B-cell maturation protein, which binds the tumor necrosis factor family members BAFF and APRIL, is dispensable for humoral immune responses

B-cell maturation protein (BCMA) is a member of the tumor necrosis factor (TNF) receptor family and is expressed in B lymphocytes. BCMA binds two TNF family members, BAFF and APRIL, that stimulate cellular proliferation. BAFF in particular has been shown to influence B-cell survival and activation, and transgenic mice overexpressing BAFF have a lupus-like autoimmune disorder. We have inactivated BCMA in the mouse germ line. BCMA(-/-) mice have normal B-cell development, and the life span of mutant B lymphocytes is comparable to that of wild-type B cells. The humoral immune responses of BCMA(-/-) mice to T-cell-independent antigens as well as high and low doses of T-cell-dependent antigens are also intact. In addition, mutant mice have normal splenic architecture, and germinal centers are formed during an ongoing immune response. These data suggest a functional redundancy of BCMA in B-cell physiology that is probably due to the presence of TACI, another TNF receptor family member that is expressed on B cells and that can also bind BAFF and APRIL.     

Development of autoimmunity in IL-14alpha-transgenic mice

Multiple genetic loci contribute to the development of systemic lupus erythematosus (SLE). In murine models for SLE, various genes on chromosome four have been implicated. IL-14 is a cytokine originally identified as a B cell growth factor. The il14 gene is located on chromosome 4. IL-14alpha is a cytokine encoded by the plus strand of the IL-14 gene using exons 3-10. The expression of IL-14alpha is increased in (NZB x NZW)F1 mice. In this study, we produced IL-14alpha-transgenic mice to study the role of IL-14alpha in the development of autoimmunity. At age 3-9 mo, IL-14alpha-transgenic mice demonstrate increased numbers of B1 cells in the peritoneum, increased serum IgM, IgG, and IgG 2a and show enhanced responses to T-dependent and T-independent Ags compared with littermate controls. At age 9-17 mo, IL-14alpha-transgenic mice develop autoantibodies, sialadenitis, as in Sj?gren's syndrome, and immune complex-mediated nephritis, as in World Health Organization class II SLE nephritis. Between the ages 14-18 mo, 95% of IL-14alpha-transgenic mice developed CD5+ B cell lymphomas, consistent with the lymphomas seen in elderly patients with Sj?gren's syndrome and SLE. These data support a role for IL-14alpha in the development of both autoimmunity and lymphomagenesis. These studies may provide a genetic link between these often related disorders.     

B cell abnormalities in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic, multisystem autoimmune disease characterized by the differentiation of short- and long-lived immunoglobulin secreting plasma cells that secrete pathogenic autoantibodies. Ectopic germinal centers and plasma cells secreting autoantibodies have been observed in lupus nephritis kidneys. Candidate genetic susceptibility loci for SLE include genes that affect differentiation and survival of plasma cells, such as those that influence activation, proliferation, cytokine and chemokine secretion/responsiveness, and apoptosis of the T and B cells that are involved in humoral immunity generated in germinal centers, as well as genes that are involved in presentation and clearance of apoptotic material and autoantigens by antigen presenting cells and other phagocytes. Emerging data have demonstrated that B lymphocytes are active participants in humoral immune responses that lead to T-dependent and T-independent differentiation of immunoglobulin-secreting plasma cells by homotypic CD154-CD40 interactions as well as continued stimulation by B cell activating factor through B cell maturation antigen, B cell activating factor receptor and transmembrane activater.     

Neutrophils Contribute to Excess Serum BAFF Levels and Promote CD4+ T Cell and B Cell Responses in Lupus-Prone Mice

Despite increased frequencies of neutrophils found in autoimmune diseases such as systemic lupus erythematosus (SLE), how they contribute to disease pathogenesis and the mechanisms that affect the accumulation of neutrophils are poorly understood. The aim of this study was to identify factors in autoantibody-mediated autoimmunity that controls the accumulation of spleen resident neutrophils and to determine whether neutrophils contribute to abnormal B cell responses. Increased levels of the cytokine BAFF have been linked to loss of B cell tolerance in autoimmunity, but the cellular source responsible for excess BAFF is unknown. B cell maturation antigen (BCMA) is a receptor for BAFF and is critical for the survival of bone marrow plasma cells. Paradoxically, BCMA deficiency exacerbates the formation of autoantibody-secreting plasma cells in spleens of lupus-prone mice and the reasons for this effect are not understood. Here we analyzed the phenotype, localization and function of neutrophils in spleens of healthy mice and congenic lupus-prone mice, and compared mice sufficient or deficient in BCMA expression. Neutrophils were found to be significantly increased in frequency and activation status in spleens of lupus-prone mice when BCMA was absent. Furthermore, neutrophils localized within T cell zones and enhanced CD4⁺ T cell proliferation and IFNγ production through the production of BAFF. Reduced BAFF and IFNγ serum levels, decreased frequencies of IFNγ-producing T cells, germinal center B cells, and autoantibody production after neutrophil depletion indicated the involvement of neutrophils in these autoimmune traits. Thus, we have identified a novel role for BCMA to control excess BAFF production in murine lupus through restraining the accumulation of BAFF-producing neutrophils. Our data suggests that devising therapeutic strategies to reduce neutrophils in autoimmunity may decrease BAFF levels and ameliorate disease.     

In vitro and in vivo activation induces BAFF and APRIL expression in B cells

B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL) play key roles in peripheral B cell survival, maturation, and differentiation. BAFF and APRIL are produced by a variety of cell types such as macrophages/monocytes and dendritic cells. Our analysis shows that BAFF mRNA is also expressed in all B cell subsets isolated from bone marrow, spleen, and peritoneal cavity of BALB/c mice. APRIL expression is restricted to early stages of B cell development in the bone marrow and the peritoneal B1 subset. Stimulation of B2 and B1 cells with LPS or CpG-oligodeoxynucleotides induced MyD88-dependent plasma cell differentiation and intracellular expression of BAFF and APRIL. Furthermore, activation of B cells up-regulated membrane expression of BAFF. The finding that in vitro activation of B cells is inhibited by the antagonist transmembrane activator and calcium modulator ligand interactor Ig, indicates that BAFF and/or APRIL are released into the culture supernatants. It shows that B cell survival, proliferation, and differentiation are supported by an autocrine pathway. In vivo activation of B cells with a T-dependent Ag- induced BAFF expression in germinal center B cells. In (NZB x NZW)F(1) mice with established autoimmune disease, marginal zone, germinal center B cells, as well as splenic plasma cells expressed high levels of BAFF. In (NZB x NZW)F(1) mice, the continuous activation of B cells and thus overexpression of BAFF and APRIL may contribute to the development of autoimmune disease.     

B cell-activating factor belonging to the TNF family (BAFF)-R is the principal BAFF receptor facilitating BAFF costimulation of circulating T and B cells

BAFF (B cell-activating factor belonging to the TNF family) is a cell survival and maturation factor for B cells, and overproduction of BAFF is associated with systemic autoimmune disease. BAFF binds to three receptors, BAFF-R, transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and B cell maturation Ag (BCMA). Using specific mAbs, BAFF-R was found to be the predominant BAFF receptor expressed on peripheral B cells, in both humans and mice, and antagonist mAbs to BAFF-R blocked BAFF-mediated costimulation of anti- micro responses. The other BAFF receptors showed a much more restricted expression pattern, suggestive of specialized roles. BCMA was expressed by germinal center B cells, while TACI was expressed predominantly by splenic transitional type 2 and marginal zone B cells, as well as activated B cells, but was notably absent from germinal center B cells. BAFF was also an effective costimulator for T cells, and this costimulation occurs entirely through BAFF-R. BAFF-R, but not TACI or BCMA, was expressed on activated/memory subsets of T cells, and T cells from BAFF-R mutant A/WySnJ mice failed to respond to BAFF costimulation. Thus, BAFF-R is important not only for splenic B cell maturation, but is the major mediator of BAFF-dependent costimulatory responses in peripheral B and T cells.     

Cutting edge: germinal centers formed in the absence of B cell-activating factor belonging to the TNF family exhibit impaired maturation and function

Germinal centers (GCs) form in B cell follicles and require specific signals for development and maintenance. B cell-activating factor belonging to the TNF family (BAFF) is a fundamental B cell survival factor and therefore may influence GC reactions and subsequent Ab responses. To test this possibility, the effect of BAFF neutralization in immunized mice was assessed. Using B cell maturation Ag-Fc, we demonstrate that BAFF blockade does not inhibit GC formation or somatic hypermutation. However, GCs in B cell maturation Ag-Fc-treated mice dissipated more rapidly than those of control mice and did not form a mature follicular dendritic cell reticulum. Examination of immunized BAFF-null mice validated the BAFF-independent nature of GC formation. Furthermore, Ab responses, including high-affinity responses, were attenuated. This is the first evidence that BAFF is required for maintenance, but not initiation, of the GC reaction, and it further hints that somatic hypermutation within the GC and selection of Ag-specific high-affinity Ab could be uncoupled.     

BAFF induces a hyper-IgA syndrome in the intestinal lamina propria concomitant with IgA deposition in the kidney independent of LIGHT

BAFF is a peripheral B cell survival factor and can mediate antibody (Ab) class switching. Over-expression of BAFF in mice results in B cell hyperplasia, elevated serum immunoglobulin (Ig), spontaneous germinal centre (GC) reactions and mild glomerulonephritis (GN). Here we show that, in addition to driving excessive levels of serum IgA, BAFF over-expression results in increased IgA levels within the intestinal lamina propria (LP) and deposition of IgA immune complexes in the renal glomerular mesangium. LIGHT has been previously shown to mediate a similar phenotype via signaling through the lymphotoxin-beta receptor (LTbetaR). We evaluated if LIGHT and BAFF cooperate in the etiology of a hyper-IgA syndrome in BAFF-overexpressing transgenic (BAFF-Tg) mice. We find that LIGHT-deficient BAFF-Tg mice exhibit similar levels of IgA in the serum, gut and kidney and develop nephritis to the same degree as LIGHT-sufficient BAFF-Tg mice. Therefore, in the context of BAFF over-expression, LIGHT is dispensable for the generation of a hyper-IgA syndrome accompanied by nephritis.     

BAFF/APRIL inhibition decreases selection of naive but not antigen-induced autoreactive B cells in murine systemic lupus erythematosus

BAFF inhibition is a new B cell-directed therapeutic strategy for autoimmune disease. Our purpose was to analyze the effect of BAFF/APRIL availability on the naive and Ag-activated B cell repertoires in systemic lupus erythematosus, using the autoreactive germline D42 H chain (glD42H) site-directed transgenic NZB/W mouse. In this article, we show that the naive Vκ repertoire in both young and diseased glD42H NZB/W mice is dominated by five L chains that confer no or low-affinity polyreactivity. In contrast, glD42H B cells expressing L chains that confer high-affinity autoreactivity are mostly deleted before the mature B cell stage, but are positively selected and expanded in the germinal centers (GCs) as the mice age. Of these, the most abundant is VκRF (Vκ16-104*01), which is expressed by almost all IgG anti-DNA hybridomas derived from the glD42H mouse. Competition with nonautoreactive B cells or BAFF/APRIL inhibition significantly inhibited selection of glD42H B cells at the late transitional stage, with only subtle effects on the glD42H-associated L chain repertoire. However, glD42H/VκRF-encoded B cells were still vastly overrepresented in the GC, and serum IgG anti-DNA Abs arose with only a slight delay. Thus, although BAFF/APRIL inhibition increases the stringency of negative selection of the naive autoreactive B cell repertoire in NZB/W mice, it does not correct the major breach in B cell tolerance that occurs at the GC checkpoint.     

BAFF augments certain Th1-associated inflammatory responses

B cell-activating factor belonging to the TNF family (BAFF; BLyS) is a critical regulator of B cell maturation and survival, and its overexpression in BAFF transgenic (Tg) mice results in the development of autoimmune disorders. BAFF also affects T cell function through binding to one of the BAFF receptors, BAFF-R. Using BAFF Tg mice, we examined a typical Th1-mediated response, the cutaneous delayed-type hypersensitivity reaction, and found a much greater degree of paw swelling and inflammation than in control mice. Importantly, delayed-type hypersensitivity scores correlated directly with BAFF levels in serum. Conversely, in a Th2-mediated model of allergic airway inflammation, BAFF Tg mice were largely protected and showed markedly reduced Ag-specific T cell proliferation and eosinophil infiltration associated with the airways. Thus, local and/or systemically distributed BAFF affects Th1 and Th2 responses and impacts on the course of some T cell-mediated inflammatory reactions. Our results are consistent with the idea that BAFF augments T cell as well as B cell responses, particularly Th1-type responses. Results in BAFF Tg mice may reflect the situation in certain autoimmune patients or virally infected individuals, because BAFF levels in blood are comparable.     

Paucity of clinical disease despite serological autoimmunity and kidney pathology in lupus-prone New Zealand mixed 2328 mice deficient in BAFF

Constitutive overexpression of B cell-activating factor belonging to the TNF family (BAFF) promotes development of systemic lupus erythematosus (SLE), and treatment of SLE mice with BAFF antagonists ameliorates disease. To determine whether SLE can develop de novo in BAFF-deficient hosts, BAFF-deficient New Zealand Mixed (NZM) 2328 (NZM.Baff(-/-)) mice were generated. In NZM.Baff(-/-) mice, spleen B cells (including CD5(+) B1a and CD5(-) B1b B cells), germinal centers, Ig-secreting cells, and T cells were reduced in comparison to NZM.Baff(+/+) mice. Serum total Ig and autoantibody levels were reduced at 4-6 mo but approached wild-type levels with increasing age, indicating that autoreactive B cells can survive and secrete autoantibodies despite the complete absence of BAFF. At least some of these autoantibodies are nephrophilic in that glomerular deposition of total IgG and IgG1 (but not of IgG2a, IgG2b, or C3) was substantial in NZM.Baff(-/-) mice by 12-13 mo of age. Despite proliferative glomerulonephritis, highlighted by widespread glomerular hyaline thrombi, being common among NZM.Baff(-/-) mice by 6-7 mo of age, severe proteinuria and mortality were greatly attenuated. These results demonstrate that the lifelong absence of BAFF does not protect NZM 2328 mice from serological autoimmunity and renal pathology. Nevertheless, the character of the renal pathology is altered, and the mice are largely spared from clinically overt disease (severe proteinuria and premature death). These observations may have profound ramifications for the use of BAFF antagonists in human SLE and related diseases.     

A role for lymphotoxin in primary Sjogren's disease

The etiology of salivary gland injury in primary Sj?gren's disease is not well understood. We have previously described a mouse model of Sj?gren's disease, IL-14α transgenic (IL14αTG) mice, which reproduces many of the features of the human disease. We now demonstrate a critical role for lymphotoxin α (LTA) in the pathogenesis of Sj?gren's disease in IL14αTG mice. IL14αTG mice express LTA mRNA in their salivary glands and spleen and produce soluble LTA protein in their salivary secretions. When IL14αTG mice were crossed with LTA(-/-) mice, the IL14αTG.LTA(-/-) mice retained normal salivary gland secretions and did not develop either lymphocytic infiltration of their salivary glands or secondary lymphomas. However, both IL14αTG and IL14αTG.LTA(-/-) mice produced similar amounts of IFN-α and had similar deposition of autoantibodies in their salivary glands. Both IL14α and IL14α/LTA(-/-) mice had similar B cell responses to T-dependent and T-independent Ags, L-selectin expression, and expression of RelA, RelB, and NF-κB2 in their spleens. These studies suggest that LTA plays a critical role in the local rather than systemic inflammatory process of Sj?gren's disease. Furthermore, local production of soluble LTA in the salivary glands of IL14αTG mice is necessary for the development of overt Sj?gren's disease. Autoantibody deposition alone is not sufficient to produce salivary gland dysfunction. We also demonstrate that LTA is increased in the salivary gland secretions and sera of patients with Sj?gren's disease, further strengthening the biological relevance of the IL14αTG model to understanding the pathogenesis of human disease.     

Mechanism of BLyS action in B cell immunity.

The B lymphocyte stimulator (BLyS), also known as BAFF, THANK, TALL-1 and zTNF4, is the most recent addition to the tumor necrosis factor family (TNF) ligands and has a unique role in B cell immunity. Its requirement for the humoral immune response is evident in mice lacking BlyS, which exhibit profound deficiencies in peripheral B cell development and maturation. It regulates the antibody response, as shown in mice overexpressing BLyS, which develop autoimmune manifestations resulting from peripheral B cell expansion and differentiation. Attenuation of apoptosis appears to underlie BLyS action in B cells. However, elucidation of the mechanism of BLyS has proven to be more challenging, because BLyS binds three different TNF receptors (TACI/BCMA/BAFF-R) and shares overlapping functions with a related TNF ligand, APRIL. The unique role of BLyS in B cell development and differentiation and the pathogenesis of autoimmune diseases, systemic lupus erythematosus (SLE) in particular, makes the study of BLyS and its downstream targets attractive in the development of novel therapies.     

B cell maturation antigen deficiency exacerbates lymphoproliferation and autoimmunity in murine lupus

Systemic lupus erythematosus and its preclinical lupus-prone mouse models are autoimmune disorders involving the production of pathogenic autoantibodies. Genetic predisposition to systemic lupus erythematosus results in B cell hyperactivity, survival of self-reactive B cells, and differentiation to autoantibody-secreting plasma cells (PCs). These corrupt B cell responses are, in part, controlled by excess levels of the cytokine BAFF that normally maintains B cell homeostasis and self-tolerance through limited production. B cell maturation Ag (BCMA) is a receptor for BAFF that, under nonautoimmune conditions, is important for sustaining enduring Ab protection by mediating survival of long-lived PCs but is not required for B cell maturation and homeostasis. Through analysis of two different lupus-prone mouse models deficient in BCMA, we identify BCMA as an important factor in regulating peripheral B cell expansion, differentiation, and survival. We demonstrate that a BCMA deficiency combined with the lpr mutation or the murine lupus susceptibility locus Nba2 causes dramatic B cell and PC lymphoproliferation, accelerated autoantibody production, and early lethality. This study unexpectedly reveals that BCMA works to control B cell homeostasis and self-tolerance in systemic autoimmunity.     

The Sjogren's syndrome-associated autoantigen Ro52 is an E3 ligase that regulates proliferation and cell death

Patients affected by Sj?gren's syndrome and systemic lupus erythematosus (SLE) carry autoantibodies to an intracellular protein denoted Ro52. Although the serologic presence of Ro52 autoantibodies is used clinically for diagnostic purposes, the function of the protein or why it is targeted as an autoantigen in several rheumatic conditions has not been elucidated. In this study, we show that the expression of Ro52 is significantly increased in PBMC of patients with Sj?gren's syndrome and SLE, and demonstrate that Ro52 is a RING-dependent E3 ligase involved in ubiquitination. Overexpression of Ro52, but not of Ro52 lacking the RING domain, in a mouse B cell line lead to decreased growth in steady state and increased cell death after activation via the CD40 pathway. The role of Ro52 in activation-mediated cell death was further confirmed as a reduction in Ro52 expression restored cell viability. These findings suggest that the increased expression of the Ro52 autoantigen in patients may be directly involved in the reduced cellular proliferation and increased apoptotic cell death observed in Sj?gren's syndrome and SLE, and may thus contribute to the autoantigenic load and induction of autoimmune B and T cell responses observed in rheumatic patients.     

TNF family member B cell-activating factor (BAFF) receptor-dependent and -independent roles for BAFF in B cell physiology

The cytokine TNF family member B cell-activating factor (BAFF; also termed BLyS) is essential for B cell generation and maintenance. Three receptors have been identified that bind to BAFF: transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI); B cell maturation Ag (BCMA); and BAFF-R. Recently, it was shown that A/WySnJ mice, which contain a dramatically reduced peripheral B cell compartment due to decreased B cell life span, express a mutant BAFF-R. This finding, together with normal or enhanced B cell generation in mice deficient for BCMA or TACI, respectively, suggested that the interaction of BAFF with BAFF-R triggers signals essential for the generation and maintenance of mature B cells. However, B cells in mice deficient for BAFF differ phenotypically and functionally from A/WySnJ B cells. Residual signaling through the mutant BAFF-R could account for these differences. Alternatively, dominant-negative interference by the mutant receptor could lead to an overestimation of the importance of BAFF-R. To resolve this issue, we generated BAFF-R-null mice. Baff-r(-/-) mice display strongly reduced late transitional and follicular B cell numbers and are essentially devoid of marginal zone B cells. Overexpression of Bcl-2 rescues mature B cell development in Baff-r(-/-) mice, suggesting that BAFF-R mediates a survival signal. CD21 and CD23 surface expression are reduced on mature Baff-r(-/-) B cells, but not to the same extent as on mature B cells in BAFF-deficient mice. In addition, we found that Baff-r(-/-) mice mount significant, but reduced, Ag-specific Ab responses and are able to form spontaneous germinal centers in mesenteric lymph nodes. The reduction in Ab titers correlates with the reduced B cell numbers in the mutant mice.     

Complementary effects of TNF and lymphotoxin on the formation of germinal center and follicular dendritic cells

The formation of germinal centers (GC) around follicular dendritic cells (FDC) is a critical step in the humoral immune responses that depends on the cooperative effects of B cells and T cells. Mice deficient in either TNF or lymphotoxin (LT) fail to form both GC and FDC network in B cell follicles. To test a potential complementary effect of TNF and LT, a mixture of bone marrow cells from TNF(-/-) mice and LT alpha(-/-) mice was transferred into irradiated LT alpha(-/-) mice or TNF(-/-) mice. Interestingly, the formation of both GC and FDC clusters in B cell follicles was restored in such chimeric mice, suggesting that TNF and LT from different cells could complement one another. To identify the exact contributions of each subset to the complementary effect of TNF and LT, different sources of T and B cells from LT alpha(-/-) mice or TNF(-/-) mice were used for reconstitution. Our study demonstrates that either T or B cell-derived TNF is sufficient to restore FDC/GC in the presence of LT-expressing B cells. However, TNF itself is not required for GC reactions if the FDC network is already intact. Thus, the development and maintenance of these lymphoid structures depend on a delicate interaction between TNF and LT from different subsets of lymphocytes.     

Disturbance of cytokine networks in Sj?gren's syndrome

The difficulty in predicting the consequences of interactions between different cytokine networks has increased with the expansion of the T helper (Th) cell universe and the discovery of numerous B lymphocyte-derived cytokines. Consequently, it is now difficult to conceptualize a straightforward view of the contribution of these disturbances to the pathogenesis of primary Sj?gren's syndrome (SS). Th1 cells, which produce interferon-γ and IL-2, and Th17 cells, which make IL-17 and TNF-α, have been cast in the leading roles of the play. However, the complex role of T-cell subsets in SS is accentuated by the reciprocal effects of Th17 cells and regulatory T cells found in salivary glands of SS patients. Furthermore, B lymphocyte polarization into type-1 B effector (Be1) and Be2 cells and B-cell modulating factors of the TNF family, most notably the B-cell-activating factor (BAFF), and their prominent role in SS are additional complicating factors. Whereas Th17 cells orchestrate autoreactive germinal centers, local BAFF would repress the generation of Th17 cells. Such new insights into interconnected cytokines in primary SS may lead to new treatments for these patients.     

Disturbance of cytokine networks in Sjögren's syndrome

The difficulty in predicting the consequences of interactions between different cytokine networks has increased with the expansion of the T helper (Th) cell universe and the discovery of numerous B lymphocyte-derived cytokines. Consequently, it is now difficult to conceptualize a straightforward view of the contribution of these disturbances to the pathogenesis of primary Sj?gren's syndrome (SS). Th1 cells, which produce interferon-γ and IL-2, and Th17 cells, which make IL-17 and TNF-α, have been cast in the leading roles of the play. However, the complex role of T-cell subsets in SS is accentuated by the reciprocal effects of Th17 cells and regulatory T cells found in salivary glands of SS patients. Furthermore, B lymphocyte polarization into type-1 B effector (Be1) and Be2 cells and B-cell modulating factors of the TNF family, most notably the B-cell-activating factor (BAFF), and their prominent role in SS are additional complicating factors. Whereas Th17 cells orchestrate autoreactive germinal centers, local BAFF would repress the generation of Th17 cells. Such new insights into interconnected cytokines in primary SS may lead to new treatments for these patients.