The TNF family members BAFF and APRIL: the growing complexity

B cell activating factor belonging to the TNF family (BAFF) and apoptosis-inducing ligand (APRIL) are two related members of the TNF ligand superfamily. Although they share two receptors, TACI and BCMA, transgenic and knockout mice in this system reveal that their functions are not redundant. BAFF is a critical survival/maturation factor for peripheral B cells and this activity is mediated through a BAFF-specific receptor, BAFF-R. Overexpression of BAFF has been linked to autoimmune disease and aspects of B cell neoplasia. APRIL appears to play a role in T-independent type II antigen responses and T cell survival, but can also induce proliferation/survival of non-lymphoid cells. Elevated expression of APRIL has been found in some tumor cell lines and in tumor tissue libraries. Therapies designed to inhibit the BAFF and APRIL pathways holds great promise for the future.     

The role of BAFF and APRIL in rheumatoid arthritis

Development and activation of B cells quickly became clear after identifying new ligands and receptors in the tumor necrosis factor superfamily. B cell–activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are the members of membrane proteins Type 2 family released by proteolytic cleavage of furin to form active, soluble homotrimers. Except for B cells, ligands are expressed by all such immune cells like T cells, dendritic cells, monocytes, and macrophages. BAFF and APRIL have two common receptors, namely TNFR homolog transmembrane activator and Ca2+ modulator and CAML interactor (TACI) and B cell–maturation antigen. BAFF alone can also be coupled with a third receptor called BAFFR (also called BR3 or BLyS Receptor). These receptors are often expressed by immune cells in the B-cell lineage. The binding of BAFF or APRIL to their receptors supports B cells differentiation and proliferation, immunoglobulin production and the upregulation of B cell–effector molecules expression. It is possible that the overexpression of BAFF and APRIL contributes to the pathogenesis of autoimmune diseases. In BAFF transgenic mice, there is a pseudo-autoimmune manifestation, which is associated with an increase in B-lymphocytes, hyperglobulinemia, anti-single stranded DNA, and anti-double-stranded DNA antibodies, and immune complexes in their peripheral blood. Furthermore, overexpressing BAFF augments the number of peripheral B220+ B cells with a normal proliferation rate, high levels of Bcl2, and prolonged survival and hyperactivity. Therefore, in this review article, we studied BAFF and APRIL as important mediators in B-cell and discussed their role in rheumatoid arthritis.     

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.     

The Design and Characterization of Receptor-selective APRIL Variants

A proliferation-inducing ligand (APRIL), a member of the TNF ligand superfamily with an important role in humoral immunity, is also implicated in several cancers as a prosurvival factor. APRIL binds two different TNF receptors, B cell maturation antigen (BCMA) and transmembrane activator and cylclophilin ligand interactor (TACI), and also interacts independently with heparan sulfate proteoglycans. Because APRIL shares binding of the TNF receptors with B cell activation factor, separating the precise signaling pathways activated by either ligand in a given context has proven quite difficult. In this study, we have used the protein design algorithm FoldX to successfully generate a BCMA-specific variant of APRIL, APRIL-R206E, and two TACI-selective variants, D132F and D132Y. These APRIL variants show selective activity toward their receptors in several in vitro assays. Moreover, we have used these ligands to show that BCMA and TACI have a distinct role in APRIL-induced B cell stimulation. We conclude that these ligands are useful tools for studying APRIL biology in the context of individual receptor activation.     

Role of the APRIL molecule in solid tumors

The APRIL molecule, produced by immune cells, their precursors, and cancer cells, is one of the important factors that influences the process of survival and proliferation of cancer cells. In the present review, we summarize the current knowledge on the effects of APRIL on human cancer development and develop a scheme demonstrating the mechanism of the action of APRIL on solid tumors. Understanding the effects of APRIL, including the intracellular signal transduction pathway, may be key for the use of this protein as a biomarker of the cancer process. The correlations observed between APRIL levels and cancer parameters (e.g., disease stage and presence of malignant phenotypes) indicate that APRIL may play an important role, not only in the diagnostic process, but also as a therapeutic target in various cancers.     

Molecular structure, expression analysis and functional characterization of APRIL (TNFSF13) gene in bat (Vespertilio superans Thomas)

A proliferation-inducing ligand (APRIL) is a novel member of the tumor necrosis factor (TNF) superfamily, which is involved in immune regulation. In the present study, the full-length cDNA of APRIL (designated bAPRIL) from bat was cloned using RT-PCR and its biological activities have been characterized. The open reading frame (ORF) of this cDNA consists of 753 bases, encoding a protein of 250 amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other, known APRIL homologs. Real-time quantitative PCR (qPCR) analysis indicated that bAPRIL mRNA was predominantly expressed in bat lymphoid tissue spleen. The SUMO-bsAPRIL was efficiently expressed in Escherichia coli BL21 (DE3) and confirmed by SDS-PAGE and Western blot analysis. Laser scanning confocal microscopy analysis showed that bsAPRIL could bind to its receptors on B cells. In vitro, MTT assays indicated that bsAPRIL could promote the survival/proliferation of mouse splenic B cells grown with anti-mouse IgM. These findings indicate that bsAPRIL plays an important role in the survival and proliferation of B cells and has functional cross-reactivity among mammalians. The present findings may provide valuable information for research into the immune system of the bat.     

Expression and purification of APRIL by auto-induction

APRIL (A proliferation-inducing ligand) is a newly-identified member of the tumor necrosis factor family that induces pleiotropic biological responses, including immunological responses, IgA class switch and cell growth. It is associated with multiple diseases such as cancer and autoimmune diseases. High levels of APRIL mRNA can be detected in transformed cell lines and several malignant tumors; heparin sulfate proteoglycans (HSPG) are also involved in the APRIL tumor cell proliferation induction response. The interaction of APRIL and HSPG occurs through an N-terminal basic region on APRIL. We successfully expressed recombinant APRIL using an auto-induction system in Escherichia coli. By using in situ cleavage, we generated the mature form of APRIL, comprising its N-terminal basic region, which has full biological activity: receptor binding capability and proliferation induction activity.     

The TNF family member APRIL promotes colorectal tumorigenesis

The tumor necrosis factor (TNF) family member APRIL (A proliferation inducing ligand) is a disease promoter in B-cell malignancies. APRIL has also been associated with a wide range of solid malignancies, including colorectal cancer (CRC). As evidence for a supportive role of APRIL in solid tumor formation was still lacking, we studied the involvement of APRIL in CRC. We observed that ectopic APRIL expression exacerbates the number and size of adenomas in Apc^Min mice and in a mouse model for colitis-associated colon carcinogenesis. Furthermore, knockdown of APRIL in primary spheroid cultures of colon cancer cells and both mouse and human CRC cell lines reduced tumor clonogenicity and in vivo outgrowth. Taken together, our data therefore indicate that both tumor-derived APRIL and APRIL produced by non-tumor cells is supportive in colorectal tumorigenesis.     

Synovial tissues concentrate secreted APRIL

Introduction  

A proliferation-inducing ligand (APRIL) from the TNF family, owing to its role in the generation and survival of plasma cells (PCs), is currently targeted for rheumatoid arthritis (RA) treatment. However, little is known about APRIL expression in RA lesions, hampering our understanding of the way APRIL may modulate this autoimmune disease.     

Molecular structure, expression analysis and functional characterization of APRIL (TNFSF 13) in goat (Capra hircus)

A proliferation-inducing ligand (APRIL) is an important member of the tumor necrosis factor (TNF) superfamily. In the present study, a novel cDNA was isolated from the spleen of goat by RT-PCR and designated as goat APRIL (gAPRIL). The open reading frame (ORF) of this cDNA covered 753 bp, encoding a protein of 250 amino acids. Sequence comparison showed that gAPRIL contains a predicted transmembrane domain, a putative furin protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF gene in mammals. The predicted three dimensional (3D) structure of soluble part of the gAPRIL (gsAPRIL) monomer analyzed by comparative protein modeling revealed that it is very similar to its counterparts. Real-time PCR analysis revealed that gAPRIL was constitutively expressed in various tissues. Recombinant gsAPRIL fused with NusA tag was efficiently produced in Escherichia coli BL21 (DE3) and then analyzed by the SDS-PAGE as well as western blot. Laser scanning confocal microscopy analysis showed gsAPRIL could bind to its receptors. In vitro, the MTT and flow cytometric methods revealed that purified gsAPRIL protein was not only able to promote survival/proliferation of goat splenocytes, but also able to stimulate survival/proliferation of mouse B cells. These results indicated that gAPRIL plays an important role in survival/proliferation of goat splenocytes and provided a basis for investigating its potential to be used as an immunoadjuvant for enhancing vaccine efficacy and as an immunotherapeutic in goats.     

To target or not to target APRIL in systemic lupus erythematosus: that is the question!

Among the cytokines that regulate B-cell homeostasis are the TNF-like ligands B-lymphocyte stimulator (BLyS; also B-cell activation factor) and a proliferation-inducing ligand (APRIL). BLyS and APRIL share two receptors; that is, B-cell maturation antigen and transmembrane activator and CAML interactor. Therapeutic approaches using biologics are limited for treatment of lupus patients. One previously approved drug is belimumab, which antagonizes the B-cell stimulator BLyS. Atacicept, another biologic inhibiting BLyS and APRIL, was terminated for serious adverse events - raising the question of whether APRIL should be neutralized in autoimmune diseases.Treamtrakanpon and coworkers analyzed B-lymphocyte stimulator (BLyS; also B-cell activation factor) and a proliferation-inducing ligand (APRIL) expression in patients with lupus nephritis and observed a correlation with renal disease activity and APRIL serum levels [1]. In addition, the authors describe that, upon treatment with immunosuppressors, nonresponding patients had higher APRIL serum levels. They thus concluded that APRIL could be a potential biomarker for predicting difficult-to-treat cases of lupus nephritis, and propose the use of APRIL antagonists such as atacicept for treatment of lupus nephritis patients with high APRIL serum levels.These conclusions might be premature, as Treamtrakanpon and coworkers have not found a correlation between the level of APRIL in kidney tissue and renal disease activity. Another hypothesis could be that APRIL has a protective effect in autoimmune diseases. Indeed, the crucial role of BLyS in B-cell maintenance became evident by the analysis of BLyS-deficient mice displaying lower numbers of mature B cells and of BLyS transgenic mice developing severe B-cell hyperplasia. Although APRIL can trigger different B-cell responses in vitro, including proliferation and survival of human and murine B cells, it is less critical than BLyS in B-cell maintenance as APRIL knockout and transgenic mice reveal no gross abnormalities in lymphoid homeostasis [2]. In fact, APRIL was found to modulate specific B-cell responses such as IgA isotype switching, increased IgM secretion and B1 cell activity.Meanwhile, BLyS is an established promoter of B-cell-triggered autoimmmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, whereas the role of APRIL in these pathologies is rather controversial. Neutralizing BLyS with the mAb belimumab displayed a modest, although statistically significant, therapeutic effect in systemic lupus erythematosus [3,4]. But blocking both BLyS and APRIL with atacicept (TACI-Fc) was associated with a pronounced reduction of immunoglobulins, and occurrence of serious infections led to a premature termination of a phase II/III trial in lupus nephritis [5]. The combination of mycofenolate mofetil with atacicept may have contributed to the decrease of immunoglobulins. However, at acicept combined with another drug such as methotrexate in patients with rheumatoid arthritis was also associated with a significant reduction of immunoglobulins (especially IgM). In this autoimmune disease, atacicept failed to demonstrate efficacy on American College of Rheumatology 20 criteria [6]. In contrast, administration of belimumab showed a modest but significant efficacy using the same evaluation criteria in rheumatoid arthritis [7].These findings suggest distinct roles for BLyS and APRIL in lupus and other B-cell-mediated autoimmune diseases. Elevated serum levels are found for both cytokines in lupus patients, and for BLyS there is a consensus in the literature that this reflects its disease-promoting activity. Elevated APRIL serum levels, however, have been - depending on the respective study - either positively or negatively correlated with disease features [8]. One possible explanation for this discrepancy could be differences in the patient cohorts analyzed. A recent study by Jacob and colleagues analyzed a murine lupus model in APRIL-deficient mice and observed elevated numbers of splenocytes, increased autoantibody production and a tendency towards increased IgG production [9]. Notably, ectopic APRIL expression does not result - in contrast to BLyS transgenic mice - in lupus-like symptoms. In fact, we found that APRIL does dampen collagen-induced arthritis, the most common mouse model for human arthritis [10].Experimental mouse models for autoimmune diseases obviously cannot entirely mimic human diseases. Nevertheless, in vivo data are accumulating that do not support a disease-supporting role for APRIL in B-cell-mediated autoimmunity. The study by Treamtrakanpon and colleagues is putting forward the need to better elucidate the role of APRIL in B-cell-driven diseases before concluding a therapeutic approach.     

Gene cloning,expression and functional characterization of a proliferation-inducing ligand (APRIL) from hedgehog (<Emphasis Type="Italic">Erinaceus europaeus</Emphasis>)

Here we describe the identification of the hedgehog Erinaceus europaeus homologue of a proliferation-inducing ligand (APRIL) of the TNF family (designated heAPRIL). Hedgehog APRIL contains two cysteine residues (Cys¹⁹⁶ and Cys²¹¹), a furin protease cleavage site and a conserved putative N-glycosylation site (Asn¹²⁴). Real-time quantitative PCR (qPCR) analysis revealed that heAPRIL could be detected in various tissues. MTT assays and flow cytometric analysis revealed that Nus-hesAPRIL and hesAPRIL could promote the survival/proliferation of splenic B cells. Laser scanning confocal microscopy analysis showed GFP-hesAPRIL could successfully bind to the APRIL receptors of lymphocytes.     

The uncertain glory of APRIL

The tumour necrosis factor (TNF) family is intimately connected to the regulation of cellular pathways. A PRoliferation-Inducing Ligand (APRIL) is a rather new member of that family, named for its capacity to stimulate the proliferation of tumour cells in vitro. Subsequent publications also called this ligand TRDL-1 or TALL-2, respectively. APRIL and B-lymphocyte stimulator (BLyS; also termed BAFF, TALL-1, THANK, zTNF4) form a new subfamily of TNF-like ligands that are expressed in haematopoietic cells. Both ligands can bind the two members of the TNF receptor family, namely the transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), as well as B-cell maturation antigen (BCMA). BLyS has recently been the subject of several reviews (for an extensive review, see Mackay et al.). The present review will thus focus on APRIL, and discuss BLyS only briefly for the sake of clarity.     

Interaction of the TNF homologues BLyS and APRIL with the TNF receptor homologues BCMA and TACI

BLyS (also called TALL-1, THANK, or BAFF) [1] [2] [3] [4] is a member of the tumor necrosis factor (TNF) gene family that stimulates proliferation and immunoglobulin production by B cells. BLyS interacts with the TNF receptor (TNFR) homologue TACI (transmembrane activator and CAML-interactor) [5], and treatment of mice with a TACI-Fc fusion protein abolishes germinal center formation after antigenic challenge [6]. Here we report a novel interaction between BLyS and another TNFR homologue, BCMA (B cell maturation antigen) [7] [8]. Further, the TNF homologue APRIL [9], a close relative of BLyS, also bound to BCMA and TACI. BLyS or APRIL activated nuclear factor-kappaB (NF-kappaB) through TACI and BCMA, and each ligand stimulated immunoglobulin M (IgM) production by peripheral blood B cells. These results define a dual ligand-receptor system that may play an important role in humoral immunity.     

Molecular mechanism of the selectivity between BAFF/APRIL and their receptors by molecular simulations

B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) belonging to the tumour necrosis factor (TNF) ligand family can bind three unusual TNF receptors (BCMA, TACI and BR3) with various binding affinities. BAFF and APRIL are regarded as promising therapeutic targets for autoimmune diseases because of their pivotal roles in cell survival and immune regulation. In this work, we carried out molecular dynamics calculations to explore the structural and chemical features responsible for ligand recognition by extracellular functional segments of TNF receptors. We found that the conserved pocket D_cons of BAFF/APRIL contacted the DxL motif of TNF receptors, while the D_spe1–3 sub-domains were responsible for their different affinities, especially D_spe1 and D_spe2. The residues at position II–V of DxL motif were wrapped into the D_cons pocket via salt-bridge and hydrophobic interactions. The hydrophobic residues of strand3 and helix1 in TNF receptors provided remarkable contributions for the affinities to BAFF/APRIL. Additionally, Arg^VI of DxL motif played a key role in the binding selectivity via salt-bridge interaction with residue D275B in BAFF. Arg27 in BCMA contributed to the high affinity for APRIL so that BCMA showed a preference for APRIL. Our studies indicated that Arg84 and Gln95 in TACI2 played an important role in the selectivity of two cysteine-rich domain segments in TACI, leading to the higher binding affinities of TACI2 than those of TACI1. The primary cause of the disability to bind APRIL was the space conflict with the rigid conformation of the C-terminus coil of BR3. These thorough understanding of the molecular mechanism for BAFF/APRIL recognition by their receptors provides new insights for guiding inhibitor design.     

Cutting edge: the dependence of plasma cells and independence of memory B cells on BAFF and APRIL

Memory B (B(MEM)) cells and long-lived bone marrow plasma cells (BM-PCs) persist within local environmental survival niches that afford cellular longevity. However, the factors supporting B(MEM) cell survival within the secondary lymphoid organs and allowing BM-PC persistence in the bone marrow remain poorly characterized. We report herein that long-lived B(MEM) cell survival and function are completely independent of BAFF (B cell-activating factor of the TNF family) or APRIL (a proliferation-inducing ligand). Thus, B(MEM) cells represent the only mature B2 lineage subset whose survival is independent of these ligands. We have previously shown that the TNFR family member receptor BCMA (B cell maturation Ag) is a critical survival receptor for BM-PC survival in vivo. We identify in this study the ligands critical for BM-PC survival and show that either BAFF or APRIL supports the survival of BM-PCs in vivo. These data define the BAFF/APRIL-dependent and -independent components of long-lived humoral immunity.     

Biologically active APRIL is secreted following intracellular processing in the Golgi apparatus by furin convertase

Tumor necrosis factor (TNF) ligand family members are synthesized as transmembrane proteins, and cleavage of the membrane-anchored proteins from the cell surface is frequently observed. The TNF-related ligands APRIL and BLyS and their cognate receptors BCMA/TACI form a two ligand/two receptor system that has been shown to participate in B- and T-cell stimulation. In contrast to BLyS, which is known to be cleaved from the cell surface, we found that APRIL is processed intracellularly by furin convertase. Blockage of protein transport from the endoplasmic reticulum to the Golgi apparatus by Brefeldin A treatment abrogated APRIL processing, whereas monensin, an inhibitor of post-Golgi transport, did not interfere with cleavage of APRIL, but blocked secretion of processed APRIL. Thus, APRIL shows a unique maturation pathway among the TNF ligand family members, as it not detectable as a membrane-anchored protein at the cell surface, but is processed in the Golgi apparatus prior to its secretion.     

Thymus-independent class switch recombination is affected by APRIL

The tumour necrosis factor (TNF) family member a proliferation-inducing ligand (APRIL) is implicated in various B-cell processes, such as class switch recombination, plasma cell differentiation and plasma cell survival. This was suggested from initial studies analysing B-cell responses in APRIL-deficient and transgenic mice, and mice deficient for the TNF receptors of APRIL, transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen. Here, we present additional evidence for the importance of APRIL in thymus-independent (TI) B-cell responses, using APRIL-deficient and transgenic mice. APRIL-deficient mice show an impaired immunoglobulin A (IgA) response towards TI B-cell antigens, whereas APRIL transgenic mice show exaggerated TI B-cell responses. Moreover, antibody titres to TI antigens were sustained in APRIL transgenic mice for a long time and even increased up to 75 days in the case of IgA against 4-hydroxy-nitrophenacetyl-lipopolysaccharide.