Scandinavian Journal of Clinical and Laboratory Investigation
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation D. J. Buttle, D. Burnett & M. Abrahamson To cite this article: D. J. Buttle, D. Burnett & M. Abrahamson (1990) Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation, Scandinavian Journal of Clinical and Laboratory Investigation, 50:5, 509-516, DOI: 10.1080/00365519009089165 To link to this article: http://dx.doi.org/10.1080/00365519009089165
Published online: 29 Mar 2011.
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Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iclb20 Download by: [University of California, San Diego]
Date: 25 February 2016, At: 21:21
Scand J Clin Lab Invest 1900; 50: 500-516
Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation
Downloaded by [University of California, San Diego] at 21:21 25 February 2016
D. J . BUTTLE, D. BURNETT,* & M. ABRAHAMSONt Department of Biochemistry, Strangeways Research Laboratory, Cambridge, *Department of Immunology, University of Birmingham, and The Lung Immunobiological Research Laboratory, The General Hospital, Birmingham, UK, $Department of Clinical Chemistry, University of Lund, University Hospital, Lund, Sweden
Buttle DJ, Burnett D , Abrahamson M. Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation. Scand J Clin Lab Invest 1990; SO: 509-516. Sputum samples from 25 patients with bronchiectasis were assayed enzymatically for myeloperoxidase, neutrophil elastase and cathepsin B, and immunologically for cystatin A , cystatin B, cystatin C, cystatin S and kininogen. High rnyeloperoxidase and neutrophil elastase levels were found in those sputum samples that were assessed visually to be purulent. These samples were also found to contain high levels of cathepsin B activity and cystatin A , but low levels of cystatin S and of the most effective cathepsin B inhibitor, cystatin C. In contrast, sputum samples that were low in myeloperoxidase and neutrophil elastase activities had low levels of cathepsin B and cystatin A, but high cystatin C and S levels. It is concluded that cathepsin B activity in sputum is positively correlated with the degree of inflammation and neutrophil recruitment. Although this may be due in part to reduced amounts of cathepsin B inhibitors, particularly cystatin C, theoretical considerations suggest that factors other than the gross level of inhibitors must be involved in the control of cathepsin B activity. Key words: a,-macroglobulin; bronchiectasis; myeloperoxidase; neutrophil degranulation David .I. Buttle, Deparlment of Biochemistry, Strangeways Research Laboratory, Worts Causeway, Cambridge CBI 4RN, UK
The presence of proteinases in lung secretions has been implicated in the pathogenesis of several lung diseases, including cystic fibrosis and emphysema [I]. It has previously been shown, for instance, that neutrophil elastase (EC 3.4.21.37) can damage the bronchial epithelium and reduce ciliary function [2], both of which are features of bronchiectasis (3, 41.
Sputum samples from patients with bronchiectasis can be assessed visually to be mucoid (colourless), mucopurulent (yellow) or purulent (yellow to green) [S]. The colour has been considered to be due to the presence of myeloperoxidase from degranulating neutrophils (61 and it has been shown previously that elastase activity is present in purulent, but
SO9
Downloaded by [University of California, San Diego] at 21:21 25 February 2016
510
D. J . Buttle et al.
not mucoid, sputum [ S , 7, 8). Thus, the formation of purulent sputum can be regarded as part of the inflammatory process [9] and the level of myeloperoxidase activity can be used as a measure of the degree of inflammation. The cysteine proteinase cathepsin B ( E C 3.4.22.1) i s normally found i n lysosomes. Cathepsin B-like activity has been detected in human bronchoalveolar lavage fluid and sputum (10- 121, and the amount has been correlated with the number of neutrophils in lavage fluid [ 111. The activity in sputum has indeed been found to be due to a form of cathepsin B, albeit with unusual physicochemical properties. the most striking being stability at neutral and mildly alkaline pH [ 131. Cathepsin B is capable of degrading extracellular matrix components such as proteoglycan [14]. It is optimally active under mildly acidic conditions; this requirement may be satisfied in the bronchial and tracheal passages around metabolically active cells [ 151. The cystatins are a superfamily of reversible, tightly-binding inhibitors of cysteine proteinases. Five representatives have so far been found in human cells and body fluids: cystatins A, B and C, several very closely related forms of cystatin S, and kininogen [16]. Of these, cystatin C is the tightest-binding inhibitor of cathepsin B, by at least one order of magnitude [16, 17). It has been suggested that the cystatins play a defensive role, protecting the organism from cysteine proteinases of invading pathogens and parasites, and from endogenous cysteine proteinases that may escape from the lysosomes [ 16). However, no direct evidence for this has been found t o date. Cysteine proteinases and cystatins have not been found in the same compartment in vivo, and the demonstration of it7 v i v o enzyme-inhibitor complexes is still awaited. We have now assayed sputum samples from 2.5 patients with bronchiectasis for myeloperoxidase, neutrophil elastase and cathepsin B activities, and for the presence of cystatins A , B, C, S and kininogen by quantitative immunoassays. The relative activities and levels havc been examined statistically and conclusions drawn regarding their relationships with inflanimation. Possible explanations for the lack of total inhibition of cathepsin B in inflamed sputum are discussed.
MATERIALS AND METHODS Collection of sputum sumples Twenty-five patients with clinical and radiological evidence of bronchiectasis were studied. None of the subjects was receiving antimicrobial o r corticosteroid therapy at the time of sputum collection. Sputum was collected in sterile plastic containers during 4 h in the morning after rising. All subjects were instructed to avoid collecting saliva with the sputum. This method of sputum collection has been shown to result in minimal contamination with saliva, as judged by levels of oro-pharyngeal bacteria [ 1x1. The samples were ultracentrifuged at SO 000 g, 3 "C for 90 min to obtain the sol phase. which was stored at -70 "C until it was analysed. My eloperoxiduse assay
This was adapted from the published method [ 191 for use in microtitre plate wells. Calibration standards consisted o f dilutions of a neutrophil lysate. The neutrophils were obtained by Percoll density centrifugation [20] in RPMI 1640 medium. The isolated cells (96'%)neutrophils) were lysed in 1 mmol/l phosphate buffer, pH 6.0, containing O.S% hexadecyltrimethylammonium bromide, by freeze-thawing and homogenization in a Dounce homogenizer. One unit was equivalent to myeloperoxidase activity from 5~ lo3 neutrophils. Myeloperoxidase activity in sputum samples was then calculated by interpolation on the linear calibration line. Neutrophil elustuse ussuy The sputum samples, appropriately diluted, were incubated in SO mmoVl Tris/HCI, 100 mmol/l NaCI, pH 8.6 at 40 "C with 2.5 mmol/l succinyl-1.-alanyl-L-alanyl-L-alanine p-nitroanilide (Peptide Institute Inc., Osaka, Japan). After 10, 30 or 60 min the enzyme reaction was stopped by the addition of 1 ml of 100 mg/l soya bean trypsin inhibitor (Type 11-S, Sigma, Poole, UK) in water. The 4-nitroaniline released was determined by measurement of A 4 , , , ( ~ = 8 800imolillcm). O n e unit of activity corresponded to the release of 1 nmol 4-nitroaniline/ min under these conditions.
Cathepsin B and inhibitors in sputum
Downloaded by [University of California, San Diego] at 21:21 25 February 2016
Cathepsin B assay
51 1
Gel permeation Chromatography
The assay for cathepsin B activity in sputum samples, and the unit of enzyme activity, have been described [13]. The assay was adapted for determining the effect of dilution on cathepsin B activity as follows. Differing amounts of sputum (0-800 pl) were placed in incubation tubes. One-hundred pl of 1 mol/l2-(N-morpho1ino)ethanesulphonic acid, 1 mmoYl E D T A , 20 mmoVl cysteine, pH 5.5 was added, and the volume made up to 975 pl with water. The tubes were incubated at 40 "C for 5 min and the enzyme reaction was started by the addition of substrate. After appropriate times, from 112 s to 30 min, the reaction was stopped by the addition of the colour reagent.
Quantitation of the cystatins Specific polyvalent antisera against human cystatins A , B, C and S a n d kininogen were used in single radial imrnunodiffusion amplified by peroxidase-labelled goat antibodies against rabbit IgG, as previously described [17]. A concentrated urine sample from a patient with proteinuria served as standard in all cystatin assays. The concentrations of cystatins in this secondary standard were obtained using solutions of pure cystatins A , B, C, S and kininogen; protein content was determined by amino acid analysis.
Statistical analyses The activities and levels measured were compared by simple linear regression, and the null hypothesis that there was no correlation was tested using the F-test.
A sample of pooled sputa with high myeloperoxidase activity was dialysed into 50 mmoVl Tris/HCI, 0.5 mol/l NaCl, 0.1% 3-[(3cholamidopropyl)-dimethylammonio]- 1-propane sulphonate (CHAPS) in 20% (v/v) glycerol, pH 7.5. It was then clarified by centrifugation and passed through a TSK-G 2000 SW (LKB) gel chromatography column (7.5 x600 mm). Eluted fractions (0.5 ml) were assayed for cathepsin B activity. Another aliquot from the same sputum sample was dialysed into 0.1 mol/l Na phosphate, pH 6.0. It was clarified by centrifugation, and dithiothreitol was added t o 1 mmol/l. The sample was incubated at 30 "C for 15 min. Under these conditions, lysosomal cathepsin B is bound to a2-macroglobulin [21]. The mixture was applied to the gel chromatography column and eluted fractions were assayed for cathepsin B activity. Purified human a2-macroglobulin (a generous gift from D r J. S. Mort) was run on the same column. Protein was monitored at
RESULTS
My eloperoxidase The levels of myeloperoxidase in the individual sputum samples varied by over three orders of magnitude (range 1.9-2050 unitdml, mean=584 unitdrnl).
Elastase and cathepsin B Neutrophil elastase and cathepsin B activities varied by over three and over two orders of
TABLE I. Correlation coefficients and confidence limits derived from linear regression analyses of the enzyme activities and cysteine proteinase inhibitor levels in individual sputum samples. Paired data were analysed by linear regression analysis, and confidence limits determined by the F-test of the anovar M yeloperoxidase ~~
Elastase Cathepsin B Cystatin A Cystatin C Cystatin S
~
Cathepsin B
Elastase
~~~~
~
r*
(P)t
0.830 0.832 0.728 -0.602 -0.436
(0.0oOl) (0.OOOl) (0.OOOl) (0.002) (0.03)
-
r
(PI
0.805 0.760 -0.585 -0.289
(0.0001) (0.0001) (0.002) (N.S.)
~~
*Correlation coefficient. ?Confidence limits obtained by use of the F-statistic.
-
0.881 -0.618
-0.532
(0.000lj (0.001j (0,006)
Downloaded by [University of California, San Diego] at 21:21 25 February 2016
512
D. J . Buftle et al.
magnitude, respectively, the ranges being 0.08565 u n i t s h l (mean=107) and 0.7- 144.5 (mean=65.8) unitslml, respectively. Linear regression demonstrated a strong positive correlation between myeloperoxidase and neutrophil elastase activities (r=O.830; p
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation D. J. Buttle, D. Burnett & M. Abrahamson To cite this article: D. J. Buttle, D. Burnett & M. Abrahamson (1990) Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation, Scandinavian Journal of Clinical and Laboratory Investigation, 50:5, 509-516, DOI: 10.1080/00365519009089165 To link to this article: http://dx.doi.org/10.1080/00365519009089165
Published online: 29 Mar 2011.
Submit your article to this journal
Article views: 23
View related articles
Citing articles: 1 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iclb20 Download by: [University of California, San Diego]
Date: 25 February 2016, At: 21:21
Scand J Clin Lab Invest 1900; 50: 500-516
Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation
Downloaded by [University of California, San Diego] at 21:21 25 February 2016
D. J . BUTTLE, D. BURNETT,* & M. ABRAHAMSONt Department of Biochemistry, Strangeways Research Laboratory, Cambridge, *Department of Immunology, University of Birmingham, and The Lung Immunobiological Research Laboratory, The General Hospital, Birmingham, UK, $Department of Clinical Chemistry, University of Lund, University Hospital, Lund, Sweden
Buttle DJ, Burnett D , Abrahamson M. Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation. Scand J Clin Lab Invest 1990; SO: 509-516. Sputum samples from 25 patients with bronchiectasis were assayed enzymatically for myeloperoxidase, neutrophil elastase and cathepsin B, and immunologically for cystatin A , cystatin B, cystatin C, cystatin S and kininogen. High rnyeloperoxidase and neutrophil elastase levels were found in those sputum samples that were assessed visually to be purulent. These samples were also found to contain high levels of cathepsin B activity and cystatin A , but low levels of cystatin S and of the most effective cathepsin B inhibitor, cystatin C. In contrast, sputum samples that were low in myeloperoxidase and neutrophil elastase activities had low levels of cathepsin B and cystatin A, but high cystatin C and S levels. It is concluded that cathepsin B activity in sputum is positively correlated with the degree of inflammation and neutrophil recruitment. Although this may be due in part to reduced amounts of cathepsin B inhibitors, particularly cystatin C, theoretical considerations suggest that factors other than the gross level of inhibitors must be involved in the control of cathepsin B activity. Key words: a,-macroglobulin; bronchiectasis; myeloperoxidase; neutrophil degranulation David .I. Buttle, Deparlment of Biochemistry, Strangeways Research Laboratory, Worts Causeway, Cambridge CBI 4RN, UK
The presence of proteinases in lung secretions has been implicated in the pathogenesis of several lung diseases, including cystic fibrosis and emphysema [I]. It has previously been shown, for instance, that neutrophil elastase (EC 3.4.21.37) can damage the bronchial epithelium and reduce ciliary function [2], both of which are features of bronchiectasis (3, 41.
Sputum samples from patients with bronchiectasis can be assessed visually to be mucoid (colourless), mucopurulent (yellow) or purulent (yellow to green) [S]. The colour has been considered to be due to the presence of myeloperoxidase from degranulating neutrophils (61 and it has been shown previously that elastase activity is present in purulent, but
SO9
Downloaded by [University of California, San Diego] at 21:21 25 February 2016
510
D. J . Buttle et al.
not mucoid, sputum [ S , 7, 8). Thus, the formation of purulent sputum can be regarded as part of the inflammatory process [9] and the level of myeloperoxidase activity can be used as a measure of the degree of inflammation. The cysteine proteinase cathepsin B ( E C 3.4.22.1) i s normally found i n lysosomes. Cathepsin B-like activity has been detected in human bronchoalveolar lavage fluid and sputum (10- 121, and the amount has been correlated with the number of neutrophils in lavage fluid [ 111. The activity in sputum has indeed been found to be due to a form of cathepsin B, albeit with unusual physicochemical properties. the most striking being stability at neutral and mildly alkaline pH [ 131. Cathepsin B is capable of degrading extracellular matrix components such as proteoglycan [14]. It is optimally active under mildly acidic conditions; this requirement may be satisfied in the bronchial and tracheal passages around metabolically active cells [ 151. The cystatins are a superfamily of reversible, tightly-binding inhibitors of cysteine proteinases. Five representatives have so far been found in human cells and body fluids: cystatins A, B and C, several very closely related forms of cystatin S, and kininogen [16]. Of these, cystatin C is the tightest-binding inhibitor of cathepsin B, by at least one order of magnitude [16, 17). It has been suggested that the cystatins play a defensive role, protecting the organism from cysteine proteinases of invading pathogens and parasites, and from endogenous cysteine proteinases that may escape from the lysosomes [ 16). However, no direct evidence for this has been found t o date. Cysteine proteinases and cystatins have not been found in the same compartment in vivo, and the demonstration of it7 v i v o enzyme-inhibitor complexes is still awaited. We have now assayed sputum samples from 2.5 patients with bronchiectasis for myeloperoxidase, neutrophil elastase and cathepsin B activities, and for the presence of cystatins A , B, C, S and kininogen by quantitative immunoassays. The relative activities and levels havc been examined statistically and conclusions drawn regarding their relationships with inflanimation. Possible explanations for the lack of total inhibition of cathepsin B in inflamed sputum are discussed.
MATERIALS AND METHODS Collection of sputum sumples Twenty-five patients with clinical and radiological evidence of bronchiectasis were studied. None of the subjects was receiving antimicrobial o r corticosteroid therapy at the time of sputum collection. Sputum was collected in sterile plastic containers during 4 h in the morning after rising. All subjects were instructed to avoid collecting saliva with the sputum. This method of sputum collection has been shown to result in minimal contamination with saliva, as judged by levels of oro-pharyngeal bacteria [ 1x1. The samples were ultracentrifuged at SO 000 g, 3 "C for 90 min to obtain the sol phase. which was stored at -70 "C until it was analysed. My eloperoxiduse assay
This was adapted from the published method [ 191 for use in microtitre plate wells. Calibration standards consisted o f dilutions of a neutrophil lysate. The neutrophils were obtained by Percoll density centrifugation [20] in RPMI 1640 medium. The isolated cells (96'%)neutrophils) were lysed in 1 mmol/l phosphate buffer, pH 6.0, containing O.S% hexadecyltrimethylammonium bromide, by freeze-thawing and homogenization in a Dounce homogenizer. One unit was equivalent to myeloperoxidase activity from 5~ lo3 neutrophils. Myeloperoxidase activity in sputum samples was then calculated by interpolation on the linear calibration line. Neutrophil elustuse ussuy The sputum samples, appropriately diluted, were incubated in SO mmoVl Tris/HCI, 100 mmol/l NaCI, pH 8.6 at 40 "C with 2.5 mmol/l succinyl-1.-alanyl-L-alanyl-L-alanine p-nitroanilide (Peptide Institute Inc., Osaka, Japan). After 10, 30 or 60 min the enzyme reaction was stopped by the addition of 1 ml of 100 mg/l soya bean trypsin inhibitor (Type 11-S, Sigma, Poole, UK) in water. The 4-nitroaniline released was determined by measurement of A 4 , , , ( ~ = 8 800imolillcm). O n e unit of activity corresponded to the release of 1 nmol 4-nitroaniline/ min under these conditions.
Cathepsin B and inhibitors in sputum
Downloaded by [University of California, San Diego] at 21:21 25 February 2016
Cathepsin B assay
51 1
Gel permeation Chromatography
The assay for cathepsin B activity in sputum samples, and the unit of enzyme activity, have been described [13]. The assay was adapted for determining the effect of dilution on cathepsin B activity as follows. Differing amounts of sputum (0-800 pl) were placed in incubation tubes. One-hundred pl of 1 mol/l2-(N-morpho1ino)ethanesulphonic acid, 1 mmoYl E D T A , 20 mmoVl cysteine, pH 5.5 was added, and the volume made up to 975 pl with water. The tubes were incubated at 40 "C for 5 min and the enzyme reaction was started by the addition of substrate. After appropriate times, from 112 s to 30 min, the reaction was stopped by the addition of the colour reagent.
Quantitation of the cystatins Specific polyvalent antisera against human cystatins A , B, C and S a n d kininogen were used in single radial imrnunodiffusion amplified by peroxidase-labelled goat antibodies against rabbit IgG, as previously described [17]. A concentrated urine sample from a patient with proteinuria served as standard in all cystatin assays. The concentrations of cystatins in this secondary standard were obtained using solutions of pure cystatins A , B, C, S and kininogen; protein content was determined by amino acid analysis.
Statistical analyses The activities and levels measured were compared by simple linear regression, and the null hypothesis that there was no correlation was tested using the F-test.
A sample of pooled sputa with high myeloperoxidase activity was dialysed into 50 mmoVl Tris/HCI, 0.5 mol/l NaCl, 0.1% 3-[(3cholamidopropyl)-dimethylammonio]- 1-propane sulphonate (CHAPS) in 20% (v/v) glycerol, pH 7.5. It was then clarified by centrifugation and passed through a TSK-G 2000 SW (LKB) gel chromatography column (7.5 x600 mm). Eluted fractions (0.5 ml) were assayed for cathepsin B activity. Another aliquot from the same sputum sample was dialysed into 0.1 mol/l Na phosphate, pH 6.0. It was clarified by centrifugation, and dithiothreitol was added t o 1 mmol/l. The sample was incubated at 30 "C for 15 min. Under these conditions, lysosomal cathepsin B is bound to a2-macroglobulin [21]. The mixture was applied to the gel chromatography column and eluted fractions were assayed for cathepsin B activity. Purified human a2-macroglobulin (a generous gift from D r J. S. Mort) was run on the same column. Protein was monitored at
RESULTS
My eloperoxidase The levels of myeloperoxidase in the individual sputum samples varied by over three orders of magnitude (range 1.9-2050 unitdml, mean=584 unitdrnl).
Elastase and cathepsin B Neutrophil elastase and cathepsin B activities varied by over three and over two orders of
TABLE I. Correlation coefficients and confidence limits derived from linear regression analyses of the enzyme activities and cysteine proteinase inhibitor levels in individual sputum samples. Paired data were analysed by linear regression analysis, and confidence limits determined by the F-test of the anovar M yeloperoxidase ~~
Elastase Cathepsin B Cystatin A Cystatin C Cystatin S
~
Cathepsin B
Elastase
~~~~
~
r*
(P)t
0.830 0.832 0.728 -0.602 -0.436
(0.0oOl) (0.OOOl) (0.OOOl) (0.002) (0.03)
-
r
(PI
0.805 0.760 -0.585 -0.289
(0.0001) (0.0001) (0.002) (N.S.)
~~
*Correlation coefficient. ?Confidence limits obtained by use of the F-statistic.
-
0.881 -0.618
-0.532
(0.000lj (0.001j (0,006)
Downloaded by [University of California, San Diego] at 21:21 25 February 2016
512
D. J . Buftle et al.
magnitude, respectively, the ranges being 0.08565 u n i t s h l (mean=107) and 0.7- 144.5 (mean=65.8) unitslml, respectively. Linear regression demonstrated a strong positive correlation between myeloperoxidase and neutrophil elastase activities (r=O.830; p
