Compositions and methods for regulating leukocyte adhesion

Abstract

Inflammatory cell recruitment to local sites of tissue injury and/or infection is controlled by many signaling processes influencing cell-to-cell interactions between vascular endothelial cells (EC) in post-capillary venules and circulating leukocytes. Here we report that the ATP-release channel Pannexin1 (Panx1) opens downstream of EC activation by tumor necrosis factor α (TNFα). This process involves activation of Type 1 TNF receptors, recruitment of Src Family Kinases (SFK), and SFK-dependent phosphorylation of Panx1. We report a previously unidentified role for Panx1 channels in promoting leukocyte adhesion and emigration through the venous wall during acute systemic inflammation. The present application further discloses that Panx IL2 peptide consisting of amino acid sequence KYPIVEQYLKYGRKKQRR (SEQ ID NO:3) or 10Panx1 peptide consisting of amino acid sequence WRQAAFVDSY (SEQ ID NO:8) are inhibitors of leukocyte adhesion.

What is claimed is: 
     
         1 . A method for treating inflammation at a site of tissue injury, disease, or infection by inhibiting activation of venous endothelial cell Pannexin1, said method comprising administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of an inhibitor of Pannexin1 activation, thereby treating said inflammation at a site of tissue injury, disease, or infection. 
     
     
         2 . The method of  claim 1 , wherein said Pannexin1 is activated by a cytokine. 
     
     
         3 . The method of  claim 2 , wherein said cytokine is Tumor Necrosis Factor alpha (TNFα). 
     
     
         4 . The method of  claim 1 , wherein said method inhibits inflammatory cell recruitment to said site of tissue injury, disease, or infection. 
     
     
         5 . The method of  claim 4 , wherein said inflammatory cell is a leukocyte. 
     
     
         6 . The method of  claim 5 , wherein said leukocyte is a monocyte. 
     
     
         7 . The method of  claim 1 , wherein said TNFα activates Pannexin1 by stimulating src family kinase (SFK) phosphorylation of Pannexin1. 
     
     
         8 . The method of  claim 1 , wherein said inhibitor is a peptide. 
     
     
         9 . The method of  claim 8 , wherein said peptide is IL2 peptide consisting of amino acid sequence KYPIVEQYLKYGRKKQRRR (SEQ ID NO:3) or  10 Panx1 peptide consisting of amino acid sequence WRQAAFVDSY (SEQ ID NO:8), or biologically active fragments or homologs thereof. 
     
     
         10 . The method of  claim 9 , wherein said inhibitor inhibits leukocyte adhesion to an endothelial cell. 
     
     
         11 . The method of  claim 10 , wherein said endothelial cell is a venous endothelial cell. 
     
     
         12 . The method of  claim 9 , wherein said inhibitor inhibits leukocyte emigration through a blood vessel wall. 
     
     
         13 . The method of  claim 12 , wherein said blood vessel is a vein. 
     
     
         14 . The method of  claim 1 , wherein said site comprises a localized focus of inflammation. 
     
     
         15 . The method of  claim 1 , wherein said tissue injury, disease, or infection is selected from the group consisting of stroke, ischemic stroke, reperfusion injury, acute kidney injury, transplantation, autoimmune disease, obesity, aortic aneurism, sepsis, inflammatory-associated hypertension, osteoarthritis, and atherosclerosis. 
     
     
         16 . The method of  claim 7 , wherein said method inhibits TNFα, activation of Pannexin1. 
     
     
         17 . The method of  claim 16 , wherein said method inhibits ATP release from said venous endothelial cell. 
     
     
         18 . The method of  claim 1 , wherein said inhibitor is directed against Pannexin1. 
     
     
         19 . A method for inhibiting inflammation and inflammatory cell recruitment at a site of tissue injury, disease, or infection wherein said inflammation and cell recruitment are associated with Pannexin1 activation in venous endothelial cells, said method comprising administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of an inhibitor of said venous endothelial cell Pannexin1 activation, thereby inhibiting inflammation and inflammatory cell recruitment to a site of tissue injury, disease, or infection. 
     
     
         20 . The method of  claim 19 , wherein said inhibitor is selected from the group consisting of a drug, peptide, mimetic peptide, and antibody. 
     
     
         21 . The method of  claim 20 , wherein said inhibitor is directed against Pannexin1. 
     
     
         22 . The method of  claim 21 , wherein said Pannexin1 is activated by a cytokine. 
     
     
         23 . The method of  claim 22 , wherein said cytokine is TNFα. 
     
     
         24 . The method of  claim 19 , wherein said inflammatory cell is a leukocyte. 
     
     
         25 . The method of  claim 24 , wherein said leukocyte is a monocyte. 
     
     
         26 . The method of  claim 19 , wherein said TNFα activates Pannexin1 by stimulating src family kinase (SFK) phosphorylation of Pannexin1. 
     
     
         27 . The method of  claim 19 , wherein said inhibitor is directed against Pannexin1. 
     
     
         28 . The method of  claim 27 , wherein said inhibitor is a peptide. 
     
     
         29 . The method of  claim 28 , wherein said peptide is IL2 peptide consisting of amino acid sequence KYPIVEQYLKYGRKKQRR (SEQ ID NO:3) or  10 Panx1 peptide consisting of amino acid sequence WRQAAFVDSY (SEQ ID NO:8), or biologically active fragments or homologs thereof. 
     
     
         30 . The method of  claim 29 , wherein said inhibitor inhibits leukocyte adhesion to an endothelial cell. 
     
     
         31 . The method of  claim 30 , wherein said endothelial cell is a venous endothelial cell. 
     
     
         32 . The method of  claim 29 , wherein said inhibitor inhibits leukocyte emigration through a blood vessel wall. 
     
     
         33 . The method of  claim 32 , wherein said blood vessel is a vein. 
     
     
         34 . The method of  claim 19 , wherein said site comprises a localized focus of inflammation. 
     
     
         35 . The method of  claim 19 , wherein said tissue injury results from a stroke. 
     
     
         36 . The method of  claim 26 , wherein said method inhibits TNFα activation of Pannexin1. 
     
     
         37 . The method of  claim 36 , wherein said method inhibits ATP release from said venous endothelial cell. 
     
     
         38 . A method for stimulating leukocyte adhesion to a venous endothelial cell in a subject in need thereof, said method comprising administering to said subject a pharmaceutical composition comprising an effective amount of a stimulator of Pannexin1, wherein said stimulator activates Pannexin1 in said endothelial cell and induces ATP release. 
     
     
         39 . The method of  claim 38 , wherein said stimulator is TNFα. 
     
     
         40 . A method for inhibiting TNFα-induced ATP release from a venous endothelial cell, said method comprising contacting said venous endothelial cell with an effective amount of an inhibitor of Pannexin1 activity. 
     
     
         41 . The method of  claim 40 , wherein said method inhibits TNFα-induced SFK activation of Pannexin1. 
     
     
         42 . The method of  claim 41 , wherein said method inhibits SFK phosphorylation of Pannexin1. 
     
     
         43 . The method of  claim 40 , wherein said inhibitor is directed against Pannexin1. 
     
     
         44 . The method of  claim 43 , wherein said inhibitor is IL2 peptide consisting of amino acid sequence KYPIVEQYLKYGRKKQRR (SEQ ID NO:3) or  10 Panx1 peptide consisting of amino acid sequence WRQAAFVDSY (SEQ ID NO:8), or biologically active fragments or homologs thereof. 
     
     
         45 . The method of  claim 40 , wherein said venous endothelial cell is contacted with at least two inhibitors of Pannexin1 activity. 
     
     
         46 . A method for identifying an inhibitor of TNFα-induced activation of venous endothelial cell Pannexin1, said method comprising contacting a venous endothelial cell comprising Pannexin1 with TNFα and a test compound and determining whether said venous endothelial cell Pannexin1 is activated by measuring at least one marker of venous endothelial Pannexin1 activation and comparing the level of said at least one marker to the level of said at least one marker before activation or to a standard level or to a level measured when an otherwise identical venous endothelial cell is contacted with TNFα but not said test compound. 
     
     
         47 . The method of  claim 46 , wherein said venous endothelial cell contacted with said TNFα and said test compound is contacted with said TNFα first. 
     
     
         48 . The method of  claim 46 , wherein said venous endothelial cell contacted with TNFα and a test compound is contacted with said test compound first. 
     
     
         49 . The method of  claim 46 , wherein a range of varying amounts of said test compound are tested and a dose-response curve is generated. 
     
     
         50 . The method of  claim 46 , wherein said marker is selected from the group consisting of amount of ATP released, phosphorylated Pannexin1, leukocyte adhesion to an endothelial cell, and leukocyte emigration.