| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of
*
Biochemistry and
Genetics, University of Wisconsin, Madison, WI 53706
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Based on our analysis of the B cell-deficient A/WySnJ mice (5, 6, 7, 8), we predicted that a B cell-specific survival factor would provide essential homeostatic controls (7). That factor has now been identified as the TNF family member BAFF (also termed BLyS, TALL-1, zTNF4, and THANK) (9). Patients with the B cell-mediated autoimmune diseases SLE (10) and Sjogren’s syndrome (11) had excessive amounts of BAFF, suggesting a possible cause and effect relationship between increased BAFF and disease. Similarly, mice expressing transgenic BAFF had SLE-like and Sjogren’s-like syndromes (10, 11, 12). In contrast, genetically engineered BAFF-null mice completely lacked FO and marginal zone (MZ) B cells (13, 14). These reports underscored BAFF’s pivotal role as a regulator of B cell homeostasis, and stimulated a search for the receptor that mediates these functions.
There is now compelling evidence that the Bcmd-1 gene we characterized as causing B cell deficiency in A/WySnJ mice encodes a mutant form of the TNFR family member, BAFF-R (also BR3), which mediates BAFF’s homeostatic functions. The A/WySnJ mice and the BAFF-null mice have similar B cell deficiencies, but the A/WySnJ mice have some splenic B cells and make IgM responses to T-dependent and T-independent Ags (5, 6), whereas the BAFF-null mice have no B cells and make no Ab responses (13, 14). We found that the Bcmd-1 gene on mouse chromosome 15 (15, 16) controlled a B cell-intrinsic life span defect in A/WySnJ B-2 B cells, but not B-1 B cells (7, 8). Thompson et. al (17) cloned the Tnfrsf13c gene encoding BAFF-R, mapped it to the human chromosome 22q13.1 region that is syntenic to the Bcmd-1 region of mouse chromosome 15, and found that the A/WySnJ Tnfrsf13c transcript had an aberrant third exon. We and our collaborators showed that the A/WySnJ B cells did not respond to BAFF in vivo (18). Together, the evidence indicates that Tnfrsf13c is Bcmd-1, the A/WySnJ mice have a loss-of-function mutation in Tnfrsf13c, and BAFF-R mediates BAFF’s essential B lymphocyte homeostatic functions.
A major challenge now is to determine the precise nature of the BAFF-R signals, and how those signals regulate peripheral B lymphocyte development (19). Newly formed (NF) B cells transit via the blood from the adult bone marrow to the marginal sinus of the spleen, where they begin a process of selection, maturation, and relocation according to their Ag receptor specificity (20), B cell-specific chemokines (21), and B cell survival signals (7, 22). Surface markers have been used to distinguish three sequential, short-lived, transitional B cell stages, transitional (T) 1, T2, and T3 (23, 24). In BAFF-null mice, splenic B cell development appeared to be blocked at the T1 to T2 transition (13, 14). Moreover, BAFF-mediated signals appeared to support T2 B cell survival (25). These results led to the suggestion that BAFF-R-mediated differentiation signals drive the T1 to T2 transition, and/or the BAFF-R-mediated survival signals maintain the T2 B cells (26). In contrast, we previously suggested that the BAFF-R-mediated survival signals maintain splenic B-2 B cells of all developmental stages, because turnover studies showed that all A/WySnJ splenic B-2 B cells had shortened life spans (7). Additionally, a recent report shows that BAFF-R can signal NF-
B activation in T1 splenic B cells (27). Thus, it is not yet certain whether BAFF-R provides survival signals to all splenic B-2 B cells, or only to B cells from the T2 stage onward, and there are differing views as to whether this receptor transmits differentiation signals driving the T1-T2 transition.
We sought to address these uncertainties concerning BAFF-R function. We cloned and sequenced the previously uncharacterized 3' untranslated region of the A/WySnJ Tnfrsf13c transcript to determine the source of the discordant sequence. We also used the most current method of transitional B cell analysis to compare the T1, T2, and T3 B cells in the spleens from the BAFF-R-mutant A/WySnJ and wild-type A/J strains, to learn whether the Tnfrsf13c mutation blocked a particular transition. Most importantly, we investigated whether provision of a survival signal could complement the Tnfrsf13c mutation and restore B-2 B cell development. The data show that enforced in vivo expression of the bcl-xL survival gene in A/WySnJ cells complemented the Tnfrsf13c mutation and restored B-2 B cell development. These data are discussed in the context of a model for BAFF-R homeostatic function.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Male and female A/J and A/WySnJ were from our specific pathogen-free mouse colony in the Department of Biochemistry, University of Wisconsin. The mice were maintained at 23°C with 40–60% humidity and 12-h light-dark cycles and used at age 7–10 wk. The protocols were approved by the Institutional Animal Care and Use Committee (protocol A00847-4-08-99).
Immunofluorescence analysis of spleen sections
Frozen sections were prepared, stained, and analyzed, as described (28). In brief, spleens embedded in OCT compound (Lab-Tek Products, Division of Miles Laboratories, Naperville, IL) were flash frozen, sectioned, air dried, acetone fixed, and blocked with normal horse serum. The splenic metallophilic macrophages were stained with rat Abs to MOMA-1, and developed with biotinylated goat Ab to rat IgG, followed by streptavidin 7-amino-4-methylcoumarin-3-acetic acid (Vector Laboratories, Burlingame, CA). The sections were washed and then stained with FITC rat Ab to mouse CD5 (clone 53-7.3; BD PharMingen, San Diego, CA) and rhodamine isothiocyanate-coupled goat Ab to mouse IgM (Southern Biotechnology Associates, Birmingham, AL). The stained sections were washed, mounted in Fluormount G (Southern Biotechnology Associates), and viewed with a Leica/Leitz CMRB fluorescence microscope with appropriate filter cubes (Chromatechnology, Battleboro, VT). Images were acquired with a C5810 series digital color camera (Hamamatsu Photonic System, Bridgewater, NJ).
B cell staining and analysis
For immunofluorescence analysis, splenocytes were dissociated into ice-cold staining buffer (5% heat-inactivated FBS and 0.1% sodium azide in PBS, pH 7.3) and depleted for RBCs. Duplicate samples (106 cells/sample) were stained (30–40 min on ice) with optimal amounts of the mAb conjugates. Reference samples from each strain were stained with fluorochrome-coupled isotype control mAb, or single-color specific mAb stains, which were used to select fluorescence gates. The B cell turnover study and the intracellular 5-bromo-2'-deoxyuridine (BrdU) staining were performed, as described (7). Stained samples were analyzed on a FACSCalibur using CellQuest software (BD Biosciences, Franklin Lakes, NJ). The streptavidin APC; streptavidin CyChrome; FITC-, PE- and biotin-coupled rat mAb to mouse CD23 (clone B3B4); PE mouse mAb to BrdU (clone 3D4); and PE mouse IgG1 isotype control were obtained from BD PharMingen. The APC goat polyclonal Ab to mouse IgM; PE and Tricolor rat mAb to mouse CD45R (B220; clone RA3B62); and FITC, PE, Tricolor, APC, and biotin rat IgG2B and IgG2A isotype control Ab were obtained from Caltag Laboratories (Burlingame, CA). The PE rat mAb to mouse IgD (clone 11-26) was obtained from Southern Biotechnology Associates. We produced, purified, and biotinylated the rat mAb to C1qRp from the PB-493 cell line, which was a generous gift from A. Rolink (Department of Immunology, University of Basel, Basel, Switzerland), and was provided to us by J. Cambier (National Jewish Medical Research Center, Denver, CO). PE rat mAb to mouse CD21 was a generous gift from J. Kearney (University of Alabama, Birmingham, AL).
Analysis of the A/WySnJ Tnfrsf13c transcript 3' end
Total A/WySnJ splenocyte RNA was extracted and purified using TRI Reagent (Molecular Research Center, Cincinnati, OH), and reversed transcribed using the reverse-transcription system (Promega, Madison, WI), according to the manufacturer’s protocols. RACE was performed with the oligonucleotide 5'-GGATCCGGTACCTCTAGATCAGTTTTTTTTTTTTTTT-3' priming the reverse-transcription step. The cDNA was PCR amplified with a PTC-100 Programmable Thermal Controller (MJ Research, Watertown, MA); 5'-ACCCTCCTCAGAAACCCCTCAT-3' was the upper primer, and 5'-GGATCCGGTACCTCTAGATCAG-3' was the lower primer. The PCR products were cloned into the pCR II-TOPO vector using the TOPO TA PCR Cloning Kit for Sequencing (Invitrogen, Carlsbad, CA). PCR amplification and cloning were done twice. Plasmid DNA for sequencing was isolated from two individual clones per PCR. Sequencing was performed at the Biotechnology Center’s DNA Sequence Laboratory (University of Wisconsin).
Construction and characterization of the MigRI.BX retroviral vector
A/J splenic cDNA was produced, as described above, and the bcl-xL cDNA was PCR amplified with high fidelity Deep Vent DNA polymerase (New England Biolabs, Beverly, MA) using 5'-TAAGTGAGCAGGTGTTTTGGAC-3' as the forward primer and 5'-GGGAGGTGAGAGGTGAGTGG-3' as the reverse primer, encompassing bases 188–944 of the bcl-xL mRNA sequence (NCBI Locus ID 12048). The amplicon was blunt-end cloned into Invitrogen’s pCR 4Blunt-TOPO vector, according to the manufacturer’s protocol. The sequence of the insert was verified, as described above. The pCR 4Blunt-TOPO vector containing the bcl-xL insert was excised with EcoRI and subcloned into the EcoRI site of the MigRI vector’s multiple cloning site (29). To test for functional Bcl-xL protein expression, the IL-3-dependent FL5.12 cell line was used (30). The FL5.12 cells were grown in IMDM (Atlanta Biologicals, Norcross, GA), containing 10% WEHI-3B-conditioned medium as an IL-3 source (31), 10% heat-inactivated FBS (HyClone, Logan, UT), and 100 U/ml penicillin/streptomycin, 2 mmol/L L-glutamine, and 50 µmol/L 2-ME, all from Invitrogen. The FL5.12 cells (3 x 106) were infected with MigRI or MigRI.BX retroviral supernatant (2 ml, described below) in medium (5 ml total volume) with Polybrene (4 µg/ml; Sigma-Aldrich, St. Louis, MO).
Immunoblotting
For Bcl-xL protein expression analysis, cells were lysed in buffer (0.1% w/v SDS, 0.5% w/v deoxycholate, 1% v/v Nonidet P-40, 0.1 mg/ml PMSF, and 20 µg/ml aprotinin) on ice for 30 min and centrifuged, and the cleared lysates were stored at -70°C. The protein concentration was determined by the bicinchoninic acid protein assay (32). Lysates (17 µg of protein per sample) were electrophoresed in 12% SDS-PAGE gels, transferred to nitrocellulose, and immunoblotted with mAb to murine Bcl-xL (clone H-5; Santa Cruz Biotechnology, Santa Cruz, CA). Bound mAb were visualized using the ECL Western Blotting Detection System, according to the manufacturer’s protocol (Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was then stripped with 0.2 M NaOH and immunoblotted for green fluorescent protein (GFP) (clone B-2; Santa Cruz Biotechnology), as above.
Production and characterization of infectious retrovirus
The infectious MigRI.BX retroviral particles were produced, and A/WySnJ bone marrow stem cells were infected, as previously described (29). Briefly, purified MigRI.BX vector DNA (Wizard Plus Maxipreps; Promega) was CaPO4 precipitated onto 80–90% confluent monolayers of the Bosc23 packaging cells grown in IMDM (supplemented as above, but without 2-ME). Retroviral supernatants were collected 48–72 h later, stored at -70°C, and titered on NIH/3T3 cells. The NIH/3T3 cells (2 x 105) were grown 24 h in 60-mm dishes, before the supplemented IMDM (without 2-ME) was replaced with 100 µl of thawed retroviral supernatant diluted in 1 ml of medium containing Polybrene (4 µg/ml; Sigma-Aldrich). A 2-ml aliquot of fresh medium was added 24 h postinfection, and flow cytometric analysis for GFP expression was performed 48 h postinfection. Retroviral supernatants that yielded >40% GFP+ NIH/3T3 cells were used for the bone marrow stem cell infections.
Production and characterization of retrovirus-infected mice
The A/WySnJ donor mice were injected i.v. with 5-fluorouracil (5 mg in 400 µl PBS; Sigma-Aldrich). Five days later, bone marrow cells were collected and cultured (10–20 x 106 cells) in IMDM (4 ml) supplemented with 15% heat-inactivated FBS, 5% WEHI-3B-conditioned medium, 100 U/ml penicillin/streptomycin, 2 mmol/L L-glutamine, plus mouse rIL-3 (6 ng/ml), mouse rIL-6 (10 ng/ml), and recombinant mouse stem cell factor (100 ng/ml; all from Preprotech, Rocky Hill, NJ). After 24 h of culture, the medium was removed, and two sequential retroviral infections separated by 24 h were performed by spinoculation exactly as described (29). The infected cells were collected 4 h after the second inoculation and suspended in PBS, and 0.5 x 106 to 1 x 106 cells were injected i.v. into each lethally irradiated A/WySnJ mouse. Lethal irradiation (2 doses of 600 rad, separated by 4 h) was done just before bone marrow cell transfer. Splenocytes were collected and analyzed after 14–16 wk of reconstitution.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The nature of the Tnfrsf13c mutation in strain A/WySnJ mice is not completely known. The A/WySnJ Tnfrsf13c gene had a 4.7-kb insertion disrupting exon 3, which was predicted to replace the final 8 aa of BAFF-R with 21 aa of unknown origin (33). We sequenced the 3' end of the A/WySnJ Tnfrsf13c transcript to identify the source of the insertion. A 3' RACE was performed. The 3' RACE upper primer encompassed the exon 3 insertion site (Fig. 1A). The results showed that beginning at base 548, the 3' end has been replaced with 299 exogenous bases, including a stop codon and an AU-rich polyadenylation site (Fig. 1B). The predicted amino acid sequence of the mutated BAFF-R (Fig. 1C) was consistent with the published sequence (33). A BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) revealed >99% homology between the exogenous bases and an intracisternal type A particle (GenBank ID 4378062; data not shown). We conclude that a retrotransposon insertion disrupted the Tnfrsf13c gene.
|
Our previous histological studies showed that the Tnfrsf13c loss-of-function mutation severely disrupted splenic architecture (7). In those studies, it was clear that well-developed follicles were largely absent in the A/WySnJ spleen, but it was not clear whether these follicles had a marginal zone, nor where the A/WySnJ T and B lymphocytes were to be found. To better understand the function of BAFF-R in B cell development, more detailed immunohistology was performed. The BAFF-R-mutant A/WySnJ spleen and the control A/J spleen show well-defined marginal zones, as judged by the metallophilic macrophages, and well-developed T cell zones (Fig. 2). However, the A/WySnJ follicles show clear deficits in the B cell zones compared with the A/J follicles. Possible differences in MZ B cells cannot be clearly discerned from the immunohistology (J. Kearney, personal communication).
|
|
|
|
It is widely accepted that a survival signal derives from BAFF-R, but there is controversy as to whether this receptor also transmits a differentiation signal. To address this important issue, we used retroviral gene transfer to provide a survival signal to the BAFF-R mutant B cells, and analyzed whether FO B cell development had been restored. We reasoned that if the mutant BAFF-R lacks an essential differentiation signal, then provision of a survival signal would extend the transitional B cell life span, yet would not enhance FO B cell development. We selected the bcl-xL gene for these experiments, because bcl-xL transgenic mice show enhanced FO B cell survival (35), bcl-xL is up-regulated in response to BAFF treatment in vitro (36), bcl-xL has well-documented antiapoptotic function (37), and bcl-xL is the most potent antagonist of the proapoptotic Bik gene (38) that we reported is overexpressed in A/WySnJ B cells (39).
The bcl-xL gene was subcloned into the MigRI retroviral vector (29) to produce the MigRI.BX vector (Fig. 5A). This vector expresses the GFP as a marker of successful gene transfer. To test the ability of MigRI.BX to support cell survival, MigRI.BX was used to infect IL-3-dependent FL5.12 cells. High level Bcl-xL expression can overcome the apoptosis that is initiated in these cells upon IL-3 withdrawal (30). Initially, 
10–15% of the FL5.12 cells became MigRI.BX infected and expressed GFP (Fig. 5B). When IL-3 was withdrawn, the percentage of GFP+ cells increased significantly in the MigRI.BX-infected group, but not in the control MigRI-infected group. This result indicated that MigRI.BX-mediated bcl-xL gene expression had enabled the infected FL5.12 cells to survive IL-3 withdrawal. Immunoblotting for GFP and Bcl-xL protein expression in FL5.12 cell lysates supported this interpretation, showing enrichment of these proteins after IL-3 withdrawal, and a good correlation between GFP and Bcl-xL protein expression (Fig. 5C).
|
The MigRI.BX vector was used to generate AW.bcl-xL mice. MigRI.BX-infected A/WySnJ bone marrow stem cells were injected into lethally irradiated A/WySnJ mice, and after reconstitution, the splenocytes were analyzed. The transfection efficiencies ranged from 15 to 50%, as judged by the GFP+ splenocyte percentage. When cell lysates from freshly isolated splenocytes were immunoblotted, there was a good correlation between GFP and Bcl-xL protein expression for individual AW.bcl-xL mice (data not shown). In addition, when freshly isolated splenocytes were cultured 2–4 days, the percentage of GFP+ splenocytes increased 2- to 3-fold, indicating that these GFP+ cells had greater longevity than the GFP- splenocytes (data not shown).
We next analyzed whether the provision of a survival signal had extended the transitional B cell life span. Continuous BrdU-labeling studies were done in vivo, as we described (7). A short 5-day labeling period was used to specifically detect an effect of bcl-xL gene expression on transitional B cell longevity (24). Because the MB cell life span is >80 days, the 5-day labeling period did not measure the longevity of the MB cells (40). The results of the BrdU-labeling study showed that in the AW.bcl-xL mice, the B220+GFP+ B cells incorporated 50% less BrdU than the B220+GFP- B cells (Fig. 6A). These results indicated that Bcl-xL expression had significantly increased the life span of the transitional GFP+ B cells compared with the control GFP- B cells. The increase in longevity was not likely due to the MigRI retroviral vector, because we found no significant effect of the empty MigRI retroviral vector on peripheral B cells (data not shown).
|
This conclusion was confirmed by a four-color flow cytometric analysis of IgM, CD23, and C1qRp surface marker expression on GFP- and GFPhigh splenocytes from AW.bcl-xL mice (Fig. 6C). In these mice, the proportion of GFP- MB cells was similar to the A/WySnJ MB cell proportion, while the proportion of GFPhigh MB cells was increased to a level that was comparable to A/J mice (Table I). We next compared the GFP- and GFPhigh transitional B cell subsets. We reasoned that if bcl-xL gene expression simply allowed MB cells to accumulate, then all transitional subsets would be expected to decrease proportionately as the MB pool increased. However, only the GFPhigh T1 B cell subset percentage decreased significantly compared with the GFP- T1 B cell subset percentage. This was similar to the decreased proportion of T1 B cells in A/J spleens compared with the T1 B cell proportion in the A/WySnJ spleens (Table I). The T2 and T3 B cell subset proportions remained comparable between A/WySnJ and A/J mice, and between GFP- and GFPhigh B cells in the AW.bcl-xL mice. The BrdU data, together with the similarity in B cell subset proportions between GFPhigh B cells in the AW.bcl-xL spleens and the A/J spleens, clearly indicated that provision of a survival signal had increased transitional B cell longevity, allowing them to differentiate and accumulate in the MB compartment. Likewise, when the A/WySnJ mice were intercrossed with C57BL/6 mice carrying a human BCL2 transgene expressed in B lineage cells (41), and B cell development was studied in the homozygous Tnfrsf13c-mutant F2 intercross progeny with and without the BCL2 transgene, the BCL2 transgene allowed the vast majority of the B cells to attain the MB phenotype (I. Amanna, unpublished observations). Thus, provision of a survival signal by BCL2 transgene expression complemented the A/WySnJ BAFF-R mutation and restored B cell development in the same way as provision of a survival signal by retroviral transduction of the bcl-xL gene.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The retrotransposon insertion event that generated the Tnfrsf13c mutation in strain A/WySnJ mice is a new example of an insertion that has generated genetic diversity in the mouse genome. The intracisternal type A particles (IAP) are noninfectious retrovirus-like structures that accumulate in the endoplasmic reticulum of rodent cells (42). The inserted IAP proviral genomes are present at 1000 copies per haploid genome, although only a few appear to be transcriptionally active in any particular cell type. Inserted IAP proviruses can profoundly affect gene function through transcriptional regulation or formation of chimeric proteins (42). The chimeric proteins can have altered function, giving rise to important phenotypic changes, as was seen previously for the Hermansky-Pudlak syndrome protein in the pale ear (ep) mutant mouse strain (43), and in this study with the BAFF-R protein in the Tnfrsf13c mutant A/WySnJ mouse strain.
Others reported that BAFF-R had no function in T1 B cells, but was essential for the T1 to T2 cell developmental transition (13, 14, 44). This view has been interpreted to mean either that BAFF-R transmits a differentiation signal driving this transition, or it transmits a survival signal to the T2 B cells (19, 22). However, these previous T1 B cell analyses did not exclude the B-1 B cells that populate the A/WySnJ spleen. The inclusion of the B-1 B cells may have led to an overestimation of the T1 B cells, and consequently, to a misinterpretation of BAFF-R function. When the B-1 B cells were rigorously excluded, a substantial negative impact of the BAFF-R mutation on the T1 B cells was apparent. It is also important to note that BAFF-R was expressed on all splenic B220+ cells (17) and that BAFF-R-stimulated activation of NF-B was seen in T1 B cells (27). The BAFF-R expression and signaling data along with our transitional B cell data support the interpretation that BAFF-R function is essential at every stage of splenic FO B cell development.
There are important and poorly understood phenotypic and functional differences between the BAFF-R-mutant A/WySnJ and the BAFF-null mice that require further investigation to precisely define the homeostatic functions of BAFF. The BAFF-null spleens had less than 0.5 million FO B cells (13, 14), whereas the A/WySnJ spleens had 3–4 million FO B cells (5, 7). In addition, the BAFF-null mice had 80% less serum IgM than their wild-type counterparts (14), whereas the A/WySnJ mice had a serum IgM concentration that was equal to their wild-type A/J counterparts (6). Finally, the BAFF-null mice did not produce an Ab response to trinitrophenyl-Ficoll (13), whereas the A/WySnJ mice produced a robust Ab response to this T-independent Ag, equal to the A/J response (6). One possible explanation for these substantial differences is to suggest that a distinct BAFF-binding receptor such as BCMA (45), TACI (12), or a novel receptor, whose function is retained in the A/WySnJ strain, supports primary IgM responses and contributes to FO B cell development. Alternatively, the mutant BAFF-R in the A/WySnJ may not be a true null receptor and may retain some homeostatic functions.
Very recent reports have begun to document the biochemical pathways that mediate the B cell homeostatic functions of the BAFF-R. BAFF binding to the BAFF-R induced the processing of NF-B2, which was implicated in the survival of the splenic B cells (27, 46). It was intriguing that this processing occurred through a noncanonical NF-B activation pathway, setting BAFF-R apart from many other TNFR family members (27). Both the T1 B cells and the combined T2/MB cells showed this NF-B2 activation, consistent with our interpretation that the BAFF-R has an essential function in promoting the survival of all FO B cells, including T1 B cells.
The severely B cell-deficient phenotype of the A/WySnJ mice is not surprising, given the disruption in BAFF-R structure that was imposed by the retrotransposon insertion. The 8 C-terminal aa residues of the BAFF-R were replaced with 21 randomly encoded residues (33), generating a form of the BAFF-R that is unresponsive to BAFF (18). The 8 truncated residues are part of a 25-aa region that is completely conserved between human and murine BAFF-R, whereas the rest of the receptor shows much less conservation (17). Intriguingly, we noted that part of this conserved BAFF-R region is highly homologous to human and murine CD40. CD40 is also a TNFR family member, and it is known to transmit survival signals to FO B lymphocytes (47). The sequence (T)-(A)-(G/A)-(P)-(E/V)-(Q) occurs in the C-terminal region of CD40 and BAFF-R. This conserved sequence includes a portion of the (P)-x-(Q)-x-(T) motif that formed the TRAF2/3 binding site of CD40 (48) and was essential for CD40-induced processing of NF-B2 (49). It is highly significant that the A/WySnJ BAFF-R lacks the (T)-(A)-(G/A)-(P)-(E/V)-(Q) sequence and also fails to induce NF-B2 activation (27, 46). We hypothesize that the (T)-(A)-(G/A)-(P)-(E/V)-(Q) sequence of the BAFF-R will be essential for coupling the receptor to the noncanonical NF-B activation pathway, perhaps via a TRAF adapter protein, and ultimately to induction of a survival gene and/or repression of a proapoptotic gene.
The simplest model to explain how the BAFF-BAFF-R complex regulates B cell homeostasis is to suggest that this complex transmits a survival signal, enabling the cell to complete a differentiation step induced by an intrinsic developmental program or an extrinsic signal such as an Ag encounter. Our evidence that provision of a survival signal in vivo complemented the BAFF-R mutation and restored B cell development strongly supports this model. This model is also consistent with previous studies showing that BAFF provided a survival signal and led to up-regulation of some antiapoptotic Bcl-2 family members (9, 36, 40). At present, it is not yet clear whether the BAFF-R transmits a differentiation signal in addition to a survival signal. The A/WySnJ mutant BAFF-R retains much of its cytoplasmic domain, and thus may retain some BAFF-R functions, for example a differentiation signal. Indeed, the retention of partial function may begin to explain the substantial differences noted above between BAFF-null mice and A/WySnJ mice. The CD40 and other TNFR family members have distinct cytoplasmic domains that interact with specific adapter proteins (48). Perhaps the most C-terminal portion of the receptor is critical for B cell survival, whereas another cytoplasmic domain promotes B cell differentiation. Further studies dissecting the signaling capabilities of the BAFF-R will be needed to address these important questions.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Colleen E. Hayes, Department of Biochemistry, University of Wisconsin, 433 Babcock Drive, Madison, WI 53706. E-mail address: hayes{at}biochem.wisc.edu
3 Abbreviations used in this paper: SLE, systemic lupus erythematosus; BrdU, 5-bromo-2'-deoxyuridine; FO, follicular; GFP, green fluorescent protein; IAP, intracisternal type A particle; M, mature; MZ, marginal zone; NF, newly formed; T, transitional.
Received for publication December 16, 2002. Accepted for publication February 19, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B2 in maturing B cells. Nat. Immun. 3:958.[Medline]
B2. Immunity 17:515.[Medline]
B2 p100 to p52. EMBO J. 21:5375.[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. Carbonatto, P. Yu, M. Bertolino, E. Vigna, S. Steidler, L. Fava, C. Daghero, B. Roattino, M. Onidi, M. Ardizzone, et al. Nonclinical Safety, Pharmacokinetics, and Pharmacodynamics of Atacicept Toxicol. Sci., September 1, 2008; 105(1): 200 - 210. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Badr, G. Borhis, E. A. Lefevre, N. Chaoul, F. Deshayes, V. Dessirier, G. Lapree, A. Tsapis, and Y. Richard BAFF enhances chemotaxis of primary human B cells: a particular synergy between BAFF and CXCL13 on memory B cells Blood, March 1, 2008; 111(5): 2744 - 2754. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. M. Spach, F. E. Nashold, B. N. Dittel, and C. E. Hayes IL-10 Signaling Is Essential for 1,25-Dihydroxyvitamin D3-Mediated Inhibition of Experimental Autoimmune Encephalomyelitis J. Immunol., November 1, 2006; 177(9): 6030 - 6037. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. O. Jacob, L. Pricop, C. Putterman, M. N. Koss, Y. Liu, M. Kollaros, S. A. Bixler, C. M. Ambrose, M. L. Scott, and W. Stohl Paucity of Clinical Disease despite Serological Autoimmunity and Kidney Pathology in Lupus-Prone New Zealand Mixed 2328 Mice Deficient in BAFF J. Immunol., August 15, 2006; 177(4): 2671 - 2680. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. P. Miller, J. E. Stadanlick, and M. P. Cancro Space, Selection, and Surveillance: Setting Boundaries with BLyS. J. Immunol., June 1, 2006; 176(11): 6405 - 6410. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Vigorito, L. Gambardella, F. Colucci, S. McAdam, and M. Turner Vav proteins regulate peripheral B-cell survival Blood, October 1, 2005; 106(7): 2391 - 2398. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zhang, K.-H. Ko, Q. L. K. Lam, C. K. C. Lo, G. Srivastava, B. Zheng, Y.-L. Lau, and L. Lu Expression and function of TNF family member B cell-activating factor in the development of autoimmune arthritis Int. Immunol., August 1, 2005; 17(8): 1081 - 1092. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhang, C.-S. Park, S.-O. Yoon, L. Li, Y.-M. Hsu, C. Ambrose, and Y. S. Choi BAFF supports human B cell differentiation in the lymphoid follicles through distinct receptors Int. Immunol., June 1, 2005; 17(6): 779 - 788. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. S. M. Rahman and T. Manser B Cells Expressing Bcl-2 and a Signaling-Impaired BAFF-Specific Receptor Fail to Mature and Are Deficient in the Formation of Lymphoid Follicles and Germinal Centers J. Immunol., November 15, 2004; 173(10): 6179 - 6188. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Sasaki, S. Casola, J. L. Kutok, K. Rajewsky, and M. Schmidt-Supprian TNF Family Member B Cell-Activating Factor (BAFF) Receptor-Dependent and -Independent Roles for BAFF in B Cell Physiology |
element allows it to be packaged into infectious particles. The 5' long-terminal repeat element drives the bicistronic transcription of GFP and a gene of interest cloned immediately upstream of the internal ribosomal entry site, which controls GFP translation. The gene of interest is under 5'-7-methylguanosine-dependent translational control. The murine bcl-xL gene was cloned into the EcoRI site, as shown. Double restriction enzyme digests with EcoRI and SalI identified clones with the proper bcl-xL gene orientation. B, Expression of functional Bcl-xL protein was tested in IL-3-dependent FL5.12 cells in vitro. Duplicate FL5.12 cell cultures were infected with MigRI or MigRI.BX retroviral particles, passaged without IL-3 for 0, 4, or 6 days, and allowed to recover in IL-3-supplemented medium for several days. Duplicate samples from each cell culture were analyzed for GFP expression by flow cytometry, and a mean was calculated. The data presented represent the mean ± SD for the duplicate cell cultures for each time point. Comparisons made with the two-tailed Student’s t test (assuming unequal variances) gave p < 0.05 (*) or p < 0.01 (**). C, Expression of Bcl-xL and GFP protein was tested by immunoblotting. The experiment was done, as described above, except that cell lysates were collected and immunoblotting was performed. Equivalent amounts of cell lysate (17 µg) were loaded for each sample. Lane 1, Uninfected FL5.12 cells; lanes 2–3, MigRI.BX-infected cells grown with IL-3; lanes 4 and 5, MigRI.BX-infected cells grown without IL-3 for 4 days; lanes 6 and 7, MigRI.BX-infected cells grown without IL-3 for 6 days; lane 8, BJAB cell lysate as a positive control for Bcl-xL protein; lanes 9 and 10, MigRI-infected cells grown with IL-3; lanes 11 and 12, MigRI-infected cells grown without IL-3 for 4 days; lanes 13 and 14, MigRI-infected cells grown without IL-3 for 6 days.