Intravenous infusion of erythromycin inhibits CXC chemokine production, but augments neutrophil degranulation in whole blood stimulated with Streptococcus pneumoniae

Marc J. Schultza,b,*, Peter Speelmanc, C. Erik Hackd, Wim A. Buurmane, Sander J. H. van Deventera and Tom van der Polla,c

a Laboratory of Experimental Internal Medicine, b Department of Intensive Care and c Department of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, University of Amsterdam, Amsterdam; d Department of Pathophysiology of Plasma Proteins, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam; e University of Maastricht: Department of Surgery, Maastricht, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Macrolides may influence the inflammatory response to an infection by mechanisms that are unrelated to their antimicrobial effect. Indeed, erythromycin and other macrolides inhibit cytokine production and induce degranulation of neutrophils in vitro. CXC chemokines are small chemotactic cytokines that specifically influence neutrophil functions. To determine the effect of a clinically relevant dose of erythromycin on the production of CXC chemokines and neutrophil degranulation, six healthy humans received a 30 min iv infusion of erythromycin (1000 mg). Whole blood obtained before and at various times after the infusion was stimulated ex vivo with heat-killed Streptococcus pneumoniae. Ex vivo production of the CXC chemokines interleukin 8 (IL-8) and epithelial cell-derived neutrophil attractant 78 (ENA-78), in whole blood obtained after erythromycin infusion, was lower than that in blood drawn before erythromycin infusion (maximum inhibition post-infusion: 32.9 ± 6.5% and 35.2 ± 12.6% decrease in production, respectively, expressed as percentage change relative to production before infusion of erythromycin, both P < 0.05). In contrast, infusion of erythromycin was associated with an enhanced capacity of whole blood to release the neutrophil degranulation products bactericidal/permeability increasing protein (BPI), human neutrophil elastase (HNE) and human lactoferrin (HLF) upon stimulation with S. pneumoniae. Effects of erythromycin were greatest 4 h after infusion was stopped, when BPI, HNE and HLF concentrations were increased by +107.6 ± 33.5%, +134.7 ± 34.8% and +205.9 ± 55.9 %, respectively (expressed as percentage change relative to production before infusion of erythromycin) (all P < 0.05). These results indicate the ability of erythromycin to reduce CXC chemokine production and to enhance neutrophil degranulation in human blood.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The inflammatory response elicited in the host as a result of bacterial infection plays an essential role in the early defence against the invading organism.13 Neutrophils are of paramount importance in this initial reaction.4 They are attracted to the site of infection by a number of complex processes, including chemotaxis towards a chemotactic gradient produced by locally synthesized chemoattractants. Chemokines, an expanding family of small cytokines, have an important role in the chemotaxis of several leucocyte subsets.57 The CXC chemokines act specifically on neutrophils and have been implicated as pivotal mediators of host defence against bacterial infection. Indeed, high concentrations of the prototypic CXC chemokine interleukin 8 (IL-8) have been found in bronchoalveolar lavage fluids and pleural empyema in patients with pneumonia.8,9 Furthermore, passive immunization against the CXC chemokine macrophage inflammatory protein 2 (MIP-2) renders mice susceptible to Klebsiella pneumoniae pneumonia10 and, conversely, mice over-expressing the mouse IL-8 homologue KC are relatively resistant to this infection.11 Once neutrophils have reached the site of infection, degranulation of activated neutrophils contributes significantly to antimicrobial defence.4

Macrolide antibiotics are used in the treatment of infections caused by many different pathogens. There is evidence that macrolides can affect the host response to infection by mechanisms that are unrelated to their antimicrobial properties. Macrolides influence cytokine production induced by endotoxin1214 or Gram-positive bacteria.15 One study reported that erythromycin inhibited IL-8 production in Pseudomonas-stimulated neutrophils but not in alveolar macrophages.16 The effect of macrolides on chemokine production induced by Gram-positive stimuli is unknown. In addition, macrolides can influence the function of phagocytes in vitro, including the degranulation of neutrophils. In particular, several 14-member ring macrolides enhance neutrophil exocytosis directly.1720 The effect of macrolides on neutrophil degranulation when administered to humans in vivo is unknown.

In the present study we sought to determine the effect of an intravenous infusion of a clinically relevant dose of erythromycin on the capacity of whole blood to produce CXC chemokines [IL-8 and epithelial cell-derived neutrophil attractant 78 (ENA-78)] upon stimulation ex vivo with heat-killed Streptococcus pneumoniae (HKSP). We also determined the effect of erythromycin infusion on neutrophil degranulation ex vivo induced by HKSP, by measuring release of the contents of azurophilic neutrophil granules [bactericidal/permeability-increasing protein (BPI) and human neutrophil elastase (HNE)] and specific granules [human lactoferrin (HLF)].


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reagents and bacteria

Erythromycin was purchased from Abbott (Amstelveen, The Netherlands). Heat-killed S. pneumoniae (HKSP) obtained from a clinical isolate (serotype D9) were cultured overnight in 1 L Todd–Hewitt broth (Difco, Detroit, MI, USA) in 5% CO2 at 37°C, harvested by centrifugation, washed twice in pyrogen-free 0.15 M NaCl, resuspended in 10 mL 0.15 M NaCl, and heat-inactivated for 60 min at 80°C. A sample of 0.5 mL on a blood agar plate did not produce any bacterial growth.

Design and whole blood stimulation

Chemokine production.
Chemokine production was studied as described before.15,2123 Briefly, blood was drawn using a sterile collection system consisting of a butterfly needle connected to a syringe (Becton Dickinson, Rutherford, NJ, USA). Coagulation was prevented using endotoxin-free heparin (Leo Pharmaceutical Products BV, Weesp, The Netherlands) (10 U/mL blood, final concentration). Whole blood, diluted in sterile RPMI-1640 (GibcoBRL Life Technologies, Paisley, UK), was stimulated with HKSP (amounts equivalent to 106 or 107 cfu/mL, final concentration) in sterile polypropylene tubes (Becton Dickinson). For measurements of chemokine production, whole blood was diluted with an equal volume of RPMI. For these experiments, polypropylene tubes were filled with 0.75 mL of RPMI 1640 with the appropriate concentrations of HKSP and erythromycin, after which 0.75 mL of heparinized blood was added. Tube contents were mixed gently and incubated for 16 h at 37°C. Plasma was then prepared by centrifugation and stored at –20°C until assays were performed.

In a first series of in vitro experiments, blood was obtained from six healthy donors to determine the capacity of HKSP to produce chemokines. Six healthy subjects, aged 32 ± 2 years (mean ± S.E.M.) received a 30 min iv infusion of erythromycin (1000 mg in 250 mL saline); blood was collected directly before the infusion, immediately after the infusion and 1, 2 and 4 h later. All samples were handled identically.

Neutrophil degranulation.
Neutrophil degranulation was studied as described before.2426 Blood samples were handled in the same way as those for chemokine measurements, except that whole blood was diluted 1:5 in sterile RPMI and stimulated with HKSP for 2 h.2426

Assays

Chemokines were measured by specific enzyme-linked immunosorbent assays (ELISAs) according to the instructions of the manufacturers of the kits, namely Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB; Amsterdam, The Netherlands) for IL-8 and R&D systems (Minneapolis, MN, USA) for ENA-78. The lower limits of detection were 1 and 15.6 pg/mL for IL-8 and ENA-78, respectively.

BPI concentrations were measured by ELISA, as described previously, using monoclonal antibody 4E3 (specific for human BPI) as coating antibody, biotinylated polyclonal rabbit anti-human BPI IgG as detecting antibody, and recombinant human BPI as standard.24 The lower limit of detection of the assay was 200 pg/mL. Concentrations of HNE were determined using a sandwich ELISA. IgG was purified from serum obtained from a rabbit hyperimmunized with human elastase (Elastin Products, Pacific, MO, USA), by protein A affinity chromatography. Immuno Maxisorp plates (Nunc, Roskilde, Denmark) were coated overnight at room temperature with this IgG fraction at 1.5 mg/L. The plates were washed with 0.2 M PBS, 0.05% Tween 20, incubated with 2% (v/v) milk in PBS as a blocking step, and washed again. HNE standard (Elastin Products) and test-samples were diluted in high-performance ELISA buffer (CLB) and incubated for 1 h at room temperature. After four washes, the wells were incubated with biotinylated rabbit anti-human elastase IgG at approximately 1 mg/L (CLB) for 1 h. Bound elastase was detected with peroxidase-conjugated streptavidin (CLB) and ortho-phenylenediamine as the substrate. The lower detection limit of the assay was 400 pg/mL. Concentrations of HLF were determined with a sandwich ELISA, identical to those described above, except that plates were coated with the IgG fraction of polyclonal rabbit anti-human HLF (1 mg/L) (CLB), and that biotinylated rabbit anti-human HLF IgG (±1 mg/L) (CLB) was used to detect bound HLF. Purified HLF (Sigma, St Louis, MO, USA) was used as standard. The lower detection limit of the assay was 1 ng/mL.

Statistical analysis

All values are given as means ± S.E.M. Two sample comparisons were done using the paired Wilcoxon test. Serial data were analysed by one-way analysis of variance (ANOVA). P < 0.05 was considered statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
CXC chemokine induction in whole blood by HKSP

Incubation of whole blood without HKSP did not result in detectable chemokine production. Incubation of whole blood with HKSP was associated with concentration- and time-dependent production of IL-8 and ENA-78. IL-8 and ENA-78 were detectable after 4 h incubation, and reached high concentrations after 16 h in stimulations with 106 or 107 cfu/mL HKSP (P < 0.05) (Table IGo). For further experiments, 16 h incubations with HKSP 107 cfu/mL were conducted.


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Table I. Concentration-dependent release of interleukin 8 (IL-8) and epithelial cell-derived neutrophil attractant 78 (ENA-78) in whole blood stimulated in vitro with Streptococcus pneumoniae
 
Induction of BPI, HNE and HLF release in whole blood by HKSP

A 2 h incubation of whole blood with 107 cfu/mL HKSP was associated with a mean 30-fold increase in HNE, a 13-fold increase in BPI and an 18-fold increase in HLF release (all P < 0.05) (Table IIGo). Incubation with HKSP 106 cfu/mL led to a mean 19-fold increase in HNE, an eight-fold increase in BPI and a seven-fold increase in HLF release, compared with control levels (all P < 0.05). Based on these data, further studies were performed with HKSP 107 cfu/mL.


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Table II. Concentration-dependent release of human neutrophil elastase (HNE), bactericidal/permeability increasing protein (BPI) and human lactoferrin (HLF) in whole blood stimulated in vitro with Streptococcus pneumoniae
 
Effect of erythromycin infusion on CXC chemokine production ex vivo

Six healthy subjects were infused with erythromycin 1000 mg (a dose given to patients with severe infections), and the capacity of whole blood drawn before and at various times after the erythromycin infusion to produce CXC chemokines after stimulation with HKSP ex vivo was determined. In these experiments, erythromycin infusion inhibited HKSP-induced production of IL-8 and ENA-78 (one way ANOVA, P < 0.05; Figure 1Go). Inhibition was maximal immediately after infusion of erythromycin for ENA-78 (35.2 ± 12.6% decrease relative to production before erythromycin infusion; P < 0.05), while inhibition of IL-8 was maximal 1 h after the end of infusion (32.9 ± 6.5% decrease; P < 0.05). Erythromycin infusion did not affect leucocyte counts or differentials. Consequently, expression of chemokine levels corrected for the number of neutrophils or monocytes yielded similar results (data not shown).



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Figure 1. Erythromycin infusion inhibits the production of (a) IL-8 and (b) ENA-78. Six healthy volunteers received a 30 min iv infusion of erythromycin 1000 mg in 250 mL of 0.9% NaCl. Blood was collected directly before and after the end of infusion, and after 1, 2 and 4 h. Whole blood diluted 1:1 in sterile RPMI 1640 was stimulated for 16 h at 37°C with HKSP (107 cfu/mL). Values are expressed as percentage change relative to production before infusion of erythromycin (mean ± S.E.M.). t = 0 corresponds to the end of the erythromycin infusion. *P < 0.05 compared with the value obtained after stimulation before erythromycin infusion. Concentrations after stimulation before erythromycin infusion were 104 ± 14 ng/mL (IL-8) and 18 ± 10 ng/mL (ENA-78).

 
Effects of erythromycin on neutrophil degranulation in whole blood ex vivo

BPI, HNE and HLF release increased after infusion of erythromycin (one-way ANOVA, P < 0.05). Non-stimulated neutrophil degranulation did not increase. Effects of erythromycin were maximal 4 h after infusion was discontinued, when BPI concentrations were 107.6 ± 33.5% of those before erythromycin infusion, HNE 134.7 ± 34.8% and HLF 205.9 ± 55.9% (all P < 0.05) (Figure 2Go).



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Figure 2. Erythromycin augments the release of (a) human neutrophil elastase, (b) bactericidal/permeability increasing protein and (c) human lactoferrin. Six healthy subjects received a 30 min iv infusion of erythromycin 1000 mg in 250 mL of 0.9% NaCl. Blood was collected directly before and directly after infusion, and 1, 2 and 4 h after the end of infusion. Whole blood, diluted 1:5 in sterile RPMI 1640, was stimulated for 2 h at 37°C with HKSP (107 cfu/mL). Values after infusion of erythromycin are given as percentage change relative to production before infusion of erythromycin (mean ± S.E.M.). t = 0 corresponds to the end of the erythromycin infusion. *P < 0.05 compared with value obtained after stimulation before erythromycin infusion. Concentrations after stimulation before infusion of erythromycin were 8.9 ± 1.6 µg/106 neutrophils (HNE), 45 ± 13 ng/106 neutrophils (BPI) and 896 ± 166 ng/106 neutrophils (HLF).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Several studies have demonstrated that erythromycin and other erythromycin A-derived macrolides can cause a decline in cytokine production1215 and stimulate neutrophil exocytosis in vitro.1720 We investigated the effect of an intravenous infusion of erythromycin in humans on the ability of peripheral blood leucocytes to produce CXC chemokines and to release proteinases upon stimulation. S. pneumoniae was used as stimulus, since erythromycin is frequently used to treat pneumococcal infections. We chose to use heat-killed bacteria rather than viable pneumococci, to study only direct effects of erythromycin on CXC chemokine production and degranulation while ruling out indirect influences (i.e. those caused by an antimicrobial effect). We used whole blood, rather than isolated peripheral leucocytes, to study the effect of erythromycin on chemokine production and neutrophil degranulation, to avoid non-specific activation of cells caused by the isolation procedure. Whole blood has been validated previously as a physiological in vitro system to investigate cytokine and chemokine production,15,2123 and to study mechanisms regulating neutrophil degranulation.2426 Our main findings were that in vivo exposure of healthy humans to erythromycin causes a decline in the capacity of whole blood to produce the CXC chemokines IL-8 and ENA-78, while augmenting HKSP-induced release of constituents of both azurophilic (BPI, HNE) and specific granules (HLF) of neutrophils.

CXC chemokines are important for neutrophil recruitment to sites of inflammation and infection, including the lung. ENA-78 was originally isolated from A549 cells; these cells were derived from type II pneumocytes, which also secrete IL-8.27 Treatment with either anti-IL-8 or anti-ENA-78 antibodies attenuated neutrophil infiltration in lungs and the associated tissue injury in models of ischaemia–reperfusion injury.28,29 Anti-MIP-2 treatment reduced neutrophil influx and impaired host defence in mice infected with K. pneumoniae.10 In addition, transgenic mice with compartmentalized overexpression of KC in the lung demonstrated increased bacterial clearance and improved survival, in association with enhanced influx of neutrophils to the lung.11 In view of these findings, erythromycin-induced inhibition of CXC chemokine production can be considered an undesired side effect of this antibiotic.

The mechanism by which macrolides induce neutrophil degranulation has been investigated in vitro. It was found that only 14-member ring macrolides, such as erythromycin, roxithromycin and dirithromycin, can induce neutrophil degranulation in a time- and concentration-dependent manner.18,19 Studies on structure–activity relationships have established that the effect of macrolides on neutrophil exocytosis is dependent on the l-cladinose at position 3 of the lactose ring.20 The presence of l-cladinose facilitates stimulation of the phospholipase d-phosphatidate phosphohydrolase transduction pathway, which is essential for neutrophil degranulation.20 Furthermore, intracellular accumulation of macrolides is probably necessary, since experimental conditions that favour macrolide uptake also favour the degranulating effect.18 It is unlikely that intragranular accumulation is a prerequisite for this effect, since macrolides that are trapped within neutrophil granules to the greatest extent (e.g dirithromycin) are not the most effective compounds in eliciting neutrophil exocytosis.18,30

Peak erythromycin concentrations (23.5 ± 0.9 mg/mL) were comparable to those reported elsewhere.31 Interestingly, peak concentrations were measured at the end of infusion of eythromycin, with a maximum effect on chemokine production at the end of infusion and 1 h after infusion of erythromycin, while degranulation intensified thereafter. We do not have a clear explanation for this discrepancy; however, one can conclude from these findings that the underlying mechanisms for the inhibition of CXC chemokine production and augmentation of neutrophil exocytosis are different.

The stimulatory effect of macrolides on neutrophil degranulation is unique: generally they have anti-inflammatory properties. For example, they attenuate the oxidative burst reaction by neutrophils20 and can inhibit the production of cytokines by various cell types stimulated with endotoxin in vitro.12,13 Recently, we found that erythromycin caused a dose-dependent reduction in HKSP-induced tumour necrosis factor-{alpha} and interleukin 6 production in human whole blood in vitro.15 Similarly, iv infusion of erythromycin into healthy subjects was associated with reduced production of these cytokines upon stimulation of whole blood ex vivo.15 The inhibition of CXC chemokine production reported here is in line with these data.

In conclusion, we report here that erythromycin inhibits CXC chemokine production and augments neutrophil degranulation in whole blood obtained from healthy subjects infused with a clinically relevant dose of erythromycin, and stimulated with S. pneumoniae ex vivo. Taken together with earlier findings showing that macrolides can inhibit cytokine production and neutrophil respiratory burst activity, these data exemplify the multiple immunomodulatory effects that erythromycin may have on host defence mechanisms activated soon after the onset of an infection.


    Acknowledgments
 
We thank H.-J. Guchelaar of the Department of Pharmacy for determining erythromycin concentrations. The study was approved by the research and ethics committees of the Academic Medical Center; written informed consent was obtained from all subjects. The study was supported by a grant from the Royal Dutch Academy of Arts and Sciences to T. v. d. P.


    Notes
 
* Correspondence address. Department of Intensive Care, Academic Medical Center C3-326, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Tel: +31-20-5669111; Fax: +31-20-6977192; E-mail: m.j.schultz{at}uva.amc.nl Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 22 November 1999; returned 25 January 2000; revised 14 February 2000; accepted 6 March 2000