© 2002 American Thoracic Society
Lactoperoxidase and Hydrogen Peroxide Metabolism in the AirwayDepartment of Cell Biology and Anatomy and Division of Pulmonary and Critical Care Medicine, University of Miami, Miami, Florida Correspondence and requests for reprints should be addressed to Gregory E. Conner, Department of Cell Biology and Anatomy, R-124, University of Miami School of Medicine, PO Box 016960, Miami, FL 33101. E-mail: gconner{at}miami.edu ABSTRACT Hydrogen peroxide (H2O2) is known to play an important role in airway homeostasis. For this reason its levels and thus its synthesis and consumption are important mechanisms for controlling airway functions. We have identified the major macromolecular consumer of H2O2 in sheep airway secretions to be lactoperoxidase (LPO), a heme peroxidase previously studied in milk and saliva. This enzyme uses H2O2 to oxidize the anion thiocyanate to an antibiotic compound that prevents growth of bacteria, fungi, and viruses. LPO was isolated from sheep airways and proved to be a major constituent comprising about 1% of the soluble protein in airway secretions. The isolated airway LPO was catalytically active and displayed the enzymatic characteristics previously described for the enzyme isolated from bovine milk. Airway LPO activity was shown to increase the rate of bacterial clearance from sheep airways. The role of this enzyme in the airway host defense strongly suggests that an active H2O2 production system exists to supply appropriate substrate for the enzyme. The identity of this H2O2 synthesis system is an important, yet unknown feature of airway oxygen radical metabolism.
Key Words: host defense • peroxidase • oxidants • antioxidants • glutathione Hydrogen peroxide (H2O2) is one of the major oxidants found in cells, tissues, and secretions. Although not the most reactive oxidant, H2O2 can be damaging and freely permeates through biologic membranes. Because H2O2 has multiple important biologic functions, local concentrations must be carefully controlled to allow correct function of the systems that depend on H2O2, while preventing unwarranted damage. Control of H2O2 levels is a complex process that involves the synthesis of its precursor, superoxide (O2-), dismutation of O2- to H2O2, and consumption of H2O2 by scavengers that include enzymes and small molecules. The relatively recent discovery of a constitutive airway peroxidase, that uses H2O2 as a substrate to generate biocidal compounds for respiratory host defense, suggests that O2- and H2O2 may be steadily produced in the airway for its use. H2O2 SCAVENGING IN THE AIRWAY Several lines of evidence suggest that H2O2 is normally present in airway secretions. First, exhaled breath condensate contains variable amounts of H2O2 (e.g., Reference 1); second, O2- is detectable in airway epithelia using morphologic methods (2); and third, airway epithelia synthesize H2O2 in vitro (e.g., Reference 3) and airway neuroepithelial cells contain nicotinamide adenine dinucleotide phosphate reduced oxidase that synthesizes O2- (e.g., Reference 4). In addition, regulation of airway H2O2 is important during allergic responses because aerosolized catalase can block allergen-induced hyper-responsiveness in sheep (5). For these reasons, we examined sheep tracheal mucus for macromolecules that might play a role in controlling airway H2O2 by acting as a scavenger. A variety of candidates for such an action were considered and included mucins and other glycoproteins by virtue of their nonspecific reaction with H2O2, catalase, or peroxidases, as well as lipids. To identify the H2O2 scavenging activity in sheep airways, tracheal secretions free of alveolar lining fluid and saliva were collected using a double-cuff endotracheal tube providing a sealed tracheal chamber that was lavaged through catheters with buffered saline. The scavenging activity of these lavages was assessed by measuring H2O2 disappearance with the phenol red assay (6) and the activity increased with increasing mucus concentration. Solvent extraction showed that the activity was independent of the presence of lipids, and chelation showed it was not due to Fe2+-catalyzed formation of OH radical by the Fenton reaction (7). The activity was heat sensitive (100°C), nondialyzable (> 10 kD), and protease sensitive, suggesting that the scavenging was mediated through a protein. Azide inhibited the scavenging activity demonstrating that it was most likely enzymatic. Both catalase and heme-containing peroxidases are inhibited by azide. However, glutathione peroxidases are not sensitive to azide, showing that glutathione peroxidases were not responsible for the observed H2O2 scavenging activity in tracheal secretions. Glutathione peroxidase and its substrate, reduced glutathione, are known to be present in high concentration in alveolar fluid and to be important in control of H2O2 in that location (e.g., References 8 and 9). Because our airway secretions were collected exclusively from the trachea, and were thus essentially free of alveolar lining fluid, these data show that large airway secretions differ significantly from alveolar fluid with regard to the mechanism of H2O2 consumption. This is consistent with the near absence of reduced glutathione in airway secretions (9). Having ruled out glutathione peroxidase, we next considered catalase and neutrophil myeloperoxidase (MPO) as potential contributors to H2O2 consumption in tracheal secretions. Catalase is not a secreted enzyme but instead is normally found in the peroxisomes inside cells—an organelle that in no way is involved in the secretory pathway (10). Thus, it was unlikely that catalase was responsible. To confirm this, anticatalase antiserum was used to detect any catalase in mucus. Only very small amounts were found and were too minor to account for the scavenging activity. In addition, anticatalase antiserum was not able to deplete the H2O2 scavenging activity by immunoprecipitation despite being able to deplete added sheep red blood cell catalase. Thus, catalase does not play an important role in scavenging H2O2 by tracheal secretions. The remaining likely candidates for enzymatic H2O2 scavenging were MPO and unidentified peroxidases such as the endogenous peroxidase activity seen by others in cytochemical studies of airway mucosa (11). Because the sheep used for collecting lavages did not have airway inflammation and were cleared of cells by centrifugation, the measured number of neutrophils in the lavage was insufficient for released MPO to account for the scavenging activity. In addition, azide was inhibitory at lower concentrations than those required for blocking MPO activity (12). These data strongly suggested that the H2O2 scavenging activity in mucus was due to the secretion of a peroxidase not previously identified in airway secretions. AIRWAY PEROXIDASE REACTIONS Mammalian peroxidases are metalloenzymes that either contain Fe (heme) such as MPO, eosinophil peroxidase (EPO) or lactoperoxidase (LPO), or selenium (Se) such as glutathione peroxidase. Because glutathione peroxidase did not appear to play a role in scavenging airway H2O2, the heme peroxidases were the most likely candidates. Heme-peroxidases consume H2O2 through a multistep process, forming first a stable intermediate (compound I), in which the heme Fe exists as FeV instead of the normal ferric state (FeIII), containing one oxygen from the H2O2. The peroxidase then reacts with halide or a pseudohalide (e.g., thiocyanate [SCN-]) to form an enzyme–hypohalite or hypothiocyanite transition state intermediate. The enzyme–substrate intermediate releases hypohalite or hypothiocyanite, which can then react with a variety of other molecules (for review see References 13 and 14). Compound I can also oxidize organic substrates (e.g., diaminobenzidine [DAB] and tetramethylbenzidine) and similar reactions are the basis of colorimetric and fluorimetric peroxidase assays. Using DAB and H2O2 to visualize the peroxidase reaction, Christensen and Hayes (15) reported peroxidase in tracheobronchial epithelium and in the cells of airway submucosal glands. Other reports have also localized peroxidase activity in epithelial cells of the airways and in nasal glands (16, 17). We made similar cytochemical observations in sheep airway mucosa, suggesting that the scavenging activity seen in mucus might reflect secretion of this peroxidase in goblet cells and submucosal glands (7). Light microscopic examination of sheep tracheal sections incubated in the presence of DAB and H2O2 showed the DAB reaction product in a subset of epithelial cells (Figure 1A) that were identified as goblet cells (Figure 1B). No reaction product was seen when the tissues were incubated with DAB in the absence of H2O2 (Figure 1C), demonstrating that H2O2 was mandatory for formation of the reaction product. The reaction conditions excluded product formation by catalase or mitochondrial enzymes and that due solely to the presence of O2-. Electron microscopy of sheep tracheal epithelium showed peroxidase reaction product in granules and lamellar structures of the secretory pathway of goblet cells in the presence of DAB and H2O2 but not in the absence of H2O2 (12). Together these studies clearly demonstrated that the activity in tracheal epithelia was due to peroxidase and not catalase and was present in the secretory granules of goblet cells and submucosal glands.
To identify the H2O2 scavenging enzyme in mucus, the activity was purified by a two-column procedure using S-Sepharose and lentil lectin Sepharose. The strategy was based on the general properties of other secreted mammalian peroxidases that have high isoelectric points (pH 8–10) and thus can be partially purified on strong cation exchangers. Based on fold purification and yield, the peroxidase was about 1% of the total protein in sheep tracheal mucus. The absorption spectrum of purified airway peroxidase exhibited the classical Soret type spectral absorption bands typical for heme-containing peroxidases. The major absorption band was found at 412 nm and this spectrum distinguishes it from MPO, which has an absorption maximum at 430 nm, and suggested that the purified peroxidase more closely resembles either LPO or EPO. The other biochemical characteristics of the purified airway peroxidase were consistent with those of LPO (enzyme activity, pH optimum, inhibitors, apparent mass [Mapp]) and inconsistent with both MPO and glutathione peroxidases (12). Final molecular identification of airway peroxidase was made by amino acid sequencing a peptide generated by cyanogen bromide (CNBr) cleavage. The sequence of the CNBr peptide was identical to bovine LPO. With the assurance that the major peroxidase activity in sheep airway secretions was a single protein, we isolated peroxidase complementary DNAs from a sheep mucosa library. We felt that a broad screen for any complementary DNA related by sequence to the family of secreted peroxidases would be informative about both the purified peroxidase and any other peroxidases potentially made by airway mucosa. Using degenerate oligonucleotides derived from conserved regions of peroxidase nucleotide and protein sequences (18), extensive polymerase chain reaction and hybridization screening of a sheep airway mucosa complementary DNA library demonstrated only complementary DNAs that encoded a protein nearly identical to bovine milk LPO and distinct from EPO and MPO. The distribution of LPO messenger RNA in the sheep respiratory tract was determined by northern blot analysis. LPO messenger RNA was present in the conducting airways but was greatly reduced in lung parenchyma. Higher levels were seen in the bronchi than in trachea, reflecting higher levels of nonepithelial tissues in the trachea. The near absence of LPO message in lung parenchyma suggests that it does not contribute to the peroxidase activity found in alveoli and previously identified by others as glutathione peroxidase (19). Thus, both biochemical and recombinant genetics approaches confirm that the major H2O2 scavenging activity in sheep airways is LPO. Human salivary peroxidase has been cloned and shown to be identical to human milk LPO (20), and our high stringency hybridization of sheep lacrimal gland complementary DNA with LPO probes indicates that lacrimal peroxidase is also likely LPO. Thus, the expression of LPO in airway, salivary, and lacrimal secretions suggests that LPO is a common host defense feature of these epithelia and perhaps of other epithelial as well (e.g., stomach and skin). That its expression appears to be constitutive on these mucosal surfaces, in contrast to the inducible expression of LPO during mammary gland milk production, suggests that LPO expression can be controlled by at least two different regulatory mechanisms. Studies of Kinbara and coworkers (21) indicate that airway peroxidase (i.e., LPO) may be regulated by still other stimuli such as bacterial adherence or bacterial products, whereas the studies of Watanabe and Harada (17) suggest that airway peroxidase is regulated by stimulation through ß-adrenergic receptors. Whether airway LPO levels can be altered in response to hormone levels as seen in mammary tissue is not clear. SCN- IN AIRWAY SECRETIONS To function, substrates for LPO must also be present in the airway lumen in addition to the secreted catalytically active LPO. H2O2 and O2-. have been detected by us and others in airways of several species including sheep (2), guinea pig (3), and humans (e.g., Reference 22). Reports of SCN- in human sputum and bronchoalveolar lavage have been conflicting and previously ascribed to saliva contamination (23). Our assays of sheep airway lavages, in which secretions undergo large dilutions during collection, demonstrated no detectable levels of SCN-. However, suctioning of airway secretions from intubated sheep, which prevents contamination with saliva, showed that undiluted secretions contained 0.16 mM SCN-. This concentration of SCN- is within the normal range of human saliva (13, 23) and is high enough to serve as a substrate for LPO (24). This demonstration of SCN- in airway secretions, together with the detection of H2O2 reported previously and the identification of airway peroxidase as LPO, shows that all components of a functional LPO enzyme system are present in airways. ROLE OF LPO IN AIRWAY HOST DEFENSE Published studies on LPO and related peroxidases in other organs and cells provide a starting point for understanding the functions of LPO in airways. LPO is the major antimicrobial agent found in milk (for review see Reference 13), and uses H2O2 to oxidize SCN-, a pseudohalide, to the biocidal compound hypothiocyanite. In addition to LPO, the formation of biocidal compounds for host defense against infection is a general function of the mammalian heme peroxidases with the exception of thyroid peroxidase. Milk LPO, MPO, and EPO have been well studied with regard to their biocidal functions (13, 25). Neutrophils undergo a respiratory burst after stimulation resulting in the production of H2O2 that is used by MPO to oxidize chloride. The product of this reaction, hypochlorite, is a potent bactericidal compound (for review see Reference 26). LPO cannot use chloride as a substrate and although hypothiocyanite produced by LPO catalysis is less potent, it is also less damaging than the hypochlorite made by MPO. Like MPO and EPO, LPO can use I- and Br- to produce OI- and OBr- that are also bactericidal though their relative availability in the airway is not known. Recent data suggest that SCN- may also be the preferred substrate for MPO as well as EPO (27, 28) and the sensitivity of bacteria to the different hypohalite products of peroxidase reactions appears to vary considerably (29). The hypothesis that the LPO system functions in vivo to maintain airway sterility was examined using experimental bacterial challenge of the sheep respiratory tract. Sheep were pretreated by aerosol with dapsone, an inhibitor of peroxidases including airway LPO (12), or treated with PBS as a control. The animals were then challenged by aerosol with Pasteurella hemolytica, a natural pathogen in sheep. After the challenge, airway fluid collected by brushing through the bronchoscope was analyzed for colony forming units. Dapsone pretreated sheep had significantly slower bacterial clearance (the number of bacteria remaining in the airway after 1 hour increased by 100-fold compared with controls) (Figure 2) .
The result of the dapsone treatment was due to inhibition of peroxidase activity and not due to depression of mucociliary velocity nor due to inhibition of MPO. The dapsone effect was reversed by adding exogenous LPO. This experiment indicated that one function of LPO is bacterial clearance from the airways. LPO's biocidal activity is well investigated (for review see References 13 and 14) and has been shown to be effective against viruses (30–32) and fungi (33, 34) as well as bacteria. We hypothesize that the LPO system is a significant contributor to the maintenance of airway sterility and that only after this first line of defense is overcome, does the organism recruit the more damaging activity of neutrophil MPO. This hypothesis is based on (1) the broad spectrum of LPO activity against infectious diseases; (2) the less damaging product of LPO catalysis whose continuous presence is more easily tolerated by epithelial cells than the hypochlorite; and (3) its apparently constitutive expression in the airway.
CONCLUSION
The observation that levels of airway LPO are high raises important questions regarding the source of the H2O2 substrate required for its activity. Although H2O2 is known to exist in airway secretions of a variety of species and to be upregulated in several airway diseases, little is known of its enzymatic source. Speculation has centered on H2O2 as a by-product of cellular metabolism or phagocyte production. In neutrophils and other phagocytes, O2- (that dismutates to H2O2) is produced by a nicotinamide adenine dinucleotide phosphate reduced oxidase system (35) specifically for the use of the peroxidases produced by those cells for host defense purposes. Both the nicotinamide adenine dinucleotide phosphate reduced oxidase and lipid-metabolizing enzymes have been implicated in O2- generation by fibroblasts and keratinocytes (36). O2- can also be produced by xanthine oxidase, lipoxygenase-like enzymes, mitochondrial respiration, cytochrome P-450 systems, and nitric oxide synthases. That LPO exists in airway secretions, and that H2O2 is a necessary substrate, suggests the existence of an airway epithelial H2O2 generating system for LPO's use, perhaps similar to that in neutrophils for MPO. The recent discovery of multiple members of the nicotinamide adenine dinucleotide phosphate reduced oxidase gene family makes attractive the possibility that airway epithelia contain an oxidase to actively produce O2- for LPO's uses. The high levels of peroxidase activity in sheep airway secretions indicate that this catalytic capability may have a major impact on metabolic events in the airway lumen. The identification of airway peroxidase as LPO and demonstration of enzyme activity indistinguishable from same species milk LPO suggested that airway peroxidase's potential functions and activities are those described previously by others for LPO including that of an antiinfective system (13, 14). For example, the peroxidase-mediated tyrosine nitration described by van der Vliet and colleagues (37) may be due to airway LPO activity rather than MPO because they have shown that nitrite, the metabolic product of nitric oxide involved in nitration, is consumed by LPO. Others have shown that LPO can use nitric oxide as a substrate and thus directly impact nitric oxide–mediated effects (38). In addition, LPO can oxidize a number of other substrates either directly or indirectly through reaction with hypothiocyanous acid (HOSCN); these include leukotrienes and nitric oxide. LPO can also catalyze the formation of disulfides and might increase mucin polymerization. Thus, in addition to its role in host defense, LPO may have other unanticipated and important functions in the metabolism of other airway constituents. Received in original form June 14, 2002; accepted in final form September 3, 2002 REFERENCES
This article has been cited by other articles:
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
