11028545

pubmed:Citation

3

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.

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IM

MEDLINE

1073-2322

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290-4

pubmed-meshheading:11028545-Apoptosis, pubmed-meshheading:11028545-Cells, Cultured, pubmed-meshheading:11028545-Cysteine Endopeptidases, pubmed-meshheading:11028545-Humans, pubmed-meshheading:11028545-I-kappa B Proteins, pubmed-meshheading:11028545-Lipopolysaccharides, pubmed-meshheading:11028545-Multienzyme Complexes, pubmed-meshheading:11028545-NF-kappa B, pubmed-meshheading:11028545-Neutrophils, pubmed-meshheading:11028545-Oligopeptides, pubmed-meshheading:11028545-Phosphorylation, pubmed-meshheading:11028545-Proteasome Endopeptidase Complex, pubmed-meshheading:11028545-Protein Biosynthesis, pubmed-meshheading:11028545-Protein Transport, pubmed-meshheading:11028545-Proteins, pubmed-meshheading:11028545-Receptors, Tumor Necrosis Factor, pubmed-meshheading:11028545-TNF Receptor-Associated Factor 1, pubmed-meshheading:11028545-Tumor Necrosis Factor-alpha

Inhibited neutrophil apoptosis: proteasome dependent NF-kappaB translocation is required for TRAF-1 synthesis.

Journal Article, Research Support, U.S. Gov't, P.H.S.