Abstract 178: The Early Resuscitation Protein PBEF Is Released from Human Neutrophils via an Unconventional Pathway
Introduction: Our previous work suggests that PBEF, encoded by the NAMPT gene, is an early blood marker inversely related to hypothermia protection and resuscitation survival in a mouse model of hemorrhagic shock. Higher mouse blood PBEF concentrations were related to acute inflammation and cardiovascular collapse. More recently, clinical work by others demonstrated a correlation between high PBEF blood concentrations and acute heart failure exacerbation. Given that neutrophils are a potential high-concentration source of blood PBEF, we studied mechanisms of 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. Neutrophil PBEF protein expression was measured by Western blot analysis.
Results: PBEF in unstimulated neutrophil supernatant was observed at 5 min and plateaued after 30 min at 37oC (8.6 ± 0.7 vs. 0.9 ± 0.3 ng/ml, p < 0.01). 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). However, PBEF secretion was attenuated by decreased temperature (4oC), suggesting a release mechanism other than passive diffusion. Indeed, glyburide an inhibitor of the ATP-binding cassette 1 (ABC 1) transporter reduced PBEF secretion by 55%. The calcium chelator EGTA completely blocked PBEF secretion. PBEF release was also dose-dependently blocked by extracellular ATP. Exogenous ATP induced the degradation of intracellular PBEF, which was blocked by pretreatment with a proteasomal inhibitor MG132, suggesting intracellular PBEF stability also regulates its secretion.
Conclusions: PBEF is constitutively released from neutrophils via an unconventional secretion pathway. Its release is regulated by the ABC1 membrane transporter, calcium, and intracellular proteasome degradation. Changes in human neutrophil microenvironment result in significant change of PBEF release within a timeframe that could account for the blood changes seen during mouse cardiovascular resuscitation.
Author Disclosures: X. Zhu: None. A. Meliton: None. J. Li: None. A. Bellino: None. M. Zhu: None. H. Wang: None. A.R. Leff: None. T.L. Vanden Hoek: None.
- © 2014 by American Heart Association, Inc.