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Published online before print November 21, 2003
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,
,¶,,,¶,1
Departments of
* Cell Biology, Neurobiology and Anatomy and
Surgery,
The Burn and Shock Trauma Institute,
Alcohol Research Program, and
¶ Immunology and Aging Program, Loyola University Medical Center, Maywood, Illinois
1 Correspondence: Department of Cell Biology, Neurobiology and Anatomy, Loyola University Medical Center, 2160 South First Avenue, Bldg. 110, Rm. 4220, Maywood, IL 60153. E-mail: ekovacs{at}lumc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-) and interleukin (IL)-6, when stimulated with lipopolysaccharide (LPS), which signals through Toll-like receptor-4 (TLR4) and requires activation of mitogen-activated protein kinases (MAPKs). We investigated whether aging is associated with alterations in TNF- and IL-6 production and MAPK expression and activation in thioglycollate-elicited peritoneal macrophages from mice. Kinetics and LPS dose-responsiveness of macrophage TNF- production did not differ by age. Unstimulated macrophages did not differ by age in their cytokine production. However, LPS-stimulated (100 ng/mL) cultures from aged mice produced 100 ± 30 pg/mL TNF- and 6000 ± 2000 pg/mL IL-6, and those from young mice produced 280 ± 50 pg/mL and 10,650 ± 10 pg/mL, respectively (P<0.05). Likewise, levels of activated MAPKs did not differ by age in unstimulated macrophages, and LPS-stimulated macrophages from aged mice had <70% activated p38 and c-jun NH2-terminal kinase (JNK) than those of young controls. Of particular interest, we observed >25% reduction of total p38 and JNK in macrophages from aged mice relative to young. In addition, surface TLR4 levels did not vary with age. We conclude that macrophages from aged mice exhibited suppressed proinflammatory cytokine production, which correlated with diminished total levels and LPS-stimulated activation of p38 and JNK. These observations suggest that decreased MAPK expression could be a mechanism responsible for age-related deterioration of the immune system.
Key Words: immunosenescence • p38 • JNK • aging • MAPK • TLR4
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Specific age-related immune alterations are well established. Aged mice exhibit diminished delayed-type hypersensitivity and splenocyte-proliferative responses [4
5
6
]. T lymphocytes demonstrate age-associated changes and likely contribute to immunosenescence. With advancing age, memory T cells increasingly replace naïve T cells [7
8
9
]. Other T cell-related observations include a decrease in interleukin (IL)-2 production and stimulated IL-2 receptor expression [10
11
12
13
] and T cell receptor and CD28 signal transduction [14
]. Although macrophages also play critical roles in cellular responses to pathogens, less is known about their function with advancing age. However, indicators of compromised macrophage function with age are growing (reviewed in ref. [15
]). For example, reports suggest that with advancing age, macrophages have decreased lipopolysaccharide (LPS)-stimulated reactive oxygen species and nitric oxide generation (rat alveolar macrophages) [16
], phagocytic activity (mouse peritoneal macrophages) [17
], protein kinase C translocation and activation (rat alveolar macrophages), and interferon-
-induced major histocompatibility complex class II expression (bone marrow-derived mouse macrophages) [18
, 19
] and have increased LPS-stimulated nuclear factor-
B activation and subsequent cyclooxygenase-2 transcription, expression and enzymatic activity, and prostaglandin E2 production [20
]. These alterations with advancing age may contribute to compromised immunity.
Macrophages produce tumor necrosis factor (TNF-) and IL-6, two major proinflammatory cytokines. These pleiotropic cytokines stimulate cellular immune responses and induce the production of acute-phase proteins for systemic inflammatory responses. TNF- and IL-6 have been reported to be elevated in healthy and diseased, aging humans [21
], and morbidity and mortality of elderly patients are associated with increased circulating levels of proinflammatory cytokines [22
]. These observations have led many to propose that a heightened, proinflammatory state with age is a potential promoter of immunosenescence.
LPS, the major component of Gram-negative bacterial cell walls, is bound by LPS-binding protein and transferred to a complex of receptors that includes CD14 and Toll-like receptor-4 (TLR4), expressed at the macrophage cell surface [23
]. Signal transduction via TLR4 involves a series of phosphorylation events that result in the activation of transcription factors [24
]. The activation of mitogen-activated protein kinases (MAPKs) is an essential step in the LPS signal-transduction cascade that results in TNF- and IL-6 transcription and translation [23
24
25
26
27
].
Reports on macrophage-derived TNF- and IL-6 initially suggested that a hyper-proinflammatory state might be a natural consequence of the aging process. By that model, immunosenescence could result, in part, from immunosuppressive effects of these abnormally elevated cytokines [28
]. However, recent studies have called this model into question [29
30
31
]. Specifically, Renshaw et al. [32
] showed that LPS-stimulated macrophages isolated from the spleens and peritoneal cavities of aged mice secrete less TNF- and IL-6 than those from young mice. They also demonstrated less TLR4 mRNA in macrophages from aged mice and suggested that decreased TLR4 was responsible for the functional alterations in TNF- and IL-6 secretion. Here, we confirm their findings that macrophages from aged mice produce less TNF- and IL-6 compared with young and present the novel finding that the age-dependent impairment in cytokine production correlates with a decrease in basal expression of p38 and c-jun NH2-terminal kinase (JNK) MAPK in macrophages from aged mice compared with young.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of macrophages
For elicitation of peritoneal macrophages, mice were given an intraperitoneal injection of 2.5 mL 3% thioglycollate as described previously [33
]. Three days later, mice were killed by CO2 inhalation and subsequent cervical dislocation, and the peritoneal cavity was flushed for peritoneal exudate cells (PECs) with phosphate-buffered saline (PBS; Invitrogen Corporation, Carlsbad, CA). The PECs were enriched for macrophages by adherence to tissue-culture plastic [96-well (200 µL) or 24-well (1.2 mL)] in culture conditions (37°C, 5% CO2) in a concentration of 1 x 106 cells/mL in RPMI medium (RPMI 1640 without phenol red, Gibco-BRL, Grand Island, NY), supplemented with L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL), and 10% heat-inactivated fetal bovine serum (FBS). After 1.5 h, the cell cultures were rinsed twice with warm (37°C) PBS and rested overnight in 2% FBS/RPMI medium in culture conditions.
Measurement of macrophage-cytokine production
After resting, the supernatants were aspirated and replaced with 200 µL fresh medium containing 0, 10, 100, or 1000 ng/mL LPS (Sigma Aldrich, St. Louis, MO). Supernatants were collected after 0.25, 6, or 16 h and were stored at -20°C until analysis of TNF- and IL-6 by enzyme-linked immunosorbent assay (ELISA; BD PharMingen, San Diego, CA, and Endogen, Cambridge, MA, respectively), according to the manufacturers’ protocols.
Assessment of MAPK expression and activation
Following overnight rest, 24-well culture supernatants were aspirated and replaced with 1.2 mL 100 ng/mL LPS in 10% FBS/RPMI medium. After 15 min, the medium was aspirated and replaced with 200 µL ice-cold lysis buffer as described previously [34
]. Pilot studies indicated that peritoneal macrophages from both age groups demonstrate similar kinetics and dose response with regard to LPS-stimulated MAPK phosphorylation. p38 and JNK were optimally phosphorylated at 15 min and returned to basal levels of phosphorylation by 1 h with a 100 ng/mL LPS stimulus. This dose was as strong a stimulus as 1000 ng/mL in both age groups (data not shown). The cell lysates were collected and stored at -20°C until analysis by Western blotting.
The protein content of cell lysates was determined by Lowry’s method with a commercially available kit (Sigma Aldrich). Cell extracts were equally loaded (15–20 µg total protein per lane) and separated by gel electrophoresis on a 10% HCl-Tris polyacrylamide gel (Bio-Rad, Hercules, CA), as described previously [35 ]. Coomassie or the more-sensitive silver staining of the gels verified equal protein loading. Proteins were electroblotted to PolyScreen membranes (DuPont Systems NEN, Boston, MA), blocked, and probed with a 1:1000 dilution of primary antibody overnight at 4°C, as described previously [36 ]. Primary antibodies used were anti-phospho-p38 MAPK (Thr180/Tyr182), anti-phospho-stress-activated protein kinase/JNK MAPK (Thr183/Tyr185), and anti-p38 MAPK and anti-JNK MAPK (Cell Signaling Technology, Beverly, MA). A 1:4000 dilution of peroxidase-conjugated sheep anti-rabbit immunoglobulin G (IgG) secondary antibody (Amersham Pharmacia Biotech, Pascataway, NJ) was applied and incubated for 1 h at room temperature. Proteins of interest were detected by application of enhanced chemiluminescence Western blotting detection reagents and exposure to HyperfilmTM (both from Amersham Pharmacia Biotech). Band density on exposed films was quantified using Ambis optical imaging system (Ambis Systems, San Diego, CA).
To confirm the total p38 data obtained by Western blotting, we used a commercially available p38 MAPK ELISA (Assay Designs, Inc., Ann Arbor, MI). Cell lysates were prepared as described above, except the lysis buffer provided in the kit was used. Lysates were diluted 1:50 and assayed according to the manufacturer’s protocol.
The biological relevance of decreased LPS-stimulated activation levels of p38 with age was determined by analyzing phosporylated MAPK-activated protein kinase-2 (MAPK-APK-2). Western blots were performed on cell lysates from the experiments above using an antibody against phospho-MAPK-APK-2 (Thr334; Cell Signaling Technology) in a 1:1000 dilution. Films were developed and quantified by densitometry (see above).
Comparison of TLR4 surface expression
Total PECs were obtained as described above. Cell viability was checked by trypan blue exclusion. Cells were resuspended in staining buffer (PBS containing 1% bovine serum albumin and 0.1% sodium azide) and nonspecific staining was blocked with anti-CD16/CD32 (FcIII/II, BD PharMingen) and whole rat IgG (Sigma Aldrich). After blocking, cells were then incubated with CyChrome5.5-conjugated anti-F4/80 (Caltag, Burlingame, CA) and phycoerythrin (PE)-conjugated anti-TLR4 (clone MTS510, eBioscience, San Diego, CA), washed twice in staining buffer, and fixed in 4% paraformaldehyde. Flow cytometric determinations were made using a Becton Dickinson (San Jose, CA) FACSCalibur flow cytometer and CellQuest Pro software.
Statistical analyses
Data are expressed as the group mean value ± SEM of one experiment representative of two to four identical experiments in which similar results were obtained. N is the number of mice per group in the representative experiment. Data were considered significant at P 
0.05, as determined by t-tests or ANOVA with post-hoc Newman-Keuls test, where appropriate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and IL-6 production upon LPS stimulation in a concentration-dependent manner (Fig. 1A
). In the absence of stimulation (0 ng/mL LPS), macrophages from young and aged mice did not produce detectable levels of TNF-. Following treatment with a low concentration of LPS (10 ng/mL), there were minimal but detectable levels of TNF- that did not significantly differ by age. Within an age group, maximum levels of TNF- were produced after LPS stimulation with a concentration of 100 ng/mL without a change in production at 1000 ng/mL LPS, and both concentrations induced levels significantly above unstimulated and 10 ng/mL doses (P<0.01). In response to LPS stimulation, the production of TNF- by macrophages from aged mice was only 
50% that of young mice at 10, 100, and 1000 ng/mL LPS, which was significant at the higher two concentrations of stimulation (P<0.01).
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production were a result of differences in the kinetics of production, we determined the concentration of TNF- in the culture supernatants after 0.25, 6, and 16 h of LPS stimulation. Macrophages from both age groups responded to LPS (100 ng/mL) with similar kinetics (Fig. 1B)
. Immediate (0 h) TNF- production was not detectable in cultures from young or aged mice. Six hours following LPS stimulation, macrophages from both age groups produced detectable levels of TNF- (P<0.01). Within each age group, TNF- production levels at 6 h did not differ from levels at 16 h. At 6 and 16 h time points, production of this cytokine by macrophages from aged mice was only about half of that from young mice (P<0.01), thus demonstrating that the lower level of TNF- production by macrophages from aged mice was not a function of length of exposure or concentration of stimulus.
In unstimulated cultures, basal macrophage IL-6 production was detectable but did not differ by age (Fig. 2
). As with TNF- induction, LPS-stimulated macrophages from young and aged mice produced significantly elevated levels of IL-6 relative to unstimulated macrophages (P<0.01), and IL-6 secretion by macrophages from aged mice was reduced by 40% of that from young mice (P<0.05).
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and IL-6 production. To determine if aged mouse macrophages expressed less TLR4 than young, peritoneal macrophages from both age groups were examined for TLR4 surface expression by flow cytometry. As shown, F4/80+ macrophages did not exhibit age-associated differences in surface TLR4 expression (Fig. 3
).
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Unstimulated macrophages from young and aged mice had low but detectable levels of phosphorylated p38 (Fig. 4A
); there was no significant age-dependent difference between these cell populations (Fig. 4B)
. Stimulation with LPS significantly increased phosphorylated p38 in both age groups; however, the level of phosphorylated p38 in macrophages from aged mice was only 70% that of macrophages from young mice (P<0.05; Fig. 4B
). It is interesting that this age-associated deficiency in levels of activated p38 was mirrored in levels of total p38 (Fig. 4C)
. Although LPS stimulation did not influence total p38 quantity, macrophages from aged mice have only 75% total p38 as those from young mice (P<0.05). An additional experiment used the use of an ELISA kit to confirm these results and found that macrophages from aged mice had only 70% the amount of total p38 as those from young mice (Fig. 4D) . The difference was significantly different (P<0.05), even after the results were standardized for total protein (data not shown).
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50% the amount of phosphorylated JNK in aged macrophages relative to young. In addition, total JNK is not affected by LPS stimulation but is also reduced 50% in macrophages from aged mice (Fig. 5C)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, Renshaw et al. [32
] provided evidence indicating that LPS-stimulated splenic and peritoneal macrophages from aged mice secreted less TNF- and IL-6 than those from young mice. Most literature previous to that report, however, suggested that circulating levels of these proinflammatory cytokines were elevated in the elderly [21
, 22
]. Our findings confirm those of Renshaw et al. [32
] using peritoneal macrophages from aged and young BALB/c female mice. Although contrary to the prevailing expectations from earlier studies, these data are in agreement with other recent reports that suggest TNF- and/or IL-6 may not be elevated as a natural consequence of aging [29
30
31
]. Instead, elevated, proinflammatory cytokine levels in elderly humans might be indicators of concomitant inflammatory disease(s) and/or poor nutritional status [39
40
41
]. Alternatively, reports of elevated, circulating TNF- and IL-6 may reflect that a source other than macrophages is producing these cytokines at appreciable levels. Earlier studies that indicated elevated inflammatory cytokines in aging humans used the SENIEUR protocol to exclude unhealthy subjects [42
, 43
]. However, recent findings used a panel of laboratory tests to exclude additional subjects who had previously undiagnosed inflammatory diseases and poor nutritional status [29
30
31
]. These more stringent screening protocols resulted in reports that varied from prior ones. Ahluwalia et al. [29
] found no difference by age in human circulating IL-2, IL-1ß, and IL-6 levels or in whole blood culture levels of IL-2, IL-1ß, and IL-6 48 h after phytohemagglutin (PHA) stimulation. A similar study concluded that elevated IL-6 and IL-8 levels in the elderly were a result of underlying disease [31
]. Beharka et al. [30
] also found that circulating IL-6 did not differ between young (20–30 years) and aged (>65 years) volunteers; neither did spontaneous nor PHA- or concanavalin A (ConA)-stimulated IL-6 secretion by peripheral blood mononuclear cells (PBMCs) at 48 h. However, PBMCs from these aged humans produced less IL-6 than those from young when cultured with autologous plasma and stimulated 48 h with ConA. Similarly, they extended these studies to C57BL/6 male peritoneal macrophages and detected no age-related difference in spontaneous or stimulated IL-6 production.
Our studies add to this newer body of evidence and show an age-related decrease in LPS-stimulated TNF- and IL-6 production from peritoneal macrophages of a different mouse strain and sex (Figs. 1
and 2) . More importantly, we demonstrate that this decreased cytokine production with advanced age is not merely a function of altered kinetics. Macrophages from young and aged mice exhibited similar time-dependent responses to LPS stimulation (Fig. 1B)
. The maximum amount of TNF- production was already realized by 6 h post-stimulation. The most likely reason the results from Beharka et al. [30
] do not demonstrate the age-related decrease in IL-6 production that ours does is differing methodologies. We used concentrations of LPS multiple orders of magnitude less than they to stimulate cells that had been rested overnight (to ensure that stimulation from thioglycollate elicitation and adherence properties did not affect stimulation [44
]; Fig. 1A ). As TNF- and IL-6 are early released cytokines, we found that 6 h was sufficient to detect production differences by age. An extended time point may complicate an interpretation of the results because of probable feedback mechanisms and responses. It is likely that Beharka et al. [30
] did not find the age-associated differences in IL-6 production that we do, as such differences were masked by a supraoptimal stimulus (5 µg/mL LPS) and an extended endpoint (48 h). However, our data do confirm the findings of Renshaw et al. [32
], who also demonstrated reduced TNF- and IL-6 production with advancing age in macrophages from male C57BL/6 mice. Together, these reports expose the need to re-evaluate the notion that a heightened inflammatory state is a natural consequence of aging. Rather, secondarily elevated levels of proinflammatory cytokines themselves might be a result of an inability to mount a proper inflammatory response at the initial bacterial invasion in aging animals.
As discussed above, macrophages are stimulated by LPS through TLR4, which results in the phosphorylation of MAPKs and subsequent expression of TNF-, IL-6, and other cytokines [23
24
25
26
27
]. As proinflammatory cytokine production upon LPS stimulation is decreased in macrophages from aged mice, at least one point in the signal transduction pathway from TLR4 expression to cytokine production should be altered. Our findings are the first to demonstrate age-dependent differences in p38 and JNK expression and LPS-stimulated activation (Figs. 4
and 5)
. Although LPS-stimulated macrophages from both age groups demonstrated increased levels of phosphorylated p38 and JNK, they were statistically significant age-dependent differences (P<0.05). As the phosphorylated form of these kinases is the active form, decreased levels of phosphorylated MAPKs in macrophages from aged mice could result in decreased expression of the proteins under their regulatory control [45
, 46
]. Indeed, these studies indicate that a direct target of p38, MAPK-APK-2, is phosphorylated less upon LPS stimulation in macrophages from aged mice relative to young (Fig. 6)
, correlating with the observed differences in MAPK expression and LPS-stimulated activation. Therefore, the diminished ability with age to secrete proinflammatory cytokines from macrophages could be explained at the levels of MAPK transcriptional and translational regulation.
Two possibilities could account for the age-related decrease in levels of phosphorylated MAPKs that we have demonstrated: A signaling event upstream of MAPK phosphorylation is differentially regulated, or phosphorylation of MAPKs is limited by less total MAPK protein available. The most distal upstream event in the LPS signaling cascade is TLR4. Renshaw et al. [32 ] showed a decrease in TLR4 mRNA and suggested that surface TLR4 was also less in macrophages from aged mice. However, our data indicate that surface TLR4 expression does not differ by age in peritoneal macrophages (Fig. 3) . This discrepancy might be accounted for by different methodologies. We refer to TLR4 on PECs that are positive for F4/80, whereas Renshaw et al. [32 ] investigated TLR4 expression on PECs that were positive for CD11b (Mac-1). CD11b (complement receptor 3) is not specific for macrophages but is also expressed on granulocytes, dendritic cells, natural killer cells, and B-1 cells in the peritoneal and pleural cavities [47 48 49 50 51 ]. It is also up-regulated quickly on activated neutrophils [52 ]. In contrast, F4/80 is expressed on mature macrophages [53 54 55 ] and probably identifies a more pure macrophage population. In addition, the discrepancy we find with the macrophage TLR4 mRNA data is that post-transcriptional events may ensure a consistent surface-protein expression. Our finding that surface TLR4 was not reduced with age indicates that reduced receptor recognition of LPS is not likely to be responsible for reduced MAPK activation in macrophages from aged mice.
Regardless of surface TLR4 expression, we show for the first time lower LPS-stimulated phosphorylation of p38 and JNK and suggest a more proximal cause of decreased cytokine production, namely, less p38 and JNK are available to be activated (Figs. 4C and 4D and 5C) . Although it is known that MAPK expression is regulated in some cell types during development, to our knowledge, there are no reports of differential expression of MAPK proteins in adults. However, our data demonstrate that macrophages from aged mice have less total p38 and JNK than those from young mice. The decreased intracellular pool of MAPKs could account for the observation that macrophages from aged mice had lower levels of activated MAPKs following stimulation with LPS. Furthermore, we found that the ratios of phosphorylated MAPK to total available MAPK did not differ by age, indicating that activation of MAPKs was proportional to total MAPKs, regardless of age. That these ratios and the surface TLR4 expression did not differ with age suggests that decreased levels of MAPK activation may be merely a function of reduced total MAPKs with age.
In conclusion, we have demonstrated that macrophages from aged mice are functionally impaired, as exhibited by their inability to respond to LPS stimulation with levels of proinflammatory cytokine production observed in young counterparts. In addition, these studies are the first to indicate that MAPK expression and activation are reduced in LPS-stimulated macrophages from aged mice relative to those from young. Therefore, we suggest that down-regulation of macrophage MAPK expression is a possible mechanism of macrophage dysfunction with age and that the possible implications on immunosenescence deserve further investigation.
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ACKNOWLEDGEMENTS |
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Received August 19, 2003; revised October 22, 2003; accepted October 28, 2003.
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