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(Chest. 2005;128:3167-3176.)
© 2005 American College of Chest Physicians

Antiprotease Function of Airway Secretions in Purulent Tracheobronchitis*

Thomas G. O’Riordan, MD; Lori B. Seischab, PhD; Qi-Long Ying, PhD; Eliseo Colon-Carreras, MD; Bilal Chughtari, BA; Lucy B. Palmer, MD, FCCP and Sanford R. Simon, PhD

* From the Departments of Pathology and Biochemistry (Drs. Seischab, Ying, and Simon, and Mr. Chughtari), and Division of Pulmonary/Critical Care Medicine (Drs. Colon-Carreras, Palmer, and O’Riordan), Stony Brook University, Stony Brook, NY.

Correspondence to: Lori B. Seischab, PhD, Department of Biology, 132 Natural Science Building, Western Carolina University, Cullowhee, NC 28723; e-mail: lseischab{at}wcu.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Study objectives: Unopposed activity of the serine protease, human leukocyte elastase (HLE), is detectable in the airways of patients with purulent tracheobronchitis. The aim of this study was to assess the compartmentalization of HLE activity in the liquid sol phase and the solid gel phase of airway secretions.

Design: Seventy samples of tracheobrochial aspirates were obtained from patients who had hypersecretion and were receiving mechanical ventilation.

Methods: Samples were separated into sol and gel ("mucous pellet") phases, and HLE activity was measured using chromogenic substrate degradation. HLE was eluted from the mucous pellet using hypertonic saline solution, 1 mol/L, or bovine pancreatic deoxyribonuclease (DNase), 16 µmol/L.

Results: HLE activity partitioned between the sol and gel phases of the secretions, with most of the activity present in the gel phase (32:1 ratio of gel to sol HLE activity). The activity of HLE was 95% inhibited when bound to the gel phase, but activity appeared to be largely restored after elution from the gel phase. The gel phase was capable of binding additional exogenous HLE, and its binding capacity for exogenous HLE was not saturated by concentrations that exceeded the highest clinically relevant HLE levels (1.1 mg/mL). Hypertonic saline solution and DNase I efficiently liberated endogenous and exogenous gel phase-bound HLE activity, suggesting that electrostatic bonds and DNA, respectively, play important roles in binding HLE to the gel phase.

Conclusions: The solid phase of airway secretions is a more important modulator of elastase-antielastase balance than has been previously recognized.

Key Words: bronchitis, chronic • leukocyte elastase • mucus


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Human leukocyte elastase (HLE) is a 29-kd serine protease that is synthesized in the neutrophil and stored in the azurophilic granules, from where it is secreted when the neutrophil is activated.1 In addition to its ability to degrade matrix proteins, HLE has also been shown to have secretagogue, chemoattractant, and proinflammatory properties.2 HLE in mammalian airways is inhibited by relatively specific inhibitors, most notably secretory leukoprotease inhibitor (SLPI) and {alpha}1-proteinase inhibitor ({alpha}1-PI). In disease states characterized by neutrophilic inflammation such as bronchiectasis, cystic fibrosis (CF), and ventilator-associated tracheobronchitis, free HLE is detectable in airway secretions. The inability of the naturally occurring inhibitors to adequately counteract HLE activity is thought to be due predominantly to an increase in the release of HLE and to a lesser extent to the degradation of {alpha}1-PI by HLE and other inflammatory products. While for many years anti-HLE agents have been suggested to be an attractive option for the treatment of conditions associated with purulent bronchial secretions, clinical trials of these agents to date have not provided conclusive evidence of efficacy.

To facilitate the design of future therapeutic approaches that use anti-HLE agents in treating purulent bronchitic conditions, we believe that it is desirable to have a more precise understanding of the relationship between HLE and purulent secretions because there is evidence that some macromolecular components of purulent secretions, namely proteoglycans,3 mucous glycoproteins, and DNA, can interact with HLE. The present study was conducted in patients with purulent tracheobronchitis associated with prolonged mechanical ventilation. The purpose of the study was to assess the compartmentalization of HLE activity in the liquid (sol) phase of airway secretions and the solid (gel) phase.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Overview of Experimental Protocol
Samples of tracheobrochial secretions were centrifuged and separated into sol and gel ("mucous pellet") phases. HLE amidolytic activities in the sol phase and the extractions of gel phase were measured using chromogenic substrates. The efficacies of hypertonic saline solution (which disrupts electrostatic interactions), human plasma gelsolin (which severs actin filaments), bovine pancreatic deoxyribonuclease (DNase) I [which severs DNA], and dithiothreitol (DTT) [which cleaves disulfide bonds] in the extraction of HLE activity from gel phase pellets were then assessed. The most reproducible results were obtained with 1 mol/L of saline solution, which was selected for subsequent experiments. In addition to extracting the gel phases of tracheal aspirate specimens, the gel phases of CF sputum specimens were also extracted for comparison.

In an effort to estimate the true endogenous HLE content of specimens, a range of doses of exogenous HLE was added to gel phase pellets and the recoverable activity was measured after treatment with 1 mol/L of saline solution. The amidolytic activity of HLE was measured while the enzyme was still bound to gel phase by using an artificial substrate (succinyl-Ala-Ala-Ala-pNA) that could penetrate the hydrophilic but insoluble gel phase pellet. The possible role of SLPI in binding HLE within the gel phase was investigated by treating the gel phase pellet with oxidants and measuring the effect on recovered HLE. Experiments with DNase at higher concentration and for longer duration were performed to assess the importance of DNA in the partitioning of HLE in the gel.

Reagents
HLE purified from purulent sputum was purchased (Elastin Products; Owensville, MO). The active site concentration of HLE was determined by titration with N-benzyloxycarbonyl-Ala-Ala-Pro-azaAla p-nitrophenyl ester (Enzyme System Products; Livermore, CA) in a manner previously described.45 All of the concentrations of HLE reported in the present study are expressed as active site concentrations, rather than concentrations of total protein. When the results are expressed as micrograms per milliliter, the molecular weight of HLE employed for calculation was 25200. Human plasma gelsolin, bovine pancreatic DNase I, DTT, and the substrates for HLE (methoxysuccinyl-Ala-Ala-Pro-Val-pNA and succinyl-Ala-Ala-Ala-pNA) were from Sigma Chemical (St. Louis, MO). N-chlorotaurine was synthesized from sodium hypochlorite and taurine by the established method.5

Tracheal Aspirate Samples
Tracheal aspirates were collected by a previously described method6 from inpatients at the Medical and Surgical Intensive Care and the Respiratory Care Units of Stony Brook University Hospital, who were receiving mechanical ventilation during scheduled suctioning for therapeutic purposes. A total of 70 tracheal aspirate specimens were collected over a period of 11 months from 31 patients (21 women and 10 men). Except for one 19-year-old patient, the ages of the patients ranged from 47 to 96 years (average, 75 years). Almost all of the samples were obtained from patients who had received mechanical ventilation via tracheostomy for > 2 weeks. The most common causes for prolonged mechanical ventilation were COPD, congestive heart failure, cerebrovascular disease, anoxic encephalopathy, peripheral neuromuscular weakness, and interstitial lung disease. All had hypersecretion as defined by the production of > 2 mL of sputum in a 4-h period without the addition of exogenous saline solution. Almost all specimens demonstrated colonization with either methicillin-resistant Staphylococcus aureus or a variety of Gram-negative organisms including Pseudomonas aeruginosa, Enterobacter spp, Escherichia coli, Serattia marcescens, Acinetobacter spp, and Burkholderia cepacia. Patients were studied regardless of whether they were receiving systemic antibiotic therapy and regardless of whether they had radiologic evidence of pneumonia. Sampling protocols and informed consent procedures were approved by the Committee on Research Involving Human Subjects, the State University of New York at Stony Brook. No saline solution was instilled, and suctioning was performed for 30 to 60 s or until no further secretions could be obtained. Specimens were stored at – 20°C until assayed, unless otherwise noted.

Extraction of HLE From Tracheal Aspirate Gel Phase
For studies of the sol phase of sputum and aspirate, a common preparation protocol is to centrifuge the samples and collect the supernatant sol phase. While this is an effective method for isolating the supernatant sol phase, it is less effective for isolating gel phase since the pellets typically contain gel phase and sol phase. Therefore, to isolate sol-free gel phase, we modified the established protocol in two ways. First, buffer was added to the samples before centrifugation, which led to better separation between supernatant sol phase and pellet gel phase. Second, the pellets were washed with buffer, which removed the remaining sol from the pellet gel phase.

Our method for isolating sol and gel phase was as follows. Tracheal aspirate samples were suspended in four volumes of Dulbecco phosphate-buffered saline solution (DPBS) [HyClone; Logan, UT] with vigorous agitation (vortex) for 3 min. One-milliliter aliquots of the suspensions were added to a series of 1.5-mL Eppendorf tubes, and the tubes were centrifuged at 4°C and 8,000g for 20 min. The supernatants were collected and hereafter referred to as sol phase. The pellets, hereafter referred to as gel phase, were resuspended in DPBS to a total volume of 1.5 mL, and the mixtures were rocked end-over-end for 30 min at 4°C and then centrifuged as before. The supernatants obtained from pellets resuspended in DPBS are hereafter referred to as "wash solution." The tubes containing the washed gel phase pellets were classified into four groups. To each group of pellets, either 6.5 mmol/L of DTT, 1 mol/L of NaCl in DPBS, 16 µmol/L of DNase I, or 0.5 µmol/L of gelsolin in Hank balanced salt solution was added to a final volume of 1 mL. The tubes were incubated at room temperature with constant rocking for 1 h, and centrifuged. The supernatants from these treated pellets are hereafter referred to as gel phase extracts. HLE amidolytic activities in the sol phase solutions, wash solutions, and gel phase extracts were assayed as described in the next section.

Standardized Procedure for HLE Assays in Sol Phase and in Gel Phase Extracts
To measure the active site concentrations of HLE in sol phase solutions and gel phase extracts, 10 µL of the samples under study were added to 990 µL of DPBS containing 500 µmol/L methoxysuccinyl-Ala-Ala-Pro-Val-pNA, 1% (volume/volume) dimethyl sulfoxide and 0.1% (weight/volume) [w/v] Triton X-100 at 25°C. Each reaction was monitored continuously at 405 nm for a period depending on the level of HLE activity in the sample. The concentrations of HLE in the samples were calculated from these measured amidolytic rates based on a linear standard curve that was generated with titrated HLE.

Additional experiments were performed to determine the robustness of the observation that HLE activity was greater in the gel phase extracts than in the sol phase, regardless of variations in specimen handling techniques from our routinely adopted procedure: storage at – 20°C and vigorous agitation for 3 min. The ratios of HLE activity in gel phase extract to HLE activity in sol phase were compared in samples subjected to the following handling conditions: fresh specimens prepared without vigorous agitation (control), fresh specimens prepared with vigorous agitation, and frozen specimens thawed and then prepared without vigorous agitation. All specimens were collected on ice. Each specimen was divided into approximately equal portions by weight and mixed with four volumes of DPBS by vigorous agitation for 3 min or gentle end-over-end rocking. The mixtures were then centrifuged at 4°C for 90 min at 50,000g. The supernatants were collected (sol phase) and the pellets were washed with DPBS as described above. The washed pellets (gel phase) were mixed with four volumes of cetyl-trimethyl-ammonium bromide (CTAB) buffer (50 mmol/L of potassium phosphate pH 6, 0.5% [w/v] hexadecyl ammonium bromide) containing 0.1% Triton X100, incubated for 1 h at 4°C with constant rocking, and centrifuged at 4°C for 90 min at 50,000g. The supernatants (gel phase extracts) were collected. HLE amidolytic activities in the sol phase and gel phase extracts were assayed as described above, and the levels of HLE activity in the sol and gel phases obtained from 1 mL of undiluted specimen were calculated from the amidolytic activities. Myeloperoxidase (MPO) activities in the sol phase and gel phase extracts were assayed using the chromogenic substrate o-dianisidine dihydrochloride. Aliquots of sol phase or gel phase extracts were added to 200 µL of substrate (0.2 mg/mL of o-dianisidine dihydrochloride, 50 mmol/L of potassium phosphate pH 6, 0.5% [w/v] of cetyl trimethyl ammonium bromide, and 30% [w/v] hydrogen peroxide) in microtiter plates. The reactions were monitored continuously at 450 nm for periods depending on the levels of MPO in the samples. MPO activities are reported for sol phase and gel phase extracts that were derived from 1 mL of undiluted specimen. Spearman rank correlation coefficients were used to assess the association between HLE concentration and MPO concentration.

Partition of Exogenous HLE between the Sol and Gel Phases of Tracheal Aspirate
A range of concentrations of purified HLE was added to tracheal aspirates that had been suspended in an equal volume of DPBS. The mixtures were incubated with constant rocking at room temperature for 30 min. Following incubation, the amidolytic activity of HLE in sol phase solutions was assayed as described above, and the amounts of exogenous HLE absorbed by the gel phase pellets were calculated.

Estimation of the Extractable Percentage of HLE in Tracheal Aspirate Gel Phase
Washed tracheal aspirate gel phase pellets were resuspended in DPBS containing 50 µg of purified HLE and then centrifuged. The exogenous HLE that was absorbed by the pellets was computed from residual activity in the supernatants. The pellets that contained the absorbed exogenous HLE were then extracted with 1 mol/L of NaCl, and the extracts were assayed for HLE amidolytic activity (ß). Endogenous HLE in control gel phase pellets to which no exogenous HLE had been added was also extracted with 1 mol/L of NaCl, and the extracts were assayed for HLE amidolytic activity ({alpha}). The fraction of HLE activity extractable with 1 mol/L of NaCl (F) was calculated for each sample as follows:

(1)
where {epsilon} is the amount of exogenous HLE added to the gel phase pellet. The amounts of endogenous HLE in gel phase pellets, Eo, were calculated as follows:

(2)
Results were expressed as HLE levels per pellet, regardless of the different dilutions. The equations are based on the assumption that the efficiencies of extraction from a gel phase pellet are the same for endogenous HLE and for newly absorbed exogenous HLE. Since tracheal aspirate is an extremely heterogeneous material, it may be argued that the gel phase contains a variety of binding sites for HLE with different affinities. HLE would first occupy the high-affinity sites and then fill the low-affinity sites. In the presence of this heterogeneity, exogenous HLE might be more easily extracted than endogenous HLE. Therefore, the equation used to calculate the amount of endogenous HLE in gel phase pellets yields an estimate of the lower limit.

Contribution of SLPI to the Binding of HLE by the Tracheal Aspirate Gel Phase
Kramps et al7 reported that abundant levels of immunoreactive SLPI were present in the tracheal aspirate gel phase. Preliminary studies in our laboratory established that 1 mol/L of NaCl had no detectable effect on the binding of SLPI to HLE. In order to examine if SLPI contributes to the binding of HLE by tracheal aspirate gel phase, gel phase pellets were isolated and incubated with 2 mmol/L of N-chlorotaurine at room temperature for 1 h. After residual N-chlorotaurine was quenched by adding 20 mmol/L of L-methionine, the capacity of the oxidized pellets to absorb exogenous HLE was tested and compared to control pellets that had not been exposed to N-chlorotaurine.

Catalytic Activity of HLE Bound by Tracheal Aspirate Gel Phase
It is not possible to measure directly the diminution in activity of endogenous elastase while it is still bound within a gel phase pellet; therefore, a known amount of exogenous HLE and a synthetic substrate, succinyl-Ala-Ala-Ala-Pro-pNA, were added to the gel pellet. This substrate was chosen for its relatively high solubility in aqueous solutions,8 so that it was unnecessary to add organic solvents that might disturb the interaction between HLE and the gel phase pellets. Moreover, the hydrophilic properties of the substrate facilitate its diffusion through the highly hydrated fibril network of the gel phase matrix. After allowing the reaction to proceed for a series of time intervals, 40 µL of glacial acid was added to the suspensions to stop the reaction. After centrifugation, the concentrations of p-nitroaniline released from the substrate in the supernatants were measured at 405 nm. The activity of HLE in the gel phase pellets was evaluated by comparison to a standard curve that was made by addition of equivalent aliquots of HLE to the same substrate under the same conditions but in DPBS.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Dissociation of HLE From Tracheal Aspirate Gel Phase
Figure 1 compares the effects of four mucolytic reagents on extraction of HLE amidolytic activity from tracheal aspirate gel phase pellets. In these experiments DTT, DNase I, and gelsolin were used to reduce the intramolecular and intermolecular disulfide bonds in mucin glycoproteins, cleave polynucleotide chains, and sever filaments of actin polymers, respectively. High salt (NaCl) concentrations were used to dissociate HLE that might be bound to the pellets by electrostatic interaction. In preliminary experiments, we found that the amounts of HLE amidolytic activity that could be extracted from gel phase pellets with NaCl reached a plateau after treatment with 0.75 to 1.25 mol/L of NaCl. Data in Figure 1 show that the action of DTT or gelsolin led to minimal dissociation of HLE amidolytic activity from the pellets. DNase I (16 µmol/L for 1 h) liberated substantial amounts of HLE, but the amounts were lower and less consistent than those extracted with 1 mol/L of NaCl. These results support the conclusion that the binding of at least a portion of HLE to the pellets is electrostatic. For the majority of additional experiments in this study, 1 mol/L of NaCl was used as the dissociation reagent because it produced more reproducible rates of extraction.



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  		Figure 1. Dissociation of HLE from tracheal aspirate gel phase. The active site concentrations of HLE were measured for the sol phases (white bars), the wash solutions after gel phase pellets were rinsed with phosphate-buffered saline solution (gray bars), and the gel phase extracts after gel phase pellets were treated with 6.5 mmol/L of DTT, 16 µmol/L of DNase I, 1 mol/L of NaCl, or 0.5 µmol/L of gelsolin (black bars).

 
Data in Figure 1 also indicate that the wash solutions contained small amounts of HLE activity, approximately 36 ± 2% of the HLE levels found in the sol phase. Further washing of the pellets with DPBS released even less HLE (data not shown), suggesting that the majority of the protease was bound tightly by the gel phase. Since the amounts of HLE that were extractable from the gel phase pellets by 1 mol/L of NaCl were frequently one order of magnitude higher than those in the sol phase, the small amounts of HLE activity dissociated from the pellets into the wash solutions were discounted in further analyses. Although the washing step was maintained in the protocol, HLE activity in the wash solutions was not routinely assayed.

In preliminary studies, the gel phase pellets of CF sputum specimens (n = 3) were extracted with NaCl, DTT, DNase I, or gelsolin in the same manner as described for tracheal aspirate specimens. The dissociation of HLE from CF sputum gel phase pellets followed the same patterns as the dissociation of HLE from tracheal aspirate gel phase pellets (data not shown). Namely, DTT and gelsolin led to minimal dissociation of HLE amidolytic activity from gel phase pellets, and DNase I liberated substantial amounts of HLE but the amounts were lower than those extracted with 1 mol/L of NaCl.

Ratios of HLE in Gel Phase Extracts to HLE in Sol Phases
We have examined a total of 70 tracheal aspirate specimens. Eight of the 70 samples lacked the characteristic green color of MPO and had zero or negligible HLE activity in both sol phases and gel phase extracts. As we have focused on the distribution of HLE activity in purulent tracheal aspirates, data from these eight specimens have been excluded from the following results. For the other 62 purulent specimens, active HLE in the sol phases ranged from 0.06 to 52 µg/mL (mean ± SD, 4.7 ± 7.8 µg/mL). In the gel phase extracts, using 1 mol/L of NaCl as described, HLE levels ranged from 0.4 to 731 µg/mL (mean, 80 ± 114 µg/mL). The ratios of HLE in the gel phase extract to HLE in the sol phase ranged from 0.52 to 107 (mean, 32 ± 28) [Fig 2 ]. Only 1 of the 62 purulent specimens had HLE activity in the gel phase extract that was less than the sol phase HLE activity. Based on these data, we conclude first that levels of HLE activity in the sol phase, which are employed in most previous studies as an index of the elastase load in inflamed lungs, represent only a fraction of the total HLE in purulent tracheal aspirates; and second, that the majority of HLE is concentrated in the gel phase, although its activity can be demonstrated only by extraction of the gel.



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  		Figure 2. Distribution of ratios of HLE in gel phase extract to HLE in sol phase. Tracheal aspirate gel phase pellets were extracted with 1 mol/L of NaCl, and the active site concentrations of HLE were measured for the sol phases and gel phase extracts. The ratios of HLE in gel phase extract to HLE in sol phase were classified into ranges (0–1, 1–11, to 101–111). The number of specimens with ratios within each range were plotted. The white bar represents the number of specimens with ratios < 1. The black bars represent specimens with ratios > 1.

 
The conclusion that the majority of HLE is the gel phase could also be extended to the distribution of the enzyme in purulent CF sputum specimens. Gel phase pellets of CF sputum specimens were extracted with 1 mol/L of NaCl in the same manner as described above for tracheal aspirate specimens. HLE levels in the CF gel phase extracts ranged from 54 to 614 µg/mL (mean, 359 ± 283 µg/mL). HLE levels in CF sol phase ranged from 14 to 315 µg/mL (mean, 165 ± 151 µg/mL). In each CF sample, the HLE activity in the gel phase extract was greater than the HLE activity in the sol phase. The ratios of HLE in the gel phase extract to HLE in the sol phase ranged from 2.0 to 3.9 (mean, 2.8 ± 1.0). Furthermore, there was a correlation between the sol phase concentration of HLE and the concentration of HLE in the gel extract (Spearman r = 0.74, p = 0.006).

We next determined whether the levels of HLE activity in gel phase extracts correlated with levels of inflammation by comparing MPO activity with HLE activity for tracheal aspirate specimens prepared without freeze-thawing or vigorous agitation. For these experiments, the gel phase pellets were extracted with CTAB buffer, which effectively extracts MPO while it inhibits other peroxidases. We found a correlation between HLE and MPO activities for both sol and gel phase extracts. For MPO and HLE activities in gel phase extracts, the Spearman r was 0.61 (p = 0.04), and for sol phases the Spearman r was 0.65 (p = 0.02). For total MPO and total HLE activity, defined as the activity in sol phase plus the activity in gel phase extract, the Spearman r was 0.73 (p = 0.007).

As described above, we observed that HLE activity in gel phase extracts was greater than HLE activity in sol phase, and that HLE activity in sol and gel extract correlated to the activity of the inflammatory marker MPO. However, additional experiments were necessary to determine whether these observations were true regardless of variations in specimen preparation techniques, namely specimen storage at – 20°C and vigorous agitation for 3 min. In tracheal aspirate specimens that were prepared without freeze-thawing or vigorous agitation, the HLE activity extracted from gel pellets using CTAB buffer was greater than the HLE activity in the sol phase for 11 of 12 samples. The mean ratio of HLE in the gel phase extract to HLE in the sol was 3.6 ± 3.9. Vigorous agitation had little effect on the amounts of HLE in the sol and gel phases, and resulted in a mean ratio of HLE in gel phase extract to HLE in sol phase of 3.2 ± 3.1. However, freeze-thawing of specimens resulted in a net increase of HLE in the sol phase and consequently a decrease in the mean ratio of gel phase extract HLE activity to sol phase HLE activity (1.4 ± 0.9). While HLE activity was greater in the gel than in the sol regardless of variations in handling techniques, freeze-thawing the specimens could result in an underestimation of ratio of HLE activity in the gel phase extract to that in the sol phase.

In addition to determining the effects of various handling techniques on HLE activity, the sol phases and CTAB extracts of gel phases were also assayed for MPO activity. Similar to the results for HLE activity, vortexing had little impact on the amount of MPO activity in the sol phases and the gel phase extracts. The mean ratios of MPO activity in the gel phase extract to MPO activity in the sol phase were 0.5 ± 0.3 for samples that were gently mixed and 0.5 ± 0.5 for samples that were vigorously agitated. Freeze-thawing the specimens resulted in a mean ratio of MPO in the gel phase extract to MPO in the sol phase of 2.0 ± 3.2.

Partition of Exogenous HLE Between the Sol and Gel Phases of Tracheal Aspirate
In order to investigate the cause of the preferential distribution of HLE into the gel phase of tracheal aspirates, we determined the partition of exogenously added elastase between the sol and gel phases. Purified HLE was added to tracheal aspirate samples, and the mixtures were incubated. After centrifugation and separation of the sol and gel phases, the sol phases were assayed for HLE activity, and amounts of exogenous HLE that had been absorbed by the gel phases were calculated. The results shown in Figure 3 indicate that as the amount of exogenous HLE increased, the amounts of exogenous HLE in both the sol phase and the gel phase increased. However, the curve for the sol phase shows a tendency toward saturation and possesses a shallow average slope, while the curve for the gel phase is approximately linear with a much steeper slope. When a total of 84 µg of purified HLE was added, 9.2 ± 1.5 µg of exogenous elastase was found in the sol phase, while 75 ± 1.5 µg was calculated to be associated with the gel phase pellets. Specifically, 89% of the exogenous elastase partitioned into the gel phase, and only 11% partitioned into the sol phase. These results reveal that in comparison to the sol phase, the gel phase possesses a much higher affinity for HLE, and that the gel phase behaves like a sink which can continue to absorb most of the exogenous HLE.



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  		Figure 3. Partition of exogenous HLE between the sol and gel phases of tracheal aspirate. Various amounts of HLE were added to tracheal aspirate specimens. The amounts of exogenous HLE that partitioned into the sol phase (closed circles) and gel phase (open circles) were determined. The gel phase has a higher affinity for HLE than the sol phase. Despite exceeding clinically relevant concentrations of exogenous HLE, the gel phase was not saturated.

 
Estimation of the Extractable Percentage of HLE in Tracheal Aspirate Gel Phase
The discovery that tracheal aspirate gel phase absorbs exogenous HLE enabled us to estimate of the amount of endogenous HLE in gel phase. The results obtained from eight tracheal aspirate specimens are listed in Table 1 . Data in the second column show that when 50 µg of HLE was added to gel phase pellets, an average of 46 µg (92%) of HLE was absorbed by the pellets even when the specimens were diluted in up to 12 volumes of DPBS. Only 31 ± 12% of the HLE activity in the gel phase pellets could be extracted with 1 mol/L of NaCl. Therefore, on average, the actual ratios of endogenous HLE in the gel phase to endogenous HLE in the sol phase could be at least 3.2 times higher than the ratios reported above. As less than one third of the endogenous HLE in the gel phase is extractable with 1 mol/L of NaCl, the remainder may associate with the gel phase through nonelectrostatic interactions.


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  		Table 1. Absorption of HLE by Gel Phase Pellets and Extraction of HLE From Pellets by 1 mol/L of NaCl*

 
Role of SLPI in the Binding of HLE by the Tracheal Aspirate Gel Phase
Since abundant levels of SLPI have been found in tracheal aspirate gel phase, it could be hypothesized that SLPI contributes to the binding of HLE to gel phase. To test this hypothesis, tracheal aspirate gel phase pellets were first treated with N-chlorotaurine to oxidize and inactivate SLPI, and then incubated with exogenous HLE. When gel phase pellets were treated with 2 mmol/L of N-chlorotaurine and then incubated with 50 µg of HLE, only 0.26 ± 0.01 µg of HLE remained in the supernatants (n = 3). In control experiments with gel phase pellets that had not been exposed to N-chlorotaurine, 0.19 ± 0.04 µg of exogenous HLE remained in the supernatants. Therefore, the absorption of exogenous HLE by treated and untreated gel phase pellets were not significantly different. Since the active site of SLPI contains an essential methionine residue that is sensitive to oxidation by N-chlorotaurine,8 these results do not support the hypothesis that SLPI is involved in the binding of exogenous HLE to gel phase pellets.

Role of DNA in the Binding of HLE by the Tracheal Aspirate Gel Phase
As shown in Figure 1, DNase I is an effective agent for dissociating HLE from tracheal aspirate gel phase. Therefore, it could be hypothesized that DNA has a role in the binding of HLE to the gel phase. To test this hypothesis, we digested gel phase pellets with a high concentration of DNase I (32 µmol/L) for up to 7 h. The results from one such experiment are shown in Figure 4 . When DNase I digestion was prolonged, the levels of HLE released from the pellets approached a maximum asymptote, in the present case, 41 ± 8 µg per pellet. When 1 mol/L of NaCl was used as the dissociation reagent, only 9 ± 1 µg of HLE per pellet was released, ie, approximately 22% of the elastase that could be released by exhaustive digestion with DNase I. In four additional experiments with different tracheal aspirate specimens, the levels of HLE extractable with 1 mol/L of NaCl were calculated to be 40%, 70%, 59%, and 29% (mean ± SD, 44 ± 20%; n = 5) of the levels released after exhaustive digestion with DNase I.



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  		Figure 4. Release of HLE from gel phase pellets by digestion with DNase I. Tracheal aspirate gel phase pellets were incubated with 32 µmol/L DNase I for up to 7 h. HLE activity released from the pellets into the supernatants were determined (open circles). The amount of HLE activity released with 1 mol/L of NaCl from the same pellets was also determined (closed circle).

 
To estimate how efficiently DNase I dissociates HLE from the gel phase pellets, we used equation 1 to calculate the percent of HLE activity that is extractable with DNase I. First, gel phase pellets were incubated with 50 µg of purified HLE, and the amounts of exogenous HLE absorbed by the pellets were determined. Then the pellets were digested with 32 µmol/L of DNase I for 6 h. From three sets of experiments, the percentages of HLE extracted from pellets using DNase I were 74%, 70%, and 69% (mean, 71 ± 2%; n = 3). These results indicate that DNase I was unable to completely dissociate HLE from the gel phase, even with prolonged digestion at high concentrations of DNase I. However, DNase I was a more effective dissociation reagent than 1 mol/L of NaCl, which extracted only 31% of HLE from gel phase pellets (Table 1). The data suggest that DNA is one of the major components involved in the binding of HLE by the gel phase, and that DNA contributes not only to electrostatic binding but also to nonelectrostatic binding.

Catalytic Activity of HLE in Tracheal Aspirate Gel Phase
The finding that tracheal aspirate gel phase binds exogenous HLE also facilitates estimation of the catalytic activity of the bound HLE. In Figure 5 , the calculated amounts of exogenous HLE bound to gel phase (open circles), are compared to the apparent amidolytic activity of HLE bound to gel phase (closed circles), expressed as the concentration of an equivalent amidolytic activity of HLE in DPBS. When 75 µg of exogenous HLE was incubated with gel phase, 74.5 ± 0.1 µg of the exogenous elastase was absorbed by the pellets. The apparent amidolytic activity of this gel-bound HLE was equivalent to the amidolytic activity of 3.3 ± 0.5 µg of HLE in DPBS. Therefore, 95% of the catalytic activity of HLE in the gel phase pellets was inhibited. Since the pellets were obtained from 1 mL of suspension of the tracheal aspirate specimen diluted into four volumes of DPBS, it can be calculated that the apparent inhibitory capacity of the original tracheal aspirate was at least 355 µg/mL, or 14 µmol/L of HLE.



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  		Figure 5. Catalytic activity of HLE in gel phase of tracheal aspirate. Gel phase pellets were incubated with various amounts of exogenous HLE. The calculated amounts of exogenous HLE that bound to the gel phase (open circles), are compared to the apparent amidolytic activity of the HLE that bound to gel phase (closed circles), expressed as the concentration of an equivalent amidolytic activity of HLE in DPBS.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
In this article, the following novel observations are made concerning the activity of HLE in the secretions of patients with ventilator-associated tracheobronchitis: (1) HLE is partitioned between the sol and gel phases of the secretions, and most of the HLE partitions into the gel phase; (2) the amidolytic activity of HLE, as measured by degradation of synthetic oligopeptide chromogenic substrates, is significantly inhibited when HLE is bound to the gel phase; (3) the amidolytic activity of HLE appears to be largely restored after elution from the gel phase; (4) the gel phase is capable of binding additional exogenous HLE; furthermore, the binding capacity of the gel for exogenous HLE does not appear to be saturated by levels that exceed the highest clinically relevant levels of HLE (1.1 mg/mL); (5) HLE binding to the gel phase appears to be independent of SLPI; moreover, in these purulent secretions, the capacity of the gel phase to bind additional exogenous elastase appears to be quantitatively greater than the binding capacity of SLPI and {alpha}1-PI79; the observation that the activity of exogenous HLE can be extracted from the gel phase rules out the possibility that the inhibition of activity of HLE while it is still within the gel phase could be due to complexation with {alpha}1-PI; and (6) DNase I (32 µmol/L) and hypertonic saline solution (1 mol/L) efficiently liberate endogenous and exogenous gel phase-bound HLE activity, while gelsolin and DTT are relatively inefficient. The effectiveness of hypertonic saline solution and DNase I suggest that electrostatic interactions and DNA, respectively, rather than complexation with antiproteases, play important roles in binding HLE to the gel phase.

Individual components of mucous plugs, including glycoproteins,10 DNA,1011 and actin,12 can bind to and inhibit HLE. In contrast to previous studies101112 of the individual components of mucous plugs, the present study examines the totality of the solid constituents of the mucous pellets. Specifically, the extractable HLE activity of the solid phase of mucous plugs was compared to the HLE activity of the soluble phase of mucous plugs. By adding known quantities exogenous HLE and soluble synthetic substrates to gel phase pellets, we estimated that 95% of the HLE activity is inhibited by the constituents of the insoluble gel phase of purulent airway secretions. This level of inhibition is higher than the previously reported inhibition of HLE activity by the individual components of the insoluble gel phase,101113 thus suggesting that the incorporation of glycoprotein, DNA, and actin into mucous fibrils may create synergistic binding and inhibition of HLE.

While the mechanism by which HLE is bound to the solid phase has not been precisely characterized, the efficacy of hypertonic saline solution in extracting HLE from this phase indicates that electrostatic bonds are important. The fact that incubation with DNase I liberated greater quantities of HLE activity than hypertonic saline solution suggests that nonelectrostatic binding of HLE to DNA is also likely to be important. The complexity of these interactions is further illustrated by the observation that even after prolonged (6 h) incubation of gel phase pellets with high concentrations of DNase I (32 µg/mL), 30% of exogenous HLE activity still could not be recovered.

When gel phase pellets were incubated with 1 mol/L of saline solution, all but one of the original 62 purulent specimens had a gel extract/sol ratio for HLE activity that was > 1, with a mean of 32 ± 28 (range, 0.52 to 107). While the finding that the gel phase has more HLE activity than the sol phase is consistent and robust, there is considerable intersample variability in ratios, probably related to variability in the contributions of electrostatic and nonelectrostatic modes of binding within the mucous fibrils that form an insoluble but hydrated network with a complex gel-liquid interface. Additional experiments confirmed the observation that gel phase contains significantly more HLE activity than the sol phase, regardless of whether the specimens were analyzed when fresh or after freeze-thawing, and regardless of whether they were kept on ice or allowed to sit at room temperature for 1 to 4 h (data not shown). While these variations in specimen handling resulted in some alterations to the relative gel extract/sol ratio of individual samples, variations in handling of samples do not account for the finding of higher HLE activity in the gel phase. Furthermore, the observation that the gel phase of fresh (nonfrozen) specimens tended to have somewhat higher extractable HLE activity than freeze-thawed specimens makes it unlikely that the freeze-thaw process liberated sufficient HLE from intact neutrophils to account for the increase in HLE activity in the gel phase. Most of the HLE activity in these specimens had apparently been liberated from neutrophils in the airway prior to specimen collection.

While it is speculative to extrapolate from in vitro interactions of HLE with the gel phase pellet to in vivo interactions of HLE in the intact human airway, it can be useful in designing future clinical studies. The potential role of the gel phase of purulent airway secretions in inhibiting HLE is likely to be a two-edged sword. Because HLE is both a destructive proteolytic enzyme and a proinflammatory agent, its inhibition by the gel phase of airway secretions may be protective of the airway. However, the inhibition appears to be reversible and thus retention of HLE-rich mucous plugs in the airway is a potential cause of delayed inflammation. Furthermore, the design of clinical trials for the administration of exogenous irreversible inhibitors of HLE should take into account the potential complicating factor that much of the target enzyme is reversibly bound to the gel phase of airway secretions. Finally, the findings of the present study are consistent with findings of previous investigators14 who raised concerns about the potential of recombinant human DNase to liberate HLE from DNA and thus worsen inflammation. Clinical studies indicate that this agent is effective and well tolerated in CF15 but appeared to be associated with a worse outcome in non-CF bronchiectasis.16 The ability of DNase to liberate HLE was dose and time dependent in the present study, and it is likely that intraluminal dose and kinetics of DNA degradation in the airway are likely to be different than in vitro. Furthermore, recombinant human DNase is usually used as an adjunct to physical clearance methods in clinical practice, and the expectoration of HLE-rich, DNA-rich mucous secretions is likely to reduce the risks of HLE liberation in the airway.


   Conclusion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
Purulent tracheobronchitis is a significant clinical problem in patients receiving prolonged mechanical ventilation. An understanding of the interaction between HLE and the solid phase of purulent airway secretions is likely to be important in the design of studies to evaluate the role of protease-antiprotease imbalance in this airway disease. We have demonstrated that most of the recoverable HLE activity in purulent secretions of patients with ventilator-associated tracheobronchitis is associated with the solid rather than the aqueous component of the secretions. HLE appears to be relatively inhibited when bound to these solid-phase secretions but when liberated regains its amidolytic activity. Thus the solid phase of secretions may serve as an inhibitory reservoir of potentially active HLE. Whether the HLE activity is eluted from the gel phase of purulent airway secretions in vivo, either spontaneously or in response to medications, is not known. While the findings presented in this study suggest that the gel phase of airway secretions may be a more important modulator of elastase-antielastase balance than has been previously recognized, the clinical significance of this observation in relation to naturally occurring and exogenous inhibitors of HLE awaits further study.


   Footnotes
 
Abbreviations: {alpha}1-PI = {alpha}1-proteinase inhibitor; CF = cystic fibrosis; CTAB = cetyl-trimethyl-ammonium bromide; DPBS = Dulbecco phosphate-buffered saline solution; DNase = deoxyribonuclease; DTT = dithiothreitol; HLE = human leukocyte elastase; MPO = myeloperoxidase; SLPI = secretory leukoprotease inhibitor; w/v = weight/volume

This work was supported by National Institute of Dental Research Grant DE-10985, and the New York State Office of Science and Technology (Biotechnology Center, State University of New York at Stony Brook).

Received for publication January 25, 2005. Accepted for publication May 13, 2005.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

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