The Inflammation Hypothesis of Aging

Molecular Modulation by Calorie Restriction

HAE YOUNG CHUNG, HYON JEEN KIM, JUNG WON KIM, AND BYUNG PAL YU
Department of Pharmacy, College of Pharmacy, Research Institute of Genetic
Engineering, Pusan National University, Gumjung-Ku Pusan 609-735, Korea

ABSTRACT: Current evidence strongly indicates that reactive oxygen species

(ROS) and reactive nitrogen species (RNS) are widely implicated in the inflam-
matory process. However, mechanistic information is not readily available on
the extent to which ROS/RNS contributes to the proinflammatory states of the
aging process. The involvement of the underlying inflammation during the ag-
ing process and the molecular delineation of anti-inflammatory action of calo-
rie restriction (CR) is described. Age-related upregulations of NF-␬B, IL-␤,
IL-6, TNF␣, cyclooxygenase-2, and inducible NO synthase are all attenuated
by CR. The suppression of the NF-␬B activation was accomplished by blocking
the dissociation of inhibitory I␬B␣ and I␬B␤ by CR. These findings provide
underlying molecular insights into the anti-inflammatory action of CR in rela-
tion to the aging process. Based on these and other available data, it is suggest-
ed that the “Inflammation Hypothesis of Aging” supports the molecular basis
of the inflammatory process as a plausible cause of the aging process.

KEYWORDS: Aging; Antiaging; Anti-inflammatory; Calorie restriction; NF-

␬〉; ROS; Inflammation; NO synthase

INTRODUCTION

Inflammation is a complex host’s normal defense reaction to insult and stress,

both physiological and nonphysiological, like chemicals, drugs, oxidants, or a vari-
ety of microbial entities. Inflammation responses, whether acute or chronic, are ac-
tivated by well-coordinated, sequential events controlled by humoral and cellular
reactions. Reactive oxygen species (ROS), such as ⋅O2−, ⋅OH, H2O2, and reactive ni-
trogen species (RNS), such as NO and ONOO−, are heavily implicated in the inflam-
matory process (FIG. 1). The deleterious effects of ROS/RNS are dependent on their
concentration and the microenvironment in which ROS/RNS are released. Overpro-
duced or uncontrolled ROS/RNS are a major causative factor in tissue inflammation.
As shown in FIGURE 1, the inflammatory responses usually proceed in the following
sequential phases: (1) intracellular activation, (2) infiltration of proinflammatory
macrophages and lymphocytes, (3) increased vascular permeability, and (4) tissue
damage and cell death.1

Address for correspondence: Dr. Hae Young Chung, Department of Pharmacy, College of
Pharmacy, Pusan National University, Gumjung-Ku Pusan 609-735, Korea. Phone: 82-51-510-
2814; fax: 82-51-510-2814.
hyjung@hyowon.pusan.ac.kr

327
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FIGURE 1. Biochemical and molecular changes leading to inflammatory responses.

The introduction of neutrophils, lymphocytes, and macrophages, which release

ROS, RNS, and lytic enzymes further amplify the inflammatory reaction.2 Further-
more, various inflammatory genes are activated by the transcription factor, NF-κB,
which is extremely sensitive to oxidants and many proinflammatory substances.3 For
instance, NF-κB activates gene expression of proinflammatory cytokines, such as
IL-1β,4 IL-6,5 and TNFα,6 as well as the proinflammatory enzymes, cyclooxygena-
se-2 (COX-2)3 and inducible nitric oxide synthase (iNOS).7 In our recent study, we
found that COX-2 and NF-κB were upregulated in rat kidney during aging,3 and was
suppressed by calorie restriction (CR). Although several pieces of evidence show
that CR can modulate NF-κB activation by its antioxidative action,8 this aspect of
CR has not received much attention to date.
The involvement of the inflammatory process in several diseases has long been
known, but until a recent proposal by McGeer and McGeer, its implication in the ag-
ing process has been less appreciated. Their “Inflammatory Hypothesis of Demen-
tia” highlights the importance of inflammation in aging.9 Other age-related
inflammatory processes are the age-related alterations of vascular endothelial and
smooth muscle cells, which lead to pathogenic expressions, as seen in
atherosclerosis10 and vascular dysfunctions of diabetes.11
The evidence accumulated to date on the beneficial, antiaging action of CR is
consistent with its anti-inflammatory property. Based on reported data and new evi-
dence obtained from our laboratory, we propose the “Inflammatory Hypothesis of
Aging,” to highlight the involvement of the underlying inflammation process during
the aging process as discussed below. In the present report, we use the molecular
mechanisms of the anti-inflammatory action of CR as the supporting basis of the
proposal.
CHUNG et al.: INFLAMATION HYPOTHESIS OF AGING 329

OXIDATIVE STRESS, PROSTAGLANDINS, AND INFLAMMATION

Vasoactive prostaglandins (PGs) are essential components in the regulation of

vascular function under normal physiological conditions,12 and their role in the in-
flammatory process has been long known. Moreover, changes in PG activities in vas-
cular cells can have a widespread impact on vascular tone and the inflammatory
response of the vasculature.
Several reports have addressed PG production during aging. Most of the data
seem to indicate that the production of inflammatory PGs such as PGE2, TXA2,
PGH2, and PGG2 increase with advancing age, whereas cytoprotective PGs such as
PGI2 decrease with age.13–16 Macrophages from old mice are reported to produce
more PGE2 than young mice, indicating the dysregulation of immune cells for proin-
flammatory conditions with age.17
The PG synthesis pathway is a major ROS-generating source during the conver-
sion of PGG2 to PGH2. Little information is available on how much of the PG path-
way contributes to the total ROS status. At our laboratory, to estimate the extent of
PG-derived ROS, ROS generation was measured in the presence of the COX inhib-
itor, indomethacin. ROS generation was measured by the fluorometric method using
dichlorofluorescin diacetate in tissue homogenates and subcellular fraction. ROS
generation in the heart,18 kidney,19 and brain20 showed a gradual increase with age.
COX-derived, indomethacin-inhibitable ROS increased with age. The COX-derived
ROS were about 30% of total ROS. 3 COX-2 mRNA and protein levels also showed
age-related increases.3
To obtain evidence that COX activity is related to ROS levels, we used Mn-
superoxide dismutase (Mn-SOD) knockout mice to increase the oxidative stress lev-
el.18 In heart postmitochondria and mitochondria fraction from these mice, ROS
generation significantly increased compared to wild-type mice (data not shown).
These increases were parallel to the marked increases in cardiac and renal COX-2
mRNA levels,18,19 providing evidence that COX-2 expression is influenced by the
ROS status. Our findings agree with the data of Feng et al.,21 who reported that rad-
ical scavengers suppress COX-2 expression induced by IL-1, TNFα, and lipo-
polysaccharide (LPS). Taken together, the data suggest that COX-derived ROS are a
major contributor to the inflammatory process.

INOS IN INFLAMMATION AND AGING

The essential role of NO in normal physiological functions is firmly established.

The enzyme responsible for NO has several isoforms. Among three of these iso-
forms, eNOS, bNOS, and iNOS, iNOS is readily induced by proinflammatory cytok-
ines and LPS.22
The ability to induce iNOS, and thereby generate large amounts of NO, suggests
that this isoform may play a major role, as one might expect in a chronic condition
such as aging. For example, Cernadas et al. reported that the vessel walls of aging
rats show an enhanced expression of eNOS and iNOS. 23 Poynter et al. also showed
that the mean daily level of urinary nitrate plus nitrite excretion by young animals
was lower than that secreted by aged animals, indicating increased iNOS.24 Our lab-
oratory recently obtained data showing that induction of iNOS gene expression in-
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FIGURE 2. Effect of age and CR on iNOS gene expression. (ABBREVIATIONS: AL, ad

libitum; CR, calorie restriction.)

creases during aging in the kidney of lupus-nephritis-prone mice.25 In addition, as

shown in FIGURE 2, iNOS gene expression was substantially increased in aged Fis-
cher 344 rat kidney, showing that dysregulated NO production causes inflammatory
reactions and autoimmune disease states.

PROINFLAMMATORY CYTOKINES IN THE AGING PROCESS

Cytokines are major communication channels that provide links within and be-
tween the immune system and other organs.26 During aging, a shift occurs in the ra-
tio of native to memory T cells, with associated changes in the cytokine profile that
favor increases in inflammatory cytokines such as TNFα, IL-1, IL-6, INFγ, and TG-
Fβ.27–30 We should note, however, that most age-related changes in cytokine levels
are investigated mainly in vitro, and the results are not consistent.28,31,32
While the production of IL-6, but not IL-1β and TNFα, by peripheral mononu-
clear cells were reported to increase in the elderly compared to young subjects,31 Ri-
ancho et al. reported an age-related increase of IL-1 production in peripheral blood
mononuclear cells in old animals.28 On the other hand, IL-1β levels were higher and
IL-6 levels were lower in the liver of old rats than young rats.33 In a recent study in
our laboratory, the levels of proinflammatory cytokines in aged and/or LPS-treated
rats were measured. We found that mRNA levels of IL-1β and IL-6 showed an in-
crease with age, which were further enhanced by LPS challenge in older rats.33 Re-
sults showed that mRNA expressions of IL-1β and IL-6 significantly increased in
LPS-treated old rats, as did the mRNA amount of TNFα in old rats. The LPS chal-
lenge that caused a significant increase in these proinflammatory proteins, especially
in old rats, indicates an enhanced sensitivity to inflammation with aging. 33 It is im-
portant to note that the gene expression of these proinflammatory proteins was
matched by levels of intracellular oxidative stress.18–20

ROLE OF NF-␬B IN THE INFLAMMATORY PROCESS

NF-κB is an ubiquitous, pleiotropic transcription factor known to be extremely

sensitive to various stimuli,34,35 particularly ROS.36 Unlike some of the other tran-
scription factors, the NF-κB complex is held in the cytoplasm as an inactive state by
the inhibitory subunit, IκB. The phosphorylation of IκB and its subsequent degrada-
tion of inhibitory IκB allows translocation of NF-κB to the nucleus.36 This NF-κB
CHUNG et al.: INFLAMATION HYPOTHESIS OF AGING 331

FIGURE 3. Diverse stimuli activating NF-κB and a range of genes induced through
NF-κB.

transactivation can be elicited by inflammatory stimuli such as TNFα, lymphotoxin,

IL-1, lipopolysaccharide, protein synthesis inhibitors, ultraviolet light, and phorbol
esters 37,38 (FIG. 3).
Many researchers have looked into the age effect on the regulation of NF-κB. Ex-
isting data show that the nuclear DNA-binding activity of the NF-κB factor increases
during aging in all tissues of the rats and mice tested.39,40 Helenius et al. found that
heart, liver, kidney, and brain tissues from old rodents expressed higher levels of NF-
κB binding activity than tissues from young adult rodents.39 Korhonen et al. report-
ed a significant upregulation of NF-κB in brain samples of rat.40 But the precise
mode of age-related NF-κB activation has not been delineated. In a recent study, we
found new information that activated NF-κB was due to an age-related IκBα de-
crease and its dissociation increase from the complex in the cytosol, allowing the nu-
clear translocation of NF-κB.3 The study further indicated that anti-inflammatory
CR action suppressed NF-κB (FIG. 4) through inhibiting the IκB dissociation by the
upregulation of cytosolic IκBα and IκBβ.

CALORIE RESTRICTION AS A REGULATOR OF INFLAMMATION

Extensive gerontological research has confirmed that CR imposed on laboratory

rodents extends their mean and maximum lifespans.41 We investigated the effect of
CR on the ROS generation specifically derived from the COX pathway of prostanoid
metabolism; the results are summarized here. We found CR suppressed COX-de-
rived ROS generation during aging. CR also blunted increases in the production of
prostanoids such as seen in TXA2, PGI2, and PGE2 generation. These findings are
332 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 4. Effect of age and CR on NF-κB activity. (ABBREVIATIONS: AL, ad libitum;

CR, calorie restriction.)

FIGURE 5. Possible active site of CR action in NF-κB activating system. (A BBREVIA-

TION : CR, calorie restriction.)

consistent with what is known about CR’s action against inflammation and oxidative
stress. It is not surprising therefore to notice that in CR rats, the age-related increase
in COX activity, PG synthesis, and COX-derived ROS generation were all sup-
pressed by CR (unpublished data). The beneficial CR effects were further extended
CHUNG et al.: INFLAMATION HYPOTHESIS OF AGING 333

to the regulation of gene expression, as CR suppressed the increase of COX-2 mRNA

and protein levels with age through the modulation of NF-κB and IκB.
In regard to CR’s anti-inflammatory action, glucocorticoids should be mentioned,
as they are well known for their anti-inflammatory property. Klebanov et al. showed
that short-term CR animals (2 months) with elevated glucocorticoid levels were able
to better manage carrageenan-induced inflammation, credited to increased glucocor-
ticoid levels.42 However, readers should be reminded that this experimentation was
a short-term CR with an acute inflammatory response. In aged animals, glucocorti-
coid levels are elevated, and yet their inflammatory responses are still aggravated,
indicating that the anti-inflammatory action of glucocorticoids might not be the ma-
jor pathway in aged animals.
In summary, our laboratory found that CR attenuates several major proinflamma-
tory proteins by preventing the dissociation of inhibitory IκBα and IκΒβ from NF-
κB complex. Other NF-κB-dependent genes—IL-1β, IL-6, TNFα, COX-2, and
iNOS—are modulated in a similar fashion by CR (FIG. 5).

CONCLUSION

The implications of ROS/RNS in the tissue inflammation during the aging pro-
cess are well demonstrated. Until recently, however, no quantitative information has
been available on the extent of ROS/RNS generation contributing specifically to in-
flammatory reactions during aging.
Our laboratory obtained evidence for the first time that the activation of age-
related NF-κB and the gene expression of several proinflammatory proteins are all
attenuated by CR. Our data further showed that the attenuation of the NF-κB activa-
tion by CR was accomplished by blocking the dissociation of inhibitory IκBα and
IκBβ. The regulation of inflammatory response by CR at molecular levels was fur-
ther exhibited by the suppression of age-related increases in proinflammatory COX-
2 gene expression and PG synthesis. A similar attenuation by CR was shown on oth-
er NF-κB-dependent genes, IL-1β, IL-6, TNFα, COX-2, and iNOS. Thus, a signifi-
cant realization is that the inflammatory process is intricately involved in the aging
process. These findings provide supportive molecular insights into the anti-inflam-
matory action of CR. Based on these findings and rationale, we propose the “Inflam-
mation Hypothesis of Aging.” Further molecular exploration is warranted for a
better delineation of molecular interactions between normal aging and the inflam-
matory process.

ACKNOWLEDGMENT

This work was suppoted by Korea Research Foundation Grant (KRF-99-005-

F00030).

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