Sphingosine 1-phospate (S1P) has been demonstrated to protect against the formation of lipopolysaccharide (LPS)-induced lung edema when administered concomitantly with LPS. In the present study, we sought to determine the effectiveness of S1P to attenuate lung injury in a translationally relevant canine model of acute lung injury (ALI) when administered as rescue therapy. Secondarily, we examined whether the attenuation of LPS-induced physiological lung injury following administration of S1P was, at least in part, due to an alteration in local and/or systemic inflammatory cytokine expression. We prospectively examined 18 one-year old male beagles in which we instilled bacterial LPS (2-4 mg/kg) intratracheally followed in one hour with intravenous S1P (85 μg/kg) or vehicle and eight hours of high tidal volume mechanical ventilation. S1P attenuated the formation of shunt fraction (32%) and both the presence of protein (72%) and neutrophils (95%) in bronchoalveolar lavage (BAL) fluid compared to vehicle controls. Although lung tissue inflammatory cytokine production was found to vary regionally throughout the LPS-injured lung, S1P did not alter the expression pattern. Similarly, BAL cytokine production was not significantly altered by intravenous S1P in this model. Interestingly, S1P potentiated the LPS-induced systemic production of three inflammatory cytokines, TNF-α (6-fold), KC (1.2-fold), and IL-6 (3-fold), without resulting in end-organ dysfunction. In conclusion, intravenous S1P reduces inflammatory lung injury when administered as rescue therapy in our canine model of LPS-induced ALI. This improvement is observed in the absence of changes in local pulmonary inflammatory cytokine production and an augmentation of systemic inflammation.Corresponding Address:

Vasoactive properties of sphingosine 1-phosphate (S1P) have been demonstrated by many investigators to vary in systemic vascular beds. These variations appear to reflect differential S1P receptor expression in the vasculature of these tissues. Although S1P has been demonstrated to enhance endothelial barrier function, induce airway hyperresponsiveness, and modulate immune responses in the lung, the pulmonary vasomotor effects of S1P remain poorly defined. In the present study, we sought to define the vasoregulatory effects of S1P in the pulmonary vasculature and to elucidate the underlying mechanisms operative in effecting the response in the intact lung. S1P (10 microM) increased pulmonary vascular resistance (PVR) by 36% in the isolated perfused mouse lung. S1P-induced vasoconstriction was reduced by 64% by concomitant administration of the Rho-kinase inhibitor Y27632 (10 microM). Similarly, the S1P response was attenuated by >50% after S1P(2) receptor antagonism (JTE-013; 10 microM) and in S1P(2) receptor null mice. In contrast, S1P(3) receptor antagonism (VPC23019; 10 microM) had no effect on the contractile response to S1P. Furthermore, we confirmed the role of Rho-kinase as an important regulator of basal vasomotor tone in the isolated perfused mouse lung. These results suggest that S1P is capable of altering pulmonary vascular tone in vivo and may play an important role in the modulation of pulmonary vascular tone both in the normal lung and under pathological conditions.

Microvascular barrier integrity is dependent on bioavailable nitric oxide (NO) produced locally by endothelial NO synthase (eNOS). Under conditions of limited substrate or cofactor availability or by enzymatic modification, eNOS may become uncoupled, producing superoxide in lieu of NO. This study was designed to investigate how eNOS-dependent superoxide production contributes to endothelial barrier dysfunction in inflammatory lung injury and its regulation. C57BL/6J mice were challenged with intratracheal LPS. Bronchoalveolar lavage fluid was analyzed for protein accumulation, and lung tissue homogenate was assayed for endothelial NOS content and function. Human lung microvascular endothelial cell (HLMVEC) monolayers were exposed to LPS in vitro, and barrier integrity and superoxide production were measured. Biopterin species were quantified, and coimmunoprecipitation (Co-IP) assays were performed to identify protein interactions with eNOS that putatively drive uncoupling. Mice exposed to LPS demonstrated eNOS-dependent increased alveolar permeability without evidence for altered canonical NO signaling. LPS-induced superoxide production and permeability in HLMVEC were inhibited by the NOS inhibitor nitro-l-arginine methyl ester, eNOS-targeted siRNA, the eNOS cofactor tetrahydrobiopterin, and superoxide dismutase. Co-IP indicated that LPS stimulated the association of eNOS with NADPH oxidase 2 (Nox2), which correlated with augmented eNOS S-glutathionylation both in vitro and in vivo. In vitro, Nox2-specific inhibition prevented LPS-induced eNOS modification and increases in both superoxide production and permeability. These data indicate that eNOS uncoupling contributes to superoxide production and barrier dysfunction in the lung microvasculature after exposure to LPS. Furthermore, the results implicate Nox2-mediated eNOS-S-glutathionylation as a mechanism underlying LPS-induced eNOS uncoupling in the lung microvasculature.

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