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. 2010 Feb;129(2):278–290. doi: 10.1111/j.1365-2567.2009.03182.x

Migration of immature and mature B cells in the aged microenvironment

Heather A Minges Wols 1,2,*, Kara M Johnson 2,3,*, Jill A Ippolito 2, Shirin Z Birjandi 2, Yan Su 2,4, Phong T Le 2, Pamela L Witte 2
PMCID: PMC2814469  PMID: 19845796

Abstract

Studies in aged mice show that the architecture of B-cell areas appears disrupted and that newly made B cells fail to incorporate into the spleen. These observations may reflect altered migration of immature and mature B cells. Using adoptive transfer, we tested the effect of the aged microenvironment and the intrinsic ability of donor B cells from aged mice to migrate to spleens of intact hosts. Spleens of aged recipients were deficient in attracting young or old donor immature B cells. In contrast, immature and mature B cells maintained an intrinsic ability to migrate to young recipient spleens, except that as the aged immature B cells matured, fewer appeared to enter the recirculating pool. CXCL13 protein, which is necessary for the organization of B-cell compartments, was elevated with age and differences in CXCL13 distribution were apparent. In aged spleens, CXCL13 appeared less reticular, concentrated in patches throughout the follicles, and notably reduced in the MAdCAM-1+ marginal reticular cells located at the follicular edge. Despite these differences, the migration of young donor follicular B cells into the spleens of old mice was not impacted; whereas, migration of young donor marginal zone B cells was reduced in aged recipients. Finally, the aged bone marrow microenvironment attracted more donor mature B cells than did the young marrow. Message for CXCL13 was not elevated in the marrow of aged mice. These results suggest that the aged splenic microenvironment affects the migration of immature B cells more than mature follicular B cells.

Keywords: aging, B cell, CXCL13, migration

Introduction

Aging impacts the homeostatic function of many complex systems in the body, including the immune system.1 Because most types of lymphocytes circulate between the tissue and blood/lymph, an important aspect of immune homeostasis is the appropriate migration of lymphocytes to specific compartments within lymphoid organs.2–4 There is little information available about the migration of B cells in aged animals; yet, several observations and unanswered questions might be explained by altered migration of B cells. One potential problem is the histology of peripheral lymphoid tissues of aged animals, which shows micro-anatomical changes including smaller follicles, reduced follicular dendritic cell networks, smaller germinal centres, and changes in the marginal zone (MZ) structure.5–11 Changes in the tissue architecture could either influence or reflect alterations in the migration of B cells. Migration of mature B cells to follicles in secondary lymphoid tissues is well-described in young animals and is clearly dependent on the production of the chemokine, CXCL13, by specific stromal reticular cell populations in the follicle.2 Although not as extreme as the situation in CXCL13-deficient mice, the reported changes in lymphoid tissues from aged normal animals resemble some features described in mice lacking CXCL13 expression. The ultimate consequences of diminished production of CXCL13 in the microenvironment are disorganization of the B lymphoid compartments and decreased B-cell migration into the follicles and between the MZ area and the follicle.12–14 Hence, altered migration of mature B cells in aged animals might be responsible for changes in the tissue microanatomy, as the result of either deficiency in maintaining the microenvironment or qualities intrinsic to the lymphocytes.

A second enigma is the finding that new immature B cells are produced in the bone marrow of many aged mice, but few newly made B cells appear in aged spleens.15–18 The kinetic data for these conclusions do not support a loss or retention of immature B cells in the marrow, leaving the possibility that their migration to the spleen is disrupted. In young mice, immature B cells immigrate exclusively to the spleen, enter the MZ area, and gain the ability to enter the follicle and become recirculating only after a brief period of maturation (1–3 days).19–21 Therefore, it is possible that disrupted micro-architecture could affect efficient migration of immature B cells into the aged spleen.

B cells from aged animals cannot be presumed to be intrinsically equivalent to those from young animals, which has been controversial. On the one hand, mature B cells in aged animals are very long-lived and not replenished by newly made cells.15–18 On the other hand, some studies of antigen response and cell surface expression of classic mature B-cell phenotype molecules (B220hi IgDhi IgMlo) and co-stimulatory molecules (CD40 CD86) report that B cells from aged mice appear to be equivalent to B cells from young mice.22–24 However, investigators have found alterations in aged B cells that yielded subtle, but important, functional changes.22,25 More recently, mechanistic studies have shown that aged splenic B cells suffer accelerated messenger RNA (mRNA) decay rates and protein degradation of molecules important to B-cell function and these cell intrinsic alterations first appear in aged B-cell precursors.26,27 It is unknown how intrinsic changes in the cell physiology may alter the ability of B cells to migrate properly.

The purpose of the study reported here was to investigate the ability of immature and mature B cells to migrate in aged animals. A secondary objective was to determine the expression and distribution of the critical B-cell chemokine, CXCL13.

Materials and methods

Animals

Female BALB/c (IgMa allotype) mice aged 2–3 and 20–22 months were purchased from Harlan Laboratories (Indianapolis, IN) through a contract with the National Institute of Aging (Bethesda, MD). Female C.B-17 (IgMb allotype) mice aged 4–9 weeks were purchased from Taconic (Germantown, NY). Upon receipt, the animals were housed at the Animal Research Facility at Loyola University Medical Center in specific pathogen-free conditions. Experiments were conducted with an approved animal protocol from Loyola University Medical Center Institute Animal Care and Use Committee. Aged mice with enlarged spleens or other visual tumours were not used in these studies.

Immature bone marrow B cells: preparation, bromodeoxyuridine incorporation, transfer and detection by flow cytometry

Newly-formed, immature B cells were labelled prior to transfer using in vivo bromodeoxyuridine (BrdU) incorporation. Donor mice received BrdU in two forms. Injections of 0·8 mg BrdU (Sigma, St Louis, MO) suspended in 0·2 ml phosphate-buffered saline (PBS) were given intraperitoneally once a day for the first 2–3 days of the labelling period to assure a synchronized labelling starting point. Additionally, donor mice received drinking water containing 0·25 mg/ml BrdU for the duration of the labelling period. The concentrations of BrdU and maximal labelling of the immature B-cell compartment in young and old mice were established as previously described.15

Bone marrow from tibias and femurs was harvested from young or old mice, depending on the experiment. Immunoglobulin D-positive (IgD+) mature B cells were depleted using Mini MACS columns (Miltenyi Biotech, Auburn, CA) with mouse anti-mouse IgDa or IgDb–biotin (clones AMS 9.1 and 217-170, respectively; BD Pharmingen, San Diego, CA), followed by anti-biotin microbeads. A sample of the negative-selected cells was immediately stained with phycoerythrin-conjugated (PE) mouse anti-mouse IgMa or IgMb (clones DS-1 and AF6-78, respectively; BD Pharmingen), rat anti-mouse B220-peridinin chlorophyll protein (PerCP; RA3-6B2; Pharmingen), streptavidin-allophycocyanin (SA-APC) to develop the anti-IgD biotin, and mouse anti-BrdU-fluorescein isothiocyanate (FITC) according to the manufacturer’s protocol (BrdU Flow Kit; BD Pharmingen). Fluorescence-activated cell sorting (FACS) analysis was performed to determine that the depletion was efficient and to determine the frequency of immature B cells in the IgDneg fraction. The number of immature (IgM+, IgDneg) BrdU+ B cells was calculated, and each recipient mouse received an intravenous injection of IgDneg bone marrow containing 1·5 × 106 immature B cells in 200 μl PBS. Recipient mice were untreated before injection.

One, 16, or 36 hr post transfer, peripheral blood was obtained by cardiac puncture immediately after killing by CO2 asphyxiation, and lymphocytes were recovered using Lymphocyte Separation Media (Cellgro, Herndon, VA). Spleens, and in some experiments lymph nodes, were harvested and cells were dispersed by gently rubbing the organs between the frosted ends of two glass slides. The cell suspensions were treated with 0·8% ammonium chloride solution and incubated at 37° for 4 min to lyse the red blood cells and were filtered through a 100-μm Nytex membrane (Sefar America, Depew, NY) to remove any large clumps.

Recipient cell suspensions were stained with allotype-specific anti-IgD-biotin/SA-APC and anti-IgM-PE, anti-B220-PerCP and anti-BrdU-FITC (described above) to distinguish between BrdU-labelled donor cells and unlabelled recipient immature B cells. Cells were analysed by flow cytometry (Becton Dickinson, San Jose, CA), and the number of donor B cells was determined by multiplying the frequency of donor cells by the total number of cells from each organ. Consistent with previous reports,28∼ 2–10% of the injected cells were recovered in recipients, regardless of the age of the recipient, at both 1 and 16 hr post-transfer.

Mature splenic B cells: preparation, transfer and detection by flow cytometry

The experimental approach was based on the work of Cyster and colleagues.12 Donor spleen cells were dispersed as described above. These splenic B cells were then purified using a magnetic antibody cell sorting (MACS) B-cell isolation kit (Miltenyi Biotech). Cells were stained with a biotin–antibody cocktail (anti-CD43, anti-CD4 and anti-Ter-119) followed by incubation with anti-biotin microbeads. Non-B cells were depleted using Mini MACS LS columns according to the manufacturer’s protocol (Miltenyi Biotech). Purity of the B-cell fraction was determined by staining with rat anti-mouse B220 FITC (RA3-6B2; Southern Biotech, Birmingham, AL) and anti-allotype IgM biotin followed by SA-PerCP and analysed by flow cytometry. Mature, B220+ IgM+ IgD+ follicular B cells comprised > 95% of the isolated fraction. Each recipient mouse received an intravenous injection of 20 × 106 isolated splenic B cells in 200 μl PBS. Recipient mice were untreated before injection.

Spleens and bone marrow from recipient mice were harvested 6 hr post-transfer. For most adoptive transfer experiments, immunoglobulin allotype differences were used to track donor B-cell migration in an intact host animal. One-third of the spleen was frozen in Tissue-Tek® O.C.T. freezing medium (Sakura, Torrance, CA) and stored at −70° for cryosectioning and immunohistochemistry staining for the donor immunoglobulin allotype. Bone marrow and the remaining two-thirds of each spleen tissue was processed for flow cytometry to determine the frequency of donor B cells (Becton Dickinson). To identify mature follicular-type donor B cells, recipient bone marrow and spleen lymphocyte cell suspensions were stained with anti-B220-FITC and anti-allotype IgM biotin or anti-allotype IgD biotin, followed by an incubation with SA-PerCP. Because only a portion of the spleen was dispersed for flow cytometry, data are reported as frequencies rather than as absolute numbers of cells per spleen. We have previously found that the numbers of mature B cells/spleen are not significantly different between young and old mice, although the total splenic cellularity is slightly greater in aged mice.15 In some experiments, donor MZ B cells in spleens were analysed by flow cytometry; recipient cell suspensions were stained with anti-B220-FITC, rat anti-mouse CD21/CD35-PE (8D9; eBioscience, San Diego, CA), rat anti-mouse CD23 PE-Cy7 (B3B4; eBioscience), and rat anti-mouse IgM PE-Cy5.5 (II/41; eBioscience), modified from Won et al.29

Detection of donor B cells in spleen sections

Frozen spleen tissue was cryosectioned (6–8 μm) and placed onto Superfrost®/Plus microscope slides (Fisher Scientific, Pittsburgh, PA). For immunohistochemistry, tissue sections were treated with 3% hydrogen peroxide, mouse serum, and avidin and biotin solutions from the HRP-DAB Mouse Tissue Staining Kit (R&D Systems, Minneapolis, MN). Sections were then stained with a cocktail of biotinylated mouse anti-mouse IgMa or -b and anti-IgDa or –b (10 μg/ml each; BD Pharmingen) to enhance the detection of donor B cells. Positive cells were revealed using the Elite ABC Peroxidase kit (Vector Labs, Burlingame, CA) with ImmPACT DAB (Vector Labs) and counterstained with Harris Hematoxylin (Surgipath Medical Industry, Richmond, IL). Slides were mounted with VectaMount (Vector Labs) and viewed and photographed using a Leitz Diaplan microscope (W. Nuhsbaum, Inc., McHenry, IL). Images were merged using Adobe Photoshop CS3 Extended (San Jose, CA) to create a complete cross-section of the spleen. Each donor cell was colour-coded as either follicular or extra-follicular before counting. Donor cells were counted from at least two non-adjacent cross-sections.

Use of CFSE to track migration of mature splenic B cells

To control for allotypic differences between strains, some experiments used carboxyfluorescein succinimidyl ester (CFSE) to label the donor mature BALB/c B cells before injection into young or aged BALB/c recipient mice. Mature B cells were purified as described above and then labelled with 1 μm CFSE (Molecular Probes, Carlsbad, CA), according to the manufacturer’s protocol. Labelled cells were transferred intravenously to BALB/c recipient mice. After 6 hr, each recipient spleen was harvested and two-thirds of it was stained for flow cytometry and one-third was snap frozen. Cryosections were processed as described above, except that sections were cut at a thickness of 20 μm as described by others.30 To identify the outer boundary of the follicles, sections were also stained with rat anti-mouse MAdCAM-1 (MECA-367; BD Pharmingen) antibody (1 : 50), which marks the cells lining the marginal sinus of the spleen,13 and visualized with 5 μg/ml goat anti-rat IgG Alexa Fluor 633® (Molecular Probes). Slides were mounted with fluorescent mounting medium (Dako, Carpinteria, CA) and viewed using a Zeiss LSM 510 confocal microscope (Carl Zeiss, Inc., Jena, Germany) that was programmed to compile photos into a complete cross-section.

Expression and localization of CXCL13

To quantify expression of CXCL13 mRNA using real-time reverse transcription polymerase chain reaction (RT-PCR), RNA was isolated from whole spleen, splenic stroma or bone marrow using the Qiagen RNeasy Kit (Valencia, CA) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized using the Amersham First Strand cDNA synthesis kit (Buckinghamshire, UK) and real-time PCR analysis was performed using a GeneAmp 2400 thermal cycler (PerkinElmer, Wellesley, MA) as described previously.31 Real-time oligonucleotides were designed using Primer Express software (Applied Biosystems, Foster City, CA) and synthesized by GibcoBRL-Invitrogen (Carlsbad, CA). Primers were as follows: CXCL13: forward 5′-AATGAGGCTCAGCACAGCAA-3′ and reverse 5′-GTAACCATTTGGCACGAGGATT-3′; hypoxanthine phosphoribosyltransferase (HPRT): forward 5′-AGCAGTACAGCCCCAAAATGG-3′ and reverse 5′-TGCGCTCATCTTAGGCTTTGT-3′. The real-time PCR thermal cycler profile was run as follows: one cycle at 50° for 2 min, one cycle at 95° for 10 min, 40 cycles of denaturing at 95° for 15 seconds, and annealing and elongation at 60° for 1 min, followed by a dissociation protocol run to test the melting temperature of the product. The data were analysed using GeneAmp 5700 SDS software (Applied Biosystems). The number of copies of transcript/μl were determined by generating an HPRT standard curve with each PCR run, using 10-fold serial dilutions ranging from 1 × 10 to 1 × 105 copies/μl of plasmids. Samples without cDNA acted as negative controls. Relative expression of CXCL13 was compared by determining the ratios of copies/μl of the transcript of CXCL13 to copies/μl of HPRT in individual samples.

An enzyme-linked immunosorbent assay (ELISA) was used to detect CXCL13 in splenic total protein extracts. To extract protein, snap-frozen portions of spleen were homogenized in protease inhibitor cocktail (Roche, Indianapolis, IN) followed by a short sonication step, centrifuged and filtered through a 1·2-μm syringe filter (Pall Life Sciences, Ann Arbor, MI). Samples were quantified using the BioRad Protein Assay Reagent, according to the manufacturer’s instructions (Hercules, CA). The CXCL13 ELISA was modified from previous descriptions.31 Plates were coated with 2 μg/ml rat anti-mouse CXCL13 (143614; R&D Systems) and biotin-conjugated goat anti-mouse CXCL13 antibody (R&D Systems) was used at 1 μg/ml for detection and developed with SA-conjugated alkaline phosphatase (Southern Biotech) and p-nitrophenyl phosphate (Sigma-Aldrich, St Louis, MO). Twofold serial dilutions of rCXCL13 (R&D Systems) were used for standard curves included on every test plate at a range of 0·01–50 ng/ml. Samples were analysed by quantifying values within the linear range of the standard curve.

Immunocytochemistry for CXCL13 was performed on sections of spleen tissue (6–8 μm) from 2- and 22-month-old mice that were fixed in acetone for 5 min, washed in PBS, and air-dried for 10 min. Slides were then blocked with 5% normal donkey serum (Jackson ImmunoLabs, West Grove, PA) and stained with 15 μg/ml goat anti-mouse CXCL13 (R&D Systems). After washing, Alexa 555-labelled donkey anti-goat IgG (Molecular Probes) was then added at 10 μg/ml for 1 hr at room temperature. Slides were mounted and viewed using the confocal microscope as described above.

Expression of CXCL12 (stromal cell-derived factor-1)

Total RNA from bone marrow was extracted and cDNA was prepared as described above. Primers were as follows: CXCL12 (stromal cell-derived factor-1α; SDF-1α): forward 5′-GGTTTGCCAGCATAAAGACACT-3′ and reverse 5′-CCTGCAAAGCCACCGTCTATA-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward: 5′-GTGAGGCCGGTGCTGAGTAT-3′ and reverse 5′-TCATGAGCCCTTCCACAATG-3′. Plasmids containing CXCL12 or GAPDH PCR products were used to generate a standard curve to calculate the number of copies of transcript/μl. Relative CXCL12 mRNA expression levels for each sample were calculated by determining the ratio of the number of copies/μl of CXCL12 to the numbers of copies/μl of GAPDH.

Statistical analysis

Statistical differences between transfer pairs were determined by unpaired (donor cells from old or young mice into young recipients) or paired (donor cells from young mice into young or old recipients) t-tests. A linear regression analysis was performed to assess differences in regression lines between the frequency of donor cell migration to the spleen and bone marrow within young versus old mice (GraphPad Prism software, La Jolla, CA). P< 0·05 was considered significant.

Results

Migration of donor immature B cells to spleens of aged mice

Previous kinetic analysis showed that new immature B cells are made in the bone marrow of aged mice, but few of these newly made B cells are subsequently detected in the spleen.15 Here we tested the possibility that migration of immature B cells into the spleen is disrupted in aged mice. First, the ability of the aged splenic environment of old mice to support the migration of immature B cells was examined. Young C.B-17 (IgHb) mice were given BrdU to label newly made B cells. Bone marrow cells containing equal numbers of BrdU+ immature B cells (depleted of mature B cells) were transferred into young or old recipient BALB/c (IgHa) mice. Cell recoveries averaged ∼ 2–10% of that injected, regardless of the recipient age or time-point post-transfer, similar to the low recovery of donor cells post-transfer in intact mice observed by Allman’s group.28 If the splenic microenvironment is defective in producing the appropriate migration signals, then fewer young donor cells should be recovered from the spleens of the old recipient mice. Figure 1(a) illustrates that approximately half as many young donor immature B cells were recovered from the spleens of the old recipient mice (black bars) when compared with the number recovered from the young donor into young recipients (open bars) at 1 and 16 hr post-transfer. Greater numbers of young donor immature B cells remained in the blood of the old recipient mice at the early time-point, and by 16 hr, few donor immature B cells could be detected in the blood of old recipients (Fig. 1b), although a relative increase was not detected in the spleen. These data suggested that the aged splenic microenvironment was less competent than the young microenvironment in attracting new immature B cells and that this deficiency was not relieved over time.

Figure 1.

Figure 1

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Extrinsic and intrinsic influences on migration of immature B cells: 1·5 × 106 immature bone marrow B cells were transferred intravenously to recipient mice. Recipient spleens (a) and peripheral blood (b) were harvested after 1 or 16 hr and the number of donor immature B cells (allotype-specific IgM+ B220lo BrdU+) was determined by flow cytometry. The young into young transfer data (open bars; n ≥ 8 mice) consist of both BALB/c donors into C.B-17 recipients and C.B-17 donors into BALB/c recipients (data pooled; no strain differences detected). Young C.B-17 donor cells were transferred into old BALB/c recipients (black bars; n ≥ 6 mice). Donor cells from old BALB/c mice were injected into young C.B-17 recipients (striped bars; n ≥ 5 mice). Data represent the averages of individual animals ± SEM obtained in two or more independent experiments. *P= 0·006, **P= 0·05, ***P= 0·016, comparing young recipients with old recipients as determined by a paired t-test.

Intrinsic ability of old versus young immature B cells to migrate to the spleen

If advanced age affects the intrinsic ability of immature B cells to migrate to the spleen, then fewer donor immature B cells from aged mice should be recovered from the spleens of young recipient mice. Equal numbers of BrdU-labelled immature B cells from the marrows of either 2- or 22-month old BALB/c mice were injected into young recipient C.B-17 mice. Figure 1(a,b) shows that similar numbers of donor immature B cells from young (open bars) and old (striped bars) donors were recovered at both the 1-hr and 16-hr time-points from recipient spleens and peripheral blood, implying that immature B cells from young and old mice were equally able to exit the blood and enter the spleen.

To determine if the newly made immature B cells that did migrate to the spleens of aged mice were able to undergo further maturation, we assessed a later time-point (36 hr) after transfer of immature B cells from old or young mice into young recipients. Other investigators have shown that immature B cells (B220lo; transitional ‘T1’) migrate from the bone marrow exclusively to the spleen and are excluded from the follicles and lymph nodes until further maturation into recirculating B cells (B220hi; ‘T2/T3’ and mature B cells).19–21Figure 2(a–c) shows that at 36 hr some of the immature donor B cells from old mice, as well as from young mice, had matured to B220hi expression and were detected in spleen, blood and lymph nodes. Presence of donor B220hi BrdU+ B cells in the lymph nodes indicated that the donor immature cells had continued to mature and acquired the ability to recirculate (Fig. 2c). Although a fraction of the aged immature B cells did mature, fewer B220hi B cells from old donors appeared in the spleen and lymph nodes (Fig. 2a,c) and a higher proportion tended to be in the blood (Fig. 2b). These results suggested that newly made, recently matured B cells from aged mice could have an intrinsic inability to recirculate.

Figure 2.

Figure 2

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Newly made B cells from aged mice mature in young recipients but do not recirculate well to the lymph nodes. Transfers were performed as described in Fig. 1 (open and striped bars). Recipient spleens (a), peripheral blood (b), and lymph nodes (c) were harvested 36 hr post-transfer and the number of donor immature B cells (allotype-specific IgM+ B220lo or hi BrdU+) was determined by flow cytometry. Open bars indicate the number of donor B220lo cells; striped bars indicate the number of donor B220hi cells. Data represent the averages ± SEM of n = 3 to n = 6 young into young and n = 4 to n = 6 old into young transfer pairs obtained from two or more independent experiments. *P= 0·003 as determined by unpaired t-test.

Taken together, the migration of immature B cells appears to be highly influenced by the age of the splenic microenvironment. While immature B cells from old mice appear to be intrinsically competent in their ability to enter and remain in the spleen, there is some indication of intrinsic deficiency as they mature or they did not mature appropriately.

The intrinsic ability of mature B cells from old versus young mice to migrate to splenic follicles of young recipients

We next determined whether mature B cells from aged mice have intrinsic defects in migration. The experimental approach was based on those of Cyster and colleagues, who showed that migration of mature donor B cells into the spleen and homing to follicles is dependent on CXCL13 and occurs within 6 hr of transfer.12 To determine the effect of age on the donor lymphocytes, 20 × 106 donor B cells isolated from the spleens of aged or young BALB/c (IgHa) mice were transferred intravenously to young C.B-17 (IgHb) mice. Six hours post-transfer, the recipient mice were killed, and two-thirds of each spleen was used to determine the frequency of donor B cells via flow cytometry using donor IgM allotype and B220+ phenotype. The other one-third of the spleen was snap frozen for immunocytochemistry. Figure 3(a) shows that mature B cells transferred from young donors to young recipient mice (open squares) comprised ∼ 1·7% of the splenic lymphocyte population after 6 hr compared with ∼ 1·1% of the recipient lymphocyte population that received aged donor B cells (shaded triangles). However, there was not a significant difference in the ability of donor cells from aged mice to home to the spleens of young recipients.

Figure 3.

Figure 3

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Intrinsic and extrinsic influences on migration of mature B cells to the spleen: 20 × 106 splenic mature B cells were transferred intravenously into recipients. Recipient spleens were harvested 6 hr later. Donor cells were tracked by immunoglobulin H (IgH) allotypic differences using BALB/c or C.B-17 donor/recipient pairs. Open squares, young BALB/c into young C.B-17 or young C.B-17 into young BALB/c; shaded triangles, old BALB/c into young C.B-17; black diamonds, young C.B-17 into old BALB/c. (a) The per cent of splenic allotype-specific IgM+ B220+ donor cells in the lymphocyte gate was determined by flow cytometry analysis. Symbols indicate individual recipient mice obtained from two to five independent experiments; the horizontal bar indicates the average of each group. No statistical differences were observed between young or old donors (P= 0·4166 as determined by unpaired t-test) or between young or old recipients (P= 0·1337 as determined by paired t-test). (b) Spleens from recipient mice were cryosectioned and stained to detect donor allotypic IgM+ IgD+ B cells (dots). The dark dots are immunoperoxidase deposits marking individual donor migrant cells in and around the follicles. The larger panel shows lower magnification of a young recipient spleen; the right-hand smaller panels represent examples of donor B cells that migrated to follicles of the indicated recipients. Sections were counterstained with haematoxylin to visualize architectural morphology. (c) Donor cells detected in the sections of each spleen were scored for localization inside or outside B-cell follicles. Data shown are the averages ± SEM of the percentage of donor cells found in the splenic follicles of young into young (open bar; n = 7), old into young (shaded bars; n = 4), or young into old (black bar; n = 6) transfer pairs. Totals of 600–5000 cells were counted/spleen section; two non-adjacent sections were scored for each spleen. No statistical differences were calculated between any of the transfer groups. (d) The per cent of donor B cells that are marginal zone B cells (B220+ CD23neg CD21hi allotype-specific IgMhi) was detected by flow cytometry analysis. Symbols indicate individual recipient mice (n = 7 young mice, n = 6 old mice); the horizontal bar indicates the average of each group. Marginal zone B cells were phenotyped in three of the independent experiments shown in (a). *P= 0·03 as determined by paired t-test.

To compare the homing of donor B cells into the follicles of the spleens from recipient mice, immunohistochemical analysis for expression of the donor IgH allotype was performed on frozen sections. Individual donor cells were scored for localization within or outside follicles (Fig. 3b,c). Analysis of young recipient spleens showed a relatively consistent proportion (58%, open bar) of young donor B cells within the follicles 6 hr after injection, and this value did not change significantly when the donor B cells were isolated from aged mice (53%, shaded bar). Donor cells from both young and aged mice appeared randomly distributed throughout the follicles (Fig. 3b). Hence, advanced age did not appear to affect the intrinsic ability of a mature B cell to migrate to the spleen and home to follicles in a young environment.

Next we assessed migration of mature B cells in the aged splenic microenvironment (Fig. 3a–c, black diamonds/bar). CXCL13 was of special interest because it is known to play an integral role in the maintenance of B-cell organization in the spleen.12–14

The impact of the aged splenic environment on B-cell migration

The vast majority of mature B cells are follicular and the chemokine CXCL13 is essential for the recruitment of B cells into the follicular areas so we first determined the status of CXCL13 expression in the spleens of aged mice. Spleens from aged mice were compared with those from young mice for CXCL13 message, protein and localization in the tissue. Figure 4(a) shows that on average CXCL13 mRNA levels were similar in old versus young spleens. This was observed even if the splenic stroma was enriched by depleting the lymphocytes (data not shown). Splenic CXCL13 protein levels, analysed by ELISA, proved to be significantly greater in aged mice than in young mice (Fig. 4b). To compare the localization of CXCL13 protein, splenic tissue sections from old and young mice were stained with anti-CXCL13 antibodies. As expected from numerous reports using young mice, CXCL13 staining was localized in an evenly distributed network, shown to be associated with follicular dendritic cells.2 Also as reported in young mice (Fig. 4c, left), CXCL13 was expressed by the ring of MAdCAM-1+ sinus-lining cells, which have also been referred to as marginal reticular cells (MRC) that encircle the follicle and appear to line the follicle/marginal sinus border.2,32 In splenic sections from aged mice (Fig. 4c, right), CXCL13 was localized to the follicles and the distribution was reticular but less even and often concentrated in random patches. More pronounced, CXCL13 was frequently absent or reduced at the perimeter of the follicle in aged tissue (Fig. 4c, right). Specifically, when MAdCAM-1 was used to mark the sinus-lining/MRC, CXCL13 was observed to be absent in those cells (Fig. 4c, right, bottom panels).

Figure 4.

Figure 4

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Elevated CXCL13 protein expression and altered distribution in the splenic follicles of old mice. CXCL13 in spleen was examined by quantitative real-time RT-PCR (a), ELISA (b), and by immunofluoresence (c). (a) Copy number/μl of RNA isolated from spleen was determined for each sample based on a standard curve performed simultaneously. Target messenger RNA levels were calculated by normalizing the number of copies of CXCL13 to the number of copies of HPRT. Symbols represent RNA isolated from individual mice: n = 5 young and n = 5 old spleen samples. (b) Equal concentrations of total protein extracted from the spleens of young and old mice were analysed for CXCL13 protein by ELISA. Data shown are the averages ± SEM for samples from n = 6 young and n = 6 old individual mice. *P= 0·0125 as determined by unpaired t-test. (c) Confocal microscopy was used to reveal CXCL13 (red) distribution in spleen sections from young (left panels) and old (right panels) mice. In young, arrows indicate CXCL13+ marginal reticular cells aligned on the outer rims of the follicles. In the old, arrows mark the reduction of CXCL13 in the outer rim of the follicles (10× magnification). The lower four images (25× magnification) show MAdCAM-1 (blue) marking the boundary of the marginal zone and follicle. Unstained or control stained serial sections from young and old mice did not exhibit non-specific binding of antibodies. Shown are examples of spleens from n = 3 young and n = 2 old mice.

To determine if the changes in CXCL13 distribution impacted migration of mature B cells in aged mice, 20 × 106 mature B cells, isolated from young C.B-17 (IgHb) mice, were transferred intravenously into young or old BALB/c mice (IgHa), which were killed 6 hr post transfer. When mature B cells from young mice were transferred into aged mice, ∼ 1·0% of the lymphocyte population in recipients was donor cells (Fig. 3a, black diamonds), as compared with ∼ 1·7% in young recipients (open squares). Although the average frequency of donor cells detected in the spleens of aged recipients was less than that in young recipients, the decrease was not significant (P= 0·1337). The percentage of donor cells that migrated into the follicles was determined by immunohistochemistry by detecting the expression of the donor IgH allotype. Whether the recipient was young or old, the same percentage of donor B cells migrated into follicles within 6 hr and were randomly distributed (Fig. 3b,c open and black bars), indicating that follicular B cells are able to migrate appropriately within aged spleens.

Additionally, we assessed migration of the small number of MZ B cells contained in the highly purified donor B-cell population. In contrast to follicular B cells, the migration of donor MZ B cells to aged spleens appeared to be significantly reduced in the same recipients (Fig. 3d), P= 0·03. It is unlikely that the mature B cells changed phenotype within this short time post-transfer into an intact host; this is supported by the observations reported by Srivastava et al.28 and Girkontaite et al.33

Consequently, despite the microscopic appearance of disorganization and the subtle changes in CXCL13 distribution in the aged spleen, young follicular B cells were able to migrate to the follicles of an aged spleen in a manner consistent with that observed in a young spleen. However, migration of MZ B cells appeared to be more affected by the aged microenvironment.

Greater frequency of mature B cells migrating to the bone marrow in aged recipient mice

Previous data have shown that the number of mature B cells (follicular phenotype) in the bone marrow becomes greater with increased age.15 It was therefore of interest to compare migration of donor mature B cells from young donors to the bone marrow of old and young recipient mice (Fig. 5a). The marrow lymphocyte-gated population in recipient young mice contained ∼ 0·36% donor B cells 6 hr after transfer (Fig. 5a). In contrast, a significant increase in migration to the marrow of aged mice was observed because ∼ 0·72% of the lymphocyte population was donor B cells (Fig. 5a, P= 0·0199). The increase in donor cells detected in the aged bone marrow is not likely to be the result of blood contamination, because the frequency of donor cells identified in the blood did not differ with the age of the recipient (young recipients = 0·39% ± 0·1; aged recipients = 0·33%± 0·09; P =0·8931). The apparent increase in migration to the bone marrow in aged mice was even more profound when the frequency of donor cells migrating to the spleen was plotted against the frequency migrating to bone marrow in individual mice (Fig. 5b). The slopes of the best-fit lines are statistically different (P < 0·0001), inferring that the aged bone marrow has improved capacity to receive recirculating mature B cells, but not at the expense of migration to the spleen (Fig. 3a). Variations of CXL13 message levels in the bone marrow were not responsible for these homing patterns (Fig. 5c) and were ∼ 100-fold less than that detected in the spleen of young or old mice. However, CXCL12 mRNA message was much greater in the marrow than CXCL13, and was significantly increased in the marrow of aged mice compared with young mice. This age-related difference in CXCL12 in the marrow may account, in part, for the increased migration observed in the aged recipients (Fig. 5c).

Figure 5.

Figure 5

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B-cell homing to the bone marrow is altered in aged recipients. (a) Bone marrow from recipient mice shown in Fig. 3 was harvested and the per cent of IgMb+ B220+ donor cells in the lymphocyte gate was determined by flow cytometry analysis. Symbols indicate individual recipient mice (n = 12 young, n = 10 old) from five independent experiments; the horizontal bar indicates the average of each group. *P= 0·0199 as determined by paired t-test. (b) Scatterplot for per cent of donor cells detected in the spleen versus per cent of donor cells detected in bone marrow cells of individual mice in (a), for young recipient mice (r= 0·83; P= 0·0015) and for old recipient mice (r= 0·92; P< 0·0001). *P < 0·0001 as determined by linear regression analysis. (c) CXCL13 and CXCL12 messenger RNA expression in the bone marrow was examined by quantitative real-time polymerase chain reaction. Data are shown as the fold-difference in copy number/μl of RNA as compared with levels of CXCL13 detected in the bone marrow of young mice. Copy number/μl was determined for each sample based on a standard curve performed simultaneously. Target messenger RNA levels were calculated by normalizing the number of copies of CXCL13 or CXCL12 to the number of copies of HPRT or GAPDH.

In summary, these data show that the aged splenic microenvironment has a large impact on the migration of immature B cells and MZ B cells, but not follicular B cells. Although longer-lived than B cells from young mice, mature B cells from old mice do not lose their intrinsic ability to migrate to the spleen or home to the follicles.

Discussion

In this study, we took a comprehensive approach to address the question of whether or not B-cell migration is impacted in aged animals. We examined both immature and mature B cells, as well as young versus old recipient microenvironments. Immature and mature follicular B cells from aged mice may be intrinsically different from their young counterparts in function,26,34 but the short-term transfer experiments shown here suggest that B cells from aged mice are intrinsically able to migrate and home just as efficiently as those from young mice. Rather, the aged tissue microenvironment is responsible for reduced migration of certain B-cell populations, with a greater impact on newly made immature B cells and MZ B cells. These populations enter the MZ region and remain for a period of time.20,21 Of note, CXCL13 was reduced in the peripheral areas of aged follicles, near the MZ. However, the vast majority of B cells appear unaffected by the age of the tissue environment in their ability to traffic into follicles, indicating that sufficient follicular CXCL13 is available in aged mice.

Alterations in peripheral B-cell homeostasis with advanced age have been suggested by several groups.15,16,35–37 Kinetics of turnover rates show that mature B cells from aged mice have a far greater half-life than B cells from young mice. Although the physiological mechanism by which an increased cell lifespan would affect B-cell function is not known, we and others have speculated that the decline in B-cell function with age is related to the failure of the compartment to replenish with newly made cells every 6–8 weeks. The first objective of the present study was examination of immature B-cell migration as one potential mechanism for the changes in the kinetics of peripheral B-cell homeostasis. The data indicate that although immature bone marrow B cells from old mice remain functional in their ability to enter the splenic environment, the spleen itself cannot support this migration as efficiently in old mice as compared with young mice. There are two interpretations of these data and how they relate to the increased lifespan of mature splenic B cells. One theory is that the production of normal migratory cues is altered with age, and newly produced immature B cells released into the blood may not receive the appropriate signals to transverse the sinus walls and enter the parenchyma of the spleen. As a consequence, the absence of competitive pressure from new incoming B cells allows the maintenance of resident mature B cells in survival niches. Their average lifespan is then, by default, increased. An alternative explanation is that the resident mature B cells in old mice are pre-programmed to be long-lived. For example, there may be a subset of B cells produced during the life of an animal that are intrinsically stronger. These cells are more competitive and hence long-lived. This subset may be small, but accumulates over the lifetime of a mouse until, by 2 years of age, the mature B-cell compartment is dominated by these cells. The increased longevity may signal to the newly made cells that the niche is full and requires no more cells. The competitive pressure is, in effect, reversed. If the immature B cells are indeed excluded from the spleen, then where do these immature B cells go? The answer is not clear, but it is unlikely that they would traffic back to the bone marrow because the low surface CXCR4 expression on immature B cells should prevent attraction to the CXCL12-rich marrow.38 Immature B cells are also prevented from entering into the lymph nodes until they have differentiated into cells with a mature phenotype.20 A caveat to the adoptive transfer approach is that by injecting donor immature B cells directly into the blood, the mechanism involved in the exit of B cells from the bone marrow is bypassed. However, earlier kinetic data show that the immature B-cell population in old mice is a dynamic population, with a constant turnover rate.15 Unless newly made immature B cells die in the bone marrow as rapidly as they are made, one can assume that immature B cells are being exported. A final possibility is that the immature B cells are failing to be retained in/attracted to the spleen because of space constraints. Artificially provided space as a result of light irradiation could directly test this idea. However, the data might be difficult to interpret, especially in an aged animal where even light irradiation could have direct effects beyond providing space in the spleen, such as the release of pro-inflammatory cytokines.

The lymphoid architecture is disrupted in aged rodents.5–7,39 Splenic tissue in a majority of aged mice is somewhat reminiscent of the altered architecture of the CXCL13 chemokine knockout mice.12,14 CXCL13 is necessary for the maintenance of proper B-cell compartment organization of secondary lymphoid tissue. Notably, in aged mice, the B-cell areas appear most affected. Therefore, it was possible that altered production of or response to CXCL13 could affect the efficient recirculation and homing of mature B cells in aged follicles. We determined the status of CXCL13 in aged mice and found that although the production of CXCL13 appeared even greater in spleens from aged mice compared with those of young, the chemokine was less evenly distributed in follicles and was reduced at the periphery of most follicles. However, these differences did not alter the migration of follicular B cells. Overall, the migration experiments suggest that homing of ‘follicular’ phenotype mature B cells to the follicles of aged spleens and the intrinsic homing ability of aged B cells appear to be intact. An unexpected observation was the reduction of migration of donor MZ B cells into the aged environment. This finding, together with the observed reduction of immature B-cell migration, suggests that the marginal zones of the aged spleen deserve further study.

A conspicuous reduction of CXCL13 in the cells at the marginal sinus/follicular border was observed in spleens from aged mice. These cells most likely represent the ‘marginal reticular cells’ described by Katakai et al.,32 which are identical to the MAdCAM-1+ MZ sinus lining cells. Because the MRC rely on stimulation through lymphotoxin-β receptor, the age-related reduction of CXCL13 expression may be part of a breakdown in the B-cell/stromal cell interaction that maintains the architecture of the B-cell areas. An interesting report also showed that lack of expression of S1P3 by marginal sinus MAdCAM-1+ MZ sinus lining cells (but not by B cells) yielded disrupted MZ architecture, although migration of donor MZ B cells was not reduced in the S1P3−/− model.33 Alternatively, it is possible that these splenic reticular stromal cells become senescent and suffer changes in chemokine/cytokine expression, which has been reported in other tissues.40

An additional observation of our studies was that a greater proportion of mature donor B cells homed to the bone marrow of aged mice than in young recipient mice. Based on estimates of average total body bone marrow cellularity in young and old mice,15,41 we calculate from our studies here that for every 100 donor B cells that migrated to a young spleen, 7·9 donor cells migrated to the bone marrow. In comparison, for every 100 donor B cells that migrated to an aged spleen, 22 donor cells migrated to the bone marrow. In some regards, this may be surprising because the aged bone marrow already has increased numbers of mature B cells and yet is able to accept new migrants. Recent studies reveal that mature B cells in the bone marrow form an anatomically and functionally unique subset.42,43 Mature B cells appear localized within perivascular clusters that depend on the presence of bone marrow dendritic cells and their production of migration-inhibitory factor.43 Other studies have shown that the accumulation of mature B cells in the bone marrow of young mice is regulated by CXCL12,44 expression of which appears to increase with age (Fig. 5). Although the mature B cells found in the bone marrow have a follicular phenotype and recirculate,43 they respond to blood-borne antigens in a manner similar to MZ B cells.42 Why is there a greater homing and accumulation of mature B cells in the aged marrow? It is possible that it has evolved as a compensatory mechanism to enhance T-cell-independent B-cell responses. Alternatively, a separate process in the bone marrow, such as increased bone marrow dendritic cells or senescence of the stromal cells, resulting in an increased secretion of migration-inhibitory factor and CXCL12, could cause accumulation of mature B cells as a bystander effect. Finally, the greater number of mature B cells migrating to the bone marrow in the aged animal may also imply that there are fewer B cells homing to secondary sites other than the spleen.

In conclusion, mature and immature B cells from old mice maintain their ability to migrate to the spleens of young mice, whereas the splenic microenvironment of old mice is inefficient at receiving the new immature immigrants. The result is a population of mature B cells with a prolonged lifespan. However, the vast majority of these long-lived B cells (follicular phenotype) retain their intrinsic capacity for migration into follicles.

Acknowledgments

We thank Linda Fox for assistance with confocal microscopy, Dr Jerry Cleland for statistical help, and Nicole Ziegler for critical reading of the manuscript. Shubin Zhang and Anand Ramadorai provided excellent technical assistance. We also thank Patricia Simms and the Loyola FACS Core Facility for assistance with flow cytometry. This work was supported by National Institutes of Health Grants R01AG13874 and T32AG031780 (P.L.W.) and the Columbia College Faculty Development Grant (H.A.M.W.).

Glossary

Abbreviations:

APC

allophycocyanin

BrdU

bromodeoxyuridine

cDNA

complementary DNA

CFSE

carboxyfluorescein succinimidyl ester

ELISA

enzyme-linked immunosorbent assay

FACS

fluorescence-activated cell sorting

FITC

fluorescein isothiocyanate

GAPDH

glyceraldehyde 3-phosphate-dehydrogenase

HPRT

hypoxanthine phosphoribosyltransferase

IgD

immunoglobulin D

MRC

marginal reticular cells

mRNA

messenger RNA

MZ

marginal zone

PBS

phosphate-buffered saline

PE

phycoerythrin

PerCP

peridinin chlorophyll protein

RT-PCR

reverse transcription–polymerase chain reaction

SA

streptavidin

SDF

stromal cell-derived factor-1α

Disclosures

The authors have no financial conflict of interest.

References

  • 1.Dorshkind K, Montecino-Rodriguez E, Signer RA. The ageing immune system: is it ever too old to become young again? Nat Rev Immunol. 2009;9:57–62. doi: 10.1038/nri2471. [DOI] [PubMed] [Google Scholar]
  • 2.Allen CD, Cyster JG. Follicular dendritic cell networks of primary follicles and germinal centers: phenotype and function. Semin Immunol. 2008;20:14–25. doi: 10.1016/j.smim.2007.12.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Campbell DJ, Kim CH, Butcher EC. Chemokines in the systemic organization of immunity. Immunol Rev. 2003;195:58–71. doi: 10.1034/j.1600-065x.2003.00067.x. [DOI] [PubMed] [Google Scholar]
  • 4.Ohl L, Bernhardt G, Pabst O, Forster R. Chemokines as organizers of primary and secondary lymphoid organs. Semin Immunol. 2003;15:249–55. doi: 10.1016/j.smim.2003.08.003. [DOI] [PubMed] [Google Scholar]
  • 5.Cheung HT, Nadakavukaren MJ. Age-dependent changes in the cellularity and ultrastructure of the spleen of Fischer F344 rats. Mech Ageing Dev. 1983;22:23–33. doi: 10.1016/0047-6374(83)90004-0. [DOI] [PubMed] [Google Scholar]
  • 6.Eaton-Bassiri AS, Mandik-Nayak L, Seo SJ, Madaio MP, Cancro MP, Erikson J. Alterations in splenic architecture and the localization of anti-double-stranded DNA B cells in aged mice. Int Immunol. 2000;12:915–26. doi: 10.1093/intimm/12.6.915. [DOI] [PubMed] [Google Scholar]
  • 7.Holmes KL, Schnizlein CT, Perkins EH, Tew JG. The effect of age on antigen retention in lymphoid follicles and in collagenous tissue of mice. Mech Ageing Dev. 1984;25:243–55. doi: 10.1016/0047-6374(84)90145-3. [DOI] [PubMed] [Google Scholar]
  • 8.Sato H, Dobashi M. The distribution, immune complex trapping ability and morphology of follicular dendritic cells in popliteal lymph nodes of aged rats. Histol Histopathol. 1998;13:99–108. doi: 10.14670/HH-13.99. [DOI] [PubMed] [Google Scholar]
  • 9.Szakal AK, Aydar Y, Balogh P, Tew JG. Molecular interactions of FDCs with B cells in aging. Semin Immunol. 2002;14:267–74. doi: 10.1016/s1044-5323(02)00059-3. [DOI] [PubMed] [Google Scholar]
  • 10.Szakal AK, Taylor JK, Smith JP, Kosco MH, Burton GF, Tew JJ. Kinetics of germinal center development in lymph nodes of young and aging immune mice. Anat Rec. 1990;227:475–85. doi: 10.1002/ar.1092270411. [DOI] [PubMed] [Google Scholar]
  • 11.Zheng B, Han S, Takahashi Y, Kelsoe G. Immunosenescence and germinal center reaction. Immunol Rev. 1997;160:63–77. doi: 10.1111/j.1600-065x.1997.tb01028.x. [DOI] [PubMed] [Google Scholar]
  • 12.Ansel KM, Ngo VN, Hyman PL, et al. A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature. 2000;406:309–14. doi: 10.1038/35018581. [DOI] [PubMed] [Google Scholar]
  • 13.Cinamon G, Zachariah MA, Lam OM, Foss FW, Jr, Cyster JG. Follicular shuttling of marginal zone B cells facilitates antigen transport. Nat Immunol. 2008;9:54–62. doi: 10.1038/ni1542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Gunn MD, Kyuwa S, Tam C, Kakiuchi T, Matsuzawa A, Williams LT, Nakano H. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization. J Exp Med. 1999;189:451–60. doi: 10.1084/jem.189.3.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Johnson KM, Owen K, Witte PL. Aging and developmental transitions in the B cell lineage. Int Immunol. 2002;14:1313–23. doi: 10.1093/intimm/dxf092. [DOI] [PubMed] [Google Scholar]
  • 16.Kline GH, Hayden TA, Klinman NR. B cell maintenance in aged mice reflects both increased B cell longevity and decreased B cell generation. J Immunol. 1999;162:3342–9. [PubMed] [Google Scholar]
  • 17.Labrie JE, III, Sah AP, Allman DM, Cancro MP, Gerstein RM. Bone marrow microenvironmental changes underlie reduced RAG-mediated recombination and B cell generation in aged mice. J Exp Med. 2004;200:411–23. doi: 10.1084/jem.20040845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Shahaf G, Johnson K, Mehr R. B cell development in aging mice: lessons from mathematical modeling. Int Immunol. 2006;18:31–9. doi: 10.1093/intimm/dxh346. [DOI] [PubMed] [Google Scholar]
  • 19.Allman D, Lindsley RC, DeMuth W, Rudd K, Shinton SA, Hardy RR. Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation. J Immunol. 2001;167:6834–40. doi: 10.4049/jimmunol.167.12.6834. [DOI] [PubMed] [Google Scholar]
  • 20.Loder F, Mutschler B, Ray RJ, Paige CJ, Sideras P, Torres R, Lamers MC, Carsetti R. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J Exp Med. 1999;190:75–89. doi: 10.1084/jem.190.1.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Lortan JE, Roobottom CA, Oldfield S, MacLennan IC. Newly produced virgin B cells migrate to secondary lymphoid organs but their capacity to enter follicles is restricted. Eur J Immunol. 1987;17:1311–6. doi: 10.1002/eji.1830170914. [DOI] [PubMed] [Google Scholar]
  • 22.Blaeser A, McGlauchlen K, Vogel LA. Aged B lymphocytes retain their ability to express surface markers but are dysfunctional in their proliferative capability during early activation events. Immun Ageing. 2008;5:15. doi: 10.1186/1742-4933-5-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Eaton SM, Burns EM, Kusser K, Randall TD, Haynes L. Age-related defects in CD4 T cell cognate helper function lead to reductions in humoral responses. J Exp Med. 2004;200:1613–22. doi: 10.1084/jem.20041395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Snow EC. An evaluation of antigen-driven expansion and differentiation of hapten-specific B lymphocytes purified from aged mice. J Immunol. 1987;139:1758–62. [PubMed] [Google Scholar]
  • 25.Yang X, Stedra J, Cerny J. Relative contribution of T and B cells to hypermutation and selection of the antibody repertoire in germinal centers of aged mice. J Exp Med. 1996;183:959–70. doi: 10.1084/jem.183.3.959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Frasca D, Van der Put E, Landin AM, Gong D, Riley RL, Blomberg BB. RNA stability of the E2A-encoded transcription factor E47 is lower in splenic activated B cells from aged mice. J Immunol. 2005;175:6633–44. doi: 10.4049/jimmunol.175.10.6633. [DOI] [PubMed] [Google Scholar]
  • 27.King AM, Van der Put E, Blomberg BB, Riley RL. Accelerated Notch-dependent degradation of E47 proteins in aged B cell precursors is associated with increased ERK MAPK activation. J Immunol. 2007;178:3521–9. doi: 10.4049/jimmunol.178.6.3521. [DOI] [PubMed] [Google Scholar]
  • 28.Srivastava B, Quinn WJ, III, Hazard K, Erikson J, Allman D. Characterization of marginal zone B cell precursors. J Exp Med. 2005;202:1225–34. doi: 10.1084/jem.20051038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Won WJ, Foote JB, Odom MR, Pan J, Kearney JF, Davis RS. Fc receptor homolog 3 is a novel immunoregulatory marker of marginal zone and B1 B cells. J Immunol. 2006;177:6815–23. doi: 10.4049/jimmunol.177.10.6815. [DOI] [PubMed] [Google Scholar]
  • 30.Mueller SN, Hosiawa-Meagher KA, Konieczny BT, Sullivan BM, Bachmann MF, Locksley RM, Ahmed R, Matloubian M. Regulation of homeostatic chemokine expression and cell trafficking during immune responses. Science. 2007;317:670–4. doi: 10.1126/science.1144830. [DOI] [PubMed] [Google Scholar]
  • 31.Minges Wols HA, Ippolito JA, Yu Z, Palmer JL, White FA, Le PT, Witte PL. The effects of microenvironment and internal programming on plasma cell survival. Int Immunol. 2007;19:837–46. doi: 10.1093/intimm/dxm051. [DOI] [PubMed] [Google Scholar]
  • 32.Katakai T, Suto H, Sugai M, et al. Organizer-like reticular stromal cell layer common to adult secondary lymphoid organs. J Immunol. 2008;181:6189–200. doi: 10.4049/jimmunol.181.9.6189. [DOI] [PubMed] [Google Scholar]
  • 33.Girkontaite I, Sakk V, Wagner M, Borggrefe T, Tedford K, Chun J, Fischer KD. The sphingosine-1-phosphate (S1P) lysophospholipid receptor S1P3 regulates MAdCAM-1+ endothelial cells in splenic marginal sinus organization. J Exp Med. 2004;200:1491–501. doi: 10.1084/jem.20041483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Riley RL, Van der Put E, King AM, Frasca D, Blomberg BB. Deficient B lymphopoiesis in murine senescence: potential roles for dysregulation of E2A, Pax-5, and STAT5. Semin Immunol. 2005;17:330–6. doi: 10.1016/j.smim.2005.05.011. [DOI] [PubMed] [Google Scholar]
  • 35.Cancro MP. B cells and aging: gauging the interplay of generative, selective, and homeostatic events. Immunol Rev. 2005;205:48–59. doi: 10.1111/j.0105-2896.2005.00272.x. [DOI] [PubMed] [Google Scholar]
  • 36.Johnson SA, Cambier JC. Ageing, autoimmunity and arthritis: senescence of the B cell compartment – implications for humoral immunity. Arthritis Res Ther. 2004;6:131–9. doi: 10.1186/ar1180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Miller JP, Cancro MP. B cells and aging: balancing the homeostatic equation. Exp Gerontol. 2007;42:396–9. doi: 10.1016/j.exger.2007.01.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Honczarenko M, Douglas RS, Mathias C, Lee B, Ratajczak MZ, Silberstein LE. SDF-1 responsiveness does not correlate with CXCR4 expression levels of developing human bone marrow B cells. Blood. 1999;94:2990–8. [PubMed] [Google Scholar]
  • 39.Pahlavani MA, Richardson A, Cheung HT. Age-dependent changes of the mesenteric lymph node of Fischer F344 rats: morphological and histometric analysis. Mech Ageing Dev. 1987;39:137–46. doi: 10.1016/0047-6374(87)90005-4. [DOI] [PubMed] [Google Scholar]
  • 40.Campisi J, d’Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nat Rev Mol Cell Biol. 2007;8:729–40. doi: 10.1038/nrm2233. [DOI] [PubMed] [Google Scholar]
  • 41.Opstelten D, Osmond DG. Regulation of pre-B cell proliferation in bone marrow: immunofluorescence stathmokinetic studies of cytoplasmic mu chain-bearing cells in anti-IgM-treated mice, hematologically deficient mutant mice and mice given sheep red blood cells. Eur J Immunol. 1985;15:599–605. doi: 10.1002/eji.1830150613. [DOI] [PubMed] [Google Scholar]
  • 42.Cariappa A, Mazo IB, Chase C, et al. Perisinusoidal B cells in the bone marrow participate in T-independent responses to blood-borne microbes. Immunity. 2005;23:397–407. doi: 10.1016/j.immuni.2005.09.004. [DOI] [PubMed] [Google Scholar]
  • 43.Sapoznikov A, Pewzner-Jung Y, Kalchenko V, Krauthgamer R, Shachar I, Jung S. Perivascular clusters of dendritic cells provide critical survival signals to B cells in bone marrow niches. Nat Immunol. 2008;9:388–95. doi: 10.1038/ni1571. [DOI] [PubMed] [Google Scholar]
  • 44.Ueda Y, Yang K, Foster SJ, Kondo M, Kelsoe G. Inflammation controls B lymphopoiesis by regulating chemokine CXCL12 expression. J Exp Med. 2004;199:47–58. doi: 10.1084/jem.20031104. [DOI] [PMC free article] [PubMed] [Google Scholar]

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