Lymphocytes home from the blood into lymph nodes during the process of lymphocyte recirculation. Homing is initiated by the rolling of lymphocytes on high endothelial venules (HEVs) within the lymph node. This step is mediated by L-selectin, a C-type lectin, which recognizes a set of carbohydrate-based ligands on HEVs. A function-blocking mAb called MECA-79 recognizes the same complex, referred to as PNAd. The ligands are sialomucins modified by GlcNAc-6-sulfate, sialyl Lewis x, and Gal-6-sulfate. We have studied double knockout (DKO) mice in which two GlcNAc-6-O-sulfotransferases that are present in HEVs have been inactivated. These mice exhibit a 75% reduction in homing to lymph nodes and the total elimination of PNAd (complete loss of MECA-79 staining) from HEVs. We will investigate the possibility that Gal-6-sulfate modifications contribute to the """"""""residual"""""""" ligand activity on HEVs in DKO mice, as well in mice in which GlcNAc-6-O-sulfotransferases are intact (Aim 1). PNAd+ blood vessels, which co-express GlcNAc-6-O-sulfotransferase, are present in joint tissues of rheumatoid arthritis (RA) patients. This finding is recapitulated in three mechanistically-distinct models of inflammatory arthritis in mouse. We have characterized a new antibody, called Clone 40, with very similar binding properties as MECA-79 but more suitable for use in animal studies. We will employ the DKO mice, together with Clone 40, in the mouse models of arthritis to study the role of the sulfotransferases and their sulfated products in disease pathogenesis (Aim 2). Since some murine models have been very predictive of efficacious human therapeutics (e.g., anti TNF-1 therapy), our work may lead to new approaches for the treatment of RA. We will also investigate a sheep model of asthma in which L-selectin appears to play a highly novel role (Aim 3). Our experiments suggest that extravasated leukocytes in the airways can utilize L-selectin to react with sulfated mucin ligands, which are expressed in inflamed airways. This interaction leads to activation of the leukocytes, which results in an increase in airway resistance and airway responsiveness, two hallmarks of asthma. To evaluate this hypothesis, we will determine whether isolated airway mucins can activate neutrophils through binding to L-selectin and cause the secretion of broncho-active substances. Finally, we have discovered that airway mucins in lungs are ligands for Siglec-8, a receptor known to bind sulfated and sialylated sugars. This Siglec is present on eosinophils and can induce apoptosis when artificially cross-linked by antibodies. We will determine whether these mucins are natural ligands for Siglec-8, serving to crosslink Siglec-8 on eosinophils and thus triggering apoptosis of these leukocytes (Aim 4). This would provide a homeostatic control mechanism for removing eosinophils that accumulate in allergic diseases, such as in asthmatic airways. Understanding this mechanism may lead to new pharmacologic approaches for dampening eosinophil responses.
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