**4. The involvement of CFTR in neutrophil adherence leading to migration**

Before describing the process leading to neutrophil adhesion, it is important to stress the complexity of studying neutrophil adhesion and migration. Blood neutrophils isolated from patients with CF are chronically exposed to pro-inflammatory cytokines, including LTB4 and IL-8, and pathogenic particles including fMLP (formyl-methionyl-leucyl-phenylalanine) and LPS, resulting in these cells being in a constant primed state. Therefore, independent of the expression of CFTR protein, neutrophils in CF may illustrate enhanced cell adherence and migration. To circumvent this dilemma and to eliminate the potential for bias towards inflammatory versus altered neutrophil adhesion due to a lack of CFTR function, an approach taken by researchers is to include neutrophils isolated from inflammatory control patients. For example, in 1998, Russell and colleagues demonstrated that L-selectin shedding is altered in patients with CF resulting in increased neutrophil adhesion in response to IL-8 and fMLP [108]. This paper reported that non-CF bronchiectasis patients did not possess alterations in Lselectin shedding, suggesting that the defect in L-selectin shedding is CF specific and could be 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 migration in CF is not inherent.

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 triggers disproportionate release of IL-8 [116].

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 neutrophils results in increased cell adhesion [123].
