An investigation into the regulatory mechanisms of neutrophil migration into lymphatic vessels in vivo

PhDNeutrophils are recognised to play a pivotal role at the interface between the innate and adaptive immune responses following their rapid recruitment to inflamed tissues and lymphoid organs. Whilst neutrophil trafficking through blood vessels has been extensively studied, the molecular mechanisms regulating their migration into the lymphatic system are still poorly understood. This thesis therefore aimed to investigate the mechanisms involved in neutrophil migration across the lymphatic endothelium during TNF- or Complete Freund’s Adjuvant + antigen (CFA+Ag)-induced inflammation of cremaster muscles in vivo. This work revealed that TNF- or CFA+Ag-stimulation induces a rapid but transient entry of tissue-infiltrated neutrophils into lymphatic vessels, a response associated with the regulation and redistribution of the lymphatic endothelial cell glycocalyx. Interestingly, antigen sensitisation resulted in the production of endogenous TNF within cremaster muscles. Using anti-TNF blocking antibodies and mice deficient in both TNF receptors (p55 and p75), endogenous TNF was demonstrated for the first time to be involved in priming and triggering the migration of neutrophils into tissue-associated lymphatic vessels upon antigen challenge. Additionally, the use of chimeric mice exhibiting neutrophils deficient in both TNFRs demonstrated that TNF directly acts on leukocytes to induce neutrophil migration into lymphatic vessels. Furthermore, the results show that TNF-induced migration of neutrophils into the lymphatic system occurs in a strictly CCR7-dependent manner; blocking CXCR4 or CXCL1 signalling does not affect this response. Finally, both TNF- or CFA+AG-stimulation induced ICAM-1 up-regulation on lymphatic vessels, allowing neutrophils to crawl along the lumen; a response that was demonstrated to be TNF-dependent. These results have provided new insights into the mechanisms that mediate neutrophil migration into lymphatic vessels and their subsequent crawling within these vessels during inflammation. In particular, a new role for TNF as a key regulator of these processes has been demonstrated. Taken together, this work has highlighted potential and effective targets to manipulate the role of neutrophils in adaptive immune responses in vivo.Institute of Bioengineering at Queen Mary University of London (QMUL) and Arthritis Research UK (ARUK