Published online before print April 2, 2007
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,1
* Fundació IrsiCaixa, HIVACAT, Hospital Universitari Germans Trias i Pujol, Badalona, Spain;
Systems Biology Department, Universitat de Vic, Vic, Spain; and
Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain
1 Correspondence: Fundació Irsicaixa, Hospital Universitari Germans Trias i Pujol, Ctra Canyet sn, 08916 Badalona, Spain. E-mail: mbofill{at}irsicaixa.es
ABSTRACT
IL-12 and IL-18 synergistically induce the production of IFN-
by resting and activated T cells. To evaluate whether this induction was affected in HIV-1-infected patients, PBMC or isolated CD4 T cells were cultured with IL-12 plus IL-18, anti-CD3 plus anti-CD28, or PHA for 72 h. Cell samples were labeled daily to assess the levels of IL-12 receptor ß1 (IL-12Rß1), IL-12Rß2, and IL-18R
. Culture supernatants were analyzed for the presence of Th1- and Th2-related cytokines by ELISA or cytometric bead array and analyzed by flow cytometry. A twofold increase in the percentage of CD4-resting T cells expressing IL-12Rß1 and IL-18R from HIV-1-infected patients was observed when compared with cells from HIV-1-negative donors. Higher IL-12Rß1 and IL-18R expression correlated (r=0.87; P<0.007) to increased production of IFN- by isolated CD4 T cells in the presence of IL-12 and IL-18. Moreover, exogenous IL-12 and IL-18 induced the up-regulation of IL-12Rß2 to twice higher in CD4 T cells from HIV-1-positive individuals compared with controls. Conversely, upon activation with anti-CD3 and anti-CD28 antibodies, only 25% of the CD4+ T cells from HIV-1 patients showed an increase in the IL-12ß2 when compared with 50% in healthy controls. Furthermore, the percentage of IL-12Rß1-positive cells correlated inversely with the CD4 nadir of patients, suggesting that deregulation of the IL-12 and IL-18 pathways may play a role in the immunopathogenesis of HIV-1 infection.
Key Words: IFN- • cytokine receptors • Th1/Th2 cells • cell activation
INTRODUCTION
One of the hallmarks of HIV-1 infection is a deregulation in the production of the cytokines IL-2 and IFN-. There is general agreement that the capacity of T cells or T cell clones from HIV-1-infected patients to produce IL-2 upon antigenic or mitogenic stimulation in vitro is diminished or impaired [1
, 2
]. Results about the production of IFN- are more controversial, as they have been reported to be increased [3
, 4
], unaltered [2
], or decreased [5
, 6
]—differences attributable to different experimental designs. In addition to CD4 Th1 T cells, other cell types, such as CD8 cells [7
] and NK cells [8
], are important sources of IL-2 and IFN-, contributing to the total amount detected in PBMC cultures or plasma of patients. Moreover, different antigenic stimuli may preferentially trigger the production of IL-2, IFN-, or both [9
, 10
]. Furthermore, CD4, CD8, and NK cells may be induced to produce IFN-, but not IL-2, under the direct and synergistic effect of IL-12 plus IL-18 in the absence of an antigenic stimulation [11
]. In addition, IL-12 and IL-18 are implicated in the differentiation of naïve Th cells to Th1 cells during antigenic priming [12
], interlinking innate and adaptative immune responses.
The receptors for IL-12 and IL-18 are composed of a cytokine-binding chain and a signaling chain IL-12 receptor ß1 (IL-12Rß1) and IL-12Rß2 [13
] and IL-18R and IL-18Rß [11
]; IL-12Rß2 and IL-18Rß represent the signaling receptor component. The presence of both chains is necessary for a functional receptor. The cytokine-binding chain of both receptors is constitutively expressed on a proportion of both CD4, CD8 memory T cells, and NK cells and is thought to be a marker for CD4 Th1 cells [14
, 15
]. The IL-12-Rß2 chain is present on NK cells but absent, or expressed at low levels, in resting T cells [15
]. IL-12Rß1, IL-12 Rß2 and IL-18R may be up-regulated upon antigenic, anti-CD3 plus anti-CD28 [16
], PHA or IL-12 plus IL-18 stimulation [17
]. It has been suggested that deregulation of the IL-12/IL-12R [18
19
20
] or IL-18/IL-18R pathways [21
, 22
] might contribute to an imbalance in the production of IL-2 and IFN- observed in HIV-1-infected patients [1
2
3
4
5
6
]. The production of IL-12 and IL-18 by PBMC and macrophages may be lower in HIV-1 infected patients than in HIV-1-uninfected individuals [18
, 21
, 23
]. Furthermore, HIV-1-infected patients may have diminished expression of IL-12R, indicated by low levels of IL-12R mRNA in PBMC, before and after mitogenic stimulation with anti-CD3 and anti-CD28, PHA, or 12-O-tetradecanoylphorbol 13-acetate [19
].
The current study investigates how HIV-1 infection may affect the capacity of CD4 T cells to respond to IL-12 and IL-18 through an antigen-dependent or -independent pathway by looking at the percentage of resting CD4 T cells expressing IL-12Rß1, IL-12 Rß2, and IL-18R and the capacity of CD4 T cells to up-regulate these receptors and to produce IFN- upon IL-12 plus IL-18 or CD3 and CD28 stimulation.
MATERIALS AND METHODS
Samples
PBMC from 38 HIV-1-uninfected donors and 90 HIV-1-infected patients were provided by the Centre de Transfusions i Banc de Teixits, Valle Hebron Hospital (Barcelona, Spain), and AIDS Unit, Hospital Universitari Germans Trias i Pujol (Badalona, Spain), after approval of the Ethical Committee of both centers. The procedures followed in the study were in accordance with the Helsinki Declaration in 1975, as revised in 1983, and all patients gave informed consent. Thirty percent of the HIV-1-positive patients had never taken antiretroviral drugs, and 70% were on highly active antiretroviral therapy during the study. Immunological and virological data of HIV-1-positive individuals are shown in Table 1
.
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Table 1. Immunological and Virological Parameters of HIV-1-Positive Individuals
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Detection of cytokine receptors
Cytokine receptors were measured in whole blood or isolated populations by a direct immunofluorescence method using four fluorochrome combinations of reagents and analyzed using a FACSCalibur cytometer (PharMingen, BD Biosciences, Cowle, Oxford, UK) [16
]. T cells were stained with anti-CD4, anti-CD45RA, or anti-CD45RO (PharMingen, Becton Dickinson, Oxford, UK) to identify naïve and memory T cells. The two subunits of the IL-12R and the chain of the IL-18R were detected by staining with anti-CD212 (IL-12Rß1, PharMingen, BD Biosciences), anti-IL-12Rß2 (PharMingen, BD Biosciences), and anti-IL-18R mAb (R&D Systems, Abington, UK). Anti-CD25 (Dako, Denmark) was used to identify the chain of the IL-2R. Chemokine receptors were measured with antibodies to CXCR3, CCR5, CCR6, CXCR4, and CCR7.
Stimulation of lymphocyte subpopulations
Mononuclear cells at a concentration of 2 x 106 cells/ml were plated in an anti-CD3-coated plate (1 µg/ml) and cultured in culture medium: RPMI 1640 (Gibco, Madrid, Spain) supplemented with 10% heat-inactivated FCS (Gibco), 100 U/ml penicillin, 100 µg/ml streptomycin, and anti-CD28 at 1 µg/ml (PharMingen, BD Biosciences) in the presence or absence of IFN- at a concentration of 100 ng/ml (R&D Systems). Cells were also stimulated with IL-12 (Peprotech, London, UK) at 100 ng/ml, IL-18 at 100 ng/ml (Peprotech) or both.
To measure the capacity of CD4 T cells to produce Th1- and Th2-type cytokines, isolated CD4 T cells at a concentration of 2 x 106 cells/ml were stimulated in the presence of PHA (0.5 µg/ml, Sigma Chemical Co., St. Louis, MO, USA) or in the presence of IL-12, IL-18, or both. Culture supernatants were collected at 48 h and at 1 week of culture and stored at –80°C until use.
Detection of cytokine levels
Culture supernatants
Cytokine (IL-2, IL-4, IL-6, IL-10, TNF- and IFN-) levels in cell supernatants were measured by cytometric bead array (CBA; human Th1/Th2 cytokine kit, Becton Dickinson, San Diego, CA, USA), as described previously [24
, 25
].
Plasma
Blood samples were collected using vacutainer tubes with K-EDTA as an anticoagulant. Plasma samples were separated by centrifugation (3000 rpm/10 min) and stored at –80°C until tested. Circulating cytokine levels were measured by the quantitative sandwich enzyme immunoassay (ELISA) for IFN- (OptEIA ELISA set, Becton Dickinson), IL-12 (R&D Systems) and IL-18 (RayBio, bioNova, Spain).
Statistical methods
Statistical analyses were conducted using SPSS Version 12.0 software and GraphPad PRISM Version 4.00 for Windows. The Kolmogorov-Smirnov goodness-of-fit test was used to check the normality assumption of the continuous variables, and those with normally distributed variables were compared by Student’s t-test when comparing two groups or by parametric ANOVA when comparing more than two groups. When normality did not hold, comparisons were performed by the nonparametrical Mann-Whitney test for two groups and the Kruskal-Wallis ANOVA for more than two groups. The post hoc Tamhane test was performed to identify significantly different group means when the ANOVA test was significant.
The study of kinetics of up-regulation of cytokine receptors involves the analysis of longitudinal data or repeated measurements, which correspond to the response variable measured at different times for each subject. The analysis of this data was performed by the so-called repeated measures ANOVA.
Cytokine production upon stimulation in vitro involves the analysis of censored data, which arise when the response variable can only be measured in a window; the present case is between 10 and 5000 pg/ml when using CBA for analysis, therefore, values greater than 5000 pg (the upper detection limit of the kit) were right-censored. Values less than 10 pg/ml were considered to be equal to zero, as this value essentially represents no cytokine production. Thus, the analysis of cytokine production was performed with statistical methods for right-censored survival data, such as the Kaplan-Meier nonparametric estimator and the log-rank test for comparing two groups.
RESULTS
Cytokine and chemokine receptor expression in IL-12Rß1+ and IL-18R+ CD4 T cells
The expression of chemokine receptors CXCR3, CCR4, CCR5, CXCR4 and CCR7 was used to further characterize the IL-12Rß1+ and IL-18R+ CD4 T cell subpopulations (Table 2
). Despite the fact that CCR3, CCR4 and CCR5 [25
26
27
] as well as IL-12Rß1 and IL-18R [14
, 15
] have been used to identify Th1 CD4 T cells, flow cytometry analysis revealed that only 50–70% of the IL-12R- and IL-18R-positive populations expressed these chemokine receptors. Likewise, CXCR4 and CCR7, involved in the homing of cell-to-lymphoid organs, were expressed in 
50% of these cells. There were no gross statistical differences in the chemokine receptor expression between HIV-1-infected patients and controls.
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Table 2. Percentages of Chemokine Receptors on CD4+, CD4+ IL-12Rß1+, and CD4+ IL-18R+ T cellsa
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(controls: 40.4±2.9; HIV-1: 57.0±1.7; Table 3
). Nevertheless, the expression of these receptors was similar between treated and untreated HIV-1 patients (data not shown), and these patients were grouped together. In resting CD4 T cells, the expression of IL-12Rß2 was found to be below the limits of detection in all individuals. |
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Table 3. Percentages of CD45RA+ and CD45RO+ CD4 T Cells Expressing the IL-12Rß1 and IL-18R
|
and CD45RA or CD45RO demonstrated an increased percentage of IL-12Rß1+ and IL-18R+ in memory and naïve CD4 subpopulations of HIV-1-positive individuals when compared with controls (Table 3)
.
Furthermore, a weak but significant, negative correlation was observed between the percentages of IL-12Rß1+ (r=–0.23, P=0.02) and IL-18R+ (r=–0.21, P=0.04) CD4+ T cells and the CD4 nadir (the lowest CD4 count measured in these patients).
Impaired capacity in HIV-1-infected individuals to up-regulate IL-12Rß2 upon anti-CD3 plus anti-CD28 stimulation
The capacity of CD4 T cells from HIV-1-uninfected and -infected individuals to up-regulate the IL-12R and IL-18R in response to IL-12 plus IL-18 and anti-CD3 and anti-CD28 stimulation was investigated. Stimulation with IL-12 and IL-18 neither affected the morphology nor the expression of CD25 in CD4 T cells. However, 50% and 20% increases in the percentage of cells expressing IL-12ß1 and IL-18R (Fig. 1
) were observed at 72 h of culture compared with unstimulated cells. Furthermore, after 72 h of culture, IL-12ß2 (absent in unstimulated cells) was up-regulated in a higher percentage of CD4 T cells from HIV-1-infected patients (mean±SEM=7.1±2.73) than in HIV-1-negative individuals (mean±SEM=2.7±0.37), although the difference between controls and patients was not statistically significant (Fig. 1)
.
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Figure 1. Effect of IL-12 and IL-18 on IL-12R and IL-18R expression. PBMC from nine HIV-1-negative (left panels) and 10 positive (right panels) individuals were cultured with medium supplemented with IL-12 and IL-18. The cells were stained with a combination of mAb to CD3, CD4 and IL-12Rß1, IL-18R, and or IL-12Rß2 after 0, 24, 48 and 72 h of culture and analyzed by flow cytometry. The figure shows the changes in percentages of CD4+ T cells expressing the different receptors at 0 and 72 h of culture and the P values refer to the statistical significance of these changes.
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to the anti-CD3- and anti-CD28-activated cell cultures restored the capacity of these cells to up-regulate IL-12Rß2 (from 26.3±5.37% to 49.8±5.83%), and cultures from HIV-1-negative individuals remained unaffected (Fig. 3)
.
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Figure 2. Up-regulation of IL-12R and IL-18R upon anti-CD3 and anti-CD28 stimulation. PBMC from HIV-1-negative (left panels) and HIV-1-positive (right panels) individuals were cultured in the presence of anti-CD3 and anti-CD28 antibodies for 72 h. The cells were stained with a combination of mAb to CD3, CD4 and IL-12Rß1 or IL-18R at time 0 (shaded histograms) or after 72 h of culture (open histograms) and analyzed by flow cytometry. The figure shows the results of a representative experiment from one control and one HIV-1-infected patient. (A–D) Dot-plot representation of the cells, where the x-axis represents the size (forward-scatter) and the y-axis, the granularity of the cells analyzed (side-scatter). The panels show the capacity of mononuclear cells from one HIV-1-negative (A and B) and HIV-1-positive (C and D) individual to increase in size upon CD3 and CD28 stimulation. (E–J) An overlay of the expression of IL-12Rß1 (E and F), IL-18R (G and H) or IL-12Rß2 (I and J) on gated CD3+ CD4+ T cells from one healthy control and one HIV-1-infected patient after 0 and 72 h of culture. Note the impaired capacity of CD4 T cells from an HIV-1-infected patient to up-regulate the IL-12Rß2 upon CD3 and CD28 stimulation (J, arrow).
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Figure 3. IL-12R and IL-18R expression upon stimulation with IFN- or anti-CD3 and anti-CD28. PBMC from nine HIV-1-negative (left panels) and 10 positive (right panels) individuals were cultured with medium or medium supplemented with IFN-, anti-CD3 (a-CD3) and anti-CD28 (a-CD28) or a combination of all for 72 h. The cells were stained with a combination of mAb to CD3, CD4, and IL-12Rß1 or IL-18R and/or IL-12Rß2 at times 0, 24, 48, and 72 h of culture and analyzed by flow cytometry. The figure shows the percentage of positive CD4 T cells expressing the different receptors. Statistical analysis showed that an increase in the percentage of positive cells for each of the receptors studied was statistically significant and higher than the cells cultured with IFN- or medium alone (P<0.01) in both groups of patients. In contrast, no statistical significance was observed when we compared the percentage of positive cells between HIV-1-negative and -positive individuals; the only exception was the up-regulation of the IL-12Rß2 chain (P<0.05).
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by CD4+ cells from HIV-1+ individuals stimulated with IL-12 plus IL-18, IL-4, and IL-10. Production of these cytokines was found to be triggered by PHA but not IL-12, IL-18, or both, except that in one sample from an HIV-1-infected patient, low levels of IL-6 and TNF- (38 and 126 pg/ml, respectively) were noted upon IL-12 and IL-18 stimulation. As expected, production of IFN- was observed in PHA and IL-12- plus IL-18-stimulated cultures, which was increased dramatically when the three stimuli were simultaneously present. Furthermore, in cell cultures from HIV-1-infected individuals, IFN- production was tenfold higher than in cells from uninfected individuals (uninfected: 2.0±1.33 ng/ml; infected: 20.6±9.24 ng/ml; P<0.01) when stimulated with IL-12 and IL-18 (Fig. 4
). (The levels of IFN- in the absence of stimulation were undetectable in HIV-1-positive and -negative individuals.)
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Figure 4. Effect of IL-12 and IL-18 on IFN- production. CD4-isolated T cells from five healthy controls (open bars) and six HIV-1-infected patients (solid bars) were cultured with medium supplemented with PHA, with IL-12 and IL-18, or with all three, as described in Materials and Methods. The production of IFN- released into the supernatant was measured after 48 h of culture. The y-axis represents the levels of IFN- expressed in pg/ml.
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in HIV-1+ individuals were likely related to the increased numbers of CD4 T cells expressing IL-12Rß1 reported above, as there was a strong correlation (r=0.85, P=0.007) between the presence of IL-12Rß1+ cells and levels of IFN- present in these cultures (Fig. 5
). In sharp contrast, the mean level of IFN- in the plasma from HIV-1-positive individuals (n=90) was 29% of that of uninfected individuals (n=38; controls: 23.7±8.9 pg/ml; HIV-1+: 6.8±2.12 pg/ml, P=0.001). In contrast with other reports [28
], lower levels of circulating IL-18 were also found in HIV-1-infected patients compared with healthy donors (controls: 0.8±0.44 ng/ml; HIV-1: 0.6±0.02 ng/ml, P<0.02; data not shown).
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Figure 5. Correlation between percentages of IL-12Rß1+ CD4 T cells and IFN- production. CD4-isolated T cells from healthy controls and HIV-1-infected patients were cultured in medium supplemented with IL-12 and IL-18 for 48 h, and the levels of IFN- released to the supernatant were measured by ELISA. The percentage of CD4 T cells expressing IL-12Rß1 before culture correlate significantly with the levels of IFN- produced by the CD4-purified cells.
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The production of IFN- by CD4 T cells can be triggered through the IL-12 and IL-18 pathway in an antigen-independent manner or through TCR upon antigenic stimulation. IL-12 and IL-18 cytokines play a pivotal role in both pathways. On the one hand, IL-12 and IL-18 can trigger the production of IFN- directly through the IL-12R and IL-18R; conversely, they are responsible for the differentiation of naïve CD4+ T cells into functional Th1 CD4+ cells during cell priming [12
]. As a result of the ongoing controversy regarding whether HIV-1 infection triggers deregulation of IFN- production [2
3
4
5
6
, 23
], these two pathways were investigated in this study for effects by HIV-1 infection.
In HIV-1-infected individuals, an increased percentage of resting CD4 T cells expressing the binding chain of the IL-12R and IL-18R was observed when compared with HIV-1-uninfected controls. These receptors are expressed exclusively in Th1-committed cells in vitro [14
, 15
] and are also increased in vivo in patients with common variable immunodeficiency disease [15
]. An increase in IL-12Rß1 and IL-18R was observed in CD4 CD45RA+ and CD45RO+ populations, despite the lack of IL-12Rß1 in the majority of CD45RA+ cells from controls. The results from this study suggest that during HIV-1 infection, there is an increased proportion of Th1 CD4 T cells with the potential to respond to IL-12 and IL-18. The results also highlight the difficulty of identifying immune cell function through the expression of cell differentiation markers. In the present study, a full overlap was not observed between cells expressing IL-12R and IL-18R and the other Th1-associated markers (CXCR3, CCR5, and CCR6) [26
, 27
, 29
] were only expressed in 50% of these cells. Whether these markers are expressed in different T cell populations or at different stages of differentiation remains to be determined. Approximately half of the CD45RA+ CD45RO– revertant memory cells identified by the lack of expression of CD62L, CD27 or CCR7 expresses the IL-12R and IL-18R [16
].
The increased percentage of positive cells for IL-12Rß1 was also reflected by an increased capacity of isolated CD4 T cells from HIV-1-infected patients to produce IFN- upon IL-12 plus IL-18 stimulation in vitro. This indicates that the IL-12R and IL-18R, upon IL-12 and IL-18 stimulation, are fully functional in HIV-1 infection, and concomitantly, the IL-12Rß2 chain (signaling chain of the IL-12R) was up-regulated in a higher proportion of cells from HIV-1-positive patients than negative individuals upon IL-12 and IL-18 stimulation (Fig. 1)
.
In contrast, CD4 T cells from HIV-1-infected patients with anti-CD3 and anti-CD28 failed to stimulate the expected up-regulation of the signaling chain of the IL-12R, as reported for stimulated mononuclear cells from HIV-1-infected patients [19 ]. Under these conditions, only 25% of the CD4+ cells from HIV-1-infected individuals acquired IL-12Rß2, compared with 50% of cells in the controls. In contrast, the up-regulation of the IL-12- and IL-18-binding chains of the two receptors was similar between cells of controls and patients.
Another important difference between the two stimulatory pathways was that IL-12 and IL-18 stimulation did not induce T cell activation, as measured by an increase in size, up-regulation of CD25 and stimulation of cell proliferation. In contrast with expansion of antigen-specific T cells, an increase in size and the up-regulation of CD25 were seen upon anti-CD3 and anti-CD28 stimulation. The T cells of two patients, who died in culture upon anti-CD3 and anti-CD28 stimulation, were excluded from the study. This anti-CD3/CD28 activation-induced cell death has been described previously [30 ].
Upon pathogen recognition, local macrophages and dendritic cells produce IL-12 and IL-18, which trigger the production of leukocyte-attracting cytokines CXCL9 and CXCL10 [25
] and possibly the production of IFN- by macrophages [31
]. CD4 T cells may respond to IL-12 and IL-18 in an antigen-independent manner. Alternatively, antigen-specific T cells up-regulate the IL-12R and IL-18R upon contact with APC, making these cells responsive to IL-12 and IL-18. The data collected in the current study suggest that the antigen-specific pathway may be affected by HIV-1 infection, and the antigen-independent pathway is enhanced.
It is interesting that we did not find any correlation between the levels of plasma IFN- and the levels of IL-12R- and IL-18R-positive cells in these patients. Unfortunately, the pathological significance of plasma levels of IFN- is difficult to understand, as total levels of IFN- reflect an overall production of several cells types, including T, NK, and CD8 T cells, in response to a wide variety of stimuli. Similarly, IL-18 is produced by a variety of cells including keratinocytes and damaged endothelium, which might account for the discrepancies in IL-18 levels reported by different groups [22
, 28
].
We also observed a negative correlation between the increased percentage of IL-12R-positive cells with the CD4 nadir (the lowest CD4 count recorded during disease progression) but not with the levels of CD4 T cells at the time of the assay. A low CD4 nadir is associated with disease [32 ], but the significance of this correlation in the pathogenesis of HIV-1/AIDS remains unclear.
In conclusion, our data suggest that the capacity of CD4 T cells to respond to IL-12 and IL-18 in the absence of antigenic stimulation is intact or enhanced during HIV-1 infection. However, it is likely that the amplification of antigen-specific responses by IL-12 and IL-18 may be at least partially impaired, thus contributing to the imbalanced IFN- production observed in HIV-1 infection.
ACKNOWLEDGEMENTS
This work was funded in part by the Fundacio Irsicaixa, the European TRIoH Consortium (LSHB-CT-2003-503480), and the Spanish MEC (BFU2006-00966) by Fundació Irsicaixa (FIS PI051897), Red Temática de Investigación en SIDA (RG GO3/173), and FIPSE. C. C. was supported by contract FIS 04/0027 from the Fundació per a la Recerca Biomédica Germans Trias i Pujol in collaboration with the Spanish Health Department. The authors thank the patients who volunteered for this study and the nurse staff who assisted at the site of the study.
Received November 27, 2006; revised March 8, 2007; accepted March 8, 2007.
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