Polymorphonuclear leukocytes (PMN) protect the lung against microbial infection; however, an excessive or prolonged influx of PMNs may cause tissue damage. Acute lung injury is characterized by widespread inflammatory changes, including diffuse PMN infiltration, with injury to both lung epithelium and endothelium. We established that the removal of sialyl residues from the surface of PMNs and endothelial cells (EC) by an endogenous PMN sialidase is critical to normal PMN trafficking. Further, we find that desialylation of the Toll-like receptor (TLR) 4 complex enhances LPS-induced cytokine production, while restoration of sialyl residues by sialyltransferases (ST) may restore the basal state. The overall hypothesis to be tested is that sialidases and STs regulate innate immune responses in the lung, and the balance achieved will dictate whether or not the innate immune response develops into acute lung injury. To address this central hypothesis, we propose the following:
In Specific Aim 1 we will define which form(s) of PMN sialidase(s) (neul and neu3) regulates adherence to and migration across human lung microvascular EC by overexpression and knockdown of specific neu proteins.
In Specific Aim 2 we will define the substrates for PMN sialidase in PMNs and ECs and assess how their sialylation status affects PMN adhesion in vitro and trafficking in vivo. Since PMNs induce signaling cascades in ECs, in Specific Aim 3 we will examine whether an """"""""appropriate"""""""" level of PMN sialidase-induced tyrosine phosphorylation of EC proteins opens the EC-EC paracellular pathway sufficiently for transendothelial migration without protein leak, while at levels that exceed this threshold, if there will be loss of EC barrier function. A """"""""two-hit"""""""" model of acute lung injury has been proposed whereby a combination of LPS and chemotactic peptide promotes lung injury.
In Specific Aim 4, we will examine the role of sialidase in sensitizing the pulmonary vasculature to circulating LPS and lung injury. The molecular mechanism(s) by which desialylation promotes signaling through the TLR4/MD2/CD14 complex will be examined, as well as the ability of STs to downregulate this response. The ability of sialidase inhibitors to reverse acute lung injury will be studied in vivo in a murine model of pulmonary leukostasis. Since inhibitors of sialidase are commercially available, these studies will provide the experimental rationale for testing a new therapeutic approach to the treatment of lung injury. ? ? ?
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