Ouabain inhibits monocyte activation in vitro: prevention of the proinflammatory mCD14⁺/CD16⁺ subset appearance and cell-size progression

Raphael C Valente,¹ Elizabeth G Araujo,² Vivian M Rumjanek<sup>1

1</sup>Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; ²Departamento de Neurobiologia, Universidade Federal Fluminense, Rio de Janeiro, Brazil

Abstract: Classically described as a potent inhibitor of the sodium-potassium adenosine triphosphatase enzyme, ouabain has been further shown to act as an effective immunomodulator in mammals. Recently, our group showed that this hormone downregulates membrane CD14 (mCD14) in human monocytes, though it is not known whether monocyte activation status could modify ouabain influence. Hence, we aimed to investigate ouabain effect during monocyte activation in vitro, analyzing mCD14, CD16 and CD69 expression in total monocytes after two periods of adhesion (2 hours and 24 hours) or in small and large monocyte subpopulations separately. Ouabain (100 nM) inhibited monocyte-size increase, characteristic of activation, only when added to cells immediately after 2 hours' adhesion. Moreover, downregulation of both mCD14 and CD16 expression by ouabain was more effective in small monocytes and in cells after 2 hours' adhesion. Since monocytes after 24 hours' adhesion showed no lack of ouabain binding and no CD69 upregulation, it seems that ouabain is somehow incapable of triggering an appropriate cell-signaling induction once monocytes become activated. Furthermore, though p38 MAPK activation was crucial for the impairment in cell-size progression induced by ouabain, its inhibition did not alter ouabain-induced CD69 upregulation, suggesting that other molecules may participate in the response to this hormone by monocytes. Our data suggest that ouabain inhibits monocyte activation in vitro, preventing both cell-size increase and the appearance of the proinflammatory mCD14⁺/CD16⁺ subpopulation. Thus, the findings suggest that individuals suffering from disorders commonly associated with high ouabain plasma levels, like hypertension, may present defective monocyte activation under inflammation or infection.

Keywords: ouabain, human monocytes, p38 MAPK, mCD14, CD16, CD69