Since vitamin E is the most effective chain-breaking, lipid-soluble antioxidant present in cell membranes, it is considered likely that it plays a major role in maintaining cell membrane integrity by limiting lipid peroxidation by ROS.
Studies conducted in humans and animals, using either states of deficiency or supra-dietary levels, suggest strongly that vitamin E is involved in maintaining immune cell function (for a review, see Meydani and Beharka, 1998). For example, Canadian 3-year-olds with the lowest serum vitamin E levels had the lowest lymphocyte proliferative responses and serum IgM concentrations (Vobecky et al., 1984). In addition, there was a positive association between plasma vitamin E levels and DTH responses and a negative association between plasma vitamin E levels and incidence of infections in healthy adults aged over 60 (see Chavance et al., 1989). Administration of vitamin E to premature infants enhanced neutrophil phagocytosis (Baehner et al., 1977; Chirico et al., 1983) but decreased the ability of neutrophils to kill bacteria
(Baehner et al., 1977); this latter effect is most probably due to a vitamin E-induced decrease in production of free radicals and related reactive species. In laboratory animals, vitamin E deficiency decreased spleen lymphocyte proliferation in response to mitogens, NK-cell activity, specific antibody production following vaccination and phagocytosis by neutrophils (for a review, see Meydani and Beharka, 1998). Vitamin E deficiency also increases susceptibility of animals to infectious pathogens (for references, see Meydani and Beharka, 1998; Han and Meydani, 1999). Vitamin E supplementation of the diet of laboratory animals enhances antibody production, lymphocyte proliferation, NK-cell activity, and macrophage phagocytosis (for references, see Meydani and Beharka, 1998). Dietary vitamin E promoted resistance to pathogens in chickens, turkeys, mice, pigs, sheep and cattle (for references, see Meydani and Beharka, 1998; Han and Meydani, 1999); some of these studies report improved immune-cell functions in the animals receiving additional vitamin E (see Han and Meydani, 1999). For example, vitamin E prevented the retrovirus-induced decrease in production of interleukin-2 (IL-2) and interferon-7 (IFN-7) by spleen lymphocytes and in NK-cell activity in mice (Wang et al., 1994).
One application of the effects of vitamin E on immune function is in the elderly. This has been investigated in both murine models and human trials. Adding vitamin E to the diet of aged mice increased lymphocyte proliferation, IL-2 production and the DTH response (Meydani et al., 1986). A high level of vitamin E in the diet (500 mg kg-1 food) also increased NK-cell activity of spleen cells from old (but not young) mice (Meydani et al., 1988). In another study, young and old mice were fed diets containing adequate (30 mg kg-1 diet) or high (500 mg kg-1 diet) levels of vitamin E for 6 weeks and infected with influenza A virus: young mice and old mice fed the high level of vitamin E had lower lung titres of virus than old mice fed the adequate vitamin E diet (Hayek et al., 1997). The high level of vitamin E caused increased production of IL-2 and IFN-7 by spleen lymphocytes from influenza-infected old mice (Han et al., 1998; Han and Meydani, 2000). Supplementation of the diet of elderly human subjects with 800 mg vitamin E day-1 for 4 weeks increased lymphocyte proliferation stimulated by concanavalin A, IL-2 production and the DTH response, but did not affect IL-1 production, the number of CD4 cells or circulating Ig concentrations (Meydani et al., 1990). In a more recent study, 60, 200 and 800 mg vitamin E day-1 increased DTH response in elderly subjects, with 200 mg day-1 having the maximal effect (Meydani et al., 1997). The two higher vitamin E doses improved antibody responses to hepatitis B, but only the 200 mg day-1 dose increased the antibody response to tetanus toxoid (Meydani et al., 1997). The authors conclude that 200 mg vitamin E day-1 represents the optimal level for the immune response. In another study, young and elderly individuals were supplemented with 800 mg vitamin E day-1 for 48 days before being asked to run down an inclined treadmill for 45 min. Vitamin E supplementation was found to eliminate the age-associated difference in exercise-induced neutrope-nia, to prevent the exercise-induced increase in IL-1 production and to inhibit IL-6 production (Cannon et al., 1991). Since these cytokines are involved in the inflammatory process and in exercise-induced muscle damage, their inhibition by vitamin E during exercise might have important implications. However, on a cautious note, studies have reported that prolonged high intakes of vitamin E (> 1000 mg day-1) can lead to inhibition of neutrophil phagocytosis (Boxer, 1986). Further research is needed to assess the optimal intake of this nutrient required to provide benefit for different groups of individuals.
Cigarette smoke contains millions of free radicals per puff, and other compounds present can stimulate the formation of other highly reactive molecules (Pryor and Stone, 1993). Serum levels of vitamin E (as well as of vitamin C and p-carotene) and lung vitamin E concentrations are significantly lower in smokers compared with non-smokers and even supplementation with 2400 mg a-tocopherol equivalents day-1 for 3 weeks failed to restore the lung vitamin E level to that found in non-smokers (Pacht et al., 1986). Circulating phagocytes from smokers produce high levels of free radicals, which probably in part accounts for the depressed immune function observed in smokers (Johnson et al., 1990), and there is some evidence that vitamin E supplementation can reduce the overproduction of ROS by phagocytic cells from smokers (Richards et al., 1990).
Reduced vitamin E status has also been reported in human immunodeficiency virus (HlV)-infected individuals. Passi et al. (1993) found that plasma vitamin E concentrations were significantly lower in a group of 200 HlV-posi-tive individuals compared with controls, but whether this is related to an inadequate intake of this vitamin is unclear. Dietary diaries from a group of 100 HIV-infected asymptomatic men did not indicate an inadequate intake of vitamin E, but plasma levels were low or marginally low in 74% of the men (Beach et al., 1992). In a study of patients who had developed acquired immune deficiency syndrome (AIDS), an inverse relationship was observed between serum vitamin E levels and severity of disease (Favier et al., 1994). A recent study of 49 HIV-infected subjects provided with vitamin E and vitamin C observed a significant reduction in oxidative stress and a trend towards a reduction in viral load after 3 months (Allard et al., 1998). These studies suggest that larger trials of these and other antioxidant nutrients in the treatment of HIV-infected persons should be encouraged, since there is a need to find alternative, cheaper, treatments than the combination therapies currently employed.
In terms of mechanisms of action, in addition to its role as a protective antioxidant, vitamin E, at higher intakes, is associated with a reduced production of prostaglandin E2 (PGE2) (e.g. Meydani et al., 1986, 1988, 1990). Since PGE2 inhibits lymphocyte proliferation and NK-cell activity, it is possible that this may be one immunomodulatory mechanism of vitamin E action. It is also possible that vitamin E and, indeed, other antioxidant nutrients can influence a variety of inflammatory processes by inhibiting the activity of a transcription factor called nuclear factor kappa B (NFkB). Transcription factors are intracellular regulators of gene expression. Once activated, the transcription factor binds to the promoter region of a specific gene within the DNA in the nucleus, resulting in that gene being 'turned on'. NFkB is required for maximal transcription of many proteins that are involved in inflammatory responses, including several cytokines, such as IL-1p, IL-2 and tumour necrosis factor (TNF)-a. NFkB is a redox-sensitive transcription factor and it is thought that the generation of ROS is a vital link in mediating NFkB activation by a variety of stimuli (Lavrovsky et al., 2000).
The carotenoids are a group of over 600 naturally occurring coloured pigments that are widespread in plants, of which only about 20 commonly occur in human foodstuffs. In nature, they serve two essential functions: as accessory pigments in photosynthesis, and in photoprotection. These two functions are achieved through the chemical structure of carotenoids (Fig. 9.1), which allows the molecules to absorb light and to quench singlet oxygen and free radicals.
Many epidemiological studies have shown an association between diets rich in carotenoids and a reduced incidence of many forms of cancer, and it has been suggested that the antioxidant properties of these compounds are a causative factor (Block et al., 1992). Since the publication of an article by Peto et al. (1981), a great deal of attention has focused on the potential role of one particular carotenoid, p-carotene, in preventing cancer. Numerous publications have described epidemiological studies, in vitro experiments, animal studies and clinical trials that suggest that this carotenoid can protect against not only cancer, but also other oxidative damage-associated disorders, listed in Table 9.1 (reviewed by Mayne, 1996). Because the immune system plays a major role in the prevention of cancer, it has been suggested that p-carotene may enhance immune cell function (Bendich and Olson, 1989). In animals, adding carotenoids to the diet prevented stress-related thymic involution, increased the number of circulating lymphocytes, enhanced lymphocyte proliferation and cytotoxic T-cell activity and increased resistance to infective pathogens (for references, see Roe and Fuller, 1993).
Several studies have examined the effect of p-carotene on human immune function. Various doses of p-carotene have been employed in these studies, ranging from 15 mg day-1, which could be achieved through the diet, up to pharmacological doses of 180 mg day-1, provided over periods of 14-365 days. These studies
Fig. 9.1. Chemical structure of some carotenoids found in the diet.
have reported increases in the numbers of CD4+ cells or in the ratio of CD4+ to CD8+ cells in the circulation, in the percentages of lymphocytes expressing the IL-2 and transferrin receptors, and in NK-cell activity (Alexander et al., 1985; Watson et al., 1991; Murata et al., 1994), particularly in elderly subjects. The potential for increasing the numbers of CD4+ cells led to the suggestion that p-carotene might be useful as an immunoenhancing agent in the treatment of HIV infection. Preliminary studies have shown a slight but insignificant increase in CD4+ numbers in response to p-carotene (60 mg day-1 for 4 weeks) in patients with AIDS (Fryburg et al., 1995), but long-term effectiveness has not been reported.
Other investigators have been unable to confirm the increase in T-cell-medi-ated immunity in healthy individuals following p-carotene supplementation. Santos et al. (1996, 1997) reported the results of two studies in the elderly: a short-term, high-dose study (90 mg day-1 for 21 days) in women and a longer-term, lower-dose trial (50 mg alternate day-1 for 10-12 years) in men. Both studies concluded that there was no significant difference in T-cell function as assessed by DTH response, lymphocyte proliferation, IL-2 and PGE2 production and composition of lymphocyte subsets (Santos et al., 1997). However, these workers also examined the effect of p-carotene supplementation on NK-cell activity in the longer-term trial with male volunteers and observed that supplementation of the diet of older males (> 65 years) with p-carotene resulted in significantly greater NK-cell activity compared with subjects of a similar age given placebo treatment (Santos et al., 1996). Since patients with Chediak-Higashi syndrome, a disorder associated with defective NK-cell function, show a higher susceptibility to tumour formation (Roder et al., 1980), and homozygous mice genetically deficient in NK cell activity grow tumours and develop leukaemia more rapidly than do heterozygous littermates with normal NK-cell function (Lotzova, 1993), the enhancing effect of p-carotene on NK-cell activity has been postulated to be a link between raised intakes of this nutrient and cancer prevention. As shown in Fig. 9.2, the study by Santos et al. (1996) highlighted both the reduction in NK-cell activity that is observed with age and the fact that the increase in NK-cell activity observed in older males (65-86 years) following p-carotene supplementation restored it to the level seen in a group of younger males (51-64 years). The mechanism for this is unclear, but it was not due to an increase in the percentage of NK cells or to an increase in IL-2 production. The authors suggest that p-carotene may be acting directly on one or more of the lytic stages of NK-cell cytotoxicity or on NK-cell activity-enhancing cytokines other than IL-2, such as IL-12.
Individuals who are repeatedly exposed to UV light show suppression of immune function (Rivers et al., 1989). Because carotenoids can provide photoprotection, several studies have assessed the ability of p-carotene to protect the immune system from UV-induced free radical damage. In one study, a group of young males were placed on a low-carotenoid diet (< 1.0 mg day-1 total carotenoids) and given either placebo or 30 mg p-carotene day-1 for 28 days prior to periodic exposure to UV light. DTH responses were significantly suppressed in the placebo group after UV treatments and the suppression was inversely proportional to plasma p-carotene concentrations in this group (Fuller et al., 1992). In contrast, no significant suppression of DTH responses was seen in the p-carotene-treated group (Fuller et al., 1992). The ability of p-carotene
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Fig. 9.2. Natural killer cell activity in different age-groups of subjects consuming placebo or p-carotene. Natural killer cell activity was determined at several effector-to-target cell ratios (E:T), using effector cells from subjects consuming placebo (n = 17 for 51-64 years and n = 13 for 65-86 years) or p-carotene (n = 21 for 51-64 years, and n =8 for 65-86 years). Data are expressed as % target-cell lysis. Reprinted from Santos etal. (1996) with permission by the American Journal of Clinical Nutrition. ©American Journal of Clinical Nutrition, American Society for Clinical Nutrition.
to protect against the harmful effects of natural UV sunlight has also been demonstrated by exposing healthy female students to time- and intensity-controlled sunlight exposure: a Berlin-based study involved taking volunteers to the Red Sea and exposing areas of their skin to the sunlight by lifting discretely placed flaps in their specially designed swimsuits (Gollnick et al., 1996)!
Since antigen-presenting cells initiate cell-mediated immune responses, one aspect of the immune-enhancing effect of p-carotene might be improved antigen-presenting cell function. A prerequisite for antigen presentation is the expression of major histocompatibility complex (MHC) class II molecules (human leucocyte antigen (HLA)-DR, HLA-DP and HLA-DQ) (Bach, 1985), which are present on the majority of human monocytes. The antigenic peptide is presented to the T-helper lymphocyte within a groove of the MHC class II molecule (Fig. 9.3). Since the degree of immune responsiveness of an individual has been shown to be proportional to both the percentage of MHC class II-positive monocytes and the density of these molecules on the cell surface (Janeway et al., 1984), it is possible that one mechanism by which p-carotene may enhance cell-mediated immune responses is by enhancing the cell surface expression of these molecules. In addition, cell-to-cell adhesion is critical for the initiation of a primary immune response, and it has been shown that the intercellular adhesion molecule 1 (ICAM-l)-leucocyte function-associated antigen-1 (LFA-1) ligand-receptor pair is also capable of co-stimulating an immune response (Springer, 1990), enhancing T-cell proliferation and cytokine production.
Adhesion MHC class molecule molecule
Adhesion MHC class molecule molecule w
Fig. 9.3. Cell surface molecules involved in the initiation of cell-mediated immune responses. LFA, leucocyte function-associated antigen; ICAM-1, intercellular adhesion molecule 1; HLA, human leucocyte-associated antigen; MHC, major histocompatibility complex.
The effect of p-carotene supplementation (15 mg day-1 for 26 days; equivalent to 150 g carrots day-1) on expression of MHC class II and adhesion molecules on the monocyte surface has been investigated. Following dietary supplementation, there were significant increases in plasma levels of p-carotene and in the percentages of monocytes expressing the MHC class II molecule HLA-DR and the adhesion molecules ICAM-1 and LFA-3 (Hughes et al., 1997). These results suggest that moderate increases in the dietary intake of p-carotene can enhance cell-mediated immune responses within a relatively short period of time, providing a potential mechanism for the anti-carcinogenic properties attributed to this compound. The increase in surface molecule expression may also, in part, account for the ability of p-carotene to prevent the reduction in DTH response following exposure to UV radiation, since the latter can inhibit both HLA-DR and ICAM-1 expression. This finding could certainly be relevant to the preventive action of p-carotene towards skin cancer (Mathews-Roth, 1989), since immunosuppressed individuals, such as renal-transplant patients, have an increased risk of skin cancer.
As well as preventing oxidative damage, it has been suggested that p-carotene, like vitamin E, can influence immune cell function by modulating the production of PGE2. This eicosanoid is the major prostaglandin (PG) synthesized by monocytes and macrophages and is known to possess a number of immunosuppressive properties (see Calder and Field, Chapter 4, this volume). It has been suggested that p-carotene might enhance immune responses by altering the activation of the arachidonic acid cascade (from which PGE2 is derived), since it has been shown to be capable of suppressing the generation of arachidonic acid products in vitro from non-lymphoid tissues (Halevy and Sklan, 1987). This possibility requires further investigation.
There have been very few studies examining the influence of carotenoids other than p-carotene on human immune function, even though there is strong epidemiological evidence to suggest that lycopene (found in tomatoes) and lutein (found in peas, watercress and other vegetables) can protect against the development of prostate and lung cancer, respectively (Le Marchand et al., 1993; Gann et al., 1999). In addition, tomato intake has been found to be inversely associated with the risk of diarrhoeal and respiratory infections in young children in Sudan (Fawzi et al., 2000). In terms of mechanisms of action, the effect of dietary supplementation with lycopene and lutein on the expression of monocyte surface molecules involved in antigen presentation has been investigated. It was found that these carotenoids appear to be less influential than p-carotene, when given at the same level in the diet (Hughes et al., 2000). In addition, enriching the diet with lycopene (by drinking 330 ml of tomato juice daily) for 8 weeks did not modify cell-mediated immune responses in the elderly (Watzl et al., 2000). In another study, performed in a group of older volunteers (over 65 years) living in Ireland, the effects of placebo, p-carotene (8.2 mg day-1) and lycopene (13.3 mg day-1) for 12 weeks on various parameters of cell-mediated immunity were examined. There were no significant changes in circulating T-cell subsets, mitogen-stimulated lymphocyte proliferation or surface molecule expression following any of these interventions, in spite of significant increases in the plasma levels of the carotenoids (Corridan et al., 2001). The authors concluded that in well-nourished, free-living, healthy individuals, supplementation with relatively low levels of p-carotene or lycopene is not associated with either beneficial or detrimental effects on cell-mediated immunity.
Other investigators have shown an opposing effect of p-carotene and lutein upon human lymphocyte proliferation (Watzl et al., 1999), emphasizing further the fact that different carotenoids might affect immune function in different ways. Therefore, in fruits and vegetables, the influence of the combination of carotenoids they contain on immune function may represent the sum total of these different effects and, indeed, the potential for synergistic effects remains to be investigated.
One possible factor to explain the different effects seen with different carotenoids might be the preferred location of these compounds within the cell and within the body. Carotenoids are lipid-soluble and thus it is thought that most will be concentrated in the lipid-rich membranes of the cell. However, their exact location may influence their effectiveness in modulating specific cellular events. Within the body, lycopene appears to be selectively taken up within the prostate, a finding that may help explain the association between higher intakes of lycopene and a reduced incidence of prostate cancer (Giovannucci, 1999). Thus, it is possible that tests on peripheral blood cells to determine immune function will not detect any localized effects, suggesting that there might be 'hidden' benefits associated with certain dietary components that we have yet to discover.
The strongest epidemiological evidence supporting a beneficial effect of carotenoids in preventing cancer is the protective effect of p-carotene intake in reducing the incidence of cancer of the lung. Carotenoid intake has been associated with a reduced lung-cancer risk in eight of eight prospective studies and in 18 of 20 retrospective studies (for a review, see Zeigler et al., 1996). As a result, three major intervention trials were initiated, examining the efficacy of p-carotene in the prevention of lung cancer (Alpha-tocopherol Beta-carotene Cancer Prevention Study Group, 1994; Hennekens et al., 1996; Omenn et al., 1996). The failure of these trials to show a protective effect, with two of the studies showing an increase in lung cancer in smokers receiving p-carotene supplementation, has been widely publicized. The mechanism for the increased lung-cancer risk associated with the supplementation is unclear, but several suggestions have been made. Since the participants in these studies could be classified as 'high-risk' for developing lung cancer (long-term smokers or previously exposed to asbestos), it is possible that many of them had undetected tumours prior to the commencement of supplementation. The stage (or stages) of carcinogenesis that p-carotene might be effective against is unclear, but, if the effect is mediated via the immune system, it is likely to occur during the promotional stages preceding the formation of a malignant tumour. A recent analysis of the Cancer Prevention Study II (CPS-II), a prospective mortality study of more than 1 million US adults, investigated the effects of supplementation with multivitamins and/or vitamins A, C and/or E on mortality during a 7-year follow-up period. The use of a multivitamin plus vitamins A, C and/or E significantly reduced the risk of cancer in former smokers and in never-smokers, but increased the risk of lung cancer in male smokers who had used a multivitamin plus vitamins A, C and/or E, compared with men who had reported no vitamin supplement use. Interestingly, in this study, no association with smoking was seen in women (Watkins et al., 2000).
One of the major unresolved dilemmas of research into p-carotene is what intake is required for optimal immune function and other health-related properties. Most studies of this compound have been undertaken at levels that are not achievable within a normal healthy diet. It is still unclear whether different intakes are associated with different outcomes or, in mechanistic studies, with different effects on various aspects of immune function. In addition, there remains the possibility that, at supra-dietary levels, p-carotene may exhibit pro-oxidant activity, particularly in the presence of high oxygen tensions, as occur in the lungs (reviewed by Palozza, 1998). Of course, the probability remains that the apparent protection of consuming a diet rich in fruits and vegetables is the result of a multifactorial effect of a number of components of these foods. In support of this, two of the prospective studies mentioned above found that higher plasma p-carotene concentrations upon entry into the trials, resulting from dietary consumption, as opposed to taking supplements, were associated with a lower risk of lung cancer (McDermott, 2000). Greater emphasis should be placed on studying the effects of enriching the diet with antioxidants via real foodstuffs rather than by supplementation.
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