a Departments of Biological Sciences and b Microbiology and Infectious Diseases, and c Biofilm Research Group, The University of Calgary, Calgary, Alberta, Canada, T2N 1N4
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Abstract |
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Introduction |
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The antimicrobial activity of the azalide azithromycin appears to be enhanced by the ability of this compound to reach high concentrations in phagocytes.12 Azithromycin has a high affinity for neutrophils, which facilitates its delivery to the site of infection.13 A number of other studies have suggested that macrolides have anti-inflammatory effects, partly because they modulate the production of pro-inflammatory cytokines.14,15 Moreover, recent findings indicate that the anti-inflammatory benefits of tilmicosin, a macrolide used in the treatment of bovine pneumonic pasteurellosis, are associated with its induction of neutrophil apoptosis and concomitant inhibition of local leukotriene B4 (LTB4) release.2
While the clinical effectiveness of azithromycin may result mainly from its favourable pharmacodynamics, it may also promote apoptosis. The aims of this study were (i) to determine the effects of azithromycin on human neutrophil apoptosis, in the presence or absence of a common respiratory pathogen, Streptococcus pneumoniae, and (ii) to assess whether such effects may be associated with altered functional properties of neutrophils, i.e. oxidative metabolism and interleukin-8 (IL-8) synthesis.
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Materials and methods |
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Azithromycin (Pfizer Canada, Montreal, Quebec, Canada), penicillin G (Sigma Diagnostics, St Louis, MO, USA) and erythromycin (Sigma) were dissolved in phosphate-buffered saline (PBS) (pH 7.1) to give a final concentration of 0.05 mg/L in each test. Dexamethasone (Schering Canada, Pointe-Claire, Quebec, Canada) was diluted in PBS (pH 7.1) to give a final concentration of 0.02 mg/L in each test.
Bacteria
S. pneumoniae 14259, a human clinical isolate obtained from Dr R. R. Read, Foothills Hospital, Calgary, Alberta, Canada, was grown on Columbia blood agar (CBA) plates at 37°C, suspended in PBS (pH 7.1) and diluted to 1.5 x 108 bacteria/mL using a 0.5 McFarland standard (Dalynn Laboratory Products, Calgary, Alberta, Canada); it was then serially diluted to determine the concentration of bacteria , and used either live or after sonication (for 4 h on ice; 85% duty cycle, output control setting 4; W-350 Sonifier, Branson Ultrasonics Corporation, Danbury, CT, USA).
Neutrophils
Blood was collected by venipuncture from healthy human volunteers and collected in sodium heparin Vacutainers (Becton Dickinson, Franklin Lakes, NJ, USA). Neutrophils were purified by density centrifugation using Histopaque 1119/1077 (Sigma) (800g, 30 min). The neutrophil layer was removed, washed in Hanks' balanced salt solution (HBSS) (Gibco BRL, Life Technologies, Grand Island, NY, USA), and centrifuged (250g, 10 min). Contaminating erythrocytes were lysed by resuspending the pellet in sterile distilled water for 30 s, and osmolarity was restored by adding 2 x HBSS. After washing in 1 x HBSS, purified cells were resuspended in either HEPES-buffered RPMI 1640 medium (Sigma) supplemented with 0.05 mg/L l- glutamine or Ca2+/Mg2+-free PBS (pH 7.1) (for nitroblue tetrazolium (NBT) assay), to a final concentration of 106 cells/mL. Cells were counted in a haemocytometer and viability was assessed by trypan blue (0.1%) exclusion. PMN purity was assessed by staining with Diff Quick (Baxter Healthcare, Miami, FL, USA) and direct microscopy differential leucocyte counts. Neutrophils were incubated (37°C, 5% CO2) for various times with PBS (pH 7.1) (controls), or with azithromycin, penicillin, erythromycin, dexamethasone or tumour necrosis factor- (TNF-
) (R&D Systems, Minneapolis, MN, USA), with or without live S. pneumoniae or a lysate from S. pneumoniae at a 10:1 bacteria:cells ratio for all assays, excepting a 4:1 ratio for annexin V labelling.
Cfu counts
S. pneumoniae was incubated with PMNs and drugs (37°C, 5% CO2) at a ratio of 10:1 for 2 and 6 h, and plated on CBA plates after serial dilutions. Colonies were counted after 24 h incubation at 37°C.
Annexin V and propidium iodide labelling
Neutrophil apoptosis and necrosis were assessed by double staining with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide using an annexin V Fluos kit (Boehringer, Mannheim, Germany) as previously described.4 Briefly, after 1 h incubation (37°C, 5% CO2) with or without drugs and with or without bacterial lysate, cells were centrifuged (Biofuge A; Heraeus Sepatech, Germany) (200g, 5 min), then washed in PBS (pH 7.1). Cells were centrifuged again, resuspended in FITC-conjugated annexin V and propidium iodide staining solution and incubated for 15 min in the dark at room temperature. Samples were then centrifuged (200g, 5 min), resuspended in PBS (pH 7.1) and observed by fluorescence microscopy (at 450 and 535 nm) (Aristoplan; Leitz, Germany) at 400x magnification and the percentages of apoptotic and necrotic cells were determined.
As a positive control for the annexin V/propidium iodide labelling system, PMNs were incubated with 5 ng/mL (approximately 100 U/mL) recombinant human TNF- in PBS (pH 7.1) with 0.1% bovine serum albumin (BSA) for 1, 2 or 3 h. TNF-
at this concentration is known to induce neutrophil apoptosis in a time-dependent manner.16
Cell death ELISA
Neutrophils (105 cells/mL) were incubated (37°C, 5% CO2) for 6 h with or without drugs and with or without live bacteria and frozen at 70°C. Samples were assayed for apoptosis with a cell death ELISA (Boehringer), which detects the histone region of mono- and oligonucleosomes formed during apoptosis. Plates were read at 405 nm (THERMOmax microplate reader; Molecular Devices, Menlo Park, CA, USA) at intervals for 58 min, starting 1 min after addition of substrate. Results were expressed as the ratio of the sample absorbance to the mean absorbance of the control, as previously.2
Nitroblue tetrazolium assay
Oxidative function of PMNs incubated (37°C, 5% CO2) for 1 h with or without drugs and with or without bacterial lysate was assessed by NBT reduction. Cells were incubated (37°C, 5% CO2) in chamber slides (Nalge Nunc International, Naperville, IL, USA) for 20 min with NBT (Sigma) in the presence or absence of 2 mg/L phorbol 12-myristate 13-acetate (PMA) (Sigma). Cells were fixed with methanol and counterstained with safranin (1%). The percentage of oxidatively active cells with blue formazan crystals in their cytoplasm was counted under oil immersion (1000x) with a light microscope (Carl Zeiss, North York, Ontario, Canada).
Interleukin-8 assay
PMNs (2 x 107 cells/mL) were incubated for 2 h with drugs (37°C, 5% CO2), then washed in PBS (pH 7.1) and incubated (24 h, 37°C, 5% CO2) with or without S. pneumoniae lysate. Cells were then centrifuged (at 800g for 10 min) and the supernatant was collected. IL-8 production was assessed by a Quantikine human IL-8 sandwich enzyme immunoassay (R&D Systems) according to the manufacturer's instructions.
Statistical analysis
Results were expressed as mean ± s.e.m. and compared by one-way analysis of variance (ANOVA) followed by Tukey's test for multiple comparison. P < 0.05 was considered significant.
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Results |
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Viability of purified PMNs incubated in PBS (pH 7.1) or RPMI 1640 was >95%, and cell purity was >98%.
Cfu counts
To verify that the pharmacological compounds used in these experiments maintained their antibacterial properties in the presence of PMNs, S. pneumoniae cfu were counted 2 and 6 h after incubation with the various drugs. After 6 h, but not after 2 h, incubation with PMNs, azithromycin, penicillin and erythromycin significantly decreased S. pneumoniae numbers compared with controls (Figure 1). After 6 h, bacterial numbers in preparations containing azithromycin were significantly lower than those exposed to other antibiotics. Incubation with PMNs and dexamethasone did not affect bacterial numbers.
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PMN apoptosis was first assessed by annexin V/propidium iodide fluorescent labelling. As this method detects early apoptotic events, i.e. the translocation of phosphatidylserine on to the outer plasma membrane leaflet, experiments were carried out after 1 h of incubation. This system allowed apoptotic cells (those labelled with FITCannexin V only) to be distinguished from necrotic cells (those labelled with both FITCannexin V and propidium iodide). TNF- induced PMN apoptosis in a time-dependent fashion (Figure 2
). In the absence of S. pneumoniae lysate, azithromycin induced PMN apoptosis at levels similar to those seen in cells incubated with TNF
for 3 h (Figure 3a
). Cells incubated with penicillin, erythromycin or dexamethasone were not different from controls. Addition of bacterial lysate inhibited the induction of apoptosis by azithromycin (Figure 3b
).
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As the results presented above showed that azithromycin induces apoptosis, additional studies determined the effects of the drugs on oxidative metabolism and IL-8 synthesis, two important parameters of PMN integrity in the context of their antibacterial function. None of the drugs affected oxidative function in resting or stimulated neutrophil populations after 1 h of incubation (Figure 6a), a time at which azithromycin-induced PMN apoptosis was already detectable (Figure 3a
). Similarly, the drugs did not affect oxidative function when incubated with bacterial lysate (Figure 6b
).
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Discussion |
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The present study shows that after 1 h of exposure to azithromycin, apoptosis can be detected in human neutrophils. Recent findings have demonstrated that induction of bovine neutrophil apoptosis by the veterinary macrolide tilmicosin may contribute to its anti-inflammatory benefits in vivo.2 This hypothesis is consistent with the observation that physiological removal of apoptotic neutrophils in the inflamed lung in vivo promotes the resolution of inflammation.10,11 Furthermore, accumulation and necrosis of neutrophils in tissues contribute to the propagation of inflammation in a number of inflammatory diseases including infectious pneumonia, whereas apoptotic neutrophils may be eliminated without spilling pro-inflammatory products in situ.17,18 The clinical significance of azithromycin-induced apoptosis thus warrants further investigation.
Annexin V labelling detects the exposure of phosphatidylserine on the outer leaflet of the cell membrane, an early event in cell death.4,19 Cells that are labelled by annexin V, but not by propidium iodide, are apoptotic. This observation, consistent with previous studies, was validated in the present experimental setting by using TNF- as a positive control.16 Human neutrophils exposed to azithromycin for 1 h in vitro were committed to apoptotic death, while erythromycin, penicillin and dexamethasone did not induce apoptosis. In addition, formation of mono- and oligonucleosomes, a late event in apoptotic cell death, was detected by ELISA after 6 h of incubation with azithromycin. Other studies suggest that erythromycin (as well as roxithromycin, clarithromycin and midecamycin) may also induce apoptosis in neutrophils in vitro.20 The fact that erythromycin-induced apoptosis was not observed in the present study may be explained by the different incubation times and antibiotic concentrations; previous studies assessed induction of neutrophil apoptosis after 24 h of incubation, and used a minimum concentration of 1 mg/L.17 Therefore, our findings suggest that azithromycin induces neutrophil apoptosis more quickly and at lower drug concentrations than erythromycin. Additional studies will help assess whether this is is a result of the well established higher affinity for neutrophils of azithromycin versus erythromycin.12 The anti-inflammatory glucocorticoid dexamethasone has been reported to inhibit neutrophil apoptosis,21 but this effect was not seen in this study. Again, as inhibition of apoptosis in human PMNs was observed previously after 24 h exposure to dexamethasone, the 1 or 6 h incubations used in the present study may not have been sufficient to inhibit apoptosis significantly.21 Nevertheless, the present study demonstrates that azithromycin induces apoptosis in human neutrophils to a greater degree than other drugs. As apoptosis limits the ability of neutrophils to damage surrounding tissues and prevents further amplification of inflammation, additional studies are needed to determine whether this mechanism may contribute to the clinical efficacy of this azalide in the treatment of infectious pneumonia.
The results suggest that the pro-apoptotic effect of azithromycin is abolished when neutrophils are incubated with S. pneumoniae lysate. Levels of necrosis were not different between samples with or without lysate, indicating that the low level of apoptosis was not a result of an increase in necrosis. The mechanisms whereby S. pneumoniae products may reverse or inhibit the apoptotic signal remain unclear. Lipopolysaccharide of Gram-negative bacteria has been shown to inhibit TNF--induced apoptosis.22 It remains to be shown whether Gram-positive bacteria have components that also inhibit apoptosis, and whether this effect may contribute to the difficulty in treating such infections. Further studies are also needed to assess whether this inhibition occurs in the presence of other bacterial species, including Haemophilus influenzae, a respiratory tract pathogen against which azithromycin is very effective.12 Interestingly, a recent report speculated that macrolides may significantly reduce the inflammatory injury associated with the presence of H. influenzae in the lower respiratory tract, via unknown mechanisms.23
Previous studies have suggested that macrolides have anti-inflammatory effects, but the mechanisms underlying this benefit remain unclear.14 A number of experiments have focused on the effects of macrolides on cytokine production.15,2327 Although still the subject of current debate, findings from these studies indicate that erythromycin, roxithromycin and clarithromycin may reduce the production of pro-inflammatory IL-1, IL-6, IL-8 and TNF. The results presented herein indicate that after 2 h of exposure to azithromycin, the capability of a given neutrophil population to release IL-8 remains unchanged, despite the concurrent commitment of some of these cells to undergo programmed cell death. In addition, erythromycin A derivatives, including azithromycin, roxithromycin and clarithromycin, may inhibit the oxidative response of neutrophils in a time- and concentration-dependent fashion.2832 Again, findings from the present study indicate that 1 h of exposure to azithromycin is insufficient to reduce oxidative metabolism in human neutrophils, despite detectable levels of apoptosis in these cells. Taken together, these observations suggest that the effects of azithromycin on both IL-8 production and oxidative metabolism are time- and concentration-dependent.
In summary, azithromycin induces apoptosis in human neutrophils more effectively than erythromycin or penicillin. The oxidative function of resting or stimulated neutrophils and the production of IL-8 may remain unchanged, indicating that these functions are not affected in the early stages of azithromycin-induced apoptosis. The pro-apoptotic properties of azithromycin are inhibited by S. pneumoniae. Future studies will assess whether the induction of neutrophil apoptosis by azithromycin contributes to the clinical efficacy of this azalide in the treatment of lower respiratory tract infections, and whether inhibition of neutrophil apoptosis by S. pneumoniae contributes to the tissue injury caused by this pathogen.
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Acknowledgments |
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Notes |
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* Corresponding author. Department of Biological Sciences, The University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4; Tel: +1-403-220-2817; Fax: +1-403-289-9311; E-mail: aburet{at}acs.ucalgary.ca
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Received 2 September 1999; returned 5 January 2000; revised 21 January 2000; accepted 22 February 2000