The ageing process and the concepts of biological age and longevity

The ageing process may be defined as a progressive and general deterioration of the functions of the organism that leads to a lower ability to adaptively react to changes and preserve homeostasis. As Strehler pointed out, four rules can define ageing. It is universal (practically all animals suffer ageing), progressive (the rate of ageing is similar at different ages after reaching the adult state), intrinsic (its cause is endogenous) and deleterious (at least for individuals since it leads to their death)⁽Reference Strehler¹⁾. Although the accumulation of adverse changes with the passing of time should not be considered a disease, it strongly increases the risk of disease, and finally results in death.

The ageing process is highly heterogeneous, and thus, there are different rates of physiological changes in the various systems of the organism and in the diverse members of a population of the same chronological age. This justifies the introduction of the concept of ‘biological ageing’, which determines the level of ageing experienced by each individual and therefore his/her life expectancy. The biological age is related to the mean longevity, which can be defined as the mean of the time that the members of a population who have been born on the same date live. Subjects of a population with a higher rate of ageing show an older biological age and have a shorter lifespan. Since chronological age fails to provide an accurate indicator of the ageing process, it is necessary to select parameters useful as biomarkers of ageing to find out the rate of ageing and therefore the probable longevity of each subject⁽Reference De la Fuente and Miquel²⁾.

An integrated theory of ageing: how, where and why of ageing

Almost 400 single-cause theories have been proposed to explain the ageing process⁽Reference Medvedev³⁾, giving only partial explanation for the causes and effects of ageing. Recently, an integrated theory has been published⁽Reference De la Fuente and Miquel²⁾ attempting to answer the three important questions of biogerontology: the ‘how’, the ‘where’ and the ‘why’ of ageing. In answer to the question ‘how’ ageing happens, it is proposed that the ageing process is a chronic oxidative stress condition (increase of oxidant compounds and decrease of antioxidant defences). Thus, it is linked to many age-related changes that affect a large number of parameters including morphology, physiology and behaviour at all levels of organization: molecular, cellular, tissue, organic and that of the whole individual. In addition, since emerging evidence shows the close link between oxidation and inflammation, and since with ageing the pro-inflammatory compounds increase to levels higher than those of the anti-inflammatory compounds, leading to inflammatory stress, an oxidative and an inflammatory state have been suggested as the cause of the loss of function that appears with senescence⁽Reference De la Fuente and Miquel²⁾. To answer ‘where’ the ageing process starts, it is proposed that this happens in the mitochondria of post-mitotic and differentiated cells. With respect to ‘why’ ageing happens, the answer seems to be found in several evolutionary theories and related concepts published a long time ago. Hence, ageing is a consequence of characteristics selected by evolution as an advantage for the young subjects of the species allowing them to reach the reproductive age in the best condition and thus preserve the species. However, these characteristics are a disadvantage for old subjects (not needed for species maintenance). An example is the expression of more pro-oxidant and pro-inflammatory genotypes, which allow the reaching of the reproductive age with more probability since the subjects are more able to survive infections. The consequence after adult age is the establishment of what has been called ‘oxi-inflamm-ageing’⁽Reference De la Fuente and Miquel²⁾.

Immunosenescence: the immune system as a marker of biological age and predictor of longevity

Ageing is associated with an impairment of physiological systems including the immune system, which has evolved to protect individuals against infections and cancers. In fact, with the passage of time there is an increase of infectious and cancerous processes, which exert a great influence on the age-related morbidity and mortality⁽Reference Wayne, Rhyne and Garry^4, Reference High⁵⁾. Indeed, the increased death rate found in aged populations is due in great proportion to infections⁽Reference High^5, Reference Castle, Uyemura and Fulop⁶⁾. The profound impact of ageing on immunity is presently accepted. Thus, although there are contradictory results, almost every component of the immune system undergoes striking age-associated re-structuring. This leads to changes that may include not only diminished, but also enhanced functions. Therefore, the changes of the immune system with ageing are termed immunosenescence. Despite the rapidly increasing amount of data on immunosenescence in the last few decades⁽Reference De la Fuente and Miquel^2, Reference Pawelec, Barnett and Forsey^7–Reference De la Fuente, Hernanz and Guayerbas¹¹⁾, the puzzle of all the changes in the different aspects of the immune function with age has not yet been solved. Nevertheless, the pronounced age-related decrease in T-cell functions is evident, specially in the T-cell helper, which affects humoral immunity and causes an impaired B-cell function⁽Reference De la Fuente and Miquel^2, Reference Pawelec, Barnett and Forsey⁷⁾. In the cells of innate immunity, the phagocytic cells show functions that decrease with ageing and others that are over activated⁽Reference De la Fuente and Miquel^2, Reference De la Fuente, Hernanz and Guayerbas^11–Reference Alonso-Fernandez, Puerto and Maté¹³⁾, whereas the anti-tumoral activity of natural killer (NK) cells, in most of the work, shows an age-related decrease⁽Reference De la Fuente and Miquel^2, Reference De la Fuente, Hernanz and Guayerbas^11, Reference Solana, Pawelec and Tarazona¹⁴⁾.

In addition, it has been demonstrated that the competence of the immune system is an excellent marker of health⁽Reference De la Fuente and Miquel^2, Reference Wayne, Rhyne and Garry^4, Reference De la Fuente, Hernanz and Vallejo^8, Reference De la Rosa, Pawelec and Peralbo^15, Reference De la Fuente¹⁶⁾ and several age-related changes in immune functions, such as low T-cell proliferative responses, IL-2 secretion and NK cell cytotoxicity, have been linked to longevity⁽Reference De la Fuente and Miquel^2, Reference De la Rosa, Pawelec and Peralbo^15, Reference De la Fuente¹⁶⁾. In previous studies, the earlier-mentioned functions and other immune functions, in lymphocytes and phagocytes, have been established as markers of biological age and therefore as predictors of longevity⁽Reference De la Fuente and Miquel^2, Reference Alonso-Fernandez, Puerto and Maté^13, Reference De la Fuente^16, Reference Viveros, Arranz and Hernanz¹⁷⁾. These functions have shown similar age-related changes in human subjects (studied from the adult age of 20 until 80, in leucocytes of peripheral blood), and in mice (throughout the life of these animals, with a mean life span of 2 years, in their peritoneal leucocytes)⁽Reference De la Fuente and Miquel²⁾. In order to identify the above parameters as markers of biological age, it is necessary to confirm that the levels shown in particular subjects reveal their real health and senescent conditions. This has been achieved in the following two ways: (A) Ascertaining that the individuals with those parameters showing levels older than those of most subjects of the same population, sex and chronological age, die before their counterparts. This can be confirmed only in experimental animals. (B) Finding that the subjects reaching a very advanced age, preserve these immune functions at levels similar to those of adults. This can be tested on both humans (centenarians) and experimental animals, such as extremely long-lived mice. While biologically older animals showing the immune competence levels characteristic of chronologically older individuals have been found to die prematurely⁽Reference De la Fuente and Miquel^2, Reference Viveros, Arranz and Hernanz¹⁷⁾, centenarians and long-lived mice exhibit a high degree of preservation of several immune functions, which may be related to their ability to reach a very advanced age in a healthy condition⁽Reference De la Fuente and Miquel^2, Reference Alonso-Fernandez, Puerto and Maté^13, Reference Puerto, Guayerbas and Alvarez^18–Reference Arranz, Lord and De la Fuente²⁰⁾. All the above results confirm that the immune system is a good marker of biological age and a predictor of longevity. Moreover, since the evolution of these immune functions is similar in mice and human subjects, it can be assumed that those human subjects showing immune parameters at the levels of older subjects have a higher biological age and a shorter longevity⁽Reference De la Fuente and Miquel²⁾.

Neuro-endocrine-immune communication in ageing. The role of the immune system in oxi-inflamm-ageing and in the age-related loss of homeostasis

The immune system does not work alone, since the three regulatory systems, namely the nervous, the endocrine and the immune systems, are intimately linked and interdependent. Thus, there is a ‘neuroendocrine–immune’ system that allows the preservation of homeostasis and therefore of health⁽Reference Wrona^21, Reference Besedovsky and Del Rey²²⁾. The communication between these systems has allowed the understanding of why situations of depression, emotional stress and anxiety are accompanied by a greater vulnerability to infections, cancers and autoimmune diseases, which agrees with the concept that the immune system is affected⁽Reference De la Fuente and Miquel^2, Reference Arranz, Guayerbas and De la Fuente^23, Reference Arranz, De Vicente and Muñoz²⁴⁾.

The impairment of physiological systems with ageing especially affects the three regulatory systems and their communication. This seems to justify the loss of homeostatic capacity and the resulting increase of morbidity and mortality that appears with ageing⁽Reference De la Fuente and Miquel^2, Reference De la Fuente¹⁶⁾. In addition, the age-related changes in the organism are linked to a chronic oxidative and inflammatory stress affecting all cells and especially those of the regulatory systems, which explains their impaired function⁽Reference De la Fuente and Miquel^2, Reference De la Fuente¹⁶⁾. Thus, immunosenescence has as its basis an oxidative and inflammatory stress situation, and the theory of oxidation–inflammation in ageing, recently proposed⁽Reference De la Fuente and Miquel^2, Reference De la Fuente¹⁶⁾, integrates the idea of ‘inflamm-aging’⁽Reference Franceschi²⁵⁾ with the oxidation theory of ageing⁽Reference De la Fuente and Miquel^2, Reference De la Fuente¹⁶⁾. According to this theory, chronic oxidative and inflammatory stress lead to the damage of cell components, including proteins, lipids and DNA, contributing to the age-related decline of physiological functions, especially in cells of the regulatory systems, including the immune system. Moreover, the immune system, due to its capacity of producing oxidant and inflammatory compounds in order to eliminate foreign agents, could be involved with the rate of ageing, increasing oxidation and inflammation, if the age-related oxidative and inflammatory stress are not well controlled⁽Reference De la Fuente and Miquel^2, Reference De la Fuente¹⁶⁾. In this context, a relationship has been found between the redox and inflammatory state of the immune cells, their functional capacity and the lifespan of a subject. Thus, when an animal shows a high-oxidative stress in its immune cells, these cells have an impaired function and that animal shows a decreased longevity. This happens in chronologically and biologically older human subjects and mice⁽Reference De la Fuente and Miquel^2, Reference Viveros, Arranz and Hernanz¹⁷⁾. On the contrary, subjects who achieve greater longevity, such as human subject centenarians and extremely long-lived mice, show a preserved redox state and immune functions⁽Reference De la Fuente and Miquel^2, Reference Alonso-Fernandez, Puerto and Maté^13, Reference Arranz, Caamano and Lord^19, Reference Arranz, Lord and De la Fuente²⁰⁾. One of the most relevant mechanisms involved in the cellular redox state is the NF-κB. This transcription factor plays a key role in regulating the expression of a wide range of oxidants and inflammatory compounds, especially in the immune cells, and increases in many chronic inflammatory diseases. In fact, it has been observed that the NF-κB activation, in resting conditions, is very high in leucocytes from old mice, but lower in extremely long-lived mice and adult animals⁽Reference Arranz, Caamano and Lord¹⁹⁾. Moreover, only old subjects with controlled basal NF-κB activation in leucocytes achieved longevity. Adults with a high basal expression of this factor, died early⁽Reference Arranz, Caamano and Lord¹⁹⁾.

In conclusion, only aged individuals who maintain a good regulation of the leucocyte redox state and consequently a good function of their immune cells, with levels similar to those of healthy adults, reach very high longevity. Thus, the immune system seems to be a good predictor of longevity⁽Reference De la Fuente and Miquel^2, Reference Arranz, Caamano and Lord^19, Reference Arranz, Lord and De la Fuente²⁰⁾.

Murine models of premature immunosenescence

Support for the above oxidation–inflammation theory of ageing and especially for the role of the immune system in ageing, may be obtained by the study of animal models in which subjects showing premature immunosenescence and a high oxidative and inflammatory stress in their immune cells (and in other cells), show decreased longevity in relation to other members of the group of the same chronological age. In this context, several murine models, such as the following, have been investigated and developed during the last few years as novel approaches to assess premature ageing and the above-mentioned idea.

Model of prematurely ageing mice

A model of premature ageing in mice based on altered stress-related behavioural response and immunosenescence has been established⁽Reference Viveros, Arranz and Hernanz¹⁷⁾. The animals are termed prematurely ageing mice (PAM), in contrast to the non-prematurely ageing mice (NPAM) of the same population, sex and chronological age, are identified by their poor response in a simple T-maze exploration test. This provides strong support for the concept that the nervous and the immune systems are closely linked. In mice showing premature ageing, it has been observed that several immune functions were similar to those of chronologically older mice. In addition to a more significant immunosenescence, the PAM exhibited high levels of anxiety and emotionality, showing a brain neurochemistry characteristic of older animals and an oxidative stress situation. Moreover, PAM also had increased baseline corticosterone and a blunted stress response when compared to NPAM. Nevertheless, the most convincing evidence that the immune function parameters studied are useful markers of biological age, is that the PAM showed a shorter lifespan than their NPAM counterparts ⁽Reference De la Fuente and Miquel^2, Reference De la Fuente^16, Reference Viveros, Arranz and Hernanz¹⁷⁾.

Effects of a diet supplemented with antioxidants in ageing and immunosenescence

Ageing cannot be ‘eliminated’, it can only be mitigated, i.e. making the process slower. Since the base of the functional longevity of each subject is health maintenance, and this depends on the genes (approximately 25%) and on the lifestyle and environmental factors (75%), it is possible to retard the rate of ageing through the modulation of these factors such as nutrition⁽Reference De la Fuente and Miquel²⁾. Among all the aspects that can be considered good nutrition, the antioxidant compounds present in the diet seem to be the most effective. As mentioned above, ageing is the result of a chronic oxidative stress with an oxidant–antioxidant imbalance due to an excess of the oxidants and a decrease or impairment of the antioxidant defences⁽Reference De la Fuente and Miquel²⁾. In fact, a confirmation of this is the age-related decrease of several antioxidants⁽Reference Arranz, Fernandez and Rodriguez^26, Reference Droge³⁵⁾ as well as the increase of longevity in animals that received these antioxidants in their diet⁽Reference Miquel and Ecónomos^36, Reference Richie, Mills and Lang³⁷⁾.

Moreover, nutritional status has a marked effect on immune response in elderly individuals⁽Reference Lesourd³⁸⁾. Since the functional state of the immune system is a marker of health, biological age and a predictor of longevity, it would be convenient to test the effects of a strategy such as diets rich in antioxidant compounds to retard the ageing process, analysing immune cell functions and their redox state as well as the mean longevity of the subjects. The administration of antioxidants could prevent the age-related imbalance of oxidants–antioxidants in the immune cells. Nevertheless, it should be considered that the immune cells need to produce oxidants to carry out their functions and thus, they may exhaust their reserves of antioxidants⁽Reference De la Fuente and Miquel²⁾. This could help to explain why, in both adult experimental animals and human subjects, the functional capacities of the immune cells improve after diet supplementation with the appropriate amount of antioxidants⁽Reference De la Fuente and Miquel²⁾. It is evident that the amount of antioxidants needed by the immune cells in old subjects is higher than that in adults, since these cells show an age-related impairment of redox regulation with a higher production of oxidants and lower levels of antioxidant defences⁽Reference De la Fuente and Miquel^2, Reference De la Fuente, Guayerbas and Catalán³⁹⁾. Thus, the administration of compounds such as vitamins C and E, polyphenols and precursors of reduced glutathione (GSH; taurine, thioproline (TP) and N-acetylcysteine (NAC), among others) in isolation, in nutritional formulations or through food rich in these compounds, has been shown to improve the immune functions and decrease the oxidative stress in leucocytes and in other cells of the organism⁽Reference De la Fuente and Miquel²⁾. These effects have been shown in adults, but especially in prematurely ageing subjects and in chronologically elderly men, women and mice⁽Reference De la Fuente and Miquel^2, Reference De la Fuente, Hernanz and Guayerbas^11, Reference De la Fuente^16, Reference Arranz, Fernandez and Rodriguez²⁶⁾. Moreover, the confirmation of the central role of the immune system in oxi-inflamm-ageing is that the administration of adequate amounts of antioxidants in the diet, increases the longevity of the subjects⁽Reference De la Fuente and Miquel²⁾. This has been observed in experimental animals such as mice with a lifespan of 2 years. Since the improvement in the immune function and redox state found with antioxidant supplementation is similar in human subjects and mice, and because these changes in mice are accompanied by an increase in lifespan, it is probable that similar effects could be observed in human subjects. These antioxidants seem to have a direct effect on the immune cells since they improve the same immune cell and redox parameters in vitro as well as ex vivo after their ingestion in the diet⁽Reference De la Fuente and Miquel²⁾.

The effects of the dietary supplementation with antioxidants on immune cell functions and their redox state have been observed in several of the models of premature ageing mentioned earlier. The results found with mice suffering anxiety, the premature ageing model previously mentioned, will be discussed in the next section. In the murine model of ovariectomy, an improvement of several immune functions by a five-week ingestion of a diet enriched (1 mg/mouse/d) in soyabean isoflavones and green tea has been observed in ovariectomised old mice⁽Reference Baeza, De Castro and Alvarado⁴⁰⁾. This agrees with the overall observation that any positive change in the diet is more effective in improving immune response in subjects of a biological older population⁽Reference De la Fuente and Miquel^2, Reference De la Fuente^16, Reference Viveros, Arranz and Hernanz¹⁷⁾.

Effects of a diet supplemented with antioxidants on a model of prematurely ageing mice

In the model of PAM the effect of a diet supplemented with appropriate amounts of antioxidants on many immune functions and oxidative stress parameters, which were previously accredited as markers of biological age, has been extensively studied⁽Reference Viveros, Arranz and Hernanz¹⁷⁾. A dietary supplementation of biscuits enriched with nutritional doses of vitamin C and E, zinc, selenium and β-carotenes, for 15 weeks, with both 5% and 20% (w/w), improves the function and redox balance of peritoneal immune cells from chronologically adult (27–31 weeks of age) and mature (48–52 weeks of age) PAM and NPAM animals. However, the effects were stronger in PAM, and 20% supplementation was more effective than 5%⁽Reference Viveros, Arranz and Hernanz^17, Reference Alvarado, Alvarez and Puerto⁴¹⁾. This supplementation with 20% of biscuits enriched with antioxidants also improved the functions and redox balance of the immune cells from chronologically young (16–20 weeks of age) PAM after only 5 weeks of ingestion⁽Reference Alvarado, Alvarez and Jimenez^42, Reference Alvarado, Alvarez and Jimenez⁴³⁾. Moreover, a supplementation with 20% (w/w) of polyphenol-rich cereals, for 5 weeks, improved the immune cell functions in young (16–20 weeks of age) PAM⁽Reference Alvarez, Alvarado and Puerto⁴⁴⁾.

The type of antioxidant supplementation most frequently studied in PAM and NPAM has been that using sulphur-containing antioxidants that are precursors of GSH⁽Reference Viveros, Arranz and Hernanz¹⁷⁾. These antioxidants have been shown to increase longevity⁽Reference Miquel and Ecónomos^36, Reference Richie, Mills and Lang³⁷⁾. GSH is the most abundant non-protein thiol in mammalian cells, being considered essential for their survival, and with an important role in many biological processes⁽Reference De la Fuente and Miquel²⁾. An increase in the levels of GSH reinforces antioxidant protection, preserves the intracellular redox state and the cellular function⁽Reference De la Fuente and Miquel^2, Reference Arranz, Fernandez and Rodriguez^26, Reference Droge³⁵⁾. Therefore, optimal immune functions, such as T-cell proliferation among others, will require proper levels of GSH⁽Reference De la Fuente and Miquel^2, Reference Arranz, Fernandez and Rodriguez^26, Reference Droge³⁵⁾. In previous studies, it has been observed that GSH stimulates many immune functions in vitro and protects cells against apoptosis⁽Reference De la Fuente and Miquel²⁾. The two antioxidant GSH precursors most often studied and used in the present work have been TP and NAC. TP is an antioxidant present in mitochondria, which can increase the levels of GSH⁽Reference Richie, Mills and Lang³⁷⁾ and thus increases the longevity⁽Reference Miquel and Ecónomos^36, Reference Richie, Mills and Lang³⁷⁾. Although this is an aspect very little studied, in previous investigations, it has been shown that TP in vitro improves several functions of immune cells from mice, as well as the activity of antioxidant enzymes⁽Reference De la Fuente and Miquel²⁾. In aged mice, the supplementation with TP (0·07 (w/w), for 5 weeks) improved several immune functions⁽Reference De la Fuente, Ferrández and Del Rio⁴⁵⁾. NAC is an antioxidant that acts as a GSH precursor⁽Reference Arranz, Fernandez and Rodriguez^26, Reference Droge³⁵⁾ and also neutralizes free radicals in a direct manner. This antioxidant action has been observed in immune cells from mice with endotoxic shock, a situation of high oxidative stress⁽Reference Victor, Rocha and Esplugues⁴⁶⁾. NAC in vitro increases several functions of peritoneal macrophages from adult mice in a similar way to GSH, as well as the activity of antioxidant enzymes⁽Reference De la Fuente and Miquel²⁾. In elderly women, the ingestion of NAC improves several immune functions and the redox state⁽Reference Arranz, Fernandez and Rodriguez²⁶⁾.

In adult PAM, the supplementation of a diet with TP (0·1% (w/w) for 5 weeks) improves the peritoneal macrophage functions⁽Reference Correa, Blanco and Del Rio⁴⁷⁾ and the same occurs with the supplementation with NAC⁽Reference Puerto, Guayerbas and Victor⁴⁸⁾. When the diet contains both TP and NAC (0·1% (w/w)), the supplementation for 4–5 weeks improves the function of immune cells in Swiss and BALB/c mice⁽Reference Guayerbas, Puerto and Ferrandez^49, Reference Guayerbas, Puerto and Alvarez⁵⁰⁾.

Effects of a diet supplemented with two sulphur-containing antioxidants (thioproline and N-acetylcysteine), precursors of reduced glutathione, on several leucocyte functions in old prematurely ageing mice of two strains

In previous studies, it has been shown that the ingestion of a diet supplemented with TP+NAC (0·1% (w/w)) by adult female Swiss and BALB/c mice for a short period of time (4–5 weeks) improves several immune functions such as chemotaxis, lymphoproliferative response to the mitogen concanavalin A, IL-2 release and NK activity in leucocytes from peritoneum, axillary nodes, spleen and thymus⁽Reference De la Fuente, Guayerbas and Catalán^39, Reference Guayerbas, Puerto and Ferrandez⁴⁹⁾. In the present work, it has been investigated whether this supplementation with 0·1% of TP+NAC for 5 weeks could be enough in chronologically old (69–73 weeks of age) PAM Swiss and BALB/c mice to improve those immune functions to the level of adult (22–26 weeks of age) animals.

Female mice of the Swiss and BALB/c strains (Harlan, Iberica, Spain) 20–24 weeks of age were maintained in sterile conditions at a constant temperature (20–24°C) on a 1212 reversed light–dark cycle and fed water and Standard Sander Mus pellets (A.04 diet; Panlab LS, Barcelona, Spain) ad libitum until the moment of starting the experiment. At 64–68 weeks of age, the exploratory behaviour of each mouse was tested in a T-shaped maze. As previously described⁽Reference Viveros, Arranz and Hernanz¹⁷⁾, the mice that completed the exploration of the first arm of the maze four times in more than 20 s (once each week for 4 consecutive weeks) are considered PAM and those that spent less than that time are NPAM. At 69–73 weeks of age, eight groups of 10 animals each were used. In each strain, 10 PAM and 10 NPAM received a diet (A.04 diet; Panlab) supplemented with 0·1% (w/w) of both TP and NAC (Sigma, San Louis, MO, USA) for 5 weeks (PAMA and NPAMA, respectively). Two other groups of 10 PAM and 10 NPAM, of both strains, received a normal diet (PAMC and NPAMC, respectively) during that time. After 5 weeks, the mice were killed by cervical dislocation according to the European Community Council Directives 86/6091 EEC and the axillary nodes, spleen and thymus were removed. In parallel, a group of adult (20–26 weeks of age) Swiss and BALB/c mice were sacrificed.

The leucocyte suspensions from organs were obtained and the functions studied were evaluated following methods previously described⁽Reference Guayerbas, Puerto and Ferrandez⁴⁹⁾. The chemotaxis was evaluated using a chamber with two compartments separated by a filter. The samples of leucocytes were deposited in the upper compartment and the chemoattractant (f-met-leu-phe, 10⁻⁸ mol/l) in the lower compartment. After 3 h incubation, the filters were fixed and stained and the number of leucocytes found in four scans of 5 mm each on the lower face of the filter was determined and nominated the chemotaxis index. The proliferation of lymphocytes was determined using a commercially available 5-bromo-2′deoxyuridine ELISA (BrdU labelling and detection kit III; Boehringer, Mannheim, Germany). The leucocytes were incubated for 48 h in the absence (basal proliferation) or presence of the mitogen concanavalin A (1 mg/ml; Sigma). The absorbance of the samples (measured at 405 nm, with a reference wavelength of 490 nm) is directly correlated with the level of 5-bromo-2′deoxyuridine incorporated into cellular DNA. The NK activity of the leucocytes was studied using an enzymatic colorimetric assay (Cytotox 96, Promega, Madison, WI, USA) for cytolysis measurements of target cells (yeast artificial chromosome-1 cells from a murine lymphoma) based on the determination of lactate dehydrogenase enzymatic activity using tetrazolium salt. After 4 h of incubation, the percentage lyses of target cells was calculated. The concentration of IL-2 was determined in culture supernatants of leucocytes from axillary nodes after 48 h of incubation with concanavalin A using an ELISA kit (R&D System, Minneapolis, MN, USA).

The results are shown in Tables 1–3 for leucocytes from axillary nodes, spleen and thymus, respectively. The chemotaxis indexes of leucocytes from axillary nodes, spleen and thymus (with similar values in both strains of mice) were in Swiss and BALB/c PAMC, smaller than in the corresponding NPAMC. In all cases, the values in old PAM and NPAM were lower than those in the adult animals. The ingestion for 5 weeks of a diet supplemented with TP+NAC increased, in general, with statistical significant differences, the chemotaxis in PAMA and NPAMA with respect to the corresponding controls (PAMC and NPAMC). Moreover, the ingestion of the antioxidants brought the levels of chemotaxis in aged animals close to those in adults.

Table 1. Effect of a diet supplemented with two sulphur-containing antioxidants (thioproline (TP) and N-acetylcysteine (NAC); 0·1% (w/w) for 5 weeks) on several functions of axillary node leucocytes from chronologically old prematurely and non-prematurely aged Swiss and BALB/c mice

(Mean values and standard deviations for 10 subjects)

NPAM, non-prematurely ageing mice; PAM, prematurely ageing mice; NPAMC and PAMC, NPAM and PAM controls; NPAMA and PAMA, NPAM and PAM with antioxidant supplementation; AC, adult controls; Con A, concanavalin A; NK, natural killer.

The data were analysed statistically by the three-way ANOVA for unpaired observations, followed by the Scheffe's F post-hoc test.

* P<0·05; **P<0·01; ***P<0·001 with respect to the corresponding values in controls (NPAMC and PAMC). ^aP<0·05; ^bP<0·01; ^cP<0·001 with the corresponding values in adults. †P<0·05; ††P<0·001 with respect to the corresponding values in NPAMC. ‡P<0·05; ‡‡P<0·01 with respect to the corresponding values in Swiss mice.

Table 2. Effect of a diet supplemented with two sulphur-containing antioxidants (thioproline (TP) and N-acetylcysteine (NAC); 0·1% (w/w) for 5 weeks) on several functions of spleen leucocytes from chronologically old prematurely and non-prematurely aged Swiss and BALB/c mice

(Mean values and standard deviations for 10 subjects)

NPAM, non-prematurely ageing mice; PAM, prematurely ageing mice; NPAMC and PAMC, NPAM and PAM controls; NPAMA and PAMA, NPAM and PAM with antioxidant supplementation; AC, adult controls; Con A, concanavalin A; NK, natural killer.

The data were analysed statistically by the three-way ANOVA for unpaired observations, followed by the Scheffe's F post-hoc test.

* P<0·05; **P<0·01; ***P<0·001 with respect to the corresponding values in controls (NPAMC and PAMC). ^bP<0·01; ^cP<0·001 with the corresponding values in adults. †P<0·05; ††P<0·01; †††P<0·001 with respect to the corresponding values in NPAMC. ‡P<0·05; ‡‡P<0·01; ‡‡‡P<0·001 with respect to the corresponding values in Swiss mice.

Table 3. Effect of a diet supplemented with two sulphur-containing antioxidants (thioproline (TP) and N-acetylcysteine (NAC); 0·1% (w/w) for 5 weeks) on several functions of thymus leucocytes from chronologically old prematurely and non-prematurely aged Swiss and BALB/c mice

(Mean values and standard deviations for 10 subjects)

NPAM, non-prematurely ageing mice; PAM, prematurely ageing mice; NPAMC and PAMC, NPAM and PAM controls; NPAMA and PAMA, NPAM and PAM with antioxidant supplementation; AC, adult controls; Con A, concanavalin A; NK, natural killer.

The data were analysed statistically by the three-way ANOVA for unpaired observations, followed by the Scheffe's F post-hoc test.

** P<0·01; ***P<0·001 with respect to the corresponding values in controls (NPAMC and PAMC). ^aP<0·05; ^bP<0·01; ^cP<0·001 with the corresponding values in adults. †P<0·05; ††P<0·01; †††P<0·001 with respect to the corresponding values in NPAMC. ‡P<0·05; ‡‡P<0·01; ‡‡‡P<0·001 with respect to the corresponding values in Swiss mice.

With respect to basal proliferation, there are differences between Swiss and BALB/c mice, the latter showing lower values. In general, the levels of proliferation in PAMC and NPAMC were lower than those in adults. The supplementation only increased the basal proliferation in cells from spleen and thymus of PAMA. Nevertheless, the proliferative response of lymphocytes to concanavalin A, which was lower in all cases in PAMC with respect to the corresponding NPAMC and also in all the old NPAMC and PAMC with respect to the values in the corresponding adults, increased after the supplementation. Moreover, the values in aged PAMA and NPAMA were generally similar to those in adults. The NK activity was, in general, smaller in PAMC with respect to NPAMC and in all cases in PAMC and NPAMC when the values were compared with those corresponding in adults. The ingestion of a supplementation of sulphur-containing antioxidants increased the NK activity in all cases bringing the values closer to those in adults although they did not reach those values. The IL-2 release in lymphocytes from axillary nodes (Table 1) was higher in cells from BALB/c mice than in those from Swiss. In both Swiss and BALB/c PAMC groups, there were significant decreases in the levels of this cytokine with respect to those in NPAMC, and in all groups of old animals (both NPAMC and PAMC) the values were lower than in adults. After the antioxidant supplementation in all the groups, there were significant increases of IL-2 concentrations bringing them close to the levels of adults, a change that was more evident in BALB/c mice.

Since all the functions studied are markers of biological age in mice and predictors of longevity⁽Reference De la Fuente and Miquel²⁾, the present data show that the ingestion of a low amount (0·1% (w/w)) of two antioxidants such as TP+NAC for a short term (5 weeks) decreases the biological age of mice making it more similar to that of chronological adults. Moreover, this effect was found in two different strains of mice, outbred Swiss and inbred BAB/c mice, and in chronologically old PAM and NPAM, but more extensively in PAM. In a previous study, it has been observed that in chronologically adult PAM and NPAM this kind of supplementation was also more effective in PAM than in NPAM, and in Swiss mice than in BALB/c mice⁽Reference Guayerbas, Puerto and Ferrandez⁴⁹⁾. However, in adult mice, the supplementation was, in general, less effective than in old animals, since the present results show that practically all the functions studied in leucocytes have been improved after the ingestion of the diet supplemented with the antioxidants (Tables 1–3). Although in chronologically old Swiss mice an ingestion of a high concentration of these antioxidants (0·3%) caused a stimulation of several of the functions studied here, this concentration in the adults decreased those functions and increased the oxidative stress of the animals⁽Reference De la Fuente, Guayerbas and Catalán³⁹⁾. In the present study, the mice were chronologically old, but they were divided into two groups with different biological age, since NPAM are always biologically younger than PAM of the same age⁽Reference Viveros, Arranz and Hernanz¹⁷⁾. Based on the present results, 0·1% (w/w) TP+NAC seems an appropriate amount to improve immune function in aged mice. Nevertheless, further research on the effects of higher amounts of those antioxidants in old PAM and NPAM should be carried out.