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Ouabain is a cardiac steroid hormone with immunomodulatory effects. It inhibits neutrophils migration induced by different stimuli, but little is known about the mechanisms involved in this effect. Thus, the aim of this study was to evaluate the ouabain effect on chemotactic signaling pathways in neutrophils. For that, mice neutrophils were isolated from bone marrow, treated with ouabain (1, 10, and 100 nM) for 2 h, submitted to transwell chemotaxis assay and flow cytometry analysis of Akt, ERK, JNK, and p38 phosphorylation induced by zymosan. Ouabain treatment (1, 10 and, 100 nM) reduces neutrophil chemotaxis induced by chemotactic peptide fMLP, but this substance did not inhibit Akt, ERK, and JNK activation induced by zymosan. However, ouabain (1 and 10 nM) reduced p38 phosphorylation in zymosan-stimulated neutrophils. These results suggest that ouabain may interfere in neutrophil migration through p38 MAPK inhibition.

Ouabain, a hormone that inhibits Na+/K+-ATPase, modulates many aspects of the inflammatory response. It has been previously demonstrated that ouabain inhibits neutrophil migration in several inflammation models in vivo, but little is known about the mechanisms underlying this effect. Thus, this work aimed to evaluate the effect of ouabain on molecules related to neutrophil migration. For this purpose, neutrophils obtained from mouse bone marrow were treated with ouabain (1, 10, and 100 nM) in vitro. Neutrophil viability was assessed by annexin V/propidium iodide staining. Ouabain treatment did not affect neutrophil viability at different times (2, 4, and 24 h). However, basal neutrophil viability was decreased after 4 h. Thus, we assessed the effect of ouabain on the adhesion molecule CD18, an integrin β2 chain protein, and on the chemokine receptor CXCR2 after 2 h of treatment. CD18 expression was reduced (by 30%) by 1 nM ouabain. However, the expression of CXCR2 on the neutrophil membrane was not affected by ouabain treatment (1, 10, and 100 nM). Moreover, ouabain (1, 10, and 100 nM) did not modulate the zymosan-induced secretion of CXCL1 (a chemokine receptor CXCR2 ligand) in macrophage cultures. These data suggest that the inhibitory effect of ouabain on neutrophil migration is related to reduced CD18 expression, indicating a novel mechanism of action.