**3. The role of CFTR in neutrophils: oxygen dependent and independent mechanisms of microbial killing**

### **3.1. Oxygen dependent killing**

demonstrated the expression of low levels of *CFTR* mRNA transcripts in non-epithelial cells, including T-lymphocytes, neutrophils, monocytes, and alveolar macrophages, all of human origin [26]. This was the first record of *CFTR* gene expression in a multitude of immune cell types, and the authors suggested an important role for CFTR Cl- transport. In agreement, it was later demonstrated that alveolar macrophages from *CFTR* deficient mice retained the ability to phagocytose and generate an oxidative burst, but exhibited defective killing of internalized bacteria due to impaired acidification of the phagosome. Of interest, in this later study CFTR protein was not detected in either murine or human neutrophils [27] and further studies have shown CFTR independent phagosomal acidification in macrophages [28, 29]. Nevertheless, the expression of *CFTR* mRNA transcripts in human macrophages was con‐ firmed by Del Porto, who published that the bactericidal capabilities of macrophages was CFTR dependent, indicating an important functional role for the CFTR protein in these

Up to this point, no connection between abnormal neutrophil function and the expression of CFTR protein in human neutrophils had been made, and whether or not neutrophils express functional CFTR was still the topic of great debate among leading scientists and clinicians [5, 8, 13, 14], with relevance for the development of CFTR-targeting pharmacotherapy. This is emanating from worldwide laboratories equally committed to the pursuit of knowledge on the cause for impaired neutrophil activity in CF and also the potential consequence of the loss of functional CFTR Cl- channels. Indeed, there is still great controversy as to the true nature of dysregulated neutrophil activity in CF and, for example, in 2010, McKeon and colleagues could only detect low levels of *CFTR* mRNA transcripts in human neutrophils by either reverse transcriptase polymerase chain reaction (RT-PCR) or real time PCR methods of amplification [31]. The authors could not detect CFTR protein expression in membrane or cytosolic fractions, or cell lysates from human neutrophils by Western blot analysis, suggesting that human neutrophils do not express CFTR protein and that the dysregulated neutrophil function in CF was due to the inflammatory status of the individual [31]. Similarly, a study by Morris *et al.,* (2005) who investigated altered phagocytosis of neutrophils in CF due to cell priming, did not

Over the years, a number of reasons for not detecting CFTR protein in neutrophil cell fractions by Western blot analysis have been put forward and include the susceptibility of CFTR protein to degradation, a lack of reliable anti-CFTR antibodies, and also boiling of cell fractions prior to electrophoresis. As these issues were addressed researchers have detected CFTR protein in human neutrophils and have established functional roles for membrane associated CFTR. In line with this concept, in 2006, Painter and colleagues demonstrated the presence of CFTR protein in human neutrophil membrane phagolysosomes by confocal microscopy, and also verified the expression of *CFTR* at a mRNA level, and at a protein level by Western blot analysis [33]. Interestingly, in this same study results demonstrated the expression of CFTR protein by Western blot analysis in a human myeloid cell line (HL-60 cells) differentiated into neutrophillike cells [33]. This is in contrast to the study by Yoshimura *et al*., who could not detect *CFTR* mRNA transcripts in HL-60 cells [24]. Further studies have identified *CFTR* mRNA transcripts in differentiated HL-60 cells and demonstrated CFTR protein localisation to the phagocytic vacuole, strengthening the similarities between HL-60 cells and human neutrophils [34].

detect CFTR protein in human neutrophils by Western blot analysis [32].

immune cells [30].

252 Cystic Fibrosis in the Light of New Research

The focus of this chapter will now turn to studies that have reported impaired neutrophil activity due to a lack of CFTR function. Of major relevance to bacterial killing in CF, reported defective killing of microbes due to conditions that prevail within the vacuole of phagocytosing neutrophils will be discussed. The process of neutrophil mediated bacterial clearance can be divided into two main processes: those that are oxygen independent and those that are oxygen dependent. With regards to oxidative mechanisms of microbial killing, the first indications that oxygen plays a role in the functionality of neutrophils was first discovered by Baldridge and Gerard [37]. By exposing canine neutrophils to bacteria they observed a significant increase in oxygen consumption [37], and later it was revealed that this increase in oxygen consumption was independent of mitochondrial respiration [38]. Following discovery of the respiratory burst, it was discovered that neutrophils from patients with chronic granulomatous disease (CGD) failed to mount a respiratory burst during phagocytosis and these individuals are characterised by an abnormality of neutrophil function and recurrent life-threatening infec‐ tions [39, 40]. CGD provides the most definitive evidence for the physiological and clinical importance of the respiratory burst, or alternatively referred to as the NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase). Indeed, patients with CGD have played a vital role in understanding the structure and mechanism of the NADPH oxidase (Figure 2). The burst of oxygen consumption upon phagocytosis and the absence of this process in CGD leading to impaired bacterial killing was understood to indicate that the oxygen consumed was converted to antimicrobial oxidants. By exposing neutrophils to opsonised bacteria, Cohen and colleagues demonstrated that 99% of oxygen consumed was converted to superoxide (O2 - ) [41] and the requirement of O2 as a precursor to hydrogen peroxide (H2O2) was confirmed [42]. It is not known what concentration of H2O2 is attained within the vacuole, with measurement from 0.01μM [42] to 100mM estimated, depending on the amount of phagocytosis and the intracellular pH [43]. These oxidants produced by neutrophils may also contribute to lung tissue damage and the shield against oxidant-modulated injury is the extracellular anti-oxidant glutathione; but this is significantly decreased in CF epithelial lining fluid [44-47]. Of major relevance is that CFTR has been linked to extracellular glutathione transport [48] and in paediatric reports of extremely high levels of protein oxidation have been detected in airway samples [49].

The neutrophil respiratory burst, or NADPH oxidase, generates superoxide (O2 - ) as the initial oxygen-derived reactive species in response to bacteria or a variety of soluble stimuli (fMLP). A) The enzyme complex is latent in quiescent unstimulated neutrophils and approximately 20% flavocytochrome b558 is found in the plasma membrane pool, and 80% in the specific granules. The active site of this enzyme is located in an integral membrane cytochrome, b558, which consists of the two subunits gp91phox and p21phox (α and β subunits, respectively). B) Stimulation of the neutrophil by fMLP induces activation and phosphorylation (P) of a number of kinases including Akt. C) p21rac is converted into the active guanosine triphosphate GTP-bound form and the phosphorylation of the cytosolic components (p67phox, p47phox, and p40phox) occurs. D) These subunits then translocate to the plasma membrane where they interact with cytochrome b558 to initiate reactive oxygen species production.

**Figure 2.** Schematic illustration of the NADPH oxidase of resting and fMLP activated cells.

H2O2 generated during the oxidative burst has limited bactericidal properties and the bestdefined function of H2O2 in the antimicrobial activities of neutrophils comes from the function of H2O2 as a substrate for myeloperoxidase (MPO) in the presence of halides (chloride (Cl- )). Neutrophil MPO was initially called verdoperoxidase, and later its name subsequently changed to MPO. It is present in exceptionally high concentrations in neutrophils, with levels estimated to be no less than 5% of the dry weight of the cell. MPO is synthesized and packaged into azurophilic, also referred to as primary granules of neutrophils, during the promyelocyte stage of granulocyte development and is present in mature resting granulocytes.

transport [48] and in paediatric reports of extremely high levels of protein oxidation have been

detected in airway samples [49].

254 Cystic Fibrosis in the Light of New Research

The neutrophil respiratory burst, or NADPH oxidase, generates superoxide (O2

**Figure 2.** Schematic illustration of the NADPH oxidase of resting and fMLP activated cells.

b558 to initiate reactive oxygen species production.

species in response to bacteria or a variety of soluble stimuli (fMLP). A) The enzyme complex is latent in quiescent unstimulated neutrophils and approximately 20% flavocytochrome b558 is found in the plasma membrane pool, and 80% in the specific granules. The active site of this enzyme is located in an integral membrane cytochrome, b558, which consists of the two subunits gp91phox and p21phox (α and β subunits, respectively). B) Stimulation of the neutrophil by fMLP induces activation and phosphorylation (P) of a number of kinases including Akt. C) p21rac is converted into the active guanosine triphosphate GTP-bound form and the phosphorylation of the cytosolic components (p67phox, p47phox, and p40phox) occurs. D) These subunits then translocate to the plasma membrane where they interact with cytochrome

H2O2 generated during the oxidative burst has limited bactericidal properties and the bestdefined function of H2O2 in the antimicrobial activities of neutrophils comes from the function of H2O2 as a substrate for myeloperoxidase (MPO) in the presence of halides (chloride (Cl-

Neutrophil MPO was initially called verdoperoxidase, and later its name subsequently


) as the initial oxygen-derived reactive

)).

Mature MPO is a 150 kDa tetramer composed of two glycosylated 59–64 kDa heavy subunits and two unglycosylated 14 kDa light subunits as a pair of protomers linked together by a disulphide bond. When the phagosome containing microorganisms fuses with cytoplasmic granules, MPO, along with the other components of the granules, is released into the vacuole. A role for MPO as a component of the antimicrobial armoury of neutrophils was proposed with the finding that MPO was strongly microbial when combined with H2O2 and a halide [50, 51]. MPO and H2O2 form an enzyme-substrate complex, which oxidises ions to the toxic agent hypohalous acid. Any of the halide ions (I- , Br- , Cl- ) can be oxidised with iodide and bromide being more effective than Cl on a molar basis [51]. It is more likely however that the neutrophil uses Cl because it is present in high concentration in biological fluids, resulting in the formation of hypochlorous acid (HOCl). There are three proposed means of Cl- transport to the phagosome: extracellular Cl- intake during phagocytosis of a pathogen, stored Cl within granules released into the phagosome upon vesicle fusion, and passive or active transport of Cl from the cytosol to the phagosome [52]. Only active or passive transport has been suggested to provide a constant supply of Cl- (Figure 3) [53] and two Cl- ion channels (ClCs), ClC-3and CFTR have been implicated in the transport of Cl- within the neutrophil and the phagosome [33, 54]. The influx of protons to the phagosomal lumen by V-ATPase has been demonstrated to facilitate the transport of Cl ions into the phagosome by ClCs including the CFTR [55] (Figure 3).

It is generally believed that HOCl is the most bactericidal oxidant known to be produced by the neutrophil. Levels of HOCl produced are based on calculations made after phorbol ester stimulation, which results in the secretion of O2 across the membrane to the surrounding supernatant and levels achieved are estimated at 80μM.

Levels produced in the phagocytic vacuole have been estimated using chlorinated fluorescein as a specific marker for HOCl production and by use of this technique it was calculated that 11% of oxygen consumed was converted to HOCl, resulting in 28μM within the phagosome [56]. HOCl is an extremely strong non-radical oxidant and bacterial targets include adenosine triphosphate (ATP)-generating systems [57], disruption of basement membranes or cell membranes, and fragmentation of proteins [58]. Chloramines are generated indirectly through the reaction of HOCl with amines, but are less reactive than HOCl but much more stable, and are therefore called "long lived oxidants." Because of the high intracellular concentration of the β-amino acid taurine, *N*-chlorotaurine is the major compound of low molecular weight long lived oxidants produced by neutrophils [59, 60]. Of major relevance to bacterial killing in CF it was reported that CF neutrophils had impaired chlorination of internalised bacteria within the phagosome when compared to healthy control neutrophils [33]. The authors highlighted the role of CFTR in facilitating Cl- flux into the phagosome of neutrophils which was impaired in CF cells. This CFTR dysfunction resulted in limited availability of phagosomal Cl- required for the generation of HOCl, resulting in reduced intravacuolar killing of *Pseudo‐*

Upon activation of the NADPH oxidase, the cytochrome takes electrons from NADPH and passes them, via FAD and haem, to O2 in the following reaction: NADPH + 2O2 → NADP+ + H+ + 2O2 - . NADPH oxidase generates an electron current which is compensated through voltage gated ion influx of protons (Voltage G-H+ ) and potassium (Voltage G-K + ). Cl transport is facilitated by CFTR and also other channels including ClC-3. The influx of H+ to the phagosomal lumen by V-ATPase facilitates Cl- transport into the phagosome by ClCs including the CFTR. O2 - is a mild oxidant and reductant with restricted biological activity. H2O2 is a slow acting oxidising agent and HOCl is a strong non-radical oxidant.

**Figure 3.** Suggested mechanism of Cl and ion transport within the neutrophil phagosome.

*monas aeruginosa*, the archetype infecting organism in CF [33, 34, 53, 61]. Moreover, new data has shown that mutant CFTR fails to target to the neutrophil vacuole resulting in impaired intraphagosomal HOCl production and neutrophil microbial killing [62].

Having described the importance of MPO mediated halogenation for adequate microbial killing, there is also a need to mention that MPO deficiency occurs with a high prevalence and patients are not clinically afflicted by serious bacterial infections [63], with infections by *Candida* species being the main difficulty. Although more slowly than healthy cells, neutrophils deficient in MPO can kill bacteria *in vitro* [64], a result incompatible with the concept that HOCl is the major mediator of neutrophil bactericidal function in man. MPO deficient cells illustrate a prolonged respiratory burst resulting in increased levels of H2O2 and this coupled with nonoxidative methods of bacterial killing has been proposed to compensate for MPO deficiency. Furthermore, despite the fact that neutrophils of CGD patients do not produce O2 and H2O2, studies have shown that neutrophils from CGD patients are capable of killing significant numbers of phagocytosed bacteria and yeast [65, 66] and the presence of oxygen-independent microbial mechanisms in neutrophils is demonstrated by the ability of these cells to kill bacteria in the absence of oxygen [67].

### **3.2. Oxygen independent microbial killing**

*monas aeruginosa*, the archetype infecting organism in CF [33, 34, 53, 61]. Moreover, new data has shown that mutant CFTR fails to target to the neutrophil vacuole resulting in impaired

and ion transport within the neutrophil phagosome.

Upon activation of the NADPH oxidase, the cytochrome takes electrons from NADPH and passes them, via FAD and

reductant with restricted biological activity. H2O2 is a slow acting oxidising agent and HOCl is a strong non-radical

 + H+ + 2O2 -

Having described the importance of MPO mediated halogenation for adequate microbial killing, there is also a need to mention that MPO deficiency occurs with a high prevalence and patients are not clinically afflicted by serious bacterial infections [63], with infections by *Candida* species being the main difficulty. Although more slowly than healthy cells, neutrophils deficient in MPO can kill bacteria *in vitro* [64], a result incompatible with the concept that HOCl is the major mediator of neutrophil bactericidal function in man. MPO deficient cells illustrate a prolonged respiratory burst resulting in increased levels of H2O2 and this coupled with nonoxidative methods of bacterial killing has been proposed to compensate for MPO deficiency.

studies have shown that neutrophils from CGD patients are capable of killing significant numbers of phagocytosed bacteria and yeast [65, 66] and the presence of oxygen-independent microbial mechanisms in neutrophils is demonstrated by the ability of these cells to kill bacteria


. NADPH oxidase generates an electron

) and potassium (Voltage G-K

to the phagosomal


and H2O2,

intraphagosomal HOCl production and neutrophil microbial killing [62].

current which is compensated through voltage gated ion influx of protons (Voltage G-H+

transport is facilitated by CFTR and also other channels including ClC-3. The influx of H+

lumen by V-ATPase facilitates Cl- transport into the phagosome by ClCs including the CFTR. O2

haem, to O2 in the following reaction: NADPH + 2O2 → NADP+

+ ). Cl-

oxidant.

Furthermore, despite the fact that neutrophils of CGD patients do not produce O2

in the absence of oxygen [67].

**Figure 3.** Suggested mechanism of Cl-

256 Cystic Fibrosis in the Light of New Research

The presence of an oxygen independent microbial mechanism in neutrophils is demonstrated by the ability of these cells to kill bacteria under anaerobic conditions. Hirsch and colleagues reported that neutrophil lysates killed bacteria and that this effect was due to a substance they called phagocytin [68]. The component responsible for bacterial killing was localized to the cytoplasmic granules which are released into the phagocytic vacuole [69]. Neutrophil-derived microbial molecules are packaged within four distinct subgroups of granules (Figure 4) and are released either into the phagocytic vacuole or to the outside of the cell upon activation. Granule biogenesis follows the granulocyte differentiation pathway. The azurophilic (also referred to as primary) granules first emerge at the stage of the promyelocytes [70] and contain MPO, the serine proteases neutrophil elastase (NE), cathepsin G and proteinase 3, defensins and bacterial permeability-increasing protein [71], and are considered as the true microbial compartment mobilized upon phagocytosis. Later in differentiation, at the metamyelocyte stage, specific granules containing lactoferrin [72], 18 kDa human cathelicidin antimicrobial protein (hCAP-18], and lysozyme emerge [73], followed by a third population termed the gelatinase granules which predominantly contain gelatinase (matric metalloprotease (MMP)-9 and MMP-2), lysozyme, and leukolysin [74, 75]. A forth type of granule, called the secretory vesicles, appears at the stage of the mature neutrophil. Movement of the cytoplasmic granules following ingestion of bacteria was first observed by Robineaux and Frederic [76], and by use of chicken leukocytes with their large dense granules, Hirsch observed by phase contrast microscopy, degranulation and release of the granule contents directly into the phagocytic vacuole, by fusion of the granule membrane with the invaginated cell membrane [77]. As for any form of intracellular vesicle transport, degranulation is a tightly regulated process. Small GTPases of the Ras superfamily are known key regulators of cellular events including vesicle transport, cell division, control of cytoskeletal rearrangements, and nuclear assembly. Secon‐ dary and tertiary granules are tethered through a small GTPase Rab27a and its effector protein Munc13-4 [78] which interacts with the soluble N-ethylmaleimide association protein receptor (SNARE), a protein complex composed of vesicle-associated membrane proteins (VAMPs) on the vesicle surface and SNAP23 and STX4 on the plasma membrane [79-81]. Once the granule is docked the granule membrane fuses with the plasma membrane enabling release of granule contents. The small GTPase Rac2 has been shown necessary for the release of primary granules [82] but of interest, a sub-population of Rab27a positive primary granules was also found [83] with different Rab27a effectors Slp1 and Munc13-4 reported necessary for primary and tertiary granules release, respectively [84].

Although the neutrophil possesses an armoury of anti-microbial proteins and peptides, individual components have been shown to exert microbial effects. For example, NE has long been regarded as the major antibacterial protein and mice made homozygous for a disrupted NE gene have demonstrated impaired resistance to *Klebsiella pneumonia* and *Escherichia coli* sepsis [85]. A target for NE is the bacterial outer-membrane protein OmpA [86]. NE degrada‐ tion of OmpA results in cell death as a result of loss of bacterial integrity by localized weakening of the cell wall followed by osmotic lysis [86]. The amino acid composition of cathepsin G shares 37% sequence homology with NE and plays a role in neutrophil responses against a

**Figure 4.** Granule and vesicle contents of the neutrophil. The most abundant organelles within the cytoplasm are the granules, which are membrane-bound organelles containing an array of antimicrobial proteins. Three major types have been identified, azurophilic, specific, and gelatinase-containing granules. A forth type of granule, called the secretory vesicles, are endocytic in nature and act as an internal reservoir of membrane/cytokine receptors.

variety of bacteria. Purified cathepsin G has been shown to inhibit the growth of several organisms including *Staphylococcus aureus, E. coli, P. aeruginosa*, and *Neisseria gonorrhoea* [87, 88]. Moreover, Tkalcevic and colleagues performed *in vivo* studies of mice deficient in cathe‐ psin G, NE, or both and demonstrated the importance of cathepsin G in the successful clearance of *Aspergillus fumigatus*. Wild type mice almost completely cleared the fungal pathogen, while the single mutants showed an intermediate phenotype between the wild type and double mutants, thus establishing a critical role for both elastase and cathepsin G in the control of fungal infection *in vivo* [89].

Examples of proteins stored in specific (secondary) and gelatinase (tertiary) granules include human lactoferrin and MMPs. Lactoferrin is a major component of the specific granules and is active against a variety of pathogens [90]. This protein binds to bacteria through its highly positively charged *N*-terminus and displays antimicrobial properties against Gram-positive and Gram-negative bacteria by limiting the availability of environmental iron. However, since iron-saturated lactoferrin is also capable of killing certain bacteria, mechanisms other than iron depletion are involved. Further studies have indicated that peptides obtained after enzymatic hydrolysis of lactoferrin are much more effective in killing bacteria than is the intact protein. It is likely that the *N*-terminal cationic domain of human lactoferrin plays an essential role in the bactericidal activity and has been shown to be highly effective against infections with antibiotic-resistant *S. aureus* [91]. MMP-9 is stored in an inactive preform that requires activation by a serine protease such as NE. Its main function is the degradation of type V collagen in the extracellular matrix to aid migration to the site of infection [92]. The importance of MMP-9 in host defence was illustrated by a higher frequency of peritoneal sepsis in MMP-9 knockout mice due to impaired migration of neutrophils to the site of infection [93].

In spite of their original role in host defence, NE and proteinase 3 have been strongly implicated in the pulmonary pathology of CF. Indeed it has been shown that NE is the main mediator of proteolysis (Figure 1) but can also cause up-regulation of expression of other proteases including MMPs and cathepsins and as a result it has been proposed that neutralisation of NE activity is central to reducing the overall protease burden [94]. In line with this thought, NE has the ability to degrade structural proteins in the lung including elastin, collagen, and fibronectin and to promote IL-8 production by bronchial epithelial cells [95], to degrade antimicrobial peptides [96], and to degrade antiproteases including alpha-1 antitrypsin, SLPI [97], and elafin [98] leading to a protease/antiprotease imbalance [99]. Moreover, it has been shown that the process of primary granule release by CF neutrophils appears altered, as greater levels of NE [100] and MPO [101] were recorded in the extracellular fluids post stimulation with either CF airway samples, TNF-alpha and IL-8, or serum-opsonised particles. Of impor‐ tance, altered cytosolic pH regulation in CF neutrophils has been demonstrated [102, 103], which could in turn influence the process of degranulation. In further support of increased primary granule release by CF neutrophils, MPO and NE levels have been reported to be present at significantly increased levels in airway samples from patients with CF compared to controls [49, 104]. Moreover, levels of NE degranulation were not significantly altered following intravenous antibiotic treatment of patients with CF, indicating continued dysre‐ gulation of neutrophil activity even with clinical improvement [105]. While the mechanism for excessive primary granule release by CF neutrophils has not been fully investigated [104, 106], new research on the cause of reduced secondary and tertiary granule release has recently been revealed [35]. Contrary to increased release of secondary and tertiary granules by neutrophils of individuals with airway disease linked to alpha-1 antitrypsin deficiency (AATD) [107], evidence was presented indicating that abnormal CFTR function contributes to impaired neutrophil killing in CF due to inadequate Rab27a activation, which regulates the release of antimicrobial proteins from secondary and tertiary granules. In this study reduced degranulation of lactoferrin of secondary granules and MMP-9 of tertiary granules from patients with CF compared to healthy control cells was observed, an effect mirrored in healthy control cells post CFTR inhibition. Collectively results revealed that CFTR inhibition or dysfunction reduces cytosolic Mg2+ levels resulting in impaired Rab27a activity, ultimately reducing the CF neutrophils ability to kill bacterial pathogens [35].

variety of bacteria. Purified cathepsin G has been shown to inhibit the growth of several organisms including *Staphylococcus aureus, E. coli, P. aeruginosa*, and *Neisseria gonorrhoea* [87, 88]. Moreover, Tkalcevic and colleagues performed *in vivo* studies of mice deficient in cathe‐ psin G, NE, or both and demonstrated the importance of cathepsin G in the successful clearance of *Aspergillus fumigatus*. Wild type mice almost completely cleared the fungal pathogen, while the single mutants showed an intermediate phenotype between the wild type and double mutants, thus establishing a critical role for both elastase and cathepsin G in the control of

vesicles, are endocytic in nature and act as an internal reservoir of membrane/cytokine receptors.

**Figure 4.** Granule and vesicle contents of the neutrophil. The most abundant organelles within the cytoplasm are the granules, which are membrane-bound organelles containing an array of antimicrobial proteins. Three major types have been identified, azurophilic, specific, and gelatinase-containing granules. A forth type of granule, called the secretory

Examples of proteins stored in specific (secondary) and gelatinase (tertiary) granules include human lactoferrin and MMPs. Lactoferrin is a major component of the specific granules and is active against a variety of pathogens [90]. This protein binds to bacteria through its highly positively charged *N*-terminus and displays antimicrobial properties against Gram-positive and Gram-negative bacteria by limiting the availability of environmental iron. However, since iron-saturated lactoferrin is also capable of killing certain bacteria, mechanisms other than iron depletion are involved. Further studies have indicated that peptides obtained after enzymatic hydrolysis of lactoferrin are much more effective in killing bacteria than is the intact protein. It is likely that the *N*-terminal cationic domain of human lactoferrin plays an essential role in the bactericidal activity and has been shown to be highly effective against infections with antibiotic-resistant *S. aureus* [91]. MMP-9 is stored in an inactive preform that requires activation by a serine protease such as NE. Its main function is the degradation of type V collagen in the extracellular matrix to aid migration to the site of infection [92]. The importance of MMP-9 in host defence was illustrated by a higher frequency of peritoneal sepsis in MMP-9

knockout mice due to impaired migration of neutrophils to the site of infection [93].

In spite of their original role in host defence, NE and proteinase 3 have been strongly implicated in the pulmonary pathology of CF. Indeed it has been shown that NE is the main mediator of proteolysis (Figure 1) but can also cause up-regulation of expression of other proteases including MMPs and cathepsins and as a result it has been proposed that neutralisation of NE activity is central to reducing the overall protease burden [94]. In line with this thought, NE

fungal infection *in vivo* [89].

258 Cystic Fibrosis in the Light of New Research
