(A) Surface expression profiling of various stable lines generated that express different levels of wild-type CD11b/CD18

(A) Surface expression profiling of various stable lines generated that express different levels of wild-type CD11b/CD18. signaling.1 Together with their ligands, integrins play central roles in many processes, including development, hemostasis, inflammation, and VEGFA immunity, and in pathologic conditions such as cancer invasion and cardiovascular disease. The 2 2 integrins, which have a common -subunit (2, CD18) but distinct -subunits (CD11a, CD11b, CD11c, and Refametinib CD11d), are critical leukocyte receptors that are important not only for the function of leukocytes but also for the development of the inflammatory response in vivo.2 Leukocytes normally circulate in the vasculature in a quiescent state but, in response to inflammatory stimuli, adhere, transmigrate across the vascular endothelium, and enter areas of tissue inflammation, where they participate in the destruction and removal of infectious agents and in amplifying the process of inflammation. The integrin CD11b/CD18 (complement receptor type 3 [CR3], Mac-1, or M2) is the predominant 2 integrin receptor in neutrophils, macrophages, and monocytes and mediates a large number of pro-inflammatory functions in these cells.3,4 CD11b/CD18 recognizes a wide variety of ligands, including the complement fragment iC3b, fibrinogen, blood-clotting factor X, CD54 (ICAM-1), the hookworm neutrophil inhibitory factor (NIF), and denatured proteins such as bovine serum albumin (BSA).5 Studies in CD11b?/? mice have shown that this integrin has Refametinib a distinct and cooperative role (with integrin CD11a) in the inflammatory process.6 In addition to the knockout mice studies, the biological importance of this integrin in maintaining immunological homeostasis has been illustrated by different pathological conditions where integrins are absent or defectiveloss of functional 2 integrins causes life-threatening infections in humans, and mutations result in leukocyte adhesion deficiency type 1, where circulating neutrophils fail to adhere to or migrate across the endothelium and the patients are susceptible to recurrent, life-threatening bacterial infections.7C9 Similarly, improper excessive activation of leukocyte integrins is also harmful, as overactivation of 2 integrins contributes to sustained inflammation, ischemia-reperfusion injury (including acute renal failure, atherosclerosis and autoimmune disorders,1,7 and tissue damage10), and the development of various autoimmune diseases.11 CD11b/CD18 is also implicated in stroke,12 neointimal thickening in response to vascular injury,2 bullous pemphigoid,13 and neonatal obstructive nephropathy.14 Thus, there is a considerable potential for agents that block the binding of CD11b/CD18 to its physiologic ligands as therapeutics for the treatment of such inflammatory conditions. Physiologic ligand binding by CD11b/CD18 is divalent cation dependent and is mediated by CD11b von Willebrand factor type A (VWFA) domain, CD11bA-domain (A-domain).15 Blocking Refametinib anti-CD11b/CD18 antibodies decreases ischemia/reperfusion injury,16 the area of myocardial infarction,17 and liver cell injuries,18 and it diminishes neointimal thickening and restenosis after balloon injury of carotid arteries19 in animal models. Refametinib These antibodies are also effective in the treatment of endotoxic challenge and hemorrhagic shock20 and autoimmune injury in various organs, including the kidney. However, antibody therapy is not ideal, as adverse effects due to nonselective blockade of various other leukocyte functions may lead to severe complications.21 Similarly, NIF, a 41-kDa glycoprotein ligand mimic, is effective in attenuating the deleterious effects of excessive neutrophil activation in animal models,22 but its large size and immunogenecity preclude its use as a therapeutic agent. In addition, although blockade of the binding sites of integrins with ligand-mimetic peptides or small molecules has proved effective in inhibiting the activities of 1 1 and 3 integrins, peptides derived either from CD11b/CD18 ligands or anti-CD11b/CD18 antibodies were not very efficacious in blocking ligand binding in vitro.23 The failure of these ligand-mimetic peptides to block the interaction between iC3b and CD11b/CD18 may be due to their improper conformation in solution or to the size of the ligand-binding sites, which may be too extensive to block with a small peptide. The identification of small molecules that selectively modulate CD11b/CD18 ligand binding, especially by targeting allosteric regulatory sites, such as the hydrophobic site-for-isoleucine (SILEN) pocket in CD11b/CD18,24 may prove to be a more promising therapeutic strategy, as has been shown by the discovery of several small-molecule antagonists targeting a similar site for the related integrin CD11a/CD18.25 Progress is being made in that direction, with 2 recent reports describing identification of a few small molecules targeting CD11b/CD18.26,27 However, current assays rely on purified proteins adsorbed to microplates, and even though these assays are compatible.