On death receptor 3 and its ligands…

The recent article in Immunology by Park et al.¹ entitled ‘Interleukin-32 enhances cytotoxic effect of natural killer cells to cancer cells via activation of death receptor 3’ is very interesting; however, I believe that non-specialist readers would benefit from a more expansive and detailed discussion of its context. The authors have omitted much of the recent literature detailing the broader biological functions of Death Receptor 3 (DR3), most of which do not relate to regulating cell death. In addition, clarification is also required with regards to the ligands of DR3 because the older nomenclature can cause confusion and is particularly pertinent to the interpretation of this study.

Towards the end of 1996 and beginning of 1997, DR3 (TNFRSF25) was reported simultaneously by a number of groups as a tumour necrosis factor receptor superfamily (TNFRSF) member with an intracellular, apoptosis-inducing death domain and was ascribed a variety of names – Apo3, LARD, TR3, TRAMP and WSL-1.^2–7 In recent years, DR3 has emerged as a major regulator of inflammatory and autoimmune disease; research using knockout and transgenic mice has revealed that DR3 plays a critical role in the development of experimental autoimmune encephalomyelitis, allergic lung inflammation, inflammatory arthritis and experimental autoimmune uveoretinitis.^8–12 Studies in vivo have also demonstrated a role in colitis and ileitis.^13–17 DR3 regulates immunity to certain bacteria,¹⁸ viruses,¹⁹ tumours²⁰ and intrinsically maintains neurological function.²¹ Research in humans has mirrored these findings, primarily showing that DR3 regulates inflammation and immunity through controlling the development of effector T cells and differentiation of myeloid subsets,^22–30 but it may also have effects on other cell types such as neurons.³¹ Local and systemic increases of its ligand are associated with multiple human inflammatory disorders.^32–35 In this respect, the designation ‘Death Receptor 3’ is a misnomer because many of the recognized functions of the gene are associated with cell expansion and differentiation, rather than death.

Park et al.¹ clearly describe an increase in cell viability of tumour cell lines following exposure to natural killer (NK) cells when DR3 expression was knocked down; results consistent with DR3 acting to trigger cell death. To my knowledge, this is the first functional demonstration of a pro-apoptotic role for DR3 in human tumour cell lines, but it is not unique as a general phenomenon. The original DR3 knockout mouse exhibited a defect in negative selection of thymocytes,³⁶ while DR3-dependent apoptosis has been described in renal inflammation in vivo³⁷ and osteoblast cell lines in vitro.³⁸ Furthermore, a role in human cancer has been implied from the discovery that the DR3 gene is disrupted in ∼ 40% of neuroblastomas.³⁹

It is in this context that clarification is useful on the nature of the DR3 ligand, as its identity is also complicated by a history of diverse nomenclature. Park et al.¹ mention two ligands in their references, Apo3L and TL1A, both of which are distinct tumour necrosis factor superfamily (TNFSF) members. Apo3L was originally named as the ligand for DR3 (i.e. Apo3)⁴⁰ and was also called TWEAK (TNFSF12). However, follow-up studies could not confirm this⁴¹ and indicated that TWEAK signalled in the absence of DR3.⁴² A second receptor for TWEAK, Fn14 (TNFRSF12A), was then identified,⁴³ and TL1A (TNFSF15 and the full-length gene product of the vascular endothelial growth inhibitor, VEGI) was found to bind DR3.⁴⁴ All-encompassing work from Bossen et al.⁴⁵ involving flow cytometric binding assays between the majority of human and murine TNFSF:Fc proteins and cell lines transfected with TNFRSF members confirmed this, i.e. that TWEAK binds Fn14, whereas TL1A binds DR3 and there is minimal cross-reactivity, findings that have been borne out in later in vivo experiments using gene knockouts.^10,11 TL1A is therefore currently the only recognized TNFSF ligand for DR3.

Park et al.¹ show quite elegantly with co-cultures and a series of small interfering RNA knockdown experiments that: (i) the NK cell line NK-92 could kill prostate and colon cancer cell lines dependent on interleukin-32 (IL-32) expression, (ii) DR3 was up-regulated on the cancer cells following co-culture, (iii) IL-32 induced Apo3L (TWEAK) expression on NK cells, and (iv) DR3 knockdown decreased susceptibility of the cancer cells to NK-92. However, their efforts to antagonize Apo3L and DR3 with antibodies demonstrate the action within their system of not one, but two distinct pathways, TWEAK/Fn14 and TL1A/DR3. The relative contribution of the two pathways, and the extent to which IL-32 triggers DR3 ligand (i.e. TL1A) release, remain areas of further research in this field.