Download Responses Mediated Downregulation of B Cell

A Molecular Mechanism for TNF-α−
Mediated Downregulation of B Cell
Responses
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of June 18, 2017.
Daniela Frasca, Maria Romero, Alain Diaz, Sarah
Alter-Wolf, Michelle Ratliff, Ana Marie Landin, Richard L.
Riley and Bonnie B. Blomberg
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Copyright © 2011 by The American Association of
Immunologists, Inc. All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2012; 188:279-286; Prepublished online 23
November 2011;
doi: 10.4049/jimmunol.1003964
http://www.jimmunol.org/content/188/1/279
The Journal of Immunology
A Molecular Mechanism for TNF-a–Mediated
Downregulation of B Cell Responses
Daniela Frasca, Maria Romero, Alain Diaz, Sarah Alter-Wolf, Michelle Ratliff,
Ana Marie Landin, Richard L. Riley, and Bonnie B. Blomberg
I
nflammation is part of the protective, biological/immunological response to infections, which is crucial for survival.
However, at the same time, many pathologic conditions,
such as autoimmune diseases, are sustained by the continuous
activation of the inflammatory process. In the past few years,
the molecular basis of inflammation has been uncovered, and now
much is known about the primary role of proinflammatory cytokines, such as TNF-a. Anticytokine therapies have been used
successfully to treat patients with autoimmune diseases, such as
rheumatoid arthritis, Crohn’s disease, and psoriasis (1, 2). Increasing understanding of the role of TNF-a in inflammation
and diseases is opening new strategies for the treatment of inflammatory-based diseases through selective targeting of cytokines
(3).
Inflammation plays an important role in the pathogenesis of
many diseases typical of old age (4). Enhanced IL-6 (5–7) and
TNF-a (5, 8) plasma levels have been associated with functional
disability and mortality of the elderly. Aging is characterized by
a dysregulation of inflammatory and anti-inflammatory networks,
which results in a low-grade chronic proinflammatory status called
inflammaging (9). The age-related increase in circulating inflam-
Department of Microbiology and Immunology, University of Miami Miller School of
Medicine, Miami, FL 33101
Received for publication December 6, 2010. Accepted for publication October 29,
2011.
This work was supported by National Institutes of Health Grants AG-17618 and AG28586 (to B.B.B.) and Grants AG-025256 and AI-064591 (to R.L.R.).
Address correspondence and reprint requests to Dr. Bonnie Blomberg, Department of
Microbiology and Immunology, University of Miami Miller School of Medicine, P.O.
Box 016960 (R-138), Miami, FL 33101. E-mail address: [email protected]
Abbreviations used in this article: ABC, age-associated B cell; AID, activationinduced cytidine deaminase; CSR, class switch recombination; FO, follicular; MZ,
marginal zone; qPCR, quantitative PCR; TTP, tristetraprolin.
Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1003964
matory mediators, such as cytokines and acute-phase proteins, are
markers of the low-grade inflammation observed with aging. Agerelated alterations in responses to immune stimulation (e.g.,
chronic T cell stimulation with viruses, such as CMV) also contribute to low-grade inflammation by increasing the level of
proinflammatory mediators, such as TNF-a (10). Production of
proinflammatory cytokines is thought to be, in part, a macrophagemediated event, but it is clear that other cell types, including
stroma (i.e., epithelium, endothelium, and fat), as well as T cells,
produce these mediators in vivo. Production of TNF-a in unstimulated B cells has not been pursued and is, in part, the subject
of this article.
B cells, through the secretion of cytokines, such as TNF-a, were
shown to contribute to immunity against infectious agents, such as
Toxoplasma gondii, Heligomosomoides polygyrus, and Pneumocystis carinii, by promoting expansion and differentiation of primary and memory Th1 cells (11) or Th2 cells (12, 13). Moreover,
the possible contribution of B cells and/or APCs to the inflammatory process supports their pathogenic role in a wide range of
autoimmune diseases (14).
We previously found that the molecular basis for E47, and
hence, activation-induced cytidine deaminase (AID) and class switch
recombination (CSR) of Ig, being lower in aged individuals, mice
(15) and humans (16), is due to decreased E47 mRNA stability
(17). This reduced E47 mRNA stability with age is mediated,
at least in part, by binding of tristetraprolin (TTP) to the 39 untranslated region (18). Because TTP also regulates inflammatory
cytokine (TNF-a, IL-6) mRNAs similarly (19, 20), our hypothesis
is that, in aging, there is a feedback mechanism of inflammatory
cytokines to downregulate further expression of these and that, in
B cells, this process inadvertently also downregulates E47 and an
optimal B cell immune response, including Ig CSR and AID.
In the current study, we investigated two questions: whether
unstimulated B cells contribute to the increased inflammatory
response in aging (inflammaging) by secreting more proinflam-
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B cell function with age is decreased in class switch recombination (CSR), activation-induced cytidine deaminase (AID), and
stability of E47 mRNA. The latter is regulated, at least in part, by tristetraprolin (TTP), which is increased in aged B cells and also
negatively regulates TNF-a. In this study, we investigated whether B cells produce TNF-a, whether this changes with age, and how
this affects their function upon stimulation. Our hypothesis is that in aging there is a feedback mechanism of autocrine inflammatory cytokines (TNF-a) that lowers the expression of AID and CSR. Our results showed that unstimulated B cells from old
BALB/c mice make significantly more TNF-a mRNA and protein than do B cells from young mice, but after stimulation the old
make less than the young; thus, they are refractory to stimulation. The increase in TNF-a made by old B cells is primarily due to
follicular, but not minor, subsets of B cells. Incubation of B cells with TNF-a before LPS stimulation decreased both young and old
B cell responses. Importantly, B cell function was restored by adding anti–TNF-a Ab to cultured B cells. To address a molecular
mechanism, we found that incubation of B cells with TNF-a before LPS stimulation induced TTP, a physiological regulator of
mRNA stability of the transcription factor E47, which is crucial for CSR. Finally, anti–TNF-a given in vivo increased B cell
function in old, but not in young, follicular B cells. These results suggest new molecular mechanisms that contribute to reduced Ab
responses in aging. The Journal of Immunology, 2012, 188: 279–286.
280
matory cytokines (i.e., TNF-a) than do B cells from young mice
and whether the proinflammatory microenvironment seen in old
mice, and specifically TNF-a produced by B cells, can reduce the
ability of B cells to respond to stimuli, such as LPS. Our results
revealed new molecular mechanisms that may contribute to reduced Ab responses in aging.
Materials and Methods
Mice and definition of phenotype
Splenic B cell enrichment
B cells were isolated from the spleens of young and old mice. Briefly, cells
were washed twice with medium (RPMI 1640; Invitrogen Life Technologies) and incubated for 20 min at 4˚C with anti-CD19 Microbeads
(Miltenyi Biotec), according to the MiniMacs protocol (Miltenyi Biotec;
20 ml Microbeads + 80 ml PBS, for 107 cells). Cells were then purified
using magnetic columns. At the end of the purification procedure, cells
were 80–85% CD19+ by cytofluorimetric analysis. After the isolation
procedure was completed, cells were maintained in PBS for 3 h at 4˚C to
minimize potential effects of anti-CD19 Abs on B cell activation. In a
preliminary series of experiments, macrophages were removed by adherence, and B cells were isolated from the nonadherent fraction. This was
initially conducted to rule out the possibility that E47 and AID mRNA
expression in B cell cultures might have been due to contaminating
macrophages, which could have stimulated B cells. We obtained similar
results for E47 and AID in B cells isolated with or without depletion of
macrophages. All data in this study were obtained with B cells isolated
from whole splenocytes, without depletion of macrophages.
We (18) and others (24) reported that the number of follicular (FO)
B cells is unaffected by aging in BALB/c mice, but there is a significant
age-related decrease in marginal zone (MZ) B cells. Conversely, in
C57BL/10 mice, FO B cells decrease and MZ B cells increase with age, as
we (D. Frasca and B.B. Blomberg, unpublished observations) and other
investigators (25) observed. Because the differences that we noted are not
in MZ/Ag-experienced cells, the results should apply to both BALB/c and
C57BL/10 mice.
B cell culture
B cells were cultured in complete medium (RPMI 1640, supplemented with
10% FCS, 10 mg/ml gentamicin, 2 3 1025 M 2-ME, and 2 mM L-glutamine). FCS was certified to be endotoxin free. B cells (106/ml) were
stimulated in 24-well culture plates for different time periods (indicated in
each figure) with 1 mg/ml Escherichia coli LPS (SIGMA L2880) or 100
ng/ml TNF-a (PMC3014 Biosource). In some experiments, a purified rat
anti-mouse TNF-a Ab (551225 BD Pharmingen) was added to the LPSstimulated B cell cultures at a concentration of 5–100 ng/ml. The Ab was
either added once at the beginning of the culture or at day 0, 2, and 4. At
the end of each stimulation period, B cells were counted in trypan blue to
evaluate viability, which was comparable in cultures of young and old
B cells (within 10%).
conjugated anti-CD23 (BD 553139) for 20 min at 4˚C and then fixed with BD
Cytofix (BD 554655). FO B cells were CD19+CD23highCD21intermediate,
whereas MZ B cells were CD19+CD23lowCD21high (26). FO B cells were
sorted on a FACSAria (BD). Cell preparations were typically .98% pure.
After sorting, mRNA was extracted from unstimulated FO B cells to evaluate
TNF-a expression. FO B cells (106/ml) were also stimulated with 1 mg/ml
LPS for 6 h (for TNF-a mRNA expression) or 7 d (for AID mRNA expression).
Recently, another mature B cell subset that accumulates with age has
been described and called age-associated B cells (ABCs), because they
represent 10–30% of the peripheral B cell pool in C57BL/6, BALB/c,
(BALB/c 3 C57BL/6) F1, and DBA/2 mice $22 mo of age (27). These
cells were shown to be CD19+AA4.12CD432CD212CD232. Another
group (28) also reported the age-related increase in these cells in C57BL/
10 mice. After these two articles were published, we sorted FO B cells
from two pairs of young and old mice, gating out CD43+ and AA4.1+
B cells to include this population but exclude transitional (AA4.1+) and B1
(CD43+) B cells. Then, FO B cells were stimulated as described above for
TNF-a and AID mRNA expression. We did not find any differential TNFa or AID mRNA expression in the FO B cells sorted in the two different
ways (data not shown). Therefore, all of our FO B cell experiments were
performed with CD19+CD23highCD21intermediate FO B cells. We also sorted
for the ABC and MZ populations and evaluated TNF-a mRNA expression
in these by quantitative PCR (qPCR).
Intracellular staining of TNF-a
Sorted FO B cells were initially fixed, washed with 13 PBS/5% FCS, and
permeabilized with 13 PBS/0.2% Tween 20, followed by cytoplasmic
staining with PE-conjugated anti-TNF-a (BD 554419). Cells were analyzed within 30 min of staining. Analysis was performed on an LSR II
fluorescence flow cytometer (BD). Gates were set on isotype control (PEconjugated IgG1; BD 555749).
Preparation of total-cell lysates
Before protein extraction, splenic B cells were counted using trypan blue.
Cells were harvested and centrifuged in a 5415C Eppendorf microfuge
(2000 rpm, 5 min). Total-cell lysates were obtained as follows. The pellet of
cultured B cells was resuspended in Mammalian Protein Extraction Reagent
(Thermo Scientific), according to the manufacturer’s instructions. The
amount of protein extracted from the same number of B cells was highly
reproducible (90%) from one experiment to another in both young and old
mice.
Western blotting
For the evaluation of specific proteins in splenic B cells, protein extracts
at equal protein concentration were denatured and then electrotransferred
onto nitrocellulose filters, essentially as previously published (18). Filters
were incubated with the following primary Abs: rabbit anti–TNF-a (1/
1000 diluted; Cell Signaling 3707), or with mouse anti–b-actin (1/8000
diluted; SIGMA A4700) as loading control, in PBS-Tween 20 containing
5% milk. After overnight incubation with the primary Abs, immunoblots
were incubated with secondary Abs for 1.5 h at 4˚C: HRP-conjugated goat
polyclonal anti-rabbit (1/50,000 diluted, 111-035-003; Jackson ImmunoResearch Laboratories) or HRP-conjugated goat anti-mouse (1/16,000
diluted, 610-1319; Rockland). Membranes were developed by enzyme
chemiluminescence and exposed to CL-XPosure Film (Pierce). Films
were scanned and analyzed using the AlphaImager Enhanced Resolution
Gel Documentation & Analysis System (Alpha Innotech, San Leandro,
CA), and images were quantitated using the AlphaEaseFC 32-bit software.
In vivo anti–TNF-a treatment
Young and old mice were injected i.p. with a rabbit anti–TNF-a polyclonal
Ab (Calbiochem 654300). The dose (100 mg/0.2 ml 13 PBS) and timing
of injection (three consecutive days before sacrifice) were determined in
a preliminary series of experiments (data not shown). Control mice were
injected with PBS or with a rabbit IgG Ab (Calbiochem 4.1590; same
isotype [IgG] as the anti–TNF-a Ab). FO B cells were sorted from the
spleens of anti–TNF-a–injected and PBS-injected mice as controls and
were stimulated for 7 d to evaluate AID mRNA.
RNA extraction and cDNA preparation
B cell staining, FO B cell sorting, and culture
Splenic B cells were stained with PerCP-conjugated anti-CD19 (BD Biosciences 551001), FITC-conjugated anti-CD21/CD35 (BD 553818), and PE-
mRNA was extracted from unstimulated or stimulated total B cells or from
FO cells, MZ cells, or ABCs (106/ml) using the mMACS mRNA isolation
kit (Miltenyi Biotec), according to the manufacturer’s protocol, eluted into
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Male and female young (2–4 mo of age) and old (24–27 mo of age) BALB/
c mice were purchased from the National Institutes of Aging and maintained in our American Association for the Accreditation of Laboratory
Animal Care-certified facility. Mice were acclimated for $7 d before
sacrifice. Mice with evidence of disease were not used in these studies. We
used young and old mice with comparable numbers of splenic B cells.
Most of the experiments were done with females, but a few experiments
were done with males. No significant differences between females and
males were seen. All studies adhered to the principles of laboratory animal
care guidelines and were approved by the Institutional Animal Care and
Use Committee.
Bone marrow cells were counted and used for flow cytometry to evaluate
the percentages of pro-B/pre-B cells, as previously described (21). A
moderately depleted phenotype corresponded to 25–80% loss in preB cells. A severely depleted old mouse corresponded to $80% loss in preB cells and 50% loss in pro-B cells compared with a young mouse (22).
With the exception of the data shown in Fig. 3, the old mice used in the
experiments had moderately or severely depleted phenotypes (which represent 80–90% of mice at 24–27 mo of age) (23).
B CELL-DERIVED TNF-a IMPAIRS B CELL FUNCTION
The Journal of Immunology
281
75 ml preheated elution buffer, and stored at 280˚C until use. Ten
microliters of mRNA (∼10 ng) was used as template for cDNA synthesis in
the reverse-transcriptase reaction.
qPCR
Two microliters of cDNA was added to 10 ml TaqMan Master mix (no.
4369016, Applied Biosystems), 1 ml forward primer, 1 ml reverse primer,
and deionized water in a final volume of 20 ml. Reactions were conducted
in MicroAmp 96-well plates (ABI no. N8010560, Applied Biosystems)
and run in the ABI 7300 machine. Calculations were made with ABI
software. Briefly, we determined the cycle number at which transcripts
reached a significant threshold (Ct) for E47, AID, TTP, and GAPDH as
control. A value for the amount of the target gene, relative to GAPDH, was
calculated and expressed as DCt. Primers for PCR amplification of
TNF-a, E47, AID, TTP and GAPDH were the following (all from ABI):
Mm01161290 (TNF-a), Mm0117557 (Tcfe2/E47), Mm00507774 (AID),
Mm00457144 (TTP), and Mn99999915 (GAPDH).
ELISA
Results
Increased intrinsic TNF-a levels in old B cells correlate with
lower LPS response
B cells from young mice were previously shown to secrete TNF-a
in response to in vivo infections (11–13) or to LPS injection (29).
However, no TNF-a production was shown after in vitro stimulation of B cells from young mice with LPS from Francisella
tularensis or E. coli (30). There are no data on how much TNF-a
is made in B cells from old versus young mice or on whether
TNF-a can be released from unstimulated B cells from young and
old mice. We measured TNF-a mRNA expression and protein
release by B cells from young and old mice in vitro stimulated
with LPS for different time periods or left unstimulated. Results in
Fig. 1A show that old unstimulated B cells made 5-fold more
TNF-a mRNA than did young B cells. The expression of TNF-a
mRNA in cultures of LPS-stimulated B cells from young mice
increased with the time of stimulation—with the peak at 6 h—and
then decreased. Conversely, in old B cells, TNF-a mRNA expression progressively decreased with the time of stimulation and,
at 6 h, was half of the level observed in young B cells. After 6 and
12 h of LPS stimulation, B cells from old mice made significantly
lower (absolute) amounts of TNF-a than did young B cells, and
the stimulation index was even more severely impaired. At later
time-points, the expression of TNF-a mRNA further decreased in
both young and old B cells, but differences were not significant.
Similar impairment in TNF-a production by old B cells was observed after 6 h of stimulation with 10 mg of LPS (qPCR values in
young versus old were 1 versus 4.5 6 1.5 [unstimulated B cell
cultures] and 4.8 6 0.4 versus 2.2 6 0.3 [stimulated B cell cultures] from three pairs of mice). Thus, it appears that ex vivo old
B cells are already stimulated and are refractory to further stimulation.
TNF-a protein expression in unstimulated old B cells is 3–4fold higher than in young B cells (Fig. 1B), but after 24 h of
stimulation with LPS was half the value of young B cells, as
evaluated by Western blotting (also confirmed by ELISA in young
and old cultures of 106 B cells: 35 6 6 pg/ml versus 165 6 20 pg/
ml in three pairs of young and old unstimulated B cells, respectively, and 120 6 11 pg/ml versus 40 6 5 pg/ml in 10 pairs of
LPS-stimulated young and old B cells, respectively). In a series of
preliminary results, the peak of TNF-a protein release in culture
FIGURE 1. Increased intrinsic TNF-a levels in old B cells correlate
with lower LPS response. A, Purified splenic B cells (106 cells/ml) were
stimulated with LPS (1 mg/ml) for 1, 3, 6, 12, 24, 48, or 96 h or were left
unstimulated. The mRNA was extracted from unstimulated B cells (t0) and
after 1–96 h in culture, and qPCR was performed. In each experiment,
values were compared with the unstimulated young B cell value, taken as 1
(for this reason, this value does not have an SE). Bars represent the DCt
values (6 SE) of TNF-a mRNA expression normalized to GAPDH. Fifteen pairs of young (white bars) and old (black bars) mice were analyzed
for all time-points, with the exception of 12 h of stimulation, for which
only four pairs of young and old mice were compared. *p , 0.05, **p ,
0.01, young versus old, two-tailed Student t test. The difference between
young 0 and young 6 h of stimulation, as well as between old 0 and old 6 h
of stimulation was significant at p , 0.01. B, Total protein extracts from
20 3 106 purified unstimulated or 24-h LPS-stimulated splenic B cells
from young and old mice were prepared and loaded in Western blotting.
A representative Western blot of four performed is shown. Densitometric
analyses 6 SE (arbitrary units) of TNF-a protein expression, normalized
to b-actin, were performed for the four pairs of young and old mice. Values
were: unstimulated young, 10 (arbitrarily assigned); unstimulated old,
35 6 4; stimulated young, 45 6 3; and stimulated old, 19 6 3. The difference between young and old mice (both unstimulated and LPS stimulated)
was significant at p , 0.05, as determined by the two-tailed Student t test.
supernatants was between 6 and 24 h of stimulation for both
young and old cells, as evaluated by ELISA (data not shown). The
level of expression of TNF-a mRNA and protein in B cells was
half of that of LPS-stimulated splenic monocyte/macrophage
cultures, which are known to be one of the primary cells making TNF-a (31) (205 6 32 pg/ml versus 110 6 16 pg/ml in three
pairs of LPS-stimulated young and old macrophages, respectively
[data not shown]), which is consistent with the data reported
by other investigators (32). Both TNF-a mRNA and proteinexpression kinetics for monocyte/macrophages were similar to
those of B cells (e.g., at 6 h young was increased 10-fold but old
was decreased 2-fold; data not shown).
FO B cells represent the major population of splenic B cells. To
evaluate whether the increased TNF-a production in aged B cells
was mainly due to FO B cells and not to minor populations, which
in some studies were shown to be altered with age, we next investigated TNF-a mRNA expression in unstimulated and stimulated FO B cells from young and old mice. Results in Fig. 2A show
that FO unstimulated B cells from old mice made 4-fold more
TNF-a mRNA than did those from young mice; however, after 6 h
of stimulation with LPS, they made less (2-fold). These results
recapitulated those obtained with the whole B cell population. To
evaluate the expression of TNF-a protein in FO B cells, we
stained sorted FO B cells for intracellular TNF-a. Results in Fig.
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TNF-a concentration in serum, plasma, and culture supernatants was determined by a mouse quantitative ELISA kit (eBioscience 88-7324-22),
according to the manufacturer’s instructions. Total IgG, IgG3, and IgA
concentration in collected supernatants of cultured B cells was determined
by mouse quantitative ELISA kits (Bethyl Labs), according to the manufacturer’s instructions.
282
B CELL-DERIVED TNF-a IMPAIRS B CELL FUNCTION
in young mice, both subsets make TNF-a mRNA, and these
amounts are comparable to those made by FO B cells. Conversely,
in old mice, FO B cells represent the major TNF-a producers in
the splenic B cell pool.
TNF-a downregulates LPS-induced B cell responses
Anti–TNF-a Ab increases LPS response in young and more
significantly in old cultured B cells
Because we showed that preincubation with TNF-a inhibited LPSinduced B cell responses, we wanted to test whether an anti–TNFa Ab added at the beginning of culture, together with LPS, would
reverse the negative effects of B cell-autonomous TNF-a. Results
in Fig. 5A show that the anti–TNF-a Ab increased, in a dosedependent manner, AID mRNA expression in B cell cultures
from old, but not from young, mice. AID mRNA expression was
further increased in old, as well as young, B cell cultures when the
Ab was added three times (at the beginning of culture and at days
2 and 4) instead of once (Fig. 5B). This effect was more pronounced in old B cells compared with young B cells. The anti–
TNF-a Ab did not have any effect when added to the cultures in
the absence of LPS stimulation (data not shown). These results
suggested that the Ab was able to counteract the effects of high
TNF-a levels of old B cells from the beginning of culture, thus
allowing old B cells to respond to LPS in a similar way as that of
young B cells.
TNF-a preincubation induces more TTP in old splenic B cells
2B show that old FO B cells expressed 3.5-fold more intracellular
TNF-a than did young FO B cells, confirming the mRNA-expression results.
We also evaluated TNF-a mRNA expression in sorted MZ cells
and ABCs from young and old mice. Results in Fig. 2C show that,
We previously showed that E47 is downregulated in old stimulated
B cells as a result of increased E47 mRNA decay and that, at least
part of the decreased stability of E47 mRNA seen in aged B cells, is
mediated by TTP, a physiological regulator of mRNA expression
and stability (18). To pursue a potential mechanism of action for
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FIGURE 2. FO B cells are the major contributors to the high TNF-a
levels in unstimulated B cells from old mice. A, Sorted FO B cells (106
cells/ml) were stimulated with LPS (1 mg/ml) for 6 h or left unstimulated.
mRNA was extracted from unstimulated and stimulated FO B cells, and
qPCR was performed. Bars represent DCt values (6 SE) of TNF-a mRNA
expression normalized to GAPDH. Values are compared with the unstimulated young B cell value, taken as 1. Five pairs of young (white bars)
and old (black bars) mice were analyzed. In these mice, the percentages of
FO B cells were 71 6 2% (young) and 73 6 3% (old), whereas those of
MZ B cells were 7 6 0.5% (young) and 2 6 0.3% (old). The young and
old mice had comparable B cell numbers; therefore, the differences in
percentages were also evident at the cell number level. *p , 0.05; ** p ,
0.01, two-tailed Student t test. B, Sorted FO B cells were fixed, permeabilized, and intracellularly stained with anti–TNF-a. Results shown
are representative of three independent experiments (among those in A).
The percentages of TNF-a+ FO B cells (means 6 SE) in young and old
mice were 20 6 5% and 72 6 6%, respectively. C, mRNA was extracted
from sorted FO cells (106), MZ cells (105), and ABCs (105), and qPCR was
performed. Bars represent the DCt values (6 SE) of TNF-a mRNA expression normalized to GAPDH from three young (white bars) and four old
(black bars) mice, which are among those in A. Values were compared with
the unstimulated young FO B cell value, taken as 1. The difference between young and old was significant only for FO B cells (p , 0.05, Mann–
Whitney U test). For ABCs and MZ cells, the p values were 0.06 and 0.63,
respectively.
We previously showed that B cell function, as measured by IgG
CSR, AID, and E47, in response to various stimuli [LPS (15), antiCD40/IL-4 (15, 16), CpG (33), and influenza vaccine (33)] decreased in aged B cells from mice and humans. We next wanted to
show a direct relationship between the inhibitory capability of
TNF-a on these measures of B cell function, as well as IgA, which
is stimulated by TNF-a, in young and old B cells from mice and
humans. B cells from young and old mice were stimulated with
LPS and TNF-a together. LPS is used as a canonical TLR/
microbial mimic stimulus. Alternatively, cultures were preincubated with TNF-a before the stimulation with LPS for 1, 3, or 12
h, over a total culture time of 24 h (E47 mRNA) or 7 d (AID
mRNA, IgG, and IgA). Results in Fig. 3 show that stimulation
with LPS and TNF-a together (given at the same time, left bars,
bold box) induced a stronger response in both young and old
B cells compared with LPS or TNF-a alone, but the response to
LPS or TNF-a alone (bars outside the bold box), as well as to LPS
+TNF-a, are reduced in old B cells. Incubation of B cell cultures
with TNF-a before the stimulation with LPS decreased both
young and old B cell responses; the inhibiting effect of preincubation was more pronounced with longer TNF-a incubation
times. The peak for E47 mRNA expression was at 24 h of LPS
stimulation; at 12 h, the amount of E47 mRNA was half of that
seen at 24 h (data not shown). Therefore, one half of the decrease
that we noted at TNF 12 h/LPS is due to the suboptimal LPS
response. These results are consistent with our hypothesis that, in
old B cells, CSR is downregulated by TNF-a, in particular by
B cell-derived (autocrine) TNF-a. In support of our hypothesis,
Fig. 4 shows that AID is negatively correlated with the levels of
TNF-a in unstimulated B cells (p = 0.0001) and that most old
B cells are low for AID and high for initial TNF-a.
The Journal of Immunology
283
TNF-a inhibiting B cell function, we next asked whether the incubation of B cells with TNF-a before the stimulation with LPS
could induce TTP and, therefore, be responsible for the reduced
response that we observed in both young and old B cells. We
hypothesized that there is a feedback mechanism of inflammatory
cytokines, especially autocrine, such as TNF-a for B cells, which
reduces these cytokines to a new challenge stimulus via decreased
mRNA stability. This mechanism also decreases E47, AID, and
CSR when B cell stimulation is induced (e.g., by TLR/Ig/
costimulatory mechanisms). Results in Fig. 6 show that LPS,
alone or together with TNF-a, induced TTP mRNA expression in
both young and old B cells, with the levels in old cells being
higher than in young B cells, as previously shown (18). Incubation
of B cells with TNF-a before the stimulation with LPS increased
TTP mRNA expression in both young and old B cells, and the
effect of TNF-a was more pronounced when TNF-a was added
for increased times before addition of LPS. In two preliminary
experiments, we showed that the degradation of TNF-a mRNA
was greater in old, than in young, LPS-stimulated B cells (data not
shown).
Anti–TNF-a in vivo increases LPS-induced AID in old, but not
in young, FO B cells
FIGURE 4. AID in stimulated B cells is negatively correlated with their
unstimulated levels of TNF-a. Purified splenic B cells (106 cells/ml) were
stimulated with LPS (1 mg/ml) for 7 d to evaluate AID or were left
unstimulated to evaluate TNF-a mRNA expression. TNF-a and AID
mRNA were normalized to GAPDH, as described in Materials and
Methods. Nineteen mice were evaluated. Pearson correlation = 20.776;
p , 0.0001 (two-tailed). Old mice, n; young mice, X. The four old mice
in the “young” category (for high AID and low TNF-a) were also “younglike” for other characteristics (i.e., pre-B cell phenotype in the bone
marrow).
Based on our in vitro data showing that an anti–TNF-a Ab increased LPS response in young and old B cells, we asked
whether the same Ab given in vivo could also improve B cell
function, in particular FO B cell function. Results in Fig. 7 indicate that injection of anti–TNF-a Ab was able to significantly
increase both LPS-induced AID mRNA expression (Fig. 7A) and
IgG3 production in culture supernatants (Fig. 7B), in old, but not
in young, FO B cells, whereas TNF-a mRNA levels were significantly decreased in unstimulated FO B cells from old, but
not from young, mice (Fig. 7C). The levels of TNF-a protein
were not significantly modified by the treatment in either young
or old B cells (data not shown), likely because a 2-fold difference in mRNA levels does not give rise to a significant difference in protein levels by flow cytometry. The relative
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FIGURE 3. TNF-a downregulates LPS-induced B cell responses. Purified splenic B cells (106 cells/ml) were stimulated with LPS (1 mg/ml) or with
TNF-a (100 ng/ml) for 1 or 7 d. Alternatively, B cells were incubated with TNF-a before the stimulation with LPS for 1, 3, or 12 h. Then, mRNA was
extracted, and qPCR was performed to evaluate expression of E47 mRNA at day 1 or AID mRNA at day 7. Supernatants were also collected at day 7 to
evaluate IgG or IgA secretion by ELISA. Bars represent the DCt values (6 SE) of E47 or AID mRNA expression normalized to GAPDH, as well as IgG or
IgA secretion in culture supernatants, from 10 pairs of young (white bars) and old (black bars) mice. Values were compared with the LPS-stimulated young
B cell value, taken as 100. For data outside of the bold box, the difference between young and old B cells was significant (p , 0.01) for LPS-induced E47,
AID, IgG, and IgA, as well as for TNF-a–induced AID, as determined by the two-tailed Student t test. For TNF-a–induced AID, the difference between
young and old may be due, in part, to a 2-fold lower level of NF-kB. For TNF-a–induced IgA, the difference between young and old B cells is significant at
p , 0.05. For data inside the bold box, the difference between young and old B cells was significant (at p , 0.01) when B cells were stimulated with LPS
and TNF-a together and was significant (at p , 0.05) for all of the other stimuli. The difference between young B cells stimulated with TNF-a and LPS
together and young B cells incubated with TNF-a for 3 h and then stimulated with LPS was significant (at p , 0.01); the difference between old B cells
stimulated with TNF-a and LPS together and old B cells incubated with TNF-a 3 h and then stimulated with LPS was significant at p , 0.05 (not
significant for AID).
284
B CELL-DERIVED TNF-a IMPAIRS B CELL FUNCTION
FIGURE 5. Anti–TNF-a Ab increases LPS response in young and more significantly in old cultured B cells. Purified splenic B cells (106 cells/ml) were
stimulated with LPS (1 mg/ml) for 7 d. Anti-TNF Ab (5–100 ng/ml) was added to B cell cultures at the beginning of culture (A) or added three times
(beginning of culture and days 2 and 4) (B). Expression of AID mRNA was evaluated by qPCR at day 7. Bars represent the DCt values (6 SE) of AID
mRNA expression normalized to GAPDH. Values are compared with the young B cell value, taken as 100. Four pairs of young (white bars) and old (black
bars) mice were analyzed. *p , 0.05, **p , 0.01, two-tailed Student t test.
creased inflammatory conditions observed in aging. Our hypothesis that the increased inflammatory response in old mice
negatively impacts B cell function was directly demonstrated in
this study in vitro, and we showed that old B cell response can be
restored, at least as measured in vitro, by anti–TNF-a given
in vivo.
Discussion
This study showed that unstimulated B cells from old mice make
more TNF-a than young B cells. This can be accounted for primarily by increased TNF-a in old FO B cells, whereas ABCs and
MZ B cells do not have a significant increase in TNF-a. Importantly, we demonstrated that there is an inverse relationship between the amount of TNF-a made by B cells and the ability of
these cells to be stimulated in vitro by this cytokine and/or other
mitogenic stimuli (e.g., LPS). Moreover, the results suggested
a connection between the defective B cell response and the in-
FIGURE 6. TNF-a stimulation induces TTP in splenic B cells. Purified
splenic B cells (106 cells/ml) were stimulated with LPS (1 mg/ml), alone
or together with TNF-a, for 3 h. Alternatively, B cells were incubated with
TNF-a before the stimulation with LPS for 1 or 2 h. Then, the mRNA was
extracted, and qPCR was performed to evaluate the expression of TTP.
Bars represent the DCt values (6 SE) of TTP mRNA expression normalized to GAPDH. Values were compared with the 3-h LPS-stimulated
young B cell value, taken as 100. Values at t0 for young and old were half
of the LPS 3 h. TNF-a alone induced 10% lower levels of TTP mRNA in
both young and old B cells compared with LPS alone at 3 h (data not
shown). As controls for the combined TNF/LPS cultures, no significant
differences were observed among LPS 1, 2, or 3 h alone in both young and
old B cells (91% and 101% versus 100% in the young and 255% and 347%
versus 310% in the old). Five pairs of young (white bars) and old (black
bars) mice were analyzed. *p , 0.05, **p , 0.01, two-tailed Student t
test.
FIGURE 7. Anti–TNF-a in vivo increases LPS-induced AID in old, but
not in young, FO B cells. Young and old mice were injected i.p. with anti–
TNF-a for three consecutive days before sacrifice. Control mice were
injected with PBS or with a rabbit IgG Ab (same isotype of the anti–TNFa Ab). Then, FO B cells were sorted, as indicated in Materials and
Methods, and stimulated for 7 d to evaluate AID mRNA. Bars represent the
DCt values (6 SE) of AID (A) or TNF (C) mRNA expression normalized
to GAPDH or IgG3 (B) from four pairs (PBS injected), two pairs (IgG
injected), and six pairs (anti–TNF-a injected) of young and old mice.
Values were compared with the LPS-stimulated young B cell value, taken
as 100. *p , 0.05, two-tailed Student t test.
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percentages of FO and MZ B cells in the spleens of both young
and old mice were not modified by the injection of anti–TNF-a Ab
(Table I). We do not yet know whether these data are relevant to an
infection/vaccine model, and we are in the process of testing that.
The model we propose for these results is shown in Fig. 8.
The Journal of Immunology
Table I.
Percentages of FO and MZ B cells after in vivo injection of anti–TNF-a
Age Group
Young
Young
Old
Old
285
In Vivo Treatment
PBS
Anti–TNF-a
PBS
Anti–TNF-a
FO Cells (% of CD19+ B Cells)
73.5
72.5
78.1
69.8
6
6
6
6
3.7
1.5
1.1
3.6
MZ Cells (% of CD19+ B Cells)
8.1
6.0
2.5
1.85
6
6
6
6
0.4
1.0
0.9
0.7
Young and old mice were injected i.p. with 100 mg of anti–TNF-a for three consecutive days before sacrifice. Control mice
were injected with PBS. FO B cells were CD19+CD23highCD21intermediate, whereas MZ B cells were CD19+CD23lowCD21high.
The young and old mice had comparable B cell numbers; therefore, the differences in percentages were also evident at the cell
number level.
FIGURE 8. Model for differential activation of CSR in B cells from
young versus old mice. Unstimulated (ex vivo) B cells from old mice (2)
make significantly more TNF-a mRNA than do B cells from younger ones
(1). In the presence of endogenous TNF-a in the old (2) and exogenous
TNF-a added to the young (3), TNF-a/LPS induces TTP, which downregulates the stability of E47. Arrows indicate relative values between
young (1) and old (2) or young (1) and TNF-a added to young (3) and may
reflect “threshold” values that need to be reached to affect further downstream effector functions.
higher in old, compared with young, stimulated B cells (18),
suggesting that TTP induction in B cells may help to downregulate the production of inflammatory cytokines and may
contribute to an autocrine negative-feedback loop to keep levels
of TNF-a, and perhaps other proinflammatory cytokines, below
toxic/cell death amounts. As a side effect, TTP reduces optimal
B cell immune responses, downregulating E47, AID, and class
switch. Because TTP levels are also higher in old, compared
with young, unstimulated B cells, the higher levels of TNF-a
mRNA in old versus young unstimulated B cells could be due to
increased transcription. Our model (Fig. 8) emphasizes that the
increased autocrine TNF-a released by aged B cells impairs
their function.
Healthy aging results from the ability to control/make less destructive inflammatory responses, as well as from the ability to
mount effective anti-inflammatory responses. If chronic inflammation prevails, frailty and common age-related pathologies may
occur (20, 45). Therefore, it is important to understand the regulation of inflammatory-signaling pathways to develop effective
therapeutic strategies to fight age-related immune and inflammatory diseases and, in particular, as we showed in this study, for
optimal B cell functional responses.
In conclusion, our results may indicate that B cells make TNF-a
and, therefore, contribute to the systemic modification of the
cellular microenvironment typical of old age. We showed that
unstimulated B cells from old mice make more TNF-a mRNA and
protein than do B cells from young mice; however, after stimulation, the old make less than the young (i.e., old B cells are
preactivated and refractory to further stimulation). Unstimulated
FO B cells, which represent the major splenic B cell subset, make
more TNF-a in old mice than in young mice; however, after
stimulation they make less, thus recapitulating the results obtained with the whole population of B cells. If B cells are incubated with TNF-a before stimulation with LPS, both young and
old B cell responses are inhibited. In fact, B cells can be induced
by TNF-a to secrete IgA but not IgG, and this response is
downregulated in old B cells, emphasizing the importance of
unique stimuli for a complete evaluation of the aged B cell response. The inhibiting effect of preincubation with TNF-a is
more pronounced with longer TNF-a incubation times. This inhibitory effect correlates with the induction of TTP, a physiological regulator of mRNA stability of the transcription factor E47,
crucial for CSR, which is downregulated in old B cells. Finally,
anti–TNF-a Ab increases the LPS response in young and more
significantly in old cultured B cells. Moreover, anti–TNF-a Ab
given in vivo increased B cell function in old, but not in young,
FO B cells stimulated in vitro. Taken together, our results showed
that inflammation and B cell function are inversely related in old
mice; these studies should help to increase our understanding of
the mechanisms leading to reduced Ab responses in aging, as well
as assist in the design of novel therapeutic approaches for agerelated immune diseases.
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Our results were obtained in BALB/c mice, which is a strain
widely used in aging studies. Because we do not perform experiments with transgenic or knockout mice, we do not have the
necessity to use C57BL/10 mice. Because the cells that we studied
are not Ag-experienced cells, which appear to be expanded in old
C57BL/10 mice (25), the data that we report should be applicable
to both BALB/c and C57BL/10 mice.
TNF-a can positively or negatively modulate immune responses. It is a potent enhancer of both T-dependent Ab responses
and T cell responses against pathogens (11–13), arguing
that the ability of B cells to produce proinflammatory cytokines
is positive, especially in younger individuals, as in response to
antigenic challenge/stimulus. TNF-a is a cytokine that can kill
tumor cells; however, it can also contribute to tumorigenesis by
mediating the proliferation, invasion, and metastasis of tumor
cells, and it is an autocrine growth factor for a wide variety of
tumors (34). In general, inflammation is a protective response of
the body to infection. However, increased plasma levels of TNF-a,
which contribute to the chronic, low-grade inflammation typical of
old age, can have deleterious effects, as are implicated in the
pathogenesis of several disabling diseases of the elderly, such as
type II diabetes mellitus (35), osteoporosis (36), Alzheimer’s
disease (37), rheumatoid arthritis (38), and coronary heart disease
(39).
We showed in this study that TNF-a stimulation of B cells
induces TTP, which is a negative regulator of the stability of
mRNA for cytokines and transcription factors (18, 40–44). We
previously showed that TTP mRNA and protein levels are
286
Acknowledgments
We thank Jim Phillips (Sylvester Comprehensive Cancer Center Flow
Cytometry Core Resource at the University of Miami Miller School of
Medicine) for help with the flow cytometer and Michelle Perez for secretarial assistance.
Disclosures
The authors have no financial conflicts of interest.
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B CELL-DERIVED TNF-a IMPAIRS B CELL FUNCTION