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1. Human bronchial lysozyme was isolated from nonpurulent secretions and studied by circular dichroism (CD) spectroscopy for its conformational properties. 2. The two negative bands at 208 and 222 nm indicated that the peptide chain adopted an alpha-helical structure in physiological conditions. 3. The molecule was stable at pH 1.0 but not at pH 12.0. 4. Increasing ionic strength by adding NaCl up to 1 M did not change the CD spectra. 5. Complete unfolding of the molecule by guanidinium chloride was obtained only at the concentration of 6 M. 6. Bronchial lysozyme was also denatured by sodium dodecyl sulphate. 7. The molecule was stable when mild reduction was performed at 37 degrees C for 30 min but was completely unfolded after heating at 100 degrees C for 3 min.

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Proteins and lipids synthesized by airway secretory cells or transudated are active components in the protection of respiratory epithelium. Proteins and ions are involved in the control of mucus hydration. Secretory proteins, such as secretory immunoglobulin A (IgA), transferrin and lysozyme, participate in the airway antibacterial defence. Other biochemical components found in secretions, such as anti-inflammatory and antioxidant agents as well as antiproteases, contribute significantly to the protection of the underlying epithelium.

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1. Mucins were isolated from sputum from a patient with chronic bronchitis and subjected to two different preparation procedures. 2. In the first procedure, CsBr density-gradient centrifugation gave rise to two well-separated fractions. Mucins recovered in the high-density fraction still contained mucus proteinase inhibitor (MPI) and lysozyme (LSZ). 3. Mucins were purified after a second step of CsBr density-gradient centrifugation or after gel-filtration chromatography with a buffer of high ionic strength, containing 0.5 M NaCl. 4. In the second procedure, trichloroacetic acid treatment of whole sputum followed by cation-exchange chromatography allowed the obtention of a non-retained fraction composed of mucins. 5. Gel-filtration in buffer containing 0.5 M NaCl, allowed the release of MPI and LSZ from mucins, thus confirming that interactions still occurred between those components. 6. The chemical compositions of the mucins isolated by the two above procedures were quite similar. 7. These data support the hypothesis of the existence of ionic interactions between basic amino acid residues of MPI and LSZ and acid residues of the carbohydrate chains of mucins in the secretions of the large airways. 8. These interactions could play a role in the protection of mucins against proteolysis and consequently in the maintenance of rheological properties of the mucus gel in disease.

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1. Using crossed immunoaffinity electrophoresis with free concanavalin A in the first dimension, we studied the glycan microheterogeneity of alpha 1-antichymotrypsin in sera from patients with giant-cell arteritis and/or polymyalgia rheumatica, and its variation in the serum of several of these patients during induction of disease remission by prednisone therapy and possible further recurrence of giant-cell arteritis and/or polymyalgia rheumatica. 2. In the serum of patients with active disease we observed increased proportions of concanavalin A nonreactive and concanavalin A weakly reactive fractions. The results were expressed as the ratio of concanavalin A non-reactive fraction plus concanavalin A weakly reactive fraction to concanavalin A reactive fraction, called R alpha 1-ACT. An R alpha 1-ACT higher than 1.8 (upper normal value) was found in 30/31 sera from patients with active disease (sensitivity 97%) and in 2/22 sera from patients with inactive disease (specificity 91%). 3. The erythrocyte sedimentation rate and the serum C-reactive protein level, common markers of biological inflammation, are usually elevated in active giant-cell arteritis and/or polymyalgia rheumatica. The two parameters are commonly used to guide the therapy during the course of the disease, but they have no specificity. An erythrocyte sedimentation rate of greater than 30 mm/h was found in 30/31 sera from patients with active disease (sensitivity 97%) and in 5/22 sera from patients with inactive disease (specificity 77%).(ABSTRACT TRUNCATED AT 250 WORDS)

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Human cationic (trypsin 1) and anionic (trypsin 2) trypsins were obtained by controlled activation of purified trypsinogens 1 and 2, respectively. The interactions of trypsin 1 and trypsin 2 with human alpha 1-proteinase inhibitor (alpha 1PI) were analysed and compared by studies in vitro. The enzymatic activity and inhibitory capacity measurements were assessed using Glp-Gly-Arg-Nan as substrate. The association rate constants showed that the inhibition of trypsin 2 occurred more than 10 times faster than that of trypsin 1. The equimolar complexes obtained between either trypsin and alpha 1PI were visualized by electrophoresis followed by immunoblotting. The inhibition of the two trypsins was temporary i.e. the complexes trypsin 1-alpha 1PI and trypsin 2-alpha 1PI broke down with time yielding inactive alpha 1PI (Mr 50,000) and active enzyme. But the stability time for trypsin 1-alpha 1PI was much larger than that of trypsin 2-alpha 1PI. In vivo, alpha 1PI is not able to control the activity of trypsin 1 except when alpha 2-macroglobulin (alpha 2M) is already saturated. According to the delay times of inhibition calculated from normal concentrations in serum, alpha 1PI inhibits trypsin 2 as fast as alpha 2M inhibits trypsin 1. These results suggest that a significant role can be assigned to alpha 1PI in the inhibition of trypsin 2 in physiological conditions and of trypsin 1 in pathological ones.

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Pneumoconiosis is defined as the disease resulting from a chronic exposure to different inorganic dusts. In order to assess the lung defence against the effects of dust exposure, we studied the bronchoalveolar lavage (BAL) fluids from 30 silicotic patients (9 of them having a diagnosis of progressive massive fibrosis (PMF)) and 8 subjects with a diagnosis of asbestosis. Total protein content, N-acetyl-beta-D-glucosaminidase activity, free elastase-like activity, immunoreactive alpha 1-proteinase inhibitor (alpha 1PI) and neutrophil elastase inhibitory capacity (NEIC) were determined, and the values obtained were compared to those of 14 control BAL fluids. In all of the patients, our data showed a significant increase of total protein content and free elastase-like activity. In contrast, N-acetyl-beta-D-glucosaminidase activities did not reach statistical significance. Values concerning immunoreactive alpha 1PI and NEIC were significantly raised only in patients with PMF and with asbestosis. When the ratio NEIC/immunoreactive alpha 1PI was calculated, a significant difference was noticed in the asbestosis group; on the other hand, this ratio was significantly reduced in the group of PMF patients. After neutrophil elastase addition, an electrophoretic study by SDS-PAGE and immunoblotting was carried out; it showed more proteolysed alpha 1PI in the BAL fluids having a lowered NEIC/alpha 1PI ratio. These facts could be explained by the presence of inhibitors of neutrophil elastase different from alpha 1PI.

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Two extraction procedures of non-purulent sputum for the isolation of human mucus proteinase inhibitor (MPI) in its free and bound forms have been assayed. The dissociating procedure involved sputum homogenization in 1M NaCl and 4% (w/v) trichloroacetic treatment. When the soluble material was applied to a CM-Trisacryl column, a non-negligible, MPI-related inhibitory activity was recovered with the highly glycosylated constituents not retained on the column; the amount of MPI released in a free form was retained and eluted from the column according to the basic character of this inhibitor. The non-dissociating procedure consisted in a high water dilution (1:12) of sputum, known to bring into solution the macromolecular, fibrillar constituents, which was followed by ultrafiltration on selected Mr cut-off membranes. All the inhibitory activity was recovered with the high Mr (greater than 100,000) fraction which was shown on SDS-PAGE to be essentially composed of strongly glycosylated material; on electrophoretic analysis under non-reducing conditions, the MPI activity was visualized as three bands which corresponded to the inhibitor released from this high Mr fraction in the presence of SDS. As mucin-type molecules are the major, highly glycosylated constituents of bronchial secretions, it is suggested that they are responsible for the entrapping of MPI within their macromolecular network; it would appear that, as well as for lysozyme, electrostatic interactions occur between the acid charges of mucins and the basic charges of MPI. The possible in vivo consequences of these interactions on MPI activity are discussed.

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In crossed immunoaffinoelectrophoresis with free concanavalin A in the first dimension, human serum alpha 1-antichymotrypsin, purified or in whole serum, exhibited four peaks in presence of 0.02 M alpha-methyl-D-glucoside added to the second-dimensional gel. alpha 1-Antichymotrypsin purified from the serum of a single healthy donor was separated by affinity chromatography into three fractions on a laboratory-prepared concanavalin A-Sepharose 4B column: a pass-through fraction, a retarded fraction and a bound fraction, eluted from the column on addition of sugar to the buffer. These three fractions were analyzed by crossed immunoaffinoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing before and after desialylation. The results of electrophoresis as well as chemical analyses indicate that these microheterogeneous forms carry glycans with decreasing degrees of branching from the concanavalin A-pass-through form to the concanavalin A-bound form. This approach represents a first step towards the elucidation of the molecular basis of the microheterogeneity of alpha 1-antichymotrypsin.

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Using crossed immunoaffinoelectrophoresis with free concanavalin A (Con A) in the first dimension and alpha-methyl-D-glucoside incorporated in the second-dimension gel, we examined the microheterogeneity of alpha 1-antichymotrypsin (alpha 1Achy) in sera from healthy donors (N) and in sera from patients with various inflammatory syndromes. We studied three groups: patients with acute inflammation (myocardial infarction: MI, septic inflammation: SI), patients with chronic inflammation (metastatic breast cancer: MBC), and patients with chronic inflammation accompanied by acute attacks (connective-tissue disease: CTD). All pathological sera had a high alpha 1Achy concentration. Compared with N, MI and SI showed an increased proportion of Con A-reactive fraction and a decreased proportion of Con A-nonreactive fraction, which was more pronounced in SI. Unlike the patients with acute inflammation, patients with CTD showed an increased proportion of Con A-nonreactive fraction. Thus alpha 1Achy microheterogeneity in crossed immunoaffinoelectrophoresis may afford a means of differentiating between various inflammatory syndromes. In particular, it can provide a simple test: if the Con A-nonreactive fraction is in a proportion less than 17%, septic origin of an acute-phase reaction may be suspected.

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The interactions of human pancreatic elastase 2 with alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin were compared by studies in vitro. The equimolar complexes obtained between the enzyme and either inhibitor were relatively stable at 25 degrees C since they could be visualized for up to 5 days by an electrophoretic method. However, in both cases, a slow dissociation occurred with release of active enzyme. As the kass. rate constants are of the same order of magnitude, with a slightly lower value for alpha 1-proteinase inhibitor when compared with alpha 1-antichymotrypsin [(5.6 +/- 1.2) X 10(5) and (8.9 +/- 1.3) X 10(5) M-1.s-1 respectively], partition of human pancreatic elastase 2 between both inhibitors in human plasma is mainly dependent on their respective concentrations. A comparative study by crossed immunoelectrophoresis of the interactions of this enzyme with the two inhibitors contained in normal human plasma and in a mimetic mixture of pure inhibitors was carried out. This allowed the visualization of complexes with either inhibitor. Formation of such a complex with alpha 1-antichymotrypsin had never been demonstrated previously. The patterns obtained are similar when working with normal plasma or with the synthetic mixture, suggesting that, in the conditions used, alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin are the main inhibitors of human pancreatic elastase 2 in the plasma sample. However, it is also shown that part of the enzyme may be taken up by alpha 2-macroglobulin, which is responsible for the remaining enzyme activity on a synthetic substrate. The present work suggests that, according to the delay times of inhibition of human pancreatic elastase 2 calculated from the normal plasma concentrations of alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin, a significant role can be assigned to both inhibitors. Moreover, the role of alpha 1-antichymotrypsin would be enhanced in alpha 1-proteinase-inhibitor deficiency.

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Human insoluble elastin was prepared from newborn lungs and digested by leucocyte elastase. The soluble fragments were compared to those obtained from adult lung elastin in a previous work (Smyrlaki et al., 1986). Gel filtration on a Bio-Gel P-100 column of newborn elastin allowed the separation of fraction F1N (Mr's 30,000-10,000) which was eluted later than the excluded fraction F1A (Mr's 80,000-30,000) previously isolated from adult elastin. The difference in the sizes of the large peptide fragments originating from both elastins was also shown on SDS-PAGE. Reversed phase HPLC was performed on a C18 column using a multi-step gradient elution procedure. Different patterns were observed for the high (F1N) and the low (F2N) molecular size fragments of newborn elastin. The same peak distribution was obtained with adult elastin. Comparison of the amino acid compositions of the most retained peaks (3, 4 and 5), derived from fractions F1N and F1A, showed analogies for the contents of the major nonpolar amino acids and crosslinks. Thus, this procedure allowed the separation of typical fragments of elastin which might be released in vivo by leucocyte elastase during pulmonary diseases.

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The interaction of human pancreatic chymotrypsin A with serum inhibitors was assessed by enzyme immunoassay, enzymatic activity and inhibitory capacity measurements and electrophoretic analyses. In normal serum, chymotrypsin A was detected in four forms: one form (Mr approximately equal to 25,000) which might be chymotrypsinogen A and three forms complexed to the main inhibitors present in serum, alpha 2-macroglobulin (alpha 2-M), alpha 1-proteinase inhibitor (alpha 1-PI) and alpha 1-antichymotrypsin (alpha 1-Achy). As chymotrypsin A remains to 90% active when bound to alpha 2-M, the chymotrypsin A/alpha 2-M complex was quantified by an enzymatic assay. The kinetic parameters of the interaction of chymotrypsin A with alpha 1-PI and alpha 1-Achy were determined. Using these data the partition of chymotrypsin A between the different inhibitors in serum was calculated. In acute pancreatitis, the chymotrypsin A plasma level follows the progression of the disease and in this case as well as in normal serum alpha 1-PI is the major antagonist of chymotrypsin A.

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The functional activity of alpha 1 proteinase inhibitor (alpha 1PI) in bronchoalveolar lavages (BAL) was determined with an automated method, using its inhibitory capacity against porcine pancreatic elastase. Immunoreactive alpha 1PI was measured by immunonephelometry-laser (INL). These two methods may generally be applied on unconcentrated samples. Precision and accuracy of both methods are studied. INL was preferably used to electroimmunodiffusion (EID) because it gave unchanged values after alpha 1PI had reacted with proteases. In BAL, for some specimens, the alpha 1PI content evaluated by EID was underestimated with respect to its measurement by INL. The degree of underestimation is related to the presence of non-functional forms of alpha 1PI.

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The solubilization of human lung elastin by leucocyte elastase and cathepsin G is described. Elastolysis kinetic studies clearly show that leucocyte elastase is more efficient in solubilizing elastin fibres than is cathepsin G. Cathepsin G can degrade elastin but at a much slower rate. Characterization of elastase and cathepsin G soluble elastin fragments, obtained after 24 h of digestion (enzyme-substrate ratio, 1:100), was first performed by isoelectric focusing. Whole digests were focused as 6 bands in a pH range 4.2 to 4.7 and were found to have no significant differences in amino acid compositions. Biogel P-100 gel filtration of the elastase digested fragments separated a major excluded fraction (Mr's: 80,000 to 30,000) and a small retained one (Mr's: 6000 to 4000). Conversely, cathepsin G digests were eluted as a minor excluded fraction and a more important retarded one (Mr's: 6000 to 4000). Only the high molecular weight fractions of both enzymes digests contain crosslinked amino acids; this assigns a role for desmosines in the resistance of elastin to these proteases. These results are discussed in comparison with the data obtained by others.

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Incubation of human serum alpha 1-antichymotrypsin with human pancreatic elastase 2 or porcine pancreatic elastase results in the complete inhibition of each enzyme as determined by spectrophotometric assays. alpha 1-Antichymotrypsin reacts much more rapidly with the human than with the porcine enzyme. The inhibitor: enzyme molar ratio, required to obtain full inhibition of enzymatic activity, is equal to 1.25/1 when alpha 1-antichymotrypsin reacts with human pancreatic elastase 2 while it is markedly higher with porcine pancreatic elastase (5.5/1). Patterns obtained by SDS/polyacrylamide gel electrophoresis of the reaction products show the formation with both enzymes of an equimolar complex (Mr near 77 000) and the release of a fragment migrating as a peptide of Mr near 5000. Moreover a free proteolytically modified form of alpha 1-antichymotrypsin, electrophoretically identical with that obtained in the reaction with cathepsin G or bovine chymotrypsin, is produced in the reaction with each elastase but in a much greater amount when alpha 1-antichymotrypsin reacts with porcine elastase than with human elastase. As a consequence of our findings, the specificity of alpha 1-antichymotrypsin, so far limited to the inhibition of chymotrypsin-like enzymes from pancreas and leukocyte origin, has to be extended to the two pancreatic elastases investigated in this work. A contribution of alpha 1-antichymotrypsin to the regulatory balance between plasma inhibitors and human pancreatic elastase 2 in pancreatic diseases is suggested.

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Interaction of human plasma alpha 1-proteinase inhibitor (alpha 1-PI) with human leukocyte cathepsin G was assessed by proteinase inhibitory capacity assays and electrophoretic analyses. Only a part of purified alpha 1-PI formed a 1 : 1 complex (Mr = 74 000) with leukocyte cathepsin G. The formation of this complex was accompanied by the appearance of a great amount of a free, proteolytically modified form of alpha 1-PI (Mr = 50 000) which had lost its inhibitory capacity. The complex was unstable with time and the only dead end product observed was a modified, inactive form of alpha 1-PI. During the breakdown of the complex, no release of active enzyme could be measured by spectrophotometric assays. In spite of the fact that the equimolarity of the complex was convincingly demonstrated, studies of the alpha 1-PI-cathepsin G reaction showed that total inhibition of this proteinase needed a large molar excess of alpha 1-PI. Nevertheless, alpha 1-PI acts as an efficient "suicide inhibitor" since no cathepsin G recovery was ever found.

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The major urinary trypsin inhibitor (UTI) was found to inhibit bovine chymotrypsin and human leucocyte elastase strongly, cathepsin G weakly. No inhibition of porcine pancreatic elastase was observed. The stoichiometry of the inhibition of bovine trypsin by UTI was determined spectrophotometrically to be 1:2 (I/E molar ratio). After incubation of UTI with this enzyme in various molar ratios, two complexes (C1 and C2) could be visualized in alkaline polyacrylamide gel electrophoresis. C1 was isolated by affinity chromatography on Con-A Sepharose. In dodecyl sulfate polyacrylamide gel electrophoresis, C1 was dissociated to give an inhibitory band with the same electrophoretic mobility as native UTI. C2 released an active inhibitory fragment with Mr near 20000. A time-course study demonstrated that at a molar ratio I/E of 1.5:1, the C2 complex appears after two hours of incubation.

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Human alpha 1-antichymotrypsin reacts with bovine chymotrypsin to form an equimolar complex and this reaction is accompanied by the formation of a free, modified form of the inhibitor. Time-course studies, performed on mixtures containing an excess of native inhibitor and kept at 0 degree C or at 25 degrees C, show that the equimolar complex dissociates spontaneously; this dissociation results in the release of inactive modified alpha 1-antichymotrypsin and of some active enzyme, which is able to recycle with active inhibitor in excess. When all the native inhibitor is used up, the released active enzyme degrades the remaining intact complex into intermediate forms. At the endpoint of the reaction only inactive modified inhibitor and some active chymotrypsin remain. Immunochemical data indicate that, in the complex, a steric hindrance of the antigenic determinants of the inhibitor prevents the formation of the precipitate with specific antiserum. Inactive modified inhibitor, which has dissociated from the complex, has retained antigenic determinants of the native alpha 1-antichymotrypsin.

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Broncho-alveolar lavage (LBA) was performed in 17 pneumoconiotics. The liquid obtained was analysed after gentle centrifugation to remove the cellular element, so that biochemical factors might be sought contribution to the evolution and progressive transformation to fibrosis. The percentage of liquid gathered was generally greater. Among the glycosidases found in all the 17 LBA analysed, the beta-D-glucuronidase, which was not detected in the LBA control subjects, was also found during the course of other pulmonary disorders. The elastolytic activity was characterized in 12 out of 17 LBA. In part it could originate from alveolar macrophages. An elevated number of macrophages (greater than 20 X 10(6) for the whole lavage) allied to the presence of elastolytic activity was found in 7 of 8 patients presenting with a pneumoconiosis and signs of progressive pulmonary disease. The collagenase and cathepsin B were present in the LBA of certain pneumoconiotics, but the significance of their presence is still unknown. The three major antiproteases of the serum exist in the LBA of pneumoconiosis. The quantity of alpha 1-antiprotease has identified a group of 6 patients whose LBA showed raised alpha 1-antiprotease, an elastolytic activity and for 5 of them progressive outcome.

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