Modification of phagocytosis and cytokine production in peritoneal and splenic murine cells by erythromycin A, azithromycin and josamycin

Elena Ortega*, M. Antonia Escobar, José Juan Gaforio, Ignacio Algarra and Gerardo Alvarez de Cienfuegos

Microbiology Unit, Department of Health Sciences, Faculty of Experimental Sciences, University of Jaén, Paraje Las Lagunillas s/n, 23071 Jaén, Spain

Received 22 July 2003; returned 29 October 2003; revised 12 November 2003; accepted 13 November 2003


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Objectives: The aim of this study was to determine whether pre-incubation of peritoneal or splenic cells with different doses of the macrolides erythromycin A (14-membered ring), azithromycin (15-membered ring) and josamycin (16-membered ring) affects their phagocytic activity or cytokine production.

Methods: Peritoneal and splenic cells from BALB/c mice were pre-incubated with different concentrations of these antibiotics, those similar to serum levels attained with the treatment schedules used in human therapy.

Results: From our observations of phagocytic activity and IL-12 production by peritoneal cells, these macrolide antibiotics seem to act mainly as immunosuppressive agents, although they induce peritoneal cells to increase IL-18 production and splenic cells IL-4 production.

Conclusions: Macrolide antibiotics can interfere with the Th1 cell-amplifying activity of IL-18 in conjunction with IL-12 and, in contrast, may induce a Th2 cell response in an IL-4-dependent manner. These results could improve their therapeutic use especially in immunosuppressed patients.

Keywords: macrolide antibiotics, immunomodulation, IL-12, IL-18, IL-4


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Knowledge of possible influences of antibiotics on the immune response is a priority in the clinical approach to therapy. Awareness of the state of non-specific host defence systems is important for the outcome of antimicrobial chemotherapy, and many experimental data have been gathered on different aspects of antibiotic/phagocytic cell interactions.1

Macrolides—polyoxygenated fungal and bacterial secondary metabolites with large ring structures—are especially effective in the treatment of infections caused by intracellular bacteria and their accumulation can alter host-cell functions.2

The aim of this study was to determine whether pre-incubation of peritoneal or splenic cells with different doses of the macrolides erythromycin A (14-membered ring), azithromycin (15-membered ring) and josamycin (16-membered ring) affects their phagocytic activity or cytokine production.


    Material and methods
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 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
Animals

Male BALB/c mice were obtained from the breeding colony of the University of Jaén. They were maintained under pathogen-free conditions, with free access to food and water. The experiments were approved by the ethics committee for animal experiments at the University of Jaén.

Antibiotics

Erythromycin A (Estedi, Barcelona, Spain), azithromycin (Pfizer, Madrid, Spain) and josamycin (Yamanouchi Pharma, Madrid, Spain) were dissolved in DMSO (Sigma Chemical Co., St Louis, MO, USA) at 10 g/L and further diluted in sterile phosphate-buffered saline (PBS; Sigma) to reach the desired concentrations. These were similar to serum levels attained with the treatment schedules used in human therapy (8, 4, 2 and 1 mg/L for erythromycin A; 3.2, 1.6, 0.8 and 0.4 mg/L for azithromycin; and 12, 6, 3 and 1.5 mg/L for josamycin).

Yeasts

C. albicans (ATCC 2091) was grown in Mueller–Hinton broth (Scharlau, S.L., Barcelona, Spain) at 37°C for 24 h, and adjusted to 40 x 106 cfu/mL in PBS.

The yeasts were inactivated by heating at 100°C for 1 h. In order to obtain opsonized C. albicans, a final concentration of 75% fetal calf serum (Flow Laboratories, Irvine, UK) was used as opsonin. The mixture was incubated at 37°C for 30 min with continuous shaking, washed at 400g and finally re-suspended in 1 mL of PBS.

Peritoneal cells

Peritoneal cell suspensions were obtained from BALB/c mice and adjusted to 1 x 106 viable cells/mL in complete medium RPMI 1640 (Sigma) supplemented with 1% penicillin G/streptomycin solution (Sigma). One millilitre of these cell suspensions was pre-incubated for 24 h in the presence or absence of the drugs (erythromycin A, azithromycin or josamycin), in plastic culture dishes at 37°C in a 5% CO2 incubator. Cells were then washed thoroughly, re-suspended in complete medium and used further in the phagocytosis or cytokine (IL-12 and IL-18) production assays.

Splenic cells

Spleens were removed aseptically from BALB/c mice and homogenized in sterile Hanks balanced salt solution (Sigma). Splenocytes were adjusted to 1 x 106 viable cells/mL in complete medium and pre-incubated in the presence or absence of the different macrolide antibiotics, as described for peritoneal cells. Cells were then washed thoroughly, re-suspended in complete medium and used further in the IL-4 production assay.

Phagocytosis assay

Phagocytosis was analysed according to the method we described previously.3 Briefly, the yeasts were stained with 7-amino-actinomycin D (Sigma), a fluorescent DNA-binding agent, and incubated for 1 h with peritoneal macrophages (10 yeasts per peritoneal cell) at 37°C in a 5% CO2 incubator with continuous shaking. After incubation, the cells were washed three times in PBS (at 100g and 4°C for 10 min) to remove uningested yeasts. Phagocytosis was arrested and the cells were fixed by the addition of cold 2% paraformaldehyde.

Fluorescence was analysed on an EPICS Elite ESP Flow Cytometer (Coulter, Hialeah, FL, USA) within 30 min of cell fixation. At least 1 x 104 events were measured for each sample. We monitored the ratio of peritoneal macrophages that had taken up Candida and the resulting values were expressed as a percentage of cells.

Cytokine production assay

In order to stimulate IL-12 and IL-18 production, peritoneal cells were co-cultured for 24 h with 20 mg/L of lipopolysaccharide from Escherichia coli serotype 026:B6 (Sigma). Splenic cells were co-cultured for 96 h with 10 mg/L of concanavalin A (Sigma) in order to stimulate IL-4 production. After incubation, supernatants were collected and stored at –80°C until quantitatively assayed.

IL-12, IL-18 and IL-4 concentrations were measured by specific enzyme-linked immunosorbent assay (ELISA) with murine anti-cytokine monoclonal antibodies [Quantikine M murine IL-12 p40 (R&D Systems, MN, USA), Mouse IL-18 ELISA kit (Medical and Biological Laboratories, Japan) and Quantikine Mouse IL-4 Immunoassay (R&D Systems), respectively]. The optical density was measured in an ELISA Reader (Whittaker Microplate Reader 2001; Anthos Labtec Instruments, Salzburg, Austria), with a test wavelength of 450 nm and a reference wavelength of 550 nm.

Statistics

All results are shown as means ± S.D. for seven mice, tested in duplicate for each sample. Statistical analysis was carried out using two-way factorial analysis of variance. Results from treated and control cells were compared with the least significant difference test. A P value of <0.05 was considered significant.


    Results and discussion
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 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
All results are shown in Figure 1 (phagocytosis of C. albicans by peritoneal cells) and Figure 2 (IL-12 and IL-18 production by peritoneal cells and IL-4 production by splenic cells). Typical FACS scattergrams of control and treated cells are shown in Figure 1.



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Figure 1. Flow cytometric analysis of peritoneal cells incubated without antibiotic (a); with erythromycin: 8.0 mg/L (b), 4.0 mg/L (c), 2.0 mg/L (d), 1.0 mg/L (e); with azithromycin: 3.2 mg/L (f), 1.6 mg/L (g), 0.8 mg/L (h), 0.4 mg/L (i); with josamycin: 12.0 mg/L (j), 6.0 mg/L (k), 3.0 mg/L (l), 1.5 mg/L (m). The percentage values represent the proportion of phagocytic cells. Mean ± S.D.: (a) = 23.35 ± 1.28; (b) = 17.00 ± 1.13; (c) = 10.98 ± 1.16; (d) = 9.78 ± 0.85; (e) = 7.81 ± 0.53; (f) = 12.03 ± 1.06; (g) = 14.65 ± 1.02; (h) = 17.74 ± 1.02; (i) = 17.45 ± 1.90; (j) = 21.28 ± 1.20; (k) = 21.21 ± 1.47; (l) = 20.81 ± 1.03; (m) = 19.47 ± 0.91. FS, forward light scatter; FL3, fluorescence of 7-amino-actinomycin D.

 


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Figure 2. In vitro effect of macrolides on cytokine production by peritoneal cells (IL-12 and IL-18) and splenic cells (IL-4). None, without antibiotic (black bars); EM, erythromycin A: 8.0 mg/L (bars with vertical lines), 4.0 mg/L (bars with horizontal lines), 2.0 mg/L (bars with diagonal lines), 1.0 mg/L (white bars); AM, azithromycin: 3.2 mg/L (bars with vertical lines), 1.6 mg/L (bars with horizontal lines), 0.8 mg/L (bars with diagonal lines), 0.4 mg/L (white bars); JM, josamycin: 12.0 mg/L (bars with vertical lines), 6.0 mg/L (bars with horizontal lines ), 3.0 mg/L (bars with diagonal lines), 1.5 mg/L (white bars).* P < 0.05.

 
Control cells

When peritoneal cells were pre-incubated with PBS and DMSO (the diluents of the antibiotics) a mean value of 23.35% of the cells were detected as phagocytic (Figure 1a). IL-12, IL-18 and IL-4 production by control cells were 11.10, 19.31 and 64.27 pg/mL, respectively (Figure 2).

Erythromycin-pre-incubated cells

Phagocytosis of C. albicans by peritoneal macrophages pre-incubated with erythromycin (Figure 1b–e) was significantly inhibited compared with the control cells (Figure 1a). Moreover, the lower the concentration of antibiotic employed, the greater the inhibition in phagocytic activity detected. Similarly, only lower concentrations of erythromycin (2 and 1 mg/L) induced inhibition of peritoneal cell IL-12 production (Figure 2).

Other authors have demonstrated previously that at low concentrations certain antibiotics can affect macrophage functions in vitro, whereas higher doses have no effect on these cells. Similar results have been described previously with azlocillin4 and ciprofloxacin.5

Azithromycin-pre-incubated cells

Pre-incubation of peritoneal cells with azithromycin (Figure 1f–i) induced dose-related inhibition of phagocytic activity. IL-12 production by peritoneal cells was also inhibited in a dose-dependent manner after pre-incubation with this macrolide antibiotic (Figure 2).

Josamycin-pre-incubated cells

Cells pre-incubated with josamycin showed: (1) phagocytic activity similar to control cells (Figure 1j–m), except for the minimum dose tested (1.5 mg/L), which induced significant inhibition, and (2) a dose-dependent decreased production of IL-12 (Figure 2).

With regard to peritoneal cell IL-18 production, as well as IL-4 production by splenocytes, the three macrolide antibiotics tested induced a dose-dependent enhancement in this immune parameter (Figure 2).

Macrolides have been described extensively as down-regulators of phagocyte functions,1,6 and Morikawa et al.7 and Sugihara8 have detailed the different immunological effects of 14-, 15- and 16-membered ring macrolides. However, in this study we demonstrate that these macrolide antibiotics induce an increase in peritoneal cell IL-18 production. It is well known that IL-18 has the potential to induce IL-4 or IFN{gamma} production depending on its surrounding cytokine circumstances.9 Thus, without IL-12 it can induce IL-4 production. In this way, these macrolide antibiotics are able to interfere with the Th1 cell-amplifying activity of IL-18 in collaboration with IL-12 and, conversely, may induce a Th2 cell response in an IL-4-dependent manner. This is demonstrated by a significant increase in IL-4 production by splenocytes stimulated with concanavalin A and pre-incubated with the three macrolide antibiotics studied, compared with control cells pre-incubated in the absence of antibiotics. Such effects by macrolide antibiotics, which indicate not only inhibition but also enhancement of immune responses, could improve their therapeutic use in immunosuppressed patients.

Nevertheless, the in vivo effects of these antibiotics on immune functions should be evaluated in clinical studies, as physiological parameters may elicit different responses.10 Understanding the influence of these antibiotics on the immune mechanism is important to establish their correct therapeutic applications.


    Acknowledgements
 
This work was supported by the Plan Andaluz de Investigación (No. CTS 0105).


    Footnotes
 
* Corresponding author. Tel: +34-953-01-20-04; Fax: +34-953-01-21-41; E-mail: eortega{at}ujaen.es Back


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results and discussion
 References
 
1 . Labro, M. T. & Abdelghaffar, H. (2001). Immunomodulation by macrolide antibiotics. Journal of Chemotherapy 13, 3–8.[ISI][Medline]

2 . Noma, T., Hayashi, M., Yoshizawa, I. et al. (1998). A comparative investigation of the restorative effects of roxithromycin on neutrophil activities. International Journal of Immunopharmacology 20, 615–24.[CrossRef][ISI][Medline]

3 . Ortega, E., Algarra, I., Serrano, M. J. et al. (2001). The use of 7-amino-actinomycin D in the analysis of Candida albicans phagocytosis and opsonization. Journal of Immunological Methods 253, 189–93.[CrossRef][ISI][Medline]

4 . Oliver, A. M. & Weir, D. M. (1985). The in vitro effect of three antibiotics on alveolar macrophages. Journal of Clinical Laboratory Immunology 17, 85–9.[ISI][Medline]

5 . Aoki, M., Ono, Y., Kunii, O. et al. (1994). Effect of newer quinolones on the extra- and intra-cellular chemiluminescence response of human polymorphonuclear leucocytes. Journal of Antimicrobial Chemotherapy 34, 383–90.[Abstract]

6 . 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]

7 . Morikawa, K., Oseko, F., Morikawa, S. et al. (1994). Immunomodulatory effects of three macrolides, midecamycin acetate, josamycin, and clarithromycin, on human T-lymphocyte function in vitro. Antimicrobial Agents and Chemotherapy 38, 2643–7.[Abstract]

8 . Sugihara, E. (1997). Effect of macrolide antibiotics on neutrophil function in human peripheral blood. Kansenshogaku Zasshi 71, 329–36.[Medline]

9 . Nakanishi, K., Yoshimoto, T., Tsutsui, H. et al. (2001). Interleukin-18 is a unique cytokine that stimulates both Th1 and Th2 responses depending on its cytokine milieu. Cytokine and Growth Factor Reviews 12, 53–72.[CrossRef][ISI][Medline]

10 . Ortega, E., Escobar, M. A. & Alvarez de Cienfuegos, G. (2002). In vitro and ex vivo effects of erythromycin A, azithromycin and josamycin on the splenic response to specific antigens and mitogens. Journal of Antimicrobial Chemotherapy 49, 1031–4.[Abstract/Free Full Text]





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