Differential effects of three antibiotics on T helper cell cytokine expression

Auriol C. Williams, Helen F. Galley*, Alison M. Watt and Nigel R. Webster

Academic Unit of Anaesthesia & Intensive Care, Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland, UK


* Corresponding author. Tel: +44-1224-552775; Fax: +44-1224-555766; E-mail: h.f.galley{at}abdn.ac.uk

Received 28 April 2005; returned 13 June 2005; revised 16 June 2005; accepted 20 June 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: To test whether ciprofloxacin, moxifloxacin and clarithromycin affect the expression of the T helper (Th) cell cytokines, interferon-{gamma} and interleukin-4. Quinolone and macrolide antibiotics are routinely used for the treatment of critically ill patients with sepsis. These antibiotics modulate some aspects of immune cell function. Alteration in the profile of Th cell cytokine expression will affect the T helper cell ratio and subsequent immune responses.

Methods: Following ethics committee approval and informed consent, mononuclear cells were isolated from 20 healthy volunteers using single density gradient centrifugation. Cells were incubated with ciprofloxacin (0–100 mg/L), moxifloxacin (0–50 mg/L) or clarithromycin (0–125 mg/L) and stimulated with phorbol myristate acetate and ionomycin. For flow cytometric analysis, cells were stained with antibodies to CD3 and CD4, prior to permeabilization with saponin and intracellular staining with anti-interleukin-4 and anti-interferon-{gamma}.

Results: Both moxifloxacin and ciprofloxacin dose-dependently decreased interferon-{gamma} and interleukin-4 expression by Th cells (both P < 0.0001). Only interleukin-4 expression however, was affected by clarithromycin (P = 0.04). There was no change in the Th1/Th2 ratio for moxifloxacin or ciprofloxacin, but the Th1/Th2 ratio increased with increasing concentrations of clarithromycin, from a median [range] of 5.3 [1.3–16.0] without antibiotic to 9.1 [1.8–18.8] at 125 mg/L (P = 0.017).

Conclusions: Moxifloxacin and ciprofloxacin had pronounced effects on both Th1 and Th2 cytokine expression, without altering Th1/Th2 ratios. However, clarithromycin decreased only interleukin-4 expression such that the Th1/Th2 ratio increased. Since a Th1 profile is considered favourable for resolution of infection, elucidation of immunomodulatory profiles of antibiotics may permit more rational antibiotic choice in future.

Keywords: lymphocytes , interferon-{gamma} , interleukin-4 , quinolones , clarithromycin , immunomodulation , macrolides , T cells


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fluorinated-4-quinolones, including ciprofloxacin and moxifloxacin, are nalidixic acid analogue antibiotics, which exert their bactericidal effect by inhibiting DNA gyrase activity. Ciprofloxacin has been shown previously to have immunomodulatory effects (recently reviewed by Dalhoff and Shalit1), and although moxifloxacin is a relatively new drug, it too has been shown to be immunomodulatory in both animals and humans.2,3 Clarithromycin is a semi-synthetic acid-stable member of the broad-spectrum macrolide family and has been shown to modulate release of several cytokines.4,5

T helper lymphocytes are important for both cell-mediated and humoral immunity.6 The particular type of immune response is determined by the differentiation of precursor naive T helper (Th0) cells into Th1 or Th2 cells, a process which is dependent upon local cytokine concentrations, antigen load and mode of antigen presentation. Each cell subset secretes a particular array of cytokines which further augment the differentiation into that subset.7,8 Th1 cells produce predominantly interferon-{gamma} (IFN-{gamma}) and favour cell-mediated immune responses, while Th2 cells secrete predominantly interleukin-4 (IL-4) and favour humoral immunity.

The effects of ciprofloxacin, moxifloxacin and clarithromycin on intracellular expression of T helper cell cytokines and the ratio of Th1/Th2 subsets have not been investigated. Since the balance of cytokines produced in response to infection will have a marked effect on T helper subset predominance and hence the nature of subsequent immune responses, we investigated whether these antibiotics had modulatory effects on the Th1 cytokine IFN-{gamma}, and the Th2 cytokine IL-4.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Following local research ethics committee approval and informed consent, blood samples were taken into lithium heparin from 20 healthy volunteers (10 men/10 women, aged 21–48 years). Peripheral blood mononuclear cells were isolated by single density gradient centrifugation as previously described9 and adjusted to a concentration of 1 x 106 cells/mL in RPMI 1640 medium with Glutamax-I (Gibco-Invitrogen, Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum (Helena Biosciences, Sunderland, UK) and 10% sodium pyruvate (Gibco-Invitrogen). Cells were incubated in 5 mL flow cytometry tubes for 4 h in a humidified atmosphere of 95% air/5% CO2 at 37°C, with 20 ng/mL (10 µL) phorbol myristate acetate (PMA; Sigma–Aldrich, Gillingham, UK), 1 µmol/mL (10 µL) ionomycin (Sigma–Aldrich) and 0.67 µL/mL (5 µL) monensin (BD Biosciences, Cowley, UK).

Ionomycin and PMA are pharmacological T cell activating agents which mimic signals generated by the T cell receptor (TCR) complex and have the advantage of stimulating T cells of any antigen specificity.10,11 Monensin is a protein transport inhibitor which retains synthesized proteins within the cell, and therefore increases the sensitivity of intracellular cytokine staining.12

Some cells were also exposed to 5, 20, 50 or 100 mg/L ciprofloxacin; 2, 10, 20 or 50 mg/L moxifloxacin, or 5, 20, 50 or 125 mg/L clarithromycin (all 100 µL). Ciprofloxacin and moxifloxacin were a gift from Bayer Plc, Newbury, UK and clarithromycin was a gift from Abbott Laboratories Ltd, Queensborough, UK. During the incubation, tubes were mixed every 30 min to maintain cells in suspension. Appropriate positive and negative controls for flow cytometer calibration and compensation were also included. Cell controls included stimulated and unstimulated cells with every combination of antibody and unstimulated cells with antibiotic. In some experiments, antibiotics were added up to 4 h after cell stimulation.

Isolated mononuclear cells were washed in staining buffer (phosphate-buffered saline containing 1% fetal calf serum and 0.1% sodium azide) and stained for 30 min with allophycocyanin (APC)-conjugated anti-CD4 (all antibodies from Caltag-Med Systems, Towcester, UK) and fluorescein isothiocyanate (FITC)-conjugated anti-CD3 plus IgG control antibodies, before washing in staining buffer and re-suspending in 4% paraformaldehyde. For intracellular staining, cells were permeabilized with 0.1% saponin, washed, and then incubated with phycoerythrin (PE)-conjugated anti-IFN-{gamma} or anti-IL-4 at 4°C for 30 min in the dark before being resuspended in 1% paraformaldehyde.

Cells were analysed using a FACScan flow cytometer (Becton Dickinson) with two lasers (488 and 580 nm) with filter settings for APC at 640 nm, FITC at 515 nm and PE at 585 nm. Following calibration, 10 000 cells were analysed from each tube and analysis gates were set on CD3- and CD4-positive lymphocytes according to both forward and side scatter profiles. IFN-{gamma}+ or IL-4+ cell populations were isolated from the gated T helper lymphocyte populations (CD3+ and CD4+) and the percentage of positive cells was determined. Data were not normally distributed and are therefore expressed as median and range and were statistically analysed using Friedman 1-way ANOVA by ranks (using {chi}2 approximation and correction for ties) to test for difference between related samples, with post-hoc testing using Wilcoxon signed ranks test. Independent data sets were compared using the Mann–Whitney U-test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The number of lymphocytes expressing either intracellular IFN-{gamma} or IL-4 increased significantly when cells were stimulated with ionomycin and PMA compared to unstimulated cells (P < 0.0001, Figure 1a–c). Analysis of variance indicated that moxifloxacin and ciprofloxacin decreased the number of cells expressing both IFN-{gamma} and IL-4 in stimulated cells (all P < 0.0001, Figure 1a and b) in a concentration-dependent manner. Post-hoc analysis showed that the number of cells expressing IFN-{gamma} was lower in those treated with 20, 50 or 100 mg/L ciprofloxacin (P < 0.05, Figure 1a) and the number expressing IL-4 was lower in cells treated with 5, 20, 50 or 100 mg/L ciprofloxacin (P < 0.05, Figure 1a). The number of cells expressing either IFN-{gamma} or IL-4 was significantly lower than in the absence of antibiotic in the cells treated with 2–50 mg/L moxifloxacin (all P < 0.05, Figure 1b).



View larger version (31K):
[in this window]
[in a new window]
 
Figure 1. The effect of ciprofloxacin (a), moxifloxacin (b) and clarithromycin (c) on the percentage of CD4-positive T cells expressing interferon-{gamma} (IFN-{gamma}) and interleukin-4 (IL-4). Box and whisker plots show medians, 25th and 75th percentiles and ranges. The P value on the right of each chart refers to Friedman analysis of variance. B, basal (cells were not stimulated with ionomycin or PMA and were not treated with antibiotic). *P ≤ 0.05 compared with stimulated cells not exposed to antibiotic (post-hoc analysis).

 
Only the percentage of cells expressing IL-4 however, was affected by clarithromycin exposure (P = 0.003, Figure 1c). The number of cells expressing IL-4 was significantly lower than without antibiotic in samples treated with 20, 50 or 125 mg/L clarithromycin (P < 0.05, Figure 1c).

Despite the changes in the numbers of cells expressing IFN-{gamma} and IL-4, there was no significant change in the ratio of the number of IFN-{gamma}-positive to IL-4-positive cells (Th1/Th2 ratio) in cells exposed to moxifloxacin or ciprofloxacin. In clarithromycin-treated cells, however, the Th1/Th2 ratio was significantly higher at the highest concentration of clarithromycin (P = 0.017, Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Effect of antibiotics on the ratio of IFN-{gamma}-positive (Th1) to IL-4-positive (Th2) cells

 
None of the antibiotics had any effect on IL-4 or IFN-{gamma} in the absence of stimulation. When antibiotics were added up to 4 h after ionomycin and PMA stimulation, there was no change in the percentage of cells expressing either IFN-{gamma} or IL-4. Cell viability determined using Trypan Blue exclusion was consistently above 95% after incubation with antibiotics with and without stimulation.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have shown in this study that both ciprofloxacin and moxifloxacin caused a concentration-related decrease in the numbers of stimulated human Th cells expressing IFN-{gamma} and IL-4, without affecting the ratio of the number of IFN-{gamma}-positive (Th1) to IL-4-positive (Th2) cells. Clarithromycin treatment, however, caused a decrease only in the number of cells expressing the Th2 cytokine, IL-4, resulting in a significant increase in the Th1/Th2 ratio.

Although cytokines are essential for the resolution of infection and injury, prolonged production may result in host tissue and organ damage. Several antibiotics, including quinolones and macrolides, have been shown to have modulatory effects on cytokine release.15 Clearly, it is important to define the immunomodulatory activity of antibiotics and other drugs used to treat patients since these properties may have clinical significance, particularly in patients who are immunodeficient or patients on the intensive care unit.

Although bacterial infection is considered to develop Th1-polarized responses, the ability of pathogens to stimulate preferentially either Th1 or Th2 responses depends on several factors including genetic background of host, type of infecting organism, stage of infection and microbial load.68 When antibacterial agents inhibit cytokine generation with variable effects on either Th1- or Th2-cytokines, they may alter or reinforce the Th response induced by microbial infection. It is believed that serious injury results in predominance of the Th2 phenotype as opposed to the Th1 phenotype, rather than generalized Th suppression.13,14 It is also generally considered that Th1 predominance is associated with cell-mediated responses which are most beneficial to recovery. A shift to Th2-dominated phenotypes increases the risk for infection, for example after burn injury.14 Major burns induce a significant shift of cytokine response in the Th2 direction.14 We have also reported that the Th1/Th2 ratio is significantly lower in patients with sepsis than both non-septic controls and healthy subjects, due to both a decrease in Th1 cells and an increase in Th2 cells.10

Polarization of CD4-positive Th cells toward either a Th1 or Th2 response can significantly influence host immunity to pathogens. IFN-{gamma} and IL-4 are the signature cytokines of Th1 and Th2 cells, respectively. At an early stage of development, Th1 and Th2 populations are heterogeneous with a reversible cytokine profile, suggesting that a number of molecular changes are needed for committed profiles of cytokine gene expression. The control of T helper cell differentiation is a function of a number of factors; the most important of which is cytokine environment. There appear to be two ‘master switches’ which control Th cytokine commitment.7,8,15

IL-4 is the principal cytokine driving naive T cells (Th0) to Th2 differentiation. IL-4 is synthesized by differentiating Th2 cells and by fully differentiated Th2 cells.15 The human IL-4 promoter consists of multiple DNA binding motifs, which bind several classes of transcription factor. The GATA-binding protein 3 (GATA-3) is a Th2-specific regulatory factor necessary to direct T helper cells to Th2 differentiation.16,17 We have shown that all three antibiotics decreased IL-4 expression by Th2 cells. The mechanisms of this are not clear. There are no reports of the effect of antibiotics on GATA-3. However, like the macrolides and quinolones, the opiate morphine has wide ranging immunomodulatory effects including effects on Th cytokines, and IFN-{gamma} and IL-4 promoter activities. Recently, morphine was also shown to increase GATA-3 expression in murine splenocytes and may suggest a similar mechanism for antibiotic effects.18 Acute expression of IL-4 by Th2 cells is also regulated by nuclear factor {kappa}B (NF-{kappa}B), nuclear factor of activated T cells (NFAT), activator protein-1 (AP-1) and interferon regulatory factor-1 (IRF1).15 Effects on activation of AP-1 and NFAT by ciprofloxacin have been described,19 although we previously found no effect of ciprofloxacin on either AP-1 or NF-{kappa}B in human endothelial cells.20 Such discrepancies are probably related to cell type, cell stimulant and co-stimulatory conditions. A recent study reported that clarithromycin repressed promoter activity in an AP-1 binding site, but not in an NF-{kappa}B-like site.21 Clarithromycin can inhibit oxidant production by neutrophils22 and both AP-1 and NF-{kappa}B are redox-sensitive transcription factors, which may provide another possible mechanism for the effect of clarithromycin on transcriptional activation. Indeed, other agents with antioxidant properties can inhibit IL-4 via this mechanism.23 The effects of clarithromycin in our study are in agreement with another recent study, which found suppressive effects of macrolides including clarithromycin, on IL-4 but not IFN-{gamma}.24

The key cytokine for promoting Th1 differentiation is IFN-{gamma}.7,8 A number of transcription factors play an important role in regulation of IFN-{gamma} including NFAT, NF-{kappa}B and IRF-1. However, Th1 lineage commitment and IFN-{gamma} expression seem to be controlled by the Th1-restricted transcription factor protein T-bet, which is able to initiate Th1 differentiation while repressing Th2 differentiation.25 The effect of antibiotics on T-bet has not been studied, although a recent study showed that morphine treatment resulted in decreased T-bet mRNA levels.18 However, it is not clear whether this is a consequence of an increase in GATA-3 expression or a direct effect of morphine acting on the signalling pathways that regulated T-bet expression.

The choice of concentration in the studies of antibiotic effects is crucial in terms of clinical relevance. Both moxifloxacin and ciprofloxacin are concentrated in cells, so it is important to study a range of concentrations.26,27 Although serum levels of clarithromycin are lower than the level at which we found an effect on Th ratios, it is important to note that concentration of this drug in peripheral leucocytes can reach around 50 mg/L and up to 400 mg/L in alveolar macrophages.28

In summary, we have shown that both quinolone (ciprofloxacin and moxifloxacin) and macrolide (clarithromycin) antibiotics have marked effects on Th cell cytokines, with implications for immune responses and recovery after severe infection. The mechanisms of such effects are not known, but since the antibiotics only exert their effects after cell stimulation, recently identified so-called ‘master switches’ for Th cell cytokine control may represent targets for future investigation of antibiotic action.


    Acknowledgements
 
We are grateful for financial support from Bayer PLC (PhD studentship to A. C. W.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1. Dalhoff A, Shalit I. Immunomodulatory effects of quinolones. Lancet Infect Dis 2003; 3: 359–71.[CrossRef][ISI][Medline]

2. Shalit I, Kletter Y, Halperin D. Immunomodulatory effects of moxifloxacin in comparison to ciprofloxacin and G-CSF in murine model of cyclophosphamide-induced leukopenia. Eur J Haematol 2001; 66: 287–96.[CrossRef][ISI][Medline]

3. Williams AC, Galley HF, Webster NR. The effect of moxifloxacin on release of interleukin-8 from human neutrophils. Br J Anaesth 2001; 87: 671P.

4. Morikawa K, Watabe H, Araake M et al. Modulatory effect of antibiotics on cytokine production by human monocytes in vitro. Antimicrob Agents Chemother 1996; 40: 1366–70.[Abstract]

5. Kikuchi T, Hagiwara K, Honda Y et al. Clarithromycin suppresses lipopolysaccharide-induced interleukin-8 production by human monocytes through AP-1 and NF-{kappa}B transcription factors. J Antimicrob Chemother 2002; 49: 745–55.[Abstract/Free Full Text]

6. Mosmann TR, Sad S. The expanding universe of T cell subsets: Th1, Th2 and more. Immunol Today 1996; 17: 138–46.[CrossRef][ISI][Medline]

7. O'Garra A, Arai N. The molecular basis of T helper 1 and T helper 2 cell differentiation. Trends Cell Biol 2000; 10: 542–50.[CrossRef][ISI][Medline]

8. Rengarajan J, Szabo SJ, Glimcher LH. Transcriptional regulation of Th1/Th2 polarization. Immunol Today 2000; 21: 479–83.[CrossRef][ISI][Medline]

9. Goode HF, Webster NR, Howdle PD et al. Nitric oxide production by human peripheral blood polymorphonuclear leucocytes. Clin Sci 1994; 86: 411–5.[ISI][Medline]

10. Ferguson NR, Galley HF, Webster NR. T helper cell subset ratios in patients with severe sepsis. Intensive Care Med 1999; 25: 106–9.[CrossRef][ISI][Medline]

11. LeCras AE, Galley HF, Webster NR. Spinal but not general anesthesia increases the ratio of T helper 1 to T helper 2 cell subsets in patients undergoing transurethral resection of the prostate. Anesth Analg 1998; 87: 1421–5.[Abstract]

12. O'Neil-Andersen NJ, Lawrence DA. Differential modulation of surface and intracellular protein expression by T cells after stimulation in the presence of monensin or brefeldin A. Clin Diagn Lab Immunol 2002; 9: 243–50.[Abstract/Free Full Text]

13. O'Sullivan ST, Lederer JA, Horgan A et al. Major injury leads to predominance of the T helper-2 lymphocyte phenotype and diminished interleukin-12 production associated with decreased resistance to infection. Ann Surg 1995; 222: 482–90.[ISI][Medline]

14. Guo Z, Kavanagh E, Zang Y et al. Burn injury promotes antigen-driven Th2-type responses in vivo. J Immunol 2003; 15: 3983–90.

15. Li-Weber M, Krammer PH. Regulation of IL-4 gene expression by T cells and therapeutic perspectives. Nat Rev Immunol 2003; 3: 534–43.[CrossRef][ISI][Medline]

16. Zheng W, Flavell RA. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 1997; 89, 587–96.[CrossRef][ISI][Medline]

17. Ouyang W, Ranganath SH, Weindel K et al. Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent mechanism. Immunity 1998; 9: 745–55.[CrossRef][ISI][Medline]

18. Roy S, Wang J, Gupta S et al. Chronic morphine treatment differentiates T helper cells to Th2 effector cells by modulating transcription factors GATA 3 and T-bet. J Neuroimmunol 2004; 147: 78–81.[CrossRef][ISI][Medline]

19. Riesbeck K. Immunomodulating activity of quinolones: review. J Chemother 2002; 14: 3–12.[ISI][Medline]

20. Galley HF, Dhillon JK, Paterson RL et al. Effect of ciprofloxacin on the activation of the transcription factors NF-{kappa}B, AP-1 and NF-IL-6, and interleukin-6 and interleukin-8 mRNA expression in a human endothelial cell line. Clin Sci 2000; 99: 405–10.[CrossRef][ISI][Medline]

21. Abe S, Nakamura H, Inoue S et al. Interleukin-8 gene repression by clarithromycin is mediated by the activator protein-1 binding site in human bronchial epithelial cells. Am J Respir Cell Mol Biol 2000; 22: 51–60.[Abstract/Free Full Text]

22. Abdelghaffar HM, Vazifeh D, Labro MT. Erythromycin A-derived macrolides modify the functional activities of human neutrophils by altering the phospholipase D-phosphatidate phosphohydrolase transduction pathway: L-cladinose is involved both in alterations of neutrophil functions and modulation of this transductional pathway. J Immunol 1997; 159: 3995–4005.[Abstract]

23. Li-Weber M, Giaisi M, Treiber MK et al. Vitamin E inhibits IL-4 gene expression in peripheral blood T cells. Eur J Immunol 2002; 32: 2401–8.[CrossRef][ISI][Medline]

24. Morikawa K, Zhang J, Nonaka M et al. Modulatory effect of macrolide antibiotics on the Th1- and Th2-type cytokine production. Int J Antimicrob Agents 2002; 19: 53–9.[CrossRef][ISI][Medline]

25. Szabo SJ, Kim ST, Costa GL et al. A novel transcription factor, T-bet, directs Th1 lineage commitment. Cell 2000; 100: 655–69.[CrossRef][ISI][Medline]

26. Garcia I, Pascual A, Salvador J et al. Effect of paclitaxel alone or in combination on the intracellular penetration and activity of quinolones in human neutrophils. J Antimicrob Chemother 1996; 38: 859–63.[Abstract]

27. Pascual A, García I, Ballesta S et al. Uptake and intracellular activity of moxifloxacin in human neutrophils and tissue-cultured epithelial cells. J Antimicrob Chemother 1999; 43: 12–5.

28. 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: 1399–402.[Abstract]





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
56/3/502    most recent
dki251v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Williams, A. C.
Articles by Webster, N. R.
PubMed
PubMed Citation
Articles by Williams, A. C.
Articles by Webster, N. R.