**4.3 Neutrophil apoptosis and clearance**

Apoptosis is the process of programmed cell death after pathogen phagocytosis and eliminating. Apoptosis facilitates following phagocytosis and elimination of the spent neutrophils from areas of inflammation by macrophages, thus helping resolution of inflammation and avoiding excessive tissue destruction [14, 19]. When phosphatidyl serine is exposed during the apoptotic process, which culminates in caspase activity, the cells are marked for uptake and elimination by macrophages. Fascinatingly, since caspases are thiol-dependent enzymes, ROS generated by active neutrophils can inactivate them. As a result, ascorbic acid may be expected to prevent the oxidant-sensitive caspase-dependent apoptotic process after activation of neutrophils. This idea is supported by in vitro experiments that show ascorbic acid can enhance the amount of *Escherichia coli*-mediated neutrophil apoptosis in human neutrophils. Reduced apoptosis was seen in peritoneal neutrophils obtained from ascorbic acid-deficient Gulo mice, who instead experienced necrotic cell death. These ascorbic acid-deficient neutrophils were not phagocytosed by macrophages in vitro, and remained at inflammatory sites in vivo. In addition, supplementation of ascorbic acid to severe infection animals reduced the levels of neutrophils in the lungs of these animals [14, 20].

Several researches have explained about reduced neutrophil apoptosis in subjects with sepsis compared with healthy individuals. The greater tissue destruction seen in individuals with severe infection is assumed to be associated to the delayed apoptosis, which seems to be linked to disease severity. Additionally, it was shown that immature "band" neutrophils produced during sepsis had extended life spans and were resistant to apoptosis. Apoptosis in healthy neutrophils was shown to be inhibited by plasma from patients with severe infections, indicating that pro-inflammatory cytokines were crucial for the elevating in vivo survival of neutrophils under inflammatory situations [14]. Interestingly, high-dose ascorbic acid supplementation has been exhibited to regulate cytokine concentrations in individuals with carcinoma and, although this has not yet been evaluated in subjects with sepsis, it is conceivable that ascorbic acid may regulate neutrophil function in these subjects through a different mechanism. The role of ascorbic acid administration on neutrophil apoptosis in severe infection subjects has only been described in one study. Ascorbic acid 0.45 g/day administered intravenously to abdominal surgery subjects with severe infections was shown to reduce caspase-3 protein concentrations and thus was thought to have an anti-apoptotic impact on peripheral blood neutrophils. However, caspase activity and apoptosis of the neutrophils after activation was not examined. Additionally, neutrophils in circulation could not accurately represent their activation level at inflammatory tissue areas. Clearly, further researches are required to determine how ascorbic acid affects neutrophil apoptosis and elimination from inflammatory sites [14, 15, 20].
