Chemokine receptors are necessary and selective coreceptors of HIV-1 entry into target cells [1,2]. Macrophage-tropic isolates of HIV-1 mainly use CC-chemokine receptor (CCR)-5 [3,4], whereas the main receptor for entry by lymphocyte-tropic viruses is CXC-chemokine receptor (CXCR)-4 . In addition, an increasing number of other chemokine receptors, such as CCR-1, CCR-2b, CCR-3 have also been described to act as HIV-1 coreceptors in vitro [1,2]. The blockade of chemokine receptors by their natural ligands, the CXC-chemokine stromal cell-derived factor (SDF)-1agr; for CXCR-4 and the CC-chemokines RANTES, macrophage inflammatory protein (MIP)-1agr; and MIP-1β for CCR-5, inhibit HIV-1 replication in vitro [1,2].
The role of chemokine and chemokine receptors in HIV infection and AIDS progression has been highlighted by different reports [6-13]. It seems clear that polymorphisms in the CCR-5 gene leading to the lack of cell surface receptor have a protective effect [6-10]. In contrast, more controversial results have been reported on CCR-2b polymorphism [9,10], and little or nothing is known about the other potential HIV-1 coreceptors. On the other hand, the protection of highly exposed haemophiliacs to HIV-1 infection correlates with high levels of CC-chemokine production . Conversely, more contradictory results have been reported on the protective effect of a mutation in the SDF-1agr; gene [12,13]. The expression of chemokines and their receptors is regulated upon activation of peripheral blood mononuclear cells (PBMC) by different stimulus, showing upregulation after interleukin (IL)-2-mediated proliferation [14,15] or downregulation after treatment with anti-CD3/anti-CD28 antibodies . Therefore, modulation of immune status in vivo could regulate HIV-1 replication by modifying the chemokine or chemokine receptor system.
Immunomodulation with IL-2 is one of the complementary treatments used in combination with antiretroviral therapy in HIV-1-infected individuals [16-18]. During HIV-1 infection, IL-2 production by PBMC is significantly reduced ; thus, administration of IL-2 is expected to improve immune responses by stimulating immunocompetent cells to proliferate . Moreover, activation of latently infected cells by administered IL-2 may favour their elimination. Consistent with these positive effects, several studies have noted that treatment with IL-2 significantly increases the number of CD4+ T lymphocytes in HIV-1-infected individuals [16-18]. However, the effects of IL-2 on the immune system may also modify the pool of HIV-1 target cells by triggering not only proliferation but also expression of chemokine receptors that serve as coreceptors for the entry of HIV into CD4+ T cells or monocytes. In vitro studies have demonstrated that IL-2 has opposite effects on the expression of these receptors in different blood populations. On the one hand, IL-2 is able to increase CXCR-4 and CCR-5 expression in CD4+ T cells ; on the other hand, IL-2 downmodulates the cell surface expression of CCR-5 and CD4 in in vitro-differentiated macrophages . Despite the existence of abundant data concerning in vitro effects of IL-2, little is known about the effect of IL-2 administration on the expression of chemokines and chemokine receptors in vivo.
In order to address this latter point we have determined the expression of chemokine receptors CXCR-4 and CCR-5 in CD4+ T cells and monocytes of HIV-1-infected individuals treated with low doses of IL-2 in combination with highly active antiretroviral therapy (HAART). The expression of other chemokine receptors (CCR-1, CCR-2b and CCR-3) and the CC-chemokines RANTES, MIP-1agr; and MIP-1β were also studied. Results were compared with a control group of patients treated with equivalent antiretroviral therapy and with values obtained from healthy donors.
Materials and methods
Twenty five patients with CD4 cell counts below 250×106/l and viral load below 500 copies/ml were included in a randomized, open-label study of the effect of low-dose subcutaneous IL-2 on HIV-1 infection. All patients responded to HAART and showed stable and low viral load (<500 copies/ml) for at least 6 months. In 12 patients, HAART was supplemented with cycles of 3×106U IL-2 once daily for 5 days, every 4 weeks, for a period of 24 weeks (six cycles). After each cycle, blood samples were collected. Eighteen out of 25 patients completed the study, eight from the IL-2-treated group and 10 from the control group. All patients showed controlled viral load and sustained increase in CD4+ T-cell counts, which were higher in the IL-2-treated group . More details on the immune status of both groups will be published elsewhere (Arnó et al., manuscript submitted).
An external group of healthy donors from the Blood Bank of the Hospital Universitari Germans Trias i Pujol was used as an additional control group.
Determination of serum levels of chemokines
Serum samples were processed immediately after blood was drawn. Briefly, blood aliquots were allowed to stand for 30min at room temperature and centrifuged at 1000g for 10min. Serum samples were stored at -80°C until use. Concentration of RANTES, MIP-1agr; and MIP-1β were determined by enzyme-linked immunosorbent assay using the respective Quantikine Immunoassay kit from R&D Systems (Minneapolis, Minnesota, USA). Sera were diluted 50-fold to determine the levels of RANTES and were used undiluted in MIP-1agr; and MIP-1β determinations.
Production of chemokines by PBMC
PBMC were obtained by Ficoll-Hypaque density gradient and cryopreserved in liquid nitrogen until use. Cells from control and treated patients were cultured at a density of 106 cells/ml in RPMI medium supplemented with 10% fetal calf serum in the presence or the absence of 3μg/ml phytohaemagglutinin (PHA; Sigma, Spain) for 48h. The production of RANTES, MIP-1agr; and MIP-1β was determined in culture supernatants as indicated above.
Measure of cell-surface expression of chemokine receptors
Cell-surface expression of chemokine receptors CXCR-4 and CCR-5 was determined by FACS analysis. Antibodies used were as follows: fluorescein isothiocyanate (FITC)-coupled anti-CD3 monoclonal antibody (MAb), phycoerythrin (PE)- or PerCP- coupled anti-CD4 MAb, PE-coupled anti-CD45RO MAb and PE-coupled anti-CD14 MAb (Becton Dickinson, Mountain View, California, USA); anti-CXCR-4 MAb 12G5, isotype IgG2a (R&D Systems); anti-CCR-5 MAb 2D7, isotype IgG2a (obtained through the AIDS Research and Reference Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health from Leukosite, Inc.); and isotype control antibody and FITC-coupled goat antimouse MAb (Southern Biotechnologies, Inc., Birmingham, Alabama, USA). For the study of chemokine receptor expression, 0.5×106 cells were washed in phosphate-buffered saline (PBS) and incubated for 30min at 4°C in a total volume of 50μl with MAb 12G5 (1μg/ml), 2D7 (0.5μg/ml) or with isotype control MAb. Bound antibodies were revealed with FITC-labelled goat antimouse MAb (1μg/ml), and cells were further incubated with PE-coupled Leu3a MAb. After incubation with each antibody, cells were exhaustively washed with ice-cold PBS, and prior to analysis, cells were fixed in PBS containing 1% formaldehyde. For each sample, 10000 events were analysed in a FACScalibur (Becton Dickinson) using the CellQuest software (Becton Dickinson).
Three-colour FACS analysis was used to study expression of CXCR-4 by CD4+ naive and memory cells and to confirm CD3 expression of CD4+ T cells and CD14 expression of monocytes. These populations were identified by CD4 staining and side-scatter, as described previously ; under these assay conditions, more than 95% of CD4+ T cells were shown to express CD3 and more than 90% of monocytes were CD14+. The expression of chemokine receptors was evaluated by measuring the percentage of positive cells and the relative fluorescence intensity, as described previously .
Reverse transcriptase polymerase chain reaction determination of chemokine receptor expression
The expression of chemokine receptors CCR-1, CCR-2b, CCR-3 and CXCR-4 was assessed by reverse transcriptase (RT) polymerase chain reaction (PCR) analysis. Total RNA was extracted from 2×106 PBMC using the one-step Trizol solution (Gibco BRL, Barcelona, Spain). Prior to reverse transcription, RNA was treated with RNase-free DNase (1U for 45min at 37°C). DNase was inactivated and RNA was denatured by heating samples at 70°C for 3min, then RNA was retrotranscribed using SUPERSCRIPT II RNaseH RT (Gibco BRL) primed with oligo(dT), according to the manufacturer‚s recommendations. For each sample, negative controls were performed by amplifying non-retrotranscribed RNA.
Chemokine receptor cDNA was amplified from retrotranscribed material (2μl) using the following primers: C1S (5′-TGGAAACTCCAAACACCACAG-3′) and C1A (5′-CCCAGTCATCCTTCAACTTG-3′), which yield a 296 base-pair band corresponding to CCR-1 cDNA ; CKR2 1A (5′-TTGTGGGCAACATG ATGG-3′) and CKR2 1Z (5′- GAGCCAACA ATGGGAGAGTA-3′), which yield a 128 base-pair band corresponding to CCR-2 cDNA ; C3S (5′-TGACAACCTCACTAGATATAGTTG-3′) and C3A (5′- CTCTTCAAACAACTC TTCAGT-3′), which yield a 540 base-pair fragment corresponding to CCR-3 cDNA ; and F2S (5′-TGACTCCATGAAGG AACCCTG-3′) and F2A (5′-CTTGGCCTCTGACT GTTGGTG-3′), which yield a 381 base-pair fragment corresponding to CXCR-4 cDNA . Primers G2S (5′-ATGGAGAAGGCTGGGGCTC-3′) and G2A (5′-AAGTTGTCATGGATGACCTTG-3′), yielding a 190 base-pair fragment, were used to amplify glyceraldehyde-3-phosphate dehydrogenase cDNA, which was used as a control . Primer-amplified products were run on 2% agarose gels, stained with ethidium bromide and quantified using the Molecular Analyst software (BioRad, Hercules, California, USA).
Statistical analysis was performed using the two-sided Student‚s t-test. P values <0.05 were considered to indicate statistical significance.
Serum levels and in vitro production of chemokines
In vitro IL-2/CD3 stimulation of both CD4+ and CD8+ T cells leads to increased production of the chemokines RANTES and MIP-1agr; . Since it could be expected that low subcutaneous doses of IL-2 act in vivo in a similar way, we determined the serum levels of RANTES, MIP-1agr; and MIP-1β in HIV-1-infected patients treated with IL-2, in the control group of HIV-1-infected patients on HAART, and in an external group of healthy uninfected donors. Fig. 1a shows the time course of serum RANTES levels in the IL-2-treated group and the control group on HAART. Values corresponding to the IL-2-treated group were similar to those obtained in the untreated group [46±21ng/ml (n=8) and 46±13ng/ml (n=10), respectively, after 24 weeks of treatment]. Moreover, these concentrations of RANTES were indistinguishable from those found in healthy donors (49±26ng/ml; n=8). The levels of MIP-1agr; and MIP-1β were also studied; however, most samples showed undetectable concentrations of these chemokines. For this reason we evaluated the number of positive samples, defined as the samples whose absorbance was 0.05U higher than negative control values. No differences were found for either MIP-1agr; or MIP-1β in the number of positive samples in IL-2-treated and control groups at 0 and 24 weeks of treatment (data not shown).
The lack of effect of IL-2 treatment on serum levels of chemokines led us to evaluate the in vitro production of chemokines by PBMC obtained from IL-2-treated and control patients at baseline and at the end of treatment. Cells were cultured in the absence or the presence of PHA in order to evaluate the potential effect of IL-2 in basal and stimulation-induced levels of chemokine production. We performed PHA stimulation rather than IL-2 stimulation, because in vivo IL-2 treatment can induce a bias in chemokine production values. In fact both groups showed significantly different levels of CD25 expression, which was higher in the IL-2-treated group (Arnó et al., manuscript submitted). The production of RANTES, MIP-1agr; and MIP-1β by unstimulated cells was not significantly modified by IL-2 treatment (Fig. 1b). The production of these chemokines by PHA-stimulated cells showed slight but no significant increases of MIP-1agr; and MIP-1β after 24 weeks of IL-2 treatment.
Expression of chemokine receptors CXCR-4 and CCR-5
Like chemokines, their counterparts, chemokine receptors, are strongly regulated by in vitro treatment of T cells or monocyte-derived macrophages with IL-2 [14,21]. In order to assess the effect of IL-2 treatment in vivo, we studied the expression of the main chemokine receptors involved in HIV-1 infection, CXCR-4 and CCR-5, in both CD4+ T cells and monocytes. Studies were performed by FACS analysis of cells stained with PE-labelled anti-CD4 Leu3a antibody and FITC indirect labelling of either CXCR-4 or CCR-5. Plotting fluorescence associated with Leu3a versus side-scattering allows easy detection of CD4+ T cells (Fig. 2), by their low side-scattering and high Leu3a binding, and monocytes, which are characterized by higher side-scattering values and lower CD4 staining .
Values of CXCR-4 expressing CD4+ T cells did not show differences at 0 and 24 weeks in the control group (43±22% versus 44±19%; Fig. 3). In contrast, IL-2-treated patients showed increased percentage of CXCR-4-expressing cells (32±24% at 0 weeks versus 54±25% at 24 weeks of treatment; P=0.0771; Fig. 3). Similarly, the percentage of CCR-5-expressing cells was not modified in the control group (64±22% at 24 weeks versus 60±12% at 0 weeks; Fig. 3). However, a slight but not significant increase was observed in IL-2-treated patients (53±27% at 24 weeks versus 43±31% at 0 weeks; P=0.2647; Fig. 3). The increase in the expression of CXCR-4 was correlated to the increase in CCR-5 expression in the IL-2-treated group. Although the data showed a poor correlation coefficient (r2=0.345), five out of eight patients showed increased expression of both CXCR-4 and CCR-5 in CD4+ T cells.
We also evaluated the intensity of labelling, by calculating the ratio between the signal associated with receptor labelling and background signal (Fig. 2, Table 1). Using this method, the increase in CXCR-4 expression was significant in CD4+ T cells (3.1±0.8 compared with 2.3±0.4 at 24 and 0 weeks, respectively; P<0.05; Table 1). However, IL-2-treated patients showed lower CXCR-4 expression at baseline compared with the control group. This difference could also favour the observed increase in CXCR-4 expression. No modifications were observed in mean fluorescence intensity (MFI) ratios of CCR-5 expression in the control and IL-2-treated groups. Moreover, the expression of both CXCR-4 and CCR-5 in monocytes, assessed by calculating the number of positive cells and MFI ratios (Table 1), was not modified in either the control or the IL-2-treated group.
The increase in CXCR-4 expression observed in the IL-2-treated group was consistent with the enhancing effect of IL-2 on the expression of chemokine receptors observed in vitro . However, given the almost exclusive expression of CXCR-4 in the naive population of T cells , this increase may also be the consequence of the changes in naive and memory CD4+ subsets. In order to evaluate this possibility, we studied the expression of CXCR-4 in these CD4+ T-cell subsets (Table 2). Our data indicated that both populations expressed CXCR-4, although naive cells showed significantly higher levels (Table 2). These data also revealed a significant increase in the expression of CXCR-4 in naive cells in the IL-2-treated group (38±17% at baseline versus 54±15% at the end of the treatment; P<0.05). This increase, which was not observed in the control group (51±27% compared with 50±12% at baseline and 24 weeks, respectively), suggests that effects of IL-2 on chemokine receptor expression by CD4+ T cells can contribute to the final observed increase in CXCR-4.
Effect of IL-2 on the expression of other chemokine receptors
Besides CXCR-4 and CCR-5, other chemokine receptors, such as CCR-1, CCR-2b and CCR-3 have been shown to act as potential coreceptors in the HIV entry process [1,2]; however, little or nothing is known about their role in HIV-1 infection in vivo. We studied the expression of these receptors by RT-PCR in both groups of patients and in PBMC from healthy donors after in vitro IL-2 stimulation. Although in vitro IL-2-stimulated cells showed a general increase in chemokine receptor expression (data not shown) when the expression of these receptors was studied in IL-2-treated patients, no clear increases were observed. Fig. 4 shows representative results obtained from IL-2-treated and control patients, showing a high variability in the pattern of chemokine receptor expression. In fact, although an increase in the expression of CCR-1, CCR-2 and CCR-3 can be observed in some of the IL-2-treated individuals, this is not a general behaviour, and can be also observed in control patients. In order to validate these results, we also studied the expression of CXCR-4 by RT-PCR and compared it with the data obtained from FACS analysis. The increase in CXCR-4 mRNA levels was clear in three IL-2-treated patients and was not observed in control patients (Fig. 4), thus confirming the effect of IL-2 on chemokine receptor expression. In contrast, no correlation was observed between increased expression of CXCR-4 and the modification in the expression of CCR-1, CCR-2b and CCR-3 receptors. Therefore, the changes observed in the expression of these chemokine receptors did not appear to be directly associated with the IL-2 treatment.
A randomized, open-label, study of low-dose (3×106 IU daily) subcutaneous IL-2 in HIV-1-infected patients was carried out in order to determine the effect of this cytokine on the immune regeneration of HIV-infected individuals successfully treated with HAART (i.e., showing <500 HIV RNA copies/ml). In addition to the beneficial effects on the immune system , we addressed the side-effects that IL-2 administration might have on the expression of chemokine and chemokine receptors, which could influence the growth/selection of different strains of HIV.
Several reports have shown that viral load does not affect plasma levels of RANTES , and only abnormally high levels of serum RANTES have been reported in progressors with CD4+ cell decline higher than 100×106/l per year . Nevertheless, the effect of IL-2 administration had not been studied previously. Our results showed that low-dose IL-2 did not cause a long-term increase in the serum levels of the CC-chemokines RANTES, MIP-1agr; and MIP-1β. In addition, production of these chemokines by unstimulated and PHA-stimulated PBMC did not show differences between groups at baseline and after treatment. Although punctual increases of chemokine production could occur during IL-2 administration cycles, no effects are observed in this long-term study.
The expression of the main coreceptors of HIV-1, CXCR-4 and CCR-5 did not increase dramatically after IL-2 treatment. However, higher levels of both the percentage of CXCR-4-expressing CD4+ T cells and the intensity of this expression were observed in the IL-2-treated group. A lower increase was also observed in CCR-5 expression. Although the virological status (i.e., low but detectable viral load) might influence expression of chemokine receptors [29,30], two main factors seem to be responsible for the variations observed in this study. First, IL-2 could induce an increase in CXCR-4 and CCR-5, as it has been described in in vitro stimulation of PBMC . Second, changes in naive and memory T-cell subsets can modify the expression of CXCR-4 and CCR-5, since these receptors appear to be associated with naive and memory phenotype, respectively . Both possibilities were evaluated by studying the expression of CXCR-4 in these subsets (Table 2), and by determining the effect of IL-2 on the expression of CXCR-4 mRNA (Fig. 4). Our data suggest that the changes in naive subset cannot completely explain the observed increase in CXCR-4 expression; therefore, effects of IL-2 on the level of CXCR-4 expression should also contribute to the final observed increase. Moreover, the increase in naive cells would induce a loss in CCR-5 expression, which is characteristic of memory cells . Such a decrease is not observed in the IL-2-treated group (Table 1). Our results also show that the expression of CCR-5 and CXCR-4 in monocytes is not modified by IL-2 treatment. This is in conflict with a recent report describing a negative effect of IL-2 on the expression of both CD4 and CCR-5 in monocyte-derived macrophages . However, as with the CD4+ T-cell population, the in vivo effect of IL-2 on monocytes (Table 1) is expected to be weaker than in vitro observations would suggest [14,21].
Regardless of IL-2 treatment, it is interesting to note the high levels of expression of CCR-5 observed in HIV-1-infected individuals. By analysing data from both control and IL-2-treated groups, 50±26% (n=18) of CD4+ T cells stained positive for CCR-5 (Fig. 3), whereas in uninfected healthy donors only 14±12% (n=8) of positive CD4+ T cells expressed CCR-5 (data not shown). This significant difference (P<0.005) might be the consequence of the continuous stress that the immune system undergoes during HIV infection. A similar observation has been recently reported by Andersson et al. , who described higher expression of CCR-5 and CXCR-4 in tonsil tissue from HIV-infected individuals compared with healthy donors, even after strong reduction of viral load during HAART. Our results suggest that high levels of CCR-5 expression could also be observed in PBMC from HIV-infected individuals after HAART. This fact has been recently confirmed by others , although a concomitant decrease in CXCR-4 expression was also reported. From our data, the number of CD4+ T cells expressing CXCR-4 in PBMC from HIV-infected individuals was not significantly different from the values observed in healthy donors [38±26% (n=18) versus 35±12% (n=8)].
Other chemokine receptors involved in HIV-1 infection, such as CCR-1, CCR-2 and CCR-3, did not undergo relevant changes in expression upon IL-2 treatment; in contrast, a significant increase in the expression of these receptors was observed when PBMC from healthy donors were stimulated in vitro with IL-2. The results obtained from control and IL-2-treated patients did not correlate to increases in CXCR-4 expression (Fig. 4). Therefore, these changes did not appear to be a direct consequence of IL-2 treatment and could be due to differences in immune status.
The increased CXCR-4 expression by CD4+ T cells might favour the in vivo replication of the more pathogenic T-cell-tropic isolates of HIV-1. However, the limited extent of this increase and the strong control of HIV replication exerted by HAART make this possibility unlikely. Taken together, our results suggest that administration of low doses of IL-2 does not induce drastic changes in the chemokine/chemokine receptor system in vivo. The production and serum level of chemokines able to block macrophage-tropic HIV isolates was not significantly modified during IL-2 treatment (Fig. 1). Similarly, the in vivo effect of IL-2 on the number of cells susceptible to HIV-1 infection was much more modest than previous data obtained using in vitro models [14,22]; the low doses of IL-2 administered in this assay could partially explain these results. However, it should be noted that the dose of IL-2 used in this study (3×106 IU once daily) seems to be sufficient to induce significantly higher CD4+ T-cell recovery in the IL-2-treated group than in the control group .
In conclusion, our data argue against the existence of strong IL-2 effects favouring HIV spread. Therefore, the efficacy of IL-2 immunomodulation in combination with HAART in the treatment of HIV infection has to be evaluated by determining the beneficial effects of IL-2 on the activation and reconstitution of the immune system.
The authors thank Ana Maria García and Arantxa Gutiérrez for excellent technical assistance.
1. D‚Souza MP, Harden VA: Chemokines and HIV-1 second receptors. Nat Med
2. Premack BA, Schall TJ: Chemokine receptors: gateways to inflammation and infection. Nat Med
3. Deng H, Liu R, Ellmeier W, et al.
: Identification of a major coreceptor for primary isolates of HIV-1. Nature
4. Dragic T, Litwin V, Allaway GP, et al.
: HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature
5. Feng Y, Broder CC, Kennedy PE, Berger EA: HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science
6. Liu R, Paxton WA, Choe S, et al.
: Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply exposed individuals to HIV-1 infection. Cell
7. Samson M, Libert F, Doranz BJ, et al.
: Resistance to HIV-1 infection in Caucasian individuals bearing mutant alleles of the CCR5 chemokine receptor gene. Nature
8. Quillent C, Oberlin E, Braun J, et al.
: HIV-1-resistance phenotype conferred by combination of two separate inherited mutations of CCR5 gene. Lancet
9. Michael NL, Louie LG, Rohrbaugh AL, et al.
: The role of CCR5
and CCR2 polymorphisms in HIV-1 transmission and disease progression. Nat Med
10. Smith MW, Dean M, Carrington M, et al.
: Contrasting genetic influence of CCR2
variants on HIV-1 infection and disease progression. Science
11. Zagury D, Lachgar A, Chams V, et al.
: C-C chemokines, pivotal in protection against HIV type 1 infection. Proc Natl Acad Sci USA
12. Winkler C, Modi W, Smith MW, et al.
: Genetic restriction of AIDS pathogenesis by an SDF-1 chemokine gene variant. Science
13. Mummidi S, Ahuja SS, Gonzalez E, et al.
: Genealogy of the locus and chemokine system gene variants associated with altered rates of HIV-1 disease progression. Nat Med
14. Bleul CC, Wu L, Hoxie JA, Springer TA, Mackay CR: The HIV coreceptors CXCR4 and CCR5 are differentially expressed and regulated on human T lymphocytes. Proc Natl Acad Sci USA
15. Loetscher P, Seitz M, Baggiolini M, Moser B: Interleukin-2 regulates CC chemokine receptor expression and chemotactic responsiveness in T lymphocytes. J Exp Med
16. Schwartz DH, Skowron GS, Merigan TC: Safety and effects of interleukin-2 plus zidovudine in asymptomatic individuals infected with human immunodeficiency virus. J Acquir Immune Defic Syndr
17. Kovacs JA, Baseler M, Dewar RJ, et al.
: Increases in CD4 T-lymphocytes with intermittent courses of interleukin-2 in patients with human immunodeficiency virus infection. A preliminary study. N Engl J Med
18. Jacobson EL, Pilaro F, Smith KA: Rational interleukin 2 therapy for HIV positive individuals: daily low doses enhance immune function without toxicity. Proc Natl Acad Sci USA
19. Westby M, Marriott JB, Guckian M, Cookson S, Hay P, Dalgleish AG: Abnormal intracellular IL-2 and interferon-gamma (IFN-γ) production as HIV-1-associated markers of immune dysfunction. Clin Exp Immunol
20. Nourse J, Firpo E, Flanagan RJ, et al.
: Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature
21. Kutza J, Hayes MP, Clouse KA: Interleukin-2 inhibits HIV-1 replication in human macrophages by modulating expression of CD4 and CC-chemokine receptor-5. AIDS
22. Ruiz L, Arnó A, Juan M, et al.
: Efficacy of low dose subcutaneous interleukin 2 in HIV-1 advanced patients with CD4 counts <250 cells/mm3 and undetectable plasma viral load. Second International Workshop on Drug Resistance and Treatment Strategies.
Lake Maggiore, Italy, June 1998 [abstract 139].
23. Mo H, Monard S, Pollack H, et al.
: Expression patterns of the of the HIV type 1 coreceptors CCR5 and CXCR4 on CD4+ T-cells and monocytes from cord and adult blood. AIDS Res Hum Retroviruses
24. Mondor I, Ugolini S, Sattenteau QJ: Human immunodeficiency virus type 1 attachment to Hela cells is CD4 independent and gp120 dependent and requires cell surface heparans. J Virol
25. Fear WR, Kesson AM, Naif H, Lynch GW, Cunningham AL: Differential tropism and chemokine receptor expression of human immunodeficiency virus type 1 in neonatal monocytes, monocyte derived macrophages and placental macrophages. J Virol
26. Clerici M, Balotta C, Trabattoni D, et al.
: Chemokine production in HIV-seropositive long-term asymptomatic individuals. AIDS
27. Weiss L, Si-Mohamed A, Giral P, et al.
: Plasma levels of monocyte chemoattractant protein-1 but not those of macrophage inhibitory protein-1agr; and RANTES correlate with virus load in human immunodeficiency virus infection. J Infect Dis
28. Zanussi S, D‚Andrea M, Simonelli C, Tirelli U, De Paoli P: Serum levels of RANTES and MIP-1agr; in HIV-positive long-term survivors and progressor patients. AIDS
29. Andersson J, Fehniger TE, Patterson BK, et al.
: Early reduction of immune activation in lymphoid tissue following highly active HIV therapy. AIDS
30. Ostrowski MA, Justement SJ, Catanzaro A, et al.
: Expression of chemokine receptors CXCR4 and CCR5 in HIV-1-infected and uninfected individuals. J Immunol