Role of CD13 (aminopeptidase-N) in the regulation of TNF-alpha induced neutrophil apoptosis

Neutrophils are special cells that circulate in the blood and play a major role in helping the body fight infection. They are highly motile and migrate to areas of infection, where they destroy bacteria. Thereafter, these cells are designed to die by a process called apoptosis, which triggers their safe removal. Failure of apoptosis results in the cells remaining within the inflamed site where they cause inadvertent damage to normal tissues. This is a particular problem in patients with inflammatory lung, kidney and joint disease, where persistence of the neutrophils drives chronic inflammation and organ damage. This project will look at how a molecule on the surface of neutrophils called CD13 can influence the ability of neutrophils to undergo apoptosis, especially at the inflamed site. The lab has shown a natural variation in the expression of CD13 between people, which affects the sensitivity of neutrophils to undergo apoptosis and that chemical inhibitors of CD13 may restore neutrophil apoptosis. These drugs may therefore have the potential to treat chronic inflammation. The project will utilize cell biology techniques using neutrophils from human blood, mice deficient in CD13 and inflammatory neutrophils derived from experimental subjects.

Neutrophil recruitment to sites of tissue inflammation is integral to the innate immune system. However, the inappropriate activation or delayed clearance of these neutrophils can lead to tissue damage as a consequence of the release of proteolytic enzymes and toxic reactive oxygen intermediates. Rapid and efficient efferocytosis of apoptotic neutrophils by macrophages promotes the resolution of inflammation and restoration of normal tissue architecture through the secretion of anti-inflammatory cytokines and growth factors such as TGF . Thus, we predict that agents that facilitate neutrophil apoptosis would be beneficial for host defence mechanisms. Once such agent is TNF , which can induce early neutrophil apoptosis. We demonstrated that this response varies in a reproducible manner between individuals and is lost if the neutrophils have been exposed to priming agents such as PAF. Furthermore, with prolonged TNF exposure, the pro-apoptotic effect is superseded by an NF- B mediated survival effect. Factors which favour the pro-apoptotic response to TNF stimulation remain to be identified but may include TNFRI phosphorylation, its localization to lipid rafts, internalization and signaling complex II (FADD/caspase 8) formation. Recently, we have identified that the ectoenzyme CD13 (aminopeptidase N) may be another potential regulator of this TNF response. Inhibition of CD13 by actinonin, bestatin or blocking peptides prevented TNF induced shedding of TNFRI and augmented TNF mediated neutrophil apoptosis. We also showed that PAF up-regulated CD13, which may account for its ability to inhibit TNF -induced neutrophil apoptosis. How CD13 affects the ectodomain shedding of TNFRI and regulates TNF signaling remains to be determined. These questions form the basis of this project.
I hypothesize that CD13 promotes ectodomain shedding of TNFRI which in turn drives TNF -TNFRI signal transduction in favour of NF- B activation, as the ligand bound receptor can no longer be internalized. Specific inhibitors of CD13 will be used to explore the role of this ectoenzyme in the shedding and internalization of cell surface TNFRI. The ability of CD13 to regulate TNF -TNFRI signaling will be addressed by comparing the downstream responses (apoptosis, MAPK and NF- B activation) of purified neutrophils from CD13 deficient mice with those of inflammatory human neutrophils, obtained from cantharidin-induced skin blisters, which have high levels of CD13 activity.
The aim of this project is to define the molecular basis and in vivo relevance of CD13-mediated regulation of TNF -induced apoptosis in the neutrophil.