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(Journal of Leukocyte Biology. 2000;68:21-30.)
© 2000 by Society for Leukocyte Biology

Age-associated increase of basal corticosterone levels decreases ED2high, NF-{kappa}Bhigh activated macrophages

Takako Kizaki*, Tomomi Ookawara{dagger}, Kazuya Iwabuchi{ddagger}, Kazunori Onoé{ddagger}, Noorbibi K. Day§, Robert A. Good§, Naoki Maruyama||, Shukoh Haga#, Nobuo Matsuura**, Yoshinobu Ohira{dagger}{dagger} and Hideki Ohno*

* Department of Hygiene, Kyorin University, School of Medicine, Mitaka, Tokyo;
{dagger} Department of Hygiene, National Defense Medical College, Tokorozawa, Saitama;
{ddagger} Section of Pathology, Institute of Immunological Science, Hokkaido University, Sapporo, Hokkaido;
|| Department of Molecular Pathology, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo;
# Institute of Health and Sport Sciences, University of Tsukuba, Tsukuba, Ibaragi;
** Department of Pediatrics, School of Medicine, Kitasato University, Sagamihara, Kanagawa;
{dagger}{dagger} Department of Physiology and Biomechanics, National Institute of Fitness and Sports, Kanoya, Kagoshima, Japan; and
§ Department of Pediatrics, All Children’s Hospital, St. Petersburg, Florida

Correspondence: Takako Kizaki, Ph.D., Department of Hygiene, Kyorin University, School of Medicine, Mitaka, Tokyo 181-8611, Japan. E-mail: kizaki{at}kyorin-u.ac.jp


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ABSTRACT
 
The proportion of cells with a high density of ED2 (ED2high cells) in peritoneal cells from old rats was significantly lower than that from young rats. The expression of major histocompatibility complex class II (MHC class II) molecules, the antigen presentation, production of interleukin (IL)-1ß and IL-6, and nuclear factor-{kappa}B activity in ED2high cells were markedly higher than those in cells with a low density of ED2 (ED2low cells), although no significant difference was observed in the expression of MHC class II molecules and the antigen presentation between ED2high cells from young and old rats. Meanwhile, basal corticosterone concentration in serum and glucocorticoid (GC) receptor mRNA expression in peritoneal cells increased significantly in old rats. The proportion of ED2high cells was increased by adrenalectomy in young rats. Furthermore, nuclear translocation of GC receptor was observed in ED2low cells, whereas GC receptor was detected in cytoplasmic extracts from ED2high cells. These results suggest that the decrease in functional ED2high macrophages with age results in the age-associated decline of immune responses, which is regulated, in part, by the basal GC concentration.

Key Words: antigen presentation • cytokines • transcription factors • immunomodulators


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INTRODUCTION
 
The cells of the monocyte/macrophage lineage are capable of expressing a large number of cell surface molecules and play a central role in the induction and regulation of both specific and nonspecific immune responses [1 , 2 ]. The expression of major histocompatibility complex (MHC) class II molecules for antigen presentation and the production of various cytokines by activated macrophages are essential requisites for the development of subsequent specific immune responses [3 ]. Studies on age-associated changes in the MHC class II expression, in the ability to present antigen, and in the production of cytokines have yielded conflicting results. For example, Rich et al. [4 ] have shown that expressions of MHC class II molecules of monocytes from elderly and young subjects are similar, but that monocytes from the elderly have a reduced accessory function for phytohemagglutinin (PHA)-stimulated T cells from young control subjects. Vetvicka et al. [5 ] also detected decreased ability in antigen presentation by macrophages from aged A/J mice. Higashimoto et al. [6 ] have reported, on the contrary, that MHC class II expression of the alveolar macrophages is greater in old mice than in young mice, suggesting that the ability to present antigen in the alveolar macrophages does not decline with age. In addition, several studies reported a significant decline of interleukin (IL)-1 and/or IL-6 production with age [7 , 8 ], whereas others reported no changes or even increases in these cytokines [9 , 10 ].

Meanwhile, ED2 molecules are expressed on bone marrow macrophages in erythropoietic clusters, thymic cortical macrophages, liver Kupffer cells, and splenic red-pulp macrophages, although they are not expressed on monocytes, dendritic cells, lymphocytes, or granulocytes [11 ]. Moreover, the ED2 antibody immunoprecipitates a molecule presumably involved in cell-cell and/or cell-matrix interactions and thus is used as a differentiation marker of macrophages [12 ]. Effect of aging on the expression of this molecule, however, is not presently elucidated.

Transcriptional activation of genes encoding these cytokines and cell-surface molecules would be critical in activation of the immune systems. Nuclear factor {kappa}B (NF-{kappa}B) plays an important role in coordinately controlling some of these gene expressions during monocyte/macrophage activation. At least 10 genes are candidates for the coordinate induction by NF-{kappa}B in monocytes/macrophages: the genes encoding IL-1, IL-6, tumor necrosis factor {alpha} (TNF-{alpha}), macrophage colony-stimulating factor (CSF), granulocyte CSF, granulocyte-macrophage CSF, tissue factor, IL-2 receptor {alpha}-chain, chemotactic protein MCP-1/JE, and nitric oxide synthase [13 ]. A major form of NF-{kappa}B is composed of a dimer of p50 and p65 subunits [14 , 15 ]. NF-{kappa}B is constitutively present in the cytoplasm but is kept inactive by association with inhibitors (the I-{kappa}B family) [13 , 16 ]. Upon exposure to inflammatory stimuli such as lipopolysaccharide (LPS), I-{kappa}B is rapidly degraded, allowing NF-{kappa}B to translocate into the nucleus and to induce transcription by binding itself to defined promoter elements.

On the other hand, glucocorticoid (GC) has been used for decades as a clinical tool to suppress both the immune response and the processes of inflammation [17 18 19 ]. GCs function by binding to specific cytoplasmic receptors, allowing the complexes to translocate into the nucleus and to affect gene transcription either positively or negatively. GCs exert their potent immunosuppressive action through inhibition of the synthesis of cytokines such as IL-1, IL-6, and TNF, and of cell surface molecules by interfering with the activity of transcription factors such as NF-{kappa}B and AP-1 [20 ]. A number of scientists have investigated the mechanisms of GC receptor-mediated transcriptional repression through the use of various cell lines and synthetic GC, dexamethasone (DEX) [21 22 23 24 25 26 27 ]. In vivo effects of circulating GCs on the immune system, however, are not fully understood.

Sapolsky [28 ] analyzed in detail the data obtained from numerous studies that examined the change of GC concentration in association with age, and concluded that there was a marked increase in circulating corticosterone concentrations with age throughout the circadian cycle. In this study, we thus investigated the effects of aging on functions and cell-surface phenotypes of peritoneal cells in monocyte/macrophage lineage. The role of GC in the age-related changes of immune functions was also examined.


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MATERIALS AND METHODS
 
Animals and in vivo treatment
Male Wistar rats were obtained from a specific-pathogen-free colony (Japan SLC, Shizuoka, Japan), and held after arrival under a specific-pathogen-free condition for at least 1 week for young rats or for 22–24 months for old rats. Experiments were performed using these rats between 8 and 10 weeks of age (young rats) or between 22 and 24 months of age (old rats). Animals were housed in groups of five or six at 25°C with a 12-h light/dark cycle. Food and water were available ad libitum. The animals were cared for in accordance with the Guiding Principles for the Care and Use of Animals approved by the Council of the Physiological Society of Japan, based upon the Declaration of Helsinki, 1964. Bilateral adrenalectomies were performed through dorsal incisions while rats were under pentobarbital sodium (Nembutal) anesthesia. Sham-operated rats were exposed to the same surgical procedure, except that the adrenal glands were left intact. Adrenalectomized (ADX) rats received 0.9% NaCl solution to drink after surgery. Peritoneal cells were collected from these rats 10 days after each surgery. Peritoneal cells were not used in the study if the donor rats appeared ill or if any pathological abnormalities were noted at the time of cell harvest.

Cell preparation and T cell proliferation assay
Peritoneal cells were harvested from young and old rats and washed three times with phosphate-buffered saline (PBS). Red blood cells (RBCs) were removed by osmotic lysis. Monocyte/macrophage population was isolated by excluding lymphocytes and granulocytes by forward and side scatter using a flow cytometer (FACStar PLUS, Becton Dickinson, Mountain View, CA). In some experiments, cells with a low density of ED2 (ED2low cells) and cells with a high density of ED2 (ED2high cells) were sorted from peritoneal monocyte/macrophage population of young and old rats. Purity of the sorted cell population was analyzed by flow cytometer (ED2high cells from young rats, ED2high cells > 96%, ED2low cells < 4%; ED2low cells from young rats, ED2high cells < 1%, ED2low cells > 99%; ED2high cells from old rats, ED2high cells > 92%, ED2low cells < 8%). These cells were suspended in PBS or in tissue culture medium consisting of RPMI 1640 (Life Technologies, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal calf serum (FCS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine (Life Technologies).

Control rats were immunized in the hind footpads with 100 µg of purified protein derivative of Mycobacterium tuberculosis (PPD, Japan BCG Laboratory, Tokyo, Japan) emulsified with CFA (Sigma Chemical, St. Louis, MO). After 10 days, popliteal draining lymph nodes were excised, and a single-cell suspension was prepared by gently passing the homogenized organ through a nylon mesh (64 µm; Abe Chemical, Chiba, Japan). Lymph node cells were then applied on a nylon wool column. Nonadherent cells were collected after incubation at 37°C for 1 h. Cell viability was assessed by a trypan blue dye exclusion test. Cells were then suspended in the tissue culture medium. Peritoneal monocytes/macrophages, ED2high cells, or ED2low cells (5 x 103) were added to the culture in which the T-enriched lymph node cells (4 x 105) from PPD immunized rats were stimulated with PPD (100 µg/mL). Three days later, 0.5 µCi/well [3H]thymidine ([3H]TdR; New England Nuclear, Boston, MA) was added for the final 8 h of culture. Counts per minute (cpm) were determined in a liquid scintillation counter (Aloka, Tokyo, Japan). Results are expressed as [3H]TdR incorporation by cells.

Immunofluorescence staining and flow cytometry
Monoclonal antibodies (mAb) used in this study include ED1 (mouse IgG1 directed against rat monocytes/macrophages [29 , 30 ]), ED2 (mouse IgG1 against rat resident macrophages [29 , 31 ]), ED3 (mouse IgG2a against rat sialoadhesin [29 , 32 , 33 ]), ED8 (mouse IgG1 against rat CD11b/CD18 [34 ]), and phycoerythrin (PE)-conjugated OX6 (mouse IgG1 against rat MHC class II [35 ]; Serotec, Oxford, UK). Flow cytometric analysis was carried out as described previously [36 ] using a FACSCalibur (Becton Dickinson). Peritoneal cells were treated with mAb (ED1, ED2, ED3, or ED8) and then stained with fluorescein isothiocyanate-labeled goat anti-mouse Ig antibody (Ab; Serotec). In some experiments, the cells stained with ED2 were treated with PE-conjugated mAb OX6 (Serotec). Data were illustrated by CELL Quest software (Becton Dickinson Immunochemistry Systems).

Monokine production assay
Cells (1 x 105) were cultured either with medium alone or with 1 µg/mL LPS (Escherichia coli 005:B5, Difco, Detroit, MI) at 37°C for 48 h. Concentrations of IL-1ß or IL-6 in these culture supernatants were quantified with a rat IL-1ß or IL-6 enzyme-linked immunosorbent assay (ELISA) kit (Bio Source International, Camarillo, CA), respectively, according to the manufacturer’s protocol.

Preparation of cytoplasmic and nuclear extracts
Cells were washed twice with ice-cold PBS, and resuspended with lysis buffer [10 mM HEPES-KOH, pH 7.8, 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.5% Nonidet P-40, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 2 µg/mL aprotinin]. The tubes were vortexed, and nuclei were sedimented by centrifugation at 5,000 rpm for 5 min. Aliquots of the supernatant were stored at -80°C (cytoplasmic extract). The nuclear pellet was washed with buffer (250 mM sucrose, 10 mM HEPES-KOH, pH 7.8, 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, and 2 µg/mL aprotinin). After centrifugation at 5,000 rpm for 5 min, the nuclear pellet was suspended in extraction buffer (50 mM HEPES-KOH, pH 7.8, 420 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 20% glycerol, 1 mM DTT, 0.5 mM PMSF, and 2 µg/mL aprotinin), and then rotated at 5°C for 30 min. The nuclear proteins were isolated by centrifugation at 15,000 rpm for 15 min. Protein concentration was determined by Bradford assay (Bio-Rad, Hercules, CA) and stored at -80°C until used.

Electrophoretic mobility shift assay (EMSA)
The NF-{kappa}B oligonucleotide probe (5’-AGT TGA GGG GAC TTT CCC AGG-3’) was purchased from Promega (Madison, WI) and the GC receptor oligonucleotide probe (5’-GAC CCT AGA GGA TCT GTA CAG GAT GTT CTA GAT-3’), the mutant NF-{kappa}B oligonucleotide probe (5’-AGT TGA GGC GAC TTT CCC AGG-3’), and the mutant GC receptor oligonucleotide probe (5’-GAC CCT AGA GGA TCT CAA CAG GAT CAT CTA GAT-3’) were from Santa Cruz Biotechnology (Santa Cruz, CA). Each probe was labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase (Bio Labs) and purified in Microspin columns (Pharmacia Biotech, Uppsala, Sweden). For EMSA, 1 µg nuclear proteins for NF-{kappa}B or 3 µg nuclear proteins for GC receptor were preincubated with EMSA buffer [10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, 4% glycerol, 1 µg poly (dI-dC)] at room temperature for 20 min and then added to the radiolabeled oligonucleotide probe. In some experiments, 100-fold molar excess of the unlabeled oligonucleotide probe was added as a competitor. The specificity of the binding reaction was examined by incubation of nuclear extracts with radiolabeled mutant NF-{kappa}B or mutant GC receptor oligonucleotide probe. The mixture was incubated at room temperature for 20 min and applied to a 7% polyacrylamide gel that had previously been electrophoresed at 100 V for 30 min. Gel was run at 100 V in TGE (5 mM Tris-HCl, pH 8.3, 38 mM glycine, 0.1 mM EDTA) followed by transference to 3MM Whatman paper, drying under vacuum at 80°C for 30 min, and quantification of the band intensities was then estimated according to an autoradiograph or analysis with a BAS 2000 imaging system (Fuji Photo Film Co., Kanagawa, Japan).

To identify the protein components of the NF-{kappa}B complex, 1 µL of specific polyclonal antibodies to p50 or p65 (Santa Cruz Biotechnology) were added to the nuclear extracts, and the mixture was then incubated at 4°C for 1 h before the radiolabeled oligonucleotide probe was added.

Western blot analysis
Cytoplasmic proteins (10 µg) or nuclear proteins (5 µg) were boiled in loading buffer [Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate (SDS), 5% glycerol, and 10% 2-mercaptoethanol], separated in 10% SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene difluoride membrane (Applied Biosystems, Foster City, CA). Membranes were blocked with 3% nonfat dried milk in Tris-buffered saline (TBS) for 1 h. Primary antibodies against NF-{kappa}B p65 and/or against I{kappa}B{alpha} (Santa Cruz Biotechnology), or against GC receptor (Affinity BioReagents, Heshanic Station, NJ) were applied at appropriate dilutions. After washing three times in TBS (25 mM Tris-HCl, 500 mM NaCl) containing 0.01% Tween 20 (TBS-T), secondary antibodies [horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, DAKO Japan, Kyoto] were applied at appropriate dilutions for analysis of NF-{kappa}B p65 or I{kappa}B{alpha}. For analysis of GC receptor, secondary antibodies (biotinylated-goat anti-mouse IgG) and HRP-conjugated streptavidin (DAKO Japan) were applied at appropriate dilutions. Membranes were washed three timese in TBS-T and incubated in enhanced chemiluminescence reagents (ECL, Amersham Life Sciences, Arlington Heights, IL).

Serum corticosterone assay
Blood samples were obtained by decapitation immediately after initial contact with the rats from 10:00 to 11:00 AM. Blood sampling from all rats was carried out within 30 min. Blood was allowed to clot for 1 h, and serum was obtained after centrifugation. Serum corticosterone concentration was determined by radioimmunoassay (Amersham Life Sciences). Serum was diluted 1:5 with borate buffer (0.02 M, pH 7.4) and incubated at 60°C for 30 min to denature corticosterone-binding proteins before the assay.

Reverse transcription polymerase chain reaction
Total cellular RNA was extracted by the guanidinium-isothiocyanate method from peritoneal exudate cells. Single-strand cDNA was synthesized with reverse transcriptase from 1 µg RNA and used for polymerase chain reaction (PCR). Primer sequences were as follows: glyceraldehyde-3-phosphate dehydrogenase (G3PDH), 5’ primer, CTC AAG ATT GTC AGC AAT GC, 3’ primer, CAG GAT GCC CTT TAG TGG GC; and GC receptor 5’ primer, TGC AGC AGT GAA ATG GGC AA, 3’ primer GGG AAT TCA ATA CTC ATG GTC. cDNAs were amplified by the PCR method under the following conditions: 94°C for 1 min, 55°C for 1.5 min, and 72°C for 1.5 min with 26 cycles for G3PDH or with 30 cycles for GC receptor. PCR products were separated by electrophoresis on a 4% acrylamide gel and visualized by ultraviolet illumination after being stained with ethidium bromide. The amplified fragments were confirmed to correspond to GC receptor mRNA by sequencing analysis.

Statistical analysis
Statistical tests were carried out on a group of young rats and a group of old rats or on replicate cultures of pooled samples derived from three to four young or old rats. When two means were compared, Student’s t test for unpaired samples was used. For more than two groups, the statistical significance of the data was assessed by analysis of variance. When significant differences were found, individual comparisons were made between groups with the use of the t statistic and adjusting the critical value according to the Bonferroni method [37 ]. Differences were considered significant at P < 0.05. Data are expressed as means ± SEM.


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RESULTS
 
Changes in functions of peritoneal monocytes/macrophages from old rats
When peritoneal cells were analyzed by forward and side scatter using a flow cytometer, no significant difference in the proportion of cells in monocyte/macrophage lineage was found between young and old rats (young, 72.1 ± 2.4%; old, 72.4 ± 1.9%). Cells of monocyte/macrophage lineage from young and old rats were analyzed for the expression of MHC class II molecules and their ability of antigen presentation. Figure 1A shows the data comparing typical profiles of the MHC class II expression on monocytes/macrophages from young and old rats. The proportion of cells stained with mAb OX6 (MHC class II+ cells) in monocytes/macrophages was significantly decreased in old rats (young, 67.5 ± 4.7%, n = 3; old rats, 41.7 ± 4.4%, n = 3, P < 0.05), suggesting that cells with the ability to present antigen to T cells decreased in old rats. Then, T-enriched lymph node cells were prepared from young rats immunized with PPD antigen and stimulated with PPD in the presence of monocytes/macrophages from young or old rats. As shown in Figure 1B , significant proliferations of T-enriched cells were observed in the presence of monocytes/macrophages from either young rats or old rats. However, the responses of T-enriched cells in the presence of monocytes/macrophages from old rats were significantly lower than those in the presence of monocytes/macrophages from young rats.



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  		Figure 1. Effects of aging on MHC class II expression and an ability of PPD antigen presentation in peritoneal cells from young and old rats. (A) Peritoneal cells from young (black line) and old (gray line) rats were stained with mAb OX6 and analyzed by flow cytometry after excluding lymphocytes and granulocytes by forward and side scatter. Negative control: dotted line, young: dotted line with wider spacing, old rat. Representative results from three rats each are shown. (B) Control rats were immunized in the hind footpads with 100 µg of PPD. After 10 days, T-enriched cells were prepared from popliteal lymph nodes with a nylon wool column. Monocytes/macrophages isolated from young or old rats (n = 3 each) by excluding lymphocytes/granulocytes with a flow cytometer were pooled and then added to the culture in which T-enriched lymph node cells were stimulated with PPD (100 µg/mL). Three days later, [3H]TdR incorporation in the final 8 h was determined for triplicate cultures. Results are expressed as mean incorporation (cpm) ± SEM; *P < 0.01.

We next examined the effects of aging on cytokine production by monocytes/macrophages. Monocytes/macrophages from young or old rats were stimulated with LPS, and the amounts of IL-1ß and IL-6 in the culture supernatants were quantitated. It is apparent from Figure 2 that monocytes/macrophages from old rats produced remarkably lower levels of IL-1ß and IL-6 upon stimulation with LPS as compared with those from young rats.



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  		Figure 2. Effects of aging on the production of IL-1ß and IL-6. Monocytes/macrophages isolated from pooled peritoneal cells of young or old rats (n = 3 each). (A) IL-1ß production by monocytes/macrophages from young and old rats. Cells were cultured in triplicate with 1 µg/mL of LPS or medium alone and IL-1ß in the culture supernatants was quantitated as described in Materials and Methods. (B) The IL-6 productions by monocytes/macrophages from young and old rats. Cells were stimulated with LPS as described above and IL-6 production in the supernatants was assayed. Results are expressed as mean concentration ± SEM; *P < 0.01.

Because it has been suggested that the state of NF-{kappa}B activity correlates with the state of monocyte differentiation [38 ], we analyzed the effects of aging on nuclear translocation of NF-{kappa}B in peritoneal cells. Nuclear proteins extracted from peritoneal cells separately harvested from each of the young and old rats were examined for the NF-{kappa}B DNA-binding activity using a radiolabeled NF-{kappa}B-specific probe. Radioactive DNA binding to each nuclear protein of three old rats was greatly reduced compared with that of three young rats (Fig. 3A ). Then, monocytes/macrophages isolated form pooled peritoneal cells of three young rats or three old rats were examined for EMSA, and similar results were obtained (Fig. 3B) . A 100-fold molar excess of unlabeled NF-{kappa}B oligonucleotides completely blocked the radiolabeled NF-{kappa}B binding (Fig. 3C , lane 2). To examine the specificity of the DNA-binding capability of the complexes generated by nuclear proteins from young rats, nuclear extract was incubated with radiolabeled mutant NF-{kappa}B oligonucleotide. As was expected, the shifted band was not observed (Fig. 3C , lane 3).



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  		Figure 3. Effect of aging on NF-{kappa}B activity in peritoneal cells from young and old rats. (A) NF-{kappa}B DNA binding activity in nuclear extracts of peritoneal cells from each of young (n = 3) and old (n = 3) rats was compared by EMSA. Each lane represents the result obtained from one rat. Representative results of three separate experiments are shown. (B) Monocytes/macrophages were isolated form pooled peritoneal cells of three young rats or three old rats and were examined for NF-{kappa}B activity. (C) The identity of NF-{kappa}B was investigated. Nuclear extracts of peritoneal cells from a young rat were incubated with radiolabeled NF-{kappa}B oligonucleotide probe in the absence (lane 1) or in the presence (lane 2) of a 100-fold molar excess of the unlabeled oligonucleotide. Nuclear extract from a young rat was also incubated with radiolabeled mutant NF-{kappa}B oligonucleotide probe (lane 3). (D) The nuclear extract was incubated with 1 µl of specific polyclonal antibodies to p50 or p65 for 1 h at 4°C before the incubation with radiolabeled oligonucleotide probe. Lane 1, without antibodies; lane 2, anti-p62; lane 3, anti-p50.

To analyze the subunit composition, EMSA was performed on nuclear extracts using NF-{kappa}B subunit-specific polyclonal antibodies. Nuclear extracts and DNA binding complex was totally retarded by anti-p50 (Fig. 3D , lane 3) and also partially by anti-p65 (lane 2). These data indicate the presence of p50 and p65 in the nuclear extracts from monocytes/macrophages from young rats and DNA complex.

Changes in cell-surface characteristics of peritoneal cells from old rats
To elucidate the mechanism underlying age-associated immunomodulation, we examined the cell-surface phenotypes of peritoneal cells from young and old rats. Figure 4 shows data comparing typical profiles of histogram of immunofluorescence staining patterns with mAb ED1, ED2, ED3, or ED8 of peritoneal cells between young and old rats. The expression pattern of cell-surface molecules stained with mAbs ED1, ED3, and ED8 appeared to be unaffected by aging. On the other hand, the profiles of peritoneal cells stained with mAb ED2 varied with aging. Two distinct cell populations (ED2high cells and ED2low cells) could be seen in both young and old groups, the proportion of the ED2high cells in peritoneal cells from old rats being approximately half of those from young rats (Fig. 4 , Table 1 ). We then stained peritoneal cells from young and old rats with both mAbs ED2 and anti-MHC class II mAb, OX6, and analyzed a correlation between the expressions of ED2 and MHC class II molecules. Two distinct cell populations (ED2highMHC class IIhigh cells and ED2lowMHC class II-/low cells) could be seen in young and old groups (Fig. 5A ). The ED2highMHC class IIhigh cell population was considerably greater in young rats than in old rats, although there was no significant difference in the mean intensity of MHC class II expression between ED2high cells from young and old rats (Table 2 ).



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  		Figure 4. Effects of aging on cell-surface characteristics of peritoneal cells. (A) Peritoneal cells from young (black line) and old (gray line) rats were stained with mAb ED1, ED2, ED3, or ED8 and analyzed by flow cytometry. Negative control: dotted line, young rats; dashed line, old rats. Representative results from four rats each are shown.


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  		Table 1. Effects of Aging on ED2high Cell Population



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  		Figure 5. MHC class II expression and PPD antigen presentation in ED2high or ED2low cells. (A) Two-color flow cytometric analysis of MHC class II and ED2 on peritoneal cells from young or old rats. Peritoneal cells were stained with mAbs ED2 and OX6 and analyzed by flow cytometry after excluding lymphocytes/granulocytes by forward and side scatter. Representative results from four rats each are shown. (B) Control rats were immunized in the hind footpads with 100 µg of purified protein derivative of PPD. After 10 days, T-enriched cells were prepared from popliteal lymph nodes using a nylon wool column. ED2high cells were prepared from three young and five old rats and were added to the culture in which T-enriched lymph node cells were stimulated with PPD (100 µg/mL). Results are expressed as mean incorporation (cpm) ± SEM; *P < 0.01. (C) ED2high or ED2low cells were prepared from peritoneal cells of four young rats and prolilferative responses of T-enriched lymph node cells to PPD were analyzed as described above. Results are expressed as mean incorporation (cpm) ± SEM; *P < 0.01.


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  		Table 2. Proportion and Mean Intensity of MHC Class II Expression of ED2low and ED2high Cells in Peritoneal Monocytes/Macrophagesa

Characterization of ED2high cells and ED2low cells
To elucidate a relationship between the depressed function and the decreased proportion of ED2highMHC class IIhigh cells in the peritoneal cells from old rats, we investigated functions of ED2high cells and ED2low cells. ED2high cells were purified from peritoneal cells of young and old rats and added to the cultures of T-enriched lymph node cells from rats immunized with PPD. Similar proliferative responses of the T-enriched cells were induced by either ED2high cells from young or old rats (Fig. 5B) . Next, ED2high cells and ED2low cells were purified from young rats and analyzed the efficiencies of antigen presentation. As shown in Figure 5C , ED2low cells did not induce considerable proliferative responses of the T-enriched cells to PPD.

Then, ED2high or ED2low cells were stimulated with LPS and the amounts of IL-1ß and IL-6 in the culture supernatants were quantitated. As shown in Figure 6 , ED2high cells produced markedly higher levels of IL-1ß and IL-6 upon stimulation with LPS than ED2low cells.



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  		Figure 6. The production of IL-1ß and IL-6 by ED2high or ED2low cells. ED2high or ED2low cells were prepared from peritoneal cells of four young rats. Representative results from two separate experiments are shown. (A) IL-1ß production by ED2high or ED2low cells. Cells were cultured in triplicate with 1 µg/mL of LPS or medium alone and IL-1ß in the culture supernatants was quantitated as described in Materials and Methods. (B) The IL-6 productions by ED2high or ED2low cells. Cells were stimulated with LPS as described above and IL-6 production in the supernatants was assayed. Results are expressed as mean concentration ± SEM; *P < 0.01.

Nuclear translocation of NF-{kappa}B in ED2high and ED2low cells was next analyzed. Radioactive DNA binding to nuclear proteins was observed to a greater extent in ED2high cells relative to ED2low cells (Fig. 7A ). The DNA binding was blocked by an unlabeled NF-{kappa}B oligonucleotide (Fig. 7A , right lane or Fig. 7B , lane 2). Radiolabeled mutant NF-{kappa}B oligonucleotide did not bind the nuclear extract (Fig. 7B , lane 3). Nuclear extracts and DNA binding complex was totally retarded by anti-p50 (Fig. 7B , lane 5) and also partially by anti-p65 (Fig. 7B , lane 6). These data are consistent with the results shown in Figure 3C and 3D .



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  		Figure 7. NF-{kappa}B activity in ED2high or ED2low cells. (A) NF-{kappa}B DNA binding activity in nuclear extracts of ED2high (H) or ED2low (L) cells was detected by EMSA. Unlabeled NF-{kappa}B oligonucleotide probe (competitor) was added (right lane). Representative results of three separate experiments are shown. (B) The identity of NF-{kappa}B was investigated. Nuclear extract of ED2high cells was incubated with radiolabeled NF-{kappa}B oligonucleotide probe in the absence (lane 1) or in the presence (lane 2) of a 100-fold molar excess of the unlabeled oligonucleotide. Nuclear extract of ED2high cells was incubated with radiolabeled mutant NF-{kappa}B oligonucleotide probe (lane 3). The nuclear extract was incubated with 1 µl of specific polyclonal antibodies to p50 or p65 for 1 h at 4°C before the incubation of radiolabeled oligonucleotide probe. Lane 4, without antibodies; lane 5, anti-p62; lane 6, anti-p50. (C) Relative amounts of cytoplasmic p65 and I-{kappa}B in ED2high or ED2low cells. Cytoplasmic extracts were prepared from whole peritoneal cells (W), ED2high cells (H), or ED2low cells (L) and subjected to Western blot analysis with anti-p65 and anti-I-{kappa}B antibodies. (D) Time course of cytoplasmic and nuclear p65 after stimulation with LPS. ED2high cells were stimulated with 1 µg/mL of LPS for 1, 3, and 24 h. Cytoplasmic and nuclear extracts were prepared and subjected to Western blot analysis with anti-p65 antibody.

We also analyzed the amount of NF-{kappa}B subunit p65 and NF-{kappa}B inhibitor I-{kappa}B in the cytoplasmic extracts by Western blotting (Fig. 7C) . The amount of cytoplasmic p65 in ED2high cells was much higher than that in ED2low cells. On the contrary, the amount of cytoplasmic I-{kappa}B in ED2high cells was rather lower than that in ED2low cells. Because genes encoding IL-1ß and IL-6 are candidates for a coordinate induction by NF-{kappa}B in monocytes/macrophages after stimulation with LPS, NF-{kappa}B activity in ED2high and ED2low cells after stimulation with LPS was analyzed. Radioactive DNA binding to nuclear proteins from ED2high cells after stimulation with LPS was much stronger than that from ED2low cells, although substantial nuclear translocation of NF-{kappa}B was seen in ED2low cells after LPS stimulation (Fig. 7A , middle lane). Meanwhile, the amount of cytoplasmic p65 decreased rapidly in ED2high cells after stimulation with LPS (Fig. 7D , top panel). On the contrary, p65 could be detected in the nuclear extracts 1 h after the stimulation (Fig. 7D , bottom panel).

Effects of GC on the proportion and function of ED2high cells
We previously demonstrated that serum corticosterone level increases with age in mice, which affects functions and populations of cells in monocyte/macrophage lineage [39 ]. We thus compared serum corticosterone concentrations between young and old rats. As anticipated, serum corticosterone concentration was significantly increased in old rats compared with that in young rats (Fig. 8A ). On the other hand, we could not find significant differences in serum levels of epinephrine and norepinephrine between young and old rats (data not shown). To elucidate whether the elevated concentrations of corticosterone influenced peritoneal cells of old rats, we analyzed the levels of GC receptor mRNA expression in the peritoneal cells. As shown in Figure 8B , the expression of GC receptor mRNA in peritoneal cells from old rats was higher than that from young rats.



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  		Figure 8. Effects of aging on serum corticosterone concentrations and on GC receptor mRNA expression of peritoneal cells. (A) Serum corticosterone concentrations in young (n = 13) and old (n = 8) rats. Results are expressed as mean concentration ± SEM; *P < 0.01. (B) Expression of GC receptor mRNA on peritoneal cells from young and old rats. The expression of GC receptor and G3PDH mRNA in peritoneal cells from young (Y) and old (O) rats were analyzed by RT-PCR.

To pursue whether or not the decrease of ED2high cells in old rats was due to the physiological effect of GC, we investigated the effect of adrenalectomy on the proportion of ED2high cells. As illustrated in Figure 9A and B , the proportion of ED2high cells was significantly increased in ADX rats. Serum corticosterone concentrations were as follows: sham-operated rats, 34.4 ± 5.4 ng/mL; ADX rats, 6.1 ± 0.5 ng/mL.



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  		Figure 9. (A) Effects of adrenalectomy on peritoneal cell populations in young rats. (A) Peritoneal cells from sham-operated (solid line) or ADX (bold line) rats were stained with mAb OX6 and analyzed by flow cytometry. Negative control: dotted line, sham; widely spaced dotted line, ADX. (B) Each bar represents the mean % of ED2high cells in peritoneal cells from sham (n = 5) and ADX (n = 5) rats ± SEM; *Significantly higher than control (P < 0.05).

GCs function by binding to specific cytoplasmic receptors, allowing the complex to translocate into the nucleus and to affect gene transcription either positively or negatively. We then examined the state of GC receptor in ED2high and ED2low cells. Figure 10A shows that translocation of GC receptor to the nucleus was induced in ED2low cells but not in ED2high cells. The radioactive DNA binding (Fig. 10B , lane a) was blocked by the unlabeled oligonucleotide (Fig. 10B , lane b). The specificity of the binding was confirmed by the result that radiolabeled mutant GC receptor oligonucleotide did not bind the nuclear extract (Fig. 10B , lane d). On the other hand, the amount of cytoplasmic GC receptor was markedly lower in ED2low cells than in ED2high cells (Fig. 10C) .



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  		Figure 10. Activation of GC receptor in ED2high or ED2low cells. (A) GC receptor DNA binding activity in nuclear extracts from ED2high (H) or ED2low (L) cells was detected by EMSA. (B) The identity of GC receptor was investigated. Nuclear extract of ED2low cells was incubated with radiolabeled GC receptor oligonucleotide probe in the absence (lanes 1 and 3) or in the presence (lane 2) of a 100-fold molar excess of the unlabeled oligonucleotide. Nuclear extract of ED2low cells was also incubated with radiolabeled mutant GC receptor oligonucleotide probe (lane 4). (C) Relative amounts of cytoplasmic GC receptor in ED2high or ED2low cells. Cytoplasmic extracts were prepared from ED2high (H) or ED2low (L) cells and subjected to Western blot analysis with anti-GC receptor antibody.


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DISCUSSION
 
In this study, we demonstrated that the proportion of cells expressing MHC class II molecules and substantial antigen-presenting ability in peritoneal monocytes/macrophages from old rats were markedly lower than those from young rats. These findings suggested that the decreased capacity of antigen presentation in peritoneal monocytes/macrophages from old rats was attributable to the low proportion of cells expressing MHC class II molecules. In addition, upon stimulation with LPS IL-1ß and IL-6 productions by peritoneal monocytes/macrophages from old rats were markedly lower than those from young rats. These findings suggested that functional monocytes/macrophages in peritoneal cells were markedly reduced in old rats.

Analysis of the cell-surface phenotype revealed that the number of ED2high cells was markedly decreased in peritoneal cells from old rats as compared with that from young rats, whereas the expression of ED1, ED3, or ED8 appeared to be unaffected by aging. We demonstrated that there was no significant difference in the expression of MHC class II molecules between ED2high cells from young and old rats and that the amount of MHC class II expression was significantly higher in ED2high cells than in ED2low cells. Efficiency of antigen presentation depends partly on the density of MHC class II molecules on the surface of APC [40 ]. Indeed, we could demonstrate that the efficiency of antigen presentation resided in ED2high cells but not in ED2low cells, although there was no significant difference in the efficiency of antigen presentation between ED2high cells from young and old rats. Because we found a great deal of difficulty in collecting a sufficient number of ED2high cells from old rats, IL-1ß and IL-6 productions and NF-{kappa}B activity were compared between ED2high cells and ED2low cells from young rats. IL-1ß and IL-6 productions by ED2high cells stimulated with LPS were apparently higher than those in ED2low cells treated with LPS. Furthermore, NF-{kappa}B activity in ED2high cells was much higher than that in ED2low cells before as well as after stimulation with LPS. It seems likely, therefore, that the reduced function observed in peritoneal monocytes/macrophages from old rats was attributable not to a decrease in their function but to a decrease in functionally active ED2high cell number.

Unfortunately, function and structure of ED2 have not been elucidated. In our preliminary experiments, in vitro phosphorylation of 96-kDa molecules immunoprecipitated with mAb ED2 was observed in immune complex kinase assay (data not shown). Thus, ED2 may be involved in signal transduction during cell differentiation or during exhibiting function(s). To elucidate the relationship between ED2 expression and several functions of macrophages, however, further studies are needed.

Cells of the immune system are highly specialized to respond rapidly to diverse unpredictable extracellular events and therefore benefit from using fast-responding, pleiotropically acting triggers at their defense programs. NF-{kappa}B may play a pivotal role in cells of the immune system because it is rapidly activated by a wide variety of pathogenic signals and functions as a potent and pleiotropic transcriptional activator. Stimulation of monocytes and macrophages with LPS leads to a rapid and transient expression of genes encoding various cytokines. The genes encoding IL-1ß and IL-6 have been shown to be good candidates for being induced with the help of NF-{kappa}B [41 42 43 ]. In the present study, we found that nuclear translocation of NF-{kappa}B in ED2high cells after stimulation with LPS was markedly higher than that in ED2low cells. By contrast, p65 was present in a large amount in the cytoplasm of ED2high cells in a resting condition, but the cytoplasmic p65 decreased rapidly after stimulation with LPS. Furthermore, p65 was detected in nuclear extract after stimulation with LPS for 1 h. These observations suggest that, in ED2high cells, a large amount of inactive NF-{kappa}B is retained in the cytoplasm and is rapidly activated after stimulation with LPS, probably leading to the rapid production of large amounts of IL-1ß and IL-6. Studies on the precise kinetics of the p65 decrease in cytoplasm and of IL-1ß and IL-6 productions should be done in the future.

Meanwhile, GCs are potent immunosuppressive agents with the potential to inhibit expression of several cytokines and adhesion molecules. Several mechanisms of DEX-mediated repression of NF-{kappa}B-dependent gene expression have been presented [21 22 23 24 25 26 27 ]. However, the role of basal GCs in the immune system also remains unclear. Numerous studies have examined whether GC concentrations change with age. However, basal circulating concentrations of corticosterone have been observed to be unchanged [44 ], increased [45 46 47 ], or decreased [48 , 49 ] with age in rodents. This discrepancy may have resulted from methodological variations, such as inconsistencies in obtaining truly basal (i.e., unstressed) samples, and /or differences in strain, sex, health, and nutritional status of the animals studied [28 ]. Our present findings that serum corticosterone concentration in old rats is significantly higher than that in young rats agree with the findings by others [28 , 45 46 47 ]. Recently, we demonstrated that the increase in GC concentrations affects macrophage functions [39 , 50 ]. Eisen et al. [51 ] reported that GC receptor protein and its mRNA levels were increased after treatment of human T cells with GC. In the present study, we have found that expression of mRNA for GC receptor is enhanced in peritoneal cells from old rats compared with young rats. It thus seems that the stimulation of GC receptors by circulating GCs is stronger in old rats than in young rats, which may affect proportions of the peritoneal cells.

Actually, when we analyzed the effect of ADX on the population of peritoneal cells in young rats, ADX led to a significant increase in the proportion of ED2high cells in the peritoneal cells. The observations obtained suggest that the proportion of ED2high and ED2low cells in peritoneal cells is regulated at least partly by GC, although synergistic effects of the other mediators have not been analyzed in the present study. It should be noted that nuclear translocation of GC receptor was observed in ED2low cells, whereas markedly higher amounts of GC receptors were retained in the cytoplasm of ED2high cells. Several reports suggest that down-modulation of NF-{kappa}B-driven genes results from a physical association between activated GC receptor and the NF-{kappa}B subunit p65 [21 , 22 , 26 ]. It seems, therefore, likely that the GC receptor activation observed in ED2low cells accounts for the repressed functions described above. These findings, however, do not directly explain the decrease in ED2high cells in old rats, since effects of adrenalectomy on old rats could not be examined. Therefore, it would not probably be denied that some other or additional immunosuppressive effect may be at work.

In this study, we have demonstrated that ED2high macrophages are at a functionally high level and that the number of ED2high cells decreases with age, probably leading to a decline in immune responses. We also suggest that the proportion of ED2high and ED2low macrophages is regulated, in part, by the serum GC concentration. The critical mechanisms that regulate the cellular immune responses characterized in this report, however, still remain to be elucidated. Further studies will be needed to clarify the underlying mechanisms by which neuroendocrine products modulate immune responses and alter disease susceptibility/severity in aged animals.


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ACKNOWLEDGEMENTS
 
This work was supported in part by a grant from Daiwa Securities Health Foundation. The authors gratefully acknowledge the excellent technical assistance of Mr. M. Segawa and the excellent secretarial assistance of Ms. M. Fujii.

Received June 14, 1999; revised February 22, 2000; accepted February 24, 2000.


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