CASPASE-3, BCL-2 AND SOLUBLE FAS-LIGAND IN WHOLE BLOOD INDICATE APOPTOSIS AS AN IMPORTANT PLAYER IN THE COURSE OF SEPSIS AND SEPTIC SHOCK

Abstract

Objective: We asked whether the analysis of pro- or antiapoptosis contribute to our understanding of immune dysfunction, hyperinflammation and/or resolution of sepsis and septic shock. Patients: Twenty intensive care unit (ICU) patients were analyzed daily: 2 had SIRS, 5 suffered from sepsis (2 died), and 13 had septic shock (5 died). Methods: EDTA-blood was lysed with cell lysis buffer containing protease inhibitors. The lysate was stored at - 20?C. Active caspase-3, soluble CD95-L (sCD178) and Bcl-2 were measured by ELISAs (R&D Systems, Chemicon International, or Bender-Systems). Alternatively, we used the CBA Apoptosis® (BD-Biosciences). Cytokines and active metallo-proteinases (MMPs) were quantified by using the bead-based Luminex technology (Beadlyte® Upstate USA). Patients' courses were documented according to (i) clinical scores, (ii) diagnostic and therapeutic interventions, and (iii) complications such as bleeding, infections, positive blood cultures, etc. Results: Data obtained from whole blood lysates as well as plasma measurements demonstrate that active caspase-3 is high when leukocyte counts consecutively decline. In 2 patients with favourable outcome, these caspase 3 peaks follow plasma peaks of sCD178 and precede elevations of Creactive protein (CRP) and IL-6. High Bcl-2 contents in blood lysates accompany increased leukocyte counts and appear to be linked to higher TNF-α. Patients with prolonged phases of septic shock lacked apoptosis markers as well as elevated cytokines. Conclusions: The present study shows that quantification of apoptosis relevant factors is feasible in whole blood on a daily basis.