Cytokine regulation of human immunodeficiency virus type 1 entry and replication in human monocytes/macrophages through modulation of CCR5 expression - PubMed (original) (raw)

Cytokine regulation of human immunodeficiency virus type 1 entry and replication in human monocytes/macrophages through modulation of CCR5 expression

J Wang et al. J Virol. 1998 Sep.

Abstract

Human macrophages express chemokine receptors that act as coreceptors for human immunodeficiency virus type 1 (HIV-1) and are major targets for HIV-1 infection in vivo. The effects of cytokines on HIV-1 infection of macrophages and on the expression of CCR5, the principal coreceptor for macrophage-tropic viruses, have now been investigated. Expression of CCR5 on the surface of freshly isolated human monocytes was virtually undetectable by flow cytometry with the monoclonal antibody 5C7. However, after culture of monocytes for 48 h in serum-free medium, approximately 30% of the resulting macrophages expressed CCR5 and the cells were susceptible to infection by macrophage-tropic HIV-1. Addition of either macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) to the cultures markedly increased both the extent of HIV-1 entry and replication as well as surface expression of CCR5. In contrast, addition of the T-helper 2 (Th2) cell-derived cytokine interleukin-4 (IL-4) or IL-13 prevented the expression of CCR5 induced by culture in medium alone, and IL-4 inhibited virus entry, replication, and cytopathicity under these conditions. IL-4 or IL-13 also prevented the stimulatory effects of M-CSF or GM-CSF on CCR5 expression as well as HIV-1 entry and replication. In addition, IL-4 reversed the increase in CCR5 expression induced by pretreatment of cells with M-CSF. Although IL-10 also inhibits HIV-1 replication in macrophages, it did not suppress surface CCR5 expression induced by colony-stimulating factors. These results indicate that the cytokine environment determines the susceptibility of macrophages to HIV-1 infection by various mechanisms, one of which is the regulation of HIV-1 coreceptor expression.

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Figures

FIG. 1

FIG. 1

Effects of M-CSF, GM-CSF, and IL-4 on HIV-1 replication and entry. Monocytes were isolated by elutriation (26), cultured for 48 h in macrophage-SFM in the absence or presence of M-CSF (20 ng/ml), GM-CSF (20 ng/ml), or IL-4 (20 ng/ml), and then subjected to infection with HIV-1 BaL (2.34 × 107 cell-free virus) (23). (A) At the indicated times after infection, culture supernatants were harvested from cells incubated in the absence or presence of M-CSF, GM-CSF, or IL-4 and were then assayed for p24 antigen by enzyme-linked immunosorbent assay (Coulter, Miami, Fla.). Data are means ± standard deviations of triplicates from a representative experiment. (B) Four hours after infection, lysates were prepared from cells incubated in medium alone or in medium containing the indicated cytokine and were analyzed by semiquantitative PCR for proviral DNA. A control lysate was prepared from cell line 8E5 (22), which contains one copy of the HIV-1 genome per cell. PCR was performed with the _gag_-specific primers SK38 (5′-ATA ATC CAC CTA TCC CAG TAG GAG AAA T-3′) and SK39 (5′-TTT GGT CCT TGT CTT ATG TCC AGA ATG C-3′), 10 μl of lysate, and Taq polymerase for 30 cycles. The PCR products were detected by hybridization with excess SK19 probe (5′-ATC CTG GGA TTA AAT AAA ATA GTA AGA ATG TAT AGC CCT AC-3′) end labeled with [γ-32P]ATP. Indicated copy numbers (1,000, 100, 10) of control HIV-1 DNA were used as positive controls. Medium, uninfected parallel macrophages treated with medium only. GAG, HIV-1 prototypic gag gene segment amplified by SK18 and SK19 primers.

FIG. 2

FIG. 2

Regulation of CCR5 expression by M-CSF, GM-CSF, and IL-4. (A) Freshly isolated monocytes were maintained at 4°C in medium (Med) or cultured at 37°C for 48 h in macrophage-SFM alone or in the presence of M-CSF (20 ng/ml) or GM-CSF (20 ng/ml) as indicated. Surface expression of CCR5 was then examined by flow cytometric analysis with monoclonal antibody 5C7 to CCR5 (62) using a FACSortflow cytometer (Becton Dickinson, Sunnyvale, Calif.); analysis was also performed with a mouse isotype-matched immunoglobulin G2a (IgG2a) control antibody (top). (B) Cells were cultured for 48 h at 37°C in medium alone or in the presence of IL-4 (20 ng/ml), after which surface expression of CCR5 and CD4 (Leu-3a) was assessed by flow cytometry; mouse IgG2a was used as an isotype-matched control antibody.

FIG. 3

FIG. 3

Inhibition of M-CSF enhancement of HIV-1 entry and replication by IL-4 and IL-13. (A) Monocytes were cultured for 48 h in macrophage-SFM containing M-CSF (20 ng/ml) in the absence or presence of IL-4 (20 ng/ml) or IL-13 (20 ng/ml) and were then subjected to infection with HIV-1 BaL, HXB2-168.1, or HXB2-168.3 isolates (13). Seven days after infection, culture supernatants were harvested and assayed for p24 antigen by enzyme-linked immunosorbent assay. Data are expressed as percentages of p24 production for cells cultured with M-CSF alone and are means ± standard deviations of triplicates from a representative experiment. (B) Four hours after HIV-1 BaL infection of cells treated as described for panel A, cell lysates were prepared and analyzed for proviral DNA by semiquantitative PCR. Indicated copy numbers (500, 50, 5) of control HIV-1 DNA were used as positive controls. No virus, uninfected parallel macrophages treated with medium only as negative control. Gag, HIV-1 prototypic gag gene segment amplified by SK18 and SK19 primers and detected by [γ-32P]ATP-end-labeled SK19 hybridization; Free Probe, SK19 end labeled with [γ-32P]ATP.

FIG. 4

FIG. 4

Effects of IL-4 and IL-13 on up-regulation of CCR5 expression by M-CSF and GM-CSF. Monocytes were cultured for 48 h in macrophage-SFM in the presence of the indicated combinations of cytokines. Surface expression of CCR5 was then examined by flow cytometry; mouse IgG2a was used as an isotype-matched control antibody (upper). Fluorescence intensity is presented on a logarithmic scale.

FIG. 5

FIG. 5

Reversal of the M-CSF-induced increase in CCR5 expression in monocytes by IL-4. Monocytes were cultured for 1 day in the presence of M-CSF and then for an additional 2 days with M-CSF alone (top) or with M-CSF plus IL-4 (middle). Alternatively, cells were cultured in the presence of M-CSF and IL-4 for 3 days (bottom). Cell surface expression of CCR5 was examined by flow cytometry with antibody 5C7 (filled histogram) at the indicated times; mouse IgG2a (open histogram) was used as an isotype-matched control antibody. Fluorescence intensity is presented on a logarithmic scale.

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