Macrophage Development Rewritten Macrophages provide protection against a wide variety of infections and critically shape the inflammatory environment in many tissues. These cells come in many flavors, as determined by differences in gene expression, cell surface phenotype and specific function. Schulz et al. (p. 86, published online 22 March) investigated whether adult macrophages all share a common developmental origin. Immune cells, including most macrophages, are widely thought to arise from hematopoietic stem cells (HSCs), which require the transcription factor Myb for their development. Analysis of Myb-deficient mice revealed that a population of yolk-sac–derived, tissue-resident macrophages was able to develop and persist in adult mice in the absence of HSCs. Importantly, yolk sac–derived macrophages also contributed substantially to the tissue macrophage pool even when HSCs were present. In mice, a population of tissue-resident macrophages arises independently of bone marrow–derived stem cells. Macrophages and dendritic cells (DCs) are key components of cellular immunity and are thought to originate and renew from hematopoietic stem cells (HSCs). However, some macrophages develop in the embryo before the appearance of definitive HSCs. We thus reinvestigated macrophage development. We found that the transcription factor Myb was required for development of HSCs and all CD11bhigh monocytes and macrophages, but was dispensable for yolk sac (YS) macrophages and for the development of YS-derived F4/80bright macrophages in several tissues, such as liver Kupffer cells, epidermal Langerhans cells, and microglia—cell populations that all can persist in adult mice independently of HSCs. These results define a lineage of tissue macrophages that derive from the YS and are genetically distinct from HSC progeny.

A lineage of myeloid cells independent of Myb and hematopoietic stem cells

The immunological synapse: A molecular machine controlling T cell activation

Immobilized chemokine fields and soluble chemokine gradients cooperatively shape migration patterns of dendritic cells

The proper function of immune surveillance requires well-coordinated mechanisms in order to guide the patrolling immune cells through peripheral tissues and into secondary lymphoid organs. Analyzing gene-targeted mice, we identified the chemokine receptor CCR7 as an important organizer of the primary immune response CCR7-deficient mice show severely delayed kinetics regarding the antibody response and lack contact sensitivity and defayed type hypersensitivity reactions. Due to the impaired migration of lymphocytes, these animals reveal profound morphological alterations in all secondary lymphoid organs, Upon activation, mature skin dendritic cells fad to migrate into the draining lymph nodes. Thus, in order to bring together lymphocytes and dendritic cells to form the characteristic microarchitecture of secondary lymphoid organs, CCR7 is required to rapidly initiate an adoptive immune response.

CCR7 Coordinates the Primary Immune Response by Establishing Functional Microenvironments in Secondary Lymphoid Organs (Reprinted from Cell, vol 99, pg 23-33, 1999)

Thymic microenvironments for T cell repertoire formation

The generation of an effective immune response relies on the coordinated migration and interaction of cells of the immune system. Interactions between leukocytes and other cells occur within secondary lymphoid organs or at sites of inflammation. These interactions are tightly policed by a family of small chemotactic cytokines, or chemokines, that drive leukocyte migration. Chemokines and their receptors are broadly divided into two functional groups, homeostatic and inflammatory. Typically homeostatic chemokines and their receptors regulate leukocyte migration to and within secondary lymphoid organs, whereas inflammatory chemokines and their receptors control migration of cells to sites of inflammation. However, this division is now blurred, as some inflammatory chemokine receptors also possess clear homeostatic roles. One such receptor is the chemokine receptor CCR2, which binds a number of inflammatory chemokines, including CCL2, CCL7 and CCL12. Further complexity is added to the chemokine biology field by the discovery of atypical chemokine receptors, which lack the capacity to signal in manner similar to conventional chemokine receptors, and are proposed to act as chemokine scavengers. The most frequently studied atypical chemokine receptor, D6, scavenges inflammatory chemokines, with a number of ligands overlapping with those of CCR2.

CCR2 has been reported to play a non-redundant role in the egress of Ly6Chi monocytes from the bone marrow (BM) during homeostasis and inflammation. However, the role of CCR2 on other cell types has not been fully characterised and it is not known how much other CCR2+ cells may contribute to the role ascribed to CCR2 in several diseases, such as collagen-induced arthritis (CIA) and atherosclerosis. In addition, there is currently limited information about the leukocytic expression of D6, which can also bind CCL2. Although more information is available about CCR2, there are several conflicts reported between surface levels of CCR2 detected by antibody and transcript levels. To gain a true understanding of the function of these receptors in diseases it is crucial to have an accurate profile of CCR2 and D6 expression in vivo and subsequently determine the function of CCR2 and/or D6 on these cells. Therefore, the primary aim of this thesis was to generate a detailed profile of CCR2 expression by leukocytes, focusing mainly on the spleen, but including lymph nodes (LNs), blood and BM.

In Chapter 3, I demonstrate that currently available anti-murine CCR2 antibodies are unreliable, as they were either unable to detect CCR2 or could only detect high levels of CCR2. I describe a novel technique using fluorescently labelled CCL2 (CCL2AF647), which substantially enhanced CCR2 detection and enabled a detailed characterisation of D6 activity. Using this method, I present a comprehensive analysis of CCR2 and D6 expression on mouse leukocytes. CCR2 expression was primarily limited to myeloid cells, dendritic cells (DCs), and natural killer (NK) cells, with lymphoid cells having low levels of CCR2. Interestingly, my data illustrates that D6 expression is not as restricted as published data suggests, as several cell populations, such as plasmacytoid DCs (pDCs) and some monocyte populations were found to express both CCR2 and D6. Furthermore, I present preliminary evidence that suggests that CCR2 might exhibit cell-type specific responses to its ligands.

I also systematically analysed the cellular composition of lymphoid organs and the blood of CCR2 knock-out (KO) mice and compared it to age- and gender-matched wild-type (WT) controls. These studies revealed that CCR2 deletion only affected the distribution of cells of monocytic origin during homeostasis. In contrast, the results described in Chapter 4 demonstrated that systemic inflammation induced by lipopolysaccharide (LPS) affected the frequency of several populations possessing CCR2 activity, including Ly6Chi monocytes and conventional DCs (cDCs). I also present data that suggests that LPS temporally regulates CCR2 and D6 activity in a cell type-specific manner.

In Chapter 5, I detail the identification of two subsets of pDCs, present in the spleen, BM, blood, and LNs that differ in their expression of CCR2 and D6. Both subsets were able to stimulate naive T cell proliferation and responded to toll-like receptor 7 (TLR7) and TLR9 stimulation, in terms of interferon alpha (IFNα) production and expression of activation markers. However, transcriptomics and flow cytometry did reveal some notable differences between these two subsets and deficiency in D6 or CCR2 led to changes in the surface immunophenotype of pDCs at certain anatomical locations. I also demonstrate that pDCs are able to migrate efficiently towards CCL2 in vitro, but that CCR2 was not required for the reported CCL2 dependent migration of pDCs to imiquimod-inflamed skin.

Taking these results together, I have established CCL2AF647 as a novel sensitive method of CCR2 detection, which facilitated the identification of two pDC subsets. Although I was unable to find a role for CCR2 on pDCs, further study might better define the function that CCR2, and D6, plays on pDCs. In Chapter 6 I discuss my findings, relating the results to published data and forming conclusions and hypotheses that can be examined further in future experiments.

Characterising receptors for the chemokine CCL2,and their differential expression on dendritic cellsubsets

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Characterising receptors for the chemokine CCL2,and their differential expression on dendritic cellsubsets