Induction of G1 cycle arrest in T lymphocytes results in increased extracellular levels of beta-chemokines: a strategy to inhibit R5 HIV-1

Abstract

The beta-chemokines RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta are the natural ligands of the HIV-1 coreceptor CCR5 and compete with the virus for receptor binding. We show that secretion of the beta-chemokines by activated lymphocytes starts before cellular DNA synthesis is detected and demonstrate that transient prolongation of the G(1) phase of the cell cycle by treatment with cytostatic drugs results in increased levels of the three chemokines in culture supernatants. Supernatants collected from peripheral blood mononuclear cells exposed to hydroxyurea, which arrests the cell cycle in late G(1), contained high levels of beta-chemokines. These supernatants were able to inhibit HIV-1 replication when added to cultures of infected lymphocytes. The observed antiviral effect likely was due to the increased levels of beta-chemokines RANTES, MIP-1alpha, and MIP-1beta because (i) supernatants greatly inhibited the replication of HIV-1 BaL, whereas they affected HIV-1 IIIb replication only slightly; (ii) neutralizing antibodies against the chemokines abrogated the antiviral effect of the supernatants; and (iii) the hydroxyurea concentrations shown to up-regulate chemokine levels were not sufficient to inhibit virus replication by depletion of intracellular nucleotide pools. Although antiviral properties have been reported previously for the cytostatic agents shown here to up-regulate beta-chemokine levels, our results provide an additional mechanism by which these drugs may exert antiviral activity. In summary, increased extracellular levels of anti-HIV-1 beta-chemokines resulting from transient prolongation of the G(1) phase of the lymphocyte cell cycle by treatment with cytostatic drugs may help to control the replication of CCR5-using strains of HIV-1.

Figure 1

Kinetics of RANTES, MIP-1α, and MIP-1β secretion in activated PBMCs. (a) PBMCs (1 × 10⁶) were cultured in 1 ml of culture medium in the presence of PHA. Cultures were maintained for 72 h. Every 24 h, the entire culture medium was collected and replaced with fresh medium containing PHA. Supernatants were assayed for chemokine content in supernatants by ELISA. DNA synthesis was measured by [³H]thymidine incorporation in PBMCs cultured in parallel under identical conditions. Results are the mean ± SD of data obtained from three different donors. (b) PBMCs from four donors were cultured in the presence of PHA for 3 days and in the presence of IL-2 afterward. Culture supernatants were assayed for β-chemokine production by ELISA on days 3, 7, and 11. Values from each donor are the mean ± SD of triplicate wells.

Figure 2

HU treatment of PHA-activated PBMCs results in increased levels of secreted β-chemokines. PBMCs were cultured in the presence of PHA for 3 days and in the presence of IL-2 afterward. HU was added at the indicated concentrations at the beginning of the experiment and added fresh every time the medium was changed. Culture supernatants collected on days 3, 8, and 14 were assayed for chemokine production. Chemokine levels in the supernatants are expressed as ng/ml (a) and as ng per 10⁶ viable cells (b); cell number was monitored by trypan blue exclusion (c). Representative values of one of four experiments, each using PBMCs from a different donor, are shown. Values are means ± SD of triplicate wells. ∗, P < 0.01; #, P < 0.05, compared with untreated control by Student's t test.

Figure 3

Treatment of activated PBMCs with cytostatic drugs inducing G₁ cell cycle arrest results in increased levels of extracellular β-chemokines. PHA-activated PBMCs were cultured in the presence of APH (a), SB (b), OL (c), or RC (d) at the indicated concentrations. Cultures were kept for 14 days, with medium changes every 3 or 4 days. Culture supernatants were tested for chemokine content by ELISA, and cell number was determined by trypan blue staining. Data show day 8 values for both APH and SB and day 3 values for RC and OL. Values are means ± SD of triplicate wells. ∗, P < 0.01; #, P < 0.05, compared with untreated control by Student's t test.

Figure 4

Cell cycle arrest in G₁, but not in G₂, results in increased extracellular levels of β-chemokines. Purified CD8 lymphocytes were activated by anti-CD3 and IL-2 treatment for 3 days. Activated cells were cultured in the presence of IL-2 medium containing HU at the indicated concentrations. After 24 and 48 h of the addition of HU, cell number was evaluated by trypan blue staining (a), newly synthesized DNA was measured by [³H]thymidine incorporation (b), percentage of cells in S phase was determined by propidium iodide staining (c), and β-chemokine levels were determined by ELISA (d). (e) Cell cycle arrest and chemokine production in the presence of nocodazole 48 h after addition of the drug. Results are single data values, representative of three independent experiments for HU and representative of two independent experiments in the case of nocodazole.

Figure 5

Supernatants collected from PBMCs exposed to HU contain suppressive factors that markedly inhibit HIV-1 BaL replication, whereas they only slightly affect the replication of HIV-1 IIIb. Activated lymphocytes from a seronegative donor were infected with HIV-1 BaL (a) or HIV-1 IIIb (b). Infected cells were cultured in IL-2 culture medium supplemented by 50% with supernatants collected from HU-treated PBMCs (CM/HU) or supernatants collected from untreated PBMCs (CM/control). In addition, a culture containing 100 μM HU in fresh medium was included. Virus replication was measured in the culture supernatant on day 7 after infection. Cell viability was assessed by the MTT assay. Data are means ± SD of triplicate wells. Representative results of one of two experiments are shown.

Figure 6

The antiviral activity of supernatants collected from HU-exposed PBMCs is reversed by neutralizing antibodies against the β-chemokines RANTES, MIP-1α, and MIP-1β. Activated lymphocytes from a seronegative donor were infected with HIV-1 BaL. Infected cells were cultured in the presence of supernatants collected from PBMCs that had been cultured for 7 days in the presence of 100 μM HU (CM/HU). CM/HU was preincubated with a mixture of neutralizing antibodies (anti-RANTES, anti-MIP1α, and anti-MIP1β; indicated as nAb) or an IgG control before addition to the culture. Fresh medium containing CM/HU and the correspondent antibodies was added again on day 3 after infection. On day 7, viral replication was measured by a p24 assay, and cell viability was assessed by the MTT assay. Representative results obtained in one of two experiments are shown. Data are mean values ± SD of duplicate wells.