed by virus infection (0.2 pg of p24/cell) in the presence of ddC. Supernatants were collected for the MIP-1alpha assay at 6 h. Alternatively, fully differentiated macrophages were pretreated with 20-µg anti-CCR5 (45523, 2D7, and 45549) and anti-CXCR4 (44717, 44708, and 12G5) (except 2D7; 10 µg/ml) at 4°C for 2 h, and then cells were infected with ADA and IIIB (0.04 pg of p24/cell) in the presence of antibodies; at 3 h supernatants were harvested for the MIP-1alpha assay. For the soluble CD4 neutralization assay, viruses were preincubated with 10 µg of sCD4/ml for 30 min at 37°C, and then cells were infected with viruses (0.2 pg of p24/cell) in the presence of sCD4; supernatants were collected at 6 h for the MIP-1alpha assay.

Treatment with biochemical inhibitors and cytotoxicity assay. Intracellular Ca2+ chelator MAPTAM [1,2-bis(o-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetoxymethyl ester] (Calbiochem, San Diego, Calif.) with concentrations up to 6 µM and translational inhibitor cycloheximide (Sigma) with various concentrations (from 10 to 100 µM) were used to pretreat macrophages prior to exposure to ADA and human T-lymphotropic virus type IIIB (HTLV-IIIB). Macrophages were exposed to viruses for 5 h, and culture supernatants were assayed for MIP-1alpha by ELISA. Cytotoxic effects of MAPTAM and cycloheximide were measured by lactate dehydrogenase (LDH) release using the CytoTox 96 Assay (Promega, Madison, Wisc.) following the manufacturer's instructions. The extent of cytotoxicity was calculated using the following formula: percent cytotoxicity = 100(c - a)/(b - a), where a is spontaneous LDH release from control macrophage, b is maximum LDH release from lysis buffer-treated macrophage, and c is LDH release from virus-exposed macrophages with various concentrations of MAPTAM or cycloheximide.

Electrophoresis and immunoblot. Purified gp120s (0.5 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4 to 15% precast gradient gel; Bio-Rad, Hercules, Calif.) and transferred by electroblotting to nitrocellulose filters (10 V, 30 min). Blots were incubated with sheep anti-gp120 (1:10,000; NIH AIDS Research and Reference Reagent Program) for 1 h at room temperature. Blots were then washed and incubated with horseradish peroxidase-conjugated anti-sheep immunoglobulin G (1:5,000; DAKO, Carpinteria, Calif.) for 1.5 h. Immunoreactive proteins were visualized with luminofor solution (100 mM Tris-HCl [pH 8.5], 2.5 mM Luminol, 400 µM p-Coumaric acid, 1:1,800 dilution of 30% hydrogen peroxide).


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

HIV-1 induction of beta -chemokine and TNF-alpha by primary macrophages is strain specific, rapid, and unlinked to productive infection. To investigate activation of macrophages, we selected a panel of HIV-1 species, some of which replicate in macrophages and others of which do not. Consistent with previous studies, the R5 viruses ADA, BaL, JR-CSF, JR-FL, NLHXADA-GP, YU-2, and R5/X4 89.6 all productively infected macrophages, while the X4 viruses HTLV-IIIB, Lai, NL4-3, NLHXADA-PG, NDK, and Z6 did not (data not shown). We then tested supernatants from some infected macrophages for levels of MIP-1alpha , a beta -chemokine essential for certain antiviral immune responses (9) which shares with HIV-1 its cell surface receptor, CCR5 (2). Supernatants were collected 24 h after viral exposure to assay immediate macrophage responses and 2 weeks after exposure to assay responses near the peak of viral expression. Within 24 h, ADA, BaL, JR-CSF, HTLV-IIIB, and NDK induced high levels of MIP-1alpha production by macrophages, from 14,000 to more than 45,000 pg per ml (Fig. 1A). JR-FL, YU-2, NL4-3, and Z6 failed to induce macrophage responses. By contrast, 2 weeks after exposure, all viruses that productively infect macrophages, ADA, BaL, JR-CSF, JR-FL, and YU-2, induced MIP-1alpha , but no X4 virus induced a response (Fig. 1B). Dual-tropic 89.6 also induced MIP-1alpha at the peak of infection (data not shown). The latter responses are consistent with previous reports (37, 42). However the immediate response of macrophages to HIV-1 exposure is novel. In addition, the panel of viral species competent to elicit this response does not conform to previous classifications based on tropism or coreceptor utilization. We repeated HIV-1 exposure to macrophages from several different donors and measured chemokine levels 24 h after treatment (Fig. 1C). With one exception, the classification of virus species was reproducible. The R5 species ADA, NLHXADA-GP, and X4 species HTLV-IIIB and Lai were active; R5 YU-2 and X4 NL4-3 and Z6 were inactive. X4 NDK was moderately active in MIP-1alpha induction. However, cells from two donors responded to ADA and HTLV-IIIB by MIP-1alpha production but failed to respond to BaL. We are investigating the source of this discrepancy.


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  		FIG. 1.   R5 and X4 HIV-1 species activate macrophages to produce beta -chemokine MIP-1alpha . MDM cells were infected with R5 (ADA, BaL, JR-CSF, JR-FL, and Yu2) and X4 (IIIB, NDK, NL4-3, and Z6) HIV-1 species, and supernatants were harvested for assay of MIP-1alpha at 24 h (A) and 14 days (B) after infection. (C) MDM cells from different donors were infected with the indicated HIV-1 species, and 24 h after infection, cell supernatants were harvested for assay of MIP-1alpha levels. Numbers in parenthesis are numbers of different donors (numerator) or independently prepared viruses (denominator). (D) Macrophages were infected with the HIV-1 strain ADA, IIIB, Yu2, or PG at the doses indicated, and 24 h after infection, supernatants were harvested for assay of MIP-1alpha . Data represent means ± standard deviations for multiple experiments in triplicate (A, B, and D). Box plot (C) symbols indicate 50% (line inside the box), 75% (box extents), 90% (capped bars), and 95% (symbol marks above or below capped bars) data.

To determine if the strain differences that we observed were dose related, we exposed macrophages to graded doses of ADA and HTLV-IIIB, which induce MIP-1alpha , and YU-2 and NLHXADA-PG, which do not (Fig. 1D). The MIP-1alpha response was dose dependent for the active species; increasing viral doses up to 0.1 pg of p24 per cell increased the amount of MIP-1alpha produced. In contrast, YU-2 and NLHXADA-PG failed to induce MIP-1alpha at any dose, the highest being a fourfold excess over that inducing the peak response by active species. We conclude that the strain specificity in MIP-1alpha induction is not a function of viral dose.

The observation that certain X4 HIV-1 species that do not productively infect macrophages were able to induce beta -chemokine synthesis suggested that this rapid response is unlinked to virus replication. To directly test this proposition, we exposed macrophages to ddC, an inhibitor of reverse transcription (31) or we pretreated viruses with recombinant soluble CD4, which inhibits virus entry into macrophages (29) prior to exposure of cells to virus. MIP-1alpha levels were measured after 6 h (Fig. 2A and B). Under conditions where HIV-1 infection of macrophages was inhibited (29), neither ddC nor soluble CD4 affected the induction of MIP-1alpha production. These findings clearly indicate that HIV-1 replication is not required for the MIP-1alpha response we observe. The ability of HIV-1 to induce synthesis of MIP-1alpha despite neutralization with soluble CD4 suggested that the major receptors for HIV-1 do not play a role in this response. To investigate the involvement of the coreceptors required for entry of the HIV-1 strains studied here, we pretreated macrophages with a panel of monoclonal antibodies to either CCR5 or CXCR4, prior to exposure to either ADA or HTLV-IIIB. MIP-1alpha expression was then measured (Fig. 2C). Activation of macrophages by either ADA or HTLV-IIIB was not inhibited by antibodies to their coreceptors. However, there was some inhibition of R5 ADA activation by one anti-CXCR4 antibody, 12G5. None of the antibodies induced MIP-1alpha secretion, although there is an indication of some synergy between HIV-1 and particular antibodies, 45523, for instance. Taken together these findings indicate that the activation of macrophages by HIV-1 observed here may not involve binding to CD4 or the chemokine receptors used for virus entry.


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  		FIG. 2.   Inhibitors of HIV-1 infection do not affect induction of MIP-1alpha . (A) Macrophages were cultured in the presence or absence of 1 µM ddC and then exposed to infectious ADA, YU-2, HTLV-IIIB, Z6, or LPS or mock infected as described in Materials and Methods. Supernatants were harvested for the MIP-1alpha assay after 6 h. (B) ADA, YU-2, HTLV-IIIB, and Z6 species were pretreated with soluble CD4 prior to exposure to macrophages as described in Materials and Methods. Supernatants were harvested for the MIP-1alpha assay after 6 h. (C) Macrophages were pretreated with monoclonal antibodies to CCR5 or CXCR4 and then exposed to ADA or HTLV-IIIB as described in Materials and Methods. Supernatants were harvested for the MIP-1alpha assay after 3 h.

To further explore activation of macrophages by both R5 and X4 HIV-1, we evaluated production of a large panel of physiologically important factors by macrophages. Cells were exposed to infectious HIV-1 and after 16 h, supernatants were collected for assay of MIP-1alpha , MIP-1beta , RANTES, MCP-1, and TNF-alpha (Fig. 3A and B). There was a relatively high spontaneous release of MCP-1 in all systems, including control macrophages. ADA, NLHXADA-GP, and HTLV-IIIB stimulated the production of MIP-1alpha , MIP-1beta , RANTES, and TNF-alpha at levels comparable to that induced by LPS. None of the other viruses tested induced the factors assayed. Introduction of the ADA V3 region of gp120 onto the background of NL4-3 to construct NLHXADA-GP (44) conferred the ability to induce beta -chemokines, suggesting that HIV-1 gp120 may be responsible for the activation of macrophages by HIV-1 observed here. On that basis we tested the macrophage response to purified recombinant envelope glycoprotein, gp120.


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  		FIG. 3.   R5 and X4 HIV-1 species activate macrophages to produce beta -chemokines and TNF-alpha . Macrophages were infected with R5 (ADA, GP, and Yu2) and X4 (IIIB, NDK, NL4-3, and Z6) HIV-1 species. After 16 h, supernatants were collected for assay of beta -chemokines, MIP-1alpha , MIP-1beta , RANTES, and MCP-1, and the cytokine TNF-alpha . LPS was used as a positive control for immune activation. (A) MIP-1alpha , MIP-1beta , and TNF-alpha levels. (B) RANTES and MCP-1 levels. Data represent means ± standard deviations for experiments in triplicate.

R5 and X4 HIV-1 gp120 induces beta -chemokine and TNF-alpha production by macrophages. Macrophages were exposed to purified, glycosylated recombinant gp120 from two R5, two X4, and one X4/R5 HIV-1 species. Cell supernatants were collected after 16 h and assayed for the levels of MIP-1alpha , MIP-1beta , RANTES, and TNF-alpha (Fig. 4A and B). HTLV-IIIB and BaL gp120 induced levels of each factor comparable to that induced by LPS, but CM235, MN, and SF2 gp120 failed to induce the factors measured. The strain specificity observed using intact HIV-1 was reproduced using purified HIV-1 gp120. Macrophages responded to BaL virus and envelope and to HTLV-IIIB virus and envelope. The dose responses to HTLV-IIIB and BaL were very similar, indicating comparable activities between X4 and R5 gp120 in these inductive events (Fig. 4C). The gp120 proteins tested migrated similarly, indicating that none was degraded (Fig. 4D). These findings suggest that the immediate response of macrophages to HIV-1 exposure is induced through binding of gp120 to cell surface receptors. However, the inability of anti-CCR5 and -CXCR4 antibodies and soluble CD4 to block the response indicates that these receptors may not be involved. With this negative information in hand, it is premature to speculate on the nature of the cell surface receptors involved.


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  		FIG. 4.   R5 and X4 HIV-1 envelope glycoprotein gp120 activates macrophages to produce beta -chemokines and TNF-alpha . Macrophages were incubated overnight with 20 nM recombinant gp120 from R5 clones BaL and CM235, X4 clones IIIB and MN, and the R5X4 clone SF2, and supernatants were harvested for the chemokine-cytokine assay. (A) MIP-1alpha and MIP-1beta levels. (B) RANTES and TNF-alpha levels. Data represent means ± standard deviations for triplicate experiments. (C) Macrophages were incubated with IIIB and BaL gp120 at the doses indicated, and after 16 h, the supernatant was harvested to assay MIP-1alpha levels. Data shown are representative of experiments in duplicate. (D) Western blot of gp120. The migration of molecular mass markers of 220 and 97 kDa is indicated.

Biochemical requirements for induction of chemokine synthesis. The secretion of beta -chemokines by macrophages in response to HIV-1 exposure may result from new synthesis of the proteins or from release of proteins from intracellular pools. To determine whether the chemokines measured in macrophage culture media were newly synthesized, cells were preincubated with graded doses of the translational inhibitor cycloheximide prior to exposure to ADA or HTLV-IIIB. After 5 h, MIP-1alpha levels and cell viability were measured (Fig. 5A and B). Cycloheximide was not toxic at any dose employed; however, it inhibited the production of MIP-1alpha even at a 10 µM concentration. We conclude that HIV-1 exposure induces new synthesis of MIP-1alpha protein in macrophages.


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  		FIG. 5.   Inhibition of HIV-1-induced MIP-1alpha production by treatment of macrophages with the translational inhibitor cycloheximide or the calcium chelator MAPTAM. Macrophages were treated for 1 h with the indicated doses of cycloheximide or MAPTAM prior to HIV-1 infection. Supernatants were harvested 5 h after infection for assay of toxicity and MIP-1alpha levels. (A) MIP-1alpha produced by cycloheximide-treated macrophages. (B) Cell death of cycloheximide-treated macrophages. (C) MIP-1alpha produced by MAPTAM-treated macrophages. (D) Cell death of MAPTAM-treated macrophages. Percent cytotoxicity was calculated as described in Materials and Methods. Data represent means ± standard deviations for experiments performed in triplicate.

Previous studies of activation of macrophages by R5 gp120 implicated Ca2+-mediated signal transduction as a proximal event. In the same study, an X4 envelope failed to stimulate Ca2+ flux (3). A similar report found that both R5 and X4 envelopes stimulated Ca2+ flux by primary macrophages (27). Therefore, we investigated whether Ca2+ mobilization was required for the chemokine production we observed. Macrophages were washed and plated in Ca2+-free medium in the presence of graded doses of the membrane permeant Ca2+ chelator MAPTAM. Cells were exposed to HIV-1, and after 5 h, MIP-1alpha levels and cell viability were measured (Fig. 5 C and D). None of the doses of MAPTAM used was toxic; however, there was a dose-dependent inhibition of production of MIP-1alpha in response to either ADA or HTLV-IIIB. We conclude that Ca2+ mobilization is required for the rapid induction of chemokine responses by HIV-1. Taken together, our findings introduce a new strain-specific response to HIV-1 resulting in coordinate and rapid synthesis of a cytokine and several beta -chemokines by primary macrophages.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Our novel results show that within hours of exposure and independent of virus replication, primary human macrophages respond to HIV-1 and its envelope glycoprotein by new synthesis of several physiologically important proteins. This response is viral strain specific, but it is unrelated to coreceptor phenotype and does not appear to involve binding of CD4, CCR5, or CXCR4.

Because HIV-1 has appropriated cellular receptors that are intimately involved in immune responses, considerable research has been devoted to elucidation of the effects of HIV-1 binding on cellular activity. As early as 1989, it was recognized that HIV induction of cytokine synthesis by macrophages could be dissociated from productive infection and attributed to binding of cell surface receptors (5, 21, 30). Our studies confirm and extend these findings to demonstrate that a panel of three beta -chemokines and TNF-alpha are induced rapidly and coordinately by HIV-1 and its envelope glycoprotein. The ability to activate macrophages using X4 HIV-1 strains unable to infect them, as well as the inability to block R5 HIV-1-mediated activation by inhibition of virus replication, indicate that HIV-1 initiates a program of activation and cellular gene expression in primary macrophages independently of productive infection. A different pathway of activation, linked to HIV-1 expression, may be involved in the synthesis of beta -chemokines at the peak of productive HIV-1 infection of macrophages, as previously reported (4, 29, 37, 42) and as shown here. However, the only viral product required for the rapid induction observed here is gp120.

The cellular response we report is viral strain specific, and the specificity resides in HIV-1 gp120. We do not yet have enough information to define the structural basis of the specificity. Both R5 and X4 HIV-1 are active; however, they appear to employ receptor(s) different from CCR5 or CXCR4, since not all R5 or X4 viruses could activate cells and the response was maintained in the presence of antibodies to CCR5 or CXCR4. Examining the strain specificity, ADA and NLHXADA-GP induced chemokine synthesis while NL4-3 did not. NLHXADA-GP carries the ADA V3 region, embedded in the HXB-2 envelope on the background of the NL4-3 viral genome (44). These isolated results point to the ADA R5 V3 region as a principal determinant of the response. However, HTLV-IIIB (HXB-2) gp120 also induced chemokine responses in our hands, while NLHXADA-PG virus, a construct nearly identical to NLHXADA-GP but carrying HXB-2 V3, failed to induce a response. On that basis, it appears that activity in chemokine induction depends both on the V3 region and on sequences in envelope outside it.

There is precedent for activation of macrophages by both R5 and X4 HIV-1 species. Fantuzzi and colleagues recently reported that R5 and X4 gp120 activate beta -chemokine synthesis by macrophages through a CD4-independent route, results consistent with those reported here (12). In addition, gp120 of both phenotypes has been shown to activate several ion channels in primary macrophages (27). Ca2+ mobilization induced by gp120 was reported in the latter study, in a study of R5 HIV-1 activation of macrophages (3), and is implicated in the chemokine synthesis we describe. However, the strain specificity we observe only partially overlaps that previously reported (12, 27). HTLV-IIIB envelope activated macrophages in previous studies (12, 27) and in those we describe. Although we found that the HIV-1 JR-FL reagent was able to induce MIP-1alpha at the peak of virus infection, it did not induce a rapid response, unlike results of the previous studies (12, 27). Some of these differences may be technical. We employ a growth factor mixture to induce macrophage differentiation; the previous studies allow differentiation through adherence. Liu et al. used 10-fold more gp120 than was employed here to activate ion channels through CCR5 and CXCR4 (27). Finally, the levels of MIP-1beta and RANTES induced in our system were more than 10-fold higher than those observed by Fantuzzi et al.

Synthesis of beta -chemokines by macrophages can be activated by other stimuli. Macrophages responded to contact with cells expressing CD40L, an activator expressed on the surface of T cells, by production of beta -chemokines at levels and with kinetics comparable to that described here (23). Another study reported that the HIV-1 protein Nef can activate macrophages to produce MIP-1alpha and MIP-1beta but not RANTES (42), all of which we observed to be synthesized in response to HIV-1 gp120. The latter study also showed that the levels of MIP-1alpha and MIP-1beta in macrophage supernatants, about 10- to 50-fold lower than we report, were sufficient to activate lymphocyte chemotaxis (42). Extrapolating from that study, it is likely that the higher levels of MIP-1alpha and MIP-1beta , as well as the other beta -chemokines produced in our experimental system in response to HIV-1, would also induce lymphocyte migration to activated macrophages.

The beta -chemokines have complex roles in HIV-1 infection. Previous studies have demonstrated that they inhibit R5 HIV-1 infection in culture and that their levels are elevated in HIV-1-infected persons who appear to control their infection (6, 7, 46). However, beta -chemokine exposure can also increase viral replication in vitro in T cells producing X4 virus (22). We speculate that the TNF-alpha and beta -chemokine responses we describe are relevant in their physiological activities. In an animal model of virally induced myocarditis, MIP-1alpha was the pivotal factor initiating antiviral responses, including cytotoxic-T-cell migration (9). We consider that the rapid response of macrophages to HIV-1 exposure we describe in vitro may constitute an innate immune response that is protective in vivo, summoning activated T cells, including HIV-specific cytotoxic T cells, to eliminate HIV-1-infected cells prior to major spread of the virus through tissue. Further studies are required to illuminate the activities of beta -chemokines and other cellular products of macrophages activated by HIV-1.


    ACKNOWLEDGMENTS

We thank P. Sova for helpful discussions, G. Bentsman for excellent technical assistance, I. Totillo for skilled document preparation, and the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID-NIH, for the following reagents: HIV-1 ADA, provided by H. E. Gendelman; HIV-1 BaL, provided by S. Gartner; HIV-1 JRFL, provided by I. Chen; CM235 gp120, provided by Protein Sciences Corp.; IIIB gp120 and MN gp120, provided by DAIDS; SF2 gp120, provided by M. Quiroga; sheep anti-gp120, provided by M. Phelan; and monoclonal antibodies 12G5, provided by J. Hoxie, 2D7, provided by Millennium Pharmaceuticals, and 44708, 44717, 45523, and 45549, provided by DAIDS.

This work was supported by PHS grants to M.J.P. and D.J.V.


    FOOTNOTES

* Corresponding author. Mailing address: Molecular Virology Division, 432 W. 58th St., New York, NY 10019. Phone: (212) 582-4451. Fax: (212) 582-5027. E-mail: mjp6{at}columbia.edu.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Journal of Virology, November 2001, p. 10738-10745, Vol. 75, No. 22
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.22.10738-10745.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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