Perturbations of Cell Cycle Control in T Cells Con
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病菌学杂志 2006年第2期
Division of Infectious Diseases and Emory Vaccine Center, Emory University, Atlanta, Georgia
Department of Biochemistry, University of Urbino, Urbino, Italy
Yerkes National Primate Research Center, Atlanta, Georgia
Division of Infectious Diseases, University of Texas Southwestern, Dallas, Texas
Department of Microbiology and Immunology, Emory University, Atlanta, Georgia
Department of Public Health, University of Messina, Messina, Italy
ABSTRACT
In contrast to human immunodeficiency virus (HIV) infection of humans and experimental simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs), SIV infection of sooty mangabeys (SMs), a natural host African monkey species, is typically nonpathogenic and associated with preservation of CD4+ T-cell counts despite chronic high levels of viral replication. In previous studies, we have shown that the lack of SIV disease progression in SMs is related to lower levels of immune activation and bystander T-cell apoptosis compared to those of pathogenic HIV/SIV infection (G. Silvestri, D. Sodora, R. Koup, M. Paiardini, S. O’Neil, H. M. McClure, S. I. Staprans, and M. B. Feinberg, Immunity 18:441-452, 2003; G. Silvestri, A. Fedanov, S. Germon, N. Kozyr, W. J. Kaiser, D. A. Garber, H. M. McClure, M. B. Feinberg, and S. I. Staprans, J. Virol. 79:4043-4054, 2005). In HIV-infected patients, increased T-cell susceptibility to apoptosis is associated with a complex cell cycle dysregulation (CCD) that involves increased activation of the cyclin B/p34-cdc2 complex and abnormal nucleolar structure with dysregulation of nucleolin turnover. Here we report that CCD is also present during pathogenic SIV infection of RMs, and its extent correlates with the level of immune activation and T-cell apoptosis. In marked contrast, naturally SIV-infected SMs show normal regulation of cell cycle control (i.e., normal intracellular levels of cyclin B and preserved nucleolin turnover) and a low propensity to apoptosis in both peripheral blood- and lymph node-derived T cells. The absence of significant CCD in the AIDS-free, non-immune-activated SMs despite high levels of viral replication indicates that CCD is a marker of disease progression during lentiviral infection and supports the hypothesis that the preservation of cell cycle control may help to confer the disease-resistant phenotype of SIV-infected SMs.
INTRODUCTION
While the majority of human immunodeficiency virus (HIV)-infected individuals will develop progressive CD4+ T-cell depletion and AIDS unless treated chronically with antiretroviral drugs, the nonhuman primates that are natural hosts for simian immunodeficiency virus (SIV) appear to be disease resistant when infected with SIV (3, 8, 10, 36, 39, 43). Importantly, the absence of AIDS in naturally SIV-infected monkeys is associated with levels of virus replication that are as high or even higher than those observed in HIV-infected humans (3, 8, 9, 36, 39, 43). A typical example of nonpathogenic SIV infection of a natural host can be observed in the sooty mangabeys (Cercocebus atys) (SMs), a west African monkey species (3, 8, 39, 43). To investigate the effects of natural SIV infection on the SM immune system, we recently described the results of a cross-sectional analysis of the lymphocyte phenotype and function in SIV-infected and uninfected SMs (43). This analysis showed that the SIV-infected SMs maintain both near-normal CD4+ T-cell numbers in blood and lymph nodes (LN) and preserved T-cell function. The expression of markers of T-cell activation, proliferation, and apoptosis indicated that SIV infection of SMs is associated with limited immune activation and T-cell apoptosis compared to HIV-infected individuals and SIV-infected rhesus macaques (RMs). In addition, we found a significant correlation between increased T-cell activation and a trend towards CD4+ T-cell depletion in the naturally SIV-infected SMs (43). These results led us to hypothesize that the absence of generalized immune activation in SIV-infected SMs is a mechanism that favors the preservation of CD4+ T-cell homeostasis. Consistent with this hypothesis are the results of another study in which we inoculated three SMs and three RMs with uncloned SIV derived from the plasma of an SIV-infected SM (42). While high levels of virus replication were observed in both species, only RMs developed chronic immune activation and increased T-cell apoptosis, with two out of three RMs sacrificed due to the development of simian AIDS within 2 years. In contrast, only minimal immune activation and T-cell apoptosis were observed in the three experimentally SIV-infected SMs that are all still alive and symptom free after 5 years (42).
The low levels of immune activation and apoptosis in SIV-infected SMs are in sharp contrast to the chronic generalized immune activation that is observed in HIV-infected individuals. The HIV-associated immune activation is thought to contribute to the AIDS-associated T-cell depletion (12, 21, 22, 25, 30, 41), and high levels of circulating activated T cells are a strong predictor of disease progression in humans (16, 17, 24, 28, 44). A biological link between chronic immune activation and increased T-cell apoptosis during HIV infection was suggested by the presence of specific perturbations of cell cycle control in T lymphocytes isolated from HIV-infected patients (4, 15, 33, 38). Lymphocytes isolated from chronically HIV-infected patients with active viral replication manifest two main perturbations of cell cycle control: (i) increased activation of the G2/M phase-associated cyclin B/p34-cdc2 complex, and (ii) the presence of multiple and enlarged argyrophilic nucleolar organizing regions with deregulation of nucleolin turnover (4, 15, 35, 38). It should be noted that unscheduled p34-cdc2 activation may induce cell death by a mechanism called mitotic catastrophe (5-7, 37). Consistent with these observations, we found that the presence of this cell cycle dysregulation (CCD) is consistently associated with increased levels of activation-induced apoptosis (4, 15, 35, 38). As such, we hypothesized that the HIV-associated CCD reflects the chronic hyperimmune activation that takes place in HIV-infected patients and is involved in the pathogenesis of T-cell depletion by lowering the apoptotic threshold in both infected and uninfected T lymphocytes (33).
To test this hypothesis we have now performed a comparative analysis of CCD in the pathogenic and nonpathogenic SIV infections of RMs and SMs, respectively. We found that abnormalities of cell cycle control are present during pathogenic SIV infection of RMs but are absent in naturally SIV-infected SMs that do not progress to AIDS. These results confirm that CCD is a marker of disease progression during lentiviral infection and suggest that the ability of T cells to properly regulate cell cycle progression may help to confer the disease-resistant phenotype of naturally SIV-infected SMs.
MATERIALS AND METHODS
Animals. Animals were selected from the colonies of SIV-infected and uninfected sooty mangabeys and rhesus macaques housed at the Yerkes National Primate Research Center of Emory University, Atlanta, GA, and were maintained in accordance with National Institutes of Health guidelines. All infected animals were studied during chronic infection. The characteristics of the studied animals are show in Table 1. Based on longitudinal serologic surveys, the majority of infected SMs studied are known to have acquired SIV by 3 to 4 years of age (14). All animal studies were approved by the Emory University Institutional Animal Care and Usage Committee.
Lymph node biopsies. For lymph node biopsies, the skin over the inguinal or axillary lymph node was prepared for aseptic surgery. A small skin incision was made over the node, and blunt dissection was used to isolate and remove the node. The subcutis and skin were then closed with absorbable sutures. Either ketamine (10 mg/kg) or Telazol (4 mg/kg) was used for anesthesia. Frequency of administration was determined by the veterinarian performing the procedure so as to maximize animal safety and comfort.
SIV viral load. Quantitative real-time reverse transcription-PCR assay to determine SIV viral load was performed as described in reference 43.
Flow cytometry for surface and intracellular markers. Lymphocytes derived from peripheral blood (PB) and LN were isolated by gradient centrifugation. Four-color flow cytometric analysis was performed according to standard procedures using a panel of monoclonal antibodies (MAbs) that were originally designed to detect human molecules but that we and others have shown to be cross-reactive with SMs and RMs (42, 43). The MAbs used included CD4-phycoerythrin (PE) (clone SK3), CD4-allophycocyanin (APC) (clone SK3), CD8-APC (clone SK1), CD25-PE (clone 2A3), CD28-PE (clone L293), CD62L-PE (clone SK11), CD20-peridinin chlorophyll protein (clone L27) (all from Becton Dickinson, San Jose, CA); Ki67-fluorescein isothiocyanate (clone B56), CD3-PE (clone SP34-2), CD69-CyChrome (clone FN50), CD95-CyChrome (clone DX2), HLA-DR-CyChrome (clone G46-6) (all from BD PharMingen, San Diego, CA); CD45RA-TC (clone MEM 56), (Caltag Laboratories, Burlingame, CA); CD16-PE (clone 3G8) (Beckman Coulter, Miami, FL); and cyclin B-fluorescein isothiocyanate (clone 333) (Santa Cruz Inc., Santa Cruz, CA). Samples used for Ki67 and cyclin B were surface stained, fixed and permeabilized using the Pharmingen CytoFix/Perm kit, and stained intracellularly with the proper MAbs and controls. Flow cytometric acquisition and analysis of samples was performed on at least 100,000 events on a FACScaliber flow cytometer driven by the CellQuest software package (Becton Dickinson). Analysis of the acquired data was performed using FlowJo software (Tree Star, Inc., Ashland, OR).
Studies of lymphocyte apoptosis in vitro. The level of apoptosis was determined in freshly isolated peripheral blood mononuclear cells (PBMCs) (baseline) and after 24-h and 48-h incubations either with no stimulus (spontaneous apoptosis) or concanavalin A (ConA; activation-induced apoptosis). Apoptosis was assessed by multicolor flow cytometry in both CD4+ and CD8+ T cells following staining with Annexin V and 7-AAD using the Annexin V-PE apoptosis detection kit from BD Pharmingen.
Western blot analysis. Cyclin B, nucleolin, and caspase expression were measured by Western blot in peripheral blood- and lymph node-derived lymphocytes. Briefly, cells were lysed for 20 min on ice with 20 mM HEPES (pH 7.9), 25% glycerol, 0.42 M NaCl, 0.2 mM EDTA, and 1.5 mM MgCl2 containing 0.5% NP-40, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 1 mM sodium fluoride, and 1 mM sodium orthovanadate. From the total protein extracted, 30 μg was fractionated on sodium dodecyl sulfate-10%polyacrylamide gel electrophoresis and electrically transferred to a nitrocellulose membrane. Blots were incubated with anti-cyclin B1 (1:200) and anti-nucleolin (1:1,000) MAbs (both from Santa Cruz Biotechnology Inc., Santa Cruz, CA) as well as anti-caspase-8 and anti-caspase-3 (both from Cell Signaling Technology, Inc., Beverly, MA) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody. Blots were treated with enhanced chemiluminescence reagents, and proteins were detected and quantitated by a ChemiDoc System (Bio-Rad, Hercules, CA). Equal protein loading was confirmed by the level of actin protein present in the membrane tested with anti-actin antibody (1:500; Sigma).
Statistical analysis. The performed analyses include the Mann-Whitney U test for comparisons between groups, while correlations involving different sets of data within the same group were analyzed using either the standard Pearson correlation coefficient or the Spearman's rank correlation test. Significance was assessed at the P < 0.01 and P < 0.05 levels. All analyses were performed using SAS software.
RESULTS
Increased intracellular levels of nucleolin and its fragments are present in T cells from SIV-infected RMs but not SIV-infected SMs. We have previously shown that T cells isolated from HIV-infected patients display an abnormal activation of the cyclin B/p34-cdc2 kinase complex that is associated with increased threonine phosphorylation of nucleolin, resulting in extranucleolar localization and fragmentation of nucleolin. Importantly, these changes in the posttranslational regulation of nucleolin are temporally associated with the presence of increased levels of T-cell apoptosis (15).
To investigate the presence of abnormalities of cell cycle control during pathogenic and nonpathogenic SIV infections of nonhuman primates, we measured by Western blot the intracellular level of nucleolin and its fragments in peripheral blood lymphocytes (PBLs) isolated from 14 experimentally SIV-infected RMs, 10 naturally SIV-infected SMs, and 10 uninfected animals for each species. All infected animals, the characteristics of which are listed in Table 1, were studied during the chronic stage of infection. Figure 1A shows the intracellular levels of nucleolin, and its fragments in one representative SIV-infected RM and SIV-infected SM. The results of this experiment show a significant increase in the level of full-length nucleolin (P < 0.05) and, more markedly, in the level of fragmented nucleolin (P < 0.01) in PBLs from SIV-infected RMs compared to uninfected RMs. In contrast, no significant difference in the level of either total or fragmented nucleolin were found between PBLs isolated from SIV-infected and uninfected SMs (Fig. 1B).
As the immune activation associated with chronic retroviral infection is thought to involve lymphoid tissues, particularly lymph nodes (2, 22, 29, 31, 40), we next measured the intracellular levels of nucleolin and its fragments in lymph node-derived lymphocytes (LNL) from five SIV-infected RMs and SMs as well as five uninfected animals for each species. This is the first time that the presence of cell cycle dysregulation during a primate lentiviral infection has been investigated in lymphocytes isolated directly from lymph nodes. We show that the intracellular level of full-length nucleolin was similar in LNLs isolated from SIV-infected and uninfected animals both in RMs and SMs, although there was a trend towards higher levels in SIV-infected RMs (Fig. 1C and D). However, the level of fragmented nucleolin was markedly increased in LNLs from SIV-infected RMs compared to those of RMs not infected with SIV. In contrast, no difference in the level of nucleolin fragments in LNLs were found between SIV-infected and uninfected SMs. In all, these results indicate that an increase in the level of nucleolin and, in particular, of its fragments is observed in PBLs and LNLs during pathogenic SIV infection of RMs but not during nonpathogenic SIV infection of SMs.
Increased intracellular levels of cyclin B in T cells from SIV-infected RMs but not SIV-infected SMs. A key feature of the HIV-associated cell cycle perturbation is the increased intracellular concentration of cyclin B, with consequent inappropriate activation of the p34-cdc2 kinase (4, 38). To determine whether SIV infection is associated with increased intracellular levels of cyclin B in T cells, we measured by flow cytometry the expression of this molecule in the animals described in Table 1. We found that the level of cyclin B was significantly increased (P < 0.05) in peripheral blood-derived CD4+ and CD8+ T cells from SIV-infected RMs compared to uninfected RMs (Fig. 2A). In contrast, no changes in the intracellular levels of cyclin B were observed in either CD4+ or CD8+ T cells from naturally SIV-infected SMs compared to uninfected animals (Fig. 2B). Similar results were found when cyclin B levels were examined by Western blot in LNLs derived from a subset of animals (five infected and five uninfected for each species), with increased cyclin B expression in SIV-infected RMs but not in SIV-infected SMs (data not shown). As shown in Fig. 2C, and similar to what has been described in HIV-infected individuals (34), no correlation was found between the fraction of cyclin B "high" CD3+ T cells and the fraction of CD3+ T cells expressing Ki67 in SIV-infected RMs. This latter finding confirms that expression of high levels of cyclin B does not simply reflect the fraction of circulating proliferating T cells (34). Similarly, no correlation was found between cyclin B and Ki67 in either CD4+ or CD8+ T cells (data not shown). In all, these results indicate that, as in HIV infection of humans, pathogenic SIV infection of RMs is associated with increased levels of cyclin B in peripheral blood- and lymph node-derived T cells. In contrast, no changes in the intracellular level of cyclin B are found in lymphocytes during the nonpathogenic SIV-infection of SMs.
The level of cell cycle dysregulation in SIV-infected RMs is correlated with low CD4+ T-cell counts, but not with the level of viral replication. We next sought to determine whether, during the course of SIV infection of RMs and SMs, the presence and extent of cell cycle dysregulation is correlated with the main markers of disease progression (i.e., high levels of viral replication and low CD4+ T-cell counts). We found that the fraction of cyclin B "high" CD4+ or CD8+ T cells did not correlate with the level of viral replication in either experimentally SIV-infected RMs or naturally SIV-infected SMs (data not shown). These data suggest that the prevailing level of viral replication is not the main determinant of the loss of proper cell cycle control in T lymphocytes and are consistent with the observed lack of correlation between viral replication and extent of cell cycle dysregulation in HIV-infected patients (34). Interestingly, in SIV-infected RMs the percent of cyclin B "high" CD4+ T cells inversely correlated (P < 0.01) with the degree of CD4+ T-cell depletion in peripheral blood (Fig. 3A). In addition, there was a negative trend observed between the percent of cyclin B "high" CD8+ T cells and the number of CD4+ T cells in peripheral blood (Fig. 3A). In contrast, in naturally SIV-infected SMs there was no relationship between the percentage of cyclin B "high" CD4+ or CD8+ T cells and the CD4+ T-cell count (Fig. 3B).
The presence of a significant inverse correlation between levels of cyclin B "high" cells and CD4+ T-cell counts during SIV infection of RMs is of interest as it supports, in a nonhuman primate model of pathogenic lentiviral infection, the presence of a direct relationship between CCD and loss of CD4+ T-cell homeostasis. This hypothesis was originally proposed based on the observation that the correction of CCD after antiretroviral therapy in HIV-infected individuals is a better predictor of immunologic response (i.e., an increase in CD4+ T-cell count) than is suppression of viral replication (34).
In SIV-infected RMs, the extent of cell cycle dysregulation correlates with the level of T-cell activation. To further test the hypothesis of a direct relationship between CCD and the level of immune activation associated with HIV/SIV infection, we next investigated whether the intracellular level of cyclin B in T cells correlates with the prevailing level of T-cell activation and proliferation in SIV-infected RMs and SMs. To this end, we first measured the fraction of CD4+ and CD8+ T cells expressing the activation markers CD25, CD69, HLA-DR, and CD95 in the SIV-infected RMs and SMs described in Table 1, and we then performed a series of linear correlation analyses to compare activated T-cell numbers with the percent of cyclin B "high" CD4+ or CD8+ T cells. In SIV-infected RMs the percent of CD4+ T cells expressing high levels of cyclin B correlated directly with the percentage of CD4+ T cells expressing the activation markers HLA-DR (P < 0.05) and CD95 (P < 0.01) (Fig. 4A); in addition, a significant correlation was found between the levels of cyclin B and the level of HLA-DR (but not CD95) in CD8+ T cells (P < 0.05) (Fig. 4B). No correlation was found between cyclin B expression and the levels of CD25 and CD69 on either CD4+ or CD8+ T cells (data not shown). In contrast, in SIV-infected SMs no correlation was found between the levels of cyclin B "high" T cells and the expression of any of the studied activation markers on either CD4+ or CD8+ T cells (data not shown). No correlation was found in either SIV-infected RMs or SMs between the fraction of cyclin B "high" CD4+ or CD8+ T cells and the percentage of cells expressing the proliferation marker Ki67 (Fig. 2D and data not shown). In all, these results suggest that during pathogenic SIV infection, but not during nonpathogenic SIV infection, there is a relationship between the level of T-cell activation and CCD.
In SIV-infected RMs, the extent of CCD correlates with increased levels of T-cell apoptosis. In previous works we have shown that the HIV-associated CCD becomes more pronounced after T-cell activation with mitogens in vitro, and its occurrence is temporally associated with increased levels of T-cell apoptosis (15, 35). Since pathogenic SIV infection of RMs is consistently associated with the presence of increased levels of cyclin B+ T cells, we next sought to determine whether, in SIV-infected RMs, the fraction of cyclin B "high" CD4+ or CD8+ T cells correlates with the in vitro susceptibility to apoptosis of these cells.
To this end we first determined the percentage of cells staining positive for Annexin V in PBLs isolated from SIV-infected and uninfected RMs, both at baseline and after 48 h of treatment with either ConA (activation-induced apoptosis) or no stimulation (spontaneous apoptosis). In contrast to what has been observed in the nonpathogenic SIV infection of SMs (43), the level of activation-induced T-cell apoptosis was significantly increased in both CD4+ and CD8+ T cells isolated from SIV-infected RMs compared to uninfected RMs (P < 0.01) (Fig. 5A). Importantly, the percentage of CD4+ T cells undergoing activation-induced apoptosis correlated directly (P < 0.01) with the percent of CD4+ T cells expressing high levels of cyclin B, but not with the levels of ongoing viral replication (Fig. 5B and C). These results indicate that during pathogenic SIV infection of RMs, the level of in vivo cell cycle disregulation (measured as the percent of cyclin B "high" cells) in CD4+ T cells correlates with the in vitro susceptibility to apoptosis of the same cells.
To further explore the relationship between CCD and T-cell apoptosis, we next investigated, by Western blot analysis, the ex vivo expression and activation level of caspase-3 and caspase-8 in lymphocytes from four SIV-infected and four uninfected RMs and SMs. In both RMs and SMs, the levels of expression and/or activation of caspase-3 (i.e., a downstream effector caspase that plays a key role in determining the apoptotic phenotype) in T cells were similar between infected and uninfected animals (data not shown). In contrast, we found that SIV infection of RMs, but not SMs, is associated with a significant increase in the intracellular levels of activated caspase-8 (i.e., an upstream caspase involved in the death receptor-mediated extrinsic apoptotic pathway). As shown in Fig. 5D and E, SIV-infected RMs exhibited a significant increase (P < 0.01) in the level of activated (i.e., cleaved fragment [CF]) caspase-8 compared to uninfected RMs both in peripheral blood- and lymph node-derived lymphocytes. In SIV-infected RMs, a significant increase (P < 0.05) was also found in the level of procaspase-8, but only in lymph node-derived lymphocytes. In contrast, there were no significant differences between SIV-infected and uninfected SMs in the level of procaspase-8 or caspase-8 CF in either peripheral blood- or in lymph node-derived lymphocytes (Fig. 5D and E).
DISCUSSION
Despite extensive research efforts, the fundamental mechanisms responsible for HIV-induced CD4+ T-cell depletion are poorly understood. The concept that the AIDS-associated CD4+ T-cell depletion is caused by the direct cytopathic effect of HIV on infected CD4+ T cells has been challenged by the observation of increased apoptosis of uninfected CD4+ and CD8+ T cells during HIV infection (1, 13, 18-20, 44). It is generally believed that this increased propensity to apoptosis is related to the hyperimmune activation that follows infection with a lymphotropic and lympholytic virus and is maintained by the continued presence of both antigen and proinflammatory/proapoptotic cytokines (21, 23, 41). In support of this theory, several studies have shown that the level of immune activation is a better predictor of CD4+ T-cell depletion than viral load (16, 28, 44, 45). Furthermore, early correction of the immune activation is a strong predictor of good immunological response in patients with sustained highly active antiretroviral therapy (HAART)-mediated viral suppression (26, 34).
Studies of SMs, a natural host for SIV, support the hypothesis that HIV-associated CD4+ T-cell depletion is more closely related to the level of immune activation than to the level of viral replication. In naturally SIV-infected SMs, where high levels of virus replication generally do not result in any significant CD4+ T-cell depletion, the levels of immune activation and T-cell apoptosis are generally low and in fact are similar to those observed in uninfected animals (43). The apparent disconnection between virus replication and disease progression in naturally SIV-infected SMs supports a model whereby the pathogenesis of the lentivirus-induced CD4+ T-cell depletion is related to the lymphopenic effects of chronic immune activation more than the direct lympholytic effect of the virus.
In a series of previous studies (4, 15, 34, 35), we showed that lymphocytes from HIV-infected individuals display a complex perturbation of cell cycle control, which we termed cell cycle dysregulation (CCD). We hypothesized that the HIV-associated CCD reflects the overall state of immune system activation and thus plays a role in determining the loss of CD4+ T-cell homeostasis in chronically HIV-infected patients. In this study we investigated the presence and extent of CCD in two comparative models of SIV infection of nonhuman primates, i.e., the experimental, pathogenic SIV infection of RMs and the natural, nonpathogenic SIV infection of SMs. We found evidence of CCD (high intracellular levels of cyclin B and increased fragmentation of nucleolin) during SIV infection of RMs but not during SIV infection of SMs. Interestingly, we found that in SIV-infected RMs the level of CCD is significantly correlated with the level of in vivo CD4+ T-cell depletion, the expression of T-cell activation markers, and the in vitro propensity to T-cell apoptosis but not with viral replication. In addition, our data suggest that the increased susceptibility to T-cell apoptosis observed during pathogenic SIV infection of RMs is dependent on caspase-8 activation, a process required in the death receptor-mediated extrinsic apoptotic pathway (11, 20, 27). Interestingly, the percentage of cyclin B "high" T cells correlates with markers of T-cell activation (CD95 and HLA-DR) and apoptosis (Annexin V) but not with the Ki67 marker of T-cell proliferation. This finding is consistent with our previous studies of CCD during chronic HIV infection (4, 34, 35). Dual staining of T cells for cyclin B and Ki67 performed during pathogenic HIV/SIV infections demonstrates the presence of both cyclin B–Ki67+ and cyclin B+Ki67– T cells (34). The cyclin B–Ki67+ population likely comprises cycling cells that are not in the G2/M phase (which is characterized by transiently high cyclin B expression), while the cyclin B+Ki67– population likely represents the bonafide CCD cells that have failed to down-regulate cyclin B after completion of the cycle. In this perspective it is perhaps not surprising that the level of CCD in SIV-infected RMs is not correlated with the fraction of cycling cells as detected by the Ki67 marker.
This direct correlation between CCD, immune activation, T-cell apoptosis, and CD4+ T-cell depletion suggests the existence of a complex perturbation of T-cell function and dynamics during pathogenic lentiviral infection, involving both CD4+ and CD8+ T cells, that encompasses more than the direct, virus-mediated killing of infected CD4+ T cells. Conversely, the lack of immune activation, CCD, and increased apoptosis in SIV-infected SMs with high viral loads indicates that CCD is not a direct consequence of viral replication.
It should be noted that several other lines of evidence suggest that the CCD is not due directly to the effects of HIV on infected cells, including that (i) CCD is present in both CD4+ and CD8+ T cells (15, 38), (ii) there is no correlation between CCD and viral load (4, 38), and (iii) CCD is present in HAART-treated immunological nonresponder patients that control viral replication but do not show a corresponding increase in CD4+ T-cell counts (34). In addition, the observation that CCD is present in SIV-infected RMs (and HIV-infected humans) but absent in SIV-infected SMs is consistent with the hypothesis that CCD is involved in determining the high levels of bystander T-cell apoptosis that is associated with pathogenic lentiviral infections of primates. In particular, the presented data suggest that CCD is a biological link between chronic immune activation and increased susceptibility to apoptosis in the pathogenesis of HIV/SIV infection. Interestingly, in these studies we also determined, for the first time, that during pathogenic SIV infection, CCD is present not only in peripheral blood lymphocytes but also in lymph node-derived T cells, thus indicating that CCD is a systemic T-cell defect that involves multiple anatomic compartments.
While the findings described in this report may help to elucidate the relationship between CCD of T lymphocytes and the development of immunodeficiency during chronic, pathogenic lentiviral infections, several important points need to be explored further. First and foremost, the molecular mechanisms responsible for the loss of cell cycle control in T cells that occurs during pathogenic HIV/SIV infections but not during nonpathogenic SIV infection of natural hosts are still unknown. Second, whether and to what extent the loss of cell cycle control is a reversible phenomenon in vivo remains to be determined. In previous in vitro studies (35), we have shown that CCD is reverted by exogenous interleukin-2 (IL-2) administration, and studies are ongoing to evaluate the effect of in vivo IL-2, IL-7, and/or small-molecule cyclin-dependent kinase inhibitors on CCD during HIV/SIV infection. Interestingly, IL-7 was recently shown to be involved in maintaining T-cell homeostasis during SIV infection of SMs (32). Finally, it remains to be determined why a decline of CD8+ T cells is not observed during pathogenic HIV/SIV infections despite the presence of significant CCD in both the CD4+ and CD8+ T-cell populations. Possible explanations include that CD8+ T cells are more resistant than CD4+ T cells to the proapoptotic effect of CCD or, alternatively, that CD8+ T cells are better than CD4+ T cells in maintaining their homeostasis in the presence of increased levels of apoptosis, likely as a result of a better regenerative potential.
In summary, these data provide further support to the hypothesis that CCD is associated with pathogenic HIV/SIV infections (and is absent during nonpathogenic, natural SIV infection of SMs) representing a molecular mechanism by which hyperimmune activation and T-cell turnover result in an increased sensitivity to apoptosis of uninfected T lymphocytes. As such, CCD may be a measurable marker for the indirect (i.e., not directly caused by HIV replication) mechanisms of T-cell depletion that are associated with pathogenic HIV/SIV infections and provide a potential therapeutic target for immune-based intervention to be used, in addition to standard HAART, to improve the immune function of HIV-infected patients.
ACKNOWLEDGMENTS
This work was supported by NIH grants R01-AI52755 to G.S. and R01-AI049155 to S.S., grants 40F.62 to G.P. and 30F.31 to M.M. from the V Programma Nazionale di Ricerca sull'AIDS, Istituto Superiore di Sanità, Rome, Italy, and the Yerkes National Primate Research Center P51 NIH-NRR-RR00165.
We thank Stephanie Ehnert, Elizabeth Strobert, and Chris Souder for their assistance with animal studies, Ann Chahroudi for critical reading of the manuscript, and the research staff of the Emory Center for AIDS Research (CFAR) Virology Core for their facilitation of this work.
These authors contributed equally to this work.
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Department of Biochemistry, University of Urbino, Urbino, Italy
Yerkes National Primate Research Center, Atlanta, Georgia
Division of Infectious Diseases, University of Texas Southwestern, Dallas, Texas
Department of Microbiology and Immunology, Emory University, Atlanta, Georgia
Department of Public Health, University of Messina, Messina, Italy
ABSTRACT
In contrast to human immunodeficiency virus (HIV) infection of humans and experimental simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs), SIV infection of sooty mangabeys (SMs), a natural host African monkey species, is typically nonpathogenic and associated with preservation of CD4+ T-cell counts despite chronic high levels of viral replication. In previous studies, we have shown that the lack of SIV disease progression in SMs is related to lower levels of immune activation and bystander T-cell apoptosis compared to those of pathogenic HIV/SIV infection (G. Silvestri, D. Sodora, R. Koup, M. Paiardini, S. O’Neil, H. M. McClure, S. I. Staprans, and M. B. Feinberg, Immunity 18:441-452, 2003; G. Silvestri, A. Fedanov, S. Germon, N. Kozyr, W. J. Kaiser, D. A. Garber, H. M. McClure, M. B. Feinberg, and S. I. Staprans, J. Virol. 79:4043-4054, 2005). In HIV-infected patients, increased T-cell susceptibility to apoptosis is associated with a complex cell cycle dysregulation (CCD) that involves increased activation of the cyclin B/p34-cdc2 complex and abnormal nucleolar structure with dysregulation of nucleolin turnover. Here we report that CCD is also present during pathogenic SIV infection of RMs, and its extent correlates with the level of immune activation and T-cell apoptosis. In marked contrast, naturally SIV-infected SMs show normal regulation of cell cycle control (i.e., normal intracellular levels of cyclin B and preserved nucleolin turnover) and a low propensity to apoptosis in both peripheral blood- and lymph node-derived T cells. The absence of significant CCD in the AIDS-free, non-immune-activated SMs despite high levels of viral replication indicates that CCD is a marker of disease progression during lentiviral infection and supports the hypothesis that the preservation of cell cycle control may help to confer the disease-resistant phenotype of SIV-infected SMs.
INTRODUCTION
While the majority of human immunodeficiency virus (HIV)-infected individuals will develop progressive CD4+ T-cell depletion and AIDS unless treated chronically with antiretroviral drugs, the nonhuman primates that are natural hosts for simian immunodeficiency virus (SIV) appear to be disease resistant when infected with SIV (3, 8, 10, 36, 39, 43). Importantly, the absence of AIDS in naturally SIV-infected monkeys is associated with levels of virus replication that are as high or even higher than those observed in HIV-infected humans (3, 8, 9, 36, 39, 43). A typical example of nonpathogenic SIV infection of a natural host can be observed in the sooty mangabeys (Cercocebus atys) (SMs), a west African monkey species (3, 8, 39, 43). To investigate the effects of natural SIV infection on the SM immune system, we recently described the results of a cross-sectional analysis of the lymphocyte phenotype and function in SIV-infected and uninfected SMs (43). This analysis showed that the SIV-infected SMs maintain both near-normal CD4+ T-cell numbers in blood and lymph nodes (LN) and preserved T-cell function. The expression of markers of T-cell activation, proliferation, and apoptosis indicated that SIV infection of SMs is associated with limited immune activation and T-cell apoptosis compared to HIV-infected individuals and SIV-infected rhesus macaques (RMs). In addition, we found a significant correlation between increased T-cell activation and a trend towards CD4+ T-cell depletion in the naturally SIV-infected SMs (43). These results led us to hypothesize that the absence of generalized immune activation in SIV-infected SMs is a mechanism that favors the preservation of CD4+ T-cell homeostasis. Consistent with this hypothesis are the results of another study in which we inoculated three SMs and three RMs with uncloned SIV derived from the plasma of an SIV-infected SM (42). While high levels of virus replication were observed in both species, only RMs developed chronic immune activation and increased T-cell apoptosis, with two out of three RMs sacrificed due to the development of simian AIDS within 2 years. In contrast, only minimal immune activation and T-cell apoptosis were observed in the three experimentally SIV-infected SMs that are all still alive and symptom free after 5 years (42).
The low levels of immune activation and apoptosis in SIV-infected SMs are in sharp contrast to the chronic generalized immune activation that is observed in HIV-infected individuals. The HIV-associated immune activation is thought to contribute to the AIDS-associated T-cell depletion (12, 21, 22, 25, 30, 41), and high levels of circulating activated T cells are a strong predictor of disease progression in humans (16, 17, 24, 28, 44). A biological link between chronic immune activation and increased T-cell apoptosis during HIV infection was suggested by the presence of specific perturbations of cell cycle control in T lymphocytes isolated from HIV-infected patients (4, 15, 33, 38). Lymphocytes isolated from chronically HIV-infected patients with active viral replication manifest two main perturbations of cell cycle control: (i) increased activation of the G2/M phase-associated cyclin B/p34-cdc2 complex, and (ii) the presence of multiple and enlarged argyrophilic nucleolar organizing regions with deregulation of nucleolin turnover (4, 15, 35, 38). It should be noted that unscheduled p34-cdc2 activation may induce cell death by a mechanism called mitotic catastrophe (5-7, 37). Consistent with these observations, we found that the presence of this cell cycle dysregulation (CCD) is consistently associated with increased levels of activation-induced apoptosis (4, 15, 35, 38). As such, we hypothesized that the HIV-associated CCD reflects the chronic hyperimmune activation that takes place in HIV-infected patients and is involved in the pathogenesis of T-cell depletion by lowering the apoptotic threshold in both infected and uninfected T lymphocytes (33).
To test this hypothesis we have now performed a comparative analysis of CCD in the pathogenic and nonpathogenic SIV infections of RMs and SMs, respectively. We found that abnormalities of cell cycle control are present during pathogenic SIV infection of RMs but are absent in naturally SIV-infected SMs that do not progress to AIDS. These results confirm that CCD is a marker of disease progression during lentiviral infection and suggest that the ability of T cells to properly regulate cell cycle progression may help to confer the disease-resistant phenotype of naturally SIV-infected SMs.
MATERIALS AND METHODS
Animals. Animals were selected from the colonies of SIV-infected and uninfected sooty mangabeys and rhesus macaques housed at the Yerkes National Primate Research Center of Emory University, Atlanta, GA, and were maintained in accordance with National Institutes of Health guidelines. All infected animals were studied during chronic infection. The characteristics of the studied animals are show in Table 1. Based on longitudinal serologic surveys, the majority of infected SMs studied are known to have acquired SIV by 3 to 4 years of age (14). All animal studies were approved by the Emory University Institutional Animal Care and Usage Committee.
Lymph node biopsies. For lymph node biopsies, the skin over the inguinal or axillary lymph node was prepared for aseptic surgery. A small skin incision was made over the node, and blunt dissection was used to isolate and remove the node. The subcutis and skin were then closed with absorbable sutures. Either ketamine (10 mg/kg) or Telazol (4 mg/kg) was used for anesthesia. Frequency of administration was determined by the veterinarian performing the procedure so as to maximize animal safety and comfort.
SIV viral load. Quantitative real-time reverse transcription-PCR assay to determine SIV viral load was performed as described in reference 43.
Flow cytometry for surface and intracellular markers. Lymphocytes derived from peripheral blood (PB) and LN were isolated by gradient centrifugation. Four-color flow cytometric analysis was performed according to standard procedures using a panel of monoclonal antibodies (MAbs) that were originally designed to detect human molecules but that we and others have shown to be cross-reactive with SMs and RMs (42, 43). The MAbs used included CD4-phycoerythrin (PE) (clone SK3), CD4-allophycocyanin (APC) (clone SK3), CD8-APC (clone SK1), CD25-PE (clone 2A3), CD28-PE (clone L293), CD62L-PE (clone SK11), CD20-peridinin chlorophyll protein (clone L27) (all from Becton Dickinson, San Jose, CA); Ki67-fluorescein isothiocyanate (clone B56), CD3-PE (clone SP34-2), CD69-CyChrome (clone FN50), CD95-CyChrome (clone DX2), HLA-DR-CyChrome (clone G46-6) (all from BD PharMingen, San Diego, CA); CD45RA-TC (clone MEM 56), (Caltag Laboratories, Burlingame, CA); CD16-PE (clone 3G8) (Beckman Coulter, Miami, FL); and cyclin B-fluorescein isothiocyanate (clone 333) (Santa Cruz Inc., Santa Cruz, CA). Samples used for Ki67 and cyclin B were surface stained, fixed and permeabilized using the Pharmingen CytoFix/Perm kit, and stained intracellularly with the proper MAbs and controls. Flow cytometric acquisition and analysis of samples was performed on at least 100,000 events on a FACScaliber flow cytometer driven by the CellQuest software package (Becton Dickinson). Analysis of the acquired data was performed using FlowJo software (Tree Star, Inc., Ashland, OR).
Studies of lymphocyte apoptosis in vitro. The level of apoptosis was determined in freshly isolated peripheral blood mononuclear cells (PBMCs) (baseline) and after 24-h and 48-h incubations either with no stimulus (spontaneous apoptosis) or concanavalin A (ConA; activation-induced apoptosis). Apoptosis was assessed by multicolor flow cytometry in both CD4+ and CD8+ T cells following staining with Annexin V and 7-AAD using the Annexin V-PE apoptosis detection kit from BD Pharmingen.
Western blot analysis. Cyclin B, nucleolin, and caspase expression were measured by Western blot in peripheral blood- and lymph node-derived lymphocytes. Briefly, cells were lysed for 20 min on ice with 20 mM HEPES (pH 7.9), 25% glycerol, 0.42 M NaCl, 0.2 mM EDTA, and 1.5 mM MgCl2 containing 0.5% NP-40, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 1 mM sodium fluoride, and 1 mM sodium orthovanadate. From the total protein extracted, 30 μg was fractionated on sodium dodecyl sulfate-10%polyacrylamide gel electrophoresis and electrically transferred to a nitrocellulose membrane. Blots were incubated with anti-cyclin B1 (1:200) and anti-nucleolin (1:1,000) MAbs (both from Santa Cruz Biotechnology Inc., Santa Cruz, CA) as well as anti-caspase-8 and anti-caspase-3 (both from Cell Signaling Technology, Inc., Beverly, MA) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated secondary antibody. Blots were treated with enhanced chemiluminescence reagents, and proteins were detected and quantitated by a ChemiDoc System (Bio-Rad, Hercules, CA). Equal protein loading was confirmed by the level of actin protein present in the membrane tested with anti-actin antibody (1:500; Sigma).
Statistical analysis. The performed analyses include the Mann-Whitney U test for comparisons between groups, while correlations involving different sets of data within the same group were analyzed using either the standard Pearson correlation coefficient or the Spearman's rank correlation test. Significance was assessed at the P < 0.01 and P < 0.05 levels. All analyses were performed using SAS software.
RESULTS
Increased intracellular levels of nucleolin and its fragments are present in T cells from SIV-infected RMs but not SIV-infected SMs. We have previously shown that T cells isolated from HIV-infected patients display an abnormal activation of the cyclin B/p34-cdc2 kinase complex that is associated with increased threonine phosphorylation of nucleolin, resulting in extranucleolar localization and fragmentation of nucleolin. Importantly, these changes in the posttranslational regulation of nucleolin are temporally associated with the presence of increased levels of T-cell apoptosis (15).
To investigate the presence of abnormalities of cell cycle control during pathogenic and nonpathogenic SIV infections of nonhuman primates, we measured by Western blot the intracellular level of nucleolin and its fragments in peripheral blood lymphocytes (PBLs) isolated from 14 experimentally SIV-infected RMs, 10 naturally SIV-infected SMs, and 10 uninfected animals for each species. All infected animals, the characteristics of which are listed in Table 1, were studied during the chronic stage of infection. Figure 1A shows the intracellular levels of nucleolin, and its fragments in one representative SIV-infected RM and SIV-infected SM. The results of this experiment show a significant increase in the level of full-length nucleolin (P < 0.05) and, more markedly, in the level of fragmented nucleolin (P < 0.01) in PBLs from SIV-infected RMs compared to uninfected RMs. In contrast, no significant difference in the level of either total or fragmented nucleolin were found between PBLs isolated from SIV-infected and uninfected SMs (Fig. 1B).
As the immune activation associated with chronic retroviral infection is thought to involve lymphoid tissues, particularly lymph nodes (2, 22, 29, 31, 40), we next measured the intracellular levels of nucleolin and its fragments in lymph node-derived lymphocytes (LNL) from five SIV-infected RMs and SMs as well as five uninfected animals for each species. This is the first time that the presence of cell cycle dysregulation during a primate lentiviral infection has been investigated in lymphocytes isolated directly from lymph nodes. We show that the intracellular level of full-length nucleolin was similar in LNLs isolated from SIV-infected and uninfected animals both in RMs and SMs, although there was a trend towards higher levels in SIV-infected RMs (Fig. 1C and D). However, the level of fragmented nucleolin was markedly increased in LNLs from SIV-infected RMs compared to those of RMs not infected with SIV. In contrast, no difference in the level of nucleolin fragments in LNLs were found between SIV-infected and uninfected SMs. In all, these results indicate that an increase in the level of nucleolin and, in particular, of its fragments is observed in PBLs and LNLs during pathogenic SIV infection of RMs but not during nonpathogenic SIV infection of SMs.
Increased intracellular levels of cyclin B in T cells from SIV-infected RMs but not SIV-infected SMs. A key feature of the HIV-associated cell cycle perturbation is the increased intracellular concentration of cyclin B, with consequent inappropriate activation of the p34-cdc2 kinase (4, 38). To determine whether SIV infection is associated with increased intracellular levels of cyclin B in T cells, we measured by flow cytometry the expression of this molecule in the animals described in Table 1. We found that the level of cyclin B was significantly increased (P < 0.05) in peripheral blood-derived CD4+ and CD8+ T cells from SIV-infected RMs compared to uninfected RMs (Fig. 2A). In contrast, no changes in the intracellular levels of cyclin B were observed in either CD4+ or CD8+ T cells from naturally SIV-infected SMs compared to uninfected animals (Fig. 2B). Similar results were found when cyclin B levels were examined by Western blot in LNLs derived from a subset of animals (five infected and five uninfected for each species), with increased cyclin B expression in SIV-infected RMs but not in SIV-infected SMs (data not shown). As shown in Fig. 2C, and similar to what has been described in HIV-infected individuals (34), no correlation was found between the fraction of cyclin B "high" CD3+ T cells and the fraction of CD3+ T cells expressing Ki67 in SIV-infected RMs. This latter finding confirms that expression of high levels of cyclin B does not simply reflect the fraction of circulating proliferating T cells (34). Similarly, no correlation was found between cyclin B and Ki67 in either CD4+ or CD8+ T cells (data not shown). In all, these results indicate that, as in HIV infection of humans, pathogenic SIV infection of RMs is associated with increased levels of cyclin B in peripheral blood- and lymph node-derived T cells. In contrast, no changes in the intracellular level of cyclin B are found in lymphocytes during the nonpathogenic SIV-infection of SMs.
The level of cell cycle dysregulation in SIV-infected RMs is correlated with low CD4+ T-cell counts, but not with the level of viral replication. We next sought to determine whether, during the course of SIV infection of RMs and SMs, the presence and extent of cell cycle dysregulation is correlated with the main markers of disease progression (i.e., high levels of viral replication and low CD4+ T-cell counts). We found that the fraction of cyclin B "high" CD4+ or CD8+ T cells did not correlate with the level of viral replication in either experimentally SIV-infected RMs or naturally SIV-infected SMs (data not shown). These data suggest that the prevailing level of viral replication is not the main determinant of the loss of proper cell cycle control in T lymphocytes and are consistent with the observed lack of correlation between viral replication and extent of cell cycle dysregulation in HIV-infected patients (34). Interestingly, in SIV-infected RMs the percent of cyclin B "high" CD4+ T cells inversely correlated (P < 0.01) with the degree of CD4+ T-cell depletion in peripheral blood (Fig. 3A). In addition, there was a negative trend observed between the percent of cyclin B "high" CD8+ T cells and the number of CD4+ T cells in peripheral blood (Fig. 3A). In contrast, in naturally SIV-infected SMs there was no relationship between the percentage of cyclin B "high" CD4+ or CD8+ T cells and the CD4+ T-cell count (Fig. 3B).
The presence of a significant inverse correlation between levels of cyclin B "high" cells and CD4+ T-cell counts during SIV infection of RMs is of interest as it supports, in a nonhuman primate model of pathogenic lentiviral infection, the presence of a direct relationship between CCD and loss of CD4+ T-cell homeostasis. This hypothesis was originally proposed based on the observation that the correction of CCD after antiretroviral therapy in HIV-infected individuals is a better predictor of immunologic response (i.e., an increase in CD4+ T-cell count) than is suppression of viral replication (34).
In SIV-infected RMs, the extent of cell cycle dysregulation correlates with the level of T-cell activation. To further test the hypothesis of a direct relationship between CCD and the level of immune activation associated with HIV/SIV infection, we next investigated whether the intracellular level of cyclin B in T cells correlates with the prevailing level of T-cell activation and proliferation in SIV-infected RMs and SMs. To this end, we first measured the fraction of CD4+ and CD8+ T cells expressing the activation markers CD25, CD69, HLA-DR, and CD95 in the SIV-infected RMs and SMs described in Table 1, and we then performed a series of linear correlation analyses to compare activated T-cell numbers with the percent of cyclin B "high" CD4+ or CD8+ T cells. In SIV-infected RMs the percent of CD4+ T cells expressing high levels of cyclin B correlated directly with the percentage of CD4+ T cells expressing the activation markers HLA-DR (P < 0.05) and CD95 (P < 0.01) (Fig. 4A); in addition, a significant correlation was found between the levels of cyclin B and the level of HLA-DR (but not CD95) in CD8+ T cells (P < 0.05) (Fig. 4B). No correlation was found between cyclin B expression and the levels of CD25 and CD69 on either CD4+ or CD8+ T cells (data not shown). In contrast, in SIV-infected SMs no correlation was found between the levels of cyclin B "high" T cells and the expression of any of the studied activation markers on either CD4+ or CD8+ T cells (data not shown). No correlation was found in either SIV-infected RMs or SMs between the fraction of cyclin B "high" CD4+ or CD8+ T cells and the percentage of cells expressing the proliferation marker Ki67 (Fig. 2D and data not shown). In all, these results suggest that during pathogenic SIV infection, but not during nonpathogenic SIV infection, there is a relationship between the level of T-cell activation and CCD.
In SIV-infected RMs, the extent of CCD correlates with increased levels of T-cell apoptosis. In previous works we have shown that the HIV-associated CCD becomes more pronounced after T-cell activation with mitogens in vitro, and its occurrence is temporally associated with increased levels of T-cell apoptosis (15, 35). Since pathogenic SIV infection of RMs is consistently associated with the presence of increased levels of cyclin B+ T cells, we next sought to determine whether, in SIV-infected RMs, the fraction of cyclin B "high" CD4+ or CD8+ T cells correlates with the in vitro susceptibility to apoptosis of these cells.
To this end we first determined the percentage of cells staining positive for Annexin V in PBLs isolated from SIV-infected and uninfected RMs, both at baseline and after 48 h of treatment with either ConA (activation-induced apoptosis) or no stimulation (spontaneous apoptosis). In contrast to what has been observed in the nonpathogenic SIV infection of SMs (43), the level of activation-induced T-cell apoptosis was significantly increased in both CD4+ and CD8+ T cells isolated from SIV-infected RMs compared to uninfected RMs (P < 0.01) (Fig. 5A). Importantly, the percentage of CD4+ T cells undergoing activation-induced apoptosis correlated directly (P < 0.01) with the percent of CD4+ T cells expressing high levels of cyclin B, but not with the levels of ongoing viral replication (Fig. 5B and C). These results indicate that during pathogenic SIV infection of RMs, the level of in vivo cell cycle disregulation (measured as the percent of cyclin B "high" cells) in CD4+ T cells correlates with the in vitro susceptibility to apoptosis of the same cells.
To further explore the relationship between CCD and T-cell apoptosis, we next investigated, by Western blot analysis, the ex vivo expression and activation level of caspase-3 and caspase-8 in lymphocytes from four SIV-infected and four uninfected RMs and SMs. In both RMs and SMs, the levels of expression and/or activation of caspase-3 (i.e., a downstream effector caspase that plays a key role in determining the apoptotic phenotype) in T cells were similar between infected and uninfected animals (data not shown). In contrast, we found that SIV infection of RMs, but not SMs, is associated with a significant increase in the intracellular levels of activated caspase-8 (i.e., an upstream caspase involved in the death receptor-mediated extrinsic apoptotic pathway). As shown in Fig. 5D and E, SIV-infected RMs exhibited a significant increase (P < 0.01) in the level of activated (i.e., cleaved fragment [CF]) caspase-8 compared to uninfected RMs both in peripheral blood- and lymph node-derived lymphocytes. In SIV-infected RMs, a significant increase (P < 0.05) was also found in the level of procaspase-8, but only in lymph node-derived lymphocytes. In contrast, there were no significant differences between SIV-infected and uninfected SMs in the level of procaspase-8 or caspase-8 CF in either peripheral blood- or in lymph node-derived lymphocytes (Fig. 5D and E).
DISCUSSION
Despite extensive research efforts, the fundamental mechanisms responsible for HIV-induced CD4+ T-cell depletion are poorly understood. The concept that the AIDS-associated CD4+ T-cell depletion is caused by the direct cytopathic effect of HIV on infected CD4+ T cells has been challenged by the observation of increased apoptosis of uninfected CD4+ and CD8+ T cells during HIV infection (1, 13, 18-20, 44). It is generally believed that this increased propensity to apoptosis is related to the hyperimmune activation that follows infection with a lymphotropic and lympholytic virus and is maintained by the continued presence of both antigen and proinflammatory/proapoptotic cytokines (21, 23, 41). In support of this theory, several studies have shown that the level of immune activation is a better predictor of CD4+ T-cell depletion than viral load (16, 28, 44, 45). Furthermore, early correction of the immune activation is a strong predictor of good immunological response in patients with sustained highly active antiretroviral therapy (HAART)-mediated viral suppression (26, 34).
Studies of SMs, a natural host for SIV, support the hypothesis that HIV-associated CD4+ T-cell depletion is more closely related to the level of immune activation than to the level of viral replication. In naturally SIV-infected SMs, where high levels of virus replication generally do not result in any significant CD4+ T-cell depletion, the levels of immune activation and T-cell apoptosis are generally low and in fact are similar to those observed in uninfected animals (43). The apparent disconnection between virus replication and disease progression in naturally SIV-infected SMs supports a model whereby the pathogenesis of the lentivirus-induced CD4+ T-cell depletion is related to the lymphopenic effects of chronic immune activation more than the direct lympholytic effect of the virus.
In a series of previous studies (4, 15, 34, 35), we showed that lymphocytes from HIV-infected individuals display a complex perturbation of cell cycle control, which we termed cell cycle dysregulation (CCD). We hypothesized that the HIV-associated CCD reflects the overall state of immune system activation and thus plays a role in determining the loss of CD4+ T-cell homeostasis in chronically HIV-infected patients. In this study we investigated the presence and extent of CCD in two comparative models of SIV infection of nonhuman primates, i.e., the experimental, pathogenic SIV infection of RMs and the natural, nonpathogenic SIV infection of SMs. We found evidence of CCD (high intracellular levels of cyclin B and increased fragmentation of nucleolin) during SIV infection of RMs but not during SIV infection of SMs. Interestingly, we found that in SIV-infected RMs the level of CCD is significantly correlated with the level of in vivo CD4+ T-cell depletion, the expression of T-cell activation markers, and the in vitro propensity to T-cell apoptosis but not with viral replication. In addition, our data suggest that the increased susceptibility to T-cell apoptosis observed during pathogenic SIV infection of RMs is dependent on caspase-8 activation, a process required in the death receptor-mediated extrinsic apoptotic pathway (11, 20, 27). Interestingly, the percentage of cyclin B "high" T cells correlates with markers of T-cell activation (CD95 and HLA-DR) and apoptosis (Annexin V) but not with the Ki67 marker of T-cell proliferation. This finding is consistent with our previous studies of CCD during chronic HIV infection (4, 34, 35). Dual staining of T cells for cyclin B and Ki67 performed during pathogenic HIV/SIV infections demonstrates the presence of both cyclin B–Ki67+ and cyclin B+Ki67– T cells (34). The cyclin B–Ki67+ population likely comprises cycling cells that are not in the G2/M phase (which is characterized by transiently high cyclin B expression), while the cyclin B+Ki67– population likely represents the bonafide CCD cells that have failed to down-regulate cyclin B after completion of the cycle. In this perspective it is perhaps not surprising that the level of CCD in SIV-infected RMs is not correlated with the fraction of cycling cells as detected by the Ki67 marker.
This direct correlation between CCD, immune activation, T-cell apoptosis, and CD4+ T-cell depletion suggests the existence of a complex perturbation of T-cell function and dynamics during pathogenic lentiviral infection, involving both CD4+ and CD8+ T cells, that encompasses more than the direct, virus-mediated killing of infected CD4+ T cells. Conversely, the lack of immune activation, CCD, and increased apoptosis in SIV-infected SMs with high viral loads indicates that CCD is not a direct consequence of viral replication.
It should be noted that several other lines of evidence suggest that the CCD is not due directly to the effects of HIV on infected cells, including that (i) CCD is present in both CD4+ and CD8+ T cells (15, 38), (ii) there is no correlation between CCD and viral load (4, 38), and (iii) CCD is present in HAART-treated immunological nonresponder patients that control viral replication but do not show a corresponding increase in CD4+ T-cell counts (34). In addition, the observation that CCD is present in SIV-infected RMs (and HIV-infected humans) but absent in SIV-infected SMs is consistent with the hypothesis that CCD is involved in determining the high levels of bystander T-cell apoptosis that is associated with pathogenic lentiviral infections of primates. In particular, the presented data suggest that CCD is a biological link between chronic immune activation and increased susceptibility to apoptosis in the pathogenesis of HIV/SIV infection. Interestingly, in these studies we also determined, for the first time, that during pathogenic SIV infection, CCD is present not only in peripheral blood lymphocytes but also in lymph node-derived T cells, thus indicating that CCD is a systemic T-cell defect that involves multiple anatomic compartments.
While the findings described in this report may help to elucidate the relationship between CCD of T lymphocytes and the development of immunodeficiency during chronic, pathogenic lentiviral infections, several important points need to be explored further. First and foremost, the molecular mechanisms responsible for the loss of cell cycle control in T cells that occurs during pathogenic HIV/SIV infections but not during nonpathogenic SIV infection of natural hosts are still unknown. Second, whether and to what extent the loss of cell cycle control is a reversible phenomenon in vivo remains to be determined. In previous in vitro studies (35), we have shown that CCD is reverted by exogenous interleukin-2 (IL-2) administration, and studies are ongoing to evaluate the effect of in vivo IL-2, IL-7, and/or small-molecule cyclin-dependent kinase inhibitors on CCD during HIV/SIV infection. Interestingly, IL-7 was recently shown to be involved in maintaining T-cell homeostasis during SIV infection of SMs (32). Finally, it remains to be determined why a decline of CD8+ T cells is not observed during pathogenic HIV/SIV infections despite the presence of significant CCD in both the CD4+ and CD8+ T-cell populations. Possible explanations include that CD8+ T cells are more resistant than CD4+ T cells to the proapoptotic effect of CCD or, alternatively, that CD8+ T cells are better than CD4+ T cells in maintaining their homeostasis in the presence of increased levels of apoptosis, likely as a result of a better regenerative potential.
In summary, these data provide further support to the hypothesis that CCD is associated with pathogenic HIV/SIV infections (and is absent during nonpathogenic, natural SIV infection of SMs) representing a molecular mechanism by which hyperimmune activation and T-cell turnover result in an increased sensitivity to apoptosis of uninfected T lymphocytes. As such, CCD may be a measurable marker for the indirect (i.e., not directly caused by HIV replication) mechanisms of T-cell depletion that are associated with pathogenic HIV/SIV infections and provide a potential therapeutic target for immune-based intervention to be used, in addition to standard HAART, to improve the immune function of HIV-infected patients.
ACKNOWLEDGMENTS
This work was supported by NIH grants R01-AI52755 to G.S. and R01-AI049155 to S.S., grants 40F.62 to G.P. and 30F.31 to M.M. from the V Programma Nazionale di Ricerca sull'AIDS, Istituto Superiore di Sanità, Rome, Italy, and the Yerkes National Primate Research Center P51 NIH-NRR-RR00165.
We thank Stephanie Ehnert, Elizabeth Strobert, and Chris Souder for their assistance with animal studies, Ann Chahroudi for critical reading of the manuscript, and the research staff of the Emory Center for AIDS Research (CFAR) Virology Core for their facilitation of this work.
These authors contributed equally to this work.
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