Available online at www.sciencedirect.com
Experimental Gerontology 43 (2008) 61–65 www.elsevier.com/locate/expgero
Mini Review
The immune system in extreme longevity P. Sansoni
b
a,*
, R. Vescovini a, F. Fagnoni b, C. Biasini a, F. Zanni a, L. Zanlari a, A. Telera a, G. Lucchini a, G. Passeri a, D. Monti c, C. Franceschi d,*, M. Passeri a
a Dipartimento di Medicina Interna e Scienze Biomediche, Universita` di Parma, Via Gramsci 14, 43100 Parma, Italy Divisione di Medicina Oncologica, IRCCS, Fondazione Maugeri, Clinica del Lavoro e della Riabilitazione, Istituto Scientifico di Pavia, Pavia, Italy c Dipartimento di Patologia e Oncologia Sperimentali,Via Morgagni 50, 50134 Firenze, Italy d Dipartimento di Patologia Sperimentale, Universita` di Bologna, Via S. Giacomo 12, 40126 Bologna, Italy
Received 14 September 2006; received in revised form 3 November 2006; accepted 26 June 2007 Available online 4 July 2007
Abstract Recent observations indicate that immunosenescence is not accompanied by an unavoidable and progressive deterioration of the immune function, but is rather the result of a remodeling where some functions are reduced, others remain unchanged or even increased. In addition, it appears that the ancestral/innate compartment of the immune system is relatively preserved during aging in comparison to the more recent and sophisticated adaptive compartment that exhibit more profound modifications. The T-cell branch displays an agedependent decline of the absolute number of total T-cells (CD3+), involving both CD4+ and CD8+ subsets, accompanied by an increase of NK cells with well-preserved cytotoxic function and by a reduction of B-cells. One of the main characteristics of the immune system during aging is a progressive, age-dependent decline of the virgin T-cells (CD95 ), which is particularly profound at the level of the CD8+ subpopulation of the oldest old subjects. The progressive exhaustion of this important T-cell subpopulation dedicated primarily to the defense against new antigenic challenges (viral, neoplastic, bacterial ones), could be a consequence of both the thymic involution and the lifelong chronic antigenic stimulation. The immune function of the elderly, is therefore weakened by the exhaustion of CD95 virgin cells that are replaced by large clonal expansions of CD28 T-cells. The origin of CD28 cells has not been completely clarified yet, but it is assumed that they represent cells in the phase of replicative senescence characterized by shortening telomers and reduced proliferative capacity. A major characteristic of the immune system during aging is the up-regulation of the inflammatory responses which appears to be detrimental for longevity. In this regard, we have recently observed a progressive age-dependent increase of type 1(IL-2, IFN-c, TNF-a) and type 2 (IL-4, IL-6, IL-10) positive CD8+ T-cells; in particular, type 1 cytokine-positive cells significantly increased, with age, in all CD8+ subsets particularly among effector/cytotoxic and memory cells. A major force able to drive a chronic pro-inflammatory state during aging may be represented by persistent viral infections by EBV and CMV. Therefore, we have determined the frequency and the absolute number of viral antigen-specific CD8+ T-cells in subjects older than 85 years, who were serologically positive for CMV or EBV. In the majority of these subjects we detected the presence of T lymphocytes positive for epitopes of CMV or EBV. In all subjects the absolute number of CMV-positive CD8+ cells outnumbered that of EBV-positive ones. In addition, the majority of CMV+ T cells were included within the CD28 subpopulation, while EBV+ T cells belonged mainly to the CD28+ subset. These data indicate that the chronic antigenic stimulation induced by persistent viral infections during aging bring about important modifications among CD8+ subsets, which are particularly evident in the presence of CMV persistence. The age-dependent expansions of CD8+CD28 T-cells, mostly positive for pro-inflammatory cytokines and including the majority of CMV-epitope-specific cells, underlines the importance of chronic antigenic stimulation in the pathogenesis of the main immunological alterations of aging and may favour the appearance of several pathologies (arteriosclerosis, dementia, osteoporosis, cancer) all of which share an inflammatory pathogenesis. Ó 2008 Published by Elsevier Inc. Keywords: Lymphocytes; Oldest old; CD8+ T-cells; Inflame-aging; CMV
*
Corresponding authors. Tel.: +39 0521 033307; fax: +39 0521 033271. E-mail address:
[email protected] (P. Sansoni).
0531-5565/$ - see front matter Ó 2008 Published by Elsevier Inc. doi:10.1016/j.exger.2007.06.008
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1. Introduction It is generally assumed that the immune system is progressively deteriorating with age (Makinodan and Kay, 1980; Murasko et al., 1986). However, it has recently been suggested that the immunosenescence is not an unavoidable and progressive decline of all immune functions, but rather the result of a continuous remodeling process where several functions are reduced, others remain unchanged, or even increased (Franceschi et al., 1995). Many years ago, we started to study the immune system of centenarians, assuming that one of the main factors of longevity could be represented by a well functioning immune system which allows the prevention of the main age-related pathologies such as infections, atherosclerosis, Alzheimer’s disease, diabetes and even cancer. The immune system may schematically be divided into an ancestral/ innate part, mainly represented by monocytes, natural killer (NK) and dendritic cells (DC), and into a phylogenetically younger part, represented by adaptive immunity (B and T lymphocytes). The aging process seems to hit both branches of the immune system, however, in different ways: the innate immunity seems to be better preserved globally, while the adaptive one, being more sophisticated and able to recognize fine antigenic specificites, manifests deep age-dependent modifications, which are often detrimental (Franceschi et al., 2000a). 2. Innate immunity The NK cells have extensively been studied during both human and murine aging and often contrasting results emerged (Facchini et al., 1987; Krishnaraj and Blandford, 1987). Nevertheless, the age-related decrease of NK activity reported by some investigators can be related to selection bias, as indicated by the fact that people selected according to strict criteria (SENIEUR Protocol) do not present such a diminution (Ligthart et al., 1989, 1990). A detailed cytofluorimetric analysis of the phenotype of peripheral blood lymphomonocytes (PBMC), from a total of 138 people aged 4 to 103 years, allowed us to demonstrate an age-related increase of cells with high NK activity (CD16+CD57 ), while cells with intermediate (CD16+CD57+) or low (CD16 CD57+) NK activity did not show any significant modification (Sansoni et al., 1993). In centenarians, the increase of NK cells with high NK activity was mirrored by very well-preserved cytotoxic activity, comparable to that of young (19–36 years) controls; surprisingly, a significant reduction of NK activity was found in apparently healthy middle-aged controls. The same pattern was observed when cytotoxicity was assessed by anti-CD16-redirected killing. In this respect, a persistently low NK activity in elderly subjects proved to be of predictive value of morbidity and mortality (Levy et al., 1991), conversely, an elevated NK activity correlated with well-preserved endocrine functions and muscular mass (Mariani et al., 1999). Therefore, the finding of a
well-preserved NK cell activity may be interpreted as a factor of longevity. Monocytes and macrophages play a central role in innate immunity (Ottaviani and Franceschi, 1997), being able to respond to a variety of antigens (bacteria, viruses, fungi, toxic substances, transformed cells) through both cell-mediated responses and the liberation of soluble factors (cytokines, chemokines), hormones (MSH, cortisol), amines and oxygen free radicals, stimulating continuously the immune system lifelong thus playing an important role in the establishment of a chronic pro-inflammatory status defined inflame-aging (Franceschi et al., 2000b), which is characterized by increased serum cytokine levels and other pro-inflammatory factors (Brambilla et al., 2001). This age-dependent pro-inflammatory imbalance seems to be of paramount importance, because the main age-related diseases (arteriosclerosis, dementia, osteoporosis, and cancer) all have an important inflammatory component. It has widely been demonstrated that aging is characterized by a progressive increase of serum concentration of IL-6 (Ershler and Keller, 2000), which may reach in centenarians values up to 10-times larger than those of young subjects (Giuliani et al., 2001). On the other hand, a soluble IL-6 receptor (sIL6R) increases progressively up to the seventh decade of age, and declines thereafter, reaching, in centenarians, levels comparable to those of young controls. The reduction of sIL6R in centenarians may be a longevity factor, perhaps by modulating the pro-inflammatory activity of IL-6. In this regard, increased serum values of sIL6R represent a negative prognostic factor for morbidity and mortality, and are in line with our observations that women with elevated sIL6R have a reduced bone mass (Giuliani et al., 2001), a parameter correlated with increased mortality (Browner et al., 1991). The monocytes–macrophages are an important source of IL-6 production in the elderly (Daynes et al., 1993; Fagiolo et al., 1993; Ershler and Keller, 2000), however, one cannot exclude that several other cell types like T-cells, endothelial cells (O’Mahony et al., 1998) or osteocytes (Manolagas and Jilka, 1995) may also be involved in the production of this cytokine. DC represent a rare and heterogeneous population of circulating cells (about 1% of PBMC) that plays a key role in initiating immune responses, being able to capture and process many antigens, and also to secrete a variety of cytokines. Using in vitro generated DC, originated from peripheral monocytes, it has been shown that DC of elderly subjects are functionally intact as regards their capacity of antigen presentation in comparison to young subjects (Steger et al., 1996). However, preliminary data of our laboratory (R. Vescovini, unpublished) indicate that the main peripheral subpopulations of DC (myeloid and lymphoid) are numerically reduced in centenarians when compared to young subjects and these observations are in line with other studies indicating a decline of circulating, interferon-producing plasmacytoid dendritic cells during human aging (Shodell and Siegal, 2002).
P. Sansoni et al. / Experimental Gerontology 43 (2008) 61–65
3. Adaptive immunity 3.1. Humoral immunity, B lymphocytes One of the first observations we made by studying the immune system of centenarians was a progressive and significant increase of some Ig classes and IgG subclasses (Paganelli et al., 1992) accompanied by the almost complete absence of organ-specific autoantibodies (anti-thyreoglobulin and anti-thyreoperoxidases) (Mariotti et al., 1992). Further studies have shown that the absence of autoantibodies can be observed not only in centenarians, but also in healthy very old subjects (Mariotti et al., 1995). Paradoxically, we also found a progressive, agedependent decrease of both total B-cells (CD19+) and CD5-positive B-cells (a subpopulation of B-cells able to synthesize autoantibodies) (Paganelli et al., 1992). In this respect, it would be of importance to analyze other sites of the B-cell production such as the bone marrow, lymphnodes, spleen and lymphoid tissue associated with the mucosa. Therefore, the absence of organ-specific autoantibodies seems to represent a longevity factor in the very old subjects, and the increased levels of IgG and IgA might confer an increased resistance against bacterial and viral infections. 3.2. T lymphocytes One of the most widely accepted paradigms is that the T-cell compartment is progressively deteriorating with advancing age. This is an inevitable consequence of the thymic involution, which starts during the puberty and becomes almost complete by the end of the sixth decade of life. These modifications accompanied also by a shrinkage of secondary lymphoid organs, are reflected by a progressive, age-dependent decrease of circulating lymphocytes (Sansoni et al., 1993). The lymphocyte reduction is also accompanied by a profound remodeling of circulating lymphocyte subsets: (1) a reduction of the absolute number of total T lymphocytes (CD3+) including both helper/inducer (CD4+) and suppressor/cytotoxic (CD8+) cells, (2) a marked decrease of B-cells (CD19+), (3) an increase of activated T-cells (CD3+HLA-DR+) as well as of cells with NK phenotype and NK-like cells (T lymphocytes expressing NK markers) (Sansoni et al., 1993; Mariani et al., 1999). Another important characteristic of immunosenescence is the progressive expansion of CD28-negative T-cells both among CD4+ and CD8+ subsets reaching a peak in the oldest old (Fagnoni et al., 1996). These cells mostly included within CD8+ lymphocytes, demonstrate a memory phenotype (CD11a+CD62L ), cytotoxic markers (CD56+CD11b+) and exhibit, in vitro, cytotoxic activity (Fagnoni et al., 1996). The detection of increased numbers of activated T-cells (HLA-DR+) together with a progressive expansion of effector/cytotoxic cells (CD8+CD28 ) suggests that the immune system
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becomes activated, with age, from unknown stimuli. An increased number CD28 T-cells has been described not only during aging, but also as a consequence of radio-chemotherapies, during autoimmune diseases, and also in HIV infection. The origin of this T-cell subpopulation has not completely been clarified, however, it is assumed that they are generated from CD28+ lymphocytes, which after repeated antigenic stimulations lose the CD28 receptor, and enter in a phase of replicative senescence as shown by the shortened telomers and the reduced proliferative capacity (Nociari et al., 1999; Globerson and Effros, 2000). In order to get further insights to the function of CD8+ T lymphocytes, we studied the intracellular cytokine profile within the virgin, memory and effector CD8+ subpopulations in a group of subjects aged 25 to 100 years. As a general trend, we observed an increase of type 1 (IL-2, IFN-c,TNF-a) and type 2 (IL-4, IL-6, IL-10) cytokines within the three CD8+ subsets in aged subjects. In particular, we showed that type 1 cytokine positive cells significantly increased, with age, in all CD8+ subpopulations, while a marked increase of type-2 producing cells was observed only in memory cells (Zanni et al., 2003). These observations give support to the hypothesis according to which aging is associated with a chronic pro-inflammatory status (Franceschi et al., 2000b), characterized by an increase of pro-inflammatory cytokines (IL1, IL6, TNFa) and of soluble factors of inflammation, which are predictors of morbidity and mortality in elderly subjects (Ferrucci et al., 1999; Harris et al., 1999). Considering the importance of virgin T-cells (i.e., cells which have never met the antigen) in the defense of the organism against external (bacterial, virus) and internal (neoplastic cells) antigenic aggressions, we studied the reservoir of virgin T-cells during aging. After having identified these cells by means of a new cytofluorimetric method (CD95-negative cells), we could observe a progressive, age-dependent reduction both among CD4+ and CD8+ T lymphocytes with an almost complete exhaustion in centenarians (Fagnoni et al., 2000). By analysing the progressive, age-dependent, decrease of virgin CD95 cells along with the increase of CD28-negative T-cells, it emerges that the decrease of the virgin T-cells is a direct consequence of the aging process, while the progressive increase of CD28 T-cells is a consequence and perhaps a compensatory mechanism (Fagnoni et al., 2000). These data on absolute numbers of CD95 naive T-cells were used in a mathematical model to obtain demographic curves that fitted well with the current mortality curves indicating that the number of CD95 virgin cells represent a hallmark of immunosenescence, being able to predict the mortality in the oldest old subjects (Luciani et al., 2001). Another immune function that has been extensively studied during aging is the proliferative capacity of lymphocytes to respond, in vitro, to antigens or polyclonal mitogens. It has generally been observed that the proliferative capacity of lymphocytes shows a progressive, age-depen-
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dent decline in response to the phytohemagglutinin (Murasko et al., 1987). Recent studies of our laboratory, while confirming this phenomenon, did not find significant differences in the proliferative capacity of young, middle-aged and centenarians when well-defined stimuli (anti-CD3 MoAb or PMA, phorbolesters) were used. Furthermore, T-cells stimulated by anti-CD3 MoAb or PMA and costimulated with anti-CD28 MoAb, did not proliferate differently among young, middle-aged and centenarians. (Sansoni et al., 1997). These data indicate that T-cell proliferative capacity is not impaired as previously thought and some important pathways of signal transduction such as CD3 activation pathway and the main costimulatory pathway (CD28) are well functioning even in the extreme human longevity. As a whole, these observations indicate that the main age-related immune modifications are represented by a progressive up-regulation of the inflammatory response (Franceschi et al., 2000b), a progressive reduction of virgin T-cells (Fagnoni et al., 2000), particularly among the CD8+ subset, accompanied by a concomitant increase of memory/effector CD28-negative T-cells (Fagnoni et al., 1996) with a senescent phenotype, and that frequently exhibit clonal expansions (Posnett et al., 1994). The age-dependent clonal type expansions of CD8+ CD28 cells might be driven by homeostatic mechanisms or rather by persistent intracellular antigens, most likely viruses. In elderly subjects it has been observed that viral persistence (CMV) induces modifications of the T-cell subpopulations and is responsible for clonal expansions of CD8+ effector T-cells that include the majority of viral antigen-specific memory cells that can constitute up to a quarter of total CD8+ cells (Khan et al., 2002). The importance of viral persistence along with other immune parameters has been evidenced in a longitudinal study of subjects above 85 years of age, where it was observed that the increased anti-CMV Ig levels correlated negatively with survival (Olsson et al., 2000). In order to evaluate the impact of the ubiquitous chronic viral infections (CMV, EBV) on the immune system during aging, we determined the frequency and the absolute number of CD8+ T-cells specific for the viral antigens in subjects above the age of 85 years, who were serologically positive for CMV and EBV. Our recent data (Vescovini et al., 2004) indicate the presence of CMV or EBV epitope-specific CD8+ T-cells in the overwhelming majority of aged subjects. In all subjects, the number of CD8+ CMV-positive cells outnumbered that of EBV-positive ones, and in several subjects remarkable expansions (more than 10% of all CD8+ cells) of CD8+ CMV-positive cells were found. In addition, the great majority of CMV-positive cells proved to be CD28-negative, while the EBV-positive ones were mainly found in the CD28+ subpopulation. Moreover, a highly variable percentage of CMV-specific CD8+ T-cells expressed intracellular perforin, indicating that only a fraction of these cells were fully cytotoxically active.
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