CSF-induced and HIV-1-mediated distinct regulation of Hck and C/EBPbeta represent a heterogeneous susceptibility of monocyte-derived macrophages to M-tropic HIV-1 infection

Abstract

Granulocyte/macrophage colony-stimulating factor (GM-CSF)-induced monocyte-derived macrophages (GM-MPhi) are permissive to M-tropic HIV-1 entry, but inhibit viral replication at posttranscriptional and translational levels, whereas M-CSF-induced macrophages (M-MPhi) produce a large amount of HIV-1. M-MPhi express a high level of Hck and a large isoform of C/EBPbeta, and HIV-1 infection increases the expression of Hck but not of C/EBPbeta. GM-MPhi express a high level of C/EBPbeta and a low level of Hck, and HIV-1 infection drastically increases the expression of a short isoform of C/EBPbeta but decreases that of Hck. Treatment of M-MPhi with antisense oligonucleotide for Hck (AS-Hck) not only suppresses the expression of Hck, but also stimulates the induction of the short isoform of C/EBPbeta and inhibits the viral replication. Treatment of GM-MPhi with a moderate amount of AS-C/EBPbeta not only inhibits the expression of the small isoform of C/EBPbeta preferentially, but also stimulates the induction of Hck and stimulates the virus production at a high rate. These results suggest that CSF-induced and HIV-1-mediated distinct regulation of Hck and small isoform of C/EBPbeta represent the heterogeneous susceptibility of tissue MPhi to HIV-1 infection, and the regulation of Hck and C/EBPbeta are closely related and these two molecules affect one another.

Figure 1.

Different susceptibility of Mo-derived MΦs to M-tropic HIV-1 infection. (A) Measurement of viral production at the indicated days in the culture supernatants by p24-ELISA. The data shown are representative one of three independent experiments. (B) Detection of viral DNA in total DNA from MΦ lysates at 2 d PI by nested PCR using a specific outer- and inner-primer for HIV-1 LTR-gag region. (C) Detection of viral DNA by a single step PCR in MΦ infected by AZT-pretreated (AZT) or not treated (−) viral innocula of HIV-1_BaL. The data shown here are representative one of three independent experiments. (D) Estimation of HIV-1_BaL DNA levels by semi-dilution PCR using a specific primer for the HIV-1 LTR region. The levels of viral DNA in infected MΦ were compared with that of NL4–3 in 8E5 cells.

Figure 2.

Immunoblot analysis and in vitro kinase assay of Hck in M-MΦ and GM-MΦ. (A) Immunoblots of Hck protein in PBMC, PHA-activated CD4⁺T cells, Mo, and Mo-derived MΦs. (B) Immuno-blots of Hck protein in HIV-1_BaL–infected M-MΦ and GM-MΦ at 2 d PI. The relative amounts of the Hck in cells were measured using NIH image software (PSL; photo stimulating luminescence, A/mm²). (C) In vitro kinase assay of Hck in HIV-1_BaL–infected Mo-derived MΦs. Hck protein was immunoprecipitated by a specific antibody against p56/59^Hck, and reacted with a kinase buffer containing the tyrosine kinase substrate p50 (Sam 68) in the presence of [γ-³²P] ATP. The samples were resolved in SDS-PAGE and autoradiography or blotted with anti-Hck antibody. The data shown here are representative one of three independent experiments.

Figure 3.

Immunoblot analysis of C/EBPβ in M-MΦ and GM-MΦ. (A) Immunoblots of C/EBPβ protein in PBMC, PHA-activated CD4⁺ T cells, Mo, and Mo-derived MΦs. (B) Immunoblots of C/EBPβ protein in HIV-1_BaL–infected M-MΦ and GM-MΦ at 2 d PI. The relative amounts of the large band to the small band (L/S ratio) of C/EBPβ were calculated using PSL values of 37 kD and 23 kD of C/EBPβ isoforms. The data shown here are representative one of three independent experiments.

Figure 4.

Translational suppression of Hck expression by the antisense oligonucleotide probe inhibits HIV-1_BaL replication in M-MΦ. (A) Immunoblot analysis of Hck in M-MΦ treated with oligonucleotide probes for Hck (2 μM) (AS, AS-Hck; NS, NS-Hck; S, S-Hck; and L, lipofectin alone) at 24 h (left) and in HIV-1_BaL–infected M-MΦ pretreated with the corresponding probes at 7 d PI, respectively (right). The data shown are representative one of three independent experiments. (B) Kinetics of the p24 levels in the culture supernatants of the oligonucleotide-pretreated HIV-1_BaL–infected M-MΦ. (C) Detection of viral DNA in the oligonucleotide-pretreated HIV-1_BaL–infected M-MΦ by nested PCR. The data shown are representative one of seven independent experiments. (D) p24 levels in AS-Hck or AS-C/EBPβ–treated PHA-activated CD4 T cells infected by HIV-1_NL4–3 (1 ng/ml) at 7 d PI. AS-Hck or AS-C/EBPβ has no effect on T-tropic HIV-1 replication in PHA-activated CD4 T cells.

Figure 5.

Translational inhibition of C/EBPβ expression by the antisense oligonucleotide probe triggers HIV-1_BaL replication in GM-MΦ. (A) Immunoblot analysis of C/EBPβ in GM-MΦ treated with oligonucleotide probes for C/EBPβ (2 μM; AS, AS-C/EBPβ; NS, NS-C/EBPβ; S, S-C/EBPβ; and L, lipofectin alone) at 24 h (left), and HIV-1_BaL–infected GM-MΦ pretreated with the corresponding probes at 7 d PI, respectively (right). The data shown are representative one of three independent experiments. (B) Kinetics of p24 levels in the culture supernatants of the oligonucleotide-pretreated HIV-1_BaL–infected GM-MΦ. (C) Detection of viral DNA in the oligonucleotide-pretreated HIV-1_BaL–infected GM-MΦ by nested PCR. The data shown are representative one of three independent experiments.

Figure 6.

Effects of different concentrations of AS-C/EBPβ on the expression of protein and mRNA of C/EBPβ and HIV-1_BaL replication in GM-MΦ. (A) Effect of different concentrations of AS-C/EBPβ on the expression of C/EBPβ protein. (B) Dependence of HIV-1_BaL replication in GM-MΦ on the alternative L/S ratio of C/EBPβ expression. (C) Effect of different concentrations of AS-C/EBPβ on the expression of C/EBPβ mRNA. GM-MΦ were treated with various concentrations (1–10 μM) of AS-C/EBPβ for 24 h, and then infected with HIV-1_BaL. Cell lysates for immunoblot and mRNA were obtained 2 d and 1 d after infection, respectively. p24 levels of the culture supernatants obtained 7 d after infection much correspond with the alternative L/S ratio of C/EBPβ expression (AS, AS-C/EBPβ; NS, NS-C/EBPβ; S, S-C/EBPβ; and L, lipofectin alone). The data shown are representative one of two experiments.

Figure 7.

Immunoblot analysis of C/EBPβ in M-MΦ treated with AS-Hck and of Hck in GM-MΦ treated with AS-C/EBPβ with or without HIV-1_BaL infection M-MΦ and GM-MΦ were treated with AS-Hck (2 μM), AS-C/EBPβ (2 μM) and lipofectin alone (L) for 24 h. These MΦs were infected with or without HIV-1_BaL, and cultured for 48 h with M-CSF and GM-CSF, respectively. Cell lysates were obtained at indicated time points after antisense oligonucleotide treatment. The data shown are representative one of two experiments.