The efficient functioning of the immune system is dependent on the coordinated movement and positioning of immune cells. These cells patrol the body and facilitate the clearance of pathogens, whilst maintaining self-tolerance and inducing adaptive immunity. The coordinated migration of cells into and within tissues is mediated by chemokines, a family of small chemotactic cytokines that are potent inducers of cellular movement. Chemokines and their cognate receptors have been shown to play key roles in health, and in a broad spectrum of diseases. After their secretion, chemokines can be controlled by proteases and interactions with atypical chemokine receptors that structurally resemble conventional chemokine receptors but cannot couple to the signaling pathways that they use. Instead, they are thought to degrade, transport or buffer extracellular chemokines to regulate their access to cells bearing conventional chemokine receptors. However, their functions in vivo remain to be fully defined. CCRL1, a member of the atypical chemokine receptor family, binds to CCL19, CCL21 and CCL25 and is proposed to be a scavenger of these chemokines. Post-translational regulation of these chemokines is important because their interactions with cognate, conventional receptors CCR7 and CCR9, are critical for the development and functioning of the immune system. The work in this thesis primarily explores the importance of CCRL1 in regulating CCR7 ligands, but also considers the impact of protease-mediated ‘clipping’ on the function of CCL21 and other extended chemokines. Whilst in vitro evidence, and a growing body of in vivo evidence, supports the idea that CCRL1 serves an important role in the chemokine control, the true biological function of CCRL1 remains unclear. Therefore, the principal aim of this thesis was to further our understanding of the biology of CCRL1, mainly through the characterisation of CCRL1-deficient mice. Firstly, the effect of CCRL1 deletion on dendritic cell (DC) migration from the skin was investigated. Four distinct subsets of migratory DCs were examined in skin-draining lymph nodes at steady state and after induction of cutaneous inflammation. Under inflammatory conditions, skin-derived DCs were identified by FITC painiting or by photoconverting the skin of Kaede transgenic mice. CCRL1 deficiency was found to result in a specific reduction in the abundance of Langerin+ skin-derived DCs in inguinal lymph nodes at rest, and although the initial inflammation-driven arrival of DCs at skin-draining lymph nodes showed no requirement for CCRL1, DCs that took longer to reach these organs were substantially reduced in CCRL1-deficient mice. All DC subsets were affected, but overall it was epidermal Langerhans cells that showed the greatest requirement for CCRL1. Defective DC arrival at lymph nodes was accompanied by DC retention in resting and inflamed skin, and these cells struggled to leave CCRL1 deficient skin in ex vivo ‘crawl out’ assays. Further experiments demonstrated that, as expected, DC arrival at lymph nodes draining inflamed skin was heavily dependent on CCR7 in the models being used, and that increased levels of bioavailable CCL19 and CCL21 accompanied CCRL1 deficiency in inflamed skin. This suggested CCRL1 regulates chemokine to prevent DCs from becoming disorientated in the skin and failing to efficiently egress this tissue. I hypothesised that dysregulation of CCL19 rather than CCL21 may be particularly significant because of its greater diffusivity, and strikingly, inflammation-driven DC migration defects observed in CCRL1 deficient mice were completely reversed by genetic deletion of CCL19 in the same animals. To place these findings in an anatomical context, expression of CCRL1 in skin and lymph node was explored using CCRL1+/gfp ‘knock-in’ receptor mice and anti-CCRL1 antibodies. In skin, CCRL1 was abundantly expressed by keratinocytes, and found on lymphatic endothelial cells (LECs) that are traversed by migrating DC as they leave the skin. In lymph nodes draining the skin, LECs in the supcapsular sinus (SCS) were strongly CCRL1+. Although some leukocytes were found to express very low levels of eGFP in CCRL1+/gfp, the data suggested that CCRL1-mediated scavenging of CCL19 by keratinocytes and LECs facilitates CCR7-driven DC migration from resting and inflamed skin. CCRL1 expression in other secondary lymphoid tissues was examined and its role in regulating leukocyte populations residing in or around the SCS was explored. As in the inguinal lymph node, CCRL1 was restricted to the SCS LECs in other lymph nodes, such as the mesenteric lymph nodes that drains the intestine. In the spleen, endothelial cells lining venules adjacent to the white pulp marginal zone specifically expressed CCRL1. The overall cellularity and microanatomy of lymph nodes and the spleen appeared normal in CCRL1-deficient mice, as was the recruitment of CCR7+ cells into the spleen two hours after adoptive transfer. However, NK cells, iNKT cells and γδ T cells, which are thought to reside in intrafollicular regions, were less abundant in CCRL1-deficient inguinal and mesenteric lymph nodes, although they were present at normal frequency in the spleen. CD169+ macrophages are intimately associated with CCRL1+ endothelial cells in the spleen. CCRL1 deficiency had no clear impact on these cells in the inguinal lymph nodes, and only resulted in a small increase in the abundance of these cells in the spleen, without obviously affecting their position. Strikingly, however, in mesenteric lymph nodes, CD169+ macrophages, and LECs with which they associate, were far more abundant when CCRL1 had been deleted and were aberrantly distributed throughout the lymph node parenchyma. The reason this phenotype is restricted to the mesenteric lymph node is unclear, but it might be related to the fact the intestine, unlike other non-lymphoid tissues, is an abundant source of CCL25. This, along with the immunological implications of these observations, requires further investigation. There is a lot of evidence that macrophages regulate lymphangiogenesis. The close association between LECs and CD169+ macrophages in lymph nodes, and the phenotype in CCRL1-deficient mesenteric lymph nodes, stimulated experiments to explore functional interactions between these cells. Inflammation in the footpad induced lymphangiogenesis in draining popliteal and inguinal lymph nodes. When clodronated liposomes were used to specifically deplete macrophages from the popliteal lymph node of WT mice, lymphangiogenesis appeared suppressed while it continued unabated in the inguinal lymph node where the CD169+ macrophage population was intact. WT and CCRL1-deficient mice responded similarly, and although preliminary, these data suggest that CD169+ macrophages play an important role in stimulating lymph node lymphangiogenesis. In addition to the CCRL1 studies above, other mechanisms that might regulate chemokines after their secretion were explored. Work published at the beginning of my PhD showed that CCL21, which carries an extended C-terminus that anchors it to the extracellular matrix, can be cleaved by bone marrow-derived DCs (BMDCs) to release a more freely diffusible version. These findings were reproduced here using in vitro-derived human or mouse DCs. DCs were far better at cleaving CCL21 than other leukocytes, and they could also cleave CCL2, a pro-inflammatory chemokine with an extended C-terminus. Interestingly, a truncated version of CCL21 was detected in mouse secondary lymphoid tissues. It was larger than the version generated by BMDCs in vitro, and the nature of its truncation is not clear, but this shows that CCL21 processing occurs in vivo. If this form of CCL21 is more diffusible than the full-length protein, then it might be more available for regulation by CCRL1. / However, neither forms of CCL21 were more abundant in CCRL1-deficient secondary lymphoid tissues than equivalent tissues from WT mice. CCR7 plays a critical role in directing DC egress from tissues, their entry into lymph nodes, and their movement within these tissues. The work presented in this thesis provides evidence of two mechanisms that regulate CCR7 ligands: CCRL1-mediated CCL19 scavenging and DC-mediated CCL21 cleavage. It reveals that, under certain circumstances, CCRL1 is critical for facilitating DC egress from peripheral tissues to the lymph nodes, and plays an indispensible role in regulating LECs and CD169+ macrophages in lymph nodes. These studies extend our understanding of CCRL1 and the chemokine networks at work in lymph nodes and other tissues, and form the foundation on which to explore the immunological importance of the regulation of extracellular chemokines.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:732713 |
| Date | January 2014 |
| Creators | Bryce, Steven Alan |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/5904/ |
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