Basal secretion of lysozyme from human airways in vitro.

The aim of this study was to examine the basal release of lysozyme from isolated human lung tissues. Measurements of lysozyme in the fluids derived from lung preparations were performed using a rate-of-lysis assay subsequent to acidification of the biological samples. Lysozyme released from bronchial preparations into fluids was greater than that observed for parenchymal tissues. The lysozyme quantities detected in bronchial fluids were not modified by removal of the surface epithelium. Furthermore, the quantities of lysozyme in bronchial fluids was correlated with the size of the bronchial preparations. These results suggest that the lysozyme was principally secreted by the human bronchi (submucosal layer) rather than by parenchyma tissues and that a greater release was observed in the proximal airways.


Introduction
Lysozyme has bee n desc ribed to be pre se nt in glandular serous cells 1 ,2 and to be sec rete d by tracheal ex plants by both glandular and epithelial ce lls. 3 Furthermore, neutrophils and macrophages that ac cumulate w ithin the airw ays may be also a sourc e of lysozyme . 3,4 Since high c onc entrations of lysozyme have be en identified in airw ay lavage fluids 5,6 and in ex pe ctorated sp utum from patients w ith asthma, 7 chronic obstructive lung disease 6 -8 and cystic fibrosis, 8 the dete ction of this e nzyme in biological fluids from patients w ith resp iratory disease has frequently be en monitored as an index of inflammation in human airw ays. How eve r, in these investigations the origin of the lysozyme w as not identified. Re ce ntly, Thompson and c ow orke rs 6 determine d the lysozyme c oncentrations in both bronchial and alveolar lavage fluid samples, but the re lative contribution of lysozyme by prox imal and distal airw ays w as not discusse d.
To investigate the possibility that this enzyme might serve as a useful marker of airw ays sec retion and to ex amine the relative contribution of the upper and low er respiratory tract to lysozyme re le ase from isolated human lung tissues, the basal re le ase of lysozyme w as me asured in the biologic al fluids derive d from prox imal and distal bronchial rings, as w ell as from parenchymal tissues.

Functional studies
Human lung tissue s w ere obtained from patie nts (six lung sample s) w ho had unde rgone surgery for lung c arc inoma. Tis sues w ere obtained from the resected lung at a distanc e from the tumor are a. Parenchymal lung tissues w ere cut as strips (2/15 mm). The subsegme ntal bronchi (first to third generation bronchi) w e re disse cted fre e from parenchymal tissue, c ut as rings of constant le ngth (2 mm, internal diamete r) and w eighe d. In some bronchial rings, the surfac e epithelium w as remove d by ge ntly rubbing the luminal surfac e of the bronchial preparations w ith a moiste ned cotton sw ab. Bronchial and pare nchymal tissue s w e re w ashe d w ith a physiological Tyrode 's solution, pH 7.4 and allow e d to e quilibrate in Tyrode 's solution for 1 h at 37°C in a humidifie d incubator (5% CO 2 / air). Bronchial rings w ere placed in 1 ml of Tyrode's solution w hile pare nchymal tissues w ere e quilibrate d in 10 ml of the Tyrode 's solution. At the e nd of this equilibration pe riod, me dia w ere ex change d. Bronchial and parenchymal tissues w ere rep lace d in identic al volumes of fresh Tyrode's solution previously w armed for 1 h at 37°C. Media de rive d from bronchial and pare nchymal tissue s w ere c ollec te d and stored at -20°C.

Assay of lysozyme
Lysozymal activity w as determine d spe ctrophotome trically by measuring the initial rate of lysis of a 3 mg / ml Mic ro co c cu s ly s o de iktic u s c ell w all susp ension (Sigma Chemic al Co., St Louis). 9 Standard curves w ere construc te d by inc ubating egg-w hite lysozyme (0.5-100 ng /ml; Sigma Chemic al Co.) in 1.5 ml of 50 mM potassium phosphate buffer, pH 7.4 containing M. ly s o de iktic u s (300 m g /ml), sodium azide (0.1%; Sigma Chemical Co.), BSA (1 mg /ml; Sigma Chemical Co.), mix ed and inc ubate d at 37°C for 2 h. In parallel, fluids (1 ml) colle cted from bronchial and pare nchymal tissues w e re dilute d in deionized w ater (1:4 v /v) and ace tic acid w as added to adjust the pH to 4.5. Samples w ere heate d at 100°C for 2 min 10 and ce ntrifuge d (3000 rpm for 10 min) to prec ipitate impurities. An aliquot of 200 m l of e ach tre ate d sample w as added to 1.8 ml of potassium phosphate buffer containing M. ly s o de ikticu s (300 m g /ml), BSA (1 mg / ml), 0.1% sodium azide and incubated at 37°C for 2 h.
Change in turbidity w as monitore d at a w avelength of 450 nm w ith potassium phosp hate buffer as blank. No change of turbidity occurred unde r the se c onditions in absenc e of e nzyme . A standard curve w as plotted as optical de nsity ve rs u s the c onc entration of lysozyme.

Calculation
Coe fficie nts of variation of the low est and the highe st value of the standard c urve w e re calculate d by dividing the standard deviation by the me an and ex pre sse d as a perce ntage . The conc entration of lysozyme detected in the fluids derived from human bronchial and parenchymal preparations are ex pre sse d as ng /ml and w e re estimated from the standard c urves. All results are means± SEM. Statistic al analysis w ere performed using the Student's t-test. A value (P< 0.05) w as taken as an indic ation of significance .

Results
The me an standard curve (n =15) for the lysozyme conc entration measurements obtaine d w ith the eggw hite lysozyme is p re sented in Fig. 1. The assay w as se nsitive since the linear part of the curve allow e d the dete rmination of lysozyme conce ntration s from 100 to 1.6 ng /ml. This assay w as also highly reproduc tive sinc e the c oe ffic ie nt of variation for the highest and the low est value w e re 3% and 6%, respe ctive ly.
Whereas no significant alte ration of the standard curve w as observe d w hen the e gg-w hite lysozyme w as ac idified and heated (data not show n), the lysozyme conc entrations determine d in treate d bronchial fluids w ere significantly inc reased compare d w ith the c ontents of untreate d fluids de rive d from the same bronchial pre parations ( Table 1).
The lysozyme quantitie s of fluids derived from bronchial rings w e re the n c ompare d w ith the conc entrations detec te d in pare nchymal fluids subse que nt to acidification and heating of the sample s. Approximatively tw o-fold highe r c onc entrations w ere measured in bronchial fluids c ompare d w ith the amounts dete cte d in fluids derived from the parenchymal tissues (data not show n). Furthermore, the mean parenchyma w et w eight w as 19-fold higher than the bronchial w et w eight, sugge sting that the lysozyme quantities dete rmined in tre ated bronchial fluids w ere largely depende nt on the lung tissue w et w eight from w hich samples derived.  Since signific ant c orrelations w ere observe d betw e en the tissue w e t w e ight and the quantitie s of lysozyme detected in fluids de rive d from either human bronchial rings (correlation coefficie nt, r= 0.86; P< 0.05) or lung pare nchyma (corre lation coeffic ie nt, r= 0.63; P< 0.05), normalization of the lysozyme fluid conte nts to tissue w e t w eight w as performe d. The lysozyme quantities de te cte d in bronchial fluids w ere significantly highe r (approx imately, 15-fold) than the quantitie s of lysozyme dete cte d in the pare nchymal fluids (Fig. 2).
No significant difference in the lysozyme quantitie s w as observe d in the fluids c ollec te d from bronchial rings w ith an intact e pithe lium compare d w ith the quantities de te c te d in fluids de rived from rings w here the surface e pithelium had bee n re moved (Fig. 3).

Discussion
These results sugge st that the me asure ments of lysozyme in the biological fluids de rived from human bronchial and pare nchymal tissue s subsequent to acidification and heating facilitated the lysozyme dete ction. A predominent rele ase of this enzyme from human bronchial rings, partic ularly from submucosal glands w as obse rved, w hereas lung parenchymal tissues re le ased markedly le ss quantitie s of lysozyme. The results also suggested that prox imal airw ays se creted higher lysozyme quantities than distal bronchi.
The de te rmination of the lysozyme c ontents in fluids collected from human bronchial rings and parenchymal tissue s w as performed using a highly se nsitive and reproduc tive spe ctrophotome tric assay based on the bacteriolytic ac tivity of this e nzyme . 9,1 1 This assay allow e d the detec tion of lysozyme levels as low as 1.6 ng /ml in biologic al fluids. How ever, strong inte rac tions betw ee n the lysozyme and muc us glyc oprote ins have bee n re ported due to their negative charge , leading to the aggregation of these p rote ins and to the diffic ulty in e valuating the lysozyme quantities ex actly in biologic al samples. 12 Se veral ye ars ago, Jollès and Pe tit 10 de sc ribe d a tre atment of salivary fluids by ac idifing and he ating samples, w hich purifie d the enzyme w ithout alte ring the lysozymal activity. 10 ,1 3 How e ve r, this observation has bee n large ly neglec te d in the methods used to e valuate the lysozyme c onc entrations in biological samples. The prese nt report clearly show ed that the lysozyme quantitie s detec te d in the treate d bronchial fluids w ere significantly incre ased c omp ared w ith the contents of untre ate d samples. These data supporte d the notion that the lysosomal ac tivity w as stable after acidification and heating of samples and sugge ste d that this tre atment w as suitable for purifying the enzyme and there by facilitated the de te rmination of the quantities detected by the rate-of-lysis assay.
The lysozyme quantitie s detected in human bronchial fluids w ere approx imatively 1 m g /ml, w hereas low er quantitie s of lysozyme (660 ng /ml) w e re dete cte d in the fluids derived from parenchymal tissues (prese nt re port). Although high c oncentrations of this enzyme have bee n dete cte d in airw ay lavages or nasal fluids from patie nts w ithout resp iratory distre ss (ap prox imative ly, 10 m g /ml), 5,6 the differe nce s in the levels of lysozyme dete cte d in bronchial fluids may be ex plained by the fac t that measure ments (prese nt report) w ere pe rforme d in a mode l of isolate d bronchial rings w hic h w ere pe riodically rinsed w ith fresh me dium. Pre vious investigations reporte d that the bronchial sample lavage fluid conce ntrations of lysozyme w e re highe r than alveolar samp le c ontents after normalization of the quantities to albumin. 6 In the prese nt study, w he n the lysozyme quantities w ere correc te d by tissues w et w e ight from w hich biological fluids w e re derived, bronchial conte nts w ere markedly highe r (approx imate ly, 15-fold) compare d w ith the p arenchymal fluid conc entrations, supp orting the previous observations. In human lung tissues, Klockars and cow orkers 4 reported that alveolar epithelium w as de void of lysozyme w hile an intense lysozyme immunohistochemic al labelling w as observe d in neutrophils and monocyte s w hich accumulate w ithin the airw ays. In view of these investigations, the present data sugge ste d that the lysozyme dete cte d in parenchymal fluids may be due to the degranulation of macrophage s and neutrophils present in the lung conne ctive tissue.
The prese nt re sults show e d that the lysozyme conc entrations w e re highly c orre late d w ith the bronchial tissues w et w eight, suggesting that the lysozyme sourc e w as gre ater in human prox imal bronchi compared to the distal airw ays. Rece nt investigations reporte d that goat airw ay bronchiole s and alveolar duc ts w ere essentially c onstitute d of six c ell typ es, among w hich glandular structures w e re not present. 14 The se data supported the notion that in the more distal portion of the lung, the glandular volume w as smalle r.
The p re sent study also de monstrated that the removal of the surface epithelium from human bronchial rings did not modify the lysozyme re lease from the se pre parations. These data suggested that the submucosal laye r w as the principal sourc e of lysozyme and supported previous immunohistochemical observations, w hich show ed that the lysozyme w as sec rete d from the bronchial glandular se rous ce lls. 1,2 In conclusion, the present report demonstrate d that a tre atment (acidific ation and heating) of biological fluids derive d from lung samples fac ilitated the dete ction of lysozyme by the spe ctrophotome tric rate-of-lysis assay. In addition, the measureme nts of lysozyme show ed that this enzyme w as release d from human bronchial rings, spec ifically from glandular ce lls of the submuc osal layer rather than from lung parenchyma. Since various investigations reporte d that lysozyme leve ls w ere elevate d in association w ith respiratory distress, such as chronic bronchitis and asthma 6 -8 w here glandular tissue volume w as high, these data suggested that the lysozyme may be a marker of the sec retory activity of glands and could be use d in the study of agents regulating the glandular se cretion.