Neutrophil Collagenase Research Articles

Lung neutrophils in the adult respiratory distress syndrome. Clinical and pathophysiologic significance.

Although neutrophils are of pathogenetic importance in various animal models of acute lung injury, their role in the adult respiratory distress syndrome (ARDS) is unclear. To study the significance of lung neutrophils in this disorder, patients with ARDS (n = 11) were evaluated by bronchoalveolar lavage within 24 h of admission to the intensive care unit. Patients with non-ARDS respiratory failure requiring mechanical ventilation (n = 4) and normal volunteers (n = 12) were also studied. Neutrophils constituted 67.6 +/- 9.8% of recovered lavage cells in patients with ARDS compared with only 4.0 +/- 2.4% of cells in mechanically ventilated control patients and 0.8 +/- 0.2% in normal volunteers (p less than 0.005, both comparisons). Furthermore, in patients with ARDS (n = 6) evaluated serially by bronchoalveolar lavage at 72-h intervals, neutrophil percentages decreased from 91 +/- 3.2% (initial lavage) to 42.8 +/- 12% (final lavage) (p less than 0.005). Lung neutrophils also predicted the severity of abnormalities in gas exchange and lung protein permeability. That is, the percentage of neutrophils correlated directly with the alveolar-arterial Po2 difference (r = 0.69, p less than 0.01) and lavage fluid total protein concentrations (r = 0.62, p less than 0.01). Because large numbers of lung neutrophils were present in these patients, ARDS lavage fluid was assayed for neutrophil mediators relevant to the pathogenesis of acute lung injury. Neutrophil elastase activity was not detected in any ARDS lavages, although elastase was antigenically present in most samples and appeared to be complexed to alpha-1-antitrypsin. In contrast to elastase, neutrophil collagenase was readily detectable in ARDS fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

The generation of utilization of chlorinated oxidants by human neutrophils

Abstract The human neutrophil uses the hydrogen peroxide-myeloperoxidase-chloride system to generate a complex mixture of chlorinated oxidants which includes both hypochlorous acid (HOCI) and a wide spectrum of nitrogen-chlorine (N-C1) derivatives. Although these species differ in their reactivity with susceptible target molecules, they are capable of mediating diverse biologic effects. These effects range from the modulation of cellular function and induction of cell lysis to the regulation of neutrophil proteoIytic enzyme activity and enzyme-inhibitor interactions. When chlorinated oxidants are used by neutrophils to mediate ceil injury, HOCI appears to be the toxic species involved. The endogenous, long-lived, N-CI derivatives generated by triggered neutrophils cause little injury to microbial or mammalian targets by virtue of their hydrophilic characteristics. However, the presence of appropriate endogenous or exogenous nitrogenous compounds in the extracellular medium can result in the generation of lipophilic N-C1 oxidants having a heightened cytotoxic potential. Chlorinated oxidants are also able to participate in the regulation of neutrophil elastase and collagenase activity during release of these enzymes from intracellular lysosomal granules. Elastase activity is indirectly up-regulated by the oxidative destruction of its primary inhibitor, α1-proteinase inhibitor, while collagenase is oxidatively converted from a latent, inactive enzyme to a fully proteolytic product. Thus, neutrophils are able to convert the short-lived, nonspecific reactivity of HOCI into highly specific and long-acting effects by modifying cellular or plasma constituents that are critical to the development of the inflammatory response.

Heterogeneity among human collagenases demonstrated by monoclonal antibody that selectively recognizes and inhibits human neutrophil collagenase.

The heterogeneity of human collagenases has been examined using a monoclonal antibody to neutrophil collagenase. This antibody inhibited collagenase activity and, when covalently coupled to Sepharose, bound both latent and active enzyme. Although human neutrophil collagenase was inhibited by the antibody, the activity of human skin and rheumatoid synovial collagenase was not significantly diminished in the presence of the antibody. Competitive inhibition studies also differentiated between these collagenases. Only human neutrophil collagenase effectively blocked the antibody in a competitive enzyme-linked immunosorbent assay while skin and rheumatoid synovial collagenase again failed to interact with the antibody. The unequivocal recognition of neutrophil collagenase as an immunologically distinct entity from other collagenases supports the hypothesis that neutrophil collagenase is a separate gene product from fibroblast or synovial collagenase.

Neutrophil collagenolytic activity in patients with Behçet disease.

Studies were made on neutrophil dysfunction in Behçet disease by examining collagenolytic activity in neutrophils. The collagenolytic activity of neutrophil was found to be significantly higher in patients during the attack phase of this disease than in patients during the remission phase, in patients with other forms of uveitis and in normal controls. Collagenase was present in the collagenolytic enzyme, which showed high activity in the attack phase. The molecular weight of this enzyme was similar to that of mammalian neutrophil collagenase.

Purification of human neutrophil collagenase and production of a monospecific antiserum

Although there is good evidence for the presence of human neutrophil (PMN) collagenase, only moderate purification has been reported. The probable explanation for this fact is that most assays used to specifically measure collagenase activity are not reliable if high levels of several different proteases are also present in the assay mixture. The PMN granule is just such a concentrated mixture. Therefore, polyacrylamide gel electrophoresis was used to identify and quantitate the alpha 1 3/4 and alpha 2 3/4 cleavage products diagnostic for mammalian collagenase. White cells (85% PMN's) were lysed in 0.34 M sucrose and granules were obtained. The granules were lysed by sonication, and the lysate was chromatographed on a Sephadex G-200 column followed by a Trasylol-Sepharose 4B column. This procedure resulted in a 1350-fold purification and a yield of 75 micrograms of enzyme/unit of blood. The collagenase was inhibited by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid but not by sulfhydryl or serine protease inhibitors. The preparation was free of elastase, which has been shown to cleave type III collagen into alpha 1 3/4 and alpha 1 1/4 pieces. The pI of collagenase was shown to be 4.7 by isoelectric focusing, and the enzyme lost activity below a pH of 6.5 if collagen was absent. Antiserum was produced by 100-micrograms injections of the purified collagenase into rabbits. Titers were measured by the enzyme-linked immunosorbent assay. For determination of the specificity, collagenase and PMN extract were isoelectrically focused and blotted onto nitrocellulose. The antibody recognized only one band of protein in the PMN extract, which comigrated with the purified collagenase.

The latent collagenase and gelatinase of human polymorphonuclear neutrophil leucocytes

Two metallo-proteinases of human neutrophil leucocytes, collagenase and gelatinase, were studied. Collagenase specifically cleaved native collagen into the TCA and TCB fragments, whereas gelatinase degraded denatured collagen, i.e. gelatin, and the TCA fragments produced by collagenase. On subcellular fractionation by zonal sedimentation, collagenase was found to be localized in the specific granules, separate from gelatinase, which was recovered in smaller subcellular organelles known as C-particles. Neither enzyme was present in the azurophil granules, which contain the two major serine proteinases of neutrophils, elastase and cathepsin G. Collagenase and gelatinase were separated by gel filtration from extracts of partially purified granules. Both enzymes were found to occur in latent forms and were activated either by trypsin or by 4-aminophenylmercuric acetate. Gelatinase was also activated by cathepsin G, which, however, destroyed collagenase. Both enzymes were destroyed by neutrophil elastase. Activation resulted in a decrease by 25 000 in the apparent mol. wt. of both latent metallo-proteinases.

COMPARISON OF COLLAGENASE ACTIVITY IN EOSINOPHIL AND NEUTROPHIL FRACTIONS FROM RAT PERITONEAL EXUDATES

The collagenase activity had been compared in extracts of eosinophils and of neutrophilss from peritoneal exudates in two groups of rats, one of which had been treated to augment the numbers of eosinophils and the other the numbers of neutrophils. The proportion of granulocytes to other cells in each preparation was increased by differential centrifugation over a continuous gradient. Collagenase was extracted from the fractions in which granulocytes were concentrated and the activity assayed by the radioactive fibril method. There was at least as much collagenase in the eosinophil-enriched extracts as in the neutrophil-enriched extracts. It is postulated that eosinophil collagenase may have a function in the remodelling of newly-synthesised collagen during the post-inflammatory phase of healing, since eosinophil leucocytes appear in significant numbers within the connective tissue during this phase. This suggests a different role for eosinophil collagenase than that for neutrophil collagenase, since neutrophil are present only in the early stages of inflammation, when collagen is being degraded.

Neutrophil leukocyte collagenase, elastase and serum protease inhibitors in human gingival crevicles.

Extra-cellular neutrophil collagenase and elastase were demonstrated by immunochemical methods in crevicular material from clinically healthy and chronically inflamed human gingivae. Collagenolytic activity was shown by viscosimetry and elastolysis by its digestive action on elastin agarose plates. Also the serum protease inhibitors, a,1-antitrypsin and a2-macroglobulin, were demonstrated in the crevicular material. The inflamed gingivae contained increased amounts of enzymes and inhibitors. The protease inhibiting capacity of a1-antitrypsin was saturated in material from inflamed as well as from healthy gingivae, but free neutrophil collagenase and elastase were found mainly in crevices of the inflamed gingivae. It was suggested that free activities of these two proteolytic enzymes might contribute to collagen break-down in gingivitis and periodontitis.