Neutrophils die by apoptosis spontaneously within 12–24 h of their release from the bone marrow. The mechanism regulating entry of neutrophils into apoptosis at the end of their life-span is currently under debate. Our data suggest that neutrophil apoptosis involves a novel mechanism of caspase 8 activation that is indirectly regulated by accumulation of reactive oxygen species. We detected early activation of caspase 8 upstream of caspase 3 activation, suggesting death receptor signalling. The CD95 DISC (death-inducing signalling complex) was detected in neutrophils, but blocking antibodies to death receptors did not inhibit apoptosis, suggesting a novel mechanism for caspase 8 activation. Death receptor clustering in ceramide-rich lipid rafts is thought to be an early event in their signalling, so we investigated the role of ceramide generated by ASM (acid sphingomyelinase) in neutrophil apoptosis. Ceramide was generated early in neutrophil apoptosis, and ASM activity was required for neutrophil apoptosis. Moreover, neutrophil apoptosis was significantly delayed in ASM −/− mice compared with their wild-type littermates. CD95 DISC components were present in lipid rafts in neutrophils, and were progressively clustered in cultured neutrophils. Generation of ceramide was blocked by desferrioxamine, suggesting that hydroxyl radicals are important for the activation of ASM. This observation was in line with our earlier observation of a precipitous drop in reduced glutathione in the aging neutrophil.
Neutrophils are very abundant, short-lived leucocytes and their death by apoptosis is central to homoeostasis and the resolution of inflammation, yet the trigger for apoptosis is still a topic of debate. Depolarization of the mitochondrial membrane has been supposed to initiate neutrophil spontaneous apoptosis, as neutrophils gradually lose the anti-apoptotic protein Mcl-1 and Bax translocates and inserts into the mitochondrial membrane. However, other reports show that caspase 8 is required for neutrophil apoptosis, suggesting the involvement of DR (death receptor) signalling. As DR ligation is not required for neutrophil apoptosis, this raises the intriguing possibility that activation of caspase 8 during neutrophil apoptosis occurs via a novel mechanism. In the present paper, we discuss the current evidence for mechanisms occurring in neutrophil apoptosis, which could trigger DR signalling in the absence of DR ligation.
Several protein toxins, including the A chain of the plant protein ricin (RTA), enter mammalian cells by endocytosis and catalytically modify cellular components to disrupt essential cellular processes. In the case of ricin, the process inhibited is protein synthesis. In order to reach their cytosolic substrates, several toxins undergo retrograde transport to the ER (endoplasmic reticulum) before translocating across the ER membrane. To achieve this export, these toxins exploit the ERAD (ER-associated protein degradation) pathway but must escape, at least in part, the normal degradative fate of ERAD substrates in order to intoxicate the cell. Toxins that translocate from the ER have an unusually low lysine content that reduces the likelihood of ubiquitination and ubiquitin-mediated proteasomal degradation. We have changed the two lysyl residues normally present in RTA to arginyl residues. Their replacement in RTA did not have a significant stabilizing effect on the protein, suggesting that the endogenous lysyl residues are not sites for ubiquitin attachment. However, when four additional lysyl residues were introduced into RTA in a way that did not compromise the activity, structure or stability of the toxin, degradation was significantly enhanced. Enhanced degradation resulted from ubiquitination that predisposed the toxin to proteasomal degradation. Treatment with the proteasomal inhibitor lactacystin increased the cytotoxicity of the lysine-enriched RTA to a level approaching that of wild-type RTA.
Previous work has demonstrated an age-related decline in neutrophil function, including a decline in phagocytic capacity, with age in healthy individuals. This decline in function may contribute to increased susceptibility to bacterial infections in the elderly population. The present study has investigated the effects of age on susceptibility to infection and neutrophil function in elderly humans following mild trauma. Specifically, we have measured neutrophil function in 44 patients, all of whom had no significant co-morbidity, were over 65 years old (mean age 82.5 years) and had sustained a fractured neck of femur. We obtained neutrophils and examined the process of microbial engulfment by phagocytosis and the bactericidal mechanism of superoxide production. In the 5-week period after trauma, almost half of the elderly trauma patients succumbed to bacterial or fungal infection, with a predominance of chest and urinary tract infections. When examining neutrophil function, a decline in superoxide production was observed in neutrophils from the elderly trauma group at the time of hip fracture when compared with those from healthy elderly controls, and this was maintained 5 weeks after trauma. This was accompanied by an age-related reduction in phagocytic function during this period. We propose that trauma and an age-related decline in neutrophil function combine to decrease the immune response to bacteria in the elderly.