NF-?B inhibition accelerates apoptosis of bovine neutrophils

Abstract

Apoptosis is one of the major events that contribute to the regulation of the immune system. For human neutrophils, evidence has been produced that the transcription factor NF-kappaB is critical in influencing the ultimate outcome of a cell's fate. However, such research has not yet been performed on bovine neutrophils. This urged us to examine the possible involvement of NF-kappaB in apoptosis of these cells. At first, we investigated whether p65 and p50, the most important members of the NF-kappaB family, are expressed in isolated blood neutrophils. The presence of both members was demonstrated on the RNA and protein level. Then the effect on bovine neutrophil apoptosis of gliotoxin, a potent and specific inhibitor of NF-kappaB, was examined. The rate of constitutive apoptosis was found to be greatly accelerated by inhibition of NF-kappaB. Furthermore, gliotoxin dramatically augmented the limited pro-apoptotic effect of TNF-alpha, an important inflammatory mediator. The results were obtained in six cows by annexin-V-FITC staining of externalized phosphatidylserine and subsequent flow cytometric analysis. Additional measurement of caspase-3/7 activity and evaluation of morphological criteria confirmed the outcome of this experiment. Finally, NF-kappaB activity was assessed under these conditions. The activity of p50 was found to be minimally affected by gliotoxin, while significantly lower active p65 values were observed. Still, the highest percentage of apoptosis, which was caused by incubation with both gliotoxin and TNF-alpha, did not correspond to the lowest activity of p65. We conclude that NF-kappaB p65 promotes the survival of bovine neutrophils by delaying the initiation of apoptosis.