Cyclin-dependent kinase (CDK) inhibitor drugs induce apoptosis in human neutrophils through regulation of critical survival proteins
Riley, Nicola Amy
Neutrophil apoptosis is an important process contributing to the resolution of inflammation. This is because it allows the neutrophil membrane to remain intact preventing it’s potentially histotoxic contents from being released into the extra-cellular milieu, a process that can contribute to the exacerbation of many inflammatory disorders such as rheumatoid arthritis. When considering the life-span of a neutrophil and how it can be augmented by various inflammatory mediators to allow it to carry out its essential protective role in the body’s innate immune defences it is also important to consider how to terminate this process when the inflammatory insult has been dealt with or when the system goes awry. It is this information that we believe may hold the key to developing novel anti-inflammatory therapies. Through exploitation of the mechanisms controlling neutrophil apoptosis, it may be possible to selectively target these cells to enter apoptosis, and therefore help aid the process of resolution, especially if used in conjunction with treatments that up-regulate phagocytosis of apoptotic cells. This is important given that the main treatment for disorders of the inflammatory response are glucocorticoids, which whilst proven to be a powerful treatment for eosinophil based diseases such as asthma where they increase eosinophil apoptosis in conjunction with enhancing phagocytic clearance of apoptotic cells, glucocorticoids have been found to have the converse affect on neutrophils, actually serving to prolong their life-span potentially exacerbating the condition. Furthermore, it has been previously shown that the transcription factor nuclear factor kappa B (NF-κB) plays a pivotal role in neutrophil apoptosis, becoming activated by inflammatory agents such as lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-α). NF-κB activation results in the transcription of many pro-inflammatory agents and anti-apoptotic proteins such as X-linked inhibitor of apoptosis (X-IAP) increasing the life-span of the neutrophil. Interestingly, it has also been demonstrated that key neutrophil survival proteins such as myeloid cell leukemia-1 (Mcl-1) are not directly regulated by NF-κB activation. Therefore it is because of the aforementioned reasons that I have chosen to investigate further neutrophil apoptosis including the role played by NF-κB. Thus, I have investigated the hypothesis that NF-κB-dependent and independent survival proteins critically regulate the rates of neutrophil apoptosis and that newly identified pro-apoptotic agents such as the cyclin-dependent kinase (CDK) inhibitor, R-roscovitine interferes with the expression of such survival proteins. It has been previously found by myself and others in our laboratory during the course of this thesis that cyclin dependent kinase inhibitor (CDKi) drugs such as R-roscovitine are powerful novel anti-inflammatory agents with the ability to up-regulate rates of neutrophil apoptosis in vitro and influence the resolution of neutrophilic inflammation in vivo. Whilst the exact mechanism of CDK inhibitor drugs on neutrophil apoptosis remains elusive, work shown in this thesis demonstrates that R-roscovitine has the ability to over-ride powerful anti-apoptotic signals from pro-inflammatory agents such as granulocyte-macrophage colony stimulating factor (GM-CSF) and LPS causing the neutrophils to enter apoptosis. Furthermore, it has been found that R-roscovitine causes a decrease in levels of the antiapoptotic protein Mcl-1 in as little as 2h and that it prevents the maintenance / protective effect that GM-CSF has on the Mcl-1 protein levels. In addition R-roscovitine may also reduce levels of the NF-κB regulated protein X-IAP. The effect of R-roscovitine on X-IAP was investigated further using an X-IAP HIV-tat construct, though results from this remain inconclusive. This is because although the X-IAP construct appeared to be extending neutrophil longevity, it was discovered that LPS contamination of the construct had occurred which could therefore pose an alternative explanation for the increase in neutrophil life-span. As X-IAP, TNF-α and LPS are all regulated by NF-κB and given that NF-κB is already known to be a key player in neutrophil biology, the effects of R-roscovitine on this important transcription factor were investigated. It was discovered that R-roscovitine does not directly activate NF-κB, since this CDK inhibitor drug does not cause degradation and loss of the cytoplasmic inhibitor of NF-κB, IκBα. This lack of NF-κB activation was confirmed since R-roscovitine did not mobilize the NF-κB subunit, p65, from the cytoplasm to the nucleus. Furthermore, R-roscovitine (unlike the NF-κB inhibitor gliotoxin) does not interfere with the ability of LPS or TNF-α to activate NF-κB. Therefore by R-roscovitine to induce apoptosis, although this does not rule out the involvement of NF-κB at a later stage. When considering a reagent for possible use as a novel anti-inflammatory agent I think it is important to assess what effects it has on the activation state of the neutrophil. Therefore the effects of R-roscovitine on the activation markers CD62L, CD11b and shape change were assessed. It was found that R-roscovitine alone did not cause any significant neutrophil activation as measured using the parameters stated above. Importantly, it was also found that R-roscovitine did not interfere with the activation states induced by the inflammatory mediators GM-CSF, LPS, TNF-α or leukotriene B4 (LTB4). Another important consideration is the effect of R-roscovitine on the removal of apoptotic cells by macrophage phagocytosis. Results demonstrated that pre-treatment of macrophages with R-roscovitine did not augment their uptake of apoptotic neutrophils. In addition Rroscovitine did not detrimentally affect the increase in phagocytosis that results from macrophage treatment with the synthetic glucocorticoid dexamethasone. The data presented in this thesis suggest that CDK inhibitor drugs such as R-roscovitine are novel powerful pro-apoptotic agents for neutrophils with the ability to over-ride antiapoptotic signals from multiple pro-inflammatory mediators. It has been discovered that Rroscovitine causes a reduction in one of the neutrophil’s most prominent anti-apoptotic proteins (Mcl-1) whilst not altering the activation state of the neutrophil and furthermore it does not interfere with the uptake of apoptotic neutrophils by macrophages or result in any alteration to the increase in phagocytosis caused by treatment with dexamethasone. In conclusion, CDK inhibitor drugs such as R-roscovitine have the potential to be promising candidates for novel anti-inflammatory agents with the ability to selectively target neutrophil apoptosis whilst not interfering with steroid induced up-regulation of phagocytosis, therefore allowing a two pronged attack to help treat neutrophil based inflammatory disorders.