Infection caused by HIV-1 is characterized by a gradual depletion of the CD4 T lymphocytes, which determines disease progression until the development of AIDS and can increase the occurrence of opportunistic infections and malignancies . Besides the decrease in CD4 T cell number, there is also functional damage to these lymphocytes, which is partly related to the virus-induced dysregulation in cytokine network [1–4].
Macrophages and T lymphocytes from HIV-infected patients are important sources of chronic cytokine production, which has great impact in clinical progression [4–7]. Pro-inflammatory cytokines, for example, can favour intense HIV replication [3–7]. Studies have shown that HIV-derived Nef protein activates macrophages to secrete a variety of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-1α, IL-12, macrophage colony-stimulating factor, granulocyte–macrophage colony-stimulating factor and tumour necrosis factor (TNF)-α, all resulting in attraction of nonspecific CD4 T cells to the area of HIV replication [4–6]. These pro-inflammatory mediators, which are elevated during HIV-1 infection, also diminish the threshold of CD4 T cell activation and, consequently, favour HIV replication as well as activation-induced cell death [2–6].
As infection proceeds, however, many patients show an abnormal cytokine pattern characterized by increased secretion of IL-4 and IL-10 and decreased production of cytokines by T helper 1 cells (Th1), such as IL-2 and interferon γ (IFN-γ) [8,9]. In this phase, the lack of Th1 cytokines makes individuals susceptible to both opportunistic infections and malignancies. Therefore, in the absence of therapy, the HIV-1-related AIDS is uniformly fatal.
The marked decrease in the prevalence of AIDS-related diseases following the introduction of HAART relates to the control of plasma viral load and an increase in peripheral CD4 cell counts . Many studies with HAART-treated HIV-infected young adults have shown that the risk for some opportunistic infection is reduced when patients achieve a certain increased CD4 cell count [11–15]. However, in addition to increasing CD4 cell count, the impact of HAART on the degree of functional immune recovery is also related to reduction of the hyperactivation state that is observed in chronically HIV-1-infected patients [16,17]. Normalization in pro-inflammatory cytokines and recovery of IFN-γ secretion has been detected in some patients following successful HAART [17,18]. However, several host variables, particularly age, have been shown to correlate with the degree of immune restoration during HAART [19,20]. As described in a recent report, the accumulative number of AIDS cases in adults over 50 years in age had a five-fold increase worldwide from 1900 to 2001 [21,22] which confirms the need for more research on this particular population. The influence of age on the numeric CD4 T cell response to HAART was examined in the EuroSIDA study with 1956 patients after 31 months of treatment . The mean increase in CD4 cells during the first 6 months on HAART was lower in patients over 50 years, despite a better virological response in this group. Immune function status in this group of AIDS patients requires further elucidation.
The present study aimed to characterize age-related immune functional events in HIV-infected patients successfully treated with HAART and their impact on viral load. Lymphoproliferation and cytokine production induced by polyclonal activators was examined in vitro in cells from adult young and aged AIDS patients and correlated with in-vitro HIV-1 replication.
The study examined a small group of 16 individuals over 55 years old (median, 58.9 years; range, 55–65) who had chronic HIV-1 infection and were successfully treated with antiretroviral therapy. A second group included 16 young HIV-1-infected patients (median, 31.1 years; range, 22–38), with similar infection characteristics. All individuals were recruited from the Centre of Epidemiology at the Hospital of the State University of Rio de Janeiro. The consent for participation was obtained from all, and the study was approved by Ethic Committee from University Hospital of the State University of Rio de Janeiro, Brazil.
Characteristics of the HIV-infected individuals were obtained from medical records, including the HAART regimens and CD4 cell count at baseline and 24 months after the beginning of antiretroviral therapy. CD4 cell counts are expressed as absolute values and the viral loads are given as log10 values in order to normalize distribution. To avoid problems concerning multidrug failure, all the participating individuals were previously naive to antiretroviral drugs. Patients with chronic clinical complications other than HIV-1 infection, such as cancer, or those in use of immunomodulating drugs were excluded.
Cell culture and stimulation
Blood samples were collected from the HIV-1-infected patients approximately 2 years after they had started HAART, and the peripheral blood mononuclear cells (PBMC) were collected by centrifugation on Ficoll–Hypaque gradients as previously described . For some experiments, the CD4 cell-depleted PBMC or purified CD4 T cells were obtained by negative selection using magnetic beads coated with anti-CD4 monoclonal antibody or a kit containing antibody mixes to antigens targeted CD8, HLA-DR/DP, CD56, CD14, anti-CD19, CD36 and CDW123 (Dynal Biotech, Great Neck, New York, USA), respectively. The efficacy of this procedure was approximately 97% as evaluated by flow cytometry (data not shown). The number of viable cells for each condition was measured by Trypan blue exclusion in a haemocytometer. The viable cells were adjusted to a concentration of 1 × 105 cells/well and were cultured in a 96-well flat bottom microtitre plates with 200 μl RPMI 1640 added with 2 mmol/l L-glutamine (GIBCO, Carlsbad, California, USA), 10% fetal calf serum, 20 U/ml penicillin, 20 μg/ml streptomycin and 20 mmol/l HEPES buffer. These cultures were stimulated with plate-bound anti-CD3 (OKT3; 5 μg/ml) in the absence or presence of recombinant human IL-2 (rhIL-2) at 20 U/ml (BD Systems, Minneapolis, Minnesota, USA). The cells were cultured for 3 days at 37°C in a humidified 5% CO2 incubator for proliferation and cytokine assays.
Approximately 1 × 105 cells/well of the cell cultures containing PBMC or CD4 T lymphocytes were activated or not with anti-CD3 (5 μg/ml), with or without rhIL-2 (20 U/ml) for 3 days. The cellular proliferation was measured after addition of [3H]-thymidine (0.5 μCi/well) for the last 8 h of incubation. The cells were harvested in glass fibre filters in an automatic cell harvester and radioactive incorporation was measured using a liquid scintillation counter. In some experiments, the [3H]-thymidine uptake by activated CD4 T cell cultures was evaluated after addition of saturating doses of anti-IL-10 (22 μg/ml; BD Systems). Results for each patient group are shown as means (±SD).
The supernatants from the different cell cultures were collected after 3 days and cytokines were measured using OptEIA enzyme-linked immunosorbent assays (ELISA) (BD Pharmigen, San Diego, California, USA), according to manufacturer's protocol. Briefly, each ELISA used pairs of monoclonal antibodies directed against human IL-1β, IL-10, IL-4, TNF, and IFN-γ. The reaction was revealed with streptavidin–horseradish peroxidase, using 3,3′,5,5′-tetramethylbenzidine as substrate. Recombinant human IL-1β, IL-4, IFN-γ, TNF and IL-10 at concentrations ranging from 10 to 500 pg/ml were used to construct standard curves.
The effect of interleukin-10 produced by CD4 T cells on in-vitro HIV-1 replication
In experiments to evaluate the impact of endogenous production of IL-10 on in-vitro HIV-1 replication, saturating doses of anti-IL-10 (22 μg/ml; BD Systems) were added in some wells at the time of stimulation of CD4 T cells cultures with anti-CD3 (OKT3; 5 μg/ml) plus rhIL-2 (20 U/ml), and the supernatants were collected 7 days later. This time was chosen because in previous experiments the peak of in-vitro HIV-1 replication occurred at this point (data not shown). As controls, some wells were incubated with control isotype (IgG2a). HIV RNA was measured in the supernatants stored at −70°C by a commercial HIV-1 RNA quantitative reverse transcriptase polymerase chain reaction (Amplicor HIV Monitor Test, Roche Molecular System, Branchburg, New Jersey, USA), with an average detection threshold of 80 copies/ml.
Student's t test was used to determine statistically differences between the two groups for a given variable. For correlation of different variables within a group, Spearman's correlation coefficient was calculated and tested for statistical significance. Significance was defined as P < 0.05.
Virological and immunological characteristics of the patients
Table 1 shows the characteristics of the two groups of patients, who all had undetectable plasma viral load. Despite the differences in baseline viral load, all patients were successfully treated with a three-drug combination. All achieved undetectable plasma HIV-1 RNA levels, defined as < 80 copies/ml, within 3 months of initiating therapy and remained below this level for at least 2 years (data not shown). Importantly, the patients started therapy with similar baseline CD4 cell counts and responded immunologically by increasing these counts following therapy.
The lymphoproliferative response was lower in aged patients successful treated with HAART than in their counterpart young HIV-1-infected individuals.
The first immune event studied was lymphoproliferative response induced by nonspecific T cell stimuli. Figure 1 shows the lymphoproliferative response to anti-CD3, which was higher in younger HIV-1-infected patients than in aged patients. As reduction in the T cell proliferation in AIDS patients is related to impairment of IL-2 secretion , rhIL-2 was added to some cultures containing anti-CD3. As demonstrated in Fig. 1, T cell lymphoproliferative response induced by anti-CD3 was elevated by exogenous IL-2 in both groups, but it remained lower in aged HIV-1-infected group.
Cytokine secretion by HAART-treated aged HIV-1-infected patients
An important immune disturbance in HIV-1-infected patients involves the persistent production of pro-inflammatory cytokines [3–7], and better immune reconstitution following HAART has been linked to normalization of the cytokine network [11,14,16,17]. PBMC from younger HIV-infected individuals spontaneously secreted higher concentrations of IL-1β and TNF-α than those from the older patients (Table 2). When these cultures were polyclonally activated, those from the younger group continued to secrete higher levels of these pro-inflammatory cytokines than did those from the older group (Table 2). While pro-inflammatory cytokines were secreted in lower amounts in cell cultures from aged patients compared with the young group, IL-10 secretion was significantly higher in the older group (Table 2). No statistical difference was observed between the two groups for IL-4 release. The most interesting result was observed when IFN-γ production was assayed. As demonstrated in Table 2, a dramatic age-related deficiency in the ability to secrete IFN-γ was detected. Although IL-2 addition significantly elevated IFN-γ secretion in anti-CD3-activated PBMC cultures from aged patients (P < 0.001), it remained lower than that detected in the younger group. Therefore, critical cytokine dysregulation following HIV-1 infection was more dramatic among the aged patients.
The role of CD4 T cells on cytokine secretion in aged HIV-1-infected patients with immunological response to HAART
To understand the involvement of the rise of peripheral CD4 cell count in the cytokine pattern observed, PBMC were treated with T cell polyclonal activators after removal of the CD4 T cells using anti-CD4-coated immunomagnetic beads. As demonstrated in Fig. 2, IL-1β and TNF-α release, in contrast to IFN-γ secretion, was not statistically modified by depletion of CD4 cells from whole PBMC in the young group. Interestingly, such depletion of PBMC from the aged group significantly elevated IL-1β and TNF-α secretion (Fig. 2). In the absence of the CD4 T cell subset, IL-10 production was reduced in the majority of activated cell cultures, an effect that was stronger in cell cultures purified from the aged individuals (Fig. 2). In this activation model system, the secretion of these cytokines was not significantly changed by depletion of CD19 cells (B lymphocytes) from PBMC cultures from either age group (data not shown). These results suggest that activated CD8 T cells may be the main source of the pro-inflammatory cytokines detected in all cell cultures. In contrast, elevated IL-10 secretion, detected mainly in the activated aged cell cultures, is probably a CD4 cell-dependent event. Finally, these results suggest that recovery of peripheral CD4 T cells following HAART had a different impact on the pattern of cytokine production in younger and older HAART-treated AIDS patients.
The impact of interleukin 10 produced by CD4 T cells on in-vitro HIV-1 replication and on cytokine secretion
As shown in the Table 2, the highest levels of IL-10 were detected in activated cell cultures from elderly patients. Furthermore, the depletion of CD4 cells from PBMC dramatically reduced the secretion of this cytokine, indicating the involvement of this T cell subset in IL-10 production (Fig. 2). Highly purified CD4 T cells were examined during polyclonal activation (Fig. 3), which induced significantly higher levels of IL-10 in aged patients than in the young group (P = 0031). The mean IL-10 production in aged and young groups was 5168 pg/ml (SD, 3557) and 1296 pg/ml (SD, 1007), respectively. Lower virus replication was also detected in supernatants from aged activated CD4 T cells cultures (Fig. 3a). Interestingly, when these cultures were sorted by their IL-10 secretion and levels of in-vitro HIV-1 replication, the highest IL-10 secretion clearly correlated with lower in-vitro virus replication in almost all patients (Fig. 3b,c). In this context, 69% (11/16) of the young patients had HIV-1 RNA > 5000 copies/ml against 25% (4/16) in the aged group. To understand the impact of this correlation, saturating doses of anti-IL-10 were added at the beginning of the culture period, and virus replication was evaluated 7 days later. As demonstrated in Fig. 4A, there is a clear correlation between the blocked of IL-10 activity and the ability of HIV to replicate. Anti-IL-10 elevated HIV-1 RNA in 88% (14/16) of the activated CD4 T cell cultures from the aged group, increasing the virus replication up to six times compared with isotype-matched controls. The direct relationship between lower virus replication with elevated IL-10 secretion was only significantly observed in the aged group (P = 0.036; (Fig. 4b).
To evaluate if the ability of IL-10 to downregulate HIV-1 replication was related to the capacity of this anti-inflammatory cytokine to reduce proliferation of CD4 T lymphocytes, [3H]-thymidine uptake was evaluated in the presence of anti-IL-10 or isotype-matched control. As shown in Fig. 5a, IL-10 neutralization did not significantly modify CD4 T lymphocytes proliferation. Many pro-inflammatory cytokines have been known to support HIV-1 replication in vitro[3–7] and so TNF-α and IFN-γ secretion was also examined in these cultures. Blockade of IL-10 significantly augmented TNF-α secretion but did not change that of IFN-γ in activated CD4 cells cultures from aged patients (Fig. 5b). These data suggested that the IL-10 secretion by CD4 T cells was involved in the downregulation of virus replication, probably by inhibiting TNF-α. Additionally, these data suggest that reduced IFN-γ secretion observed in cultures from aged HIV-1-infected patients was not directly related to their elevated IL-10 production.
The pharmacological treatment of AIDS by HAART is associated with decreasing plasma viral load and increasing CD4 T cells counts . However, the success of immunological reconstitution following HAART can be less effective in some patients, particularly in older individuals. In our study, functional cellular immune recovery was significantly lower in the older HIV-1-infected patients even when they had augmented their CD4 cell counts to similar levels to those observed in younger patients. Many studies with HAART-treated young HIV-infected adults have stated that once patients have achieved a certain CD4 cell count, the risk for some opportunistic infections is accepted as low [11–13]. Our results, however, suggest that the interruption of prophylaxis for opportunistic agents following increase in CD4 cell counts after HAART may not be a merely mathematical clinical decision.
In our study, the T cell proliferative response to polyclonal activators was higher in the HAART-treated young HIV-1-infected patients than in the older group. This difference was also observed when we evaluated the specific lymphoproliferative response to tetanic toxoid in those in both groups who had been previously immunized against Clostridium tetani (data not shown). Some authors have previously shown that HIV-1-infected young adults treated with IL-2 had a marked increase in proliferative response mediated by CD4 and CD8 T cells to mitogens and recall antigens . However, in other HIV-1-infected patients, particularly older ones, this immune approach could contribute less to improve the immune status.
Many of the immune disturbances described in individuals with chronic HIV-1 infection are related to the dysregulation in cytokine network induced by the virus [25,26], and the magnitude of functional immune recovery is related in part to the degree to which immune hyperactivation is controlled . In this context, spontaneous and activated IL-1β and TNF-α secretions were lower in the PBMC cultures from older patients than from younger ones. The analysis of the cell subsets involved in the cytokine pattern observed in our system suggested that the main source of these pro-inflammatory cytokines in CD3-activated cell cultures from both groups was probably from CD8 T cells. It has been known that, as HIV disease progresses, specific and nonspecific chronically activated CD8 T cells contribute to a generalized state of immune activation by secreting high levels of IL-1β and TNF-α; patients successfully treated with HAART tend to reduce this immune dysregulation .
The immune hyperresponsiveness observed in individuals with chronic HIV-1 infection is paradoxically associated with a decrease in IFN-γ-producing CD4 T cells upon antigenic stimulation , and the ability of HAART to restore IFN-γ production seems to depend on endogenous IL-2 release [18,29,30]. In our study, even after IL-2 addition, severe dysfunction in IFN-γ production by polyclonally activated T cell cultures was observed in the aged AIDS patients, despite their good numerical CD4 T cell recovery following HAART. A similar IFN-γ-release deficiency was also observed in cultures of PBMC from aged AIDS subjects during restimulation with tetanic toxoid compared with PBMC from younger patients (data not shown). These results suggest that, in older patients, HAART does not efficiently restore the ability to mount an adequate cellular immune response. Furthermore, these novel observations raise important issues for immunotherapeutic approaches such as subsubcutaneous IL-2 injection to improve immune function in aged AIDS patients.
In our study, there was a higher tendency of activated CD4 T cells to produce IL-10 in aged AIDS patients. Furthermore, elevated IL-10 production was also detected in the anti-CD3-activated CD4 T cells from healthy aged subjects (data not shown), suggesting that this phenomenon is age related and not AIDS associated. Excessive production of IL-10 in HIV-infected patients has been suggested to cause deleterious effects by contributing to decrease the production of Th1 cytokines, which are implicated in promoting resistance to different pathogens . In our system, the blockade of IL-10 secreted by activated CD4 T lymphocytes increased the TNF-α secretion but did not alter the IFN-γ production in aged subjects, in contrast to young HIV-1-infected patients. In healthy aged individuals, however, blockade of IL-10 did elevate IFN-γ production (data not shown). These findings are in agreement with the theory that IFN-γ-producing cells are the group most prone to destruction in the course of HIV infection [1,5,8].
Our experiments have shown that the ability of CD4 T cells from elderly AIDS patients to secrete higher IL-10 is directly associated with low in-vitro HIV-1 replication. Anti-IL-10 elevated significantly the number of RNA copies in activated CD4 T cell cultures from older AIDS patients. This phenomenon was not related to any change in CD4 T lymphocytes proliferation, because anti-IL-10 did not significantly alter lymphocyte polyclonal expansion. However, blockade of IL-10 in our model enhanced significantly TNF-α release by activated CD4 T cells from the aged AIDS patients. Weissman et al. demonstrated that IL-10 blocked HIV-induced TNF-α and IL-6 release and inhibited virus replication in monocyte-derived macrophage cultures. Therefore, the good virological response to HAART in older patients observed by physicians and described by some authors [33,34] could be explained, at least in part, by the IL-10 produced in high levels in this age group, which reduces the TNF-α secreted by some immune cells such as CD8 T lymphocytes.
In conclusion, our results reveal a complex immune dysfunction in aged HIV-1-infected patients, even in those successfully treated with HAART. In our system, the IL-10 produced by CD4 T cells had an antiviral effect by diminishing HIV-1 replication, probably by decreasing TNF-α production. However, the low IFN-γ secretion in older AIDS patients does not appear to be directly related to the high levels of IL-10. A better characterization of this IL-10-secreting CD4 cell group could provide valuable information that might help in the design of better immunotherapeutic tools for this older group of patients with AIDS.
We gratefully acknowledge assistance from Miercio Perrin in reviewing the manuscript.
Sponsorship: This work was supported by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).
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