Hurson, Catherine Eileen
Expression and function of the atypical chemokine receptor CCX-CKR.
PhD thesis, University of Glasgow.
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The ability to clear infections and repair injury is dependent on the coordinated migration of immune cells, or leukocytes. These cells can directly destroy invading pathogens and also produce a variety of bioactive factors that promote pathogen clearance. Interactions between immune cells occur both at the site of inflammation and in specialised lymphoid organs throughout the body. The efficiency and specificity of these interactions relies on the production of a family of molecules called chemotactic cytokines, or chemokines, that drive leukocyte migration. Cells express specific profiles of chemokine receptors to ensure they are directed to the appropriate location to exert their immunological function. The field of chemokine biology, already complex, has been further complicated by the discovery of a subfamily of receptors, the atypical chemokine receptors. These molecules lack the ability to couple to signal transduction pathways used by the other chemokine receptors, and are proposed to act as chemokine scavengers or transport molecules.
The atypical chemokine receptor CCX-CKR was discovered more than a decade ago but its function in vivo remains unclear. At the beginning of my project, information about this molecule was very limited. The murine receptor binds the CC chemokines CCL19, CCL21 and CCL25, which have well-characterised and critical roles in the development and homeostasis of the immune system as well as in the immune response to infection. Thus, identification of this new receptor, which unlike classical receptors does not induce cell migration in response to ligand binding, presented some exciting possibilities as to how these processes might be regulated in vivo. Reports describing the pattern of expression of CCX-CKR have thus far provided only limited and sometimes contradictory information. Additionally, while in vitro studies from our lab have provided some important clues as to the potential role of the receptor, published in vivo studies were, at the time of commencing this work, limited to one report describing an unvalidated EGFP reporter knock-in transgenic mouse and a conflicting online resource detailing data generated using a LacZ reporter mouse. To understand the true function of this molecule, it is critical to know where it is expressed in vivo and to explore its function on these cells. In this project I set out with the aim of identifying murine tissues and cells expressing CCX-CKR, as well as examining its potential as an in vivo scavenger of chemokine. Related to this, I hoped to uncover any impact of deletion of CCX-CKR on lymphoid tissue cellularity and/or function, both in resting and inflamed conditions.
In chapter 3, I present data that identify lymphoid tissues and “barrier” tissues as sites of robustly detectable CCX-CKR mRNA expression. I describe how I have established a novel fluorescent chemokine tetramer-based protocol for the detection of CCL19 receptors, with emphasis on the application of this protocol to identify CCX-CKR activity on specific cell subsets. Using this method, I present evidence that some CD11b+ CD11c+ myeloid subsets in the inguinal lymph node exhibit CCX-CKR dependent internalisation of chemokine. I also describe attempts to fractionate tissues to identify cell populations responsible for the detected whole-tissue expression of CCX-CKR mRNA.
The results described in chapter 4 provide support for the hypothesis that CCX-CKR regulates levels of its ligands in vivo, with alterations in chemokine levels in serum and inguinal lymph nodes in the absence of CCX-CKR. I also present evidence demonstrating that deletion of the receptor can influence mRNA levels of the related receptor CCR7. Following on from this, chapter 5 details my analysis of the impact of CCX-CKR on the cellularity of various lymphoid compartments. I present evidence that CCX-CKR influences lymphocyte populations in the peritoneal cavity, with both innate-like and conventional lymphocytes significantly overrepresented in this compartment. The cellularity of the inguinal lymph node, but not the spleen, is subtly altered by deletion of the receptor. Splenic leukocyte cellularity is not affected, either in number or in localisation.
In chapter 6, I turn my attention to the possible role of CCX-CKR during the inflammatory response by examining various experimental parameters during a short-term model of induced cutaneous inflammation. This study shows that CCX-CKR deletion alters the cellularity of the myeloid compartment in the draining lymph node and again highlights myeloid subsets as displaying CCX-CKR dependent chemokine internalisation. Finally, I present preliminary data suggesting a protective effect of CCX-CKR deletion during a long-term model of inflammation-driven tumorigenesis.
Taken together, my data provide tentative support for the theory that CCX-CKR acts as a chemokine scavenger in vivo. They further indicate that CCX-CKR is involved in regulating cellularity of various lymphoid compartments both at rest and during induced inflammation. In chapter 7 I discuss in detail the implications of my findings in the context of work published since my project began, and highlight growing evidence to suggest a role for CCX-CKR in regulating immune function.
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