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Annals o f Clinical & Laboratory Science, vol. 30, no. 2, 2000

145

Review: Free Radicals, Antioxidants, and the Immune System

Joseph A. Knight Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah

Abstract. Oxygen-derived free radicals are important in both natural and acquired immunity. Neutrophil and macrophage phagocytosis stimulates various cellular processes including the “respiratory burst” whereby increased cellular oxygen uptake results in the production of the potent oxidant bactericidal agents, hypochlorous acid and hydroxyl radical. In addition, nitric oxide, a gaseous radical produced by macrophages, reacts with superoxide to form peroxynitrite, also a potent bactericidal agent. Conversely, oxidative stress may be detrimental in acquired immunity by activation of nuclear factor kappa B, which governs gene expression involving various cytokines, chemokines, and cell adhesion molecules, among others. However, antioxidant supplementation essentially reverses several age-associated immune deficiencies, resulting in increased levels of interleukin-2, elevated numbers of total lymphocytes and T-cell subsets, enhanced mitogen responsiveness, increased killer cell activity, augmented antibody response to antigen stimulation, decreased lipid peroxidation, and decreased prostaglandin synthesis.

Keywords: Free radicals, oxidants, antioxidants, immune, immunity, lipid peroxidation

Introduction

Two general types of immunity are recognized; innate (natural) and acquired (adaptive). The cellular component of natural immunity is composed of monocytes (mononuclear phagocytes), neutrophils (polymorphonuclear leukocytes, PMN), and natural killer cells (NKC). These cells use the complement cascade as the primary protein effector mechanism, as well as various recognition proteins such as C-reactive protein and amyloid protein, among others. These proteins are able to bind carbohydrate structures present on bacteria but not on eukaryotic cells. NKC, the first line of defense against viruses, not only increase with age but remain functionally active [1]. Acquired immunity involves several lymphocyte subtypes and utilizes antibody as the effector protein. The T-cell receptor and antibody are the recognition molecules.

Address correspondence to Joseph A. Knight, M .D., Department o f Pathology, University o f Utah School o f Medicine, 50 North Medical Drive, Salt Lake City, U T 84132; tel 801 581 4516; fax 801 585 2463; e-mail joe_knight@hlthsci.med.utah.edu. This paper was presented at the Fall Meeting o f the Association of Clinical Scientists in La Jolla, California, on 3-7 November 1999.

B-lymphocytes recognize carbohydrate, protein, and some relatively simple chemical structures while T-lymphocytes recognize only peptides. In recent years it has become increasingly apparent that free radicals play a critical role in a variety of normal regulatory pathways. Yet, dysregulation may play an important role in inflammation. Thus, oxidant- antioxidant balance is critical for immune cell function because it maintains cell membrane integrity and functionality, cellular proteins, and nucleic acids. Moreover, this balance is important in controlling signal transduction and gene expression. Immune cells are particularly sensitive to oxidative stress because of the high percent of polyunsaturated fatty acids in their plasma membranes and a higher production of reactive oxygen species (ROS), which is part of their normal function [2]. Moreover, membrane-related signaling and gene expression are critical in maintaining normal function of immune cells and their ability to defend against various foreign antigens. These functions are, however, highly sensitive to ROS. As a result, it seems appropriate that immune system cells generally have higher concentrations of antioxidant micronutrients than other cells [3,4].

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Age-related decline in immune function

It is widely recognized that a progressive age-related

decline in immune function is common in many mammals, including humans [5,6]. Moreover, the immune system is protective of some malignancies, while an altered or misdirected immune response may result in an autoimmune disorder. As a result, the immune system occupies a critical position between aging and disease [7]. Thus, aging has been associated with (a) decreased total lymphocyte count; (b) decreased T-cell subsets [CD3, CD4, CD8]; and (c) decreased response to concanavalin A and phyto­

hemagglutinin [8]. In addition, Nagel et al [9] reported

a decrease in both the number and percentage of

T-cell subsets in elderly humans. Levels of the cytokines interleukin-2 (IL-2) and interleukin-6 (IL-6) are also altered with age. Thus, IL-2, a critical growth factor for maintaining T-cell proliferation, is commonly decreased in the elderly [10]. Conversely, IL-6 levels generally increase with age [11]. Although usually not measurable in healthy young adult serum, IL-6 is readily measured in most healthy elderly individuals. Its production may also be related to increased oxidative stress [11,12]. IL-6 dysregulation has been associated with the following [13]: (a) mediation of the acute inflammatory response; (b) increased autoantibody production (paraprotein­ emia is common in the elderly); (c) age-associated malignant B-cell tumors (ie, multiple myeloma, chronic lymphocytic leukemia, lymphoma); (d) alteration of amyloid protein precursor; and (e) stimulation of post-menopausal bone resorption. A lthough aging is associated w ith general functional impairment of the immune system, the impaired progression differs significantly among aging individuals, suggesting that genetic, environmental, and other factors, especially nutrition, play an important role. Thus, a wide range of cellular and molecular changes are age-associated. However, exactly which of these are primary, and therefore age-specific, and which are secondary to other causes, is not fully understood. For example, Mysliwska and associates [14] reported that increased IL-6 and decreased IL-2 production during the aging process are both influenced by an individual’s health status, a topic that will be discussed in later sections of this review.

Hirokawa [15] discussed various methods that might be used to restore some immune functions in the elderly. These include (1) grafting of cells/tissues from younger individuals; (2) caloric restriction and nutritional supplementation; (3) administration of free radical scavengers and antioxidants; (4) thymic peptide treatment; (5) endocrine manipulation; and (6) physical activity. Several of these are interrelated, especially with respect to the role of free radicals. In this regard, increased oxidative stress has been repeatedly demonstrated in aging individuals [16-19]. Moreover, caloric restriction, use of antioxidants/free radical scavengers, and exercise are, to a significant degree, related to various oxidative influences that will be discussed in subsequent sections of this review.

Innate immunity and phagocytosis

The emigration of leukocytes at an inflammatory site results in phagocytosis with the release of enzymes from both macrophages and neutrophils. This phagocytic process consists of three distinct but interrelated steps [20]: (a) recognition of bacteria or other foreign matter that has been coated with opsonins, such as the Fc fragments of immunoglobulin G (IgG) and/or the complement fragment C3b, the opsonic component of C3; (b) engulfment of bacteria or other material followed by leukocyte degranulation; and (c) bacterial killing and degradation of other digested material. Phagocytosis stimulates various independent processes, especially the so-called “respiratory burst” which results from activation of NADPH oxidase, an enzyme normally inactive in resting cells. It also includes several major chemical events including glycogenolysis, increased glucose oxidation, and the production of potent ROS [21-23]. The generation of ROS begins with the rapid uptake of oxygen and activation of NADPH oxidase and the production of

the superoxide free radical anion (0 2 *)•

2 0 2 + NADPH

oxidase> 2 0 2_* + NADP+ + H +

Superoxide is then rapidly converted to hydrogen peroxide (H2O 2) by superoxide dismutase (SOD).

2 0 2~* + 2 H +

SQD

> H 20 2 + 0 2

These reactive oxygen species can act by either of two oxygen-dependent mechanisms with the destruction of microorganisms or other foreign matter.

Myeloperoxidase-halide-Fl202 mechanism. The neutrophile cytoplasmic granules contain the enzyme myeloperoxidase (MPO). In the presence of the ubiquitous chloride ion, hydrogen peroxide is converted to hypochlorous acid (HOC1), a potent oxidant and antimicrobial agent [22,23].

cr +H 20 2 +H +

MPO > HOC1 +h 2o

Myeloperoxidase-independent system. Although not as important as the MPO-dependent system, the MPO-independent mechanism is still essential and is particularly pertinent in mature macrophages which lack MPO. This system also requires oxygen; ROS are generated from superoxide and hydrogen peroxide produced via the “respiratory burst.” Thus, the hydroxyl radical (HO*), the most potent of the biological oxygen-derived free radicals, is produced by (a) Fenton and/or (b) Haber-Weiss chemistry [24],

(a)

H20 2 + Fe2+ ------- s- HO*+ OH- + Fe3+

(b)

0 2~* + H 20 2

ironcatalyst>

HO*+ O H " + 0 2

Support for the “respiratory burst” mechanisms for microbial killing includes (a) the demonstration that phagocytic cells deprived of oxygen engulf but cannot efficiently kill some bacteria [25,26], and (b) the recognition in humans of an X-linked inherited disorder, chronic granulomatous disease (CGD). CGD exists when membrane-bound flavoprotein cytochrome b-245 NADPH oxidase is absent or abnormal [27-29]. The deficiency results in failure to form H 20 2 following phagocytosis and the oxygen burst. As a result, affected individuals are susceptible to various fungi and bacteria, especially Staphylococcus [30], which possesses catalase, an enzyme that inactivates hydrogen peroxide, preventing formation of hypochlorous acid and the hydroxyl radical.

2H20 2

catakse

>

2 H 20

+ 0 2

Free radicals and immune system 147

Oxygen-independent system. It should be noted that bacteria can be killed in the absence of ROS by the following mechanisms: (a) the hydrogen ion, derived from increased lactic acid production, lowers the local pH to about 4.0, a level that prevents most bacterial growth; (b) neutrophilic granules contain bactericidal permeability increasing protein (BPIP), a highly cationic protein that lyses the outer bacterial membrane [31]; (c) lysozyme, a leukocyte granule-containing enzyme, hydrolyzes the muramic acid—N-acetyl-7- glucosamine bond which is present in the bacterial glycopeptide membrane; (d) lactoferrin, an iron- containing protein in leukocyte granules, is bactericidal; and (e) eosinophil cationic protein has m inor bactericidal activity and is toxic for many parasites [32].

Reactive nitrogen species and immunity

Reactive nitrogen species are also important in some infectious processes. For example, various studies have shown that the free radical nitric oxide (NO*), commonly known as endothelium-derived relaxing factor (EDRF), is produced from a terminal guanido nitrogen atom of L-arginine (L-Arg) by nitric oxide synthase (NOS).

L-Arg + 0 2 + NADPH

NO* + L-citrulline

This enzyme exists in three isoforms: two constitutive synthases (cNOS) and an inducible synthase (iNOS). iNOS, a calcium-independent enzyme, is expressed in many different cells following challenge with immunologic or inflammatory stimuli. These cells include macrophages, neutrophils, mast cells, endothelial, and vascular smooth muscle cells [33].

of continuously

Im portantly, iNOS is capable

producing large amounts of nitric oxide. In activated immune cells, it acts “as a killer molecule” [33]. That is, although the direct toxicity of nitric oxide is modest, toxicity is greatly increased when it reacts with superoxide to form peroxynitrite, a very strong oxidant.

NO* + 0 2_* ------> ON OO-

Among other toxic reactions, peroxynitrite reacts with aromatic amino acid residues to form nitrotyrosine,

In

which can lead to enzyme inactivation [34,35].

148 Annals o f Clinical & Laboratory Science

addition, peroxynitrite, produced by macrophages, kills

E. coli in direct proportion to its concentration [36]. In addition, N-methyl-L-arginine inhibits nitric oxide synthesis and thereby reduces the bactericidal and

tum oricidal

Furthermore, Gregory et al [38] demonstrated in mice that increased nitric oxide production during primary infection with Listeria moncytogenes suppresses host defenses by diminishing the proliferation of immune cell populations. Nitric oxide is also an important cytotoxic effector molecule in defense against tumor cells, various protozoa, fungi, helm inths, and mycobacteria [39,40].

activities of macrophages [37].

Antioxidants and the immune system

Lipid peroxidation is an autocatalytic free radical reaction whereby polyunsaturated fatty acids and phospholipids undergo degradation by a chain reaction that results in the formation of lipoperoxides, various aldehydes (eg, malondialdehyde, 4-hydroxynonenal), and short chain hydrocarbons (eg, ethane, propane, pentane), among others [41]. Inasmuch as lipid

peroxidation damages the bi-lipid cell membrane, the result is an altered immune system, since a functioning

m em brane is necessary for norm al membrane

metabolic activity as well as antigen reception, secretion

of lymphokines and antibodies, contact cell lysis, and

lymphocyte transformation. Lipid peroxidation may also result in increased prostaglandin levels which are strong immunomodulators.

Cell membrane-dependent functions are affected by the membrane fluidity and physical state, both of which are determined by the membrane lipid acyl chain profile. Importantly, these acyl chains are modified by ROS. Recognizing these features, Eze [42] proposed that increased endogenous ROS present in aging and various disease states affect integral membrane function, including the cell-mediated immune reaction involving phagocyte membrane NADPH oxidase which depends on triggering by membrane-bound protein kinase C to produce superoxide. Depressed immunocompetence associated with aging, various diseases, and poor nutrition may result in down- regulation of these two enzymes by excess ROS. However, various antioxidants may prevent and/or improve immune dysfunction (Table 1).

Table 1. Antioxidants and immunity

Intracellular enzymes and co-factors Copper-zinc superoxide dismutase Catalase Glutathione peroxidase Reduced glutathione (GSH) Dietary or oral supplements Vitamin C Vitamin E Beta-carotene Zinc Selenium

Antioxidant enzymes

The major antioxidant enzymes are superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR). SOD, of which there are two enzymes (mitochondrial MnSOD and cytoplasmic Cu-ZnSOD), inactivates O2 *' by converting it to H2O2. This reaction is then followed by CAT inactivation of H2O2 by converting it to water and oxygen; GPx, in the presence of reduced glutathione (GSH), also converts H2O2 to water. In this latter reaction, GSH is oxidized to GSSG; GSH is then regenerated from GSSG by the enzyme GR. Umeki et al [43] measured blood SOD and O 2 * levels in compromised patients with and without pneumonia. They found that although SOD levels were decreased in compromised hosts w ithout pneumonia, they were further significantly decreased in compromised hosts with pneumonia. Conversely, although0 2 * levels were above normal in both groups, they were significantly higher in the compromised group with pneumonia compared to compromised individuals without pneumonia. Others [44] measured serum CAT activity in allogeneic bone marrow transplant recipients. Their results showed that CAT activity was five-fold greater in patients with acute graft-vs-host disease (GVHD) compared to patients with non-GVHD-related complications.

Glutathione

Glutathione (GSH), a ubiquitous tripeptide, is involved in many cellular functions. These include

protein and DNA synthesis; enzyme activation; amino acid transport; and cell protection from the negative effects of radiation, oxygen radicals, and other reactive oxygen intermediates [45], such as the GPx-catalyzed inactivation of hydrogen peroxide. Immunoregulation studies involving cysteine- delivery agents (eg, 2-mercaptoethanol, N-acetyl- cysteine, 2-oxothiazolidine-4-carboxylate) increase intracellular GSH levels and thereby enhance the mitogenic responses of lymphocytes. For example, Fidelus and Tsan [46] found that depletion of intra­ cellular GSH inhibited [^H ]-thymidine incorporation into DNA by mitogen (ie, concanavalin A) -stimulated cells. On the other hand, enhancement of intracellular GSH with either 2-oxothiazolidine-4-carboxylate or 2-mercaptoethanol stimulated the [^H]-thymidine incorporation by mitogen-stimulated cells. In addition, Smyth [47] reported that 2-mercaptoethanol enhanced IL-2-induced proliferation ofhuman CD3+ and CD3- lymphocytes, while others [48] demonstrated that N-acetylcysteine increases intracellular GSH levels, potentiates the activity of lymphokine-activated killer cells, and increases lymphocyte mitogen responsiveness. There is considerable evidence that survival and virulence of various parasites depend on endogenous antioxidant defense systems. For example, although the plasmodial antioxidant defense is not completely understood, it evidently depends on GSH and is related to mammalian hydroperoxide metabolism. This inadequately studied topic, including various parasite antioxidant systems, has been recently reviewed [49].

Nutrition and immunity

Zinc. Zinc (Zn) is an essential trace element, being a co-factor for about 200 human enzymes, including the cytoplasmic antioxidant Cu-ZnSOD. In addition, Zn (a) competes directly with copper and iron, thereby decreasing hydroxyl radical formation via Fenton and/ or Haber-Weiss chemistry; (b) protects protein sulfhydryl groups from oxidation; and (c) stimulates the immune system. Zn deficiency is associated with lymphoid atrophy, decreased thymic hormone activity, decreased dermal delayed-hypersensitivity response, and delayed homograft rejection [50]. Plasma Cu-ZnSOD activity correlates inversely with age [51], and plasma Zn levels are commonly

Free radicals and immune system 149

decreased in aging mice [52] and elderly humans [53]. Zn supplementation reportedly stimulates both B- and T-cell activity. For example, Duchateau and associates [54] supplemented an elderly group aged over 70 years with daily oral Z nSO ^ Compared with a non­ supplemented control group, those receiving Zn daily

showed significant improvement in the following areas:

(a) increased number of circulating T cells; (b)

improved delayed-hypersensitivity response; (c) increased antibody response to tetanus vaccine; and

(d) improved lymphocyte mitogen responsiveness.

Moreover, Fortes et al [55] reported that with Zn supplementation, a healthy group of elderly people

showed significantly increased levels of CD4+DR+

T cells and cytotoxic T cells.

Zn deficiency is also very common in children of developing countries. Here, it contributes significandy to diarrheal illnesses, which in turn contribute to growth retardation and early death [56]. In this regard, Sazawal et al [56] reported that in infants and young children with acute diarrhea, Zn supplementation

significantly reduced the severity and duration of diarrhea. Others [57] noted that Zn supplementation improved T cell-mediated responses, which are critical for host protection against parasitic infections.

Selenium. Se is an essential trace element, being a co­ factor for two enzymes, type 1 iodothyronine deiodinase and glutathione peroxidase (GPx). This

H 2O 2 in the

latter antioxidant enzyme inactivates

presence of GSH, thereby preventing the formation

of the hydroxyl radical (HO«) [58].

H 20 2

+2GSH

-----> 2H20

+ GSSG

Decreased plasma Se levels are reportedly common in older people, as is GPx activity [59]. Keshan disease, an endemic dilative cardio­ myopathy initially described in the Keshan province of China, can be prevented by Se supplementation. However, since the disease is seasonal, an infectious co-factor may be required. Beck [60] studied a mouse model of coxsackievirus B3 (CVB3)—induced myocarditis and showed that Se-deficient mice were more susceptible to the virus than Se-supplemented mice. Beck also found that a normal benign strain of CVB became virulent in Se-deficient mice, which

150 Annals o f Clinical & Laboratory Science

suggested that ROS may be an important factor. Another study [61] confirmed these findings and also demonstrated that vitamin E deficiency resulted in increased CVB3 virulence. Interestingly, Se and vitamin E have been shown to influence each other’s antioxidant requirements [62].

Meeker et al [63] demonstrated depressed natural killer cell activity and depressed T cell-mediated cyto­ toxicity by either Se or vitamin E deficiency, or their combination. Se deficiency in mice has been shown to produce a decreased lymphocyte response to conconavalin A (Con A) [64]. In this study, lympho­ cyte proliferation was directly related to Con A when

Se was adequately administered, whereas Se deficiency

resulted in decreased lymphocyte proliferation. These findings correlated with the concentration of

lymphocyte lipoperoxides. In Con A-stimulated cells with adequate Se, lymphocyte lipid peroxides were decreased and cell proliferation was increased

Antioxidant vitamins: vitamin E. Vitamin E is an efficient lipid soluble antioxidant that functions as a “chain breaker” in lipid peroxidation of cell membranes and various lipid particles (eg, LDL). Like other critical micronutrients, vitamin E is commonly deficient in the elderly. Thus, low vitamin E levels lead to unstable immune cell membranes, which lead to enhanced production ofimmunosuppressors (eg, prostaglandins); however, supplementation readily corrects this problem [65]. More specifically, vitamin E supplementation enhances cell-mediated immunity. For example, Meydani et al [66], in a double-blind, placebo- controlled study of healthy people > 60 years old, reported that vitamin E supplementation resulted in the following improvements compared to the placebo group: (a) increased plasma and mononuclear cell vitamin E levels; (b) increased positive antigen response

to delayed-type hypersensitivity skin tests; (c) increased

IL-2 production; (d) increased mitogenic response to Con A; (e) decreased synthesis of prostaglandin

(PGE2); and (f) decreased plasma lipoperoxide levels. In a vitamin E dose-response study [67], these researchers also showed increased T cell-mediated functions; the immune response was most effective at 200IU/day. Others [68] reported that optimal vitamin

E supplementation results in significantly improved

blood clearance of-E coli in cases of bacteremia.

Antioxidant vitamins: vitamin C. Vitamin C (ascorbic acid) is a water-soluble free radical scavenger [69]; it also regenerates vitamin E in cell membranes and maintains LDL particle integrity [70,71]. This vitamin is important in neutrophil functions, which perhaps explains its normally high concentrations in circulating leukocytes. In an in vitro study, Anderson and Lukey [72] showed that extracellularly released ROS are mutagenic, immunosuppressive, and autotoxic to phagocytes. Ascorbate efficiently neutralized phagocyte-derived extracellular oxidants; intracellular antimicrobial oxidants remained unchanged. They noted that ascorbate causes a dose-related inhibition of lucigenin-enhanced chemiluminescence of neutrophils activated by the leuko-attractants N-form yl-m ethionyl-leucyl-phenylalanine and cytochalasin B, as well as by a cell free xanthine/ xanthine oxidase superoxide generating system. An additional protective effect of vitamin C was shown to be at least partially mediated by its ability to reduce circulating levels of glucocorticoids [73]. Vitamin C was shown to alleviate the glucocorticoid suppressive effect on neutrophil function in cattle [74]. Gross and associates [75] found that ascorbate supplementation in chicks with E coli pericarditis significantly reduced mortality compared to non-supplemented controls.

Antioxidant vitamins: carotenoids. Beta-carotene, lycopene, lutein, and other structurally related carotenoids are important antioxidants. Lycopene and beta-carotene effectively neutralize singlet oxygen while the latter also quenches peroxyl radicals [76]. Numerous experimental animal studies have demonstrated that carotenoids modulate host defense systems. For example, Chew [77] observed that beta- carotene supplementation increased the (a) total number of circulating mononuclear cells; (b) number ofhelperT cells; (c) natural killer cell cytotoxicity; and (d) tumor necrosis factor alpha and IL-1. In humans, Alexander et al [78] reported that beta-carotene supplementation showed an increase in both the total lymphocytes and percent of T-helper cells (CD4+) without affecting the percentage ofT-cell subsets. More recently, others [79] studied the effects of beta-carotene on photosuppression of the immune response induced by ultraviolet light. The beta-carotene-supplemented

group showed no significant change in delayed-type hypersensitivity (DTH) response to ultraviolet light, while the placebo group experienced a significant decrease in DTH response.

Micronutrient combinations. Penn and co-workers [80] supplem ented a group of healthy elderly individuals with vitamins A, C, and E and compared

the results with an age-matched placebo group. The supplemented group showed the following improved responses: (a) increased total number of circulating

T cells; (b) increased number of helper cells (CD4+);

(c) increased helper to cytotoxic cell ratio (ie, CD4+

to CD8+); and (d) increased lymphocyte response to

phytohemagglutinins. Chandra [81] randomly assigned a group of healthy elderly people to receive placebo or a multivitamin/trace-element supplement. Their nutrient status and several immunologic variables were assessed at baseline and after 6 and 12 months. Compared to the placebo group, those receiving the supplement showed the following: (a) increased T-cell subsets; (b) increased number of natural killer cells; (c) increased killer cell activity; (d) increased IL- 2 levels; and (e) improved antibody response to an antigenic stimulus. In addition, the supplemented group had fewer infectious sick days during the year. In a similar study, Bogden and associates [82] compared delayed-type hypersensitivity skin test (DHST) responses in elderly subjects who received a

placebo to those who received a m icronutrient supplement. The micronutrient-supplemented group showed a significant increase in DHST responses to a

panel of seven recall antigens; there were no changes

in the placebo group.

Reviews regarding the effects of nutrition and antioxidants on the immune system in elderly people have been published [83,84].

Caloric restriction and immunity

Caloric restriction (CR) is the only known method whereby life span has been significantly prolonged. This applies not only to small laboratory animals, but also to non-human primates, and presumably to humans [85,86]. A major mechanism for this phenomenon is apparently related to a reduction in oxidative stress [87].

Free radicals and immune system 151

CR attenuates various age-related immunologic changes in rodents, such as thymic involution, decreased immune response capacities, and changes in lymphocyte subsets [88,89]. Kim and associates [90] reported that in a primate model, oxidative stress is probably important in the age-related increase in various cytokine levels. Here, adult-onset CR partially ameliorated this alteration such that in circulating mononuclear cells, CR reduced both IL-6 and mRNA levels induced by the xanthine/xanthine oxidase system which generates superoxide. Others [91] noted that, in addition to IL-6, tumor necrosis factor-alpha (TNF- alpha) is also increased in the sera of aged humans and mice. These researchers studied the effects of age and CR on the constitutive production ofTNF-alpha and IL-6, the concentrations of which were significantly higher in old versus young mice. Long-term CR in old mice resulted in cytokine serum levels that were comparable to those of young mice.

Oxidative stress and viral infections

Although the role of oxidants in viral infections is not fully understood, oxidants are, nevertheless, very important. In an early study, Belding et al [92] recognized that oxidants are involved in viral inactivation. Subsequent reports have shown that ROS and antioxidant micronutrients have significant roles in numerous viral infections, including measles, influenza, human immunodeficiency virus (HIV), and virus-related hepatitis [93-95]. Semba [96] recently discussed the occurrence of “severe measles” in vitamin A-deficient children and its association with pneumonia and high mortality rate. He stressed the im portance of vitam in A supplementation for hospitalized infants and children with this disease. In addition, Beck [61] reported that vitamin E deficiency increased the virulence of coxsackievirus B3 and reviewed the effect of antioxidant nutrients on viral infections [62]. N-acetylcysteine, an analogue and presursor for the antioxidant GSH, has been used for many years as a mucolytic drug. De-Flora et al [97] recently reported that administration of N-acetylcysteine during the winter significantly attenuated the symptomatology of influenza and influenza-like episodes, especially in older, high-risk patients.

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In vitro studies have shown that increased production of ROS results in enhanced H IV replication, possibly by activation of the transcription factor nuclear factor-kappa B (NF-KB), as well as tumor necrosis factor-alpha (TNF-alpha). Mycoplasmas are also able to enhance HIV replication, possibly by increasing oxidative stress, since they (as well as influenza and paramyxoviruses) directly activate macrophages to produce ROS. Some mycoplasmas also produce hydrogen peroxide [94]. As a result, HIV and mycoplasma coinfection may result in release of hydrogen peroxide from T cells [98]. Humans with HIV infection have repeatedly been shown to be under chronic oxidative stress. For example, increased byproducts of lipid peroxidation (ie, breath alkanes and serum malondialdehyde) have been reported in HIV patients [94,99]. In addition, the perturbations in a persons antioxidant defenses include significant changes in various antioxidants (eg, vitamins C and E, carotenoids, selenium, GSH) and superoxide dismutase [94,99]. In this regard, vitamin E supplementation may be useful in the treatment of patients with AIDS [100]. Further evidence of the role of oxidative stress comes from an established increased synthesis of interferon-gamma in HIV infections. This cytokine enhances macrophage production of ROS, as well as neopterin and its reduced form, 7,8-dihydroneopterin; both compounds play significant roles in free radical—mediated processes, which can lead to apoptosis [95].

Autoimmunity and oxidative stress

The im m une system is norm ally tolerant to components of itself (ie, it exhibits self-recognition). However, if this system becomes dysregulated, autoantibodies and autoantigen-specific T cells may develop. Weimann and Weiser [101] suggested that autoimmune disorders may be related to ROS, which modify various cellular structures, especially the mitochondria and nuclear and plasma membranes. To test this hypothesis, they studied the effects of various antioxidants (ie, vitamins C and E, beta-carotene, and Se) on a disorder that mimics systemic lupus erythematosus in mice. Compared with a placebo group, the antioxidant-supplemented mice showed: (a) increased mean survival; (b) absence of extensive

lymphoproliferation; (c) significantly lower IgG levels; and (d) significantly reduced elevation in anti-dsDNA antibody titer. In agreement with others [102], the authors concluded that diets deficient in antioxidants may induce lupus-like syndromes. Inflammatory joint diseases with neutrophil infiltration may be induced by bacteria, crystal deposition, or immune complexes. These infiltrating leukocytes become “activated” and produce ROS which can degrade joint hyaluronic acid and other proteoglycans [103]; ROS may also inactivate protease inhibitors while activating latent neutrophil collagenase, and thereby accelerate joint deterioration [104]. In rheumatoid arthritis (RA), neutrophil phagocytosis of immune complexes releases ROS, which may then react not only with the immune complexes but with a plasma component to produce a chemotactic agent that attracts additional neutrophils to the inflamed site [105]. Synovial fluid from normal subjects does not contain significant levels of antioxidant enzymes (ie, catalase, glutathione peroxidase, superoxide dismutase) [106]. However, the iron levels are increased in fluid from RA patients [107], which increases the possibility of hydroxyl radical production by Fenton and/or Haber-Weiss chemistry. In addition, Biemond et al [108] reported that superoxide stimulates the release of iron from ferritin; importantly, in RA patients, synovial fluid ferritin concentrations are three to eight times normal levels [109]. Furthermore, the frequent presence of erythrocytes in the synovial fluid of patients with RA (and other joint disorders) results in further ROS, since hydrogen peroxide stimulates iron release from hem oglobin [110]. Another mechanism of ROS production in RA is that xanthine oxidase, formed from xanthine dehydrogenase in ischemic and inflammatory sites, results in increased production of superoxide. This leads to further iron release from ferritin and the production of hydrogen peroxide and hydroxyl radicals [111]. As a final note, nitric oxide (NO*) may also play a role in autoimmune phenomena. Weinberg and associates [112] reported significantly increased levels of NO* in MRL-lpr/lpr mice which spontaneously developed arthritis, vasculitis, and immune complex glomerulonephritis. This research group, using electron paramagnetic resonance spectra (EPRS), also

reported significantly increased concentrations of nitrosyl hemoglobin in blood samples from these mice, compared to control mice [113]. Moreover, EPRS of the kidneys exhibited a signal characteristic of a dinitrosyl-iron-dithiolate complex. Importantly, nitrosylated non-heme protein is associated with glomerulonephritis in the autoimmune mice. Others [114] reported that NO* reacts with thiol-containing proteins to form S-nitrosoproteins, which are increased in the synovial fluid of patients with RA.

Prostaglandins/isoprostanes and immunity

Isoprostanes (IsoP) are prostaglandin (PG) F2—like compounds formed in vivo from the free radical peroxidation of polyunsaturated fatty acids, including arachidonic acid [115]. Unlike PGs, however, their formation does not require cyclooxygenase. Some isoprostanes (eg, 8-iso-PGF2 and 8-iso-PGE2) are vasoconstrictors and modulate platelet function [116]. As a result, the measurement of Iso-P has been proposed as a sensitive and specific method to measure oxidative stress in scleroderma and hepatorenal syndrome [117]. With respect to a role for prostaglandins in the immune system, macrophages from old mice produce more PGE2 than macrophages from young mice; PGE2 also suppresses T cell-mediated function. Beharka et al [118] confirm ed that increased macrophage PGE2 production contributes to the age- related decline in T-cell function. They also reported that the addition of PGE2 to cell cultures, at concentrations produced by macrophages from old mice, decreased IL-2 levels and lymphocyte proliferation by young T cells. However, vitamin E supplementation improved T-cell responsiveness in old mice, primarily by reducing macrophage-produced PGE2; this effect was in addition to a direct effect of vitamin E on T cells.

Nuclear factor-kappa B (NF-KB) and immunity

Chronic inflammatory disorders such as RA, asthma, psoriasis, and inflammatory bowel disease result in the production of several cytokines that recruit activated inflammatory and immune cells to the involved site and thereby may amplify and perpetuate the inflammatory process [119]. Although the causes of

Free radicals and immune system 153

these diseases remain unknown, our understanding of the molecular mechanisms has increased significantly in recent years. For example, gene-specific factors that regulate the transcription of target genes by binding to specific recognition elements have been identified [120]. Although some of these transcription factors are cell-specific, others, such as NF-KB, are ubiquitous. Importantly, NF-KB governs expression of genes that encode cytokines, chemokines, cell adhesion molecules, growth factors, and some acute phase proteins in health and in some diseases. NF-KB, a heterodimer, consists of a 50-kDa protein (p50) and a 65-kDa protein (p65). It is bound in the cytoplasm to an inhibitory protein (IkB) which inhibits its entry into the nucleus [121]. Numerous agents, including various cytokines (ie, IL-1 and tumor necrosis factor-alpha), inhaled particles, ultraviolet radiation, oxyradicals, and viruses, readily activate NF- KB (Table 2) [122,123]. For example, Wang and associates [124] recently reported that superoxide radicals are the major species responsible for TNF- alpha—induced NF-KB activation.

Table 2. Agents that activate NF-KB [122,123]

Oxidants Hydrogen peroxide Hydroxyl radical Ozone Cytokines Interleukin-1 (IL-1) Tumor necrosis factor-alpha (TNF-alpha) Viruses Rhinovirus Influenzaevirus Epstein-Barr virus Others Miscellaneous Protein kinase C Lipopolysaccharides Ultraviolet light Irradiation Phorbol myristate acetate Lymphocyte mitogens

154 Annals o f Clinical & Laboratory Science

Beg et al [125] provided in vivo evidence that NF- KB activation occurs through phosphorylation of I- KB, which is then proteolytically degraded or processed by proteasomes and other proteases. This proteolytic process releases NF-KB, which is then free to enter the nucleus where it can initiate and/or regulate gene transcription. These authors reported that the loss of I-KB and subsequent activation of NF-KB can both be inhibited in the presence of antioxidants. Importantly, antioxidant prevention of NF-KB activation may be beneficial in suppressing toxic/septic shock, acute inflammatory responses, grafit-vs-host reactions, and radiation damage [126]. The role of NF-KB in chronic inflammatory disorders has been recently reviewed [122,127,128].

Summary

A major role of the immune system is to protect the host from infectious agents by phagocytosis and destruction by phagocyte-mediated ROS. In addition, the immune system has various regulatory functions that are ultimately dependent upon an oxidant/ antioxidant balance. Thus, increased ROS is associated with various decreased immune responses, mainly T-cell—mediated functions. Although the exact mechanisms of these latter phenomena are not fully understood, oxidative stress activates NF-KB, which leads to increased production of various cytokines which may be harmful. However, antioxidant supplementation stimulates IL-2 production, T-cell proliferation, natural killer cell activity, mitogen responsiveness, and antibody response following

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