Urinary B-cell-activating factor of the tumour necrosis factor family (BAFF) in systemic lupus erythematosus.

INTRODUCTION: We examined the clinical relevance of urinary concentrations of B-cell-activating factor of the tumour necrosis factor family (BAFF) and a proliferation-inducing ligand (APRIL) in systemic lupus erythematosus (SLE). METHODS: We quantified urinary BAFF (uBAFF) by enzyme-linked immunosorbent assay in 85 SLE, 28 primary Sjögren syndrome (pSS), 40 immunoglobulin A nephropathy (IgAN) patients and 36 healthy controls (HCs). Urinary APRIL (uAPRIL) and monocyte chemoattractant protein 1 (uMCP-1) were also quantified. Overall and renal SLE disease activity were assessed using the Systemic Lupus Erythematosus Disease Activity Index 2000. RESULTS: uBAFF was detected in 12% (10/85) of SLE patients, but was undetectable in HCs, IgAN and pSS patients. uBAFF was detectable in 28% (5/18) of SLE patients with active nephritis vs 5/67 (7%) of those without ( p = 0.03), and uBAFF was significantly higher in active renal patients ( p = 0.02) and more likely to be detected in patients with persistently active renal disease. In comparison, uAPRIL and uMCP-1 were detected in 32% (25/77) and 46% (22/48) of SLE patients, respectively. While no difference in proportion of samples with detectable uAPRIL was observed between SLE, HCs and IgAN patients, both uAPRIL and uMCP-1 were significantly detectable in higher proportions of patients with active renal disease. CONCLUSIONS: uBAFF was detectable in a small but a significant proportion of SLE patients but not in other groups tested, and was higher in SLE patients with active renal disease.

The BAFF receptor TACI controls IL-10 production by regulatory B cells and CLL B cells.

Interleukin (IL)-10-producing B cells (B10 cells) have emerged as important regulatory elements with immunosuppressive roles. Chronic lymphocytic leukemia (CLL) B cells also secrete IL-10 and share features of B10 cells, suggesting a possible contribution of CLL B cells to immunosuppression in CLL patients. Factors controlling the emergence of B10 cells are not known. B-cell-activating factor of the tumor necrosis factor (TNF) family (BAFF) is critical for B-cell maturation and survival, and is implicated in the development and progression of CLL. We sought to investigate the role of BAFF in the emergence of IL-10-producing regulatory B cells in healthy donors and CLL patients. Here, we report that BAFF signaling promotes IL-10 production by CLL B cells in a mouse model of CLL and in CLL patients. Moreover, BAFF-mediated IL-10 production by normal and CLL B cells is mediated via its receptor transmembrane activator and cyclophilin ligand interactor. Our work uncovered a major targetable pathway important for the generation of regulatory B cells that is detrimental to immunity in CLL.

Cytokine-driven loss of plasmacytoid dendritic cell function in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5(+)CD19(+) B cells in the peripheral blood, and in primary and secondary lymphoid organs. A major complication associated with CLL is severe recurrent infections, which are often fatal. Vulnerability to infection is due to a wide variety of immunological defects, yet the initiating events of immunodeficiency in CLL are unclear. Using CLL patient samples and a mouse model of CLL, we have discovered that plasmacytoid dendritic cells (pDCs), which underpin the activity of effector immune cells critical for anti-viral immunity and anti-tumor responses, are reduced in number and functionally impaired in progressive CLL. As a result, the levels of interferon alpha (IFNα) production, a cytokine critical for immunity, are markedly reduced. Lower pDC numbers with impaired IFNα production was due to the decreased expression of FMS-like tyrosine kinase 3 receptor (Flt3) and Toll-like receptor 9 (TLR9), respectively. Reduced Flt3 expression was reversed using inhibitors of TGF-ß and TNF, an effect correlating with a reduction in tumor load. Defects in pDC numbers and function offer new insight into mechanisms underpinning the profound immunodeficiency affecting CLL patients and provide a potentially novel avenue for restoring immunocompetency in CLL.

Association of serum B cell activating factor from the tumour necrosis factor family (BAFF) and a proliferation-inducing ligand (APRIL) with central nervous system and renal disease in systemic lupus erythematosus.

INTRODUCTION: The objective of this study is to determine whether serum concentrations of B cell activating factor from the tumour necrosis factor family (BAFF) and/or a proliferation-inducing ligand (APRIL) are associated with clinical manifestations of systemic lupus erythematosus (SLE). METHODS: BAFF and APRIL concentrations were quantified using a commercial ELISA in serum samples obtained at the time of clinical assessment in 98 patients, and on 245 samples from 75 of these patients followed prospectively. RESULTS: Serum BAFF was significantly increased, and APRIL decreased, in patients with either renal or central nervous system (CNS) lupus. In contrast, in cross-sectional analysis, there was no correlation between disease activity (SLEDAI-2k) and serum BAFF or APRIL. In longitudinal follow-up, there was no association between changes in serum BAFF or APRIL and changes in SLEDAI-2k, or between baseline serum BAFF or APRIL and subsequent changes in SLEDAI-2k. However, between-visit changes in BAFF were significantly different in patients with increases in SLEDAI-2k ≥ 4, compared to patients whose SLEDAI-2k did not change. CONCLUSIONS: Although neither serum BAFF nor APRIL correlated with disease activity in the overall population, elevated serum BAFF and reduced APRIL may be markers of renal and CNS disease in SLE patients.

Maturation of marginal zone and follicular B cells requires B cell activating factor of the tumor necrosis factor family and is independent of B cell maturation antigen.

B cells undergo a complex series of maturation and selection steps in the bone marrow and spleen during differentiation into mature immune effector cells. The tumor necrosis factor (TNF) family member B cell activating factor of the TNF family (BAFF) (BLyS/TALL-1) plays an important role in B cell homeostasis. BAFF and its close homologue a proliferation-inducing ligand (APRIL) have both been shown to interact with at least two receptors, B cell maturation antigen (BCMA) and transmembrane activator and cyclophilin ligand interactor (TACI), however their relative contribution in transducing BAFF signals in vivo remains unclear. To functionally inactivate both BAFF and APRIL, mice transgenic for a soluble form of TACI were generated. They display a developmental block of B cell maturation in the periphery, leading to a severe depletion of marginal zone and follicular B2 B cells, but not of peritoneal B1 B cells. In contrast, mice transgenic for a soluble form of BCMA, which binds APRIL, have no detectable B cell phenotype. This demonstrates a crucial role for BAFF in B cell maturation and strongly suggests that it signals via a BCMA-independent pathway and in an APRIL-dispensable way.

Induction of germinal centers by MMTV encoded superantigen on B cells.

It has not been established whether an endogenous superantigen (SAg) expressed on B cells can induce germinal centers (GCs). An interesting model is that of mammary tumor virus encoded viral SAgs, which induce vigorous T cell proliferation and are predominantly expressed on activated B cells. We have used this model to analyze the possibility that direct stimulation of Mtv7+ DBA/2 B cells by vSAg-responsive (Vbeta6+) BALB/c T cells can give rise to GCs. Injection of BALB/c SCID mice i.v. with 2 x 10(6) DBA/2 B cells, together with LPS, followed by 2 x 10(6) BALB/c T cells induces numerous large splenic GCs within 3-5 days. The GCs are still large on day 7, but are very much reduced by day 10. B cell activation with LPS is needed for this effect. These GCs form in spite of the apparent absence of follicular dendritic cells (FDCs) as judged by staining for several FDC surface markers. Control mice receiving either BALB/c T or DBA/2 B cells + LPS alone or DBA/2 T + B cells + LPS fail to exhibit any GCs on days 3-7. Numerous small clusters of PNA+ cells, but few large GCs are observed when TNF-R(p55)-Ig is also injected, whereas LTbetaR-Ig treatment impeded the formation of aggregations of these cells even further, leaving scattered PNA+ single cells and very small clumps throughout the white pulp of the spleens. Anti-TNFalpha had no effect. These results suggest that endogenous vSAg mediated GC formation is independent of antigen trapping by FDCs.

BAFF mediates survival of peripheral immature B lymphocytes.

B cell maturation is a very selective process that requires finely tuned differentiation and survival signals. B cell activation factor from the TNF family (BAFF) is a TNF family member that binds to B cells and potentiates B cell receptor (BCR)-mediated proliferation. A role for BAFF in B cell survival was suggested by the observation of reduced peripheral B cell numbers in mice treated with reagents blocking BAFF, and high Bcl-2 levels detected in B cells from BAFF transgenic (Tg) mice. We tested in vitro the survival effect of BAFF on lymphocytes derived from primary and secondary lymphoid organs. BAFF induced survival of a subset of splenic immature B cells, referred to as transitional type 2 (T2) B cells. BAFF treatment allowed T2 B cells to survive and differentiate into mature B cells in response to signals through the BCR. The T2 and the marginal zone (MZ) B cell compartments were particularly enlarged in BAFF Tg mice. Immature transitional B cells are targets for negative selection, a feature thought to promote self-tolerance. These findings support a model in which excessive BAFF-mediated survival of peripheral immature B cells contributes to the emergence and maturation of autoreactive B cells, skewed towards the MZ compartment. This work provides new clues on mechanisms regulating B cell maturation and tolerance.

BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population.

The tumor necrosis factor (TNF) family member B cell activating factor (BAFF) binds B cells and enhances B cell receptor-triggered proliferation. We find that B cell maturation antigen (BCMA), a predicted member of the TNF receptor family expressed primarily in mature B cells, is a receptor for BAFF. Although BCMA was previously localized to the Golgi apparatus, BCMA was found to be expressed on the surface of transfected cells and tonsillar B cells. A soluble form of BCMA, which inhibited the binding of BAFF to a B cell line, induced a dramatic decrease in the number of peripheral B cells when administered in vivo. Moreover, culturing splenic cells in the presence of BAFF increased survival of a percentage of the B cells. These results are consistent with a role for BAFF in maintaining homeostasis of the B cell population.

Impaired prion replication in spleens of mice lacking functional follicular dendritic cells.

In scrapie-infected mice, prions are found associated with splenic but not circulating B and T lymphocytes and in the stroma, which contains follicular dendritic cells (FDCs). Formation and maintenance of mature FDCs require the presence of B cells expressing membrane-bound lymphotoxin-alpha/beta. Treatment of mice with soluble lymphotoxin-beta receptor results in the disappearance of mature FDCs from the spleen. We show that this treatment abolishes splenic prion accumulation and retards neuroinvasion after intraperitoneal scrapie inoculation. These data provide evidence that FDCs are the principal sites for prion replication in the spleen.

Mice transgenic for BAFF develop lymphocytic disorders along with autoimmune manifestations.

The cause of many autoimmune and inflammatory diseases is unresolved, although dysregulated production of tumor necrosis factor (TNF) family members appears to be important in many cases. BAFF, a new member of the TNF family, binds to B cells and costimulates their growth in vitro. Mice transgenic for BAFF have vastly increased numbers of mature B and effector T cells, and develop autoimmune-like manifestations such as the presence of high levels of rheumatoid factors, circulating immune complexes, anti-DNA autoantibodies, and immunoglobulin deposition in the kidneys. This phenotype is reminiscent of certain human autoimmune disorders and suggests that dysregulation of BAFF expression may be a critical element in the chain of events leading to autoimmunity.

BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth.

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family, designated BAFF (for B cell activating factor belonging to the TNF family), which is expressed by T cells and dendritic cells. Human BAFF was mapped to chromosome 13q32-34. Membrane-bound BAFF was processed and secreted through the action of a protease whose specificity matches that of the furin family of proprotein convertases. The expression of BAFF receptor appeared to be restricted to B cells. Both membrane-bound and soluble BAFF induced proliferation of anti-immunoglobulin M-stimulated peripheral blood B lymphocytes. Moreover, increased amounts of immunoglobulins were found in supernatants of germinal center-like B cells costimulated with BAFF. These results suggest that BAFF plays an important role as costimulator of B cell proliferation and function.

Both the lymphotoxin and tumor necrosis factor pathways are involved in experimental murine models of colitis.

BACKGROUND & AIMS: Membrane lymphotoxin (LT) alpha/beta, a member of the tumor necrosis factor (TNF) family of immune regulatory molecules, is involved both in the development of secondary lymphoid tissues and the maintenance of organized lymphoid tissues in the adult. Defects observed in the mucosal immune system in animals with a genetically disrupted LTalpha/beta pathway coupled with the expression of LTalpha/beta in activated T cells motivated an examination of the importance of this pathway in experimental colitis. METHODS: Soluble LTbeta receptor (LTbetaR) immunoglobulin fusion protein was used to inhibit the LTalpha/beta/light axis in two independent rodent models of colitis: CD45RBhi CD4(+)-reconstituted SCID mice and bone marrow-transplanted tg26 mice (BM --> tg26). RESULTS: Treatment with LTbetaR immunoglobulin attenuated the development of both the clinical and histological manifestations of the disease in these two murine models of colitis. Given the success of TNF inhibitors in the treatment of human Crohn's disease, the effects of LTbetaR immunoglobulin have been compared with antibody to TNF in the BM --> tg26 model, and both treatments were equally efficacious. CONCLUSIONS: The LT pathway plays a role in the development of colitis as important as that of the TNF system and, therefore, represents a potential novel intervention point for the treatment of inflammatory bowel disease.

Lymph node genesis is induced by signaling through the lymphotoxin beta receptor.

We investigated lymphotoxin (LT) and TNF function in lymph node genesis and cellular organization by manipulating LTbeta-R and TNF-R signaling. Lymph nodes developed in LTalpha-/- mice treated in utero with agonist anti-LTbeta-R monoclonal antibody. Thus, LTbeta-R signaling mediates lymph node genesis. Surprisingly, mucosal lymph nodes that can develop independently of LTalphabeta/LTbeta-R interaction were generated. Normal mice treated in utero with LTbeta-R-Ig and TNF-R55-Ig or anti-TNF lacked all lymph nodes, indicating that TNF signaling contributes to lymph node genesis. Lymph nodes generated in LTalpha-/- mice had disrupted cellular organization. Therefore, LTbeta-R signaling during gestation is not sufficient to establish normal cellular microarchitecture. We conclude that LT and TNF play critical roles in the genesis and cellular organization of lymph nodes.

The sequential role of lymphotoxin and B cells in the development of splenic follicles.

The transfer of lymphocytes into severe combined immunodeficiency (SCID) mice induces a series of histological changes in the spleen, including the appearance of mature follicular dendritic cells (FDCs). Studies were undertaken to clarify the role of lymphotoxin (LT) in this process. The results show that SCID mice have a small and partially differentiated white pulp containing marginal zone and interdigitating dendritic cells, but lacking FDCs. Transferred spleen cells can segregate into T and B cell areas shortly after their injection to SCID mice. This ability is dependent on signaling through LT-beta receptor (LT-betaR), since blocking ligand-receptor interaction in recipient SCID mice ablates the capacity of the transferred cells to segregate. A week after lymphocyte transfer, host-derived FDCs appeared in the reconstituted SCID mice. This induction of FDCs is dependent on LT-betaR signaling by B cells since LT-alpha-/- B cells are incapable of inducing development of FDCs in SCID mice, even after cotransfer of LT-alpha+/+ T cells. Therefore, LT plays at least two discrete roles in splenic organization. First, it appears that LT induces the differentiation of the white pulp to create sites for lymphocyte segregation. Second, LT expression by B cells drives the maturation of FDCs and the organization of B cell follicles.

Cytotoxic activities of recombinant soluble murine lymphotoxin-alpha and lymphotoxin-alpha beta complexes.

Human lymphotoxin-alpha (LT alpha) is found in a secreted form and on the surface of lymphocytes as a complex with a second related protein called lymphotoxin-beta (LT beta). Both secreted human LT alpha and TNF have similar biological activities mediated via the TNF receptors, whereas the cell surface LT alpha beta complex binds to a separate receptor called the LT beta receptor (LT beta R). The murine LT alpha and LT beta (mLT alpha and mLT beta) proteins have never been characterized. When recombinant mLT alpha was produced by either of several methods, the protein had a very low specific activity relative to that of human LT alpha in the conventional WEHI 164 cytotoxicity bioassay. The weak activity observed was inhibited by a soluble murine TNF-R55 Ig fusion protein (mTNF-R55-Ig), but not by mLT beta R-Ig. Coexpression of both mLT alpha and a soluble version of mLT beta in insect cells led to an LT alpha beta form that was cytotoxic in the WEHI 164 assay via the LT beta R. To determine whether natural mLT alpha-like forms with cytotoxic activity comparable to that of secreted human LT alpha were secreted from primary spleen cells, splenic lymphocytes were activated in various ways, and their supernatants were analyzed for cytotoxic activity. Using specific Abs to distinguish between mTNF and mLT, a TNF component was readily detected; however, there was no evidence for a secreted mLT alpha cytotoxic activity using this assay. Combined, these observations suggest that secreted mLT alpha may not play a role in the mouse via interactions with TNF-R55, and the ramifications of this hypothesis are discussed.

Lymphotoxin but not tumor necrosis factor functions to maintain splenic architecture and humoral responsiveness in adult mice.

To compare the function of the tumor necrosis factor (TNF) and lymphotoxin (LT)alpha/beta systems in the mature immune system, these two pathways were blocked with soluble receptor-immunoglobulin (R-Ig) fusion proteins in normal adult mice. Inhibition of LT alpha/beta signaling using LT betaR-Ig or a blocking monoclonal antibody against murine LT beta had profound effects. The spleen lacked discrete B cell follicles and the marginal zone was altered. Less marked changes were detected in lymph nodes. LT alpha/beta inhibition also prevented germinal center formation in the spleen and impaired Ig production in response to sheep red blood cells (SRBC) immunization. These results show that the LT alpha/beta system is required for the maintenance of splenic architecture and normal immune responses, and not simply for the development of peripheral immune organs during ontogeny. In contrast, inhibition of the TNF/LT alpha pathway with TNF-R55-Ig did not affect the splenic architecture or the anti-SRBC response. Splenic defects and impaired antibody responses are seen in TNF-deficient mice, suggesting that TNF is important during development. Therefore relative to TNF, the LT system has the dominant influence on splenic organization and anti-SRBC Ig formation in the adult mouse.

Surface lymphotoxin alpha/beta complex is required for the development of peripheral lymphoid organs.

For more than a decade, the biological roles and the apparent redundancy of the cytokines tumor necrosis factor (TNF) and lymphotoxin (LT) have been debated. LT alpha exists in its soluble form as a homotrimer, which like TNF only binds the TNF receptors, TNF-R55 or TNF-R75. The cell surface form of LT exists as a heteromer of LT alpha and LT beta subunits and this complex specifically binds the LT beta receptor (LT beta-R). To discriminate the functions of the LT and TNF systems, soluble LT beta-R-immunoglobulin (Ig) or TNF-R-Ig fusion proteins were introduced into embryonic circulation by injecting pregnant mice. Exposure to LT beta-R-Ig during gestation disrupted lymph node development and splenic architecture in the progeny indicating that both effects are mediated by the surface LT alpha/beta complex. These data are the first to identify a cell surface ligand involved in immune organ morphogenesis. Moreover, they unambiguously discriminate the functions of the various TNF/LT ligands, provide a unique model to study compartmentalization of immune responses and illustrate the generic utility of receptor-Ig fusion proteins for dissecting/ordering ontogenetic events in the absence of genetic modifications.

Lymphotoxin beta receptor triggering induces activation of the nuclear factor kappaB transcription factor in some cell types.

NFkappaB is a pleiotropic transcription factor capable of activating the expression of a great variety of genes critical for the immunoinflammatory response. Tumor necrosis factor alpha (TNFalpha) and lymphotoxin alpha (LTalpha, originally TNFbeta) are potent nuclear factor kappaB (NFkappaB) activators in various cell types. The LTalpha molecule, in addition to being secreted as a soluble trimer, can also form membrane-anchored heterotrimers with the LTbeta chain, another member of the TNF family. The LTalpha1beta2 heterotrimer binds a specific receptor, called the LTbeta receptor (LTbeta-R), which is also a member of the TNF receptor family. Here, we show that engagement of LTbeta-R with a soluble form of LTalpha1beta2 or with a specific anti-LTbeta-R agonistic monoclonal antibody CBE11 quickly induces activation of NFkappaB in HT-29 and WiDr human adenocarcinomas. LTbeta-R triggering activates NFkappaB and induces proliferation in WI-38 human lung fibroblasts. No NFkappaB activation is observed in human umbilical vein endothelial cells, correlating with the inability of LTbeta-R activation to induce expression of NFkappaB-dependent cell surface adhesion molecules. Thus, like several other members of the TNF receptor family, the LTbeta-R can activate NFkappaB following receptor ligation in some but not all LTbeta-R-positive cells.