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Oxidative Stress in Phagocytic Cells: Changes with Age and Effect of Melatonin

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Phagocytosis is an important element of the nonspecific immune response because it is a fundamental mechanism of defense against infectious agents. Phagocytic cells first ingest the target (antigen) and then destroy it by the action of enzymes by means of a series of redox reactions known as the respiratory burst. In this process, several aggressive chemical species are formed, including superoxide anion, hydrogen peroxide, the hydroxyl radical, and hypochlorite to destroy the invasive microorganisms. Once their work is done, the free radicals are scavenged or eliminated by the antioxidant mechanisms that the phagocytes have available, thereby ensuring the integrity of these cells. The effect of melatonin on phagocytosis has been studied in depth. Melatonin at pharmacological concentrations increases both the chemoattractive capacity (chemotaxis) of these cells and their capacity to phagocytose antigen particles and reduces the intensity of the respiratory burst. The changes in plasma melatonin levels were found to be positively correlated with phagocytosis and negatively with the oxidative metabolism. Studies showed daily oscillations of the levels of this hormone in young animals and a decline in plasma levels with advancing age. The decline is accompanied by a loss of the daily rhythm, with no significant differences being found between nocturnal and diurnal values in old animals. Given that melatonin acts by sending information to the organism on its temporal organization, this hormone could be an important pharmacological agent for the attenuation of age-related changes in the immune system, circadian organization, sleep, and other disorders accompanying old age.
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Chapter 31
Oxidative Stress in Phagocytic Cells: Changes
with Age and Effect of Melatonin
C. Barriga, M.P. Terrón, S.H. Parvez, J. Cubero, D. Narciso,
S.D. Paredes, S. Sánchez, and A.B. Rodríguez
INTRODUCTION
The immune system
The immune system contributes to the maintenance of the integrity of the individual by
eliminating foreign substances or infectious agents to which the individual is exposed. This
function sets in motion two categories of processes: the specific (acquired) and nonspecific
(innate) immune responses. Both act via cells (cellular immunity) and via the molecules
that the cells release (humoral immunity). The nonspecific immune response is defined
by its spontaneous character, i.e. absence of memory, plasticity, and adaptation to the
pathogenic agents it faces. On the contrary, the specific immune response is induced by the
organism’s first contact with the antigen, and possesses memory. One must nevertheless not
forget that the immune system responds as a unit, so that this division is more theoretical
than real. In a very general and abridged form, the following are some brief commentaries
about the immune system.
In the nonspecific immune response, the most important humoral immunity system
is the complement, a system that, when activated, provokes the destruction of bacteria
by perforation of their membranes. The phagocytic cells (monocytes, macrophages, neu-
trophils, eosinophils, and basophils) and the natural cytotoxic (NK, natural killer) cells are
responsible for putting this nonspecific response into action. To carry out the phagocytosis,
the cells of this system migrate toward the focus of the infection, attach to the bacteria,
and then phagocytose them. The NK cells produce cytotoxic substances that provoke the
destruction of tumor cells.
The B-lymphocytes synthesize the immunoglobulins IgM, IgA, IgG, IgE, and IgD, also
known as antibodies. These cells are activated by antigens – substances that are for-
eign to the organism, and present in infectious agents. These substances carry out the
specific humoral response. The specific cellular response is mediated by the activation,
proliferation, and differentiation of the T lymphocytes, of which there exist various sub-
populations: helper T lymphocytes that enhance the response of other immune cells;
Oxidative Stress and Neurodegenerative Disorders 737 © 2007 Elsevier B.V.
Edited by G. Ali Qureshi and S. Hassan Parvez All rights reserved.
738 C. Barriga et al.
cytotoxic T lymphocytes that destroy both infected and tumor cells, and suppressor
T lymphocytes with a suppressory role in the regulation of immune response.
Part of the immune response is carried out by the cytokines. These hormones are
proteins formed and secreted by both the immune cells and other types of cells, above all
nerve cells. They participate in the signaling between different cells during the immune
response, being capable of acting on many different cell types. In the case of neurons,
they inform the brain of the appearance and growth of inflammation in peripheral tissues,
so that the central nervous system is able to coordinate the organism’s response to the
aggression.
Phagocytic cells
The phagocytic process as shown in (Fig. 1), is triggered when an aggressor agent over-
comes the organism’s natural barriers, such as the integument, the mucosa, and the
secretions. It is the process of recognizing and engulfing microorganisms or the waste
tissue that accumulates during infection, inflammation, or the repair of an injury (1). In
order to be able to carry out the function of phagocytosing the invader agent, the phagocyte
needs to leave the capillary by means of its capacity of adhering to the vascular endothe-
lium, freeing itself from the blood vessel by a process called diapedesis. The immune cells
that have phagocytic capacity are: monocytes, macrophages, neutrophils, eosinophils, and
basophils.
Once outside the capillary, the phagocyte adheres to tissue substrates and migrates
by means of spontaneous movements or attracted by chemotactic substances toward the
site of the infection or damage. This process is called chemotaxis. The phagocytic cell
moves on pseudopods, which are transitory prolongations of the cytoplasm that permit
the guided movement of the cell. When it reaches the site of infection, it is able to
bind the antigen cells or particles to its membrane, and subsequently ingest and destroy
Blood vessel
Adherence
Chemotaxis
Antigen
Antibody
Antigen binding
Opsonization Ingestion
Phagosome
Degranulation
Phagolysosome
Digestion
Fig. 1. The phagocytic process.
Oxidative Stress in Phagocytic Cells 739
them intracellularly. This ingestion is essential for the defense of the host. It occurs
when an invader microorganism is recognized by specific receptors on the surface of
the phagocyte, and requires multiple, successive interactions between the phagocyte and
the invader. Each of these interactions results in a transduction signal which is confined
to the membrane and cytoskeleton bound to the receptor, and which is required for the
phagocytosis to be successful (1–3).
Once the phagocytic cell, thanks to its membrane receptors, has established contact with
the membrane of the germ that is to be phagocytosed, the phagocyte emits cytoplasmic
prolongations in the form of pseudopods in the zone of union with the germ. These
pseudopods surround the germ. The phagosome or digestive vacuole thus formed then
separates off from the cell membrane, and moves centripetally towards the interior of the
cell, carrying out the ingestion.
As soon as the phagocytic vacuole is formed, movements within the cytoplasm are
activated, and, as a consequence, azurophil and specific granules approach the common
membrane and pour their enzyme content into the interior of the phagosome. This con-
stitutes the phagolysosome, where the processes begin that are to destroy and digest the
phagocytosed germ or particle. This process is called degranulation. The movement of
the granules toward the phagosome also involves the interaction of microtubules and
microfilaments (4).
Following this internal degranulation, a series of chemical events or processes occur
that provoke the death and digestion of the ingested material.
Destruction of the ingested material
After the microorganism has been ingested, a series of events is set off in the phagocyte
that leads to the microorganism’s death and digestion. The chemical processes constituting
the microbicidal mechanism and involving the destruction of the ingested material can
be categorized into two broad groups: the oxygen-independent and the oxygen-dependent
systems. The former is a set of compounds that act in the absence of oxygen, while the
latter comprises a set of redox (oxidation–reduction) reactions that lead to the so-called
“respiratory burst” that accompanies phagocytosis, and in which there occurs a great
consumption of oxygen.
Oxygen-dependent microbicidal process
The microbicidal processes consist of myeloperoxidases and cofactors (halides, thio-
cyanate, thyroxine, and triiodothyroxine), hydrogen peroxide, superoxide anions,
hydroxyl radicals, and oxygen singlets. These systems, via redox reactions, are responsi-
ble for the destruction and digestion of the phagocytosed particles. After activation, the
phagocytic cells increase oxygen consumption in a process known as the “respiratory
burst.” This takes place within those cells after their exposure to a certain stimulus that
occurs by means of the activation of an enzyme, NADPH (nicotinamide adenine dinu-
cleotide phosphate) oxidase. In some species, NADPH oxidase is associated with the
membrane (5), which effectively constitutes an electron transport chain in which NADPH
740 C. Barriga et al.
dehydrogenase, flavin adenine dinucleotide (FAD), ubiquinone, and cytochrome-b par-
ticipate. These form part of tertiary granules that bind to the cell membrane during the
formation of the endosome, and are necessary for the activation of the oxidase (6).
Formation of Superoxide Anion. By far most of the oxygen consumed during phagocy-
tosis is transformed into superoxide anion, an intermediary in the formation of hydrogen
peroxide, with the intervention of an oxidase according to the reaction:
2O2+NAD(P)H−→ 2O
2+NAD(P)++H+
The O2thus receives an additional electron. When it reaches certain concentrations,
this superoxide anion has great germicidal power (7). Since superoxide is highly toxic,
the organism quickly eliminates it by the action of superoxide dismutase (SOD) (8).
Formation of Hydrogen Peroxide. The reaction by which hydrogen peroxide, also a
powerful germicide, is formed is enabled by the presence of the SOD:
2O
2+2H+SOD
H2O2+O2
Since this hydrogen peroxide perfuses into the cytoplasm, where it can be toxic for
the phagocytic cells, it is degraded by the glutathione-peroxidase/glutathione-reductase
system. The hydrogen peroxide is thus reduced to H2O, avoiding any harmful damage to
the cytoplasm of these cells.
Activation of Halogens. The germicidal activity of hydrogen peroxide can be consider-
ably enhanced in the presence of halogens and a peroxidase. The different intracellular
halogen ions, Br,Cl
, and I, especially the last two, are activated by the presence of
hydrogen peroxide and myeloperoxidase (MPO).
H2O2+halogen +H+Myeloperoxidase
−−−−−−−−halogen +H++H2O
It seems that the principal product formed by the myeloperoxidase system is the
hypochlorite ion, which is characterized by its great toxicity.
H2O2+Cl Myeloperoxidase
−−−−−−−−HOCl +OH
Decarboxylation of Amino Acids. The basic reaction is:
R-CHNH2-COOH R-CHO +CO2+NH3
This reaction is controlled partly by myeloperoxidase. Many of the amino acids of the
bacterial membrane are degraded, causing the death of the germ.
Oxidative Stress in Phagocytic Cells 741
Formation of Hydroxyl Radicals. The hydroxyl radicals are very unstable, and react
rapidly with any organic matter, thus playing an important germicidal role (5). The reaction
in this case is:
O
2+H2O2OH+OH+O2
Formation of Oxygen Singlets. As a consequence of the consumption of oxygen dur-
ing phagocytosis, besides the production of hydroxyl radicals, oxygen singlets (1O2)
are also formed. These are electronically excited states of oxygen that emit light upon
being formed and are very unstable, as they are constantly trying to revert to the triplet
or normal form of atmospheric oxygen. This electronic alteration of singlet oxygen
molecules gives them great chemical reactivity, especially on compounds that have a
double bond. In consequence, they have the capacity to interfere with and alter many
biological systems (9).
Melatonin
Melatonin (Fig. 2) is an extremely old molecule with a degree of conservation and ubiquity
that is highly conspicuous in a biological molecule. Such conservation in a biomolecule,
without apparent metabolic or structural function, is difficult to match in nature.
The presence of melatonin, identical in structure in all vertebrates (10), has been
described in unicellular organisms, fungi, plants, and invertebrates (11). In plants, the
biological role of melatonin is yet to be clarified, but its role in animals has always been
related to the transduction of seasonal and circadian photoperiod information (11).
According to its chemical properties, the melatonin present in an organism should be
destroyed by light but would stay relatively stable in darkness. Its effective concentration
would have undergone, in evolutionary terms, oscillations coinciding with the light–dark
cycle. This alternation could be seen as the primary reason for melatonin, somewhat later
on, to have been adopted as a mediator of information on darkness. Also, melatonin
has a powerful scavenging effect on reactive oxygen species, which also cycle with rest
and activity. This is an important function that could have been used by the ancestors of
modern-day organisms whose cells – especially their retinal and pineal photoreceptors –
would have had to be protected from oxygen free radicals. The role of melatonin, therefore,
N
H
H
H
HHH
CC CCH3
N
O
Fig. 2. Chemical structure of melatonin.
742 C. Barriga et al.
may have evolved polyphyletically on the basis of the molecule’s exceptional properties
and availability. In a last evolutionary step, melatonin synthesis could have been coupled
to a circadian oscillator, a connection that would have had the advantage of the melatonin
rhythm being a predictor of oscillations in levels of both light and free oxygen radicals (11).
For the animal, the oscillating levels of melatonin would serve to determine with great
precision both the time of day (the melatonin clock) and the season of the year (the
melatonin calendar), allowing it to make the physiological and behavioral changes needed
to anticipate the dramatic fluctuations in the environment (12).
As mammals have reached a high degree of independence from environmental factors,
the importance of melatonin and circadian clocks has been somewhat reduced in this
group. However, it still retains some important functions: (1) as a clock, a marker of
the dark; (2) as a calendar in some species, regulating seasonal reproductive function;
and (3) as a mild hypnotic in diurnal animals. However, considering the interest of this
report, focused on the activity of melatonin upon sleep in human beings, only the clock and
hypnotic properties of melatonin will be reviewed. Indeed, the chronobiotic (time keeping)
properties of melatonin are a fundamental part of process C, the circadian regulatory
process of sleep, while the hypnotic ones form part of the S process, that is, homeostatic
regulation.
Functional connection between the pineal organ and the immune system
Interpretation of the melatonin message within the body is essential for the physiological
functions of an animal to adapt to environmental conditions and needs, an adaptation
that would increase the probability of survival. Immune system activity is one of the
physiological capabilities most responsible for the survival of an individual animal, and
the reproductive system function guarantees the survival of the species.
There is no longer any doubt about the interaction between the immune, nervous,
and endocrine systems (13–15) (Fig. 3). The efficacy of the immune system function in
defending against harmful microorganisms, foreign molecules, or tumor cells requires it
to be coordinated into periods of about 24 h. As do other body functions, the activity
of the immune system undergoes circadian changes, and reciprocal synchrony is of great
importance to homeostasis. Illnesses, however, can alter these rhythms and modify their
temporal coordination (16).
Corticosteroids were the first humoral factors recognized as regulators of the daily
rhythm of the immune system (13). There exists clear evidence, however, that certain
parameters and some immune cells fluctuate differentially over a 24-h period and exhibit
different phase relationships with circulating corticosteroid levels (16). The implication is,
hence, that there are one or more other factors involved in regulating the circadian rhythm
of the immune function, and one of the main candidates would seem to be the pineal gland
and its secretion of melatonin. There are three reasons for this statement: (1) the circadian
and seasonal periodicity of the pineal gland function; (2) the strong dependence of the
circadian (17) rhythm of melatonin synthesis on light conditions; and (3) the participation
of melatonin in the control of different biological rhythms, including those associated
Oxidative Stress in Phagocytic Cells 743
PSYCHIC
Emotion
Stress
Abstract thought
AMBIENTALES
Light
Temperature
Magnetism
Infections
Tumors
Autoantigens
CONDITIONING REPRODUCTION
HOMEOSTATION
IMMUNE
RESPONSE
CHALLENGES
MLT
M
MLT
CORTEX
C S N
Hypothalamus
Hypophysis
ENDOCRINE
SYSTEM
Hormones
thyroid
adrean
PINEAL
COMPLEX
opioids
opioids B
IMMUNE
SYSTEM
T
opioids
opioids
opioids
OPS
opioids
Fig. 3. The pineal gland and the circadian secretion of melatonin within the signal of
translation between environmental influences and the immune, nervous, and endocrine
systems. MLT =melatonin and OPS =opioids.
with aging and with affective and psychosomatic diseases, which, in turn, are related to
an increased incidence of infections, autoimmune disorders, and cancer (13).
There have been few studies published on the functional connection between melatonin
and the immune system of poikilotherms, birds, and mammals. A chronohematological
study was performed on the blood of pinealectomized and sham-operated lizards over
a 48-h time period. The removal of the pineal significantly inhibited leukopoiesis and
erythropoiesis (as reflected in the reduced number of cells in the circulation) and led
744 C. Barriga et al.
to hypoglycemia. In the sham-operated lizards, however, there was an evident circadian
rhythm in the white and red blood cell and glucose levels (17). In mammals and birds,
developmental and age-related changes in pineal function appear to be at least partially
related to immune system efficiency. The mechanisms by which melatonin influences
immune system function are complex but are known to involve the participation of such
mediators as endogenous opioids, cytokines, hormones, etc. As melatonin is a highly
lipophilic compound, it may easily penetrate immune cells without the mediation of
any specific receptors and act within the cells as a potent free-radical scavenger and
as an anti-aging and oncostatic factor. The immune system may in turn, via the syn-
thesis and secretion of soluble factors, i.e. cytokines, influence pineal gland function,
thereby closing the information loop to maintain homeostasis in order to face the harmful
environment (13).
PINEALECTOMY, BURSECTOMY, AND THE IMMUNE SYSTEM
Immunomodulatory action of melatonin
General observations suggest that the effects exerted by melatonin on aspects of the func-
tion of the immune system not only depend on the species, age, and sex, but also on the
experimental protocol (including the season), melatonin dose, and the route of admin-
istration. One of the most interesting relationships between melatonin and the immune
system is represented by the season-dependent changes in immunity observed in wild-
living animals both in nature and under laboratory conditions, where the animals can be
kept under different lighting regimes (18).
It is surprising that there have been no studies of the influence of melatonin on the
immune system of fish, given the great applied interest in knowledge of the possi-
ble immunostimulatory role of melatonin for fish farming. Abundant indirect evidence,
however, supports such a role. Environmental factors, particularly temperature and
photoperiod, are known to be immunomodulatory in the lower vertebrates such as
fish (19). With respect to nonspecific immune mechanisms (e.g. phagocytosis), in the
tench Tinca tinca, a warm-water cyprinid, the different stages of the phagocytic process
of blood granulocytes show the highest level of activity during winter. It remains high in
the spring and declines in the summer, when the lowest level of activity is found (20).
The complement system is one of the main routes by which the inflammatory process is
effected. There are two different but convergent pathways of complement activation: the
immunoglobulin-dependent or classical pathway, and the immunoglobulin-independent or
alternative pathway. In Tinca tinca, it was found that the alternative complement path-
way activity was greater in winter in both males and females than in the other three
seasons (21). When the seasonal variations in specific immunity in this fish were ana-
lyzed through the mitogen-induced proliferative response of lymphocytes, it was found
that the lowest levels of this response to the different mitogens (phytohemagglutinin,
concavalin A, Escherichia coli lipopolysaccharide, and pokeweed mitogen) occurred dur-
ing winter and the highest during summer (22). Hence, one can presume that the high
Oxidative Stress in Phagocytic Cells 745
winter melatonin concentrations activate the nonspecific immune response, doubtless to
counteract the stress-mediated winter suppression of the specific immune response, i.e.
as a compensatory mechanism for the activity of the specific immune system, which
appears to operate principally in summer. In sum, while there has been no study directly
approaching the influence of melatonin on the immune response in fish, it is probable that
the seasonal changes in melatonin levels affect that response.
In birds, melatonin has been shown to modulate several immune functions, namely,
antibody production, lymphocyte proliferation, antibody-dependent cell-mediated cyto-
toxicity (ADCC) activity, natural killer (NK) cell cytotoxicity, cytokine synthesis and
release, etc. (13). It is known that melatonin enhances mitogen-induced T-cell blood lym-
phocyte and T-cell and B-cell splenocyte proliferation in male broiler chicken (23). It has
also been suggested that melatonin inhibits phytohemagglutinin (PHA)-stimulated chicken
lymphocyte proliferation in vitro (24).
In chicken, the circadian rhythm of different immune parameters was found to be
strongly dependent on the presence of an intact pineal gland (25). In 7-week-old chicken
immunized three times at 9-day intervals with T-dependent porcine antigen (26), the
diurnal serum melatonin concentration increased after the second antigen challenge. Also,
studies (27) have shown that exogenous melatonin can reconstitute a deficient cellular and
humoral immune response in pinealectomized Japanese quail.
Melatonin added to avian lymphocyte cultures over a wide range of concentrations
did not influence cell proliferation, as measured by 3H-thymidine incorporation. How-
ever, when the culture was stimulated with mitogens, the addition of melatonin generally
diminished cell proliferation. Also, when melatonin was added to splenocytes pretreated
with T-cell mitogens, blast formation was almost completely blocked. This effect was best
seen in cells isolated from the youngest (5 days old) group of studied chicken (28).
Finally, and as will be commented on in the following section, the effect has also been
evaluated in vitro of melatonin on cells of the nonspecific immune response. Thus, in
human neutrophils, low doses of melatonin (in the range of 10 nM) result in an increase
of the respiratory burst in response to PMA (phorbol 12-myristate 13-acetate) (29,30),
while 2 mM of melatonin inhibits the respiratory burst. Apparently, melatonin modulates
this function in a dose-dependent manner. Figure 4 shows a summary of melatonin’s
effects on the immune system.
Melatonin and reactive oxygen species: antioxidant role
The N-acetyl-5-methoxytryptamine molecule, commonly known as melatonin, is a prod-
uct that all vertebrates synthesize in the pineal gland as well as in other organs. This
indolamine was initially known for its function in mediating circannual reproductive
rhythms (31) as well as circadian rhythms (32). It was then shown that it had onco-
static (33), immunostimulatory (29), and anti-inflammatory (34) effects. More recently,
melatonin has been identified as a potent free radical scavenger (35) and indirect antiox-
idant (36,37). Particularly noteworthy is its great efficacy in protecting against reactive
oxygen species (ROS) and reactive nitrogen species (RNS). This field of research has seen
explosive growth in the last 20 years or so, and, although all the mechanisms of the effects
746 C. Barriga et al.
Phagocytic
activity
MEL
N
BL
CD19+
Th1
CD3+
CD4+
ADCC
M
IO CD16+
M
CD14+
CD4+
NO
ROI
TF
TNF
IL-1
IL-2 IL-6
IF
N
+
+
+
+
+
+
+
+
+
+
GM-
CFU
+
++
+
+
+
Respiratory
burst
Circulation of
lymphocytes
and monocytes
Circulation of
neutrophyls
NK activity
Ephithelial
cell
Thymus
Thymosin a1
Thymulin
Thymocyte
Thymosin a1
Bone marrow
Lipoxygenase
Fig. 4. Hypothetical outline of the regulation of the immune system by melatonin. Mela-
tonin modulates the activity of the monocytes (CD14+/CD4+cells) and T cells, resulting
in increases in the NK-cell and ADCC (antibody-dependent cytotoxic cell) activities and
the production of GM-CFU (granulocyte-macrophage colony forming units). Indepen-
dently, it represses the expression of 5-lipoxygenase by the B cells, and regulates the
activity of the neutrophils. Melatonin also acts on the thymus to increase both thymosin
alpha-1 and thymulin through thymus epithelial cells and thymocytes (29).
of melatonin as a scavenger of free radicals and affine products are yet to be identified,
there is no doubt concerning the antioxidant role of this hormone.
The antioxidant functions of melatonin include both direct and indirect actions: (1) direct
scavenging of free radicals; (2) stimulation of antioxidant enzymes; (3) enhancing the
efficiency of mitochondrial oxidative phosphorylation and reduction of the electrons;
and (4) enhancing the effectiveness of other antioxidants. Melatonin could also have
other as yet undiscovered functions that would increase its capacity to protect against
Oxidative Stress in Phagocytic Cells 747
molecular damage by ROS and RNS. Several in vitro and in vivo studies have docu-
mented melatonin’s capacity at both physiological and pharmacological concentrations
to protect against free radical destruction (38) and to moderate the molecular damage
produced by toxic oxygen and RNS (39,40).
It has been seen that there are various pathways through which melatonin operates in
reducing oxidative stress. There is experimental evidence for its action as a direct free
radical scavenger (41–43), as an indirect antioxidant in stimulating antioxidant enzymes
(37,44), in stimulating the synthesis of glutathione (an essential intracellular antioxidant)
(45), in enhancing the activity of other antioxidants (or vice versa) (46), in protect-
ing antioxidant enzymes from oxidative damage (47,48), and in increasing efficacy of
mitochondrial electron transport chain (40,49).
Melatonin as an antioxidant in phagocytic cells
Phagocytosis is an important element of the nonspecific immune response and represents
a fundamental mechanism of defense against infection. Phagocytic cells engulf their tar-
get (antigen) and then destroy it by the action of enzymes that form oxygen-derived free
radicals by means of a series of redox reactions, which lead to what is known as the
“respiratory burst.” In this process, various chemically aggressive species are formed,
such as superoxide anions, hydrogen peroxide, hydroxyl radicals, and hypochlorite. Their
function is to destroy the invading microorganism. The presence of free radicals in phago-
cytes is beneficial for the organism, since it is thanks to their formation within those cells
that pathogenic microorganisms are destroyed. It is clearly an effective adaptation and
solid defense adopted by the organism in its natural habitat. What would really be an
advantage is, if the radicals, once they have fulfilled their goal, were then sequestered
and/or eliminated from the phagocytes, as this would have the effect of guaranteeing the
integrity of those cells.
Rodríguez (50) observed a decrease in superoxide anion levels (O
2)in heterophils
of Streptopelia risoria after the phagocytosis of inert particles when the phagocytes had
been incubated with pharmacological doses of melatonin. Also, in the same species,
Rodríguez (51) found that incubation with pharmacological doses of melatonin led to the
disappearance of the antigen-produced rise in the activity of SOD, a metalloenzyme that
catalyzes the dismutation of superoxide anion into oxygen and hydrogen peroxide. These
workers also observed that the same melatonin dose induced an increase in the concen-
tration of myeloperoxidase stored in heterophils, this being the major component of the
bactericidal armory of phagocytes (52) and a decrease in the production of malonaldehyde
(MDA), an indicator of induced oxidative damage to lipid membranes (53). All these data
confirm the existence of a negative correlation between serum melatonin levels over a
24-h period and the superoxide anion levels in heterophils, with minimum and maximum
levels coinciding with the diurnal oscillations of melatonin (54). In addition, Terrón (55)
observed that melatonin acts as an antioxidant in phagocytic cells even at physiological
doses, favoring phagocytic activity at the same time as neutralizing free radical levels
after the digestion of the antigen.
The effect of melatonin on phagocytosis has also been studied in the ringdove
(Streptopelia risoria) using isolated heterophils (56). Melatonin, at pharmacological
748 C. Barriga et al.
concentrations, enhanced both the chemoattractant capacity of these cells and their capac-
ity to phagocytose antigen particles, and reduced the intensity of the respiratory burst (50).
It also modulated the superoxide dismutase activity in the same species (51) and increased
the concentration of myeloperoxidase, an enzyme used as an indicator of the bactericidal
capacity of heterophils (52). These results were confirmed when the circadian changes in
plasma melatonin were found to be positively correlated with the phagocytic capacity of
the heterophils and negatively with their oxidative metabolism (54).
In this same line, Terrón (57) studied the in vitro effect of the physiological mela-
tonin concentrations found in young and mature ringdoves (300 pg/ml as the maximum
nocturnal concentration and 50 pg/ml as the minimum diurnal concentration) on the het-
erophils obtained from old animals, evaluating the capacity for ingestion and destruction
of Candida albicans and the oxidative metabolism associated with phagocytosis by deter-
mining the superoxide anion levels. Melatonin induced a dose-dependent increase in both
phagocytosis and candidicide index. Also, a decline in superoxide anion levels was found
after incubation with both concentrations. These results thus confirm the physiological
effects of melatonin on phagocytic function.
In sum, melatonin is a significant endogenous antioxidant for bird heterophils as, even at
physiological concentrations, it is an effective free-radical scavenger, yielding protection
from the oxidative stress that accompanies phagocytosis.
Changes in melatonin secretion with age: possible causes
The concentration of melatonin in the blood, which in mammals is primarily a consequence
of secretion by the pineal gland, shows a clear circadian rhythm with low values during the
day and a 10- to 15-fold increase during the night (58,59). In humans, this rhythm develops
at around the sixth month of life, and the greatest levels are reached at between 4 and
7 years of age. At around maturity, there might be a fall in melatonin concentrations, and its
levels diminish gradually from then on (60). In many individuals above 65 years, the day–
night rhythm is practically absent (Fig. 5) (59,60). The amplitude of nocturnal melatonin
secretion is believed to be determined genetically and shows large differences between
individuals (61). Hence, some individuals produce significantly less melatonin during their
lifetime than do others, which could have significance with respect to aging (62).
It is also well established that, in humans and other species, melatonin secretion follows
a circadian pattern, with low levels during the day and high levels at night. Nonetheless,
the amounts that are secreted, and consequently the amplitude of these circadian rhythms,
can vary considerably in adults. Approximately 1–5% of the human population has very
low levels of melatonin, with no evidence for a circadian pattern of release (63,64). The
reason why some adults do not produce melatonin is not clear. Hypersecretion of melatonin
in normal adults seems to be uncommon. It was seen by Meyer et al. (65), although
further confirmation is required. In spite of these individual differences, the amplitude
of melatonin’s rhythm is highly consistent from one day to another (63,64). Although
there are no data available on the intrauterine production of melatonin in the pineal gland
of the fetus (66,67), there is evidence that the free transport of melatonin between the
Oxidative Stress in Phagocytic Cells 749
180
160
140
120
100
80
60
40
20
08 10 12 14 16 18 20 22 24 02 04
[
hours
]
2040 years
5060 years
MELATONIN (pg/ml)
Over 70 years
510 years
06 08
0
Fig. 5. Diurnal profiles of the serum concentrations of melatonin at various ages (62).
maternal and fetal compartments probably exposes the fetus to similar circadian variations
of melatonin as its mother (68).
Several groups have studied the nocturnal and diurnal serum melatonin levels in chil-
dren (69,70). From these data, it seems that the diurnal level is low and does not change
appreciably in the first year of life. The nocturnal levels are similar to the diurnal levels –
i.e. they are low or undetectable – during the first 2 or 3 months. They increase gradually
during the following months. This indicates that melatonin’s circadian rhythm is absent
after birth. The patterns of secretion begin at approximately 3 months of age, with an
increase in the amplitude of melatonin. These findings were confirmed by studies of the
excretion of 6-hydroxy-melatonin (6-OH-MLT), melatonin’s principal metabolite (69,70).
Kennaway et al. demonstrated a very low and arrhythmic excretion of 6-OH-MLT in
children of 9 to 12 weeks of age. Also, the onset of melatonin’s circadian rhythm cor-
responds to the development of other circadian variables, such as the sleep–wake cycle,
body temperature, cortisol secretion, and TSH.
Studies of individual nocturnal and diurnal serum samples in normal subjects (70,71)
have found the highest levels of melatonin during the night in children aged 1–3 years.
750 C. Barriga et al.
Mean levels of melatonin descend steadily by 80% from infancy to adolescence. This
decline during childhood could be explained by the increase in body weight. While there is
a 500 to 800% increase in the size of the human body from infancy to adolescence, the data
on the size of the pineal (72), the HIOMT (hydroxyindole-O-methyl transferase) content
in the pineal (73), and the production of melatonin (74) indicate only small changes after
infancy. In sum, the descent in melatonin concentrations during childhood seems simply
to be the result of there being a constant range of production of the hormone against the
increasing volume of distribution of the hormone during growth. There is also support for
this concept in different animal models (75,76).
The serum concentrations of nocturnal melatonin also descend significantly during
adulthood (age groups of 70–90 years vs. age groups of 20–35 years) (71,75). Nonetheless,
the difference in the mean values was only 10% with respect to the maximum values mea-
sured in many young individuals. Most of this additional decline occurs during senescence
(Fig. 6). This could explain why some workers who examined adults within a narrow age
range (72,77) were unable to detect any dependence of melatonin concentrations on age,
FEMALE
MALE
AGE
y
ears
908070605040302015
10
100
200
300
400
500
600
MELATONIN (pg/ml)
700
800
131197531
Fig. 6. Nocturnal serum concentrations of melatonin in 367 subjects of ages between
3 days and 90 years (70).
Oxidative Stress in Phagocytic Cells 751
while others who compared young and elderly subjects (78) found the lowest melatonin
levels in the latter group. Thus, the age-dependent decline in nocturnal serum melatonin
levels after infancy consists of a sharp descent from early childhood to adolescence, and
a moderate descent in adulthood (62,79).
A small additional decline in melatonin in elderly subjects could be the result of degen-
eration of the pineal body with age, a characteristic frequently found in other endocrine
glands. Nevertheless, other possible causes have been suggested for the age-dependent
alterations in serum melatonin, including a reduction in the metabolism or population of
pinealocytes (70).
Melatonin, oxidative stress, and age: relationship with the immune system
As indicated above, there are several theories concerning the events that occur in cellular
aging. One of these theories is that the lifelong accumulation of free radicals is the cause
of the degenerative processes. Free radicals are atoms or molecules that have an unpaired
electron (O
2,HO
,H
2O2). They are highly reactive, and hence have short lifetimes.
They can cause damage to cells, and are capable of provoking cancers and mutations.
There are many (enzymatic and nonenzymatic) systems in cells that protect the organism
from the harmful effects of free radicals. These defense systems eliminate or reduce the
production of free radicals, and are normally used in preventing or delaying aging and age-
related diseases (80,81). The best-known free radical scavengers are tocopherol, ascorbic
acid (vitamin C), and glutathione, to which has been added in the last few years the
proposed antioxidant role of melatonin. Melatonin seems to act as a potent scavenger of
hydroxyl radicals, with results similar to those induced by glutathione or mannitol (82,83).
Oxygen free radicals play an important part in the phagocyte-mediated immune response
for the destruction of toxic bacteria or pathologically altered cells. The activation of
macrophages has been shown to synthesize another type of free radical, nitric oxide,
which, when the destruction of bacteria has ended, activates the generation of new reac-
tive hydroxyl radicals. Free radicals are used by the immune cells for the destruction of
pathogenic germs, but they unfortunately also attack lymphocytes and phagocytes. The
level of free radicals depends on the presence of antioxidant agents, including the intra-
cellular levels of zinc, and it has been observed that melatonin can modulate the volume
of zinc (84). It has also been seen that melatonin possesses a direct antioxidant action in
phagocytes (85).
The damage to deoxyribonucleic acid (DNA) in particular can be highly significant in
old organisms (86). Tan et al. (87) showed clearly that melatonin is a potent protector of
DNA against oxidative damage. The hormone, whose synthesis and secretion diminish
sharply with age (88), may be correlated with free radical neutralization (89).
Besides aging, a variety of age-related diseases have been linked to the damage caused
by free radicals (90,91). In particular, cancer, which is provoked in its onset by damage
to the DNA, may be reduced in part if melatonin is maintained throughout life (89).
Neurodegenerative disorders are generally associated with free radical damage, at least in
certain areas of the brain (91), so that the potential benefit of melatonin with respect to
neurodegenerative alterations seems obvious (92). Melatonin is taken up by the brain (93),
752 C. Barriga et al.
and quickly acts in promoting the neuronal activity of glutathione peroxidase, an enzyme
essential to the defense system of the central nervous system (CNS).
One of the most important observations about the pineal gland came from research into
the decline, both in the amplitude of the rhythm of melatonin and in the immune function
in old age (62,79). The defects in the synthesis of melatonin with age may exist at various
levels. Environmental sensors, for instance, lose keenness over the course of the aging
process, cutting off the input signal. The pineal itself may lose something of its activity to
synthesize melatonin in old age. Also, there is evidence that the primary defect with age
is at the level of the CNS, since transplants of the CNS of fetuses to old hamsters restores
this capacity (58). The effects of melatonin on the endogenous opiates (β-endorphin,
methionine-enkephalin, leukin-enkephalin, and dynorphin) promote the stimulation of the
immune system (94).
Grad and Rozecwaig (95) proposed the hypothesis that aging is a consequence of
pineal failure. Thus, aging would be a syndrome of the relative deficiency of melatonin
accompanied by a decrease in the ratio melatonin/5HT (5-hydroxy-l-tryptophan). This
would be prejudicial to different aspects of the individual’s neurophysiology, and would
cause the aging process.
Armstrong and Redman (96) observed that melatonin had anti-aging properties. As the
organism ages, there is a fall in melatonin production and in the existence of melatonin
receptors. Pinealectomy in the mouse leads to acceleration in the aging process as well as
to states of hypertension and diabetes, induction of rapid eye movement in sleep, raised
blood cholesterol and alkaline phosphatase activity, and altered prostaglandin synthesis.
The administration of melatonin can counteract some of these effects (58).
As was noted above, melatonin can be a potent endogenous antioxidant, and, being
a highly lipophilic compound, it can easily pass through morphophysiological barriers
and protect all portions of the cell against free radicals. As the organism ages, there is
an accumulation of free radicals at the same time as a decline in the rate of production
of melatonin. This provokes, besides other factors, senescence of the efficiency of the
immune system (94).
Work by Amstrong and Redman (96) showed that pinealectomy shortens life in rats,
and that pineal extracts inhibit age-dependent processes. Studies on Swiss mice (97)
found a 20% prolongation of life in the animals administered melatonin nocturnally,
and suggested that the results were due to the stimulation of the immune system and
to the antistress action of melatonin. In particular, the mice that received melatonin
every night in their drinking water lived a mean of 931 ±80 days, compared to a mean
of 752 ±80 days for the untreated animals. Also, the pinealectomized animals, which
presented a notable melatonin deficiency, died long before the animals with the pineal
intact.
Finally, it can in general be indicated that the pineal gland, through its hormone mela-
tonin, may directly or indirectly retard aging or inhibit some age-related diseases. Some
studies have demonstrated that melatonin presents potentially beneficial effects on cer-
tain neurodegenerative disorders such as Parkinson’s (40) or Alzheimer’s (98) diseases.
A consequence of these results is that melatonin has come to be regarded as an anti-aging
and as a juvenilizing hormone. If these predictions and their experimental support can be
verified with time, one could take the pineal gland to be the authentic “fountain of youth.”
Oxidative Stress in Phagocytic Cells 753
Certainly, the data that have been accumulated up to now support the hypothesis that a
supplementary melatonin treatment might be beneficial during aging (36,39).
Melatonin as immunostimulator and antioxidant in phagocytic cells: changes with age
Phagocytosis is an important element of the nonspecific immune response, being a funda-
mental mechanism of defense against infectious agents. The phagocytic cells first ingest
the target (antigen), and then destroy it by the action of enzymes that form free radicals
derived from oxygen by means of a series of redox reactions known as the “respiratory
burst.” In this process, several aggressive chemical species are formed, including super-
oxide anion, hydrogen peroxide, the hydroxyl radical, and hypochlorite. Their function
is to destroy the invasive microorganisms. The presence of free radicals in the phago-
cytes is beneficial for the organism, since it is due to their formation within the cells that
pathogenic microorganisms can be destroyed. This clearly represents a solid defense that
the organism adopts in its natural habitat, and is really advantageous when, once their
work is done, the radicals are scavenged or eliminated by the antioxidant mechanisms
that the phagocytes have available, thereby ensuring the integrity of these cells.
The effect of melatonin on phagocytosis has been studied in depth by our research
group on the ringdove (Streptopelia risoria) using isolated heterophils (56). Melatonin at
pharmacological concentrations increases both the chemoattractive capacity (chemotaxis)
of these cells and their capacity to phagocytose antigen particles, and reduces the intensity
of the respiratory burst (51). Thus, we found in this species (50) that incubating the
heterophils with pharmacological doses of melatonin increased the activity of SOD, a
metalloenzyme that catalyzes the dismutation of the superoxide anion into oxygen and
hydrogen peroxide. It also increases the concentration of myeloperoxidase, an enzyme
that is used as an indicator of heterophils’ germicidal capacity (52). These results were
confirmed when the changes in plasma melatonin levels were found to be positively
correlated with phagocytosis, and negatively with the oxidative metabolism (54).
In this same bird species, we have observed daily oscillations of the levels of this
hormone in young animals, and a decline in plasma levels with advancing age. The latter
is accompanied by a loss of the daily rhythm, with no significant differences being found
between nocturnal and diurnal values in old animals (57) (Fig. 7). It has been shown that
the decline in nocturnal levels of melatonin during aging affects the integrity of circadian
structures, and is a precursor of disease states. Hence, melatonin could have both direct
and indirect beneficial effects on the degenerative processes of aging, and slows the
development of such processes as tumor growth or the immunodeficiency associated with
age, both of which contribute to reducing life expectancy (79).
Our studies have confirmed that old ringdove presents a decline in the function of the
heterophils, which could be due, at least in part, to the absence of the daily rhythm of
melatonin (57,99,100). In this sense, we focused our research on the age-related changes
in the levels of melatonin, and on the effect of administering the hormone on possible
changes in phagocytic activity (phagocytosis and oxidative metabolism) during aging.
Melatonin administered to old ringdove increased the differences between the nocturnal
and diurnal plasma levels of the hormone and, in parallel, enhanced phagocytosis and
reduced the levels of the superoxide radical in the heterophils. Also, in old animals, with
754 C. Barriga et al.
02 am 6 am 10 am
Time of da
14 pm
Young
Mature
Old
18 pm 22 pm
50
100
150
Serum melatonin (pg/ml)
200
300
250
Fig. 7. Plot of the circadian rhythm of melatonin in serum from ringdove (Streptopelia
risoria) of different age groups (57).
the oral administration of melatonin, there was a clear positive correlation between the
levels of the hormone and the values of the phagocytosis index. It was found that, as
the plasma levels of melatonin in old animals rises, so does the capacity of their blood
heterophils to phagocytose latex beads (100). These findings confirmed earlier results of a
positive melatonin–phagocytosis correlation in young ringdove (54), and of an increase in
the phagocytic activity in vitro of heterophils from mature and old animals after incubation
of the cells with both physiological and pharmacological doses of melatonin (57,101).
Therefore, our studies show that, in heterophils from young individuals of this species
of bird, melatonin has a dose-dependent stimulatory effect on phagocytosis at the same
time as neutralizing the oxidative stress deriving from this immune function. This effect,
however, was not observed in the heterophils from old animals, which could reflect, at least
in part, their absence of any circadian rhythm in melatonin (99). The increase observed
in both the plasma levels of melatonin and in the phagocytic activity of the heterophils
of old animals treated orally with the hormone is of special interest, since the melatonin
deficiency of old age is related to suppression of the immune function (62). In this sense,
Cardinali et al. (102) found that treatment in vivo with melatonin not only restores the
immune circadian rhythm in aging, but also that pharmacological levels of the hormone
can overstimulate the immune system and cause an increase in autoimmune processes.
The claim that melatonin is an anti-aging hormone is also based on studies indicating that
it might have immunomodulatory and tumor-suppressing effects due to its being a powerful
free radical scavenger (82). There are certainly various age-related diseases, particularly of
the brain in which free radicals are believed to be involved in the pathological processes.
Antioxidants would be of potential benefit in these degenerative conditions. Various studies
Oxidative Stress in Phagocytic Cells 755
have demonstrated, as have our own (57,99–101), both in vivo and in vitro, that melatonin
has immunostimulatory properties and is able to modulate certain immune functions at
the same time as attenuating the reactions of oxidation (56,79,103,104). Also, as we
had observed previously in young animals with a clear circadian pattern of melatonin,
including high nocturnal levels of the hormone (54), in vivo studies of the administration
of melatonin to old animals showed a clear negative correlation between superoxide anion
levels and plasma melatonin levels (100).
Again with respect to the oxidative metabolism of phagocytes, melatonin promotes the
activity of antioxidant enzymes, also reducing oxidative damage. Indeed, in the ring-
dove, we had demonstrated in vitro that, at pharmacological concentrations in ringdove
heterophils, melatonin controls superoxide anion levels by modulating the activity of the
antioxidant enzyme superoxide dismutase (51), and at the same time inducing the sup-
pression of both basal lipid peroxidation as measured by the levels of MDA, and the
antigen-induced levels of lipid peroxidation (53). In addition, our results have indicated
that physiological concentrations of melatonin also reduce the levels of peroxidation in
heterophils of young-mature animals (55). Recently (100), we have shown that, in general
and independently of the age of the animals, the lowest MDA levels are found follow-
ing incubation of the heterophils with melatonin. The heterophils of old ringdove present
greater basal and antigen-induced concentrations of MDA than those of young animals,
in which the lowest levels of peroxidation are observed at 02:00 (the time of night at
which the plasma levels of melatonin are highest). Also, incubation of heterophils from
old animals with the physiological concentrations of melatonin observed in young animals
reduced the levels of MDA, with the effect being dependent on the concentration of the
hormone and on the incubation time (100).
In recent years, several studies have shown that melatonin is a broad-spectrum antioxi-
dant due to its ability to scavenge free radicals and to stimulate antioxidant enzymes (105).
It has also been shown that melatonin in vitro is an effective scavenger both of free rad-
icals and of other reactive oxygen species, and that it reduces oxidative stress in vivo at
both physiological and pharmacological concentrations (106).
Melatonin administered at physiological levels has major immunoreconstitutive effects
on phagocytes (55), and causes a decrease in the production of MDA, an indicator of
oxidative damage induced in lipid membranes (53). These data confirm the existence of
a negative correlation between the serum levels of melatonin over a 24-h period and the
superoxide anion levels in heterophils, which have minima and maxima coinciding with
the circadian oscillations of melatonin (54).
In sum, melatonin can be described as a powerful endogenous antioxidant in the het-
erophils of birds, even at physiological concentrations, and as an effective free radical
scavenger, protecting cells from the oxidative stress that accompanies phagocytosis.
Melatonin receptors in immune cells
Melatonin acts on immune cells at various levels, both directly and indirectly. In non-
mammalian vertebrates, melatonin receptors can be divided into three subtypes, Mel 1a,
Mel 1b, and Mel 1c, according to their DNA and amino acid sequence. High-affinity
melatonin binding sites have been found in the thymus, bursa of Fabricius, and spleen of
756 C. Barriga et al.
several birds and mammals, as well as in bone marrow Th (T helper) cells, and in humans
in the membrane of peripheral blood lymphocytes. Human monocytes express melatonin
receptors depending on their state of maturity. Although much remains to be discovered
about the connection between the activation of melatonin binding sites in lymphocytes
and the effects of melatonin on lymphocyte functions, it seems that transduction occurs
by means of membrane G-protein-coupled receptors of different regions of the brain.
As was noted above, its lipophilic nature enables melatonin to cross the cell membrane
and bind to intracellular sites. Nuclear receptors have been described in human and murine
immunocompetent cells. It seems that it is these nuclear receptors that are mainly involved
in melatonin’s effect on cytokine production in human peripheral blood mononuclear cells,
although it is not yet known whether the hormone’s action is on the expression of the
cytokine genes or is only at a post-transcriptional level. Nuclear receptors for melatonin
are found in the brain and blood leukocytes.
Besides acting via specific receptors, melatonin can influence the activity of the intracel-
lular proteins involved in activating immune cells. It can affect, for instance, intracellular
calmodulin-dependent phosphodiesterase activity. The variety of possible direct effects
as intracellular signals, the existence of different types of melatonin receptors, and the
fact that the lymphocytes themselves produce melatonin in response to certain stimuli, all
point to a physiological role of melatonin as a modulator in the paracrine, autocrine, or
even intracellular immune systems.
Melatonin receptor genes were first cloned in immortalized Xenopus melanophore cell
lines, and since then many receptors and fragments of receptors have been cloned. The
mRNA sequences of Mel 1a and Mel 1b are present in all vertebrates, while the mRNA
of Mel 1c is only present in nonmammalian vertebrates. The expression of the membrane
melatonin receptor mRNA has been detected in rat thymus and spleen lymphocytes, which
makes one think that the same receptor found in zones of the brain is responsible for the
specific binding of melatonin in lymphocytes.
CONCLUSIONS: FUTURE HORIZON
In sum, although aging is a multifactor process, the age-related decline in melatonin
secretion seems to be one of the most important of these factors, since this hormone
is recognized as a substance whose actions are potentially beneficial in working against
aging. The direct consequences of its loss with age are related to problems in the capacity
for effective sleep, dysregulation of the circadian rhythm, reduction of antioxidant pro-
tection, depression of the immune function, and other disorders (61). There is a growing
literature indicating the existence of a close relationship between the circadian rhythm of
melatonin and the changes related to age in physiology and behavior. Given that melatonin
acts by sending information to the organism on its temporal organization, this hormone
could be an important pharmacological agent for the attenuation of age-related changes
in the immune system, circadian organization, sleep, and other disorders accompanying
old age.
The possible therapeutic and physiopathological implications of melatonin’s immuno-
stimulatory properties have as yet been insufficiently investigated. In general, further
Oxidative Stress in Phagocytic Cells 757
research is required for there to be a consensus on proposing melatonin as a “replacement
therapy” to reduce the incidence, and we are in full agreement with this thought.
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