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Keywords:

  • alveolar macrophage;
  • azithromycin;
  • chronic obstructive pulmonary disease;
  • monocyte-derived macrophage;
  • phagocytosis

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Background and objective:  Chronic inflammation and reduced airways integrity in chronic obstructive pulmonary disease (COPD) potentially results from secondary necrosis as a result of impaired phagocytosis of apoptotic material by airway macrophages, and increased bacterial colonization. We have previously shown that administration of low-dose azithromycin to subjects with COPD improved macrophage phagocytosis of apoptotic airway epithelial cells, reduced inflammation and increased expression of macrophage mannose receptor.

Methods:  We firstly investigated whether there were defects in the ability of both alveolar (AM) and monocyte-derived macrophages (MDM) to phagocytose bacteria in COPD, as we have previously reported for phagocytosis of apoptotic cells. We then assessed the effects of administration of low-dose azithromycin to COPD patients on the ability of AM and MDM to phagocytose bacteria. Azithromycin (250 mg orally daily for 5 days then 2× weekly (total 12 weeks)) was administered to 11 COPD subjects and phagocytosis of fluorescein isothiocyanate-labelled Escherichia coli assessed by flow cytometry.

Results:  COPD subjects had a significant defect in the ability of both AM and MDM to phagocytose bacteria that was significantly improved by administration of low-dose azithromycin

Conclusions:  The data provide further support for the long-term use of low dose azithromycin as an attractive adjunct treatment option for COPD. Improved clearance of both apoptotic cells and bacteria in the airway may have a dual effect; reducing the risk of secondary necrosis and release of toxic cell contents that perpetuate inflammation as well as contributing to a reduction in the rate of exacerbations in COPD.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

In contrast to the normal lung, the airways and lungs in chronic obstructive pulmonary disease (COPD) have chronic inflammation and emphysema resulting from loss of structural integrity from the large airways to the alveolar walls. This damage is thought to result from an inability of the normal repair processes to maintain structural homeostasis with ongoing noxious insults including cigarette smoke. Even patients who stop smoking are left with reduced physiological functioning and are susceptible to acute deterioration in the face of new insults such as infection. We have previously shown an accumulation of apoptotic material and impaired clearance of this material by macrophages (a process termed ‘efferocytosis’) in the airways in COPD.1–3 We have shown that the resultant net increase in apoptotic material has the potential to perpetuate airways inflammation via secondary necrosis.4

COPD is also characterized by increased bacterial colonization and an increased frequency of bacterial exacerbations. It has been suggested that the increased colonization contributes to chronic airway inflammation and to the progression of COPD.5 Consistent with our findings of reduced efferocytosis of apoptotic cells,1,3 other groups have reported defects in the ability of alveolar macrophages (AM) or monocytes to phagocytose bacteria in COPD. Berenson et al. reported that phagocytosis of non-typeable H. influenzae was impaired in COPD; interestingly, no phagocytic defect was noted for peripheral blood monocytes, suggesting a compartmentalized immunological defect.6 In contrast, Prieto et al. showed defective ingestion of Escherichia coli by COPD monocytes.7 More recently, Taylor et al. noted a defect in the ability of both AM and monocyte-derived macrophages (MDM) to phagocytose bacteria.8 We have previously shown that administration of the macrolide antibiotic, azithromycin, to COPD subjects at low doses, improved the ability of AM to phagocytose apoptotic cells, associated with a significant reduction in local and systemic inflammation.9 Whether azithromycin also improved the ability of AM to phagocytose bacteria is unknown, but likely given the findings of studies that have reported a reduction in exacerbation rates following long-term macrolide therapy in COPD patients.10

We therefore investigated whether there were defects in the ability of AM and MDM from COPD patients to phagocytose E. coli, as we have previously shown for phagocytosis of apoptotic cells. We then assessed the effects of 12 weeks treatment of COPD patients with low-dose azithromycin on the ability of AM and MDM to phagocytose E. coli.

METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

The following antibodies and reagents were used. For identification of macrophages CD14[PE-CY5], CD33[PE-CY5], CD71[fluorescein isothiocyanate] (BD Biosciences, San Jose, CA, USA); for efferocytosis: Mitotracker red (Molecular Probes, Eugene, OR, USA) and CD33[PE-CY5].

Bronchoscopy sampling and subject selection

Bronchoalveolar lavage was obtained via flexible bronchoscopy from volunteers as we have previously reported.1–3,9,11 Informed consent was obtained and the study protocol was approved by our institutional Research Ethics Committee. Bronchoalveolar lavage was collected from 10 COPD subjects (four current and six ex-smokers ≥ 1 year; diagnosed using the Global Initiative for Chronic Obstructive Lung Disease criteria (forced expiratory volume in 1 s/forced vital capacity < 70%) with clinical correlation). Ten healthy adult volunteers no history of asthma or allergy and normal lung function were recruited as controls. We included four healthy ex-smokers in the control group as we have previously shown that efferocytosis returns to within normal levels upon smoking cessation in the absence of COPD disease.

Administration of azithromycin to COPD subjects

The COPD subjects were included in a previously reported study of the effects of azithromycin on efferocytosis in COPD.9 After screening, bronchoscopy was performed, baseline samples collected, then treatment was commenced with azithromycin (‘Zithromax’, Pfizer) 250 mg orally daily for five days then twice weekly for a total of 12 weeks as we have previously described.9 A repeat bronchoscopy was performed during weeks 12–13 (while still taking azithromycin).

Preparation of bronchoalveolar lavage

Bronchoalveolar lavage was prepared, total cells and macrophages counted and AM purified by adhesion to plastic as previously described.1–3

Preparation of MDM

Venous blood was collected into tubes containing 10 U/mL preservative-free sodium heparin (DBL, Sydney, Australia). Blood differential cell counts were performed using a CELL DYN 4000 (Abbott Diagnostics, Sydney, Australia). Mononuclear cells were isolated by density gradient centrifugation within 4 h with Ficoll-Isopaque gradient (Lymphoprep 1.077 mg/mL; Nycomed Pharma, Oslo, Norway). Cells were re-suspended in culture medium.

Monocytes were isolated from PBMC by adherence to plastic. Cells were washed with phosphate buffer saline and re-suspended in ISCOVE's medium (+100 IU/mL penicillin, 100 µg/mL streptomycin) at a density of 4 × 106/mL. One millilitre aliquots of cell suspension were transferred into 24-well plates and incubated at 37°C for 1 h. The cells were washed four times with warmed HBBS and the adherent monocyte layer incubated with ISCOVE's medium containing 10% autologous donor serum. Cells were re-fed with autologous serum every 48 h and differentiated for 7 days. Differentiation was assessed by monitoring expression of CD71, CD14 and flow-cytometry scatter patterns.

Efferocytosis of apoptotic bronchial epithelial cells

Efferocytosis of apoptotic bronchial epithelial cells by AM was quantified using a flow-cytometric assay as previously reported.1,3

Phagocytosis of polystyrene beads

Phagocytosis of fluorescein isothiocyanate-labelled polystyrene microbeads (1.7 µm in diameter) was performed as we have reported previously.1

Phagocytosis of fluorescein-labelled E. coli

Phagocytosis of heat-killed, fluorescein isothiocyanate-labelled E.coli K-12 BioParticles (obtained from Invitrogen, Carlsbad, CA, USA) was measured using a Vybrant phagocytosis kit (Invitrogen) with modifications that allowed analysis by flow cytometry. Briefly, after adding the labelled E. coli to triplicate aliquots of AM or MDM in 96-well plates, the non-adhered fluid was removed and cells dislodged using ice-cold phosphate buffer saline and gentle pipetting. Cells were washed with phosphate buffer saline, and stained with PE-Cy5 conjugated CD33 as previously described,1,3 then rewashed with phosphate buffer saline. Quenching with crystal violet was performed as instructed by the manufacturer and dual-staining CD33+ fluorescein isothiocyanate+ events acquired immediately using a FACSCalibur flow cytometer (BD Biosciences). Results were expressed as the percentage of macrophages that had ingested bacteria as previously reported for phagocytosis of apoptotic cells and beads.1,3

Statistical analyses

Differences between groups were assessed using Kruskal–Wallis and Mann–Whitney U-tests, or Wilcoxon signed ranks test as appropriate and SPSS software (SPSS Inc., Chicago, IL, USA). P < 0.05 considered significant.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

Patient demographics

Patient demographics are presented in Table 1.

Table 1.  Demographic characteristics of the population studied
SubjectsControlCOPD
  • P < 0.05 compared with never-smoker controls.

  • Data presented as median ± data range.

  • BAL, bronchoalveolar lavage; curr, current smoker; ex, ex-smoker; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; WCC, total leukocyte count.

Subjects (n)1010
Age (years)50 (21–71)63 (57–72)*
Gender (male/female)5/59/1
Inhaled steroids (yes/no)0/102/8
Smoker (curr/ex)0/44/6
Smoking (pack-year)13 (0–40)66 (30–179)*
FEV1 % pred92 (82–108)66 (39–101)*
FEV1 % FVC82 (70–103)50 (28–65)*
BAL WCC0.173 (0.15–0.21)0.206 (0.02–0.45)
Macrophages (%)86 (70–98)67 (10–98)

Phagocytosis of apoptotic cells, E. coli and polystyrene beads by AM and MDM

We firstly showed that there was a significant reduction in the ability of MDM from subjects with COPD to phagocytose E. coli or apoptotic cells versus controls (Fig. 1) A similar defect was noted for AM (not shown). There were no changes in the phagocytosis of polystyrene beads versus controls (not shown).

image

Figure 1. Phagocytosis of (a) Mitotracker red-labelled apoptotic bronchial epithelial cells and (b) fluorescein-labelled E. coli by MDM from chronic obstructive pulmonary disease (COPD) and control subjects. *Significant (P < 0.05) decrease versus controls.

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Comparison of the effects of 12 weeks of azithromycin therapy on phagocytosis by AM and MDM in COPD

Low-dose azithromycin treatment for 12 weeks significantly increased the ability of AM and MDM from COPD subjects to phagocytose E. coli (Fig. 2). Azithromycin significantly improved the ability of AM to phagocytose apoptotic cells (as previously reported3,9) with a trend (P = 0.139) for an improvement in MDM. Azithromycin had no significant effect on phagocytosis of beads (Fig. 2).

image

Figure 2. Effect of azithromycin treatment on phagocytosis of (a) Mitotracker red-labelled apoptotic bronchial epthelial cells, (b) fluorescein-labelled E. coli, (c) fluorescent-labelled polystyrene beads by alveolar macrophages (AM) and monocyte derived macrophages (MDM) from patients with chronic obstructive pulmonary disease. *Significant (P < 0.05) improvement in phagocytosis following azithromycin treatment (Wilcoxon signed rank test). AM data in panel (a) has in part been presented previously.9

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Use of MDM for longitudinal evaluation of treatment effects

Analysis of the ability of MDM to phagocytose apoptotic cells was assessed for six COPD subjects at four weekly intervals for the duration of azithromycin treatment and 12 weeks following cessation of treatment. We noted a gradual increase in phagocytic ability to 12 weeks; in 5/6 patients, increased phagocytic ability was still detectable 12 weeks after cessation of therapy (Fig. 3).

image

Figure 3. Time course of changes in phagocytosis of Mitotracker red-labelled apoptotic bronchial epthelial cells by MDM after 12 weeks of azithromycin treatment in individual patients with COPD. Each plotted line represents data from an individual COPD patient tested prior to administration of azithromycin (Week 0), during the treatment phase (weeks 4 and 12) and 12 weeks following cessation of treatment (24 weeks). Note that improved phagocytic ability is still significantly improved 12 weeks following cessation of therapy. *Significant (P < 0.05) improvement in phagocytosis following azithromycin treatment (Wilcoxon signed rank test).

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

The reasons why some COPD patients are prone to frequent viral or bacterial exacerbations of their disease have not been established. We have previously reported dysregulated ‘efferocytosis’ (phagocytosis of apoptotic cells) in the airways.1,3,9 The resultant net increase in apoptotic material and secondary necrosis of this material is thought to contribute to chronic inflammation and ineffective repair of the injured epithelium in COPD. We now show a reduced ability of macrophages to phagocytose bacteria in COPD, consistent with other reports.6,8 There has been a great deal of interest in the use of macrolide antibiotics as therapeutic agents beyond their role as antimicrobials in chronic lung diseases. In our previous studies we have shown that the 15-member macrolide azithromycin, used at low, ‘sub-bactericidal’ dose, improved efferocytosis of apoptotic airway epithelial cells by AM both in vitro and following oral administration to a small group of COPD subjects.3,9 In the present study we evaluated the effect of oral administration of low-dose azithromycin on phagocytosis of E. coli by both AM and MDM. The uncontrolled, open-label study was primarily focused on comparing objective biological responses obtained from both the bronchoscopy samples taken and from blood-derived MDM. A cross-over format was not possible for patient acceptability reasons as this would require three bronchoscopy procedures. Nevertheless the present study enabled us to follow the subjects for an additional 12 weeks after the treatment phase and second bronchoscopy, during which time MDM were prepared at four weekly intervals.

We noted a significant increase in the ability of both MDM and AM to phagocytose bacteria in COPD patients following 12 weeks of azithromycin treatment. Azithromycin also increased the ability of both MDM and AM to phagocytose apoptotic cells (AM data has been previously reported9), although no significant changes in the ability to ingest polystyrene beads was noted. Taken together the data suggests that macrolides may possibly be exerting at least some anti-bacterial effects by changes to phagocytic functions that are independent of bacterial killing. This is supported by the report by Berenson et al. who showed that phagocytosis of non-typeable H. influenzae was impaired in COPD, but that there was no impairment in intracellular bacterial killing.6 No phagocytic defect was noted for peripheral blood monocytes, suggesting a compartmentalized immunological defect.

We then showed that MDM are suitable for longitudinally evaluating the effects of candidate phagocytosis-modulating treatments as a surrogate for AM. When we assessed the ability of MDM to phagocytose apoptotic cells during and following azithromycin treatment we showed a significant increase in phagocytic ability of MDM even after 4 weeks of therapy; this effect was significantly maintained for 12 weeks after cessation of azithromycin. The reason for this persistent effect is not clear but it may be an indirect systemic manifestation of a persisting anti-inflammatory pulmonary effect of azithromycin. The latter is likely explained by the pharmokinetics of azithromycin that are characterized by rapid uptake by AM with high, sustained levels in tissues.12,13 These effects may be amplified by cigarette smoking, as smokers have increased intrapulmonary uptake of antibiotics that are highly concentrated in AM.14

We applied heat-killed, fluorescein isothiocyanate-labelled E.coli for our experiments, based on the findings of Taylor et al. who noted similar defects in phagocytosis of both killed E. coli and live bacteria in COPD.8 We do acknowledge that further studies that incorporate pathogens more relevant to COPD, including pneumococcus and haemophilus, would be of interest, but given the common mechanisms involved in bacterial phagocytosis we predict similar findings. The presence of defects in clearance of both bacteria and apoptotic cells suggest that underlying defects that are common to both processes may play a role in this disease. These defects may be highlighted by further investigations of receptors and opsonin families that can mediate phagocytosis of both apoptotic cells and bacteria (including collectins, scavenger receptors, complement and their receptors). Interestingly, we have previously reported that administration of low-dose azithromycin to COPD subjects for 12 weeks improved expression of mannose receptor, a scavenger receptor that is expressed at significantly reduced levels on AM in COPD, and involved in both phagocytosis of apoptotic cells and bacteria.9 We have also shown a trend for improved levels of collectins in the airway of COPD subjects following azithromycin therapy, suggesting that the pro-phagocytic effects of azithromycin could be at least partially linked to changes in this system.9

COPD patients may suffer recurrent exacerbations with a reduction in lung function and worsening of symptoms that may not be recovered in some patients. Moreover, exacerbations are associated with an impaired quality of life, reduced survival and a high health-care expenditure. A recent study10 showed that administration of erythromycin (although not at low dose) for 12 months reduced the rate of exacerbations in COPD. Our findings suggest that this may be at least partially explained by a macrolide-mediated promotion of phagocytosis of bacteria in the airway of these subjects.

The results of this study provide further support for the use of long-term use of low dose azithromycin as an attractive adjunct treatment option for COPD. The data provide additional insights into the biological basis of macrophage dysfunction in COPD. Improved clearance of both apoptotic cells and bacteria in the airway may have a dual effect; reducing the risk of secondary necrosis and release of toxic cell contents that perpetuate inflammation as well as contributing to a reduction in the rate of exacerbations in COPD.

ACKNOWLEDGEMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES

The authors thank Ms Jessica Ahern for her excellent technical assistance. This study was supported by a National Health and Medical Research Council Project Grant and Career Development Award and Practitioner Fellowship.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. REFERENCES
  • 1
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    Direct Link:
  • 2
    Hodge S, Hodge G, Ahern J et al. Smoking alters alveolar macrophage recognition and phagocytic ability: implications in chronic obstructive pulmonary disease. Am. J. Respir. Cell Mol. Biol. 2007; 37: 74855.
  • 3
    Hodge S, Hodge G, Brozyna S et al. Azithromycin increases phagocytosis of apoptotic bronchial epithelial cells by alveolar macrophages. Eur. Respir. J. 2006; 28: 48695.
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    Wilkinson TMA, Patel IS, Wilks M et al. Airway bacterial load and FEV1 decline in patients with chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 2003; 167: 10905.
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    Berenson CS, Garlipp MA, Grove LJ et al. Impaired phagocytosis of nontypeable Haemophilus influenzae by human alveolar macrophages in chronic obstructive pulmonary disease. J. Infect. Dis. 2006; 194: 137584.
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    Hodge S, Hodge G, Jersmann H et al. Azithromycin improves macrophage phagocytic function and expression of mannose receptor in chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 2008; 178: 13948.
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    Seemungal TA, Wilkinson TM, Hurst JR et al. Long-term erythromycin therapy is associated with decreased chronic obstructive pulmonary disease exacerbations. Am. J. Respir. Crit. Care Med. 2008; 178: 113947.
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    Hodge S, Matthews G, Dean MM et al. Therapeutic role for mannose binding lectin in cigarette smoke-induced lung inflammation? Evidence from a murine model. Am. J. Respir. Cell Mol. Biol. 2010; 42: 23542.
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    Patel KB, Xuan D, Tessier PR et al. Comparison of bronchopulmonary pharmacokinetics of clarithromycin and azithromycin. Antimicrob. Agents Chemother. 1996; 40: 23759.
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    Rodvold KA, Gotfried MH, Danziger LH et al. Intrapulmonary steady-state concentrations of clarithromycin and azithromycin in healthy adult volunteers. Antimicrob. Agents Chemother. 1997; 41: 1399402.
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    Hand WL, Boozer RM, King-Thompson NL. Antibiotic uptake by alveolar macrophages of smokers. Antimicrob. Agents Chemother. 1985; 27: 425.