Peripheral blood monocytes are a heterogeneous population of circulating leukocytes. Using a murine adoptive transfer system to probe monocyte homing and differentiation in vivo, we identified two functional subsets among murine blood monocytes: a short-lived CX(3)CR1(lo)CCR2(+)Gr1(+) subset that is actively recruited to inflamed tissues and a CX(3)CR1(hi)CCR2(-)Gr1(-) subset characterized by CX(3)CR1-dependent recruitment to noninflamed tissues. Both subsets have the potential to differentiate into dendritic cells in vivo. The level of CX(3)CR1 expression also defines the two major human monocyte subsets, the CD14(+)CD16(-) and CD14(lo)CD16(+) monocytes, which share phenotype and homing potential with the mouse subsets. These findings raise the potential for novel therapeutic strategies in inflammatory diseases.

Parenchymal microglia are the principal immune cells of the brain. Time-lapse two-photon imaging of GFP-labeled microglia demonstrates that the fine termini of microglial processes are highly dynamic in the intact mouse cortex. Upon traumatic brain injury, microglial processes rapidly and autonomously converge on the site of injury without cell body movement, establishing a potential barrier between the healthy and injured tissue. This rapid chemotactic response can be mimicked by local injection of ATP and can be inhibited by the ATP-hydrolyzing enzyme apyrase or by blockers of G protein-coupled purinergic receptors and connexin channels, which are highly expressed in astrocytes. The baseline motility of microglial processes is also reduced significantly in the presence of apyrase and connexin channel inhibitors. Thus, extracellular ATP regulates microglial branch dynamics in the intact brain, and its release from the damaged tissue and surrounding astrocytes mediates a rapid microglial response towards injury.

SUMMARY Mononuclear phagocytes, including monocytes, macrophages and dendritic cells, contribute to tissue integrity, as well as innate and adaptive immune defense. Emerging evidence for labour division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organisation of the cellular network are not well-defined. Here we report a fate mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX3CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue resident macrophage populations, including liver Kupffer cells, lung alveolar, splenic and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that the short-lived Ly6C+ monocytes constitute obligatory steady state precursors of blood-resident Ly6C− cells and that the abundance of Ly6C+ blood monocytes dynamically controls the circulation life span of their progeny.

Monocytes and macrophages are critical effectors and regulators of inflammation and the innate immune response, the immediate, pre-programmed arm of the immune system. Dendritic cells initiate and regulate the highly pathogen-specific adaptive immune responses, and are central to the development of immunologic memory and tolerance. Recent in vivo experimental approaches in the mouse have unveiled new aspects of the developmental and lineage relationships among these cell populations. Despite this, the origin and differentiation cues for many tissue macrophages, monocytes, and dendritic cell subsets in mice, and the corresponding cell populations in humans, remain to be elucidated.White blood cells or leukocytes are a diverse group of cell types that mediate the body's immune response. They circulate through the blood and lymphatic system and are recruited to sites of tissue damage and infection. Leukocyte subsets are distinguished by functional and physical characteristics. They have a common origin in hematopoietic stem cells and develop along distinct differentiation pathways in response to internal and external cues. The mononuclear phagocyte system represents a subgroup of leucocytes originally described as a population of bone marrow-derived myeloid cells that circulate in the blood as monocytes and populate tissues as macrophages in the steady state and during inflammation (1). In different tissues they can show significant heterogeneity with respect to phenotype, homeostatic turnover and function. The discovery of dendritic cells (DCs) as a distinct lineage of mononuclear phagocytes, specialized in antigen presentation to T cells and the initiation and control of immunity (2), revealed additional roles of these cells in shaping the immune response to pathogens, vaccines and tumors, as well as additional heterogeneity. Whereas a detailed map of the relationship between monocytes, DCs and their progenitors begins to emerge, other areas like the origin and renewal of tissue macrophage subsets remain less defined. (Fig. 1A) circulate in the blood, bone marrow, and spleen and do not proliferate in a steady state (3,4). Monocytes represent immune effector cells, equipped with chemokine Monocytes

A multitude of leukocyte migration events is needed to accomplish immunosurveillance in vertebrate organisms. Blood monocytes derived from central hematopoietic organs continuously seed the periphery with sentinels specialized in antigen uptake. Antigen encounter results in the mobilization of antigen-presenting cells (APC) to afferent lymphatics and their recruitment to secondary lymphoid organs, where they trigger T-cell responses. Long-distance migration of leukocytes is accomplished via blood and lymph circulation and thus requires transendothelial migration through vessel walls. The interaction of leukocytes with vascular endothelial cells during extravasation at sites of inflammation is a highly regulated process. After an initial, predominantly selectin-mediated "rolling" step, engagement of G-protein-coupled chemokine receptors leads to activation of integrins and the establishment of firm arrest, followed by diapedesis (2, 3).Recently a novel chemokine named fractalkine (FKN) (neurotactin [NTN]) was identified (1, 15) and shown to have unique properties. FKN has a CX 3 C chemokine domain and thus constitutes, according to the current chemokine nomenclature based on the spacing of N-terminal cysteines, its own CX 3 C family. Unlike any other known chemokine, the CX 3 C module was found to exist in two isoforms; one is membrane anchored and presented on an extended mucin-like stalk, and the other is a soluble form resulting from membrane-proximal proteolytic cleavage of FKN. In addition to its classical function as a chemoattractant, high-affinity interaction of FKN with its specific receptor CX 3 CR1 (8) mediates leukocyte arrest under flow conditions (4). In vitro data show that this firm adhesion is signaling independent and does not involve integrin activation, and may thus represent a novel mechanism in leukocyte trafficking (4, 7). FKN has been shown to be expressed on activated endothelial cells (1, 15), dendritic cells (DC) (9, 16), and neurons (6, 14). The FKN receptor, CX 3 CR1 (formerly V28 [18]), is a typical seven-transmembrane G-protein-coupled receptor. CX 3 CR1 is expressed on human monocytes and undefined subsets of NK and T cells (8). Expression of FKN and CX 3 CR1 in neurons and microglia, respectively, has fostered speculations that the receptor-ligand pair might be crucial for neuronal-glial cross talk (6,14).To investigate the in vivo role of FKN-CX3CR1 interactions, we generated a mouse mutant that lacks the FKN receptor. Our strategy was to replace the murine CX 3 CR1 gene with the gene encoding the enhanced green fluorescent protein (EGFP; Clontech). This approach allowed not only the generation of a mutant CX 3 CR1 locus but also the examination of the CX 3 CR1 expression pattern and migration of cells that normally express this receptor. MATERIALS AND METHODSMolecular cloning and generation of CX 3 CR1 mutant mice. Genomic fragments of the murine CX 3 CR1 locus were isolated from a 129/Sv phage library (Stratagene, La Jolla, Calif.) by hybridization with a human CX 3 CR1 cDNA pr...