Bronislava Shteyngart

Synergistic Effect of Hydrogen Peroxide and Elastase on Elastic Fiber Injury In Vitro

This laboratory has previously shown that hyperoxia enhances airspace enlargement in a hamster model of elastase-induced emphysema. To further understand the mechanism responsible for this finding, the effect of oxidants on elastase activity was studied in vitro, using a radiolabeled elastic fiber matrix derived from rat pleural mesothelial cells. Matrix samples were treated with either 0.1%, 1%, 3%, or 10% hydrogen peroxide (H2O2) for 1 hr, then incubated with 1.0 μg/ml porcine pancreatic elastase for 2 hrs. Radioactivity released from the matrix was used as a measure of elastolysis. Results indicate that sequential exposure to H2O2 and elastase markedly enhanced elastolysis compared to enzyme treatment alone. A 22% increase in elastolysis was seen with 0.1% H2O2 (325 vs. 396 cpm; P < 0.05), whereas samples pretreated with 1%, 3%, and 10% H2O2 showed increases of 53% (274 vs. 420 cpm; P < 0.05), 71% (381 vs. 653 cpm; P < 0.01), and 38% (322 vs. 443 cpm; P < 0.01), respectively. Exposure to various concentrations of H2O2 alone (0.1% to 10%) produced only minimal elastolysis (<20 cpm). However, 1% H2O2 was capable of degrading peptide-free desmosine and isodesmosine, suggesting that exposure to this oxidant may reduce the stability of the elastic fiber matrix. With regard to lung diseases such as emphysema, H2O2 and other oxidants derived from inflammatory cells or the environment could possibly act as priming agents for elastase-mediated breakdown of elastic fibers, resulting in amplification of lung injury.

Preferential binding of lysozyme to elastic fibres in pulmonary emphysema

BACKGROUND Lysozyme is increased in inflammatory reactions and is a component of the extracellular matrix, but its possible role in lung diseases such as emphysema and interstitial fibrosis has not been investigated. METHODS To characterise differences in lysozyme content among normal, emphysematous, and fibrotic human lungs, tissue sections obtained from necropsy specimens were immunostained with rabbit polyclonal anti-human lysozyme antibody using the labelled streptavidin-biotin peroxidase method. The immunostained sections were evaluated semi-quantitatively (grading the degree of immunostaining on a scale of 0–4). To determine if degradation of the extracellular matrix affects lysozyme binding, hyaluronidase-treated normal lung tissues were incubated with egg white lysozyme, immunostained with the lysozyme antibody, which crossreacts with egg white lysozyme, and evaluated for degree of staining. RESULTS Lysozyme immunostaining was significantly increased in lungs with pulmonary emphysema compared with normal or fibrotic tissues (3.4 versus 1.6 and 1.9, respectively; p<0.05) and was preferentially associated with interstitial elastic fibres. Hyaluronidase-treated lung tissues incubated with lysozyme showed increased immunostaining for this protein compared with untreated controls (1.9 versus 1.2; p<0.05). CONCLUSIONS The results suggest that damage to elastic fibres and/or the surrounding extracellular matrix increases lysozyme binding. It is hypothesised that attachment of lysozyme to elastic fibres may interfere with their repair and possibly enhance the progression of pulmonary emphysema.

The Effect of Lysozyme on Elastase-Mediated Injury

Previous studies by this laboratory demonstrated that lysozyme is increased in human pulmonary emphysema, and that it preferentially binds to elastic fibers, which undergo degradation in this disease. In the current investigation, the relationship between lysozyme and elastic fiber injury was further examined, both In vitro and In vivo. The effect of exogenously administered egg-white lysozyme on pancreatic elastase-induced injury was determined using a biosynthetically radiolabeled extracellular matrix preparation mainly composed of elastic fibers. Although matrix treated with lysozyme showed attachment of the protein to elastic fibers, there was no significant increase in elastolysis compared with untreated controls following exposure to either 1 μg/ml or 100 ng/ml of pancreatic elastase. However, lysozyme did Impair the ability of hyaluronan (HA) to prevent elastase injury to elastic fibers. Matrix samples sequentially treated with lysozyme and HA, then incubated with 1 μg/ml or 100 ng/ml of pancreatic elastase, showed significantly Increased elastolysis compared with those treated with HA alone. Since HA is closely associated with elastic fibers In vivo, the ability of lysozyme to enhance elastolysis was further tested in an animal model of emphysema induced by intratracheal administration of porcine pancreatic elastase. Animals exposed to aerosolized lysozyme prior to elastase administration showed significantly increased airspace enlargement. The mean linear intercept of the lysozyme-treated animals was 123 μm compared with 75 μm for controls receiving aerosolized water (P < 0.0001). These findings suggest that lysozyme may not be an innocuous component of the inflammatory response associated with pulmonary emphysema, but may actually play a role in the pathogenesis of the disease.

Does lysozyme play a role in the pathogenesis of COPD

Elastic fiber injury is an important process in the pathogenesis of chronic obstructive pulmonary disease (COPD), particularly with regard to the development of pulmonary emphysema. Damage to these fibers results in uneven distribution of mechanical forces in the lung, leading to dilatation and rupture of alveolar walls. While the role of various enzymes and oxidants in this process has been well-documented, we propose that a previously unsuspected agent, lysozyme, may contribute significantly to the changes in elastic fibers observed in this disease. Studies from our laboratory have previously shown that lysozyme preferentially binds to elastic fibers in human emphysematous lungs. On the basis of this finding, it is hypothesized that the attachment of lysozyme to these fibers enhances their susceptibility to injury, and further impairs the transfer of mechanical forces in the lung, leading to increased alveolar wall damage and enhanced progression of COPD. The hypothesized effects of lysozyme are predicated on its interaction with hyaluronan (HA), a long-chain polysaccharide that is found in close proximity to elastic fibers. By preventing the binding of HA to elastic fibers in COPD, lysozyme may interfere with the protective effect of this polysaccharide against enzymes and oxidants that degrade these fibers. Furthermore, the loss of the hydrating effect of HA on these fibers may impair their elastic properties, greatly increasing the probability of their fragmentation in response to mechanical forces. The proposed hypothesis may explain why the content of HA is significantly lower in the lungs of COPD patients. It may also contribute to the design of clinical trials involving the use of exogenously administered HA as a potential treatment for COPD.