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,
, and, , , 5
Departments of * Nutritional Sciences, Animal Health and Biomedical Sciences, ** Biostatistics, Medicine, University of Wisconsin-Madison, Wisconsin Regional Primate Research Center and Veterans Administration-Geriatric Research, Education and Clinical Center, Madison, WI 53705
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
We previously reported that energy restriction (ER) of mice attenuated age-associated increases in serum levels of interleukin-6 (IL-6). Here, we studied peripheral blood mononuclear cells (PBMC) from male rhesus monkeys to investigate the following: 1) the production of IL-6 and other cytokines become dysregulated with aging; 2) ER influences cytokine production and mRNA expression; and, 3) oxidative stress, as induced in vitro by xanthine and xanthine oxidase (X/XOD), influences cytokine mRNA and protein levels. Two types of comparisons were made as follows: 1) between normally fed young (6-9 y) and old monkeys (22-33 y); and 2) between middle-aged monkeys (15-21 y) fed either a normal energy intake or subjected to ER (for 5.5 y at 30% less than base-line intake). IL-6 protein levels and X/XOD-induced IL-6 mRNA levels in PBMC from old monkeys were significantly greater than those in PBMC from young animals. In contrast, interleukin-1
(IL-1) and interleukin-8 mRNA levels were not strongly influenced by advancing age. X/XOD, which increased levels of protein carbonyls (indicative of oxidative damage) in PBMC, induced the expression of all three cytokines. ER reduced IL-6 protein and mRNA levels induced by X/XOD and the unstimulated mRNA levels of IL-1. These results indicate that, in a nonhuman primate model, oxidative stress may contribute to age-associated increases in the levels of certain cytokines and that adult-onset ER partially ameliorates this alteration.
INTRODUCTION
Energy restriction (ER)6 extends maximum life span and attenuates the progression of hundreds of age-associated biological changes in laboratory mice and rats (McCay et al. 1935
, Weindruch and Walford 1988, Yu 1994). ER, which is usually accomplished by a 30-50% reduction of energy intake while avoiding malnutrition, increases average and maximum life span and delays the onset and/or progression of many age-related diseases (Weindruch and Walford 1988, Weindruch 1996). Although the effectiveness of ER at postponing aging processes has been well documented, nearly all of these studies have used rodent models. Initial findings from long-term trials of ER in rhesus monkeys suggest that ER may have beneficial effects on glucoregulation (Kemnitz et al. 1994).
One proposed mechanism for the retardation of aging as a result of ER is a reduction in oxidative stress (Sohal and Weindruch 1996). Increased generation of reactive oxygen species (ROS), which include superoxide anion: O·
2, hydrogen peroxide: H2O2 and hydroxyl radical: ·OH, decreased antioxidant defense and reduced capacity to repair oxidatively damaged molecules all can contribute to increased oxidative stress. The rate of mitochondrial ROS generation increases with age in several different organs from C57BL/6NNia mice (Sohal et al. 1994). Lipid peroxidation, protein oxidation and DNA oxidative damage also increase with age and have been implicated in several major age-related diseases including certain cancers, Parkinson's and Alzheimer's diseases, among others (Ames 1993, Beal 1996, Sohal and Weindruch 1996). Studies examining the effect of age on antioxidant defenses point toward tissue-specific alterations (Matsuo 1993), and little is known about the efficiency of repair and removal of oxidatively damaged molecules. ER reduces mitochondrial production of ROS and lowers age-related increases in oxidative damage (Sohal et al. 1994), supporting the hypothesis that ER may retard aging by reducing oxidative stress.
With age, there is increased susceptibility to infection, autoimmune diseases, cancers and other diseases associated with an assortment of changes in the immune system (Miller 1996). In rodents, ER attenuates several age-associated immunologic changes including thymic involution, decreased immune response capacities and shifts in lymphocyte subsets (Spear-Hartley and Sherman 1994, Weindruch and Walford 1988). Proinflammatory cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1) and interleukin-8 (IL-8) play important roles in immune regulation. These molecules are produced by diverse cell types and have multiple actions including the regulation of cell proliferation and hematopoiesis (IL-6; Akira et al. 1993), T- and B-cell activation (IL-1; Dinarello 1996) and chemotaxis (IL-8; Baggiolini et al. 1995). IL-6 protein levels increase with age in plasma from humans (Wei et al. 1992) and in serum from mice (Volk et al. 1994) as does mitogen-stimulated IL-6 production by mouse splenocytes (Zhou et al. 1993) and by human peripheral blood mononuclear cells (PBMC; Fagiolo et al. 1993). We have shown that ER initiated at middle age (12 mo) in mice attenuates the age-associated increase in serum IL-6 (Volk et al. 1994). Age-associated increase (Riancho et al. 1994) or decrease (McLachlan et al. 1995) of IL-1 protein levels is also reported in PBMC and monocytes, respectively. IL-8 protein levels in supernatants from cultured monocytes are lower and lipopolysaccharide (LPS)-induced levels are higher in older men compared with those from young subjects (Clark and Peterson 1994). Increased levels of these cytokines have been found to occur in certain age-associated diseases including lymphoma, osteoporosis and Alzheimer's disease (Ershler et al. 1994).
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Table 1. Diet composition1 |
Studies examining human tissues indicate that levels of some cytokines increase in response to oxidative stress. For example, plasma IL-6 protein levels (Urabanski et al. 1990) are increased after exposure of humans to ultraviolet light (an oxidative stress) as are IL-6 levels in supernatants from keratinocytes (Kirnbauer et al. 1991). Further, LPS-stimulated production of IL-6 and IL-1 by PBMC can be inhibited by antioxidants (Eugui et al. 1994). IL-8 production by PBMC can be induced by paraquat, and this response can be inhibited by a hydroxyl radical scavenger (Horiguchi et al. 1993). These data imply that oxidative stress may play a role in upregulating cytokine levels. Accordingly, we hypothesize that oxidative stress may contribute to the age-associated increases in IL-6 levels, and perhaps, in other cytokines, resulting in geriatric immune dysregulation. Further, ER may preserve certain immune functions with aging by reducing oxidative stress.
In this study, we examined influences of aging on IL-6 protein and IL-6, IL-1 and IL-8 mRNA levels in PBMC from male rhesus monkeys. We also investigated whether oxidative stress, as induced in vitro by xanthine (X) and xanthine oxidase (XOD), influences PBMC expression of IL-6, IL-1 and IL-8. The effect of adult-onset ER on these measures was also examined.
MATERIALS AND METHODS
for details of experimental design and animal maintenance). These control and ER monkeys were given a purified diet (#85387, Teklad, Madison, WI) for 5.5 y. It is a pellet diet containing 16 kJ energy, 150 g protein, 100 g fat and 50 g fiber per kilogram diet (Table 1). The ER animals were gradually restricted by 10%/mo for 3 mo to reach 70% of each monkey's predetermined base-line intake. The controls were given their base-line intake for 8 h/d. The ER monkeys were further restricted after 18 mo to reestablish a 30% difference between groups because the voluntary food intake of the control monkeys had decreased (Kemnitz et al. 1993). The protocol was in compliance with the Guide for the Care and Use of Laboratory Animals (NRC 1985).
). The PBMC (5 × 109/L) were resuspended in culture medium (RPMI 1640 supplemented with 10% fetal calf serum, 2 mmol/L L-glutamine, 1 × 105 U/L penicillin, and 100 mg/L streptomycin) and incubated (37°C, 5% CO2) in a culture tube in the presence or absence of stimulation. LPS (5 mg/L) was used as a strong inducer of IL-6 production. All reagents were purchased from Sigma Chemical (St. Louis, MO). Cell culture supernatants and cells were harvested and frozen at 
80°C until assay.
Oxidative stress. X/XOD was used to produce oxidative stress in the cell culture medium. X/XOD in the presence of oxygen produces O·
2 and H2O2 (Fridovich 1970). X (0.1 nmol/L) and XOD (800 mU/L) were dissolved in culture medium and filtered through 0.45-µm filters before use. Assay of two samples by cytochrome c reduction (Kuthan et al. 1982) indicated that X/XOD produced 0.86 nmol of O·2/min in the presence of 106 PBMC. No difference in the rate of cytochrome c reduction was seen in cultures containing PBMC from young or old monkeys (data not shown). The absence of endotoxin in X/XOD preparations was confirmed by the Limulus amebocyte lysate assay (Biowhittaker, Walkersville, MD). X/XOD treatment did not affect cell number, viability (determined by trypan blue exclusion) and DNA synthesis rate (incorporation of tritiated thymidine; data not shown).
ELISA.
The concentration of IL-6 protein in the cell culture supernatants was measured by ELISA at 24 h of culture. A 96-well microtiter plate was coated with 0.5 mg/L monoclonal mouse anti-human IL-6 antibody (R & D Systems, Minneapolis, MN) at 4°C overnight in Tris-buffered saline (TBS; 25 mmol/L Tris, 0.15 mol/L NaCl, pH 7.6, Pierce, Rockford, IL). After blocking the wells with 200 µL of 1 × Blocker bovine serum albumin in TBS (Pierce) at room temperature for 2 h, either recombinant human IL-6 standard (R & D Systems) or test supernatant was added and incubated overnight at 4°C. Plates were sequentially incubated with 200 µL of 4000-fold diluted polyclonal goat anti-human IL-6 antibody (R & D Systems) and 2500-fold diluted alkaline phosphatase-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 2 h. The wells were washed five times with 0.2% Tween 20 in TBS between each step. Phosphatase substrate (200 µL, Sigma) was added and absorbance was measured at 405 nm.
RNase protection assay. To determine mRNA levels of IL-6, IL-1
and IL-8, RNase protection assay was used. PBMC (5 × 109/L) were cultured in the presence or absence of X/XOD for 2 h; then, total RNA was extracted using RNeasy Total RNA kits (QIAGEN, Chatsworth, CA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), IL-6, IL-1 and IL-8 mRNA levels were detected by RNase protection assay using the HybSpeed RPA kit (Ambion, Austin, TX). GAPDH cDNA was obtained from skeletal muscle of a rhesus monkey by reverse transcriptase polymerase chain reaction using primers based on human sequences. cDNA sequencing analysis showed that rhesus GAPDH cDNA was homologous to nucleotides 862-1037 of the human sequence. Rhesus monkey cytokine cDNA clones were kindly provided by Dr. Francois Villinger (Emory University, Atlanta, GA). Radiolabeled RNA was generated by in vitro transcription of 1 µg of linearized plasmid using SP6 RNA polymerase (MAXIscript kit, Ambion). Total RNA (1-2 µg) and 32P-labeled riboprobes (2-4 × 104 cpm) were combined in each reaction. The sample was then precipitated, size fractionated on a 5% PAGE gel and mRNA levels were quantitated by phosphorimager analysis (Molecular Dynamics, Sunnyvale, CA).
(IL-1) and interleukin-8 (IL-8) in peripheral blood mononuclear cells (PBMC) from young and old monkeys. PBMC (5 × 109/L) were cultured in the presence or absence of X/XOD (X:100 pmol/L, XOD: 0.8 U/L) for 2 h. Total RNA (1 µg) was analyzed by RNase protection assay as described in the text. Panel A: Unstimulated PBMC from a young (Y, lane 2), an old(O, lane 3) and X/XOD-treated PBMC from a young (Y, lane 4) and an old (O, lane 5) monkey show 170 nucleotide (nt), 235 nt and 287 nt protected fragments for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), IL-8 and IL-1 transcripts, respectively. Lane 1 shows a negative control in which yeast RNA was hybridized with GAPDH, IL-8 and IL-1 probes. Panels B and C: IL-1 (B) and IL-8 (C) mRNA levels were normalized using GAPDH as an internal control. Each bar represents the mean ± SEM of five monkeys. Wilcoxon sign-rank test was used for statistical analysis. +P < 0.003, the main effect of X/XOD treatment compared with unstimulated.
(IL-1) and interleukin-8 (IL-8) in peripheral blood mononuclear cells (PBMC) from energy-restrcted (ER) and control monkeys. PBMC (5 × 109/L) were cultured in the presence or absence of X/XOD (X:100 pmol/L, XOD: 0.8 U/L) for 2 h. Total RNA (1 µg) was analyzed by RNase protection assay as described in the text. Panel A: Unstimulated PBMC from a control (lane 1), an ER (lane 2) and X/XOD-treated PBMC from a control (lane 3) and an ER (lane 4) monkey show 170 nucleotide (nt), 235 nt and 287 nt protected fragments for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), IL-8 and IL-1 transcripts, respectively. Panels B and C: IL-1 (B) and IL-8 (C) mRNA levels were normalized using GAPDH as internal control. Each bar represents the mean ± SEM of six monkeys. Wilcoxon sign-rank and rank-sum tests were used for comparison between treatments in each animal group and between ER and control groups within treatments, respectively. *P < 0.05, unstimulated vs. X/XOD treatment. #P < 0.05, control vs. ER monkeys.
Enzyme assays. The pelleted cells (5 × 106) were resuspended in 25 µL of 0.1% Triton X-100 in TBS and disrupted by freezing and thawing three times. The suspensions were centrifuged (9000 × g) for 20 s at 4°C and the supernatants were used for enzyme and protein assays. Glutathione peroxidase (GPx: EC 1.11.1.9) activities were measured in 5 µL as described by Flohé and Gunzler (1984)
with hydrogen peroxide as the substrate. Absorbance was monitored at 340 nm at 37°C for 2 min with a Shimadzu UV-2101PC spectrophotometer (Kyoto, Japan). GPx activity is presented as nanomoles of NAPDH oxidized per minute per milligram protein. Total superoxide dismutase (SOD: EC 1.15.1.1) activity was measured in 15 µL (Sun and Zigman 1978) as the inhibition of epinephrine autoxidation (monitored at 320 nm). One unit of SOD was defined as the amount of activity that causes 50% inhibition of epinephrine oxidation. Protein content in PBMC lysates was measured by the Bradford method using a protein assay kit (Bio-Rad, Hercules, CA).
Protein oxidation.
Protein carbonyl content was determined as described by Reznick and Packer (1994) with slight modifications. All reagents were purchased from Sigma. Briefly, pelleted cells (5-10 × 106) were resuspended in 5 mmol/L phosphate buffer containing 0.1% Triton X, aprotinin (0.5 mg/L), leupeptin (0.5 mg/L) and pepstatin (0.7 mg/L). Samples were incubated with 7 mmol/L streptomycin sulfate at room temperature for 15 min and centrifuged at 2900 × g for 10 min to remove DNA. The supernatants were incubated with 10 mmol/L dinitrophenyl hydrazine (DNPH) in 2 mol/L HCl or with 2 mol/L HCl alone as a blank for 1 h at room temperature, precipitated with 612 mmol/L trichloroacetic acid for 10 min on ice and then centrifuged at 16,000 × g. The pellet was washed three times with ethanol/ethyl acetate (1:1, v/v) and resuspended in 6 mol/L guanidine HCl. The difference in absorbance between the DNPH-treated and the HCl-treated samples was determined at 366 nm; the results were expressed as nanomoles of carbonyl groups per milligram of protein using the extinction coefficient of 22.0 mmol/(L·cm). Protein concentration was determined using the Pierce BCA protein kit (Rockford, IL).
Statistical analysis.
Results are expressed as means ± SEM. Statistical comparisons were made using SAS (SAS/STAT Version 6, SAS Institute, Cary, NC). Differences between treatment conditions (unstimulated vs. X/XOD or LPS) for each animal group were tested by Mann-Whitney for IL-6 protein levels and Wilcoxon signed-rank tests for cytokine mRNA levels, protein carbonyl content and antioxidant enzyme activities (Woolson 1987). Within-treatment condition, comparisons between young and old monkeys and comparisons between ER and control monkeys were made by Wilcoxon rank-sum test (Woolson 1987).
RESULTS
and IL-8.
To determine whether other cytokine mRNA levels were also increased with age, IL-1 and IL-8 mRNA levels were measured from the same samples. IL-1 mRNA levels were not significantly different in unstimulated PBMC from young and old monkeys (Fig. 5). X/XOD treatment elevated IL-1 mRNA levels ~400% in both young and old animals. X/XOD treatment significantly elevated IL-1 mRNA levels over unstimulated control levels (P < 0.003). The increase in IL-1 mRNA levels by X/XOD was not significantly different between young and old monkeys. IL-8 mRNA levels in unstimulated PBMC from young and old monkeys were not significantly different (Fig. 5). X/XOD treatment elevated IL-8 mRNA levels ~500% in both young and old animals. X/XOD treatment significantly increased IL-8 mRNA levels over unstimulated control levels (P < 0.003). The increase in IL-8 mRNA levels by X/XOD was not significantly different between young and old monkeys.
mRNA level was observed in unstimulated PBMC from control compared with ER monkeys (P < 0.05, Fig. 6). IL-1 mRNA levels were 180% higher in X/XOD-treated PBMC from both ER (P < 0.04) and control monkeys (P < 0.04) compared with their own unstimulated controls. mRNA levels of IL-1 in X/XOD-treated PBMC were not significantly different between ER and control monkeys. IL-8 mRNA levels in unstimulated PBMC were not influenced by ER (Fig. 6). X/XOD treatment increased IL-8 mRNA levels by 200-300% in ER and control monkeys (P < 0.04). IL-8 mRNA levels in X/XOD-treated PBMC were not significantly different between ER and control monkeys.
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Table 2. Influence of age and xanthine (X) and xanthine oxidase (XOD) on antioxidant enzyme activities in peripheral blood mononuclear cells from rhesus monkeys1,2 |
DISCUSSION
) and lymphocyte IL-6 production (Ershler et al. 1993). Therefore, elevated cellular IL-6 production leading to higher circulating levels appears to be a common feature of senescence among several animals including mice (Zhou et al. 1993), rats (Foster et al. 1992), monkeys (present study) and humans (Fagiolo et al. 1993, Wei et al. 1992) and could contribute to the pathogenesis of certain age-associated diseases.
and IL-8 was not strongly influenced by age, and only IL-1 mRNA levels in unstimulated PBMC were lowered by ER (Fig. 5, 6). Unlike some other studies, our data do not suggest age-related changes in mRNA levels of these two cytokines. Spontaneous and mitogen-stimulated IL-1 protein levels in PBMC were found to be higher in cells harvested from older persons (Fagiolo et al. 1993, Riancho et al. 1994). LPS-induced IL-1 protein levels in monocytes either decreased (McLachlan et al. 1995) or increased (Born et al. 1995) in older individuals. Clark and Peterson (1994) observed that spontaneous IL-8 protein production in human monocytes was lower, but LPS-stimulated IL-8 protein production increased in older men. Considered together, the nature and the magnitude of age-related changes in IL-1 and IL-8 vary depending on different stimulants, cell types, end product measured (protein vs. mRNA) and different species studied.
and IL-8 mRNA levels. This interpretation is supported by the observation that the expression of these cytokines is induced by oxidative stress from different ROS-generating sources such as X/XOD (this study), UV light (IL-6; Urabanski et al. 1990), H2O2 (IL-8; DeForge et al. 1992) and paraquat (IL-8; Horiguchi et al. 1993). Further, some antioxidants can attenuate either mitogen- or ROS-induced cytokine production (DeForge et al. 1992, Eugui et al. 1994, Horiguchi et al. 1993). In addition, we observed that the ROS-induced increase in IL-6 expression was more pronounced in old monkeys, which suggests that PBMC from old animals could be more susceptible to oxidative stress.
), only recently have cells of the immune system been investigated in this context (Grigolo et al. 1994, Niwa et al. 1993, Tian et al. 1995). High levels of protein oxidative damage (as manifested by protein carbonyl groups) are found in plasma and splenocytes of old rats (Tian et al. 1995), and our results also tend to support these findings. Although statistical significance was not obtained in our study because of the small number of animals studied for protein carbonyl content, X/XOD-treated PBMC from old monkeys tended to show higher values than those derived from young animals.
). We observed that total SOD activity was higher in unstimulated PBMC from old monkeys whereas the relative increase in SOD activity induced by X/XOD was greater in young monkeys (Table 2). GPx activity of PBMC decreased in response to X/XOD in old but not in young monkeys (Table 2). Studies with human peripheral blood lymphocytes derived from donors of diverse ages revealed little change with age in the basal activities of SOD and a decrease in GPx activity in old individuals (Grigolo et al. 1994, Niwa et al. 1990 and 1993). These enzyme activities in peripheral blood lymphocytes could be induced by paraquat and the relative induction was greater in cells from young than from old humans (Niwa et al. 1990 and 1993). Also, increased SOD activities in peripheral blood lymphocytes have been reported in patients with Parkinson's disease (in which ROS may be casually involved [Beal 1996]) compared with age-matched normal subjects (Kushleika et al. 1996). These findings, although not completely consistent, do imply that antioxidant enzyme activities in lymphocytes are variably altered with aging and certain age-related diseases. Further, the augmentation of antioxidant enzyme activities in response to ROS is lower in old animals. Considered together, immune cells of old animals, exposed to relatively severe oxidative stress in vitro, are likely to accrue comparatively greater oxidative damage than those of young animals. Accumulation of oxidative damage, or an increased intracellular oxidative tone with aging, may be an important cause of the dysregulation of IL-6 and other expressions of immune senescence.
, Weindruch and Walford 1988, Yu 1994). However, similar to the lack of studies on the possible involvement of oxidative stress in immunologic aging, little is also known in the context of ER. Our data demonstrate that adult-onset ER in rhesus monkeys lessens both the X/XOD-induced elevation of IL-6 protein and mRNA levels in PBMC and reduces IL-1 mRNA levels in unstimulated cells. Splenocytes from Fischer 344 or (Fischer 344 × Brown Norway)F1 rats subjected to ER show less overt decline in immune response capacity and alterations in ROS metabolism compared with cells from controls (Byun et al. 1995, Tian et al. 1995). For example, the rats show age-associated decreases in mitogen-induced proliferative responses and IL-2 production, whereas age-associated increases occur in indicators of oxidative damage such as levels of protein carbonyls and lipid peroxides (Byun et al. 1995, Tian et al. 1995). All four of these aging changes were ameliorated by ER (Byun et al. 1995, Tian et al. 1995). In Wistar rats, the age-dependent impairment of splenocyte proliferation was partially corrected by either ER or by the addition of an antioxidant (reduced glutathione) into the culture medium (Pieri et al. 1993). Collectively, these data imply that ER in rodents may preserve several immune system changes by reducing oxidative stress. Further, a lowering of oxidative stress would likely have broad effects on cellular function because diverse processes such as apoptosis, signal transduction and gene expression are all modulated by levels of ROS (Payne et al. 1995).
ACKNOWLEDGMENTS
FOOTNOTES
, interleukin-1; IL-6, interleukin-6; IL-8, interleukin-8; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cells; ROS, reactive oxygen species; SOD, superoxide dismutase; TBS, Tris-buffered saline; X/XOD, xanthine and xanthine oxidase.
Manuscript received 30 June 1997. Initial reviews completed 5 August 1997. Revision accepted 28 August 1997.
LITERATURE CITED
overproduction of IL-8 in elderly males in response to lipopolysaccharide.
Mech. Ageing Dev.
1994;
77:127-139
[Medline]
, IL-1, and IL-6 by human peripheral blood mononuclear cells.
Int. Immunol.
1994;
6:409-422
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