Inhibition of Neutrophils by Hypertonic Saline Involves Pannexin-1, CD39, CD73, and Other Ectonucleotidases

Abstract

Hypertonic saline (HS) resuscitation has been studied as a possible strategy to reduce PMN activation and tissue damage in trauma patients. HS blocks PMNs by ATP release and stimulation of A2a adenosine receptors. Here we studied the underlying mechanisms in search of possible reasons for the inconsistent results of recent clinical trials with HS resuscitation. Purified human PMNs or PMNs in whole blood were treated with HS to simulate hypertonicity levels found after HS resuscitation (40 mM beyond isotonic levels). ATP release was measured with a luciferase assay. PMN activation was assessed by measuring oxidative burst. The pannexin-1 (panx1) inhibitor 10panx1 and the gap junction inhibitor carbenoxolone (CBX) blocked ATP release from PMNs in purified and whole blood preparations, indicating that HS releases ATP via panx1 and gap junction channels. HS blocked fMLP-induced PMN activation by 40% in purified PMN preparations and by 60% in whole blood. These inhibitory effects were abolished by 10panx1 but only partially reduced by CBX, which indicates that panx1 has a central role in the immunomodulatory effects of HS. Inhibition of the ectonucleotidases CD39 and CD73 abolished the suppressive effect of HS on purified PMN cultures but only partially reduced the effect of HS in whole blood. These findings suggest redundant mechanisms in whole blood that may strengthen the immunomodulatory effect of HS in vivo. We conclude that HS resuscitation exerts anti-inflammatory effects that involve panx1, CD39, CD73, and other ectonucleotidases, which produce the adenosine that blocks PMNs by stimulating their A2a receptors. Our findings shed new light on the immunomodulatory mechanisms of HS and suggest possible new strategies to improve the clinical efficacy of hypertonic resuscitation.

Immunomodulatory Effect of Hypertonic Saline Solution in Traumatic Brain-Injured Patients and Intracranial Hypertension

“…8,45 The activation of the TLRs regulates the chemokine receptors as well as stimulates A2a receptors that are linked to cAMP signaling cascade. And by this, both pathways regulate neutrophil functions, 8,41 and HSS modifies the TLR-4 pathway, leading to the immunomodulation of the effects mediated by PMN cells. 8 In vitro studies indicate that shock-mediated MODS is caused by the liberation of ROS, proteases, hydrolytic enzymes, and inflammatory cytokines, as well as by the neutrophil sequestration in vital organs.…”

“…15,40 In these cases, it is the immunomodulatory effect of HSS that causes a reduction in neutrophil recruitment and activation, which might improve patient outcomes and prognosis. 41 HSS resuscitation therapy has been found to reduce the priming and activation of the PMN by activating intracellular signaling cascades related to the cAMP (cyclic adenosine monophosphate) mediated pathways that suppress the cell activation processes. 28 Neutrophils suppression by the HSS also happens by means of reduction in the expression of adhesion molecule and production of ROS, as well as attenuation of the oxidative stress.…”

Exciting and not so exciting roles of pannexins

“…Because Panx1 channels are permeable to ATP [43, 60] they contribute to purinergic signaling including that involved in the propagation of intercellular calcium waves between astrocytes [34, 61], communication between satellite glia and sensory neurons [29, 62], neutrophil activation and immune defense [63, 64], and vascular tone [54]. Several studies indicate the involvement of Panx1 in certain pathophysiological conditions (ischemia, seizures, innate immune response, cell death, HIV infection, neuropathic and inflammatory pain, spinal cord injury and cardiac arrhythmias) [65–71].…”

Purinergic Signaling and the Immune Response in Sepsis: A Review

“…19 In addition to its beneficial effects on hemodynamic functions, blood viscosity, and capillary blood flow, hypertonic saline resuscitation can suppress excessive neutrophil activation. 20-23 It was shown that hypertonic saline regulates immune cell functions by inducing the release of cellular ATP into the extracellular environment. 24 In the early 1980s, Chaudry and colleagues reported beneficial effects of ATP-MgCl 2 infusions in experimental models of ischemia 25 , hemorrhagic shock 26 , and sepsis 27,28 .…”