Lactoperoxidase and Hydrogen Peroxide Metabolismin the AirwayGregory E. Conner, Matthias Salathe, and Rosanna Forteza

Department of Cell Biology and Anatomy and Division of Pulmonary and Critical Care Medicine, University of Miami, Miami, Florida

Hydrogen peroxide (H2O2) is known to play an important role in catalase can block allergen-induced hyper-responsiveness inairway homeostasis. For this reason its levels and thus its synthesis sheep (5). For these reasons, we examined sheep tracheal mucusand consumption are important mechanisms for controlling airway for macromolecules that might play a role in controlling airwayfunctions. We have identified the major macromolecular consumer H2O2 by acting as a scavenger. A variety of candidates for suchof H2O2 in sheep airway secretions to be lactoperoxidase (LPO), a an action were considered and included mucins and other glyco-heme peroxidase previously studied in milk and saliva. This enzyme proteins by virtue of their nonspecific reaction with H2O2, cata-uses H2O2 to oxidize the anion thiocyanate to an antibiotic com- lase, or peroxidases, as well as lipids.pound that prevents growth of bacteria, fungi, and viruses. LPO was To identify the H2O2 scavenging activity in sheep airways,isolated from sheep airways and proved to be a major constituent

tracheal secretions free of alveolar lining fluid and saliva werecomprising about 1% of the soluble protein in airway secretions.collected using a double-cuff endotracheal tube providing aThe isolated airway LPO was catalytically active and displayed thesealed tracheal chamber that was lavaged through catheters withenzymatic characteristics previously described for the enzyme iso-buffered saline. The scavenging activity of these lavages waslated from bovine milk. Airway LPO activity was shown to increaseassessed by measuring H2O2 disappearance with the phenol redthe rate of bacterial clearance from sheep airways. The role of thisassay (6) and the activity increased with increasing mucus con-enzyme in the airway host defense strongly suggests that an activecentration. Solvent extraction showed that the activity was inde-H2O2 production system exists to supply appropriate substrate forpendent of the presence of lipids, and chelation showed it wasthe enzyme. The identity of this H2O2 synthesis system is an impor-

tant, yet unknown feature of airway oxygen radical metabolism. not due to Fe2�-catalyzed formation of OH radical by the Fentonreaction (7). The activity was heat sensitive (100�C), nondialyz-

Keywords: host defense; peroxidase; oxidants; antioxidants; gluta- able (� 10 kD), and protease sensitive, suggesting that the scav-thione enging was mediated through a protein. Azide inhibited the

scavenging activity demonstrating that it was most likely enzy-Hydrogen peroxide (H2O2) is one of the major oxidants foundmatic. Both catalase and heme-containing peroxidases are inhib-in cells, tissues, and secretions. Although not the most reac-ited by azide. However, glutathione peroxidases are not sensitivetive oxidant, H2O2 can be damaging and freely permeatesto azide, showing that glutathione peroxidases were not responsi-through biologic membranes. Because H2O2 has multiple im-ble for the observed H2O2 scavenging activity in tracheal secre-portant biologic functions, local concentrations must be care-tions. Glutathione peroxidase and its substrate, reduced glutathi-fully controlled to allow correct function of the systems thatone, are known to be present in high concentration in alveolardepend on H2O2, while preventing unwarranted damage.fluid and to be important in control of H2O2 in that locationControl of H2O2 levels is a complex process that involves the(e.g., References 8 and 9). Because our airway secretions weresynthesis of its precursor, superoxide (O2

�), dismutation ofcollected exclusively from the trachea, and were thus essentiallyO2

� to H2O2, and consumption of H2O2 by scavengers thatfree of alveolar lining fluid, these data show that large airwayinclude enzymes and small molecules. The relatively recentsecretions differ significantly from alveolar fluid with regard todiscovery of a constitutive airway peroxidase, that uses H2O2the mechanism of H2O2 consumption. This is consistent with theas a substrate to generate biocidal compounds for respiratorynear absence of reduced glutathione in airway secretions (9).host defense, suggests that O2

� and H2O2 may be steadilyHaving ruled out glutathione peroxidase, we next consideredproduced in the airway for its use.

catalase and neutrophil myeloperoxidase (MPO) as potentialcontributors to H2O2 consumption in tracheal secretions. Cata-H2O2 SCAVENGING IN THE AIRWAYlase is not a secreted enzyme but instead is normally found in

Several lines of evidence suggest that H2O2 is normally present the peroxisomes inside cells—an organelle that in no way isin airway secretions. First, exhaled breath condensate contains involved in the secretory pathway (10). Thus, it was unlikely thatvariable amounts of H2O2 (e.g., Reference 1); second, O2

� is catalase was responsible. To confirm this, anticatalase antiserumdetectable in airway epithelia using morphologic methods (2); was used to detect any catalase in mucus. Only very smalland third, airway epithelia synthesize H2O2 in vitro (e.g., Refer- amounts were found and were too minor to account for theence 3) and airway neuroepithelial cells contain nicotinamide scavenging activity. In addition, anticatalase antiserum was notadenine dinucleotide phosphate reduced oxidase that synthe- able to deplete the H2O2 scavenging activity by immunoprecipita-sizes O2

� (e.g., Reference 4). In addition, regulation of airway tion despite being able to deplete added sheep red blood cellH2O2 is important during allergic responses because aerosolized catalase. Thus, catalase does not play an important role in scav-

enging H2O2 by tracheal secretions.The remaining likely candidates for enzymatic H2O2 scaveng-

ing were MPO and unidentified peroxidases such as the endoge-(Received in original form June 14, 2002; accepted in final form September 3, 2002) nous peroxidase activity seen by others in cytochemical studiesCorrespondence and requests for reprints should be addressed to Gregory E. Conner, of airway mucosa (11). Because the sheep used for collectingDepartment of Cell Biology and Anatomy, R-124, University of Miami School of lavages did not have airway inflammation and were cleared ofMedicine, PO Box 016960, Miami, FL 33101. E-mail: gconner@miami.edu

cells by centrifugation, the measured number of neutrophils inAm J Respir Crit Care Med Vol 166. pp S57–S61, 2002

the lavage was insufficient for released MPO to account for theDOI: 10.1164/rccm.2206018Internet address: www.atsjournals.org scavenging activity. In addition, azide was inhibitory at lower

S58 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 166 2002

concentrations than those required for blocking MPO activity(12). These data strongly suggested that the H2O2 scavengingactivity in mucus was due to the secretion of a peroxidase notpreviously identified in airway secretions.

AIRWAY PEROXIDASE REACTIONS

Mammalian peroxidases are metalloenzymes that either containFe (heme) such as MPO, eosinophil peroxidase (EPO) or lacto-peroxidase (LPO), or selenium (Se) such as glutathione peroxi-dase. Because glutathione peroxidase did not appear to play arole in scavenging airway H2O2, the heme peroxidases were themost likely candidates.

Heme-peroxidases consume H2O2 through a multistep pro-cess, forming first a stable intermediate (compound I), in whichthe heme Fe exists as FeV instead of the normal ferric state(FeIII), containing one oxygen from the H2O2. The peroxidasethen reacts with halide or a pseudohalide (e.g., thiocyanate[SCN�]) to form an enzyme–hypohalite or hypothiocyanite tran-sition state intermediate. The enzyme–substrate intermediatereleases hypohalite or hypothiocyanite, which can then reactwith a variety of other molecules (for review see References 13and 14). Compound I can also oxidize organic substrates (e.g.,diaminobenzidine [DAB] and tetramethylbenzidine) and similarreactions are the basis of colorimetric and fluorimetric peroxi-dase assays.

Using DAB and H2O2 to visualize the peroxidase reaction,Christensen and Hayes (15) reported peroxidase in tracheobron-chial epithelium and in the cells of airway submucosal glands.Other reports have also localized peroxidase activity in epithelialcells of the airways and in nasal glands (16, 17). We made similarcytochemical observations in sheep airway mucosa, suggestingthat the scavenging activity seen in mucus might reflect secretionof this peroxidase in goblet cells and submucosal glands (7). Lightmicroscopic examination of sheep tracheal sections incubated inthe presence of DAB and H2O2 showed the DAB reaction prod-

Figure 1. Sheep trachea was excised, fixed in aldehyde, and incubateduct in a subset of epithelial cells (Figure 1A) that were identifiedin DAB in the presence (panel A and B ) or absence (panel C) of H2O2. Afteras goblet cells (Figure 1B). No reaction product was seen whenincubation samples were postfixed in osmium tetraoxide, embedded inthe tissues were incubated with DAB in the absence of H2O2plastic resin, and sectioned for direct visualization with interference(Figure 1C), demonstrating that H2O2 was mandatory for forma-contrast microscope (panels A and C ) or were stained with Mallory’stion of the reaction product. The reaction conditions excludedstain (panel B) for identification of goblet cells. Bars are equal to 15 �m.product formation by catalase or mitochondrial enzymes and(Reprinted with permission from Reference 12.)

that due solely to the presence of O2�. Electron microscopy of

sheep tracheal epithelium showed peroxidase reaction productin granules and lamellar structures of the secretory pathway ofgoblet cells in the presence of DAB and H2O2 but not in the [Mapp]) and inconsistent with both MPO and glutathione peroxi-absence of H2O2 (12). Together these studies clearly demon- dases (12). Final molecular identification of airway peroxidasestrated that the activity in tracheal epithelia was due to peroxi- was made by amino acid sequencing a peptide generated bydase and not catalase and was present in the secretory granules cyanogen bromide (CNBr) cleavage. The sequence of the CNBrof goblet cells and submucosal glands. peptide was identical to bovine LPO.

To identify the H2O2 scavenging enzyme in mucus, the activity With the assurance that the major peroxidase activity in sheepwas purified by a two-column procedure using S-Sepharose and airway secretions was a single protein, we isolated peroxidaselentil lectin Sepharose. The strategy was based on the general complementary DNAs from a sheep mucosa library. We feltproperties of other secreted mammalian peroxidases that have that a broad screen for any complementary DNA related byhigh isoelectric points (pH 8–10) and thus can be partially puri- sequence to the family of secreted peroxidases would be informa-fied on strong cation exchangers. Based on fold purification and tive about both the purified peroxidase and any other peroxi-yield, the peroxidase was about 1% of the total protein in sheep dases potentially made by airway mucosa. Using degeneratetracheal mucus. The absorption spectrum of purified airway per- oligonucleotides derived from conserved regions of peroxidaseoxidase exhibited the classical Soret type spectral absorption nucleotide and protein sequences (18), extensive polymerasebands typical for heme-containing peroxidases. The major ab- chain reaction and hybridization screening of a sheep airwaysorption band was found at 412 nm and this spectrum distin- mucosa complementary DNA library demonstrated only com-guishes it from MPO, which has an absorption maximum at 430 plementary DNAs that encoded a protein nearly identical tonm, and suggested that the purified peroxidase more closely bovine milk LPO and distinct from EPO and MPO.resembles either LPO or EPO. The other biochemical character- The distribution of LPO messenger RNA in the sheep respira-istics of the purified airway peroxidase were consistent with those tory tract was determined by northern blot analysis. LPO mes-

senger RNA was present in the conducting airways but wasof LPO (enzyme activity, pH optimum, inhibitors, apparent mass

Conner, Salathe, and Forteza: Airway Lactoperoxidase S59

greatly reduced in lung parenchyma. Higher levels were seen inthe bronchi than in trachea, reflecting higher levels of nonepithe-lial tissues in the trachea. The near absence of LPO message inlung parenchyma suggests that it does not contribute to theperoxidase activity found in alveoli and previously identified byothers as glutathione peroxidase (19). Thus, both biochemicaland recombinant genetics approaches confirm that the majorH2O2 scavenging activity in sheep airways is LPO.

Human salivary peroxidase has been cloned and shown tobe identical to human milk LPO (20), and our high stringencyhybridization of sheep lacrimal gland complementary DNA withLPO probes indicates that lacrimal peroxidase is also likely LPO.Thus, the expression of LPO in airway, salivary, and lacrimalsecretions suggests that LPO is a common host defense featureof these epithelia and perhaps of other epithelial as well (e.g.,stomach and skin). That its expression appears to be constitutiveon these mucosal surfaces, in contrast to the inducible expressionof LPO during mammary gland milk production, suggests thatLPO expression can be controlled by at least two different regu-latory mechanisms. Studies of Kinbara and coworkers (21) indi-cate that airway peroxidase (i.e., LPO) may be regulated by stillother stimuli such as bacterial adherence or bacterial products,whereas the studies of Watanabe and Harada (17) suggest that

Figure 2. Experimental bacterial challenge of sheep airways. Sheepairway peroxidase is regulated by stimulation through �-adrener-were either pretreated with 3 ml of 10�3 M dapsone in PBS (filled squares)gic receptors. Whether airway LPO levels can be altered in re-or with PBS alone (filled circles). Control sheep (n � 6) and pretreatedsponse to hormone levels as seen in mammary tissue is not clear.animals (n � 4) were challenged with 106–109 P. hemolytica (ATCC29698) in 3 ml of PBS. Immediately after, 30 minutes, 1 hour, and 3SCN� IN AIRWAY SECRETIONS hours, after challenge, samples of tracheal surface fluid were collectedand quantitative bacterial cultures used to determine colony-formingTo function, substrates for LPO must also be present in theunits. Values, normalized to the initial value after challenge, are plottedairway lumen in addition to the secreted catalytically active LPO.as means � SE. Control sheep showed rapid clearance of inhaled bacte-H2O2 and O2

�. have been detected by us and others in airwaysria. Dapsone treatment significantly inhibited bacterial clearance at 60of several species including sheep (2), guinea pig (3), and humansand 180 minutes (both p 0.05). Treatment with 5 mg bovine milk(e.g., Reference 22). Reports of SCN� in human sputum andLPO reversed the impaired clearance in dapsone treated animals (filled,bronchoalveolar lavage have been conflicting and previouslyinverted triangles). As an additional control, treatment with 5 mg ofascribed to saliva contamination (23). Our assays of sheep airwayLPO alone did not significantly improve clearance of bacteria (filled

lavages, in which secretions undergo large dilutions during col- triangles) Asterisks indicate p values less than 0.05. (Reprinted withlection, demonstrated no detectable levels of SCN�. However, permission from Reference 18.)suctioning of airway secretions from intubated sheep, which pre-vents contamination with saliva, showed that undiluted secre-tions contained 0.16 mM SCN�. This concentration of SCN� iswithin the normal range of human saliva (13, 23) and is high hypochlorite made by MPO. Like MPO and EPO, LPO can useenough to serve as a substrate for LPO (24). This demonstration I� and Br� to produce OI� and OBr� that are also bactericidalof SCN� in airway secretions, together with the detection of though their relative availability in the airway is not known.H2O2 reported previously and the identification of airway peroxi- Recent data suggest that SCN� may also be the preferred sub-dase as LPO, shows that all components of a functional LPO strate for MPO as well as EPO (27, 28) and the sensitivity

of bacteria to the different hypohalite products of peroxidaseenzyme system are present in airways.reactions appears to vary considerably (29).

The hypothesis that the LPO system functions in vivo toROLE OF LPO IN AIRWAY HOST DEFENSEmaintain airway sterility was examined using experimental bacte-

Published studies on LPO and related peroxidases in other or- rial challenge of the sheep respiratory tract. Sheep were pre-gans and cells provide a starting point for understanding the treated by aerosol with dapsone, an inhibitor of peroxidasesfunctions of LPO in airways. LPO is the major antimicrobial including airway LPO (12), or treated with PBS as a control.agent found in milk (for review see Reference 13), and uses The animals were then challenged by aerosol with PasteurellaH2O2 to oxidize SCN�, a pseudohalide, to the biocidal compound hemolytica, a natural pathogen in sheep. After the challenge,hypothiocyanite. In addition to LPO, the formation of biocidal airway fluid collected by brushing through the bronchoscopecompounds for host defense against infection is a general func- was analyzed for colony forming units. Dapsone pretreatedtion of the mammalian heme peroxidases with the exception of sheep had significantly slower bacterial clearance (the numberthyroid peroxidase. Milk LPO, MPO, and EPO have been well of bacteria remaining in the airway after 1 hour increased bystudied with regard to their biocidal functions (13, 25). Neutro- 100-fold compared with controls) (Figure 2).phils undergo a respiratory burst after stimulation resulting in The result of the dapsone treatment was due to inhibition ofthe production of H2O2 that is used by MPO to oxidize chloride. peroxidase activity and not due to depression of mucociliaryThe product of this reaction, hypochlorite, is a potent bacteri- velocity nor due to inhibition of MPO. The dapsone effect wascidal compound (for review see Reference 26). LPO cannot use reversed by adding exogenous LPO. This experiment indicatedchloride as a substrate and although hypothiocyanite produced that one function of LPO is bacterial clearance from the airways.

LPO’s biocidal activity is well investigated (for review see Refer-by LPO catalysis is less potent, it is also less damaging than the

S60 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 166 2002

H2O2 generating system for LPO’s use, perhaps similar to thatin neutrophils for MPO. The recent discovery of multiple mem-bers of the nicotinamide adenine dinucleotide phosphate re-duced oxidase gene family makes attractive the possibility thatairway epithelia contain an oxidase to actively produce O2

� forLPO’s uses.

The high levels of peroxidase activity in sheep airway secre-tions indicate that this catalytic capability may have a majorimpact on metabolic events in the airway lumen. The identifica-tion of airway peroxidase as LPO and demonstration of enzymeactivity indistinguishable from same species milk LPO suggestedthat airway peroxidase’s potential functions and activities arethose described previously by others for LPO including that ofan antiinfective system (13, 14). For example, the peroxidase-

Figure 3. Components of the LPO antibacterial system. LPO is synthe- mediated tyrosine nitration described by van der Vliet and col-sized by serous cells of the submucosal glands and by goblet cells. SCN�

leagues (37) may be due to airway LPO activity rather than MPOis transported from the plasma compartment and concentrated in the because they have shown that nitrite, the metabolic product oflumen of the airway. H2O2 is made by epithelial cells and perhaps other nitric oxide involved in nitration, is consumed by LPO. Othersresident cells of the airway. The three components are mixed in the

have shown that LPO can use nitric oxide as a substrate andairway lumen to form a functional LPO system that consumes the H2O2 thus directly impact nitric oxide–mediated effects (38). In addi-and produces the biocidal compound hypothiocyanite OSCN.tion, LPO can oxidize a number of other substrates either di-rectly or indirectly through reaction with hypothiocyanous acid(HOSCN); these include leukotrienes and nitric oxide. LPOcan also catalyze the formation of disulfides and might increaseences 13 and 14) and has been shown to be effective againstmucin polymerization. Thus, in addition to its role in host de-viruses (30–32) and fungi (33, 34) as well as bacteria. We hypothe-fense, LPO may have other unanticipated and important func-size that the LPO system is a significant contributor to the main-tions in the metabolism of other airway constituents.tenance of airway sterility and that only after this first line of

defense is overcome, does the organism recruit the more damag-Referencesing activity of neutrophil MPO. This hypothesis is based on (1)

the broad spectrum of LPO activity against infectious diseases; 1. Jobsis Q, Raatgeep HC, Schellekens SL, Hop WC, Hermans PW, deJongste JC. Hydrogen peroxide in exhaled air of healthy children:(2) the less damaging product of LPO catalysis whose continuousreference values. Eur Respir J 1998;12:483–485.presence is more easily tolerated by epithelial cells than the

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