Exhaled Nitric Oxide and Bronchoalveolar Lavage Nitrite/Nitrate in Active Pulmonary Sarcoidosis DEARBHAILE M. O’DONNELL, JOHN MOYNIHAN, GERALDINE A. FINLAY, VERA M. KEATINGS, CLARE M. O’CONNOR, PAUL MCLOUGHLIN, and MUIRIS X. FITZGERALD Department of Medicine and Therapeutics, University College Dublin, St. Vincent’s Hospital; and Department of Physiology, University College Dublin, Dublin, Ireland

Increased exhaled nitric oxide (NO) may reflect respiratory tract inflammation in untreated asthmatics. We compared exhaled NO and bronchoalveolar lavage (BAL) nitrate/nitrite (NO32/NO22) in 10 patients who had untreated, active pulmonary sarcoidosis with those of normal control subjects. Exhaled NO concentrations, determined by chemiluminescence, were similar in patients and control subjects (peak NO concentration of patients [mean 6 SD]: 13.6 6 5.9 parts per billion [ppb], peak NO concentration of control subjects: 11.2 6 5.7 ppb, p 5 0.32; mean alveolar NO concentration of patients: 7.8 6 4.4 ppb, mean alveolar NO concentration of control subjects: 7.1 6 4.2 ppb, p 5 0.70; end-tidal NO concentration of patients: 6.9 6 4.5 ppb, end-tidal NO concentration of control subjects: 6.6 6 4.0 ppb, p 5 0.60). BAL NO22 was assayed using a modified Griess reaction after reduction of NO32 to NO22. There was no significant difference in mean BAL NO22 concentrations, expressed as nanomoles per milliliter of epithelial lining fluid (patients: 544 nmol/ml, control subjects: 579 nmol/ ml, p 5 0.81) or as nanomoles per milliliter of BAL fluid (patients: 6.7 nmol/ml, control subjects: 5.7 nmol/ml, p 5 0.41). These data suggest that excess NO generation does not accompany the respiratory tract inflammation of pulmonary sarcoidosis. O’Donnell DM, Moynihan J, Finlay GA, Keatings VM, O’Connor CM, McLoughlin P, Fitzgerald MX. Exhaled nitric oxide and bronchoalveolar lavage nitrite/nitrate in active pulmonary sarcoidosis. AM J RESPIR CRIT CARE MED 1997;156:1892–1896.

Nitric oxide (NO) is produced endogenously in the human respiratory tract, where it can act as a dilator of bronchial and vascular smooth muscle, a neurotransmitter, and an immune response mediator. It is generated by the enzyme nitric oxide synthase through the oxidation of L-arginine and reacts readily with O2 to form the stable bio-active oxidation end products nitrate (NO32) and nitrite (NO22) (1). It has been proposed that the increased NO in the exhaled air of untreated asthmatic patients (2) reflects induction of the inducible form of nitric oxide synthase (iNOS) in the inflammatory microenvironment of the asthmatic respiratory tract (3). This view is supported by the immunocytochemical detection of iNOS in the airway epithelium of asthmatic patients (4) and by its in vitro induction in alveolar macrophages and epithelial cells after the addition of pro-inflammatory stimuli, including cytokines such as interferon-g (IFN-g), tumor necrosis factor-a (TNF-a), and interleukin-1 (5, 6). Whether increased exhaled NO is unique to asthma or is a nonspecific result of many forms of pulmonary inflammation is not clear. Although some studies have reported increased

exhaled NO among subjects with bronchiectasis (7), values are normal or low in patients with cystic fibrosis (8, 9). The interpretation of the cystic fibrosis results is complicated by the presence of copious airway secretions that might absorb or degrade any excess NO produced by the inflammatory process. No published studies to date have investigated exhaled NO in interstitial lung disease although alveolar macrophages are a potential source of iNOS (10). In the alveolitis of active pulmonary sarcoidosis, alveolar macrophages are present in increased absolute numbers along with the characteristic increase in activated T lymphocytes. Furthermore, the iNOS-inducing cytokines mentioned previously, i.e., IFN-g and TNF-a, have been associated with the inflammatory milieu of sarcoid alveolitis (11, 12). We therefore hypothesized that respiratory tract NO production and detectable exhaled NO might be elevated in this disorder. The aim of this study was to examine exhaled NO and the concentration of its stable metabolites NO32 and NO22 in bronchoalveolar lavage fluid (BALF) in a group of untreated sarcoidosis patients with active alveolitis.

METHODS Subjects (Received in original form May 6, 1997 and in revised form July 29, 1997) Supported by a grant from the Health Research Board of Ireland. Correspondence and requests for reprints should be addressed to Dr. Paul McLoughlin, Department of Physiology, University College Dublin, Earlsfort Terrace, Dublin 2, Ireland. Am J Respir Crit Care Med Vol 156. pp 1892–1896, 1997

Study protocols were approved by the local ethics committee, and all subjects gave written informed consent. Ten patients (eight male) were studied at the time of their first evaluation for suspected active pulmonary sarcoidosis. All were Irish (Caucasian), and the mean age of the group was 35.5 yr (range: 22 to 63 yr). All subjects were symptomatic at the time of entry into the study. The major presenting complaint in six patients was dyspnea or cough; in two patients, granulomatous

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TABLE 1 PULMONARY FUNCTION TESTS OF ALL SUBJECTS

FEV1, % predicted FVC, % predicted DLCO, % predicted

Patients

Control Subjects (Exhaled NO)

89 (59–111) 91 (72–114) 76 (52–98)

111 (94–129)* 106 (94–125)* 92 (75–112)†

Control Subjects (BAL NO32/NO22) 102 (96–108)† 100 (95–109) 97 (88–105)*

Each subject’s results were expressed as the percentage of predicted values for age and sex. Data are shown as mean (range). Where values for control groups are significantly different from those for the sarcoidosis group, the following symbols are used: * p , 0.01, †p , 0.05.

uveitis; in one patient, nonspecific chest pain with fatigue, fever, and weight loss; and in one patient, wheeze and progressive peripheral lymphadenopathy. In all cases, the chest X-ray (CXR) was abnormal (4 5 Siltzbach Stage 1, 3 5 Stage 2, 2 5 Stage 3, 1 5 Stage 4). Six patients had an elevated serum angiotensin converting enzyme (sACE) concentration1 (mean sACE for the group: 50 U/ml; range: 31 to 220 U/ml). None had hypercalcemia or renal or hepatic impairment. No patient had ever received oral or inhaled glucocorticoid or other systemic immune-suppressive therapy. In all cases, the diagnosis of sarcoidosis was confirmed by transbronchial or lymph node biopsy. Control subjects for the exhaled NO measurement (n 5 12; 8 male; mean age: 28.2 yr; range: 22 to 44 yr; all Irish) were healthy nonsmokers with no history of asthma or allergic rhinitis and who had no upper or lower respiratory tract symptoms during the month before testing. Control BAL samples were obtained from nonsmokers (n 5 5; all male; mean age: 47.8 yr; range: 40 to 58 yr; all Irish) with no history of respiratory disease and a normal CXR who were undergoing elective surgery. Pulmonary function tests of all subjects are displayed in Table 1.

Exhaled NO Measurement Exhaled NO was measured using a rapidly responding chemiluminescence analyzer (LR 2000; Logan Research, Medway, UK), which has an infrared CO2 analyzer in sequence and is accurate to within 1 part per billion (ppb) NO. Ambient NO on all occasions was , 3 ppb. Subjects exhaled with maximal effort via a narrow-bore Teflon™ tube against a fixed resistance. During this maneuver, a sample of exhaled gas was aspirated continuously into the analyzer at a rate of 250 ml/ min through a side-port on the Teflon tube close to the subject’s mouth. NO, CO2, mouth pressure, and cumulative volume signals (25 Hz per channel) were digitized and stored directly on a computer hard disk for later analysis. Each subject performed three consecutive tests. The difference between the response times of the NO and CO2 analyzers was examined by presenting abruptly to the instrument a gas mixture containing CO2 and NO in O2 and N2. The time taken to reach 90% of the steady-state value for CO2 and NO was determined, and the difference computed. The mean difference in response times was 2.44 s. Before analyzing the data from all patients, the NO and CO2 records were realigned by delaying the CO2 record by 2.44 s. This allowed the delineation of three exhaled NO parameters (see Figure 1 and its legend for definitions) based on the phase of expiration. The final value for each parameter in each subject represents the mean of the values obtained in the three tests performed.

Bronchoalveolar Lavage BAL was performed using a flexible fiberoptic bronchoscope wedged in a segmental bronchus of the right middle or right lower lobe. In the sarcoidosis patients, local anesthesia and intravenous sedation were used. In the control subjects, BAL was performed immediately after intubation for an elective surgical procedure. Three 60-ml aliquots of normal saline were instilled and withdrawn in turn. The returned fluid was stored on ice and processed immediately. It was centrifuged at

1

Laboratory reference range for sACE: 0 to 85 U/ml.

Figure 1. Three parameters were used in analyzing exhaled NO concentrations, as illustrated in this real-time curve of NO (ppb) and CO2 (%) recorded during expiration by one of the control subjects. Peak exhaled NO was defined as the highest value of NO recorded in exhaled air during expiration. Mean alveolar NO was defined as the mean of all NO values recorded during the plateau phase of the CO2 curve (beginning when the rapid rise in CO2 had ended and CO2 had reached at least 80% of its peak value, ending with the peak CO2). End-tidal NO was defined as the mean of all NO values occurring during the last 2 s before the peak CO2.

1,000 rpm for 6 min at 48 C, and the supernatant stored at 2708 C. The cell pellet was resuspended in RPMI medium (Life Technologies, Paisley, Scotland) and reserved for differential cell counting. When an increased percentage of lymphocytes was present, samples were incubated with fluorochrome-conjugated monoclonal antibodies to CD4 and CD8 (Becton Dickinson, Oxford, UK) and the ratio of CD4 to CD8 lymphocytes was determined using flow cytometry (FACScan®; Becton Dickinson).

Measurement of BAL NO32/NO22 Total concentration of NO32 and NO22 was determined in thawed BAL supernatant by a modified Griess reaction method (13). Triplicate samples of BAL were incubated for a minimum of 3 h at 208 C with glucose-6-phosphate (500 mmol/L), glucose-6-phosphate dehydrogenase (160 U/L), NADPH (1 mmol/L), and nitrate reductase (20 U/L) in phosphate buffer (80 mmol/L, pH 7.5). The Griess reaction was then initiated by addition of sulfanilamide to a final concentration of 0.5% (wt/vol), orthophosphoric acid (1.25%, vol/vol), and N-(1 naphthyl)ethylenediamine hydrochloride (0.05%, wt/vol). All reagents were obtained from Sigma (Poole, Dorset, UK). After a further incubation at 208 C for 10 min, the absorbance of each sample mixture was measured at 540 nm and corrected for opacity by measuring the absorbance at 750 nm. The corrected absorbance was interpolated in a standard curve of absorbance plotted versus concentration in order to find the concentration of NO22 in the sample. As all NO32 had already been reduced to NO22 by the use of nitrate reductase, this represented the combined concentration of NO32 and NO22 in the BALF. Results of the NO22 assay were expressed as NO22 concentration in nanomoles per milliliter of BALF. The volume of epithelial lining fluid (ELF) recovered was determined (14) as follows: BAL urea mg ⁄ ml × BAL volume ELF volume ( ml ) = --------------------------------------------------------------------------------plasma urea mg ⁄ ml and NO22 was also expressed as NO22 concentration in nanomoles per milliliter of ELF. A suitable plasma sample for this determination was not available in one of the 10 patients studied.

Statistical Analysis Data for exhaled NO parameters and BALF NO32/NO22 (expressed as nanomoles of NO22 per milliliter of BALF or nanomoles of NO22 per milliliter of ELF) were expressed as either mean value 6 SD or as mean value (range). Comparisons between patient and control groups

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AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE TABLE 2 BAL CHARACTERISTICS OF SARCOIDOSIS PATIENTS AND CONTROL SUBJECTS

Total cell count, 3 105/ml BALF Macrophages, % total cells Lymphocytes, % total cells Lymphocyte CD4/CD8 ratio

Patients

Control Subjects (BAL NO32/NO22)

2.48 (1.45–4.30) 42 (15–65) 55 (32–83) 4.4 (1.1–13.8)

0.98 (0.47–2.32)* 89 (86–95)* 9 (4–13)* —

Data are shown as mean (range). The symbol * denotes a significant difference where p , 0.01.

were made using an unpaired Student’s t test. Correlation was examined using standard linear regression. A p value , 0.05 was accepted as statistically significant.

RESULTS BAL Total and Differential Cell Counts

All BAL samples from sarcoidosis patients were highly cellular and contained a significantly higher percentage of lymphocytes than those from control subjects. Control BAL samples displayed absolute and relative cell counts within the normal range (Table 2). Exhaled NO

The scatter of values and the mean 6 SD of each group for the 10 patients and 12 control subjects are shown in Figure 2. Peak exhaled NO, mean alveolar NO, and end-tidal NO concentrations were not significantly different in the sarcoidosis patients compared with the values recorded in the normal subjects. There was no significant correlation between any exhaled NO parameter and the sACE value, BAL leukocyte counts, or DLCO (data not shown). Peak NO values for the six patients with interstitial infiltration on CXR (14.0 6 5.1 ppb) were no higher than those in the patients with hilar adenopathy only (13.0 6 8.0 ppb, p 5 0.80). Similarly, there was no significant difference for mean alveolar NO (6.8 6 2.5 versus 9.2 6 6.6 ppb, p 5 0.44) or end-tidal NO (6.7 6 2.4 versus 8.9 6 6.8 ppb, p 5

Figure 2. Exhaled NO in sarcoidosis patients and control subjects. This figure shows the scatter of NO values and the mean value and SD for all parameters in each group. There was no significant difference in peak exhaled NO (p 5 0.32), mean alveolar NO (p 5 0.70), or end-tidal NO (p 5 0.60) between the two groups.

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0.46) between these two subgroups of patients. Comparing each of the two subgroups separately with the control group for every parameter again showed no significant differences (p . 0.4 for all comparisons). Although peak exhaled NO values were higher than mean alveolar and end-tidal values in all subjects, there was a strong correlation between all parameters (peak versus end-tidal NO, r 5 0.88; peak versus mean alveolar NO, r 5 0.86; mean alveolar versus end-tidal NO, r 5 0.99). BALF NO22

The scatter of these values is shown in Figure 3. The concentration of NO22 in the BALF of the patients expressed as nanomoles of NO22 per milliliter of BALF or as nanomoles of NO22 per milliliter of ELF was not significantly different from that of control subjects. There was no statistically significant correlation between the BALF NO22 concentrations and the sACE values, BAL leukocyte counts, or DLCO. BALF NO22 concentrations did not differ significantly between the group of patients with radiographic interstitial infiltration and those without (7.2 6 2.8 versus 7.1 6 2.8 nmol/ml BALF, p 5 0.96) or between either of those two subgroups and control values (p . 0.4 for all such comparisons).

DISCUSSION No difference was observed between patients with active pulmonary sarcoidosis and normal subjects for any measure of exhaled NO or BALF NO22. Factors that might influence the results are patients’ smoking habits, disease activity at time of testing, and the methods used to measure exhaled NO. One of our patients was a regular smoker, but analysis performed omitting her results did not change our findings. It is clear from the total and differential cell counts in BAL that all patients had active sarcoid alveolitis at the time of exhaled NO testing. Exhaled NO values may vary according to the technique used for measurement, the maneuver performed by the subject, and the parameters used for analysis. On-line measurement of a sample continuously aspirated into the analyzer, as in our study, is the technique generally used in recently published clinical studies (8, 15) as it causes less NO loss than the collection of expired air into a reservoir for later analysis (3). Our use of the maximal effort maneuver, made possible by the relatively rapid analyzer response time, was based on a twofold rationale. First, we found in preliminary studies that it was a maneuver easily understood and reproduced by patients and normal subjects alike. Second, we wished to minimize contamination of measured NO by nasal NO (16, 17). Closure of the soft palate is the most straightforward method of reducing diffusion of nasal NO into the pharynx and thus into expired air. It occurs involuntarily when mouth pressure during expiration exceeds 4 cm H2O, as always occurred during the forced vital capacity maneuver against resistance performed by our subjects. This simple, reproducible maneuver therefore minimizes nasal contamination. It did not interfere with the detection of increased exhaled NO. Testing untreated asthmatics using our protocol, we have obtained high peak NO values (range: 32 to 84 ppb). The most usually reported parameter in other studies is the peak NO, a value that in normal and asthmatic subjects is usually observed in the early part of the expiratory curve. Conscious that increases in exhaled NO in patients with alveolitis might be detectable only in gas arising from the alveoli, we analyzed two other NO values (see Figure 1) occurring during the plateau phase of the CO2 curve, i.e., mean alveolar and end-tidal NO. In fact, these “alveolar” values correlated very

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Figure 3. NO22 concentrations in BALF. This figure shows the concentration of NO22 in BALF in control subjects and in sarcoidosis patients and the mean and SD for each group. There was no significant difference in NO22 concentrations between the two groups, whether expressed as nanomoles per milliliter of recovered BALF (p 5 0.81) (left panel) or nanomoles per milliliter of ELF (p 5 0.41) (right panel).

closely with the peak NO (see RESULTS) and, even using these indices, values obtained in the patient group were not significantly different from those recorded in control subjects (see Figure 2). There are two possible reasons why patients with active pulmonary sarcoidosis have normal exhaled NO concentrations. The first is that excess NO production and, by inference, iNOS induction, does not accompany the alveolar inflammation of sarcoidosis; the second, that excess NO, though produced, is not detectable in the exhaled air. The latter explanation is certainly possible, as alveolar NO would react readily with heme in the red blood cells of the pulmonary capillaries. However, in biologic systems, the half-life of NO is between 0.1 and 5 s and the presence of NO at air-aqueous interfaces results in the generation of intermediate nitrogen oxides and the stable end products of NO metabolism, i.e., NO32 and NO22 (1). Thus, increased alveolar NO production, even if not reflected in exhaled air, would be likely to cause some detectable increase in BALF NO32/NO22. However, we found no difference in their concentration in the BALF of patients with sarcoidosis when compared with normal values. Furthermore, the range of values obtained among the sarcoidosis patients corresponds to that reported by other investigators in normal BALF (18). The absence of a detectable increase in either exhaled NO or BALF NO32/NO22 in active sarcoidosis suggests that excess NO production does not accompany the pulmonary inflammation in this disorder. Before discussing the implications of these findings, the limitations of this study must be considered. First, our patients were all Irish. Therefore, we cannot answer the question whether excess NO generation occurs in black sarcoidosis patients, whose disease tends to follow a course with much more frequent end-organ damage than that of Caucasian patients (19). Second, despite the presence of a high percentage of lymphocytes in BAL, not all our patients had radiologic evidence of interstitial pulmonary disease. The question thus arises whether there is any difference in pulmonary NO generation between those patients with hilar adenopathy alone and those with interstitial shadowing. Although we did not find any significant difference in exhaled NO or BALF NO32/NO22 between these two subgroups, the numbers in each (n 5 4 and n 5 6, respectively) are small and a study of larger groups is required to test that specific hypothesis. A third issue is whether respiratory tract concentrations of NO and its metabolites continue to be low among patients who progress to long-term pulmonary in-

flammation and tissue damage. As we examined our patients at a single time-point, this question remains. Nonetheless, the overall results suggest that excess NO generation is not part of the respiratory tract inflammation that occurs in pulmonary sarcoidosis. The reasons why this might be so are not immediately apparent. Both airway epithelial cells (4, 10, 20) and alveolar macrophages (AMs) (10, 21) from humans can express iNOS. The granulomatous response to several infectious agents in animal species includes iNOS induction in macrophages (22, 23), although there is less evidence for this in human disease. We are not aware of any studies in humans addressing the question of exhaled NO concentrations in histoplasmosis or brucellosis or the role of iNOS in these infections. However, one study has clearly demonstrated increased expression of active iNOS in AMs from patients with clinically active Mycobacterium tuberculosis infection (21), and a recent abstract reported that exhaled NO concentrations were higher in patients with tuberculosis than in control subjects (24). In addition, excess NO is generated in the gastrointestinal tract of patients with Crohn’s disease, a noninfectious granulomatous disorder (25). Taken together, these lines of evidence suggest that iNOS would be expressed in the human airway in the granulomatous inflammation of sarcoidosis. Furthermore, TNF-a (11, 26) and IFN-g (12, 27), both of which promote iNOS expression, are part of the inflammatory alveolar milieu of sarcoidosis. Perhaps some other mechanism, specific to sarcoidosis, is preventing AM iNOS expression in this apparently favorable cytokine milieu. One possibility is the additional presence of transforming growth factor-b, which has recently been described (28) in macrophages from patients with sarcoidosis and which in a murine system (29) blocks TNF-a–mediated amplification of IFN-g–induced iNOS expression. Another is that iNOS expression is highest in fully mature AMs (30), whereas in sarcoidosis many macrophages display a phenotype intermediate between monocytes and mature AMs (31) and hence may not yet be capable of NO production. Several animal studies suggest that NO has a lymphocytostatic as well as a pro-inflammatory function. NO produced by AMs seems to be one mediator contributing to suppression of T-cell proliferative responses to antigens in the lung (30). NO inhibits the expansion of cloned T-helper 1 (Th1) lymphocytes (32) and iNOS knockout mice develop a preferential Th1 expansion following antigen challenge (23). In this context, our finding of apparently normal respiratory tract NO production

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in association with a lymphocytic alveolitis in which T lymphocytes display a Th1 polarization (27) is even more intriguing. Perhaps low respiratory tract NO generation in a pro-inflammatory environment itself favors the development of an inappropriate lymphocytic alveolitis. The control of iNOS expression and activity and the exact role of NO in the human respiratory tract are not fully understood. We have shown that exhaled NO and BALF NO22/ NO32 are normal in sarcoidosis despite the presence of active alveolitis. These results suggest that excess NO generation is not a universal by-product of chronic cytokine-mediated pulmonary inflammation but depends upon the precise inflammatory milieu and the nature of the inflammatory stimulus. Acknowledgment : This study was assisted by the Health Research Board of Ireland. The writers wish to thank Geraldine Lawless and Carol Delahunty for performing the pulmonary function tests.

15.

16.

17.

18.

19. 20.

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