Delayed neutrophil apoptosis in sepsis is associated with maintenance of mitochondrial transmembrane potential and reduced caspase-9 activity.
OBJECTIVE
The resolution of neutrophil (PMN)-mediated inflammation occurs through the apoptosis, or programmed cell death, of the neutrophil. PMN apoptosis is inhibited by a variety of inflammatory stimuli; moreover, PMN from critically ill septic patients show profoundly delayed rates of apoptosis in vitro. Since apoptosis is effected through the activity of intracellular cysteine proteases (caspases), we evaluated caspase expression and activity in neutrophils from septic patients and compared them with caspase expression and activity of resting or lipopolysaccharide-activated neutrophils from healthy volunteers.


METHODS
Prospective observational cohort study.


METHODS
Tertiary level intensive care unit and associated research laboratory.


METHODS
Thirty-six intensive care unit patients with sepsis; ten healthy laboratory controls.


METHODS
Collection of up to 10 mL of whole blood for in vitro study of rates of apoptosis, expression and activity of caspases-1, -3, and -9, activation of nuclear factor-kappaB, and change in mitochondrial transmembrane potential.


RESULTS
Following 24 hrs of in vitro culture, 52 +/- 7.8% of control neutrophils, but only 29 +/- 5.4% of lipopolysaccharide-stimulated (1 microg/mL) PMN, showed nuclear changes of apoptosis. Only 6.2 +/- 1.1% of neutrophils from septic patients were apoptotic after 24 hrs. Significant nuclear translocation of nuclear factor-kappaB was evident in septic PMN, and inhibition of apoptosis was partially abrogated by prevention of nuclear factor-kappaB dissociation with pyrrolidine dithiocarbamate. Caspase-3 transcription and catalytic activity were significantly reduced in both patients' and lipopolysaccharide-treated PMN; caspase-1 transcription and activity were increased by lipopolysaccharide but reduced in septic patients. In contrast, caspase-9 transcription and activity were reduced in septic patients but not in lipopolysaccharide-treated PMN. Decreased caspase-9 activity was associated with sustained maintenance of mitochondrial transmembrane potential and reduced translocation of cytochrome c from the mitochondria to the cytosol.


CONCLUSIONS
Apoptosis of circulating neutrophils from patients with clinical sepsis is profoundly suppressed, through a mechanism that involves activation of nuclear factor-kappaB that is associated with reduced activity of caspases-9 and -3 and maintenance of mitochondrial transmembrane potential and that differs in important respects from the inhibitory effects seen following the exposure of healthy neutrophils to inflammatory stimuli.
Taneja
Ravi
R
Department of Surgery, The Toronto General Hospital, University Health Network, University of Toronto, Ontario, Canada.
Parodo
Jean
J
Jia
Song Hui
SH
Kapus
Andras
A
Rotstein
Ori D
OD
Marshall
John C
JC
D018806:APACHE; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D017209:Apoptosis; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D017052:Hospital Mortality; D006801:Humans; D007362:Intensive Care Units; D007902:Length of Stay; D008297:Male; D008564:Membrane Potentials; D008875:Middle Aged; D008928:Mitochondria / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology / Q000502:physiology*; D011446:Prospective Studies; D018805:Sepsis / Q000145:classification / Q000201:enzymology / Q000503:physiopathology*
D016328:NF-kappa B; D020169:Caspases