Acute peripheral inflammation initiates after inflammatory signals recognition (e.g., infection and tissue injury) by resident cells, such as mast cells and macrophages. This recognition promotes mediators release (e.g., vasoactive amines and prostaglandins), which stimulates rapid effects on the vasculature, including vasodilation and fluid extravasation (i.e., increased vascular permeability; Medzhitov, 2008). One of the first reports associating ouabain and inflammation revealed that this steroid suppresses vascular permeability in the sheep skin and pleural cavity induced by the irritant agent turpentine (Lancaster and Vegad, 1967). Later, our group demonstrated that ouabain given intraperitoneally decreases zymosan-induced plasma extravasation in mice peritoneal cavity (Leite et al., 2015) and reduces the mouse paw edema stimulated by several phlogistic agents (de Vasconcelos et al., 2011). However, Gonçalves-de-Albuquerque et al. (2014) showed that intratracheal administration of ouabain induces lung edema formation in mice. It is important to consider that ouabain effect on lung edema must be related to Na⁺/K⁺-ATPase inhibition in alveolar cells (Gonçalves-de-Albuquerque et al., 2014), while ouabain effects demonstrated by our group may be associated with cell signaling mechanisms in immune cell (e.g., P-p38 and NF-κB activity inhibition; Mascarenhas et al., 2014; Leite et al., 2015).

Vasodilation and vascular permeability are events tune regulated by vasoactive amines. Histamine, which plays a critical role among these vasoactive molecules, is released by perivascular mast cells together with other mediators (e.g., newly synthesized cytokines and tryptases) during inflammation (Nathan, 2002). Different ouabain effects on histamine secretion by mast cells have been described. Okazaki et al. (1976) reported that ouabain inhibits antigen-induced histamine release on guinea-pig mast cells. On the other hand, several studies revealed that ouabain increased histamine secretion induced by different agents on rat mast cells (Frossard et al., 1983; Amellal et al., 1984; Knudsen et al., 1992; Lago et al., 2001), while no ouabain effect on human mast cells (Senol et al., 2007) and basophils (Magro, 1977) were reported. Different protocols and species variation in the ouabain sensitivity of Na⁺/K⁺-ATPase (Abeywardena et al., 1984; Herzig and Mohr, 1984; Wang et al., 2001) could explain this discrepant ouabain effects on mast cell degranulation.

Upon initiation of acute inflammation, circulating leukocytes are able to recognize molecules (e.g., selectins, integrins, and chemokines) on activated vascular endothelium and, after rolling and adhesion steps, they cross blood vessel barrier and reach inflamed tissue (Ley et al., 2007; Vestweber, 2015; Kourtzelis et al., 2017). Neutrophils are the first cells recruited to the injured site, followed by other inflammatory cells such as monocytes (Kolaczkowska and Kubes, 2013; Wang and Arase, 2014). These polymorphonuclear leukocytes are important not only to eliminate microorganisms but they also play a key role in inflammation resolution (Mayadas et al., 2014; Sugimoto et al., 2016). Moreover, neutrophils role in chronic inflammation has been described and they are pointed as a target to emerging therapeutic strategies (Soehnlein et al., 2017). Considering this, blocking neutrophil recruitment appears to be a crucial way to avoid inflammation maintenance.

Ward and Becker (1970) initially described ouabain inhibitory effect on rabbit neutrophil migration toward bacterial chemotactic factors in vitro. In agreement with this study, our group revealed that ouabain pretreatment reduces mice neutrophil migration induced by zymosan, a component of the cell wall of yeast Saccharomyces cerevisiae, (Leite et al., 2015) and by Leishmania amazonensis (Jacob et al., 2013). These data provide clear evidence that ouabain inhibits neutrophil recruitment induced by microbial agents. This ouabain effect was also demonstrated in peritoneal inflammation induced by mitogen concanavalin A (de Vasconcelos et al., 2011). Furthermore, in airway allergic inflammation model, ouabain has an anti-migratory effect (Galvão et al., 2017). Ray and Samanta (1997) have also demonstrated that ouabain impairs in vitro human neutrophil migration, by interfering with IL-8 receptor recycling. Additionally, other studies have demonstrated that ouabain also decreases lung cancer cells migration (Liu et al., 2013), possibly by reducing the expression of molecules related to cell adhesion (e.g., integrins and ICAM) (Takada et al., 2009; Ninsontia and Chanvorachote, 2014) and cell migration (e.g., Src, Akt, and FAK) (Pongrakhananon et al., 2013; Shin et al., 2015).

However, the inhibitory effect of ouabain on cell migration seems to depend on the presence of a previous inflammatory stimulus, since ouabain itself given by inhalation (Feng et al., 2011) or intratracheally (Gonçalves-de-Albuquerque et al., 2014) causes acute lung inflammation with increased neutrophil migration. This proinflammatory effect was followed by LTB₄ and PGE₂ high levels, both lipid mediators associated with cell migration. Moreover, it has been demonstrated that ouabain at high concentrations induces VCAM-1 expression (an adhesion molecule) in murine endothelial cells (Bereta et al., 1995). Na⁺/K⁺-ATPase inhibition may be, at least partially, responsible for this ouabain effect (Lacroix-Lamandé et al., 2012; Gonçalves-de-Albuquerque et al., 2014), but Na⁺/K⁺-ATPase-dependent activation of signaling cascades (e.g., ERK and p38 MAPK) cannot be ruled out (Bereta et al., 1995; Feng et al., 2011). Indeed, Leu et al. (1973) demonstrated that ouabain stimulates guinea-pig alveolar and peritoneal macrophages migration independent of the sodium pump.

A different pattern of cytokines is present since the inflammation onset until resolution phase. These soluble proteins are secreted by a variety of cells (e.g., immune e non-immune cells) and allow intercellular communication, mediating and regulating inflammatory process. An imbalance in proinflammatory (e.g., TNF-α) and anti-inflammatory (e.g., IL-10) cytokine production could entail inflammatory disorders (Tayal and Kalra, 2008; Sugimoto et al., 2016). Monocytes/macrophages are immune cells that act as a key source of cytokines because of their plasticity ability (i.e., change their pattern of cytokines and functions when exposed to different signals; Gordon and Taylor, 2005; Mantovani et al., 2014). It has been described that ouabain itself can stimulate human monocytes to secrete cytokines such as IL-1α, IL-1β, IL-6, and TNF-α (Foey et al., 1997; Matsumori et al., 1997, 2000; Teixeira and Rumjanek, 2014). Some different results are related to IL-6 and TNF-α production, which could be associated with individual variability of human donors. Indeed, critically ill patients with high levels of ouabain had higher serum concentrations of these proinflammatory cytokines and other inflammatory markers, such as C-reactive peptide and serum amyloid A, when compared to patients with low ouabain concentrations (Berendes et al., 2003). Interestingly, ouabain enhances interleukin-10 levels in human monocytes (Teixeira and Rumjanek, 2014). Recently, Kobayashi et al. (2017) demonstrated that ouabain effect on IL-1β release, in both macrophages and cardiac tissue, is related to NLRP3 inflammasome activation, which in turn is mediated through K⁺ efflux. It is noteworthy that in this study the authors used high doses of ouabain both in vitro and in vivo. This contrasts with other studies that show a reduction of a different pattern of cytokines, including IL-1β (Leite et al., 2015) with lower ouabain doses in presence of inflammatory stimulus (Jacob et al., 2013; Galvão et al., 2017).

Despite the well-established proinflammatory activities of some cytokines, such as TNF-α and IL-6, some studies have demonstrated their anti-inflammatory role (Liu et al., 1998; Zakharova and Ziegler, 2005; Masli and Turpie, 2008; Scheller et al., 2013). In this regard, use low concentrations of ouabain as cytokine immunoregulator could be useful in different clinical conditions. In fact, ouabain at low doses reverses sepsis-induced immunoparalysis by increase TNF-α, IFN-γ, and GM-CSF levels and improve mice survival (Dan et al., 2014).

Pain is another cardinal sign of inflammation, which is also modulated by ouabain. de Vasconcelos et al. (2011) demonstrated that intraperitoneal administration of ouabain reduces nociceptive behavior in mice model of inflammatory pain (i.e., acetic acid induced writhing test). This steroid also induces supraspinal antinociceptive activity, related to opioid mechanisms, since naloxone, an opioid antagonist, inhibits its effect. In addition, other studies revealed that ouabain intracerebroventricular (i.c.v.) (Calcutt et al., 1971) and intratechal (i.t.) (Zeng et al., 1999) injections at relative high doses (micrograms) produce central antinociception and potentiate morphine and clonidine central antinociceptive effect, mainly by enhancement of cholinergic transmission at the spinal cord level (Zeng et al., 1999, 2007). In contrast, it has also been shown that low doses (nanograms) of ouabain (i.c.v.) antagonize opioid receptor agonists (Masocha et al., 2003, 2016; Gonzalez et al., 2012) and that ouabain (i.t.) itself did not cause antinociception (Horvath et al., 2003). This pain modulation by ouabain, which depends on the dose and administration route used, suggests that it can modulate events in the central nervous system such as neuroinflammation.

Some studies have described ouabain role in the central nervous system (CNS). However, the effects of this steroid on neuroinflammation can be controversial. In a study with rat hippocampus, ouabain anti-inflammatory effect against neuroinflammation induced by LPS was observed. Acute intraperitoneal pre-treatment with this steroid reduced iNOS and IL-1β mRNA levels. In addition, ouabain also reduced p65 subunit NF-κB translocation and IκB degradation, both mechanisms important to inflammatory process (Kinoshita et al., 2014). However, when ouabain is administrated by intrahippocampal route in a concentration that does not inhibit Na⁺/K⁺-ATPase, it induces activation of NF-κB and increases iNOS and TNF-α mRNA levels (Kawamoto et al., 2012). As well as in peripheral inflammation, the anti-inflammatory effect of ouabain on hippocampus was only observed after an inflammatory stimulus. This could explain the different effects regarding NF-κB activation. Additionally, another study demonstrated the ability of ouabain to restore the lipid composition of rat hippocampal membranes in neuroinflammation induced by LPS (Garcia et al., 2015).

In rat cerebellar cell culture, ouabain at high concentrations induced NF-κB activation and consequent TNF-α and IL-1β cytokines increase through NMDA-Src-Ras pathway in absence of inflammatory stimulus (de Sá Lima et al., 2013). However, ouabain decreases IL-1β release in LPS-stimulated astrocytes (Forshammar et al., 2011). In spite of that, ouabain did not modulate IL-1β release in LPS-stimulated microglia, while increased TNF-α release at low concentration (Forshammar et al., 2013). Therefore, ouabain role in cytokine production at CNS level depends on cell type and concentration used. Besides that, ouabain effects on CNS could be associated with its role in bipolar and depressive disorders (Goldstein et al., 2006; Tonin et al., 2014). The interaction between neuroinflammation and cardiac steroids is more substantially detailed by Orellana et al. (2016).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.