Background and objectives Sites of chronic inflammation, such as rheumatoid arthritis synovial tissue, are often characterised by the formation of tertiary lymphoid structures, which show characteristic features of lymphoid organs, like high endothelial venules (HEV) and sometimes even true germinal centres. Ligation of the lymphotoxin (LT)-β receptor (LTβR) results in activation of both canonical and non-canonical NF-κB signalling in endothelial cells (ECs) and plays a crucial role in lymphoid neogenesis. Non-canonical NF-κB signalling in ECs promotes inflammation-induced angiogenesis and triggers the development of the cuboidal HEV appearance. However, the relative contribution of the individual pathways to the acquisition of leukocyte traffic-regulating properties by ECs is less well understood. We therefore aimed to identify the molecular pathways by which LTβR drives activation of ECs and interactions with leukocytes.
Methods Primary human ECs were stimulated with LTβ or LIGHT to activate LTβR. Activation of downstream signalling pathways was assessed by western blot analysis. Levels of cytokines secreted by LTβ-treated ECs were measured in conditioned media using antibody arrays. To block canonical NF-κB signalling pathway, a small molecule inhibitor of IKKβ was used. Inactivation of non-canonical NF-κB signalling was obtained with a small molecule inhibitor of NIK, and siRNAs targeting NIK or NFκB2. EC interactions with leukocytes were determined by an adhesion assay, whereas EC monolayer permeability was assessed by a permeability assay.
Results Treatment of ECs with LTβ or LIGHT activated both canonical and non-canonical NF-κB signalling pathways. Exposure of ECs to LTβ induced the expression of several inflammatory cytokines (IL-8, IL-6, CXCL1, CXCL5, MCP-1, GM-CSF, MIP3α), and this response was mediated mainly by the canonical NF-κB pathway. Consistent with the inflammatory activation of ECs, LTβR ligation induced adhesion of T cells and monocytes to activated endothelium in a predominantly canonical NF-κB-dependent manner. Of note, LTβ was more potent at inducing adhesive properties of ECs than LIGHT. Finally, LTβR ligation by LTβ induced permeability across EC monolayers.
Conclusions LTβR ligation on ECs drives inflammation-induced angiogenesis and differentiation into HEV via the non-canonical NF-κB pathway, as well as inflammatory activation of the endothelium and leukocyte adhesion through canonical NF-κB signalling, implicating ECs as immune cell mobilizers during the formation of tertiary lymphoid structures in the course of autoimmune diseases. Further molecular dissection of these pathways may help to better understand the function of tertiary lymphoid structures and potentially to develop therapeutics that modulate the formation of these structures and reduce inflammation.