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 im m unity 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 M eeting o f the Association o f
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
im portant 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
p art o f their norm al 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].

0091-7370/00/0200-0145 $3.50; © 2000 by the Association of Clinical Scientists, Inc.

146 Annals o f Clinical & Laboratory Science
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
w ith (a) decreased total lymphocyte count; (b)
decreased T-cell subsets [CD3, CD4, CD 8]; 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
m alignant B-cell tumors (ie, m ultiple 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 nu tritio n , 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-Fl20 2 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 im portant as the M PO-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 o f the
biological oxygen-derived free radicals, is produced by
(a) Fenton and/or (b) Haber-Weiss chemistry [24],
(a) H 20 2 + Fe2+ -------s- HO*+ O H - + 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.
2H 20 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
m any different cells following challenge w ith
immunologic or inflammatory stimuli. These cells
include m acrophages, neutrophils, m ast cells,
endothelial, and vascular smooth muscle cells [33].
Im portantly, iN O S is capable o f continuously
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_ * ------> O N O O -
Among other toxic reactions, peroxynitrite reacts with
aromatic amino acid residues to form nitrotyrosine,
which can lead to enzyme inactivation [34,35]. In
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
tu m oricidal activities o f m acrophages [37].
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].
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 m em brane
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 m em brane
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 m ajor antioxidant enzymes are superoxide
dism utase (SO D ), catalase (CAT), glutathione
peroxidase (GPx), and glutathione reductase (GR).
SOD, of which there are two enzymes (mitochondrial
M nSOD and cytoplasmic Cu-ZnSOD), inactivates
O 2 *' by converting it to H 2O 2. 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 H 2O 2 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 com prom ised 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
G lutathione (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 of human 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 n S O ^ Compared with a non­
supplemented control group, those receiving Zn daily
showed significant improvement in the following areas:
(a) increased num ber o f 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 o f 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
latter antioxidant enzyme inactivates H 2O 2 in the
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
m odel o f coxsackievirus B3 (CV B 3)—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 im portant 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 w ith the con cen tratio n 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 vitam in 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 th a t extracellularly released ROS are
mutagenic, immunosuppressive, and autotoxic to
phagocytes. A scorbate efficiently neutralized
phagocyte-derived extracellular oxidants; intracellular
antimicrobial oxidants remained unchanged. They
noted that ascorbate causes a dose-related inhibition
of lucigenin-enhanced chem ilum inescence o f
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 supplem entation 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 D TH 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
phytohem agglutinins. C handra [81] random ly
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 im m unity
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-hum an primates, and presumably to
hum ans [85,86]. A m ajor 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 thym ic 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 of TNF-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 th at 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 o f vitam in A
supplementation for hospitalized infants and children
with this disease. In addition, Beck [61] reported that
vitam in 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.
152 Annals o f Clinical & Laboratory Science
In vitro studies have shown that increased
p ro d u ctio n o f 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 H IV
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
T he im m une system is norm ally to le ran t 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 m im ics 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.
Inflamm atory 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 jo in t hyaluronic acid and o ther
proteoglycans [103]; ROS may also inactivate protease
inhibitors while activating laten t n eutrophil
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, glu tath io n e 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]. A nother
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 o f 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].
W ith 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-m ediated function.
Beharka et al [118] confirm ed th at 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
m ice, decreased IL-2 levels and lym phocyte
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 am plify and p erpetuate 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 in h ib ite d in the presence o f antioxidants.
Im portantly, antioxidant prevention o f 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 harm ful. However, antioxidant
supplementation stimulates IL-2 production, T-cell
proliferation, natural killer cell activity, mitogen
responsiveness, and antibody response following
antigen stimulation. W hen this process is more
completely understood, we may be able to control the
immune system, prevent disease, and improve health.
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