| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Published online before print January 3, 2005
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,1
* Department of Microbiology, Immunology, and Molecular Genetics and
Sanders Brown Center on Aging, University of Kentucky, Lexington
1 Correspondence: Department of Microbiology, Immunology, and Molecular Genetics and Sanders Brown Center on Aging, 329A Sanders-Brown Building, University of Kentucky, Lexington, KY 40536-0230. E-mail: bondada{at}uky.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) function with aging makes mice less responsive to bacterial capsular polysaccharides, such as those present in the pneumococcal polysaccharide vaccine, a model of thymus independent (TI) antigen (Ag). Using trinitrophenol (TNP)-lipopolysaccharide (LPS) and TNP-Ficoll, two other well-studied TI Ag, we studied the mechanistic basis of reduced M function in the aged. We show that aged mice are profoundly hyporesponsive to these TI Ag. As a result of a requirement for M, highly purified B cells from young-adult mice do not respond to TI Ag. When purified, young B cells were immunized with TNP-Ficoll, the antibody production from those cultures reconstituted with M from aged mice was significantly lower than that seen with young M. Consequently, this unresponsiveness can be overcome by a mixture of interleukin (IL)-1ß and IL-6. Upon stimulation with LPS, in comparison with young M, aged M secreted reduced amounts of IL-6, tumor necrosis factor 
, IL-1ß, and IL-12, cytokines necessary for B cells to respond to TI Ag. LPS also induced aged M to produce an excess of IL-10. Neutralization of IL-10 enhanced the production of proinflamatory cytokines by M upon LPS stimulation and also induced Ab production by aged splenocytes. Thus, the inability of aged M to help the B cell response appears to be caused by an excess of IL-10. As aged M have a reduced number of cells expressing Toll-like receptor 4 and CD14, the imbalance in cytokine production might be partly a result of fewer cells expressing key components of the LPS receptor complex.
Key Words: B lymphocytes • cytokines • LPS • TNP-Ficoll • TNP-LPS
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
One consequence of the age-associated decline in the immune system is the increased susceptibility to infection with encapsulated bacteria, such as Streptococcus pneumoniae and Hemophilus influenzae [10 ]. Thus, as individuals advance in age, incidence of pneumonia and bacteremia caused by S. pneumoniae increases, and this is accompanied by a rising rate in mortality [11 ]. This increased susceptibility is a result of the inability of the elderly to generate opsonic Ab to pneumococcal capsular PS, which facilitate phagocytosis by cells of the reticuloendothelial system and neutrophils [12 13 14 ].
Vaccines made up of purified bacterial capsular PS have been available for many years and are effective in young adults. However, the overall efficacy against invasive pneumococcal disease among persons 65 years of age and older is only 75% and appears to decrease with advancing age [13 , 15 ]. Pneumococcal capsular PS are thymus-independent (TI) Ag, and therefore, the generation of mature, Ab-secreting B cells occurs in the absence of major histocompatibility complex class II-restricted T cell help [12 , 16 ]. TI Ag are divided into two types, based on whether they induce immune responses in CBA/N or neonatal wild-type (WT) mice. CBA/N mice, which have an X-linked, recessive immune defect, and neonatal WT mice respond to TI-1 Ag but not to TI-2 Ag [16 , 17 ]. Another difference is that at high doses, TI-1 Ag, unlike TI-2 Ag, become mitogenic and invoke a polyclonal response from B cells [13 , 18 ].
Although TI Ag stimulate B cells without cognate T cell-B cell interactions, the responses of B cells to TI Ag are dependent on the presence of other cells, such as macrophages (M) and T cells. Thus, rigorous depletion of T cells and M will virtually eliminate the response of young-adult B cells to all TI Ag. However, this response can be reconstituted by supplementing highly purified B cells with purified M or M- or T cell-derived cytokines, suggesting that a function of M in the TI-Ag response is to secrete B cell stimulatory cytokines [13
, 18
19
20
21
].
B cells of the elderly do not mount an effective Ab response to these PS Ag. Consequently, they do not develop an effective immune response to the bacterial capsule nor to the pneumococcal PS vaccines [12
]. Just as with humans, mice also exhibit an age-associated decline in Ab responses to TI Ag [22
23
24
25
]. We have previously shown that splenocytes from aged mice responded poorly to the pneumococcal PS vaccine in vitro and in vivo. Moreover, M from young-adult mice or M-derived interleukin (IL)-1ß restored the pneumococcal vaccine response of aged splenocytes [26
]. This finding suggested that M had a part to play in the hyporesponsiveness of aged mice to TI Ag.
The immune system of the neonatal mice (as well as human infants) is also unresponsive to infection with encapsulated bacteria such as S. pneumoniae as a result of an inability to respond to TI Ag. Using highly purified, young-adult B cells, we recently showed that although M from young-adult mice could restore the response of young-adult B cells to TI Ag, M from neonatal mice failed to do so [27
]. The inability of neonatal M to help the adult B cells to mount an Ab response against TI Ag is most likely a result of the fact that they were defective in secreting a variety of B cell stimulatory cytokines upon stimulation with a TI Ag.
As IL-1ß could restore the response of aged splenocytes to TI Ag and as neonatal M could not support an Ab response against TI Ag, we decided to further investigate the role played by aged M in the hyporesponsiveness of aged splenocytes to TI Ag. In this study, we show that aged M play a role in the unresponsiveness of aged mice to TI Ag; that is, aged, splenic M could not restore the TI response of highly purified, young-adult B cells. Moreover, this appears to be a result of a decrease in IL-1 production by the aged M. The dysregulated IL-10 production by aged M may be related to a reduction in the expression of Toll-like receptor (TLR)-4, a key component of the lipopolysaccharide (LPS) receptor complex and a key mediator of M activation by gram-negative bacteria.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
were obtained from BD Biosciences (San Diego, CA), and the polyclonal goat anti-mouse IL-10 neutralizing Ab was obtained from R&D Systems (Minneapolis, MN). Fluorochrome-conjugated Ab to CD11b (or Mac-1) was purchased from Caltag (Burlingame, CA), and those to CD14 and CD86 were obtained from BD Biosciences. The Ab to TLR-2 (Clone 6C2) and TLR-4 (Clone MTS510) were obtained from eBioscience (San Diego, CA).
Cell enrichment and culture
B cells (80–90% IgM+) were isolated from spleens of unimmunized mice, processed, and enriched by panning on anti-IgM-coated plates, according to the protocol described previously [19
]. M were purified by positive selection from unfractionated splenocytes using the magnetic cell sorter (MACS) CD11b microbeads from Miltenyi Biotec (Bergisch Gladbachuor, Germany). The enriched M were routinely found to be 
95% Mac-1+. For in vitro immunization, cultures were set up in 1 ml IMDM + Ham’s F12 (+10% fetal bovine serum) in 48-well plates (Costar, Corning, NY) with TNP-Ficoll (16 ng/ml), TNP-LPS (0.25–4.00 µg/ml), splenocytes (0.5–4.0x106 per culture), B cells (1x106 per culture), or M (0.75x106 per culture) for 4 days in 5% CO2 and at 37°C [28
]. The number of IgM anti-TNP-secreting cells was determined using a glass-slide version of the Ab-forming cell (AFC) assay as described earlier [19
].
Cytokine analysis
M prepared by enriching with Mac-1 MACS microbeads (0.25 or 0.50x106) were cultured in duplicate for 1 day in medium or in 1 µg/ml LPS. Various cytokines in the supernatant were estimated in duplicate using enzyme-linked immunosorbent assay (ELISA). IL-12, IL-10, and tumor necrosis factor (TNF-) were estimated with OptEIA kits (PharMingen, San Diego, CA) and IL-1ß with the Quantikine M ELISA kit (R&D Systems). IL-6 was measured with a matched-pair Ab set (Clones MP5-20F3 and MP5-32C11) from BD Biosciences. The optical densities were read on an HTS 7000 (Perkin Elmer, Norwalk, CT). Results are presented as mean of four measurements ± SE.
Flow cytometry
The FcR was blocked by incubating with 1 µg normal rat IgG per 0.25 x 106 M for 20 min. Without washing, the cells were further incubated with 1 µg Ab for 30 min on ice. The cells were then washed with Hanks’ balanced saline solution (+0.1% bovine serum albumin). Where necessary, the cells were further incubated with allphycocyanin-conjugated streptavidin for 20 min. The cells were then washed and immediately analyzed using a FACSCalibur (BD Instruments, San Jose, CA).
Statistical analysis
Statistical significance of differences between averages was determined by the Student’s t-test. We considered the two samples to be compared to be of equal variance and determined the significance for a two-tailed distribution.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
-TNP response to TNP-Ficoll at this cell density [19
]. Splenocytes from young mice produced a robust response when cultured with TNP-Ficoll for 4 days, but aged splenocytes failed to respond (Fig. 1B)
.
M from aged mice failed to reconstitute the response of young-adult B cells to TNP-Ficoll
Previously, we discovered that the unresponsiveness of the aged splenocytes to the pneumococcal PS vaccine was not a result of an intrinsic B cell defect or of T cell-mediated immunosuppression but resulted from an accessory cell deficiency [29
]. Therefore, we decided to determine if such a deficiency in accessory cells could also be demonstrated with a more potent TI Ag such as TNP-Ficoll. Purified B cells from young-adult mice were stimulated with TNP-Ficoll in the presence of M from aged or young-adult mice. As expected, highly pure, young-adult B cells failed to respond to TNP-Ficoll and instead, produced a background response of 18 ± 21 AFC/culture (Fig. 2
). As seen previously [27
], supplementing the cultures with M from young-adult mice reconstituted the response (P<0.005), and M from aged mice failed to help young-adult B cells to respond (P>0.100; Fig. 2 ). Even when the number of M added to the B cell cultures was titrated above 0.75 x 106 cells per culture, M from aged mice were unable to help B cells to respond to TNP-Ficoll (data not shown).
|
produced reduced amounts of proinflammatory cytokines and excess IL-10-derived cytokines, such as IL-1ß and IL-6 [13
, 19
]. Recently, we showed that the defective responses of neonatal mice to TI Ag were also a result of defects in M function, which was, in turn, a result of a reduction in cytokines secreted by neonatal M known to be required for B cell response to TI Ag [27
]. Based on these studies, we investigated if the unresponsiveness of aged splenocytes to TI Ag was also a result of the inability of aged M to secrete the right mixture of cytokines needed to help B cells to mount an Ab response to TI Ag.
Aged M were stimulated with 1 µg/ml LPS, a well-characterized TI Ag [16
], for 24 h, and then the levels of the cytokines were measured in the culture supernatant by ELISA. Within 1 day of stimulating with LPS, M from aged and young-adult M secreted IL-1ß, IL-6, IL-10, IL-12, and TNF- (Fig. 3
). However, aged M secreted about two times less IL-1ß (P<0.010; Fig. 3A
), four times less IL-6 (P<0.001; Fig. 3B
), and two times less IL-12 (P<0.001; Fig. 3C
). Aged M consistently produced 20% less TNF- (P<0.050; Fig. 3D
; data not shown), an effect that was more pronounced when lower concentrations of LPS were used. In contrast to these four cytokines, production of IL-10, an anti-inflammatory cytokine, was enhanced in activated, aged M (P<0.010; Fig. 3E
).
|
was not restricted to BALB/c, adherent cells were obtained from C57BL/6 and stimulated with LPS for 1 day. Like BALB/c mice, aged C57BL/6 M produced reduced amounts of IL-6 (twofold; P<0.001) and TNF- (1.4-fold; P<0.001; data not shown). Just as with BALB/c, the age-associated reduction in TNF- was less than twofold, but it was nevertheless statistically significant.
The kinetics of IL-10 and IL-6 secretion is altered in aged M
As we were particularly interested in the fact that activated, aged M secreted significantly more IL-10 than young-adult M (Fig. 3E)
, we determined if the kinetics of LPS-induced IL-10 and IL-6 production was altered in aged M. Within 6 h, aged and young M had started to secrete IL-10, and by 9 h, aged M had produced about threefold more IL-10 (P<0.010; Fig. 4A
). The levels of IL-10 present in the culture supernatant for aged and young-adult M did not change significantly between 12 h and 36 h. Within 6 h of stimulation with LPS, aged and young-adult M also began to secrete IL-6, and by as early as 9 h, young-adult M cultures contained about twofold more IL-6 (P<0.001; Fig. 4B ). Although the amount of IL-6 in aged M cultures leveled off by 12 h, young-adult M continued to secrete IL-6 until the experiment was terminated at 72 h (Fig. 4B)
.
|
and reduce the production of IL-6 in LPS-activated, young-adult M [30
, 31
]. We therefore determined if neutralizing IL-10 in cultures would induce aged M to secrete cytokines at amounts comparable with adult M. As previously seen (Fig. 3B)
, stimulating aged M with LPS resulted in a reduced amount of IL-6 (P<0.050; Fig. 5A
). However, neutralizing IL-10 by supplementing the cultures with anti-IL-10 resulted in aged M producing comparable amounts of IL-6 as young-adult M (P>0.100). It is interesting that anti-IL-10 also enhanced the production of IL-6 by young-adult M. Providing aged M with anti-IL-10 also enhanced the production of IL-12 and TNF-, such that they were comparable to levels produced by young-adult M (P>0.100; Fig. 5
, B and C). As seen with IL-6, neutralization of IL-10 also enhanced the production of IL-12 and TNF- by young-adult M. In this particular experiment, aged macrophages did not produce measurable levels of IL-12 when incubated with only LPS, unlike in Figure 3C
. This is probably because of the inherent variability in the absolute levels of cytokines produced from experiment to experiment (compare the levels of TNF or IL-6 in Fig. 3
vs. Fig. 5
). It is important that the pattern was always the same (high IL-10 and low IL-6 and TNF- in the aged).
|
have fewer TLR-4+ cells could be explained in part by a defective expression of the LPS receptor complex, Mac-1+ cells were analyzed by flow cytometry for surface expression of CD14 and TLR-4, components of the LPS receptor complex [32
]. Aged M, which were harvested and kept on ice, had significantly higher amounts of surface expression of Mac-1 than young M (P<0.050; Table 1
). When M were placed in culture for 18 h, they down-regulated the surface expression of Mac-1. However, the decrease in aged M was more dramatic, resulting in unstimulated, aged M now having lower levels of surface Mac-1. When M from both age groups were cultured with LPS, the drop in Mac-1 was not seen.
|
had slightly fewer CD14+ cells (Table 1 ; data not shown). Upon placing the cells in culture, there was an increase in CD14+ cells in both age groups. Stimulating with LPS resulted in a larger amount of CD14+ cells in the young-adult cultures (P<0.05). MTS510 detects the functional complex of TLR-4 and MD-2 but not TLR-4 alone [33
]. Using MTS510, we showed that harvested, aged Mac-1+ cells had a slightly reduced proportion of TLR-4/MD-2+ cells (Table 1)
. After overnight culture in medium alone, the aged M still had fewer numbers of TLR-4/MD-2+ cells (Table 1
; data not shown). Stimulation with LPS increased the number of TLR-4/MD-2+ cells in both groups of M, but young-adult M cultures had slightly higher proportions of TLR-4/MD-2+ cells (P<0.05; Table 1
). However, the surface density of TLR-4/MD-2 on aged M was comparable with that of young-adult M.
As B7.2 is an activation marker for M [34
], we also looked at B7.2 expression. Harvested, aged M had a fewer proportion of B7.2+ cells (Table 1
; data not shown). Placing M in culture caused a dramatic increase in the proportion of B7.2+ cells in the young-adult M cultures (P<0.05), but aged M cultures failed to increase the proportion of B7.2+ cells (P<0.05; Table 1
; data not shown). Stimulating with LPS caused an up-regulation in the numbers of B7.2+ M in both age groups, but aged M cultures continued to have fewer B7.2+ cells than the young. M were also analyzed for surface expression of TLR-2 (the receptor for peptidoglycan and lipoteichoic acid) and intercellular adhesion molecule-1 (an adhesion molecule). However, there were no significant differences between aged and young-adult M (data not shown).
IL-1ß and IL-6 enabled aged splenocytes to respond to TNP-Ficoll
Previously, we had shown that IL-1ß would induce neonatal splenocytes to respond to TNP-Ficoll [19
]. Therefore, we supplemented cultures with IL-1ß and measured the response to TNP-Ficoll. In the presence of IL-1ß, aged splenocytes responded to TNP-Ficoll, and the level of response was comparable with the adult response in the absence of IL-1ß (P>0.100; Fig. 6A
).
|
Neutralization of the effect of IL-10 enhances TNP-LPS response of the aged splenocytes
Having shown that increased IL-10 levels in aged mice might contribute to the inability of aged M to respond to TI Ag, we wondered if aged splenocytes could be induced to respond to TI Ag upon neutralization of IL-10. Cultures of splenocytes were incubated with TNP LPS in the presence of an Ab to the IL-10R. This Ab neutralizes the effect of IL-10 by reacting with the extracellular region of the IL-10R and blocks the binding of IL-10 [34
]. Indeed, the TNP-LPS response of aged splenocytes was significantly enhanced three- to fourfold by the inclusion of the anti-IL-10R Ab in the culture (Fig. 7
). However, the magnitude of the response was still less than that of the young adult (400 PFC/culture; see Fig. 1A
), presumably because of the importance of IL-10 for B cell activation [30
, 35
].
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
restored an appropriate Ab response to PS Ag, the cause of the age-related unresponsiveness to PS Ag did not lie solely with the B cell compartment [19
, 26
, 27
, 35
36
37
]. We recently showed that the inability of neonatal M to be properly activated by TI Ag is a critical component in the unresponsiveness of neonates to TI Ag. Therefore, we investigated the role of M in the hyporesponsiveness to PS TI Ag in splenocytes from aged mice. Splenocytes from aged mice are unable to mount an Ab response against pneumococcal capsular PS, a TI-2 Ag [38 ], TNP-Ficoll (Figs. 1B and 6A) , a much more potent TI-2 Ag, and even to TNP-LPS (Fig. 1A) [39 ], an example of a robust TI-1 Ag. The inability of aged splenocytes to respond to a number of TI Ag suggests a systemic, underlying cause.
The immune system of neonates is functionally immature. In contrast, as a result of the process of immunosenescence, the ability of aged mice to respond to various Ag gradually breaks down. It is interesting that despite being at two ends of the age spectrum, the splenocytes of neonatal and aged mice behave remarkably similar in that their hyporesponsiveness to TI Ag could be overcome simply by providing the cultures with IL-1ß (Fig. 6A and ref. [27
]). Moreover, highly purified B cells of aged mice, like those of young adults and neonates, respond in vitro, when provided with IL-1ß and IL-6. As the level of response of aged B cells was comparable with that of young-adult B cells, this would indicate that aged B cells were not intrinsically defective in mounting an Ab response. Using highly purified, young-adult B cells as responders, we see that unlike young-adult M, aged M could not reconstitute the Ab response (Fig. 2)
.
We recently showed that the unresponsiveness in neonates against TI Ag was partly a result of an inability of neonatal M to respond appropriately to LPS [27
]. Unlike young-adult M, neonatal M secreted little or no IL-1ß, IL-12, and TNF-, reduced amounts of IL-6, and excess amounts of IL-10. Just like neonatal M, aged M secreted reduced amounts of IL-6 and excess of IL-10. Conversely, unlike neonatal M, aged M secreted detectable levels of IL-1ß, IL-12, and TNF-, but the amounts were all reduced in comparison with young-adult M. However, despite the ability to secrete measurable levels of IL-1ß, IL-6, IL-12, and TNF-, albeit at reduced amounts, aged M were as ineffective as neonatal M in reconstituting a young-adult B cell response to TI Ag. This could imply that there was yet another cytokine(s) that aged M did not secrete. However, it is also possible that the defect in aged M is a result of a combined reduction in two or more of the proinflammatory cytokines measured. Experiments are under way to determine if supplementing cultures with one or more of these cytokines could enhance the ability of aged M to reconstitute the Ab response of B cells to TI Ag.
By 6 h after onset of stimulation with LPS, aged M, in contrast to those from young mice, were secreting twice as much IL-10 as IL-6 (Fig. 4A)
. As the expression of the IL-10 gene upon LPS stimulation only occurs after the initiation of TNF- and IL-1 transcription [40
], it is probable that in aged M, the initiation of IL-10 transcription might actually coincide with that of the inflammatory cytokines. As IL-10 suppresses the production of TNF-, IL-1ß, IL-6, and IL-12 in young-adult M, it is not surprising that this early burst of IL-10 keeps in check the ability of aged M to secrete TNF-, IL-1ß, IL-6, and IL-12. As a result of conflicting reports, there is no clear consensus if inhibition of nuclear factor (NF)-B or mitogen-activated protein kinases (MAPKs) is involved in IL-10-mediated suppression of LPS-induced activation [40
]. However, IL-10 does induce Bcl-3 and protein disulfide isomerase in M, both of which have been shown to be involved in IL-10-mediated suppression of LPS-induced inflammatory cytokines [40
, 41
].
It cannot be ruled out that this early burst in IL-10 production may be a result of a defect in aged M to secrete sufficient quantities of the proinflammatory cytokines tested. Previously, when we were investigating neonatal M, we used IL-10–/– mice to answer this question [27
]. However, as IL-10–/– mice begin to develop colitis within 3–6 months of age [42
], these mice were not a suitable model to address this question in aged M. Therefore, to determine if excess IL-10 production was the cause or the effect of the cytokine imbalance in aged M, we stimulated aged BALB/c M with LPS in the presence of anti-IL-10. Neutralizing IL-10 allowed aged M to secrete levels of inflammatory cytokines in quantities comparable with that of LPS-stimulated, young-adult M. This would indicate that the excess production of IL-10 was the cause of the cytokine imbalance. It is important to note that as LPS also induces young-adult M to secrete IL-10, it is not the secretion of IL-10 per se that causes the imbalance in aged M but rather the excess production of IL-10 at early time-points. A small amount of IL-10 is definitely required for proper functioning of the immune system. The reason for having just a critical amount of IL-10 may have to do with the nature of the cytokine. Although IL-10 is well-known to suppress M function, it also plays a role in enhancing B cell function [43
]. Thus, in the young adult, the amount of IL-10 produced is sufficient to induce B cell activation but not enough to severely inhibit the ability of M to secrete other B cell stimulatory cytokines.
These results with IL-10-dependent down-regulation of IL-1 and IL-6 predict that inhibition of IL-10 signaling should restore the TI Ag responses. However, this situation is slightly more complex because of the positive effects of IL-10 on B cell differentiation, as noted above. Despite this complexity, we were able to obtain a partial restoration of the TNP-LPS response in the aged splenocytes when anti-IL-10R Ab was included in the culture (Fig. 7) . Thus, increased secretion of IL-10 by aged macrophages is in part responsible for the decreased response of the aged to TI Ag.
B7.2 is an important costimulatory molecule for T cell activation and an activation marker of M [44
]. As IL-10 has been implicated in down-regulating B7 [40
], it is not surprising that aged M had fewer B7.2+ M than young adults. Allowing them to adhere did not up-regulate B7.2 expression. As B7 has been implicated in the in vivo Ab response against pneumococcal capsular PS in mice immunized with S. pneumoniae bacteria [45
], this would imply that aged M are not as competent to recruit T cells into this Ab response. This is also consistent with decreased Ag-presenting function in T cell responses to the influenza vaccine in the aged [46
].
Although the defect in aged M is a result of excess IL-10 production, the question still remains as to the cause of the dysregulation in IL-10 production as the mouse increases in age. We looked at the levels of expression of CD14 and TLR-4/MD-2, critical components of the LPS receptor complex. We did not observe a reduced amount of CD14 in aged M. Although Renshaw et al. [47
] showed a reduction in TLR-4 on aged M, we, like Boehmer et al. [48
], did not see a reduction in surface TLR-4 expression per cell. This difference in levels of TLR-4 per cell could be a result of the difference in methodologies used to obtain M. Renshaw et al. [47
] used adherence on plastic to obtain M, and our procedure did not involve adherence. We noticed that adherence on plastic predisposed M to die when they were further enriched with Mac-1 microbeads to increase the purity of our cells. We then modified our procedure to enrich M by selecting for Mac-1+ cells directly from the freshly obtained, unadhered splenocytes. The number of Mac-1+ cells isolated was similar, and these cells had a greater purity. Although we didn’t observe a reduction in the level of surface expression of the components of the LPS receptor complex, we did detect significantly fewer aged M, which were positive for CD14 or TLR-4. In other words, fewer aged M expressed components of the LPS receptor complex, but those that did had amounts comparable with that of young-adult M. However, this reduction in LPS receptor-positive cells is not likely to be the major reason why aged M are functionally altered. Fewer cells with the LPS receptor complex should translate into lower amounts of LPS-induced cytokines, not an imbalance in cytokine production. Thus, the clue to this imbalance most likely lies with the signaling upon ligation of LPS. In keeping with this hypothesis, Boehmer et al. [48
] showed that there was a reduced expression of p38 and c-jun NH2-terminal kinase MAPK in M from aged mice.
Differential modulation of IL-10 and the proinflammatory cytokines in the aged M suggests that NF-B activity may be impaired in the aged, and activity of specificity protein-1, a transcription factor critical for IL-10 expression [49
], may be maintained or up-regulated in the aged. Changes in the transcription factors could be influenced by regulators such as suppressor of cytokine signaling (SOCS)-1, transforming growth factor-ß (TGF-ß), and cyclic adenosine monophosphate (cAMP), which are known to affect cytokine gene expression in M [50
51
52
53
]. Effects of age on production of SOCS-1 and TGF-ß are not yet well-studied, and cAMP has been observed to increase with aging [54
].
Previous studies by some have shown that serum levels of IL-6 and TNF- were higher in the elderly and aged mice [55
]. Our preliminary experiments confirm a significantly higher level of IL-6 in sera of aged mice (26±9 pg/ml for aged vs. 0±0 pg/ml for young; P<0.001). However, our data from splenic M are in agreement with that of Renshaw et al. [47
], who also noted that LPS-stimulated, splenic M of aged mice secrete reduced IL-6 and TNF- upon LPS stimulation. Possibly, these increased levels of circulating IL-6 and TNF- detected in the serum are produced by cells other than splenic M such as T cells and macrophages from other tissues.
In conclusion, we have demonstrated that M from aged mice are functionally impaired, as exhibited by their inability to help young-adult B cells to mount an Ab response against TI Ag. We believe that as a result of a dysregulation in IL-10 production, the levels of inflammatory cytokines needed for a proper B cell response are suppressed. Our studies suggest that efforts to enhance vaccine responses in the aged should probably include agents that can restore normal macrophage function.
|
ACKNOWLEDGEMENTS |
|---|
Received August 10, 2004; revised December 6, 2004; accepted December 10, 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
production in macrophages Blood 102,4123-4129 and cAMP-elevating drugs Int. Immunol. 7,517-523This article has been cited by other articles:
|
|
 |
|
  E. J. Albert and J. S. Marshall Aging in the absence of TLR2 is associated with reduced IFN-{gamma} responses in the large intestine and increased severity of induced colitis J. Leukoc. Biol., April 1, 2008; 83(4): 833 - 842. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B.-C. Chiu, V. R. Stolberg, C. M. Freeman, and S. W. Chensue Mononuclear Phagocyte-Derived Interleukin-10 Suppresses the Innate Pulmonary Granuloma Cytokine Response in Aged Mice Am. J. Pathol., September 1, 2007; 171(3): 829 - 837. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Chelvarajan, D. Popa, Y. Liu, T. V. Getchell, A. J. Stromberg, and S. Bondada Molecular mechanisms underlying anti-inflammatory phenotype of neonatal splenic macrophages J. Leukoc. Biol., August 1, 2007; 82(2): 403 - 416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Chelvarajan, Y. Liu, D. Popa, M. L. Getchell, T. V. Getchell, A. J. Stromberg, and S. Bondada Molecular basis of age-associated cytokine dysregulation in LPS-stimulated macrophages J. Leukoc. Biol., June 1, 2006; 79(6): 1314 - 1327. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Sen, Q. Chen, and C. M. Snapper Immunization of Aged Mice with a Pneumococcal Conjugate Vaccine Combined with an Unmethylated CpG-Containing Oligodeoxynucleotide Restores Defective Immunoglobulin G Antipolysaccharide Responses and Specific CD4+-T-Cell Priming to Young Adult Levels Infect. Immun., April 1, 2006; 74(4): 2177 - 2186. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
