BackgroundMonocyte subpopulations distinguished by differential expression of chemokine receptors CCR2 and CX3CR1 are difficult to track in vivo, partly due to lack of CCR2 reagents.Methodology/Principal FindingsWe created CCR2-red fluorescent protein (RFP) knock-in mice and crossed them with CX3CR1-GFP mice to investigate monocyte subset trafficking. In mice with experimental autoimmune encephalomyelitis, CCR2 was critical for efficient intrathecal accumulation and localization of Ly6Chi/CCR2hi monocytes. Surprisingly, neutrophils, not Ly6Clo monocytes, largely replaced Ly6Chi cells in the central nervous system of these mice. CCR2-RFP expression allowed the first unequivocal distinction between infiltrating monocytes/macrophages from resident microglia.Conclusion/SignificanceThese results refine the concept of monocyte subsets, provide mechanistic insight about monocyte entry into the central nervous system, and present a novel model for imaging and quantifying inflammatory myeloid populations.

Microglial cells are difficult to track during development due to the lack of specific reagents for myeloid sub-populations. To further understand how myeloid lineages differentiate during development to give rise to microglial cells, we investigated CX3CR1 and CCR2 transcription unit activation in Cx3cr1+/GFPCCR2+/RFP knock-in fluorescent protein reporter mice. The principal findings include: 1) CX3CR1+ cells localized to the AGM region, and visualized at E9.0 in the yolk sac and neuroectoderm, 2) At E10.5 CX3CR1 single positive microglial cells were visualized penetrating the neuroepithelium, 3) CX3CR1 and CCR2 distinguished infiltrating macrophages from resident surveillant or activated microglia within tissue sections and by flow cytometric analyses. Our results support the contribution of the yolk sac as source of microglial precursors. We provide a novel model to monitor chemokine receptor expression changes in microglia and myeloid cells early (E8.0-E10.5) in development and during inflammatory conditions, which have been challenging to visualize in mammalian tissues.

Embryonic development is controlled by a small set of signal transduction pathways, with vastly different phenotypic outcomes depending on the time and place of their recruitment. How the same molecular machinery can elicit such specific and distinct responses, remains one of the outstanding questions in developmental biology. Part of the answer may lie in the high inherent genetic complexity of these signaling cascades, as observed for the Wnt-pathway. The mammalian genome encodes multiple Wnt proteins and receptors, each of which show dynamic and tightly controlled expression patterns in the embryo. Yet how these components interact in the context of the whole organism remains unknown. Here we report the generation of a novel, inducible transgenic mouse model that allows spatiotemporal control over the expression of Wnt5a, a protein implicated in many developmental processes and multiple Wnt-signaling responses. We show that ectopic Wnt5a expression from E10.5 onwards results in a variety of developmental defects, including loss of hair follicles and reduced bone formation in the skull. Moreover, we find that Wnt5a can have dual signaling activities during mouse embryonic development. Specifically, Wnt5a is capable of both inducing and repressing β-catenin/TCF signaling in vivo, depending on the time and site of expression and the receptors expressed by receiving cells. These experiments show for the first time that a single mammalian Wnt protein can have multiple signaling activities in vivo, thereby furthering our understanding of how signaling specificity is achieved in a complex developmental context.

In vitro studies have implicated chemokine receptors in consumption and clearance of specific ligands. We studied the role that various signaling chemokine receptors play during ligand homeostasis in vivo. We examined the levels of ligands in serum and CNS tissue in mice lacking chemokine receptors. Compared with receptor-sufficient controls, Cx3cr1 Ϫ/Ϫ mice exhibited augmented levels of CX3CL1 both in serum and brain, and circulating levels of CXCL1 and CXCL2 were increased in Cxcr2 Ϫ/Ϫ mice. CCR2-deficient mice showed significantly increased amounts of circulating CCL2 compared with wild-type mice. Cxcr3 Ϫ/Ϫ mice revealed increased levels of circulating and brain CXCL10 after experimental autoimmune encephalomyelitis ( IntroductionActions of chemokines through chemokine receptor signaling leads to an array of diverse functions in different tissue compartments. 1,2 Such functions go beyond the original assigned roles of chemokines in leukocyte chemoattraction to inflamed tissues, and involve physiological trafficking to localize surveillant populations in noninflamed tissues, cellular activation, proliferation, adhesion, phagocytosis, apoptosis, and angiogenesis. [3][4][5][6] Chemokine/ chemokine receptor interactions exhibit defined roles during inflammation, atherosclerosis, autoimmunity, viral pathogenesis, cancer, and neurodegeneration. [7][8][9][10][11] Even though the system exhibits apparent redundancy, modulation of chemokine function via chemokine receptor blockade is a challenging area of considerable interest for therapeutic purposes.Among the chemokine receptors, CCR2 and its ligand CCL2 (MCP-1) have been extensively studied, and their role in regulating monocyte and T-cell infiltrations is well established. In 2002, Tylaska et al showed that CCR2-knockout mice manifested extremely high levels of CCL2 at sites of alloinduced inflammation, and in vitro studies confirmed that clearance of ligand was mediated by CCR2. 12 Our group reported that CCL2 is consumed by CCR2 ϩ migrating cells in a human blood-brain barrier model using peripheral blood mononuclear cells from healthy donors. 13 These results suggest an important biologic role of chemokine receptors as scavenger molecules involved in clearance of specific ligands.Chemokine receptor-deficient mouse strains have been instrumental in understanding chemokine biology in health and disease states. 14 In the present study, we evaluated levels of chemokines in 4 receptor-deficient mice, including those lacking CC, CXC, and CX3C receptors. Both circulating and brain tissue levels were studied. Brain was selected for study as a distinct tissue compartment in which chemokines may be produced under physiological and pathological conditions. We found that the levels of circulating and-in some instances-tissue chemokines are dramatically increased in healthy chemokine receptor-deficient mice. Reconstitution with wild-type bone marrow cells restored chemokine homeostasis. Importantly, for chemokines that signal to more than one receptor, absence of one recep...

Fractalkine, a chemokine anchored to neurons or peripheral endothelial cells, serves as an adhesion molecule or as a soluble chemoattractant. Fractalkine binds CX3CR1 on microglia and circulating monocytes, dendritic cells and NK cells. The aim of this study is to determine the role of CX3CR1 in the trafficking and function of myeloid cells to the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE). Our results show that in models of active EAE Cx3cr1–/– mice exhibited more severe neurological deficiencies. Bone marrow chimeric mice confirmed that CX3CR1-deficiency in bone marrow enhanced EAE severity. Notably, CX3CR1 deficiency was associated with an increased accumulation of CD115+Ly6C–CD11c+ dendritic cells into EAE affected brains which correlated with enhanced demyelination and neuronal damage. Furthermore, higher IFN-γ and IL-17 levels were detected in cerebellar and spinal cord tissues of CX3CR1-deficient mice. Analyses of peripheral responses during disease initiation revealed a higher frequency of IFN-γ and IL-17 producing T cells in lymphoid tissues of CX3R1-deficient as well as enhanced T cell proliferation induced by CX3CR1-deficient DCs. In addition, adoptive transfer of MOG35-55 reactive wild type T cells induced substantially more severe EAE in CX3CR1-deficient recipients when compared to wild type recipients. Collectively, the data demonstrate that besides its role in chemoattraction, CX3CR1 is a key regulator of myeloid cell activation contributing to the establishment of adaptive immune responses.

Mycoplasma mobile is a bacterium that uses a unique mechanism to glide on solid surfaces at a velocity of up to 4.5 μm/s. Its gliding machinery comprises hundreds of units that generate the force for gliding based on the energy derived from ATP; the units catch and pull sialylated oligosaccharides fixed to solid surfaces. In this study, we measured the stall force of wild-type and mutant strains of M. mobile carrying a bead manipulated using optical tweezers. The strains that had been enhanced for binding exhibited weaker stall forces than the wild-type strain, indicating that stall force is related to force generation rather than to binding. The stall force of the wild-type strain decreased linearly from 113 to 19 picoNewtons after the addition of 0-0.5 mM free sialyllactose (a sialylated oligosaccharide), with a decrease in the number of working units. After the addition of 0.5 mM sialyllactose, the cells carrying a bead loaded using optical tweezers exhibited stepwise movements with force increments. The force increments ranged from 1 to 2 picoNewtons. Considering the 70-nm step size, this small-unit force may be explained by the large gear ratio involved in the M. mobile gliding machinery.

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X-ray diffraction measurements on the Cr-H system were made using synchrotron radiation at high hydrogen pressures and high temperatures, and the phase diagram was determined up to p(H 2 ) = 5.5 GPa and T 1400 K. Three solid phases were found to exist; a bcc phase (α) of low hydrogen concentrations, x = [H]/[Cr] 0.03 existing at low hydrogen pressures ( 4.4 GPa), and two high-pressure phases, an hcp (ε) phase at lower temperatures and an fcc (γ ) phase at higher temperatures, both having high hydrogen concentrations x ∼ 1. A drastic reduction of the melting point is caused by dissolution of hydrogen. A gradual lattice contraction observed in the fcc phase indicates the formation of superabundant Cr-atom vacancies (vacancy-hydrogen clusters). Thermal desorption measurements after recovery from high p(H 2 ), T treatments revealed several desorption stages including those due to the release from vacancy-hydrogen clusters and from hydrogen-gas bubbles, and allowed determination of relevant trapping energies.