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

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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.

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