Increased neutrophil adenosine a3 receptor expression is associated with hemorrhagic shock and injury severity in trauma patients

Abstract

Hypertonic saline (HS) has been investigated as an immune modulator following hemorrhagic shock and sepsis. The polymorphonuclear neutrophil (PMN) response to HS is regulated by the release of ATP, which is converted to adenosine and activates adenosine receptors. Binding to A3 adenosine receptors promotes PMN activation, and inhibition of A3 receptors improves the efficacy of HS resuscitation. A3 receptor expression of PMNs has not been previously evaluated in injured patients. Whole blood was obtained from 10 healthy volunteers and 60 injured patients within 2 h of injury. Inclusion criteria were blunt or penetrating injury with evidence of hypovolemic shock (systolic blood pressure [SBP] ≤90 mmHg and base deficit ≥6 mEq/L or need for blood transfusion) or evidence of severe traumatic brain injury including initial Glasgow Coma Scale score of 8 or less or evidence of traumatic brain injury on head computed tomography scan (head Abbreviated Injury Score ≥3) or intubation in the field or emergency department. A3 receptor expression was assessed by flow cytometry. Polymorphonuclear neutrophils were also exposed to fMLP or HS (20-40 mM) in vitro. Clinical data were collected including admission physiology, injury severity (Injury Severity Score [ISS]), development of multiple organ failure, and survival. In normal volunteers, less than 1% of PMNs expressed A3 receptors on the cell surface. A3 receptor expression was significantly higher in injured patients, and the level of expression correlated with the severity of injury (ISS ≥25: A3 positive PMN 36.6% vs. ISS <25: 16.2%; P = 0.019) and degree of hypovolemic shock (SBP ≤90 mmHg: A3 positive PMN 43.8% vs. SBP>90 mmHg: 20.6%; P = 0.008). Stimulation with fMLP or HS increased A3 expression in normal volunteers, but only in patients with ISS of less than 25 or without hypovolemic shock. A3 receptor expression on the surface of PMNs is upregulated by injury, and increased expression levels are associated with greater injury severity and hypovolemic shock. Hypertonic saline increases A3 expression of PMNs from healthy volunteers and less severely injured patients.

Figures

Fig. 1

Polymorphonuclear neutrophil A3 receptor expression in whole blood from healthy volunteers. Polymorphonuclear neutrophil A3 receptor expression was evaluated by flow cytometry on whole-blood samples obtained from healthy volunteers (n = 10). Baseline A3 receptor expression was very low and was significantly increased by in vitro exposure to HS (20 or 40 mM) or fMLP.

Fig. 2

Polymorphonuclear neutrophil A3 receptor expression in injured patients. Polymorphonuclear neutrophil A3 receptor expression was evaluated by flow cytometry on whole-blood samples obtained from injured patients within 2 h of arrival in the ED (n = 60). Patients were stratified based on severity of injury (ISS score) or evidence of hypovolemic shock (SBP

Fig. 3

Effect of HS on A3 receptor expression from injured patients. Polymorphonuclear neutrophil A3 receptor expression was evaluated by flow cytometry on whole-blood samples obtained from injured patients within 2 h of arrival in the ED (n = 60). Samples were exposed to HS in vitro. Patients were stratified based on severity of injury (ISS score) or evidence of hypovolemic shock (SBP <90 mmHg or arterial base deficit ≥6 mM/L). A, Results based on stratification by the ISS score. B, Results based on stratification by hypovolemic shock (SBP ≤90 mmHg). C, Results based on stratification by metabolic acidosis (arterial base deficit ≥6 mM/L).

Fig. 3

Effect of HS on A3 receptor expression from injured patients. Polymorphonuclear neutrophil A3 receptor expression was evaluated by flow cytometry on whole-blood samples obtained from injured patients within 2 h of arrival in the ED (n = 60). Samples were exposed to HS in vitro. Patients were stratified based on severity of injury (ISS score) or evidence of hypovolemic shock (SBP <90 mmHg or arterial base deficit ≥6 mM/L). A, Results based on stratification by the ISS score. B, Results based on stratification by hypovolemic shock (SBP ≤90 mmHg). C, Results based on stratification by metabolic acidosis (arterial base deficit ≥6 mM/L).

Fig. 3

Effect of HS on A3 receptor expression from injured patients. Polymorphonuclear neutrophil A3 receptor expression was evaluated by flow cytometry on whole-blood samples obtained from injured patients within 2 h of arrival in the ED (n = 60). Samples were exposed to HS in vitro. Patients were stratified based on severity of injury (ISS score) or evidence of hypovolemic shock (SBP <90 mmHg or arterial base deficit ≥6 mM/L). A, Results based on stratification by the ISS score. B, Results based on stratification by hypovolemic shock (SBP ≤90 mmHg). C, Results based on stratification by metabolic acidosis (arterial base deficit ≥6 mM/L).

Fig. 4

Relationship between A3 receptor expression and outcome in injured patients. Polymorphonuclear neutrophil A3 receptor expression was evaluated by flow cytometry on whole-blood samples obtained from injured patients within 2 h of arrival in the ED (n = 60). Patients were stratified based on mortality or the development of ARDS or MODS after injury.