Macrolide activities beyond their antimicrobial effects: macrolides in diffuse panbronchiolitis and cystic fibrosis

Marcus J. Schultz1,2,*

1 Laboratory of Experimental Internal Medicine and 2 Department of Intensive Care Medicine, C3-329, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands


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Diffuse panbronchiolitis (DPB) is a pulmonary disease characterized by chronic inflammation of the bronchioles and chronic infiltration of inflammatory cells in the lungs. DPB has several features in common with cystic fibrosis (CF). Clinical trials in patients with DPB or CF suggest a potential role for maintenance (long-term and low-dose) macrolide therapy in the treatment of these chronic pulmonary conditions. Indeed, these studies demonstrate improved clinical and physiological states with macrolide therapy. The beneficial effects of long-term low-dose macrolides are not related to their antimicrobial properties, since levels of macrolides with low-dose treatment are too low to have sufficient antimicrobial effects. Data indicate that macrolides may have immunomodulatory activities: (1) in vitro and ex vivo studies clearly show that macrolides can influence cytokine production by several cell types; (2) furthermore, macrolides can alter polymorphonuclear cell functions in vitro and ex vivo. Although immunomodulation may serve as one explanation for the beneficial effects of macrolides in patients with chronic pulmonary inflammation, the effect of low-dose macrolide therapy on biofilm-formation may form a second explanation for the positive effects of long-term low-dose macrolide therapy. In the present paper, the clinical trials on maintenance macrolide therapy in patients with DPB or CF are reviewed. This is followed by a discussion on the immunomodulating effects of macrolides, and the effects of macrolides on biofilm formation.

Keywords: erythromycin , azithromycin , clarithromycin , innate immunity , alveolar macrophages , polymorphonuclear cells , cytokines , biofilms


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Diffuse panbronchiolitis (DPB) is a condition of unknown aetiology, usually diagnosed between the second and fifth decade. DPB is an important cause of progressive obstructive lung disease in the Far East (mainly in Japan and Korea), but a rare disease in non-Asian populations. The onset of DPB resembles bronchial asthma, chronic bronchitis, and pulmonary emphysema, because it is clinically manifested by common symptoms such as dyspnoea, cough, sputum, coarse crackles and wheeze. The chest radiograph often shows hyperinflation. Pulmonary function tests show decreased vital capacity (VC), and forced expiratory volume in 1 s (FEV1). Lung histology reveals a cellular bronchiolitis, mononuclear cell proliferation and foamy macrophages involving the bronchiolar walls, adjacent alveolar ducts and alveoli. Narrowing and constriction of respiratory bronchioles may develop in advanced stages. Advanced disease is associated with chronic mucoid Pseudomonas aeruginosa infection. Death is usually secondary to respiratory failure and/or P. aeruginosa pneumonia. For a long time, the long-term prognosis of DPB was poor, but a key report by Kudoh et al.1 clearly suggested spectacular improved survival with the introduction of macrolide therapy.

DPB shares many similarities with cystic fibrosis (CF). The triad of chronic obstructive pulmonary disease, pancreatic exocrine deficiency and abnormally high sweat electrolyte concentrations is present in most CF patients. The clinical and pathological findings are attributable to genetic defects, leading to an abnormality in the chloride channel. Pulmonary complications usually dominate the course of the disease, although clinical manifestations may not appear until weeks, months or years of age. Bronchiectasis is present in most patients by the end of the second decade of life. Lung infections due to Staphylococcus aureus and P. aeruginosa are extremely common and cause additional lung damage. Until now, the therapeutic approach of CF patients included physiotherapy, DNase and antibiotics. Successful treatment strategies with macrolides in DPB patients have inspired researchers to introduce macrolides as immunomodulating agents in CF patients, showing some improvements in morbidity.2,3

To summarize current evidence on the effectiveness of macrolides during treatment of DPB or CF, I carried out a systematic search in the medical literature. Here, I will review the current literature on macrolide therapy in patients with DPB or CF. Thereafter, mechanisms by which macrolides may achieve their beneficial effects are discussed, focusing on the immunomodulating effects of macrolides, and their influence on biofilm formation.


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A search of the PubMed (National Library of Medicine, USA at www.pubmed.org) for unlimited citations using the words ‘DPB’ OR ‘diffuse panbronchiolitis’ found a total of 708 publications. At the end of March 2004, a limited search using the items ‘macrolides’ AND ‘DPB OR diffuse panbronchiolitis’ found 164 papers; a limited search using the items ‘erythromycin OR azithromycin OR clarithromycin’ AND ‘DPB OR diffuse panbronchiolitis’ listed 154 papers. Limiting these searches for ‘human’ and ‘clinical trial’, listed 10 identical papers. Only seven papers were about macrolide therapy in DPB patients, and only three of them were in the English language.46 The reference lists of these publications, and several reviews on this topic,79 were used to find additional papers on macrolide therapy in DPB. This resulted in identification of 17 additional papers.1,1025

A search of the PubMed database for unlimited citations using the words ‘CF’ OR ‘cystic fibrosis’ found a total of 31 600 publications. At the end of March 2004, using limited search items ‘macrolides’ AND ‘CF OR cystic fibrosis’ identified 325 papers; for a limited search using the items ‘erythromycin OR azithromycin OR clarithromycin’ AND ‘CF OR cystic fibrosis’ 125 papers were listed. Limiting these searches for ‘human’ and ‘clinical trial‘, listed eight and 14 papers, respectively. Finally, after carefully reading the abstracts, only five papers were about macrolide therapy in CF patients, and only three of them were in the English language.2628 Reviews on the topic of macrolide therapy in chronic lung disease2,3,29 identified two additional papers.30,31


    Macrolide therapy in patients with DPB (Table 1)
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The first papers reporting amelioration of signs and symptoms of DPB by long-term low-dose macrolide therapy were all published in Japanese journals.1012 Kudoh et al.10 were the first to describe the beneficial effects of maintenance erythromycin therapy in patients with DPB. Takeda et al.11 showed improvement in the symptoms of patients with DPB with long-term clarithromycin therapy. These publications were followed by a paper by Yamamoto et al.,12 who summarized the long-term therapeutic effects of erythromycin or a new quinolone on DPB in 101 patients. In this study, DPB patients treated with erythromycin showed a significant improvement in dyspnoea on exertion and sputum production. Treatment with the new quinolone did not result in any improvement. Unfortunately, these papers were in the Japanese language.


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Table 1. Studies on macrolides in diffuse panbronchiolitis

 
Since these publications, several reports in the English medical literature have confirmed the effectiveness of long-term macrolide therapy in patients with DPB. In most of these studies, erythromycin was used.1,4,1318,21,23,25 Seven papers reported on the effectiveness of clarithromycin,6,11,19,25 azithromycin,20 and roxithromycin,5,23 respectively. The vast majority of publications reported on open, non-randomized trials evaluating the clinical effectiveness of macrolides, with special emphasis on lung function tests.6,16,17,21,23,25 All cited studies indeed showed improved VC and FEV1 with use of macrolides. Other studies demonstrated improvements in the diffusion capacity of lungs (DLCO),16 and arterial oxygenation.6,17,23 Some studies also showed improvement of radiographic findings.4,12,13

Tamaoki et al.19 carried out the one and only randomized controlled trial with macrolides in patients with DPB. In this study, change in sputum production with macrolide therapy was determined.19 For this, patients were divided into two groups: one group received clarithromycin (100 mg twice a day for 8 weeks), the control group received placebo. In evaluating airway secretion, the daily amounts of expectorated sputum, solid composition, and sputum microbiology were assessed. Clarithromycin decreased sputum production from 51 ± 6 to 24 ± 3 g/day after treatment, whereas placebo had no effect. Bacterial density and sputum flora were unaltered. These results are in line with results from other studies.13,17,20

The largest report on macrolide therapy in patients with DPB is the paper by Kudoh et al.1 In this retrospective, though important analysis on almost 500 DPB patients, a significant improvement in survival was found to be associated with long-term low-dose erythromycin treatment. Patients were clustered in three consecutive groups: group 1 included patients diagnosed between 1970 and 1979 (the historical control patients); group 2 included patients diagnosed between 1980 and 1984; group 3 included patients diagnosed between 1984 and 1990. In the last group, a subgroup of patients were on long-term erythromycin therapy. Whereas mortality rates in the first groups were approximately 70% and 50%, respectively, the last group showed a survival rate of approximately 90%. Survival of patients in the subgroup receiving long-term macrolide therapy showed a better survival, compared with patients without erythromycin therapy. Although several other mechanisms may be responsible for the dramatic change in survival over time, the comparison between the subgroups in group 3, showing less mortality with erythromycin therapy, strongly suggests that maintenance erythromycin therapy is, at least in part, responsible for the improved survival of DPB patients.


    Macrolide therapy in patients with CF (Table 2)
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Based on the similarities between DPB and CF, several investigators have tried to determine the efficacy of macrolides in CF patients.2628,30,31


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Table 2. Studies on macrolides in cystic fibrosis

 
To determine whether macrolide antibiotics improve pulmonary function and decrease airway inflammation in CF, Ordonez et al.31 treated 10 patients with 3 weeks of placebo, followed by 6 weeks of clarithromycin (500 mg twice a day) in a single-blind prospective study. Although there was an 11% improvement in pulmonary function, differences were not statistically significant, nor were there significant differences in any of the inflammatory indices measured. However, this lack of significance may have been caused by the small number of patients studied (type 2 error). In addition, Ordonez and colleagues gave a short course of therapy at a high dose and therefore extrapolation of results may be difficult especially as the macrolides can initially increase the inflammatory response. In contrast, however, Jaffe et al.30 showed that long-term azithromycin for a period of 3 months or longer, resulted in improvement of lung function in a small group of young CF patients, suggesting that treatment of just 6 weeks, as used by Ordonez et al. may be too short to have a clinical effect.

Wolter et al.26 determined if azithromycin improved clinical parameters and reduced inflammation in patients with CF. In their prospective randomized double-blind, placebo-controlled study, 3 months of azithromycin (250 mg/day) was compared with placebo in 60 adults. Monthly assessment included lung function, weight and quality of life. Blood and sputum collection assessed systemic inflammation and changes in bacterial flora. Respiratory exacerbations were treated according to the policy of the CF unit. The main results of this study were that FEV1 and forced VC were maintained in the study group, whereas in the placebo group there was a decline in both parameters. In addition, fewer courses of intravenous antibiotics were used in study patients. Finally, quality of life improved over time in study patients, and remained unchanged in patients receiving placebo.

Equi et al.27 carried out a 15-month randomized double-blind, placebo-controlled cross-over trial in 41 children with CF. Patients received either azithromycin (bodyweight ≤40 kg: 250 mg/day, >40 kg: 500 mg/day) or placebo for 6 months. Treatments were crossed over after 2 months of washout. Median relative difference in FEV1 between azithromycin and placebo was 5.4% (95% CI 0.8–10.5). Thirteen of 41 patients improved by more than 13% and five of 41 deteriorated by more than 13%. Forced VC did not significantly change. Seventeen of 41 patients had 24 fewer oral antibiotic courses when on azithromycin than when taking placebo. Sputum bacterial densities, inflammatory markers, exercise tolerance and subjective well-being did not change.

To determine whether an association between azithromycin use and pulmonary function exists in patients with CF, Saiman et al.28 carried out a multicentre, randomized, double-blind, placebo-controlled trial at 23 CF care centres in the United States. Of 251 screened CF patients, 185 patients chronically infected with P. aeruginosa were randomized to receive either azithromycin 3 days a week for 6 months (bodyweight <40 kg: 250 mg/day; ≥40 kg: 500 mg/day) (n=87) or placebo (n=98). Groups were well-matched. Although there was a marked variation in individual responses, treatment benefit was found: the study group had a greater increase in FEV1 compared with the placebo group. Furthermore, study patients had less risk of experiencing an exacerbation than placebo patients, with a 40% reduction in infective exacerbations with azithromycin therapy.


    Immunomodulating properties of macrolides
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While the exact mechanisms are unknown, anti-inflammatory rather than antimicrobial properties of macrolides seem to be responsible for the beneficial effects in patients with DPB or CF. Indeed, macrolides may affect several components of pulmonary host defence. Several papers in the medical literature reported data from in vitro and ex vivo studies. In addition, some of the papers cited above have focused on immunomodulating properties of macrolides as well.

Neutrophil cell infiltration and function

Macrolides can influence several neutrophil cell functions. In numerous in vitro and ex vivo studies, it was shown that macrolides inhibit oxidant production.3237 In addition, macrolides promote neutrophil cell degranulation in vitro,38,39 and ex vivo.40 Furthermore, macrolides reduce neutrophil cell phagocytosis ex vivo.37 Additionally, macrolides increase neutrophil cell migration in healthy volunteers,41 and in patients with persistently abnormal neutrophil cell chemotaxis.42

Macrolides have been found to have anti-inflammatory effects in vivo in several animal models. A series of animal investigations have studied the effects of macrolides on pulmonary host defence: in one study, the effect of erythromycin on intrapulmonary accumulation of neutrophil cells after intra-tracheal challenge with interleukin (IL)-8 or lipopolysaccharide (LPS) was studied in mice.15 Intrapulmonary neutrophil cell accumulation was significantly suppressed after intraperitoneal administration of 5 mg erythromycin per animal 2 h before intra-tracheal injection of IL-8 or LPS. However, administration of erythromycin for 1 week did not alter the intrapulmonary neutrophil cell accumulation. In another animal model, LPS-induced vascular leakage and neutrophil cell accumulation in rat trachea, both roxithromycin and erythromycin reduced neutrophil cell accumulation.43

Long-term macrolide treatment of patients with DPB gave lower neutrophil cell counts in bronchoalveolar lavage fluids in numerous studies.5,1417,2123,25 Although attenuation of inflammation by macrolides was not demonstrated in patients with CF, it must be mentioned that Ordonez et al.31 gave a short course of therapy at a high dose. Therefore extrapolation of results from this study may be difficult especially as the macrolides can initially increase the inflammatory response.

The mechanisms underlying the effects of macrolides on neutrophil cell functions are largely unclear. It has been suggested that inhibition of activation of protein kinase A is responsible for the inhibition of oxidant production.36 Another study suggests that macrolides influence the phospholipase D–phosphatidate phosphohydrolase transduction pathway, which is essential for neutrophil cell degranulation.44

Cytokines and chemokines

Several studies on the effect of macrolides on production of cytokines/chemokines have been carried out with diverse cell types and different stimuli, including pro-inflammatory cytokines (IL-1), bacteria (Streptococcus pneumoniae and P. aeruginosa), or bacterial products (LPS). Macrolides suppressed IL-6 and IL-8 production by IL-1-stimulated bronchial epithelial cells.45,46 Similarly, erythromycin suppressed IL-8 production by human neutrophil cells in vitro.47 These data are in line with several in vitro and ex vivo studies in whole blood stimulated with S. pneumoniae or P. aeruginosa, in which erythromycin dose-dependently inhibited the production of tumour necrosis factor {alpha} (TNF), IL-6, IL-8 and IL-10.40,48,49 Comparable results have been obtained in studies with LPS, in which macrolide antibiotics attenuated the production of cytokines by bronchial epithelial cells or mononuclear cells.50,51 Interestingly, one of these studies showed that clarithromycin decreased TNF and IL-1 production, while it increased IL-6 and IL-10 production by LPS-stimulated mononuclear cells,51 which is different from the previously cited studies, in which the production of all cytokines was attenuated. But there is more inconsistency: in an ex vivo study in mice, a short course of erythromycin or roxithromycin was associated with increased production of TNF and IL-1 by murine cells stimulated ex vivo,5255 while administration of roxithromycin for a longer period was associated with a decrease in IL-1 production.55

In a chronic inflammation model in mice, the effect of macrolides on cytokine production was determined.56 During chronic P. aeruginosa infection, concentrations of TNF and IL-1 were measured serially in lungs until 60 days after infection. Compared to baseline, higher cytokine levels were found in the lungs during the course of the disease. In this model, clarithromycin significantly inhibited pulmonary production of TNF and IL-1, and treatment with anti-TNF antibodies significantly reduced the number of lymphocytes in the lung, but did not change the number of viable bacteria. In another report, the same group showed a significant decrease in the number of viable bacteria in an experimental murine model of chronic P. aeruginosa pneumonia.57

In clinical studies focusing on cytokine levels in lung fluids, long-term macrolide therapy resulted in reduced IL-1ß and IL-8 in patients with DPB.21,22,58 There are no data available on the effects of maintenance macrolide therapy on cytokine production in patients with CF.

Studies on the mechanisms underlying the effect of macrolides on cytokine production have produced inconsistent data. Since macrolide antibiotics exhibit their antimicrobial activity by interfering with the protein production of microorganisms, interference with protein production may be one mechanism by which cytokine production is influenced by macrolide antibiotics. Inhibition of cytokine production may also result from a modulation of gene expression. Indeed, clarithromycin inhibited TNF-mediated IL-8 gene expression by human bronchial epithelial cells.59 Similarly, erythromycin inhibited the transcriptional activity of nuclear factor {kappa}B (NF{kappa}B) in T cells,60 and clarithromycin inhibited the activation of NF{kappa}B by TNF and staphylococcal enterotoxin A.61 In airway epithelial cells, erythromycin increases cAMP levels.62 Elevation of cAMP has a marked effect on cytokine production induced by LPS, which includes inhibition of TNF and up-regulation of IL-6 and IL-10.63,64 Hence, a possible increase in cellular cAMP levels by erythromycin would only partly explain the inhibitory effects of macrolides on cytokine production.


    Macrolides and biofilms
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Biofilm-forming bacteria, such as P. aeruginosa, resist phagocytosis by host immune cells and the actions of antimicrobial agents. P. aeruginosa recovered from the lungs of patients with DPB or CF is often encased in mucoid-alginate biofilm. Biofilms bind cells and organic and inorganic materials to each other. Their tightly-formed structure reduces antimicrobial activity, promotes bacterial adhesion to lung epithelia, and prevents bacterial dehydration. Interference with biofilm formation may play a part in the effect of macrolides in patients with DPB or CF.

The major component of biofilm is alginate. Alginate induces a continuous antigen–antibody reaction on the surface of small airways. Through inhibition of alginate production, macrolides decrease the viscosity of media containing P. aeruginosa.6567 Alginate production is inhibited in a dose-dependent manner by erythromycin, clarithromycin or azithromycin, through inhibition of enzymes in the alginate system that activate the alginate production inside P. aeruginosa. Importantly, alginate production is inhibited by macrolides at concentrations well below the MIC. Perhaps of more importance is the effect of macrolides on flagellin-induced inflammation. Flagellin induces a stronger inflammatory response, and macrolides have been found to inhibit twitching motility of P. aeruginosa.68,69

The interaction of neutrophil cells with P. aeruginosa biofilms treated with macrolides is markedly enhanced compared with the untreated biofilms.67 As with the effect of macrolides on alginate production, this effect is also dose-dependent.

These findings indicate that biofilm is involved in impairment of phagocytosis of P. aeruginosa, and inhibiting its formation with macrolides may enhance clearance of bacteria by the patient's own pulmonary host defence. Next to the above-mentioned effects of macrolides on neutrophil cell function and cytokine production, attenuation of biofilm formation may be responsible for the beneficial effects of macrolides in patients with DPB or CF.


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In conclusion, in nearly all reports, long-term low-dose macrolide therapy resulted in, sometimes dramatic, improvements in pulmonary function in patients with DPB. Before introduction of maintenance macrolide therapy, the 10-year survival rate was below 50%. After introduction of macrolide therapy, the 10-year survival rate improved impressively, and is now above 90%. Although almost all studies were not randomized controlled trials, data presented in the peer-reviewed journals are convincing enough to justify recommendations for the general use of long-term macrolide therapy in DPB.

Similar to DPB, there seems to be some role for macrolides in the treatment of CF. However, data presented in peer-reviewed journals are sparse, and there seems to be considerable variation in response. At present, it is recommended to use maintenance macrolide therapy for individual patients who do not respond to conventional therapy.3


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* Tel: +31-20-5669111; Fax: +31-20-5669568; Email: m.j.schultz{at}amc.uva.nl


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33 . Labro, M. T., el Benna, J. & Babin-Chevaye, C. (1989). Comparison of the in vitro effect of several macrolides on the oxidative burst of human neutrophils. Journal of Antimicrobial Chemotherapy 24, 561–72.[Abstract]

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37 . Wenisch, C., Parschalk, B., Zedtwitz-Liebenstein, K. et al. (1996). Effect of single oral dose of azithromycin, clarithromycin, and roxithromycin on polymorphonuclear leukocyte function assessed ex vivo by flow cytometry. Antimicrobial Agents and Chemotherapy 40, 2039–42.[Abstract]

38 . Abdelghaffar, H., Mtairag, E. M. & Labro, M. T. (1994). Effects of dirithromycin and erythromycylamine on human neutrophil degranulation. Antimicrobial Agents and Chemotherapy 38, 1548–54.[Abstract]

39 . Abdelghaffar, H., Vazifeh, D. & Labro, M.T. (1996). Comparison of various macrolides on stimulation of human neutrophil degranulation in vitro. Journal of Antimicrobial Chemotherapy 38, 81–93.[Abstract]

40 . Schultz, M. J., Speelman, P., Hack, C. E. et al. (2000). Intravenous infusion of erythromycin inhibits CXC chemokine production, but augments neutrophil degranulation in whole blood stimulated with Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 46, 235–40.[Abstract/Free Full Text]

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