Abstract
Neutral endopeptidase (NEP) is a cell surface enzyme found in normal human lung and which hydrolyzes small bioactive peptides, some of which act as growth factors for normal and malignant airway epithelial cells. Expression of NEP varies widely in human lung tissue from different individuals. NEP is often expressed at low or undetectable levels in both small-cell and non-small–cell lung cancer, and inhibits the growth of lung cancer cell lines. Variation in the expression of NEP could be a factor in susceptibility to lung cancer. We hypothesized that NEP could be measured in bronchoalveolar lavage fluid (BALF) and that airway levels of NEP would be low in lung cancer patients as compared with normal controls. We measured NEP and total protein in cell-free BALF supernatant, and expressed the respective concentrations as a ratio. NEP levels showed wide variation in BALF of healthy volunteers. Most patients with lung cancer had no NEP detectable in BALF. The mean NEP/total protein ratio was significantly lower in patients with lung cancer (0.87 ± 0.7 ng NEP/mg protein) than in normal healthy subjects (14.0 ± 4.3, p < 0.0003). We conclude that NEP levels are highly variable in BALF of normal volunteers, and are low or undetectable in most BALF specimens from patients with lung cancer. Low NEP levels in the airways may be a factor in the pathogenesis of carcinoma of the lung.
Neutral endopeptidase (NEP, CD 10, common acute lymphoblastic leukemia antigen [CALLA], E.C. 3.4.24.11) (1-5) hydrolyzes multiple bioactive peptides, such as the bombesinlike peptides (BLP) gastrin-releasing peptide (GRP) and neuromedin B (NMB) (6). Because NEP is located on the cell membrane and inactivates bioactive neuropeptides, it can regulate their availability at the cell surface. NEP has been described in most types of cells including endothelial cells, epithelial cells, and fibroblasts. For example, NEP has been shown to modulate BLP-mediated fetal lung development (7, 8). Inhibition of NEP increases local concentrations of substance P in the lung, potentiating bronchoconstriction, and modifies neutrophil physiology (9-13). Recently, targeted disruption of the NEP locus in mice was shown to enhance lethality from endotoxin shock, with a pronounced gene dosage effect (14), demonstrating an important role for NEP in the modulation of septic shock. In addition, NEP null mice were found to have lower blood pressure than control mice with widespread basal plasma extravasation in postcapillary venular endothelia that was reversed by recombinant NEP (15).
NEP is expressed at low levels or is absent in many small-cell lung cancer (SCLC) (6, 16) and non-small–cell lung cancer (NSCLC) cell lines and tumors (16, 17). Inhibition of NEP potentiates peptide-induced calcium flux (16), and recombinant NEP reduces SCLC and NSCLC cell growth in vivo and in vitro (6, 18). NEP is overexpressed in idiopathic diffuse hyperplasia of pulmonary neuroendocrine cells (19), respiratory bronchiolitis–interstitial lung disease (20), and eosinophilic granuloma (21).
BLP, produced in most SCLC cell lines and tumors, are autocrine growth factors for many SCLC (22) and some NSCLC cell lines (23). BLP are potent mitogens for fibroblasts (7, 24), alveolar type II cells, and bronchial epithelial cells (25, 26). BLP are believed to be important growth factors in several diseases. For example, concentrations of BLP are increased in open lung biopsies of patients with the smoking-associated disorders respiratory bronchiolitis–interstitial lung disease (27) and eosinophilic granuloma (24). BLP are also increased in the bronchoalveolar lavage fluid (BALF) (28) and urine (29) of a subset of smokers without obvious lung disease, as well as in the urine of smokers with chronic obstructive pulmonary disease (COPD) or lung cancer, as compared with smokers who do not develop these disorders (30). These data suggest that increased BLP levels may be a susceptibility factor for some smoking-related lung diseases. Low expression of NEP might be one mechanism for increased BLP levels.
We hypothesized that airway concentrations of NEP would be low in lung cancer patients as compared with normal controls. We performed bronchoalveolar lavage (BAL) as a minimally invasive technique for measurement of airway NEP. This was done in an initial study in which we demonstrated that NEP is present in normal subjects, and present at low levels in lung cancer. Additional studies are needed to elucidate the exact role of age, smoking status, COPD, or lung cancer in determining airway levels of NEP.
The study population consisted of patients and healthy volunteers enrolled in the Colorado Multiple Institution Review Board (COMIRB)- approved Biomarkers Clinical Protocol, which is supported by a National Cancer Institute Specialized Program of Research Excellence (SPORE) in Lung Cancer. All cancer patients were assessed clinically, radiographically, and with pulmonary function testing. Informed consent was obtained from each patient and from the healthy volunteers enrolled in the study. Subjects were designated as current smokers, ex-smokers, or never-smokers. Two groups of subjects were studied (Table 1). The first group consisted of patients with the histopathologic diagnosis of lung cancer. The second group consisted of healthy volunteers. All subjects underwent fiberoptic bronchoscopy with BAL. Health questionnaires were completed by all study participants.
| Demographic data | Patients with Lung Cancer (n = 10) | Healthy Volunteers (n = 9) | ||
|---|---|---|---|---|
| Mean age | 68 | 31 | ||
| Men/women | 8/2 | 4/5 | ||
| Race of patient, Caucasian/ African American/ Hispanic/Native American | 7/1/2/1 | 8/1/0/0 | ||
| Smoking history | ||||
| Current smoker | 3 | 0 | ||
| Ex-smoker | 7 | 0 | ||
| Never-smoker | 0 | 9 |
Information regarding the course and details of the illness were obtained from an admission questionnaire completed by the patient and from the initial history. All lung cancer patients underwent either a bronchoscopic biopsy and/or a resection for tissue diagnosis of the tumor. The tissue was embedded in paraffin and cut into 4-μm-thick slices that were placed on glass slides, and stained with hematoxylin and eosin (H&E), and a specific diagnosis of lung cancer was made by a pathologist.
All patients and healthy subjects underwent BAL after being admitted to the study. The BAL was performed as previously described, with small modifications (31). Intramuscular atropine and codeine were used for premedication, with nebulized 4% lidocaine to the oropharynx and 1% lidocaine to the trachea to obtain local anesthesia. Bronchoscopy was performed with subjects in the supine position, with supplemental oxygen (2 to 5 L/min) given during and for 30 min after the procedure. After inspection of the segmental orifice, the tip of the bronchoscope was wedged into a subsegment of the right middle lobe or lingula. A segment away from the lung tumor was always selected for this. Sterile saline at room temperature was instilled in 40 ml aliquots to a total of 120 ml, with harvest of fluid immediately by gentle hand suction on a syringe. The recovered lavage fluid was pooled, mixed, and cooled immediately to 4° C. The samples of lavage fluid were centrifuged at 480 × g for 5 min, and the cell-free supernatants were removed and stored at −70° C.
In the case of all healthy subjects and some cancer patients, lung tissue was obtained by bronchoscopic biopsy at two or more sites, usually from the right upper lobe and from the left upper lobe orifices.
Cell and tissue lysates were evaluated for NEP protein with 96-well enzyme-linked immunosorbent assay (ELISA) plates as previously described (16). The plates were coated overnight with 100 ng per well MEK 5, a monoclonal anti-NEP antibody (Khepri Pharmaceuticals, South San Francisco, CA) that captures NEP from solution. Samples and recombinant NEP standards were added to each plate in duplicate in 100-μl volumes (allowing for at least eight wells as reference blanks) and incubated, with shaking, for 2 h at 25° C. All patients' samples were run at the same time. The monoclonal antibody solution used for detection of NEP, consisting of 100 μl of a 1:5,000 dilution of horseradish peroxidase (HRP)-conjugated MEK-7 (Khepri Pharmaceuticals), was added to each well, and the plate was incubated with shaking for 1 h. The plates were then washed, and 100 μl of HRP substrate, consisting of O-phenylenediamine I in sodium citrate buffer (Sigma Chemical Co., St. Louis, MO), was added to each well. After a 30-min incubation in the dark, 50 μl of 4.5 N H2SO4 was added to each well to quench the reaction. The plate was then read with a Thermomax plate reader. Quantitation of NEP in the samples was derived from a standard curve generated with recombinant NEP (2 to 100 ng/ ml), using software supplied by Molecular Devices (Sunnyvale, CA). Final results were expressed as ng NEP/mg total protein.
Student's t test or the Mann-Whitney U test for rank sums was used to test differences between two groups of patients. Spearman's correlation was performed to test relationships between variables. Summary statistics for normally distributed variables are presented as mean ± SEM, and medians and ranges are reported for variables with skewed distributions.
We studied 10 patients with lung cancer and 10 healthy nonsmoking volunteers. The demographic and clinical data for the study subjects are outlined in Table 1. Physiologic parameters in lung cancer patients were consistent with a mild obstructive disorder in four of the 10 patients. The remainder of the patients did not demonstrate airway obstruction, and a normal mean FEV1/FVC ratio of 69.8 (normal ratio > 69) was found for the entire lung cancer cohort. Three of the 10 lung cancer patients were current smokers, and the remainder were ex-smokers. The mean pack-year history of the cancer patients was 82 yr. Histopathologic analysis revealed a diagnosis of SCLC in two of 10 patients, adenocarcinoma of the lung in four of 10, and of squamous cell carcinoma of the lung in four of 10. The bronchial biopsies of the healthy subjects were normal in six cases; one subject had mild dysplasia, and there was reserve-cell hyperplasia in two cases. One healthy subject showed changes of bronchitis on biopsy, and was removed from this study. The removal of this subject's data did not significantly change the mean NEP/protein ratio of the control group.
Analysis of BALF was done for all 10 healthy nonsmoking volunteers (one of whom was excluded because of a biopsy showing bronchitis) for comparison with the lung cancer patients. NEP was detectable in all nine of the healthy subjects whose BALF was assayed, and showed a wide range of values. The mean NEP/protein ratio in the healthy subjects was 14.82 ± 4.3 ng NEP/mg protein.
NEP was undetectable in eight of the 10 patients with lung cancer (Table 2). The mean NEP/protein ratio in lung cancer patients was 0.87 ng NEP/mg protein, which was significantly less than the normal subjects (p < 0.0021). Total protein in the lavage fluid of the lung cancer patients (median = 117.5 ng NEP/mg protein) was not significantly different from that of the normal subjects (median = 82 ng NEP/mg protein, p = 0.2775, Mann-Whitney U test). The percentage of lavage return was significantly lower in the lung cancer patients (34%) than in the healthy subjects (63%) (p = 0.0001, paired t test). This difference in lavage return is similar to that in previous reports for healthy subjects and patients with airways disease (31).
| NEP/protein (ng NEP/mg protein) | Histology | |||
|---|---|---|---|---|
| Normal subjects | ||||
| 1 | 13.5 | |||
| 2 | 16.7 | |||
| 3 | 1.7 | |||
| 4 | 7.3 | |||
| 5 | 12.0 | |||
| 6 | 17.1 | |||
| 7 | 5.4 | |||
| 8 | 6.9 | |||
| 9 | 45.5 | |||
| Lung cancer patients | ||||
| 1 | 0 | Squamous | ||
| 2 | 0 | SCLC | ||
| 3 | 0 | Adenocarcinoma | ||
| 4 | 0 | Adenocarcinoma | ||
| 5 | 0 | Adenocarcinoma | ||
| 6 | 0 | SCLC | ||
| 7 | 0 | Squamous | ||
| 8 | 0 | SCLC/Squamous | ||
| 9 | 1 | Squamous | ||
| 10 | 7.8 | Adenocarcinoma |
In the cancer group there was no correlation (Spearman's correlation) between the BALF NEP/protein ratio and age, FEV1, current smoking status, gender, BALF return, or cancer cell type. In the normal volunteers there was no correlation between the BALF NEP/protein ratio and sex, BALF return, or age.
We have shown that NEP is present in the BALF of normal humans. NEP levels in our normal volunteers varied greatly, indicating a wide range of baseline NEP levels in the population. The BALF NEP/protein ratio was low or absent in the small cohort of lung cancer patients studied. The explanation for this observation is not evident. A low NEP/protein ratio in lung cancer could be due to global inactivation of NEP in the majority of airway epithelial cells, perhaps by humoral mediators secreted by the tumor. The finding that NEP is expressed at low levels in the airways of cancer patients suggests that the loss of its expression could be a mechanism for potentiation of cell growth and development of lung cancer. It is unlikely that the low NEP levels observed in the cancer patients in our study were due to cellular changes of COPD. First, only four of our 10 cancer patients demonstrated mild airways obstruction, and the group FEV1/FVC ratio was normal. Furthermore, low levels of NEP have not been reported in COPD patients. Smoking could potentially affect the levels of NEP in BALF, although this has not so far been extensively studied in humans. In one animal study, cigarette smoke solution inhibited tracheal NEP in a concentration-dependent manner, an inhibiting effect that was felt to be due to the gas phase of the smoke and not to the nicotine (32). However, in a recent human study, NEP activity in bronchial biopsy specimens did not differ significantly in the biopsies of smokers and nonsmokers (33), supporting the contention that NEP levels in humans are not affected by smoking. NEP in nonsmokers' biopsy specimens was 669 fmol, versus 617 fmol in smokers' biopsies (33). In our cancer patients, moreover, there was no relationship between smoking status (current versus ex-smoker) and NEP level. Age may also affect BALF NEP or protein levels, but this has not yet been studied. The subjects in our control group were younger than the cancer patients, and one could speculate that age could somewhat affect NEP levels. However, evidence against this contention is that in our normal volunteer group, with subjects aged between 20 and 52 yr, NEP levels did not correlate with the age of the subject. In addition, the differences in NEP levels in our two study groups were so dramatic as to be unlikely to be explained by age difference alone.
In our normal subjects, there was wide variation in the NEP levels in BALF. We have previously described that histologically normal-appearing lung tissue from resections in patients with lung cancer has shown wide variation in NEP levels (16). In these patients there were three clusters in NEP expression, suggestive of genetic determination by one or a small number of loci (16). In addition, levels of BLP, a substrate of NEP, show considerable variation in BALF (28) and urine (29), and may be a marker of susceptibility to lung cancer (30). Recently, Siegfried and colleagues demonstrated variation in BLP receptor expression in bronchial epithelial cells, which may be the result of genetic or environmental factors (34). In view of these data, we speculate that genetically determined variability in NEP expression could be a factor influencing susceptibility to tobacco-induced lung diseases such as lung cancer. A physiologically relevant polymorphism affecting expression of another peptidase, angiotensin converting enzyme, has already been shown to be associated with susceptibility to cardiovascular disease (35). In summary, NEP levels are variably expressed in the BALF of humans. There is an association between lung cancer and low BALF levels of NEP.
The authors thank Joy Folkvord and Christina Hartmann for technical assistance. We especially acknowledge the patients and subjects who volunteered for this study and the physicians who participated in their care. We thank Shelia Brown for preparation of the manuscript.
Supported by the Canadian Lung Association (A.J.C.), the Medical Research Council of Canada (A.J.C.), the American Lung Association (A.J.C.), American Physiological Society Giles Filley Memorial Award (A.J.C.), the Royal College of Physicians and Surgeons of Canada (A.J.C.), NCI Specialized Program of Research Excellence (SPORE) in Lung Cancer CA58187 (Y.E.M., W.F., A.J.C.), and Veterans Affairs Merit Review Grant (Y.E.M.).
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The data presented here have been previously published in abstract form in the American Journal of Respiratory and Critical Care Medicine 1996;153:A674.
