Full text of DOI:10.1093/gerona/56.2.B81

Journal of Gerontology: BIOLOGICAL SCIENCES
001, Vol. 56A, No. 2, B81–B88
In the Public Domain
2
Interleukin-6 Production Does Not Increase With Age
1
1
1
1
1
Alison A. Beharka, Mohsen Meydani, Dayong Wu, Lynette S. Leka, Ahou Meydani, and
Simin Nikbin Meydani1
,2
1Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging and
Department of Pathology, Sackler Graduate School of Biomedical Science, Tufts University, Boston, Massachusetts.
2
Investigators have reported an increase, decrease, or no effect of age on interleukin-6 (IL-6) production. Differences
in experimental conditions and the health status of subjects may explain these contradicting results. Because the
subjects used in most of the previous studies were not carefully screened for health, we investigated the effect of
age on IL-6 production in healthy young and elderly subjects. Twenty young (aged 20–30 years) and 26 elderly
(
Ͼ65 years) men completed the study. Each subject was screened for good health, undergoing physical examina-
tions and laboratory tests. Circulating IL-6 levels were not significantly different between young and elderly subjects.
A subgroup of subjects representing both young and elderly volunteers had high (Ͼ1000 pg/ml) circulating levels
of IL-6. However, circulating IL-6 levels were low (Ͻ100 pg/ml) in the majority of subjects in both age groups.
Peripheral blood mononuclear cells (PBMC) were cultured for IL-6 production in the presence or absence of
phytohemagglutinin (PHA) or concanavalin (Con)A for 48 hours. Unstimulated secretion of IL-6 by PBMC cul-
tured in autologous plasma (AP) or fetal bovine serum (FBS) was detectable in the majority of cultures. Age did
not influence this spontaneous secretion of IL-6. PBMC stimulation with PHA or ConA significantly increased
IL-6 production, but age did not affect the ability of PBMC to secrete IL-6 after stimulation when cultured in
FBS. IL-6 production by PBMC cultured in AP and stimulated with PHA was not affected by age. However,
when stimulated with ConA, PBMC from the elderly subjects produced less IL-6 than PBMC from the young
subjects. Because IL-6 has been suggested to contribute to the age-related increase in prostaglandin (PG)E and
2
nitric oxide (NO) production, we investigated the effect of age on the production of IL-6 by murine peritoneal
macrophages (M␾) as well as the effect of IL-6 on the production of other M␾ inflammatory products. Similar to
the findings in humans, mouse age did not influence the level of IL-6 produced by M␾. These data suggest that in
healthy subjects, increased production of IL-6 is not a normal consequence of aging. Previously reported higher
IL-6 levels in elderly subjects might reflect an underlying, undiagnosed disease state. PGE and NO production
2
were not affected by the addition of IL-6 to M␾ from young mice or anti-IL-6 antibody to M␾ from old mice.
Thus, IL-6 does not appear to influence the M␾ production of selected inflammatory molecules.
GING is associated with dysregulation of the immune
and inflammatory responses, including changes in the
clear cells (PBMC) production in a carefully screened popu-
lation of healthy human subjects. Additionally, because mac-
rophages (M␾) are key producers of IL-6 (4,5) and because it
has been suggested that any age-related change in IL-6 pro-
duction is due to changes in M␾, to determine age-related
changes in IL-6 secretion we used purified in vitro cultures of
primary resident murine peritoneal M␾.
A
regulation and production of cytokines. Cytokines are essen-
tial for normal immune function. However, certain cytokines,
when produced at inappropriate levels, can initiate and me-
diate many of the metabolic and pathological changes ob-
served in infection, trauma, chronic inflammatory diseases,
and autoimmune conditions. One such cytokine is interleukin-6
It has been hypothesized that many of the changes in im-
mune function seen with advanced age may be the conse-
quence of increased IL-6 production (1). M␾ production of
prostaglandin E (PGE ) and nitric oxide (NO) has been
(
IL-6), which is produced by both lymphoid and nonlym-
phoid cells. It is a multifunctional cytokine that plays a role
in a wide range of immune responses including acute-phase
reactions and hemopoiesis. IL-6 production is normally low,
and its serum levels are often undetectable in the absence of
disease. High levels of IL-6 have been reported in the plasma
and serum of aged mice and humans (1,2). Additionally, in
vitro production of IL-6 by unstimulated splenocytes was
reported to be higher in old mice than in young mice (1).
These reports have led to the conclusion that dysregulation of
IL-6 production is a normal consequence of aging. However,
most human studies have used volunteers that reported them-
selves to be healthy, whereas murine studies in general have
discarded animals with obvious lesions. IL-6, arguably more
than any other cytokine, is a potential marker for a variety of
diseases, the incidence of which increase with age (3). There-
fore, we investigated the effect of age on IL-6 serum levels
and constitutive and stimulated peripheral blood mononu-
2
2
shown to increase with age and to contribute to the decline
in T-cell function with age (6–9). IL-6 has been reported to
regulate in vitro PGE production by selected cell lines (10).
2
Sawada and colleagues (11) reported that expression of in-
ducible NO synthase, the enzyme that catalyses the synthe-
sis of NO in M␾, was transcriptionally activated by IL-6 in
murine myeloid M1 cells. Therefore, the second specific
aim of this study was to determine the effect of IL-6 on the
production of PGE and NO by resident peritoneal M␾.
2
MATERIALS AND METHODS
Human Subjects
Subjects were recruited from an existing pool of volun-
teers via advertisement and telephone interviews. Accept-
B81

B82
BEHARKA ET AL.
able volunteers were (i) those with no acute or chronic dis-
eases, (ii) those not receiving prescription medication or
nonsteroidal anti-inflammatory drugs on a regular basis, or
the care and use of laboratory animals. Mice were sacrificed
via CO asphyxiation. Mice exhibiting visible tumors were
2
excluded from the study.
(
iii) those not taking vitamin or mineral supplements for the
last 3 months. Volunteers who met these criteria were invited
to the Jean Mayer U.S. Department of Agriculture Human
Nutrition Research Center on Aging (JM USDA-HNRCA)
at Tufts University for a day of screening procedures. Each
subject underwent a standard physical examination that in-
cluded a chest radiograph and a 12-lead electrocardiogram,
clinical chemistry profile, complete blood cell and differen-
tial counts, and routine urine analysis before being admitted
to the study. The study was approved by the Tufts Univer-
sity/New England Medical Center Human Investigation Re-
view Committee. Twenty young men aged 20 to 30 years
Peritoneal M␾ Isolation
Peritoneal exudate cells were obtained by peritoneal lav-
ϩϩ
ϩϩ
age (13) with ice-cold Ca and Mg free HANKS media
(Gibco). Peritoneal exudate cells were enriched for M␾ by
adherence (14). Cells were plated at 5 ϫ 10 or 1 ϫ 10
cells per well on 24-well tissue culture plates (Falcon Lab-
ware, Lincoln Park, NJ) in endotoxin-free RPMI 1640 me-
dia supplemented with 5% fetal calf serum, 25 mmol/l
HEPES, 2 mmol/l glutamine, 100 units penicillin per ml,
and 100 ␮g streptomycin per ml. Peritoneal M␾ were al-
5
6
lowed to adhere for 2 hours at 37ЊC in a 5% CO incubator
2
(
26.6 Ϯ 2.7; mean Ϯ SE) and 26 elderly men aged 65 to 85
at which time nonadherent cells were removed by vigorous
washing. Cells were cultured in complete RPMI with and
without lipopolysaccharide (LPS; 5 ␮g/ml for 48 hours).
Cell-free supernatants were collected and stored at Ϫ70ЊC
for later analysis.
years (73.0 Ϯ 4.8) completed the study.
Blood Collection and PBMC Separation
Blood was collected from subjects at different times of
the year to eliminate seasonal effects. Subjects were asked
to fast for 14 hours prior to having their blood drawn. Blood
was collected in foil-wrapped vacutainer tubes with sodium
heparin and was maintained at room temperature until pro-
cessed. Blood was drawn from each subject once a week for
Effect of IL-6 on M␾ Production of PGE and NO
2
After enrichment, peritoneal M␾ from old mice were in-
cubated in medium containing either anti-IL-6 antibody (1,
5, or 10 ␮g/ml) (PharMingen, San Diego, CA) or isotype
control antibody (1, 5, or 10 ␮g/ml) (Genzyme) with and
without LPS (5 ␮g/ml). Next, M␾ from young mice were
incubated in medium containing recombinant murine IL-6
(Genzyme) with and without LPS (5 ␮g/ml). After 24
hours, supernatants were harvested, and cell-free superna-
tants were stored at Ϫ70ЊC until analysis for IL-6, PGE2,
and NO.
3
weeks. Results obtained from the three blood draws were
averaged to account for day-to-day variation in cytokine
levels.
PBMC were isolated from whole blood by Ficoll-paque
(Pharmacia Biotech, Piscataway, NJ) density-gradient cen-
trifugation as previously described (12). PBMC were cul-
tured in endotoxin-free RPMI 1640 (Sigma, St. Louis, MO)
media supplemented with 25 mmol/l HEPES, 2 mmol/l
glutamine (BioWhittaker, Walkerville, MD), 100 units pen-
icillin per ml, and 100 ␮g streptomycin per ml (Gibco BRL,
Grand Island, NY) (complete RPMI). Additionally, com-
plete RPMI was supplemented with 10% fetal calf serum
or autologous heat-inactivated plasma. To determine the
IL-6, PGE , and NO Production
2
Human (Genzyme) and murine (PharMingen) IL-6 was
quantitated by ELISA. Cytokine concentration was deter-
mined by comparison with a standard curve generated from
serial dilutions of purified recombinant mouse (PharMin-
gen) or human (Genzyme) IL-6.
9
amount of lL-6 produced by PBMC, cells (1.0 ϫ 10 cells/l)
were cultured in the presence or absence of phytohemagglu-
tinin (PHA; 10 mg/l) or concanavalin A (ConA; 10 mg/l)
for 48 hours. IL-6 levels in cell-free supernatants were mea-
sured by enzyme-linked immunosorbent assay (ELISA,
Genzyme, Cambridge, MA).
PGE was analyzed by radioimmunoassay. The method
2
for PGE analysis was based on the procedure described by
2
McCosh and colleagues (15). The details of antibody speci-
ficity and cross-reactivity have been published (16).
NO production was determined by measuring the stable
end product of NO metabolism, nitrite, by the colorimetric
Griess reaction (17). Culture supernatant (100 ␮l) was
mixed with an equal volume of Griess reagent and incu-
bated at room temperature for 15 minutes. The absorbance
was measured at 550 nm and compared with a standard
Mice
Over a 2-year period, 40 young (6-month-old) and 40
old (24-month-old) specific pathogen-free, male C57BL/
6
NCrlBR mice (Charles River, Wilmington, MA) fed Pu-
rina Mouse Chow (Ralston Purina, St. Louis, MO) and 40
young (6-month-old) and 40 old (24-month-old) C57BL/
curve generated from serial dilutions of NaNO in complete
RPMI. The standard curve was linear from 3 to 24 ␮mol/l.
2
6
NCrlBR mice fed a semipurified diet containing National
Research Council-recommended levels of all nutrients were
used in four separate experiments. Mice were individually
housed in microisolator cages maintained in a controlled en-
vironment with a 12/12 hour light/dark cycle. Food and wa-
ter were provided ad libitum. All conditions and handling of
the animals were approved by the Animal Care and Use
Committee of the JM USDA-HNRCA at Tufts University
and followed the National Institutes of Health guidelines for
Statistical Analysis
Human data were tested for normality with the Wilk-Sha-
piro test. Data from the three separate blood draws were av-
eraged, and their mean was used in the analysis. Because
the data were not normally distributed, the two-sample Wil-
coxon Mann-Whitney rank sum test was used to determine
differences in IL-6 production between age groups. Data are
reported as median and range or mean Ϯ SE. Significance

AGING AND IL-6
B83
was set at p Ͻ .05. SAS Statistical System 7.0 (Cary, NC)
was used for these calculations. NQuery 2.0 (Statistical So-
lutions, Saugus, MA) was used to determine the power of
the tests to detect differences in IL-6 levels between young
and elderly subjects with a Wilcoxon Mann-Whitney rank
sum test with a .05, two-sided significance level.
The murine data addressing the age effect on IL-6 pro-
duction was analyzed by analysis of variance (ANOVA)
where an experimental date was used to block the data. Be-
cause an experiment by outcome (IL-6 level) interaction did
occur, each of the four independent experiments was ana-
lyzed for age differences in IL-6. Additionally, the mean IL-6
values from all four experiments were tested for differences
between age groups by Student’s t test for normally distrib-
uted parameters and by Wilcoxon signed rank test for data
that was not normally distributed using the SYSTAT statis-
tical package (Systat, Inc, Evanston, IL). The mouse data
Figure 1. Circulating interleukin-6 (IL-6) levels in healthy male
elderly subjects divided into four age groups. Serum was analyzed for
IL-6 by enzyme-linked immunosorbent assay. Numbers in paren-
theses represent the number of subjects in each group. Values are
means Ϯ SE.
addressing the influence of IL-6 on PGE or NO production
2
were first analyzed to confirm whether age differences in
IL-6, PGE , and NO did or did not occur. This was done us-
Effect of Age on Human PBMC IL-6 Production
2
ing a two-factor ANOVA. The effect of treatment within an
age group was determined by Student’s t test. Data are re-
ported as mean Ϯ SE. Significance was set at p Ͻ .05.
Unstimulated secretion of IL-6 by PBMC cultured in au-
tologous plasma (AP) was low, with a mean production of
75 Ϯ 108 and 19 Ϯ 23 pg/ml for PBMC from young and
elderly subjects, respectively (Figure 2). Culturing PBMC
in fetal bovine serum (FBS) resulted in low IL-6 production
RESULTS
(
280 Ϯ 45 and 220 Ϯ 25 pg/ml). However, FBS cultures se-
creted levels of IL-6 that were approximately 4-fold (p Ͻ
02) and 10-fold (p Ͻ .03) higher for young and elderly sub-
Effect of Age on Serum IL-6 Levels of Humans
.
The subjects who participated in the study had no chronic
or acute diseases and were judged healthy by the study phy-
sician on the basis of a standard physical examination that
included a chest radiograph and a 12-lead electrocardio-
gram, clinical chemistry profile, complete blood cell and
differential counts, and routine urine analysis. In addition,
subjects were not receiving prescription medication or non-
steroidal anti-inflammatory drugs on a regular basis and had
not consumed vitamin, mineral, or other dietary supple-
ments for the 3 months prior to participation. There was no
difference in the mean (457 Ϯ 231 pg/ml in young subjects
vs 173 Ϯ 86 pg/ml in elderly subjects) or median (Table 1)
serum IL-6 levels between young and elderly healthy sub-
jects. While both young and elderly subjects had a wide
range of serum IL-6 levels, the majority of young and el-
derly subjects exhibited low serum IL-6 levels (Ͻ100 pg/
ml) (Table 1). When serum IL-6 levels from elderly subjects
were plotted against the subject age, individuals with higher
than average IL-6 levels did not fall into a specific age
group (Figure 1).
jects, respectively, than those found in the AP cultures of
the same age group. Subject age did not influence unstimu-
lated secretion of IL-6 regardless of culture conditions (Fig-
ure 2).
ConA or PHA stimulation of PBMC cultured in AP or
FBS (Figures 3A and 3B, respectively) significantly (p Ͻ
.
05) increased the levels of IL-6 secreted into the culture
medium when compared with the appropriate unstimulated
controls from the same experiment (Figure 2). PBMC from
elderly subjects were able to produce IL-6 after PHA stimu-
lation at levels similar to those from young subjects. Addi-
Table 1. Serum Interleukin-6 Levels in Young and Elderly Subjects
Figure 2. Effect of age on unstimulated interleukin-6 (IL-6) pro-
duction by human peripheral blood mononuclear cells cultured in
complete RPMI medium containing either autologous plasma (AP)
or fetal bovine serum (FBS) for 48 hours. Cell-free supernatants were
analyzed for IL-6 by enzyme-linked immunosorbent assay. Values
are means Ϯ SE; n ϭ 21 (young subjects) and n ϭ 26 (elderly sub-
jects).
Young
n ϭ 21)
Elderly
(n ϭ 26)
(
Median (pg/ml)
Range (pg/ml)
41.5
5–2300
30
54.5
5–2100
38.5
%
Over 100 pg/ml

B84
BEHARKA ET AL.
Figure 4. Effect of age on interleukin-6 (IL-6) production by mu-
rine macrophages cultured in complete RPMI medium with and with-
out lipopolysaccharide (LPS) (5 ␮g/ml) stimulation. The bars in A
represent the combined results from all four experiments; n ϭ 80. B,
Results from the one unrepresentative experiment that demonstrated
a significant age effect; n ϭ 30. Cell-free supernatants were analyzed
for IL-6 by enzyme-linked immunosorbent assay. Values are means Ϯ
SE. *Significant difference between age groups at p Ͻ .05.
Figure 3. Effect of age on interleukin-6 (IL-6) production by hu-
man peripheral blood mononuclear cells cultured in complete RPMI
medium containing either autologous plasma (A) or fetal bovine se-
rum (B) stimulated with concanavalin A (ConA) (5 ␮g/ml) or phyto-
hemagglutinin (PHA) (5 ␮g/ml) for 48 hours. Cell-free supernatants
were analyzed for IL-6 by enzyme-linked immunosorbent assay. Val-
ues are means Ϯ SE; n ϭ 21 (young subjects) and n ϭ 26 (elderly sub-
jects). *Significant difference between age groups by Wilcoxon
Mann-Whitney rank sum test; p Ͻ .05.
ure 4B), while no age-related differences were found in the
other three. The level of unstimulated IL-6 secreted in this
particular experiment was higher than that of the other three
experiments. Although mice were excluded if lesions were
observed at the time of sacrifice, a more extensive histolog-
ical examination of the old mice revealed that the majority
of mice used to determine IL-6 production in the experi-
ment that showed an increased IL-6 level had a higher than
normal lesion load compared with a control population (19).
Concurrent with the high level of spontaneous IL-6 produc-
tionally, subject age did not affect the ability of PBMC cul-
tured in FBS and stimulated with ConA to produce IL-6
(
Figure 3B). However, IL-6 production was significantly
lower in PBMC from elderly subjects than in PBMC from
young subjects when they were cultured in AP and stimu-
lated with ConA (Figure 3A).
Effect of Age on IL-6 Secretion by M␾ From Mice
Macrophages are a major source of IL-6 in freshly iso-
lated immune cell preparations (5,18). To determine whether
M␾ demonstrated age-related changes in IL-6 secretion, we
examined purified in vitro cultures of primary resident peri-
toneal M␾ from mice. Over a 2-year period, four indepen-
dent experiments were done. When the results from all ex-
periments were combined, mouse age did not affect
unstimulated (control) IL-6 production by M␾ (Figure 4A).
However, when data from each independent experiment
were analyzed separately, unstimulated IL-6 production was
higher (p Ͻ .05) by resident peritoneal M␾ from old com-
pared with young mice in one of the four experiments (Fig-
tion seen in this study, spontaneous production of PGE was
2
also high. Thivierge and Rola-Pleszczynski (20) have shown
that PGE can increase the production of IL-6 by M␾.
2
LPS stimulation induced M␾ to secrete even higher lev-
els of IL-6 (Figures 4A and 4B); however, there was no sta-
tistically significant difference in LPS-stimulated IL-6 pro-
duction between age groups from any of the experiments.
Effect of IL-6 on Murine M␾ Production of Other
Inflammatory Molecules
In concurrence with other investigators, we have reported
that M␾ production of PGE and NO increases with age
2

AGING AND IL-6
B85
(
8,9,21). It has been suggested that age-related increases in
Table 3. Effect of Exogenous Interleukin (IL)-6
on Prostaglandin (PG)E and Nitric Oxide (NO) Production
by Macrophages (Mø) From Young Mice
IL-6 levels contribute to this finding. To test this hypothe-
sis, we added antibody against IL-6 to M␾ cultures from old
mice to determine the effect of lowering the level of IL-6 on
2
PGE
2
Production
(ng/ml)
NO Production
PGE production in old mice. Additionally, we added re-
2
Treatment
(␮mol)
combinant IL-6 to M␾ cultures from young mice to deter-
Medium†
Medium ϩ IL-6
LPS (5 ␮g/ml)
LPS ϩ IL-6 (500 pg/ml)
LPS ϩ IL-6 (1000 pg/ml)
2.0 Ϯ 1.3
2.8 Ϯ 1.1
12.5 Ϯ 3.1
15.3 Ϯ 8.1
17.9 Ϯ 6.5
Ͻ1.0 Ϯ 1.0
Ͻ1.0 Ϯ 0.5
3.1 Ϯ 2.2
3.0 Ϯ 2.0
2.2 Ϯ 1.9
mine the effect of increasing IL-6 levels on PGE produc-
2
tion of young mice. Similar to previously published data by
our laboratory and others, murine M␾ from either old (Ta-
ble 2) or young (Table 3) mice produced low levels of PGE2
and NO in the absence of stimuli (medium control). In the
absence of an additional stimulus, addition of recombinant
IL-6 to M␾ from young mice (Table 3) or neutralizing anti-
body against IL-6 to M␾ from old mice (Table 2) did not
‡
Notes: Data are presented as mean Ϯ SE; n ϭ 10. LPS ϭ lipopolysaccha-
ride.
†
2
PGE and NO production by Mø from old mice cultured in medium was 2.7 Ϯ
0.2 ng/ml and Ͻ1.0 Ϯ 1.0 ␮mol, respectively.
‡
2
PGE and NO production by Mø from old mice stimulated with LPS was
change (p Ͼ .05) this low level of unstimulated PGE or NO
2
2
1.1 Ϯ 4.0 ng/ml and 8.2 Ϯ 2.9 ␮mol, respectively, which was significantly
production by M␾. As expected, the addition of LPS to the
culture medium stimulated M␾ from old (p Ͻ .05) (Table
higher than that from young mice at p Ͻ .05.
2
) and young (p Ͻ .05) mice (Table 3) to secrete increased
levels of PGE and NO compared with cultures containing
2
medium only. Confirming our previous findings, LPS-stim-
ulated M␾ production of PGE and NO was higher in M␾
DISCUSSION
2
from old compared with M␾ from young mice (21.1 Ϯ 4 vs
Immune function changes with aging, resulting in an in-
creased incidence of age-related diseases and susceptibility
to infections. Several factors contribute to these changes.
Intrinsic and functional age-related changes have been re-
ported in all cell types of the immune system, with those in
the T-cell population being the most pronounced. Addition-
ally, alterations in the tightly regulated communication net-
work of cytokines and hormones that control immune cells
have been reported (22,23).
Changes in the regulation and/or production of the cy-
tokine IL-6 is one alteration that may have a great impact
on immunity due to the wide variety of functions and cell
types this cytokine affects (24). IL-6 has been shown to in-
duce fever, regulate the acute-phase response, and promote
differentiation and/or activation of T cells, B cells, and M␾
(3,5,24). Dysregulation of IL-6 expression has been impli-
cated in the pathogenesis of various diseases, including
multiple myeloma, lymphoma, rheumatoid arthritis, and
cardiac myxoma. In most conditions involving dysregu-
lated IL-6 production, the mechanism(s) are not known
1
2.5 Ϯ 3.1 ␮mol/l for PGE , p Ͻ .05, and 8.2 Ϯ 2.9 vs 3.1 Ϯ
2
2
.2 ␮mol/l for NO, p Ͻ .05 in old and young mice, respec-
tively) (Tables 2 and 3).
To test whether lowering the level of IL-6 found in the
culture medium of M␾ from old mice would affect the age-
related changes in PGE and NO production, IL-6 neutraliz-
2
ing antibody was added to cultures containing old M␾. Ad-
dition of the IL-6 neutralizing antibody to LPS-stimulated
M␾ from old mice decreased (p Ͻ .05) IL-6 levels by 50%
to 70% (data not shown). Lowering the level of IL-6, how-
ever, had no effect on PGE (p Ͼ .05) or NO (p Ͼ .05) pro-
duction (Table 2).
In a parallel experiment, addition of rIL-6 to LPS-stimu-
lated M␾ from young mice increased (p Ͻ .05) the IL-6 lev-
els in the stimulated cultures between 20% (low) and 40%
2
(
high) (data not shown) (Table 3) but did not affect PGE or
2
NO production in young mice (Table 3).
(
3,5,24).
Because of its central role in modulating immunity, IL-6
Table 2. The Effect of Neutralizing Interleukin (IL)-6
production is tightly regulated, and multiple mechanisms of
IL-6 gene regulation exist (3). Therefore, under steady-state
conditions, circulating IL-6 levels are low. In this study, the
majority of subjects in both age groups had low IL-6 levels.
Other researchers have hypothesized that age-related
changes in the endocrine system somehow “relax” the strict
control over IL-6 expression, resulting in its increased pro-
duction with age (1,2,25). However, we found no difference
in the average circulating levels of IL-6 between the healthy
young and elderly human subjects. While these data agree
with those of Peterson and colleagues (26), they contradict
other human studies, which have reported higher levels of
plasma/serum IL-6 in elderly subjects compared with young
controls (1,2,4). Nor did we find an effect of age on consti-
tutive IL-6 production by PBMC from the same individuals.
Fagiolo and colleagues (24) also reported no effect of age
on unstimulated PBMC IL-6 secretion in humans. However,
on Prostaglandin (PG)E and Nitric Oxide (NO) Production
2
by Macrophages (Mø) From Old Mice
PGE Production
NO Production
2
Treatment
(ng/ml)
(␮mol)
Medium†
Medium ϩ anti-IL-6
LPS (5 ␮g/ml)‡
LPS ϩ anti-IL-6 (50% inhibition)
LPS ϩ anti-IL-6 (75% inhibition)
LPS ϩ anti-IL-6 (90% inhibition)
2.7 Ϯ 0.2
3.2 Ϯ 1.1
21.1 Ϯ 4.0
18.9 Ϯ 7.3
19.6 Ϯ 1.4
18.3 Ϯ 1.8
Ͻ1.0 Ϯ 1.0
Ͻ1.0 Ϯ 0.5
8.2 Ϯ 2.9
9.0 Ϯ 2.0
7.9 Ϯ 2.2
8.3 Ϯ 2.4
Notes: Data are presented as mean Ϯ SE; n ϭ 10. LPS ϭ lipopolysaccha-
ride.
†PGE
.0 Ϯ 1.3 ng/ml and Ͻ1.0 Ϯ 1.0 ␮mol, respectively.
2
and NO production by Mø from young mice cultured in medium was
2
‡
PGE
5 ␮g/ml) was 12.5 Ϯ 3.1 ng/ml and 3.1 Ϯ 2.2 ␮mol, respectively, which was
significantly lower than that from old mice at p Ͻ .05.
2
and NO production by Mø from young mice stimulated with LPS
(

B86
BEHARKA ET AL.
Daynes and colleagues (1) reported higher constitutive IL-6
production by PBMC in elderly than in young subjects. One
of the largest studies to examine the effect of age on consti-
tutive IL-6 production by PBMC is that of Roubenoff and
colleagues (27). They reported lower unstimulated IL-6 pro-
duction by PBMC from young subjects compared with
those of older subjects. However, the higher IL-6 levels in
elderly subjects correlated with higher C-reactive protein
draws, thus validating the result. Other studies (1,2) re-
ported that almost all the young individuals tested had low
levels of IL-6 in their serum. It is possible that this greater
variability in IL-6 levels within the young group might have
reduced the statistical detectability of a difference between
the two age groups. However, exclusion of the two subjects
with high IL-6 levels did not change the results. Our conclu-
sion is supported by the finding that in contrast to the results
found in the young group, the median IL-6 serum levels
from the elderly subjects in this study were lower than those
previously reported (1). The majority of the elderly subjects
had very low IL-6 levels, and only 38% had IL-6 levels
greater than 100 pg/ml, indicating that in healthy elderly
persons, IL-6 is not necessarily high.
(
CRP) levels, an indicator of inflammation. Furthermore,
the age difference in IL-6 was more pronounced in those
with higher CRP levels. It is possible that higher IL-6 values
lead to higher CRP levels. Roubenoff and colleagues (27)
suggested that IL-6 elevation is related to both age and in-
flammation and may not be solely a phenomenon of ad-
vancing age.
Similar to findings in humans, an age-related increase in
IL-6 production by murine cells has been reported (1).
However, our mouse M␾ data concurs with and expands
upon the human data: unstimulated production of IL-6 by
peritoneal M␾ was not affected by mouse age. Although the
majority of immune cells can produce IL-6, M␾ are report-
edly the cell type that becomes dysregulated with age. Data
to support this hypothesis are limited. Daynes and col-
leagues (1) reported that removing the adherent cell popula-
tion from splenocytes of old mice resulted in reduced IL-6
production. However, in the current study, which utilized
purified peritoneal M␾, no effect of age on constitutive IL-6
production was found. IL-6 was higher in old compared
with young mice only in a subgroup of mice with high le-
sion load (19). It should be noted that this group of mice ap-
peared to be physically healthy, and mice with obvious le-
sions had already been eliminated from the study (about
20%). However, further lesions were noted after necropsy.
Few studies conduct extensive pathological examinations of
the mice from which samples are collected, yet the majority
of C57BL/6NIA mice over 24 months of age do exhibit
some type of lesion when examined (19). Further studies are
needed to determine the contribution of age-associated pa-
thologies to the reported age-related increase in IL-6 pro-
duction.
In addition to health differences, there are two other, al-
beit less plausible, reasons for differences in findings
among studies: the methods used to measure IL-6 and the
sex of subject/animal. Transport of IL-6 in blood is funda-
mental to the biology of this cytokine. Typically, IL-6 pro-
duced locally at sites of tissue damage makes its way into
the bloodstream where it then elicits a systemic response.
Recent evidence suggests that this transport is much more
complex than previously thought. Multiple binding proteins
can complex with IL-6 resulting in distinct classes of chap-
eroned IL-6 in human blood that appear to vary in their bio-
logical availability (29). Little is known about the effect of
age or disease on IL-6 transport. Commercial ELISA and
commonly utilized bioassays vary in their ability to distin-
guish free and bound IL-6 (29).
Our data support this conclusion. The main difference be-
tween the current study and others is the health of the sub-
jects. The elderly subjects in our study were stringently se-
lected for good health. Although all the volunteers in the
study by Roubenoff and colleagues (27) were ambulatory,
the authors indicated that subjects taking medications and
those with a variety of chronic illnesses were not excluded.
In the study by Daynes and colleagues (1), because subjects
were considered healthy by “self-report,” their true health
status cannot be ascertained. In the study by Mysliwska and
colleagues (28), health status was graded according to the
criteria of the Senieur protocol. Whereas increased levels of
nonstimulated IL-6 were reported in this study, the level of
IL-6 strictly correlated with individual health status; those
with the best health status produced the lowest IL-6 (28). In-
terestingly, of the 50 elderly volunteers (men and women)
who participated in this study, only 8 (incidentally, all fe-
male) fit the profile for “healthy.” Thus, IL-6 may be ele-
vated in most elderly persons due to the presence of disease.
A second possible explanation for the lack of age differ-
ence in this study is that an effect of age would have been
demonstrated had more subjects, particularly young sub-
jects, been used. There were 21 subjects in the young group
and 26 subjects in the elderly group. Power analysis for a
Wilcoxon (Mann-Whitney) rank-sum test with a .05 two-
sided significance level was performed using the nQuery
Advisor 2.0. Because the nQuery software could not com-
pute the power for unequal samples sizes, we computed the
effect size for equal sample sizes of 21 and 26 that can be
detected with a power of .80; the actual effect size will
clearly be between the lower and upper effect sizes. For
sample size 21 in each group, the results indicate 80%
power to detect a probability of .75 that an observation in
the young group is less than an observation in the elderly
group; for a sample size of 26 in each group, the results in-
dicate 80% power to detect a probability of .724 that an ob-
servation in the young group is less than an observation in
the elderly group. Because sample sizes are unequal, the ac-
tual effect size (i.e., the probability that an observation in
the young group is less than an observation in the elderly
group) is somewhere between .724 and .75. In the current
study, 30% of the healthy young subjects had greater than
Barrat and colleagues (30) reported that the onset, magni-
tude, and kinetics of the age-related changes in cytokine
production are parity and sex dependent. In late adulthood,
murine IL-6 levels were higher in multiparous females than
in males or females that had not reproduced (30). Therefore,
the sex of the subjects could influence the levels of IL-6 re-
1
00 pg/ml of IL-6 in their serum. Two young individuals
had very high (Ͼ1000 pg/ml) IL-6 levels. These individuals
maintained their high IL-6 levels throughout the three blood

AGING AND IL-6
B87
ported. Additionally, Cheleuitte and colleagues (31) re-
ported that women receiving hormone replacement therapy
had lower unstimulated IL-6 production by bone marrow
than did age-matched controls. In the current study, all sub-
jects were males, while other studies have used mixed sex
populations. However, some studies in which mixed sex
populations were used report no influence of sex on IL-6
production (28,32), but the lack of sex effect in these studies
could be due to small sample size and thus low power to de-
tect sex differences.
IL-6 is readily induced by infectious agents, LPS, and a
variety of cytokines (5). Mice that can no longer produce
IL-6 are more susceptible to bacterial infection. Elderly hu-
mans are also more susceptible to microbial infections than
young humans. This declining resistance may be due to the
inability of M␾, when challenged, to produce cytokines at
an appropriate level. In the current study, murine macro-
phages stimulated with LPS and human PBMC cultured in
FBS and stimulated with PHA retained the capacity to re-
spond to an exogenous stimulus. Only PBMC cultured in
autologous serum and stimulated with ConA were influ-
enced by donor age, with PBMC from the elderly subjects
producing less IL-6 than PBMC from the young subjects.
The lower IL-6 production by PBMC from the elderly sub-
jects compared with the young subjects in the presence of
AP and not FBS might be due to the presence of protein(s)
in the plasma of elderly persons, which can bind IL-6 and
reduce its detectability by ELISA. Although the majority of
studies reported no effect of age on IL-6 production by
stimulated immune cells, Effros and colleagues (33) re-
ported that IL-6 production by monocytes begins to decline
once mice reach 24 months of age.
Acknowledgments
This material is based on work supported by the U.S. Department of Ag-
riculture (USDA), under agreement No. 58-1950-9-001 and National Insti-
tute on Aging Grant R01AG09140-08. Any opinions, findings, conclu-
sions, or recommendations expressed in this publication are those of the
author(s) and do not necessarily reflect the view of the USDA. We thank the
staff of the Metabolic Research Unit at the JM USDA-HNRCA for their as-
sistance with recruitment and screening procedures for this study, the staff
of Comparative Biology and Medicine for care of the animals, and Joanne
Meegan for preparation of this manuscript.
Address correspondence to Simin Nikbin Meydani, DVM, PhD, Nutri-
tional Immunology Laboratory, Jean Mayer USDA Human Nutrition
Research Center on Aging, Tufts University, 711 Washington Street, Bos-
ton, MA 02111. E-mail: smeydani@hnrc.tufts.edu
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Received May 13, 1999
Accepted May 15, 2000
Decision Editor: Jay Roberts, PhD