Arata Azuma, Respiratory Medicine of Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. Email: email@example.com
Abstract: Diffuse panbronchiolitis is characterized by chronic sinobronchial infection and diffuse bilateral centrilobular lesions consisting of peribronchial infiltration of inflammatory cells. At present, it is known that diffuse panbronchiolitis is relatively restricted to East Asia. This uneven distribution is suspected to be highly associated with genetic predisposition located between human leucocyte antigen-A and -B loci. Low-dose, long-term macrolide therapy for the disease was suggested from a detailed observation of a single case that significantly improved by erythromycin therapy. Otherwise simple bactericidal activity of macrolides has been assumed as a candidate because of their clinical effect on the pathogenesis. In the last 10 years, the possible mechanism underlying the effectiveness of macrolide therapy has been dynamically investigated. To understand the pathological features and potential targets for macrolides in diffuse panbronchiolitis, the authors introduce the incidence of diffuse panbronchiolitis in East Asia, the profile of the disease and then trace the history of macrolide therapy in this review. The proposed mechanism of action includes the inhibition of excessive mucus and water secretion from the airway, the inhibition of neutrophil, and sometimes of lymphocyte and macrophage accumulating in the airway, the inhibition of transcription factors expressing several cytokines and the attenuation of bacterial virulence. Intracellular mechanisms of the action of macrolide are a hot topic of interest in research. The anti-inflammatory activity of macrolides is independent of their bactericidal effect, and a new anti-inflammatory analogue without antimicrobial activity should be developed to minimize the emergence of macrolide-resistant microorganisms and to maintain the safety of this treatment.
In the 1960s, a new clinicopathological entity distinguished from other COPDs was established by a Japanese group of clinicians and lung pathologists. Homma and coworkers gathered cases of this disease and reviewed the clinical, radiological and pathophysiological features.1 Yamanaka designated this new disease entity as diffuse panbronchiolitis (DPB), whose characteristic pathological features were numerous micronodular pulmonary lesions composed of chronic inflammatory cells infiltrating the walls of the respiratory bronchioles.2
A nationwide survey was subsequently conducted and the clinical features of DPB were identified.3 Until the first comprehensive report of DPB was published in Western countries in 1983,4 DPB had been unaccepted internationally as it was a disease unique to Asians. Most patients with DPB have a long history of sinusitis. Chronic sinobronchial infection therefore is a common feature of the disease. Since Pseudomonas aeruginosa occurred in the advanced stage, the prognosis was thought to be bleak. However, since the introduction of long-term treatment with 14-membered ring macrolides reported by Kudoh in the mid-1980s,5 the prognosis of DPB has been dramatically improved, and now it is regarded as a curable disease. Although the aetiology of DPB is still unknown, recent advances in cellular and molecular biology have enabled people to narrow the possibility of the susceptibility gene down to a location in the human leucocyte antigen (HLA) locus.
In this review, the authors update the epidemiology, pathophysiology and diagnosis of DPB. The role of anti-inflammatory agents in the management of patients with DPB is discussed within the current treatment strategies available to the practitioner. Areas of current research and the potential of treatments undergoing investigation are also discussed.
In the first nationwide survey in Japan conducted by Izumi and Homma in 1980 with the support of the Ministry of Health and Welfare of Japan, more than 1000 probable cases of DPB were collected.3,6 The subsequent clinicopathological conferences extracted 319 clinically definite cases and 82 histologically proven cases. There was no remarkable difference in the male-to-female ratio as shown in the ratio of 1.4:1, respectively. Two-thirds of the patients were non-smokers. There was no particular history of inhalation of toxic fumes and biomass. According to another previous population-based survey in 1980, the prevalence of physician-diagnosed DPB was 0.00011 among 70 000 employees in the Japanese national railway corporation.7 Recently, however, the incidence rate appears to have decreased.
Diffuse panbronchiolitis was also described in other East Asian populations such as Chinese and Koreans in the 1990s, and currently a number of case reports are found in these countries.8–16 In contrast, outside Asia, the number of reported cases was limited.17–26 Of those patients reported in Western countries, approximately half were Asian immigrants. In Asian countries, it was revealed that the incidence of patients with DPB is not common (Fig. 1). DPB was also reported to be widely distributed in China, but a nationwide survey needs to be conducted to confirm this in the near future.27 Currently, it is reasonable to conclude that DPB is a chronic airway disease predominantly affecting East Asians.
AETIOLOGY AND GENETICS
Although the aetiology of the disease remains unknown, recent advances in molecular genetics have shed considerable light on the genetic mechanisms of the disease. DPB is not a single genetic disorder, but is considered to be a multifactorial disease in adulthood. In fact, HLA-B54, an ethnic antigen unique to East Asians, was strongly associated with the disease in Japan.28 This association has been recently confirmed at the nucleotide sequence level in a larger case–control study;29 the odds ratio was 3.4 (95% confidence interval: 1.7–7.0). In contrast, Korean patients with DPB showed a positive association with another HLA class I antigen, HLA-A11.30 Because there is a close relation between the Japanese and Korean HLA profiles and their genetic background, these observations have raised the possibility that a major disease susceptibility gene is located between the HLA-A and HLA-B loci (Fig. 2). It is possible that different historical recombination events around the disease locus have resulted in DPB associations with HLA-B54 in Japanese, and HLA-A11 in Korean, populations. Together with Keicho the authors hypothesized that the major disease susceptibility gene exists between the two HLA loci on chromosome 6 and then analysed genetic markers in the most likely region.31
Cystic fibrosis (CF), a Mendelian genetic disorder in Caucasians is often compared with DPB.32 However, there is neither pancreatic insufficiency, nor obvious abnormalities of the electrolytes in sweat;33 and there is no reproductive failure in men with DPB. A large amount of sputum generated by hypersecretion in the inflamed airway is a typical feature of DPB. In fact, the most common mutation in CF, delta F508 of the CFTR gene on chromosome 7, was not found in patients with DPB.34 Nevertheless, chronic sinopulmonary infection followed by superinfection with P. aeruginosa in the advanced stage is a phenotype common to the two diseases, and neutrophil-dominated inflammatory reaction in the airway is commonly observed in both.35,36 Therefore, minor mutations in the CFTR gene have not been ruled out as having a possible pathogenic contribution to DPB.37
Another genetic disorder, bare lymphocyte syndrome type I, closely resembles DPB in its clinical features with diffuse granular shadows revealed on a chest CT scan.38–40 This rare disease is characterized by a deficient processing of HLA class I antigens caused by a defect in the transporter associated with antigen processing-1 or -2.41,42 A Japanese patient with bare lymphocyte syndrome type I was successfully treated with macrolides.43 Interestingly, CFTR and antigen processing are members of the ATP-binding cassette transporter superfamily,44 which translocates a variety of substrates across extra- or intracellular membranes.
Fine yellowish nodules in the parenchymal area are shown in cut surfaces of autopsied lung tissue in DPB.45 These nodules consist of thickened walls of the respiratory bronchioles with lymphofollicules and infiltrations of lymphocyte, plasma cells and histiocytes (Fig. 3). A large amount of sputum from hyperplasia of the goblet cells in the bronchiole comes from the increased expression of MAC5AC and aberrant MAC5B mRNA.46 These inflammatory changes extend to peribronchiolar tissues, whereas the alveolar walls are not affected. Accumulation of foamy histiocytes in the walls of the respiratory bronchioles, adjacent to the alveolar ducts, is a prominent pathological finding, although to a lesser extent in the adjacent alveolar area.47 Diffuse bronchiectasis of the proximal bronchioles is a secondary occurrence, which is indistinguishable from primary diffuse bronchiectasis.
More than 80% of the patients have a history of or suffer from chronic paranasal sinusitis.3,6 Even if patients have felt asymptomatic, most of them show roentgenographic signs of chronic sinusitis. Chronic cough with copious purulent sputum is usually present. Exertional dyspnoea subsequently develops. Auscultation reveals crackles, wheezes, or both. In the review of 81 histologically proven cases in 1980, 44% had Haemophilus influenzae in their sputum at presentation and 22% had P. aeruginosa.3,6 The detection rate of P. aeruginosa increases to 60% after 4 years.
Laboratory findings suggest immunological abnormalities and reflect chronic bacterial infection.33 The titre of cold haemagglutinin is continuously raised in most patients without evidence of Mycoplasma infection.48 Other laboratory abnormalities suggest non-specific inflammation.3,6
Pulmonary function measurements show significant airflow limitation and a relative resistance to bronchodilators.49 Decreased FEV1/FVC (less than 70%), decreased VC (less than 80% predicted), and RV (greater than 150% predicted) are usually affected.3,6 Hypoxaemia (PaO2 less than 80 mm Hg) subsequently develops.
In the advanced stage, there is frequent infection by P. aeruginosa and the capacity for gas exchange is reduced, which causes progression of hypoxaemia, and later, hypercapnia. Pulmonary hypertension develops and is associated with the development of cor pulmonale. Therefore, the cause of death is chronic respiratory failure in most cases. The 10-year survival rate with P. aeruginosa infection was only 12%, compared with 73% for those who remained uninfected before the introduction of erythromycin (EM) therapy.6
A plain CXR film reveals bilateral, diffuse, small nodular shadows predominantly in the lower field of the lung with hyperinflation. In advanced cases, ring-shaped or tram-line shadows indicating bronchiectasis appear.50 High-resolution CT is extremely useful for the detection of characteristic pulmonary lesions of DPB (Fig. 4).50–52 In the advanced stage, multiple cystic lesions predominate in the lower lung fields and are accompanied by dilated proximal bronchi showing the appearance of extensive bronchiectasis. These findings indicate that the inflammatory lesions extend from the respiratory bronchioles to the proximal airways.
Recent diagnostic criteria proposed in 1998 by a working group of the Ministry of Health and Welfare of Japan are as follows:53
• Persistent cough, sputum and exertional dyspnoea
• Past history of or current chronic sinusitis
• Bilateral diffuse small nodular shadows on a plain CXR film or centrilobular nodular shadows on chest CT images
• Coarse crackles
• FEV1/FVC less than 70% and PaO2 less than 80 mm Hg
• Titre of cold haemagglutinin equal to or higher than 64
Definite cases should fulfil the first three criteria, and at least two of the last three criteria.
Before 1980, DPB was a fatal disease despite the available therapy. Oral glucocorticosteroids and antibiotics such as beta-lactams against H. influenzae and other bacteria failed to change the prognosis. Mucolytic agents and bronchodilators did not remarkably improve the airflow limitation composed of bronchiolar inflammation.49
In 1982, Kudoh in the Tokyo Metropolitan Hospital discovered a DPB patient who had discontinued the previous treatment, and surprisingly his condition and CXR shadow were greatly improved. From his prescription records, it was assumed that 600 mg of EM administered every day for 2 years had been effective. To confirm this, the first open trial of low-dose, long-term EM therapy was started immediately.5 After 6 months−3 years of treatment with 600 mg of EM, symptoms and clinical parameters were markedly improved in 18 patients. FEV1 and PaO2 were significantly increased on average from 1.61 to 2.17 L (P < 0.01) and from 65.2 to 75.1 mm Hg (P < 0.01), respectively. Small nodular shadows on CXR films disappeared in more than 60% of the cases.
These favourable effects were quickly confirmed by other investigators.54–57 In all of their reports, clinical efficacy was satisfactory. Sputum volume was reduced and body weight increased. Dyspnoea was reduced. FEV1 and VC in pulmonary function tests and PaO2 were significantly increased, and RV/TLC was decreased. Radiological abnormalities reflecting centrilobular nodular lesions in DPB were improved simultaneously with changes in pulmonary function (Fig. 5).58–60 Bacterial species in the sputum were not replaced with P. aeruginosa during the treatment. Even in cases of superinfection with P. aeruginosa, EM treatment improved airflow limitation and gas exchange abnormalities.57,61 Regardless of bacterial clearance, general conditions improved. The clinical efficacy of EM therapy for 3 months was superior to that of a fluoroquinolone55 or ampicillin35 for the same duration in retrospective studies. A prospective double-blind, placebo-controlled study revealed the beneficial effect.62 Survival rates of patients treated with or without EM were compared only in a retrospective study.63 In the 1970s before EM, the overall 5-year survival rate was 63%. Between 1980 and 1984, fluoroquinolones were started for treating P. aeruginosa and the survival rate was limited to 72%. After 1985 when EM therapy was introduced, the 5-year survival rate was significantly improved to 91% (Fig. 6a). In a subgroup of 24 patients treated with other antibiotics (even after 1985), the survival rate was significantly lower than that of a subgroup of 63 patients treated with EM (Fig. 6b).
New 14-membered ring macrolides other than EM have also been used for treatment and obtained similar clinical benefits.64–66 These new macrolides were sometimes effective even when EM was ineffective.64 Azithromycin, a 15-membered ring macrolide had been in limited use in Japan until it was properly available in 2001. It appears to have similar effects on DPB, although people do not yet have sufficient experience to confirm this unequivocally.
Recommended treatment protocol
The Diffuse Lung Disease Committee members of the Ministry of the Health and Welfare of Japan proposed clinical guidelines on macrolide therapy for DPB in 2000, based mainly on evidence from the earlier-mentioned historical study,63 observational studies and expert opinion:67
1Macrolides should be started soon after the diagnosis is made, because the clinical response is better in the earlier stage.
2Choice of drugs and dose per day:
• The first choice: EM 400 or 600 mg orally
When it is ineffective or should be stopped because of its adverse events or drug-to-drug interactions,
• The second choice: clarithromycin 200 or 400 mg orally or roxithromycin 150 or 300 mg orally
Note: 16-membered ring macrolides appear to be ineffective.
3Assessment of response and duration of treatment:
• Although clinical response is usually obtained within 2 or 3 months, the treatment should be continued for at least 6 months and then the overall response should be evaluated
• It should be completed after 2 years when clinical manifestations, radiological findings and pulmonary function evaluations are improved or stable without significant loss of daily activity
• It should be restarted if symptoms relapse after the cessation
• When it is effective in advanced cases with extensive bronchiectasis or respiratory failure, it should be continued for more than 2 years
THE ROLE OF ANTI-INFLAMMATORY EFFECTS OF EM
Mechanisms of action of macrolides
Erythromycin therapy reduced the number of both Haemophilus and Pseudomonas organisms and induced reversion to normal flora. Because chronic airway infection begins with abnormalities such as a defect in the airway defence mechanisms and is accompanied by an inflammatory process, which results in the ‘vicious circle’ described by Cole,68 it is believed that the efficacy of bactericidal treatment with antibiotics for chronic airway infection is usually insufficient.
Presently, it is believed that EM cuts this vicious circle in chronic airway infection by inhibiting the inflammatory process, as shown in Figure 7.
The mechanism of action of low-dose, long-term EM treatment is recognized to have non-bactericidal effects, as indicated by the following issues. First, DPB can improve without the elimination of bacteria. Second, improvement can be found even in patients with P. aeruginosa infection. Third, the maximal concentration of EM in serum or sputum is lower than the minimum inhibitory concentration (MIC) for major species of bacteria.56 It has recently been found that even sub-MIC of 14-membered ring macrolides exhibits inhibitory effects on biofilm formation and the expression of virulence factors (pyocyanine, elastase, proteases) of P. aeruginosa. Recent Japanese investigations of EM have focused on bacterial virulence and cellular effects on the airway inflammation of DPB, including airway epithelial cells, neutrophils, lymphocytes and macrophages.
Therefore, the anti-inflammatory effects of EM are bidirectional, towards host defence mechanisms and bacterial activities.
Inhibition of hypersecretion
Reduction of sputum volume is the most sensitive parameter of DPB disease activity, as shown previously by a double-blind study. Goswami et al. first reported that EM dose-dependently inhibited mucus secretion in vitro with the use of a glycoconjugate marker.69 EM inhibited ion transport in epithelial cells in vitro in a dose-dependent fashion when it attached to the serosa.70 This inhibition depends on the blockade of chloride channels that subsequently inhibits the water secretion.
Inhibition of neutrophil activity
The most advanced research concerns the effect of EM on neutrophil accumulation and activity in inflamed tissues of the airway. After EM treatment, neutrophil elastase levels decrease in both sputum71 and BAL fluid.72 The percentage of neutrophils in BAL markedly decreased after EM therapy, highly associated with attenuated neutrophil chemotaxis.36,72 The level of IL-8 in BAL fluid markedly decreases along with the neutrophil number and its elastase concentration.73 EM dose-dependently inhibited IL-8, IL-6 and granulocyte-macrophage colony-stimulating factor secretion from epithelial cells in vitro using a human airway epithelial cell line.74,75 Recently, it was revealed that EM suppressed the activation of nuclear factor-kappa B and activator protein-1 in human bronchial epithelial cells and subsequently inhibited IL-8 mRNA expression on epithelial cells.76
Effects on lymphocytes and macrophages
In the peribronchiolar areas, chronic inflammation with lymphocytes, plasma cells and foamy macrophages is the characteristic pathological feature of DPB. These foci disappeared after EM treatment. A study of BAL fluid from a patient with DPB found that the number of memory T cells and activation of CD8+ cells, mainly consisting of cytotoxic T cells, were significantly increased in DPB but decreased after EM treatment.77 An increase in the number of lymphocytes was inhibited by EM in a dose-dependent manner.78 This inhibited T-cell proliferation associates with decreased T-cell responses to IL-2 in the later activation process.
Furthermore, EM has been found to accelerate both differentiation and proliferation of monocyte-macrophage system cells.79 EM actually inhibited LPS-induced production of tumour necrosis factor-alpha in human monocytes in vitro.80
However, human T-cell lymphocyte virus type-1-associated bronchiolitis, which mimics the pathological features of DPB, has been reported to poorly respond to macrolide therapy.81 The roles of EM in eliminating the inflammatory process in the respiratory bronchioles need to be further clarified in the near future.
Points of action of EM in the treatment of airway inflammation
The action points of EM in the treatment of airway inflammation are summarized in the schematic diagram in Figure 8, which is based on recent papers. First, EM inhibits hypersecretion due to the inhibition of mucus and water secretion from epithelial cells. Second, EM inhibits neutrophil accumulation at sites of inflammation due to the inhibition of adhesion and migration of neutrophils into inflamed regions from capillary vessels, secretion of IL-8 and leucotriene B4 from the epithelial cells and from neutrophils.82 These effects subsequently reduce the levels of injurious substances, such as elastase and superoxide anion,83 and clearly play important roles in the improvement of airway inflammation, although controversies exist concerning the effects of EM on neutrophil activity itself84–87 and on lymphocytes and macrophages.
As the authors mentioned above, interest in the macrolide's point of action is reaching transcriptional factors.76 In a recent report, macrolide antibiotics modulated extracellular regulation kinase (ERK ) phosphorylation resulting in the inhibition of IL-8 and granulocyte/macrophage colony-stimulating factor (GM-CSF) production by human bronchial epithelial cells.88 The authors revealed that new macrolide derivative, EM703, inhibited Smad phosphorylation in the transforming growth factor (TGF)-β-induced fibrogenic process.89
Effects on bacteria
Another direction of the anti-inflammatory effect of macrolide is towards bacteria. Sub-MIC of macrolide can reduce the infectibility of bacteria, which includes virulence factor production and bacterial activity itself. Sub-inhibitory levels of EM, clarythromycin (CAM) and azythromycin (AZM) enhance the susceptibility of P. aeruginosa to serum bactericidal activity by altering the cell membrane structure.90
The sub-MIC effect of macrolides on the production of verotoxin by Escherichia coli O157 was reported. The production of verotoxin 1 was suppressed up to 10 h in incubation with 1/100 of MIC of CAM.91
Pseudomonas aeruginosa infection is preceded by the selective adhesion of bacteria to the host target cells via adhesins, including lectins. The production of both lectins and many of the virulence factors is positively controlled by transcription activators including signalling autoinducers (N-acyl-l-homoserine lactones). Autoinducers are key factors in the bacterial cell-to-cell communication system promoting defence activities of the bacteria towards the human immune system, so called ‘quorum-sensing’.92 Sofer et al. reported that EM at sub-MIC concentrations suppressed the production of P. aeruginosa haemagglutinins (including lectins).93 In contrast, sub-MICs of AZM strongly suppressed the synthesis of elastase, proteases, lecithinase and DNAse.94 CAM and EM were far less effective. In these virulence factors, pyocyanine, a pigment, suppresses superoxide anion production from neutrophils, differentiation and proliferation of lymphocytes, cilliary beating of bronchial epithelial cells, and nitrogen intermediate and cytokine production from alveolar macrophages. EM suppresses the production of pyocyanine dose-dependently in vitro.95
These sub-MIC inhibitory effects of macrolide induce convergence of chronic airway inflammation.
Potential adverse effects
Although there is widely spreading evidence for the clinical efficacy of macrolides in chronic inflammatory diseases, attention must be paid to possible adverse events. Gastrointestinal disturbances are sometimes experienced as an adverse effect of macrolides, because EM and its derivatives have motilin-like activity and stimulate gastrointestinal motility.96 Other adverse effects including hepatotoxicity and allergic skin eruptions are less common.97 Rarely, macrolides have been reported to cause cardiac arrhythmias, including QT prolongation with ventricular tachycardia,98 although the authors have not experienced such serious adverse effects in the low-dose macrolide treatment of patients with DPB.99 EM and other 14-membered macrolides have been shown to potentiate the effects of many drugs by interfering with the cytochrome P450-mediated pathway in the liver.97 Newer 15-membered ring macrolides, such as azithromycin, appear to have less potential for drug interactions.
The frequent use of any antibiotic used in animal husbandry increases the risk of emerging bacterial resistance. To the best of the authors’ knowledge, there have been no reports of patients treated with long-term macrolides who experienced life-threatening infectious disease with macrolide-resistant bacteria. Nevertheless, an increase in bacteria such as drug-resistant Streptococcus pneumoniae should be avoided as much as possible.100 Therefore, if alternative therapies are available, the application of other new anti-inflammatory macrolides without antimicrobial activity should be developed. For that purpose, an appropriate screening system for drug design is necessary. It is hoped that modern technologies will identify a target molecule to clarify the core mechanism of the macrolide's anti-inflammatory action.
More than 30 years have passed since DPB was first reported in Japan. Studies on the aetiology of DPB have progressed in the context of a genetic predisposition unique to Asians. Knowledge of the disease characteristics of DPB needs to be spread widely in the Asian region for accurate recognition of this disease.
The introduction of macrolide therapy has remarkably changed the prognosis of the disease, and the beneficial effects are grouped in other chronic inflammatory diseases. Although many researchers have attempted to elucidate the mechanism of macrolide action, the essential mechanisms of the anti-inflammatory activities of macrolides and how different they are from those of other anti-inflammatory drugs such as corticosteroids remain unknown. The authors hope that this review detailing their experiences in East Asia will be helpful for identifying new patients with DPB and for the introduction of appropriate treatment for them in the near future.