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Molecular basis of age-associated cytokine dysregulation in
LPS-stimulated macrophages
R. Lakshman Chelvarajan,*, Yushu Liu, Diana Popa,* Marilyn L. Getchell,*,
Thomas V. Getchell,*,, Arnold J. Stromberg, and Subbarao Bondada*,,1
*Sanders Brown Center on Aging and Departments of Microbiology, Immunology & Molecular Genetics, Statistics,Anatomy and Neurobiology, and Physiology, University of Kentucky, Lexington
Abstract: Aged humans and rodents are suscepti-ble to infection with Streptococcus pneumoniaebacteria as a result of an inability to make antibod-ies to capsular polysaccharides. This is partly aresult of decreased production of proinflammatorycytokines and increased production of interleukin(IL)-10 by macrophages (M) from aged mice. Tounderstand the molecular basis of cytokine dys-regulation in aged mouse M, a microarray anal-ysis was performed on RNA from resting and lipo-polysaccharide (LPS)-stimulated M from agedand control mice using the Affymetrix Mouse Ge-nome 430 2.0 gene chip. Two-way ANOVA analy-sis demonstrated that at an overall P < 0.01 level,853 genes were regulated by LPS (169 in only theyoung, 184 in only the aged, and 500 in both).Expression analysis of systematic explorer re-vealed that immune response (proinflammatorychemokines, cytokines, and their receptors) andsignal transduction genes were specifically reducedin aged mouse M. Accordingly, expression of Il1and Il6 was reduced, and Il10 was increased, con-
firming our previous results. There was also de-creased expression of interferon-. Genes in theToll-like receptor-signaling pathway leading to nu-clear factor-B activation were also down-regu-lated but IL-1 receptor-associated kinase 3, a neg-ative regulator of this pathway, was increased inaged mice. An increase in expression of the genefor p38 mitogen-activated protein kinase (MAPK)was observed with a corresponding increase in pro-tein expression and enzyme activity confirmed byWestern blotting. Low doses of a p38 MAPK inhib-itor (SB203580) enhanced proinflammatory cyto-
kine production by M and reduced IL-10 levels,indicating that increased p38 MAPK activity has arole in cytokine dysregulation in the aged mouseM. J. Leukoc. Biol. 79: 13141327; 2006.
Key Words: inflammation microarray p38 MAPK
INTRODUCTION
Function of the immune system is decreased with age, leading
to increased susceptibility of the elderly to infections [1, 2],
and infections with influenza viruses and Streptococcus pneu-
moniae, which lead to pneumonia, are a major cause of mor-
bidity and mortality in the elderly. Previous studies have
suggested that influenza virus-specific T cell responses are
decreased in the aged, and that it is in part a result of defects
in antigen presentation [3]. The increased incidence of pneu-
mococcal infections is a result of a defect in the production of
antibodies to the capsular polysaccharide antigens, which are
critical for killing of the bacteria by the phagocytic cells [4].
The antibody response to polysaccharides is dependent on Bcells and macrophages (M), and our recent studies suggest
the defects in the aged are in part a result of deficiencies in
M function [5].
Pure polysaccharide vaccines are less effective in aged
individuals compared with young adults [4, 6]. Neonates are
also unresponsive to polysaccharide antigens or vaccines based
on pure polysaccharides. Although the cellular basis of this
unresponsiveness in neonates has been attributed to B cell
immaturity, we have found that neonatal M are defective in
supporting B cell responses from young mice to polysaccharide
antigens [79]. In the aged, where mature B cells are present
and are only marginally affected in their responses to B cell
receptor signaling, the reason for reduced responsiveness to
polysaccharide vaccines is not known. The pneumococcal
polysaccharides have been classified as thymic-independent
antigens, as they do not elicit antigen-specific helper T cells,
which can interact with B cells in a cognate manner [10].
However, B cells and M are required for generating an
immune response to polysaccharide antigens. We have focused
on the spleen, as it is known that spleen is critical for immune
responses against polysaccharide-encapsulated bacteria [11].
B cells and marginal zone M from the spleen have a role in
the antibody response to polysaccharide antigens [12, 13].
Recently, using a mouse model system, we have shown that
splenic M from young but not the aged mice are able tosupport young mouse B cells to produce antipolysaccharide
antibodies [1]. In this system, we showed that the main function
of M is to produce the cytokines interleukin (IL)-1 and IL-6,
which are needed for B cell differentiation. In fact, in the
presence of added IL-1 and IL-6 B cells from the young and the
1 Correspondence: Sanders Brown Center on Aging, Room 329, University of
Kentucky, Lexington, KY 40536. E-mail: [email protected]
Received January 12, 2006; revised February 11, 2006; accepted February
20, 2006; doi: 10.1189/jlb.0106024.
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aged produced equivalent levels of antipolysaccharide re-
sponses. A major reason for the inability of M from the aged
to support B cell responses to polysaccharide antigens was a
result of a defect in secretion of IL-1 and IL-6. However, the
cytokine secretion defect was not limited to IL-1 and IL-6, as
other proinflammatory cytokines, such as IL-12 and tumor
necrosis factor (TNF-) were also produced at lower levels
by M from the aged in comparison with young mice. It is
interesting that the M from the aged were not defective in
IL-10 production but produced more of this cytokine than M
from the young. Thus, the cytokine production was dysregu-
lated in M from the aged.
Similar defects in production of IL-1, IL-6, TNF-, M-
inflammatory protein (MIP)-1 and MIP-1 by M from aged
mice have been noted by several other researchers [1417].
Defects in M function, in particular, a reduction in secretion
of vascular endothelial growth factor and expression of cell
adhesion molecules, are thought to contribute to the delay in
wound healing in the aged [18]. In contrast, peritoneal M
from aged mice have been shown to produce more cyclooxy-
genase-2 (COX-2) and prostaglandin E2 (PGE2) in response to
lipopolysaccharide (LPS) stimulation [19]. Renshaw et al. [15]
found that expression of a variety of Toll-like receptors (TLRs),
including TLR4, was decreased in the aged, which could be the
reason for a decreased response of M from aged mice to LPS.
However, this does not explain increased IL-10 production in
the ageda cytokine not examined by Renshaw et al. [15].
Conversely, Boehmer et al. [20, 21] found a decrease in pro-
duction of TNF- and IL-6 by thioglycollate-elicited peritoneal
M or splenic M from the aged but did not find a reduction
in TLR4 expression. Instead, they attributed the reduced cy-
tokine production to a decrease in c-jun N-terminal kinase
(JNK) and p38 mitogen-activated protein kinase (MAPK) ac-
tivation in M from the aged. Few studies have measured the
effect of age on M in a comprehensive manner.To determine if cytokine dysregulation in the splenic M
from aged mice extends to other proinflammatory cytokines and
chemokines and to understand the molecular basis of the
altered function of M from aged mice, we performed a mi-
croarray analysis of genes expressed in M from young and
aged mice stimulated with LPS. Despite many microarray
studies on M [2226], few studies focused on splenic M,
and none have compared the transcriptional profile of M as a
function of age. Our study is thus unique in that it provides
novel information about gene expression in splenic M from
young and the aged mice. This allowed us to test several
hypotheses to interpret the age-associated dysfunction in M.This analysis confirmed and extended to the gene level our
findings about cytokine dysregulation and showed that several
other proinflammatory cytokines and chemokines were de-
creased in the aged, suggesting that decreased TLR signaling
may be involved in the M defect. It is most interesting that
the microarray data showed that the expression of the gene
encoding for p38 MAPK was increased dramatically in the
M from aged mice, which was confirmed by Western blot
analysis. Moreover, the increased p38 MAPK activity appeared
to have a causal role in the cytokine dysregulation phenotype
of M from aged mice.
MATERIALS AND METHODS
Mice
Female young (4 months old) and aged (2022 months old) BALB/c mice were
obtained from the colonies of the National Institute on Aging, National Insti-
tutes of Health (NIH; Bethesda, MD). The mice were maintained in the animal
facility at the Department of Laboratory Animal Research on a 12:12 h
light/dark cycle and were given food and water ad libitum. All protocols were
implemented in accordance with NIH guidelines and approved by the Univer-
sity of Kentucky Institutional Animal Care and Use Committee (Lexington).
Reagents
SB203580, the inhibitor of p38 MAPK, was obtained from Calbiochem (San
Diego, CA). Antibodies against the MAPKs were obtained from Cell Signaling
Technologies (Beverly, MA), and the monoclonal antibody against -actin was
obtained from Sigma Chemical Co. (St. Louis, MO). Gel-purified LPS (Esche-
richia coli 055:B5) was obtained from Sigma Chemical Co., and fluorochrome-
conjugated antibody to CD11b (membrane-activated complex-1 or Mac-1) was
purchased from Caltag (Burlingame, CA).
Cell preparation
CD11b-positive cells were purified from spleens of mice using CD11b anti-
body-coupled magnetic beads (Miltenyi Biotec, Bergisch Gladbachuor, Ger-
many). The purified cells were found to be routinely 9095% CD11b-positive
when tested by flow cytometry. For each experiment, the CD11b-positive cellswere pooled from two young and two aged mice. However, for the microarray
study, the CD11b-positive cells were pooled from 10 young and five aged mice.
Cell culture
For the microarray study, M (10106) were cultured in triplicate in Iscove/
F12 medium 10% fetal calf serum at 37C in 5% CO2 at a density of 1
106/ml. Cultures were stimulated with 1 g/ml LPS or left unstimulated for 6 h.
RNA isolation
The cells were spun down in the plate, the growth medium removed by
pipetting, and the cultures were incubated for 5 min in TRI Reagent (Molec-
ular Research Center, Inc., Cincinnati, OH). Total RNA was extracted under
RNase-free conditions and was further purified using the Qiagen RNeasy mini
kit, according to the manufacturers protocol (Qiagen, Valencia, CA). TotalRNA yield and purity were assessed with a spectrophotometer and with the
Model 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA); all samples
had two sharp peaks corresponding to 18S and 28S RNA on the bioanalyzer
electropherograms. The RNA was then transcribed into cRNA by the microar-
ray facility and was hybridized to the whole mouse genome chips (Affymetrix
Mouse Genome 430 2.0) using one chip for RNA from each culture, resulting
in a total of 12 gene chips.
Microarray data analysis
From each sample, 4 g total RNA was used for the amplification and labeling
reactions. Out of this, 20 g labeled cRNA was used in the hybridization
reaction. The Affymetrix Mouse Genome 430 2.0 has 45,101 probe sets, and
the probe sets with absent calls across all samples and unannotated genes were
removed to reduce the multiple testing problem. These steps resulted in 18,627
probe sets. Two-way ANOVA tests were carried out to identify differentially
expressed genes. For each probe set, the model yijk i j ij
ijk was applied, where yijk is the log-transformed expression level of the kth
chip in the ith age and the jth LPS. Furthermore, represents the grand mean
expression, i is the effect as a result of age [aged (A) and young (Y)], j is the
effect as a result of the LPS [medium (M) and LPS (L)], ij is the interaction
effect between age and LPS, and ijk is an error term, which is assumed to be
normally distributed with mean 0 and variance 2. Applying an overall P value
of 0.01, ANOVA analysis indicated that only 9894 out of the 18,627 probe
sets were significantly regulated for age, treatment, or both. To find the genes
with biological significance, we applied the intensity, fold-change, and pair-
wise P values as additional filters. Probe sets with mean intensities 200
across all four treatments or fold changes that were less than twofold and
pair-wise P value (intensity with LPS vs. that for medium) 0.01 were not
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analyzed further. Application of these filters resulted in 1115 probe sets. As
the gene chip has a large number of genes detected by multiple probe sets, to
produce the Venn diagram (see Fig. 2), principal component analysis was
applied to resolve genes with multiple probe sets that appear in more than one
region of the Venn diagram. After principal component analysis, the number of
unique genes regulated by LPS was further reduced to 853.
The gene-tree diagram was constructed using the GeneSpring 7.2 (Agilent
Technologies) program. Expression analysis of systematic explorer (EASE) was
used to facilitate the biological interpretation of gene lists derived from the
results of the microarray and was accessed online from the Database for
Annotation, Visualization and Integrated Discovery website (http://
apps1.niaid.nih.gov/david/) from the National Institute of Allergy and Infec-
tious Disease, NIH.
Real-time reverse transcriptase-polymerasechain reaction (RT-PCR)
For RT-PCR, the 12 RNA samples used for Microarray plus a further six
samples were reverse-transcribed using the High Capacity cDNA Archive kit
(Applied Biosystems, Foster City, CA). The cDNA was then amplified by
real-time PCR using TaqMan primers, probes, and TaqMan Master Mix on an
ABI Prism 7000 sequence detection system (Applied Biosystems). The Taq-
Man primers and probes used were designed by Applied Biosystems to cover
the intron-exon junction of the respective genes and were tested further by the
manufacturer to prove their specificity for the genes for which they were
designed. The standard curve was done in duplicate from cDNA derived from
P388D1 cells stimulated with LPS. The experimental cDNA was run in
triplicate and normalized to 18S RNA, and the no-template controls were donein duplicate.
Cytokine analysis
M (0.25106) were cultured in duplicate for 1 day in 1 g/ml LPS, various
doses of SB203580, or medium only. Various cytokines in the supernatant were
estimated in duplicate using enzyme-linked immunosorbent assay (ELISA).
IL-12, IL-10, and TNF- were estimated with OptEIA kits (PharMingen, San
Diego, CA). IL-6 was measured with a matched-pair antibody set (Clones
MP5-20F3 and MP5-32C11) from BD Biosciences (San Jose, CA). The optical
densities (OD) were read on an HTS 7000 (Perkin Elmer, Norwalk, CT).
Results are presented as mean SE of four measurements.
Western blotting
Approximately 1.5 106 M were cultured per well per 24-well plate. After
allowing the cells to rest for at least 90 min, the cells were stimulated with LPS
for 15 min. Cells were lysed in the plate using the lysis buffer provided by Cell
Signaling Technologies. An aliquot of the lysate was subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western
blot analysis. The blots were analyzed by probing the membrane using various
primary antibodies followed by horseradish peroxidase-conjugated secondary
antibodies (Santa Cruz Biotechnologies, CA). The blots were developed with
the Pico Chemiluminescence substrate (Pierce Biotechnology, Rockford, IL)
and exposed to Kodak X-Omat film, which was then scanned by a Kodak Image
Station 2000RT (Eastman Kodak, New Haven, CT). For reprobing, membranes
were stripped using a solution containing 62.5 mM tris(hydroxymethyl)amino-
methane-HCl, 2% SDS, and 100 mM -mercaptoethanol at 65C for 5 min. The
relative integrated OD of the protein bands was estimated using the Kodak
Image Station. Band intensities were normalized by dividing the intensity of
phosphorylated protein by that of total protein [e.g., for extracellular signal-regulated kinase (ERK)1/2] or by dividing protein of interest by that of-actin
(e.g., for p38 MAPK).
Statistical analysis
Students t test was used to evaluate the significance of the differences among
means in Western blots, ELISA data, and PCR data.
RESULTS
Definition of LPS and aging signatures
The Affymetrix Mouse Genome 430 2.0 whole mouse genome
gene chip, which was used in this study, has 45,101 probe sets.
After removing the probe sets for unannotated and control
genes and expression sequence tags, a two-way ANOVA anal-
ysis determined that 9894 probe sets were regulated differen-
tially between the two age groups (overall P value 0.01). To
get a broad overview of the gene expression pattern changes
with age and/or LPS stimulation, a gene tree was created using
GeneSpring, which then revealed a number of patterns of gene
expression (Fig. 1). Region A indicates a set of genes that were
induced upon stimulation with LPS in M from the aged and
young. These genes had low constitutive levels of expression
and upon stimulation, were of comparable intensities in M
from young and aged. Genes, which were present at low inten-
sities in unstimulated M but were induced by LPS in only
Fig. 1. A gene tree of the probe sets from the overall P 0.01 list. A
microarray analysis was performed on M from young and aged mice cultured
for 6 h with LPS. For each treatment, RNA was obtained from triplicate M
cultures (each culture was processed separately) and hybridized onto separate
gene chips. Depicted here are the 9894 probe sets, which were identified to be
differentially expressed when the overall P value was 0.01 (two-way ANOVA
analysis). Each column represents the expression levels for a given gene chip.
Using the GeneSpring computer program, we applied a hierarchical method to
cluster these probe sets. Pearson correlation coefficient was used as the
distance measure. The analysis shows clusters of genes regulated by age only,
LPS only, or by both, identified as Regions AF, and explained in the text. The
color-coding is for the intensity of expression of each probe set, and red is the
highest; green, the lowest.
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M from young mice or from aged mice, are depicted in
Regions B and E, respectively. The expression of genes de-
picted in Regions C and D were not modulated by LPS. Those
in Region C were significantly higher in M from young mice,
and those in Region D were higher in M from the aged.
Collectively, these genes would constitute an aging signature
and include pellino 1 (Peli1; Region C) and chemokine-binding
protein 2 (Ccbp2), Mapk14, and IL-1 receptor (IL-1R)-associ-
ated kinase (Irak)3 and several genes related to proliferation
(Region D). Genes in Region F were expressed constitutively at
high levels in both age groups but are dramatically down-
regulated upon LPS stimulation [e.g., peroxisome proliferator-
activated receptor- (Ppar) and CC chemokine ligand 24
(Ccl24)].
Analysis of the effect of age on genes affectedby LPS stimulation
To begin the task of identifying the key genes involved in the
differential response between M from aged and young, probe
sets with low mean intensities200 in all four treatments (i.e.,
youngLPS and agedLPS) were eliminated, and then a cri-
terion of at least a twofold change was used for all pair-wise
comparisons (i.e., LPS and/or age). The remaining probe setswere then divided further into those that were uniquely regu-
lated in M from the aged, young, or both. As a number of
genes are detected by multiple probe sets on the Affymetrix
Mouse Genome 430 2.0 gene chip, the probe set numbers were
converted to the numbers of genes. Where genes were detected
by probe sets with conflicting patterns of regulation, principal
component analysis was used to assign these genes to one
pattern. The numbers of genes regulated by LPS were then
summarized in a Venn diagram (Fig. 2). A total of 853 genes
was affected by LPS stimulation. A comparable number of
genes were uniquely regulated in M from the young and aged
(169 and 184 genes, respectively), and the remainder (500) wasregulated in both ages by LPS. It is interesting that more genes
were down-regulated than up-regulated by LPS in either age
group: In all, 500 genes (or 60%) were down-regulated in one
age group or the other or both age groups, and expression of the
remaining 40% was up-regulated by LPS (Fig. 2). The common
subset (i.e., genes affected by LPS in both age groups) was
divided further into four groups, of which 194 were increased
in M from young and aged, and 297 were decreased in both
groups of M. Another interesting feature of the response to
LPS is that in the common subset, there were few genes (nine)
that were cross-regulated (i.e., up in the age and down in the
young and vice versa).
Validation of microarray data by ELISA
We had previously shown by ELISA that aged M secreted
lower amounts of the proinflammatory cytokines IL-1, IL-6,
IL-12, and TNF- but higher amounts of IL-10 than M from
the young [1]. These cytokines were also affected similarly in
the microarray analysis (i.e., reduced amounts of mRNA for
IL-1, IL-6, IL-12, and TNF- and increased levels of IL-10
mRNA). Thus, the altered pattern of cytokine secretion ob-
served with M from the aged is also reflected at the steady-
state levels of mRNA for these cytokines and therefore, vali-
dating the microarray dataset for these key pro- and anti-
inflammatory cytokines (Fig. 3 and data not shown). It is to be
noted that ELISA data show that IL-6 is the most abundant
cytokine, and IL-1 is produced at the lowest level in M from
either age group. However, this is not reflected by the intensity
data from the microarray (IL-1 gives the highest signal), which
is because the hybridization efficiencies are different across
the various probe sets on the chips.
EASE: cytokine and chemokine subset
To identify functional categories of genes that could be respon-
sible for the difference in cytokine secretion, we divided theprobe sets from the overall P 0.01 list into those that were
at least twofold higher or lower (aged vs. young) upon LPS
stimulation. We then performed an EASE of the LPS-regulated
genes identified by the microarray data [27]. EASE calculates
EASE scores to identify over-represented gene categories
within lists of genes. Using a cut-off value of an EASE score
0.001 for significance, we have found that a variety of im-
mune-response genes (including cytokines and chemokines)
and signal transduction genes (TLR and MAPK pathways) was
over-represented among the probe sets that were lower in M
from aged when compared with M from the young (data not
shown). We then analyzed the expression of the genes encoding
cytokines and chemokines from our microarray data (Table 1).Although a majority of the cytokines and chemokines depicted
is lower in M from the aged, a few, such as Ccbp2, are
expressed at considerably higher amounts in M from the
aged, before and after LPS stimulation. A number of genes
[e.g., CXC chemokine ligand 16 (Cxcl16)] were higher in M
from the young, and this difference remains even upon LPS
stimulation. A number of genes, such as Ccl24, were actually
turned off by LPS.
The microarray data show that in addition to the cytokines
identified by us by ELISA assays, IFN-, CSF-1, GM-CSF, and
bone morphogenetic protein-1 (BMP-1; Ifng, Csf1, Csf2, and
Fig. 2. A Venn diagram of the genes regulated by LPS in M from young and
aged mice. Only probe sets with a pair-wise P 0.01 and an LPS-induced fold
change of at least two (up- and down-regulation) were considered. Where there
were multiple probe sets for the same gene in more than one region of the Venn
diagram, principal component analysis, a dimension-reduction technique, was
used to reduce the number of probe sets to unique genes.
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Bmp1) were also reduced in the aged, and IL-16 (Il16) and
IL-1f6 (Il1f6) were increased (Table 1). IL-16 is known to
promote proinflammatory cytokine production, but the mecha-
nisms are not fully understood [28]. IL-1f6 is a new IL-1 family
member, whose effects are not fully known but does not appear
to replace the function of reduced IL-1 in the aged [29].
Increased expression of these cytokines needs to be verifiedfurther by ELISA or RT-PCR. Many of the chemokines that
were reduced in the aged are involved in innate immunity and
inflammation. Thus, chemokines CCL4, CXCL1 (or keratino-
cyte-derived chemokine), CCL6, CCL9, and CCL24 (or
eotaxin-2; gene symbols are Ccl4, Cxcl1, Ccl6, Ccl9, and
Ccl24), as well as the receptors CC chemokine receptor 3
(CCR3) and CCR5 (Ccr3 and Ccr5), involved in chemotaxis of
neutrophils, M, and eosinophils, are reduced in the aged
(Table 1), which goes well with a reduction in the overall
inflammatory response in spleens from the aged. CCBP2
(Ccbp2), which is up-regulated in the aged, is a protein that has
no signaling domain, suggesting that it could further inhibit
chemokine function [30]. In addition, a variety of chemokines
and receptors (CXCL9, CXCL10, CXCL11, CCR7), which af-
fect CD4 and CD8 T cell migration and T helper cell type 1
(Th1) development, are reduced in the aged. Reduction of some
of these chemokines (IP-9, IP-10, monokine induced by IFN-)
could be secondary to the reduction in IFN-, as they are allinduced by IFN-. This is in agreement with an age-associated
decrease in T cell function and in particular, Th1 cell function.
TLR signaling is reduced in the LPS-stimulatedM from the aged
As stimulation with LPS led to a differential regulation of
cytokine genes in M from aged and young mice, we decided
to further scrutinize the TLR pathway and its regulation. Upon
LPS stimulation, a majority of the genes in the TLR pathway
had higher levels of expression in M from the young than
those from the aged [e.g., TNF-receptor-associated factor 6
Fig. 3. Microarray data validate previous data ob-
tained by ELISA. The RNA extracted from M
stimulated for 6 h with LPS was analyzed by mi-
croarray; data are shown for pro- and anti-inflam-
matory cytokines (left column). The corresponding
amounts of IL-1, -6, -10, and -12 protein secreted
into culture supernatants after stimulation with LPS
for 1 day, as determined by ELISA, are shown in the
right column. Means identified by the same symbol
are statistically different (P
0.05). The ELISAdata are representative of at least six independent
experiments.
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(Traf6), Cd14, Rel, and Relb; Table 2]. However, a relatively
small group of TLR components was up-regulated considerablyin M from aged mice, resulting in higher levels of expression
than in M from young (e.g., Mapk14). Table 2 contains a
partial list of key components of the TLR pathway affected in
M from the aged. The p100, p105, and p65 subunits of
NF-B (Nfkb2, Nfkb1, and Rela) together with other compo-
nents of the pathway were also reduced in M from the aged
stimulated with LPS.
As a defect in the TLR pathway could account for an
alteration in not only a response to LPS but also to other TLR
ligands, the expression of a number of key components of the
TLR pathway was validated by RT-PCR. Thus, the reduction of
myeloid differentiation primary-response protein 88 (MyD88)
and TRAF-6 (Myd88 and Traf6), adaptor molecules that arecritical for activation of the NF-B pathway, in M from the
aged was validated further by RT-PCR (Fig. 4 and Table 2). In
addition to a decrease in the several components required for
activation of the NF-B pathway, there was also an increase in
a few components of the pathway, such as IRAK-M and TIRAP
(Irak-3 and Tirap; Fig. 4 and Table 2). IRAK-M negatively
regulates TLR signaling, and TIRAP is a critical upstream
adaptor molecule for TLR2 responses, and its increased ex-
pression may have a role in the altered response of M from
the aged to LPS [31]. As these two genes did not fit the general
pattern of TLR genes (i.e., reduction in M from the aged),
TABLE 1. Expression of Key Cytokine and Chemokine Genes
Gene title Symbol AliasAged LPS/Young LPS Pair-wise P
Chemokine-binding protein 2 Ccbp2 24.5 6.E-05Chemokine (CC motif) ligand 24 Ccl24 Eotaxin-2 0.4 6.E-03Chemokine (CC motif) ligand 4 Ccl4 MIP-1 0.4 1.E-08Chemokine (CC motif) ligand 6 Ccl6 MRP-1 0.3 9.E-05Chemokine (CC motif) ligand 9 Ccl9 MIP-1 /MRP-2 0.3 1.E-09Chemokine (CC motif) receptor 3 Ccr3 MIP-1RL2 0.2 3.E-05Chemokine (CC motif) receptor 5 Ccr5 0.5 2.E-03Chemokine (CC motif) receptor 7 Ccr7 0.4 5.E-09Chemokine (CC motif) receptor-like 2 Ccrl2 0.4 9.E-09Colony-stimulating factor 1 (macrophage) Csf1 M-CSF 0.3 2.E-04Colony-stimulating factor 2 (granulocyte-macrophage) Csf2 GM-CSF 0.2 4.E-06Chemokine (CXC motif) ligand 1 Cxcl1 Gro1 0.5 6.E-05Chemokine (CXC motif) ligand 10 Cxcl10 IP-10 0.4 5.E-08Chemokine (CXC motif) ligand 11 Cxcl11 0.1 6.E-07Chemokine (CXC motif) ligand 16 Cxcl16 SR-PSOX 0.4 7.E-04Chemokine (CXC motif) ligand 9 Cxcl9 0.1 5.E-07Growth differentiation factor 15 Gdf15 MIC-1 0.4 2.E-06Interferon ( and ) receptor 1 Ifnar1 0.5 2.E-04Interferon ( and ) receptor 2 Ifnar2 0.5 1.E-06Interferon- Ifng 0.1 1.E-07Interleukin 1 Il1b IL-1 0.6 7.E-08
Interleukin 1 receptor antagonist Il1rn IL-1RA 0.5 4.E-06Interleukin 12a Il12a 0.4 2.E-11Interleukin 13 receptor, 1 Il13ra1 0.4 1.E-03Interleukin 15 Il15 0.2 2.E-12Interleukin 15 receptor, chain Il15ra 0.2 2.E-09Interleukin 16 Il16 3.5 2.E-07Interleukin 1 Il1a 0.2 7.E-10Interleukin 1 family, member 6 Il1f6 2.5 3.E-08Interleukin 1 receptor-like 1 Il1rl1 0.5 3.E-03Interleukin 1 receptor antagonist Il1rn 0.4 4.E-06Interleukin 23, subunit p19 Il23a 0.5 2.E-05Interleukin 2 receptor, chain Il2ra 0.4 5.E-08Interleukin 4 Il4 0.2 7.E-06Interleukin 6 Il6 0.4 3.E-10Leukemia inhibitory factor Lif 0.3 4.E-05
Transforming growth factor- 1 Tgfb1 TGF- 0.9 2.E-02Tumor necrosis factor Tnf 0.5 1.E-10Tumor necrosis factor receptor superfamily, member 1a Tnfrsf1a TNF-R1 0.5 5.E-06Tumor necrosis factor receptor superfamily, member 1b Tnfrsf1b TNF-R2 0.5 4.E-06Tumor necrosis factor (ligand) superfamily, member 14 Tnfsf14 LIGHT 0.3 3.E-04
Data generated from microarray analysis. Fold-change represents the ratio of hybridization intensity of any gene in the aged to that in the young M treated
with LPS. Only genes with a statistical significance at the level of P 0.01 are shown. Although we routinely used a fold-change filter of 2.0 (or 0.5), this table
includes some biologically relevant genes that do not meet this criterion. MRP-1, Migration inhibitory factor-related protein-1; GM-CSF, granulocyte macrophage-
colony stimulating factor; IP-10, interferon (IFN)-inducible protein 10; SR-PSOX, scavenger receptor for phosphatidylserine and oxidized lipoprotein; MIC-1,
macrophage-inhibitory cytokine-1; IL-IRA, IL-1R agonist; TGF-, transforming growth factor-; LIGHT, homologous to lymphotoxin, exhibits inducible
expression, competes with herpes virus glycoprotein D for herpes virus entry mediator on T cells.
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TABLE 2. Expression of Key Components of the TLR Pathway
Gene title Symbol AliasAged LPS/Young LPS Pair-wise P
Adaptors & TLR-interacting proteinsHeat shock protein 1A Hspa1a 0.3 8.E-07Lymphocyte antigen 96 Ly96 0.6 6.E-06Myeloid differentiation primary response gene 88 Myd88 0.6 9.E-09Pellino 1 Peli1 0.4 5.E-04Peptidoglycan recognition protein 1 Pglyrp1 4.3 4.E-08
Receptor (TNFRSF)-interacting serine-threonine kinase 2 Ripk2 0.4 6.E-08Toll-like interleukin 1 receptor (TIR) domain containing adaptor protein Tirap 1.9 4.E-03
EffectorsCaspase 8 Casp8 0.6 3.E-05Interleukin-1 receptor-associated kinase 1 Irak1 1.9 3.E-04Interleukin-1 receptor-associated kinase 2 Irak2 0.6 2.E-05Interleukin-1 receptor-associated kinase 3 Irak3 IRAK-M 2.1 2.E-06Mitogen-activated protein kinase kinase kinase 7-interacting protein 2 Map3k7ip2 Tab2 0.6 7.E-07TNF receptor-associated factor 6 Traf6 0.5 3.E-03
IRF pathwayChemokine (CXC motif) ligand 10 Cxcl10 IP-10 0.4 5.E-08Interferon regulatory factor 1 Irf1 0.3 4.E-10Interferon regulatory factor 7 Irf7 0.4 4.E-06TANK-binding kinase 1 Tbk1 0.5 1.E-03
MAPK pathwayMitogen-activated protein kinase kinase 4 Map2k4 MEK4 0.6 5.E-05Mitogen-activated protein kinase 14 Mapk14 p38 MAPK 2.9 1.E-03Mitogen-activated protein kinase 1 Mapk1 Erk-2 1.3 1.E-03Mitogen-activated protein kinase 3 Mapk3 Erk-1 1.4 3.E-06Mitogen-activated protein kinase 8 Mapk8 JNK1 1.1 3.E-01Mitogen-activated protein kinase 9 Mapk9 JNK2 0.8 1.E-03
NF/IL-6 pathwayProstaglandin E synthase Ptges mPGES-1 0.4 1.E-06Prostaglandin-endoperoxide synthase 2 Ptgs2 Cox-2 0.3 4.E-08
NF-B pathwayInhibitor of B kinase Ikbkg NEMO 0.6 3.E-03Interleukin-12a Il12a 0.4 2.E-11Mitogen-activated protein kinase kinase kinase 14 Map3k14 Nik 3.2 1.E-04Mitogen-activated protein kinase kinase kinase kinase 4 Map4k4 0.5 7.E-05Ubiquitin-conjugating enzyme E2D 3 (UBC4/5 homolog, yeast) Nfkb1 p50/p105 0.3 7.E-06Nuclear factor of light polypeptide gene enhancer in B cells 2, p49/p100 Nfkb2 p52 0.5 2.E-07Nuclear factor of light-chain gene enhancer in B cells inhibitor, Nfkbib I-B 0.5 5.E-10Nuclear factor of light polypeptide gene enhancer in B cells inhibitor, Nfkbie IKBE 0.4 2.E-08Reticuloendotheliosis oncogene Rel c-Rel 0.6 1.E-05v-Rel reticuloendotheliosis viral oncogene homolog A (avian) Rela p65 0.5 1.E-06Tumor necrosis factor Tnf TNF- 0.5 1.E-10Tumor necrosis factor receptor superfamily, member 1a Tnfrsf1a TNF-R1 0.5 5.E-06
Regulation of adaptive immunity
CD80 antigen Cd80 B7-1 0.6 3.E-03
Toll-like receptorsLymphocyte antigen 64 Ly64 Muc13 0.5 1.E-03Toll-like receptor 2 Tlr2 0.4 1.E-07Toll-like receptor 4 Tlr4 1.0 8.E-01Toll-like receptor 6 Tlr6 0.6 5.E-08Toll-like receptor 9 Tlr9 1.0 9.E-01
Data generated from microarray analysis. Fold-change represents the ratio of hybridization intensity of any gene in M from the aged to that in those from young
mice treated with LPS. Only genes with a statistical significance at the level of P 0.01 are shown. Although we routinely used a fold-change filter of 2.0 (or
0.5), this table includes some biologically relevant genes that do not meet this criterion. TNFRSF, TNF superfamily; Tab2, TGF -activated kinase-1-binding
protein 2; IRF, IFN-regulatory factor; TANK, TRAF family member-associated nuclear factor (NF)-B activator; MEK4, MAPK kinase 4; MPGES-1, membrane
PGE synthase-1; NEMO, NF-B essential modulator; NIK, NF-inducing kinase; 1-B, inhibitor of -B.
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they were analyzed further by real-time RT-PCR, which vali-
dated the microarray data (Fig. 4).The reduction in the com-
ponents of NF-B signaling pathway explains the reduction in
production of several proinflammatory cytokines and chemo-
kines in LPS-stimulated M from the aged, as this pathway is
known to be critical for the production of such soluble medi-
ators. This is consistent with a global defect in the responses ofM from the aged, not only to LPS but also to TLR2 ligands
such as peptidoglycan and S. pneumoniae bacteria and TLR9
ligand CpG (unpublished observations).
Effect of aging on the MAPK pathway
LPS-induced cytokine secretion from M has been shown to
be dependent on activation of the MAPKs: ERK1/2 (Mapk3
and Mapk1), JNK1/2 (Mapk8 and Mapk9), and p38 MAPK
(Mapk14). The microarray data did not find significant changes
in the ERK and JNK levels, but the level of p38 MAPK
mRNA was enhanced significantly in resting and LPS-stimu-
lated M from the aged (Table 2 and Fig. 5A). To determine
if the increased levels of mRNA reflect an increased level of
p38 MAPK protein, a Western blot analysis was performed on
lysates from M. The Western blot demonstrated that levels of
total p38 MAPK protein (normalized to -actin levels to correct
for differences in protein loading) were increased in M from
the aged (Fig. 5, B and C). We then verified that M from theaged also had increased amounts of phospho-p38 MAPK, the
functionally active form of p38 MAPK (Fig. 5, B and D). In this
figure, phospho-p38 levels were normalized to -actin, which
was similar in both age groups rather than to total p38 MAPK,
as total p38 MAPK was different in the two age groups.
The microarray study indicated that dual-specificity phospha-
tase (DUSP)-10 (or MAPK-activating protein kinase 5; gene sym-
bol is Dusp10), a newly discovered DUSP, was present in higher
amounts in the M from the aged (data not shown). As a knockout
of DUSP-10 had increased p38 MAPK activity [32], we verified by
real-time RT-PCR that M from aged mice did indeed have an
Fig. 4. Validation of microarray data by real-time
RT-PCR. Multiple samples of mRNA, including
those not analyzed by microarray, were transcribed
into cDNA and analyzed by real-time PCR. RNA
was extracted from three or more cultures of M
and transcribed separately into cDNA. PCRs for
Toll-IL-1R translation initiation region domain-
containing adaptor protein (Tirap), Myd88, Irak3,
and Traf6 were done in triplicate for each sample of
cDNA. Intensities derived from each PCR were
normalized to the corresponding values for 18SRNA, also obtained from real-time PCR. Means
identified by the same symbol were statistically
different (P0.05).
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increased expression ofDusp10 (data not shown). To determine if
the increased phospho-p38 MAPK could play a role in the altered
pattern of cytokine secretion of M from the aged SB203580, a
well-characterized inhibitor to p38 MAPK was used. Inhibiting
p38 MAPK in M from the aged resulted in reduced production
of IL-10 (Fig. 6A). It is surprising that there was an increase in
the production of IL-6, IL-12, and TNF- at low doses of
SB203580 (Fig. 6, B and C, and data not shown). This increase
was found in cultures of M from both age groups. The dose-
response curve showed a steady inhibition of IL-10 synthesis withan increasing dose of SB203580, and TNF- and IL-12 were
enhanced at low doses of the inhibitor but were suppressed at
higher doses. This is consistent with the hypothesis that a thresh-
old level of p38 MAPK is required for the synthesis of all cyto-
kines, but after a certain level, it is inhibitory for the pro- but not
anti-inflammatory cytokines.
In contrast to p38 MAPK, which is elevated in the aged, our
microarray data showed only a marginal change in ERK1/2
mRNA (Table 2). Western blot analysis also showed that M
from the aged had slightly reduced amounts of total protein
levels of ERK1/2 (Fig. 7, A and B). However, when these
blots were probed for phosphorylated forms of ERK, there was
a dramatic reduction in phoshpho-ERK1/2 in M from the
aged before and after stimulation with LPS (Fig. 7, A and C).
This reduction in ERK may also contribute to the decreased
cytokine production in the aged.
DISCUSSION
The primary goal of this microarray study was to understandthe molecular basis of cytokine dysregulation in M from aged
mice, which was in part responsible for the decreased antibody
response of aged mice to pneumococcal polysaccharides. The
major findings from this analysis are that there is a more
extensive dysregulation in cytokine production and TLR and
MAPK signaling in M from the aged than previously appre-
ciated and that the phenotype of M from the aged does not fit
into the known patterns of M heterogeneity.
As noted in previous studies, LPS stimulation not only
induces expression of many genes but also represses many
genes that are constitutively expressed in both age groups [25,
Fig. 5. M from the aged have higher amounts of p38 MAPK. Data from microarray show that M from the aged have more p38 MAPK mRNA (A). M were rested
at 37C for 90 min and then stimulated in duplicate with LPS for 15 min. The cells were lysed, subjected to SDS-PAGE, transferred to polyvinylidene difluoride, and then
probed for phospho-p38 MAPK; stripped and then probed for total p38 MAPK; and stripped and then probed for -actin (B). Total p38 MAPK was normalized to -actin
(C), and phosphorylated p38 MAPK was normalized to -actin (D). In this panel, the data points come from two different experiments, yielding a total of six values foreach treatment with the exception of M from the aged LPS, which only had four values. Means identified by the same symbol are statistically different (P0.05).
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33]. It is surprising that the numbers of LPS-repressed genes in
both age groups are rather large and greater than those induced
by LPS. Some of these repressed genes include PPAR-,
CCL24, and CCR1 (gene symbols are Ppar, Ccl24, and Ccr1,
respectively), although these were suppressed by LPS to the
same extent in both age groups. CCL24 (or eotaxin-2) is a
chemokine involved in recruitment of eosinophils and ba-
sophils to the sites of inflammation, and its suppression may
control the type of inflammatory response to be induced. Sim-
ilarly, PPAR- has been shown to inhibit production of several
inflammatory mediators such as TNF-, IL-1, IL-6, and induc-
ible nitric oxide synthase (NOS) in M, and its suppression by
LPS may be a prerequisite for the induction of the LPS-induced
inflammatory phenotype [34, 35]. However, the suppression of
PPAR- was similar in both age groups, eliminating it as a
possible candidate for the differential production of cytokines
by M from the young and aged mice. Moreover, the microar-
ray study has allowed us to eliminate the possibility that
elevated production of inhibitory cytokines, including TGF-
(Tgfb), suppressor of cytokine signaling family molecules, or
IL-1RA (Il1rn; Table 1 and data not shown), is responsible for
the anti-inflammatory phenotype in M from the aged, as none
of these genes was expressed at elevated levels in M from
aged in comparison with young mice [3638].
M heterogeneity has been recognized recently, and an
imbalance in M subsets could be a reason for the difference
between the young adult versus the aged. First, M have been
subdivided into M-1 and M-2 phenotypes depending on their
ability to produce NO and proinflammatory cytokines (M-1type) or anti-inflammatory agents such as IL-1RA and arginase
(M-2 type), suggesting a possibility that one of these types of
M accumulates in the spleens of the aged [39]. Our gene
expression analysis has shown this to be unlikely, as NOS-2
and arginase, respectively, unique to M-1 and M-2 M, were
reduced in M from the aged (data not shown). Second,
resident alveolar M are known to be anti-inflammatory as a
result of constitutive production of IL-10, but the effect of
IL-10 is overcome by TLR agonists [40]. The splenic M from
the aged are unlike the alveolar M from young adult mice, as
they do not produce IL-10 constitutively, and TLR4 ligands do
not overcome their defects in proinflammatory cytokine pro-duction. Third, they are also distinct from the anti-inflamma-
tory M from the intestine, which neither express CD11b (and
many other M cell surface receptors) nor produce IL-1,
IL-10, and IL-12, whereas the M from the aged are
CD11bve and produce IL-10 in excess [41]. Fourth, it has
also been shown that M can be alternatively activated by
IL-4, leading to suppression of proinflammatory cytokines and
enhanced expression of major histocompatibility complex class
II (MHC II) genes as well as IL-1RA [42]. As the aged have
been shown to have an increased incidence of Th2 T cells [43],
it was conceivable that the M in the aged have markers of
IL-4 activation. Our cytokine expression pattern and the gene
expression analysis have shown that splenic M from the ageddo not have this alternative activation phenotype (no increase
in IL-1ra or MHC II; data not shown). Fifth, Mosser and
colleagues [44, 45] have shown that LPS immune complexes
can induce yet another activation pattern, resulting in in-
creased IL-10 and decreased production of IL-12 with no effect
on TNF- production. As the aged have been shown to have
increased autoantibodies, it is plausible that M from the aged
are responding as if they have encountered immune complexes.
This is unlikely, as our microarray study has shown that M
from the aged exhibit decreased expression of most proinflam-
matory cytokines and chemokines, such as Il1b, Il6, and Tnf
Fig. 6. Inhibiting p38 MAPK enhances production of proinflammatory cyto-
kines and suppresses production of anti-inflammatory cytokines. M were
cultured in duplicate with LPS and various amounts of S203580, a p38 MAPKinhibitor, for 24 h. The supernatant was then assayed in triplicate by ELISA for
IL-10 (A), TNF- (B), and IL-12 (C). Students t-test was performed with
means identified by the same symbol. These data are representative of three
independent experiments.
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(Table 1). Thus, the studies from our laboratory have identified
a uniquely hyporesponsive M in spleens from the aged,
which has profound influences on immune responses to poly-saccharide antigens and may affect the overall ability of the
aged to generate an inflammatory response necessary to contain
infections.
Although the response of M from the aged to LPS (i.e., a
TLR ligand) is altered significantly, the expression of several
TLR members (Tlr4, Tlr6, and Tlr9), with the exception ofTlr2,
is comparable with that of M from the young (Table 2). This
finding is consistent with previous reports from Boehmer et al.
[20, 21], who also found no decrease in these receptors, but
disagrees with that of Renshaw et al. [15]. However, our finding
that the downstream signaling components, such as the adaptor
molecule MyD88, and several members of the NF-B pathway,
such as Rel-a, Rel-b, NF-B p50 and p52, and TRAF6(Myd88, Rela, Relb, Nfkb1, Nfkb2, and Traf6), were also re-
duced in the aged suggests that the TLR-dependent pathway is
working at a significantly reduced efficiency. It is interesting
that the NF-B-independent pathway is also reduced, as com-
ponents of this pathway, such as TANK-binding kinase 1 and
IRF-1 (Tbk1 and Irf1), are reduced in LPS-stimulated M
(Table 2). Superimposed on the reduction of these TLR signal-
ing pathway intermediates needed for positive signaling, levels
of IRAK-M (Irak3), a known negative regulator of this pathway,
are enhanced in M from the aged. Thus, there is an overall
reduction in the TLR signaling pathway, which may account for
the generalized decrease in the proinflammatory cytokine se-
cretion from M from the aged. Presently, it is unclear why so
many components of the TLR signaling pathway intermediatesare reduced in the aged (or increased in the case of the
negative regulator IRAK-M), as they are not known to be on the
same chromosome, ruling out a coordinated regulation of sev-
eral of these genes in the aged.
The most surprising finding of our microarray study is the
significant increase in the levels of p38 MAPK in M from
the aged, independent of LPS stimulation. It is of interest that
age-associated changes in the mRNA levels for other major
MAPKs, such as ERK1/2 and JNK1/2, were minimal or un-
changed. This was confirmed at the protein level for p38
MAPK and ERK1/2 by Western blot analysis (Figs. 5 and 7).
Not only was there an increase in the total p38 MAPK level,
but the level of the functionally active, phosphorylated form ofthe enzyme was also elevated in M from the aged before and
after stimulation with LPS. It is notable that Iwasa et al. [46]
found that senescent fibroblasts express higher levels of acti-
vated p38 MAPK, and this elevated phospho-p38 MAPK has a
causal role in the senescent phenotype of the fibroblast cells.
Thus, inhibition of the p38 MAPK activity enhanced the pro-
liferation capability of fibroblasts, whereas expression of con-
stitutively active MEK (MKK)6, an activator of p38 MAPK,
induced a senescent phenotype in fibroblasts from young [46].
Our findings about an increase in p38 MAPK in the aged M
are in contrast to the results of Boehmer et al. [20, 21], who
Fig. 7. M from the aged have dramatically reduced
amounts of phospho-ERK1/2. The lysates used in Figure
5 were also probed for total ERK1/2 and phospho-
ERK1/2 (A). (B) Amount of total ERK1/2 was normalized
to -actin. For this determination, duplicate cultures
were pooled. (C) The amounts of phospho-ERK1/2 nor-
malized to total ERK1/2 are shown. Means identified by
the same symbol are statistically different (P0.05).
These data are representative of two independent exper-
iments.
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found a decrease in the total and phosphorylated forms of p38
MAPK. The discrepancy could be a result of the use of thio-
glycollate-induced peritoneal M in one study by these au-
thors [20]. Although splenic M were examined in the second
study, the Western blots in both studies were not normalized to
a housekeeping protein such as actin for equal loading. This
may affect the interpretation of the data.
Consistent with the published reports about the need for p38
MAPK for IL-10 gene expression, inhibition of p38 MAPK
activity with SB203580 led to a dose-dependent inhibition of
LPS-induced IL-10 production in both age groups. However,
p38 MAPK has also been implicated in the production of
several proinflammatory cytokines, such as TNF-, IL-1, and
IL-6 in numerous studies and in many different cell types such
as monocytes, M, and dendritic cells (DC) [47, 48]. More-
over, inhibition of p38 MAPK reduced inflammation and sepsis
in some animal models [49, 50]. In contrast, Li et al. [51]
recently reported that p38 MAPK is crucially involved in
osteoclast production but not cytokine production by bone
marrow-derived M. It turns out that most studies that use
p38-specific inhibitors in vitro have used various cell lines
such as RAW264.7, THP-1, and 70Z/3 transfected with CD14,
and few of them have performed detailed, dose-response stud-ies. Although the in vivo data clearly establish the anti-inflam-
matory effects of the p38 MAPK inhibitors, it is difficult to
know the critical cell that is affected. Thus, our data, showing
that at low doses, the p38 MAPK inhibitor SB203580 enhances
production of the proinflammatory cytokines TNF-, IL-12,
and IL-6 (Fig. 6 and data not shown), are rather unique and
ascribe a negative and a positive role for p38 MAPK in causing
an inflammatory phenotype. This dose response may explain
why in the literature, there are conflicting reports about the
requirement of p38 MAPK for the synthesis of pro- and anti-
inflammatory cytokines [44, 52, 53].
The ability of low doses of the p38 inhibitor to enhanceproinflammatory cytokines is consistent with our finding that
total and phospho-p38 levels are enhanced in the aged. Our
data, suggesting that at higher doses, the p38 MAPK inhibitor
reduces proinflammatory cytokines, are consistent with the
published literature. We hypothesize that certain minimal lev-
els of this enzyme are required for production of these cyto-
kines, but at higher levels, p38 MAPK may actually inhibit
production of IL-6 and IL-12. As it is well known that the
expression of many cytokine genes (TNF- and IL-1 among
others) is regulated at the transcriptional level and at the level
of mRNA stability, it is conceivable that the low and high
concentrations of active phospho-p38 MAPK influence these
two processes differently [50, 52, 54]. In contrast, p38 MAPKdoes not appear to have any negative effect on IL-10 produc-
tion, as IL-10 levels were decreased in a dose-dependent
manner with the p38 inhibitor.
We have shown previously that the altered pattern of cyto-
kine production in M from aged mice was a result of an
excess production of IL-10 [1]. The neutralization of IL-10
resulted in enhanced production of proinflammatory cytokines,
to levels comparable with control M from young mice. In this
study, we see that by partially suppressing p38 MAPK activity
in M from aged mice, a reduction in IL-10 occurred, with a
concomitant enhancement of proinflammatory cytokine produc-
tion, similar to what was seen when IL-10 was neutralized [1].
This would lead us to postulate that the increased level of
LPS-induced IL-10 seen in M from aged mice is a result of
the higher amount of p38 MAPK activity in M from aged
mice.
Unlike p38 MAPK, levels of ERK1/2 were similar in M
from both age groups. However, levels of phosphorylated
ERK1/2 were reduced significantly in M from the aged. ERK
has also been shown to be important for LPS-induced secretion
of cytokines such as IL-1, IL-6, and TNF- and for LPS
immune complexes, induced production of IL-10 from M [44,
55, 56]. Conversely, Dillon et al. [57] found that ERK activa-
tion inhibits production of IL-12 by inducing c-fos in DC and
that c-fos-negative DC have elevated levels of IL-12 [58]. A
negative role for ERK in LPS-induced M production of IL-12
has not been described so far. Data presented here suggest that
ERK may not be having such a negative role in production of
IL-1, IL-6, IL-12, or TNF-, as all of these cytokines are
reduced in M from the aged, which have dramatically re-
duced levels of active ERK. Presently, we have not tested if
ERK has a negative role in IL-10 production. Our results
suggest that a balance between functionally active ERK and
p38 MAPK is required for a pattern of cytokine productionsuch as that seen in LPS-stimulated M from young mice and
that a loss of this balance leads to the cytokine-dysregulated
phenotype of the aged. To further clarify the mechanism of
age-associated cytokine dysregulation, we will also be assess-
ing the levels of functionally active JNK in M from the aged,
as JNK has been shown to have positive and negative roles in
cytokine secretion by M [47, 59]. Another question that
remains is about the mechanisms that are responsible for p38
MAPK up-regulation in M from the aged, as in most inflam-
matory responses, the levels of these enzymes are not changed,
but their functional activities are regulated by various stimuli
[47].In addition to the direct effects of aging on p38 MAPK and
ERK expression as well as activation, some of the signaling
proteins that regulate the MAPK pathway are also affected by
aging. Thus DUSP-10, an enzyme known to inhibit MAPK
signaling, is elevated in aged M before and after LPS stim-
ulation in comparison with the young M. Mice in which
DUSP-10 is deleted have an increase in the production of
proinflammatory cytokines, in part, as a result of an increase in
activities of p38 and JNK MAPK, but the cellular source of
these cytokines was not identified [32]. Presently, we are
investigating the contribution of DUSP-10 to the cytokine-
dysregulated phenotype of the aged M.
In summary, our microarray analysis has shown that Mfrom aged mice have a global defect in the TLR signaling
pathway and in production of proinflammatory cytokines and
chemokines, and the anti-inflammatory cytokines are in-
creased, such that the splenic M in the aged have an anti-
inflammatory phenotype. We find that the aged mouse M
have a unique phenotype, which is distinct from the currently
known modes of M regulation. The aging signature for M
includes an elevation of proliferation-specific genes. Our pre-
liminary, immunohistochemistry studies with bromodeoxyuri-
dine labeling show that indeed, there are more proliferating
cells in the spleens of aged mice. Currently, we are investigat-
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ing the relation between this proliferation phenotype and the
cytokine dysregulation phenotype in M from the aged mice.
Finally, we have shown that the cytokine dysregulation is a
result of an imbalance in MAPK activation (increased p38
MAPK) and that inhibition of p38 MAPK partially restores
production of cytokines such as IL-6 and IL-12 in M from the
agedcytokines that are important for B cell responses.
ACKNOWLEDGMENTS
This work was supported in part by NIH Grants AG05731 and
CA 92372 to S. B., AG-16824 to T. V. G., and P20-RR16481
to A. J. S. Supplementary data for this article are available at
National Cancer Institutes caArray data portal (http://
caarray.nci.nih.gov). Our thanks are to Dr. Alan Kaplan
for critical reading of this manuscript, Ms. Radhika Vaishnav
for her help with developing the protocol for RNA extraction,
and to Ms. Donna Wall and Dr. Kuey-Chu Chen for their expert
help with the microarray analysis.
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