Alpha-1-proteinase inhibitor activity can be compromised by the choice of assay conditions.
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Aged↗
Biomedical subjects
T D Tetley
Publications and source records attributed to T D Tetley.
New perspectives on basic mechanisms in lung disease. 6. Proteinase imbalance: its role in lung disease.
The hypothesis, some 30 years ago, that NE was the sole proteolytic agent responsible for the development of emphysema seems naive in retrospect. The availability of technology to measure NE facilitated the early research into the relationship between NE and lung disease. Despite an abundance of information on the activity of NE in the lung, it will probably require prospective studies in man with specific NE inhibitors or control at the gene level to establish a causal relationship between NE and lung disease. Parallel research has resulted in the isolation and characterisation of NE inhibitors other than PI and, indeed, alternative proteolytic enzymes that might contribute to lung disease. It is perhaps impossible now to think that a single proteinase, however omnipotent it may be, causes lung diseases as diverse as emphysema and fibrosis. An important aspect that is emerging is the interrelationship between proteolytic enzymes produced by different, or sometimes the same, cells that could potentiate tissue proteolysis. The evidence suggests that there is likely to be coordinated action between neutrophils, macrophages, and possibly mesenchymal proteinases which can activate or inactivate each other. In addition, one class of proteinases often appears able to proteolytically inactivate inhibitors of the opposite class, which presumably could amplify proteolysis if it occurred in vivo. Although the work on this aspect of proteinase activity is in its infancy, one suspects that part of the normal regulation of proteinase activity might include compartmentalisation. For example, the neutrophil stores proteinases before appropriate release and can inactivate PI to enable proteolytic action pericellularly, whereas degradation of extracellular matrix by macrophages requires interaction between the cell and matrix which is facilitated by cell receptor bound uPA. Disintegration of these "compartments" due to oedema, proteolysis, or for mechanical reasons could, firstly, expose further extracellular matrix substrates to inflammatory and damaged cell proteinases but, secondly, might enhance proteinase potential by the cooperative action of these enzymes. It seems increasingly likely that, where proteinases play a part, there is a cocktail of proteinases that is characteristic of the injury that develops (fig). What remains unclear is why only a proportion of those susceptible, such as smokers or those with acute lung injury, develop irreversible lung disease. This suggests that there are other factors acquired or inherited that need to be considered.
Endopeptidases↗
Isolation and characterization of sheep alpha 1-proteinase inhibitor.
Sheep plasma proteinase inhibitor, analogous to human alpha 1-proteinase inhibitor (alpha 1 PI), was isolated to homogeneity. Purification was achieved by using (NH4)2SO4 precipitation, concanavalin A-Sepharose chromatography, Mono Q ion-exchange chromatography and PAGE. Sheep alpha 1 PI had an Mr of 56,000, inhibited human leucocyte elastase, pig pancreatic elastase and bovine trypsin on a 1:1 molar basis and had a plasma concentration of 1.6 +/- 0.21 g/l (mean +/- S.D.). Amino acid/carbohydrate composition (15% glycosylated) was similar to that of human alpha 1 PI (16% glycosylated); N-terminal analysis to 31 residues revealed 48-52% identity between the human and sheep proteins. Sheep alpha 1 PI was susceptible to oxidative inactivation by chloramine-T. Re-activation with the use of methionine sulphoxide peptide reductase and dithiothreitol indicated the presence of a methionine residue at the active site. These results establish that sheep alpha 1 PI has functional and structural characteristics close to those of human alpha 1 PI.
Amino Acid Sequence↗
Urinary desmosine, elastolysis, and lung disease.
Desmosine is an amino acid specific to elastin. Animal studies suggest that urinary desmosine (UD) represents endogenous elastin degradation. Therefore, UD has previously been used to investigate endogenous elastolysis, but was not elevated in subjects with chronic obstructive airways disease (COAD), although accelerated pulmonary elastolysis is thought to contribute to COAD. We have investigated whether this reflects large day-to-day and between-subject variation in UD and whether, in man, dietary desmosine contributes significantly to that in urine. Mean 24-hour UD output (over 5 consecutive days) from 10 asymptomatic subjects (5 males) was higher in males than females (77.4 +/- 9.6 and 40.2 +/- 5.0 nmol/24 hours, respectively; mean +/- SD, P less than .001), but not significantly different when expressed in terms of creatinine (micrograms desmosine/100 mg creatinine: males, 2.5 +/- 0.4; females, 3.1 +/- 0.8; mean +/- SD). The lowest between-subject variation was observed when the mean of 5 days' 24-hour UD values was analyzed on the basis of gender (coefficient of variation [CV], 12.5%); when gender was not considered, the least between-subject variation was found for the mean of 5 days' desmosine/creatinine analysis (CV, 24.5%). Approximately 1% of dietary desmosine (ingested as [3H]elastin and [3H] desmosine) was excreted in the urine within 24 hours, contributing approximately 15% of UD while on a normal diet. Although ingestion of a low elastin diet (less than 1/10 desmosine/24 hours than a normal diet) resulted in lower within-subject variation in 24-hour UD excretion (mean CV decreased from 31.5% to 20.2%), the between-subject CV and UD levels did not alter.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
The blotchy mouse, lung desmosine, and emphysema.
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Animals↗
An assay for the assessment of lipocortin 1 levels in human lung lavage fluid.
The physiological function of the lipocortins, proteins which are thought to be glucocorticoid-regulated, is unclear. An improved assay for lipocortins might help to elucidate their role. A rapid and specific sandwich enzyme-linked immunosorbent assay (ELISA) for lipocortin 1 with a working range of 1-2000 ng/ml and an interrun coefficient of variation of less than 10% is described and used in this pilot study to quantify human lipocortin 1 for the first time in acellular bronchoalveolar lavage fluid (BALF), and in media conditioned by BAL cells, from control patients and those with pulmonary sarcoidosis. Using this assay a statistically significant relationship, not previously observed in man, has been demonstrated between concentrations of lipocortin 1/ml of BALF and serum cortisol levels (n = 10, rs = 0.6939, P less than 0.05). Although lipocortin 1 levels in acellular BALF were the same in control and sarcoid patients, significantly more lipocortin 1 was released from sarcoid BAL cells in culture (median 21.6, range 8.1-45.4 ng lipocortin/10(6) cells/h in culture) than from control cells (2.5, 1.5-7.6 ng lipocortin/10(6) cells/h in culture). The possible clinical significance of these data is discussed, but remains to be established.
Annexins↗
Type II pneumocytes in mixed cell culture of human lung: a light and electron microscopic study.
Alveolar Type II epithelial cells dedifferentiate rapidly in vitro. Studies with animal tissue suggest that cell-cell and extracellular matrix-cell interactions are important in the retention of Type II cell morphology in vitro. Thus, in this study with human tissue, alveolar Type II cells, alveolar macrophages, and spindle cells were prepared from the same sample of lung (obtained following lobectomy for cancer, n = 3), cocultured on glass cover slips or tissue culture plastic, and studied by light microscopy with scanning (SEM) and transmission (TEM) electron microscopy for 8 days. The primary cell isolates contained approximately 45% Type II cells; the remainder were macrophages or unidentifiable cells. Clusters, made up of a single layer of cuboidal Type II cells around a central core of connective tissue (largely collagen and some elastic tissue), formed above a monolayer of spindle cells. The Type II cells were morphologically similar to those seen in vivo. The cells were still cuboidal at 8 days but had lost their lamellar bodies, which were released into the medium via the apical surface. The clusters increased in size with time (area, microns 2: day 1, 29(5-143) x 10(2); day 8, 63(10-311) x 10(2); mean(range); p less than 0.02) without changing in number per culture, suggesting Type II cell proliferation. This may have been due to factors produced by the other cells and adherence to the extracellular matrix (ECM); (free collagen fibers, present in the original preparation, spindle cells, and/or Type II cells could be responsible for presence of ECM). We propose this as a useful model for the study of human Type II epithelial cells in vitro.
Cell Differentiation↗
Detection of lipocortin 1 in human lung lavage fluid: lipocortin degradation as a possible proteolytic mechanism in the control of inflammatory mediators and inflammation.
Lipocortins are structurally related, glucocorticoid-inducible proteins that inhibit phospholipase A2 (PLA2), thereby reducing the liberation of arachidonic acid from phospholipids and so limiting the synthesis of eicosanoid inflammatory mediators. This study is the first demonstration of one lipocortin, lipocortin 1 (Lc 1; 37 kDa), in human lung lavage supernatants. In lavage fluid from healthy volunteers, a higher percentage (greater than 70%) of the detected Lc 1 was in its native form, compared to that from patients with abnormal lungs. In patients' lavage fluids, Lc 1 was more likely to be partially degraded (34 kDa). In abnormal bronchoalveolar lavage fluid (BALF), the more polymorphonuclear neutrophils (PMN)/lavage, the lower the proportion of Lc 1 in the native (37 kDa) form (n = 7 pairs, rs = -0.8214, p less than 0.05). Furthermore, when BALF cells were cultured and the harvested conditioned media incubated with pure human recombinant Lc 1, degradation of the 37 kDa form increased with the percentage of PMN (n = 10 pairs, s = -0.7200 after 1 hr; n = 6 pairs, rs = -0.9241 after 6 hr). These results suggest that factors released from the PMN are responsible for Lc 1 degradation in man. When recombinant human Lc 1 was incubated with human neutrophil elastase, the enzyme degraded Lc 1 in a dose-dependent way, suggesting that neutrophil elastase may be one such factor. Since PMNs are ubiquitous at sites of inflammation, it is possible that Lc 1 degradation is a permissive mechanism, which ensures that sufficient inflammation occurs to destroy the provocative stimulus. However, it is equally possible that, in some circumstances, the mechanism may be pathological and that the inactivation of Lc 1 leads to chronic, uncontrolled inflammation.
Adult↗
The acute effect of cigarette smoking on the neutrophil elastase inhibitory capacity of peripheral lung lavage from asymptomatic volunteers.
Cigarette smoke-induced emphysema is thought to involve reduction of antielastolytic capacity, resulting in elevated elastase activity and lung tissue damage. Peripheral lavage collected from ten asymptomatic subjects immediately before and 20 min after smoking two high tar cigarettes was analysed for neutrophil elastase (NE) inhibitory capacity (IC), alpha 1-proteinase inhibitor (PI) function, elastolytic activity and immunoreactive levels of PI and bronchial inhibitor (BI). The only change found was a small fall in mol immunoreactive PI/mol albumin after smoking (approximately 17%, p less than 0.05) which did not affect NEIC, since PI contributed less than 50% of the NEIC. There was often more NEIC than mol BI + functional PI, suggesting the presence of other NE inhibitors. Thoracic computerized tomography scans of eight of these subjects highlighted two with emphysematous regions of lung; lavage from these two subjects contained either undetectable BI or inactive BI and this suggests a protective role for BI in emphysema.
Adult↗
Emphysema in the Blotchy mouse: a morphometric study.
Inheritance of the Blotchy allele at the X-chromosomal Mottled locus of mice results in changes in the lung which resemble emphysema. Previous studies using measurements of mean linear intercept have recognized emphysema in the hemizygous, Blo/Y, male and homozygous, Blo/Blo, female, but not in the heterozygous, Blo/+, female. The aim of this study was to develop a rapid, accurate method for analysis of emphysema and to establish whether it would identify emphysema in the heterozygote. Lungs from wild type and outbred Blo mice were inflated with fixative at a pressure of 25 cm and then sectioned and stained. Transect lengths across air spaces were measured using computerized image analysis and the results were plotted as histograms. Data were also expressed as cumulative frequencies (ogives) and subjected to statistical analysis by the non-parametric Kolmogorov-Smirnov test. Emphysema was shown by significantly increased (P less than 0.001) transect lengths in the Blo/Blo female and Blo/Y male compared with the wild type controls. The heterozygous Blo/+ mice form an intermediate group significantly different (P less than 0.001) from the wild and blotchy mice. The sensitivity of the method is illustrated by the fact that the Blo/+ female formed an intermediate group between wild type and the Blo/Y male and Blo/Blo female.
Animals↗
Comparison of elastolytic activity in lung lavage from current, ex- and non-smokers.
Cigarette smokers have an increased risk of developing a wide variety of lung diseases compared to non-smokers, and there have been many studies of the possible biochemical changes underlying this increased susceptibility. However, although epidemiological and physiological studies have shown that in the ex-smoker, the risk of smoking-related lung diseases falls between that of current and non-smokers, the biochemical and cellular mechanisms responsible for this intermediate status have not been investigated. In the present study, therefore, the extracellular elastolytic activities in lung lavage fluid from current, ex- and non-smokers have been compared. Elastolytic enzyme activity was investigated, since it is significantly elevated in lung lavage from smokers, and because it has been implicated in the development of pulmonary emphysema. In the subjects studied, extracellular elastolytic activities in lung lavage from ex-smokers were intermediate between those from current and non-smokers. There was no correlation between the time of abstinence from smoking, or the number of pack-years smoked and the extracellular elastolytic activities in ex-smokers' lung lavage. Elastolytic activity may remain elevated in ex-smokers' lungs for a number of reasons, including the persistence of particulate matter which may activate phagocytic cells on the lung surface. The possible significance of the raised elastolytic activity remains to be fully determined.
Adolescent↗
Is neutrophil elastase associated with elastic tissue in emphysema?
To test the role of elastase in the pathogenesis of emphysema human neutrophil elastase (HNE) was localised by electron microscopy using an immunogold staining technique. Specific localisation of HNE to elastic tissue in emphysema did not occur, but non-specific binding of immunoglobulin G (IgG) to elastic tissue in emphysematous and normal lung tissue, which was completely blocked by the non-immune serum that was homologous to the gold labelled second antibody, was found. HNE was also present, however, in the granules of neutrophils in the same sections. Non-specific labelling associated with elastin was probably due to binding of IgG to the high numbers of hydrophobic and charged regions known to be present in this molecule, and it is concluded that our findings do not support the existence of high concentrations of elastase in association with elastin in emphysematous lung tissue.
Aged↗
Comparison of human lung surface protein profiles from the central and peripheral airways sampled using two regional lavage techniques.
This study describes two new techniques of lung lavage which selectively remove material from the central airways, or from the lung below the seventh generation. Bronchograms confirmed that discrete regions of the lung were washed by central lavage (CL; maximum airway diameter approximately 6.5 mm) and peripheral lavage (PL; maximum airway diameter approximately 1.3 mm), and that both could be clearly distinguished from conventional bronchoalveolar lavage (BAL). These techniques were used to establish whether or not large-airway proteins made a major contribution to the protein profile of BAL. Twenty consecutive patients undergoing routine fibreoptic bronchoscopy were investigated. More bronchial mucus proteinase inhibitor per unit albumin and per unit total measured antiproteinase was present in CL than PL or BAL. In contrast alpha 1-proteinase inhibitor per unit albumin and as a percentage of total measured antiproteinase was lower in CL than in other lavage types. There were no differences in elastase activity, irrespective of the way in which the data were expressed. As no differences were found between BAL and PL for any of the variables measured, it was concluded that in the subjects studied the contribution of CL proteins to BAL was minimal.
Adult↗
The elastase: anti-elastase profile in lung lavage from sarcoidosis patients.
One feature of pulmonary sarcoidosis is an increase of lymphocyte numbers in bronchoalveolar lavage fluid (BALF). In some patients the number of polymorphonuclear neutrophils (PMN) also rises. It has been suggested that the prognosis for the latter subjects is likely to be worse than that for the former, but the damaging agent (or agents) released by the PMN have not yet been identified. Therefore, in the current study, the activity of one neutrophilic enzyme, elastase, which is known to damage the pulmonary interstitium, has been assessed in sarcoid BALF and compared to BALF from subjects matched for age, sex and smoking status. As BALF also contains locally-produced and serum-derived inhibitors of elastase, levels of which may change in subjects with sarcoidosis, the serine protease inhibitory capacity and the levels of three anti-elastases have been measured in the samples. Levels of the serum-derived antiproteases, alpha 1 proteinase inhibitor (alpha 1PI) and alpha 2 macroglobulin (alpha 2M) were found to be significantly increased. However, alpha 1PI/albumin and alpha 2M/albumin ratios were unchanged, suggesting that the increased levels were due to an increased permeability of the alveolar-capillary barrier. The total protease inhibitory capacity was elevated and this could be entirely explained by the raised levels of alpha 1PI and alpha 2M. Levels of the locally-produced inhibitor were unaltered. The elastolytic capacity of sarcoid BALF was unchanged. Thus, the elastase: anti-elastase balance was shifted against elastolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Effects of cigarette smoking and drugs on respiratory tract proteases and antiproteases.
Increased pulmonary proteolytic (elastolytic) activity is thought to be the primary cause of emphysema and may also play a rôle in the pathology of bronchitis. These diseases are common amongst tobacco smokers. Serum-derived alpha 1-proteinase inhibitor (alpha 1PI) and locally produced protease inhibitors normally protect the pulmonary epithelium from proteolytic attack, but tobacco smoke can inactivate these antiproteases by oxidative and non-oxidative mechanisms. Bronchoalveolar lavage fluid (BALF) samples lung surface components and most studies show that there is elevated elastolytic activity in smokers' BALF. Whether antiproteolytic capacity is reduced in these samples remains debatable. A selective lavage technique is described which independently samples central and peripheral epithelium from the same subject. Analysis demonstrates a protease-antiprotease imbalance which can differ in central and peripheral lavage and which could be significant in the development of obstructive airways disease. Therapeutic approaches include augmenting antiprotease potential using genetically engineered, oxidant-resistant alpha 1PI or synthetic peptide inhibitors.
Epithelium↗
Acid-stable low molecular mass proteinase inhibitors in human lung lavage.
An acid-stable, low molecular mass proteinase inhibitor, bronchial mucus proteinase inhibitor (BMPI), has been isolated from sputum and partially characterised. A single band with a modal molecular mass of 18 700 was observed following SDS-polyacrylamide gel electrophoresis. BMPI inhibited human leukocyte elastase, cathepsin G, trypsin and chymotrypsin, but not porcine pancreatic elastase. Although BMPI had a molecular mass close to the similarly isolated inhibitor of Girard et al. (Girard, F., Tournier, J.M., Polu, J.M. & Sadoul, P. (1980), Bull. Eur. Physiopathol. Respir. 16 (Suppl.) 237-245), and although it showed immunological cross reactivity to the low molecular mass inhibitor of Kramps et al. (Kramps, J.A., Franken, C., Meyer, C.J.L.M. & Dijkman, J.H. (1981) J. Histochem. Cytochem. 29, 712-719), it was found to have an amino-acid profile different to any previously described inhibitor. BMPI was detectable in bronchoalveolar lavage fluid collected from healthy and diseased human lungs. The median molar ratio of BMPI/alpha 1-proteinase inhibitor (alpha 1 PI) observed in these lavage samples was 0.7, which is generally higher than those derived from the data of other authors. This suggests that BMPI is a different protein to those previously described, although its exact relationship to other low molecular mass proteinase inhibitors remains to be determined.
Amino Acids↗
Effects of cigarette smoke on antiproteases at the human lung surface.
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Albumins↗