Alison A. Beharka

Macrophage prostaglandin production contributes to the age-associated decrease in T cell function which is reversed by the dietary antioxidant vitamin E

The aging process is associated with a decline in T cell-mediated immunity, including decreased interleukin (IL)-2 production and mitogen-induced T cell proliferation. Because macrophages (M phi) from old mice have higher production of prostaglandin (PG) E2 than young mice, and PGE2 has been shown to suppress T cell-mediated function, we hypothesized that increased production of PGE2 would contribute to decreased T cell function with aging and that decrease in PGE2 production by dietary antioxidants would enhance T cell-mediated function. Experiments were conducted in which combinations of purified M phi and T cells (> 95% pure) from young or old C57BL/6N1A mice were cultured together. Co-cultures containing T cells and M phi from old mice had reduced ConA-stimulated proliferation and IL-2 secretion than those consisting of T cells and M phi from young mice. Addition of M phi from old mice suppressed proliferation and IL-2 secretion by T cells from young mice. Likewise, T cells from old mice secreted more IL-2 when cultured with M phi from young mice compared to those cultured with M phi from old mice. Addition of PGE2, at concentrations produced by old M phi, decreased proliferation and IL-2 production by young but not old T cells. Neither addition of H2O2 at physiological levels, nor catalase changed the response of cultures from young or old mice. However, addition of indomethacin and the antioxidant nutrient vitamin E, both of which decreased PGE2 production, improved T cell proliferation and IL-2 production. These experiments demonstrate that increased production of PGE2 by M phi contributes to the age-associated decline in T cell function. Vitamin E improves T cell responsiveness in old mice mostly by reducing M phi PGE2 production, although a direct effect of vitamin E on T cells was also observed.

Age-associated increase in PGE2 synthesis and COX activity in murine macrophages is reversed by vitamin E

We previously showed that increased macrophage and PGE2 production with age is due to enhanced cyclooxygenase (COX) activity and COX-2 expression. This study determined the effect of vitamin E supplementation on macrophage PGE2 synthesis in young and old mice and its underlying mechanism. Mice were fed 30 or 500 parts per million vitamin E for 30 days. Lipopolysaccharide (LPS)-stimulated macrophages from old mice produced significantly more PGE2 than those from young mice. Vitamin E supplementation reversed the increased PGE2 production in old mice but had no effect on macrophage PGE2production in young mice. In both LPS-stimulated and unstimulated macrophages, COX activity was significantly higher in old than in young mice at all intervals. Vitamin E supplementation completely reversed the increased COX activity in old mice to levels comparable to those of young mice but had no effect on macrophage COX activity of young mice or on COX-1 and COX-2 protein or COX-2 mRNA expression in young or old mice. Thus vitamin E reverses the age-associated increase in macrophage PGE2 production and COX activity. Vitamin E exerts its effect posttranslationally, by inhibiting COX activity.We previously showed that increased macrophage and PGE2 production with age is due to enhanced cyclooxygenase (COX) activity and COX-2 expression. This study determined the effect of vitamin E supplementation on macrophage PGE2 synthesis in young and old mice and its underlying mechanism. Mice were fed 30 or 500 parts per million vitamin E for 30 days. Lipopolysaccharide (LPS)-stimulated macrophages from old mice produced significantly more PGE2 than those from young mice. Vitamin E supplementation reversed the increased PGE2 production in old mice but had no effect on macrophage PGE2 production in young mice. In both LPS-stimulated and unstimulated macrophages, COX activity was significantly higher in old than in young mice at all intervals. Vitamin E supplementation completely reversed the increased COX activity in old mice to levels comparable to those of young mice but had no effect on macrophage COX activity of young mice or on COX-1 and COX-2 protein or COX-2 mRNA expression in young or old mice. Thus vitamin E reverses the age-associated increase in macrophage PGE2 production and COX activity. Vitamin E exerts its effect posttranslationally, by inhibiting COX activity.

The Effect of Long-term Dietary Supplementation with Antioxidantsa

ABSTRACT: The impact of diet and specific food groups on aging and age‐associated degenerative diseases has been widely recognized in recent years. The modern concept of the free radical theory of aging takes as its basis a shift in the antioxidant/prooxidant balance that leads to increased oxidative stress, dysregulation of cellular function, and aging. In the context of this theory, antioxidants can influence the primary “intrinsic” aging process as well as several secondary age‐associated pathological processes. For the latter, several epidemiological and clinical studies have revealed potential roles for dietary antioxidants in the age‐associated decline of immune function and the reduction of risk of morbidity and mortality from cancer and heart disease. We reported that long‐term supplementation with vitamin E enhances immune function in aged animals and elderly subjects. We have also found that the beneficial effect of vitamin E in the reduction of risk of atherosclerosis is, in part, associated with molecular modulation of the interaction of immune and endothelial cells. Even though the effects of dietary antioxidants on aging have been mostly observed in relation to age‐associated diseases, the effects cannot be totally separated from those related to the intrinsic aging process. For modulation of the aging process by antioxidants, earlier reports have indicated that antioxidant feeding increased the median life span of mice to some extent. To further delineate the effect of dietary antioxidants on aging and longevity, middle‐aged (18 mo) C57BL\6NIA male mice were fed ad libitum semisynthetic AIN‐76 diets supplemented with different antioxidants (vitamin E, glutathione, melatonin, and strawberry extract). We found that dietary antioxidants had no effect on the pathological outcome or on mean and maximum life span of the mice, which was observed despite the reduced level of lipid peroxidation products, 4‐hydroxynonenol, in the liver of animals supplemented with vitamin E and strawberry extract (1.34 ± 0.4 and 1.6 ± 0.5 nmol/g, respectively) compared to animals fed the control diet (2.35 ± 1.4 nmol/g). However, vitamin E‐supplemented mice had significantly lower lung viral levels following influenza infection, a viral challenge associated with oxidative stress. These and other observations indicate that, at present, the effects of dietary antioxidants are mainly demonstrated in connection with age‐associated diseases in which oxidative stress appears to be intimately involved. Further studies are needed to determine the effect of antioxidant supplementation on longevity in the context of moderate caloric restriction.

Vitamin E supplementation increases T helper 1 cytokine production in old mice infected with influenza virus

Compared with young mice, old mice infected with influenza virus have significantly higher pulmonary viral titres, although these can be reduced significantly with dietary vitamin E supplementation. T helper 1 (Th1) cytokines, especially interferon‐γ (IFN‐γ), play an important role in defending against influenza infection. However, there is an age‐associated loss of Th1 cytokine production. Prostaglandin E2 (PGE2) production, which increases with age, can modulate the T helper cell function by suppressing Th1 cytokine production. To investigate the mechanism of vitamin E supplementation on reduction of influenza severity in old mice, we studied the cytokine production by splenocytes, and PGE2 production by macrophages (Mφ), in young and old C57BL mice fed semipurified diets containing 30 (control) or 500 parts per million (ppm) (supplemented) vitamin E for 8 weeks, and then infected with influenza A/PC/1/73 (H3N2). Old mice fed the control diet had significantly higher viral titres than young mice; old mice fed the vitamin E‐supplemented diet had significantly lower pulmonary viral titres than those fed the control diet (P = 0·02 and 0·001 for overall age and diet effect, respectively). Following influenza infection, interleukin (IL)‐2 and IFN‐γ production was significantly lower in old mice than in young mice. Vitamin E supplementation increased production of IL‐2 and IFN‐γ in old mice; higher IFN‐γ production was associated with lower pulmonary viral titre. Old mice fed the control diet showed significantly higher lipopolysaccharide (LPS)‐stimulated Mφ PGE2 production than old mice fed the vitamin E diet or young mice fed either diet. There was no significant age difference in IL‐6, IL‐1β, or tumour necrosis factor‐α (TNF‐α) production by splenocytes. Young mice fed the vitamin E‐supplemented diet had significantly lower IL‐1β (day 7) and TNF‐α production (day 5) compared with those fed the control diet. Old mice fed the vitamin E‐supplemented diet had significantly lower TNF‐α production (day 2) than those fed the control diet. Our results indicate that the vitamin E‐induced decrease in influenza viral titre is mediated through enhancement of Th1 cytokines, which may be the result of reduced PGE2 production caused by vitamin E.

Mechanism of vitamin E inhibition of cyclooxygenase activity in macrophages from old mice: role of peroxynitrite.

Vitamin E inhibits cyclooxygenase activity in macrophages from old mice by reducing peroxynitrite production. PGE(2) is a proinflammatory mediator that has been linked to a variety of age-associated diseases such as cancer, arthritis, and cardiovascular disease. Furthermore in the aged, increased cyclooxygenase (COX)-2-mediated PGE(2) production contributes to decline in T-cell-mediated function. Previously we reported that increased macrophage PGE(2) production in the aged is due to higher COX-2 activity and that supplementation with vitamin E significantly reduced the age-associated increase in macrophage PGE(2) production posttranslationally without changing COX-2 expression. Peroxynitrite, a product of nitric oxide (NO) and superoxide (O(-)(2)), increases the activity of COX without affecting its expression. Thus, we investigated if vitamin E inhibits COX activity through decreasing peroxynitrite formation. Macrophages from old mice had higher PGE(2) levels, COX activity, and NO levels than those from young mice, all of which were significantly reduced by vitamin E. When added individually, inhibitors of NO and O(-)(2) did not significantly reduce COX activity; however, when the inhibitors were combined, COX activity was significantly reduced in macrophages from old mice fed 30 ppm vitamin E. Increasing NO levels alone using SNAP or O(-)(2) levels, using X/XO, had no effect; however, increasing peroxynitrite levels using Sin-1 or X/XO + SNAP significantly increased COX activity in macrophages from old mice fed 500, but not those fed 30 ppm vitamin E. These data strongly suggest that peroxynitrite plays an important role in the vitamin E-induced inhibition of COX activity. These findings have important implications for designing interventions to reverse and/or delay age-associated dysregulation of immune and inflammatory responses and diseases associated with them.

VITAMIN E STATUS AND IMMUNE FUNCTION

Evidence from animal and human studies indicates that vitamin E plays an important role in the maintenance of the immune system. Even a marginal vitamin E deficiency impairs the immune response, while supplementation with higher than recommended dietary levels of vitamin E enhances humoral and cell-mediated immunity. The current RDA level of vitamin E prevents clinical deficiency syndrome but in some situations, especially in older subjects or in a disease state, fails to maintain optimal host defense. The immunological parameters reviewed are all sensitive to changes in the availability of vitamin E and, therefore, may reflect the vitamin E status of a given individual more accurately than conventional methods.

Vitamin E improves bone quality in the aged but not in young adult male mice

It is generally viewed that with advancing age, humans and other animals including mice experience a gradual decline in the rate of bone formation. This, in part, may be due to the rise in oxygen-derived free radical formation. Vitamin E, a strong antioxidant, functions as a free radical scavenger that potentially can suppress bone resorption while stimulating bone formation. Although the effects of vitamin E on immune functions are well documented, there is a paucity of information on its effect on skeletal health in vivo. The purpose of this study was to explore the influence of vitamin E supplementation on bone in young adult and old mice. Six and twenty-four month-old male C57BL/6NIA mice each were divided into two groups and fed a diet containing either adequate (30 mg/kg diet) or high (500 mg/kg diet) levels of vitamin E. Thirty days later, mice were killed and bones were removed for analyses including biomechanical testing using three-point bending and mRNA expressions of insulin-like growth factor-I (IGF-I), osteocalcin, and type 1alpha-collagen using Northern blot. In old but not the young adult mice, high-dose vitamin E enhanced bone quality as evident by improved material and structural bone properties in comparison with adequate. This improved quality was accompanied by increases in bone dry weight, protein, and mRNA transcripts for osteocalcin, type Ialpha-collagen, and IGF-I. These data demonstrate that high-dose vitamin E has pronounced effects on bone quality as well as matrix protein in old mice by augmenting bone matrix protein without reducing bone mineralization as evidenced by unaltered bone density.

In vitro supplementation with different tocopherol homologues can affect the function of immune cells in old mice

Alpha-tocophorel (T) is the most common form of vitamin E inplasma and tissues. Alpha-T is also believed to be superior to its homologues beta-T, gamma-T, and delta-T in antioxidant activity. Biological activity of alpha-T has been intensively studied in a number of bodily systems. In contrast, the other homologues have received little attention beyond the evaluation of their relative antioxidant activity. We as well as others have previously shown that alpha-T can enhance cell- mediated immune function of aged animals and humans. Gamma-T is a principal form of vitamin E in the American diet and some cooking oils contain substantial amount of beta-T and delta-T. Thus it is of public health interest to compare their biological effects with than of alpha-t in various systems. In this study, we used an in vitro supplementation protocol to determine immunologic effects of these T homologues on murine splenocytes. The results showed that all four T homologues enhance both spontaneous and mitogen-stimulated lymphocyte proliferation (LP) and the maximal enhancement produced by them was of the same magnitude. The dose range to produce maximal enhancement varied with different homologues. The efficiency was in the order of beta-T approximately delta-T > alpha-T. Interestingly, at 50 (optimal for alpha-T) and 150 micromol/L, while alpha-T enhanced LP, all the other homologues inhibited LP. This inhibition was found to be due to their cytotoxicity at these levels. T homologues had a differential effect on interleukin (IL)-2 and prostaglandin (PG)E(2) production. IL-2 production by mouse splenocytes was not affected by alpha-T or beta-T, but was increased by gamma-T and delta-T. All T homologues, except for beta-T, inhibited PGE(2) alpha-T. Thus, all the T homologues enhance LP. However, the dose required to reach maximal enhancement varies among the homologues. On the other hand, they have a differential effect on IL-2 and PGE(2) production. The difference in nature and magnitude of the effect on immune function does not correlate with their reported relative antioxidant activity and might be due to minor differences in their structure important to their other biological activities.

Long-term dietary antioxidant supplementation reduces production of selected inflammatory mediators by murine macrophages

Although macrophage (Mo)-derived compounds, including proinflammatory cytokines, prostaglandins (PG), and nitric oxide (NO), play important roles in host defense, excessive or inappropriate production of them has been implicated in the pathogenesis of a variety of inflammatory diseases. Aging is also associated with increased production of Mo compounds involved in inflammation. Short term dietary supplementation with selected antioxidants, such as glutathione (GSH) and vitamin E (E), has been shown to reduce age-related changes in Mo production of inflammatory compounds, but information is limited regarding the effects of long-term supplementation. Nor is information available on other less studied compounds with antioxidant properties, such as melatonin, strawberry extract, or combinations of compounds such as E + GSH. Therefore, we sought to determine if long term, from middle to old age, dietary supplementation of C57BL/6NCrlBR mice with E, GSH, E + GSH, melatonin or strawberry extract could modify age related changes in Mo production of pro- and anti-inflammatory compounds. Eighteen-month-old C57BL/6NCrlBR mice were fed one of the following six semi-synthetic diets for 6 months: 1) control (30 ppm vitamin E), 2) vitamin E supplemented (control + 470 ppm vitamin E), 3) glutathione (GSH) supplemented (control + 0.5% glutathione), 4) vitamin E and glutathione supplemented (control + 470 ppm vitamin E + 0.5% glutathione), 5) melatonin supplemented (control + 11 ppm melatonin), or 6) strawberry extract supplemented (control + 1% strawberry extract). Supplementation with vitamin E or melatonin, but not GSH, E + GSH, or strawberry extract, reduced (p<0.05) unstimulated IL-6 and PGE2 production by peritoneal Mo. Because PGE2 has been shown to modulate IL-6 production, the reduction in PGE2 production by vitamin E and melatonin may be partially responsible for the reduction in unstimulated IL-6 production. LPS stimulation resulted in production of IL-1β, IL-6, IL-10, IL-12, IL-15, NO, PGE2, and TNF-α by peritoneal or bone marrow (BM)-derived Mo. Of these, only NO production was modulated by antioxidant supplementation and then significantly by vitamin E only. Vitamin E supplementation reduced the age-related increase in inducible NO production by peritoneal Mo (p<0.05), but not by BM Mo. In conclusion, vitamin E modulated production of at least three, and melatonin modulated production of at least two, molecules produced by Mo, which are involved in a variety of age-associated chronic and acute inflammatory diseases. Further research is needed to determine the mechanisms and clinical benefits of E and melatonin-induced reduction in Mo inflammatory mediator production.