INTRODUCTION

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Diffuse panbronchiolitis (DPB) is a pulmonary disease of unknown origin with chronic inflammation in the respiratory bronchioles, progressive obstructive respiratory dysfunction and diffuse micronodular shadows on the chest roentgenogram (1). This disease is largely confined to individuals of Japanese, Korean, or Chinese descent. In Europe and the United States, several cases of DPB have been reported in the literature (2, 3). Patients typically present with a discharge of abundant mucus in the airway, recurrent sinusitis, dyspnea, wheezing, and hypoxemia. In patients with DPB, morphologic and functional abnormalities of the mucociliary transport system have been documented (4). Although the pathogenesis remains unknown, low doses of erythromycin, an accepted empirical therapy, improve the survival of patients with DPB (5).

High concentrations of nitric oxide (NO) are found in nasally exhaled air of healthy subjects (6, 7). The high concentration of nasal NO may play a critical role in the pathophysiology of the respiratory system because NO has multiple functions, including the regulation of the tone of the vascular and bronchial systems, host defense, and mucociliary clearance (8, 9). Two pulmonary diseases are known to reduce nasal NO: primary ciliary dyskinesia (PCD) syndrome (10) and cystic fibrosis (CF) (12). Although the precise causes for the reduced nasal NO in PCD and CF are unknown, a common manifestation of these diseases is chronic airway infection resulting from mucociliary dysfunction. Furthermore, clinical features of DPB have many similarities to those of PCD and CF, including chronic sinusitis and bronchiectasis (15, 16). In the present study we hypothesized that patients with DPB have abnormal concentrations of nasal NO that may contribute to the disease's pathogenesis. To test the hypothesis we measured the concentrations of nasal NO in DPB patients.

	METHODS

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Subjects

The study was approved by the ethics committee of Asahikawa Medical College, and informed written consent was obtained from patients. We measured nasally exhaled NO in 15 healthy control subjects (all nonsmoking males) and eight patients with DPB (all nonsmokers, 7 males and 1 female). The control subjects were recruited from hospital colleagues and were taking no prescription medications. DPB was diagnosed by transbronchial lung biopsy or open-lung biopsy according to established criteria (17). The diagnosis of chronic paranasal sinusitis was based on the clinical history and examination with X-ray or computed tomographic scanning. None of the patients had severe infections of either the paranasal sinuses or the lower respiratory tract at the time of the study. None of the patients was taking inhaled or oral glucocorticoids, but all were being treated with low doses of erythromycin (400 to 600 mg/d). In four of 15 control subjects, measurements of nasal NO were performed before and after the administration with daily 600 mg of oral erythromycin for 14 d.

Pulmonary Function Test

Spirometry was performed with auto-spirometer (Chestac 65V; CHEST, Japan). Lung volume was measured by helium dilution technique. Diffusing capacity of the lungs for carbon monoxide (DL_CO) was measured by single-breath method and expressed as a permeability index (DL_CO/VA, where VA is alveolar volume).

Measurements of Nasal NO

A side-to-side ventilation technique was used to measure nasal NO (18). Subjects rested in a sitting position during the measurement of nasal NO. A polystyrene tube was securely attached into the vestibulum of one nostril; in the other nostril, we connected the same tube to supply NO-free room air. NO concentration was continuously measured by a chemiluminescence analyzer (NOA 270B; Sievers, CO) at the proximal site of the nasal orifice, while CO₂ concentration was monitored with a gas analyzer (MG-360; Minato Medical Science, Japan). Each sampling rate was 0.3 L/min and 0.15 L/min, respectively. The signals were transferred to a data acquisition system (MacLab; AD Instrument, Australia) for real-time recordings and later analysis. To isolate the nasal airway from the lower airway, the subject was instructed to take a shallow breath spontaneously while watching a monitor displaying the CO₂ concentration. When the CO₂ concentration decreased to zero, the nasal NO concentration reached a stable plateau. These maneuvers were found to increase nasal NO concentration by closure of the soft palate. The plateau levels of nasal NO were registered on the data acquisition system. The measurement was taken for the right nostril and then for the left. Each measurement was repeated three times. Nasal NO production (NO) was calculated by multiplying nasal NO concentration (parts per billion [ppb]) times sampling flow rate (L/min). An average value was calculated from the right and left values.

Statistical Analysis

All data were expressed as mean ± SD. Comparisons between the two groups were made by unpaired Student's t tests. Comparisons between pre- and posterythromycin treatment in control subjects were made by analysis of variance (ANOVA) for repeated measures, followed by a post hoc t test with Bonferroni's correction. A p value < 0.05 was considered statistically significant.

	RESULTS

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Characteristics of the Subjects

The ages of control subjects and patients with DPB were 48 ± 17 and 51 ± 14 yr, respectively. Patients with DPB had significantly lower vital capacity (VC), lower FEV₁/FVC ratio, and higher ratio of residual volume to total lung capacity (RV/ TLC) compared with control subjects (VC: 82 ± 20 versus 103 ± 14% predicted normal; FEV₁/FVC: 61 ± 14 versus 96 ± 8%; RV/TLC: 45 ± 6 versus 30 ± 7%, p < 0.01, respectively). DL_CO/VA was not different between DPB patients and control subjects (6.1 ± 1.2 versus 5.8 ± 1.5 ml/min/mm Hg/L). All patients with DPB had mild to moderate degrees of chronic paranasal sinusitis.

Nasal NO Levels

Three of eight patients with DPB exhibited essentially undetectable concentrations of nasal NO (Figure 1). The mean value of NO was significantly lower by 88% in patients with DPB than in control subjects (69 ± 70 versus 556 ± 87 nl/min; p < 0.001), and no overlap was found between the two groups (Figure 1).

View larger version (9K): [in this window] [in a new window]   Figure 1.   Nasal NO in control subjects and patients with diffuse panbronchiolitis (DPB). Bar = mean value. Nasal NO is significantly decreased in patients with DPB compared with control subjects (p < 0.001).

Nasal NO before treatment with erythromycin was not significantly different from that after the treatment in four control subjects (530 ± 96 versus 548 ± 104 nl/min).

	DISCUSSION

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We found that nasal NO in patients with DPB was 88% lower than that in control subjects. Thus, DPB becomes the third pulmonary disease with low nasal NO. Several investigators have shown that nasal NO in PCD and CF patients is 55 to 98% lower than that in normal individuals (10). Nasal NO originates mainly from the paranasal sinuses. Lundberg and coworkers (7) have shown that the NO concentration in the air aspirated directly from the maxillary sinuses is very high, and epithelial cells lining the sinuses express an inducible NO synthase (NOS). Besides paranasal sinuses, nasal mucosa is thought to contribute to the nasal NO because strong immunostaining activity of NOS is localized to normal nasal mucosa (19).

Several explanations have been proposed to account for the lowered nasal NO. One is that the activity of NOS in the superficial epithelium of nasal airways is reduced. Morphologic and functional impairments of mucociliary transport system in both the upper and lower airways have been documented in patients with DPB (4, 20). Moreover, DPB has clinicopathologic similarities to PCD and CF, including impaired mucociliary function, chronic airway infection, bronchiectasis, and recurrent sinusitis (15, 16). NO plays an important role in antiviral and antibacterial host defenses (8), and impaired NO production may enhance susceptibility to airway infection. Ciliated epithelial cells normally contain NOS (21), which upregulates ciliary motility (22). Reduced nasal NO correlates with impaired mucociliary function (9), and reduced NO synthesis may contribute to the chronic airway infections and the mucociliary dysfunction noted in patients with DPB, PCD, and CF. In patients with PCD and CF, NO concentrations remained low despite stimulation with L-arginine, the substrate of NOS, suggesting that NOS activity is reduced (12). In addition, inducible NOS expression is reduced in airway epithelial cells of patients with CF compared with normal subjects (23). Both PCD and CF are genetic diseases, and DPB is associated with the human leukocyte antigen (HLA) class I alleles (24, 25). We therefore speculate that the mechanism for the reduction in nasal NO may be linked with genetic profiles, although we did not investigate the relationship between nasal NO concentrations and HLA typing in the present subjects.

The second possible reason for lowered nasal NO is that, whatever the pathogenesis of a disease, chronic sinusitis per se can potentially decrease nasal NO concentrations because of the widespread epithelial damage in paranasal sinuses resulting from recurrent inflammation. Indeed, nasal NO in patients with chronic sinusitis is reduced by 23 to 59% compared with healthy individuals (11, 26). However, this does not fully explain the 88% drop in nasal NO in patients with DPB.

An alternative explanation could be that the erythromycin therapy contributed to the low nasal NO production in patients with DPB because erythromycin inhibits lung inflammation and suppresses NO synthesis in an experimental inflammatory lung model (27). However, erythromycin treatment had no effect on nasal NO concentrations in healthy subjects in the present study.

In conclusion, we found that nasal NO production was significantly reduced in patients with DPB. This finding makes DPB the third pulmonary disease with low nasal NO. We propose that impaired NOS activity in the upper airway may play a role in the pathogenesis of DPB, and nasal NO measurements, which are noninvasive and easy to perform, may be a useful clinical test in the diagnosis of DPB.