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Immunologic neutrophil elastase levels in sequential aliquots of bronchoalveolar lavage Elsser, Kimberley Anne 1992

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IMMUNOLOGIC NEUTROPHIL ELASTASE LEVELS INSEQUENTIAL ALIQUOTS OF BRONCHOALVEOLAR LAVAGEbyKimberley Anne ElsserB.Sc. (Microbiology), University of Manitoba, 1980A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinThe Faculty of Graduate StudiesDepartment of BiologyWe accept this thesis as conforming to the required standardThe University of British ColumbiaSeptember 1992© Kimberley Anne Elsser, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of Biology      The University of British ColumbiaVancouver, CanadaDate     DE-6 (2/88)iiAbstractPulmonary emphysema is a destructive disease of the peripheral lung that causes progressive lossof functional alveoli. It is considered that proteolytic enzymes, particularly neutrophil elastase(NE), play an important pathogenetic role in the development of emphysema.In order to evaluate the contribution of bronchial lining fluid to immunologic NE levels inbronchoalveolar lavage (BAL), NE levels and cell counts in sequential aliquots of BAL in 44subjects were determined. The subjects were comprised of healthy volunteer smokers (n=20)and smokers, nonsmokers and ex-smokers who were patients undergoing diagnosticbronchoscopy for localized disease (n =24). Lavage was performed through a fiberopticbronchoscope wedged into a segmental bronchus, using 5 x 50 ml aliquots of saline. The firsttwo aliquots were analyzed separately while aliquots 3 to 5 were pooled for analysis.NE levels in unconcentrated BAL were assayed using a sensitive double-sandwich enzyme linkedimmunosorbent assay (ELISA). Mean NE levels were highest in aliquot 1 (2.16 ng/ml) andlowest in aliquot 3-5 (0.46 ng/ml). The percentage of neutrophils was highest in the first aliquotof BAL (10.6%) and lowest in the pooled aliquots 3-5 (4.4%). A significant correlation wasestablished between the number of neutrophils present and the NE levels in the firstiiialiquot of BAL in smokers. These data indicate that the number of neutrophils and NE levelsare highest in aliquot 1 which reflects bronchial lining fluid, while NE levels in aliquots 3-5 ofBAL, which reflect alveolar lining fluid and may be more important in the pathogenesis ofemphysema, were lower.ivTABLE OF CONTENTSPageABSTRACT 	 iiTABLE OF CONTENTS 	 ivList of Tables 	 viList of Figures 	 viiACKNOWLEDGEMENTS 	 viiiINTRODUCTION 	 1METHODSI. Study Population 	 7II. Recovery & Analysis of Bronchoalveolar Lavage1. Bronchoscopy & BAL 	  102. Handling of BAL 	  10III. Assay for NE by ELISA 	  12IV. Preparation of Antibody & Reagents for NE ELISA1. Affinity Chromatography 	  172. Ion Exchange Chromatography 	  183. Preparation of NE Affinity Purified Antibody 	  19VTABLE OF CONTENTS (continued)Page4. Conjugation with Alkaline Phosphatase 	  20V. Statistics 	  21RESULTS 	 23DISCUSSION 	 41REFERENCES 	 47viLIST OF TABLESTable	 Page1. Characteristics of the volunteers and patients studied	 82. Comparison of neutrophil elastase concentration and neutrophil countsin sequential aliquots of bronchoalveolar lavage of all subjects	 243. Comparison of neutrophil elastase concentration and neutrophil countsaccording to smoking status 	 264. Neutrophil elastase concentration and neutrophilcounts comparingvolunteers and patients	 275.	 Comparison of neutrophil elastase levels in broncho-alveolar lavageand macrophage extracts in aliquots 1, 2, and 3-5 in 8 subjects 	 40viiLIST OF FIGURESFigure	Page1. Duplicate standard curves for neutrophil elastase ELISA 	 162. Comparison of NE concentrations in smokers with normal andabnormal lung function	 293. Correlation of neutrophil numbers and NE in patient smokers,aliquot 1	 324. Relationship of neutrophil numbers and NE in patient smokers,aliquot 2	335. Relationship of neutrophil numbers and NE in patient smokers,aliquot 3	 346. Correlation of neutrophil numbers and NE in volunteer smokers,aliquot 1	 357. Relationship of neutrophil numbers and NE in volunteer smokers,aliquot 2	 368. Relationship of neutrophil numbers and NE in volunteer smokers,aliquot 3	 37viiiACKNOWLEDGEMENTSThe guidance and supervision of Dr. R.T. Abboud throughout my research is gratefullyacknowledged. I would like to thank him for making space available to me in his laboratory andfor supplying the specimens and materials for this study.I would also like to thank Anna Bonga and Dr. A.F. Ofulue for technical assistance, DebraHenry for help with statistical analysis, and Allan Hustad for computer advice. My husband,Peter, and infant son, Philip, have been patient with me during the long hours and tensemoments which have been given to this thesis. Without their devotion, I could not have beensuccessful.This thesis is dedicated to the staff of the Microbiology Laboratory, Vancouver GeneralHospital, who continually expressed their support and encouragement in all my academicendeavors.INTRODUCTIONPulmonary emphysema is a disease of the lungs caused by cigarette smoking, where thereis the destruction of lung tissue, with hyperinflation of the lungs and air trapping. Thedisease is defined in pathologic terms as the abnormal permanent enlargement of airspacesdistal to the terminal bronchiole accompanied by destruction of their walls (1). This is oftenassociated with a chronic inflammatory process in the airways.In 1963, Laurell and Ericksson reported their observation of an association betweenemphysema and a genetic deficiency of a plasma protein, alpha-l-antitrypsin (2), morerecently termed alpha-l-protease inhibitor. This led to the current hypothesis of thepathogenesis of emphysema that it results from an imbalance between proteases andantiproteases in the lung (3). According to this hypothesis, emphysema develops when thelevel of proteases exceeds the antiprotease protection, allowing for proteolytic destructionof the elastic meshwork that provides the support for the alveolar walls.Elastase is the protease that can attack elastin; if released in the lung, it is capable ofdigesting lung elastic tissue. It is also a broad-spectrum enzyme, capable of cleaving otherprotein components of connective tissue. The major source of elastase release in the lungis the neutrophil which has a high content of elastase (3,4). Neutrophils originate fromblood capillaries and then migrate into the alveolar air spaces and airways (3). Macrophages2are another potential source of elastase (5,6). Lung macrophages are derived from bloodmonocytes which differentiate into interstitial lung macrophages from which the alveolarmacrophages originate.The composition of all multicellular organisms includes materials of strength and rigidity aswell as components that can stretch and undergo recoiling when required. Elastin iscomposed of polypeptide chains which are cross-linked to generate an elastic meshwork thatenables tissues such as the lung to deform and stretch without damage. Up to 28% of thehuman lung may be composed of elastin (7). For this reason, degradation by proteolyticenzymes can be highly destructive. Large air spaces form in the areas surrounding thedamaged elastic fibers. As these foci of emphysema become more widespread, they willeventually cause respiratory difficulty. This is partly due to a loss of part of the gasexchanging surface of the lung, and partly due to the loss of the tethering support to thesmall airways, given by the lung elastic fibers, which makes ventilation more difficult.There is direct confirmation that leukocyte elastase can produce emphysema in laboratoryanimals. The first experimental evidence that emphysema may develop as a result ofproteolytic lung injury was the report of Gross and co-workers that rats developedemphysema as a result of intratracheal injection of papain, which has elastolytic activity (8).Dogs injected intratracheally with purified human elastase, developed emphysematous lesionsin their lungs (9). Hamsters which had human leukocyte elastase injected into their tracheasand survived, developed severely damaged lungs and emphysema (4). Some studies on blood3neutrophil elastase levels in humans have shown a relationship between increased elastaselevels and chronic obstructive lung disease (10).The major inhibitor of neutrophil elastase (NE) is alpha-l-protease inhibitor (a-PI). Onlya small proportion of emphysema patients (1-2%), have levels of a-PI which are severelydeficient. These patients are particularly susceptible to development of emphysema (2).They tend to develop emphysema at an earlier age (in their forties) than persons with normallevels, presumably because of inability to inhibit released NE (11). In persons with normallevels of a-PI who develop emphysema, the disease may result from possible inactivationof a-PI, or from an excessive load of elastase in the lung (3,12,13).Smoking is strongly associated with the development of emphysema in the generalpopulation. Smoking may predispose to protease-antiprotease imbalance in the lung bydecreasing the functional activity of antiproteases and by increasing the amount of elastasereleased. Smoking may inactivate a-PI directly by chemicals or oxidants in the tobaccosmoke or by causing the release of oxidants from macrophages and neutrophils (13). Thismay lead to an acquired defect in which elastase inhibitory capacity is decreased. Smokinginduces elevated neutrophil counts in the lung (14), which in turn may lead to an increasedrelease of oxidants and elastase. Smoking also increases the number of macrophages in thelung (15), which also may increase oxidant production and elastase release (13).There is controversy surrounding the inactivation of a-PI in healthy smokers (16). However,4recent studies in patients with chronic bronchitis and emphysema indicate that these patientshave significantly lower levels of active antiprotease available to neutralize elastase inbronchoalveolar lavage (12). It is to be noted that a-PI is not the only elastase inhibitor ofthe human respiratory tract. A low molecular weight inhibitor termed the bronchial orsecretory leukocyte protease inhibitor, is the major inhibitor in bronchial lining fluid and isactive against elastase released by neutrophils (17).Another source of elastase in the lung is the alveolar macrophage. Studies havedemonstrated that although the level of macrophage elastase activity is low, this elastase isnot inhibited by a-PI (18) and thus may be important in inducing damage to lung elastin.Alveloar macrophages are recovered in highest concentrations from alveolar lining fluidwhile neutrophil counts are highest in the airways (19). Alveolar macrophages can bind andinternalize NE (6). They can then release it at a later time into connective tissue targets inthe lung. Thus they may contribute to airway damage in two ways: by secreting their ownhighly resistant degradative enzyme, and by serving as a vector for the more abundant NE(3)-Smoking may lead to a potential increase in the elastase load in the lung since inhaled smokestimulates alveolar macrophages to produce chemoattractants for neutrophils (20), which mayinduce neutrophils to release elastase. Studies have shown neutrophil counts in the lungs ofsmokers to be significantly higher than those in nonsmokers (14). These neutrophils mayrelease elastase which can lead to damage of lung elastic tissue. Smoking also leads to an5increase in the number of macrophages in the lung (15), which secrete their own elastase(18).Levels of elastase release have been evaluated in healthy volunteers and in patients. Abboudand co-workers compared immunologic elastase levels in plasma of healthy smokers withthose of nonsmokers and found that short-term intense smoking leads to an increase inplasma elastase concentration, suggesting an in vivo release of NE (21). They alsodemonstrated that acute smoking increases the levels of NE in bronchoalveolar lavage (22).Studies of bronchoalveolar lavage (BAL) from smokers with chronic bronchitis andpulmonary emphysema reported that the greater the severity of emphysema, the higher theconcentration of released NE (12).These previous studies were performed on pooled BAL specimens. Since it has been foundthat the airways contain higher numbers of neutrophils than alveolar lining fluid (19), thisinvestigation sought to compare NE values in sequential aliquots of lavage.Two main evaluations were undertaken:1. A comparison between NE levels in bronchial lining fluid and NE levels in alveolarlining fluid. This was done by separating BAL into aliquots in the order in which they wereinstilled during the lavage procedure. The initial aliquots reflected a large component ofbronchial fluid while the final aliquots contained mainly alveolar lining fluid.62. Examination of NE levels and their correlation with neutrophil counts in the BALaliquots. Two different groups of subjects were studied - healthy volunteers in their thirtiesand forties who were smokers, and older subjects who were undergoing diagnosticbronchoscopy for localised disease. Results were compared in these 2 groups of subjects todetermine if age and cigarette consumption affected the results.7METHODSI. Study PopulationMy study of NE levels in sequential aliquots of BAL was done in conjunction with ongoingstudies of BAL in Dr. Abboud's laboratory which were evaluating the relationship betweenmacrophage elastase and plasminogen activator, protease inhibitor levels, and thedevelopment of emphysema in smokers. I evaluated NE levels in BAL in 44 subjects.Thirty-two of them were smokers, ranging in age from 27 to 67 years. Most of them hadsmoked cigarettes for a long time, and many had smoked the equivalent of forty pack years.One pack year is the equivalent of smoking one pack per day for a year. Seven nonsmokers(who had never smoked) were studied as well as five ex-smokers. Table 1 summarizes thecharacteristics of the study population. The subjects fell into two groups. There were 20healthy volunteers and there were 24 patients undergoing diagnostic bronchoscopy forlocalized disease.None of the patients studied had active or previous history of tuberculosis or respiratoryinfection at the time of the study. Lung function studies were performed on the subjects toevaluate airflow obstruction and/or the presence of abnormalities indicating emphysema (23).A standardized technique expressing results as percent of predicted values was used (23).Volunteers studied were generally healthy smokers with no recent respiratory infection and8TABLE 1 Characteristics of the volunteers and patients studiedVolunteers Patients# of Subjects 20 24Male 11 11Female 9 13# of Smokers 19 13# of Nonsmokers 1 6# of Ex-smokers 0 5Pack Years Smoked0-20 3 420 - 40 15 5>40 1 5Age<30 2 130 - 40 5 240 - 50 11 350 - 60 2 460 - 70 0 14DiagnosisNormal Lung Function 13 21Evidence of Airflow Obstruction 7 3 *Cancer 0 7 ** Some of the subjects counted for both airflow obstruction and cancer9were recruited through advertisements in the university and hospital communities. Thesmokers may have had mild cough and sputum production but did not have evidence ofrespiratory infection.10II. Recovery and Analysis of Bronchoalveolar Lavage1. Bronchoscopy and Bronchoalveolar LavageEach subject underwent a bronchoalveolar lavage (BAL). This is a safe method to recovercells and lining fluid from the lung, mainly from the alveoli (24). It involved passing afiberoptic bronchoscope transnasally or orally into the subject's airways. The instrument waswedged into a segment or subsegmental bronchus of the lingula or right middle lobe or ofan uninvolved lobe if there was localized disease. The tip was wedged into the bronchus inorder to avoid contamination of the lavage fluid by secretions from more proximal airways.Two hundred and fifty ml of sterile saline were instilled in five, 50-ml aliquots with a plasticsyringe. Each aliquot of saline was instilled with the syringe and immediately aspirated bygentle suction with the syringe and then kept on ice. The total return of lavage fluid fromeach subject averaged about 150 ml. The first two aliquots were analyzed separately whilealiquots 3-5 were pooled for analysis. Previous studies suggest that the initial part of thelavage sampled cells and proteins from the peripheral bronchi while alveolar componentswere recovered in subsequent portions of lavage (25). Thus, aliquot 1 reflected a largerproportion of bronchial lining fluid than aliquot 2, while aliquots 3-5 sampled alveolarsecretions.2. Handling of Bronchoalveolar LavageThe volume, cell count and percentage of different cells were determined for each sample.The lavages were centrifuged at 500g for 10 minutes at 4 degrees C to obtain cell free11supernatants. These were subjected to high speed centrifugation (15,000g for 30 min.) tosediment cellular or subcellular debris. Finally, the supernatants were frozen in smallaliquots at -70 degrees C until analyses were performed. Prior to freezing, 1% rabbitplasma was added to guard against loss of neutrophil elastase in storage.Cell counts were obtained from each aliquot; the recovered fluid was immediately loaded ina counting chamber and the macrophages were numbered. Smears for determining thepercentage of different cells (differential counts) were prepared using a cytocentrifuge. Thecytospin preparations were stained with a modified Wright's stain and 400 cells were countedto obtain the percentage of neutrophils.To check whether released elastase from neutrophils was being taken up by macrophages andwas leading to lower NE levels in aliquots 3-5, purified macrophages were preparedseparately from aliquots 1, 2, and 3-5 and then assayed for NE content. The cellssedimented from aliquots 1, 2, 3-5 were suspended in Dulbecco's modified minimal essentialmedia at a density of 1 million cells/ml and then plated in culture flasks or petri dishes. Themacrophages were allowed to adhere for 2 hours at 37 degrees C in an incubator with 5%carbon dioxide. After 2 hours of adherence, the media and nonadherent cells were removed.The adherent cells, which were macrophages without significant contamination byneutrophils, were scraped and suspended in a mixture of phosphate buffered saline (PBS),bovine serum albumin (BSA) and 0.1% Triton-X-100 at a concentration of 2-5 millioncells/ml. Then they were subjected to three periods of sonication for 30 seconds each to12break down cell membranes and release elastase. These preparations were then frozen at -70degrees C until assays could be performed comparing their elastase levels to those of thelavage supernatants. NE levels in macrophages were expressed in ng/ml of original lavagefluid from which the macrophages were obtained, for comparison with NE levels in thelavage fluid itself.III. Assay for Neutrophil Elastase by ELISANeutrophil elastase was measured by a sensitive enzyme-linked immunosorbant assay(ELISA). The assay was based on the double-sandwich method. The main components ofthis method include:1. adsorption of NE antibody to a solid phase,2. addition of lavage samples, followed by incubationand then washing,3. addition of alkaline phosphatase labelled NE antibody,again followed by incubation and washing,4. addition of alkaline phosphatase substrate,5. determination of end point, and reproducibility oftest measurements (26).The solid phase used was a polystyrene microtiter plate (CoStar brand) containing 96 wells.13This brand gave consistent readings throughout the plate and low zero readings whencompared with other brands that were tested.The method followed was developed by Ochnio, Abboud, et al in this laboratory (27). Inbrief, the procedure calls for the ELISA microtitre plates to be coated with affinity purifiedNE-antibody in carbonate buffer, pH 9.6. 0.1 ug of NE-antibody in 100 ul of buffer wasdispensed into each well, except for a column of four wells which served as blanks.The coated plates were wrapped in plastic and kept for 30 minutes at room temperature andthen left in the refrigerator overnight to allow the antibody to bind to the plates. The nextday they were washed three times with phosphate buffered saline containing Tween-20 (PBS-Tween) in a final concentration of 0.05%. Free binding sites were blocked by a 1% solutionof BSA in PBS-Tween.A set of NE standards from 0 - 2.0 ng/ml was prepared for each plate. The dilutions weremade with PBS-Tween containing 1 % BSA and 1% non-immune rabbit serum. The rabbitserum was added to guard against nonspecific binding and loss of NE (28). The standardswere prepared on ice and loaded onto the plate immediately.Smokers' and nonsmokers' samples were also diluted with PBS-Tween with 1% BSA and1 % rabbit serum. Samples expected to contain high amounts of NE were diluted 2X, 4Xand 8X for the first aliquot of lavage and 2X and 4X for the second and third aliquots.14Undiluted assays were also performed on the second and third lavage aliquots. To thesamples to be tested without dilution, I added PBS containing 10% rabbit serum, 10% BSAand 1% Tween-20 in a volume of 1/10 of the sample to achieve final concentrations of 1%for both BSA and rabbit serum. The samples were also prepared on ice and loaded onto theplate immediately. Each sample or standard was applied to 4 wells of the plate with avolume of 100 ul per well.The plates were incubated for 2 hours at 37 degrees C, washed with PBS-Tween and thenthe second antibody in the sandwich was applied. NE antibody conjugated with alkalinephosphatase was added at a concentration of 1:1000 diluted in PBS-Tween/1% BSA. Eachwell was loaded with 100 ul of the conjugate. This was followed by a 2-hour incubation at37 degrees C. After another washing, 100 ul of a solution of disodium p-nitrophenylphosphate (alkaline phosphatase substrate, Sigma Chemical Co., St. Louis, Mo)diluted in diethanolamine buffer, pH 9.8, was added to each well. The plate was thenincubated at 37 degrees C and analyzed in half hour increments for 2 hours. Alkalinephosphatase substrate is a clear solution which in the presence of the enzyme, turns yellow.The intensity of the color in each well, which is proportional to the concentration of NEoriginally added to the well, was determined spectrophotometrically at a wavelength of 405nm, in an automated microplate ELISA reader (Model EL 311, Biotek Instruments,Winooski, Vt). The first reading at half hour incubation was for the samples with highlevels of NE, and the 2-hour readings were for samples with NE levels less than 1-2 ng/ml.15The virtues of the ELISA, which include low cost, reagent stability, safety, sensitivity andease of procedure (29), require that the tests be rigidly controlled by multiple assays withreproducible standards. Many duplicate standard curves were run to ensure consistency andaccuracy. Figure 1 illustrates a sample of a duplicate standard curve which was run to checkreproducibility of dilutions and pipetting technique.Good ELISA results also involve the use of pure and active antibody and enzymepreparations. NE antibody was purified by affinity chromatography. Part of the antibodywas conjugated with alkaline phosphatase, checked for activity and stored in the refrigerator.The activity was not affected by storage for periods of up to 1 year. During this time, 48ELISA's were run.0 	 1 	 1 	 ,I 	 10.00 	 0.20 	 0.40 	 0.60 	 0.60 	 1.00 	 1.20 	 1.40 	 1.60Neutrophil Elastase ng/ml1.7016Figure 1: Duplicate standard curves for neutrophil elastase ELISA.Obtained after incubation with alkaline phosphatase substrate at 37C.There was good agreement between the standard curves.17IV. Preparation of Antibody and Reagents for NE ELISAHuman NE was extracted from white blood cell (WBC) suspensions by the methods ofBaugh and Travis (30) and Martodam and co-workers (31). These WBC suspensions wereobtained by a cell separator from patients with chronic granulocytic leukemia. Two stepswere involved in purifying the NE from the crude extract of the WBC suspensions:1. affinity chromatography and,2. ion exchange chromatography.1. Affinity Chromatography with Sepharose-TrasylolThis step takes advantage of the fact that Trasylol (aprotinin or bovine Kunitz basic trypsininhibitor) is a weak proteinase inhibitor of elastase and elastase will bind to it. Sepharose,obtained from Sigma, was bound with Trasylol. The WBC extract was equilibrated with0.1M TRIS/0.15M NaCl buffer, pH 8.0, and applied to the Sepharose-Trasylol gel. Afterincubation to bind proteases, the gel was packed into a chromatography column. Thecolumn was then washed with the same buffer to remove unbound protein, while elastase andCathepsin G remained bound to the column (30,31). Thirty 5-ml fractions were collectedand the OD 280 measured. When the OD of the eluate dropped below 0.020, it indicatedthat most of the unbound components were eluted and only elastase and chymotrypsin werebound. A change of buffer to 0.05M sodium acetate/1.0M NaC1 buffer, pH 5.0 broke theTrasylol-elastase bonds and NE was eluted. It was collected in 6 fractions with OD 28018measuring approximately 0.60. These fractions were combined and dialyzed against 3changes of 0.05M sodium acetate/0.1M NaC1 buffer, pH 5.5. At this point the NE was onlypartially purified and required separation from another protease, Cathepsin G. This wasachieved by purifying NE from the dialyzed solution by using an ion exchange column.2. Ion Exchange Chromatography with CM-CelluloseThe NE containing solution from the previous step was applied to a column of CM-SephadexC-50 which had been equilibrated against a 0.02M sodium acetate/0.15M NaCl, pH 5.5buffer. After washing with the same buffer and collecting eight 5-ml fractions, a low OD280 below 0.020 was achieved. Then the buffer was changed to 0.02 M sodiumacetate/0.45M NaC1, pH 5.5, to displace the NE. These fractions containing the NE werepooled and dialyzed. (The cathepsin G was eluted later when the NaCl concentration of thebuffer was raised to 1.0M.) The NE was checked for purity and activity by SDSpolyacrylamide gel electrophoresis and by kinetic assays for elastase activity related to themeasurement of protein by OD 280.193. Preparation of NE Affinity Purified AntibodyThe NE isolated by the above two steps was used to prepare a Sepharose-NE column topurify NE antibody from crude rabbit antiserum by affinity chromatography. First, the NEwas inactivated by treatment with phenylsulfonylmethylfluoride. Then 4.4 mg was linkedto 1.1 g of cyanogen bromide activated Sepharose-4B (supplied by Sigma), using a couplingbuffer of 0.1M sodium bicarbonate/0.5M NaC1, pH 8.3. This was kept mixing on a rotatorfor 4 hours. According to measurements of OD 280 on supernatant before and after binding,only about 25% of the NE remained unbound. 0.1M TRIS/0.5M NaC1 was added to thegel/NE mixture to block empty binding sites. The mixture was washed with 4 cycles ofsodium acetate buffer (0.1M sodium acetate/0.5M NaC1, pH 4.0) alternating with couplingbuffer (0.1M NaHCO3/0.5M NaC1, pH 8.3) on a sintered funnel apparatus. The change inpH served to ensure that nonspecific binding was eliminated. The gel mixture wassuspended in 2 ml of PBS with azide.Rabbit antiserum to NE, previously prepared in the laboratory by immunizing rabbits withthe purified human NE, according to the technique described by Feinstein and Janoff (32),provided the crude antibody for the next step. About 6 ml of this rabbit antiserum wasadded to the NE-gel mixture. Binding took place on a rotator at 4 degrees C overnight.This mixture was then poured into a column and washed with PBS (to remove unboundproteins) until a low OD 280 was reached. Next, 0.1M glycine-HC1 buffer, pH 2.5, wasused to elute the antibody. Nine 2-ml fractions were collected. Each fraction was20immediately neutralized with 2 drops of saturated TRIS and the OD 280 was measured.When the OD dropped below 0.02, 0.6M guanidine-HC1 buffer, pH 3.1, was added to elutethe rest of the antibody bound to the column.The glycine fractions, containing the NE antibody, were pooled and dialyzed extensivelyagainst phosphate buffered saline. They were concentrated by ultra-filtration through amembrane (Amicon), to a volume of approximately 1 ml. Double ouchterlony plates againstNE were used to determine that antibody activity was high in the glycine fraction.4. Conjugation with Alkaline PhosphataseThe final concentration of the NE antibody eluted in the glycine fractions was 4.43 mg/ml.Half of this preparation was stored in a vial at 4 degrees C to be used as the coating antibodyin the bottom part of the ELISA sandwich. A portion of the antibody, 1.0 mg (226 ul), wasconjugated with 2.5 mg (325 ul) of alkaline phosphatase (supplied by Sigma PO405 Lot11H8130, concentration = 7.7mg protein/m1) using glutaraldehyde.(33) Sterile PBS, pH7.2, was added to make the volume up to 1 ml, and then 8.0 ul of 25% glutaraldehyde wasadded to give a final concentration of 0.2% glutaraldehyde. The mixture was stirred in thedark for 2 hours. This was followed by 48 hours of dialysis, initially against PBS, pH 7.4and finally 0.05M TRIS, pH 8.0. The conjugate was then diluted to a volume of 2.0 mlwith 0.05M TRIS, pH 8.0 containing 1% BSA and 0.02% sodium azide, achieving a finalconcentration of 400 ug of antibody/ml . This solution was stored in the dark at 4 degrees21C and constituted the source of the detecting antibody for the top part of the ELISAsandwich. The use of the same antibody from the same species for both layers of the ELISAhas been documented in literature (29,34) and previously used in studies with BAL(27,35,36).The process of obtaining affinity purified NE antibody was time consuming and technicallydemanding. However, the end result was an active purified antibody, specific to the NE,which was required for the ELISA. This specificity was indicated by low background zerovalues for the standard curves.V. StatisticsThe subjects consisted of 2 groups, volunteers and patients undergoing diagnosticbronchoscopy. They were analyzed both separately and grouped together according tosmoking status. Comparisons were also made between those who had evidence of abnormallung function and those who did not. Between-group comparisons were made by analysisof variance and comparisons of different aliquots of the same subjects were made by repeatedmeasures of analysis of variance. Least-squares linear regression analysis and the Pearsoncorrelation coefficient were used to evaluate variables that were strongly associated with highNE levels. Statistical analysis was done using a Quattro Pro Statistics program. A p valueof <0.05 was considered as statistically significant.22RESULTSThe values for neutrophil elastase concentration and neutrophil numbers recovered from thelavages for all subjects combined are summarized in Table 2. All data in this and subsequenttables are shown as the mean value + standard deviation. The NE concentration in aliquot1, which was similar for all three groups of subjects - smokers, nonsmokers and ex-smokers,averaged 2.16 + 0.46 ng/ml. When all the subjects were tested as a group (n=44), thislevel was significantly higher than the level of 0.86 + 0.10 ng/ml in the second aliquot(p<0.001), which was significantly higher than the level of 0.46 + 0.08 ng/ml in the pooled3rd to 5th aliquot (p <0.005). As discussed in the next paragraph, when the subjects weredivided into smoking and nonsmoking categories, the same trend was evident. To correctfor the variation contributed by dilution via the instilled saline during the lavage, NEconcentration was divided by the albumin concentration for each aliquot, since albumin inthe BAL depends on the bronchoalveolar lining fluid recovered in the lavage. The NEresults showed a similar decreasing pattern of concentration from aliquot 1 to aliquots 3-5when expressed per mg of albumin. The percentage of neutrophils in the first aliquot ofBAL (10.6 + 2.6%) was significantly higher (p <0.02) than in the second or subsequentaliquots (5.4 + 2.1%; 4.4 + 1.5%). The calculated neutrophil count (x 1000/m1) in thefirst, second and subsequent aliquots were 12.2 +8.5, 12.6 + 11.3, and 7.7 + 5.2respectively. Although values tended to be higher in the first aliquot, the differences werenot statistically significant.23TABLE 2 Comparison of neutrophil elastase concentration and neutrophil counts in sequentialaliquots of bronchoalveolar lavage of all subjects (n=44)Aliquot # NE ng/mL NEng/Albuminmg# ofNeutrophils x1000/mL% Neutrophils1 2.2 + 0.5 102.7 + 110.3 12.2 + 8.5 10.6 ± 2.62 0.9 + 0.1 25.6 + 25.8 12.6 ± 11.3 5.4 + 2.13-5 0.5 + 0.08 16.2 + 14.6 7.7 ± 5.2 4.4 + 1.524Table 3 sub-divides the data by smoking history. Again, a similar trend of high elastaselevels in aliquot 1 diminishing to significantly lower levels in the last aliquot is observed foreach smoking category. The NE:albumin ratios indicated that the first aliquot from eachsmoking group yielded a much higher value than subsequent aliquots. These values,however, had a relatively higher standard of deviation than NE levels expressed per ml.This indicates that adjusting NE levels for albumin concentrations does not improve thevariability in the NE data, probably because albumin concentrations in the recovered lavagedepend not only on the dilution effect but also are affected by variability in albumin levelsin bronchoalveolar lining fluid due to inflammation.Neutrophil counts per ml and neutrophils expressed as percent of total cells were highest inaliquot 1 and lowest in aliquots 3-5 for smokers and nonsmokers. Neutrophil counts in ex-and nonsmokers, of which all but one were patients, were similar to the smokers. Theselevels reflect the fact that these patients had symptoms of cough or lesions for which theywere having diagnostic bronchoscopy. Therefore, the neutrophils likely indicate airwayTABLE 3 Comparison of neutrophil elastase concentration andneutrophil counts in sequential aliquots of bronchoalveolar lavageaccording to smoking statusAliquot NE Conc. ng/mL NE ng/Albuminmg#Neutrophils x1000 /mL%NeutrophilsSmokers (n=32)1 2.1 + 1.9 117.0 + 166.5 16.0 + 17.2 6.7 + 4.52 0.8 + 0.9 24.3 + 26.9 17.2 + 21.1 2.3 + 1.23-5 0.4 + 0.4 12.5 + 14.6 10.6 + 11.9 2.5 + 2.9Nonsmokers (n=7)1 2.7 + 2.0 129.1 + 129.5 13.3 + 6.5 13.8 + 9.22 1.0 + 0.6 19.1 + 28.7 6.8 ± 2.5 6.0 + 2.93-5 0.6 + 0.2 12.6 + 18.0 5.4 ± 1.7 6.7 + 3.9Ex-smokers (n=5)1 1.6 + 1.0 62.0 + 34.9 7.1 + 1.9 11.3 + 2.72 0.8 + 0.2 33.3 ± 21.8 13.2 + 10.3 7.9 + 5.03-5 0.5 ± 0.1 23.6 + 11.1 7.1 + 1.9 4.1 + 0.826TABLE 4 Neutrophil elastase concentration and neutrophil counts insequential aliquots of bronchoalveolar lavage comparing volunteers andpatientsVolunteer Smoker(n=19)Patient Smoker(n=13)Patient Non/Ex-smoker (n=11)Aliquot 1NE ng/mL 2.1 + 1.9 2.3 + 2.0 2.1 + 1.7% Neutrophils 6.9 + 3.8 7.7 + 5.1 12.6 + 7.9# Neutrophils x1000/mL14.8 + 8.7 10.2 + 6.3 11.1 + 6.1Aliquot 2NE ng/mL 0.9 + 1.0 0.7 + 0.6 0.9 + 0.4% Neutrophils 3.1 + 3.0 2.8 + 2.0 7.0 + 4.0# Neutrophils x1000/mL13.0 + 8.0 11.8 + 8.7 9.1 ±7.2Aliquot 3-5NE ng/mL 0.4 + 0.4 0.3 + 0.3 0.5 + 0.2% Neutrophils 2.2 + 2.7 2.1 + 1.4 6.1 + 3.4# Neutrophils x1000/mL8.5 + 5.1 8.7 + 9.3 5.5 + 1.827inflammation. The percentage of neutrophils was higher in non and ex-smokers than insmokers because the total number of macrophages which make up the majority of cells waslower in the ex and nonsmokers.The percentage of neutrophils in the three aliquots was compared by analysis of variance.The mean percentage of neutrophils in aliquot 1 was significantly higher than that of aliquots2 and 3-5. Other researchers have also found significant differences, with the first aliquotcontaining the greatest percentage of neutrophils (19). When the smokers were analyzedseparately from nonsmokers, the highest percentage of neutrophils was again found in thefirst aliquot for each group.Table 4 presents data separating the smokers into the 19 volunteers and the 13 patientsundergoing diagnostic bronchoscopy. The average NE levels in aliquot 1 were similar, 2.05+ 1.88 ng/ml and 2.25 + 2.02 ng/ml respectively. Results are compared with non and ex-smokers who were patients except for one volunteer nonsmoker. In the non and ex-smokinggroup (with the volunteer excluded), the NE concentration in aliquot 1, 2.08 + 1.66 ng/ml,did not differ significantly from the two groups of smokers. The pattern of decreasing NEconcentration from aliquot 1 to aliquots 3-5 was similar in all three groups.28Figure 2 compares the NE levels in smokers, divided into volunteers and patients, withrespect to the presence or absence of abnormal lung function. There was no significantdifference in NE levels in the patients with abnormal lung function compared with those withnormal lung function. The mean NE levels in aliquot 1 were 2.55 + 1.71 ng/ml and 2.23+ 2.23 ng/ml respectively. The NE levels decreased from aliquots 2 through 3-5, withabnormal patients having slightly higher corresponding values. In the volunteers, those withabnormal lung function tended to have elevated levels of NE in all three aliquots but theselevels were not statistically significantly different from the NE levels of volunteers withnormal lung function. Mean levels in aliquots 1, 2, 3-5 were 2.41 + 1.81; 1.18 + 1.50;0.55 + 0.50 respectively in volunteers with abnormal lung function vs. 1.79 + 1.82, 0.65+ 0.47, 0.26 + 0.19 in normal volunteers.A large body of research has attempted to correlate relationships between neutrophilnumbers, neutrophil elastase levels, and smoking, since evidence suggests that the mainsource of elastase in the lung is the neutrophil (3,27), and that this may be of pathogeneticimportance in emphysema. One of the objectives of this study was to compare neutrophilnumbers with NE concentrations. Linear regression analysis was performed on the data ofthe patient and volunteer smokers. There was a significant correlation between neutrophilnumbers and NE levels in the first aliquot of BAL but not in the last two aliquots. Figure3 shows the relationship29Figure 2: Comparison of neutrophil elastase concentrationsin sequential aliquots of bronchoaveolar lavage in smokerswith normal and abnormal lung function32.5-0.5-0Aliquot 1\ abnormal pt as normal pt1 	 rAliquot 2normal vol. [22 abnormal vol.Aliquot 3-5"E.cocii.)0Lo2ED:ET).24-.mU)z1.530between neutrophil numbers and NE concentrations in aliquot 1 of BAL from patientsmokers, which had a significant correlation, r=0.835 (p <0.001). Aliquots 2 and 3-5 didnot show a correlation between neutrophil numbers and NE concentrations (Figures 4 & 5).In analyzing the relationship between NE and neutrophil counts, there was one subject whohad very high neutrophil counts and very high NE levels in the BAL. Analysis of the datafor all 3 aliquots showed a significant correlation if his values were included, but thecorrelation was no longer significant when his values were excluded. The subsequentanalyses are shown excluding his data. The relationship between neutrophil counts and NElevels in aliquot 1 in the volunteer smokers is shown in Figure 6. There were 2 outlyingpoints in the data, one with a very high NE level and one with a high neutrophil count. Asignificant correlation ( r=0.498, p =0.05) was obtained by excluding these 2 points. Thecorrelation was no longer significant if these were included. In aliquots 2 and 3-5, as seenin Figures 7 & 8, there was no correlation at all between neutrophil numbers and NE.Linear regression analysis of the same variables in the non and ex-smoking group resultedin a similar pattern. The first aliquot showed a significant correlation (r=0.708, p <0.01)while the other aliquots did not.Regression analyses indicated that there was no correlation between NE levels in aliquot 1and age of subjects, total amount smoked (number of pack years) or current smoking incigarettes per day. Comparison of results in male and female smokers indicated no significantdifferences in levels of NE and neutrophil counts (Students t test of independent means).31Table 3 showed a large decrease in NE concentration between aliquot 1 and aliquots 3-5.The NE decline is greater proportionately than the percentage of neutrophil decrease betweenthe aliquots. It seemed possible that released NE was being taken up by alveolarmacrophages, which were more numerous in aliquots 3-5 of the lavage, resulting in110 	I20 3 	I0	 400 5032FIGURE 3: Correlation of neutrophil numbers and neutrophilelastase levels in patient smokers, BAL aliquot 1 (r = 0.835; p<0.001)Neutrophils x 1000/m133Figure 4: Relationship of neutrophil numbers and neutrophilelastase levels in patient smokers, BAL aliquot 2 (lack of correlation)32.7-aiU) 1.8-cr)cci'LE2Z sas-0.6-0.3-00• •••10 	 20 	 30 	 410 	 510 	 610Neutrophils x 1000/m170100 60500.4-0.2- • •• •1 	 1 	 i20 	 30 	 40Neutrophils x 1000/m1034Figure 5: Relationship of neutrophil number and neutrophilelastase levels in patient smokers, BAL aliquot 3 (lack of correlation)21.8-1.6-0 5 30 401 	 1 	 1 	 l10 	 15 	 20 	 25Neutrophils x 1000/m135Figure 6: Correlation of neutrophil numbers and neutrophilelastase levels in volunteer smokers, BAL aliquot 1 (r = 0.498; p = 0.05)There was a significant correlation if the 2 outlying points were not included.21.8-1.6- ••A••A•0.4- A • • 	 ••0.2-I 	 I 	 r 	 I 	 1 	 110 	 15 	 20 	 25Neutrophils x 1000/m100 5 30 35 4036Figure 7: Relationship of neutrophil numbers and neutrophilelastase levels in volunteer smokers, BAL aliquot 2 (lack of correlation)37Figure 8: Relationship of neutrophil numbers and neutrophilelastase levels in volunteer smokers, BAL aliquot 3 (lack of correlation)10.8-••••0.2-• ••••00	5	10Neutrophils x 1000/m12538lower NE levels in the BAL supernatant. To determine if this was the case, NEconcentration was measured in the sonicated macrophage extracts from aliquots 1, 2 and 3-5of BAL of 8 subjects. The amount of elastase present in the macrophage extracts wasexpressed as the equivalent in the original lavage volume from which the macrophages wereobtained. Macrophage released NE was compared with NE recovered from the lavage fluid.These results are shown in Table 5. Macrophage extracts from aliquot 1 contained anaverage of 23.4 + 7.9% of the total NE, while in aliquot 2 the yield was 11.9 + 7.2% andin aliquots 3-5 it was 4.9 + 3.0%. This indicates that the relatively low levels of supernatantNE in aliquots 3-5 of BAL is not due to uptake by the increased population of macrophages.The NE levels were lower because there was less NE released. A certain amount of erroris inherent in these values since the levels of NE potentially available for analysis were low,in some cases approaching the lower limit of detection by the ELISA.39TABLE 5 Comparison of neutrophil elastase levels in bronchoalveolar lavage andmacrophage extracts in aliquots 1, 2, and 3-5 in 8 subjectsSubject#Aliquot#MacrophageExtractng/mL originalBALBALSupernatantng/mLTotal ofBAL &Macrophagesng/mLMacrophage% ofTotalN98 1 0.52 0.82 1.34 392 0.009 0.23 0.24 43-5 0.003 0.10 0.10 3N101 1 0.19 0.72 0.91 212 0.024 0.40 0.42 63-5 0.003 0.17 0.17 2N102 1 0.54 1.64 2.18 252 0.13 1.04 1.17 113-5 0.005 0.13 0.14 4N107 1 1.80 6.56 8.36 222 0.27 4.72 4.99 53-5 0.037 1.6 1.64 2N109 1 0.30 1.56 1.86 162 0.034 0.22 0.25 143-5 0.003 0.07 0.07 4N110 1 0.43 2.5 2.93 152 0.24 1.26 1.50 163-5 0.07 0.66 0.73 10N111 1 0.86 1.98 2.84 302 0.24 0.67 0.91 263-5 0.04 0.40 0.44 9N112 1 0.10 0.42 0.52 192 0.009 0.06 0.069 133-5 0.001 0.02 0.021 540DISCUSSIONThe results of this study demonstrate that NE concentration is significantly greater in aliquot1 of BAL than in the subsequent aliquots. This was true when NE concentrations werecorrected for the dilution effect of the instilled saline and expressed as ratios to albumin.Previous studies have found that the first aliquot of lavage reflects a greater contribution ofbronchial lining fluid than subsequent aliquots which reflect alveolar lining fluid (19,24).Therefore the data suggest that NE is present in greater concentrations in the bronchial liningfluid than in alveolar lining fluid.This finding could have an impact on emphysema research involving measurements of NEin BAL, particularly trials of drugs to decrease elastase release in the lung. In the 2 studiesof Cohen and co-workers (37,38) evaluating the effect of colchine on elastase levels in BAL,the aliquots of BAL (5 x 50 ml) were pooled for analysis. This may not be a goodindication of what is happening in the lung periphery. My results indicate that a significantproportion of the NE is contributed by aliquots 1 and 2 as compared to aliquots 3-5. Aliquot1 contributed 35.4% + 14.3 % of the total NE, aliquot 2 contributed 23.9% + 9.5% andthe combined last three aliquots yielded 40.7 % + 14.5%. Pooling all the aliquots togetherresults in a distorted indication of the NE concentration in the lung periphery. It would bemore helpful for evaluation of pathogenetic effects in emphysema to separate the initialaliquots from the remaining ones. In the study of Fujita and coworkers (12), the first aliquotwas discarded but lavage was done using 5 x 20 ml aliquots of saline. These smaller41volumes of fluid used, 20 ml per aliquot instead of 50 ml, may still be affected by bronchiallining fluid even after discarding the first aliquot (39). However, it should be noted thatNE levels in aliquot 1 of BAL may be important in contributing to airway damage (40).The data indicates that the elastase levels recovered in aliquot 1 (mainly from airways) ofthe lavage samples was significantly correlated with neutrophil counts. NE is released fromthe neutrophil when it encounters objects to be phagocytosed, when it dies, or when it isexposed to chemotactic factors or toxic products such as tobacco smoke (3). The releasedelastase may then attack and hydrolyse proteins in connective tissue (3). However, NElevels in aliquots 3-5 were low, relatively lower compared with aliquot 1 than the numberof neutrophils, and there was no correlation between NE levels and neutrophil counts inaliquots 3-5. The lack of correlation between neutrophil numbers and NE in the latterportions of lavage indicate that NE levels in aliquots 3-5 of BAL did not simply depend onneutrophil numbers. In the alveolar spaces other factors become important. One possibleexplanation is NE uptake by alveolar macrophages. Macrophages have been shown to havemembrane receptors for binding NE which is then internalized by the macrophage (5). Mymeasurement of NE in macrophage extracts excludes the possibility that the lower levels ofNE in aliquots 3-5 were due to uptake by macrophages. Only 5% of the total NE in aliquot3-5 was present in the macrophages. The remaining 95% was recovered in the supernatant,presumably as a result of NE release.In this regard it should be noted that the NE enzyme can remain active, internalized by the42macrophage for as long as 5 days (5). The alveolar macrophages can then release the NEinto connective tissue targets against which they are tightly apposed. This macrophage-delivered elastase may remain uninhibited by (3), thus contributing to lung damage. In thisway, macrophages can serve as a vector for the NE, carrying it and releasing it later atconnective tissue sites. The critical area of NE release in regard to the pathogenesis ofemphysema is the lung interstitium, but this is difficult to sample, so instead, because of itsease, BAL is used to access the alveolar spaces.Another possible explanation for the lack of correlation between neutrophils and NE in theperiphery of the lung is that the concentration of cigarette smoke is lower in the lungairspaces than in the airways. Smoke contains oxidants and other products which mayinduce elastase release from neutrophils (41). The airways, which receive more intensesmoke, are therefore more vulnerable to the effect of tobacco smoke on the release of NE,while lung periphery, the effects of tobacco smoke may be less, resulting in less elastaserelease.Evidence in literature that indicates that neutrophils are present in increased numbers in thelungs of cigarette smokers (3,9,10,19). It is thought that smoking may release a chemotacticfactor to which alveolar macrophages and neutrophils respond (20). The subjects I studiedwho were non and ex-smokers were relatively few in number and had high neutrophil countssimilar to the patient smokers. This reflects that these subjects were patients, who wereundergoing diagnostic bronchoscopy for lesions or unexplained cough; therefore, they were43likely to have airway inflammation and increased numbers of neutrophils, even though theywere non or ex-smokers. In order to properly evaluate the effects of smoking, healthynonsmoking volunteers would have to be studied for comparison with the healthy volunteersmokers who comprisedthe majority of the subjects in the smoking group. Volunteer nonsmokers were not beingstudied in Dr. Abboud's laboratory at the time my research was carried out, because thestudies on volunteers were designed specifically to determine the relationship betweenmacrophage enzyme activity and lung function abnormalities in smokers. To study volunteernonsmokers for the purposes of this thesis would have been too costly and too timeconsuming.The comparisons made between subjects with evidence of abnormal lung function and thosewho were normal suggest that volunteers with abnormal lung function had higher NE levelsin aliquots 3-5, than the volunteers with normal lung function. This is consistent with thehypothesis that increased NE levels in smokers may lead to peripheral lung damage. In thepatient smokers, there was no association between NE levels and abnormal lung function.The lack of a difference between those with normal and abnormal lung function may be dueto the relatively small number of patient smokers studied and to the presence of lung lesionsor other respiratory problems for which they were undergoing diagnostic bronchoscopy. Itshould be noted that BAL studies on patients with chronic bronchitis and emphysemaundergoing bronchoscopy specifically for research purposes, reported that NE levels werehigh in subjects with abnormal lung function (12,19).44The overall lack of relationship between a lifetime of heavy smoking (number of pack years)and high NE levels suggests that the current smoking pattern rather than total amountsmoked may be more important in inducing NE release in BAL. One individual who hadsmoked the equivalent of 100 pack-years had NE levels of only half of the mean forsmokers. For this reason, the current smoking pattern of the subjects (cigarettessmoked/day) was tested against NE levels; there was no correlation. However, when resultsof smokers of 40 pack-years and more (n =8) were analyzed separately from the remainingsmokers (n=24), their NE levels were generally higher. This was especially true for NEconcentrations in the second and third to fifth aliquots which were 1.47 + 1.54 ng/ml and0.70 + 0.62 ng/ml respectively as compared to the mean of 0.58 + 0.46 ng/ml and 0.24+ 0.16 ng/ml. The lack of correlation for the group as a whole could be a result of otherfactors such as age, health and volume of lavage fluid recovered. Although subjects wereadvised to abstain from smoking on the day of lavage, some of them may have smoked oneor two cigarettes an hour or more prior to the procedure and this could introduce anothersource of variability.The NE levels of my study compare favourably with the levels obtained by workers in thislaboratory using the same ELISA technique four years ago (27). They found healthy youngsmokers to have an average value of 0.69 ng/ml in the pooled lavage of aliquots 1-5, whilemy calculated average over the five aliquots was 1.10 ng/ml. For healthy volunteernonsmokers, they obtained a level of 1.15 ng/ml compared with my 1.43 ng/ml innonsmokers undergoing diagnostic bronchoscopy.45In conclusion, this study has evaluated NE levels in sequential aliquots of BAL and hasfound them to be highest in the first aliquot and lowest in the pooled third to fifth aliquots.The first aliquot reflects bronchial lining fluid while the latter aliquots sample alveolar liningfluid. The percentages of neutrophils also decreased significantly from the first aliquot tothe last three but the neutrophil numbers correlated with the NE concentrations only in thefirst aliquot. There was a suggestion of an association between NE levels in aliquots 3-5 andlung function in the volunteer smokers indicating that higher NE levels in the lung peripherymay be related to development of lung damage in smokers.46REFERENCES1. Snider, G., Kleinerman, J., Thurlbeck, W., Bengali, Z. The definition of emphysema.Report of a National Heart, Lung and Blood Institute, Division of Lung Diseases Workshop.Am Rev Respir Dis 1985; 132: 182-185.2. Laurel!, C., Ericksson, S. The electrophoretic alpha-1-globulin pattern of serum inalpha-1-antitrypsin deficiency. Scand J Clin Lab Invest 1963; 15: 132-140.3. Janoff, A. Elastases and emphysema. Am Rev Resp Dis 1985; 132: 417-433.4. Senior, R., Tegner, H., Kuhn, C., Ohlsson, K., Starcher, B., Pierce, J. The inductionof pulmonary emphysema with human leukocyte elastase. Am Rev Resp Dis 1977; 116:469-475.5. McGowan, S., Stone, P., Snider, G., Franzblau, C. Alveolar macrophage modulationof proteolysis by neutrophil elastase in extracellular matrix. Am Rev Respir Dis 1984; 130:734-739.6. Campbell, E., Wald, M. Hypoxic injury to human alveolar macrophages acceleratesrelease of previously bound neutrophil elastase. Am Rev Respir Dis 1983; 127: 631-635.477. Starcher, B. Elastin and the lung. Thorax 1986; 41: 577-585.8. Gross, P., Pfitzer, A., Tolker, E., Babyalc, M., Kaschalc, M. Experimental emphysema:its production with papain in normal and silicotic rats. Arch Environ Health 1965; 11: 50-58.9. Janoff, A., Sloan, B., Weinbaum, G., Damiano, V., Sandhaus, R., Elias, J., Kimbel,P. Experimental emphysema induced with purified human neutrophil elastase: tissuelocalization of the instilled protease. Am Rev Resp Dis 1977; 115: 461-477.10. Rodriguez, J., Seals, J., Radin, A., Lin, J., Mandl, I., Turino, G. Neutrophillysosomal elastase activity in normal subjects and in patients with chronic obstructivepulmonary disease. Am Rev Resp Dis 1979; 119: 409-417.11. Travis, J., Fritz, H. Potential problems in designing elastase inhibitors for therapy. AmRev Respir Dis 1991; 143(6): 1412-1415.12. Fujita, J., Nelson, N., Daughton, D., Dobry, C., Spurzem J., Shozo, I., Rennard, S.48Evaluation of elastase and antielastase balance in patients with chronic bronchitis andpulmonary emphysema. Am Rev Respir Dis 1990; 142: 57-62.13. Janoff, A. Biochemical links between cigarette smoking and pulmonary emphysema.J Appl Physiol Respirat Environ Exercise Physiol 1983; 55(2): 285-293.14. Hunninghake, G., Crystal, R. Cigarette smoking and lung destruction. Am Rev RespirDis 1983; 128: 833-838.15. Pratt, S., Finley, T., Smith, M., Ladman, A. A comparison of alveolar macrophagesand pulmonary surfactant obtained from lungs of human smokers and non-smokers byendobronchial lavage. Anat Rec 1969; 163: 497-506.16. Abboud, R., Fera, T., Richter, A., Tabona, M., Johal, S. Acute effect of smoking onthe functional activity of alpha-1-protease inhibitor in bronchoalveolar lavage. Am RevRespir Dis 1985; 131: 79-85.17. Laurent, P., Rabaud, D., Bieth, J. Inhibition of free elastin bound human pancreaticelastase by human bronchial inhibitor. Biochem Pharmacol 1987; 36: 765-767.18. Chapman, H., Stone, 0. Comparison of live human neutrophil and alveolar49macrophage elastolytic activity in vitro. J Clin Invest 1984; 74: 1693-1700.19. Martin, T., Raghu, G., Maunder, R., Springmeyer, S. The effects of chronicbronchitis air-flow obstruction on lung cell populations recovered by bronchoalveolar lavage.Am Rev Respir Dis 1985; 132: 254-260.20. Hunninghake, G., Gadek, J., Crystal, R. Human alveolar macrophage neutrophilchemotactic factor: stimuli and partial characterization. J Clin Invest 1980; 66: 473-483.21. Abboud, R., Tharwat, F., Johal, S., Richter, A., Gibson, N. Effect of smoking onplasma neutrophil elastase levels. Clin Lab Med 1986; 108: 294-300.22. Fera, T., Abboud, R., Richter, A., Johal, S. Acute effect of smoking on elastase-likeesterase activity and immunologic neutrophil elastase levels in bronchoalveolar lavage fluid.Am Rev Respir Dis 1986; 133(4): 568-573.23. Morrison, N., Abboud, R., Ramadan, F., Miller, R., Gibson, N., Evans, K., Nelems,B., Muller, N. Comparison of single breath carbon monoxide diffusing capacity andpressure-volume curves for detecting emphysema. Am Rev Respir Dis 1989; 139: 1179-1187.24. Morrison, H., Kramps, J., Dijkman, J., Stockley, R. Comparison of concentrations50of two proteinase inhibitors, porcine pancreatic elastase inhibitory capacity, and cell profilesin sequential bronchoalveolar lavage samples. Thorax 1986; 41: 435-441.25. Merrill, W., O'Hearn, E., Ranking, J., Naegl, G., Matthay, R., Reynolds, H. Kineticanalysis of respiratory tract proteins recovered during a sequential lavage protocol. Am RevRespir Dis 1982; 126: 617-20.26. McLaren, M., Lillywhite, J., Au, A. Indirect enzyme linked immunosorbent assay(ELISA); practical aspects of standardization and quality control. Medical LaboratorySciences 1981; 38: 245-51.27. Ochnio, J., Abboud, R., Smyrnis, E., Johal, S. A sensitive double-sandwich ELISAfor neutrophil elastase: assay results in unconcentrated bronchoalveolar lavage fluid. AmRev Respir Dis. 1991; 143: 61-65.28. Meurman, 0. Detection of antiviral IgM antibodies and its problems. Curr TopMicrobiol Immunol 1983; 104: 101-31.29. Voller, A., Bidwell, D., Bartlett, A. Enzyme-linked immunosorbent assay. In: Rose,N., Friedman, H., ed. Manual of clinical immunology. Washington, DC: American Societyfor Microbiology 1980; 339-71.5130. Baugh, R., Travis, J. Human leukocyte granule elastase: rapid isolation andcharacterization. Biochemistry 1976; 15(4): 836-841.31. Martodam, R., Baugh, R., Twumasi, D., Liener, I. A rapid procedure for the largescale purification of elastase and cathepsin G from human sputum. Preparative Biochemistry1979; 9(1): 15-31.32. Feinstein, G., Janoff, A. Rapid method of purification of human granulocyte cationicneutral proteases. Purification and further characterization of human granulocyte elastase.Biochim Biophys Acta 1975; 403: 493-505.33. Avrameas, S. Coupling of enzymes to proteins with gluteraldehyde. Use of theconjugates for the detection of antigens and antibodies. Immunochemistry 1969; 6: 43-52.34. Ferrua, B., Vincent, C., Revillard J., et al. A sandwich method of enzymeimmunoassay. III. Assay for human B2-macroglobulin compared with radioimmunoassay.J Immunol Methods 1980; 36: 149-58.35. Kramps, J., Franken, C., Dijkman, J. ELISA for quantitative measurement of lowmolecular weight bronchial protease inhibitor in human sputum. Am Rev Respir Dis 1984;129: 959-63.5236. Out, T., Jansen, H., van Stemwijk, K., de Nooijer, M., van de Graaf, E.,Zuijderhardt, F. ELISA of ceruloplasmin and a2-macroglobulin in paired bronchoalveolarlavage fluid and serum samples. Clin Chim Acta 1987; 165: 277-288.37. Cohen, A., Girard, W., McLarty, J., Starcher, B., Davis, D., Stevens, M.,Rosenbloom, J., Kucich, U. A controlled trial of colchicine to reduce the elastase load inthe lungs of ex-cigarette smokers with chronic obstructive pulmonary disease. Am RevRespir Dis 1991; 143: 1038-1043.38. Cohen, A., Girard, W. McLarty, J. A controlled trial of colchicine to reduce theelastase load in the lungs of cigarette smokers with chronic obstructive pulmonary diseaseCOPD). Am J Respir Dis 1990; 142: 63-72.39. Abboud, R., Tsang, A., Taller, M. Relative contribution of bronchial leucocyteprotease inhibitor (BI) and alpha-l-protease inhibitor (al-PI) to neutrophil elastase inhibitingcapacity (NEC) in sequential aliquots of bronchoalveolar lavage (BAL). (Abstract) Am RevRespir Dis 1990; 141: A110.40. Snider, G., Lucey, E., Christensen, T., Stone, P., Calore, J., Catanese, A., Franzblau,C. Emphysema and bronchial secretory cell metaplasia induced in hamsters by humanneutrophil products. Am Rev Resp Dis 1984; 129: 155-160.5341. Blue, M., Janoff, M. Possible mechanisms of emphysema in cigarette smokers.Release of elastase from human polymorphonuclear leukocytes by cigarette smoke condensatein vitro. Am Rev Resp Dis 1978; 117: 317-325.

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