Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0106024 on April 7, 2006

Published online before print April 7, 2006
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(Journal of Leukocyte Biology. 2006;79:1314-1327.)
© 2006 by Society for Leukocyte Biology

Molecular basis of age-associated cytokine dysregulation in LPS-stimulated macrophages

R. Lakshman Chelvarajan*,{dagger}, Yushu Liu{ddagger}, Diana Popa*, Marilyn L. Getchell*,§, Thomas V. Getchell*,§,¶, Arnold J. Stromberg{ddagger} and Subbarao Bondada*,{dagger},1

* Sanders Brown Center on Aging and Departments of
{dagger} Microbiology, Immunology & Molecular Genetics,
{ddagger} Statistics,
§ Anatomy and Neurobiology, and
Physiology, University of Kentucky, Lexington

1Correspondence: Sanders Brown Center on Aging, Room 329, University of Kentucky, Lexington, KY 40536. E-mail: bondada{at}uky.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aged humans and rodents are susceptible to infection with Streptococcus pneumoniae bacteria as a result of an inability to make antibodies to capsular polysaccharides. This is partly a result of decreased production of proinflammatory cytokines and increased production of interleukin (IL)-10 by macrophages (M{Phi}) from aged mice. To understand the molecular basis of cytokine dysregulation in aged mouse M{Phi}, a microarray analysis was performed on RNA from resting and lipopolysaccharide (LPS)-stimulated M{Phi} from aged and control mice using the Affymetrix Mouse Genome 430 2.0 gene chip. Two-way ANOVA analysis demonstrated that at an overall P < 0.01 level, 853 genes were regulated by LPS (169 in only the young, 184 in only the aged, and 500 in both). Expression analysis of systematic explorer revealed that immune response (proinflammatory chemokines, cytokines, and their receptors) and signal transduction genes were specifically reduced in aged mouse M{Phi}. Accordingly, expression of Il1 and Il6 was reduced, and Il10 was increased, confirming our previous results. There was also decreased expression of interferon-{gamma}. Genes in the Toll-like receptor-signaling pathway leading to nuclear factor-{kappa}B activation were also down-regulated but IL-1 receptor-associated kinase 3, a negative regulator of this pathway, was increased in aged mice. An increase in expression of the gene for p38 mitogen-activated protein kinase (MAPK) was observed with a corresponding increase in protein expression and enzyme activity confirmed by Western blotting. Low doses of a p38 MAPK inhibitor (SB203580) enhanced proinflammatory cytokine production by M{Phi} and reduced IL-10 levels, indicating that increased p38 MAPK activity has a role in cytokine dysregulation in the aged mouse M{Phi}.

Key Words: inflammation • microarray • p38 MAPK


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 pneumoniae, which lead to pneumonia, are a major cause of morbidity 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 pneumococcal 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 B cells and macrophages (M{Phi}), and our recent studies suggest the defects in the aged are in part a result of deficiencies in M{Phi} 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{Phi} are defective in supporting B cell responses from young mice to polysaccharide antigens [7 8 9 ]. 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{Phi} 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{Phi} 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{Phi} from young but not the aged mice are able to support young mouse B cells to produce antipolysaccharide antibodies [1 ]. In this system, we showed that the main function of M{Phi} 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 aged produced equivalent levels of antipolysaccharide responses. A major reason for the inability of M{Phi} 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 {alpha} (TNF-{alpha}) were also produced at lower levels by M{Phi} from the aged in comparison with young mice. It is interesting that the M{Phi} from the aged were not defective in IL-10 production but produced more of this cytokine than M{Phi} from the young. Thus, the cytokine production was dysregulated in M{Phi} from the aged.

Similar defects in production of IL-1, IL-6, TNF-{alpha}, M{Phi}-inflammatory protein (MIP)-1{alpha} and MIP-1ß by M{Phi} from aged mice have been noted by several other researchers [14 15 16 17 ]. Defects in M{Phi} 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{Phi} from aged mice have been shown to produce more cyclooxygenase-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{Phi} from aged mice to LPS. However, this does not explain increased IL-10 production in the aged—a cytokine not examined by Renshaw et al. [15 ]. Conversely, Boehmer et al. [20 , 21 ] found a decrease in production of TNF-{alpha} and IL-6 by thioglycollate-elicited peritoneal M{Phi} or splenic M{Phi} from the aged but did not find a reduction in TLR4 expression. Instead, they attributed the reduced cytokine production to a decrease in c-jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) activation in M{Phi} from the aged. Few studies have measured the effect of age on M{Phi} in a comprehensive manner.

To determine if cytokine dysregulation in the splenic M{Phi} from aged mice extends to other proinflammatory cytokines and chemokines and to understand the molecular basis of the altered function of M{Phi} from aged mice, we performed a microarray analysis of genes expressed in M{Phi} from young and aged mice stimulated with LPS. Despite many microarray studies on M{Phi} [22 23 24 25 26 ], few studies focused on splenic M{Phi}, and none have compared the transcriptional profile of M{Phi} as a function of age. Our study is thus unique in that it provides novel information about gene expression in splenic M{Phi} from young and the aged mice. This allowed us to test several hypotheses to interpret the age-associated dysfunction in M{Phi}. This analysis confirmed and extended to the gene level our findings about cytokine dysregulation and showed that several other proinflammatory cytokines and chemokines were decreased in the aged, suggesting that decreased TLR signaling may be involved in the M{Phi} defect. It is most interesting that the microarray data showed that the expression of the gene encoding for p38{alpha} MAPK was increased dramatically in the M{Phi} 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{Phi} from aged mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Female young (4 months old) and aged (20–22 months old) BALB/c mice were obtained from the colonies of the National Institute on Aging, National Institutes 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 University 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 ß-actin was obtained from Sigma Chemical Co. (St. Louis, MO). Gel-purified LPS (Escherichia 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 antibody-coupled magnetic beads (Miltenyi Biotec, Bergisch Gladbachuor, Germany). The purified cells were found to be routinely 90–95% CD11b-positive when tested by flow cytometry. For each experiment, the CD11b-positive cells were 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{Phi} (10x106) were cultured in triplicate in Iscove/F12 medium + 10% fetal calf serum at 37ºC in 5% CO2 at a density of 1 x 106/ml. Cultures were stimulated with 1 µg/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 (Molecular 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 manufacturer’s protocol (Qiagen, Valencia, CA). Total RNA 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 microarray 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 µg total RNA was used for the amplification and labeling reactions. Out of this, 20 µg 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 = µ + {alpha}i + ßj + {gamma}ij + {epsilon}ijk was applied, where yijk is the log-transformed expression level of the kth chip in the ith age and the jth LPS. Furthermore, µ represents the grand mean expression, {alpha}i is the effect as a result of age [aged (A) and young (Y)], ßj is the effect as a result of the LPS [medium (M) and LPS (L)], {gamma}ij is the interaction effect between age and LPS, and {epsilon}ijk is an error term, which is assumed to be normally distributed with mean 0 and variance {sigma}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 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.


Figure 2
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  		Figure 2. A Venn diagram of the genes regulated by LPS in M{Phi} 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.

 
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 Infectious Disease, NIH.

Real-time reverse transcriptase-polymerase chain 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 TaqMan 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 done in duplicate.

Cytokine analysis
M{Phi} (0.25x106) were cultured in duplicate for 1 day in 1 µg/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- {alpha} 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 x 106 M{Phi} 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)aminomethane-HCl, 2% SDS, and 100 mM -mercaptoethanol at 65°C 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 ß-actin (e.g., for p38 MAPK).

Statistical analysis
Student’s t test was used to evaluate the significance of the differences among means in Western blots, ELISA data, and PCR data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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 analysis determined that 9894 probe sets were regulated differentially 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{Phi} from the aged and young. These genes had low constitutive levels of expression and upon stimulation, were of comparable intensities in M{Phi} from young and aged. Genes, which were present at low intensities in unstimulated M{Phi} but were induced by LPS in only M{Phi} from young mice or from aged mice, are depicted in Regions B and E, respectively. The expression of genes depicted in Regions C and D were not modulated by LPS. Those in Region C were significantly higher in M{Phi} from young mice, and those in Region D were higher in M{Phi} 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)-associated 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-{gamma} (Ppar{gamma}) and CC chemokine ligand 24 (Ccl24)].


Figure 1
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  		Figure 1. A gene tree of the probe sets from the overall P < 0.01 list. A microarray analysis was performed on M{Phi} from young and aged mice cultured for 6 h with LPS. For each treatment, RNA was obtained from triplicate M{Phi} 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 A–F, 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.

 
Analysis of the effect of age on genes affected by LPS stimulation
To begin the task of identifying the key genes involved in the differential response between M{Phi} from aged and young, probe sets with low mean intensities ≤200 in all four treatments (i.e., young±LPS and aged±LPS) were eliminated, and then a criterion of at least a twofold change was used for all pair-wise comparisons (i.e., LPS and/or age). The remaining probe sets were then divided further into those that were uniquely regulated in M{Phi} 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{Phi} from the young and aged (169 and 184 genes, respectively), and the remainder (500) was regulated 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{Phi} from young and aged, and 297 were decreased in both groups of M{Phi}. 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{Phi} secreted lower amounts of the proinflammatory cytokines IL-1ß, IL-6, IL-12, and TNF-{alpha} but higher amounts of IL-10 than M{Phi} from the young [1 ]. These cytokines were also affected similarly in the microarray analysis (i.e., reduced amounts of mRNA for IL-1ß, IL-6, IL-12, and TNF-{alpha} and increased levels of IL-10 mRNA). Thus, the altered pattern of cytokine secretion observed with M{Phi} from the aged is also reflected at the steady-state levels of mRNA for these cytokines and therefore, validating 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{Phi} 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.


Figure 3
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  		Figure 3. Microarray data validate previous data obtained by ELISA. The RNA extracted from M{Phi} stimulated for 6 h with LPS was analyzed by microarray; data are shown for pro- and anti-inflammatory cytokines (left column). The corresponding amounts of IL-1ß, -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 ELISA data are representative of at least six independent experiments.

 
EASE: cytokine and chemokine subset
To identify functional categories of genes that could be responsible for the difference in cytokine secretion, we divided the probe 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 immune-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{Phi} from aged when compared with M{Phi} 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{Phi} from the aged, a few, such as Ccbp2, are expressed at considerably higher amounts in M{Phi} from the aged, before and after LPS stimulation. A number of genes [e.g., CXC chemokine ligand 16 (Cxcl16)] were higher in M{Phi} from the young, and this difference remains even upon LPS stimulation. A number of genes, such as Ccl24, were actually turned off by LPS.


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  		Table 1. Expression of Key Cytokine and Chemokine Genes

 
The microarray data show that in addition to the cytokines identified by us by ELISA assays, IFN-{gamma}, CSF-1, GM-CSF, and bone morphogenetic protein-1 (BMP-1; Ifng, Csf1, Csf2, and 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 mechanisms 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-1ß in the aged [29 ]. Increased expression of these cytokines needs to be verified further 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 keratinocyte-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{Phi}, 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 affect 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-{gamma}) could be secondary to the reduction in IFN-{gamma}, as they are all induced by IFN-{gamma}. 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-stimulated M{Phi} from the aged
As stimulation with LPS led to a differential regulation of cytokine genes in M{Phi} 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{Phi} from the young than those from the aged [e.g., TNF-receptor-associated factor 6 (Traf6), Cd14, Rel, and Relb; Table 2 ]. However, a relatively small group of TLR components was up-regulated considerably in M{Phi} from aged mice, resulting in higher levels of expression than in M{Phi} from young (e.g., Mapk14). Table 2 contains a partial list of key components of the TLR pathway affected in M{Phi} from the aged. The p100, p105, and p65 subunits of NF-{kappa}B (Nfkb2, Nfkb1, and Rela) together with other components of the pathway were also reduced in M{Phi} from the aged stimulated with LPS.


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  		Table 2. Expression of Key Components of the TLR Pathway

 
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 are critical for activation of the NF-{kappa}B pathway, in M{Phi} 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-{kappa}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 expression may have a role in the altered response of M{Phi} from the aged to LPS [31 ]. As these two genes did not fit the general pattern of TLR genes (i.e., reduction in M{Phi} from the aged), they were analyzed further by real-time RT-PCR, which validated the microarray data (Fig. 4) .The reduction in the components of NF-{kappa}B signaling pathway explains the reduction in production of several proinflammatory cytokines and chemokines in LPS-stimulated M{Phi} from the aged, as this pathway is known to be critical for the production of such soluble mediators. This is consistent with a global defect in the responses of M{Phi} 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).


Figure 4
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  		Figure 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{Phi} 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 18S RNA, also obtained from real-time PCR. Means identified by the same symbol were statistically different (P<0.05).

 
Effect of aging on the MAPK pathway
LPS-induced cytokine secretion from M{Phi} has been shown to be dependent on activation of the MAPKs: ERK1/2 (Mapk3 and Mapk1), JNK1/2 (Mapk8 and Mapk9), and p38{alpha} MAPK (Mapk14). The microarray data did not find significant changes in the ERK and JNK levels, but the level of p38{alpha} MAPK mRNA was enhanced significantly in resting and LPS-stimulated M{Phi} 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{Phi}. The Western blot demonstrated that levels of total p38 MAPK protein (normalized to ß-actin levels to correct for differences in protein loading) were increased in M{Phi} from the aged (Fig. 5B and 5C) . We then verified that M{Phi} from the aged also had increased amounts of phospho-p38 MAPK, the functionally active form of p38 MAPK (Fig. 5B and 5D) . In this figure, phospho-p38 levels were normalized to ß-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.


Figure 5
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  		Figure 5. M{Phi} from the aged have higher amounts of p38 MAPK. Data from microarray show that M{Phi} from the aged have more p38 MAPK mRNA (A). M{Phi} were rested at 37°C 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 ß-actin (B). Total p38 MAPK was normalized to ß-actin (C), and phosphorylated p38 MAPK was normalized to ß-actin (D). In this panel, the data points come from two different experiments, yielding a total of six values for each treatment with the exception of M{Phi} from the aged + LPS, which only had four values. Means identified by the same symbol are statistically different (P<0.05).

 
The microarray study indicated that dual-specificity phosphatase (DUSP)-10 (or MAPK-activating protein kinase 5; gene symbol is Dusp10), a newly discovered DUSP, was present in higher amounts in the M{Phi} 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{Phi} from aged mice did indeed have an increased expression of Dusp10 (data not shown). To determine if the increased phospho-p38 MAPK could play a role in the altered pattern of cytokine secretion of M{Phi} from the aged SB203580, a well-characterized inhibitor to p38 MAPK was used. Inhibiting p38 MAPK in M{Phi} 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-{alpha} at low doses of SB203580 (Fig. 6 B and C , and data not shown). This increase was found in cultures of M{Phi} from both age groups. The dose-response curve showed a steady inhibition of IL-10 synthesis with an increasing dose of SB203580, and TNF-{alpha} 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 threshold level of p38 MAPK is required for the synthesis of all cytokines, but after a certain level, it is inhibitory for the pro- but not anti-inflammatory cytokines.


Figure 6
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  		Figure 6. Inhibiting p38 MAPK enhances production of proinflammatory cytokines and suppresses production of anti-inflammatory cytokines. M{Phi} were cultured in duplicate with LPS and various amounts of S203580, a p38 MAPK inhibitor, for 24 h. The supernatant was then assayed in triplicate by ELISA for IL-10 (A), TNF-{alpha} (B), and IL-12 (C). Student’s t-test was performed with means identified by the same symbol. These data are representative of three independent experiments.

 
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{Phi} from the aged had slightly reduced amounts of total protein levels of ERK1/2 (Fig. 7A and 7B ). However, when these blots were probed for phosphorylated forms of ERK, there was a dramatic reduction in phoshpho-ERK1/2 in M{Phi} from the aged before and after stimulation with LPS (Fig. 7A and 7C) . This reduction in ERK may also contribute to the decreased cytokine production in the aged.


Figure 7
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  		Figure 7. M{Phi} 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 ß-actin. For this determination, duplicate cultures were pooled. (C) The amounts of phospho-ERK1/2 normalized to total ERK1/2 are shown. Means identified by the same symbol are statistically different (P<0.05). These data are representative of two independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The primary goal of this microarray study was to understand the molecular basis of cytokine dysregulation in M{Phi} 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{Phi} from the aged than previously appreciated and that the phenotype of M{Phi} from the aged does not fit into the known patterns of M{Phi} 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 , 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-{gamma}, CCL24, and CCR1 (gene symbols are Ppar{gamma}, 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 basophils to the sites of inflammation, and its suppression may control the type of inflammatory response to be induced. Similarly, PPAR-{gamma} has been shown to inhibit production of several inflammatory mediators such as TNF-{alpha}, IL-1, IL-6, and inducible nitric oxide synthase (NOS) in M{Phi}, 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-{gamma} was similar in both age groups, eliminating it as a possible candidate for the differential production of cytokines by M{Phi} from the young and aged mice. Moreover, the microarray study has allowed us to eliminate the possibility that elevated production of inhibitory cytokines, including TGF-ß (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{Phi} from the aged, as none of these genes was expressed at elevated levels in M{Phi} from aged in comparison with young mice [36 37 38 ].

M{Phi} heterogeneity has been recognized recently, and an imbalance in M{Phi} subsets could be a reason for the difference between the young adult versus the aged. First, M{Phi} have been subdivided into M-1 and M-2 phenotypes depending on their ability to produce NO and proinflammatory cytokines (M-1 type) or anti-inflammatory agents such as IL-1RA and arginase (M-2 type), suggesting a possibility that one of these types of M{Phi} 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{Phi}, were reduced in M{Phi} from the aged (data not shown). Second, resident alveolar M{Phi} 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{Phi} from the aged are unlike the alveolar M{Phi} from young adult mice, as they do not produce IL-10 constitutively, and TLR4 ligands do not overcome their defects in proinflammatory cytokine production. Third, they are also distinct from the anti-inflammatory M{Phi} from the intestine, which neither express CD11b (and many other M{Phi} cell surface receptors) nor produce IL-1, IL-10, and IL-12, whereas the M{Phi} from the aged are CD11b+ve and produce IL-10 in excess [41 ]. Fourth, it has also been shown that M{Phi} 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{Phi} in the aged have markers of IL-4 activation. Our cytokine expression pattern and the gene expression analysis have shown that splenic M{Phi} from the aged do 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 increased IL-10 and decreased production of IL-12 with no effect on TNF-{alpha} production. As the aged have been shown to have increased autoantibodies, it is plausible that M{Phi} from the aged are responding as if they have encountered immune complexes. This is unlikely, as our microarray study has shown that M{Phi} from the aged exhibit decreased expression of most proinflammatory cytokines and chemokines, such as Il1b, Il6, and Tnf (Table 1) . Thus, the studies from our laboratory have identified a uniquely hyporesponsive M{Phi} in spleens from the aged, which has profound influences on immune responses to polysaccharide antigens and may affect the overall ability of the aged to generate an inflammatory response necessary to contain infections.

Although the response of M{Phi} 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 of Tlr2, is comparable with that of M{Phi} 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-{kappa}B pathway, such as Rel-a, Rel-b, NF-{kappa}B p50 and p52, and TRAF6 (Myd88, Rela, Relb, Nfkb1, Nfkb2, and Traf6), were also reduced in the aged suggests that the TLR-dependent pathway is working at a significantly reduced efficiency. It is interesting that the NF-{kappa}B-independent pathway is also reduced, as components of this pathway, such as TANK-binding kinase 1 and IRF-1 (Tbk1 and Irf1), are reduced in LPS-stimulated M{Phi} (Table 2) . Superimposed on the reduction of these TLR signaling pathway intermediates needed for positive signaling, levels of IRAK-M (Irak3), a known negative regulator of this pathway, are enhanced in M{Phi} 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 secretion from M{Phi} from the aged. Presently, it is unclear why so many components of the TLR signaling pathway intermediates are 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 several of these genes in the aged.

The most surprising finding of our microarray study is the significant increase in the levels of p38{alpha} MAPK in M{Phi} 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 unchanged. 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 of the enzyme was also elevated in M{Phi} 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 activated 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 proliferation capability of fibroblasts, whereas expression of constitutively 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{Phi} are in contrast to the results of Boehmer et al. [20 , 21 ], who found a decrease in the total and phosphorylated forms of p38 MAPK. The discrepancy could be a result of the use of thioglycollate-induced peritoneal M{Phi} in one study by these authors [20 ]. Although splenic M{Phi} 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-{alpha}, IL-1, and IL-6 in numerous studies and in many different cell types such as monocytes, M{Phi}, and dendritic cells (DC) [47 , 48 ]. Moreover, 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{Phi}. 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 studies. Although the in vivo data clearly establish the anti-inflammatory 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-{alpha}, 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 enhance proinflammatory 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 levels of this enzyme are required for production of these cytokines, 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-{alpha} 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 MAPK does not appear to have any negative effect on IL-10 production, as IL-10 levels were decreased in a dose-dependent manner with the p38 inhibitor.

We have shown previously that the altered pattern of cytokine production in M{Phi} 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{Phi} from young mice. In this study, we see that by partially suppressing p38 MAPK activity in M{Phi} from aged mice, a reduction in IL-10 occurred, with a concomitant enhancement of proinflammatory cytokine production, 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{Phi} from aged mice is a result of the higher amount of p38 MAPK activity in M{Phi} from aged mice.

Unlike p38 MAPK, levels of ERK1/2 were similar in M{Phi} from both age groups. However, levels of phosphorylated ERK1/2 were reduced significantly in M{Phi} 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-{alpha} and for LPS + immune complexes, induced production of IL-10 from M{Phi} [44 , 55 , 56 ]. Conversely, Dillon et al. [57 ] found that ERK activation 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{Phi} 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-{alpha}, as all of these cytokines are reduced in M{Phi} from the aged, which have dramatically reduced 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 production such as that seen in LPS-stimulated M{Phi} 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 assessing the levels of functionally active JNK in M{Phi} from the aged, as JNK has been shown to have positive and negative roles in cytokine secretion by M{Phi} [47 , 59 ]. Another question that remains is about the mechanisms that are responsible for p38 MAPK up-regulation in M{Phi} from the aged, as in most inflammatory 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{Phi} before and after LPS stimulation in comparison with the young M{Phi}. 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{Phi}.

In summary, our microarray analysis has shown that M{Phi} from 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 increased, such that the splenic M{Phi} in the aged have an anti-inflammatory phenotype. We find that the aged mouse M{Phi} have a unique phenotype, which is distinct from the currently known modes of M{Phi} regulation. The aging signature for M{Phi} includes an elevation of proliferation-specific genes. Our preliminary, immunohistochemistry studies with bromodeoxyuridine labeling show that indeed, there are more proliferating cells in the spleens of aged mice. Currently, we are investigating the relation between this proliferation phenotype and the cytokine dysregulation phenotype in M{Phi} 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{Phi} from the aged—cytokines that are important for B cell responses.


   ACKNOWLEDGEMENTS
 
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 Institute’s 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.

Received January 12, 2006; revised February 11, 2006; accepted February 20, 2006.


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 DISCUSSION
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