Inhibited neutrophil apoptosis: Proteasome dependent NF-kappa B 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 bee n implicated as mediators of apoptosis; however, the signaling pathways for their production in stimulated PMN are unclear. We hypothesize that NF-kap paB translocation is necessary for the induction of TRAF-1 in PMNs with pro longed survival. Neutrophils were isolated from the blood of healthy volunt eers by Ficoll gradient centrifugation and red blood cell sedimentation. Ne utrophil NF-kappaB was inhibited with a proteasome inhibitor, PSI-I. Cells were treated with PSI-I (30 muM) or vehicle (DMSO 0.2%) for 50 min then inc ubated over an 18-h time course with LPS (10 to 1000 ng/mL), tumor necrosis factor alpha (TNF alpha) (2 to 20 ng/mL) or control media. in vitro apopto sis was quantified by propidium iodide FAGS 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 blo t analysis for nuclear p65. Means and standard errors were calculated; data were analyzed by ANOVA. Lipopolysaccharide (LPS) and TNF alpha increased P MN nuclear p65 and steady state TRAF-1 mRNA. Apoptosis was inhibited by TNF alpha and LPS at 12 and 18 h (P < 0.01). Incubation of cells in the NF-<ka ppa>B inhibitor PSI-I blocked LPS and TNF alpha -induced inhibition of apop tosis (P < 0.05) and the induction of both nuclear p65 and TRAF-1 mRNA. The se data demonstrate that inhibition of PMN apoptosis and TRAF-1 induction b y LPS and TNF<alpha> is NF-kappaB dependent.