Mohammad A. Pahlavani

Influence of aging and caloric restriction on activation of Ras/MAPK, calcineurin, and CaMK-IV activities in rat T cells.

The signaling cascade mediated by Ras (p21ras) and MAPK (mitogen-activated protein kinase) and calcium/calmodulin regulating enzymes, calcineurin (CaN) and CaMK-IV, are considered to be essential for T-cell growth and function. In the present study, the effect of aging and caloric restriction (CR) on the induction of Ras and MAPK activation by concanavalin A (ConA) was studied. Splenic T cells were isolated from young (4-6 months) and old (22-24 months) rats that had free access to food (control group), and from caloric restricted old (22-24 months) rats that beginning at 6 weeks of age were fed 60%(40% caloric restriction) of the diet consumed by the control rats. We found that the induction of Ras activity in T cells isolated from control old rats was lower (P<0.001) than that in control young rats. However, the levels of Ras activity in T cells isolated from CR old rats were similar to the levels in the age-matched control rats. The induction of MAPK activity in T cells isolated from control old rats and CR old rats was significantly less than in T cells isolated from control young rats, and caloric restriction significantly (P<0.05) reduced the age-related decline in MAPK activation. We also measured the induction of CaN and CaMK-IV activities by ConA in T cells from control young and old and CR old rats. The induction of both CaN and CaMK-IV activity decreased with age. Caloric restriction significantly (P<0.05) reduced the age-related decline in CaN activity, but had no significant effect on CaMK-IV activity. The changes in Ras/MAPK activation and in CaN and CaMK-IV activity with age or with CR were not associated with alterations in their corresponding protein levels. Thus, caloric restriction has a differential effect on the activation of the upstream signaling molecules that are altered with age.

IN VITRO EFFECTS OF MELATONIN ON MITOGEN-INDUCED LYMPHOCYTE PROLIFERATION AND CYTOKINE EXPRESSION IN YOUNG AND OLD RATS

Melatonin (MLT) treatment in vivo has been shown to have immunomodulatory and anti-immunosenescent effects in the mouse model. In the present report, the in vitro effect of MLT on mitogen-induced lymphocyte proliferation and cytokine expression was evaluated in a rat model. Splenic lymphocytes were isolated from young (6 months) and old (24 months) F344 rats and were incubated with MLT in the presence or absence of mitogens. The proliferative response to concanavalin A (ConA) or PMA plus ionomycin was measured in splenocytes or T cells isolated from young and old rats. In addition, the induction of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production was measured in MLT-treated and untreated lymphocytes isolated from young and old rats. The ConA-induced lymphocyte proliferation and IL-2 expression were significantly lower and induction of IFN-gamma production was significantly higher in splenocytes and purified T cells isolated from old rats compared to splenocytes and T cells isolated from young rats. Treatment of lymphocytes with MLT did not significantly alter ConA-induced lymphocyte proliferation or IL-2 or IFN-gamma expression in lymphocytes isolated from either young or old rats. On the basis of these data, we conclude that in vitro MLT treatment had no immunomodulatory effect on lymphocytes from rats.

The age-related changes in DNA binding activity of AP-1, NF-κB, and OCT-1 transcription factors in lymphocytes from rats

The effect of aging on the induction of the ubiquitous transcription factors AP-1, NF-κB, and OCT-1 by concanavalin A (conA) was studied in nuclear extracts from spleen lymphocytes isolated from young (6 mo) and old (24 mo) male Fischer 344 rats. The induction of AP-1, NF-κB, and OCt-1 DNA binding activities were significantly lower (35 to 60%) in nuclear extracts from spleen lymphocytes isolated from old rats compared to nuclear extracts from spleen lymphocytes isolated from young rats. Because the transcription factor AP-1 consists of heterodimers of Fos and Jun oncoproteins, the induction of c-fos and c-jun expression (mRNA levels) by conA stimulated lymphocytes was measured using northern blot analysis. ConA induction of c-fos mRNA but not c-jun mRNA decreased approximately 70% with age. Therefore, the age-related decrease in the induction of AP-1 transcription factor appears to arise from alterations in c-fos expression.

Effect of dehydroepiandrosterone on mitogen-induced lymphocyte proliferation and cytokine production in young and old F344 rats

The steroid hormone intermediate, dehydroepiandrosterone (DHEA), has been proposed as a therapeutic agent for the treatment of immunosenescence in mouse model. In the present study, the in vitro effect of DHEA on mitogen-induced lymphocyte proliferation and cytokine production was evaluated in a rat model. Spleen lymphocytes were isolated from young (4-6 months) and old (24-26 months) F344 rats and were incubated with DHEA for 30 min. The induction of lymphocyte proliferation, interleukin-2 (IL-2), and interferon-gamma (IFN-gamma) production by concanavalin A (Con A) was measured in a culture medium supplemented with either fetal calf serum (FCS) or with serum-free medium (Nutridoma-SR, N-SR). The induction of lymphocyte proliferation and IL-2 production by Con A decreased significantly with age, whereas induction of IFN-gamma increased with age. Treatment of lymphocytes with DHEA did not significantly alter Con A-induced proliferation or the production of IL-2 or IFN-gamma by spleen lymphocytes isolated from either young or old rats. These data indicate that in vitro DHEA treatment appears to have no immunomodulatory effect on the age-related changes in mitogen-induced proliferation or cytokine production in rat lymphocytes.

Melatonin fails to modulate immune parameters influenced by calorie restriction in aging Fischer 344 rats.

The aim of this study was to determine if long-term treatment with melatonin (MEL), a purported anti-aging agent, was as effective as calorie restriction (CR) in modulating immune parameters in aging Fischer 344 male rats. Splenic lymphocytes were isolated from 17-month-old rats that, beginning at 6 weeks of age, were treated with MEL (4 or 16 μg/ml in drinking water) and from 17-month-old rats fed ad libitum (AL) or rats fed a CR diet (55% of AL intake). The number of splenic T cell populations and T cell subsets was measured by flow cytometry, the proliferative response of splenocytes to Concanavalin A (Con A) and lipopolysaccharide (LPS) was measured by [3H]thymidine incorporation, and the induction of cytokine production (IL-2 and IFN-γ) was measured by ELISA assay. In addition, the level of the natural killer (NK) cell activity was assessed by fluorimetric assay. CR rats had a higher number of lymphocytes expressing the naïve T cell marker (CD3 OX22) than AL rats (P < 0.05). CR rats also showed greater induction of proliferative response, IL-2 and IFN-γ levels following Con A simulation, and NK cell activity than AL rats (P < 0.05). MEL-treated rats did not differ from AL rats in any of these parameters or in any other measurement. These results indicate that MEL treatment is unable to modulate immune function in a manner comparable with that of CR.

Intervention in the aging immune system: Influence of dietary restriction, dehydroepiandrosterone, melatonin, and exercise.

The decline in immunologic function with age is associated with an increase in susceptibility to infections and the occurrence of autoimmune diseases and cancers. Hence, the restoration of immunologic function is expected to have a beneficial effect in reducing pathology and maintaining a healthy condition in advanced age. A number of therapeutic strategies have been employed to intervene in the aging immune system. This article reviews the effect of dietary restriction (DR), dehydroepiandrosterone (DHEA) treatment, melatonin (MLT) therapy, and exercise on modulating the immune responses and retarding/reducing immunosenescence. DR has been subject to intensive research and is known to be the most efficacious means of increasing longevity, reducing pathology and enhancing immune function.The circulatory levels of the androgenic hormone DHEA and the pineal hormone MLT decrease with increasing age, and this decrease has been correlated with the age-related decline in the immune system. Therefore, the observation that immunosenescence is associated with low levels of DHEA and MLT has provided a rationale for therapeutic intervention. DHEA treatment and MLT therapy both exhibit immunostimulatory actions and preliminary reports indicate that hormonal (DHEA or MLT) substitution therapy reverses immunosenescence in mice. Similarly, exercise in some studies has been shown to enhance the immune response. However, these findings have not been confirmed by other laboratories. Thus, at the present time, it is difficult to draw any definitive conclusions on the efficacy of DHEA, MLT, and exercise on reversing or restoring the aging immune system.

Thoughts on the evolutionary basis of dietary restriction

The dramatic effect of nutrition on the life span of rodents was shown initially by McCay’s laboratory in the 1930’s (McCay et al., 1935, McCay et al., 1939). They found that both the median and maximum survival of rats were increased significantly when the diet of weanling rats was restricted severely and growth retarded. This phenomenon became known as dietary or calorie restriction, and subsequent studies have demonstrated that the life span of rats could be extended significantly using less severe restriction regimens; e.g., a 30 to 50% restriction of calories generally results in a 20 to 50% increase in life span (mean and maximum survival) (Masoro, 1985, Masoro, 1988, Masoro, 1992a, Weindruch & Walford, 1988). At the present time, it is generally accepted that dietary restriction extends the life span of laboratory rodents by retarding the aging process because dietary restriction increases the maximum survival of rodents and alters most physiological and pathological processes that change with increasing age (Richardson & McCarter, 1991; Masoro, 1992a). Therefore, dietary restriction offers gerontologists a unique system for studying the aging process.

Normal immune function in young and old DNA polymerase-β deficient mice

Abstract The effect of the DNA polymerase-β (β-pol) deficiency on mitogenic response and cytokine production was studied in spleen lymphocytes from 4–5- and 20–22-month-old β-pol −/+ mice and their age-matched wild-type littermates. The proliferative response of lymphocytes to Concanavalin A (Con A) and lipopolysaccharide (LPS) was measured by [ 3 H]thymidine incorporation, and the induction of cytokine production (interleukin (IL)-2, IL-4, and interferon necrosis factor (IFN)-γ) was assessed by enzyme-linked immunosorbent assay. There was no significant difference in Con A- or LPS-induced proliferation or cytokine production in young β-pol −/+ mice compared with young wild-type littermates or in old β-pol −/+ mice compared with old wild-type littermates. However, mitogen-induced proliferation and cytokine production changed significantly with age. The proliferative response to Con A and to LPS, and the IL-2 production was significantly lower, and IL-4 and IFN-γ levels were significantly higher in lymphocytes from old β-pol −/+ mice and old wild-type mice than in lymphocytes from young β-pol −/+ mice and young wild-type littermates. In addition, flow cytometric analysis showed no significant differences between young β-pol −/+ mice and young wild-type littermates or between old β-pol −/+ mice and old wild-type littermates in the proportion of B- and T-cell populations, and T-cell subsets. However, the number of lymphocytes expressing CD4 + phenotype slightly decreased and the proportion of lymphocytes expressing CD44/Pgp-1 (memory) phenotype increased with age. Thus, we found no evidence for alteration in immune function in DNA polymerase-β deficient mice, although they exhibit a decline in immunologic function with age.