Abstract
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.
Highlights
TNF family ligands are type 2 membrane-bound proteins that form non-covalent trimers through an extracellular, carboxyterminal domain of about 150 amino acid residues, coined the TNF homology domain [1]
Stimulation of the same cells with LPS induced ERK and IkBa phosphorylation, this was achieved with kinetics different from those observed for TACI-Fc and BAFFR-Fc
We showed that the agonist activity of TACI-Fc and BAFFR-Fc in different primary cells and cell lines was related to the presence of high molecular mass oligomers that formed the active fraction
Summary
TNF family ligands are type 2 membrane-bound proteins that form non-covalent trimers through an extracellular, carboxyterminal domain of about 150 amino acid residues, coined the TNF homology domain [1]. BAFF (B cell Activating Factor of the TNF Family) is mainly expressed by myeloid cells and by radiation-resistant stromal cells [2,3,4]. It is synthesized as a membrane-bound protein that can be cleaved at a furin consensus sequence to release a soluble form of the cytokine. We have previously shown that TACI stimulation in primary mouse B cells is inefficient using soluble trimeric BAFF or APRIL, but requires higher-order multimeric forms of the ligands that probably mimic the membrane-bound ligand [10]. T-cell priming in vivo requires TACI-expressing B cells, and B cells can be replaced by TACI-Fc in this context [15]
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