ORIGINAL ARTICLE CA 94301, USA. Clin Microbiol Infect 2002; 8: 26 30

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1 ORIGINAL ARTICLE Effect of moxifloxacin on secretion of cytokines by human monocytes stimulated with lipopolysaccharide F. G. Araujo 1, T. L. Slifer 1 and J. S. Remington 2 1 Research Institute, Palo Alto Medical Foundation, and 2 Stanford University Medical School, Palo Alto, CA 94301, USA Objective To determine the effect of moxifloxacin on secretion of cytokines by human monocytes stimulated with lipopolysaccharide (LPS) or Pansorbin. Methods Monocytes obtained from 10 healthy volunteer donors were stimulated with LPS or Pansorbin and exposed or not to different concentrations of the fluoroquinolone antibiotic moxifloxacin. At 3, 6 and 24 h, the amounts of interleukin-1a (IL-1a), IL-1b, IL-6, IL-10, IL-12 (p70) and tumour necrosis factor-a (TNF-a) were measured in the supernatants of the monocyte cultures using enzyme-linked immunosorbent assay. Results Stimulation of human monocytes with either LPS or Pansorbin resulted in a significant increase in secretion of each of the cytokines examined. Treatment of LPSstimulated monocytes with moxifloxacin significantly inhibited (P < 0.01) secretion of IL- 1a by monocytes of each of 10 human donors; the secretion of TNF-a was significantly inhibited (P < 0.01) in monocytes from six of 10 donors. In general there was a trend towards inhibition of secretion of IL-6, IL-10 and IL-12 (p70), but the inhibitory effect was not statistically significant. Secretion of cytokines by Pansorbin-stimulated monocytes was not significantly inhibited by moxifloxacin. Conclusions Moxifloxacin has immunomodulatory activity through its capacity to alter the secretion of IL-1a and TNF-a by human monocytes. Keywords moxifloxacin, cytokines, monocytes Accepted 9 October 2001 Clin Microbiol Infect 2002; 8: INTRODUCTION A number of antibiotics have been found to have significant immunomodulatory properties both in vitro and in vivo in animal models [1 4]. Such properties may have clinical significance for the modulation of the immune response of patients, including those who are immunodeficient, and those who have inflammatory diseases including septic shock. Thus, antibiotics may play a dual role in infections, an antimicrobial effect and an effect on either the beneficial or the detrimental Corresponding author and reprint requests: J. S, Remington, Research Institute, Palo Alto Medical Foundation, Ames Building, 795 El Camino Real, Palo Alto, CA 94301, USA Tel.: þ Fax: þ host response per se [1,5]. The increasing evidence for the role of cytokines and chemokines in the severity of, or even death due to, infection [6 8] indicates the importance of defining the immunomodulatory activity of antibiotics and other drugs used to treat patients with infection. Among the mechanisms by which the immune system combats invasion by pathogens, the role played by multiple cytokines working in concert is becoming increasingly recognized. It has also become apparent that in the infected host, excessive or unrestricted activity of certain cytokines can be detrimental as, for example, occurs in toxic shock syndrome. Potentially harmful effects of T cells and macrophages can be prevented by down-regulatory, antiinflammatory, cytokines [6]. Lack of such control, as in mice with targeted disruption of interleukin-10 (IL-10) and Toxoplasma gondii infection, can result in a cytokine storm that causes tissue injury and ß 2002 Copyright by the European Society of Clinical Microbiology and Infectious Diseases

2 Araujo et al Moxifloxacin and secretion of cytokines by human monocytes 27 death of the host [9,10]. The fluoroquinolone moxifloxacin (1-cyclopropyl-7-(2,8-diazabicyclo [4.3.0]nonane)-6- fluoro-8-methoxy-1,4-di-hydro- 4-oxo-3-quinoline carboxylic acid hydrochloride) has potent antimicrobial activity against both Gram-negative and Gram-positive bacteria [11 13]. Our interest in the role of cytokines in the immunopathogenesis of infection [10,14], as well as our demonstration of the capacity of certain antibiotics to modulate the production of cytokines by human monocytes [3,4,15], prompted us to study the effects of moxifloxacin on the in vitro production of cytokines by purified human peripheral blood monocytes (PBMC). METHODS Reagents Moxifloxacin (Bay , batch ) was obtained from Bayer Corp., West Haven, CT. The drug stock solution and further dilutions were made in RPMI-1640 medium. Lipopolysaccharide (Escherichia coli O26:B6, LPS) was purchased from Difco Laboratories, Detroit, MI. Pansorbin, heatkilled Staphylococcus aureus Cowan strain I (SAC) cells were purchased from Calbiochem-Behring Co., La Jolla, CA. Isolation of monocyte-enriched PBMC Blood was obtained by venepuncture from seven male and three female healthy donor volunteers (ages from 20 to 57 years). Written authorized consent was obtained from each donor. PBMC were obtained and cultured as previously described [15]. Briefly, cells were separated on Ficoll Paque (Pharmacia Biotech AB, Uppsala, Sweden) density gradients, washed twice with calcium- and magnesium-free phosphate-buffered saline (PBS; Mediatech, Inc., Herndon, VA) and fractionated by centrifugation over discontinuous Percoll (Pharmacia Biotech AB, Uppsala, Sweden) gradients. The monocyte-enriched cell fraction was collected, washed with calcium- and magnesium-free PBS, and resuspended in RPMI-1640 (with 25 mm HEPES, L-glutamine; Mediatech, Inc., Herndon, VA) containing 10% fetal bovine serum (FBS; GIBCO BRL Products, Grand Island, NY) at a cell density of cells/ml. Characterization of the monocytes was with the Naphthol AS-D Chloroacetate Esterase and a- Naphthyl Acetate Esterase kit (Sigma Diagnostics, St Louis, MO) to demonstrate the a-naphthyl acetate esterase enzyme within monocytes. Under the conditions of the assay, the enzyme is detected primarily in monocytes, macrophages and histiocytes and is virtually absent in granulocytes [16]. Lymphocytes may occasionally exhibit weak activity [17]. At least 90% of the cells used in each experiment in the present study were identified as monocytes. In vitro cytokine assays Cells were seeded into 24-well plates (Costar Corporation, Cambridge, MA.) at a cell density of cells/ml (1 ml per well) and incubated in the presence of 100 ng/ml of LPS or % (wt/vol.) of Pansorbin with or without 1, 5, or 10 mg/l of moxifloxacin for 3, 6, or 24 h at 37 8Cin a5%co 2 incubator. Cell-free supernatants were recovered by centrifugation and stored at 20 8C until assayed. The concentration of each cytokine [IL-1a, IL-1b, IL-6, IL-10, IL-12 (p70), tumour necrosis factor-a (TNF-a)] was determined by enzyme-linked immunosorbent assay (ELISA) with commercially available reagents (PharMingen, San Diego, CA). The concentration of IL-1b was determined by ELISA with matched antibody pairs and supporting reagents from ENDOGEN (Weburn, MA). Quantification was performed on the basis of a standard curve derived by linear dilution of the cytokine standards included in the respective kits. The detection limit for IL-1a and IL-10 was 8 pg/ml, for IL-6 and TNF-a was 20 pg/ ml, and for IL-1b was 3.9 pg/ml. In reproducibility assays for each cytokine the coefficient of variation (%CV) was <12% in replicate assays from the same sample. Cytokine assays were performed in quadruplicate, using the supernatant samples or appropriate dilutions of the supernatants as determined in preliminary studies. Cellular toxicity assay Toxicity of moxifloxacin for the purified monocytes was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide cytotoxicity assay using Cell Titer 96 Kit (MTT assay, Promega Corp., Madison, WI). Briefly, cells were plated in quadruplicate wells at cells/ well. Following a 4-h incubation at 37 8C ina5% CO 2 incubator, moxifloxacin in various concentrations was added. Four hours before the time-point

3 28 Clinical Microbiology and Infection, Volume 8 Number 1, January 2002 at 24 h, 16 ml of the dye indicator solution was added. Following an additional 4-h incubation, 100 ml of the solubilization/stop solution was added to each well. One hour later, the plates were read for absorbency at 570 nm in an automatic ELISA plate reader. The results were quantified as the optical density at 570 nm of cultures exposed to moxifloxacin or exposed to the diluent alone. Statistics All values were expressed as means standard deviations. Welch s test, which does not assume a similar distribution of variances, was used to determine statistical differences. A P 0.05 was considered statistically significant. RESULTS Figure 1 In vitro secretion of IL-1a (a) and TNF-a (b) by LPS-stimulated or -unstimulated human monocytes at 3, 6 or 24 h. LPS stimulation resulted in a significant increase in the secretion of these cytokines by monocytes of all 10 donors. The figures show the combined results of monocytes from three representative donors. Individual variation in the secretion of each cytokine was observed following stimulation of the monocytes with LPS or Pansorbin. Treatment of monocytes with LPS resulted in a significant (P < 0.001) increase in accumulation of each of the cytokines in the cell culture supernatants. Figure 1(a,b) shows the combined representative results of monocytes from three donors with IL-1a and TNF-a, respectively. Secretion of IL-1a (Figure 1a) increased progressively over the 24-h study period whereas the highest amount of TNF-a (Figure 1b) was observed at 6 h. Secretion of IL-1b followed the same pattern as IL-1a, IL-6 was highest after 6 and 24 h of exposure and IL-4, IL-10 and IL-12 were secreted at each of the three time periods (data not shown). Treatment of monocytes with Pansorbin also resulted in a significant increase in the secretion of each cytokine (data not shown). Exposure of LPS-stimulated monocytes to the three concentrations of moxifloxacin resulted in significant (P < 0.01) inhibition of secretion of IL- 1a in all 10 donors, of IL-1b in one of 10 donors (P < 0.05) and of TNF-a in six of 10 donors (P < 0.05). Secretion of IL-4, IL-6, and IL-12 (P70) by LPS-stimulated monocytes was not significantly inhibited by treatment with any of the concentrations of moxifloxacin. Inhibition of secretion of IL-1a (Figure 2a) occurred 6 and 24 h following treatment with each concentration of moxifloxacin, whereas inhibition of secretion of TNF-a (Figure 2b) occurred mainly after 6 h of exposure to the antibiotic. The highest concentration of moxifloxacin (10 mg/l) significantly (P < 0.05) inhibited TNF-a at each time period. In LPS-treated monocytes of four donors, secretion of TNF-a was not significantly inhibited. Treatment with moxifloxacin of LPS-stimulated monocytes of two of the latter donors resulted in a significant (P < 0.05) increase in the secretion of TNF-a (data not shown). Although the pattern of inhibition of secretion of IL-1b was similar to that observed for IL-1a, the differences in the amounts of IL-1b

4 Araujo et al Moxifloxacin and secretion of cytokines by human monocytes 29 inhibition of secretion of TNF-a in monocytes from only one of 10 individuals. Secretion of IL-a, IL-1b, IL-6, IL-10 and IL-12 (P70) by Pansorbin-stimulated monocytes was not significantly affected by exposure to any of the concentrations of moxifloxacin. Toxicity of moxifloxacin for the cultured monocytes was not noted at any of the concentrations used as determined by the MTT assay. DISCUSSION Figure 2 In vitro secretion of IL-1a (a) and TNF-a (b) by human monocytes exposed for different time periods to LPS alone or to LPS plus different concentrations of moxifloxacin. Combined results of monocytes from the six responsive donors are shown. secreted by LPS-stimulated monocytes and by LPS-stimulated monocytes treated with moxifloxacin reached statistical significance in the monocytes from only one donor (data not shown). Treatment of Pansorbin-stimulated monocytes with moxifloxacin resulted in significant (P < 0.05) Monocytes were employed in our studies because of their critical role in the early cytokine response to a number of pathogens and/or their products which serve as major mediators of the acute inflammatory response [18,19]. Our results revealed that stimulation of monocytes with LPS alone or with Pansorbin alone resulted in a significant increase in accumulation of each of the examined cytokines in the supernatants of the cultured monocytes. Although treatment of LPSstimulated monocytes with moxifloxacin inhibited secretion of most cytokines, the same treatment did not have a demonstrable effect on cytokine secretion by Pansorbin-stimulated monocytes. This observation may be explained by the fact that LPS and Staphylococcus aureus, Cowan strain, use different intracellular pathways to induce cytokine production by human monocytes [20]. It is also possible that moxifloxacin may directly interact with LPS, its receptors and/or its stimulatory pathway to inhibit cytokine secretion. The inhibitory effect of moxifloxacin was most pronounced for IL-1a and TNF-a. In general, there was a trend towards inhibition of secretion of each of the cytokines but statistical significance was achieved only for these two cytokines. This is in contrast to previous observations [15] with another fluoroquinolone, trovafloxacin, which significantly inhibited secretion of IL-1a, IL-1b, IL-6, IL-10, granulocyte macrophage colony-stimulating factor and TNF-a by monocytes stimulated either with LPS or Pansorbin. The reason for this difference is unclear. In humans, a single dose of 50 mg or 800 mg of moxifloxacin results in plasma levels of 0.29 mg/l and 4.73 mg/l, respectively, after h of administration [11]. Concentrations within this range were examined in this study. Moxifloxacin significantly inhibited secretion of TNF-a in LPS-stimulated monocytes of 6 of 10 donors. Inhibition occurred at each of the time

5 30 Clinical Microbiology and Infection, Volume 8 Number 1, January 2002 periods but was most pronounced at the peak of secretion of this cytokine, i.e. 6 h of exposure. The reason for the failure of the drug to significantly inhibit TNF-a secretion by monocytes of the other four donors is unclear. This observation also differs from what was noted with trovafloxacin [15], with the streptogramin synercid [4] and with the macrolide antibiotics azithromycin and clarithromycin [3] which strongly inhibited the secretion of TNF-a in a dose response manner. Similar to results with azithromycin and clarithromycin [3], moxifloxacin did not have a demonstrable inhibitory effect on the secretion of TNF-a by Pansorbin-stimulated monocytes. In contrast, both trovafloxacin [15] and synercid [4] significantly inhibited TNF-a by Pansorbin-stimulated monocytes. Our results indicate that moxifloxacin has an immunomodulatory effect through its capacity to inhibit the secretion of IL-1a and TNF-a by human monocytes. ACKNOWLEDGMENTS This work was supported by U.S. Public Health Service grant AI REFERENCES 1. Stevens DL. Immune modulatory effects of antibiotics. Curr Opin Infect Dis 1996; 9: Labro MT. Immunological effects of macrolides. Curr Opin Infect Dis 1998; 11: Khan AA, Slifen TR, Araujo FG, Remington JS. Effect of clarithromycin and azithromycin on production of cytokines by human monocytes. Int J Antimicrob Agents 1999; 11: Khan AA, Slifen TR, Araujo FG, Remington JS. Effect of Quinupristin/Dalfopristin on Production of Cytokines by Human Monocytes. J Infect Dis 2000; 182: Van Vlem B, Vanhobler R, De Paepe P, Vogelaers D, Ringoir S. Immunomodulating effects of antibiotics: literature review. Infection 1996; 24: Remick DG, Friedland JS. Cytokines in Health and Disease. 2nd. edn. New York: Marcel Dekker, Inc., 1997; Opal SM, Wherry JC, Grint P. Interleukin-10: potential benefits and possible risks in clinical infectious diseases. Clin Infect Dis 1998; 27: Cohen J, Abraham E. Microbiologic findings and correlations with serum tumor necrosis factor-a in patients with severe sepsis and septic shock. J Infect Dis 1999; 180: Gazzinelli RT, Wysocka M, Hieny S. In the absence of endogenous IL-10, mice acutely infected with Toxoplasma gondii succumb to a lethal immune response dependent on CD-4þ T cells and accompanied by overproduction of IL-12, IFN-g and TNFa. J Immunol 1996; 157: Neyer LE, Grunig G, Fort M, Rennick D, Hunter CA. Role of interleukin 10 in regulation of T-cell dependent and T-cell independent mechanisms of resistance to Toxoplasma gondii. Infect Immun 1997; 65: Stass H, Dalhoff A, Kubitza D, Schuhly U. Pharmacokinetics, Safety, and Tolerability of Ascending Single Doses of Moxifloxacin, a New 8-Methoxy Quinolone, Administered to Healthy Subjects. Antimicrob Agents Chemother 1998; 42: Wilson R, Kubin R, Ballin I. Five day moxifloxacin therapy compared with 7-day clarithromycin therapy for the treatment of acute exacerbations of chronic bronchitis. J Antimicrob Chemother 1999; 44: Fogarty C, Grossman C, Williams J, Haverstock D, Church D. Efficacy and Safety of Moxifloxacin vs Clarithromycin for Community-Acquired Pneumonia. Infect Med 1999; 16: Hunter CA, Suzuki Y, Subauste CS, Remington JS. Cells and Cytokines in Resistance to Toxoplasma gondii. In: Gross U, ed. Current Topics in Microbiology and Immunology: Toxoplasma gondii. Basel: Springer- Verlag, 1996; Khan AA, Slifer T, Remington JS. Effect of Trovafloxacin on Production of Cytokines by Human Monocytes. Antimicrob Agents Chemother 1998; 42: Yam LT, Li CY, Crosby WH. Cytochemical Identification of Monocytes and Granulocytes. Am J Clin Pathol 1971; 55: Kaplow LS. Cytochemical Identification of Mononuclear Macrophages. In: Herscowitz HB, et al. eds. Manual of Macrophage Methodology. Basel: Marcel Dekker, Inc., 1981; Braumann H, Gauldie J. The acute phase response. Immunol Today 1994; 15: Astiz M, Saha D, Lustbacter D, Lin R, Rackow E. Monocyte responses to bacterial toxins, expression of cell surface receptors, and release of anti-inflammatory cytokines during sepsis. J Lab Clin Med 1996;128: Rabehi L, Irinopoulou T, Cholley B, Haeffner- Cavaillon N, Carreno MP. Gram-Positive and Gram-NegativeBacteria Do Not Trigger Monocytic Cytokine Production through Similar Intracellular Pathways. Infect Immun 2001; 69: