Role of Antioxidants in the Maintenance of Immune Functions

IV Immunity and Infection 15 Role of Antioxidants in the Maintenance of Immune Functions ADRIANNE BENDICH

I. Introduction Free radicals have been associated with damage to cellular membranes and enzymes as well as to the DNA in the cell nucleus. Leukocyte (white blood cell) function is dependent on cell membrane integrity, and thus free radical damage to membrane constituents is often associated with impaired immune responses. However, free radicals are used by cells of the immune system to destroy pathogens and malignant cells. Antioxidants have the ability to stabilize highly reactive, potentially harmful free radicals. The ability of antioxidants to destroy highly reactive free radicals serves to protect the structural integrity of immune cells and prevents the loss of essential functions. Therefore, it is critical for optimal immune responses that there be a balance between free radical generation and antioxidant protection. The objective of this review is to examine the effects of antioxidants on immune responses, with emphasis on data from human intervention studies in which the population groups are at risk for increased exposure to free radicals, compromised immune function, and/or lowered antioxidant status.

II. Free Radicals and Antioxidants Free radicals are generated during normal cellular metabolism, are ingested or inhaled from the environment, can be generated during the metabolism of certain Natural Antioxidants in Human Health and Disease

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drugs or xenobiotics, and are produced at significantly higher than normal rates as a consequence of many diseases and/or degenerative conditions. The human body has three antioxidant systems that are highly effective in neutralizing free radicals. These are (1) antioxidant vitamins (vitamin C, E, and (3-carotene) and other low-molecular-weight compounds (such as glutathione and uric acid), (2) antioxidant enzymes, e.g., copper-zinc- or manganese-containing superoxide dismutases (SODs), iron-containing catalase (CAT), and seleniumcontaining glutathione peroxidase (GSH-Px), and (3) metal-binding proteins (such as transferrin, ferritin, and ceruloplasmin). There are also a number of therapeutic agents that have the capacity to reduce the oxidative damage that results from an imbalance between the concentration of free radicals and the antioxidant defenses (Machlin and Bendich, 1987).

III. Immune Responses Two major functions of the immune system are to ward off infections and to prevent cancer. Immune responses involve a series of interactions that include cellto-cell contacts; generation of immunoreactive molecules (including cytokines, antiviral interferons, prostaglandins, and Ieukotrienes); lymphocyte proliferation; synthesis and secretion of immunoglobulins; cytotoxicity; and, among other functions, the expression of cell surface markers not found on resting lymphocytes and mononuclear cells (Bendich, 1992).

A. Nonspecific Immune Responses Many of these activities are involved with prevention of infectious disease via nonspecific immune responses. For instance, the skin blocks the movement of infectious organisms into the body. Pathogenic organisms that are present in the food we eat and the air we breathe are usually curtailed from entering the blood by an intact basement membrane, which forms the natural barrier. Within the blood, the circulating immunoglobulins (antibodies) and complement factors protect against systemic infections. Interferons are secreted by immune and other cells in response to viral infection. Many of the nonspecific factors secreted during responses to pathogens include free radicals and other reactive oxygen species, such as hydrogen peroxide. The most abundant circulating white blood cells involved in killing pathogens are the neutrophils. These cells respond to chemical signals, including oxidation products, released at sites of infection, and move toward the infection. When stimulated, neutrophils have the capacity to take up molecular oxygen and generate oxygen-containing free radicals and other reactive molecules. This is often called the oxidative burst. Free radicals and singlet oxygen, along with other reactive molecules, can kill bacterial pathogens. Neutrophils can also generate highly toxic halogenated molecules (e.g., hypochlorous acid) when the myeloperoxidase halide enzyme system is activated during the oxidative burst. The halogenated species can also lyse the phagocytized pathogen. In addition to killing, these reactive molecules

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can be released into the surrounding area and can cause host cell mutations, lyse normal cells (including other neutrophils), cause inflammation of surrounding tissues, inactivate protective enzymes (such as antiproteases), and inhibit lymphocyte proliferation (Bendich, 1990).

B. Specific Immune Responses In addition to the responses of the nonspecific arm of the immune system following pathogen invasion, lymphocytes proliferate rapidly and then develop specific antibodies, as well as specific cytotoxic and memory lymphocytes that coordinate in the elimination of the pathogen. Recognition of foreign, pathogenic organisms is accomplished by the cooperation between macrophages, T (thymicassociated) lymphocytes, and B (bursal- or bone marrow-associated) lymphocytes. A critical index of overall specific immune function is the delayed-type hypersensitivity (DTH) skin test responses. DTH responses are the culmination of cooperative activities of all immune and inflammatory cells involved in memory of prior exposure to specific antigens. Lack of DTH, or anergy, is predictive of increased morbidity and mortality risk (Wayne et al., 1990). The capacity of the immune system to recognize specific foreign organisms as "nonself" permits immune cells to destroy any cell, tissue, or organ that is characterized as alien. Products of free radical reactions are utilized in the killing of invading organisms as well as in the destruction of cells and tissues considered by the immune system as nonself. Because the immune system can destroy tissues within the body, organ transplants of nonself donor tissues must be preceded by immunosuppressive therapy. Autoimmune diseases, such as rheumatoid arthritis, are thought to result from inappropriate identification of self components as nonself, followed by an attempt by immune cells to eliminate the affected tissue. Immunosuppressive drugs are also used to control autoimmune diseases. With regard to its role in cancer prevention, it is hypothesized that the immune system recognizes tumor-forming cells as nonself. The three types of immune cells that have the capacity to lyse tumor cells are the cytotoxic T lymphocytes, macrophages, and natural killer cells. In addition, factors, including free radicals and oxidation products, that are secreted by immune cells can lyse tumor cells. The optimal functioning of specific immune responses is thus important for protection against infections and many types of cancer.

IV. Risk Factors for Increased Exposure to Free Radicals, and/or Lowered Antioxidant Status: Effects on Immune Responses Risk factors can be divided into those that are mainly dependent on environmental factors and those that are inherited. Of course, environmental factors can affect the expression of genetic predispositions; likewise, inherited traits can impact on the effects of environmental stresses. One of the most obvious environmental factors that could reduce antioxidant status is a dietary antioxidant deficiency. Jacob et al. (1991), in a unique clinical experiment, determined the effects of marginal

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vitamin C deficiency on indices of oxidative stress and immune function. They found that low intake of vitamin C (5 to 20 mg/day for 32 days) resulted in significant depression in DTH responses, but no changes in lymphocyte proliferation, in healthy young men whose diets otherwise contained recommended levels of all other nutrients, including essential micronutrients. DTH responses were not returned to baseline, even when vitamin C intake was increased to 250 mg/day for 4 weeks. Concurrently, blood levels of glutathione decreased and there was evidence of oxidative damage to DNA and an increased production of certain oxidatively derived mutagens. These data document the measurable effects of short-term reductions in a single antioxidant on clinically important immune responses.

A. Environmental Factors Environmental factors include dietary components and life-style choices that can tip the balance between free radical generation and antioxidant protection, resulting in oxidative damage. Among the many environmental factors that can affect the balance, the role of dietary fat, exposure to cigarette smoke, ultraviolet (UV) light, and strenuous exercise, and the effects of HIV infection are highlighted. In each instance, data from human studies suggest that higher than normal intakes of antioxidants can prevent certain of the free radical-mediated adverse effects on immune function that are associated with the damaging environmental factor.

/. Fat Content of the Diet Although the majority of the activities involved in the generation of an immune response involves the activation of genes and the consequent production of newly synthesized molecules, the initiation of the immune response is considered to occur at the level of the cell membrane (Roitt, 1979). The composition of the lipid bilayer of the cell membrane reflects the dietary intake of fats. The potential for free radical damage is dependent in large part on the level of potentially oxidizable fatty acids, mainly polyunsaturated fatty acids (PUFAs), in the diet. High levels of dietary PUFAs have been shown to be immunodepressive. Dietary fats may undergo free radical-mediated oxidation prior to ingestion, as can occur when foods are fried. Mice fed oxidized lipids show marked atrophy of the thymus as well as T lymphocyte dysfunction (Mertin and Hunt, 1976; Erickson et al., 1980; Newberne, 1981; Gurr, 1983). One mechanism suggested for the immunosuppressive effects of high-fat diets involves a decrease in membrane fluidity. The fluidity of immune cell membranes is dependent on the degree of unsaturation of its fatty acids. As the level of PUFAs is increased, the potential for free radical-generated lipid peroxidation is also increased. Lipid peroxidation causes a decrease in membrane fluidity (Meade and Mertin, 1978). In addition, metabolites of lipid peroxidation can adversely affect immune responses and have been shown to be cytotoxic (Mertin and Hughes, 1975; Erickson et al., 1983). Loss of membrane fluidity has also been directly related to the decreased ability of lymphocytes to undergo proliferation when stimulated (Fountain and Schultz, 1982). The type of unsaturated lipids incorporated into leukocyte membranes can also

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affect the capacity of the immune system to respond to challenges. Linoleic acid, the major unsaturated fatty acid present in vegetable oils, is the precursor of arachidonic acid. Metabolites of arachidonic acid include immunosuppressive prostaglandins (Meydani et al., 1990a). The formation of prostaglandins (PGs) from the oxidation of arachidonic acid by cells and tissues involved in immune responses is reflective of the level and type of fat in the diet as well as the level of dietary antioxidants (Meydani et al. 1988). Increasing the level of PUFAs in diets increases the potential for PG formation (Erickson et al., 1983). Increasing the amount of dietary antioxidants such as vitamin E in the diet results in PG decreases in serum. Fish oils contain higher concentrations of n-3 PUFAs than the predominantly n-6 PUFAs contained in vegetable oils. Fish oil PUFAs are highly unsaturated (20:5, 22:6) and thus more prone to autoxidation than vegetable oil PUFAs (18:2, 20:4). In clinical studies, the use of fish oil supplements has been found to decrease interleukin-l and -2 production and lymphocyte proliferation (Kramer et al., 1991). In a placebo-controlled, double-blind study, healthy adult males were given 15 g/ day of either placebo oil or fish oil. Lymphocyte proliferative responses were significantly depressed in the fish oil group. Subsequently, the fish oil group was given 200 mg/day of vitamin E and the control group was given a matching placebo. Lymphocyte proliferative responses of those given vitamin E were restored to the level seen in the placebo group (Kramer et al., 1991). Vitamin E is thought to protect fish oils from oxidation; the oxidation products are known to suppress immune responses.

2. Cigarette Smoking Cigarette smoke contains millions of free radicals per puff. In addition, several other harmful products in cigarette smoke can stimulate the formation of highly reactive molecules that further increase the free radical burden (Church and Pryor, 1985). Recent studies have shown that the concentration of pentane exhaled in the breath reflects the level of in vivo lipid peroxidation. Hoshino et al. (1990) demonstrated that smokers have significantly higher breath pentane levels compared to nonsmokers. Supplementation with 800 IV of vitamin E for 2 weeks resulted in a significant decrease in breath pentane levels in smokers. Cigarette smokers have depressed immune responses compared to nonsmokers (Holt, 1987; McSharry and Wilkinson, 1986), which may, in part, be due to the overproduction of immunosuppressive free radicals by phagocytic neutrophils and macrophages in the lungs of smokers. The lung of the healthy nonsmoker contains very few neutrophils (Pacht et al., 1986). Cigarette smoke attracts these cells to the lung and activates them to generate free radicals. Pacht et al. (1986) found that killing of lung tissue cells was greatest of those smokers with the lowest lung vitamin E concentration. Richards et al. (1990) found that the circulating phagocytes of smokers produce high levels of free radicals. Administration of 900 IV/day of vitamin E for 6 weeks to smokers significantly reduced the overproduction of oxidant radicals by circulating phagocytic cells. Tobacco use is linked to more than 70% of oral cancers, and lung, head and

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neck, esophageal, and stomach cancers are all associated with cigarette smoking. Cigarette smokers and tobacco chewers have significantly lower serum levels of ~-carotene, vitamin C, and several other micronutrients, compared to nonsmokers who consume comparable levels of the micronutrients. However, smokers with the foods showed a significant reduction in risk of highest intake of ~-carotene-rich these cancers in a number of studies (Block, 1992). In a placebo-controlled, doublefor 14 weeks had a significant blind study, smokers given 20 mg/day of ~-carotene reduction in abnormal cells in their sputum, but no change in the level of DNA damage in circulating white blood cells (van Poppe I et al., 1992). Abnormal sputum cells may be an early sign of precancerous oral, bronchial, or lung lesions, which are precursors of cancer. r3-Carotene supplementation (30 mg/ day for 2 months) reduced the progression of oral precancerous lesions in the mouths of 70% of at the same time, it enhanced smokers in one intervention trial (Garewal, 1992)~ natural killer cell receptors and in vitro killing of tumor cells. Lymphocyte proliferation is also depressed in smokers and has been shown to be increased following supplementation of 20 mg/day of r3-carotene for 14 weeks (van Poppel et al., 1993). 3. Exposure to UV Light Exposure to UV light has been shown to depress human immune responses and also increase the risk of skin cancer. UV exposure causes a significant reduction in circulating total lymphocytes and helper T lymphocytes, resulting in an inversion of the helper:suppressor ratio (Hersey et al., 1983) Furthermore, UV exposure decreases antioxidant enzyme levels in the skin while increasing lipid hydroperoxide levels (Shindo et al., 1993). White et al. (1988) reported a significant decrease in circulating plasma carotenoids following exposure to UV light. Carotenoids containing nine or more conjugated double bonds can quench the high energy from UV light and block the is not only a precursor of subsequent formation of singlet oxygen. ~-Carotene vitamin A, but is also a potent quencher of singlet oxygen and an antioxidant (Burton and Ingold, 1984). supplementation (30 mg/ day for 7 In a placebo-controlled trial, ~-carotene weeks) blocked the UV-induced depression in overall immune responses, as determined by DTH skin tests (Fuller et al., 1992). In the placebo group, the number of DTH responses was significantly decreased, but returned to baseline by the end of the study. In contrast, the vigor of the response, as measured by the induration diameter, was still significantly reduced in the placebo group at the end of the study. Ultraviolet light, either from lamps or sunlight, can activate human HIV gene expression in transgenic mice (Morrey et al., 1992). The immune system of HIVinfected patients is severely compromised because the virus attacks helper T lymphocytes (see section on HIV infection). Exposure to UV could adversely affect HIV-infected patients in two ways: by activating the viral genetic material and by further reducing immune responses due to increased exposure to reactive oxygen species.

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4. Strenuous Exercise Skeletal muscle has one of the highest rates of oxidative metabolism of all human tissues (Kohen et al., 1988). Exercise, by its very nature, increases oxygen consumption and thus, indirectly, enhances the potential for the formation of oxygen-containing free radicals. Strenuous exercise increases free radical attack on muscle fiber lipids and results in higher levels of lipid peroxidation products such as malondialdehyde, ethane and pentane, lipid alcohols, conjugated dienes, and epoxy-fatty acids (Bendich, 1991b; see also Chapter 20, this volume). Moderate exercise also increases the concentration of breath pentane. Supplementation of adult males with 1200 mg/ day of vitamin E for 2 weeks lowered the baseline pentane exhalation as well as the level exhaled following exercise (Dillard et al., 1978). During strenuous exercise, skeletal muscle undergoes severe physical stresses, including shearing forces, often becoming inflamed (Jackson, 1990). Neutrophils are found in injured and/ or inflamed muscle tissues and certain products of muscle membrane lipid peroxidation are chemoattractants for neutrophils. Strenuous exercise has been shown to cause a marked decline in T and B lymphocyte activities, reduced complement levels, and impaired neutrophil function (Newsholme et al., 1991). Recently, Peters et al. (1993) reported that marathon runners have a significantly higher rate of upper respiratory tract (URT) infections following races than do nonrunning controls. When runners were given placebos or supplements of vitamin C (600 mg/day) for 3 weeks prior to and following the race, 68% of the placebo group reported symptoms of URT infections compared to 33% of the supplemented group (p < 0.01). Nonrunning controls taking vitamin C reported significant reductions in duration and symptoms of URT infections compared to their matched placebo group (p < 0.05). A possible explanation for the efficacy of vitamin C in this study comes from the findings of Maeda and Akaike (1991) that oxygen free radicals are the primary pathogenic factors induced by viral infections in the lungs. 5. HIV Infection HIV infection is characterized by a destruction of the helper T lymphocytes and the consequent loss of immune responses to pathogens and an increased risk of certain cancers. In addition, there are secondary effects of HIV infection that cause significant decreases in serum levels of many vitamins and minerals (Bogden et al., 1990). The potential that free radical damage is also involved in the pathogenesis of HIV, and that antioxidants may reduce infection, is an area of intense interest (Staal et al., 1993; see also Chapter 17, this volume). Recently, a placebo-controlled, double-blind cross-over study examined the effects of high-dose f3-carotene supplementation on the immune cells of HIVinfected patients. Supplementation with 180 mg/ day for 1 month caused a significant increase in total white blood cells and the percentage change in helper T lymphocytes, resulting in a significant improvement in the helper:suppressor ratio (Coodley et al., 1993). In earlier, noncontrolled studies, HIV-infected patients were

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given lower doses of ~-carotene and no improvements in helper:suppressor ratios were seen, but there were increases in natural killer cells and in other markers of lymphocyte activation in one study, a decrease in CD8 suppressor cells in another study, and an improvement in well-being in a third study (as defined by responses to a standardized quality-of-life questionnaire). The differences in the studies may reflect the decreased intestinal absorption of many micronutrients found in HIV infection. HIV-infected patients may require more than 60-120 mg/day of ~-carotene to elevate helper T lymphocyte levels (reviewed in Bendich, 1993). Of importance is the recent finding by Ward et ale (1993) that HIV-infected patients had significantly decreased serum vitamin A levels. Vitamin A supplementation significantly increased the helper T lymphocyte levels in vitamin A-deficient children and increased the helper:suppressor ratio from 0.97 to 1.32. Low serum vitamin A levels were associated with low helper T lymphocyte levels and increased risk of mortality in intravenous-drug-using, HIV-infected adults in the United States supplementation (Semba et al., 1993). It is possible, therefore, that ~-carotene increased vitamin A levels as well as ~-carotene levels, and the resulting immune cell changes were due to vitamin A. On the other hand, Loya et ale (1992) showed that the carotenoid halocynthiaxanthin could block the HIV reverse transcriptase enzyme without affecting human cells. The use of multivitamins and/or single-entity supplements of vitamin E or iron significantly delayed the progression of HIV infection to AIDS (Abrams et al., 1993). Total intake from dietary sources and supplements of vitamin A, riboflavin, or thiamin or the use of daily multivitamin supplements were associated with a reduced risk of low T helper cell counts. There is a growing awareness of the importance of nutrition in HIV-infected patients; the role of antioxidants is being explored by more and more investigators.

B. Genetic Conditions Inherited conditions can result in impaired immunity, which is associated with increased free radical production and! or reduced antioxidant status. Of the numerous genetic defects, six are discussed here. These include inherited neutrophil dysfunctions, sickle cell disease and other conditions that increase the risk of infections in children, and asthma. In addition, there is a detailed discussion of rheumatoid arthritis. The more general immunological effects of aging and cancer are also reviewed because of the growing interest in preventing age-associated, immune-mediated diseases by increasing antioxidant intakes of the elderly.

I. Inherited and Other Neutrophil Dysfunctions Vitamin C supplementation has been shown to normalize the reduced chemotactic and bactericidal activities of neutrophils from individuals with inherited phagocytosis disorders (Weening et al., 1981; Gallin, 1981) as well as from newborn infants whose neutrophil responses are often suboptimal (Vohra et al., 1983). In several instances, clinical improvements were found. The concentration of vitamin C in neutrophils is approximately 150 times the concentration in plasma (Moser and Bendich, 1991).

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Anderson et al. (1990) have shown that vitamin C can decrease the damaging effects of the products of the oxidative burst without decreasing the intracellular concentration of reactive, bactericidal molecules. During the oxidative burst, neutrophils take up vitamin C (Moser and Weber, 1983) and, following activation, the intracellular vitamin C concentration is reduced (Hemila et al., 1985; Oberritter et al., 1986). Vitamin C also protects o.-l-prorease inhibitor (a protective enzyme found in the lung) from inactivation by the free radicals generated during the neutrophil oxidative burst (Theron and Anderson, 1985). Miller (1979) found that the bactericidal and chemotactic capacity of neutrophils from premature infants is impaired compared to full-term infants. Chirico et al. (1983) demonstrated that high-dose vitamin E administration to preterm infants during the first week of life enhanced the phagocytic activity of their neutrophils. Baehner et al. (1977) also found (in adults) that vitamin E supplementation (1600 IV /day for 2 weeks) increased the phagocytic capacity of neutrophils and decreased the release of hydrogen peroxide while maintaining the level of superoxide radical production. However, they showed that the ability of the neutrophils to kill bacteria in vitro was lower in the vitamin E-supplemented group. Chirico et al. (1983) did not report a decrease in bactericidal capacity following high-dose vitamin E supplementation in their study of preterm infant phagocytic cells.

2. Children with Sickle Cell Disease and Others at Risk for Infections The genetic defect, sickle cell disease (SCD), results in an alteration in the shape of the red blood cells (erythrocytes) and the production of highly reactive oxygen-containing free radicals within the erythrocytes (Hebbell et al., 1982). Increased hemolysis of sickled erythrocytes activates neutrophils and increases the oxidative damage to cells and tissues of SCD patients. Additionally, several studies have shown that the levels of circulating antioxidant vitamins are significantly lower in children with SCD compared to normal children consuming similar levels of the vitamins (Sindel et al., 1990a). A major cause of childhood morbidity and mortality in SCD is bacterial infection. One possible explanation for the severity of the infections is a defect in neutrophil function. Neutrophils have less killing capacity in SCD, which is correlated with the vitamin E status (Baliga et al., 1989; Sindel et al., 1990b). Additionally, the serum of SCD patients contains higher than normal levels of oxidative products. Vitamin E supplementation (800 IV/day for 6 months) resulted in a significant decrease in the excessive levels of free radicals generated in the blood of SCD children (Sindel et al., 1990b). A marginal vitamin E status in young children may, in part, lower the resistance to childhood infectious diseases. In a small clinical study of eight children with a history of respiratory tract infections, supplementation with 20 mg/kg body weight for 6 weeks resulted in an improvement in the clinical presentation, with six out of eight children remaining healthy during the supplementation period. Concomitantly, those children with initially low T lymphocyte helper:suppressor ratios had a normalization of T helper cell numbers (Skopinska-Rozewska et al., 1987).

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In a study of 513 apparently healthy Canadian children (approximately 3 years old), those with the lowest serum vitamin E levels had significantly decreased lymphocyte proliferative responses, indicating impaired immunity. Serum IgM concentration, the immunoglobulin associated with protection from bacterial infections, was also significantly lower in children with low serum vitamin E levels (Vobecky et al., 1984). 3. Asthma Asthma has been categorized as an inflammatory disorder of the airways. Recent evidence points to increased production of free radicals in asthmatics, which is exacerbated in cases of severe asthma. Oxidized lipids were significantly higher in the serum of asthmatics compared to controls, suggesting that peroxidative damage is not localized but systemic (Owen et al., 1991). Floersheim (1990) gave 19 adults with asthma 900 mg/ day of vitamin E for several months and reported a decrease in symptom severity and reduction in use of steroid medications. 4. Rheumatoid Arthritis Rheumatoid arthritis (RA) is thought to begin when a substance (an antigen) triggers an inappropriate immune response in the articular joints (e.g., elbow, knee, shoulder). The responsible factor is unknown, but viruses, nonbiodegradable products of bacteria, or antibodies directed against structures within the joint have all been implicated. At some point, the initial insult and inflammation becomes a chronic process; the change from acute to chronic inflammation appears to result from a specific immune reaction directed at the persisting antigen (Zvalifer, 1988). The rate of progression of the disease depends on both the intensity and the duration of the inappropriate immune responses (Harris, 1985). At the cellular level, the earliest recorded change during the days to weeks following the first symptoms of RA is damage to the lining of the small blood vessels in the joints. At the same time, infiltration of the fluid-filled joint space (synovium) with inflammatory neutrophils occurs and structural changes begin to appear in the synovium, forming an inflamed layer called a pannus. The synovial fluid of the inflamed rheumatoid joint also contains numerous neutrophils. Oxidative products reduce the viscosity of the synovial fluid by oxidizing lipids in synovial fluid, making them more hydrogenated (comparable to vegetable oils oxidized to margarine), hindering joint movement further (Halliwell et al., 1988). The longer term consequences of these joint changes include erosion of cartilage, bone, ligaments, and tendons in the area (Zvalifer, 1988). Several components of the immune system contribute to the development of RA. The B lymphocytes produce antibodies that form the components of rheumatoid factors. Rheumatoid factors are immunoglobulins (IgM) that inappropriately react with other immunoglobulins (IgG) to form immune complexes. T lymphocytes also generate inappropriate immune responses that result in the increased production of inflammatory cytokines and interferons. Neutrophils and macrophages synthesize and release free radicals, prostaglandins, and immunostimulatory molecules, all of which enhance the inflammatory response in RA. Neutrophils are the major mediators of tissue-destructive events in RA (Harris, 1985).

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Some of the adverse immunological consequences result from the alteration of antibodies by free radical reactions. Lunec and Hill (1984) reported that certain immunoglobulins not only underwent free radical denaturation but that the altered immunoglobulins stimulated neutrophils to generate free radicals. Animal models have documented the increased production of pentane in arthritic animals; vitamin E supplementation lowered the pentane exhalation as well as joint swelling (Tappel and Summerfield, 1984). Humad et al. (1988) showed that breath pentane concentrations correlated with the severity of symptoms in RA patients. Coltro et al. (1987) found that clinical improvement was associated with a significant decrease in breath pentane in a separate group of RA patients. Kowsari et at. (1983) assessed the baseline nutritional status of arthritis patients in a prospective study. Data indicated that many arthritis patients were at least marginally inadequate in selected nutrients, including vitamin E and vitamin C. Thurnham et at. (1987) conducted a study to assess tocopherol and ascorbic acid antioxidant capacity in RA patients by utilizing the total radical-trapping antioxidant parameter ("Trap") assay. A high Trap value indicates a good antioxidant status. Twenty patients with RA were matched by age and sex to 20 healthy controls. The Trap values in the patients with RA were significantly lower than those in the control subjects. Serum concentrations of tocopherol and ascorbic acid were also significantly lower in the patients compared to the controls. Serum and plasma selenium (Se) concentrations in RA patients are much lower than those of healthy controls. In a cohort study of 87 RA patients, serum Se level was inversely related to the severity of the disease (Tarp et al., 1985). Superoxide anion production by neutrophils and superoxide dismutase enzyme activities (SODI and SOD2) were assessed in RA patients and matched healthy controls. After 30 minutes, neutrophils from individuals with RA produced more superoxide than did those from healthy controls. Levels of SODI were similar in RA and controls. However, RA patients had 1/30 the level of SOD2 compared to controls (Pasquier et al., 1984). Imadaya et al. (1988) studied the activities of antioxidant enzymes in erythrocytes isolated from patients with RA and patients with osteoarthritis (controls) of the knee joints. The activities of SOD, GSH-Px, and CAT were significantly decreased in patients with RA. Biemond et al. (1984) measured SOD, CAT, GSH-Px, and ceruloplasmin (Cp) levels in synovial fluid obtained from 7 patients with traumatic knee lesion (controls) and 17 patients with RA. The authors found similar concentrations of SOD in synovial fluid of both groups. Although CAT and GSH-Px levels were increased in the synovial fluid in the RA subjects, the activity of these enzymes was reduced. Cp, an acute-phase protein with chelating and antioxidant capacities, was elevated in RA synovial fluid. Since 1985, there have been seven double-blind, placebo-controlled studies of the effects of vitamin E supplementation in patients with arthritis (Bendich and Cohen, 1994). Vitamin E supplementation, at levels from 100 to about 600 Il.l/day, produced significant pain relief in all of these studies. There was also a consistent report of antiinflammatory activities in these and other nonplacebo-controlled trials.

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The effects of supplementation with Se were studied in six patients with severe, active RA and in six healthy controls (Tarp et al., 1987). Initial concentrations of Se and the activity of GSH-Px were significantly lower in RA patients compared with controls. During 26 weeks of Se supplementation (250 p.g Se-enriched yeast tablets daily), the differences in Se levels and in GSH-Px activity between the two groups disappeared; however there was no report of clinical improvement. Gold compounds represent one of the oldest therapies for RA, and there is abundant evidence of their efficacy. One gold compound, auranofin, is an effective quencher of singlet oxygen, a reactive oxygen intermediate (Corey et al., 1987). Penicillamine treatment has been reported to reduce rheumatoid factor titers (Ruddy, 1985). This compound is a chelating agent, and can reduce the concentration of reactive metals that can increase free radical formation. Strom and AhnfeltRonne (1989) reported that penicillamine is also a direct scavenger of free radicals. In summary, there are sufficient positive data to suggest that a well-controlled clinical trial of antioxidant supplementation in RA is warranted. 5. Aging The cumulative effects of free radical damage throughout the life span are graphically seen in the free radical-generated pigmented age spots and increased circulating levels of lipid hydroperoxides (as measured by TBA-reactive substances) found in the elderly (Meydani et al., 1990a). As cells and tissues age, there is frequently an alteration in cell membrane components. There is a measurable increase in the "senescent cell antigen" in the cell membranes of old versus young subjects; of interest, cells from vitamin E-deficient animals have significantly more of the antigen than cells from vitamin E-sufficient animals (Kay et al., 1986). The cell-mediated immune responses, involving T lymphocyte functions (cytotoxicity, interleukin-2 production, proliferation), are the most sensitive to the agerelated decline in immune responses. Laboratory studies suggest that some of the immunosuppression seen in aging can be reversed with antioxidant supplementation (Meydani and Blumberg, 1993). The accumulating data from epidemiological and intervention studies suggest that antioxidant status affects clinically relevant immune parameters in the elderly. Chavance et ale (1984), in an epidemiological study, found a significant association between high plasma vitamin E levels and a lower number of subsequent infections in healthy adults over the age of 60. An important clinical index of immune function is the DTH to skin test antigens. DTH are significantly diminished in the elderly. Anergy, a complete lack of skin test responses, is seen in those most immunosuppressed. Recent studies have shown that DTH can be used as a predictor of morbidity and mortality in the elderly; i.e., elderly patients who are anergic had twice the mortality risk as elderly patients with DTH responses (Wayne et al., 1990). Recently, several placebo-controlled, double-blind studies have shown that supplementation with vitamin E alone or intake of multivitamin supplements containing higher than recommended levels of antioxidants significantly enhanced DTH responses, T cell subpopulations, proliferative responses, and/or interleukin-2 activities in the elderly.

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In a carefully controlled, placebo double-blind study, vitamin E supplementation (800 Il.l/day) for 1 month resulted in significantly increased DTH responses of the healthy elderly (Meydani et al., 1990b). Lymphocyte vitamin E levels increased over threefold (from 0.12 to 0.39 nmol/LO? cells) with supplementation and were correlated with enhanced interleukin-2 production. Beneficial effects included enhanced lymphocyte proliferation, decreased production of immunosuppressive prostaglandin E 2 , as well as decreased levels of serum lipid hydroperoxides (as assessed by specific high-performance liquid chromatography determination of malondialdehyde and other aldehydes). In a second well-controlled study, intake of a high-potency multivitamin/ mineral supplement for 16 months also significantly enhanced DTH in healthy elderly patients (Bogden et al., 1990). In a separate placebo-controlled intervention trial, the elderly were given a multivitamin daily for 1 year; the supplement contained approximately eight times the standard level of intake of ~-carotene (16 mg). The supplemented group had significantly less infections than the placebo group. Responses to vaccines were also improved in the supplemented group. In an unblinded study, supplementation of healthy elderly individuals for 2 months with 45 or 60 mg/ day of f)-carotene increased the indices of T lymphocyte and natural killer cell activation (Chandra, 1992). Another study included elderly patients who were hospitalized for at least 2 months following a stroke. A supplement of 8000 IV of vitamin A, 50 mg of vitamin E, and 100 mg of vitamin C taken for 28 days increased total T lymphocyte numbers and T helper cell markers as well as enhanced lymphocyte proliferation (Penn et al., 1991). DTH was enhanced following oral supplementation with 10 g/day of vitamin C in one study (Panush et al., 1982) in young adults, and in an elderly population following injections of 500 mg/ day of vitamin C in another study (Kennes et al., 1983). Oral supplementation with vitamin C (2 g/ day) in an elderly population enhanced in vitro lymphocyte proliferative responses but did not affect delayed hypersensitivity responses in another study (Delafuente et al., 1986). The data strongly point to a beneficial effect of antioxidant vitamin supplementation on immune responses in the elderly.

6. Cancer Conditions that depress immune functions increase the risk of certain cancers. Conversely, factors that can enhance immunity may lower the risk of cancer (Roitt, 1979). Evidence that immunosuppression increases risk of cancer comes from many laboratory studies as well as from clinical investigations. For example, patients undergoing kidney transplants are given immunosuppressive drugs to prevent the rejection of the transplant. The incidence of skin cancer, especially squamous cell carcinoma, is significantly higher in these patients. Skin cancer cells are normally strong stimulators of immune responses that destroy the transformed cells. However, in the immunosuppressed patient, the loss of tumor immunity is considered an increased risk factor for the development of skin cancer (Streilein, 1991).

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Epidemiological evidence has pointed to an inverse relationship between dietary intake of foods rich in antioxidant nutrients and the incidence of cancer of the lung and other organs (Ziegler and Subar, 1991). Studies suggest that (3-carotene and vitamin E can prevent certain types of animal tumors and reduce tumor frequency as well as enhance tumor regression (Bendich, 1991a; see also Chapters 5-9, this volume). Several clinical studies have shown that f3-carotene supplementation enhances many aspects of tumor immunity, especially in populations with compromised immune responses. (3-Carotene supplementation (30 mg/ day for 3-6 months) caused complete or partial remission of lesions in 71 % of smokers with oral leukoplakia, the precancerous lesion which precedes oral cancer (Garewal, 1992). Significant increases in natural killer cell number and function were documented at the same time that the regression or stabilization of the leukoplakia was seen. Natural killer cell activity is usually depressed in smokers. Lymphocyte proliferation is also depressed and has been shown to be improved when smokers were given 20 mg/day of (3-carotene for 14 weeks (van PoppeI et al., 1993).

v.

Summary and Conclusions

The studies reviewed point to consistent negative immunological effects of higher than normal exposure to free radicals. Increased free radical damage and/or reduced antioxidant status can be a consequence of genetic, environmental, and lifestyle factors. As summarized in Tables I and II, dietary fat, cigarette smoking, UV light, strenuous exercise, and HIV infection, as well as certain inherited diseases, resulted in increased free radical production that was detected as either increased exhalation of breath pentane, increased levels of serum lipid hydroperoxides (as indicated by increased serum malondialdehyde concentrations), and/or decreased antioxidant status. Immune parameters that were adversely affected included decreased specific responses, such as DTH and lymphocyte proliferation, which may be the cause of the increase in URT infections. Nonspecific immune responses to bacterial pathogens were reduced by the majority of the genetic conditions reviewed, and the rates of infections and inflammatory responses were increased. Vitamins E, C, and (3-carotene, at levels above those found in normal diets, showed multiple immunoenhancing effects. Both nonspecific and specific aspects of immune function were enhanced in several intervention trials in which vitamin E, C, or f3-carotene supplementation was used. Because of recent recommendations for adults to increase dietary PUFAs, it is important to determine whether vitamin E and other dietary antioxidants can alter the potential immunosuppressive effects of higher PUFA diets. This may be of critical importance in populations that are known to have lower immune responses as well as antioxidant status, such as the elderly, smokers, strenuous exercisers, and people with sickle cell disease and HIV infection. With regard to arthritis, the efficacy of gold compounds, based on their capacity to quench singlet oxygen, brings to light another possible strategy for

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Table I Effects of Antioxidants on Overcoming Environmental Factors That Increase Free Radical Exposure and Reduce Immune Functions a

Factor

Increased free radicals

Decreased immune functions

Effects of antioxidants

Dietary fat

Increased lipid hydroperoxide/MDA levels

Increased PG formation, thymic atrophy; decreased IL-l and IL-2 production; decreased lymphocyte proliferation

Vitamin E-enhanced lymphocyte proliferation

Cigarette smoking

Increased breath pentane, serum oxidant radicals; decreased serum antioxidant levels

Increased numbers of neutrophils in lungs, increased URT infection; decreased lymphocyte proliferation

Vitamin E-decreased breath pentane, serum oxidant radicals; r3-carotene-increased lymphocyte proliferation

UV light

Decreased skin antioxidants, serum 13carotene levels

Reduced numbers of circulating lymphocytes, T helper cells; decreased DTH; activated HIV gene

~-Carotene-no

Strenuous exercise

Increased serum MDA levels, breath pentane

Decreased T and B lymphocyte activities, complement, neutrophil function; increased URT infections

Vitamin E-decreased breath pentane Vitamin C-decreased symptoms and duration of URT infections

HIV infection

Decreased levels of glutathione in infected cells

T helper cells destroyed; increased cancer risk; decreased vitamin A status

~-Carotene-enhanced

DTH

decrease

T helper cell activity; multivitamins and vitamin E-slower HIVto-AIDS progression

aDTH, Delayed type hypersensitivity; IL, interleukin; MDA, malondialdehyde; PG, prostaglandin; URT, upper respiratory tract.

treatment. ~-Carotene is one of the most potent singlet oxygen-quenching comin the pounds known; therefore, an evaluation of the clinical efficacy of ~-carotene prevention and/or treatment of arthritis warrants serious consideration. The current interest in the potential cancer prevention role of antioxidants leads to an increased

emphasis to determine the mechanisms of cancer prevention, including immunoenhancement. Although each of the factors were discussed individually in this review, there are instances when there may be additive or synergistic effects that further exacerbate the imbalance between free radical exposure and antioxidant defenses. For example, strenuous exercise in air-polluted outdoor environments exposes the ath-

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Table II Effects of Antioxidants in Overcoming Genetic Factors That Increase Free Radical Exposure and Reduce Immune Functions a

Factor

Increased free radicals

Decreased immune functions

Effects of antioxidants

Inherited neutrophil dysfunction

Nonspecific

Reduced killing of bacteria

Vitamin C-reduced nonspecific oxidative damage

Premature infants

Nonspecific; increased hydrogen peroxide production by neutrophils

Reduced killing of bacteria

Vitamin E-increased neutrophil phagocytosis; decreased hydrogen peroxide production

Sickle cell disease

Oxidative hemolysis of erythrocytes; low serum antioxidant levels; increased serum MDA levels

Reduced killing of bacteria; decreased lymphocyte proliferation

Vitamin E-reduced serum levels of MDA; increased neutrophil phagocytosis

Asthma

Increased serum MDA levels

Reduced killing of bacteria

Vitamin E-reduced asthma symptoms

Rheumatoid arthritis

Increased serum MDA and breath pentane; low serum levels of antioxidants and enzymes; low Trap serum levels

Oxidized RA factors; increased nonspecific inflammatin

Vitamin E-reduced pain, antiinflammatory effects; seleniumnormalized enzyme levels; gold-reduced pain; penicillaminereduced RA factor titers

aMDA, Malondialdehyde; RA, rheumatoid arthritis.

lete to higher concentrations of the oxidative pollutants over a shorter period of time and possibly higher levels of UV light compared to the sedentary individual. On the other hand, reduction in dietary fat intake can reduce potential oxidation stress. However, the greatest documented benefit to health is cessation of smoking, as cigarette smoke significantly increases free radical burden.

In conclusion, humans are placed in many life situations wherein their immune systems would benefit from increased antioxidant intakes. Because the immune system is pivotal in the prevention of many of the serious diseases and disabilities that affect human morbidity and mortality, it seems to be critical that the immune system's balance between antioxidants and free radicals is maintained.

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