Inhibited neutrophil apoptosis: proteasome dependent NF-kappaB translocation is required for TRAF-1 synthesis.
Neutrophil (PMN) apoptosis regulates local and systemic inflammation during sepsis. Tumor necrosis factor receptor-associated factors (TRAFs) have been implicated as mediators of apoptosis; however, the signaling pathways for their production in stimulated PMN are unclear. We hypothesize that NF-kappaB translocation is necessary for the induction of TRAF-1 in PMNs with prolonged survival. Neutrophils were isolated from the blood of healthy volunteers by Ficoll gradient centrifugation and red blood cell sedimentation. Neutrophil NF-kappaB was inhibited with a proteasome inhibitor, PSI-I. Cells were treated with PSI-I (30 microM) or vehicle (DMSO 0.2%) for 50 min then incubated over an 18-h time course with LPS (10 to 1000 ng/mL), tumor necrosis factor alpha (TNFalpha) (2 to 20 ng/mL) or control media. In vitro apoptosis was quantified by propidium iodide FACS analysis. Total cellular TRAF-1 was detected by Western blot analysis of cell lysates. Steady state TRAF-1 mRNA was detected by RPA. NF-kappaB activity was determined by Western blot analysis for nuclear p65. Means and standard errors were calculated; data were analyzed by ANOVA. Lipopolysaccharide (LPS) and TNFalpha increased PMN nuclear p65 and steady state TRAF-1 mRNA. Apoptosis was inhibited by TNFalpha and LPS at 12 and 18 h (P < 0.01). Incubation of cells in the NF-kappaB inhibitor PSI-I blocked LPS and TNFalpha-induced inhibition of apoptosis (P < 0.05) and the induction of both nuclear p65 and TRAF-1 mRNA. These data demonstrate that inhibition of PMN apoptosis and TRAF-1 induction by LPS and TNFalpha is NF-kappaB dependent.
Nolan
B
B
Department of Surgery, University of Massachusetts Medical School, Worcester 01655, USA.
Kim
R
R
Duffy
A
A
Sheth
K
K
De
M
M
Miller
C
C
Chari
R
R
Bankey
P
P
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D020672:I-kappa B Proteins / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects; D009842:Oligopeptides / Q000494:pharmacology; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D021381:Protein Transport; D011506:Proteins / Q000187:drug effects / Q000235:genetics; D018124:Receptors, Tumor Necrosis Factor / Q000096:biosynthesis; D047990:TNF Receptor-Associated Factor 1; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D020672:I-kappa B Proteins; D008070:Lipopolysaccharides; D009097:Multienzyme Complexes; D016328:NF-kappa B; D009842:Oligopeptides; D011506:Proteins; D018124:Receptors, Tumor Necrosis Factor; D047990:TNF Receptor-Associated Factor 1; D014409:Tumor Necrosis Factor-alpha; C090535:benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex