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Published ahead of print on March 6, 2003, doi:10.1165/rcmb.2002-0152OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 206-212, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0152OC

Lactoperoxidase and Human Airway Host Defense

Corinne Wijkstrom-Frei, Souheil El-Chemaly, Radia Ali-Rachedi, Cynthia Gerson, Miguel A. Cobas, Rosanna Forteza, Matthias Salathe and Gregory E. Conner

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

Address correspondence to: Gregory E. Conner, Ph.D., University of Miami School of Medicine, P.O. Box 016960 (R124), Miami, FL 33101. E-mail: gconner{at}miami.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The lactoperoxidase (LPO) antibiotic system is a well-characterized component of mammary and salivary gland secretions. Because LPO has been shown to function in ovine airways, human airway tissue and secretions were examined for the presence of LPO and its substrate, the anion thiocyanate (SCN-). In addition, human airway secretions were tested for LPO-mediated antibacterial activity, and LPO's activity was assessed against some human airway pathogens. The data showed that normal human airway secretions contained LPO enzyme activity (0.65 ± 0.09 µg/mg secreted protein; n = 17), and Western blots of secretions demonstrated bands of the expected sizes for LPO. LPO mRNA was detected in trachea by sequencing PCR-amplified cDNA. SCN-, LPO's substrate, was present in undiluted airway secretions at concentrations sufficient for LPO catalysis (0.46 ± 0.19 mM; n = 8), and diluted secretions contained antibacterial activity with LPO-like properties. Immunocytochemistry localized LPO to submucosal glands in human bronchi. Finally, as expected based on the known antibacterial spectrum of the LPO system, airway secretions showed LPO-dependent activity against Pseudomonas aeruginosa. In addition, the airway LPO system was shown to be effective against Burkholderia cepacia and Haemophilus influenzae. Thus, a functional LPO system exists in human airways and may contribute to airway host defense against infection.

Abbreviations: lactoperoxidase, LPO • myeloperoxidase, MPO • phosphate-buffered saline, PBS • tetramethylbenzidine, TMB


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Inhalation of pathogens is the major route for infections of the respiratory tract. It has been estimated that an individual in an urban environment may inhale between 104–105 bacteria per day. Thus, ready and potent airway host defenses are important for protection against airway colonization and infection, a fact supported by diseases that result from failing airway host defense such as primary ciliary dyskinesia, immunoglobulin deficiency, and cystic fibrosis. The variety of pathogenic mechanisms encountered in these diseases suggests that airway sterility is not maintained by a single system but instead points toward a complex and multi-factored airway host defense. Successful defense relies on mucociliary clearance, epithelial cooperation with the adaptive immune system, secretion of epithelial defense molecules, and many other mechanisms. The diversity of approaches used by the airways to prevent infection may reflect the variety of infectious agents that can challenge the respiratory tract through inhalation. How the diverse epithelial activities interact to form a complete airway host defense is still not completely understood. For example, the host defense defect(s) in cystic fibrosis are still not fully understood, and a significant portion of bronchiectasis remains idiopathic.

Epithelial cell secretion of macromolecules that are effective against bacterial infection is clearly a key feature of innate airway host defense. These macromolecules include lysozyme (1–4), defensin (5–8) and cathelicidin peptides (9), lactoferrin (1, 10, 11), and secreted leukocyte protease inhibitor (1, 12, 13). In addition to these proteins, we showed that ovine airway peroxidase is lactoperoxidase (LPO) and that inhalation of an LPO inhibitor delays clearance of bacteria from sheep airways (14). Airway peroxidase found in airway secretions of other species (15, 16) might also be LPO.

The LPO antibiotic system is very well explored in other organ systems. It works to preserve milk sterility (17) and contributes to antibiotic activity of saliva (18) in several species. LPO uses H2O2 to catalyze oxidation of the anion SCN- to the antibiotic HOSCN (19).


The LPO system is known to be effective against staphylococci (20), streptococci, E. coli, and pseudomonads (20–22), all organisms that are significant respiratory pathogens. Because the LPO host defense system was important in bacterial clearance from sheep airways, and because it is known to be effective against some pathogens of the human respiratory tract, we examined human airways for the presence of a functional LPO host defense system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Unless noted otherwise, all reagents were purchased from Sigma Chemical (St. Louis, MO).

Collection of Secretions
All studies of human secretions were conducted according to local IRB-approved protocols.

Diluted human airway secretions were obtained from intubated adult patients undergoing ambulatory outpatient elective surgery. Patients were selected for not having respiratory disease. Saline (3 ml) was injected into the tracheal tube shortly after intubation and immediately suctioned back into a trap. Recovered secretions were spun at 16,000 rpm for 20 min at 4°C. The supernatant was aliquoted and stored at –80°C for later analysis.

Undiluted secretions were obtained from acutely (< 24 h) intubated adult patients in the medical ICU by direct suctioning of secretions accumulated at the tip of endotracheal tubes. These patients were mechanically ventilated for reasons other than respiratory disease and had no clinical signs of respiratory infection. Secretions were typically very viscous and were cleared by ultracentrifugation to generate a soluble phase (100,000 x g, 30 min), aliquoted, and stored at –80°C.

Western Blotting and Immunocytochemistry
Anti-LPO antiserum was prepared by immunizing rabbits with chromatographically purified sheep airway LPO (14) that was further enriched by excision and elution from SDS gels (23). Affinity purification of LPO-specific antibodies was performed using bovine milk LPO coupled to CNBr-activated Sepharose.

Proteins in secretions were concentrated by 80% acetone precipitation, and electrophoresed on 8% acrylamide SDS gels as described previously (14). Gels were transferred to Immobilon-P paper (Millipore Corp., Bedford, MA) and blocked with 5% bovine serum albumin in phosphate-buffered saline (PBS) containing 0.2% Tween-20 for 1 h at room temperature. Anti-LPO antibody was diluted to 0.3 µg/ml in normal human serum. As a specificity control, antibodies were preincubated with 120 µg/ml bovine milk LPO before application to blots. Second antibody was affinity purified goat anti-rabbit IgG coupled to alkaline phosphatase (Kirkegaard and Perry Laboratories, Gaithersburg, MD).

Rings of bronchi were collected from human lungs obtained from the Life Alliance Organ Recovery Agency at the University of Miami according to IRB approved protocols, and then fixed in 4% formaldehyde and embedded in paraffin. Sections were rehydrated and treated with 3% H2O2 for 30 min to inactivate endogenous peroxidase activity. Sections were then blocked with goat serum for 2 h, incubated with antisera (1:100) overnight at 4 C°, followed by anti rabbit IgG antibodies coupled to HRP for 1 h. Immunoreactivity was visualized using diaminobenzidine as a substrate.

Biochemical Assays
Thiocyanate was assayed using the ferric nitrate and nitric acid procedure (24). LPO activity was measured by oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence or absence of 0.1 mM dapsone to distinguish LPO from myeloperoxidase (MPO) (25, 26), because 90% of the LPO activity and only 5% of the MPO activity is sensitive to dapsone at this pH. The assay conditions were 50 mM sodium acetate pH 5.2, 0.15 mM H2O2, 1.3 mM TMB at 37°C for 30 min. After the reaction was stopped by adding HCl to 400 mM, product was measured by changes in absorbance at 405 nm. The concentration of active LPO was determined by comparison to a standard curve using bovine milk LPO.

PCR
Human tracheal mRNA was purchased from Clontech (Palo Alto, CA). Double-stranded cDNA was made using the SuperScript kit from Life Technologies (Bethesda, MD) and size fractionated. After directional cloning into pSPORT1, transformation into E. coli DH10{alpha} gave 5 x 105 independent colonies. Degenerate oligonucleotides were designed from conserved regions of mammalian heme peroxidases (14) and were 5'-GAGCACAACCGNCTGGCC-3' and 5'-GTTYTCCCACCARAACC-3'. Annealing temperature was 52°C.

Assay of Antibacterial Activity
Pseudomonas aeruginosa (ATCC 27,853) and Burkholderia cepacia (ATCC 17,616) were grown in LB broth. Haemophilus influenzae (ATCC 8,149) was grown in brain heart infusion broth. Cultures were grown overnight at 37°C in a rotary shaker and bacteria were collected in the stationary phase of growth and further diluted in LB broth to 5–6 x 104/ml. After adding glycerol to 15%, aliquots of bacteria were stored at –80°C. Exponentially growing bacteria have been reported to be more sensitive to LPO, and use of cells from stationary phase cultures are therefore expected to be more resistant to LPO (27). Thus, frozen aliquots of bacteria were used to provide easily reproducible numbers of cells for the assays with the expectation that exponentially growing cells are equally or more sensitive to the LPO system.

Antibacterial activity was assayed using 560 µl of sample, pH adjusted to 5.7, 6.2, or 6.8, and containing 2,400–3,600 bacteria. Growth of bacteria was not affected by addition of LPO (<= 6.5 µg/ml), H2O2 (<= 10-5 M), or SCN- (<= 5 x 10-4 M) singly or in pairs. However, addition of all three reconstituted a functional LPO system that was bactericidal. Mixtures were sampled immediately and 4 h after the start of incubation at room temperature. Colony-forming units (cfu) were determined by plating 50 µl in triplicate on Luria-Bertani or brain heart infusion agar followed by growth overnight at 37°C. Antibacterial activity was expressed as a ratio of cfu after 4 h to the starting cfu in the sample to control for slight differences in the starting number of bacteria. In some experiments, H2O2 consumption was measured during the incubations using the phenol red assay (28), and additional H2O2 was added to restore the concentration to 10-5 M. Assays were performed at room temperature to slow the LPO reaction and bacterial growth that allowed easier detection of changes in cell viability.

Statistics
Mean relative cfu were compared using the unpaired Student's t test. To compare more than two groups ANOVA followed by the Tukey-Kramer honestly significant difference test was used. A P value of < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPO System in Human Airways
We have previously shown that the LPO system is important for maintaining airway sterility in sheep (14). To determine whether a complete LPO system was also present in the human respiratory tract, we examined airways and airway secretions for LPO and its required substrate SCN-. The presence of H2O2, the other required component of the LPO system, is well documented in human airways (29–34). Airway secretions were collected, with negligible contamination by saliva (that also contains LPO), by instillation of saline into cuffed endotracheal tubes of intubated surgical patients and immediate suctioning into traps. Assays (25, 26) of these secretions were conducted in the presence and absence of 10-4 M dapsone, which inhibits 90% of LPO but only 5% MPO activity under the assay conditions (25, 26). The assays demonstrated peroxidase activity that was 82 ± 3% (mean ± SE, n = 17) sensitive to dapsone. Based on the relative dapsone sensitivity of LPO and MPO in this assay and the assumption that all of the secreted peroxidase activity is either LPO or MPO, 91% of the peroxidase activity, measured in the absence of dapsone, is LPO and not MPO (25, 26). Thus, using bovine LPO as a standard, the secretions contained 0.65 ± 0.09 µg LPO/mg secreted protein (mean ± SE, n = 17). The average total protein concentration of the secretions was 0.35 ± 0.03 mg/ml, suggesting that the secretions were diluted, by the saline used for collection, between 15- and 50-fold based on an estimate of 10 mg/ml in airway secretions in vivo. The LPO concentrations in airway secretions are therefore expected to be between 3 and 12 µg/ml. Western blot analysis demonstrated the presence of anti-LPO immunoreactive bands (Figure 1) , equivalent in MWapp to that reported for human milk (35) and salivary LPO (36), and thus confirmed the presence of LPO in secretions.



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  		Figure 1. LPO in human airways. Aliquots of seven different secretion samples equivalent to 100 ng of LPO as determined by the TMB assay and 30 ng of bovine milk LPO, were electrophoresed on two separate SDS gels and electro-blotted. Incubation of the blot with anti-LPO antibodies followed by goat anti-rabbit IgG coupled to alkaline phosphatase demonstrated two immunoreactive bands in human airway secretions (A and B). The number and size of the anti-LPO reactive bands in the human secretions was similar to that reported for LPO in human milk (35, 36), where two different forms of LPO were reported that may differ in extent of glycosylation (35). In contrast, only a single form is observed in bovine and ovine milk (14). Pre-incubation of the primary antibodies with excess soluble bovine LPO blocked detection of the same bands as well as bovine LPO (C). A Coomassie-stained gel of the same samples is shown for comparison along with a larger amount of bovine LPO (1 µg) (D). Molecular weight standards were Bio-Rad (Cambridge, MA) wide-range prestained markers.

 
To demonstrate that the LPO detected by enzymatic assays and Western blots was made by the airway, human tracheal mRNA was used to construct a cDNA library. Two degenerate oligonucleotides complementary to conserved regions of mammalian heme peroxidases (14) were used to screen the library by PCR and identified a partial cDNA (GenBank: AF027971) identical in nucleotide sequence to human salivary (GenBank: U39573) and milk LPO (GenBank: M58151). Immunocytochemistry confirmed the localization of LPO to bronchial submucosal glands (Figure 2) . This suggested that the previous endogenous airway peroxidase activity shown by cytochemistry in airways of sheep (26) and other animals (37) was due to endogenous production of LPO as well. Together, these data suggested that human airways synthesized and secreted enzymatically active LPO into the airway lumen.



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  		Figure 2. Human Airway LPO in submucosal glands. Human bronchial sections were stained as described in MATERIALS AND METHODS. A shows a section stained with rabbit anti-sheep airway LPO antiserum. B shows an adjacent section stained with preimmune serum obtained from the same rabbit. A subset of cells in the submucosal glands were specifically stained indicating the intracellular presence of LPO. Bar represents 50 µm.

 
To function as an antibiotic system, LPO requires SCN- in addition to H2O2 as a substrate. SCN- has not been conclusively demonstrated in human airways before (38). Because the large dilutions associated with bronchoalveolar lavage in sheep resulted in false negative [SCN-] (14), we measured SCN- in human tracheo-bronchial secretions from intubated patients, which were collected without instillation of saline and thus without any dilution. SCN- assays (24) revealed the presence of 0.46 ± 0.19 mM SCN- (mean ± SE, n = 8). These were adult patients, who were intubated using an endotracheal tube having an expanded cuff, thus preventing salivary secretions from entering the airways. This SCN- concentration was within the ranges reported in saliva and gastric secretions, suggesting that saliva contamination could not solely account for the measured SCN-. The measured [SCN-] was adequate to serve as a substrate for LPO to generate antibacterial levels of OSCN- (39–41). Thus, together with the presence of LPO and H2O2 (29–34), an intact LPO antibiotic system is expressed in human airways.

Antibacterial Activity of Human Airway LPO
The enzymatic and antibacterial activity of LPO in human salivary secretions has been extensively studied (e.g., 18, 42). The activity of the system increases with decreasing pH, and is dependent on the concentrations of SCN- and H2O2 (41). To demonstrate that the LPO system has antibacterial functions in human airways, we tested tracheobronchial secretions collected by saline instillation from patients who were undergoing outpatient elective surgeries. These secretions provided more material for analysis than undiluted secretions collected by direct suctioning. Although we measured LPO activity in secretions collected by saline installation, no H2O2 was detectable in the samples (< 10-7 M). LPO is expected to consume any existing H2O2 shortly after collection (43) and production of more H2O2 is not expected in these secretions after collection because its source is thought to be epithelial in origin. Others have detected between 10-8 M and 10-6 M H2O2 in breath condensate that is frozen or measured immediately after expiration (e.g., 44–46). Dilutions of breath condensate are estimated to be between 100- and 1,000-fold (47, 48), and thus the airway surface liquid H2O2 concentrations are expected to be between 10-6 M and 10-4 M, a concentration that can easily support LPO activity (29–34). For this reason, samples were supplemented with 10-5 M H2O2 to explore their LPO-dependent antibacterial activity.

To test secretions for their ability to prevent growth of bacteria in vitro, secretion samples or PBS as a control were adjusted to pH 5.7 and 10-5 M H2O2 and then incubated with P. aeruginosa for 4 h at room temperature. These experiments were performed at pH 5.7, a pH closer to the optimum for LPO catalytic activity, to compensate for the dilution of LPO and SCN- during sample collection. This pH is below that expected in normal airways, but allows more experiments to be performed given the small volumes of secretions collected from patients. With PBS alone, bacteria increased in cell number by a factor of 2.1 ± 0.3 (mean ± SE; n = 6; Figure 3) . Addition of neither H2O2 to 10-5 M nor dapsone to 10-3 M had any measurable effect on cell growth (1.9 ± 0.3, P > 0.05; n = 6). However, when the airway secretions of six different patients were used in the presence of 10-5 M H2O2, cell growth was completely inhibited (0.98 ± 0.16, n = 6, P < 0.05 compared with PBS control). Inclusion of dapsone, a potent inhibitor of LPO, significantly blocked the H2O2-dependent bacteriostatic properties of the airway secretions (1.68 ± 0.31, n = 6, P < 0.05 compared with no dapsone; Figure 3). The H2O2 dependence and dapsone sensitivity of the bacteriostatic activity supports the conclusion that the LPO system functions in human airway secretions as an antibacterial defense. Assays of these six secretion samples using TMB as a substrate showed that they contained primarily LPO (72 ± 9% of the total peroxidase activity sensitive to dapsone, n = 6).



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  		Figure 3. Antibacterial activity of human airway secretions. P. aeruginosa were incubated for 4 h with either PBS or human airway secretions (n = 6) previously adjusted to pH 5.7, 10-5 M H2O2, with (+) or without (-) 10-3 M dapsone. Relative cfu (cfu after 4 h/starting cfu) are shown. Significant antibacterial activity was seen in human secretions and this activity was inhibited by dapsone. Each bar represents the mean of three separate experiments (± SE). *P < 0.05.

 
To demonstrate that the LPO system functions in a pH range that is physiological in the airway (49–52), we performed experiments at pH 5.7, 6.2, and 6.8 using bovine milk LPO as a model for the airway LPO. Concentrations of the LPO system components were selected to be within the measured ranges in human airways. Bovine milk LPO (6.5 µg/ml), SCN- (0.4 mM), and H2O2 (10-5 M) at all pHs tested effectively blocked the growth of P. aeruginosa and Burkholderia cepacia, which frequently colonize cystic fibrosis airways. At pH 5.7 and 6.2, the activity was bactericidal, whereas at pH 6.8 it was bacteriostatic (Figure 4) . Additional controls showed that the antibacterial activity was dependent on the presence of SCN- and H2O2. It was sensitive to heating the LPO to 100°C before use and was inhibited by the peroxidase inhibitors dapsone (10-3 M) or salicyl hydroxamic acid (10-4 M). All of these characteristics were consistent with LPO-mediated antibacterial activity.



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  		Figure 4. pH dependence of LPO antibacterial activity. Bovine LPO, SCN-, and H2O2 were mixed with P. aeruginosa (A) or Burkholderia cepacia (B) at the indicated pH and mean (± SE) relative cfus were plotted. Closed circles represent LPO containing mixtures. Closed square represents the PBS control without LPO or H2O2.

 
Because the pK of the LPO product HOSCN/OSCN- is ~ 5.3, the loss of bactericidal activity at higher pH could be due to the lower concentration of HOSCN. To increase the product, we compensated for the enzymatic consumption of H2O2 during experiments at pH 6.8 by measuring the consumption of H2O2 at 0.5- or 1-h intervals and replenishing to restore it to 10-5 M (Figure 5) . Under these conditions the LPO system was bactericidal at pH 6.8, suggesting that increased LPO activity is required for bactericidal activity. Thus, we expect that a constant supply of H2O2 in the airway for LPO's use results in an effective antibacterial system against P. aeruginosa and B. cepacia.



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  		Figure 5. H2O2 dependence of LPO activity. Bovine LPO or PBS were mixed with P. aeruginosa at pH 6.8 containing SCN- with or without 10-5 M H2O2. Samples containing H2O2 were assayed for remaining H2O2 after each 1 h or 0.5 h of incubation during the 4-h incubation period and supplemented to restore the initial concentration. Relative cfu were plotted. PBS (-H2O2), and LPO samples are means (± SE) of three separate experiments. PBS control with H2O2 replenished at 0.5 h and 1 h are single experiments with means (± SE) of triplicate plates. *P < 0.05 compared with PBS controls.

 
Antibacterial Activity of LPO Depends on Thiocyanate and Bacterial Concentrations
The efficacy of the LPO antibacterial activity is known to depend not only on the concentration of H2O2 but also on the concentration of thiocyanate and bacteria. To confirm that the LPO system shows an identical dependence in the range of concentrations of LPO, H2O2, and SCN- expected to be present in the airway, bovine milk LPO (6.5 µg/ml) and H2O2 (10-5 M) were tested against different initial concentrations of P. aeruginosa and B. cepacia and at different concentrations of SCN- (Figure 6) . At these estimated airway concentrations of LPO and H2O2, the LPO antibacterial activity depended on the initial concentration of bacteria (Figure 6), suggesting that in vivo, the LPO system can be overwhelmed if challenged with high levels of bacteria. In addition, the effectiveness of the LPO system at the same initial concentration of bacteria was dependent on the initial [SCN-]. Thus, the system is, as expected, dependent on [H2O2] (Figure 5) and [SCN-] (Figure 6) as well as the concentration of bacteria.



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  		Figure 6. Dependence of LPO antibacterial activity on initial concentration of bacteria and SCN-. Bovine LPO and H2O2, as described in MATERIALS AND METHODS, were mixed with different numbers of P. aeruginosa at pH 5.7 and either 0.015 mM SCN- (closed squares) or 0.4 mM SCN- (closed circles). After 4 h, relative mean (± SE) cfu were plotted. The effectiveness of the LPO system decreased with increasing bacteria and lower [SCN-].

 
LPO Has Antibacterial Activity against Haemophilus influenzae
LPO purified from various sources has been shown to be effective against both gram-positive and gram-negative organisms, including Pseudomonas (various species), Staphylococcus aureus, Escherichia coli, and Streptococcus (various species). To test the efficacy of another pathogen that infects the respiratory tract, we examined H. influenzae for sensitivity to bovine milk LPO activity at pH 5.7. The complete LPO system was bactericidal toward this bacterium (Figure 7) . Without treatment, H. influenzae grew 1.48-fold ± 0.19 (n = 12) in 4 h, whereas in the presence of LPO for the same period, the number of bacteria decreased (0.76 ± 0.05 [n = 12]), demonstrating LPO's bactericidal activity toward H. influenzae. Incubation with H2O2 alone did not significantly alter the growth of the bacteria.



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  		Figure 7. Sensitivity of H. influenzae to LPO antibacterial activity. H. influenzae were incubated for 4 h with PBS alone, bovine LPO in the presence of SCN- and H2O2, or SCN- and H2O2 without LPO. Relative mean (± SE) cfu were plotted. The LPO system was effective in killing H. influenzae compared with controls. *P < 0.05 compared with control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The experiments presented here show that the LPO antimicrobial system, not previously recognized as a factor in human respiratory host defense, is functional in human airway secretions in vitro. We have shown that two of the three necessary components, LPO and SCN-, are present in concentrations that support antibacterial activity, and others have shown that the third component, H2O2, is present in airway secretions at concentrations that support LPO activity (29–34, 44–46). The observed sequence identity, enzymatic characteristics, and apparent MW of the peroxidase found in human airway secretions indicates that it is the product of the LPO gene that is also expressed in milk and saliva. As secretions were obtained from patients without known respiratory disease, the data suggest that LPO is a normal constituent of airway secretions. The apparent molecular weight of the immunoreactive protein, the dapsone sensitivity of the measured enzyme activity, and the use of secretions from normal individuals rule out the possibility that MPO activity was the major contributor of the peroxidase activity.

Our data showed that SCN- is present in ample concentrations to support in vivo LPO-catalyzed antibiotic activity, in agreement with published data regarding LPO activity in saliva and milk (17, 19). Others have detected H2O2 in exhaled breath condensate of normal individuals (29–34, 44–46) and, although the exact concentration of H2O2 in airway surface liquid is unknown because dilutions of exhaled breath condensates are difficult to measure, estimates range from 10-6–10-4 M. These concentrations are capable of supporting LPO activity. The presence of LPO and SCN- in secretions might explain the inability to detect H2O2 in secretions collected by suctioning, because most if not all of it should be rapidly consumed by LPO after collection (53). Because H2O2 is potentially toxic to epithelial cells (e.g., 54), the presence of LPO, in addition to its antibacterial role, may serve other important functions such as control of the [H2O2] in the airway lumen (43). Conversely, production of H2O2 may be the rate-limiting factor for LPO activity in normal airways. Similarly, increases in [H2O2], known to follow a variety of inhaled challenges to the airway, may serve specifically to upregulate LPO enzymatic activity in anticipation of needed extra anti-infection defenses.

As expected from earlier in vivo studies of ovine airways (14) and based on the enzymatic properties and antibacterial function of LPO isolated from other secretions, human airway secretions prevented the in vitro growth of P. aeruginosa in an LPO-dependent fashion. Although H2O2 can be antibacterial by itself if present in sufficient concentration, it was unable to moderate growth of bacteria at the concentration used in the in vitro assays. Instead, a significant portion of the antibacterial properties of airway secretions was dependent on LPO enzymatic activity, as demonstrated by its sensitivity to dapsone. These data strongly support, but do not directly demonstrate, that LPO contributes to in vivo host defense against infection of human airways. The LPO system is known to be effective in killing S. aureus, E. coli, P. aeruginosa, and Bacillus cereus (20–22, 40), and we have shown here that B. cepacia and H. influenzae are also susceptible to LPO.

The LPO antibacterial system increases in activity with decreasing pH. For this reason we performed most experiments at pH 5.7 to conserve human samples. Importantly, however, we showed that the LPO system, when supplied with adequate H2O2, is effective at pH 6.8, the estimated pH of airway surface liquid in vivo (51, 55). Any lowering of airway secretion pH by bacterial growth, however, would increase LPO-mediated antibacterial activity and thus increase its effectiveness in protecting the airway from infection or colonization. The LPO system also was shown to be dependent on [SCN-]. Thus, any alteration in normal airway concentrations of the substrates would be expected to change the activity of the LPO system with possible deleterious effects.

In summary, human airways, like ovine airways, contain a complete and functional LPO antibacterial system that is effective against a variety of respiratory pathogens and thus may offer new approaches to boosting airway host defense in individuals with increased exposure or susceptibility to respiratory infection.


   Acknowledgments
 
The authors thank Dr. Adam Wanner for his thoughtful advice and strong support throughout this work. This work was supported in part by grants from the American Lung Association of Florida (G.E.C.) and the NIH (HL-60644 & 67206 to M.S.; and HL-66125 to G.E.C.).

Received in original form August 12, 2002

Received in final form February 17, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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