Abstract
Introduction: Our previous work suggests that PBEF is an early blood marker inversely related to hypothermia protection and resuscitation survival in a mouse model of hemorrhagic shock. Given that neutrophils are a potential high-concentration source of blood PBEF, we studied mechanisms of PBEF release from isolated human neutrophils that could help design novel resuscitation strategies. Methods: Human blood neutrophils were isolated by Percoll density gradient. PBEF secretion into neutrophil supernatant was measured by ELISA. Results: Non-stimulated neutrophils (i.e. exposed to serum-starved conditions) released PBEF. PBEF in supernatant was observed at 5 min and plateaued after 30 min at 37oC. This release was not due to cell injury since LDH was unchanged, nor was it blocked by the classical secretion inhibitors, brefeldin A (blocks ER-Golgi transport) or nocodazole (blocks microtubule polymerization). As PBEF product NAD+ is consumed by multiple intra- and ecto-NADases, we determined whether inhibition of NADase activity impairs PBEF release. Nicotinamide, a global inhibitor of all NADases, blocked PBEF secretion by 73%, suggesting NADases promote PBEF secretion. To identify the specific NADase in PBEF secretion, neutrophils were treated with inhibitors for PARP, sirtuin, CD38 or mono-ADP-ribosyltransferase. We found that inhibition of CD38 by tannic acid or apigenin was able to block PBEF secretion in neutrophils (10.2 ± 2.8 ng/ml for control vs 1.8 ± 0.2 ng/ml for tannic acid at 100 μM, P < 0.01), but not by inhibitors of the other NADases. This observation indicated that CD38 glycohydroase activity is required for PBEF secretion. To further support the role of tannic acid in CD38 inhibition, tannic acid was shown to inhibit ε-ADP ribose production when neutrophils were treated with ε-NAD+. Finally, PBEF secretion was significantly enhanced by oxidative stress using H2O2 exposure. Conclusions: PBEF is constitutively released from neutrophils and this release is regulated by CD38 NAD glycohydrolase activity. Changes in human neutrophil microenvironment, especially oxidative stress, result in significant PBEF release within a timeframe that could account for the blood changes seen during mouse cardiovascular resuscitation.
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