chelvarajan - leukocyte biol 06 - aging micro array

8/6/2019 Chelvarajan - Leukocyte Biol 06 - Aging Micro Array

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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.

1314 Journal of Leukocyte Biology Volume 79, June 2006 0741-5400/06/0079-1314 Society for Leukocyte Biology


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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

Chelvarajan et al. Macrophages from aged mice are defective and have increased p38 MAPK 1315


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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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chelvarajan - leukocyte biol 06 - aging micro array

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