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Published ahead of print on April 17, 2003, doi:10.1164/rccm.200208-829OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 192-198, (2003)
© 2003 American Thoracic Society

Original Article

Sputum Sol Neutrophil Elastase Activity in Bronchiectasis

Differential Modulation by Syndecan-1

Stanley C. H. Chan, Daisy K. Y. Shum and Mary S. M. Ip

Departments of Biochemistry and Medicine, Faculty of Medicine, University of Hong Kong, Hong Kong, China

Correspondence and requests for reprints should be addressed to D. K. Y. Shum, Ph.D., Department of Biochemistry, University of Hong Kong, 21 Sassoon Road, Hong Kong, China. E-mail: shumdkhk{at}hkucc.hku.hk


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The persistently dominant activity of neutrophil elastase in bronchial secretions replete with antielastases is crucial to the pathogenesis of bronchiectasis. We hypothesize that components in the bronchial secretions bind neutrophil elastase and compromise the inhibitory efficiency of prevailing antielastases. Zymographic analysis of sputum sols from patients with bronchiectasis found elastase activity in a polydisperse, alcian blue-stained zone of high molecular mass. This suggested that neutrophil elastase was complexed with polyanionic partners. Western blot analysis found not only the polyanionic partner, heparan sulfate/syndecan-1, but also the physiological antielastases, secretory leukoproteinase inhibitor and {alpha}1-antitrypsin, in the complex. Both dissociative density gradient ultracentrifugation and heparin displacement revealed that elastase dissociated from heparan sulfate/syndecan-1 was fully inhibited by the endogenous antielastases. This contrasts with the effects of exogenous antielastases on sputum neutrophil elastase activity—that of {alpha}1-antitrypsin was limited, but that of secretory leukoproteinase inhibitor was facilitated. Similarly, complexed elastase on blots of sputum sol zymographs was bound and inhibited by exogenous secretory leukoproteinase inhibitor but not by exogenous {alpha}1-antitrypsin. Taken together, the results bring a new focus to heparan sulfate/syndecan-1 complexed with neutrophil elastase in inflamed bronchial secretions as a target for modulating elastase susceptibility to physiological antielastases.

Key Words: {alpha}1-antitrypsin • heparan sulfate proteoglycans • proteinase • secretory leukoproteinase inhibitor

Bronchiectasis is a chronic pulmonary disease characterized by irreversibly dilated, thick-walled bronchi that are inflamed and colonized by bacteria. In patients with bronchiectasis, pulmonary inflammation is dominated by neutrophils (1). Although neutrophils are primarily recruited to the airways in response to an initial trigger that may be infective or otherwise, the airway epithelium and neutrophils can become sources of cytokines that sustain the chemotactic recruitment of neutrophils (2–4). We showed that neutrophils recruited in bronchiectasis could be locally activated to damage bronchial tissues in their vicinity (5). Indeed, the expectorated bronchial secretions of these patients contain not only neutrophil elastase (NE) activity, but also proinflammatory mediators that can promote the pericellular proteolytic action of neutrophils (5, 6). It is, however, unclear how neutrophil-derived proteinase activity can remain dominant when the inflamed bronchial environment is replete with antiproteinases (7, 8).

Among the neutrophil-derived proteinases, human NE is potent in the stimulation of airway secretion, acceleration of airway inflammation, and destruction of the airway mucosal tissue in both acute and chronic pulmonary diseases (9–13). The mature 29-kD enzyme is produced in granulocyte precursor cells, stored in azurophilic granules of neutrophils, and released on surface activation, phagocytosis, or cell death (14, 15). NE activity in inflamed pulmonary tissue is counterbalanced essentially by secretory leukoproteinase inhibitor (SLPI), a 12-kD polypeptide product of the bronchial epithelium and submucosal glands (16–18). With increased microvascular permeability, the role of the plasma-derived antiproteinase, {alpha}1-antitrypsin ({alpha}1-AT), becomes important (19). {alpha}1-AT is a 52-kD plasma glycoprotein synthesized mainly by hepatocytes (20). Local production by lung-derived epithelial cells (21) and mononuclear phagocytes (22) has also been reported, but the antiproteinase effect of these latter cell types is readily overwhelmed in the event of a major neutrophil degranulation. Kinetic studies with purified products showed rapid binding of both {alpha}1-AT and SLPI to NE and consequent inhibition of elastase activity; polyanions introduced into the reaction mixture, however, differentially modulated the rates of inhibition such that the antielastase effect of SLPI was accelerated but that of {alpha}1-AT was depressed (23–26).

Heparan sulfate proteoglycans (HSPGs) represent a family of polyanions associated with human bronchial epithelium. Syndecan-1 is a transmembrane HSPG found expressed on surfaces of bronchial epithelial cells whereas perlecan is a pericellular HSPG found in the basement membrane (27, 28). The diminished immunostaining of HS in tissue sections of lung biopsy samples from patients with pulmonary emphysema (29) suggests shedding of the proteoglycans into inflammatory respiratory fluids. HS-substituted proteoglycan fragments have been reported in skin wounds and synovial fluids, where the fragments were found to participate in regulating proteinase–antiproteinase balance (30) and modulating the bioavailability of soluble effectors (31). We hypothesize that in secretions of chronically inflamed bronchi, similar HS–proteoglycan moieties bind NE and compromise the inhibitory efficiency of the prevailing antielastases. We report the novel finding that in bronchial secretions of patients with bronchiectasis, NE indeed exists not as free monomers, but as supramolecular complexes with HS/syndecan-1, and thus becomes differentially susceptible to inhibition by SLPI and {alpha}1-AT.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects and Sputum Samples
Patients with bronchiectasis were recruited from the Bronchiectasis Clinic, Queen Mary Hospital, University of Hong Kong. Inclusion criteria were as follows: bronchiectasis documented on high-resolution computerized tomography of chest, idiopathic etiology of bronchiectasis, chronic sputum production with daily sputum greater than 10 ml, absence of asthma (according to American Thoracic Society guidelines) and other major pulmonary diagnoses, and a steady state as defined by absence of change of symptoms noted by the patient over the past 3 weeks. Exclusion criteria were as follows: bronchiectasis with defined etiology, for example, posttuberculous, primary ciliary dyskinesia; common variable immunodeficiency (sweat test has not been performed to exclude cystic fibrosis because of the known rarity of cystic fibrosis among Chinese and the lack of suggestion of multisystem disease in any of the patients); maintenance use of oral or nebulized antibiotics; and use of antibiotics within the previous 3 weeks. Patients taking inhaled bronchodilators or inhaled corticosteroids were advised to omit these drugs for at least 12 hours before sputa were collected. Sputum samples were collected over a maximum of 4 hours in sterile pots and were then immediately centrifuged at 50,000 x g for 1.5 hours (4°C); the supernatant sol phase of each sample was collected, aliquoted, and stored at –70°C until use (5). The study has been approved by the University of Hong Kong Ethics Committee, and patients gave informed consent before sputum collection.

Determination of NE Activity in Sputum Sol
The NE activity of sputum sol was estimated with methoxysuccinyl-L-alanyl-L-alanyl-prolyl-L-valyl-p-nitroanilide as substrate at 2 mM in 0.2 M Tris-HCl, 0.5 M NaCl, pH 8.0; the hydrolytic release of p-nitroaniline was monitored at 410 nm, 37°C (E410 = 8,800) (32). An equivalent activity of purified human NE (Sigma, St. Louis, MO) was titrated with increasing concentrations of the irreversible inhibitor, methoxysuccinyl-L-alanyl-L-alanyl-prolyl-L-valyl-chloromethylketone (Calbiochem-EMD Biosciences, San Diego, CA) and residual activities were measured to determine the active site concentration (33). The concentrations of {alpha}1-AT (Sigma) and SLPI (R&D Systems, Minneapolis, MN) were then standardized against the active site-titrated human NE (34). Both sputum sol samples and purified human NE were separately incubated with standard additions of {alpha}1-AT, SLPI, or eglin C (Sigma) for 15 minutes at 37°C and residual NE activities were then measured. Individual results were expressed as percentages of activities of corresponding controls to which no inhibitor was added; subtraction of the percentage residual activity from 100% yielded the percentage inhibition.

Casein Zymography
Casein zymography of sol samples was performed under nonreducing conditions (35). Sol samples that had been incubated (37°C, 90 minutes) with heparin were similarly analyzed. The gels were stained for protein with Coomassie Brilliant Blue R-250 (0.5%). Nonstaining regions of the gel indicated proteinase activity. The protein-stained gel was further stained for glycosaminoglycans with alcian blue (1%).

Western Blot Analysis
Sodium dodecyl sulfate-polyacrylamide gels of sol samples were blotted on polyvinylidene fluoride membranes (Schleicher & Schuell BioScience, Keene, NH) (36) and probed in turn with antibodies against NE (diluted 1:200, mouse anti-human NE, AHN-10; BD Biosciences Pharmingen, San Diego, CA), HS (diluted 1:200, mouse anti-human HS; Roche Molecular Biochemicals, Indianapolis, IN), syndecan-1 (diluted 1:200, mouse anti-human CD138; Serotec, Raleigh, NC), perlecan (diluted 1:200, rat anti-mouse EHS tumor HSPG; Chemicon International, Temecula, CA), {alpha}1-AT (diluted 1:200, rabbit anti-human {alpha}1-AT; Sigma), and SLPI (diluted 1:200, goat anti-human SLPI; R&D Systems) and the relevant secondary antibodies conjugated with horseradish peroxidase. Visualization was enhanced with a chemiluminescence kit (ECL; Amersham Biosciences, Piscataway, NJ). Bound antibodies were stripped from the membrane by incubation for 30 minutes at 50°C with 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate, 62.5 mM Tris-HCl, pH 6.7, before reprobing with another primary antibody.

Western Ligand Blot
After 1 hour in blocking buffer (36), each blot was incubated (1 hour, 4°C) with 10 µM human plasma {alpha}1-AT or 10 µM recombinant human SLPI. Bound SLPI and {alpha}1-AT were probed with the relevant antibodies as described above.

Casein Gel Overlay Assay
After incubation with excess {alpha}1-AT or SLPI as described above, each blot was pressed against a casein–agarose gel (1.5% in 50 mM Tris-HCl [pH 8.0], 0.15 M NaCl, 0.2 M EDTA, casein [1 mg/ml]) and incubated in a humid chamber (37°C, 16 hours). Proteinase activity was visualized as described under CASEIN ZYMOGRAPHY.

Dissociative Ultracentrifugation of Sputum Sol Samples
Sol samples were mixed 1:1 (vol/vol) with 0.1 M sodium acetate, pH 5.8, containing guanidine-HCl (8 M) and then supplemented with {epsilon}-aminocaproic acid (0.1 M), benzamidine-HCl (0.005 M), and EDTA (0.01 M). The mixture was centrifuged (100,000 x g, 48 hours) in a cesium chloride gradient at an initial density of 1.72 g/cm3. The resultant fractions were dialyzed against 0.025 M sodium acetate (pH 5.8) containing 0.4 M guanidine-HCl and macromolecules in the retentate were recovered by ethanol precipitation.

Statistical Analysis
The INSTAT statistical software package (GraphPad, San Diego, CA) was used for statistical analyses. The Mann–Whitney U test was used to compare means. Statistical significance was accepted at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Profile
Ten patients (five men and five women), mean age of 46 years, were recruited as reported earlier (5). All the patients had diffuse bronchiectasis (involvement of more than one lobe of lung) documented on high-resolution computerized tomography of the thorax, and all were nonsmokers. Five of the patients had been prescribed regular treatment with inhaled corticosteroids: four with inhaled bronchodilators and one with theophylline. Six of the samples indicated Pseudomonas aeruginosa. Despite possible quantitative differences among sputum samples due to the effects of corticosteroids and P. aeruginosa, the observations described below applied to all 10 samples studied.

Sputum Sol NE Differs from Purified Human NE in Responses to Exogenous Anti-elastases
In all the sputum sol samples tested, NE activities fell within an active site-titrated concentration range of 0.9–1.2 µM. Exogenous inhibitors introduced at 1.2 µM would be expected to fully inhibit NE activity in the test samples. Exogenous {alpha}1-AT introduced at 1.2 µM was sufficient to inhibit 36% of the NE activity in the sputum sol samples. The inhibition could reach only 62% with exogenous {alpha}1-AT added to reach 12 µM, 10-fold the molar equivalence of NE activity in the sputum sol samples (Figure 1A) . However, complete inhibition was observed when purified human NE was treated with a molar equivalence of {alpha}1-AT. The full inhibitory potential of exogenous {alpha}1-AT was therefore not reached in the sputum sols.



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  		Figure 1. Inhibition of sputum sol neutrophil elastase (NE) activity by exogenous {alpha}1-antitrypsin ({alpha}1-AT), secretory leukoproteinase inhibitor (SLPI), and NE-specific eglin C. (A) Standard additions of {alpha}1-AT to sputum sol samples achieved increasing but less than maximal inhibition of NE activity; complete inhibition was, however, achieved in a parallel study with purified human NE of activities comparable to those in the sputum sol samples. (B) Standard additions of SLPI to sputum samples achieved percent inhibition significantly higher than that achieved in a parallel study with purified human NE. (C) Standard additions of the NE-specific tripeptide inhibitor, eglin C, achieved comparable percent inhibition of activity in both the sol samples and the purified human NE preparation. Results are shown as means ± SD of data from 10 different sputum samples (open bars) (active site-titrated NE concentration range, 0.9–1.2 µM) and from similar concentrations of a purified human NE preparation (solid bars), each assayed in triplicate. *p < 0.001.

 
Similar experiments performed with exogenous SLPI yielded different results. The activities of both sputum sol NE and purified human NE could be completely inhibited by standard addition of 1.2 µM SLPI. With standard addition of 0.6 µM SLPI, 60% inhibition of active-site titrated purified human NE was achieved, contrasting with complete inhibition of sputum sol NE activity (Figure 1B). With standard addition of 0.3 µM SLPI, the percentage inhibition of purified human NE activity was, similarly, lower than that of sputum sol NE activity (Figure 1B). This suggests that the sputum sol samples contain factors that enhance the inhibitory potential of exogenous SLPI.

We then tested for inhibition of NE activity by the eglin C peptide (fragment 60–63), a specific inhibitor of human NE. Sputum sol NE was not different from purified human NE when presented with standard additions of eglin C peptide. This contrasted with the differential inhibition seen when sputum sol NE was presented with exogenous antielastases that are proteins in nature. The persistently dominant NE activity in bronchial secretions of patients with bronchiectasis was thus not simply due to an imbalance between elastase and antielastases. Rather, the results suggested that sputum sol NE was associated with macromolecular sputum components that limited the inhibitory effect of {alpha}1-AT and facilitated the inhibitory effect of SLPI.

Sputum Sol NE Remains Proteolytically Active when Complexed with Polyanions
To find NE activity among the sputum sol components, we performed casein zymography on the sol samples. In all samples studied, we observed a Coomassie blue-clear zone that extended from the origin to a front of nominally 90 kD (Figure 2A , lane 1). The polydisperse nature was not due to overloading as dilution of the sputum sol decreased the clarity without any significant effect on the zonal spread (Figure 2A, lanes 2 and 3). Neither was it due to storage as freshly prepared sol samples gave similar profiles. Heating of the sol sample resulted in null activity (Figure 2A, lane 4). Incubation of the zymographic gel with NE-specific eglin C peptide (37) also resulted in null activity (Figure 2B), thus indicating that the caseinolytic activity was due entirely to NE. Metalloproteinase activity was not involved as incubations were performed in the presence of 0.2 M EDTA. Separate casein zymography of sputum sol with exogenous collagenase-1 (matrix metalloproteinase-1, 100 µM or less) showed that 0.2 M EDTA was sufficient to inhibit caseinolytic activity due to the added matrix metalloproteinase-1 (result not shown). Western blot of a parallel casein gel found that the caseinolytic zone at high molecular mass was immunopositive for human NE protein (Figure 2D). This contrasts with the immunopositive band (about 30 kD) shown by the purified human NE preparation (Figure 2E). The results suggested that NE in sputum sol existed not as free monomers, but in polydisperse associations across a range of molecular sizes and, as such, it remained proteolytically active.



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  		Figure 2. Sputum sol contains proteolytically active NE in association with polyanionic macromolecules. The casein zymogram of aliquots of a sol sample is shown with a Coomassie blue-stained background. The Coomassie blue-clear zone at 90–220 kD indicates NE activity (A), which could be inhibited by preincubation of zymograms with the NE-specific inhibitor peptide, eglin C (B). Further staining of the Coomassie blue-stained zymogram with alcian blue reveals stained polyanions in the NE-active region (C). Western blot of a parallel zymogram found immunopositivity for the human NE protein in the NE-active region (D), contrasting that at about 30 kD for a commercial preparation of purified human NE (E). Results shown are representative of all 10 sputum samples studied. (A–D) Lane 1, undiluted sol sample; lane 2, 1:2 dilution of sol sample; lane 3, 1:4 dilution of sol sample; lane 4, heat-inactivated sol sample. (E) Human NE (Sigma); lane 1, 200 µg/ml; lane 2, 50 µg/ml. Positions of reference molecular mass markers are shown on the left.

 
Sputum Sol NE is Complexed with Syndecan-1
The polydisperse caseinolytic profile of sputum sol NE suggested complexation of NE with polyanionic partners. This was evidenced by alcian blue staining at the Coomassie blue-clear zone of the zymogram (Figure 2C). To identify the polyanionic partner(s) in question, we probed the Western blots of sputum sol samples with monoclonal antibodies against the target molecules in addition to that against human NE. Blots probed with anti-HS revealed an immunopositive range of 97–220 kD (Figure 3B) , similar to that of the NE-positive staining in the same blot (Figure 3A). This suggested that the polyanionic macromolecules that associated with sputum sol NE comprised HS glycoforms. To find the core proteins of the HS glycoforms, blots were probed in turn with antibodies against syndecan and perlecan. The 97- to 220-kD zone was found positive for syndecan-1 (Figure 3C) but negative for perlecan (results not shown). This suggested that soluble syndecan-1 fragments bearing HS substituents were complexed with NE in the sputum sol of patients with bronchiectasis.



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  		Figure 3. Sputum sol NE is associated with heparan sulfate (HS), syndecan-1, SLPI, and {alpha}1-AT. The Western blot of a sol sample was probed in turn with antibodies against human NE (A), HS (B), syndecan-1 (C), SLPI (D), and {alpha}1-AT (E). Western blot analysis indicates immunopositive NE colocalized with HS/syndecan-1, SLPI, and subpopulations of {alpha}1-AT at 90–220 kD in the sputum sol. Results shown are representative of all 10 sputum samples studied. Positions of reference molecular mass markers are shown on the left.

 
Sputum Sol NE–Syndecan-1 Complex Includes Physiologic Antielastases SLPI and {alpha}1-AT
To determine whether physiological antielastases are included in the polydisperse association of NE and HS/syndecan-1, blots were further probed with monoclonal antibodies against human SLPI and {alpha}1-AT. An SLPI-positive zone was observable in the 97- to 220-kD range (Figure 3D), similar in range to immunopositivities observed for human NE, HS, and syndecan-1 (Figures 3A–3C). In contrast, bands immunopositive for {alpha}1-AT were not only distinguishable in the 97- to 220-kD range, but also as far down as 34 kD (Figure 3E). Although the colocalization of NE and {alpha}l-AT positivities in the 97-kD (or greater) range may suggest associations between the two, {alpha}l-AT-positive components in the 97-kD (or less) range are considered not directly involved in the associations. Clearly, in the 97- to 220-kD range, despite the colocalization of SLPI and {alpha}1-AT with NE and HS/syndecan-1, proteolytic activity was preserved.

Sputum Sol NE Associations Are Responsible for the Differential Inhibitory Effects of {alpha}1-AT and SLPI
To confirm that it was sputum sol NE in the complexed form that bound and became inhibited by the exogenous antielastases, transblots of sputum sol were incubated with either {alpha}1-AT or SLPI and then tested for caseinolytic activity by a casein gel overlay method. Incubation of the transblot with {alpha}1-AT did not result in any significant difference in the caseinolytic pattern when compared with the control (Figure 4A , top). Western ligand blot with the antibody against {alpha}1-AT indicated significant additional binding of {alpha}1-AT at bands of 33–77 kD (Figure 4A, bottom), where these were immunopositive neither for NE nor HS/syndecan-1. Not much additional binding of {alpha}1-AT was observed in the 97- to 220-kD region, where NE and HS/syndecan-1 were located and where caseinolytic activity was demonstrated. The difference between the {alpha}1-AT binding and the zymographic patterns indicated that {alpha}1-AT was prevented from both binding and inhibiting NE when NE was complexed with HS/syndecan-1. In contrast, incubation of the sputum sol transblot with recombinant SLPI eliminated all signs of caseinolytic activity observable in the gel overlays (Figure 4B, top). Western ligand blot with the antibody against SLPI indicated significant additional binding of SLPI only to the 97- to 220-kD region, where caseinolytic activity had been observed (Figure 4B, bottom). The binding of SLPI to the region immunopositive for both human NE and HS/syndecan-1, together with the observed inhibition of NE activity, confirmed that the NE–HS/syndecan-1 complex in sputum sol was accessible to effective inhibition by exogenous SLPI.




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  		Figure 4. Sputum sol NE associations are responsible for the differential inhibitory effects of {alpha}1-AT and SLPI. The Western blot of a sol sample was performed in duplicate sets. One set was incubated with one of the exogenous inhibitors, {alpha}1-AT (A) or SLPI (B), and the duplicate set was blank-incubated as control. In (A), the gel overlay assay revealed caseinolytic activity at 97–220 kD not only after control incubation, but also after test incubation with {alpha}1-AT. The corresponding Western ligand blot indicated preferential binding of {alpha}1-AT at 34–77 kD, rather than at 97–220 kD. In (B), the gel overlay assay revealed caseinolytic activity at 97–220 kD after control incubation but null activity after test incubation with SLPI. The corresponding Western ligand blot indicated selective binding of SLPI at 97–220 kD. Sputum sol samples: lane 1, undiluted; lane 2, 1:2 dilution; lane 3, 1:4 dilution; lane 4, heat inactivated. Results shown are representative of all 10 sputum samples studied. Positions of reference molecular mass markers are shown on the left.

 
Dissociation of NE from the Sputum Sol Complex Results in Effective Inhibition by SLPI
We attempted to separate the proteoglycans from NE and other associations by dissociative density gradient ultracentrifugation of sputum sol samples. Only the fraction of highest density (1.74 g/ml, fraction 6) indicated significant caseinolytic activity and this was observable in the 97-kD (or greater) range (Figure 5A , lane 4). The corresponding Western blot revealed that only fraction 6 was immunopositive for human NE protein in a similar size range; lower density fractions (fractions 3–5) showed immunopositivity for human NE protein at 97 kD (Figure 5B, lanes 1–3), where little or no caseinolytic activity was observable (Figure 5A, lanes 1–3). Interestingly, only fraction 6 indicated HS/syndecan-1 immunopositivities colocalized with both NE protein and activity (Figures 5A–5D, lanes 3 and 4). Although fraction 5 was also immunopositive for HS/syndecan-1, this was not colocalized with the human NE protein (Figures 5C and 5D versus Figure 5B, lanes 3). Fractions 3–5 therefore revealed NE subpopulations dissociated from polydisperse associations with HS/syndecan-1. The lack of caseinolytic activity in these fractions suggested that NE subpopulations dissociated from HS/syndecan-1 were effectively inhibited by the endogenous inhibitor proteins. Indeed, when probed for SLPI and {alpha}1-AT blots of fractions 3–5 showed colocalization of SLPI and NE immunopositivities at 90 kD (Figures 5B and 5E, lanes 1–3), distinct from the {alpha}1-AT immunopositivity at 50 kD (Figure 5F, lanes 1–3). Thus, only where the NE protein remained in polydisperse association with HS/syndecan-1 was NE activity preserved.



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  		Figure 5. Sputum sol NE is in part freed from association with syndecan by dissociative density gradient ultracentrifugation. Fractions recovered from dissociative density gradient centrifugation of a sputum sol sample were analyzed by casein zymography (A) and Western blotting, probed in turn for human NE (B), HS (C), syndecan-1 (D), SLPI (E), and {alpha}1-AT (F). (A–F) Lanes 1–4, fractions 3–6 (in order of increasing density) recovered from dissociative density gradient centrifugation of a representative sputum sol sample. NE remained active where it was colocalized with HS/syndecan-1 (A–D, lane 4) but became inactive otherwise (A–E, lanes 1–3). Results shown are representative of all 10 sputum samples studied. Positions of reference molecular mass markers are shown on the left.

 
Heparin Displaces NE and SLPI from Sputum Sol Complex
To determine whether the HS moiety contributed to the association of the HSPG with NE, heparin was used to displace NE from the association in sputum sol. In Western blots of heparin-treated sol samples, we found immunopositive NE not only in the range of 97–220 kD but also at 50 kD; this latter was not observed in the untreated control (Figure 6B , lanes 2–4 versus lane 1). The NE immunopositivity increased both in intensity at 50 kD and in dispersal in the 97- to 220-kD range with increasing doses of heparin. However, only NE in the 97- to 220-kD range remained colocalized with syndecan-1 (Figure 6C). The displacement of NE so revealed supports the notion that heparin-like domains in the HS moiety play a major part in the binding of NE.



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  		Figure 6. Sputum sol NE is in part displaced from association with HS/syndecan-1 by incubation with heparin. Heparin-treated sol samples were analyzed by casein zymography (A) and Western blotting, probed in turn for human NE (B), syndecan-1 (C), {alpha}1-AT (D), and SLPI (E). Heparin-dependent decrease both in the spread and intensity of the NE-active zone (A) correlates with the increase in intensity of displaced NE at about 50 kD (B) where displaced {alpha}1-AT (D) and SLPI (E) could also be detected. Results shown are representative of all 10 sputum samples studied. (A–E) Lane 1, untreated sol sample; lanes 2–4, heparin treatment of sol sample, at 100, 10, and 1 µg/ml, respectively. Positions of reference molecular mass markers are shown on the left.

 
As NE displaced by heparin remains higher in molecular mass than that of purified human NE, it is likely that NE remains associated with sputum sol components. The lack of caseinolytic activity of the displaced NE (Figure 6A, lanes 2–4) suggested its association with the endogenous inhibitor SLPI and/or {alpha}1-AT. Western blots of heparin-treated sol samples were therefore reprobed with antibodies against {alpha}1-AT and SLPI. Indeed, the blots revealed colocalization of NE-, {alpha}1-AT-, and SLPI-positive materials at 50 kD (Figures 6B, 6D, and 6E, lanes 2–4). The results therefore confirmed our finding that NE activity was preserved where the NE protein remained in polydisperse association with HS/syndecan-1. Those dissociated from the complex represented subpopulations that were bound and inhibited by the endogenous antielastases.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first report that NE in bronchial secretions of patients with idiopathic bronchiectasis exists in the inflamed environment not as the free enzyme, but as a proteolytically active member of a supramolecular complex that includes HS/syndecan-1 and the physiological antielastases SLPI and {alpha}1-AT. Exogenous SLPI can bind the complexed NE and fully inhibit residual NE activity in the complex; exogenous {alpha}1-AT can, however, exert limited inhibition, being less accessible to the complexed enzyme. This contrasts with the full inhibition achievable by both the antielastases on a purified preparation of NE. Our results not only suggest that complexation with HS/syndecan-1 renders sputum NE resistant to inhibition by {alpha}1-AT, but also offer HS/syndecan-1 as a new target to resolve the long-standing problem of the relative ineffectiveness of {alpha}1-AT defenses against progressive airway destruction in bronchiectasis and other conditions characterized by chronic mucous production.

Syndecan ectodomains are shed from epithelial cell surfaces by actions of released NE and activated metalloproteinases triggered in inflammatory environments (38–40). The ectodomains bear HS substituents that contribute heparin-like sulfated sequences to the binding of NE and SLPI (27, 41). Our finding that heparin can displace both NE and SLPI from the sputum complex confirms this. We also found that exogenous SLPI could inhibit sputum NE activity at a level higher than that due to the same standard additions to purified NE. This can be attributed to a mediating role of HS, not only in the binding of the candidate ligands, but also in allowing the bound forms to interact. Indeed, heparin-like moieties have been reported to promote NE–SLPI interaction (25, 26) due to a heparin-induced conformational change of SLPI (42, 43). In contrast, NE bound to purified syndecan ectodomains was found to be reduced in its affinity for {alpha}1-AT (30), similar to our observation of limited {alpha}1-AT inhibition of NE activity in the sputum complex on standard additions of {alpha}1-AT.

As proteinases in inflammatory fluids coexist with their physiological inhibitors, proteinase–antiproteinase balance has been considered a priori the means to achieving optimal activity for host defense or tissue repair (8). We suggest that when the local production of SLPI is overwhelmed and plasma-derived {alpha}1-AT becomes the major defense, complexed forms of NE become a limiting factor in the extent of inhibition possible from {alpha}1-AT, thus tipping the balance toward misregulated tissue injury. Indeed, these can take the form either of NE complexed with HS/syndecan in bronchial secretions, as we found in this study, or of NE complexed with alveolar macrophage-derived lipids in bronchoalveolar lavage fluids of patients with pneumonia (44). This is in addition to the reported consequences of oxidative or proteolytic inactivation of {alpha}1-AT in bronchial secretions of patients with chronic obstructive pulmonary diseases (45, 46). Coadministration of heparin and SLPI into the airways was found to ameliorate the situation in a sheep model of asthma, as shown by significant reductions of bronchoconstriction and bronchial hyperreactivity with treatment (25). In inflammatory synovial fluids of rheumatoid arthritis, where SLPI is not a native constituent, NE is found complexed with plasma-derived {alpha}1-AT (47, 48) and {alpha}2-macroglobulin (49); the issue of unopposed NE activity remains as yet unresolved.

In bronchiectasis, a condition in which both persistent bacterial colonization and inflammatory response coexist, the implications of syndecan-1 shedding and benefits of restoration of cell surface syndecan-1 would be much more complex than in sterile inflammatory conditions such as asthma and rheumatoid arthritis. The common occurrence of bacteria, especially P. aeruginosa, in the airways of patients with bronchiectasis poses a significant concern in view of the report that P. aeruginosa activates shedding of syndecan-1 from the lung epithelium and exploits the shed syndecan-1 to enhance its own virulence (50). As this did not involve direct interaction with the bacteria, the shed syndecan ectodomain was postulated to act by interfering with host defense (50).

In the context of our finding of differential modulation of SLPI and {alpha}1-AT in bronchiectasis, it is possible that in patients with bronchiectasis and cystic fibrosis with P. aeruginosa, HSPG shedding is contributing to the rapidly downhill course of lung function decline and persistent microbial infection, via both interference with antielastase action of {alpha}1-AT and enhancement of P. aeruginosa virulence. The fact that frank invasion of P. aeruginosa seldom occurs in bronchiectasis may still allow consideration in favor of replenishing airway HSPG, especially in those subjects without P. aeruginosa colonization, or supplementing the airways with HS glycoforms and SLPI, concomitantly with energetic administration of anti-P. antibiotics. Hence, whether or not the unopposed elastase activity observed in the bronchial secretions of patients with chronic airway inflammation can be resolved by targeting the differential modulation of proteolytic inhibitors by HSPG remains to be investigated. This seems worthy of further work in light of the need to achieve optimal proteolytic activity for normal tissue repair.


   FOOTNOTES
 
Supported by grants from the Committee on Research and Conference Grants, University of Hong Kong, and the Hong Kong Research Grants Council.

Received in original form August 9, 2002; accepted in final form April 13, 2003


   REFERENCES
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 RESULTS
 DISCUSSION
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