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A Molecular Mechanism for TNF-α− Mediated Downregulation of B Cell Responses This information is current as 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 References Subscription Permissions Email Alerts This article cites 45 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/188/1/279.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 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- Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 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. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 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 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 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. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 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. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 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). 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