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Basic and Clinical Immunology, University of California, Irvine, CA 92697
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Programmed cell death (apoptosis) is essential for normal development and for maintenance of cellular homeostasis in multicellular organisms (8, 9). In lymphocytes, apoptosis plays an important role in maintaining T cell repertoire and deletion of autoreactive T and B lymphocytes, thus limiting immune responses (reviewed in 10 . Apoptosis is regulated tightly by a number of gene products that promote cell death or extend cell survival (reviewed in Refs. 11–13). The Fas (CD95) surface receptor is a member of the TNF receptor/nerve growth factor receptor family (14, 15, 16, 17) and mediates apoptosis in a wide variety of cell types (18, 19, 20). Fas ligand (FasL),2 which is expressed predominantly in activated T cells (21, 22, 23), is a type II membrane protein belonging to the TNF/(nerve growth factor) family (24, 25). FasL mediates cell death by cross-linking Fas receptor in apoptosis-sensitive Fas+ cells (26, 27, 28, 29). The susceptibility of T cells to undergo apoptosis is also controlled by the family of Bcl-2 homologues (reviewed in 30 . An overexpression of Bcl-2 and the long form of its alternatively spliced homologue, Bcl-xL, enhances the survival of T cells that are induced to undergo apoptosis (31, 32, 33). In contrast, Bax, a 21-kDa protein, homodimerizes or heterodimerizes with Bcl-2 or Bcl-xL to counter their anti-apoptotic effect and promotes apoptosis (34, 35).
Cytokines are known to regulate apoptosis (36, 37). Because aging is associated with alterations in cytokine production along with lymphopenia, we hypothesized that there is an increased programmed cell death in lymphocytes from aging caused by altered expression of genes and their products regulating apoptosis. Although some initial reports suggest alterations in activation-induced apoptosis of lymphocytes in aging (38, 39), however, no detailed studies of genes regulating apoptosis and Fas-mediated apoptosis in aging have been reported. Our data show an increased expression of Fas/FasL and Bax, and decreased expression of Bcl-2 in T cells and T cell subsets that is associated with greater susceptibility of T cell subsets to undergo anti-Fas-induced apoptosis in aging humans.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood was obtained from healthy young (20–29 yr; n = 15, males) and aged (65–95 yr; n = 15, males) volunteers. The protocol was approved by Institutional Review Board, University of California, Irvine. The aging group is defined as those 65 yr or over.
Reagents
Antibodies. Anti-Fas IgM mAb (CH-11) that induces apoptosis (19, 40, 41) was purchased from Upstate Biotechnology Inc. (Lake Placid, NY). Isotype IgM control was purchased from Caltag Laboratories (South San Francisco, CA). FITC-labeled anti-CD95 (UB2) and isotype control were purchased from Kamiya Biomedical Corp. (Thousand Oaks, CA). FITC-labeled anti-Bcl-2 mAb and isotype control were obtained from Dako Corp. (Carpenteria, CA). Per-CP-labeled anti-CD4, anti-CD8, PE-labeled anti-CD45RA, and anti-CD45RO mAbs and their isotype controls were purchased from Becton Dickinson (San Jose, CA). HRP-conjugated goat anti-mouse Ab, polyclonal Ab against Bcl-xL, and HRP-conjugated anti-rabbit IgG were purchased from Transduction Laboratories (Lexington, NY). Anti-Bax Ab was raised in rabbit and kindly provided by Drs. S. Kitada and J. Reed, Burnham Institute (La Jolla, CA). mAb against FasL, raised in hamster (42), was kindly provided by Dr. S. Nagata (Osaka, Japan). FITC-labeled goat anti-hamster Ab and hamster IgG control Abs were purchased from Caltag Laboratories. Anti-CD3 mAb (OKT3) was purchased from Ortho Diagnostics (Raritan, NJ).
Primers. Primers used for RT-PCR were synthesized from Life Technologies (Gaithersburg, MD). Their sequences were as follows: fas, sense, 5'-ATG CTG GGC ATC TGG ACC CTC CTA-3', and antisense, 5'-TCT GCA CTT GGT ATT CTG GGT CCG-3' (43); bcl-2, sense, 5'-CGA CGA CTT CTC CCG CCG CTA CCG C-3', and antisense, 5'-CCG CAT GCT GGG GCC GTA CAG TTC C-3' (44); bax, sense, 5'-ATG GAC GGG TCC GGG GAG CAG CCC-3', and antisense, 5'-GGT GAG CAC TCC CGC CAC AAA GAT 3' (45); and bcl-xL, sense, 5'-TTG GAC AAT GGA CTG GTG GA-3', and antisense, 5'-GTA GAG TGG ATG GTC AGT G-3' (31).
Chemicals.
Propidium iodide, PHA, PMA, ionomycin, and Hoechst 33342 dye were
purchased from Sigma Chemical Co. (St. Louis, MD).
[
-32P]dATP was obtained from DuPont (Wilmington,
DE). RT-PCR kits were purchased from Perkin-Elmer/Cetus (Branchburg,
NJ). Metalloprotease inhibitors TIMPI and TIMPII were obtained from
Calbiochem (San Diego, CA). rIL-2 was purchased from R&D Systems
(Indianapolis, IN).
Cell culture
PBMC were separated from whole blood by Ficoll-Hypaque density centrifugation. Cells were resuspended in RPMI 1640 medium containing 10% FCS, penicillin (100 U/ml), streptomycin (100 µg/ml), and L-glutamine (1 mM) at 2 x 106 cells/ml, and subsequently cultured in the presence of anti-CD3 mAb (25 ng/ml) for 48 h, following which cells were maintained in IL-2 (10 ng/ml)-containing medium. For anti-Fas-induced apoptosis, cells were washed on day 6 of culture and restimulated with anti-Fas (1 µg/ml) mAb or its isotype (IgM) control Ab in the presence of human rIL-2 (10 ng/ml) for an additional 16 h (46, 47).
Flow cytometry
The expression of Fas, FasL, and Bcl-2 proteins was determined using flow cytometry.
Fas expression
MNC were washed twice with PBS and incubated for 45 min on ice with FITC-conjugated anti-Fas mAb and PE-conjugated anti-CD4 or anti-CD8 mAbs. For triple-color analysis, FITC-conjugated anti-Fas, Per-CP-conjugated anti-CD4, or anti-CD8- and PE-conjugated anti-CD45RA or anti-CD8 mAbs were used. FITC-labeled, PE-labeled, and Per-CP-labeled mouse IgG were used as isotype-matched background controls. Cells were washed with PBS, and 5000 cells were acquired using FACScan. Dual- or triple-color analysis was performed using Consort 30 and FACScan Research software, respectively (Becton Dickinson).
Bcl-2 expression
Bcl-2 is an intracellular protein (48), and therefore, cells were washed and sequentially fixed with 1% paraformaldehyde for 15 min at room temperature and by 70% methanol for 45 min at 4°C (49, 50). Cells were washed and incubated with FITC-labeled anti-Bcl-2 and PE-labeled anti-CD4 or anti-CD8 mAbs. For triple-color analysis, Per-CP-labeled anti-CD4 or anti-CD8- and PE-labeled anti-CD45RA or anti-CD45RO mAbs were used. FITC-labeled, PE-labeled, and Per-CP-labeled mouse IgG were used as isotype-matched background controls. Following incubation, the cells were washed twice with PBS, and 5000 cells were acquired using FACScan. Lymphocytes were gated, and percentage of double-positive cells (CD4+Bcl-2+ and CD8+Bcl-2+) expressing Bcl-2 and fluorescence intensity, as measured by mean fluorescence channel (MFC) number, was determined using Consort 30 software.
FasL expression
Because FasL is expressed on activated human T cell subsets (21, 22, 23), MNC (1 x 106 cells/ml) were activated in the presence of PHA (5 µg/ml) and IL-2 (10 ng/ml) for 2 days. Cells were washed and stimulated with PMA (10 ng/ml) and ionomycin (500 ng/ml) for an additional 16 h in the presence of metalloprotease inhibitors, TIMP1 (51) and TIMP2 (51, 52), to inhibit the release of FasL from the cell surface. Cells were then washed and incubated with anti-FasL mAb (raised in hamster) or hamster IgG as isotype control for 30 min on ice. Cells were washed with PBS containing 1% FBS and 0.1% sodium azide and incubated further with FITC-labeled goat anti-hamster Ab for another 30 min on ice. Cells were washed and counterstained with PE-labeled anti-CD4 or anti-CD8 mAbs or their isotype-matched controls for 45 min on ice. Five thousand cells were acquired, and FasL-expressing T cell populations were determined by dual-color analysis using FACScan and Consort 30 software.
Western blotting
The expression of Bcl-2, Bcl-xL, and Bax at the protein level was determined by Western blotting. Cells were lysed in a buffer containing 142.5 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.2), 1 mM EGTA, 0.2% Nonidet P-40, 0.2 mM PMSF, 0.2 trypsin inhibitory U/ml aprotinin, 0.7 µg/ml pepstatin, and 1 µg/ml leupeptin. Cells were homogenized and centrifuged at 1000 rpm for 8 min to precipitate cell debris. The supernatants were centrifuged at 30,000 x g for 45 min to precipitate membrane fractions, and 25 µg protein (or increasing amounts of proteins for limiting dilution analysis) was loaded onto 4 to 20% Tris-glycine gels and electrophoresed. The proteins were then transferred onto nitrocellulose membrane. The blots were blocked with PBS containing 3% dry milk and 0.1% Tween-20 and probed with anti-Bcl-2, anti-Bcl-xL, or anti-Bax Abs (dilution of 1/1000) for 3 h at 37°C. The blots were incubated with HRP-conjugated goat anti-mouse or HRP-conjugated goat anti-rabbit (1/2000 dilution) Ab for 1 h at room temperature and developed using enhanced chemiluminescence method (Amersham, Arlington Heights, MA). The blots were quantified using densitometric analysis using ImageQuant software (Molecular Dynamics, Sunnyvale, CA) and data represented as OD.
Quantitative PCR
Total cellular RNA was extracted from unstimulated MNC from
young and aging subjects. cDNA was synthesized using 200 ng total
cellular RNA and 100 ng random hexamers in 20 µl of a solution
containing 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2, 10
mM DTT, 500 µM each of dNTPs, and 10 U reverse transcriptase. PCR was
conducted with graded amounts of cDNA (0, 1, 2, 4, and 8 µl) using 1
U of Amplitaq polymerase and PCR reaction kit (Perkin-Elmer/Cetus).
Each cycle of PCR included 1 min of denaturation at 94°C, 1 min of
primer annealing at 60°C, and 2 min of extension/synthesis at 72°C.
Fas-, bcl-2-, bcl-xL-, and
bax-specific primers yield 384-, 780-, 318-, and 615-bp
primer products, respectively. Primers for ß-actin were
used as internal controls. Each primer was added at 37.5 pmol per
reaction. For quantitation, 2 µCi (1 Ci = 37 GBq) of
[-32P]dATP was added to each reaction mixture, and
experiments were performed in triplicates. PCR was conducted with
Thermal Cycler (Perkin-Elmer/Cetus). PCR products were separated on 6%
TBE gels and stained with ethidium bromide or exposed to x-ray films
(X-OMAT; Sigma Chemical Co.) for 2 h and developed. Bands
corresponding to each specific primer were excised from stained gels,
and the amount of radioactivity incorporated was determined by liquid
scintillation.
Propidium iodide staining
MNC (1 x 106) were washed with PBS and incubated in the presence of FITC-labeled anti-CD4 or anti-CD8 mAbs and corresponding isotype controls for 45 min on ice. Following incubation, cells were washed twice with PBS and resuspended in 70% ethanol overnight at -20°C. The cells were washed twice with PBS and incubated in sodium citrate buffer (0.1%) containing 0.1% Triton X-100, 50 µg/ml RNase A, and 50 µg/ml propidium iodide for 30 min at room temperature in the dark. Ten thousand cells were acquired, and percentage of cells undergoing apoptosis was determined by dual-color analysis, using FACScan.
TUNEL assay
TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay was performed to study DNA fragmentation (53, 54, 55, 56). Cells (5 x 105) were washed in Dulbecco’s PBS (DPBS) and incubated in the presence of PE-labeled anti-CD4 or anti-CD8 mAbs for 45 min on ice. Following incubation, cells were washed twice in DPBS containing 0.1% BSA and 0.1% NaN3, and fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were washed at 4°C and permeabilized with sodium citrate buffer containing 0.1% Triton X-100 for 2 min on ice. After washing, cells were incubated with FITC-conjugated dUTP in the presence of terminal deoxynucleotidyl transferase enzyme solution for 1 h at 37°C using cell death detection kit (Boehringer Mannheim Corp., Indianapolis, IN). Following incubation, the cells were washed with DPBS, and 5000 cells were acquired and analyzed by dual-color analysis using FACScan.
DNA fragmentation
DNA fragmentation was assessed by gel electrophoresis. Cells were harvested and centrifuged at 500 x g for 5 min and washed twice with PBS. The cell pellet was lysed in 400 µl of lysing buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.1% SDS, and 0.2% Triton X-100 and proteinase K (0.1 mg/ml) at 50°C for 16 h, followed by incubation with 50 µg/ml RNase A for an additional hour at 50°C. DNA was extracted once with phenol-chloroform-isoamyl alcohol (25:24:1) and twice with chloroform-isoamyl alcohol (24:1). The aqueous phase was precipitated with 2 vol of 100% ethanol at -20°C overnight. The precipitates were rinsed with 70% ethanol, air dried, dissolved in TE buffer (10 mM Tris buffer, pH 7.5, and 1 mM EDTA), and electrophoresed in 1.8% agarose gel with loading buffer. Gel was stained with 5 mg/ml ethidium bromide for 30 min, destained overnight, and photographed under UV transilluminator.
Morphologic determination of apoptosis
Changes in nuclear structure were studied after staining cells with DNA-binding fluorescent dye Hoechst 33342 (57). MNC (1 x 106) were washed and resuspended in 5 µg/ml Hoechst dye in PBS and incubated at 37°C for 30 min. Following incubation, cells were cytospun onto microscopic slides using Cytospin 3 (Shandon, Cheshire, UK) and visualized under fluorescent microscope at 365 nm excitation and 420 nm long pass emission.
Statistical analysis
All studies were done in pairs, i.e., each experiment was done using an equal number of aging subjects and young subjects simultaneously. Statistical analysis was performed using two Student sample t tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MNC from 10 subjects, each from aging and young groups, were
analyzed for Fas expression with mAbs using dual-color analysis by
FACScan. Figure 1
shows a significantly
higher (p < 0.001) proportion of
CD4+ and CD8+ T cells from aging subjects
expressed Fas (percentage of mean ± SD: CD4+, 45
± 7; CD8+, 56 ± 7) as compared with young subjects
(percentage of mean ± SD: CD4+, 29 ± 5;
CD8+, 35 ± 5). Furthermore, there was a significantly
(p < 0.002) higher proportion of
CD8+ T cells expressing Fas in each age group as compared
with CD4+ T cells (aging CD8+ vs
CD4+, 56 ± 7 and 45 ± 7; young CD8+
vs CD4+, 35 ± 5 and 29 ± 5).
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A, inset).
As seen in Figure 2A, there was a significant increase
(p < 0.001) in cpm ratio for
fas/ß-actin in lymphocytes from aging (mean ± SD cpm
for 1 and 2 µl fas and 1 µl ß-actin:
1.06 ± 0.03 and 2.4 ± 0.05) as compared with young
(mean ± SD cpm for 1 and 2 µl fas and 1 µl
ß-actin: 0.45 ± 0.03 and 1.05 ± 0.04),
demonstrating an increase of fas expression at mRNA level in
aging. A representative gel photograph is shown in Figure 2B.
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summarizes the data obtained from five
aging and five young subjects. A significantly higher
(p < 0.001) percentage of memory
(CD45RO+) as well as naive (CD45RA+) cells in
both CD4+ and CD8+ subsets expressed Fas in
aging subjects as compared with their memory and naive counterparts in
young subjects. Furthermore, in both of the age groups, a higher
proportion of memory cells (CD4+CD45RO+ and
CD8+CD45RO+) expressed Fas as compared with
naive cells (CD4+CD45RA+ and
CD8+CD45RA+) (p <
0.001 and p < 0.002, respectively).
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, significantly higher
(p < 0.001) proportions of both
CD4+ and CD8+ T cell subsets in aging subjects
expressed FasL (percentage of mean ± SD: CD4+,
24 ± 4; CD8+, 28 ± 5) as compared with young
controls (percentage of mean ± SD: CD4+, 8 ± 3;
CD8+, 10 ± 2).
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Fas and Bcl-2 are regulated differentially in various cell types
(60, 61, 62). Therefore, we examined the levels of Bcl-2 protein in aging
and young T cell subsets. Bcl-2 is a membrane-bound protein found in
mitochondrial membrane (48) that is expressed in almost all
CD4+ and CD8+ T cells. Therefore, the
differences in Bcl-2 protein levels were analyzed by changes in MFC
intensity/numbers assessed by flow cytometry and Western blotting. We
found that Bcl-2 levels in aging (n = 10) T cell
subsets were significantly (p < 0.001) lower
(MFC, mean ± SD: CD4+, 513 ± 18;
CD8+, 476 ± 21) as compared with young
(n = 10) controls (MFC, mean ± SD:
CD4+, 570 ± 12; CD8+, 536 ± 20).
Figure 4A shows representative
FACS plots for Bcl-2 staining for CD4+ (middle
panel) and CD8+ (right
panel) T cells in each young (bottom
panel) and aging (top panel)
subject. Markers were set for the cells positive for Bcl-2 and CD4 or
CD8 following staining with isotype control Abs (left
panel). Furthermore, Western blotting analysis was
performed on three different subjects from each aging and young. Figure 4B shows that in each of the three aging subjects, there was
a decreased Bcl-2 expression (OD mean ± SD: 0.3 ± 0.02) as
compared with young controls (OD mean ± SD: 0.43 ± 0.1).
Linearity of the chemiluminiscent method is shown in the bottom
panel.
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A
(inset). As seen in Figure 5A, a
significantly (p < 0.001) lower cpm ratio for
bcl-2:ß-actin was observed in aging (1 and 2 µl cDNA for
bcl-2, and 1 µl for ß-actin: mean ± SD
cpm, 0.14 ± 0.01 and 0.3 ± 0.03) as compared with young
controls (1 and 2 µl cDNA for bcl-2, and 1 µl for
ß-actin: mean ± SD cpm, 0.29 ± 0.02 and
0.56 ± 0.02), demonstrating a lowered bcl-2 expression
in aging. A representative gel photograph using 2 µl cDNA is shown in
Figure 5B.
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, a decrease in Bcl-2 expression was
observed in memory cell populations (CD45RO+) of both
CD4+ and CD8+ T cell subsets from aging as
compared with memory cell populations of CD4+ and
CD8+ T cell subsets from young subjects
(p < 0.003). Interestingly, decreased
expression of Bcl-2 was also observed in naive cell subpopulation
(CD45RA+) of CD4+ and CD8+ T cells
from aging as compared with CD4+ and CD8+ T
cell subsets from young subjects (p <
0.001).
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B) and
at the mRNA level (Fig. 7A;
mean ± SD cpm ratio, 1 µl bax:1 µl
ß-actin: 0.62 ± 0.03, and 2 µl bax:1
µl ß-actin: 1.4 ± 0.13) as compared with that from
young subjects at the protein level (OD mean ± SD, 0.37 ±
0.07; Fig. 6B) and at the mRNA level (Fig. 7A; mean ± SD cpm ratio, 1 µl bax:1 µl
ß-actin: 0.46 ± 0.02, and 2 µl bax:1
µl ß-actin: 0.91 ± 0.04). The limiting dilution
for Western blotting was conducted using increasing amounts of proteins
(Fig. 6A), and for quantitative PCR was conducted
using increasing amounts of cDNA (0, 1, 2, 4, and 8 µl) to
demonstrate the linearity (Fig. 7A,
inset). Representative gel photographs for Western
blotting and quantitative PCR are shown in Figures 6B
(inset) and 7B, respectively. On
the other hand, there was no significant difference
(p > 0.05) in Bcl-xL expression in
lymphocytes between aging and young both at the protein level (Fig. 8B; aging OD mean ± SD,
0.23 ± 0.02; young OD mean ± SD, 0.22 ± 0.02) and at
the mRNA level (Fig. 9A; aging
mean ± SD cpm ratio, 1 µl bcl-xL:1 µl
ß-actin: 0.35 ± 0.05, and 2 µl
bcl-xL:1 µl ß-actin: 0.68 ±
0.02; young mean ± SD cpm ratio, 1 µl
bcl-xL:1 µl ß-actin: 0.32 ±
0.03, and 2 µl bcl-xL:1 µl ß-actin:
0.71 ± 0.04). Representative graphs for limiting dilution for
Western blotting and quantitative PCR are shown in Figures 8A and 9A (inset),
respectively. Representative gel photographs for Western blotting and
quantitative PCR are shown in Figures 8B
(inset) and 9B, respectively.
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To determine whether differential expression of Fas, FasL, Bcl-2,
and Bax correlates with differential susceptibility of T cells to
undergo anti-Fas-induced apoptosis between aging and young, we
cultured MNC from aging and young subjects for 6 days in
IL-2-containing medium following an initial stimulation with
anti-CD3 (25 ng/ml) mAb for 48 h. Following culture, cells
were treated with anti-Fas (1 µg/ml) mAb or its isotype-matched
control for an additional 16 h, and susceptibility of T cells to
undergo apoptosis was determined using propidium iodide staining and
TUNEL assay. Table III summarizes data
from 10 aging and 10 young subjects. An increased proportion of
CD4+ and CD8+ T cell subsets from aging
underwent anti-Fas-induced apoptosis as compared with
CD4+ and CD8+ T cells from young controls
(p < 0.001). Figure 10, A and B,
shows representative histograms obtained for percentage of cells in
TUNEL assay and propidium iodide staining undergoing apoptosis
following anti-Fas Ab treatment (Fig. 10, A and
B, bottom panel). No significant apoptosis
was observed following treatment with isotype control Ab (Fig. 10, A and B, top panel). In
addition, in each aging and young group, a significantly increased
proportion of CD4+ T cells underwent apoptosis as compared
with their CD8+ T cell subsets (p
< 0.05).
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C shows
that following anti-Fas treatment, there was an increased DNA
fragmentation in lymphocytes from aging as compared with young
controls. Furthermore, an increase in proportions of cells showing
apoptotic bodies in aging MNC was observed with Hoechst dye as compared
with young controls. A representative photomicrograph of cells stained
with Hoechst dye following anti-Fas treatment is shown in Figure 10D. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Fas receptor cross-linking results in the death of Fas+ T cells upon ligation with FasL (26, 27, 28, 29). This interaction helps terminate an ongoing immune response by inducing apoptosis in activated lymphocytes. Our data show an increased expression of Fas at basal levels in CD4+ and CD8+ T cell subsets from aging humans as compared with young controls. Furthermore, the fas expression was also increased at mRNA level in MNC from aging. Naive cells express little or no Fas, which can be induced upon activation, whereas memory cells have increased Fas (26, 59, 60). Interestingly, we observed a significant increase in Fas expression in both naive and memory subpopulations of CD4+ and CD8+ T cell subsets in aging as compared with young. This would suggest that an increase in Fas expression observed in CD4+ and CD8+ T cells in aging is not exclusively due to a shift from naive to memory phenotype T cells. Our observations are in agreement with the findings of Shinohara et al. (62), who also reported an increased Fas expression in memory CD4+ or CD8+ T cells in elderly human population (mean age: 51 yr). However, in this report, Fas expression was not determined on naive subpopulations of CD4+ and CD8+ T cell subsets, and therefore, an increased Fas expression observed could be due to an increase in the number of memory cells, as data were not normalized for increased memory cell numbers in the elderly population. In contrast, studies on aged mice show a decrease in Fas expression on T cells (19). The reason for this discrepancy of Fas expression levels in humans and mice is not known, but could be related to species differences. Furthermore, we observed that following in vitro activation, a significantly higher proportion of CD4+ and CD8+ T cells from aging expressed FasL as compared with young. There are no published data on FasL expression in lymphocytes from aging humans.
To examine whether the increased expression of Fas and FasL in aging lymphocytes correlated with an increased susceptibility to apoptosis, we compared anti-Fas-induced apoptosis in T cell subsets between aging and young humans. Freshly isolated T cells are resistant to anti-Fas-induced apoptosis; however, they become sensitive upon prolonged in vitro activation in culture (46). Following anti-Fas treatment of cultured lymphocytes, an increased proportion of CD4+ and CD8+ T cells from aging underwent apoptosis as compared with young subjects. We also observed that following an initial stimulation with anti-CD3, a higher proportion of CD4+ and CD8+ T cells in aging underwent apoptosis as compared with young; however, the number of dying cells was very few (data not shown). Furthermore, there was no effect of IgM treatment on T cell apoptosis in both aging and young. Recently, Herndon et al. (38) have demonstrated an increased apoptosis of naive (CD3+CD45RO-) T cells in aging humans as compared with young following in vitro culture of fresh lymphocytes. Phelouzat et al. (39) have also reported an increased susceptibility of aging lymphocytes to undergo activation-induced apoptosis. However, these authors have compared susceptibility of lymphocytes between aging and young following treatment with suprapharmacologic concentrations of PMA and ionomycin. Furthermore, determination of apoptosis was done on MNC using ELISA, which quantitates the DNA fragments released following cell lysis, a relatively late phase event in apoptosis, which does not differentiate between cell types undergoing necrosis and apoptosis. We have quantitated the proportions of CD4+ and CD8+ T cell subsets undergoing apoptosis using TUNEL assay, which is specific for apoptosis and quantitates the nicks in DNA undergoing apoptosis within 4 h of initiation of the apoptotic signal. Furthermore, we observed a preferential death of CD4+ T cell subsets upon anti-Fas treatment as compared with CD8+ T cells in both aging and young groups. Using a different cell system of CD4 and CD8 clones, Zheng et al. (63) have also shown a preferential death of CD4+ T cells as compared with CD8+ T cell subsets following anti-Fas treatment. The data on apoptosis of lymphocytes in aging mice are inconclusive. Chrest et al. (64) have shown an increased death of Go T cells from spleen of aging C57BL/6 female mice upon stimulation with anti-CD3 mAb, using flow cytometry. In contrast, Zhou et al. (65) have demonstrated a decreased susceptibility of lymph node T cells from CD1-aged mice to undergo FasL-mediated apoptosis as compared with young mice. The differences between the reports from Chrest et al. and Zhou et al. could be due to differences in the strain of experimental animals, lymphoid compartments (spleen vs lymph nodes), or the method of stimulation (anti-CD3 vs FasL) used.
Bcl-2 plays an important role in the fate of cells committed to undergo apoptosis (66). We therefore compared the expression of Bcl-2 in lymphocytes from aging and young subjects. We observed a decreased Bcl-2 expression in aging both at the protein and at the mRNA level as compared with young subjects. Decreased Bcl-2 expression in aging was observed in both CD4+ and CD8+ T cell subsets. Furthermore, decreased Bcl-2 expression was seen in naive and memory cells of both CD4+ and CD8+ T cells in aging as compared with young controls. Shinohara et al. (62) have also observed decreased Bcl-2 expression in memory cell subpopulations of CD4+ and CD8+ T cell subsets in aging; however, Bcl-2 expression on naive T cells was not analyzed. The role of Bcl-2 in the regulation of anti-Fas-mediated apoptosis is controversial and may depend on the cell types and stimulus used. Iwai et al. (67) and Yoshino et al. (68), using activated T lymphocytes, have shown that Bcl-2 blocks anti-Fas Ab-induced apoptosis in mitogen-activated T cells. In contrast, some reports show that Bcl-2 is not involved in anti-Fas-induced apoptosis in lymphocytes (69, 70). However, in these reports, changes in Bcl-2 levels were studied following anti-Fas treatment. In our study, we have compared the basal expression levels of Bcl-2 (both at the protein and at the mRNA level) between aging and young and have suggested that lower Bcl-2 expression in T cells from aging may play a role in higher susceptibility of T cells to undergo anti-Fas-induced apoptosis. Differences in Bcl-2 expression in aging following anti-Fas treatment remain to be seen. It has been shown that in vitro activation of T cells is associated with down-regulation of Bcl-2 and up-regulation of Fas/FasL (22, 23, 71, 72). Therefore, it is possible that prolonged in vivo antigenic activation of T cells in aging may be one of the mechanisms of low Bcl-2 and increased Fas/FasL expression in aging.
Overexpression of Bcl-xL, the long form of an alternatively
spliced homologue of Bcl-2, has been shown to be involved in inhibition
of apoptosis (73). On the other hand, Bax, a pro-apoptotic Bcl-2
family member, can heterodimerize with either Bcl-2 or
Bcl-xL to nullify their anti-apoptotic properties. The
ratios of Bcl-2:Bax and Bcl-xL:Bax appear to be important
determinants for apoptosis: higher ratios favor cell survival, whereas
higher Bax:Bcl-2 and Bax:Bcl-xL ratios promote cell death
(66, 74). We did not find any significant differences in
Bcl-xL levels between young and aging using Western
blotting; however, because of increased level of Bax, the ratio of
Bax:Bcl-xL was higher in aging as compared with young
subjects (Fig. 11).
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One of the questions remains unanswered as to whether the cells expressing high levels of pro-apoptotic molecules and low levels of anti-apoptotic molecules represent a subset of lymphocytes that are normally present in young subjects, and aging represents an expansion of such a population. If this is true, increase in this population during aging would be as a consequence of increased production of this population or decreased death of a subset that does not express high levels of Fas/FasL and/or decreased level of Bcl-2. This latter possibility is unlikely because a subset of lymphocytes that expresses high levels of Fas/FasL and decreased level of Bcl-2 would undergo cell death earlier than the other subsets. Furthermore, it is unlikely that there is preferential increased production of this population in aging, since thymus during aging is almost completely involuted. Therefore, it is likely that lymphocytes with increased pro-apoptotic and decreased anti-apoptotic molecules may represent a prolonged in vivo activation as a reflection of age.
In summary, T cell deficiency in aging appears to be, at least in part, due to an increased apoptosis associated with increased expression of pro-apoptotic molecules and decreased expression of anti-apoptotic molecules.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: FasL, Fas ligand; DPBS, Dulbecco’s phosphate-buffered saline; HRP, horseradish peroxidase; MFC, mean fluorescence channel; MNC, mononuclear cell; PE, phycoerythrin; Per-CP, peridinin chlorophyll; TBE, Tris-borate-EDTA; TUNEL, terminal deoxytidyl transferase-mediated dUTP nick end labeling.
Received for publication July 7, 1997. Accepted for publication October 27, 1997.
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) and young (•).
B, Increased fas mRNA expression in lymphocytes
from aging. Representative gel photograph for fas mRNA
expression using 2 µl cDNA is shown. PCR reactions were conducted in
triplicate, and PCR products were electrophoresed on 6% TBE gel. Gel
exposed to x-ray films for 2 h shows increased fas
expression (top panel) in aging as compared
with young. Primers specific for ß-actin were used as
internal controls (bottom panel).
