BAFF, a Splice Isoform of BAFF, Opposes Full-Length BAFF Activity In Vivo in Transgenic Mouse Models1
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免疫学杂志 2005年第13期
Abstract
BAFF is a novel splicing isoform of the regulator B cell-activating factor (BAFF, BLyS), a TNF family protein with powerful immunoregulatory effects. Overexpression of BAFF leads to excessive B cell accumulation, activation, autoantibodies, and lupus-like disease, whereas an absence of BAFF causes peripheral B cell immunodeficiency. Based on the ability of BAFF to multimerize with full-length BAFF and to limit BAFF proteolytic shedding from the cell surface, we previously proposed a role for BAFF in restraining the effects of BAFF and in regulating B lymphocyte homeostasis. To test these ideas we generated mice transgenic for BAFF under the control of human CD68 regulatory elements, which target expression to myeloid and dendritic cells. We also generated in parallel BAFF transgenic mice using the same expression elements. Analysis of the transgenic mice revealed that BAFF and BAFF had opposing effects on B cell survival and marginal zone B cell numbers. BAFF transgenic mice had reduced B cell numbers and T cell-dependent Ab responses, but normal preimmune serum Ig levels. In contrast, BAFF transgenic mice had extraordinarily elevated Ig levels and increases in subsets of B cells. Unexpectedly, both BAFF and BAFF appeared to modulate the numbers of B-1 phenotype B cells.
Introduction
The TNF family-related cytokine B cell-activating factor (BAFF),3 also known as BLyS, TALL-1, THANK, ZTNF4, and TNF13B, has a nonredundant function in B cell survival. Mice genetically or pharmacologically deficient in BAFF or the key BAFF receptor, BAFF-R3, have similar phenotypes, characterized by hypogammaglobulinemia and a severe loss of follicular B cells, the major B cell type (1, 2, 3, 4). BAFF is continually required for most B cells starting at an early stage of peripheral B cell development (5, 6). In contrast, overexpression of BAFF by transgenesis or direct injection of recombinant BAFF promotes a lupus-like dysregulation involving hypergammaglobulinemia, splenomegaly, and autoantibody production (2, 7, 8, 9, 10, 11). It has been suggested that BCR signaling synergizes with BAFF in promoting Ab secretion, B cell developmental progression, and B cell anergy (12, 13, 14). BAFF expression levels are correlated with rheumatic diseases such as lupus and Sjogren’s syndrome (15, 16, 17). These findings suggest that BAFF activity is important in regulating immunity and tolerance.
We have recently identified a natural pathway for the negative regulation of BAFF, which involves the coexpression of a receptor nonbinding splice isoform BAFF, the protein product of which can comultimerize with full-length BAFF poisoning function (18). In addition, BAFF, unlike BAFF, is not efficiently cleaved from cell membranes, and so may regulate the biodistribution of BAFF (18). Furthermore, BAFF can form homotrimers, at least when overexpressed in cell lines (18), leaving open the possibility that BAFF3 may exist naturally and play additional roles. Finally, BAFF has been shown to form heterotrimers with the related cytokine APRIL (a proliferation-inducing ligand) in human cells (19), and an APRIL isoform analogous to BAFF has been described (20), raising the possibility that additional mixed heteromers may form with unknown functions.
BAFF is known to be expressed by cells of the myeloid lineage, including macrophages and dendritic cells (8, 21, 22). In mice, a major source of BAFF is generated by radioresistant cells of unknown phenotype, as demonstrated by the substantial reconstitution of B cell survival in bone marrow radiation chimeras consisting of BAFF-deficient hemopoietic cells and BAFF-sufficient hosts (23). These BAFF-producing cells may also be myeloid in origin, or may represent some other cell type. We have found BAFF expressed in all cellular sources in which BAFF itself is expressed, including normal macrophages and myeloid cell lines of mouse and human (18), and in human peripheral blood (our unpublished results).
In the present study, we probed the possible functions of BAFF by overexpression in transgenic mice. As controls, we also generated a series of BAFF transgenic mice.
Materials and Methods
Generation of transgenic expression constructs
The cDNAs encoding either a Flag-tagged mouse BAFF, or c-myc-tagged mouse BAFF were inserted into the blunt end repaired XbaI sites in the human CD68 promoter vector (24). The constructs were purified away from vector sequences using KpnI and BsaAI and were microinjected into CBA/C57BL/6 x C3H/C57BL/6 fertilized eggs before transfer into pseudopregnant foster mothers. Thirteen founders for BAFF, and thirteen founders for BAFF transgenic lines were identified. Seven of seven male founders of the BAFF transgenic lines overexpressed BAFF as assessed by increased IgA serum levels. Four of seven male founders of the BAFF transgenic lines overexpressed BAFF protein in bone marrow macrophage lysates in Western blot analysis. Three lines of BAFF overexpressing mice (DBF20, DBF33, and DBF40), and three lines of BAFF overexpressing mice (MB3, MB21, and MB29) were characterized in detail. All lines were backcrossed on the C57BL/6 background at least three generations. In some cases, BAFF-deficient mice (2) were bred to the BAFF transgenic line DBF20 for analysis. These studies have been reviewed and approved by The Scripps Research Institute Institutional Animal Care and Use Committee.
Cell culture
Bone marrow-derived macrophages were cultured using L cell-conditioned medium essentially as described (25) although IMDM was used instead of RPMI 1640. In some instances, LPS (1 μg/ml) was added to the cultures for varying time points before harvest. Whole cell lysates from bone marrow-derived macrophages were generated using 1% Nonidet P-40 lysis buffer containing 10 mM Tris, 150 mM NaCl, 0.1 mM EDTA, and 1 mM PMSF. Lysates were subjected to SDS-PAGE under reducing conditions, followed by Western blotting and detection using a polyclonal rabbit anti-BAFF Ab.
Serum Ig analysis
Six- to 10-wk–old animals were immunized with either 20 μg of trinitrophenyl (TNP)-Ficoll (83/1; Biosearch Technologies) or with 100 μg of TNP-hemocyanin (15/1) in alum at day 0, then again at day 21 without alum. Standard ELISAs were performed on serum from naive or immunized animals. Diluted sera from immunized animals were incubated on TNP-BSA-coated ELISA plates (15 μg/ml), and bound Ab was detected using isotype-specific Abs: IgG1 (A85-1), IgM (R6-60.2), IgG3 (R-40-82). Resting Ig levels were detected from titrations of sera using pairs of isotype-specific Abs. The first listed Ab clone was coated on the plate and the second Ab was biotinylated for: IgM, 11/41 and R6-60.2; IgG1, A85-3 and A85-1; IgG2a, R11-89 and R19-15; IgG2b, R9-91 and R12-3; and IgA, C10-3 and C10-1. Bound biotinylated Abs were detected using streptavidin-HRP (BD Biosciences). Ig levels were quantified either with purified isotype standards (resting serum levels) or by log10 of the dilution of sera in which half the maximal signal was observed. All Abs were obtained from BD Biosciences.
Flow cytometry analysis
Lymphocytes from spleen, pooled lymph nodes, bone marrow, and peritoneal cavity from 6- to 12-wk-old mice were washed with PBS containing 0.5% BSA (w/v), 0.01% NaN3, and 10 mM HEPES and analyzed on a FACSCalibur using a variety of Abs obtained from BD Biosciences or eBioscience, namely, B220-FITC, B220-allophycocyanin, IgM-biotin, IgD-FITC, CD21-FITC, CD5-FITC, CD23-PE, CD11b-PerCP Cy5.5, CD11c-allophycocyanin, and F4/80 allophycocyanin, CD43-PE, CD1d-PE, and CD9-biotin in the presence of Fc block. Biotinylated Abs were detected using streptavidin-PerCP Cy5.5 (BD Biosciences). Anti-IgM (clone M41) (26) was purified from hybridoma supernatant and coupled to Cy5 according to the manufacturer’s instructions (Amersham Biosciences). AA4.1-allophycocyanin was purchased from eBioscience. B cell subsets were defined as mature (B220+IgD+), immature (B220intIgM+), B1 subsets (B220intIgM+CD43+CD5+/–), transitional 1 (B220+AA4.1+IgM+CD23–), transitional 2/3 (B220+AA4.1+IgM+CD23+), marginal zone (MZ) (B220+CD23–IgM+CD21+) or in some cases (B220+IgM+CD1d+CD9+). Statistics were calculated using the standard Student t test function in Microsoft Excel, assuming equal variance between samples. In the case of analyzing transitional B cell numbers, the paired sample t test was applied. The detection of transgene expression was performed by extracellular staining as previously described, followed by fixation with 4% paraformaldehyde and permeabilization of cells using 0.1% saponin. Transgenic expression was then determined by intracellular staining using biotinylated FLAG (M2) detecting BAFF and biotinylated Myc (9E10) detecting BAFF, followed by streptavidin-PE or streptavidin-allophycocyanin.
Assessment of BAFF isoform RNA expression
Total RNA was harvested from bone marrow-derived macrophages from wild-type C57BL/6J mice and cDNA was generated from 5 μg of total RNA using oligo(dT) and SuperScript First Strand Synthesis System for RT-PCR (Invitrogen Life Technologies) according to manufacturer’s instructions. cDNA reaction (1–2 μl) was used as template for the amplification of BAFF using PLATINUM Pfx DNA polymerase enzyme (Invitrogen Life Technologies) according to manufacturer’s instructions. Both mouse BAFF isoforms are amplified from cDNA using primers 5'-TCGACCCCACAACTCCAGCCGCGGC and 5'-CAGCTCGTCCCCAAAGACGTGTAC. Actin was amplified from the cDNA using the following primers 5'-GCATTGCTGACAGGATGCAG and 5'-CCTGCTTGCTGATCCACATC. Amplification products were analyzed by 4% agarose electrophoresis. Negative images of ethidium bromide-stained gels were scanned and analyzed by densitometry.
Results
Generation of BAFF and BAFF transgenic mice
To overexpress BAFF or BAFF in vivo in myeloid cells, we generated transgenic mice carrying the DNA constructs shown in Fig. 1A. Epitope-tagged cDNAs of BAFF and BAFF were placed in an expression vector with promoter, polyadenylation signal, and splicing signal. We chose to use the human CD68 promoter (24, 27) because its tissue distribution of expression is believed to be similar to that of BAFF (although knowledge of the expression pattern of BAFF is still evolving). The human CD68 promoter has been successfully used in the generation of transgenic mice (24). We generated BAFF transgenic mice carrying a human c-myc epitope tag and BAFF transgenic mice carrying a FLAG (28) epitope tag in the N-terminal cytoplasmic tail. To assess transgene expression biochemically, bone marrow macrophages from different transgenic lines were expanded in M-CSF supplemented medium, and BAFF protein expression was determined by Western blotting (Fig. 1B). The results indicate that transgene-derived BAFF and BAFF were produced by the cells, but the levels of expression varied widely between founders. Endogenous BAFF protein expression was not detectable in nontransgenic cell preparations by immunoblotting (Fig. 1B, Non Tg). However, BAFF and BAFF mRNA is detectable by PCR in the nontransgenic cells and in other sources of both mouse and human myeloid cells (18). We conclude that the BAFF and BAFF transgenic constructs drive (over)expression of BAFF proteins in vivo, at least in myeloid cells.
Assessment of transgenic protein expression and tissue distribution in lymphoid organs
BAFF and BAFF transgene expression levels were assessed by flow cytometry, taking advantage of their epitope tags to quantitate intracellular protein. All transgenic lines expressed well in the splenic F4/80+ compartment, which is enriched in macrophages (Fig. 2, first row). A subset of CD11c+/CD11b+ dendritic cells also appeared to express (Fig. 2, second and third rows), whereas B cells had negligible expression levels (Fig. 2, fourth row). No expression was detected on T lineage cells (data not shown). We conclude that transgenic mice express in most macrophages and in a subset of dendritic cells.
Effects on serum Ig
To assess the biological activity of BAFF and BAFF transgene expression, serum Ig levels of transgenic mice were measured. Data shown in Fig. 3 represent pooled data from two BAFF transgenic lines (Fig. 3A) and three BAFF transgenic lines (Fig. 3B). Compared with littermate controls, BAFF transgenic mice had over 10-fold elevated IgM levels and IgA levels that were 1000-fold increased (Fig. 3A). In contrast, BAFF transgenic mice had no significant changes in basal serum Ig levels (Fig. 3B). Thus, serum Ig levels of mice not intentionally immunized were elevated in BAFF transgenic mice, but not in BAFF transgenic mice.
Effects on peripheral B lymphocytes
To evaluate the effects of BAFF and BAFF transgenes on peripheral B cells, lymphoid tissues were analyzed by flow cytometry. These experiments focused on the same two independent BAFF transgenic lines and two independent BAFF transgenic lines shown to have clear intracellular expression. As shown in Figs. 4A and 5, BAFF transgenic mice had significant decreases in mature B cell numbers in spleen, lymph nodes, and bone marrow, whereas in BAFF transgenic mice these populations were expanded (Figs. 4B and 5). Striking changes were found in number and percentages of splenic and lymph node B cells, which were elevated in BAFF transgenic mice and reduced in BAFF transgenic mice (Fig. 5). Levels of recirculating B cells in bone marrow mirrored those in the periphery. These results confirmed the bioactivity of BAFF transgenes on B cell numbers, and revealed a reciprocal, B cell reducing biological effect of BAFF that was consistent with the prediction that BAFF activity directly opposes that of BAFF.
Transitional B cell subsets
As BAFF has been proposed to exert its effect on B cells starting at the transitional stages in the spleen (12, 29, 30), we assessed the effects of BAFF and BAFF transgenes on transitional B cell subsets (Fig. 5). The T1 population is AA4.1+/CD23–/IgMhigh, the T2 population is AA4.1+/CD23+/IgMhigh, and the T3 population is AA4.1+/CD23+/IgMlow. Because the distinction between T2 and T3 populations was neither clear-cut nor consistent between experiments, the observed numbers of cells in the T2 and T3 populations were pooled for analysis. As shown in Fig. 5, BAFF transgenic mice had surprisingly little change in transitional B cell numbers. In contrast, BAFF transgenic mice had little change in the T1 population and a significant reduction in T2/3 cell numbers. These data indicate that the reductions in B cell numbers seen in BAFF transgenic mice occur at the T2 and later developmental stages, the same stages at which BAFF is believed to have a biological effect.
Analysis of B-1 cells
More detailed flow cytometric analysis was conducted to assess the effects of BAFF and BAFF on other peripheral B cell subsets. Unexpectedly, BAFF transgenic mice had increased numbers of peritoneal B-1 cells, whereas in BAFF transgenic mice B-1 cell numbers (IgM+CD43+gate) were reduced significantly (Figs. 4 and 5). To further address the role of BAFF on B-1 cells BAFF knockout mice were also analyzed (Fig. 6). B-1 cells as gated with IgM and CD5 (Fig. 6A, upper inset right) were CD43+ and B220int, as expected. BAFF- and heterozygous-deficient mice had subnormal numbers of peritoneal B-1 cells in comparison to wild-type mice (Fig. 6B). We conclude that BAFF may augment B-1 B cell numbers, but is not absolutely required for their survival.
MZ B cells
A detailed flow cytometry study of changes in splenic MZ cell number and phenotype was made in the transgenic mice. MZ B cells are CD23low/CD21high/CD1d+/CD9+ cells, however, it has been reported that BAFF levels can modulate the expression of CD23 and CD21 (31). As shown in Fig. 7A, BAFF transgenic B cells indeed had elevated CD23 levels. Conversely, and significantly, splenic B cells of BAFF transgenic mice had lower CD23 levels. MZ B cell numbers were assessed using conventional markers, including CD23 (Fig. 7B, upper panels) or using an alternative strategy using the markers B220, IgM, CD1d, and CD9 (Fig. 7B, lower panels). These approaches revealed that BAFF transgenic mice had elevated MZ B cell numbers (Fig. 7B, right panels). Conversely, in BAFF transgenic mice fewer MZ B cells were found (Fig. 7B and Fig. 5, bottom) again indicating a growth or survival promoting effect of BAFF and a contrary effect of BAFF.
Induced Ab responses
We next assessed the possible effects of BAFF overexpression on T cell-dependent and T cell-independent Ab responses. Mice were immunized with TNP-hemocyanin or TNP-Ficoll, and the serum Ab responses quantified (Fig. 8). At all time points tested, BAFF transgenic mice had a subnormal response to the T cell-dependent Ag (Fig. 8, cf open with filled symbols). Subnormal T cell-dependent Ab responses were evident in both the IgM and IgG1 responses. By contrast, responses to the type 2 T cell-independent Ag TNP-Ficoll were not changed significantly in BAFF transgenic mice. We conclude that under the conditions of immunization BAFF reduced T cell-dependent, but not T cell-independent immune responses.
Induced changes in BAFF to BAFF mRNA ratios in primary macrophages
To test the notion that in nontransgenic myeloid cells the relative amounts of BAFF and BAFF splice isoforms may be regulated, we stimulated bone marrow macrophages from wild-type C57BL/6J mice with LPS and performed RT-PCR analysis. By design, this PCR amplifies both BAFF and BAFF isoforms, yielding products that differ 57 bp in size, corresponding to the missing exon in BAFF. As shown in Fig. 9, quiescent macrophages express both BAFF and BAFF isoforms. Upon LPS stimulation, the levels of BAFF changed little, whereas BAFF was reduced over time, particularly at 7 h poststimulation. We previously showed that myeloid cell lines can also alter their BAFF to BAFF ratios upon induction (18). We conclude that changes in the BAFF to BAFF ratio can be induced in primary myeloid cells.
Assessing the effects of BAFF transgenes on the BAFF–/– background
Finally, to try to determine whether BAFF has a function independent of BAFF, we crossed BAFF transgenic mice to the BAFF–/– background and assessed mice for changes in peripheral B lymphocyte numbers (Fig. 10). Flow cytometry experiments revealed that on BAFF+/+ and BAFF+/– backgrounds the frequency of total IgD+ and MZ B cells in BAFF transgenic mice was consistently lower compared with littermate controls. However, the residual B220+/IgD+ cell numbers of BAFF–/– mice were not further reduced or rescued by the presence of the BAFF transgene. We conclude that the residual B cells in BAFF–/– mice are not affected by BAFF expression.
Discussion
BAFF opposes BAFF activity
In the present study we have tested the notion that the natural splice variant BAFF plays a role in opposing BAFF activity, a role that may be important in B cell homeostasis. BAFF is known to be expressed by cells of the myeloid lineage, including macrophages, monocytes, and dendritic cells (8, 21, 22, 32). We show that BAFF is indeed able to oppose endogenous BAFF function in an in vivo setting. We find that when BAFF is overexpressed under the control of a human CD68 promoter a significant phenotype is obtained in independent transgenic lines, with reduced follicular and MZ B cell numbers. However, the suppressive effects of transgene-expressed BAFF were partial (a 30% reduction in B cell subsets), most likely because the promoter used, human CD68, failed to drive BAFF expression in all cells that produce endogenous BAFF. Because BAFF/BAFF heteromers and homomultimers form extensive disulfide bonds between subunits, it is unlikely that complexes can form in the circulation after synthesis and release. A recent study showing that radioresistant cells make a major contribution to the BAFF levels present in vivo indicates that a major source may include long lived, radioresistant myeloid cells or nonhemopoietic cell types (23). It is unknown whether these radioresistant cells express CD68, or macrosialin. As the human CD68 promoter directs expression to myeloid and dendritic cells (24), our results are consistent with the possibility that CD68-expressing cell types make a contribution to in vivo BAFF activity, and that BAFF expression in the same cells limits BAFF activity. It is unlikely that incomplete suppression of BAFF function by transgenic BAFF was due to insufficient expression in myeloid cells because immunoblotting suggested protein amounts that greatly exceeded those of wild-type BAFF or BAFF. The fact that the phenotypes of our BAFF transgenic mice were fairly uniform, despite wide variations in apparent levels of expression, indicates that the tissue distribution of BAFF expression, rather than its level, was limiting.
Selective effects of BAFF on T cell-dependent Ab responses
A surprising finding in our study was that transgenic expression of BAFF preferentially suppressed T cell-dependent Ab response, but had no discernible effect on the T cell-independent response. Prior studies had indicated that BAFF overexpression could augment both responses (10). Our results are consistent with the possibility that although BAFF can augment T cell-independent responses and promote T cell-independent class switch recombination (10, 22), other cytokines typically play a more dominant role. Also consistent with this notion was the lack of influence of BAFF transgene expression on serum Ig levels in unimmunized mice. The lack of reduction in serum Ig levels in BAFF transgenic mice is not surprising given the phenotype of BAFF-R+/– mice, in which B cell numbers are reduced, but serum Ig is not significantly affected (33). One interpretation of these findings is that during a T cell-dependent immune response, T cells may actually stimulate BAFF expression in APCs, thereby enhancing secretion. Support for this possibility comes from data suggesting that CD40L up-regulates BAFF expression from myeloid cells (22, 34 ). Alternatively, BAFF may act directly on T cells in ways that affect the immune response (35, 36). In humans, BAFF can form heterotrimers with APRIL (19). It is unlikely that BAFF works primarily by suppressing APRIL bioactivity because, if this result were so, the phenotype of our BAFF transgenic mice should tend to mimic the phenotype in APRIL-deficient mice. However, this effect is not the case. APRIL-deficient mice have elevated T cell-dependent immune responses, defective responses to T cell-independent type 1 Ags, such as hapten-LPS conjugates, and normal responses to T cell-independent type 2 Ags such as Ficoll conjugates (37).
Given that BAFF can interact with both BAFF-R and TACI in mice, it is significant that the phenotype of BAFF transgenic mice resembles closely that of mice that have reduced BAFF-R levels, such as BAFF-R+/– mice (33, 36, 38) and mice heterozygous for the spontaneous BAFF-R mutation in A/WySnJ mice (5). These mutants have reduced B cell numbers and normal serum Ig levels. In contrast, B cell responses in BAFF transgenic mice do not resemble those of mice that lack TACI, which have impaired type 2 T cell-independent responses, normal or elevated B cell numbers, and normal T cell-dependent Ab responses (39, 40). These considerations suggest that BAFF principally suppresses BAFF signaling through the BAFF-R, rather than TACI.
An additional finding in our study was that BAFF transgenic mice had normal T cell-independent type 2 Ab responses despite their reduced levels of B-1 and MZ B cells. Although type 2 T cell-independent responses are believed to be derived from B-1 or MZ B cells (41, 42, 43, 44), mutations that diminish B-1 or MZ B cell numbers often do not reduce T cell-independent Ab responses (45, 46). This implies that certain induced responses, such as to TNP-Ficoll, may be mainly derived from B-2 type cells.
BAFF overexpressing mice
In our experiments, we have generated a new series of BAFF transgenic mice using the human CD68 promoter, a strategy that was designed to overexpress BAFF from cells that are specialized in BAFF expression. Compared with previously studied BAFF transgenic mice, we find both similarities and differences (7, 9, 47). Prior studies used promoters directing expression to liver (7), B cells themselves (47), or ubiquitously (9). Given these differences, the results have been remarkably consistent, including evidence of increased peripheral B cell numbers, expansion of the MZ B cell compartment, hypergammaglobulinemia, particularly elevated levels of IgA, and evidence of systemic autoimmunity. These findings are consistent with a role for BAFF predominantly as a soluble, rather than membrane bound, cytokine.
In our study, we find expansion of B-1 cells in BAFF overexpressing mice, whereas in two of four prior studies no such expansion was described. B-1 cells were elevated in mice in which Ig promoter and enhancer elements drove BAFF expression (47) or in the salivary glands of mice expressing BAFF under 1 antitrypsin promoter/apoE enhancer regulatory elements (48). BAFF is known to be nonessential for B-1 cell development and maintenance (2), however, our study shows 50% reduction in B-1 cell numbers in BAFF-deficient animals. Gross et al. (1) noted a similar 50% decrease in B-1 cells in an independent line of BAFF–/– mice, though given the variance, the change in that study was not judged to be statistically significant. Thus, it appears that B-1 cells can be modulated by BAFF levels, although not to the same extent as B-2 cells (>90% reduced in BAFF-deficient animals). As CD68 is not highly expressed in B cells, our results suggest the possibility that local, high level expression of BAFF in myeloid cells may nevertheless augment B-1 cell survival, expansion, or differentiation.
Modulation of BAFF to BAFF ratios
The levels of BAFF expressed, as mRNA, or as protein on cell membranes or released from cells, can vary widely depending on cellular stimulation with mitogens, microbial products, or cytokines (21, 32). However, prior studies did not take into account the differing amounts and functions of BAFF and BAFF isoforms. We find that in addition to overall levels of BAFF, the BAFF to BAFF ratio can also be modulated in myeloid cells by cell stimulus. The relative RNA levels of the two BAFF isoforms are sufficiently similar to one another in that this type of regulation is likely to have major consequence on BAFF activity. As BAFF appears to be resistant to cleavage from cell membranes, it can possibly form multimers with BAFF (or APRIL) that retain activity, but are restricted to act locally by cell contact. The in vivo data presented in this study suggest changing the BAFF to BAFF ratio toward excess BAFF would lead to less bioactive BAFF and consequently less B cell survival.
BAFF has been implicated in the exacerbation or cause of systemic autoimmunity and in regulating immune tolerance thresholds (13, 14, 49), but is also required for B cell survival (2, 5), suggesting that the balance between these activities is crucial. BAFF likely plays a key role in this regulation because it can directly influence BAFF activities through its three modes of action (18): 1) consuming BAFF mRNA by alternative splicing, 2) forming nonfunctional BAFF1/BAFF2 heterotrimers, and 3) preventing release from the plasma membrane of heteromultimerized molecules, possibly including forms with two or more active BAFF subunits. However, it is not excluded that BAFF may have unique, independent functions. In transfection experiments using 293 host cells, BAFF can form cell surface bound homomultimers, but it is unclear whether this isoform alone has an independent in vivo function. To date, we have been unable to find any obvious differences between BAFF–/– mice and BAFF–/– carrying BAFF transgenes.
Acknowledgments
We thank Min Lim for excellent technical assistance and Louise McHeyzer Williams for reading the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grants RO1GM44809, RO3AR050017 (to A.L.G.), and PO1AG01743 from the National Institutes of Health (to D.N.).
2 Address correspondence and reprint requests to Dr. David Nemazee, Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: nemazee@scripps.edu
3 Abbreviations used in this paper: BAFF, B cell-activating factor belonging to the TNF family; MZ, marginal zone; TNP, trinitrophenyl.
Received for publication February 3, 2005. Accepted for publication April 20, 2005.
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BAFF is a novel splicing isoform of the regulator B cell-activating factor (BAFF, BLyS), a TNF family protein with powerful immunoregulatory effects. Overexpression of BAFF leads to excessive B cell accumulation, activation, autoantibodies, and lupus-like disease, whereas an absence of BAFF causes peripheral B cell immunodeficiency. Based on the ability of BAFF to multimerize with full-length BAFF and to limit BAFF proteolytic shedding from the cell surface, we previously proposed a role for BAFF in restraining the effects of BAFF and in regulating B lymphocyte homeostasis. To test these ideas we generated mice transgenic for BAFF under the control of human CD68 regulatory elements, which target expression to myeloid and dendritic cells. We also generated in parallel BAFF transgenic mice using the same expression elements. Analysis of the transgenic mice revealed that BAFF and BAFF had opposing effects on B cell survival and marginal zone B cell numbers. BAFF transgenic mice had reduced B cell numbers and T cell-dependent Ab responses, but normal preimmune serum Ig levels. In contrast, BAFF transgenic mice had extraordinarily elevated Ig levels and increases in subsets of B cells. Unexpectedly, both BAFF and BAFF appeared to modulate the numbers of B-1 phenotype B cells.
Introduction
The TNF family-related cytokine B cell-activating factor (BAFF),3 also known as BLyS, TALL-1, THANK, ZTNF4, and TNF13B, has a nonredundant function in B cell survival. Mice genetically or pharmacologically deficient in BAFF or the key BAFF receptor, BAFF-R3, have similar phenotypes, characterized by hypogammaglobulinemia and a severe loss of follicular B cells, the major B cell type (1, 2, 3, 4). BAFF is continually required for most B cells starting at an early stage of peripheral B cell development (5, 6). In contrast, overexpression of BAFF by transgenesis or direct injection of recombinant BAFF promotes a lupus-like dysregulation involving hypergammaglobulinemia, splenomegaly, and autoantibody production (2, 7, 8, 9, 10, 11). It has been suggested that BCR signaling synergizes with BAFF in promoting Ab secretion, B cell developmental progression, and B cell anergy (12, 13, 14). BAFF expression levels are correlated with rheumatic diseases such as lupus and Sjogren’s syndrome (15, 16, 17). These findings suggest that BAFF activity is important in regulating immunity and tolerance.
We have recently identified a natural pathway for the negative regulation of BAFF, which involves the coexpression of a receptor nonbinding splice isoform BAFF, the protein product of which can comultimerize with full-length BAFF poisoning function (18). In addition, BAFF, unlike BAFF, is not efficiently cleaved from cell membranes, and so may regulate the biodistribution of BAFF (18). Furthermore, BAFF can form homotrimers, at least when overexpressed in cell lines (18), leaving open the possibility that BAFF3 may exist naturally and play additional roles. Finally, BAFF has been shown to form heterotrimers with the related cytokine APRIL (a proliferation-inducing ligand) in human cells (19), and an APRIL isoform analogous to BAFF has been described (20), raising the possibility that additional mixed heteromers may form with unknown functions.
BAFF is known to be expressed by cells of the myeloid lineage, including macrophages and dendritic cells (8, 21, 22). In mice, a major source of BAFF is generated by radioresistant cells of unknown phenotype, as demonstrated by the substantial reconstitution of B cell survival in bone marrow radiation chimeras consisting of BAFF-deficient hemopoietic cells and BAFF-sufficient hosts (23). These BAFF-producing cells may also be myeloid in origin, or may represent some other cell type. We have found BAFF expressed in all cellular sources in which BAFF itself is expressed, including normal macrophages and myeloid cell lines of mouse and human (18), and in human peripheral blood (our unpublished results).
In the present study, we probed the possible functions of BAFF by overexpression in transgenic mice. As controls, we also generated a series of BAFF transgenic mice.
Materials and Methods
Generation of transgenic expression constructs
The cDNAs encoding either a Flag-tagged mouse BAFF, or c-myc-tagged mouse BAFF were inserted into the blunt end repaired XbaI sites in the human CD68 promoter vector (24). The constructs were purified away from vector sequences using KpnI and BsaAI and were microinjected into CBA/C57BL/6 x C3H/C57BL/6 fertilized eggs before transfer into pseudopregnant foster mothers. Thirteen founders for BAFF, and thirteen founders for BAFF transgenic lines were identified. Seven of seven male founders of the BAFF transgenic lines overexpressed BAFF as assessed by increased IgA serum levels. Four of seven male founders of the BAFF transgenic lines overexpressed BAFF protein in bone marrow macrophage lysates in Western blot analysis. Three lines of BAFF overexpressing mice (DBF20, DBF33, and DBF40), and three lines of BAFF overexpressing mice (MB3, MB21, and MB29) were characterized in detail. All lines were backcrossed on the C57BL/6 background at least three generations. In some cases, BAFF-deficient mice (2) were bred to the BAFF transgenic line DBF20 for analysis. These studies have been reviewed and approved by The Scripps Research Institute Institutional Animal Care and Use Committee.
Cell culture
Bone marrow-derived macrophages were cultured using L cell-conditioned medium essentially as described (25) although IMDM was used instead of RPMI 1640. In some instances, LPS (1 μg/ml) was added to the cultures for varying time points before harvest. Whole cell lysates from bone marrow-derived macrophages were generated using 1% Nonidet P-40 lysis buffer containing 10 mM Tris, 150 mM NaCl, 0.1 mM EDTA, and 1 mM PMSF. Lysates were subjected to SDS-PAGE under reducing conditions, followed by Western blotting and detection using a polyclonal rabbit anti-BAFF Ab.
Serum Ig analysis
Six- to 10-wk–old animals were immunized with either 20 μg of trinitrophenyl (TNP)-Ficoll (83/1; Biosearch Technologies) or with 100 μg of TNP-hemocyanin (15/1) in alum at day 0, then again at day 21 without alum. Standard ELISAs were performed on serum from naive or immunized animals. Diluted sera from immunized animals were incubated on TNP-BSA-coated ELISA plates (15 μg/ml), and bound Ab was detected using isotype-specific Abs: IgG1 (A85-1), IgM (R6-60.2), IgG3 (R-40-82). Resting Ig levels were detected from titrations of sera using pairs of isotype-specific Abs. The first listed Ab clone was coated on the plate and the second Ab was biotinylated for: IgM, 11/41 and R6-60.2; IgG1, A85-3 and A85-1; IgG2a, R11-89 and R19-15; IgG2b, R9-91 and R12-3; and IgA, C10-3 and C10-1. Bound biotinylated Abs were detected using streptavidin-HRP (BD Biosciences). Ig levels were quantified either with purified isotype standards (resting serum levels) or by log10 of the dilution of sera in which half the maximal signal was observed. All Abs were obtained from BD Biosciences.
Flow cytometry analysis
Lymphocytes from spleen, pooled lymph nodes, bone marrow, and peritoneal cavity from 6- to 12-wk-old mice were washed with PBS containing 0.5% BSA (w/v), 0.01% NaN3, and 10 mM HEPES and analyzed on a FACSCalibur using a variety of Abs obtained from BD Biosciences or eBioscience, namely, B220-FITC, B220-allophycocyanin, IgM-biotin, IgD-FITC, CD21-FITC, CD5-FITC, CD23-PE, CD11b-PerCP Cy5.5, CD11c-allophycocyanin, and F4/80 allophycocyanin, CD43-PE, CD1d-PE, and CD9-biotin in the presence of Fc block. Biotinylated Abs were detected using streptavidin-PerCP Cy5.5 (BD Biosciences). Anti-IgM (clone M41) (26) was purified from hybridoma supernatant and coupled to Cy5 according to the manufacturer’s instructions (Amersham Biosciences). AA4.1-allophycocyanin was purchased from eBioscience. B cell subsets were defined as mature (B220+IgD+), immature (B220intIgM+), B1 subsets (B220intIgM+CD43+CD5+/–), transitional 1 (B220+AA4.1+IgM+CD23–), transitional 2/3 (B220+AA4.1+IgM+CD23+), marginal zone (MZ) (B220+CD23–IgM+CD21+) or in some cases (B220+IgM+CD1d+CD9+). Statistics were calculated using the standard Student t test function in Microsoft Excel, assuming equal variance between samples. In the case of analyzing transitional B cell numbers, the paired sample t test was applied. The detection of transgene expression was performed by extracellular staining as previously described, followed by fixation with 4% paraformaldehyde and permeabilization of cells using 0.1% saponin. Transgenic expression was then determined by intracellular staining using biotinylated FLAG (M2) detecting BAFF and biotinylated Myc (9E10) detecting BAFF, followed by streptavidin-PE or streptavidin-allophycocyanin.
Assessment of BAFF isoform RNA expression
Total RNA was harvested from bone marrow-derived macrophages from wild-type C57BL/6J mice and cDNA was generated from 5 μg of total RNA using oligo(dT) and SuperScript First Strand Synthesis System for RT-PCR (Invitrogen Life Technologies) according to manufacturer’s instructions. cDNA reaction (1–2 μl) was used as template for the amplification of BAFF using PLATINUM Pfx DNA polymerase enzyme (Invitrogen Life Technologies) according to manufacturer’s instructions. Both mouse BAFF isoforms are amplified from cDNA using primers 5'-TCGACCCCACAACTCCAGCCGCGGC and 5'-CAGCTCGTCCCCAAAGACGTGTAC. Actin was amplified from the cDNA using the following primers 5'-GCATTGCTGACAGGATGCAG and 5'-CCTGCTTGCTGATCCACATC. Amplification products were analyzed by 4% agarose electrophoresis. Negative images of ethidium bromide-stained gels were scanned and analyzed by densitometry.
Results
Generation of BAFF and BAFF transgenic mice
To overexpress BAFF or BAFF in vivo in myeloid cells, we generated transgenic mice carrying the DNA constructs shown in Fig. 1A. Epitope-tagged cDNAs of BAFF and BAFF were placed in an expression vector with promoter, polyadenylation signal, and splicing signal. We chose to use the human CD68 promoter (24, 27) because its tissue distribution of expression is believed to be similar to that of BAFF (although knowledge of the expression pattern of BAFF is still evolving). The human CD68 promoter has been successfully used in the generation of transgenic mice (24). We generated BAFF transgenic mice carrying a human c-myc epitope tag and BAFF transgenic mice carrying a FLAG (28) epitope tag in the N-terminal cytoplasmic tail. To assess transgene expression biochemically, bone marrow macrophages from different transgenic lines were expanded in M-CSF supplemented medium, and BAFF protein expression was determined by Western blotting (Fig. 1B). The results indicate that transgene-derived BAFF and BAFF were produced by the cells, but the levels of expression varied widely between founders. Endogenous BAFF protein expression was not detectable in nontransgenic cell preparations by immunoblotting (Fig. 1B, Non Tg). However, BAFF and BAFF mRNA is detectable by PCR in the nontransgenic cells and in other sources of both mouse and human myeloid cells (18). We conclude that the BAFF and BAFF transgenic constructs drive (over)expression of BAFF proteins in vivo, at least in myeloid cells.
Assessment of transgenic protein expression and tissue distribution in lymphoid organs
BAFF and BAFF transgene expression levels were assessed by flow cytometry, taking advantage of their epitope tags to quantitate intracellular protein. All transgenic lines expressed well in the splenic F4/80+ compartment, which is enriched in macrophages (Fig. 2, first row). A subset of CD11c+/CD11b+ dendritic cells also appeared to express (Fig. 2, second and third rows), whereas B cells had negligible expression levels (Fig. 2, fourth row). No expression was detected on T lineage cells (data not shown). We conclude that transgenic mice express in most macrophages and in a subset of dendritic cells.
Effects on serum Ig
To assess the biological activity of BAFF and BAFF transgene expression, serum Ig levels of transgenic mice were measured. Data shown in Fig. 3 represent pooled data from two BAFF transgenic lines (Fig. 3A) and three BAFF transgenic lines (Fig. 3B). Compared with littermate controls, BAFF transgenic mice had over 10-fold elevated IgM levels and IgA levels that were 1000-fold increased (Fig. 3A). In contrast, BAFF transgenic mice had no significant changes in basal serum Ig levels (Fig. 3B). Thus, serum Ig levels of mice not intentionally immunized were elevated in BAFF transgenic mice, but not in BAFF transgenic mice.
Effects on peripheral B lymphocytes
To evaluate the effects of BAFF and BAFF transgenes on peripheral B cells, lymphoid tissues were analyzed by flow cytometry. These experiments focused on the same two independent BAFF transgenic lines and two independent BAFF transgenic lines shown to have clear intracellular expression. As shown in Figs. 4A and 5, BAFF transgenic mice had significant decreases in mature B cell numbers in spleen, lymph nodes, and bone marrow, whereas in BAFF transgenic mice these populations were expanded (Figs. 4B and 5). Striking changes were found in number and percentages of splenic and lymph node B cells, which were elevated in BAFF transgenic mice and reduced in BAFF transgenic mice (Fig. 5). Levels of recirculating B cells in bone marrow mirrored those in the periphery. These results confirmed the bioactivity of BAFF transgenes on B cell numbers, and revealed a reciprocal, B cell reducing biological effect of BAFF that was consistent with the prediction that BAFF activity directly opposes that of BAFF.
Transitional B cell subsets
As BAFF has been proposed to exert its effect on B cells starting at the transitional stages in the spleen (12, 29, 30), we assessed the effects of BAFF and BAFF transgenes on transitional B cell subsets (Fig. 5). The T1 population is AA4.1+/CD23–/IgMhigh, the T2 population is AA4.1+/CD23+/IgMhigh, and the T3 population is AA4.1+/CD23+/IgMlow. Because the distinction between T2 and T3 populations was neither clear-cut nor consistent between experiments, the observed numbers of cells in the T2 and T3 populations were pooled for analysis. As shown in Fig. 5, BAFF transgenic mice had surprisingly little change in transitional B cell numbers. In contrast, BAFF transgenic mice had little change in the T1 population and a significant reduction in T2/3 cell numbers. These data indicate that the reductions in B cell numbers seen in BAFF transgenic mice occur at the T2 and later developmental stages, the same stages at which BAFF is believed to have a biological effect.
Analysis of B-1 cells
More detailed flow cytometric analysis was conducted to assess the effects of BAFF and BAFF on other peripheral B cell subsets. Unexpectedly, BAFF transgenic mice had increased numbers of peritoneal B-1 cells, whereas in BAFF transgenic mice B-1 cell numbers (IgM+CD43+gate) were reduced significantly (Figs. 4 and 5). To further address the role of BAFF on B-1 cells BAFF knockout mice were also analyzed (Fig. 6). B-1 cells as gated with IgM and CD5 (Fig. 6A, upper inset right) were CD43+ and B220int, as expected. BAFF- and heterozygous-deficient mice had subnormal numbers of peritoneal B-1 cells in comparison to wild-type mice (Fig. 6B). We conclude that BAFF may augment B-1 B cell numbers, but is not absolutely required for their survival.
MZ B cells
A detailed flow cytometry study of changes in splenic MZ cell number and phenotype was made in the transgenic mice. MZ B cells are CD23low/CD21high/CD1d+/CD9+ cells, however, it has been reported that BAFF levels can modulate the expression of CD23 and CD21 (31). As shown in Fig. 7A, BAFF transgenic B cells indeed had elevated CD23 levels. Conversely, and significantly, splenic B cells of BAFF transgenic mice had lower CD23 levels. MZ B cell numbers were assessed using conventional markers, including CD23 (Fig. 7B, upper panels) or using an alternative strategy using the markers B220, IgM, CD1d, and CD9 (Fig. 7B, lower panels). These approaches revealed that BAFF transgenic mice had elevated MZ B cell numbers (Fig. 7B, right panels). Conversely, in BAFF transgenic mice fewer MZ B cells were found (Fig. 7B and Fig. 5, bottom) again indicating a growth or survival promoting effect of BAFF and a contrary effect of BAFF.
Induced Ab responses
We next assessed the possible effects of BAFF overexpression on T cell-dependent and T cell-independent Ab responses. Mice were immunized with TNP-hemocyanin or TNP-Ficoll, and the serum Ab responses quantified (Fig. 8). At all time points tested, BAFF transgenic mice had a subnormal response to the T cell-dependent Ag (Fig. 8, cf open with filled symbols). Subnormal T cell-dependent Ab responses were evident in both the IgM and IgG1 responses. By contrast, responses to the type 2 T cell-independent Ag TNP-Ficoll were not changed significantly in BAFF transgenic mice. We conclude that under the conditions of immunization BAFF reduced T cell-dependent, but not T cell-independent immune responses.
Induced changes in BAFF to BAFF mRNA ratios in primary macrophages
To test the notion that in nontransgenic myeloid cells the relative amounts of BAFF and BAFF splice isoforms may be regulated, we stimulated bone marrow macrophages from wild-type C57BL/6J mice with LPS and performed RT-PCR analysis. By design, this PCR amplifies both BAFF and BAFF isoforms, yielding products that differ 57 bp in size, corresponding to the missing exon in BAFF. As shown in Fig. 9, quiescent macrophages express both BAFF and BAFF isoforms. Upon LPS stimulation, the levels of BAFF changed little, whereas BAFF was reduced over time, particularly at 7 h poststimulation. We previously showed that myeloid cell lines can also alter their BAFF to BAFF ratios upon induction (18). We conclude that changes in the BAFF to BAFF ratio can be induced in primary myeloid cells.
Assessing the effects of BAFF transgenes on the BAFF–/– background
Finally, to try to determine whether BAFF has a function independent of BAFF, we crossed BAFF transgenic mice to the BAFF–/– background and assessed mice for changes in peripheral B lymphocyte numbers (Fig. 10). Flow cytometry experiments revealed that on BAFF+/+ and BAFF+/– backgrounds the frequency of total IgD+ and MZ B cells in BAFF transgenic mice was consistently lower compared with littermate controls. However, the residual B220+/IgD+ cell numbers of BAFF–/– mice were not further reduced or rescued by the presence of the BAFF transgene. We conclude that the residual B cells in BAFF–/– mice are not affected by BAFF expression.
Discussion
BAFF opposes BAFF activity
In the present study we have tested the notion that the natural splice variant BAFF plays a role in opposing BAFF activity, a role that may be important in B cell homeostasis. BAFF is known to be expressed by cells of the myeloid lineage, including macrophages, monocytes, and dendritic cells (8, 21, 22, 32). We show that BAFF is indeed able to oppose endogenous BAFF function in an in vivo setting. We find that when BAFF is overexpressed under the control of a human CD68 promoter a significant phenotype is obtained in independent transgenic lines, with reduced follicular and MZ B cell numbers. However, the suppressive effects of transgene-expressed BAFF were partial (a 30% reduction in B cell subsets), most likely because the promoter used, human CD68, failed to drive BAFF expression in all cells that produce endogenous BAFF. Because BAFF/BAFF heteromers and homomultimers form extensive disulfide bonds between subunits, it is unlikely that complexes can form in the circulation after synthesis and release. A recent study showing that radioresistant cells make a major contribution to the BAFF levels present in vivo indicates that a major source may include long lived, radioresistant myeloid cells or nonhemopoietic cell types (23). It is unknown whether these radioresistant cells express CD68, or macrosialin. As the human CD68 promoter directs expression to myeloid and dendritic cells (24), our results are consistent with the possibility that CD68-expressing cell types make a contribution to in vivo BAFF activity, and that BAFF expression in the same cells limits BAFF activity. It is unlikely that incomplete suppression of BAFF function by transgenic BAFF was due to insufficient expression in myeloid cells because immunoblotting suggested protein amounts that greatly exceeded those of wild-type BAFF or BAFF. The fact that the phenotypes of our BAFF transgenic mice were fairly uniform, despite wide variations in apparent levels of expression, indicates that the tissue distribution of BAFF expression, rather than its level, was limiting.
Selective effects of BAFF on T cell-dependent Ab responses
A surprising finding in our study was that transgenic expression of BAFF preferentially suppressed T cell-dependent Ab response, but had no discernible effect on the T cell-independent response. Prior studies had indicated that BAFF overexpression could augment both responses (10). Our results are consistent with the possibility that although BAFF can augment T cell-independent responses and promote T cell-independent class switch recombination (10, 22), other cytokines typically play a more dominant role. Also consistent with this notion was the lack of influence of BAFF transgene expression on serum Ig levels in unimmunized mice. The lack of reduction in serum Ig levels in BAFF transgenic mice is not surprising given the phenotype of BAFF-R+/– mice, in which B cell numbers are reduced, but serum Ig is not significantly affected (33). One interpretation of these findings is that during a T cell-dependent immune response, T cells may actually stimulate BAFF expression in APCs, thereby enhancing secretion. Support for this possibility comes from data suggesting that CD40L up-regulates BAFF expression from myeloid cells (22, 34 ). Alternatively, BAFF may act directly on T cells in ways that affect the immune response (35, 36). In humans, BAFF can form heterotrimers with APRIL (19). It is unlikely that BAFF works primarily by suppressing APRIL bioactivity because, if this result were so, the phenotype of our BAFF transgenic mice should tend to mimic the phenotype in APRIL-deficient mice. However, this effect is not the case. APRIL-deficient mice have elevated T cell-dependent immune responses, defective responses to T cell-independent type 1 Ags, such as hapten-LPS conjugates, and normal responses to T cell-independent type 2 Ags such as Ficoll conjugates (37).
Given that BAFF can interact with both BAFF-R and TACI in mice, it is significant that the phenotype of BAFF transgenic mice resembles closely that of mice that have reduced BAFF-R levels, such as BAFF-R+/– mice (33, 36, 38) and mice heterozygous for the spontaneous BAFF-R mutation in A/WySnJ mice (5). These mutants have reduced B cell numbers and normal serum Ig levels. In contrast, B cell responses in BAFF transgenic mice do not resemble those of mice that lack TACI, which have impaired type 2 T cell-independent responses, normal or elevated B cell numbers, and normal T cell-dependent Ab responses (39, 40). These considerations suggest that BAFF principally suppresses BAFF signaling through the BAFF-R, rather than TACI.
An additional finding in our study was that BAFF transgenic mice had normal T cell-independent type 2 Ab responses despite their reduced levels of B-1 and MZ B cells. Although type 2 T cell-independent responses are believed to be derived from B-1 or MZ B cells (41, 42, 43, 44), mutations that diminish B-1 or MZ B cell numbers often do not reduce T cell-independent Ab responses (45, 46). This implies that certain induced responses, such as to TNP-Ficoll, may be mainly derived from B-2 type cells.
BAFF overexpressing mice
In our experiments, we have generated a new series of BAFF transgenic mice using the human CD68 promoter, a strategy that was designed to overexpress BAFF from cells that are specialized in BAFF expression. Compared with previously studied BAFF transgenic mice, we find both similarities and differences (7, 9, 47). Prior studies used promoters directing expression to liver (7), B cells themselves (47), or ubiquitously (9). Given these differences, the results have been remarkably consistent, including evidence of increased peripheral B cell numbers, expansion of the MZ B cell compartment, hypergammaglobulinemia, particularly elevated levels of IgA, and evidence of systemic autoimmunity. These findings are consistent with a role for BAFF predominantly as a soluble, rather than membrane bound, cytokine.
In our study, we find expansion of B-1 cells in BAFF overexpressing mice, whereas in two of four prior studies no such expansion was described. B-1 cells were elevated in mice in which Ig promoter and enhancer elements drove BAFF expression (47) or in the salivary glands of mice expressing BAFF under 1 antitrypsin promoter/apoE enhancer regulatory elements (48). BAFF is known to be nonessential for B-1 cell development and maintenance (2), however, our study shows 50% reduction in B-1 cell numbers in BAFF-deficient animals. Gross et al. (1) noted a similar 50% decrease in B-1 cells in an independent line of BAFF–/– mice, though given the variance, the change in that study was not judged to be statistically significant. Thus, it appears that B-1 cells can be modulated by BAFF levels, although not to the same extent as B-2 cells (>90% reduced in BAFF-deficient animals). As CD68 is not highly expressed in B cells, our results suggest the possibility that local, high level expression of BAFF in myeloid cells may nevertheless augment B-1 cell survival, expansion, or differentiation.
Modulation of BAFF to BAFF ratios
The levels of BAFF expressed, as mRNA, or as protein on cell membranes or released from cells, can vary widely depending on cellular stimulation with mitogens, microbial products, or cytokines (21, 32). However, prior studies did not take into account the differing amounts and functions of BAFF and BAFF isoforms. We find that in addition to overall levels of BAFF, the BAFF to BAFF ratio can also be modulated in myeloid cells by cell stimulus. The relative RNA levels of the two BAFF isoforms are sufficiently similar to one another in that this type of regulation is likely to have major consequence on BAFF activity. As BAFF appears to be resistant to cleavage from cell membranes, it can possibly form multimers with BAFF (or APRIL) that retain activity, but are restricted to act locally by cell contact. The in vivo data presented in this study suggest changing the BAFF to BAFF ratio toward excess BAFF would lead to less bioactive BAFF and consequently less B cell survival.
BAFF has been implicated in the exacerbation or cause of systemic autoimmunity and in regulating immune tolerance thresholds (13, 14, 49), but is also required for B cell survival (2, 5), suggesting that the balance between these activities is crucial. BAFF likely plays a key role in this regulation because it can directly influence BAFF activities through its three modes of action (18): 1) consuming BAFF mRNA by alternative splicing, 2) forming nonfunctional BAFF1/BAFF2 heterotrimers, and 3) preventing release from the plasma membrane of heteromultimerized molecules, possibly including forms with two or more active BAFF subunits. However, it is not excluded that BAFF may have unique, independent functions. In transfection experiments using 293 host cells, BAFF can form cell surface bound homomultimers, but it is unclear whether this isoform alone has an independent in vivo function. To date, we have been unable to find any obvious differences between BAFF–/– mice and BAFF–/– carrying BAFF transgenes.
Acknowledgments
We thank Min Lim for excellent technical assistance and Louise McHeyzer Williams for reading the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grants RO1GM44809, RO3AR050017 (to A.L.G.), and PO1AG01743 from the National Institutes of Health (to D.N.).
2 Address correspondence and reprint requests to Dr. David Nemazee, Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: nemazee@scripps.edu
3 Abbreviations used in this paper: BAFF, B cell-activating factor belonging to the TNF family; MZ, marginal zone; TNP, trinitrophenyl.
Received for publication February 3, 2005. Accepted for publication April 20, 2005.
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