Cell experiment:

Supernatant cytokines are quantified after stimulation and culture for 18 h (monocytes) or 24 h (NK cells). Except for bead-based stimulation, all experiments are conducted using 100 μL cells in a 96-well plate format. NK cell stimuli are added as follows: (1) Syk inhibitor (1 μM), (2) Leukadherin-1 or dimethylsulphoxide (DMSO) (vector control) (7.5 μM). Shown to induce 82% of maximum response with negligible off-target effect, (3) anti-CD210 or isotype control (5 μg/mL), (4) 30-45 min after Leukadherin-1 NK cells are stimulated with combinations of IL-12 (10 ng/mL), IL-15 (30 ng/mL) or IL~18 (10 ng/mL): either IL-12 + IL-15 or IL-12 + IL-18. Monocytes are stimulated using pam3csk4 (TLR-2 agonist, 300 ng/mL) or R848 (TLR-7/8 agonist, 2 μg/mL). Supernatants are stored at –80oC for < 1 month before quantification. To exclude non-specific Leukadherin-1-mediated cytotoxicity, the cell viability is assayed at 24 h using the CellTitre-Glo reagent. No significant loss of viability in comparison with the DMSO control is seen, concurring with published data in other cell types.

Leukadherin 1 is an activator of CD11b/CD18 [1].

CD11b/CD18 is a cell surface adhesion receptor that mediates immune functions and leukocyte migration and is expressed primarily in the cells of the innate immune system.

Leukadherin 1 is a CD11b/CD18 activator. Leukadherin 1 increased CD11b/CD18-dependent cell adhesion to fibrinogen with EC50 value of 4 μM [1]. Leukadherin 1 increased CD11b/CD18-dependent cell adhesion to its ligand ICAM-1. In K562 cells, leukadherin 1 increased the binding of CD11b/CD18 to ICAM-1 through the formation of long membrane tethers [2].

In the acute peritonitis mice model, leukadherin 1 significantly reduced neutrophil accumulation by 40% [1]. In a MHC mismatched orthotopic kidney transplantation mouse model, leukadherin 1 reduced interstitial leukocyte infiltration, glomerular damage, neointimal hyperplasia and prolonged survival from 48.5% (CsA only) to 100% (CsA and LA1) [3]. In newborn rats exposed to hyperoxia (85% O2), leukadherin 1 reduced both macrophage and neutrophil infiltration in the lungs [4].

References:
[1].  Maiguel D, Faridi MH, Wei C, et al. Small molecule-mediated activation of the integrin CD11b/CD18 reduces inflammatory disease. Sci Signal, 2011, 4(189): ra57.
[2].  Celik E1, Faridi MH, Kumar V, et al. Agonist leukadherin-1 increases CD11b/CD18-dependent adhesion via membrane tethers. Biophys J, 2013, 105(11): 2517-2527.
[3].  Khan SQ, Guo L, Cimbaluk DJ, et al. A small molecule b2 integrin agonist improves chronic kidney allograft survival by reducing leukocyte recruitment and accompanying vasculopathy. Front Med (Lausanne), 2014, 1: 45.
[4].  Jagarapu J, Kelchtermans J, Rong M, et al. Leukadherin-1 Attenuates Hyperoxia-induced Lung Injury in Neonatal Rats. Am J Respir Cell Mol Biol, 2015.