Small Molecule–Mediated Activation of the Integrin CD11b/CD18 Reduces Inflammatory Disease (1)
The migration and recruitment of leukocytes is essential for their normal immune response to injury and infection and for various inflammatory and autoimmune disorders. Leukocyte functions are modulated by β2 integrins, including the highly abundant integrin CD11b/CD18 (also known as Mac-1 and CR3), which is a heterodimer of the αM (CD11b) and β2 (CD18) subunits. Blocking CD11b/CD18 and its ligands and ablation of the genes encoding CD11b or CD18 decrease the severity of inflammatory responses in many animal models.
leukadherin-1 (LA1), leukadherin-2 (LA2), and leukadherin-3 (LA3) showed enhanced activity in vitro. LA1, LA2, and LA3 separately increased CD11b/CD18-dependent cell adhesion to fibrinogen with 50% effective concentration (EC50, the effective concentration for a 50% increase in adhesion) values of 4, 12, and 14 μM, respectively. LA1, LA2, and LA3 did not inhibit cell adhesion in the presence of the agonist Mn2+, suggesting that they are true agonists. We also identified a structurally related compound, leukadherin-control (LA-C), which showed no effect on CD11b/CD18-dependent cell adhesion. Increased adhesion of CD11b/CD18-expressing cells, induced by Mn2+ and LA1, LA2, or LA3, was blocked by the monoclonal antibodies (mAbs) IB4 and 44a, which are specific for CD18 and CD11b, respectively. LA1, LA2, and LA3 also increased the binding of wild-type, but not CD11b−/−, neutrophils to immobilized fibrinogen. LA1, LA2, and LA3 (but not Mn2+) selectively increased the binding of K562 E320A cells to fibrinogen. LA1 and LA2 increased the binding of the wild-type αA domain to immobilized ligand.
The chemoattractant peptide N-formyl-Met-Leu-Phe (fMLP) treatment of the cells with LA1, LA2, or LA3 resulted in a substantial decrease in lateral migration and migration velocity. Although the cells treated with LA1, LA2, or LA3 showed some movement toward the chemoattractant, they displayed reduced directional persistence and reduced mean square displacement (MSD). leukadherins reduced the efficiency of transendothelial migration (TEM) by THP-1 cells across a layer of human umbilical vein endothelial cells (HUVECs) activated by tumor necrosis factor–α (TNF-α) in vitro by increasing cell adhesion to the HUVEC layer.
In thioglycolate-induced peritonitis model in mice, intraperitoneal injection of thioglycolate resulted in substantial accumulation of neutrophils in the peritoneum compared to that in mice injected with saline. Administration of LA1 30 min before injection with thioglycolate significantly reduced the amount of neutrophil accumulation by 40% compared to that in mice pretreated with vehicle, whereas LA2 reduced neutrophil accumulation by 65%, and LA3 reduced it by 55%. LA1, LA2, and LA3 did not substantially reduce the number of neutrophils recruited to the peritoneum of thioglycolate-treated CD11b−/− mice. The number of peritoneal neutrophils increased 4 hours after administration of thioglycolate in animals pretreated with vehicle, peaked after 12 hours, and declined thereafter. In LA1-treated animals, neutrophil accumulation was substantially reduced at 4 hours after administration of thioglycolate and stayed reduced at 12 hours, suggesting that leukadherins substantially inhibited neutrophil recruitment.
In an arterial balloon injury model in rats, which were administered LA1 or vehicle (DMSO) to Fischer male rats 30 min before injury and continued injections every other day for 3 weeks, injured arteries of the LA1-treated rats developed significantly reduced neointimal thickening. A significant reduction in the number of macrophages in the arteries of LA1-treated animals.
A mouse model of kidney disease, anti–glomerular basement membrane (anti-GBM) nephritis is characterized by neutrophil infiltration that mediates urinary protein loss, including that of albumin. Induction of disease in mice led to a peak influx of neutrophils into the kidney and maximal proteinuria at day 3. LA1 produced a maximal decrease in both the number of infiltrating neutrophils and the extent of proteinuria in treated mice.
CD11b/CD18-mediated leukocyte rolling and firm adhesion to the vascular wall lead to leukocyte transmigration across the vasculature and accumulation in the tissue as part of a multistep infiltration process. Antibodies against integrins, such as the M1/70 mAb, block the binding of integrins to ligands found on the vascular wall, thus reducing the infiltration of leukocytes into the tissue. An alternative approach to inhibiting leukocyte migration by enhancing the activation of integrins with small molecules is highly effective in reducing leukocyte infiltration and subsequent inflammation in vivo. CD11b/CD18 agonists better preserve organ function upon inflammatory injury.
