CXCR-4 is expressed by primary macrophages and supports CCR5-independent infection by dual-tropic but not T-tropic isolates of human immunodeficiency virus type 1 - PubMed (original) (raw)

CXCR-4 is expressed by primary macrophages and supports CCR5-independent infection by dual-tropic but not T-tropic isolates of human immunodeficiency virus type 1

Y Yi et al. J Virol. 1998 Jan.

Abstract

Primary macrophages are infected by macrophage (M)-tropic but not T-cell line (T)-tropic human immunodeficiency virus type 1 (HIV-1) strains, and CCR5 and CXCR-4 are the principal cofactors utilized for CD4-mediated entry by M-tropic and T-tropic isolates, respectively. Macrophages from individuals homozygous for an inactivating mutation of CCR5 are resistant to prototype M-tropic strains that depend on CCR5 but are permissive for a dual-tropic isolate, 89.6, that can use both CCR5 and CXCR-4, as well as CCR2b, CCR3, and CCR8. Here we show that 89.6 entry into CCR5-deficient macrophages is blocked by an anti-CXCR-4 antibody and by the CXCR-4-specific chemokine SDF but not by the ligands to CCR2b or CCR3. Reverse transcription-PCR demonstrated expression of CXCR-4 but not CCR3 or CCR8 in macrophages, while CCR2b was variable. Macrophage surface expression of CXCR-4 was confirmed by immunofluorescence staining and flow cytometry. Thus, CXCR-4 is expressed by primary macrophages and functions as a cofactor for entry by dual-tropic but not T-tropic HIV-1 isolates, and macrophage resistance to T-tropic strains does not result from a lack of the T-tropic entry cofactor CXCR-4. Since CXCR-4 on macrophages can be used by some but not other isolates, these results indicate that HIV-1 strains differ in how they utilize chemokine receptors as cofactors for entry and that the ability of a chemokine receptor to mediate HIV-1 entry differs, depending on the cell type in which it is expressed.

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Figures

FIG. 1

FIG. 1

HIV-1 replication in normal and CCR5-deficient macrophages. Monocytes were isolated from individuals homozygous for the wild-type CCR5 allele or ccr5Δ32, plated at 2 × 105 per well in 48-well plastic tissue culture plates, and allowed to differentiate into macrophages in vitro. After 7 days in culture they were infected overnight with 20 ng of p24 antigen of M-tropic (SF162 or ADA), T-tropic (NL43), or dual-tropic (89.6) strains. Cultures were then washed, and the supernatant was sampled periodically for p24 antigen. Cells infected with 89.6 were incubated for 1 h prior to and during infection with the anti-CD4 MAb no. 19 or the control MAb B33.1 (each at 10 μg/ml), which was then maintained in the medium throughout the experiment.

FIG. 2

FIG. 2

RT-PCR detection of chemokine receptor expression in macrophages. RNA was extracted from 7-day-old macrophage cultures and subjected to reverse transcription and PCR amplification for CCR5, CXCR-4, CCR3, CCR8 (chemR1), and CCR2b, as well as CD4. Products were separated on agarose gels and stained with ethidium bromide. Macrophages from a CCR5 wild-type-homozygous donor are shown, and the same pattern was seen with cells from _ccr5Δ32_-homozygous donors. Amplification was done following reverse transcription with (+) or without (−) reverse transcription enzyme present. m, molecular size standards.

FIG. 3

FIG. 3

Macrophage surface expression of CXCR-4 by flow cytometry. Seven-day-old cultures of monocyte-derived macrophages (MDM) were detached and stained with MAbs for major histocompatibility complex class I as a positive control (w6/32), a T-cell marker (OKT3), a macrophage marker (OKM1), CD4 (#19), and CXCR-4 (12G5). Cells were then analyzed by flow cytometry with a minimum of 104 cells. Specific MAb profiles are indicated by the black histogram, and the negative control antibody is shown as the shaded histogram in each graph. Macrophages from a CCR5 wild-type-homozygous donor are shown and are representative of cells from six different donors. The same patterns were seen with cells from _ccr5Δ32_-homozygous donors. The SUP-T1 cell line was stained in parallel.

FIG. 4

FIG. 4

Inhibition of HIV-1 entry into normal and CCR5-deficient macrophages. Macrophages were isolated from individuals homozygous for the wild-type CCR5 allele (w/w) or the ccr5Δ32 allele (Δ/Δ). After 7 days in culture they were infected with 20 ng of p24 antigen of the dual-tropic isolate 89.6, the T-tropic strain NL43, or the M-tropic strain SF162. The indicated wells were incubated for 1 h prior to and throughout the infection with the anti-CD4 MAb no. 19 (#19), the anti-CXCR-4 MAb 12G5, or the control antibody B33.1 or with SDF, MCP-1 and MCP-3, or eotaxin. Three days after infection the cells were lysed and subjected to PCR amplification with primers that detect conserved regions of the HIV-1 LTR, followed by Southern blotting. Infections were done in duplicate and amplified in independent PCR reactions, both of which are shown. Amplification with β-globin primers showed a positive signal in all wells (data not shown). HIV plasmid DNA was used as a positive control (+) for amplification. Data are representative of replicate experiments with cells from three _ccr5Δ32_-homozygous donors.

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