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Published as doi: 10.1096/fj.07-103721.
 (The FASEB Journal. 2008;22:2142-2150.)
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Nuclear factor-{kappa}B-dependent reversal of aging-induced alterations in T cell cytokines

Mei-Chuan Huang*, Jia-Jun Liao*, Stephen Bonasera*, Dan L. Longo{dagger} and Edward J. Goetzl*,1

* Department of Medicine, University of California, San Francisco, California, USA; and

{dagger} National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA

1Correspondence: University of California, Room UB8B, 533 Parnassus at 4th Ave., San Francisco, CA 94143-0711, USA. E-mail: edward.goetzl{at}ucsf.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immunosenescence is characterized by decreases in protective immune responses and increases in inflammation and autoimmunity. The T helper (Th)17 subset of cluster-of-differentiation (CD)4 T cells, which is identified by its generation of interleukin (IL) -17, is implicated in autoimmune pathogenesis. To elucidate immunosenescent changes in Th17 cell cytokines, splenic CD4 T cells from 22- to 24-month-old (old) mice and 6- to 10-wk-old (young) mice were incubated on anti-CD3 plus anti-CD28 (anti-T cell antigen receptor) antibodies. After 96 h, T cells of old C57BL/6 and CBA mice generated up to 20-fold more IL-17 and up to 3-fold more IL-6 than those of young mice; T cells of young mice generated up to 5-fold more IL-21 than those of old mice; and no difference was found for IFN-{gamma}. At 24 h, cytokine mRNA levels paralleled 96 h cytokine concentrations. Naive CD4 T cells from old mice incubated on anti-T cell antigen receptor antibodies with transforming growth factor-β, IL-1, IL-6, and IL-23 to induce de novo differentiation of Th17 cells had more IL-17 mRNA and produced more IL-17 than those of young mice. BAY11-7082 and the phytochemicals triptolide and butein suppressed nuclear concentrations of nuclear factor-{kappa}B and secreted levels of IL-17, IL-21, and IFN-{gamma} in parallel, with greater potency in Th17 cells from young than old mice. Pharmacological correction of altered generation of Th17 cell cytokines in immunosenescence represents a novel therapeutic approach to aging-induced inflammatory diseases.—Huang, M.-C., Liao, J.-J., Bonasera, S., Longo, D. L., Goetzl, E. J. Nuclear factor-{kappa}B-dependent reversal of aging-induced alterations in T cell cytokines.


Key Words: immunosenescence • inflammation • autoimmunity • phytochemicals


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DETERMINANTS AND CONSEQUENCES of mammalian immune system aging, termed immunosenescence, have not been elucidated at a level that permits effective countermeasures. Some functions of innate immune cells are impaired in elderly humans and in mice having genetic defects known to predispose them to accelerated aging (1 2 3) . However, most data suggest that the principal abnormalities of immunosenescence consist of various deficiencies in T cell-dependent adaptive immune responses and concurrent increases in generation of inflammatory cytokines by immune cells.

The number and intrinsic helper functions of mouse naive cluster-of-differentiation (CD)4 T cells decrease with age and CD4 T cells of 22- to 24-month-old (old) mice are less effective at forming immune synapses with antigen-presenting cells (4 , 5) . The increase in memory CD4 T cells and concomitant decrease in naive CD4 T cells in old mice is due in part to the lymphoid environment, as well as reduced thymic generation of naive T cells. Old and 6- to 10-wk-old (young) mice reconstitute their CD4 T cell population equally well after antibody-mediated obliteration and after lymphoid irradiation followed by bone marrow cell infusion (6) . In both models of post-thymic CD4 T cell recovery, lymphoid environmental control of T cell expansion is suggested by the significantly higher ratio of memory to naive CD4 T cells in old than young mice. The drop in number of naive CD4 T cells in old mice is accompanied by a parallel reduction in generation of interleukin (IL)-2 after CD4 T cell stimulation (7) . In contrast, the number of CD8 T cells may rise slightly in old mice, and their functions, such as responses to tumors, are normal when costimulation is adequate (8) . The number and efficiency of suppressor T cells of the CD4+25+Foxp3+ subset rise and other suppressor T cells in the CD4+25–Foxp3+ subset appear in old mice, which may contribute to T cell immunodeficiency (9 10 11) . The human immune profile of old age resembles that of aged mice with higher levels of CD8 T cells and lower levels of CD4 T cells, naive CD4 T cells, and natural killer (NK) cells in blood, and decreased CD4 T cell proliferation and IL-2 generation after stimulation relative to young subjects (12) .

Most investigations of alterations in concentrations of inflammatory cytokines with aging have been conducted in humans. In aged contrasted with young humans, the serum concentrations of TNF-{alpha} and soluble IL-2 receptor but not IFN-{gamma} are significantly higher in association with a 2-fold increase in lymphocyte expression of the late activation marker human leukocyte antigen-DR but not early activation markers (13) . Serum concentrations of the inflammatory cytokines IL-18 and IL-6 also are higher in aged than in young humans, in parallel with higher levels of C-reactive protein, but the increase in free IL-18 may not be as pronounced as that of total IL-18 due to elevated levels of IL-18 binding protein with aging (14 15 16 17) .

The greater prevalence of morbidity and higher cardiovascular risk factors in aged humans also may contribute to elevated levels of inflammatory cytokines in addition to a fundamentally altered immune system (16) . The IL-18 concentration as a critical determinant of aging was suggested by the finding that individuals expressing a genetic polymorphism in the human IL-18 gene, leading to lower plasma concentrations of IL-18, have better exercise scores and other higher indices of physical function than those with normal levels of IL-18 (18) . The plasma concentration of IL-6 is considered to be a major variable in the human immune profile of old age, which reflects the role of high levels of inflammatory cytokines in shortening life span (15 , 19) . Although blood levels of T cell-derived cytokines increase with aging, T cell nuclear levels of the nuclear factor-{kappa}B (NF-{kappa}B) master switch for cytokine transcription are lower in aged than young healthy human subjects, which raises questions about the nature of cytokine control mechanisms in aging (20 21 22) .

Data presented here show that the major age-related changes in immune cytokines in mice center on the T helper (Th)17 inflammatory subset of CD4 T cells with selective increases in generation of IL-17 and IL-6, along with decreases in generation of IL-21, as a result of altered levels of transcription. This is an intrinsic property of aged Th17 cells and not simply a reflection of increased numbers because newly-differentiated Th17 cells of old mice generate more IL-17 and less IL-21 than an equal number from young mice. The age-related increase in generation of IL-17 is reversed by diverse inhibitors of NF-{kappa}B activation, which may potentially improve prognosis in aged humans with an unfavorable immune risk phenotype (15 , 19) .


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and assay kits
Murine IL-6 and IL-1β, human transforming growth factor (TGF)-β1 (Peprotech, Inc., Rocky Hill, NJ, USA), murine IL-23 (R&D Systems Inc., Minneapolis, MN, USA), guinea pig purified anti-mouse CD3{epsilon}, Syrian hamster monoclonal anti-mouse CD28, and fluorescein-conjugated anti-mouse CD4 (clone Rm 4-4, rat IgG2b{kappa}) and CD62L (clone MEL 14, rat IgG2a{kappa}) antibodies (Abs), and their isotype controls (BD-PharMingen, San Diego, CA, USA), triptolide and the BAY 11-7082 inhibitor of I{kappa}B-{alpha} phosphorylation and thus of NF-{kappa}B activation (Biomol, Plymouth Meeting, PA, USA), and butein (Alexis Biochemicals, San Diego, CA, USA) were obtained from the suppliers cited.

Lymphocyte isolation and incubation
C57BL/6 mice and CBA mice, young and old, were obtained, respectively, from Simonsen Laboratories (Gilroy, CA, USA) or Charles River Laboratories (Wilmington, MA, USA) and from colonies of the National Institute on Aging (Baltimore, MD, USA). Splenic mixed CD4 T cells were purified by immunobead-magnetic column positive-selection chromatography, as described (23) , and naive (CD62L-high) CD4 T cells were purified by sequential immunobead-magnetic column negative-selection chromatography for removal of non-CD4 T cells followed by positive-selection of CD62L-high CD4 T cells (Miltenyi Biotec, Auburn, CA, USA). Replicate 0.6 ml suspensions of 1–2 x 106 CD4 T cells or CD62L-high CD4 T cells/ml of RPMI 1640 with 10% fetal bovine serum (FBS), 100 U/ml of penicillin G, and 50 µg/ml of streptomycin were cultured in 12-well plates that had been precoated with 2 µg each of anti-mouse CD3 and anti-mouse CD28 Abs. For studies of differentiation of naive CD4 T cells, medium was supplemented with 10 ng/ml of IL-1β, 10 ng/ml of IL-6, 1 ng/ml of TGF-β1, and 10 ng/ml of IL-23.

Flow cytometry
Splenic CD4 T cells were washed twice in PBS-1% (v:v) FBS and incubated with anti-fragment crystallizable (Fc) receptor Abs (rat IgG2b{kappa} anti-CD16/CD32, anti-Fc{gamma} III/II; clone 2.4G2, BD-PharMingen) for 5 min at 4°C. After 1 additional wash, CD4 T cells were incubated for 30 min at 4°C with fluorescently labeled anti-CD4 or anti-CD62L Abs. Fluorescent characteristics of labeled T cells were analyzed with a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) and a FlowJo software program (Tree Star, Ashland, OR, USA).

Reverse transcription-real time polymerase chain reaction (RT-PCR) analyses
The levels of mRNA encoding murine IL-17, IL-21, IL-6, and ROR{gamma}T in total RNA extracted from splenic CD4 T cells were quantified by Taqman RT-PCR. Total RNA was extracted using Trireagent (Molecular Research Center, Cincinnati, OH, USA) treated with RNase-free DNase I (Qiagen, Valencia, CA, USA) and isolated on RNeasy columns (RNeasy Kit, Qiagen). Taqman RT-PCR was performed with an ABI 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA), using specific primers and 5'-FAM (F)/3'-TAMRA (T) labeled probes:

IL-17: forward, 5'-ccacgtcaccctggactctc-3'; reverse, 5'-ctccgcattgacacagcg-3'; probe, F-cctctgtgatctgggaagctcagtgcc-T

IL-21: forward, 5'-atcctgaacttctatcagctccac-3'; reverse, 5'-gcatttagctatgtgcttctgtttc-3'; probe, F-aagccatcaaaccctggaaacaataagaca-T

IL-6: forward, 5'-acaagtcggaggcttaattacacat-3'; reverse, 5'-aatcagaattgccattgcacaa-3'; probe, F-tcttttctcatttccacgatttcccagagaa-T

ROR{gamma}T: forward, 5'-ccgctgagagggcttcac-3'; reverse, 5'-tgcaggagtaggccacattaca-3'; probe, F-aagggcttcttccgccgcagccagcag-T

The relative level of expression of each mRNA was reflected in its optimized threshold cycle (Ct), which was normalized with reference to the Ct value for the standard housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) (User Bulletin 2 of ABI Prism 7700 Sequence Detection System). The Taqman conditions were: 48°C for 30 min, 99°C for 7 min, and 95°C for 3 min in cycle 1 and 95°C for 15 s and 60°C for 1 min in cycles 2–41. The relative quantity of mRNA in each sample was calculated by the comparative Ct method, based on the formula 2 – {Delta}{Delta}Ct, where {Delta}{Delta}Ct = {Delta}Cts – {Delta}Ctr. Here, {Delta}Cts is the Ct value for any sample, normalized to the endogenous housekeeping gene HPRT, and {Delta}Ctr is the Ct value for the reference sample, also normalized to HPRT.

Cytokine ELISAs and ELISpot assay
Cytokines in media harvested on days 1–7 were quantified in duplicate at dilutions of 1:100 to 1:5. The ELISA kits were from eBiosciences (IL-17A, IL-6, and IFN-{gamma}; San Diego, CA, USA) and from R&D Systems (IL-21). Color intensity was determined in an ELISA plate reader (MRX Revelation; Dynex Technologies, Chantilly, VA, USA). For ELISpots, suspensions of CD4 cells were fixed, permeabilized, and labeled sequentially with rat monoclonal anti-IL-17 antibody and goat alkaline phosphatase-conjugated anti-rat IgG prior to substrate color development and microscopic counting, as described (24) .

Quantification of nuclear NF-{kappa}B levels
Nuclear proteins were extracted from splenic CD4 T cells with an NE-PER kit (Pierce Chemical Co., Rockford, IL, USA) and quantified by the BCA method (Pierce Chemical Co.). The TransAM plate method for quantification of multiple members of the NF-{kappa}B family of transcription factors is based on their highly specific interactions with immobilized synthetic oligonucleotide substituents of the NF-{kappa}B binding domain followed by detection with labeled Abs to each immobilized factor (Active Motif, Inc., Carlsbad, CA, USA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of aging on cytokine generation by splenic CD4 T cells
Patterns of generation of T cell cytokines implicated in inflammation and fever were strikingly related to mouse age. For C57BL/6 mice, the most prominent quantitative differences were in IL-17 with very significantly higher production by splenic CD4 T cells from old than young mice after both 24 and 96 h of T cell antigen receptor (TCR) stimulation (Fig. 1 ). ELISpot assays of IL-17 in splenic CD4 T cells from old mice in some experiments showed a mean of 42% stained positively as contrasted with 23% for CD4 T cells from young mice (n=3, 96 h, P<0.05). These data suggest that increased secretion of IL-17 by splenic CD4 T cells from old mice is attributable to both higher numbers of Th17 cells and greater production of IL-17 per cell. CD4 T cells from old mice also generated significantly more IL-6 than those of young mice after 96 h of TCR stimulation. In contrast, another product of Th17 cells designated IL-21 was generated at significantly higher concentrations by CD4 T cells from young than old mice after 96 h of TCR stimulation (Fig. 1) . No age-related differences were observed for TCR-stimulated CD4 T cell production of IFN-{gamma}, which mediates reactions of classical cellular immunity rather than inflammation.


Figure 1
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  		Figure 1. Alterations of the splenic CD4 T cell cytokine profile in aged C57BL/6 mice. Each column and bar depicts the mean ± SD of results of 3–6 studies of groups of 3–4 young (open columns) and old (cross-hatched columns) C57BL/6 mice. TCR = stimulation of the TCR by adherent monoclonal antibodies to CD3 plus CD28. The significance of differences between cytokine levels for young and old mice was calculated by a 2-tailed, 2-sample Student’s t test. +P = 0.05; *P < 0.01; **P < 0.002.

A similar set of studies was conducted with splenic CD4 T cells from CBA mice to examine any influences of strain differences on the apparent effects of aging. As for the CD4 T cells from old C57BL/6 mice, those of old CBA mice produced significantly more IL-17 after 24 and 96 h of TCR stimulation and less IL-21 after 96 h than those of young CBA mice (Fig. 2 ). The absence of any differences between production of IFN-{gamma} by CD4 T cells from young vs. old CBA mice under any conditions also was the same as for CD4 T cells of C57BL/6 mice. Unlike the cytokine profile of CD4 T cells from C57BL/6 mice, no difference was seen in IL-6 generation between those from young vs. old CBA mice (Fig. 2) .


Figure 2
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  		Figure 2. Alterations of the splenic CD4 T cell cytokine profile in aged CBA mice. Each column and bar depicts the mean ± SD of results of 3 studies of groups of 2–3 young (open columns) and old (cross-hatched columns) CBA mice. TCR = stimulation of the TCR by adherent monoclonal antibodies to CD3 plus CD28. The significance of differences between cytokine levels for young and old mice was calculated by a paired-sample Student’s t test. +P < 0.05; *P < 0.01.

The levels of CD4 T cell cytokine mRNAs after 24 h of stimulation generally paralleled concentrations of the corresponding cytokine proteins after 96 h (Fig. 3 ). The levels of IL-17 mRNA in CD4 T cells from old mice of both strains were significantly higher than those of young mice after 24 h without and with TCR stimulation. CD4 T cell content of mRNA encoding the ROR{gamma}T transcription factor, implicated as a requirement for differentiation of Th17 cells (25) , also was significantly higher in those from old than young mice of both strains at 24 h without TCR stimulation (Fig. 3) . After 24 h of TCR stimulation, the ROR{gamma}T mRNA level in CD4 T cells also was higher for old than young C57BL/6 mice, but levels for both age groups had fallen too low for accurate quantification in CBA mice. No consistent differences were observed between the levels of IL-21 mRNA in CD4 T cells from old vs. young mice of either strain under any condition. It is possible that levels of IL-21 mRNA may be lower in CD4 T cells of old than young mice at times earlier than 24 h or that effects of aging on IL-21 protein generation may involve post-transcriptional mechanisms.


Figure 3
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  		Figure 3. Differences between splenic CD4 T cell cytokine mRNAs of young and aged mice. Each column and bar depicts the mean ± SD of results of 4–6 studies of samples from C57BL/6 mice (A) and of 3 studies of samples from CBA mice (B). TCR = stimulation of the TCR by adherent monoclonal antibodies to CD3 plus CD28 for 24 h. The significance of differences between cytokine levels for young and old mice was calculated by a 2-tailed, 2-sample Student’s t test. +P < 0.05; **P < 0.002.

Contrasting cytokine profiles of Th17 cells derived from naive CD4 T cells of old and young C57BL/6 mice
CD4 T cells of old mice have a lower percentage of naive subset (CD62L-high) and a higher percentage of memory subset than CD4 T cells of young mice (Table 1 ). In unstimulated CD4 T cells of old mice, naive CD4 T cells ranged from nearly 26% of the total in C57BL/6 mice to just under 60% in CBA mice. To examine the contributions of intrinsic age-related alterations in cytokine production by Th17 cells to the observed differences in composite cytokine profiles (Figs. 1 and 2) , equal numbers of naive (CD62L-high) CD4 T cells from old and young mice were incubated for up to 5 days on adherent anti-TCR Abs with low concentrations of TGF-β, IL-1, IL-6, and IL-23 known to induce conversion to Th17 cells. After 3, 4, and 5 days, differentiated CD4 T cells of old mice expressed higher levels of IL-17 mRNA than those of young mice (Fig. 4 , top panel). Levels of ROR{gamma}T Th17 cell differentiation factor mRNA also were higher in CD4 T cells of old than young mice on days 3 and 4. In contrast, the levels of IL-21 mRNA were higher on days 3 and 4 in differentiated CD4 T cells of young than old mice. In the same protocol, levels of IL-17 protein detectable by ELISpot assay were in a mean of 8% and 17% of CD4 T cells from young and old mice, respectively, on day 3 and in a mean of 14% and 35% of CD4 T cells from young and old mice on day 5. Secreted cytokines were only marginally detectable on day 1. The later concentrations of secreted cytokines showed the same relationships to mouse age as mRNA levels, with significantly higher concentrations of IL-17 and lower concentrations of IL-21 from Th17 cells of old than young mice, and differences for IL-6 that paralleled those of IL-17 (Fig. 4 , bottom panel). Thus, the age-altered profile of cytokine concentrations generated by the total population of CD4 T cells is attributable to properties inherent in each Th17 cell subset as well as to differences in their numerical representation.


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  		Table 1. Age-dependence of naïve (CD62L-high) CD4 T cell prevalence in mouse spleen


Figure 4
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  		Figure 4. Courses of development of cytokine mRNAs (top panel) and proteins (bottom panel) in differentiating naive (CD62L-high) splenic T cells of young and old C57BL/6 mice. Each column in the top panel shows the mean, and each column and bar in the bottom panel depicts the mean ± range of results of 2 studies conducted in duplicate. Numbers of days = days of incubation. The significance of differences between cytokine protein levels for young and old mice was calculated by a paired-sample Student’s t test. +P < 0.05; *P < 0.01.

Roles of NF-{kappa}B in the generation of cytokines by Th17 cells of old and young C57BL/6 mice
Mouse CD4 T cell nuclear concentrations of substituent components of NF-{kappa}B were quantified with a panel of ELISAs, using adherent oligonucleotide constituents of the NF-{kappa}B binding site and an antibody probe specific for each bound NF-{kappa}B component. The level of each component of NF-{kappa}B was higher in nuclear extracts of both resting and TCR-stimulated CD4 T cells from young than old mice (Table 2 ). Further studies focused on nuclear p50, as it showed the greatest increase to the highest level in young mice and one of the two greatest increments in old mice. Nuclear concentrations of p50 were significantly lower in CD4 T cells from old than young mice after 24 and 96 h of TCR stimulation for both strains (Fig. 5 ).


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  		Table 2. Age-dependence of splenic CD4 T Cell expression of NF-{kappa}B components


Figure 5
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  		Figure 5. Nuclear concentrations of NF-{kappa}B (p50) in splenic CD4 T cells of young and old C57BL/6 mice. Each column and bar depicts the mean ± SD of results of 3 or 4 studies of samples from C57BL/6 mice (A) and of 3 studies of samples from CBA mice (B). TCR = stimulation of the TCR by adherent monoclonal antibodies to CD3 plus CD28. The significance of differences between p50 levels for young and old mice was calculated by a paired-sample Student’s t test. +P < 0.05; *P < 0.01.

As NF-{kappa}B transcriptional activity is a central requirement for generation of T cell cytokines, the effects of several structurally-distinct inhibitors of NF-{kappa}B activation were investigated for their capacity to suppress cytokine generation. Optimal concentrations of the I{kappa}B kinase inhibitor Bay 11-7082, the polyphenol butein (26) , and the diterpene triepoxide triptolide (27 , 28) all suppressed nuclear concentrations of p50 by 65% or more in CD4 T cells of young mice and by 50% or more in those of old mice (Fig. 6 A). At these same concentrations, the inhibitors nearly totally eliminated generation of IL-17 and very significantly suppressed generation of IL-21 and IFN-{gamma} (Fig. 6) . Results of analyses of the triptolide concentration-dependence of inhibition of NF-{kappa}B activation demonstrated a greater sensitivity of CD4 T cells from young than old mice to suppression of nuclear levels of p50 (Fig. 7 A). A higher susceptibility of CD4 T cells from young than old mice to triptolide inhibition of cytokine generation, similar to that observed for NF-{kappa}B signaling, was most evident in the greater suppression of their production of IL-17 by all concentrations of triptolide (Fig. 7B ). At 3 nM and 10 nM triptolide, the suppression of CD4 T cell generation of IL-17 is significant for young but not old mice, whereas suppression of IL-17 generation by 30 nM triptolide is more significant for young than old mice. Although the differences are less prominent for IFN-{gamma} generation, suppression by triptolide is evident only for CD4 T cells of young mice at 3 nM and is more significant for those of young mice at 30 nM triptolide (Fig. 7C ). No mechanism has been elucidated yet to explain the greater sensitivity of CD4 T cells from young than old mice to triptolide inhibition of NF-{kappa}B activation and cytokine generation.


Figure 6
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  		Figure 6. Selective suppression of C57BL/6 mouse CD4 T cell generation of inflammatory cytokines by NF-{kappa}B-directed inhibitors. Each column and bar depicts the mean ± SD of results of 3–5 studies of effects of 100 nM triptolide (Tr), 100 µM butein (Bu), and 30 µM Bay 11-7082 (Bay) on nuclear concentrations of NF-{kappa}B (p50) (A) and the concentrations of secreted cytokines (B) after 96 h of stimulation. The significance of suppression in A and B by each inhibitor relative to control values in the absence of an inhibitor was calculated by a 2-tailed, 2-sample Student’s t test. +P < 0.05; *P < 0.01; **P < 0.002.


Figure 7
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  		Figure 7. Triptolide concentration-dependence of suppression of C57BL/6 mouse splenic CD4 T cell nuclear level of NF-{kappa}B and concentrations of secreted cytokine proteins. Each column and bar depicting the percentage of control nuclear level of NF-{kappa}B (p50) (A), and the percentage of control concentration of secreted IL-17 (B) and IFN-{gamma} (C) represents the mean ± SD of results of 3 studies conducted in duplicate after 24 h. Mean respective control levels for CD4 T cells of young and old mice in the absence of an inhibitor (100%) were 0.533 and 0.419 relative ELISA units for NF-{kappa}B (p50), 614 and 1306 pg/106 T cells for IL-17, and 3207 and 2989 pg/106 T cells for IFN-{gamma}. The significance of suppression in A, B, and C by each concentration of triptolide relative to control values in the absence of triptolide was calculated by a 2-tailed, 2-sample Student’s t test. +P < 0.05; *P < 0.01; **P < 0.002.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The distinctive profile of CD4 T cell cytokines from old mice of two strains results from greater development and activation of Th17 cells than in young mice. Distinguishing characteristics of the spectrum of Th17 cell cytokines generated by CD4 T cells of old mice relative to young mice are significantly more IL-17 and IL-6 but less IL-21 (Figs. 1 and 2) . The age-determined differences in IL-17 but not IL-21 appear to reflect changes in transcriptional events because the relative ratios of IL-17 mRNAs in stimulated CD4 T cells of old and young mice paralleled those of the secreted cytokines (Fig. 3) .

The prevalence of naive and memory subsets of CD4 T cells changes with increasing age, which can affect cytokine profiles (4 , 6 , 7) . This result was verified for the C57BL/6 and CBA strains in which the respective percentages of naive CD4 T cells in old mice were ~30 and 60% of total CD4 T cells (Table 1) . Although IL-17 and IL-21 are signature cytokines of Th17 cells, IL-6 comes from a broader distribution of sources, necessitating further examination of its relationship here to Th17 cells. To address these issues, naive CD4 T cells were isolated and incubated with adherent anti-TCR Abs and a mixture of cytokines known to induce differentiation of Th17 cells. After 3–5 days of incubation, the old:young mouse CD4 T cell ratios of Th17 cytokine mRNAs, Th17 cell obligatory differentiation factor ROR{gamma}T mRNA (25) , and Th17 cell cytokine proteins were the same for newly differentiated Th17 cells as for established Th17 cells in splenic CD4 T cells (Fig. 4) . The quantitative relationships of IL-6 to IL-17 secreted by newly differentiated Th17 cells were similar to those for TCR-stimulated splenic CD4 T cells, indicating that the capacity to generate IL-6 developed in parallel with Th17 cell differentiation.

IL-21 generation by mouse Th17 cells usually appears developmentally and after immune stimulation concurrently with that of IL-17, when the Th17 cells are induced in a TGF-β environment by a mixture of cytokines including the terminal differentiation factors IL-23 (29) or sphingosine 1-phosphate (24) . IL-21 itself appears to be an autocrine inducer of Th17 cell differentiation, which may explain its frequently contemporaneous appearance (30) . In aging and some other circumstances, however, Th17 cells are capable of full generation of IL-17 with little or no IL-21. For example, exposure of CD4 T cells to anti-TCR Abs, TGF-β, and the immune-associated neuromediator vasoactive intestinal peptide is one such condition that elicits generation of IL-17 without IL-21. It has not been established whether this dissociation is attributable to differential stimulation of IL-17 or selective suppression of IL-21 generation. The mechanisms responsible for lower levels of IL-21 in old age also are not elucidated but have immune functional implications. Il-21 has broad stimulatory and inhibitory effects on T, B, and NK cells (31) . Like other members of the IL-2 family of cytokines, IL-21 stimulates and costimulates CD8 T cell proliferation and cytokine generation but is alone in its ability to drive CD8 T cells to apoptosis (32) . Thus, lower levels of IL-21 in the aging immune system may contribute both to the observed higher levels of CD8 T cells, due to reduced apoptosis, and to their lower efficiency in host defense against microbes. Age-determined decreases in IL-21 also may lessen its effectiveness as a potent antitumor factor.

The diminished levels of NF-{kappa}B activation in aged T cells, suggested by the lower levels of p50 and other components in T cell nuclei of old than young mice (Table 2 , Fig. 5 ), may have profound effects on host defense and contribute to the increased inflammation of aging. It is not clear what level of activation of NF-{kappa}B is optimal for host defense against infections and tumors, but decreases in several settings appear to increase susceptibility to infections. Compensatory mechanisms may supervene when classical NF-{kappa}B pathways are physiologically significantly reduced. Such is the case in estrogen treatment where nuclearization of some NF-{kappa}B elements is inhibited but NF-{kappa}B-dependent cytokine generation increases because the B cell lymphoma factor 3 or bcl3 becomes a substitute partner for p50 (33) . No information is yet available about these mechanisms in aging. One means by which decreases in NF-{kappa}B might actually enhance inflammation is through paradoxically increased generation of IL-1β (34) . A decrease in NF-{kappa}B-dependent inhibition of protease processing of pro-IL-1β may thereby significantly increase IL-1β levels and its consequent inflammatory effects.

Two plant-derived complex organic compounds, triptolide and butein, inhibit activation of NF-{kappa}B to a similar extent as the Bay 11-7082 synthetic inhibitor of I{kappa}B-{alpha} phosphorylation, reflected in significant decreases in nuclear levels of p50 in CD4 T cells of young and old mice (Fig. 6) . Optimal concentrations of each inhibitor also significantly suppress generation of IL-17 and IL-21, as well as IFN-{gamma}, by CD4 T cells of young and old mice. The triptolide concentration-NF-{kappa}B inhibition relationships demonstrate a greater sensitivity of CD4 T cells from young than old mice (Fig. 7) . The same differentially higher susceptibility of CD4 T cells from young than old mice is evident in the greater suppression of their generation of IL-17 by low concentrations of triptolide.

Suppression of NF-{kappa}B activation by topical and systemic inhibitors decreases inflammation and the resultant tissue injury in several settings (35 , 36) . However, impaired activation of NF-{kappa}B also may have deleterious effects on innate and adaptive immunity (37) . Even modest reductions in nuclear levels of NF-{kappa}B may decrease immune functions significantly (38) . Therefore, it will be important to find dose schedules of NF-{kappa}B inhibitors for each disease setting that allow the most selective immunosuppressive effects. Triptolide and related compounds will be of more value as specific and safe anti-inflammatory drugs in aging if they suppress products of Th17 cells without altering host defense cytokines of Th1 cells.


   ACKNOWLEDGMENTS
 
This research was supported by grants from the U.S. National Institutes of Health to E.J.G. (RO-1 HL31809) and the Intramural Research Program of the National Institute on Aging to D.L.L. The authors are grateful to Y. Kong for expert technical assistance and to R. Chan for skillful production of graphic illustrations. M.-C.H. designed and performed research; J.-J.L. performed research; S.B. assisted in research design; D.L.L. assisted in research design and wrote sections of the manuscript; and E.J.G. designed and performed research and wrote most of the manuscript.

Received for publication November 30, 2007. Accepted for publication January 10, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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