Lactoferrin is an iron-binding glycoprotein (Mw 80-kDa) and as such it exists in both iron-replete and iron-depleted forms. The iron-depleted form of lactoferrin is its more biologically active form. Lactoferrin is a cationic protein with an isoelectric point (pI) of 8.7 and is widespread in human secretions. It is found in high concentrations in breast milk (~3–7 mg/ml), tear fluid (1–4 mg/ml), vaginal secretions, gut-lining fluid, cervical mucus plugs, saliva, exocrine secretions and respiratory secretions (0.1–1 mg/ml). Lactoferrin is released from neutrophil secondary (specific) granules at areas of inflammation. Like many innate immunity proteins, lactoferrin is highly cationic (calculated pI = 8.5). Lactoferrin is both antimicrobial and anti-inflammatory and contributes to host defense both systemically and at mucosal surfaces. Its antimicrobial effects include being directly bactericidal and bacteriostatic and more recently it has been shown to inhibit Pseudomonas biofilm formation by a separate mechanism 12. Lactoferrin exhibits antibacterial effects on Gram-negative bacteria by means of two mechanisms. Firstly, by binding iron, it limits the amount of free iron (an essential growth factor for microorganisms) available. For example, in one study Streptococcus mutans and Vibrio cholerae were killed by incubation with purified human apolactoferrin, the iron-depleted form of the protein. Concentrations of lactoferrin below that necessary for total inhibition resulted in a marked reduction in viable colony-forming units. This bactericidal effect was contingent upon the metal-chelating properties of the lactoferrin molecule 13. Lactoferrin contains two high-affinity ferric iron binding sites facilitating such iron sequestration from pathogenic microbes. Secondly, lactoferrin can destabilise the outer membrane of gram-negative bacteria by binding to it resulting in altered permeability that leads to microbial injury and death. This activity has been attributed to the 17 amino acid N-terminal portion of lactoferrin. This portion is rich in arginine residues that give the molecule its highly cationic nature and has been termed lactoferricin. The related iron-binding protein transferrin lacks these arginine residues and is less cationic (pI of 5–5.5) 14. Lactoferrin has been shown to kill clinical strains of E.coli, S. aureus and mucoid P. aeruginosa isolated from CF airways 15. Lactoferrin acts synergistically with other innate immunity proteins such as lysozyme and SLPI in bacterial killing 1617. It has also been shown that lactoferrin enhances the antimicrobial effects of some antibiotics 18. Lactoferrin reduces the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of doxycycline for Burkholderia cepacia and P. aeruginosa strains with MICs for B. cepacia falling from highly resistant to clinically achievable levels 19. By binding iron, lactoferrin can also act as an anti-oxidant since iron bound to the protein is unable to participate as a catalyst for the generation of free hydroxyl radicals via the Haber-Weiss reaction 202122.

Lactoferrin has recently been shown to inhibit Pseudomonas biofilm formation 12. The inhibition of biofilm formation is a property unique to lactoferrin and thus it plays a pivotal role in host defense against this highly destructive mode of bacterial growth. Biofilm bacteria are notoriously resistant to host killing and antibiotics. They may be up to one thousand times more resistant to antibiotics than their free-swimming, planktonic counterparts 2324. In the presence of lactoferrin, free iron levels decreased inducing a twitching motion in bacteria. This twitching ensured that the bacteria wandered across a surface rather than stopping to form microcolonies, clusters and subsequent pillar and mushroom-shaped mature biofilms. Furthermore, Singh et al demonstrated increased susceptibility of biofilm bacteria to tobramycin in the presence of lactoferrin. We have shown previously that lactoferrin levels are depleted in BAL from Cystic Fibrosis patients with active Pseudomonas infections compared to those with no active Pseudomonas infection and that this depletion results in impaired ability to prevent Pseudomonas biofilm formation 25.

Lactoferrin also has been shown to be active against a number of viruses including human immunodeficiency virus (HIV) and cytomegalovirus (CMV) 2627. Lactoferrin is known to prevent replication of hepatitis B and C viruses and is being investigated clinically together with interferon as a potential therapeutic agent in these conditions 2829. A recent study elucidated the potential of oral administration of lactoferrin to attenuate pneumonia in influenza-virus-infected mice through the suppression of infiltration of inflammatory cells in the lung 30. In addition to antiviral properties, lactoferrin also possesses anti-fungal properties: it has been shown to be active against Candida species and is being investigated as a potential treatment for oral candidiasis 31.

Lactoferrin is also a key anti-inflammatory protein. It possesses two basic cradles at residues 1 to 5 and 28 to 34 of the N-terminal end that can bind anionic molecules such as lipopolysaccharide (LPS), heparin and heparin sulfates. Lactoferrin has been shown to inhibit the LPS-induced expression and proteoglycan binding ability of Interleukin-8 in human endothelial cells 32. Lactoferrin protects against sublethal doses of LPS in mice and germfree piglets. Animals that received pre-treatment with lactoferrin showed minimal effects when given an intra-peritoneal injection of LPS whereas the control group of animals that did not receive pre-treatment with lactoferrin succumbed rapidly to septic shock 33. Lactoferrin has also been shown to lower the expression of adhesion molecules E-selectin and ICAM-1 on endothelial cells further modifying the immune response 34. Another immunomodulatory function of lactoferrin stems from its ability to bind unmethylated CpG motifs, which are bacterial DNA products capable of stimulating various innate and acquired immune responses in human and murine models 20. A schematic of lactoferrin's many antimicrobial and immunomodulatory roles is shown in figure 1.

SLPI is a 11.7-kDa non-glycosylated protein that is expressed by macrophages, neutrophils and the mucosal surface of epithelial cells 3536. It is a highly basic (pI > 9.5), acid-stable but alkaline-labile protein 37. SLPI is the third most abundant innate immunity protein of respiratory secretions after lysozyme and lactoferrin. It is estimated that SLPI is present at concentrations of 0.1 to 2 ug/ml in airway lavage fluid 3839 and 2.5 ug/ml in nasal secretions40. SLPI through its C terminal domain is a serine protease inhibitor and provides significant protection against neutrophil elastase, a powerful elastolytic enzyme released from neutrophils during degranulation at areas of infection and inflammation. SLPI also inhibits the serine protease cathepsin G as part of its anti-protease effects in the lung 41. In addition to its potent anti-protease activity, SLPI has important anti-bacterial, anti-viral and anti-inflammatory properties. The N-terminal domain of SLPI has modest antimicrobial activity in vitro against Gram-negative and Gram-positive bacteria 42. SLPI has been shown to inhibit human immunodeficiency virus (HIV) infectivity of monocytes by blocking viral DNA synthesis. Saliva is rich in SLPI and it is felt that this accounts for the low viral transmission rates via saliva 4344. Prior administration of SLPI to rats attenuated pulmonary recruitment of neutrophils in an immunoglobulin G (IgG) immune complex model of acute lung injury 45. Pre-treatment with SLPI greatly reduced inflammation in both liver and lungs in a mouse model of hepatic ischaemia/reperfusion injury 46. SLPI can inhibit LPS-induced NF-κB activation by inhibiting degradation of IRAK, IκBα and IκBβ 47 and can also impair lipoteichoic acid (LTA) and LPS induced pro-inflammatory gene expression in monocytes and macrophages in vitro 3648. In addition to its antiprotease activity, SLPI has been shown to exhibit anti-inflammatory properties, including down-regulation of tumor necrosis factor alpha expression by lipopolysaccharide (LPS) in macrophages and inhibition of nuclear factor (NF)-kappaB activation in a rat model of acute lung injury. SLPI has recently been shown to enter cells, becoming rapidly localized to the cytoplasm and nucleus where it affects NF-kappaB activation by binding directly to NF-kappaB binding sites in a site-specific manner 47. However once oxidised, the anti-inflammatory and anti-elastase effects of SLPI are diminished. Greene et al. demonstrated cleavage of SLPI in infected lobes in community acquire pneumonia resulting in impaired anti-NE activity. SLPI was inactivated by cleavage, oxidation and complex formation paving the way for free neutrophil elastase to exacerbate pulmonary parenchymal inflammation and tissue damage 49. SLPI is susceptible to protease cleavage. Cathepsins B, L, and S have been shown to cleave and inactivate SLPI. Analysis of epithelial lining fluid samples from individuals with emphysema indicated the presence of active cathepsin L and cleaved SLPI 50. Serine and cysteine proteases produced by the house dust mite in asthma have been shown to cleave SLPI and may increase the susceptibility of patients with allergic inflammation to infection 36.

Lysozyme is a 14-kDa enzyme directed against the β 1→4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid residues that make up peptidoglycan, the cell wall material that gives bacteria their shape. Lysozyme is a basic protein with a pI of 10.5 51. It is stored in both primary and secondary neutrophil granules. In addition to enzymatic lysis of bacterial cell walls, lysozyme can also kill bacteria by a non-enzymatic mechanism 52. Lysozyme is highly active against many Gram-positive species, including Bacillus megaterium, Micrococcus luteus, and many streptococci. Lysozyme also has an important role against Gram-negative organisms. Its ability to kill Gram-negative organisms may be influenced by ionic concentration, osmolarity, and the presence of synergistic cofactors 21517.

As lactoferrin and lysozyme are present together in high levels in mucosal secretions and neutrophil granules, it is probable that their interaction contributes to host defense 17. Lactoferrin in concert with other cofactors presumably disrupt the outer membrane of Gram-negative bacteria and allow lysozyme access to the sensitive peptidoglycan layer. Lysozyme is a component of both phagocytic and secretory granules of neutrophils and is also produced by monocytes, macrophages, and epithelial cells. Both lysozyme and lactoferrin arise in the lower respiratory tract within the airways and their levels are elevated in association with chronic bronchitis suggesting that lactoferrin and lysozyme may contribute to the modulation of airway inflammation in chronic bronchitis. Lysozyme is about tenfold more abundant in the initial "airway" aliquot than in subsequent aliquots of bronchoalveolar lavage 53, and its concentration correlates poorly with neutrophil concentrations, suggesting that, in general, airway epithelium and its glands are the major sources of lysozyme in airway secretions. Lysozyme is present at concentrations of between 0.1 and 1 mg/ml in respiratory secretions. In one study to assess the role of lysozyme in pulmonary host defense in vivo, transgenic mice expressing rat lysozyme cDNA in distal respiratory epithelial cells were generated. Two transgenic mouse lines were established in which the level of lysozyme protein in bronchoalveolar lavage (BAL) fluid was increased 2- or 4-fold relative to that in wild type (WT) mice. Lysozyme activity in BAL was significantly increased (6.6- and 17-fold) in 5-wk-old animals from each transgenic line. Killing of group B streptococci was significantly enhanced (2- and 3-fold) in the mouse transgenic lines at 6 h following infection and was accompanied by a decrease in systemic dissemination of pathogen. Killing of Pseudomonas aeruginosa was also enhanced in the transgenic lines (5- and 30-fold). Twenty-four hours after administration of Pseudomonas aeruginosa, all transgenic mice survived, whereas 20% of the WT mice died. The authors concluded that increased production of lysozyme in respiratory epithelial cells of transgenic mice enhanced bacterial killing in the lung in vivo, and was associated with decreased systemic dissemination of pathogen and increased survival following infection 54. A recent in vivo study of lysozyme derived from submucosal glands in ferret trachea demonstrated that lysozyme-depleted secretions were much less effective at inhibiting bacterial growth than mock-depleted samples, suggesting that lysozyme is partially responsible for the antibacterial activity of the glandular airway secretions. Furthermore, depletion of lysozyme from human nasal secretions also reduced antibacterial activity by 50% 55.

Defensins are 3- to 5-kDa peptide members of a widely distributed family with characteristic three-dimensional folding with six cysteine-three disulfide patterns. Defensins have broad cytotoxic activity against bacteria, fungi, parasites, viruses and even host cells 56. Defensins are subdivided into different classes that include alpha and beta defensins. Alpha defensins are also known as human neutrophil peptides (HNPs). HNP-1 to -4 are found in azurophil granules of neutrophils where they constitute up to 50% of the total protein present 57. HNP-5 and -6 have been identified in Paneth cells in the crypts of the small intestinal mucosa and also in the female reproductive tract. The alpha defensins have a wide variety of actions including mitogenic and chemotactic activities 5859. Alpha defensins contribute to epithelial repair in the lung by enhancing epithelial cell proliferation 60. The more recently identified human beta defensins (HBDs) 1–4 differ slightly from classical alpha defensins in the spacing and connectivity of their cysteines 2. Beta defensins have five to eight positively charged residues resulting in quite similar (calculated) isoelectric points of 8.9 to 9.5 61. HBD-2 and -3 are secreted in response to LPS and cytokines (TNFα, interleukin-1 beta) and are active against Gram-positive (HBD-3) and Gram-negative (HBD-1, -2 and -3) bacteria whilst HBD-1 is upregulated by interferon-gamma (IFN-γ) 626364. We have previously demonstrated that human lung epithelial cells express Toll-like receptor four (TLR4) on their surface and that stimulation of these cells with Pseudomonas LPS results in increased HBD2 gene and protein expression 65. All three beta defensins have been identified in lung and they have been shown to act synergistically with other innate immunity proteins in bacterial killing 15. Individual beta defensins have differential antimicrobial activity. Staphylococcus aureus is resistant to killing by HBD-1 and HBD-2 but even strains of this organism that are multi-drug resistant are susceptible and sensitive to killing by HBD-3 66. We have shown previously that HBD 2 and 3 are susceptible to proteolytic cleavage by cysteinyl cathepsins that are present at elevated concentrations in the Cystic Fibrosis and COPD airways 49. New families of defensins and individual defensins are being discovered. A novel family of antimicrobial peptides termed "theta defensins" has been described in monocytes and macrophages of macaque monkeys. Theta defensins are naturally produced by a unique ligation of two truncated alpha defensins 67. Whilst they are important in their own right, β-defensins are present at much lower concentrations than lysozyme, SLPI or lactoferrin and thus their overall contribution to host defense must be taken in context 15.

Cathelicidins are a family of antimicrobial proteins found in neutrophil specific granules of which LL-37 is the only human member identified to date 96869. LL-37 is also present in certain lymphocytes, testicular tissue and airway epithelium 968. Cathelicidins are stored as inactive pro-peptides precursors and require processing to become active peptides 70. LL-37 is activated when proteinase 3 cleaves its precursor, hCAP-18. Some cathelicidin genes possess upstream DNA motifs (eg. NF-κB) predicted to convey inducibility during acute phase responses 71. LL-37 has been shown to reduce bacterial load by Pseudomonas when over-expressed in murine models 72. Furthermore it conveys improved survival following administration of lethal doses of LPS 72. LL-37 also reduced production of the pro-inflammatory cytokine TNF-α from macrophages stimulated with LPS and may be responsible for the migration of immune cells to areas of inflammation and infection 73. Protegrin, the porcine equivalent of LL-37 is currently being evaluated as an anti-inflammatory/antimicrobial agent in patients with mucositis post chemotherapy 3. LL-37 activity may be impaired in Cystic Fibrosis where anionic filaments of F-actin and DNA bind to it and inhibit its bactericidal action. Addition of the actin filament-fragmenting protein gelsolin frees LL-37 from this binding and restores antimicrobial activity 72.

BPI is a 55-kDa protein that is predominantly active against Gram-negative bacteria 274. It is stored in neutrophil primary granules and exerts its effects through three distinct mechanisms: firstly, it is directly cytotoxic via its effects on bacterial membranes; secondly, it acts as an opsonin to enhance neutrophil phagocytosis and thirdly, it can neutralise bacterial LPS 75. In common with many innate immunity proteins, BPI possesses a highly cationic N-terminal end which contains its bactericidal and endotoxin neutralising zones 7576. As with many of these defensive proteins, BPI acts synergistically with other members of the innate immune system such as cathelicidins and defensins in bacterial killing. It also acts in concert with the complement system 76. BPI is thought to have a role in down-regulating the pro-inflammatory effects of gram-negative bacteria and endotoxins in vivo 77.

Pulmonary surfactant is a lipoprotein complex that is synthesised by type II pneumocytes and by airway Clara cells. It is secreted into the epithelial lining fluid where it modulates surface tension. More recently surfactant has been shown to play a role in host defense against infection and inflammation. Surfactant proteins belong to the collagen-like-lectin or collectin family that also includes mannose binding protein bovine coglutinin and CL-43 7879. Collectins share an N-terminal collagen-like domain and a C-terminal lectin or Carbohydrate recognition domain (CRD) domain capable of binding carbohydrates in a calcium-dependent manner. These C-type lectin domains can bind oligosaccharides found on bacterial, non-encapsulated fungal as well as viral envelope surfaces 79. Surfactant consists of the four surfactant proteins 1–4 bound to phospholipids and is responsible for reducing surface tension at the air liquid interface within the alveoli in lung. Surfactant proteins are key opsonins facilitating phagocytosis of bacteria and viruses by macrophages and monocytes 80 Both SP-A and SP-D have been shown to be directly bactericidal against E.coli, while SP-A and SP-D are fungicidal against Histoplasma Capsulatum. SP-A and D deficient mice were unable to kill this fungus 81. Both SP-A and SP-D have the capacity to modulate multiple leucocyte functions 7882. The addition of SP-A to cultures of Mycoplasma. pneumoniae markedly attenuated the growth of the organism assessed by colony formation, metabolic activity, and DNA replication. The bacteriostatic effects of SP-A were reversed by dipalmitoylphosphatidylglycerol. These findings demonstrate that human SP-A can play a direct role in antibody-independent immunity to M. pneumoniae by interacting with lipid ligands expressed on the surface of the organism and implicate SP-A in the immediate host response to the bacteria 83.

Recent research has focused on the proteolytic cleavage of SP-A and D by various proteases in the lung. Proteolytic damage to surfactant protein by neutrophil elastase and cathepsin G was demonstrated in bronchoalveolar lavage fluid of cystic fibrosis patients 84. The bacterial protease, Pseudomonas aeruginosa elastase was shown to degrade SP-A and SP-D 85. Furthermore, cleavage of SP-D by this enzyme results in failure of the surfactant to bind or aggregate bacteria that are aggregated by intact SP-D. Thus, cleavage eliminates many of SP-D's normal immune functions 86.

Early animal studies showed airway mucosa secretes an enzyme known as peroxidase that was active in preventing infection of the airway87. Gerson et al subsequently demonstrated production of the biocidal compound hypothiocyanite in vitro by airway lactoperoxidase (LPO). They also showed in vivo inhibition of airway LPO in sheep leads to a significant decrease in bacterial clearance from the airways. Their data suggest that the LPO system is a major contributor to airway defenses 88. The airway LPO system may provide additional protection against viral 899091 and fungal infections 9293. LPO was subsequently demonstrated in human airways airway secretions with activity against Pseudomonas aeruginosa, Burkholderia cepacia and Haemophilus influenzae 94.

Chemokine ligand 20 (CCL20) shares similar structural and functional properties with human beta-defensins (HBDs) Airway epithelial cells have been shown to express CCL20 95. The inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) upregulate expression of this protein via the nuclear factor (NF)-κB pathway 9697. CCL20 is also produced by neutrophils 98. It has been shown to exert antimicrobial activity against a wide spectrum of mainly Gram-negative bacteria 99. Starner et al demonstrated that CF BAL contains elevated concentrations of CCL20 compared to normal BAL and that CCL20 exhibited salt-sensitive bactericidal activity 95.