Immunoproteasomes and immunosenescence

Ageing Research Reviews 2 (2003) 419–432 Review Immunoproteasomes and immunosenescence Michele Mishto a , Aurelia Santoro a , Elena Bellavista a , Massimiliano Bonafé a , Daniela Monti c , Claudio Franceschi a,b,∗ a c Department of Experimental Pathology, University of Bologna, Via San Giacomo, 12, Bologna IT-40126, Italy b Italian National Research Center on Aging, Ancona, Italy Department of Experimental Pathology and Oncology, University of Florence, Florence, Italy Received 28 April 2003; accepted 29 April 2003 Abstract Aging is a complex process which is accompanied with the decline and the reshaping of different functions of the body. In particular the immune system is characterized, during ageing (immunosenescence) by a remodeling of innate immunity (well preserved, up-regulated) and clonotypical immunity (severely altered) and by the occurrence of a chronic inflammatory process (inflammaging) which are, at least in part, genetically controlled. In this scenario, it can be anticipated that a crucial role is played by age-related structural and functional alterations and modifications of proteasomes and immunoproteasomes, the last being a key component of antigen processing and MHC class I antigen presentation. A variety of experimental data are available, suggesting that proteasomes are affected by age, and that in centenarians they are relatively preserved. On the contrary, few data are available on immunoproteasomes, likely as a consequence of the poverty of suitable cellular models. Lymphoblastoid cell lines from EBV immortalized B cells from old donors is envisaged as a possible model for the study of immunoproteasomes in humans and their changes with age. Thus, basic questions such as those related to possible consequences, for immune responses in infectious diseases and cancer, of age-related alterations of antigen processing and presenting, change with age of self-antigen repertoire, and the genetic basis of immunoprotesome activity and its change with age, remain largely unanswered. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Aging; Immunosenescence; Proteasomes; Immunoproteasomes; Antigen presentation; Centenarians ∗ Corresponding author. Tel.: +39-051-2094743; fax: +39-051-2094747. E-mail address: [email protected] (C. Franceschi). 1568-1637/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S1568-1637(03)00030-8 420 M. Mishto et al. / Ageing Research Reviews 2 (2003) 419–432 1. Remodelling and inflammaging: the major characteristics of immunosenescence in human In the last 10 years we have thoroughly investigated the immunology and genetics of human longevity and two major findings emerged from such studies. The first is that the ageing of the immune system (immunosenescence) does not affect equally all the components of the immune system and that an apparent dichotomy appears to exist (Franceschi et al., 2000c). Indeed the innate immunity, which is the most ancestral and present from invertebrates to mammals, appears to be much less affected with age than clonotypical immunity, the most sophisticated and evolutionary recent but likely the most frail. The second is that a basic defense mechanism such as inflammation, deeply related to innate immunity, is apparently chronically activated in elderly subjects. We conceptualized all these phenomena and the “remodelling hypothesis of immunosenescence” and the new concept of “inflammaging” were proposed to fully account for this age related changes of immune response and defense mechanisms (Franceschi et al., 1995; Franceschi et al., 2000a; Franceschi et al., 2000b). We also proposed that the major driving force behind the remodelling of immune system with age and inflammaging was the chronic antigenic load which impinges upon the immune system throughout life. The major characteristic of clonotypical immune senescence is the accumulation of expanded clones of memory cells and the concomitant decrease of virgin T cells. In elderly humans up to 10–15% of peripheral T cells have been shown to be specific for protein epitopes of common viral infections such as cytomegalovirus (CMV) (Khan et al., 2002). On the other hand we have shown that CD8+ virgin T cells decrease dramatically with age and that their number is extremely reduced in centenarians (Fagnoni et al., 2000). It is conceivable that the immune system of humans has evolved to fully cope with an overall amount of antigenic stimulation which likely could impact on the immune system for 30–50 years, a life span which was probably most common in humans until the past two centuries during which life expectancy roughly doubled. The result is that for the first time in the history a large number of humans live until 80 years or more. Accordingly, the immune system is stimulated for several additional decades, and this antigenic stimulation was probably not foreseen by evolution. We have mathematically modeled these phenomena and two major findings emerged from these models: (1) a stochastic model for CD8+ T cell dynamics is capable of fitting the experimental data concerning the change of virgin T cells concentration over age in humans, and at the same time to reproduce survival curves similar to the demographic ones; (2) the extension of this approach to historical curves, starting from 1750 until present days, showed that the quality of the fit of the historical demographic data improves as we approach the recent, quantitative and qualitative decrease of chronic antigenic load (Luciani et al., 2001; Mariani et al., 2003). The almost linearity of the increased life span and in the decrease of the noise fluctuation amplitude (a term related to chronic antigenic load) within historical period suggests that the improvement of life condition has steadily lowered the intensity of chronic antigenic load and restricted the variability which results from the interaction between the individuals and the immunological environment. On the whole, this approach allowed us to appreciate when and how immunosenescence has likely started to impact on survivorship and to predict an increasing crucial role of immunosenescence and it is related phenomena (inflammaging) in explaining the reduction of human mortality in hygienized economically developed societies. In M. Mishto et al. / Ageing Research Reviews 2 (2003) 419–432 421 any case the peculiar inflammatory status which occurs with age and that we called inflammaging is probably responsible for major age related diseases such as cardiovascular diseases, diabetes, neurodegeneration and dementia and is involved in ageing processes, such as the loss of muscle mass and strength (sarcopenia) (Ferrucci et al., 1999). A variety of experimental data suggest that all these pathological conditions share an inflammatory pathogenesis and that inflammaging could explain large part of frailty, morbidity and mortality in aged humans (Yashin et al., 2001). Recently, our studies of centenarians allowed us to collect experimental data which suggest that inflammaging has a genetic basis. Indeed, association studies of candidate genes involved in inflammaging indicate that the frequency of functional polymorphisms related to high plasma levels of pro-inflammatory cytokines is significantly decreased in exceptionally old individuals and that, conversely, functional polymorphisms related to high production of anti-inflammatory cytokines are significantly over-represented in centenarians (Bonafe et al., 2001; Lio et al., 2002a and b). At a molecular level all tissues and organs in the body, including cells and organs of the immune system, undergo a variety of changes which affect informational macromolecules such as DNA. Consequently, mutation accumulation in DNA and in proteins is a well-recognized characteristic of ageing process. However, a large number of changes occurs in proteins, such as oxidation, glycation and conjugation with lipid peroxidation products. The changes occurring with age at the protein level have received a particular attention for decades. In the recent past, proteasomes, owing to their role in the degradation of altered and obsolete proteins, turned out to be an important topic in the field of aging research. 2. Proteasomes Proteasomes are multicatalytic enzyme complexes that are responsible for the turnover of most cellular proteins and also for the generation of the bulk of the antigenic peptide transporters (TAP) associated with the antigen presentation and presented by MHC class I molecules (Rock et al., 1994). 26S proteasomes consist of the catalytic 20S core and either the 19S or 11S regulatory complexes. 20S proteasomes are a four-ring structure with seven different subunits in each ring, arrayed as ␣7 ␤7 ␤7 ␣7 (Voges et al., 1999). The regulatory complexes provide the specificity of the polypeptide recognition. They also open the gated channel formed by the outer ring of 20S proteasomes to control substrate access to the catalytic chamber (Groll et al., 2000). Substrates of 26S proteasomes are largely targeted to 19S regulatory subunits by the addition of the polyubiquitin chains (Hershko and Ciechanover, 1998). Three of the ␤ subunits (␤1, ␤2, ␤5) of 20S constitutive proteasomes are known to possess protease activity. Exposing cells to few stimuli such as IFN-␥, TNF-␣ and LPS induces the synthesis of other catalytic subunits (respectively, LMP2, MECL-1 and LMP7) that together are incorporated into alternative proteasome form (Kloetzel, 2001). These iso-forms, known as immunoproteasomes which have an enhanced capacity to generate peptides bearing hydrophobic and basic amino acids at their C-termini, and a reduced capacity to produce peptides bearing acidic residues at their C-terminus (Rock et al., 1999; Kloetzel, 2001). Consequently, the spectrum of the produced peptides is shifted towards peptides which associate with MHC class I molecules with increased affinity (Früh et al., 1994) (Fig. 1). 422 M. Mishto et al. / Ageing Research Reviews 2 (2003) 419–432 Fig. 1. After IFN-␥, TNF-␣ and LPS stimulation constitutive proteasomes are changed in immunoproteasomes, through the substitution, in the 20S core, of ␤1, ␤2, ␤5 subunits with respectively LMP2, MECL-1 and LMP7. Immunoproteasomes have similar activity regarding short-life and damaged proteins, while they have an increase production of epitope proper for the MHC class I antigen presentation. 3. Proteasomes and ageing Aging is a complex process which is accompanied by the decline of different functions of an organism throughout its life. This progressive and irreversible phenomenon is controlled by genetic and environmental elements. Proteins are of particular interest since they are crucial cellular aging factors; indeed one important feature of the aging process is the accumulation of damaged cellular proteins (Berlett and Stadtman, 1997; Beckman and Ames, 1998). In particular, levels of oxidized proteins, that are generally less active and often exhibit an alteration of the secondary and/or tertiary structure (Ferrington et al., 2001), have been reported to increase significantly with age in human dermal fibroblasts, human keratinocytes, human erythrocytes and human brain (Petropoulos et al., 2000; Levine and Stadtman, 2001). The impairment of important enzymes and the age-related accumulation of damaged proteins are believed to affect cellular integrity. As an example, oxidatively damaged protein accumulation has been associated with age-related diseases, such as Parkinson’s and Alzheimer’s diseases, amyotrophic lateral sclerosis and rheumatoid arthritis (Berlett and Stadtman, 1997). Furthermore ubiquitin-protein conjugates have been found to accumulate with age in different tissue, besides pathological situations such as Parkinson’s and Alzheimer’s diseases (Keller et al., 2000; Jenner, 2001). The age-related accumulation of oxidized and ubiquitinated proteins and the slowing down of protein turnover raise the possibility that proteasome degradation is impaired with age. Indeed, oxidized proteins are preferentially degraded by 20S proteasomes while ubiquitination marks M. Mishto et al. / Ageing Research Reviews 2 (2003) 419–432 423 Fig. 2. Ageing induces several alterations related to proteasome activity, structure and content, that change in base to the studied tissues. On the contrary, immunoproteasomes, that are not been studied further, seem to not be influenced by ageing. proteins for 26S proteasome degradation (Carrard et al., 2002a). It is worth noting that the oxidized proteins have, themselves, an inhibitory effect on the proteasome activity (Bulteau et al., 2002) (Fig. 2). In fact, proteasome activity has been reported by different groups to decline with age in a variety of tissues, while other studies have shown that impairment of proteasome function may not be universal. Different studies demonstrated variable decline in the trypsin, chymotrypsin and PGPH (PeptidylGlutamyl-Peptide Hydrolyzing) activities with age, depending on the tissue analyzed. From the different reports on age-related alterations of proteasome peptidase activities, only the PGPH activity has consistently been shown to decline with age, while other peptidase activities were found either to increase, decrease or not change (Carrard et al., 2002a). In addition, impairment of proteasome function has been recently documented in Parkinson’s and Alzheimer’s diseases (Keller et al., 2000; Jenner, 2001). This finding is particular interesting in the light of the numerous studies showing that proteasome inhibition is sufficient to induce neuronal cell death by triggering such events as caspase activation, cytochrome c release, elevated p53 expression and chromatin fragmentation (Ding and Keller, 2001) (Fig. 2). In recent studies with human epidermal cells and rat myocardiac cells, it has been observed that the accumulation of oxidatively modified proteins is associated with a decreased proteasome activity and content, suggesting that proteasome expression is down-regulated 424 M. Mishto et al. / Ageing Research Reviews 2 (2003) 419–432 with age (Bulteau et al., 2002; Petropoulos et al., 2000). Furthermore, the proteasome activity decline is directly associated with the decrease in the NF-␬B activation after TNF␣ treatment in T cells from elderly compared to young donors. It has been inferred that this association is due to the turnover regulation of proteasomes on IkB-␣, the cytoplasmatic inhibitor of transcription factor NF-␬B (Ponnappan et al., 1999). It is worth noting that the susceptibility to TNF-␣ induced-apoptosis is increased with age, as described in two independent studies (Aggarwal et al., 1999; Mishto et al., 2002), suggesting that, at least in part, the decline in the IkB-␣ degradation (by proteasomes) might account for the increased susceptibility to TNF-␣ induced-apoptosis (Fig. 2), because NF-␬B is known to prevent apoptosis (Mattson and Camandola, 2001). The aging seems to act on the proteasome activity also inducing post-translational modifications, such as oxidation, ubiquitination, glycation and conjugation with lipid peroxidation product (e.g. 4-hydroxy-2-nonenal or HNE), in the proteasome subunits. Indeed a recent study showed that the number of modified proteasome subunits increases with age (Carrard et al., 2002b). Therefore, depending on the tissue or cellular system investigated, the age-related decline of the proteasome activity appears to be the result of the combined effects of at least: (1) a decreased proteasome expression; (2) structural modification and/or replacement of proteasome subunits and (3) inhibitory damaged proteins (Carrard et al., 2002a) (Fig. 2). Of course more should be known to delineate the underlying causes of these age-related processes but often the studies in this field are limited to the restricted availability of human biological material. 4. Proteasomes and longevity: the centenarian model Centenarians are the best example of successful aging: they are people who escaped major common diseases, cancer included, and reached the extreme limits of human life-span. So they offer an intriguing model to better understand the molecular and genetic factors that permit life-span extension (Bonafe et al., 2002). In order to analyze the state of proteasomes in these individuals the laboratory of Statis Gonos in Athens, in collaboration with our laboratory, performed the first study on this topic: healthy centenarian fibroblast cultures were examined testing several subunits RNA expression levels, one proteolytic activity and the oxidized protein levels. The results revealed that centenarian cultures exhibit proteasome subunit expression levels and activity close to those found in young donor cultures, whilst fibroblasts from aged people showed the expected decrease in the proteasome function, favoring the hypothesis that the sustained activity of the proteasome level in centenarians might have contributed to their longevity (Chondrogianni et al., 2000). In fact it can be assumed that, de facto, centenarians escaped age-related diseases as cancer. In this regard, despite epidemiological data showing that the majority of cancer occurs in patients over the age of 65 years (DePinho, 2000), demographic studies show a leveling off around 85–90 years of age, followed by a plateau, or even a decline in the last decades of life (Piantanelli, 1988; Smith, 1999). Consistently, data on Italian centenarians indicate that some of these exceptional individuals had been affected by cancer in their life, but they survived, in an historical period when cancer treatment was not as developed as in the M. Mishto et al. / Ageing Research Reviews 2 (2003) 419–432 425 present days. Thus, it is reasonable to conclude that centenarians are people endowed with a peculiar resistance to cancer and that their proteasomes might play a role in the “war” against cancer. Indeed, the ubiquitin proteasome pathway is responsible not only for the degradation of long lived proteins, but also of tumor suppressor proteins (p53, p21, p27, etc.), transcription factors (NF-␬B) and cell cycle proteins. Altered degradation of these proteins is thought to promote cancer growth and spread. By contrast, inhibition of proteasomes would lead to cell arrest and ultimately apoptosis in many different solid tumor types in vitro and in vivo. The role of proteasome inhibition in cancer therapy lies in its ability to overcome chemoresistance and enhance the effectiveness of chemotherapeutic regimens through different mechanisms (NF-␬B, MDR, bcl-2, anti-angiogenetic factors, etc.) (Shah et al., 2001). 5. Immunoproteasomes in ageing The age-dependent effects on immunoproteasome activity and expression have not been further investigated, likely because difficulty to identify a human ex vivo cellular model that permit to study specifically the immunoproteasomes. Lee et al. published three different papers where they analyzed, through high density oligonucleotide microarrays, the gene expression in aging mice heart (Lee et al., 2002), brain (Lee et al., 1999) and skeletal muscle (Lee et al., 2000). In the brain of aged mice it has been reported, regarding proteasome expression, only a decrease of ␤2-subunit (constitutive proteasomes) and an increase expression of MHC class I ␤2-microglobulin TAP and immunoproteasome subunits after IFN-␥, LPS and TNF-␣ stimuli. In the heart of aged mice they identified an increased expression of PA28, while in the skeletal muscle they reported a decrease of ␤2 and LMP7 proteasome subunits. Thus, the proteasome expression profile in these tissues suggest a general decrease in proteasome content (with no difference between constitutive and immuno-proteasomes), even if the PA28, the regulatory subunit often associated with immunoproteasomes, is significantly over expressed. In agreement with these conclusions are the results obtained by Carrard et al. on peripheral blood mononuclear cells (PBMCs). They compared spots from two D-gel of LMP2 and LMP7, and constitutive subunit counterparts (␤1 and ␤5) in young and elderly donors and no significant difference was observed (Carrard et al., 2002b) (Fig. 2). Hence, literature data suggest that the ratio proteasomes/immunoproteasomes does not changes significantly with age. No data on the immunoproteasome activity in ageing has been reported. Likely the experimental difficulties did not permit the studies in this field that might be important to better understand the onset of age-related diseases (autoimmune pathologies, inflammatory disease and cancers). Indeed the immunoproteasome, besides the well described activity in the short life, damaged or obsolete protein degradation, plays a pivotal role in the antigen presentation pathway: indeed 20S immunoproteasomes are more adept at producing peptides with hydrophobic and positively charged COOH-terminal residues, which are the fragments preferred by class I MHC (Rock and Goldberg, 1999). These antigenic peptides are transported by TAP in the endoplasmatic reticulum (ER), where they bind the MHC class I molecules and are presented on the cell surface. Then qualitative and quantitative alterations of the immunoproteasome activity 426 M. Mishto et al. / Ageing Research Reviews 2 (2003) 419–432 with age might have a strong influence on the quality and quantity of immunodominant epitopes presented to T cell receptor (TcR) of CD8+ lymphocytes. Immunosenescence induces an immune remodeling where the ancestral, innate immunity is preserved, while recent, clonotypical immunity deteriorates (including CD8+ lymphocyte response). Hence, the immunoproteasome-dependent alteration in antigen presentation might lead to a consistent modification of the immune response against antigens (tumor, viral or self antigens). In this scenario they might play a role in cancer development, in the impaired reaction against viral insult (or in the vaccine effectiveness) and in the anti-self immunity (Fig. 3). Therefore close examination of the immunoproteasome modification activity with age should help to clarify the immunosenescence scenario. In particular the capability of different age-donor Fig. 3. Qualitative/quantitative changes in immunoproteasome activity in elderly might lead to alterations of epitopes (e.g. self, tumor or viral epitopes) presentation. This effect, into the immunosenescence scenario, might be in part responsible for the appearance in elderly of autoimmune diseases and tumor, besides the minor vaccine effectiveness or the anti-virus response. M. Mishto et al. / Ageing Research Reviews 2 (2003) 419–432 427 immunoproteasomes to produce specific tumor, viral and self-immunodominant epitopes, tested in the polypeptide digestion in vitro assay, might offer insight regarding the development of specific tumor, viral and autoimmune diseases in aged people. 6. The study of immunoproteasomes in aged humans: in search of a reliable model The first step to deepen this field is to identify cellular models that express immunoproteasomes and permit the study of their modification with age. The immunoproteasomes are basically expressed in the thymus, APC and B activated lymphocytes besides cells stimulated with LPS, TNF-␣ and IFN-␥. Studies on human subjects are therefore difficult. Indeed, the first strategy used to compare proteasome and immunoproteasome activity has been to purify proteasomes from spleen (immunoproteasomes) and skeletal muscle (constitutive proteasomes) (Cascio et al., 2001), but this approach for human ageing studies has evident limitations: indeed the small availability of the material does not easily permit to realize population studies in ZD gel assay. Possible solutions to resolve this material’s problem are to select and expand APC, to treat cells (e.g. lymphocytes) with IFN-␥ or to transfect them with LMP2, LMP7 and MECL-1 genes. This last strategy has been used, for example, in recent studies, in which proteasome and immunoproteasome activities have been compared regarding their abilities to produce a specific immunodominant epitope (Morel et al., 2000; Chen et al., 2001; Kuckelkorn et al., 2002; Schultz et al., 2002; Lautscham et al., 2003). It is unlikely, however, a model that maintains the features of an aged immunoproteasome can be obtained by the expression of the transfected immunoproteasome subunits genes. The expansion of activated APC or others cell type which express mainly immunoproteasomes, could present problems regarding the availability of enough material. On the contrary, the treatment with cytokines such as IFN-␥ and TNF-␣ should permit a high percentage of immunoproteasomes conserving the age-feature in a potentially large amount of cells. The ideal solution would be the identification of a human cell line model that maintains the age-dependent proteasome/immunoproteasome features. In order to study specifically the immunoproteasome one useful model might be the Lymphoblastoid cell Lines (LcLs), i.e. B lymphocytes immortalized with EBV (Fig. 4). Indeed LcLs have a basal up regulation of immunoproteasomes, through NF-␬B pathway activation, as reported by Frisan et al. (Frisan et al., 1998, 2000) and confirmed in our 2D gel assay (Mishto et al., manuscript submitted). Besides this effect, the EBV acts on the LcLs immunoproteasomes with its EBNA1 that, however, have only a cis-inhibition of immunoproteasomes, thus not altering their activity (Dantuma et al., 2002). The LcLs can be obtained from different age donors; then they could be used for the immunoproteasome activity assay, and therefore they could be investigated for the effect of aging in the presentation of EBV (Lautscham et al., 2003; Kuzushima et al., 2003) and tumor antigens (Kubuschok et al., 2002; Gavioli et al., 2002). Thus, combining in vitro immunoproteasome activity assays (e.g. EBV and tumor antigen polypeptides digestion) and ex vivo capability of specific epitope presentation and CTL activation, in LcLs from donors with different ages, we might obtain an overview of the ageing in the Ag presentation. Of 428 M. Mishto et al. / Ageing Research Reviews 2 (2003) 419–432 Fig. 4. Studies of ageing and immunoproteasomes presents limitations in material findings. In this figure we presents the main strategies that could be used to challenge the topic, and their advantages and disadvantages. course in order to validate this model it is necessary to check whether the immunoproteasome activity is influenced by the donor age both in the LcLs and in the autologous B activated lymphocytes. 7. Is there a genetic control of immunoproteasome activity that alters the life’s expectation? In the last years few laboratories have investigated the possible association of the known polymorphisms of the immunoproteasome subunits with tumor, treatment and autoimmune diseases. In this regard the association of the LMP2 and LMP7 polymorphisms with pathologies such as Graves’ disease, juvenile and adult ankylosing spondylitis, insulin-dependent diabetes mellitus (IDDM) and interferon response in patients with chronic hepatitis C has been reported (Heward et al., 1999; Maksymowych et al., 1997; Deng et al., 1995; Vinasco et al., 1998; Sugimoto et al., 2002). Our research group has been investigating the possible association of LMP2 codon 60 and LMP7 nucleotide 145 polymorphisms with age. Our first comparison between young and extreme long-lived people did not show any significant differences in the frequencies of the polymorphisms (Mishto et al., 2002). Hence, though if the LMP2 and LMP7 polymorphisms are associated with several diseases they apparently do not affect longevity in humans. M. Mishto et al. / Ageing Research Reviews 2 (2003) 419–432 429 Intriguingly, we described for the first time the influence of LMP2 codon 60 polymorphism on the susceptibility of PBMCs to TNF-␣-induced apoptosis. It is worth to note that this effect was evident only in the elderly donors (Mishto et al., 2002). A similar phenomenon was observed in a study on a genetic control of IL-6 plasma levels in longevity (Bonafe et al., 2001; Olivieri et al., 2002), where the influence of the IL-6 polymorphism (−174 C/G locus) was manifest only in the elderly subjects. Thus, we may infer that in an immunosenescence scenario, characterized by the remodeling of several immunology balances, a genetic control of Immunity pathways might emerge. 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