**5. The involvement of CFTR in neutrophil apoptosis**

Timely and effective neutrophil programmed cell death is essential for the resolution of inflammation [124, 125] and abnormal neutrophil apoptosis is associated with decreased antimicrobial defences [126], incomplete microbial clearance, and sustained inflammation [127, 128]. Three different pathways are involved in the regulation of apoptosis and particular attention will be given to signalling pathways that are relevant to the neutrophil as it has been recognised that neutrophils have unique mechanisms of cell death due to their short half-life and phagocytic activity (Figure 5). Essential to the regulation and execution of apoptosis are the caspases, a family of cytosolic proteases or cysteine-dependent aspartate-directed proteas‐ es, involved in all three pathways. The first pathway described is the mitochondrial or intrinsic pathway. This pathway responds to physical and chemical stress signals, including growth factor withdrawal, DNA damage, and endoplasmic stress, and is transduced by members of the Bcl-2 family, which ultimately trigger the mitochondrial outer membrane permeabilisation (MOMP). As a result, several pro-apoptotic mitochondrial proteins, the most important being cytochrome C, are released into the cytosol. Subsequently cytochrome C, apoptotic protease activating factor-1 (Apaf-1) and caspase-9 form the apoptosome [129-131], this results in the activation of caspase-9 which thereafter activates caspase-3 and initiates the execution of apoptosis. In addition, caspase-8 can cleave Bid into truncated Bid (TBid), which ultimately triggers MOMP, thus providing a link from the intrinsic pathway to the extrinsic pathway.

a result of defective CFTR rather than the inflammatory status of the individual [108]. Indeed, CFTR expression in human neutrophils has provoked the idea that altered neutrophil migra‐ tion and adhesion in CF could be caused by an intrinsic defect. Counteracting this concept however, Pohl *et al*. (2014) did not demonstrate impaired neutrophil migration in healthy control cells exposed to the CFTR inhibitor, CFTR(inh)-172, suggesting that altered neutrophil

Circulating neutrophils generally adhere and migrate in response to pro-inflammatory mediators including TNF-alpha and pathogenic components including N-formyl peptides produced by bacteria. Lipid mediators involved in neutrophil adhesion and chemotaxis include LTB4 [109, 110], with significantly increased levels quantified in sputum of patients with CF [111]. The chemokine IL-8 is the main neutrophil chemo-attractant involved in CF lung neutrophil infiltration [112], and increased levels of IL-8 have been detected in bronchial lavage fluid and sputum of patients with CF [113]. IL-8 is produced by a number of cells, including fibroblasts [114], epithelial cells [115], and by neutrophils themselves [116]. Inter‐ estingly, neutrophils isolated from children with CF demonstrate increased migration to IL-8 [117], and also release significantly increased levels of the chemokine when compared to the blood neutrophils of the same donor, suggesting that the environment that the cell is found

Neutrophils migrate in a multistep process consisting of rolling, tight binding, diapedesis, and migration. Initially, E-selectin and P-selectin are upregulated on the epithelium cell surface, and reversibly bind to L-selectin found on the neutrophil cell surface. In turn, L-selectin is shed from the neutrophil membrane resulting in up-regulation of integrins LFA-1 (CD11a/CD18) and MAC-1 (CD11b/CD18) which can both bind to ICAM-1 on the epithelium. Research studies have demonstrated that activation of integrins is in part mediated by IL-8 [118], and this is particularly relevant in CF as studies involving infants and children have demonstrated increased expression of IL-8 and ICAM-1, possibly indicating intrinsic inflammatory changes at a very early stage in disease progression supporting cell adhesion [119, 120]. This latter study is reinforced by data indicating that CF neutrophils show higher migratory responsiveness to IL-8 [117] supporting elevated numbers of neutrophils migrating to the airways. Moreover, neutrophil activation results in increased cytosolic Ca2+ levels triggering activation of calpain, a calcium dependent protease. Calpain has been demonstrated to liberate adhesion molecules CD11b and CD18, facilitating cell adhesion through the tight binding of integrins to epithelium cell surfaces [121]. Of major importance, studies have shown that CF neutrophils possess increased calpain activity, affecting cleavage of the cholesterol transporter caveolin-1, thereby modulating cholesterol trafficking to the plasma membrane [122]. Interestingly, and of major relevance to CF, Solomkin *et al*. (2007) demonstrated that cholesterol depletion in human

Timely and effective neutrophil programmed cell death is essential for the resolution of inflammation [124, 125] and abnormal neutrophil apoptosis is associated with decreased

migration in CF is not inherent.

260 Cystic Fibrosis in the Light of New Research

triggers disproportionate release of IL-8 [116].

neutrophils results in increased cell adhesion [123].

**5. The involvement of CFTR in neutrophil apoptosis**

The second pathway, the extrinsic or external death receptor pathway, is activated in response to extracellular signals such as FasL and TNF-alpha and is mediated by the binding of these members of the tumour necrosis factor family to death receptors on the cell surface (e.g., Fas, TNFR). The binding of the FasL and TNF-alpha to its cognate receptor results in the multi‐ merisation of the death receptor and the formation of the death inducing signal complex (DISC), containing multiple adaptor molecules such as the Fas associated death domain (FADD) or TNF-R1-associated death domain (FADD) or TNF-R1-associtaed death domain (TRADD). The respective adaptor death domain then interacts with caspase-8 [132, 133], which in turn leads to the autolytic activation from pro-caspase-8 to caspase-8. After activation and release of its active subunit (p18), caspase-8 then activates caspase-3, which finally executes apoptosis by releasing caspase-activated DNase (CAD) from its inhibitor (ICAD) with DNA fragmentation as a consequence [134]. The third pathway is the ER stress pathway. This pathway is thought to involve the activation of caspases, increase in cytosolic calcium and proapoptotic transcription factors in response to stress signals such as hypoxia, accumulation of unfolded protein, and alteration in calcium homeostasis within the ER [135, 136].

In the neutrophil the enzymatic activity of active caspase-3, -7, and -9 can be selectively inhibited by X-linked inhibitor of apoptosis (XIAP), a member of the conserved inhibitor of apoptosis (IAP) family of proteins [137]. Moreover, there are a number of key distinguishing features of neutrophil apoptosis. Firstly, the prominent role of the Bcl-2 homologue, Mcl-1, as a survival protein is central to the neutrophil's ability to undergo rapid apoptosis and may therefore limit the neutrophil's lifespan [138].

Mcl-1 is an unusual bcl-2 protein and can be rapidly turned over in the proteasome giving it a short half-life (2h) in the cell and is in contrast to the essential pro-apoptotic bcl-2 homologues, which are known to persist in the cell beyond 12 h. A critical excess of pro- over anti-apoptotic homologues decides the fate of the neutrophil and this eventually leads to loss of mitochondrial membrane potential and the progression of apoptosis [139]. Secondly, the involvement of

**Figure 5. Summary schematic of apoptosis pathways in the neutrophil.** [1] Intrinsic mitochondrial pathway (red): mito‐ chondria release cytochrome c (Cyt C) in response to cellular stress. Together with apoptotic protease activating factor-1 (Apaf-1) and procaspase-9, Cyt C will form the apoptosome complex. This results in the proteolytic activation of the pro‐ caspase. Mature caspase-9 can then proteolytically activate caspase-3. [2] Extrinsic/ligand death-receptor pathway (black): death factors such as tumour necrosis factor alpha (TNF-alpha) and Fas ligand (FasL) trigger apoptosis by binding on death receptors such as tumour necrosis factor receptor 1 (TNFR1) and Fas. The death receptors recruit procaspase-8 by means of an adaptor protein, TNF-R1-associated death domain (TRADD), or Fas associated death domain protein (FADD). After cleavage the mature caspase-8 can then directly activate caspase-3. [3] Endoplasmic reticulum (ER) stress pathway (yellow bolt): the ER can also induce apoptosis as a reaction to ER stress by releasing intracytosolic calcium (Ca2+) there‐ by activating effector caspases or through the expression of pro-apoptotic transcription factors such as CHOP. These dif‐ ferent initiation pathways converge further downstream into activation of caspase-3. The effector caspase-3 cleaves ICAD (inhibitor of CAD) and releases it from CAD (caspase-activated DNase). CAD translocates from the cytoplasm to the nu‐ cleus and can now act as active endonuclease and fragment DNA. Inhibitors of apoptosis such as myeloid leukaemia cell differentiation protein (Mcl-1) and X-linked inhibitor of apoptosis (XIAP) act upstream against pro-apoptotic Bcl-2 fami‐ ly members at the mitochondria and downstream, directly inhibit caspases, respectively.

reactive oxygen species (ROS) is very specific to neutrophil apoptosis. The observation that neutrophils isolated from CGD patients which are known to be NADPH oxidase defective display a significant delayed spontaneous cell death relative to that of neutrophils from healthy donors is important [140]. This suggests that activation of the NADPH oxidase, with conse‐ quent production of ROS, is involved in spontaneous apoptosis and in regulating the pro‐ grammed cell death of neutrophils during phagocytosis.

In contrast to the reported accelerated apoptosis in neutrophils from patients with airways disease associated with AATD [141], prolonged neutrophil survival has been reported in people with CF independent of infectious state and mutation type [142]. In support of this concept, Moriceau *et al*. (2010) illustrated that neutrophils isolated from heterozygous asymp‐ tomatic parents of people with CF exhibited delayed apoptosis [143, 144]. Moreover, the expression of the apoptosis-inducing membrane receptor Fas and its ligand have been reported reduced on CF neutrophils [145] while in addition, NE can degrade the phosphatidylserine receptor on macrophages, further delaying the removal of apoptotic neutrophils in CF [146].
