Small Molecule Antagonists of Complement Receptor Type 3 Block Adhesion and Adhesion-Dependent Oxidative Burst in Human Polymorphonuclear Leukocytes

Abstract

The leukocyte integrin complement receptor type 3 (CR3, Mac-1, CD11b/CD18) is the predominant ␤ 2 integrin receptor of polymorphonuclear leukocytes (PMNs). This cell surface receptor plays a central role in innate immunity against pathogens as well as being a major cellular effector of inflammation and tissue injury. Two small molecules, compounds 1 and 2, have been identified, that interact with CR3 and prevent CR3 from binding to its natural ligand, C3bi. Compounds 1 and 2 have IC 50 values of 0.14 and 0.33 M, respectively, for the inhibition of binding of monomeric C3bi-alkaline phosphatase to immobilized CR3. Both compounds also inhibit binding of CR3 to biotinylated sheep red blood cells opsonized with C3bi, with IC 50 values in the micromolar range. Inhibition of ligand binding by the compounds is not easily reversed and requires light, suggesting the formation of a covalent adduct through photoactivation. Compounds 1 and 2 also inhibit adhesion of human PMNs to fibrinogen in response to tumor necrosis factor (TNF) or PMA, with IC 50 values of 2.5 to Ͼ10 M. They block the adhesion-dependent production of H 2 O 2 stimulated by TNF or phorbol 12-myristate 13-acetate (PMA) with IC 50 values of 0.2 to 0.8 M and 1 to 3 M, respectively. Limited structure-activity relationship studies based on compound 2 indicate the importance of the two benzothiazole rings, an ethyl side chain, and the length of the carbon chain linking the rings. Further modification of these groups may help in making compounds appropriate for in vivo studies.Polymorphonuclear leukocytes (PMNs) represent an important constituent of the innate immune system. Under normal circumstances, they circulate in the vasculature in a quiescent state. However, in response to inflammatory stimuli, they adhere, transmigrate across the vascular endothelium, and enter areas of tissue inflammation where they participate not only in the destruction and removal of pathogens but also amplify the process of inflammation (Kishimoto et al., 1999). Migration of PMNs and phagocytosis of bacteria are tightly controlled, and adhesion receptors present on the cell surface of PMNs are crucial for initiating these responses. Upon activation of PMNs in response to stimuli such as TNF, the adhesion receptors, members of the integrin superfamily, are activated, and additional receptors present in intracellular granules translocate to the cell surface (Carlos and Harlan, 1994). The interaction of integrins with ligands present on vascular endothelial cells, stromal cells, and bacteria promotes adhesion and its sequelae.The major adhesion-promoting receptor present on the cell surface of activated PMNs is complement receptor type 3 (CR3), also called Mac-1, CD11b/CD18, or ␣ M ␤ 2 (Kishimoto et al., 1999). CR3 is a heterodimer belonging to the ␤ 2 integrin subfamily. The two subunits, ␣ M (M r 165,000) and ␤ 2 (M r 95,000), are held together by noncovalent interactions. Studies have been done on the structure and functions of ␤ 2 integrins and have been reviewed extensiv...

Critical role for complement receptor 3 (CD11b/CD18), but not for Fc receptors, in killing of Streptococcus pyogenes by neutrophils in human immune serum

“…These conditions do not readily translate to the ex vivo conditions used here, where simultaneous signaling through other receptors could make the requirement for FcR stimulation redundant. Thus, immobilization of the neutrophils on matrix proteins can induce oxidative burst in neutrophils, particularly in the presence TNF-a [29,30]. Clearly, in the bactericidal assay with growing bacteria there are a number of ways through which such costimulatory pathways can be induced.…”

Gelatinase-mediated migration and invasion of cancer cells

“…These integrins are also clinically highly relevant. Consequently, antagonists of the leukocyte integrins α L β 2 and α M β 2 are being developed for the treatment of various autoimmune diseases and inflammatory conditions (Bansal et al, 2003;. The formation of the adhesive contacts is regulated by members of the Rho subfamily of GTPases, including Cdc42, Rac and Rho (Hall, 1994).…”

Identification of novel agonists of the integrin CD11b/CD18

“…Progress towards identifying small molecules that selectively target integrin CD11b/CD18 has been slow, with only a few reported discoveries,10, 11 primarily due to the lack of good high-throughput screening (HTS) assays. We recently described a simple, no-wash cell-adhesion based HTS assay in the 384-well plate format that is ideal for the discovery of small molecules against the integrin CD11b/CD18 12.…”