Visit our Expo - Redox and Inflammation signaling 2012

Session X : Cell death in cancer Poster X, 52
Caspase 3 activation, Bcl-2 contents and soluble Fas-ligand are appear to be
independent of the inflammatory marker profile in patients with sepsis and septic shock
Fabian Kriebel1, Silke Wittemann2, Hsin-Yun Hsiu2, Thomas Joos2, Manfred Weiss3,
E. Marion Schneider1
Manfred.weiss.ulm@online.de
1Sektion Experimentelle Anaesthesiologie, Universitaetsklinikum Ulm, Ulm, Germany;
2NMI Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen
, Germany; 3Abteilung Klinische Anaesthesiologie, Universitaetsklinikum Ulm, Ulm,
Germany.
Objective: In order to extend treatment and diagnostics in intensive care patients suffering
from systemic inflammatory response syndrome (SIRS), sepsis and/or septic shock, we asked
whether apoptosis plays a role in hyperinflammation.
Patients: Twenty intensive care unit (ICU) patients were analyzed daily: 2 had systemic
inflammatory response syndrome (SIRS), 5 suffered from sepsis (2 died), and 13 had septic
shock (5 died).
Methods: EDTA-blood was lysed on ice with Cell Lysis Buffer (BD-Germany; 10mM Tris-
Hcl, 10mM NaH2PO4/NaHPO4, 130mM NaCl, 1% Triton X-100, 10mM PPi, protease
inhibitor cocktail of 800 µg/ml benzamidine- HCL, 500 µg/ml o-phenanthroline, 500 µg/ml
aprotinin, 500 µg/ml leupeptin, 500 µg/ml pepstatin A, 50nM PMSF). The lysate was
centrifuged at 14,000rpm for 10min and stored at –20˚C. Active caspase-3 was measured with
the R&D Quantikine Active Caspase-3 Immunoassay (R&D Systems, Hamburg, Germany).
Cytokines and active metalloproteinases (MMPs) were quantified by the Beadlyte(
Multiplex Cytokine Detection System (Upstate USA, Lake Placid, NY, USA).
Metalloproteinases (MMPs) were quantified using Luminex assisted Beadlyte Assays (Qiagen
LiquiChip, Hilden, Germany). Soluble FAS-Ligand (sCD178) was determined by ELISA
(MBL, purchased via Beckman-Coulter, Krefeld, Germany).
Results: Laboratory data obtained by plasma and cell lysate analysis demonstrate that active
caspase-3 was identified in defined samples of whole blood lysates, (but never in EDTAplasma)
covering (e.g. 5/7, 8/18, 6/11) consecutive days during the patients’ stay on the ICU.
Caspase-3 antigen contents were not found to be related to any of the inflammatory markers,
including the inflammatory cytokines (IL-1, IL-6, IL-8, TNF-a, etc.) the metalloproteinases
(MMPs 1, 3, 7, 9, 13) and C-reactive protein (CRP). However, when comparing the kinetics
of active caspase 3 and Bcl-2 protein quantified in whole blood lysates with plasma
concentrations of soluble Fas-ligand (sCD178-L), all 3 parameters were found to be elevated
either simultaneously or in close time window.
Conclusions: We conclude that the activation of apoptosis can be determined in whole blood
by active caspase-3 and by Bcl-2. Interestingly, the upregulation of caspase 3 coincides with
high Bcl-2 contents and a consecutive peak of plasma soluble FAS-ligand in a patient with a
successful reconstitution after septic shock. We therefore conclude that pro- and antiapoptotic
effects during sepsis may not be related to inflammatory markers may indicate
modelling of leukocyte subpopulations and may play an important role in immune
reconstitution.
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