Innate Airway Defenses

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Airway secretions provide critical elements of the innate, i.e., nonantigen specific, defense of the respiratory tract against inhaled microorganisms. Such a system is required to maintain sterility of the lungs in the settings of constant exposure to inhaled debris and potential pathogens and of their proximity to the oral and nasopharyngeal cavities, both of which are colonized with a wide range of bacteria. Structural aspects of the bronchopulmonary tree, especially frequent branchpoints and changes in orientation of bronchi, favor deposition of inhaled particles onto airway surfaces (1). These are removed from the upper two-thirds of the airways by retrograde propulsion via the mucociliary escalator and cough reflex. Secreted mucus and the glycocalyx, which directly binds microorganisms, form a physical barrier protecting the epithelial surface. In addition to these structural and physical defenses, innate immunity in the lung relies upon a complex mixture of antibacterial enzymes, peptide antimicrobials, and professional phagocytic cells. The role of these cells, largely airway macrophages and polymorphonuclear leukocytes, as well as their regulatory cytokine network, has been reviewed in detail elsewhere (2). In the event that bacteria do contact the epithelial surface, nuclear factor-κB–dependent pathways generate both mucin (3) and interleukin-8 (4) expression with subsequent recruitment of granulocytes to the airway. This pathway can be activated by a variety of pulmonary pathogens through a calcium-dependent mitogen-activated protein kinase–signaling cascade (5). However, it is clear that normal airway defenses are remarkably efficient at preventing inhaled bacteria from reaching and activating epithelial cytokine responses. The recent discovery of human epithelial defensins (6) and their potential role in the pathogenesis of infection in cystic fibrosis has generated renewed interest in the composition and antimicrobial activity of airway surface fluid. Other components of respiratory tract secretions, which include immunoglobulins, complement factors, lipopolysaccharide- binding protein, lysozyme, lactoferrin, and others, exert antimicrobial activities either via direct mechanisms or by facilitating interactions between phagocytes and pathogens (Figure 1). In this issue, Gerson and colleagues demonstrate for the first time the antibacterial properties of lactoperoxidase (LPO), a major component of the antimicrobial armamentarium in the airway, thus defining another factor in the complex system of innate immunity in the airway surface fluid (7).

Fig. 1. Overview of innate defense mechanisms in the airway.

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Antimicrobial Activity of Peroxidases

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LPO is present in a variety of secretions including tears, saliva, and milk and, as is demonstrated in this issue, airway surface fluid. LPO is a member of the family of mammalian peroxidases, which utilize hydrogen peroxide to oxidize thiocyanate to hypothiocyanate (Figure 2), and are active in a variety of anatomic sites (8). Other members of this group include myeloperoxidase (MPO) (present in neutrophils and monocytes), eosinophil peroxidase, and thyroid peroxidase. Studies of sequence homology have demonstrated the close evolutionary relationship among members of this family (9). The antibacterial potential of peroxidases was first described by Agner in 1941 (10) who noted that the “verdoperoxidase” (later renamed myeloperoxidase) from leukocytes was capable of inactivating diphtheria toxin. The complete LPO system (enzyme plus substrates) was characterized in milk in 1963 (11). Since then a broad spectrum of antimicrobial activity has been described for the peroxidase system with activity against gram-positive and gram-negative bacteria, as well as viruses and fungi (12). Peroxidases are important for inhibition of formation of dental caries, control of microorganisms in milk from lactating animals, and cell-mediated pathogen killing.

Fig. 2. Lactoperoxidase catalyses the reaction of hydrogen peroxide with thiocyanate, producing hypothiocyanate.

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The relative antimicrobial activity of peroxidases depends on the ion acting as the electron donor. Both LPO and MPO can utilize iodide or thiocyanate as substrates, and MPO can use chloride as well. The generation of hypochlorite by MPO leads to potent bacterial killing, usually within the setting of the phagolysosome. However, hypochlorite can be toxic to host tissue, including epithelial cells. Thiocyanate, which is necessary for the antibacterial activity of LPO, is present in significant amounts in saliva, milk, and airway secretions (13). The other substrate utilized by LPO, hydrogen peroxide, can be generated by resident bacteria in sites such as the oral cavity or generated in situ by cells in the respiratory tract. While H₂O₂ can be bacteriocidal or bacteriostatic, experimental evidence indicates that inhibition of bacterial growth is far more efficient in the presence of the complete LPO system. The product of the reaction catalyzed by LPO is hypothiocyanate, a weak anion in equilibrium with HOSCN. HOSCN is thought to be the mediator of bacterial killing, as it is cell-permeable and can inhibit glycolysis as well as nicotinamide adenine dinucleotide (NADH)/nicotinamide adenine dinucleotide phosphate (NADPH)–dependent reactions in bacteria (13). Bacterial resistance to LPO-mediated killing can occur via a “reversal enzyme,” which inactivates HOSCN, pigment generation, or anaerobic metabolism.

LPO deficiency in humans has not been well characterized, but it is known that genetic lack of MPO activity leads to an increased incidence of malignant tumors but not an appreciable susceptibility to infectious diseases (14). This underscores the importance of multiple, overlapping systems for pathogen surveillance. In fact, the one subset of MPO-deficient patients with a documented increase in infections is those with concomitant diabetes mellitus. LPO functions well in concert with other biocidal systems. It has synergistic activity with secretory immunoglobulins against oral streptococcal species (15) and increases activity of H₂O₂ against several species of oral pathogens (16).

Peroxidases in Airway Defense

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The study by Gerson and associates is an important continuation of the work in this field. The action of peroxidases in the airway is not a new idea, as granulocytes, which act as a secondary line of defense against pulmonary pathogens, utilize MPO as a part of their antimicrobial defenses. In addition, nearly two decades ago Christensen and coworkers histochemically demonstrated a secreted peroxidase in rodent airways (17), but the significance of this finding in relation to airway protection was not appreciated. The observation that sheep airway secretions could potently scavenge hydrogen peroxide was initially thought to serve a distinct purpose (18), namely to protect the respiratory tree from potentially harmful reactive oxygen species. The peroxidase involved in this reaction, originally referred to as airway peroxidase (APO), was found to be abundant (about 1% of total protein) in airway secretions and distinct from a known selenium-dependent glutathione peroxidase found in airway secretions. The study presented in this issue demonstrates by purification of protein from the airway cloning of complementary DNA from a sheep tracheal library and by DNA and amino-acid sequence comparison that the previously described airway peroxidase is, in fact, LPO. In the present study by Gerson and colleagues, the availability of sufficient thiocyanate to act as substrate in the airway was demonstrated, as was the presence of hypothiocyanate, an indicator of enzymatic activity. The generation of hypothiocyanate by airway LPO was inhibited using aerosolized dapsone, and increased susceptibility to a pulmonary challenge with Pasteurella hemolytica was shown. Although the activity of pulmonary LPO against other pathogens remains to be established, data from other systems, such as the activity of LPO against Pseudomonas species in milk, are entirely consistent with these results. The function of LPO in the airway is logical; LPO generates the weak oxidizing agent hypothiocyanate but not the more toxic hypochlorite species produced by MPO. Thus, it may provide less damaging antimicrobial activity than the recruitment of granulocytes to decontaminate the airway mucosa.

Future Directions

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The addition of a single enzyme to the coincident activities of lysozyme, defensins, immunoglobulins, and a host of other factors does not significantly change current concepts regarding the pathogenesis of airway infection. However, several factors specific to LPO indicate that it is likely to be especially important in the maintenance of airway sterility. By sheer bulk alone (roughly 1% of total protein from airway lavage), it is easy to imagine that LPO might have important activity in the respiratory tree. Presence in such a significant amount, in addition to its known propensity for acting in concert with other antibacterial systems, makes LPO a potentially significant factor in airway defense. Its localization to the bronchi, with reduced amounts present in the trachea and at the epithelial surface, is consistent with an antimicrobial system that functions mainly on deposited pathogens. In addition, the correlation of LPO inhibition and decreased ability to clear inspired bacteria is intriguing. It is possible that disease states resulting in chronic bacterial contamination of airways, such as cystic fibrosis or bronchiectasis, might be affected by derangements of the LPO system as a part of their pathogenesis, either through lack of production, direct inactivation, or changes in substrate availability. The importance of the broad antimicrobial activity of LPO at other sites, such as milk and saliva, is well described. Further analysis of LPO in the lung, including demonstration of activity in other species, better characterization of the antimicrobial spectrum against human pathogens, and direct evidence (i.e., using knockout or antisense technology) of the link between LPO inhibition and infection are certainly indicated.