Molecular Characterization of Equine APRIL and its Expression Analysis During the Adipogenic Differentiation of Equine Adipose-Derived Stem Cell In Vitro

A proliferation inducing ligand (APRIL) is a member of the TNF superfamily. It shares two receptors with B-cell activating factor (BAFF), B-cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI). Herein, the equine APRIL was identified from equine adipose-derived stem cell (ASC), and the protein expression of APRIL and its related molecules were detected during the adipogenic differentiation of equine ASC in vitro. The equine APRIL gene was located on chromosome 11, spans 1852 base pairs (bp). Its open reading frame covers 753 bp, encoding a 250-amino acid protein with the typical TNF structure domain. During the two weeks’ adipogenic differentiation of equine ASC, although the protein expression of APRIL and TACI had an insignificant change, that of BCMA increased significantly. Moreover, with the addition of recombinant protein His₆-sAPRIL, a reduced differentiation of equine ASC toward adipocyte was detected. These results may provide the basis for investigating the role of APRIL in ASC adipogenic differentiation.     

Raised intrathecal levels of APRIL and BAFF in patients with systemic lupus erythematosus: relationship to neuropsychiatric symptoms

Introduction  

The tumour necrosis factor (TNF) family ligands BAFF (B-cell activating factor of TNF family) and APRIL (a proliferation-inducing ligand) are essential for B-cell survival and function. Elevated serum levels of BAFF and APRIL have been reported earlier in patients with systemic lupus erythematosus (SLE). Since autoantibody formation in the central nervous system (CNS) is a distinct feature of neuropsychiatric SLE (NPSLE), we have investigated whether NPSLE is associated with an enhanced intrathecal production of APRIL and BAFF.     

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.     

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.     

Multiple Novel Classes of APRIL-specific Receptor-blocking Peptides Isolated by Phage Display

A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) ligand superfamily and has a proliferative effect on both normal and tumor cells. The TNF family receptors (B-cell maturation antigen (BCMA), transmembrane activator and CAML-interactor (TACI), and BAFF receptor-3 (BR3)) for APRIL and the closely related ligand, B-cell activating factor of the TNF family (BAFF), bind these ligands through a highly conserved six residue DXL motif ((F/Y/W)-D-X-L-(V/T)-(R/G)). Panning peptide phage display libraries led to the identification of several novel classes of APRIL-binding peptides, which could be grouped by their common sequence motifs. Interestingly, only one of these ten classes consisted of peptides containing the DXL motif. Nevertheless, all classes of peptides prevented APRIL, but not BAFF, from binding BCMA, their shared receptor. Synthetic peptides based on selected sequences inhibited APRIL binding to BCMA with IC₅₀ values of 0.49-27 μM. An X-ray crystallographic structure of APRIL bound to one of the phage-derived peptides showed that the peptide, lacking the DXL motif, was nevertheless bound in the DXL pocket on APRIL. Our results demonstrate that even though a focused, highly conserved motif is required for APRIL-receptor interaction, remarkably, many novel and distinct classes of peptides are also capable of binding APRIL at the ligand receptor interface.     

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.     

The Expression of BAFF, APRIL and TWEAK Is Altered in Eczema Skin but Not in the Circulation of Atopic and Seborrheic Eczema Patients

The TNF family cytokines BAFF (B-cell activating factor of the TNF family) and APRIL (a proliferation-inducing ligand) are crucial survival factors for B-cell development and activation. B-cell directed treatments have been shown to improve atopic eczema (AE), suggesting the involvement of these cytokines in the pathogenesis of AE. We therefore analyzed the expression of these TNF cytokines in AE, seborrheic eczema (SE) and healthy controls (HC). The serum/plasma concentration of BAFF, APRIL and a close TNF member TWEAK (TNF-like weak inducer of apoptosis) was measured by ELISA. The expression of these cytokines and their receptors in skin was analyzed by quantitative RT-PCR and immunofluorescence. Unlike other inflammatory diseases including autoimmune diseases and asthma, the circulating levels of BAFF, APRIL and TWEAK were not elevated in AE or SE patients compared with HCs and did not correlate with the disease severity or systemic IgE levels in AE patients. Interestingly, we found that the expression of these cytokines and their receptors was altered in positive atopy patch test reactions in AE patients (APT-AE) and in lesional skin of AE and SE patients. The expression of APRIL was decreased and the expression of BAFF was increased in eczema skin of AE and SE, which could contribute to a reduced negative regulatory input on B-cells. This was found to be more pronounced in APT-AE, the initiating acute stage of AE, which may result in dysregulation of over-activated B-cells. Furthermore, the expression levels of TWEAK and its receptor positively correlated to each other in SE lesions, but inversely correlated in AE lesions. These results shed light on potential pathogenic roles of these TNF factors in AE and SE, and pinpoint a potential of tailored treatments towards these factors in AE and SE.     

The crystal structure of a proliferation-inducing ligand, APRIL

A proliferation-inducing ligand (APRIL) is a TNF-like cytokine that stimulates tumor cell growth. Within the TNF ligand superfamily, APRIL is most similar to B-cell activation factor (BAFF) with which it shares 30% sequence identity. APRIL binds the receptors B-cell maturation antigen (BCMA) and TACI with high affinity; both of these receptors have also been shown to bind BAFF, although BCMA has significantly higher affinity for APRIL than BAFF. Determination of the crystal structure of APRIL from three crystallization conditions at resolutions of 1.8-2.4A over a pH range from 5.0 to 8.5 reveals a compact trimeric ligand with a backbone fold very similar to that of BAFF (1.1A RMSD over 122 structurally equivalent Calpha atoms), with the exception of differences in the AA' and DE loop regions. Whereas BAFF has been shown to form 20-trimer assemblies under certain conditions, the molecular determinants required for BAFF-like assemblies are absent in the APRIL structure. No crystal packing suggestive of the formation of higher-order assemblies is seen in any of the crystal forms nor does the structure vary significantly between pH 5.0 and 8.5. Modeling of the APRIL-BCMA complex shows the resulting interface is in agreement with mutagenesis data.     

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.     

APRIL-deficient mice have normal immune system development

APRIL (a proliferation-inducing ligand) is a member of the tumor necrosis factor (TNF) superfamily. APRIL mRNA shows high levels of expression in tumors of different origin and a low level of expression in normal cells. APRIL shares two TNF receptor family members, TACI and BCMA, with another TNF homolog, BLyS/BAFF. BLyS is involved in regulation of B-cell activation and survival and also binds to a third receptor, BR3/BAFF-R, which is not shared with APRIL. Recombinant APRIL and BLyS induce accumulation of B cells in mice, while BLyS deficiency results in severe B-cell dysfunction. To investigate the physiological role of APRIL, we generated mice that are deficient in its encoding gene. APRIL(-/-) mice were viable and fertile and lacked any gross abnormality. Detailed histological analysis did not reveal any defects in major tissues and organs, including the primary and secondary immune organs. T- and B-cell development and in vitro function were normal as well, as were T-cell-dependent and -independent in vivo humoral responses to antigenic challenge. These data indicate that APRIL is dispensable in the mouse for proper development. Thus, BLyS may be capable of fulfilling APRIL's main functions.     

Differential effects on BAFF and APRIL levels in rituximab-treated patients with systemic lupus erythematosus and rheumatoid arthritis

The objective of this study was to investigate the interaction between levels of BAFF (B-cell activation factor of the tumour necrosis factor [TNF] family) and APRIL (a proliferation-inducing ligand) and B-cell frequencies in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) treated with the B-cell-depleting agent rituximab. Ten patients with SLE were treated with rituximab in combination with cyclophosphamide and corticosteroids. They were followed longitudinally up to 6 months after B-cell repopulation. Nine patients with RA, resistant or intolerant to anti-TNF therapy, treated with rituximab plus methotrexate were investigated up to 6 months after treatment. The B-cell frequency was determined by flow cytometry, and serum levels of BAFF and APRIL were measured by enzyme-linked immunosorbent assays. BAFF levels rose significantly during B-cell depletion in both patient groups, and in patients with SLE the BAFF levels declined close to pre-treatment levels upon B-cell repopulation. Patients with SLE had normal levels of APRIL at baseline, and during depletion there was a significant decrease. In contrast, patients with RA had APRIL levels 10-fold higher than normal, which did not change during depletion. At baseline, correlations between levels of B cells and APRIL, and DAS28 (disease activity score using 28 joint counts) and BAFF were observed in patients with RA. In summary, increased BAFF levels were observed during absence of circulating B cells in our SLE and RA patient cohorts. In spite of the limited number of patients, our data suggest that BAFF and APRIL are differentially regulated in different autoimmune diseases and, in addition, differently affected by rituximab treatment.     

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.     

Molecular Cloning,Recombinant Expression,and Funtional Characterization of APRIL (TNFSF13) in Cat (Felis catus)

A proliferation-inducing ligand (APRIL) is a critical member of the tumor necrosis factor (TNF) superfamily, which is involved in immune regulation. In the present study, the cDNA of cat APRIL (cAPRIL) was successfully amplified. Sequence analysis showed that the open reading frame (ORF) of cAPRIL contains a putative furin protease cleavage site (R-R-K-R), a conserved putative N-glycosylation site (Asn¹²⁴), and two conservative cysteine residues (Cys¹⁹⁶ and Cys²¹¹). Real-time quantitative PCR (qPCR) analysis revealed that cAPRIL could be detected in various tissues. The phylogenetic analysis and predicted three dimensional (3D) structure revealed that it is similar to its counterparts. The extracellular soluble domain of the cAPRIL (csAPRIL) fragment was cloned into the expression vector pET43.1a. SDS-PAGE and Western blotting analysis indicated a high-level expression of csAPRIL protein in Escherichia coli BL21 (DE3). MTT assays revealed that purified recombinant csAPRIL protein was able to stimulate proliferation of mouse B-cells. These findings indicate that cAPRIL plays an important role in proliferation of B-cells and provide the basis for investigation on the roles of APRIL in this important domestic species.     

Characterisation of monoclonal antibodies to the TNF and TNF receptor families

Tumour necrosis factor (TNF) family ligands and their corresponding receptors play important roles in the immune system and are involved in immune regulation such as lymphoid development, cell proliferation, differentiation, activation and death. Antibodies against these ligands and receptors together with Fc-fusion proteins, have been particularly useful as immunological tools in addressing the underlying involvement of these proteins in these contexts and furthermore, have given us hope in using them as potential therapeutic agents. Over last few years, there have been many additions to these ever-growing TNF family ligands and their receptors. Here, we have generated and characterised a set of monoclonal antibodies, together with mAbs from the HLDA workshop, against DcR1, DcR2, DR4, DR5, TRAIL, APRIL, BAFF, BAFF-R, BCMA, and TACI, which may be useful in phenotypic and functional studies of the role of TNF and TNF receptor family in immune function and regulation in relation to health and disease.     

APRIL and BAFF promote increased viability of replicating human B2 cells via mechanism involving cyclooxygenase 2

Of relevance to both protective and pathogenic responses to Ag is the recent finding that soluble molecules of the innate immune system, i.e., IL-4, B cell-activation factor of the TNF family (BAFF), and C3, exhibit significant synergy in promoting the clonal expansion of human B2 cells following low-level BCR ligation. Although IL-4, BAFF, and C3dg each contribute to early cell cycle entry and progression to S phase, only BAFF promotes later sustained viability of progeny needed for continued cycling. The present study sought to further clarify the mechanisms for BAFF's multiple functions. By comparing BAFF and a proliferation-inducing ligand (APRIL) efficacy at different stages in the response (only BAFF binds BR3; both bind transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation Ag, the early role was attributed to BR3, while the later role was attributed to TACI/B cell maturation Ag. Importantly, BAFF- and APRIL-promoted viability of cycling lymphoblasts was associated with sustained expression of cyclooxygenase 2 (COX-2), the rate-limiting enzyme for PGE2 synthesis, within replicating cells. Supernatants of cultures with BAFF and APRIL contained elevated PGE2. Although COX-2 inhibitors diminished daughter cell viability, exogenous PGE2 (1-1000 nM) increased the viability and recovery of lymphoblasts. Increased yield of viable progeny was associated with elevated Mcl-1, suggesting that a BAFF/APRIL --> TACI --> COX-2 --> PGE2--> Mcl-1 pathway reduces activation-related, mitochondrial apoptosis in replicating human B2 cell clones.     

No Evidence That Soluble TACI Induces Signalling via Membrane-Expressed BAFF and APRIL in Myeloid Cells

Myeloid cells express the TNF family ligands BAFF/BLyS and APRIL, which exert their effects on B cells at different stages of differentiation via the receptors BAFFR, TACI (Transmembrane Activator and CAML-Interactor) and/or BCMA (B Cell Maturation Antigen). BAFF and APRIL are proteins expressed at the cell membrane, with both extracellular and intracellular domains. Therefore, receptor/ligand engagement may also result in signals in ligand-expressing cells via so-called “reverse signalling”. In order to understand how TACI-Fc (atacicept) technically may mediate immune stimulation instead of suppression, we investigated its potential to activate reverse signalling through BAFF and APRIL. BAFFR-Fc and TACI-Fc, but not Fn14-Fc, reproducibly stimulated the ERK and other signalling pathways in bone marrow-derived mouse macrophages. However, these effects were independent of BAFF or APRIL since the same activation profile was observed with BAFF- or APRIL-deficient cells. Instead, cell activation correlated with the presence of high molecular mass forms of BAFFR-Fc and TACI-Fc and was strongly impaired in macrophages deficient for Fc receptor gamma chain. Moreover, a TACI-Fc defective for Fc receptor binding elicited no detectable signal. Although these results do not formally rule out the existence of BAFF or APRIL reverse signalling (via pathways not tested in this study), they provide no evidence in support of reverse signalling and point to the importance of using appropriate specificity controls when working with Fc receptor-expressing myeloid cells.     

Plasma levels of BAFF and APRIL are elevated in patients with asthma in Saudi Arabia

B-cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) are members of the tumor necrosis factor superfamily of cytokines and can induce B cell activation, differentiation, and antibody production via interaction with their receptors, including transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI), B-cell maturation antigen (BCMA), and B-cell activating factor receptor (BAFF-R). Herein, we assessed the plasma protein levels of BAFF and APRIL in patients with asthma to determine whether their expression is correlated with total IgE production and examined the surface expression of BAFF/APRIL receptors on B cells. Blood samples were collected from 47 patients with controlled asthma symptoms and 20 healthy normal controls, and plasma levels of APRIL, BAFF, and total IgE protein were quantified by corresponding ELISA assays. Furthermore, lymphocytes were isolated and B cells were analyzed for the presence of BAFF-R, BCMA, and TACI receptors using flow cytometry. Our results showed that IgE, BAFF, and APRIL plasma levels were markedly increased in patients with asthma compared with healthy controls. Moreover, expression of BAFF-R and BCMA, but not that of TACI, was significantly increased in patients with asthma compared with healthy controls. Overall, the findings suggest BAFF and APRIL as key mediators of asthma, and determination of their plasma levels may be useful in monitoring asthma symptoms and treatment response.     

Structures of APRIL-receptor complexes: like BCMA, TACI employs only a single cysteine-rich domain for high affinity ligand binding

TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function. TACI binds two ligands, APRIL and BAFF, with high affinity and contains two cysteine-rich domains (CRDs) in its extracellular region; in contrast, BCMA and BR3, the other known high affinity receptors for APRIL and BAFF, respectively, contain only a single or partial CRD. However, another form of TACI exists wherein the N-terminal CRD is removed by alternative splicing. We find that this shorter form is capable of ligand-induced cell signaling and that the second CRD alone (TACI_d2) contains full affinity for both ligands. Furthermore, we report the solution structure and alanine-scanning mutagenesis of TACI_d2 along with co-crystal structures of APRIL.TACI_d2 and APRIL.BCMA complexes that together reveal the mechanism by which TACI engages high affinity ligand binding through a single CRD, and we highlight sources of ligand-receptor specificity within the APRIL/BAFF system.     

Molecular cloning, genomic organization and expression analysis of the gene encoding bovine (Bos taurus) B-cell activating factor belonging to the TNF family (BAFF)

A novel bovine cDNA has been isolated by EST assembly and subsequently confirmed by using RT-PCR and designated bovine B-cell activating factor belonging to TNF family (bBAFF). The open reading frame (ORF) of this cDNA covers 843 bp, encoding 280 amino acids. The functional soluble part of bBAFF (bsBAFF) shows 96% and 91% identity with its pig and human counterparts, respectively, at the level of the primary protein structure. The bBAFF genomic sequence consists of six exons and five introns, is approximately 30 kb in size, and maps to bovine chromosome 12q. Southern blotting analysis indicated that the bBAFF gene is a single copy gene. Real-time quantitative PCR (qPCR) analysis revealed that bBAFF is predominantly expressed in bovine lymphoid tissues PBLs and spleen. The predicted three dimensional (3D) structure of the bsBAFF monomer analyzed by "comparative protein modeling" revealed that it is very similar to its human counterpart. In western blotting analysis, His6-tagged bsBAFF protein expressed in E. coli could be recognized not only by an anti-His6.tag mAb but also by an anti-human sBAFF mAb, indicating immunological cross-reactivity occurs between bovine and human sBAFF protein.