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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Immunol. Author manuscript; available in PMC Jul 3, 2013.
Published in final edited form as:
PMCID: PMC3700394
NIHMSID: NIHMS336624
A molecular mechanism for TNF-α-mediated down-regulation of B cell responses1
Daniela Frasca, Maria Romero, Alain Diaz, Sarah Alter-Wolf, Michelle Ratliff, Ana Marie Landin, Richard L Riley, and Bonnie B. Blomberg2
Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL 33101, USA
2Corresponding author: Bonnie Blomberg, Department of Microbiology and Immunology, University of Miami Miller School of Medicine, P.O. Box 016960 (R-138), Miami, FL 33101, USA, Tel.: 305-243 6040, Fax: 305-243 4623, bblomber/at/med.miami.edu
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-α. Here, we investigate whether B cells produce TNF-α, 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-α) that lowers the expression of AID and CSR. Our results show that unstimulated B cells from old BALB/c mice make significantly more TNF-α mRNA and protein than B cells from young mice, but after stimulation the old make less than young, thus they are refractory to stimulation. The increase in TNF-α made by old B cells is primarily due to follicular, but not minor subsets of B cells. Pre-incubation of B cells with TNF-α before LPS stimulation decreases both young and old B cell responses. Importantly, B cell function was restored by adding anti-TNF-α antibody in cultured B cells. To address a molecular mechanism, we found that pre-incubation of B cells with TNF-α, before LPS stimulation, induces tristetraprolin, a physiological regulator of mRNA stability of the transcription factor E47, crucial for CSR. Finally, anti-TNF-α given in vivo was able to increase follicular B cell function in old but not in young follicular B cells. These results suggest new molecular mechanisms which contribute to reduced antibody responses in aging.
Keywords: aging, B cells, inflammation, TNF-α, tristetraprolin
Inflammation is part of the protective, biological/immunological response to infections which is crucial for survival. At the same time, however, 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 pro-inflammatory cytokines such as TNF-α. Anti-cytokine therapies have been used to successfully treat patients with autoimmune diseases, such as rheumatoid arthritis, Crohn’s Disease and psoriasis (1, 2). Increasing understanding of the role of TNF-α 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 (57) and TNF-α (5, 8) plasma levels have been associated with functional disability and mortality of the elderly. Aging is characterized by a disregulation of inflammatory and anti-inflammatory networks, which results in a low grade chronic pro-inflammatory status called inflammaging (9). The age-related increase in circulating inflammatory mediators such as cytokines and acute phase proteins are markers of the low-grade inflammation observed with aging. Age-related alterations in responses to immune stimulation, for example chronic T cell stimulation with viruses such as CMV, also contribute to low-grade inflammation by increasing the level of pro-inflammatory mediators such as TNF-α (10). Production of pro-inflammatory cytokines is thought to be in part a macrophage-mediated event, but it is clear that other cell types, including stroma (i.e. epithelium and endothelium and fat), as well as T cells, produce these mediators in vivo. Production of TNF-α in unstimulated B cells has not yet been pursued and is in part the subject of this article.
B cells, through the secretion of cytokines such as TNF-α, have been shown to contribute to immunity against infectious agents, such as Toxoplasma gondii, Heligomosomoides polygyrus or Pneumocystis carinii by promoting expansion and differentiation of primary and memory Th1 (11) or Th2 cells (12, 13). Moreover, the possible contribution of B cells and/or antigen presenting cells to the inflammatory process supports their pathogenic role in a wide range of autoimmune diseases (14).
We have previously found that the molecular basis for E47, and hence AID (activation-induced cytidine deaminase) and class switch recombination (CSR) of immunoglobulin (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 3′UTR (18). As TTP also regulates inflammatory cytokine (TNF-α, IL-6) mRNAs similarly (19, 20), our hypothesis is that in aging there may be a feedback mechanism of inflammatory cytokines to down-regulate further expression of these, and that in B cells this process inadvertantly also downregulates E47 and an optimal B cell immune response, including Ig CSR and AID.
In the present study, we investigate two questions: 1) whether unstimulated B cells may contribute to the increased inflammatory response in aging (inflammaging) by secreting more pro-inflammatory cytokines i.e. TNF-α than B cells from young mice and 2) whether the pro-inflammatory microenvironment seen in old mice, and specifically TNF-α produced by B cells, can reduce the ability of B cells to respond to stimuli such as LPS. Our results reveal new molecular mechanisms which may contribute to reduced antibody responses in aging.
Mice, definition of phenotype
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 AAALAC-certified facility. Mice were acclimated for at least 7 days before sacrifice. Mice with evidence of disease were not used in these studies. In the experiments herein we used young and old mice with comparable numbers of splenic B cells. Most of the experiments have been done with females. A few experiments have been 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 IACUC approved.
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 pre-B cells. A severely depleted old mouse corresponded to 80% or more loss in pre-B cells and 50% loss in pro-B cells, as compared to young (22). Except for Fig. 3, the old mice used in the experiments herein had the moderately or severely depleted phenotypes (which represent 80–90% of mice at 24–27 months of age (23).
Figure 3
Figure 3
TNF-α down-regulates LPS-induced B cell responses
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 μl Microbeads + 80 μl PBS, for 107 cells). Cells were then purified using magnetic columns. At the end of the purification procedure, cells were 80–85% CD19-positive by cytofluorimetric analysis. After the isolation procedure was ended, cells were maintained in PBS for 3 h at 4°C to minimize potential effects of anti-CD19 antibodies on B cell activation. In a preliminary series of experiments, macrophages were removed by adherence and B cells were isolated from the non-adherent 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 on E47 and AID in B cells isolated with or without depletion of macrophages. All data herein have been obtained with B cells isolated from whole splenocytes, without depletion of macrophages).
We (18) and others (24) have published that in BALB/c mice the number of follicular (FO) B cells is unaffected by aging, but there is a significant age-related decrease in marginal zone (MZ) B cells. In C57BL/10 mice, conversely, FO B cells decrease and MZ B cells increase with age, as we (unpublished) and others (25) have observed. As the differences that we see are not in MZ/antigen-experienced cells, the results herein should apply to both BALB/c and C57BL/10.
B cell culture
B cells were cultured in complete medium (RPMI 1640, supplemented with 10% FCS, 10 μg/ml gentamicin, 2 × 10−5 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-points (indicated in each figure) with 1 μg/ml of LPS (from E. coli, SIGMA L2880). Alternatively, B cells were stimulated in 24-well culture plates for different time points with 100 ng/ml of TNF-α (PMC3014 Biosource). In some experiments, a purified rat anti-mouse TNF-α antibody (551225 BD Pharmingen) was added to the LPS-stimulated B cell cultures at the concentration of 5–100 ng/ml. The antibody was either added once at the beginning of the culture, or it was added every day. At the end of each stimulation time, B cells were counted in trypan blue to evaluate viability which was found comparable in cultures of young and old B cells (within10%).
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-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 FACS Aria(BD). Cell preparations were typically >98% pure. After sorting, mRNA was extracted from unstimulated FO B cells to evaluate TNF-α expression. FO B cells (106/ml) were also stimulated with 1 μg/ml of LPS for 6 h (for TNF-α mRNA expression) or 7 days (for AID mRNA expression).
Recently, another mature B cell subset that accumulates with age has been described and called age-associated B cells (ABC) as they represent 10–30% of the peripheral B cell pool in C57BL/6, BALB/c, (BALB/c x C57BL/6) F1 and DBA/2 mice 22 months of age or older (27). These cells have been shown to be CD19+AA4.1-CD43-CD21-CD23-. Another group (28) has also shown the age-related increase of these cells in C57BL/10 mice. After these two papers were published, we sorted FO B cells from 2 pairs of young and old mice gating out CD43+ and AA4.1+ B cells in order 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-α and AID mRNA expression. We didn’t find any differential TNF-α or AID mRNA expression in the FO B cells sorted in the 2 ways (data not shown). Therefore, all the FO B cell experiments reported herein have been performed with CD19+CD23highCD21intermediate FO B cells. We also sorted for the ABC and the MZ populations and evaluated TNF-α mRNA expression in these by qPCR.
Intracellular staining of TNF-α
Sorted FO B cells were initially fixed, washed with 1X PBS/5% FCS, permeabilized with 1X PBS/0.2%Tween 20, followed by cytoplasmic staining with PE-conjugated anti-TNF-α (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 (PE-conjugated 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 (2,000 rpm, 5 min). Total cell lysates were obtained as follows. The pellet of cultured B cells was resuspended in M-PER (Mammalian Protein Extraction Reagent, Thermo Scientific), according to the manufacturer’s instructions. The amount of protein extracted from the same number of B cells is highly reproducible (90%) from one experiment to another in both young and old mice.
Western blotting (WB)
For the evaluation of specific proteins in splenic B cells, protein extracts at equal protein concentration were denatured and then electro-transferred onto nitrocellulose filters, essentially as previously published (18). Filters were incubated with the following primary antibodies: rabbit anti-TNF-α (1/1000 diluted, Cell Signaling 3707), or with mouse anti-β-actin (1/8000 diluted, SIGMA A4700) as loading control, in PBS-Tween 20 containing 5% milk. After overnight incubation with the primary antibodies, immunoblots were incubated with the following secondary antibodies: 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) for 1.5 h at 4°C. 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-α treatment
Young and old mice were injected i.p. with a rabbit anti-TNF-α polyclonal antibody (Calbiochem 654300). The dose (100 μg/0.2 ml 1X PBS) and timing of injection (3 consecutive days before sacrifice) were determined in a preliminary series of experiments (not shown). Control mice were injected with PBS or with a rabbit IgG antibody (calbiochem 4.1590, same isotype (IgG) as the anti-TNF-α antibody). FO B cells were sorted from the spleens of anti-TNF-α-injected and PBS-injected mice as controls and were stimulated for 7 days to evaluate AID mRNA.
RNA extraction and cDNA preparation
mRNA was extracted from unstimulated or stimulated total B cells, or from FO, MZ or ABC (106/ml) using the μMACS mRNA isolation kit (Miltenyi Biotec), according to the manufacturer’s protocol, eluted into 75 μl of preheated elution buffer, and stored at −80°C until use. Ten μl of mRNA (approximately 10 ng) were used as template for cDNA synthesis in the reverse transcriptase reaction.
Quantitative PCR (qPCR)
Two μl of cDNA were added to 10 μl of Taqman Master mix (Applied Biosystems no.4369016), 1 μl of forward primer, 1 μl of reverse primer, and deionized water in a final volume of 20 μl. Reactions were conducted in MicroAmp 96-well plates (Applied Biosystems, ABI no.N8010560), 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 ΔCt. Primers for PCR amplification of TNF-α, E47, AID, TTP and GAPDH were the following (all from ABI): Mm01161290 (TNF-α), Mm0117557 (Tcfe2/E47), Mm00507774 (AID), Mm00457144 (TTP), Mn99999915 (GAPDH).
Enzyme-linked immunosorbent assay (ELISA)
TNF-α 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.
Increased intrinsic TNF-α levels in old B cells correlate with lower LPS response
B cells from young mice were previously shown to secrete TNF-α in response to in vivo infections (1113) or to LPS injection (29). No TNF-α production, however, was shown after in vitro stimulation of B cells from young mice with LPS from F. tularensis or E. coli (30). Currently there are no data on how much TNF-α is made in B cells from old versus young mice or on whether TNF-α can be released from unstimulated B cells from young and old mice. We measured TNF-α mRNA expression and protein release by B cells from young and old mice in vitro stimulated with LPS for different time-points or left unstimulated. Results in Fig. 1A show that old unstimulated B cells make 5-fold more TNF-α mRNA than young B cells. The expression of TNF-α mRNA in cultures of LPS-stimulated B cells from young mice increases with the time of stimulation, the peak being at 6 h of stimulation, and then decreases. In old B cells, conversely, TNF-α mRNA expression progressively decreases with the time of stimulation and at 6 h is half the level observed in young B cells. After 6 and 12 h of LPS stimulation, B cells from old mice make not only significantly lower (absolute) amounts of TNF-α than young B cells, but also the stimulation index is even more severely impaired. At later time-points, the expression of TNF-α mRNA further decreases in both young and old B cells, but differences are not significant. Similar impairment in TNF-α production by old B cells was observed after 6 h stimulation with 10 μg of LPS (qPCR values in young versus old were: 1 versus 4.5±1.5, unstimulated and 4.8±0.4 versus 2.2±0.3, stimulated B cell cultures from 3 pairs of mice). Thus, it appears that ex vivo old B cells are already stimulated and are refractory to further stimulation.
Figure 1
Figure 1
Increased intrinsic TNF-α levels in old B cells correlate with lower LPS response
TNF-α protein expression in unstimulated old B cells is 3–4-fold higher than in young B cells (Fig. 1B), but after 24 h stimulation with LPS is half the value of young B cells, as evaluated by WB (also confirmed by ELISA in young and old cultures of 106 B cells, 35±6 pg/ml versus 165±20 pg/ml in 3 pairs of young and old unstimulated B cells, respectively, and 120±11 pg/ml versus 40±5 pg/ml in 10 pairs of LPS-stimulated young and old B cells, respectively). We found in a series of preliminary results that the peak of TNF-α protein release in culture supernatants is between 6 and 24 h stimulation for both young and old, as evaluated by ELISA (data not shown). The level of expression of TNF-α mRNA and protein in B cells was half of that of LPS-stimulated splenic monocyte/macrophage cultures which are known as one of the primary cells making TNF-α (31) (205±32 pg/ml versus 110±16 pg/ml in 3 pairs of LPS-stimulated young and old macrophages, respectively, consistent with what others have also published, data not shown, (32). Both TNF-α mRNA and protein expression kinetics for monocyte/macrophages were similar to those of B cells (e.g., at 6 h young was increased 10X but old was decreased 2X, data not shown).
FO B cells represent the major population of splenic B cells. In order to evaluate whether the increased TNF-α production in aged B cells was mainly due to FO B cells and not to minor populations which in some studies have been shown to be altered with age, we next investigated TNF-α 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 make 4-fold more TNF-α mRNA than those from young mice, but after 6 h stimulation with LPS make less (2-fold). These results recapitulate those obtained with the whole B cell population. To evaluate the expression of TNF-α protein in FO B cells, we stained sorted FO B cells for intracellular TNF-α. Results in Fig. 2B show that old FO B cells express 3.5-fold more intracellular TNF-α than young FO B cells, confirming the mRNA expression results.
Figure 2
Figure 2
FO B cells are the major contributors to the high TNF-α levels in unstimulated B cells from old mice
We also evaluated TNF-α mRNA expression in sorted MZ and ABC from young and old mice. Results in Fig. 2C show that in young mice both subsets make TNF-α mRNA and these amounts are comparable to those made by FO B cells. In old mice, conversely, FO B cells represent the major TNF-α producers in the splenic B cell pool.
TNF-α down-regulates LPS-induced B cell responses
We have previously shown that B cell function, as measured by IgG CSR, AID and E47, in response to various stimuli [LPS (15), anti-CD40/IL-4 (15, 16), CpG (33) and influenza vaccine (33)] decrease in aged B cells. We next wanted to show a direct relationship between the inhibitory capability of TNF-α on these measures of B cell function, as well as IgA which is stimulated by TNF-α, in young and old B cells. B cells from young and old mice were stimulated with LPS and TNF-α together. LPS is used as a canonical TLR/microbial mimic stimulus. Alternatively, cultures were pre-incubated with TNF-α before the stimulation with LPS for 1, 3 or 12 h, over a total time of culture of 24 h (E47 mRNA) or 7 days (AID mRNA, IgG, IgA). Results in Fig. 3 show that stimulation with LPS and TNF-α together (given at the same time, left bars, bold box) induced a stronger response in both young and old B cells, as compared to LPS or TNF-α alone, but the response to LPS or TNF-α alone (bars outside the bold box) as well as to both LPS+TNF-α are reduced in old B cells. Pre-incubation of B cell cultures with TNF-α before the stimulation with LPS decreases both young and old B cell responses, the inhibiting effect of pre-incubation being more pronounced with longer TNF-α incubation times. The peak for E47 mRNA expression is at 24 h of LPS stimulation; at 12 h, the amount of E47 mRNA is half of that seen at 24 h (data not shown); therefore of the decrease we see 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 down-regulated by TNF-α, in particular by B cell-derived (autocrine) TNF-α. In support of our hypothesis, Fig. 4 shows that AID is negatively correlated with the levels of TNF-α in unstimulated B cells (p=0.0001) and that most old B cells are low for AID and high for initial TNF-α.
Figure 4
Figure 4
AID in stimulated B cells is negatively correlated with their unstimulated levels of TNF-α
Anti-TNF-α antibody increases LPS response in young and more significantly in old cultured B cells
Because we have shown above that pre-incubation with TNF-α inhibits LPS-induced B cell responses, we wanted to test whether an anti-TNF-α antibody added at the beginning of culture together with LPS would reverse the negative effects of B cell autonomous TNF-α. Results in Fig. 5A show that the anti-TNF-α antibody was able to increase in a dose-dependent manner AID mRNA expression in B cell cultures from old but not from young mice. AID mRNA expression was further increased not only in old but also in young B cell cultures when the antibody was added 3 times (at the beginning of culture, at day 2 and at day 4) instead of once (Fig. 5B). This effect was more pronounced in old B cells as compared to young B cells. The anti-TNF-α antibody did not have any effect when added to the cultures in the absence of LPS stimulation (not shown). These results suggest that the antibody was able to counteract the affects of high TNF-α 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.
Figure 5
Figure 5
Anti-TNF-α antibody increases LPS response in young and more significantly in old cultured B cells
TNF-α pre-incubation induces more TTP in old splenic B cells
We have previously shown that E47 is down-regulated in old stimulated B cells, due to increased E47 mRNA decay, and that at least part of the decreased stability of E47 mRNA seen in aged B cells is mediated by tristetraprolin (TTP), a physiological regulator of mRNA expression and stability (18). In order to pursue a potential mechanism of action for TNF-α inhibiting B cell function, we next asked whether the pre-incubation of B cells with TNF-α before the stimulation with LPS can induce TTP and therefore be responsible for the reduced response we have observed in both young and old B cells. Our hypothesis is that there is a feedback mechanism of inflammatory cytokines, especially autocrine, such as TNF-α 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-α, induces TTP mRNA expression in both young and old B cells, the levels in old being higher than in young B cells, as previously shown (18). Pre-incubation of B cells with TNF-α before the stimulation with LPS increases TTP mRNA expression in both young and old B cells and the effect of TNF-α is more pronounced when TNF-α is added for increased times before addition of LPS. In two preliminary experiments we showed that the degradation of TNF-α mRNA is greater in old than in young LPS-stimulated B cells (data not shown).
Figure 6
Figure 6
TNF-α stimulation induces TTP in splenic B cells
Anti-TNF-α in vivo increases LPS-induced AID in old but not in young FO B cells
Based on our in vitro data showing that an anti-TNF-α antibody increases LPS response in young and old B cells, we asked whether the same antibody given in vivo could also improve B cell function, and in particular FO B cell function. Results in Fig. 7 indicate that injection of anti-TNF-α antibody was able to significantly increase both LPS-induced AID mRNA expression (A) and IgG3 production in culture supernatants (B), in old but not in young FO B cells, whereas TNF-α mRNA levels were significantly decreased in unstimulated FO B cells from old but nor from young mice (C). The levels of TNF-α protein were not significantly modified by the treatment in either young or old B cells (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 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-α antibody (Table 1). We do not yet know if these data will be relevant to an infection/vaccine model, and we are in the process of testing that (for a subsequent paper).
Figure 7
Figure 7
Anti-TNF-α in vivo increases LPS-induced AID in old but not in young FO B cells
Table 1
Table 1
Percentages of FO and MZ B cells after in vivo injection of anti-TNF-α
The present paper shows that unstimulated B cells from old mice make more TNF-α than young B cells. This can be accounted for primarily by increased TNF-α in old FO B cells, while ABC and MZ B cells do not have a significant increase in TNF-α. Importantly, we demonstrate here that there is an inverse relationship between the amount of TNF-α 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 herein suggest a connection between the defective B cell response and the increased inflammatory conditions observed in aging. Our hypothesis that the increased inflammatory response in old mice negatively impacts B cell function has been directly demonstrated here in vitro and we have shown that old B cell response can be restored, at least as measured in vitro, by anti-TNF-α given in vivo.
Our results have been obtained in BALB/c mice which is a strain widely used in aging studies. Because we do not perform experiments with transgenic or KO mice, we do not have the necessity to use C57BL/6 mice. As the cells studied here are not antigen-experienced cells, which appear to be expanded in old C57BL/10 (25), the data we report here should be applicable to both BALB/c and C57BL/10.
TNF-α can positively or negatively modulate immune responses. It is a potent enhancer of both T-dependent antibody responses and T cell responses against pathogens (1113), arguing that the ability of B cells to produce pro-inflammatory cytokines is positive, especially in younger individuals, as in response to antigenic challenge/stimulus. TNF-α has been discovered as a cytokine that could kill tumor cells, but 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-α, 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 here show that TNF-α stimulation of B cells induces TTP, which is a negative regulator of the stability of mRNA for cytokines and transcription factors (18, 4044). We have previously shown that TTP mRNA and protein levels are higher in old as compared to young stimulated B cells (18), suggesting that TTP induction in B cells may help to down-regulate the production of inflammatory cytokines, and may contribute to an autocrine negative feedback loop to keep levels of TNF-α and perhaps other pro-inflammatory cytokines below toxic/cell death amounts. As a side effect, TTP reduces optimal B cell immune responses, down-regulating E47, AID and class switch. Because TTP levels are also higher in old as compared to young unstimulated B cells, the higher levels of TNF-α mRNA in old versus young unstimulated B cells can be due to increased transcription. Our model, shown in Fig. 8, emphasizes that the increased autocrine TNF-α released by aged B cells impairs their function.
Figure 8
Figure 8
Model for differential activation of CSR in B cells from young versus old mice
Healthy aging results not only from the ability to control/make less distructive inflammatory responses, but also 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 in order to develop effective therapeutic strategies to fight age-related immune and inflammatory diseases and in particular, as we have shown here, for optimal B cell functional responses.
In conclusion, results herein may indicate that B cells make TNF-α and therefore contribute to the systemic modification of the cellular microenvironment typical of old age. We show that unstimulated B cells from old mice make more TNF-α mRNA and protein than B cells from young mice, but after stimulation the old make less than the young, i.e. old B cells are pre-activated and refractory to further stimulation. Unstimulated follicular B cells, which represent the major splenic B cell subset, make more TNF-α in old than in young mice, but after stimulation they make less, thus recapitulating the results obtained with the whole population of B cells. If B cells are pre-incubated with TNF-α before stimulation with LPS, both young and old B cell responses are inhibited. B cells can in fact be induced by TNF-α to secrete IgA but not IgG, and this response is down-regulated in old B cells, emphasizing the importance of unique stimuli for a complete evaluation of the aged B cell response. The inhibiting effect of pre-incubation with TNF-α is more pronounced with longer TNF-α 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, down-regulated in old B cells. Finally, anti-TNF-α antibody increases the LPS response in young and more significantly in old cultured B cells. Moreover, anti-TNF-α antibody given in vivo increases B cell function in old but not in young FO B cells stimulated in vitro. Taken together, our results show that inflammation and B cell function are inversely related in old mice and these studies should help further understanding of the mechanisms leading to reduced antibody responses in aging. These will also help to design new possibilities for novel therapeutic approaches for age-related immune diseases.
Acknowledgments
We thank Jim Phillips (Sylvester Comprehensive Cancer Center Flow Cytometry Core Resource), for help with the Flow Cytometer; and Michelle Perez, for secretarial assistance.
Footnotes
1This work is supported by NIH AG-17618 and AG-28586 (BBB) and by NIH AG-025256 and AI-064591 (RLR).
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