Daniela Frasca

Genes, immunity, and senescence: looking for a link

Summary: Aging is under the control of a small number of regulatory genes. Mice genetically selected for high immune responses, in most cases, exhibit longer life span and lower lymphoma incidence than do mice selected for low responses. The link between immunity and aging is further evidenced by the age‐related alterations of the immune system, mostly of the T‐cell population, in terms of replacement of virgin by memory cells, accumulation of cells with signal transduction defects, and changes in the profile of Thl and Th2 type cytokines. Also, B cells exhibit intrinsic defects, and natural killer (NK) cell activity is profoundly depressed by aging. In vitro experiments indicate that IL‐2, IFN‐γ, and IL‐4 production by mouse spleen cells changes with aging and may be upregulated by recombinant cytokines. These findings suggest possible cytokine interventions to prevent or treat age‐related immune disorders, as they may affect the duration and the biological quality of life.

Regulation of cytokine production in aging: use of recombinant cytokines to upregulate mitogen-stimulated spleen cells

We investigated the production of IL-2 and IFN-gamma (Th1 type) and IL-4 (Th2 type) cytokines by mitogen-activated spleen cells from young, adult and old mice. Cytokine production was evaluated in culture supernatants by CTLL proliferation (IL-2), ELISA (IFN-gamma), CT4.S proliferation (IL-4) and in mRNA extracted from activated CD4+ cells by RT-PCR (IL-2, IFN-gamma and IL-4). Results show that the production of IL-2, as protein and mRNA, is profoundly depressed by aging, whereas that of IFN-gamma, as protein and mRNA, firstly declines and then increases with age. The production of IL-4, as protein, monotonically declines with aging whereas, as mRNA, firstly decreases and then increases above the level in young mice. Spleen cells in culture were also incubated with mitogens and with a recombinant cytokine (IL-1 beta, IL-2, IL-3, IL-4, IL-12 or IFN-gamma) at various concentrations. It was found that recombinant cytokines by and large enhance cytokine production when the level induced by mitogens only is low. This conclusion applies to IL-2 and IFN-gamma production as protein and mRNA. The addition of recombinant cytokines also increases the production of IL-4 at the protein level in spleen cells from old mice but, at the mRNA level, only in spleen cells from young mice. This finding suggests age-related changes in IL-4-specific mRNA transcription rate and post-transcriptional half-life as well as translation kinetics.

Hormone replacement therapy affects various immune cell subsets and natural cytotoxicity

The effects of hormone replacement therapy (HRT) on lymphocytes and granulocytes have never been determined in detail. Ten healthy menopausal women (age 49-51 years; menopause less than 2 years) were treated for 6 months by administering transdermal estradiol (100 micrograms/day for 21 consecutive days) and oral medroxyprogesterone acetate (10 mg/day from day 10 to day 21). Days 22-28 were therapy-free. All subjects were examined during the first and the last month of treatment: evaluations were carried out on days 0, 8, 21 and 28. CD4+CD45RO+ cells were found to be significantly reduced on day 8. CD56+ cells and CD8+CD11b+ cells were decreased on day 21 and recovered basal level on day 28. Natural killer cell function was transiently increased on day 8 and greatly reduced on day 21. During the first month of therapy, the expression of Leu8 and CD11b antigens on granulocyte membranes was significantly affected by HRT. Taken together, the results indicate that HRT selectively affects various immune cell subsets.

Role of mRNA stability in the different patterns of cytokine production by CD4+ cells from young and old mice

CD4+ cells from young (3 months) and old (19 months) mice were stimulated by plate‐bound anti‐CD3 monoclonal antibody (mAb) alone or also by soluble anti‐CD28u2003mAb. Supernatants were analysed by enzyme‐linked immunosorbent assay (ELISA) to determine cytokine concentrations. Total RNA was extracted from cells, reverse transcribed and the cDNA amplified by polymerase chain reaction (PCR) to evaluate the amount of specific mRNA. The results indicate that anti‐CD3 alone is not sufficient to induce interleukin‐2 (IL‐2) production in CD4+ cells from both young and old mice. However, anti‐CD28, together with anti‐CD3u2003mAb, induces a much higher production of IL‐2 in CD4+ cells from young as compared with old mice. Conversely, interferon‐γ (IFN‐γ) production is also induced by anti‐CD3 alone and is higher in CD4+ cells from old as compared with young mice. Upon addition of anti‐CD28u2003mAb, IFN‐γ production increases in both groups, but it remains much higher in old than in young mice. Also the production of IL‐4 and IL‐10 is induced by anti‐CD3u2003mAb but it is increased by the addition of anti‐CD28u2003mAb. CD4+ cells from old mice produce more IL‐4 and IL‐10 as compared with cells from young mice. The amounts of cytokine specific mRNA in CD4+ cells from young and old mice parallel the cytokine levels in culture supernatants. Results on the mRNA turnover indicate that when CD4+ cells are stimulated by anti‐CD3 or costimulated also by anti‐CD28u2003mAb, the IFN‐γ, IL‐4 and IL‐10 specific mRNAs are more stable in old than in young mice, suggesting that mRNA stability has a relevant role in the different patterns of cytokine production.

Effect of age on DNA binding of the ku protein in irradiated human peripheral blood mononuclear cells (PBMC)

DNA binding of the ku protein was investigated in peripheral blood mononuclear cells (PBMC) from 24 subjects of different ages (20-89 years old) displaying age-related changes in DNA repair, mitotic responsiveness, and cytokine production. Ku is an heterodimeric protein composed of two subunits of 70 and 80 kDa, which is involved in the earliest steps of DNA damage recognition. DNA binding of ku 70/80 was found unchanged in normal PBMC from aging subjects but progressively declined in x-ray-irradiated PBMC from young to adult, and elderly subjects. This finding was concomitant with the age-related fall of DNA repair in the whole population.

Effect of synthetic thymic humoral factor (THF‐γ2) on T cell activities in immunodeficient ageing mice

Immunodeficient ageing (C57BL/10 × DBA/2)F1 mice were treated by a single injection of synthetic thymic hormones and 4 days later their thymus and spleen cells were assayed in vitro for T cell activities. A few nanograms of THF–γ2 were found to raise the frequency of mitogen‐responsive T cells in thymus and spleen cell populations as well as the frequency of cytokine‐producing splenic T cells, up to the levels observed in young mice. Moreover, injection of THF‐γ2 was found to restore T cell growth factor (TCGF) production by mitogen‐stimulated spleen cells. Also, the helper activity of spleen cells was enhanced by this treatment and increased with increasing theTHF‐γ2 dose over a wide range. Similarly, the effects of thymopentin and thymosin–α1 on T helper cell activity increased with increasing the injected dose, but the efficiencies of THF‐γ2 and thymopentin were, respectively. 400‐fold and eight‐fold greater than that of thymosin–α1.

Expression and DNA binding activity of the Ku heterodimer in bladder carcinoma

The Ku protein is a tightly associated heterodimer, comprised of 70‐kilodalton (kD) and 86‐kD subunits, that forms the DNA‐dependent protein kinase (DNA‐PK) complex together with the 470‐kD DNA‐PKcs catalytic subunit, and is involved mainly in DNA double‐strand breaks (DSBs) repair. The objective of the current study was to investigate the expression and DNA‐binding activity of the Ku protein in fresh tissues from patients with bladder carcinoma and to compare it with that in nontumor tissues obtained from the same organ. Moreover, the DNA‐binding activity of Ku was assessed after exposure of the tumor cells to 1 or 2 grays (Gy) of X‐rays. Furthermore, the level of phosphorylated Ku was analyzed in both the nuclear and cytoplasmic compartment of normal tissue after exposure to 2 Gy of X‐rays.

Aging of the recipients but not of the bone marrow donors enhances autoimmunity in syngeneic radiation chimeras

Young and old mice have been lethally irradiated and injected with syngeneic bone marrow cells from young or old donors to investigate whether self reactivity in old mice results from age-related damage of the radioresistant stromal cells and/or of the bone marrow hematopoietic cells. Thymus and spleen cell repopulations and mitotic responses at 3 months after irradiation are lower in old than in young recipients, suggesting age-related accumulation of stromal cell damage in the thymus as well as in other central and peripheral lymphoid tissues. The same efficiency of bone marrow cells from young and old donors to repopulate the thymus and spleen in recipients of equal age rules out the detrimental effects of aging on stem cells as well as T and B cell precursors. The serum concentration of auto-antibody and glomerular lesions at 3 and 9 months after irradiation were more pronounced in old than in young recipients and displayed no difference in recipients of equal age, regardless of the age of the bone marrow cell donors. These findings support the possibility that age-related damage of stromal cells induces disregulation of the immune system leading to autoimmune phenomena.

Cell proliferation and ku protein expression in ageing humans

Previous studies on DNA repair in ageing have demonstrated increased frequencies of single and double strand breaks in lymphocytes from elderly subjects and, as a consequence, decreased efficiency in DNA replication. We have investigated the relationship between cell proliferation and the nuclear expression of ku protein in a human population of 43 subjects of different ages. Ku is an heterodimeric protein composed of two subunits of 70 and 80 kDa, which is involved in the early steps of DNA damage recognition. In the present study, PBL from subjects of different ages were PHA-activated to evaluate the stimulation index and the production of Th1- and Th2-type cytokines. Moreover, nuclear extracts were obtained from activated lymphocytes to evaluate by a gel retardation assay the presence and the functional activity of the heterodimer ku 70/80. Our results indicate that ageing affects the mitotic responsiveness and cytokine production to a significant extent, but only marginally the expression of ku 70/80. These findings suggest that the age-related impairment in DNA repair mechanisms are only in part related to the reduced expression of ku protein able to recognize DNA damage.

Regulation of Cytokine Production in Aging Mice

The thymus plays a central role in the development and maintenance of immunity and tolerance, as it provides the microenvironment for T-cell maturation and selection. The intrathymic differentiation results mainly from cell-to-cell interactions between T-cell precursors and accessory (epithelial and dendritic) cells. Moreover, the thymus apparently secretes several hormone-like products that influence T-cell differentiation..3 Several extracts have been prepared from the thymus and their chemical characterization has led to the identification of the active peptides, some of which have been sequenced and synthesized.-l? The synthetic peptides more extensively investigated are: thymosin a,, (28 aa), thymopentin (5 aa), thymulin (9 aa), whose activity is strictly dependent on the presence of zinc, and thymic humoral factor (THF)-y2 (8 aa). These synthetic peptides differ in amino acid sequence and biologic properties.I3 In our previous studies, we demonstrated that injection of thymosin a, thymopentin, or THF-y2 into aging mice can increase T-cell functions when spleen cells were tested in vitro for T helper cell activity, proliferative responses to T-cell mitogens, cytokine production, and expression of cytokine receptors. The effect of injected synthetic thymic peptides on T helper cell activity increased with an increase in the injected dose, but the efficiencies of THF-y2 and thymopentin were 400-fold and 8fold greater than that of thymosin aI, respectively. More recently, we investigated the production of cytokines, namely, interleukin2 (IL-2) and IL-4, by mitogen-stimulated spleen cells from aging mice when cultured with THF-y2 or a recombinant murine cytokine (IL-lp, IL-2, IL-3, IL-4, or IFN-y). Briefly, 4 x lo5 spleen cells from aging (C57BW10 x DBN2)Fl mice were stimulated in culture with mitogens (concanavalin A, 0.5 p.g/well and phorbol myristate acetate, 4 ng/well) alone or with mitogens and THF-y2 or a given cytokine in various concentrations. Each culture was set up in triplicate, in a final volume of 200 p.1. Spleen cell proliferation was assessed as follows. After 18 hours of incubation, cells in culture were seeded by centrifugation, and the supernatants were collected and substituted with medium containing tritiated thymidine (0.5 p. Ci/20 p.1, spec. act. 1.739 GBq/mmol). Four hours later, cultures were sacrificed with an automated cell harvester. Radioactivity was expressed as cpm/culture.