The effect of fosfomycin on neutrophil function

Robert Krausea,*, Sanda Patrutab, Florian Daxböcka,,b, Petra Fladerera and Christoph Wenischa

a Division of Infectious Diseases, Department of Medicine, Karl-Franzens University Graz, Auenbruggerplatz 15, A-8036 Graz, Austria; b Division of Infectious Diseases, Department of Internal Medicine I, University Hospital Vienna, Austria


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fosfomycin (cis-1,2-epoxypropyl phosphonic acid) is a cell wall synthesis-inhibiting antibiotic. We investigated the effect of fosfomycin on several indices of neutrophil function. Neutrophil phagocytosis was analysed by flow cytometry. Cytosolic calcium kinetics were assessed fluorometrically and neutrophil bactericidal ability was assessed by fluorescence microscopy. Intracellular reactive oxygen intermediate (ROI) production was analysed by flow cytometry and extracellular ROI by cytochrome c reductase assay. After fosfomycin incubation, phagocytosis was unaffected as assessed by the FACS assay. Fosfomycin incubation resulted in enhanced bactericidal ability, in increased intracellular calcium concentrations, elevated extracellular ROI production and decreased chemotaxis but it did not affect intracellular ROI production and chemokinesis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Neutrophils are the leucocytes that respond most rapidly to invasion by pathogens. Suppression of phagocyte number or function may result in impaired host defence.13 Impaired neutrophil function is found in various diseases, for example hereditary disorders like chronic granulomatous disease, diabetes mellitus, renal impairment, sepsis and certain physiological conditions, such as in elderly patients and after strenous exercise.46 The effects of antibiotics on the host response has become of increasing interest.710

Fosfomycin (cis-1,2-epoxypropyl phosphonic acid) is a bactericidal cell wall synthesis-inhibiting non-ß-lactam antibiotic. Its bactericidal activity is exerted through inhibition of the enzyme pyruvyl transferase, which is important for the first step in cell wall synthesis. Fosfomycin is a broad- spectrum agent active against Staphylococcus aureus, Staphylococcus epidermidis, enterococci and Escherichia coli.

Fosfomycin has been shown to suppress immunoglobulin secretion by antibody-producing B cells in vitro, but it had no effect on the role of B cells in antigen presentation.11 Fosfomycin suppresses T-cell function via a specific reduction of IL-2 production,12 and affects cytokine production by monocytes.13

In this study we investigated the effect of fosfomycin on neutrophil phagocytosis, intra- and extracellular reactive oxygen intermediate (ROI) production, neutrophil bactericidal ability, intracellular calcium kinetics, chemokinesis and chemotaxis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacteria

Escherichia coli ATCC 25922 was used for the intracellular killing and FACS assays. For the intracellular killing assay E. coli was grown overnight in 3 mL of Columbia broth (Life Technologies, Paisley, UK). After harvesting and washing with Hanks' buffer, 108 E. coli per mL were opsonized by incubation with 1:10 volume of autologous serum for 30 min at 37°C. For the FACS phagocytosis assay a 108 cells/mL suspension was heat-killed and labelled with fluorescein isothiocyanate (FITC) as described previously.14

Antibiotic

Fosfomycin (Biochemie, Kundl, Austria) was diluted with phosphate-buffered saline (PBS, pH 7.4) to final concentrations of 20, 60, 90 and 150 mg/L to correspond to fosfomycin tissue concentrations.15

Neutrophil isolation

Heparinized whole blood (60 mL) was obtained from five healthy volunteers (one female, four male; mean age 27.8 years, range 25–32 years), and the neutrophils isolated.14,16 Ten millilitres of Ficoll-Paque (Pharmacia LKB Biotechnology, Uppsala, Sweden) were overlaid with 10 mL of whole blood anticoagulated with 10 µL of heparin sodium (Liquemin Roche iv, Hoffmann-La Roche, Basel, Switzerland). After 45 min, most of the erythrocytes sedimented into the Ficoll layer. Granulocytes, monocytes, lymphocytes and thrombocytes in the plasma supernatant were separated on a Percoll (Pharmacia) density gradient. After isolation of the neutrophil band, cells were washed twice with buffer and counted under a microscope. The viability of the neutrophil population obtained by this procedure was >95%, as determined by the trypan blue (Sigma, St Louis, MO, USA) exclusion test. Before all functional assays the blood samples were incubated with the respective concentration of fosfomycin for 30 min at 37°C in a shaking water bath and for an additional 30 min in the FACS assay.

Intracellular killing (microscopy assay)

Intracellular killing of opsonized E. coli was performed as described by Moiola et al.17 A 400 µL volume containing 2 x 106 neutrophils was incubated with 2 x 107 E. coli for 30 min at 37°C. Thereafter, 1 mL of ice-cold Hanks' buffer was added, centrifuged for 7 min at 160g, and the supernatant removed. The pellet was stained by the addition of 200 µL acridine orange (1.44 mg/100 mL Hanks' buffer) for 1 min. After addition of 1 mL ice-cold Hanks' buffer, 500 µL of this suspension was centrifuged in a cytocentrifuge for 5 min at 10000 rpm and the supernatant removed. Intracellular killing was evaluated under the fluorescence microscope: the DNA of killed E. coli cells reacts with acridine orange and fluoresces. All tests were performed in duplicate. Data are expressed as the percentage of bacteria killed by neutrophils per total intracellular bacteria.

Phagocytosis and oxidative metabolism (FACS analysis)

Phagocytosis and intracellular ROI production by neutrophils were determined by flow cytometry according to Wenisch et al.5 Phagocytic capacity was assessed by adding 10 µL of pre-cooled FITC-labelled E. coli to 100 µL of heparinized whole blood and incubating for 10 min at 37°C. Thereafter samples were washed twice in PBS pH 7.4. Finally, 2 mL of FACS lysing solution (Becton Dickinson, San Jose, CA, USA) was added. After 20 min the samples were washed again, resuspended in 100 µL of PBS containing propidium iodide (PI) at a concentration of 50 µg/mL for DNA staining and kept on ice until analysis. For analysis of ROI production, blood samples were stimulated with 25 µL of 108 cells/mL E. coli at 37°C. After 10 min 25 µL of the dihydrorhodamine (DHR) solution was added. After 10 min at 37°C, 2 mL of FACS lysing solution was added and incubated for 20 min at room temperature. Thereafter samples were washed with PBS and resuspended with 100 µL of PBS containing PI at a final concentration of 50 µg/mL for DNA staining. Cells were analysed on a standard FACScan flow cytometer (Becton Dickinson). For each measurement, 10000 events were collected. To exclude cell debris and non-phagocytosed bacteria, a live gate was set on PI-stained leucocytes during acquisition in FL2. For analysis of the ROI production, the shift to the right in FL1 (green) was determined. The amount of cleaved substrate was estimated by the mean fluorescence using the statistical option of the FACScan software. Similarly, the amount of phagocytosed bacteria was assessed by a shift in mean fluorescence to the right (FL1). The mean fluorescence of both assays was compared with unstimulated controls. Daily alignment and calibration of the instrument was performed using fluorescence beads (Calibrite, Becton Dickinson). The beads were inserted into the same histogram channel every day. All tests were performed in duplicate.

Oxidative metabolism (cytochrome c reductase assay)

Extracellular ROI production by neutrophils was determined by measuring superoxide dismutase-inhibitable reduction of cytochrome c according to Nauseef et al.16 Neutrophils were resuspended in Hanks' buffer at a concentration of 2 x 106 cells/mL. A 250 µL cell suspension per tube (5 x 105 cells) was preincubated for 5 min at 37°C. Thereafter 10 µL buffer or phorbol myristate in a final concentration of 1.3 x 10–8 mol/L was added at 37°C for 15 min. Cytochrome c (50 µL of 80 µM) and 150 µL Hanks' buffer were added and further incubated for 30 min at 37°C in a shaking water bath. The tubes were centrifuged at 300g for 15 min at 4°C. The supernatant was decanted and read at 550 nm. Data are expressed as nmol cytoC (O2) produced by 5 x 105 cells per 15 min. The calculation was made using the molar extinction coefficient of 21.1 x 103/mol/cm. All tests were performed in duplicate.

Intracellular Ca2+ concentrations (fluorometer assay)

The basal levels of intracellular calcium ([Ca2+]i) in neutrophils were measured with Fura-2 AM, using a Perkin-Elmer fluorometer, model LS 5B (Perkin-Elmer, Norwalk, CT, USA). The details of this method have been reported by Alexiewicz et al.18 The dissociation constant for Ca2+–Fura-2 was assumed to be 225 mM, and [Ca2+]i was calculated using the Grynkiewicz equation.19 Data are expressed as nmol/L. All tests were performed in duplicate.

Chemokinesis and chemotaxis (agarose assay)

Neutrophils were resuspended in Hanks' buffer at a concentration of 106 cells per 10 µL. N-FMLP (Sigma) (10–6 M) was used as a chemo-attractant. The assay was performed using the under-agarose method.20 The agarose plates were incubated for 100 min at 37°C, then the cells were fixed with methanol and paraformaldehyde and stained with Giemsa. The distance the cells migrated under the agarose was measured under the microscope. All tests were performed in duplicate.

Statistical analysis

Differences were calculated using the Student's t-test. Pearson's correlation was used. All the analyses were two-sided and differences with a P value of <0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Neutrophil phagocytosis and bactericidal ability

Table IGo shows the effect of fosfomycin on neutrophil phagocytosis and bactericidal ability. After fosfomycin incubation neutrophil phagocytosis was unaffected as assessed by FACS analysis. In contrast, fosfomycin incubation resulted in an increased bactericidal ability (+7.4% at a concentration of 150 mg/L; P < 0.05).


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Table I. Effect of fosfomycin on phagocytic ability (FACS assay) and bactericidal ability (microscopy assay)
 
Reactive oxygen production

There was no significant difference in neutrophil intracellular ROI production between control samples and samples after fosfomycin incubation as assessed by flow cytometry. In contrast, extracellular ROI production increased after fosfomycin incubation (12-fold at a concentration of 150 mg/L; P < 0.001) (Table IIGo). After stimulation and fosfomycin incubation extracellular ROI increased in a concentration-dependent manner (+30% at a concentration of 150 mg/L; P = 0.094).


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Table II. Effect of fosfomycin on intracellular (FACS assay) and basal and stimulated extracellular ROI production (cytochrome c reductase assay)
 
Intracellular calcium concentration

The effect of fosfomycin on [Ca2+]i is shown in Table IIIGo. Fosfomycin incubation (150 mg/L) resulted in a significant increase in [Ca2+]i in resting neutrophils (+74% at a concentration of 150 mg/L; P < 0.001). In stimulated neutrophils [Ca2+]i was not significantly increased after fosfomycin incubation (+41%; P = 0.078). Fosfomycin incubation did not result in a significant increase in calcium influx (+37.2%; P = 0.076).


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Table III. Effect of fosfomycin on intracellular calcium concentrations ([Ca2+]i) in resting and stimulated neutrophils and on calcium influx (fluorometer assay)
 
Chemokinesis and chemotaxis

There was no significant difference in chemokinesis between control samples and samples after fosfomycin incubation, but chemotaxis was significantly decreased at a concentration of 150 mg/L (–19%; P = 0.031) (Table IVGo).


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Table IV. Effect of fosfomycin on chemokinesis and chemotaxis (agarose assay)
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Suppression of phagocyte number or function may result in impaired host defence,13 and there are a number of antibiotics with known intracellular accumulation with adverse effects on important neutrophil functions.4 For instance, tetracyclines are seven-fold accumulated within neutrophils and inhibit chemotaxis as well as phagocytosis and oxidative metabolism of neutrophils. Tobramycin does not affect phagocytosis, but impairs neutrophil killing.2123

In neutrophils from patients with chronic granulomatous disease, fosfomycin was shown to improve the killing ability up to normal levels and this effect was not concentration dependent at concentrations between 16 and 200 mg/L.4 This effect was believed to be restricted to neutrophils with bactericidal deficiency. However, after pretreatment of S. aureus for 10 min with 4 x MIC of fosfomycin an enhanced leucocytic killing was observed.24 This corresponds to our study with neutrophils from healthy donors demonstrating a slight enhancement of intracellular bactericidal activity (+7.4%) by fosfomycin. This effect was concentration dependent at concentrations between 20 and 90 mg/L, but there was no significant difference at concentrations between 90 and 150 mg/L. These concentrations are well within therapeutically achievable serum (86–284 mg/L) and tissue concentrations (30–178 mg/g).15,25

Fosfomycin incubation resulted in elevated [Ca2+]i levels in resting neutrophils. An increase in calcium influx into cells by itself is not adequate to cause elevated [Ca2+]i levels because cells are endowed with mechanisms that directly (Ca2+ ATPase) or indirectly (Na+,K+ ATPase) extrude calcium from the cells.26 Impairment of these calcium transport mechanisms contributes to elevated [Ca2+]i in diabetic rats and in platelets of diabetic patients.27,28 Fosfomycin is taken up into cells by ATP-dependent transport mechanisms.4 The subsequently increased intracellular Na+ levels due to the uptake of fosfomycin (1 g fosfomycin contains 14.5 nmol Na+) may cause a decreased Na+,K+ ATPase-dependent Na+–Ca2+ exchange which results in elevated [Ca2+]i levels.28,29 Elevated intracellular calcium in resting neutrophils is associated with impaired phagocytosis.30 Normally, neutrophil activation is dependent on a rapid, phospholipase C-mediated increase in intracellular calcium levels from <50 nmol/L to micromolar concentrations.31 We found elevated cytosolic calcium levels in resting neutrophils without alteration of neutrophil phagocytosis because calcium influx into stimulated neutrophils was unaffected after fosfomycin incubation. This corresponds with the results of a previous study where the phagocytosis of Candida guillermondi by neutrophils was unaffected by fosfomycin.32

IL-6 was shown to ‘prime’ neutrophils, enhancing superoxide production by PMA-treated neutrophils by 54%.33 Similarly, TNF was shown to ‘prime’ neutrophils to kill S. aureus by an oxygen-dependent mechanism.34 In LPS-primed monocytes in vitro fosfomycin suppressed the synthesis of TNF-{alpha}, IL-1, IL-1 receptor antagonist and granulocyte–macrophage colony stimulating factor in a concentration-dependent manner (between 1.6 and 40 mg/L) and increased the synthesis of IL-6 and IL-10.35 In our study, fosfomycin increased extracellular ROI production and had no effect on intracellular ROI production. These findings could be related to an increase in IL-6 and a decrease in TNF during priming (i.e. preincubation with/ without fosfomycin and PMA or bacteria, respectively). In neutrophils, fosfomycin was demonstrated to reduce the production of leukotriene B4 in a concentration-dependent manner, which was related to a reduced expression of IL-8 mRNA in monocytes.13 In this respect, the concentration-dependent reduced chemotaxis in our study could be related to a diminished priming effect of IL-8, which is a potent neutrophil chemo-attractant.

In vivo fosfomycin was shown to lower the peak serum levels of TNF-{alpha} and IL-1 in mice.36 In a gut-derived sepsis caused by Pseudomonas aeruginosa in mice fosfomycin increased survival (from 30% for control mice to 80% for treated mice) despite the lack of a specific antimicrobial effect against P. aeruginosa. Again, this effect was related to a reduction in pro-inflammatory cytokines in serum.37

In summary fosfomycin increased intracellular bactericidal activity, intracellular calcium concentrations and extracellular ROI production. Fosfomycin did not affect neutrophil phagocytosis, intracellular ROI production and chemokinesis, whereas chemotaxis was decreased.


    Notes
 
* Corresponding author. Tel: +43-316-385-2274; Fax: +43-316-385-3062; E-mail: robert.krause{at}kfunigraz.ac.at Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received 5 June 2000; returned 16 August 2000; revised 11 September 2000; accepted 9 October 2000





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