Introduction
Hydroxyurea (HU) has been used for more than 40 years in the hematology field for the treatment of myeloproliferative disorders. It acts by inhibiting ribonucleoside diphosphate reductase, which catalyzes an essential step in DNA synthesis (conversion of ribonucleotides to deoxyribonucleotides) [1] [2] [3] [4] [4] [2] [5–8] [1,2,9,10] [11]
Methods
Proliferation assay
Peripheral blood mononuclear cells (PBMC) from healthy donor samples were separated on Ficoll density gradients (Hypaque, Pharmacia Biotech, Upsala, Sweden). Cells were counted by trypan blue (Eurobio, Les Ulis, France) exclusion and subsequently stained with carboxyfluorescein diacetate succinimidyl ester (CFSE: Molecular Probes, Eugene, Origon, USA). CFSE is a fluorescein-based dye that covalently attaches to the cytoplasmic component of cells, providing a uniform bright fluorescence. Upon cell division, the dye is distributed equally between daughter cells, allowing the resolution of up to five or six cycles of cell division by flow cytometry. Cells were resuspended in phosphate-buffered saline (PBS: Gibco-BRL, Paisley, UK) at 37 °C to a final concentration of 1 × 1010 cells/l, to which an identical volume of CFSE at 37 °C was added. After staining with CFSE, cells were resuspended in complete medium [CM: RPMI 1640 (Gibco-BRL) supplemented with 20% fetal bovine serum (Gibco-BRL), 2 mmol/l glutamine, and 100 U/ml penicillin (Eurobio)] to a final concentration of 2.5 × 109 cells/l. Cells were then treated with three different concentrations of HU: 10, 50, and 100 μmol/l, while a control sample was left untreated. After incubation for 1 h at 37 °C, cells were stimulated with 5 μg/ml phytohemagglutinin [from PHA-M stock 5 mg/ml stored at −20 °C (Sigma Aldrich, St Louis, Missouri, USA)] and 20 IU/ml interleukin (IL) 2 [from stock 20 000 IU/ml stored at −20 °C (Boehringer-Mannerheim, Mannerhein, Germany)]. At day 2, cells were washed to remove PHA, while IL-2 was continued for 5 additional days. At day 7, 1 × 106 cells/l were washed and resuspended in 100 μl PBS in 1.5 ml vials. The CFSE-labeled lymphocytes were stained for dual color analysis by (Epics XL; Beckman-Coulter, Milan, Italy) together with CD4–phycoerythrin (PE) or CD8 –PE–cyanin 5.1 (Beckman-Coulter). For data analysis, the mitotic index (M) was used, calculating the sum of mitotic events at each proliferation cycle [12]
where n is the number of cycles and (x n T )) is the number of acquired events per cycle.
T cell activation assay
PBMC were separated as above, treated with 100 μmol/l HU for 1 h, and then stimulated for 48 h with PHA/IL-2 in the presence of HU. Cells were harvested and washed in PBS containing 1% bovine serum albumin (BSA; Sigma) then 5 × 105 cells were labeled with the following antibody combinations: CD69, CD25, and HLA-DR as activation markers, and CD8 and CD4 as subtype markers. These populations were gated on total lymphocytes (all monoclonal antibodies were obtained from Caltag, Burlingame, California, USA). Cells were analyzed utilizing a Becton Dickinson cytofluorimeter (FACSCalibur).
Detection of intracellular cytokines after non-specific stimulation of T cells
PBMC were obtained as described previously and resuspended at 2.5 × 109 cells/l in CM and incubated for 1 h at 37 °C, under 5% carbon dioxide in the absence or presence of 100 μmol/l HU. To induce preferential production of T helper (Th) type 1 cytokines, cells were then stimulated with PHA 5 μg/ml and IL-2 20 IU/ml for 72 h (in the presence of HU for the treated samples). To stimulate production of Th2 cytokines, cells were stimulated with PHA 5 μg/ml, IL-2, 20 IU/ml, and IL-420 ng/ml (Boehringer-Mannerheim) for 72 h (in the presence of HU for treated samples), followed by 48 h incubation with IL-2 and IL-4 (in the presence of HU for treated samples) at the same concentration as before. Cells were then harvested, washed in PBS, and incubated for an additional 4 h in CM in the presence of PMA 5 ng/ml, CIA2387 250 ng/ml and Brefeldin A 2 μg/ml (all Sigma). Subsequently, cells were collected, washed twice with PBS, labeled for 1 h at 4 °C with CD3–phenocyanin TC (TC) antibody, successively permeabilized for 15 min on ice with 4% paraformaldehyde and 0.2% saponin (Sigma). After washing with PBS/1% BSA/0.1% saponin, cells were resuspended in PBS/1% BSA/0.2% saponin and labeled with antibodies to one of the following human cytokines, all labeled with PE: IL-2, interferon-γ (IFN-γ), IL-10, and IL-4 (Caltag). After labeling for 1 h at 4 °C, cells were washed with PBS/1% BSA and analyzed on a Becton Dickinson FACSCalibur cytofluorimeter.
Non-human primates
Seventeen rhesus macaques (Macaca mulatta ) were infected with SIVmac251 by mucosal (intrarectal) inoculation. All animals had seroconverted before treatment was initiated (6 weeks after challenge). Plasma viremia in all animals had reached a plateau, with an average of 200 000–300 000 copies/ml, before treatment was started. The animals were randomized into three groups. One group (five animals) served as an untreated control. The other two groups (six animals each) were treated for 3 weeks, one group with (R )-9-(2-phosphonylmethoxypropyl)adenine (PMPA) (20 mg/kg body weight, once daily subcutaneously) and didanosine (10 mg/kg, once daily intravenously), and the other group with PMPA, didanosine and HU (15 mg/kg, once daily intravenously). Peripheral blood and lymph node (LN) biopsies were collected before therapy start and after 3 weeks of treatment. PBMC and cells isolated from lymph node were stained with the following combinations of antibodies: CD3–fluoroscein isothiocyanate (FITC), CD4–PE plus CD8 –TC; CD4–FITC plus CD69–TC or CD8 –FITC plus CD69–TC; CD8 –FITC plus CD25–TC or CD4–FITC plus CD25–TC; CD4–FITC plus HLA-DR–TC or CD8 –FITC plus HLA-DR–TC (Caltag). The cells were then analyzed with a Beckman Coulter cytofluorimeter.
SIVmac251 mRNA copies were quantified by polymerase chain reaction with a threshold of detection of 200 copies/ml [13]
Detection of intracellular cytokines after antigen-specific stimulation of T cells
The rhesus macaque trial described above was extended to carry out three cycles of structured treatment interruption, one cycle being 3 weeks off and 3 weeks on therapy, for a total period of 21 weeks. Three weeks after final treatment, PBMC samples were separated as described previously and plated in round-bottom 96-well microtiter plates (Costar, Cambridge, Massachusetts, USA) at 0.5 × 106 cells/well in 0.1 ml CM containing 5 μg zinc-finger-inactivated SIVmac239 (kindly provided by Jeff Lifson, NCI, Frederick, Maryland, USA) or without zinc-finger inactivated SIVmac239 (control media) and with 50 IU/ml recombinant human IL-2 (gift from Hoffman La Roche, Nutley, New Jersey, USA). Cells were cultured for 15 h and treated with Brefeldin A at 10 μg/ml for an additional 3 h. Cells were collected and divided into samples of 0.5 × 106 cells/test tube. After washing once with 2 ml PBS/1% BSA, cells were suspended in 0.1 ml PBS/1% BSA and stained with human antibodies CD8 –PE–cyanine 5 (Immunotech, USA) and CD3–FITC (BD PharMingen, San Diego, CA, USA) for 15 min at room temperature. After washing, cells were fixed with 2% paraformaldehyde pH 7.4, for 10 min and washed with PBS/1% BSA before permeabilized with 0.1 ml 0.1% saponin in PBS/1% BSA for 5 min and stained with IFN-γ–PE (BD PharMingen) for 15 min at room temperature. After intracellular staining, cells were washed twice with 1 ml PBS and resuspended in a 0.5 ml PBS/1% paraformaldehyde buffer. Samples were analyzed by FACS. Specific responses were calculated as differences between the zinc-finger-inactivated SIVmac239 and the control media values.
Results
Effects of hydroxyurea on lymphocyte proliferation and activation in vitro
HU reduced cell proliferation of both CD4 and CD8 T lymphocytes in a linear dose-dependent manner (Fig. 1 a). The average M values in the untreated controls were 31.79 (±6.35), 34.4 (±11.65) and 36.66 (±9.90) for total lymphocytes, CD4 T lymphocytes, and CD8 T lymphocytes, respectively. HU at 10 μmol/l, representing the trough blood concentration after administration of the most commonly used 1000 mg total daily dose of the drug, was sufficient to decrease the value of M compared with the untreated controls. HU at 50 and 100 μmol/l, representing median and peak blood concentrations, respectively, further reduced the value of M. Overall, CD4 T lymphocytes showed a slightly higher sensitivity to the cytostatic effect of HU than the CD8 subpopulation.
Fig. 1: Effects of hydroxyurea (HU) on peripheral blood mononuclear cells. (a) T cell proliferation after 7 days in the presence of three different concentrations of HU: 10 (light gray columns), 50 (gray columns), and 100 μmol/l (black columns), untreated control sample remained untreated (white columns). The mitotic index was calculated for total lymphocytes and for CD8 and CD4 cell subsets. (b,c) Cell activation in vitro using expression of CD69, CD25, and HLA-DR as activation markers and CD8 (b) or CD4 (c) as subtype markers: untreated controls (white circles) and samples treated with 100 μmol/l HU (black circles).
No relevant difference between treated and untreated controls was observed regarding activation markers. The activation profiles of total T lymphocytes and CD8 and CD4 T cell subsets were monitored for the expression of three surface activation markers: CD69, CD25, and HLA-DR (Fig. 1 b,c). CD69 is an activation marker expressed on CD4 and CD8 T lymphocytes early after activation. In the CD8 subset, average expression of CD69 was 23.18% (±5.65) for untreated controls and 27% (±6.97) for samples treated with 100 μmol/l HU. The CD4 subpopulation exhibited an average CD69 expression of 42.76% (±6.22) in the untreated controls and 38.87% (±7.08) in the samples treated with 100 μmol/l HU. CD25 is the IL-2 receptor and its expression is increased under activated conditions, expression peaks at 18 h after activation. The average expression of this surface marker was 20.24% (±4.02) in the untreated control and 20% (±5.18) in the CD8 T lymphocytes treated with 100 μmol/l HU; the CD4 subset exhibited an average CD25 expression of 40.6% (±5.8) and 36.87% (±6.56) for untreated and treated conditions, respectively. HLA-DR is a late activation marker, its expression during activation is delayed compared with the previous markers. In the CD8 T lymphocyte subpopulation, an average HLA-DR expression of 16.6% (±11.6) and 15.5% (±7.33) was found in the untreated control and in samples treated with 100 μmol/l HU, respectively, whereas HLA-DR expression was 15.93% (±11.75) in CD4 T lymphocytes from the untreated control and 18.17% (±12.12) in the samples treated with 100 μmol/l HU.
Effects of hydroxyurea on cytokine production in vitro
The effect of HU on the ability of T lymphocytes to synthesize cytokines after mitogen stimulation was calculated as the ratio between the percentage of lymphocytes positive to a specific cytokine in the presence of 100 μmol/l HU and that in its absence. IL-2 and IFN-γ expressions were analyzed from cells stimulated under Th1 conditions, whereas IL-2, IL-10 and IL-4 expressions were analyzed from cells stimulated under Th2 conditions. T lymphocytes stimulated in the presence of HU were not inhibited in their capacity to produce specific cytokines. On the contrary, cytokine production was enhanced by HU under both Th1 and Th2 stimulations (Fig. 2 ). For all cytokines analyzed, the ratio was >1 in almost all the experiments and, in some experiments, there was almost a three-fold enhancement of cytokine expression.
Fig. 2: Effect of hydroxyurea (HU) on in vitro cytokine production in lymphocytes under conditions favoring interleukin (IL) 2 and interferon-gamma (IFN-γ) production (Th1 stimulation) or IL-2, IL-10 and IL-4 production (Th2 stimulation). The positive cells ratio is the ratio of the percentage of cells, for a certain cytokine in the presence or absence of 100 μmol/l.
Effects of hydroxyurea on T cell activation and lymphocytes count in vivo
In the non-human primate study, there was a blunted increase of peripheral CD4 T cells in the HU-treated arm (average increase: 562 × 106 cells/l) compared with the group not treated with HU (average increase: 1181 × 106 cells/l) (Fig. 3 a). This was not explained by differences in viral load (Fig. 3 b), but it was consistent with a cytostatic effect of HU on T cells. CD4 counts marginally changed from an average (±SD) of 562 × 106 cells/l (±364) at the end of the treatment to 505 × 106 cells/l (±250) at week 24 (not significant). This lack of CD4 fluctuations during treatment interruptions is an interesting feature of HU-containing regimens, and it is consistent with previous observations in the same animal model during intermittent HU, didanosine and PMPA treatment [14] [15] in vivo expression in PBMC and lymph nodes of the T cell activation markers CD69, CD25, and HLA-DR, also used for the in vitro analysis, was consistent with other data published in the literature [16] Fig. 4 ) and showed no statistically significant difference among groups in any of the analyses performed. In the CD8 T lymphocyte population from animals receiving therapy, there was a tendency for a decrease of HLA-DR-positive cells in the lymph nodes, regardless of HU treatment (Fig. 4 a). An opposite tendency was observed in peripheral blood. No difference in the CD69 and CD25 expression was observed among groups, both in peripheral blood and lymph nodes. No clear-cut difference was observed in the CD4 T lymphocyte population (Fig. 4 b).
Fig. 3: CD4 T cell count (a) and viral load (b) in SIV-infected monkeys. NT, untreated; HU+, treated with (R )-9-(2-phosphonylmethoxypropyl)adenine (PMPA), didanosine and hydroxyurea; HU−, treated with PMPA and didanosine.
Fig. 4: Effects of hydroxyurea (HU) on T cell activation in vivo CD8 T cells (a) and CD4 T cells (b) in peripheral blood and lymph nodes of SIV-infected monkeys untreated (white columns), treated with (R )-9-(2-phosphonylmethoxypropyl)adenine (PMPA) and didanosine (gray columns) and treated with PMPA, didanosine and HU (black columns).
Effect of hydroxyurea on virus-specific responses in vivo
Data from the cytofluorimetric analysis of IFN-γ on the CD8 T lymphocyte subset from the three groups of Rhesus macaques after SIV-specific stimulation is shown in Fig. 5 . In the untreated controls, no difference in the average IFN-γ-specific response was observed between baseline (0.2%) and the last observation (0.3%; 3 weeks after last treatment withdrawal). The group treated with HU showed a slight increment in IFN-γ production over the time course, from a baseline average of 0.2% to 0.9% after last treatment withdrawal. In Fig. 5 b–d, representative animals from each treatment group are shown. Dot plots represent IFN-γ production on CD8 T cells gated on CD3. The data acquired show a discrepancy between SIV-specific immune response and control media for each group of treatment at the baseline and last stop time points.
Fig. 5: Effects of hydroxyurea (HU) on antigen-specific activation and interferon-γ (IFN-γ) production in vivo CD8 T lymphocytes from Rhesus macaques untreated (NT), treated with (R )-9-(2-phosphonylmethoxypropyl)adenine (PMPA) and didanosine (HU−), and treated with PMPA, didanosine and hydroxyurea (HU+) at baseline (white circles) and 3 weeks after final treatment withdrawal (black circles). Each symbol represents the specific response calculated as differences between the zinc-finger-inactivated SIVmac239 and the control media value. (b–d) Representative dot plots for IFN-γ production to CD8 subpopulation gated on CD3 T lymphocytes for a representative animal from each treatment group: animal 812 in NT group (b); animal 776 in HU− group (c); and animal 191555 in HU+ group (d). For each animal, control media and SIV-specific stimulation are represented at baseline and the last stop time point.
Discussion
A cytostatic effect does not necessarily correlate with a state of immune suppression [1] in vivo and in vitro results support this hypothesis. In fact, no important differences in the expression of activation surface markers were noticed in T lymphocytes in vitro or in the peripheral blood and in the lymph node of the animals. On the contrary, a prolonged G1–S state might provide favorable conditions for the continued expression of cytokines involved in the immune response, as has been shown here. Similarly, a recent study has demonstrated that the transient prolongation of the G1 phase of the cell cycle by treatment with cytostatic drugs, including HU, resulted in the accumulation of β-chemokines (RANTES, macrophage inflammatory protein 1α and 1β) [17] in vitro and preclinical data, a recent study conducted by McElrath et al . [18] Candida ) and HIV-specific (p24, gp160) responses [18] [19]
The lack of an immunosuppressive effect by a cytostatic drug like HU may be explained by comparison with the mechanisms of action of an immunosuppressive drug such as rapamycin, an agent that inhibits T cell proliferation. Rapamycin induces a profound T cell anergy, reduces IL-2 production and blocks Il-3 and IFN-γ production. In contrast, HU does not cause anergy. This difference may be explained by the fact that rapamycin inhibits the cell division process at the late G1 phase, whereas HU blocks the cycle more distally (early S phase), thus permitting anergy rescue [20]
If HU decreases the number of HIV targets without blunting immune responses, one would expect a lower viremia while the drug is used and a delayed viral rebound when the drug is interrupted. Both assumptions have been confirmed with clinical data. The RIGHT 901 clinical protocol has shown that during structured treatment interruption the use of HU decreased the amplitude of viral rebound and led to more potent viral load suppression after therapy restart [15] [21]
There are other theoretical and practical advantages associated with the use of a cytostatic drug in HIV infection. It is becoming increasingly evident that the overactivation of the immune system is a key component of HIV pathogenesis, maybe the most important one [2,14,22–33] [34] P = 0.004) compared with a group treated with the same antiretroviral therapy without HU [35]
The results presented here show that although HU exerts a cytostatic effect it does not have a suppressive effect on T lymphocytes immunological functionality. Moreover, the recent dose-finding study (RIGHT 702) demonstrated that decreasing the dose of HU reduced toxic effects when it was combined with a nucleoside reverse transcriptase inhibitor [11] et al. unpublished data). At the optimal dosage of 600 mg daily, HU in combination with didanosine represents a durable ‘virostatic’ (antiviral and cytostatic) drug combination characterized by an excellent resistance profile [36] [15]
These new findings should generate renewed interest in HU, which remains a therapeutic agent with unique characteristics for the treatment of HIV-1 infection and could prove to be a valuable treatment option in a variety of clinical conditions, for example, as maintenance therapy with didanosine, as recently proposed [37]
Acknowledgements
We are grateful to Laurene M. Kelly for technical support and to Sylva Petrocchi for editorial assistance.
Sponsorship: This study was in part supported by a Istituto Superiore di Sanità grant (ISS 30D.46).
Note: Luca Lova and Antonella Groff contributed equally to the research described.
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