CCL2 increases X4-tropic HIV-1 entry into resting CD4+ T cells

Abstract

During human immunodeficiency virus type 1 (HIV-1) infection, there is a strong positive correlation between CCL2 levels and HIV viral load. To determine whether CCL2 alters HIV-1 infection of resting CD4(+) T cells, we infected purified resting CD4(+) T cells after incubation with CCL2. We show that CCL2 up-regulates CXCR4 on resting CD4(+) T cells in a CCR2-dependent mechanism, and that this augmentation of CXCR4 expression by CCL2 increases the ability of these cells to be chemoattracted to CXCR4 using gp120 and renders them more permissive to X4-tropic HIV-1 infection. Thus, CCL2 has the capacity to render a large population of lymphocytes more susceptible to HIV-1 late in the course of infection.

FIGURE 1.

CCL2 induces up-regulation of surface CXCR4 expression on resting CD4⁺ T cells through a CCR2-dependent mechanism that does not require de novo protein synthesis. A, purified resting CD4⁺ T cells from HIV-negative subjects were incubated with increasing concentrations of CCL2. After 4 h, cells were harvested and stained for surface CXCR4. CCL2 induced a dose-dependent increase in surface expression of CXCR4. Data are expressed as means ± S.E. of the mean fluorescence intensity calculated from three independent experiments. B, purified resting CD4⁺ T cells were pretreated with 20 μm RS102895 and cultured for 4 h in the presence of 2 nm CCL2 (black line histogram) or with vehicle control (solid gray histogram). Cells were then harvested and stained for surface CXCR4. Histograms are shown for a representative donor. C, purified resting CD4⁺ T cells were incubated with 2 nm CCL2 (black line histogram) or with vehicle control (solid gray histogram) for 4 h. Cells were then stained for surface CXCR4 or permeabilized and stained for total CXCR4. Histograms are shown from a representative donor. D, CXCR4 mRNA content was evaluated using real time PCR after 4 h of incubation with 2 nm CCL2. ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001.

FIGURE 2.

Effect of CCL2 on CXCR4 internalization and on plectin content. A, CXCR4 internalization was quantified by flow cytometric assessment of anti-CXCR4 antibody binding following acid stripping of cell surface-bound antibody. Acid-resistant binding (internalized CXCR4) is expressed as a fraction of total antibody binding over 1 h of incubation at 37 °C. CCL2 reduced CXCR4 internalization rates by 42% across four experiments (^**, p = 0.03). B, effect of CCL2 on plectin expression. Western blot was used to quantify plectin levels in resting CD4⁺ T cells cultured for 4 h in the presence of 0 (U) or 2 nm CCL2 (C). Parallel determination of β-actin verified equivalent protein loading. C, densitometric analysis of the protein bands revealed that CCL2 conditioning of resting CD4⁺ T cells significantly increased plectin content (^*, p = 0.01).

FIGURE 3.

CD4⁺ T cell migration induced by 200 nm gp120 protein alone or with prior conditioning with 1 nm CCL2 and/or inhibitors. Bars represent the mean ± S.E. number of migrated cells of three independent experiments performed in triplicate. Cells not pretreated with an inhibitor or antibody are represented by white bars. In some instances the cells were also treated with 20 μm RS102895 (black bars), 1 μm AMD3100 (gray bars), 1 μg/ml PTX (white striped bars), or 10 μg/ml anti-CD3 antibody (gray striped bars). Experiments were performed three times in triplicate. Chemokinesis was minimal when equal concentrations of gp120 were added to both the apical and basal chambers and has been subtracted from these histograms. X4 gp120_MN induced chemotaxis of CD4⁺ T cells and was augmented by preconditioning with 1 nm CCL2. The augmentation was abrogated in the presence of RS102895 (p < 0.0001). Treatment with AMD3100, PTX, or anti-CD3 antibody inhibited X4 gp120_MN induced chemotaxis. R5 gp120_SF162 induced minimal chemotaxis and was unaffected by all inhibitor and stimulus treatments. ^*, p < 0.0001.

FIGURE 4.

CCL2 augments X4 gp120_MN-induced F-actin polymerization in resting CD4⁺ T cells. Using FITC-phalloidin as a probe for intracellular F-actin, the effects of CCL2 pre-conditioning on F-actin polymerization in resting CD4⁺ T cells was assessed by flow cytometry. Results show the kinetics of F-actin polymerization following stimulation of unconditioned cells with R5 gp120_SF162 (open circles) or X4 gp120_MN (open squares). The change in F-actin content is expressed as a relative fold change in the median fluorescence intensity, when the base-line fluorescence intensity before addition of ligand (time 0) is expressed as 100%. CCL2 preconditioning of resting CD4⁺ T cells significantly increased the content of X4 gp120-induced F-actin (solid squares; ^*, p = 0.022) but had no effect on R5 gp120-induced F-actin (solid circles; p = 0.89). AMD3100 inhibited the increase in X4 gp120-induced F-actin content (solid triangles). All data presented are representative of three independent experiments.

FIGURE 5.

CCL2 augments X4 gp120-induced Erk 1/2 phosphorylation in primary resting CD4⁺ T cells. Primary resting CD4⁺ T cells were stimulated with gp120 for 2 min, fixed, permeabilized/ and stained with a fluorescently tagged antibody specific for phosphorylated Erk 1/2 and then analyzed by flow cytometry. A, Erk 1/2 phosphorylation induced by X4 tropic gp120_MN. Histograms shown are from a representative donor. Solid gray histogram in each image corresponds to cells not stimulated with 200 nm gp120_MN. Light gray line corresponds to unconditioned cells stimulated with 200 nm gp120_MN. Black line corresponds to pretreatment with either 1 nm CCL2, 1 μm AMD3100, or 1 μg/ml PTX. B, Erk 1/2 phosphorylation induced by R5-tropic gp120_SF162. Histograms shown are from a representative donor. Solid gray histogram in each image corresponds to cells not stimulated with 200 nm gp120_SF162. Light gray line corresponds to unconditioned cells stimulated with 200 nm gp120_SF162. Black line in -CCL2 corresponds to pretreatment with 1 nm CCL2. C, bars represents the mean ± S.E. of the relative change in fluorescence intensity indicative of Erk 1/2 phosphorylation after stimulation with X4 gp120_MN (black bars) or R5 gp120_SF162 (gray bars) from three independent donors carried out in triplicate. The change in Erk 1/2 phosphorylation is expressed as the relative fold change in the mean fluorescence intensity, with the base-line fluorescence intensity before the addition of gp120 expressed as 1.00. CCL2 preconditioning of resting CD4⁺ T cells significantly increased X4 gp120-induced phosphorylation of Erk 1/2 (p < 0.0001) but had no effect on R5 gp120-induced events. AMD3100 and PTX inhibited Erk 1/2 phosphorylation. ^*, p < 0.0001.

FIGURE 6.

CCL2 augments X4 HIV-1 infection of primary resting CD4⁺ T cells. A, purified resting CD4⁺ T cells were conditioned with 1 nm CCL2, 1 nm CXCL12, or left unconditioned for 4 h before infection with X4-tropic HIV-1_Lai (black bars) or the R5-tropic HIV-1_93In905 (gray bars) at a multiplicity of infection of 0.01 for 3 h. Cells were then cultured for 14 h without further stimulation. Entry of HIV-1 was evaluated by real time PCR using primers specific for the LTR R/U5 region as described under “Experimental Procedures” (means ± S.E.; n = 3). CCL2 conditioning significantly augmented X4-tropic HIV-1_Lai infection of resting CD4⁺ T cells (^*, p < 0.05) and had no effect on R5-tropic HIV-1_93In905 infection. B, cells infected with X4-tropic HIV-1_Lai as described above were further cultured in the presence of 20 units/ml IL-2. After 96 h, supernatants were collected from unconditioned (gray bars) and CCL2-conditioned (white bars) infected cells and p24-quantified. CCL2 conditioning significantly augmented X4-tropic HIV-1_Lai infection of resting CD4⁺ T cells (^*, p < 0.05).