Zinc improves the development of human CD34+ cell progenitors towards NK cells and increases the expression of GATA-3 transcription factor in young and old ages.

Aim of this study was to evaluate the effect of zinc on the kinetic of development of CD34(+) cell progenitors towards NK cells in young and old ages. CD34(+) cells were purified from peripheral blood of healthy subjects and cultured in medium supplemented with interleukin-15, interleukin-7, Flt 3 ligand, and stem cell factor. The number of cells developed in culture was significantly lower in old than in young subjects. CD34(+) cells progressively lost CD34 antigen with a faster kinetics in old than in young donors. The percentage of primitive double positive CD34(+)CD133(+) cells inside the purified CD34(+) cells was greatly lower in old than in young subjects. These cells progressively decreased in cultures from young subjects whereas they remained at very low levels in old donors. Cells developed in culture acquired a NK phenotype mainly characterized by CD56(+)CD16(-) cells in young subjects and CD56(-)CD16(+) cells in old donors. These NK cells exerted a lower cytotoxic activity in old than in young subjects. The supplementation with zinc greatly increased the number of cells in culture and the percentage and the cytotoxic activity of NK cells both in young and old ages. In zinc supplemented cultures, a 3.6-fold and a 4.1-fold increased expression of GATA-3 transcription factor was observed in young and old donors, respectively. Our data demonstrate that zinc influences the proliferation and differentiation of CD34(+) progenitors both in young and old ages.

Zinc improves the development of human CD34+ cell progenitors towards Natural Killer cells and induces the expression of GATA-3 transcription factor.

The Natural Killer cell maturation from CD34(+) hematopoietic cell precursors is a complex process that requires the synergistic effect of different cytokines and growth factors. Although there have been a number of important advances in our understanding of the Natural Killer differentiation, the developmental step leading to mature Natural Killer cells is still poorly defined. We evaluated the effect of two zinc concentrations (10 and 20microM) on the kinetic of development of CD34(+) cell progenitors towards Natural Killer cells. CD34(+) cells were purified from peripheral blood and cultured in medium supplemented with interleukin-15, interleukin-7, Flt 3 ligand, and stem cell factor. CD34(+) cells underwent proliferation and progressively lost CD34 antigen and acquired a CD56(+) phenotype. These CD56(+) cells exerted cytotoxic activity and expressed the CD94 inhibitory receptor. The supplementation with zinc greatly increased both the number of cells in culture and the absolute number of CD56(+) cells. Zinc induced higher levels of cytotoxic activity and a higher number of perforin-producing and of CD94-bearing CD56(+) cells in comparison with zinc unsupplemented cultures in early stages of Natural Killer cell development. The zinc-induced changes in CD34-derived CD56(+) cells were associated with an increased expression of GATA-3, a zinc-finger transcription factor providing for maturation and activity of T and Natural Killer cells. The increase was related to a higher CD56(+) cell number (10microM zinc), or to an increased GATA-3 mRNA transcription in CD56(+) cells (20microM zinc). Our data demonstrate that zinc influences the proliferation and differentiation of CD34(+) progenitors.

Psychological, neuroendocrine and immune measures in non spousal carers of disabled elderly in Italy.

OBJECTIVES: The purpose of this study was to determine whether psychological well being as well as metabolic, neuroendocrine and immune functions were different in non spousal primary caregivers of disabled elderly than in controls. SETTING AND DESIGN: We randomly recruited 38 primary family carers of over 65 year old recipients of health home care services and 37 controls stratified according to sex and age. METHOD: Data were collected on psychological wellbeing (including anxiety, depression and self-perceived quality of life), on neuroendocrine and immune conditions (haemanalysis and metabolic signs, plasma ACTH, cortisol, prolactin, intra-lymphocyte content of beta-endorphins, NK cell activity and number), as well as on the incidence and severity of influenza disease during previous winter. RESULTS: Caregivers showed greater anxiety, although mean scores did not reach pathological levels. Neither depression nor satisfaction on quality of life did differ significantly, nor differences in haemanalisis and metabolic signs were found, apart from leukocyte and lymphocyte number, which was significantly lower in carers. Plasma levels of ACTH, cortisol and prolactin, the intra-lymphocyte content of beta-endorphins as well as the NK cell number and cytotoxicity did not show significant differences. Incidence and severity of influenza episodes was also similar, whereas the duration of influenza disease showed to be significantly longer. CONCLUSIONS: Non spousal caregivers of disabled elderly suffer from only slight alterations of psychological, endocrine and immune parameters, and do not respond very differently to influenza disease. This does not support therefore any generic privilege for them in the allocation of public support or respite services.

Zinc, immune plasticity, aging, and successful aging: role of metallothionein.

The capacity of the remodeling immune responses during stress (immune plasticity) is fundamental to reach successful aging. We herein report two pivotal models to demonstrate the relevance of the immune plasticity in aging and successful aging. One model is represented by the circadian rhythms of immune responses; the other one is the immune responses during partial hepatectomy/liver regeneration (pHx). The latter is suggestive because it mimics the immunosenescence and chronic inflammation 48 hours after partial hepatectomy in the young through the continuous production of IL-6, which is the main cause of immune plasticity lack in aging. The constant production of IL-6 leads to abnormal increments of zinc-bound metallothionein (MT), which is, in turn, unable in zinc release in aging. As a consequence, low zinc ion bioavailability appears for thymic and extrathymic immune efficiency, in particular, of liver NKT cells bearing TCR gd. The remodeling during the circadian cycle and during pHx of zinc-bound MT confers the immune plasticity of liver NKT gamma delta cells and NK cells in young and very old age, not in old age. Therefore, zinc-bound MT homeostasis is crucial in conferring liver immune plasticity with subsequent successful aging.

The variations during the circadian cycle of liver CD1d-unrestricted NK1.1+TCR gamma/delta+ cells lead to successful ageing. Role of metallothionein/IL-6/gp130/PARP-1 interplay in very old mice.

NKT cells derive from the thymus and home to the liver. Liver NKT cells can be divided in two groups: 'classical' and 'non-classical'. The first is CD1d-restricted, the second is CD1d-unrestricted. NKT cells (classical and non-classical) co-express T-cell receptor (TCR) and NK-cell marker (NK1.1), display cytotoxicity and produce IFN-gamma under IL-12 stimulation affecting, thereby, Th1 response and innate immunity. NK1.1(+)TCR alpha/beta(+) cells belong to both groups. NK1.1(+)TCR gamma/delta(+) cells belong to the second group. Anyway, both NKT cell subtypes, via IFN-gamma production, protect against viruses and bacteria from early in life. Immune variations as well as zinc rhythmicity during the circadian cycle confer the immune plasticity, which is essential for successful ageing. Liver NK1.1(+)TCR gamma/delta(+) cells, rather than TCR alpha/beta(+), from young and very old mice display 'in vitro' (under IL-12 stimulation) nocturnal peaks in cytotoxicity and IFN-gamma production. The acrophase of liver NK1.1(+)TCR gamma/delta(+) cells is present in young and very old mice, not in old. The interplay among zinc-bound metallothionein (MT)/IL-6/gp130/poly(ADP-ribose) polymerase-1 (PARP-1) may be involved in conferring plasticity to liver NK1.1(+)TCR gamma/delta(+) cells. IL-6, via sub-unit receptor gp130, induces MTmRNA. At night, gene expressions of MT, IL-6, gp130 are lower in very old mice than old and young MT-I transgenic mice (MT-I*). In very old mice, this phenomenon allows limited sequester of intracellular zinc from MT leading to good free zinc ion bioavailability for immune efficiency and zinc-dependent PARP-1 activity. Indeed (1) in vitro, high IL-6 provokes strong accumulation of MT, impaired cytotoxicity and low zinc ion bioavailability in liver NK1.1(+)TCR gamma/delta(+) cells exclusively from old and MT-I* mice. (2) The ratio total/endogen PARP-1 activity is higher in very old than in old and MT-I* mice, suggesting a higher capacity of PARP-1 in base excision DNA-repair in very old age thanks to low zinc-bound MT. Cytotoxicity and IFN-gamma production from liver NK1.1(+)TCR gamma/delta(+) cells are thus preserved leading to successful ageing. In conclusion, MT/IL-6/gp130/PARP-1 interplay may confer plasticity to liver CD1d-unrestricted NK1.1(+)TCR gamma/delta(+) cells, where MT, IL-6, gp130 are the main upstream protagonists, and PARP-1 is the main downstream protagonist in immunosenescence.

Metallothionein (I+II) confers, via c-myc, immune plasticity in oldest mice: model of partial hepatectomy/liver regeneration.

Because of its similarity to ageing in impaired immune efficiency 48 h after surgical procedures on young partially hepatectomised mice, partial hepatectomy/liver regeneration (pHx) provides a good model for the study of inflammation in ageing. In old age, high metallothionein (I+II) (MT) sequesters a substantial number of intracellular zinc ions consequently leading to low zinc ion bioavailability for an adequate immune response. Corticosterone and IL-6 affect MTmRNA induction in inflammation and after pHx against oxidative damage. The aim of this study was to investigate the role played by MT in conferring immune plasticity in ageing and in very old age using the pHx model. 48 h after their partial hepatectomy, the crude zinc balance was negative in young, old and very old mice coupled with increased MT, corticosterone, sIL-6R and IL-6. Concomitantly, Natural Killer (NK) cell activity and IL-2 production decreased. Complete restoration of the nutritional-endocrine-immune parameters occurred 15 days from the surgical procedures in young and very old mice, but not in old or transgenic mice overexpressing MT. A significant positive or inverse correlation among nutritional-endocrine-immune parameters exists in young and very old mice, but not in old mice during liver regeneration. Since MT also affects c-myc, the gene expression of c-myc declines from 48 h to days 7 and 15 after pHx in young and very old mice, but remains constantly high in old pHx mice for the same days. This circumstance leads to the appearance of tumours in the long run in old pHx mice and survival times that are shorter than old sham controls. Because complete remodelling also occurs in IL-6 and in sIL-6R in very old mice during liver regeneration, the pre-existing inflammation is not detrimental in very old age. As such, very old mice are still responsive to large inflammation, such as pHx, thanks to correct MT homeostasis. Correct MT homeostasis, via c-myc, is therefore pivotal in both suitable liver regeneration and in conferring immune plasticity with subsequent successful ageing. High MT plays an extremely harmful role in ageing: on one hand it lowers zinc ion bioavailability levels required for immune efficiency and on the other hand it increases c-myc expression. The combination of immune depression and enhanced c-myc, via high MT, may trigger the appearance of age-related degenerative diseases.

Interrelationships among brain, endocrine and immune response in ageing and successful ageing: role of metallothionein III isoform.

Metallothionein-III (MT-III) a brain-specific member of metallothionein family contributes to zinc neuronal homeostasis, and zinc is an important regulator of many brain functions, including the activity of hormone realising factors by hippocampus. Among them, somatostatin is pivotal because affecting thyroid hormones turnover and consequently thymic and peripheral immune efficiency (Natural Killer, NK) cell activity. Somatostatin is in turn affected by somatomedin-C, which is also zinc-dependent. Therefore, somatomedin-C may be a marker of somatostatin status in the hippocampus. MTs sequester and release zinc in transient stress, as it may occur in young age, to protect cells by reactive oxygen species. In order to accomplish this task, MTs are induced by IL-6 for a prompt immune and anti-inflammatory response. During ageing, MTs are high with a role of sequester of zinc, but with very limited role in zinc release because stress-like condition and inflammation is persistent. Therefore, high MTs may become to protective in young age to harmful during ageing leading to low zinc ion bioavailability for many body homeostatic mechanisms, including brain function. As a consequence, an altered physiological cascade from the brain (upstream) to endocrine and immune system (downstream) may occur. The aim of this work is to study the role of MT-III in the interrelationships among brain-endocrine-immune response in ageing and successful ageing. The main results are: (1) MT-III and IL-6 gene expressions increase in the hippocampus from old mice, in comparison with young and very old mice. (2) Somatomedin-C plasma levels decrease in old mice in comparison with young and very old mice. (3) Low zinc ion bioavailability (tested by the ratio total thymulin/active thymulin) is coupled with altered thyroid hormone turnover and depressed IL-2 in old mice in comparison with young and very old mice. (4) 'In vitro' experiments display more increments on NK cells activity by adding zinc-bound active thymulin than T3 alone. In conclusion, low MT-III in the hippocampus from young and very old mice leads to good zinc ion bioavailability that it is upstream coupled with normal hippocampal function affecting downstream normal thyroid hormones turnover and satisfactory NK cell activity, via complete saturation of zinc-bound active thymulin molecules. Therefore, a correct MTs homeostasis is pivotal for brain-endocrine-immune response in order to reach successful ageing.

Metallothioneins/PARP-1/IL-6 interplay on natural killer cell activity in elderly: parallelism with nonagenarians and old infected humans. Effect of zinc supply.

Metallothioneins (MTs) play pivotal role in zinc-related cell homeostasis because of their high affinity for this trace element which is in turn relevant against oxidative stress and for the efficiency of the entire immune system, including natural killer (NK) cell activity. In order to accomplish this role, MTs sequester and/or dispense zinc during stress and inflammation to protect cells against reactive oxygen species. MTs gene expression is affected by IL-6 for a prompt immune response. Concomitantly, MTs release zinc for the activity of antioxidant zinc-dependent enzymes, including poly(ADP-ribose)polymerase-1(PARP-1), which is involved in base excision DNA-repair. This role of MTs is peculiar in young adult-age during transient stress and inflammation, but not in ageing because stress-like condition and inflammation are persistent. This may lead MTs to turn-off from role of protection in young age to deleterious one in ageing with subsequent appearance of age-related diseases (severe infections). The aim is to study the role played by MTs/IL-6/PARP-1 interplay on NK cell activity in elderly, in old infected patients (acute and remission phases by bronchopneumonia infection) and in health nonagenarian/centenarian subjects. MTmRNA is high in lymphocytes from elderly people coupled with high IL-6, low zinc ion bioavailability, decreased NK cell activity and impaired capacity of PARP-1 in base excision DNA-repair. The same trend in this altered physiological cascade during ageing also occurs in old infected patients (both acute and remission phases) with more marked immune damage, inflammatory condition and very impaired PARP-1 in base excision DNA-repair. By contrast, centenarian subjects display low MTmRNA, good zinc ion bioavailability, satisfactory NK cell activity and higher capacity of PARP-1 in base excision DNA-repair. These findings clearly demonstrate that the sequester of zinc by MTs in ageing is deleterious because leading to low zinc ion bioavailability with subsequent impairment of PARP-1 and NK cell activity and appearance of severe infections. Physiological zinc supply (12 mg Zn(++)/day) for 1 month in elderly and in old infected patients (remission phase) restores NK cells activity with values observed in health centenarians. Therefore, the zinc ion bioavailability by zinc-bound MTs homeostasis is pivotal to reach health longevity and successful ageing.

Metallothioneins (I+II) and thyroid-thymus axis efficiency in old mice: role of corticosterone and zinc supply.

Thymic atrophy or thymus absence causes depressed thyroid-thymus axis (TTA) efficiency in old, young propyl-thiouracil (PTU) (experimental hypothyroidism) and in young-adult thymectomised (Tx) mice, respectively. Altered zinc turnover may be also involved in depressed TTA efficiency. Zinc turnover is under the control of zinc-bound metallothioneins (Zn-MTs) synthesis. Thyroid hormones, corticosterone and nutritional zinc affect Zn-MT induction. Zn-MT releases zinc in young-adult age during transient oxidative stress for prompt immune response. In constant oxidative stress (ageing and liver regeneration after partial hepatectomy), high liver Zn-MTs, low zinc ion bioavailability and depressed TTA efficiency appear. This last finding suggested that MT might not release zinc during constant oxidative stress leading to impaired TTA efficiency. The aim of this work/study is to clarify the role of Zn-MTs (I+II) in TTA efficiency during development and ageing. The main results are (1) Old and PTU mice display high corticosterone, enhanced liver MTmRNA, low zinc and depressed TTA efficiency restored by zinc supply. Increased survival and no significant increments in basal liver Zn-MTs proteins occur in old and PTU mice after zinc supply. (2) Lot of zinc ions bound with MT in the liver from old mice than young (HPLC). (3) Young-adult Tx mice, evaluated at 15 days from thymectomy, display high MTmRNA and nutritional-endocrine-immune damage restored by zinc supply or by thymus grafts from old zinc-treated mice. (4) Young-adult Tx mice, but evaluated at 40 days from thymectomy, display natural normalisation in MTmRNA and nutritional-endocrine-immune profile with survival similar to normal mice. (5) Stressed (constant dark for 10 days) mice overexpressing MT display low zinc, depressed immunity, reduced thymic cortex, high corticosterone, altered thyroid hormones turnover showing a likeness with old mice. These findings, taken altogether, show that corticosterone is pivotal in MTs induction under stress. MTs bind preferentially zinc ions in constant oxidative stress, but with no release of zinc from MT leading to impaired TTA efficiency. Zinc supply restores the defect because zinc has no interference in affecting pre-existing Zn-MTs protein concentrations in old and PTU mice. Therefore, free zinc ions are available for TTA efficiency after zinc supply. Thymus from old zinc-treated mice induces the same restoring effect when transplanted in Tx recipients. However, Tx mice display natural normalisation in MTmRNA and in nutritional-endocrine-immune profile in the long run. Therefore, Zn-MTs (I+II) are crucial in zinc homeostasis for endocrine-immune efficiency during the entire life assuming a role of potential and novel 'biological clock of ageing'.

MtmRNA gene expression, via IL-6 and glucocorticoids, as potential genetic marker of immunosenescence: lessons from very old mice and humans.

Metallothioneins (MTs) are involved in metal-related cell homeostasis because of their high affinity for metals forming clusters. The main functional role of MTs is to sequester and/or dispense zinc participating in zinc homeostasis, which is relevant in normal immune response. Consistent with this role, MTs gene expression (MTmRNA) is transcriptionally induced by a variety of stressing agents to protect cells from reactive oxygen species. In order to accomplish this task, MTs gene expression is affected by glucocorticoids and IL-6 for a prompt immune response. This protection is peculiar in young-adult age during transient stress and inflammatory condition, but not in ageing because stress-like condition and inflammation are constant for the whole circadian cycle. This may lead MTs to turn-off from role of protection in young age to deleterious one in ageing. The aim is to suggest MTmRNA as potential genetic marker of immunosenescence. Liver MTmRNA, IL-6 and glucocorticoids levels are high, whereas the bioavailability of zinc ions is low and natural killer cells activity is depressed in old and very old mice during the light period as compared to young in the same period. An inversion of nutritional-endocrine-immune profile exclusively occurs in young mice during the night showing the existence of immune plasticity. No inversion occurs in old mice during the night. As a consequence, no immune plasticity in old mice ensues. By contrast, very old mice remodel the altered MTmRNA and immune-endocrine profile during the night up to values of young ones observed during the light period. Therefore, the remodelling of MTmRNA may be involved in the maintenance of immune plasticity with subsequent successful ageing. Thus, MTmRNA, via IL-6 and glucocorticoids, may be potential genetic marker of immunosenescence. This assumption is reinforced by low MTmRNA in lymphocytes of nonagenarians and young-adult people in comparison with elderly and Down's syndrome subjects.