Thorax 1998;53:193–196 193

Short paper Thorax: first published as 10.1136/thx.53.3.193 on 1 March 1998. Downloaded from

Preferential binding of to elastic fibres in pulmonary emphysema

Bronislava Shteyngart, Surachat Chaiwiriyakul, John Wong, Jerome O Cantor

Abstract prompted by earlier studies indicating that lys- Background—Lysozyme is increased in ozyme is increased in a number of inflamma- inflammatory reactions and is a compo- tory conditions and is specifically associated nent of the , but its with extracellular matrix constituents.1–3 These possible role in lung diseases such as findings suggested that the amount of lysozyme emphysema and interstitial fibrosis has in the lung might be altered in diseases such as not been investigated. emphysema or interstitial fibrosis where there Methods—To characterise diVerences in is damage to various matrix components. lysozyme content among normal, emphy- To determine if lysozyme is involved in pul- sematous, and fibrotic human lungs, tis- monary disease, tissue sections from normal, sue sections obtained from necropsy fibrotic, and emphysematous human lungs specimens were immunostained with rab- were evaluated for diVerences in lysozyme con- bit polyclonal anti-human lysozyme anti- tent. An increase in extracellular lysozyme was body using the labelled streptavidin-biotin specifically observed in lung tissues with peroxidase method. The immunostained pulmonary emphysema, and the protein was sections were evaluated semi- preferentially associated with elastic fibres quantitatively (grading the degree of im- which undergo breakdown in this disease. munostaining on a scale of 0–4). To Since this laboratory and other investigators have previously shown that and determine if degradation of the extracel- 45 lular matrix aVects lysozyme binding, other polysaccharides surround elastic fibres, http://thorax.bmj.com/ normal lung tissues were treated with hyaluro- hyaluronidase-treated normal lung tis- nidase and examined for their ability to bind sues were incubated with egg white lys- exogenously administered lysozyme. Such ozyme, immunostained with the lysozyme treatment resulted in increased attachment of antibody, which crossreacts with egg white lysozyme, suggesting that degradation of extra- lysozyme, and evaluated for degree of cellular matrix components, as occurs in staining. pulmonary emphysema, may expose binding Results—Lysozyme immunostaining was sites for lysozyme on elastic fibres, possibly significantly increased in lungs with pul- altering matrix interactions and influencing the on September 26, 2021 by guest. Protected copyright. monary emphysema compared with nor- repair process in this disease. mal or fibrotic tissues (3.4 versus 1.6 and 1.9, respectively; p<0.05) and was prefer- entially associated with interstitial elastic Methods St Luke’s Roosevelt fibres. Hyaluronidase-treated lung tissues Institute for Health PROCUREMENT OF NECROPSY LUNG TISSUES Sciences, New York, incubated with lysozyme showed in- The necropsy files of The Brooklyn Hospital NY 10019 and The creased immunostaining for this protein Pathology Department were reviewed to obtain Brooklyn Hospital compared with untreated controls (1.9 tissues from normal (n = 7), emphysematous Center, Brooklyn, NY versus 1.2; p<0.05). (n = 7), and fibrotic lungs (n = 6). Tissues des- 11201, USA —The results suggest that B Shteyngart Conclusions ignated normal were obtained from patients S Chaiwiriyakul damage to elastic fibres and/or the sur- with no lung complications at the time of J Wong rounding extracellular matrix increases death. Multiple slide sections (three or more) J O Cantor lysozyme binding. It is hypothesised that of the lungs were examined by a pathologist attachment of lysozyme to elastic fibres (JOC) to confirm the findings and immunohis- Correspondence to: Dr J O Cantor, Department may interfere with their repair and poss- tochemical studies were then performed on of Pathology, The Brooklyn ibly enhance the progression of pulmo- sections of a representative paraYn-embedded Hospital Center, 121 nary emphysema. tissue block from each case. DeKalb Avenue, Brooklyn, (Thorax 1998;53:193–196) NY 11201, USA. IMMUNOHISTOCHEMICAL STAINING FOR Received 18 August 1997 Keywords: lysozyme; pulmonary emphysema; elastic Returned to authors fibres LYSOZYME 6 October 1997 The labelled streptavidin-biotin peroxidase Revised version received method was used to label lysozyme. Tissue 6 November 1997 Accepted for publication This study examined the possible role of sections were deparaYnised in xylene, hy- 21 November 1997 lysozyme in lung injury. The investigation was drated through graded alcohols, then rinsed in 194 Shteyngart, Chaiwiriyakul, Wong, et al

0.05 M Tris-HCl buVer, pH 7.6. Following 4 inhibition of endogenous peroxidase with 3% p < 0.05 Thorax: first published as 10.1136/thx.53.3.193 on 1 March 1998. Downloaded from hydrogen peroxide, the tissue sections were washed with Tris-HCl buVer and incubated with 2% fetal bovine in Tris-HCl for 20 3 minutes to block non-specific protein binding. They were then incubated for 30 minutes with a polyclonal rabbit anti-human lysozyme anti- body (BioGenex Laboratories, San Ramon, 2 California, USA) and separately treated for 15 minutes with biotin-labelled link antibody and peroxidase-labelled streptavidin. Immuno- staining was completed with a 20 minute incu- 1 bation in 3-amino-9-ethylcarbazole (AEC) Degree of immunostaining buVer solution. The tissues were then counter- stained with Mayer’s haematoxylin. 0 A positive control specimen of normal Normal Fibrosis Emphysema human tonsil accompanied each staining run Figure 1 Mean (SE) immunostaining for lysozyme in to verify proper functioning of all reagents pulmonary emphysema compared with both normal and involved in the immunostaining procedure. fibrotic lungs. Negative control specimens for each test case were also performed, substituting the anti- incubated with the antibody and labelled as human lysozyme primary antibody with a non- described above. immune rabbit serum negative control reagent To determine the eVect of hyaluronidase to determine the degree of non-specific stain- treatment alone on lysozyme immunostaining, ing. tissue sections (n = 7) from normal lungs were incubated with the or with buVer alone, as described above. The tissues were EVALUATION OF IMMUNOSTAINING then immunostained without prior incubation The tissue sections were coded and independ- with egg white lysozyme. ently graded for the degree of extracellular lys- ozyme immunostaining by a pathologist (SC) STATISTICAL ANALYSIS unfamiliar with the design of the experiment. DiVerences in immunostaining among normal, The entire area of each tissue section was emphysematous, and fibrotic lung tissues were examined and only extracellular staining of the analysed for statistical significance (p<0.05) lung parenchyma was evaluated, since inflam- with the Kruskal-Wallis multiple comparison z matory cells (monocytes, macrophages, and value test. The Wilcoxon signed rank test was http://thorax.bmj.com/ neutrophils) showed intense staining which used to evaluate diVerences between could obscure diVerences in lysozyme content hyaluronidase-treated and untreated tissues. related to interstitial connective tissue changes. Slide sections were given a score between 0 and 4. Intermediate scores were averaged (e.g. 1–2 Results Emphysematous lungs showed a significant = 1.5). increase in extracellular immunostaining for lysozyme compared with both normal and

HYALURONIDASE-LYSOZYME TREATMENT OF fibrotic lung tissues (mean (SD) 3.4 (0.5) on September 26, 2021 by guest. Protected copyright. LUNG TISSUES versus 1.6 (0.8) and 1.9 (1.0), respectively; Tissue sections (n = 9) from normal lungs were p<0.05; fig 1). In all three groups there was used in these studies (in some cases more than preferential staining of interstitial, vascular, and one paraYn block per lung was utilised). The pleural elastic fibres. However, the emphysema- sections were deparaYnised and coated with tous lungs showed particularly intense staining aliquots of a solution of bovine testicular of these fibres, especially in areas where there hyaluronidase (Wyeth Laboratories, Philadel- was airspace dilatation and attenuation of the phia, Pennsylvania, USA) at a concentration of alveolar septa (fig 2). The immunostained elas- 150 U per ml phosphate buVered saline. The tic fibres associated with alveolar distention tissues were treated for two hours at 37°C. often appeared fragmented (fig 3). Control sections from the same tissue blocks Tissue sections from normal lungs treated were coated with phosphate buVered saline with bovine testicular hyaluronidase and incu- solution alone under similar conditions. Both bated with egg white lysozyme showed a groups were then incubated with a 1% solution significant increase in immunostaining for lys- of egg white lysozyme (Sigma Chemical Com- ozyme compared with controls not exposed to pany, St Louis, Missouri, USA) in phosphate hyaluronidase (1.9 (0.8) versus 1.2 (0.7), buVered saline for one hour at room tempera- respectively; p<0.05). This result was not due ture. After extensive rinsing, the tissues were to increased immunostaining of endogenous immunostained for lysozyme. lysozyme, since tissue sections treated with The rabbit antibody, prepared with lysozyme hyaluronidase but not incubated with lysozyme isolated from the urine of patients with mono- showed no significant increase in immuno- cytic leukaemia, was tested for crossreactivity staining compared with controls (0.9 (0.2) ver- with egg white lysozyme. Samples of human sus 0.6 (0.2), respectively; p>0.05). neutrophil lysozyme and egg white lysozyme The anti-human lysozyme antibody used in were spotted onto nitrocellulose paper, then these studies reacted positively when tested Preferential binding of lysozyme to elastic fibres in pulmonary emphysema 195 Thorax: first published as 10.1136/thx.53.3.193 on 1 March 1998. Downloaded from

Figure 2 (A) Emphysematous lung showing prominent immunostaining (grade 4) with anti-lysozyme antibody. (B) Fibrotic lung showing focal immunostaining (grade 1) with anti-lysozyme antibody. (C) Normal lung showing focal immunostaining with anti-lysozyme antibody (grade 0), only associated with mononuclear inflammatory cells. (D) Normal lung control not treated with anti-lysozyme antibody showing complete lack of immunostaining. Streptavidin-biotin labelling counterstained with Mayer’s haematoxylin. Original magnification ×800. thermore, it has been shown that injury to car- tilage results in a parallel release of lysozyme and chondroitin sulphate, indicating a possible biochemical relationship between these two constituents.6 Since neither of these matrix components is specific to cartilage, similar http://thorax.bmj.com/ interactions could occur in other tissues. While the mechanism responsible for the observed aYnity of lysozyme for elastic fibres is unclear, it is possible that lysozyme may bind to specific carbohydrate residues in elastic fibres. N-acetyl-D-glucosamine, a component of bac- Figure 3 Disrupted elastic fibres (arrowheads) in terial cells susceptible to degradation by

emphysematous lung showing prominent immunostaining lysozyme, has also been found in glycoproteins on September 26, 2021 by guest. Protected copyright. with anti-lysozyme antibody. Streptavidin-biotin labelling associated with elastic fibres.7 Injury to elastic counterstained with Mayer’s haematoxylin. Original 8 magnification ×2000. fibres, as occurs in pulmonary emphysema, may expose such residues, thereby facilitating against both human neutrophil lysozyme and lysozyme binding. egg white lysozyme. A study from this laboratory has previously shown that intratracheally administered hy- Discussion aluronic acid preferentially binds to pulmonary Determining the significance of any changes in elastic fibres and reduces alveolar injury lung lysozyme content is complicated by the experimentally induced by human neutrophil fact that this protein has no recognised elastase.4 Hyaluronidase treatment of lung physiological function beyond its role in bacte- tissues may therefore increase lysozyme bind- riolysis. The increased levels of lysozyme which ing by degrading hyaluronic acid and exposing accompany inflammation may only reflect specific attachment sites on the elastic fibre. non-specific release from . Further- With regard to pulmonary emphysema, the more, the association of lysozyme with in- presence of lysozyme on elastic fibres might flamed tissues may be due to its strongly disrupt the normal interactions between these cationic nature, reflecting electrostatic binding fibres and other matrix components and poss- to other tissue components rather than ibly result in more rapid progression of the substrate-specific interactions. disease. Nevertheless, the presence of lysozyme in a The presence of increased lysozyme in wide variety of tissues suggests that it may have emphysematous tissues also lends further sup- a physiological function. In cartilage, for exam- port to the concept that the disease is caused by ple, lysozyme is present as a non-lysosomal the release of from neutrophils and matrix protein capable of interacting with sur- monocytes. Since lysozyme is secreted by both rounding polyanionic tissue components. Fur- neutrophils and monocytes, its presence on 196 Shteyngart, Chaiwiriyakul, Wong, et al

elastic fibres may serve as a “footprint” denot- 4 Cantor JO, Cerreta JM, Armand G, et al. Further investiga- ing the proximity of these cells to the fibres. tion of the use of intratracheally administered hyaluronic acid to ameliorate elastase-induced emphysema. Exp Lung Thorax: first published as 10.1136/thx.53.3.193 on 1 March 1998. Downloaded from Whether or not lysozyme facilitates the access Res 1997;23:229–44. of these cells to elastic fibres or alters the repair 5 Baccarani-Contri M, Vincenzi D, Cicchetti F, et al. Immunocytochemical localization of proteoglycans within of the fibres remains to be determined. normal elastin fibers. Eur J Cell Biol 1990;53:305–12. 6 Greenwald RA, Cantor JO, Schwartz CE, et al.EVects of acute cartilaginous injury on serum and cartilage lysozyme 1 Pruzanski W, Saito S, Orgryzlo M. The significance of levels. Arthritis Rheum 1975; :139–44. lysozyme (muramidase) in rheumatoid arthritis. Arthritis 18 Rheum 1970;13:389–99. 7 Amaya J. The eVect of steroids on organ-cultured porcine 2 Mera SL, Lovell CR, Jones RR, et al. Elastic fibres in normal trabecular meshwork: an ultrastructural, biochemical, and and sun-damaged skin: an immunohistochemical study. Br lectin histochemical study. Acta Soc Opthalmol Jap J Dermatol 1987;117:21–7. 1995;99:995–1004. 3 Kahn HJ. “Aberrant elastic” in elastofibroma: an immuno- 8 Senior RM, Kuhn C III. The pathogenesis of emphysema. histochemical and ultrastructural study. Ultrastruct Pathol In: Fishman AP, ed. Pulmonary diseases and disorders. 2nd 1995;19:45–50. ed. New York: McGraw-Hill, 1982:1209–18. http://thorax.bmj.com/ on September 26, 2021 by guest. Protected copyright.