Hydrolytic Enzyme of the Alveolar Macrophage in Diffuse Pulmonary Interstitial Disease

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Respiratory Medicine (1996) 90, 159-166

Hydrolytic enzyme of the alveolar macrophage in diffuse pulmonary interstitial disease

J. L. EREZ-ARELLANO*, M. N. BARRIOS*, T. MARTIN*, M. L. SANCHEZ*, A. JIMBNEZ* AND J. M. GONZALEZ-BUITRAGO? *Departamentos de Medicina y Bioquimica y Biologia Molecular, Universidad de Salamanca, and tServicio de Bioquimica, Hospital Universitario, Salamanca, Spain

Hydrolytic enzymes [acid phosphatase, /?-glucuronidase, p-D-N-acetyl glucosaminidase @-D-NAGA), lysozyme and angiotensin-converting enzyme (ACE)] are the major constituents of alveolar macrophages (AM). These enzymes play a crucial role in the pathogenesis of interstitial lung diseases. Cell-associated activity of several enzymes in alveolar macrophages obtained from control subjects (n=5) and patients suffering five representative types of interstitial pulmonary diseases [sarcoidosis (n = lo), extrinsic allergic alveolitis (n=5), idiopathic pulmonary fibrosis (n=5), neoplastic infiltration of the lung (n= 5) and Pneumocystis carinii pneumonia (n=5)] were evaluated. Cells were obtained by bronchoalveolar lavage and isolated by Ficoll-Hypaque gradient. Enzymatic activity was assessed by standardized tests. Bronchoalveolar lavage (BAL) lymphocyte counts were significantly elevated in the patients with active sarcoidosis (median: 57%), allergic extrinsic alveolitis (median: 51%) and neoplasic infiltration (median: 31%) as compared with the other groups, whereas BAL neutrophil and eosinophil counts were significantly elevated in the patients with idiopathic pulmonary fibrosis (neutrophil median: 29%; eosinophil median: 3%). The highest alveolar macrophage enzymatic activities were obtained in the active sarcoidosis group (median ACE: 23.38 PKat 10 ~ 6 AM; median lysozyme: 8.64 nKat 10 - 6 AM; median P-glucuronidase: 324.22 U 10 - 6 AM; median acid phosphatase: 0.78 nKat 10 ~ 6 AM; median /3-D-NAGA: 1.85 nKat 10 - 6 AM) which was significantly greater than in the control group (median ACE: 6.69pKat lop6 AM; median lysozyme: 1.95 nKat 10 - 6AM; median P-glucuronidase: 39.88 U 10 - 6 AM; median acid phosphatase: 0.38 nKat 10 - 6 AM; median P-D-NAGA: 044 nKat 10 - 6 AM). However, intracellular lysosomal enzymatic activities of alveolar macrophages from patients with allergic extrinsic alveolitis, a disease in which the degree of alveolar macrophage activation is maximal, were similar to those of the control group. These findings demonstrated a different pattern of expression of alveolar macrophage’s hydrolytic enzymes in lymphocytic diffuse pulmonary interstitial disease. In sarcoidotic patients, hydrolytic enzymes were increased whereas in allergic extrinsic alveolitis, hydrolytic enzyme activities were similar to control groups. Indirect data suggest that the release of lysosomal enzymes by alveolar macrophages during allergic extrinsic alveolitis may be a factor involved in the pulmonary lesions appearing in this disease.

Introduction response, alveolar macrophages release large num- The pulmonary interstitial-alveolar region is con- bers of soluble mediators (i.e. hydrolytic enzymes, oxygen free radicals, cytokines, arachidonic acid tinuously attacked by antigens from the exterior and from the pulmonary circulation. Alveolar macro- metabolites) which can injure the lung parenchyma. phages play a crucial role in the induction and Hydrolytic enzymes are the major constituents of resolution of inflammatory pulmonary injury. In alveolar macrophages and have been shown to be involved in many aspects of the inflammatory pulmo- order to participate adequately in the inflammatory nary response, in addition to their better known role

Received 15 November 1994 and accepted in revised form 10 July in bactericidal processes (1). Although it is known 1995. that in most diffuse interstitial pulmonary diseases tAuthor to whom correspondence should be addressed at: Servicio there is recruitment and activation of macrophages de Bioquimica, Hospital Universitario de Salamanca, Salamanca, (2), no systematic study has been conducted on the E-37007 Spain. catalytic activity of hydrolases in these diseases.

0954-6111/96/030159+08 $12.00/O 0 1996 W. B. Saunders Company Ltd 160 J. L. Pkrez-Arellano et al.

The aim of this study was to evaluate the cell- extrinsic allergic alveolitis patients had an acute form associated activity of the enzymes: acid phosphatase of the disease (less than 1 week of evolution) and the (E.C. 3.1.3.2) P-glucuronidase (E.C. 3.2.1.31), P-D- other patient had a subacute form. N-acetyl glucosaminidase (E.C. 3.2.1.30), lysozyme (E.C. 3.2.1.17) and angiotensin-converting enzyme Group 4 (idiopathic pulmonary jibrosis) (E.C. 3.4.15.1) in alveolar macrophages obtained This group consisted of five patients (three women from patients suffering from interstitial pulmonary and two men; mean age 67 years). The diagnosis diseases. of idiopathic pulmonary fibrosis was based on the following (6): (a) pulmonary biopsy specimen dem- onstrating fibrosis of the alveolar wall with inflam- Methods matory changes in the absence of granulomas; (b) PATIENTS AND CONTROLS interstitial infiltrates demonstrated by chest radio- A total of 35 subjects were subjected to broncho- graph; (c) a restrictive functional respiratory pattern; alveolar lavage (BAL) for diagnostic purposes. All and (d) the absence of known agents capable of were examined clinically and a basic complementary causing interstitial pulmonary disease. All idiopathic study was also carried out, covering the essential pulmonary fibrosis patients had a severe alveolitis analytical parameters, such as simple chest X-ray. documented by BAL (> 10% of neutrophils) and high None of the subjects smoked and none had been resolution computed tomography. treated for at least 2 months prior to study. All were informed of the technical aspects of the pro- Group 5 (neoplastic infiltration of the lung) cedure and written consent was obtained prior to the This group comprised five women (mean age 58 bronchoalveolar lavage. years) with pathologically proven breast adenocarci- Subjects were divided into six groups: noma and metastatic infiltration of the lung. In all cases, there was no evidence of infection due to Group 1 (control) opportunistic bacteria, congestive heart failure or This group consisted of five subjects (four men and other associate factors. one woman; mean age 47 years) selected according to the criteria of Reynolds et al. (3). All of them Group 6 (Pneumocystis carinii pneumonia) displayed unexplained dry cough. No bronchoscopic This group included five HIV( +) intravenous drug anomalies were observed in the pulmonary area in users. All patients were men with a mean age of 29 which the BAL was performed. Normal subjects were years. The diagnosis of P. carinii pneumonia was not included in this study for ethical reasons. based on the demonstration of parasite by Diff- Quik@, Grocott and direct immunofluorescence Group 2 (sarcoidosis) stains. All patients displayed moderate hypoxaemia This group consisted of 10 patients (seven women and a restrictive functional respiratory pattern. and three men; mean age 49 years). The diagnosis of sarcoidosis was based on a compatible clinical pic- BRONCHOALVEOLAR LAVAGE ture, the presence of non-caseating granulomas of the Bronchoalveolar lavage (BAL) was performed as liver, lymphatic ganglia and/or pulmonary tissue and described (7). Transnasal fibre optic bronchoscopy exclusion of other diseases (4). Patients with pulmo- and BAL were performed after premeditation with nary sarcoidosis were classified into two subgroups. atropine and topical anaesthesia with lidocaine. Six patients had active sarcoidosis according to three Three 50-ml aliquots of sterile 0.9% saline solution criteria (bronchoalveolar lymphocytes >28%, an were instilled and immediately withdrawn into a increased serum angiotensin-converting enzyme con- subsegmental bronchus of the right middle lobe or centration and a pathological uptake of 67Ga in lingula. After each instillation, fluid was recovered by pulmonary parenchyma) and the other four patients aspiration, collected in sterile polystyrene tubes and had inactive sarcoidosis. immediately transported, on ice, to the laboratory for examination. The BAL fluid obtained was quantified Group 3 (allergic extrinsic alveolitis) and filtered through two layers of sterile surgical These five subjects (four men and one woman; gauze. Mean recovered volume was 60-70% of mean age 54 years) fulfilled the criteria of Salvaggio instilled fluid. Following this, it was centrifuged at and Karr for this syndrome (5). The final diagnoses 500 X g for 10 min at room temperature into poly- were bird fancier’s lung in two cases, farmer’s lung in styrene tubes, the cell pellet washed twice with two cases and plasterer’s lung in one case. Four phosphate-buffered saline (PBS, pH 7.4) and the cells Enzymes in pulmonary interstitial diseases 161

resuspended at a concentration of lo6 cells mll i. saminidase catalytic activity was measured at 37°C Cell viability, determined by Trypan blue dye ex- using an automated procedure according to the clusion was >92% in all cases. The total number of method of Iimura (14). cells was counted in a Neubauer chamber. Using All measurements were carried out on the same cytocentrifuge preparations stained with Diff-Quik day and under the same conditions to avoid (American Scientific, McGaw Park, IL), a conven- interassay variability. tional differential count of 200 cells was performed by two independent observers. The identity of STATISTICAL ANALYSIS macrophages was confirmed by non-specific esterase Data are expressed as medians f interquartilic staining. Mononuclear cells were separated immedi- range. Statistical analysis was performed using non- ately by centrifugation over a Ficoll-Hypaque gradi- parametric tests (Kruskal-Wallis test for global ent, and after two washings were resuspended comparison, Mann-Whitney test for two-groups in sucrose-EDTA buffer (sucrose 0.25 M, EDTA comparison and Spearman rank correlation test) 0.002 M) and stored at - 70°C until required. Lym- using the Statworks and Statview software program phocytes and macrophages were not separated from for an Apple Macintosh@ computer. For compari- each other for two reasons: (a) lysozyme and ECA sons, P values co.05 were adopted as significant. enzyme content of lymphocytes is nil1 (18); and (b) separation methods can modify the enzymatic content of the cells by inducing release of enzymes (9). Results Homogenates were prepared using mechanical dis- The bronchoalveolar lavage total and relative ruption at 0°C (Polytron Typ PT 10135 Kinematic, (expressed by relation to BALF volume) cell counts Switzerland) and three freeze-thawing cycles. Cell are shown in Table 1. Patients with allergic extrinsic lysis was verified microscopically with Trypan blue alveolitis and P. carinii pneumonia had significantly dye. An aliquot was centrifuged at 800 x g for 10 min increased total and relative bronchoalveolar lavage and the supernatant was used for enzyme assays cell counts compared with the other groups. Differ- (lysozyme and lysosomal hydrolases). Determination ential cells counts are shown in Table 2. The pro- of angiotensin-converting enzyme was performed on portion of bronchoalveolar lavage lymphocytes the non-centrifuged homogenates. was significantly elevated in the patients with active sarcoidosis, allergic extrinsic alveolitis and neoplasic ENZYMATIC ACTIVITY MEASUREMENTS infiltration as compared with the other groups. The Angiotensin-converting enzyme catalytic activity proportions of bronchoalveolar lavage neutrophils was measured at 37°C by the rate of hydrolysis of and eosinophils were significantly elevated in the hypurylglicylglycine (Boehringer Mannheim SA kit patients with idiopathic pulmonary fibrosis. 78 9011) according to the method of Lieberman (10). All alveolar macrophage lysates displayed enzy- Activity was expressed in nkat per lo6 alveolar matic activity for all the enzymes studied. The mean macrophages. Lysozyme catalytic activity was meas- specific activity of each of the groups of patients is ured kinetically at 25°C by monitoring the decrease in shown in Figs l-3. The highest macrophage lysozyme the absorbance of a Micrococcus luteus suspension at activity was in the active sarcoidosis group, which 450 nm on a Spectronic 3000 (Bausch & Lomb) was significantly greater than in the other groups according to a standardized method (11). Micro- (Fig. 1). Angiotensin-converting enzyme activity was coccus luteus and lysozyme were purchased from also significantly greater in this group (Fig. 2). Boehringer Mannheim (cat 107387 and cat 107255). Lysosomal acid hydrolase activity was greatest in Activity was expressed in nKat per lo6 alveolar the group of patients with active sarcoidosis, par- macrophages. Acid phosphatase catalytic activity ticularly j%glucuronidase activity and P-D-N-acetyl was measured at 37°C by a calorimetric kinetic assay glucosaminidase activity, the mean values of which modified by Hillman (12) using a standardized test were 4.5- and 8-fold greater than those in the control (Boehringer Mannheim 287539). Activity was group (Fig. 3). Macrophage acid phosphatase activ- expressed in nKat per lo6 alveolar macrophages. ity was also significantly elevated in the group of P-glucuronidase catalytic activity was measured using patients with sarcoidosis, albeit to a lesser extent. a Sigma kit (no 325) based on the procedure Finally, P-glucuronidase activity was significantly described by Fishman et al. (13) modified by using higher in the group of patients with infiltration due to an incubation temperature of 56°C instead of 37°C. adenocarcinoma than in the control group (Fig. 3). Activity was expressed in Sigma units per lo6 A significant positive correlation was found alveolar macrophages. Finally, p-D-N-Acetyl gluco- between the percentage of lymphocytes and both 162 J. L. PPrez-Arellano et al.

Table I Total and relative cell numbers in bronchoalveolar lavage fluid (BALF)

Cell concentrations Total cells ( x 106)* ( x lo6 ml-’ BALF)* Median IQRt Median IQR

Control (n=5) 15 9-16 0.20 0.13-0.37 Unactive sarcoidosis (n=4) 25 8-35 0.31 0.09-0.47 Active sarcoidosis (n = 6) 28 8-51 0.36 0.07-0.41 Extrinsic allergic alveolitis (n= 5) 45t. 32-99 0.721 0.58-1.08 Idiopathic pulmonary fibrosis (n=5) 15 7-31 0.30 0.18-0.40 Neoplasic infiltration of the lung (n=5) 17 12-25 0.34 0.160.49 Pneumocystis carinii pneumonia ‘3% 59-61 0.60$ 0.58-0.90 (HIV patients) (n= 5)

Table 2 Differential cell counts in bronchoalveolar lavage fluid

Cell populations (%)

Macrophages* Lymphocytes* Neutrophils* Eosinophils* Median IQRt Median IQR Median IQR Median IQR

Control (n= 5) 89 86-94 7 3-10 3 2-3 0 Ml Unactive sarcoidosis (n=4) 741 7G-83 21 1425 1 l-2 3 l-2 Active sarcoidosis (n = 6) 413 34-51 573 4663 1 l-2 1 o-1 Hypersensitivity pneumonitis 34$ 19-68 513 29-59 2 1-13 2 G-6 (n=5) Idiopathic pulmonary fibrosis 543 36-65 8 5-12 291 20-50 31 3-8 (n=5) Neoplasic infiltration of the lung 67$ 48-86 311: 12-47 1 l-3 0 O-1 (n=5) Pneumocystis carinii pneumonia 711 33-86 24 lo-39 5 2-26 2 1-2 (HIV patients) (n = 5)

Unactive Active Extrinsic Idiopathic NeoplasCc Pneumocystis cp%?’n sarcoidosis sarcoidosis allergic pulrril;;ry m~ltr$on CC.Wl,IlLZ ” (n=4) (n=6) alveolitis pn~~i)nia (n=5) (n=5) n n

Fig. 1 Lysozyme, acid phosphatase and /I-D-N-acetyl-glucuronidase activities in alveolar macrophages from diffuse interstitial pulmonary diseases. *Significant differences (P~0.05) in comparison with the control group. Solid bars, p-D-N-acetyl glucuronidases; open bar, lysozyme; stippled bar, acid phosphatase.

. *

Unactive Active Ea;;+$ Idiopathic Neoplastic Pmum~~stis c(pztg’ sarcoidosis sarcoidosis pu&on3sry mfiltratlon ClWL7LZl (n=4) (n=6) alveolitis (n=5) pn yrnn)nia (n=5) &5=5) n

Fig. 2 Angiotensin-converting enzyme activity in alveolar macrophages from diffuse interstitial pulmonary diseases. *Significant differences (PcO.05) in comparison with the control group. hydrolases (angiotensin-converting enzyme, lysozyme striking feature was the ‘normal’ lysosomal activity and lysosomal acid hydrolases) in various pathogenic detected in the alveolar macrophages from patients types of human diffuse interstitial pulmonary dis- with allergic extrinsic alveolitis. eases. The highest alveolar macrophage enzymatic The results of the present work confirm previous activities were in the active sarcoidosis group. A observations (8) showing that human alveolar 164 J. L. Perez-Arellano et al.

d Control Unactive Active Extrinsic Idiopathic Neoplastic Pneummystis (r&=5) sarcoidosis sarcoidosis allergic ~I.I~~I~~~ infiltration COlWLll ” (n=4) (n=6) alveolitis (n=5) pneumonia (n=5) (n=5) (n=5) Fig. 3 p-glucuronidase activity in alveolar macrophages from diffuse interstitial pulmonary diseases. ,*Significant differences (P~0.05) in comparison with the control group.

macrophages display angiotensin-converting enzyme peptidoglucan moiety of the bacterial cell wall, fun- activity. Alveolar macrophage angiotensin- gus opsonization and modulation of the lymphocyte converting enzyme activity has mainly been related response (20). The authors have observed that in all to the development of granulomatous lesions in the types of diffuse interstitial pulmonary diseases there lung, rises having been reported in serum (15), bron- is a tendency for intramacrophage lysozyme activity choalveolar lavage fluid (16) and alveolar macro- to increase, although with strong intra-group vari- phages (17) of patients with sarcoidosis. The present ability. However, lysozyme activity was significantly work showed that the elevation of angiotensin- increased in the alveolar macrophages from patients converting enzyme activity was highest in active with active sarcoidosis. This finding can explain the sarcoidosis. There were also rises in angiotensin- elevated serum lysozyme values observed in pulmon- converting activity in some cases of allergic extrinsic ary sarcoidosis. The explanation for this finding is alveolitis, a disease in which granulomas are formed. not obvious since it is generally considered that the This had previously been demonstrated indirectly cells of the phagocyte mononuclear system synthesize by Larsson et al. in patients with subclinical forms of and release lysozyme constitutively (20) and activa- allergic extrinsic alveolitis (18). However, there was tion decreases the release. However, this phenom- considerable intra-group variability, probably related enon is not completely true since, although several to the stage of development of the disease. The classic stimulants (phagocytosis of bacteria, phorbol- positive correlation between angiotensin-converting myristate, C5a, y-interferon) do not increase the enzyme activity and the percentage of lymphocytes production of lysozyme by alveolar macrophages, suggests a role of lymphocyte cytokines in the induc- other agents (tobacco smoke, Calmette-Guerin bacil- tion of the enzyme. In this sense, positive feedback lus, glucans and TNFa) increase synthesis, intra- was established between macrophage-derived cellular concentration and release of lysozyme (1,21). cytokines (i.e. IL-I, IL-6) and lymphocyte-derived It is possible that antigen(s) involved in the patho- cytokines (i.e. y-IFN) in the granuloma formation genesis of sarcoidosis or certain cytokines released during active sarcoidosis (19). by helper T-lymphocytes are responsible for this Alveolar macrophages are the body cells with the phenomenon. highest lysozyme activity (20). The main biological Lysosomal acid hydrolases form a group of activities of this protein include hydrolysis of the enzymes whose function is to degrade the material Enzymes in pulmonary interstitial diseases 165 ingested in the phagocytic vacuole. However, lyso- References somal enzymes can sometimes gain access to the 1. Goldstein E. Hydrolytic enzymes of alveolar macro- extracellular medium where they exert a lytic action phage. Rev Infect Dis 1983; 5: 107881092. by degrading collagen and proteoglycans under acid 2. Fantone JC, Ward PA. Mechanisms of lung parenchy- pH conditions (as happens at the focus of inflamma- ma1 injury. Am Rev Respir Dis 1984; 130: 484491. tion) (22). The results of the present study suggest 3. Reynolds HY, Fulmer JD, Kazmierowski JA, Roberts there is an important increase in the intracellular WC. Frank MM. Crvstal RG. 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