INHIBITED NEUTROPHIL APOPTOSIS: PROTEASOME DEPENDENT NF-κB... : Shock (original) (raw)

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INHIBITED NEUTROPHIL APOPTOSIS: PROTEASOME DEPENDENT NF-κB TRANSLOCATION IS REQUIRED FOR TRAF-1 SYNTHESIS

Nolan, Brian; Kim, Robin; Duffy, Andrew; Sheth, Ketan; De, Mita; Miller, Carol; Chari, Ravi; Bankey, Paul

Department of Surgery, University of Massachusetts Medical School, Worcester, Massachusetts 01655

This publication was supported by NIH Grant 525753. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.

Presented at the 20th Annual Meeting of the Surgical Infection Society, April 27-29, 2000, Providence, RI.

Address reprint requests to Paul Bankey, MD, PhD, FACS, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655.

Received 28 Apr 2000; first review completed 5 May 2000; accepted in final form 6 Jun 2000

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Abstract

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-κB 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-κB was inhibited with a proteasome inhibitor, PSI-I. Cells were treated with PSI-I (30 μM) 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 (TNFα) (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-κB activity was determined by Western blot analysis for nuclear p65. Means and standard errors were calculated; data were analyzed by ANOVA. Lipopolysaccharide (LPS) and TNFα increased PMN nuclear p65 and steady state TRAF-1 mRNA. Apoptosis was inhibited by TNFα and LPS at 12 and 18 h (P < 0.01). Incubation of cells in the NF-κB inhibitor PSI-I blocked LPS and TNFα-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 TNFα is NF-κB dependent.