Drotrecogin alfa (activated) inhibits NF-kappa B activation and MIP-1-alpha release from isolated mononuclear cells of patients with severe sepsis.
OBJECTIVE
Non-anticoagulant biological activities, such as anti-inflammatory and anti-apoptotic mechanisms of action, have been suggested for recombinant human activated protein C (rhAPC; drotrecogin alfa (activated)). However, these mechanisms are much less characterized and understood than rhAPC's anticoagulant activity. Aim of the study was to determine the effect of rhAPC on the activity of the pro-inflammatory transcription factor nuclear factor kappa B (NF-kappaB) in mononuclear cells isolated from septic patients and to characterize an effect downstream from NF-kappaB activation, such as the release of the NF-kappaB-controlled chemokine Macrophage Inflammatory Protein-1-alpha (MIP-1-alpha).


METHODS
Peripheral blood was obtained from 13 septic patients and from 8 healthy controls.


METHODS
Mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation and were incubated with or without rhAPC (10 microg/ml) for 2 h for the measurement of NF-kappaB activity in cell lysates or alternatively for 6 h for the determination of MIP-1-alpha levels in supernatants. NF-kappaB activity was measured by an ELISA-based assay directed against the p50 and the p65 subunit of NF-kappaB.


RESULTS
RhAPC, at supra-pharmacological concentration (10 microg/ml), significantly inhibited NF-kappaB activity and the release of MIP-1-alpha ex vivo in isolated mononuclear cells from patients with severe sepsis. In mononuclear cells of healthy subjects, however, rhAPC did not change NF-kappaB activity. Basal NF-kappaB activity early in severe sepsis was not predictive for survival.


CONCLUSIONS
RhAPC at supra-pharmacological concentration (10 microg/ml) inhibits the activity of NF-kappaB in ex vivo isolated mononuclear cells of septic patients as well as the release of MIP-1-alpha, a proinflammatory chemokine regulated by NF-kappaB. These findings may represent immunomodulatory pathways by which rhAPC exerts specific anti-inflammatory activity in vitro in addition to its known anticoagulant and profibrinolytic activity and should be further investigated in an in vivo setting.
Brueckmann
M
M
1st Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, 68167, Mannheim, Theodor-Kutzer-Ufer 1-3, Germany. martina.brueckmann@med.ma.uni-heidelberg.de
Hoffmann
U
U
Dvortsak
E
E
Lang
S
S
Kaden
J J
JJ
Borggrefe
M
M
Haase
K K
KK
D000328:Adult; D000368:Aged; D000890:Anti-Infective Agents / Q000494:pharmacology; D054407:Chemokine CCL4; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism*; D019402:Macrophage Inflammatory Proteins / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D016328:NF-kappa B / Q000378:metabolism*; D011486:Protein C / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology*; D018805:Sepsis / Q000097:blood / Q000188:drug therapy*; D013997:Time Factors
D000890:Anti-Infective Agents; D054407:Chemokine CCL4; D019402:Macrophage Inflammatory Proteins; D016328:NF-kappa B; D011486:Protein C; D011994:Recombinant Proteins; C421124:drotrecogin alfa activated