Faculty Bibliography

The chemokine receptor CXCR4 is expressed in the embryonic and mature CNS, yet its normal physiological function in neurons remains obscure. Here, we show that its cognate chemokine, stromal cell-derived factor-1 (SDF-1), promotes the survival of cultured embryonic retinal ganglion cell neurons even in the absence of other neurotrophic factors. This survival effect is mediated primarily through a cAMP-dependent pathway that acts through protein kinase A and MAP kinase. Addition of SDF-1 to a human neuronal cell line induces phosphorylation of p44/p42 MAP kinase and GSK3beta. Mouse embryos lacking the CXCR4 receptor have a reduced number of retinal ganglion cells. The ligand of CXCR4, SDF-1, may therefore provide generalized trophic support to neurons during their development and maturation

Altering the concentrations of cyclic nucleotides within nerve cells can dramatically change their responses to axonal guidance cues, but the physiological signals that might induce such alterations are unknown. Here we show that the chemokine stromal cell-derived factor 1 (SDF-1) reduces the repellent activities of slit-2 on cultured retinal ganglion cell axons, of semaphorin 3A on dorsal root ganglion sensory axons, and of semaphorin 3C on sympathetic axons. This is a modulatory effect because SDF-1 has no detectable attractive or repellent effects on retinal or DRG axons by itself. This modulation is mediated through CXCR4, the receptor of SDF-1, and a pertussis toxin-sensitive G-protein-coupled signaling pathway that induces an elevation of cAMP. The spinal cords of CXCR4 mutant mice contain hyperfasciculated and aberrantly projecting axons. These results suggest that SDF-1 plays an essential role in modulating axonal responsiveness to various known guidance cues through a cyclic nucleotide-dependent signaling pathway

We studied whether circulating activated platelets and platelet-leukocyte aggregates cause the development of atherosclerotic lesions in apolipoprotein-E-deficient (Apoe(-/-)) mice. Circulating activated platelets bound to leukocytes, preferentially monocytes, to form platelet-monocyte/leukocyte aggregates. Activated platelets and platelet-leukocyte aggregates interacted with atherosclerotic lesions. The interactions of activated platelets with monocytes and atherosclerotic arteries led to delivery of the platelet-derived chemokines CCL5 (regulated on activation, normal T cell expressed and secreted, RANTES) and CXCL4 (platelet factor 4) to the monocyte surface and endothelium of atherosclerotic arteries. The presence of activated platelets promoted leukocyte binding of vascular cell adhesion molecule-1 (VCAM-1) and increased their adhesiveness to inflamed or atherosclerotic endothelium. Injection of activated wild-type, but not P-selectin-deficient, platelets increased monocyte arrest on the surface of atherosclerotic lesions and the size of atherosclerotic lesions in Apoe(-/-) mice. Our results indicate that circulating activated platelets and platelet-leukocyte/monocyte aggregates promote formation of atherosclerotic lesions. This role of activated platelets in atherosclerosis is attributed to platelet P-selectin-mediated delivery of platelet-derived proinflammatory factors to monocytes/leukocytes and the vessel wall

T lymphocytes differentiate in discrete stages within the thymus. Immature thymocytes lacking CD4 and CD8 coreceptors differentiate into double-positive cells (CD4(+)CD8(+)), which are selected to become either CD4(+)CD8(-)helper cells or CD4(-)CD8(+) cytotoxic cells. A stage-specific transcriptional silencer regulates expression of CD4 in both immature and CD4(-)CD8(+) thymocytes. We show here that binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and that different Runx family members are required to fulfill unique functions at each stage. Runx1 is required for active repression in CD4(-)CD8(-) thymocytes whereas Runx3 is required for establishing epigenetic silencing in cytotoxic lineage thymocytes. Runx3-deficient cytotoxic T cells, but not helper cells, have defective responses to antigen, suggesting that Runx proteins have critical functions in lineage specification and homeostasis of CD8-lineage T lymphocytes

An intronic silencer within the CD4 gene is the critical cis regulatory element for T cell subset-specific expression of CD4. We have combined transfection studies with gene targeting in mice to identify several key sequences within the silencer core that are required for gene silencing during thymocyte development. In mice, mutations in individual sites resulted in variegated, but heritable, derepression of CD4 in mature CD8(+) T lymphocytes, whereas compound mutations resulted in full derepression. These results indicate that there is partial redundancy in recruiting a chromatin remodeling machinery that results in epigenetic silencing. Mutations in single sites also resulted in partial derepression of CD4 in immature double-negative thymocytes, but there was no apparent variegation. These findings suggest two distinct modes of CD4 silencer function at different developmental stages: active repression in CD4(-)CD8(-) thymocytes, in which silencing must be reversible, and epigenetic gene silencing upon differentiation to the CD8(+) cytotoxic T cell lineage

This is the first study reporting the inactivation of a member of the mouse gene family of toxin-related ecto-ADP-ribosyltransferases (ARTs). Transfer of the ADP-ribose moiety from NAD onto extracellular arginine residues on T-cell membrane proteins is mediated by glycosylphosphatidylinositol-linked cell surface ARTs. Exposure of T cells to ecto-NAD blocks T-cell activation and induces T-cell apoptosis. To determine a possible role of ecto-ART2.1 and ART2.2 in these processes, we generated ART2.1/ART2.2 double-knockout mice. ART2-deficient mice were healthy and fertile and showed normal development of lymphoid organs. ART2-deficient T cells showed a dramatically reduced capacity to ADP-ribosylate cell surface proteins, indicating that most if not all ART activity on the T-cell surface can be attributed to the ART2s. Moreover, ART2-deficient T cells were completely resistant to NAD-induced apoptosis and partially resistant to NAD-mediated suppression of proliferation. These results demonstrate that the ART2 ectoenzymes are an essential component in the regulation of T-cell functions by extracellular NAD, e.g., following release of NAD upon lysis of cells in tissue injury and inflammation

The dentate gyrus is the primary afferent pathway into the hippocampus, but there is little information concerning the molecular influences that govern its formation. In particular, the control of migration and cell positioning of dentate granule cells is not clear. We have characterized more fully the timing and route of granule cell migration during embryogenesis using in utero retroviral injections. Using this information, we developed an in vitro assay that faithfully recapitulates important events in dentate gyrus morphogenesis. In searching for candidate ligands that may regulate dentate granule cell migration, we found that SDF1, a chemokine that regulates cerebellar and leukocyte migration, and its receptor CXCR4 are expressed in patterns that suggest a role in dentate granule cell migration. Furthermore, CXCR4 mutant mice have a defect in granule cell position. Ectopic expression of SDF1 in our explant assay showed that it directly regulates dentate granule cell migration. Our study shows that a chemokine is necessary for the normal development of the dentate gyrus, a forebrain structure crucial for learning and memory

Cytotoxic T lymphocytes (CTL) respond to antigenic peptides presented on MHC class I molecules. On most cells, these peptides are exclusively of endogenous, cytosolic origin. Bone marrow-derived antigen-presenting cells, however, harbor a unique pathway for MHC I presentation of exogenous antigens. This mechanism permits cross-presentation of pathogen-infected cells and the priming of CTL responses against intracellular microbial infections. Here, we report a novel diphtheria toxin-based system that allows the inducible, short-term ablation of dendritic cells (DC) in vivo. We show that in vivo DC are required to cross-prime CTL precursors. Our results thus define a unique in vivo role of DC, i.e., the sensitization of the immune system for cell-associated antigens. DC-depleted mice fail to mount CTL responses to infection with the intracellular bacterium Listeria monocytogenes and the rodent malaria parasite Plasmodium yoelii