Lymphatic vascular cell traffic from the periphery to lymph nodes occurs during inflammatory responses as well as neoplasia. In both cases, unique chemokines (chemotactic cytokines) produced by the lymphatic endothelium play central roles in driving the transit of either innate immune cells, such as dendritic cells, or tumor cells. This proposal examines the genetic importance of unique complex carbohydrates (glycans) in mediating chemokine-dependent cell traffic in the lymphatic microenvironment. We focus on heparan sulfate (HS), composed of sulfated glycans tethered to unique cell-surface bound as well as secreted proteoglycan core proteins. HS interacts with basic amino acid-rich domains of several chemokines. The focus herein is on HS produced by the lymphatic endothelium, as preliminary work suggests that lymphatic HS may mediate two critical chemokine functions: (1) scaffolding of major lymphatic chemokines (such as CCL21 and CXCL12) onto peri-lymphatic spatial gradients, and (2) unique clustered presentation of chemokines to cognate receptors on trafficking cells, wherein secreted lymphatic HS proteoglycans may serve as chemokine co-receptors. This proposal addresses the hypothesis that targeting lymphatic glycan biosynthesis will alter the ability of dendritic cells or tumor cells to migrate toward lymphatic vessels and traffic to lymph nodes in a chemokine-dependent manner. The goals are to: (1) Examine the effects of genetically altering lymphatic HS on cell trafficking in the lymphatic microenvironment in vivo. Migration of dendritic cells and tumor cells from peripheral tissue to regional lymph nodes will be examined in mice bearing gene defects in lymphatic HS biosynthesis. T cell responses in antigen-driven trafficking studies will also be examined. Mutants will be characterized for lymphatic HS structure and proteoglycan expression. (2) Determine the role of HS in establishing lymphatic chemokine gradients. HS chains will be purified and tested for their ability to bind major lymphatic chemokines. In lymphatic endothelial matrix-based assays, the effects of genetically altering HS biosynthesis on chemokine gradient formation in static and flow conditions will be examined. Chemokine distribution in the mutant state will also be examined in vivo. (3) Characterize the importance of lymphatic HS as a co-receptor for chemokinedependent cell migration and signaling. The ability of dendritic cells to migrate toward lymphatic endothelium bearing mutations in HS biosynthesis will be determined, and conditioned medium from mutant lymphatic endothelia will be tested for its ability to oligomerize (cluster) lymphatic chemokines and activate chemokine-dependent migration signaling in dendritic cells. Finally, the effect of lymphatic HS mutation on the association between lymphatic chemokines and cognate receptors on trafficking cells will be examined in vivo. Collectively, this work may uncover novel mechanisms for how the actions of multiple chemokines may be controlled by glycans in the lymphatic microenvironment. It may also establish a basis for novel therapeutic discovery.
Chemokines are 'sensing' molecules that drive migration of specific cells toward a chemokine source. In states of disease such as inflammation or cancer, chemokines produced by blood or lymph vessels may drive the transit of inflammatory or tumor cells with deleterious consequences. Examples include the promotion of transplant rejection by the trafficking of specialized ('dendritic') immune cells from the donor organ, the priming of inflammation by activated dendritic cells in autoimmune disease, and the spread of tumor cells as well as tolerance-promoting dendritic cells to lymph nodes in cancer. Preliminary work shows that a class of complex sugar molecules (glycans) may underlie or mediate the actions of chemokines that drive such cell traffic via the lymphatic system. Herein, we block the production of such complex sugars in the living cells that make up lymphatic vessels using state-of-the-art genetic tools, and examine the effects on pathologic cell traffic. This work may lead to novel therapy that may inhibit disease-promoting cell traffic in both pathologic inflammatory states as well as cancer.
