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(Journal of Leukocyte Biology. 2000;68:318-323.)
© 2000 by Society for Leukocyte Biology

Chemokine signaling and HIV-1 fusion mediated by macrophage CXCR4: implications for target cell tropism

Ronald G. Collman*, Yanjie Yi*, Qing-Hua Liu{dagger} and Bruce D. Freedman{dagger}

* Department of Medicine, University of Pennsylvania School of Medicine; and
{dagger} Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia

Correspondence: Ronald Collman, 807 Abramson Bldg., 34th & Civic Center Blvd., Philadelphia, PA 19104-4399. E-mail: collmanr{at}mail.med.upenn.edu


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ABSTRACT
 
To better understand CXCR4 function on macrophages and the relationship between coreceptor use and macrophage tropism among diverse HIV-1 isolates, we analyzed macrophage pathways involved in Env-mediated fusion, productive HIV-1 infection, and chemokine-elicited signaling. We found that both CXCR4 and CCR5 transduced intracellular signals in monocyte-derived macrophages, activating K+ and Cl- ion channels and elevating intracellular calcium in response to their chemokine ligands stromal cell-derived factor-1{alpha} and macrophage inflammatory protein-1ß, respectively. The prototype T-tropic X4 strain IIIB infected macrophages poorly, and this was associated with failure of the IIIB Env to fuse efficiently with target macrophages despite functional CXCR4. In contrast, several primary X4 isolates mediated efficient CXCR4-dependent fusion and productive macrophage infection. Several R5X4 strains could fuse with and infect macrophages through both CCR5 and CXCR4. Thus, macrophages express functional CXCR4 and CCR5 but primary and prototype X4 isolates differ in their ability to utilize macrophage CXCR4. Isolates classified as X4 based on coreceptor use may be phenotypically either T-tropic or dual-tropic and, conversely, phenotypically dual-tropic isolates may be either R5X4 or X4 based on coreceptor use.

Key Words: gp120 • gp160 • envelope • chemokine receptor • ion channel • calcium


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INTRODUCTION
 
HIV-1 strains are broadly classified as macrophage (M)-tropic, T cell line (T)-tropic, and dual-tropic. T-tropic strains infect CD4+ T cell lines and primary lymphocytes but not primary macrophages, M-tropic strains infect macrophages and lymphocytes but not T cell lines, and dual-tropic strains infect all three target cells. Although tropism is defined in terms of virus replication patterns in vitro, it is an important determinant of pathogenesis in vivo, including person-to-person transmission, disease progression, and neurological, pulmonary, and other sequelae [1 , 2 ]. HIV-1 biology and genetics are most frequently studied using prototype strains that display distinctive replication patterns representative of each category. T-tropic prototype strains like IIIB replicate very efficiently in cell lines and are highly restricted in macrophages, but these strains are lab-adapted due to serial passage in vitro. On the other hand, prototype M-tropic strains like JRFL are used because they are especially reliable in establishing very efficient replication in macrophages. Since prototype strains have been widely adopted because of their particularly distinctive features, however, they may not be fully representative of primary isolates that exist in vivo.

HIV-1 tropism is determined initially by fusion and entry, mediated by the viral envelope glycoprotein (Env) and the cellular coreceptors comprised of CD4 plus a chemokine receptor [1 ]. Prototype M-tropic strains use the chemokine receptor CCR5 for entry, prototype T-tropic strains use CXCR4, and dual-tropic strains use either coreceptor. This pattern of coreceptor usage initially suggested that the cellular determinants of tropism would be linked to the selective expression on macrophages of CCR5 but not CXCR4, CXCR4 but not CCR5 on T cell lines, and both on lymphocytes. In fact, we and others have shown that macrophages do express CXCR4 and that some strains can use macrophage CXCR4 for infection [3 4 5 6 ].

In this study we sought to better understand CXCR4 function on macrophages and the relationship between coreceptor usage and macrophage tropism. Because the normal function of chemokine receptors is to transduce intracellular signals in response to chemokines, we examined the response in macrophages to CXCR4 stimulation, and compared the signals activated through CXCR4 with those activated through CCR5. Because membrane fusion is a critical initial step in virus entry and a major determinant of tropism, we examined the function of CXCR4 and CCR5 in Env-mediated fusion through the use of a primary macrophage-based fusion assay. Finally, we tested the coreceptors used on macrophages for productive infection. These studies show that CXCR4 in macrophages is functional for chemokine signaling, and supports Env-mediated fusion and infection by some primary but not prototype HIV-1 isolates.


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MATERIALS AND METHODS
 
Macrophage isolation
Monocytes were isolated from peripheral blood mononuclear cells of healthy donors by selective adherence as previously described [7 ]. For infection and fusion studies, cells were plated at 2 x 105 cells per well in 48-well plates. For signaling studies, 106 cells were plated on 22-mm poly-D-lysine-coated glass coverslips. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10% horse serum, glutamine, antibiotics, and macrophage colony-stimulating factor (M-CSF; 100 U/mL; Genetics Institute) for 5–7 days before study to allow differentiation into monocyte-derived macrophages (MDM). To address macrophage CCR5 function, cells were obtained from donors homozygous for the non-functional CCR5 {Delta}32 allele as identified by polymerase chain reaction (PCR) [8 ]. To define the role of macrophage CXCR4, cells were treated with the specific antagonist AMD3100 [9 ] for 1 h before fusion (10 µg/mL) or infection (1 µg/mL) experiments, or immediately before agonist application (1 µg/mL).

Signaling studies
To measure current responses to chemokine stimulation, MDM on coverslips were placed into a temperature-controlled recording chamber on the stage of an inverted fluorescence microscope, and voltage clamp experiments were performed using standard whole-cell recording techniques as previously described [10 , 11 ]. Bath solution contained 140 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4), and the pipette solution contained 145 mM KCl, 0.5 mM EGTA, 0.5 mM MgCl2, and 5 mM HEPES (pH 7.4). The chemokines macrophage inflammatory protein-1ß (MIP-1ß) and stromal cell-derived factor-1{alpha} (SDF-1{alpha}; 1 µg/mL; Peprotech, Rocky Hill, NJ) were applied directly onto cells through the use of a pressure-controlled micropipette. The ionic nature of elicited currents were characterized using instantaneous current reversal potentials at the point of maximal current activation (300-ms ramp depolarizations applied from -100 to 75 mV) and ion-substituted solutions as previously described [10 , 11 ], and the pharmacological inhibitors NPPB (Calbiochem, San Diego, CA) and charybdotoxin (Alomone Laboratories, Jerusalem, Israel).

Primary macrophage fusion studies
Effector 293T cells were infected with recombinant vaccinia viruses expressing the IIIB, 89.6 and DH12 env genes [12 , 13 ], transfected with a plasmid encoding luciferase under control of the T7 promoter, and incubated overnight at 32°C in medium containing rifampicin (100 µg/mL). Target MDM were infected with T7 polymerase-expressing recombinant vaccinia virus vTF7.3 and incubated at 32°C overnight in medium supplemented with rifampicin. Env-expressing effector cells and macrophages were then mixed in the presence of both rifampicin and Ara-C (0.1 µM). Cell-cell fusion was quantified by measuring luciferase activity in cell lysates 6 h later. For strains UG021, UG024, and JRFL, effector 293T cells were infected with the T7 polymerase-expressing recombinant vaccinia virus vP11T7gene1, co-transfected with plasmids encoding env under control of the T7 promoter [14 ] and luciferase under control of the SP6 promoter, and incubated at 32°C overnight. Target MDM were infected with recombinant vaccinia virus vSIMBE/L, which expresses the SP6 RNA polymerase, and incubated at 32°C overnight in medium with rifampicin. Env-expressing effector cells were then mixed with target macrophages in the presence of rifampicin and Ara-C, and luciferase activity measured 6 h later. Details of the recombinant vaccinia viruses, plasmids, and macrophage fusion assays have been described previously [6 , 12 13 14 15 ].

Infections
MDM were infected overnight using 20 ng of p24 antigen of each virus, washed, and p24 antigen levels in supernatant were measured periodically. HIV-1 isolates tested included the T-tropic X4 prototype IIIB, M-tropic R5 prototype JRFL, R5X4 strains DH12 and 89.6 [12 , 13 ], and X4 primary isolates UG021 and UG024 ([14 ], obtained from the NIH AIDS Reagent Program).


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RESULTS
 
Macrophage-expressed CXCR4 and CCR5 support cell signaling
We and others recently showed that primary human macrophages express immunoreactive CXCR4 even though they are not permissive for prototype CXCR4-dependent HIV-1 strains [3 , 16 ]. We therefore sought to determine whether macrophage CXCR4 was expressed in a form that was functionally intact as well. Although HIV has subverted the G protein-coupled chemokine receptors for its own use as an entry coreceptor, their normal role is to activate intracellular signals in response to chemokines. Because G protein-coupled receptors are linked to ionic signaling pathways in many cell types [17 ], we used single-cell patch-clamp recordings to determine whether ionic conductances were elicited by SDF-1{alpha}, the chemokine ligand for CXCR4, as well as MIP-1ß, the most specific of several chemokines that signal through CCR5.

Both SDF-1{alpha} and MIP-1ß activated a transient outward current in macrophages, followed by a more slowly developing and sustained inward current (Fig. 1A ). The initial outward current was elicited by MIP-1ß in all macrophages, whereas SDF-1{alpha} evoked the current in about half. In contrast, the subsequent inward current was activated somewhat more frequently by SDF-1{alpha} than by MIP-1ß (70 vs. 40% of cells). To confirm that current activation was specifically mediated by CXCR4 and CCR5, we used macrophages lacking surface CCR5 derived from donors homozygous for the CCR5 {Delta}32 deletion allele, and a specific inhibitor of CXCR4, AMD3100. As shown in Figure 1B , macrophages lacking CCR5 failed to signal after MIP-1ß stimulation but responded normally to SDF-1{alpha}, with activation of inward, or inward and outward, currents. On the other hand, blocking CXCR4 prevented current activation by SDF-1{alpha} but had no effect on the response to MIP-1ß (Fig. 1C) . Thus, both CXCR4 and CCR5 are functionally active in MDM and mediate chemokine-induced signaling.



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  		Figure 1. Chemokine signaling through CCR5 and CXCR4 on primary macrophages. Currents were recorded in MDM stimulated with MIP-1ß (left) and SDF-1{alpha} (right). The arrows indicate the time of agonist application. (A) Wild-type MDM were stimulated without any blocking agent present. (B) The role of CCR5 was tested using CCR5-deficient macrophages, obtained from donors homozygous for the CCR5 {Delta}32 allele. (C) Signaling mediated by CXCR4 was tested by adding the CXCR4 antagonist AMD3100 to wild-type MDM immediately before chemokine application.

K+ and Cl- channel activation through CXCR4 and CCR5 in macrophage
We next determined the ionic nature of these chemokine-elicited macrophage currents from the membrane potentials at which each current reversed direction (ER), the sensitivity of the ER to ion substitution, and the effects of pharmacological inhibitors (Table 1 ). We found that the initial outward currents elicited by SDF-1{alpha} and MIP-1ß reversed direction at approximately -75 mV, which is characteristic of a potassium current. This current was also blocked by charybdotoxin (100 nM), a peptidyl blocker of K+ channels. In contrast, the slower-developing inward current evoked in macrophages by SDF-1{alpha} and MIP-1ß reversed direction at approximately 0 mV, which, under the conditions of these measurements, is consistent with a chloride current. The identity of the inward current was confirmed by replacing Cl- in the extracellular bath with the impermeant anion gluconate, which shifted this reversal potential toward the predicted Cl- reversal potential (ER = 45 mV). The inward current was also blocked by the Cl- channel inhibitor NPPB (10 µM). Therefore, both K+ and Cl- channels are activated by CXCR4 and CCR5 in macrophages upon stimulation by SDF-1{alpha} and MIP-1ß, respectively. Although modest quantitative differences in the frequency of current activation were seen, the overall patterns of ion channel activation were similar for CXCR4 and CCR5. We also found that both SDF-1{alpha} and MIP-1ß elevated intracellular calcium levels in macrophages, as measured using calcium indicator Fura-2/AM [10 , 11 ] (data not shown).


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  		Table 1. Currents Activated by Chemokines in Monocyte-Derived Macrophages

Macrophage CXCR4 supports Env-mediated fusion with primary but not prototype X4 isolates
HIV-1 tropism is determined initially at the level of entry, so we next addressed the roles of macrophage CXCR4 and CCR5 in fusion mediated by diverse types of HIV-1. Effector cells expressing Env from several prototype and primary X4, R5X4, and R5 strains were tested for fusion with primary macrophages in which the functions of CXCR4, CCR5, or both coreceptors were selectively abrogated.

As shown in Figure 2 , Env from the prototype X4 T-tropic strain IIIB failed to fuse with primary macrophages, indicating that it is restricted in macrophages at the level of entry. In contrast, efficient fusion was seen with the R5 prototype JRFL. JRFL fusion was not affected by blocking CXCR4 but was eliminated if CCR5 was absent (Fig. 2B) , confirming that CCR5 served as its exclusive fusion pathway in macrophages. Two dual-tropic R5X4 strains, 89.6 and DH12, fused with macrophages as long as either CXCR4 or CCR5 was available, and fusion was prevented only if both pathways were blocked, as in CCR5-negative macrophages treated with a CXCR4 antagonist (Fig. 2A and 2B ). Thus, these strains could use either CXCR4 or CCR5 for fusion with macrophages.



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  		Figure 2. Fusion mediated by HIV-1 Env and CCR5 or CXCR4 on macrophages. Primary MDM targets were derived from donors homozygous for the CCR5 wild-type or {Delta}32 deletion allele, and AMD3100 was added 1 h before cell mixing. (A) Fusion was tested using effector 293T cells that were infected with recombinant vaccinia viruses expressing Env from the prototype X4 T-tropic strain IIIB or dual-tropic R5X4 strains DH12 and 89.6. (B) Fusion was tested using effector 293T cells that were infected with recombinant vaccinia virus expressing T7 polymerase and then transfected with plasmids encoding T7-driven env genes from the prototype M-tropic R5 strain JRFL or two X4 primary isolates, UG021 and UG024. The difference in scales for panels A and B is because recombinant vaccinia-expressed Envs give higher levels of fusion than T7-driven Envs.

Two primary X4 isolates were then tested, UG021 and UG024 [14 ]. We confirmed that these Envs used CXCR4 but not CCR5 in non-human cells transfected with CD4 and coreceptor plasmids (data not shown). In contrast to IIIB, Env from UG021 and UG024 mediated fusion with both wild-type and CCR5-negative macrophages (Fig. 2B) . Blocking CXCR4 prevented UG021 and UG024 fusion with macrophages regardless of whether CCR5 was present. These results indicated that these X4 isolates used CXCR4 but not CCR5 for fusion with primary macrophages.

Productive macrophage infection supported by CXCR4 or CCR5
To characterize pathways of productive macrophage infection, wild-type and CCR5-negative macrophages were infected, with or without CXCR4 blockade, using the panel of HIV-1 strains examined above for fusion (Fig. 3 ). As expected, JRFL replicated in wild-type macrophages but not in those lacking CCR5, and blocking CXCR4 had no effect on CCR5-mediated JRFL infection. IIIB did not establish productive macrophage infection, consistent with its well-defined T-tropic phenotype. However, very low levels of supernatant p24 antigen were often seen with IIIB, which were nevertheless several orders of magnitude less than those produced by permissive strains. This illustrates that macrophage tropism, and the restriction in macrophages to T-tropic strains, are relative rather than absolute features. There was no difference between wild-type macrophages and those lacking CCR5 in p24 level produced by IIIB, indicating that the absence of CCR5 did not lead to enhanced IIIB infection.



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  		Figure 3. Infection of macrophages via CCR5 or CXCR4. MDM from CCR5 wild-type or {Delta}32 homozygous blood donors were infected with the prototype M-tropic R5 strain JRFL, prototype T-tropic X4 strain IIIB, dual-tropic R5X4 strains DH12 and 89.6, and primary X4 isolates UG021 and UG024. The CXCR4 inhibitor AMD3100 was added 1 h before infections and maintained throughout. Viral p24 antigen was measured periodically in supernatant.

In contrast to JRFL and IIIB, the R5X4 strains DH12 and 89.6 infected macrophages, whether or not CCR5 was expressed (Fig. 3) . Blocking CXCR4 inhibited infection by these strains only if CCR5 was absent, but had no clear effect if CCR5 was present. Thus, DH12 and 89.6 can use either CCR5 or CXCR4 for productive macrophage infection. We then tested UG021 and UG024, the two X4 primary isolates that used macrophage CXCR4 for fusion. These strains also replicated in both wild-type and CCR5-negative macrophages. Unlike the R5X4 strains, however, blocking CXCR4 prevented UG021 and UG024 from infecting macrophages regardless of whether CCR5 was expressed. The somewhat lower replication in Figure 3 by UG021 in CCR5-negative compared with wild-type macrophages was not consistent and likely reflects donor and experimental variability rather than real differences. Thus, UG021 and UG024 used CXCR4 exclusively for productive macrophage infection, and neither fused with nor infected macrophages through CCR5.


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DISCUSSION
 
We show here that primary human monocyte-derived macrophages express both CXCR4 and CCR5 in a form that is functional for cell signaling and for HIV-1 Env-mediated fusion and infection. The prototype T-tropic X4 strain IIIB was unable to utilize macrophage CXCR4 for fusion and infection even though it efficiently uses CXCR4 in lymphocytes and cell lines, whereas several other X4 and R5X4 primary isolates did use macrophage CXCR4. This indicates that the HIV-1 entry coreceptor activity of a chemokine receptor does not simply result from its expression in conjunction with CD4, but involves cell-specific and virus-specific determinants that enable fusion and productive infection.

HIV-1 isolates have long been classified by target cell tropism based on replication patterns in vitro. The T-tropic designation applies to isolates that replicate in T cell lines and lymphocytes but not macrophages, M-tropic strains replicate in macrophages and lymphocytes but not in cell lines, and dual-tropic strains replicate in all three target cells. A valuable biological classification system was recently introduced and is now widely used that categorizes HIV-1 isolates based on major coreceptor selectivity [18 ]. Because there is a close association among prototype strains between M-tropism and CCR5 use, T-tropism and CXCR4 use, and dual-tropism and use of both coreceptors, the coreceptor classification R5, X4, R5X4 is now often employed as a surrogate for describing target cell tropism. However, our studies show that some X4 strains are restricted to infection of T cell lines and lymphocytes and therefore have the T-tropic phenotype (IIIB and other X4 prototypes), but that other X4 strains infect T cell lines, lymphocytes, and macrophages through CXCR4 and so are dual-tropic in phenotype (UG021 and UG024). As a corollary, some dual-tropic strains are R5X4 in coreceptor use but others are exclusively X4. Thus, coreceptor usage can complement but does not substitute for target cell tropism in HIV-1 phenotyping, and X4 viruses should be further classified as T- or dual-tropic, whereas dual-tropic viruses should be characterized as X4 or R5X4 (Table 2 ).


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  		Table 2. Relationship Between Tropism and Coreceptor Selectivity Among HIV-1 Prototype and Primary Isolates

In this study we focused on HIV-1 isolates that use macrophage CXCR4 very efficiently because we wanted to highlight this pathway’s function [3 , 6 ], and CXCR4-mediated macrophage infection has been confirmed by others as well [4 , 5 ]. However, among primary isolates there exists a broad range in how efficiently macrophage CXCR4 is used, and some X4 isolates infect macrophages relatively poorly. For example, Lathey et al. recently analyzed a panel of 12 primary HIV-1 isolates [19 ]. Consistent with the results reported here, all strains replicated in macrophages regardless of coreceptor use. However, the mean peak titers for the CCR5-using isolates as a group were considerably higher than for those restricted to CXCR4. Thus, even though there was overlap for individual strains, CCR5 appeared in general to provide a more efficient pathway for macrophage infection. A broad survey of primary X4 isolates (or primary R5X4 isolates tested in CCR5-negative macrophages) will be needed to determine what proportion of strains use macrophage CXCR4 efficiently, and whether this correlates with particular aspects of pathogenesis.

Macrophages in our study were cultured with M-CSF, which can up-regulate CXCR4 expression [16 ], but infection of macrophages by X4 strains in the absence of M-CSF has also been reported [4 , 5 ]. Nevertheless, it is possible that cytokines within local microenvironments may modulate chemokine receptor expression and regulate permissiveness for HIV-1 in vivo. In addition, freshly isolated monocytes express higher levels of CXCR4 and lower levels of CCR5 than differentiated macrophages, and the low CCR5 level is one factor in monocytes’ long-recognized resistance to infection by M-tropic R5 strains [20 , 21 ]. Despite their higher CXCR4 level, however, we found that monocytes are also resistant to infection by strain 89.6 (data not shown), which can use CXCR4 on macrophages. This indicates that coreceptor expression is not the only restriction to monocyte infection [22 ], and the higher level of CXCR4 expression on monocytes does not result in permissiveness for X4 HIV-1 variants.

It is not known what determines cell-specific and virus-specific coreceptor function. Macrophage CXCR4 and CD4 levels are relatively low, but this does not appear to underlie differential utilization by primary and prototype X4 strains [23 ]. Another potential mechanism for differential coreceptor utilization in macrophages is intracellular signals elicited through the chemokine receptors [24 , 25 ]. Our results do not resolve this question because we found that both CXCR4 and CCR5 activated ionic currents and elevated intracellular calcium in macrophages in response to their chemokine ligands. Alternatively, differences in coreceptor function may result from cell-specific differences in posttranslational modification, association with CD4, or co-association with other molecules that affect the ability of a chemokine receptor to function for some but not other viral strains [26 , 27 ]. Defining the factors in addition to CD4/chemokine receptor expression that are required for coreceptor activity will provide insight into the mechanisms of HIV-1 entry, and may suggest strategies to interfere with coreceptor function.


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ACKNOWLEDGEMENTS
 
This work was supported by NIH grants to R. G. C. and B. D. F. We thank E. De Clercq and D. Schols for AMD3100, M. Cho for strain DH12, D. Williams for excellent technical assistance, and blood donors who generously provided cells.


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