The Effect of Adenosine A2a Receptor Activation on Leukocyte Adhesiveness
Shuali, Annika, Biomedical Engineering - School of Engineering and Applied Science, University of Virginia
Lawrence, Michael, Department of Biomedical Engineering, University of Virginia
Neutrophils infiltrate inflamed tissues and become activated in response to a variety of inflammatory stimuli. Once activated neutrophils release cytokines, migrate towards chemoattractants, increase their expression of integrins(e.g. CD11b), shed L-selectin and undergo processes that aid in the destruction of pathogens. The migration of leukocytes to inflamed tissues is a multi-step process that involves specific ligand-receptor interactions between adhesion molecules expressed on the surface of both endothelial cells and the leukocytes. Adenosine A2A receptors have been shown to exert anti-inflammatory effects in animal models of inflammation, however their effect on neutrophils is unknown.
The aim of the present work is to investigate the effects of Adenosine A2A receptor stimulation on the expression of neutrophil activation markers and LFA-1 integrin-mediated adhesion to the ligand ICAM-1 using the agonist ATL313.
Flow cytometry was used to assess the expression of neutrophil activation markers(CD11b, L-selectin, F-actin) in cells stimulated with Phorbol 12-myristate 13-acetate(PMA) with and without 1 μM ATL313 stimulation. PMA-stimulation led to a 2.26-fold increase in neutrophil CD11b expression, a 5.25-fold decrease in L-selectin expression, and a 1.49-fold decrease in F-actin polymerization that was statistically significant compared to unstimulated cells. ATL313 incubation with and without PMA stimulation was not statistically different from untreated cells or cells treated with PMA alone, respectively.
PMA-stimulated neutrophil adhesion to ICAM-1 was examined using a flow chamber with and without ATL313 incubation. PMA stimulation increased cell spreading on ICAM-1 16.7-fold in comparison to unstimulated cells. Stimulation with both ATL313 and PMA led to a 10.6-fold decrease in cell spreading on ICAM-1 compared to PMA-stimulated neutrophils. Furthermore, incubation with ATL313 resulted in a 60 to 90% decrease in the average percent of cells that remained firmly adhered to ICAM-1 after 30 seconds of shear stress at 1 dyne/cm2.
In conclusion this study demonstrated that ATL313 stimulation is unable to abolish Protein Kinase-C (PKC)-mediated changes in the expression of neutrophil activation markers but can robustly alter the adhesion of neutrophils to ICAM-1. Occupancy of A2A receptors with drugs such as ATL313 may exert their anti-inflammatory effects on neutrophils through inhibition of LFA-1-mediated adhesion and migration.
MS (Master of Science)
Adenosine, Neutrophils, Adhesion, Integrins, Leukocyte
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