Gemifloxacin Inhibits Cytokine Secretion by Lipopolysaccharide

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ORIGINAL ARTICLE 10.1111/j.1198-743X.2004.00824.x

Gemiﬂoxacin inhibits cytokine secretion by lipopolysaccharide stimulated human monocytes at the post-transcriptional level F. Araujo1, T. Slifer1,S.Li1, A. Kuver1, L. Fong1 and J. Remington1,2

1Palo Alto Medical Foundation, Palo Alto and 2Stanford University Medical School, Stanford, California, USA

ABSTRACT The fluroquinolone gemifloxacin was examined for its capacity to modulate secretion of cytokines by human monocytes stimulated with lipopolysaccharide (LPS). Monocytes from six male and two female healthy volunteers were stimulated with LPS, exposed to gemifloxacin and the amounts of secreted IL- 1a, IL-1b, IL-6, IL-10 and TNF-a measured at 3, 6 and 24 h. The results revealed that LPS alone increased secretion of each cytokine significantly. Treatment of the LPS-stimulated monocytes with gemifloxacin resulted in a significant inhibition (p < 0.01) of secretion of each of the cytokines from monocytes of the eight volunteers. Nuclear extracts of the human monocyte cell line, THP-1, were used in the electrophoretic mobility shift assay to determine whether gemifloxacin affects nuclear factor-jB (NF-jB) activation. In addition, RNA from THP-1 cells was used in Northern blots to determine whether inhibition of secretion of IL-1b and TNF-a by gemifloxacin occurred at the transcription or translation level. Whereas LPS induced a rapid increase in NF-jB activation, gemifloxacin alone did not. Gemifloxacin did not affect the kinetics or decrease the extent of activation. Northern blots indicated that the inhibitory activity of gemifloxacin occurred post-transcription. Thus, gemifloxacin may modulate the immune response by altering secretion of cytokines by human monocytes. Although the concentrations of gemifloxacin used were higher than those observed in the serum of human volunteers treated with the dose under clinical development, it should be taken into consideration that concentrations at tissue and intracellular levels may be considerably higher than serum concentrations. Keywords Cytokine secretion; gemifloxacin; human monocytes; lipopolysaccharide Original Submission: 5 December 2002; Revised Submission: 8 April 2003; Accepted: 30 April 2003 Clin Microbiol Infect 2004; 10: 213–219

addition, treatment with clindamycin [2], trovafl- INTRODUCTION oxacin and the fluoroquinolones, ciprofloxacin A number of antibiotics have been found to have and tosufloxacin [1] protected mice from early significant immunomodulatory properties both death following intravenous injection of a lethal in vitro and in animal models in vivo [1,2]. Such dose of LPS. Thus, fluoroquinolones may play a properties may have clinical significance for dual role in infections by having an antimicrobial modulation of the immune response of patients effect and an effect on either the beneficial or the [3–5]. Previous work has shown that the fluoro- detrimental host response per se [4,8,9]. quinolones trovafloxacin and moxifloxacin inhibit An interest in the role of cytokines in the secretion of cytokines significantly, particularly immunopathogenesis of infection [10], together IL-1a and TNF-a, by human monocytes stimula- with previous work demonstrating the capacity of ted in vitro with lipopolysaccharide (LPS) [6,7]. In certain antibiotics to modulate the production of cytokines by human monocytes [6,7,11,12], prompted an examination of the effects of gemi- Corresponding author and reprint requests: J. S. Remington, floxacin on the in-vitro production of cytokines by Head, Department of Immunology and Infectious Diseases, human monocytes stimulated with LPS. Gemi- Research Institute, Palo Alto Medical Foundation, 795 El Camino Real, Palo Alto, California 94301 floxacin is a highly active fluoroquinolone which E-mail: [email protected] exhibits broad-spectrum activity, with particular

2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases 214 Clinical Microbiology and Infection, Volume 10 Number 3, March 2004 potency against penicillin-susceptible and -resist- immunosorbent assay (ELISA) using commercially available ant strains of Streptococcus pneumoniae [13]. The reagents (PharMingen, San Diego, CA, USA). Quantification was performed by means of a standard curve derived by linear MIC90 for 602 isolates of S. pneumoniae was dilution of the cytokine standards included in the respective 0.06 mg ⁄ L, compared to 0.25 mg ⁄ L for trovafl- reagent kit. The detection limit for IL-1a and IL-10 was oxacin, 0.12 mg ⁄ L for grepafloxacin, and 1 mg ⁄ L 8pg⁄ 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 for levofloxacin [14]. The MIC90 range of gemifl- % % oxacin for 80 isolates of Escherichia coli was 0.002– coefficient of variation ( CV) was < 12 in replicate assays from the same sample. Cytokine assays were performed in 128 mg ⁄ L, compared to 0.002–128 mg ⁄ L for ci- quadruplicate, using the supernatant samples or appropriate profloxacin, 0.015–64 mg ⁄ L for levofloxacin, and dilutions of the supernatants, as determined in preliminary 0.004–128 mg ⁄ L for trovafloxacin [15]. When the studies. results revealed that gemifloxacin strongly inhibited secretion of cytokines, the cellular level of this Cellular toxicity assay inhibitory activity was examined by determining Toxicity of gemifloxacin for the purified monocytes was the effect of gemifloxacin on activation of the determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- transcription factor NF-jB and regulation of tetrazolium bromide cytotoxicity assay with a Cell Titer 96 Kit (MTT assay; Promega, Madison, WI, USA). Briefly, cells were levels of cytokine mRNA. 3 plated in quadruplicate wells at 1 · 10 cells ⁄ well. Following incubation for 4 h at 37 °CinaCO2 5% v ⁄ v incubator, gemifloxacin concentrations of 0.001 mg ⁄ L, 0.005 mg ⁄ Lor MATERIALS AND METHODS 0.010 mg ⁄ L were added. After incubation for 20 h, 16 lL of the Reagents dye indicator solution was added. Following an additional 4-h incubation, 100 lL of the solubilisation stop solution was Gemifloxacin was obtained from SmithKline Beecham (Col- added to each well. The results were quantified by ELISA as legeville, PA, USA). Stock solutions and further dilutions were the optical density at 570 nm of cultures exposed to gemifl- made in RPMI 1640 medium (Gibco BRL, Grand Island, NY, oxacin or exposed to the diluent alone. USA). LPS (from Escherichia coli O26:B6) was purchased from Difco (Detroit, MI, USA). Statistics Isolation of monocyte-enriched peripheral blood All values were expressed as means ± two standard devia- mononuclear cells tions. Welch’s modified t-test was used in a computer software package (InStat 2.0; GraphPad Software, San Diego, CA, USA) Blood was obtained from six male and two female healthy to determine, for each donor, whether the differences in volunteers (aged 20–57 years). Written authorised consent was cytokine secretion between LPS-stimulated monocytes treated obtained from each volunteer. Mononuclear cells (PBMC) were or not treated with gemifloxacin were statistically significant cultured as described previously [6,7]. Briefly, cells were (p 0.05). separated on Ficoll-Paque (Pharmacia Biotech, Uppsala, Swe- den) density gradients, washed twice with calcium and magnesium-free phosphate-buffered saline (PBS; Mediatech, Electrophoretic mobility shift assay and Northern blot Herndon, VA, USA), and fractionated by centrifugation over analysis discontinuous Percoll (Pharmacia Biotech) gradients. The To determine whether inhibition of cytokine secretion was an monocyte-enriched cell fraction was collected and washed effect of gemifloxacin on gene transcription, the activation of with PBS free of calcium and magnesium. Thereafter, the cells NF-jB and the steady state cytokine mRNA levels were were resuspended in RPMI 1640 (with 25 mM HEPES, measured. LPS-induced production of several cytokines, inclu- L-glutamine; Mediatech) containing fetal bovine serum (FBS) ding IL-1a and TNF-a, is dependent on NF-jB activation and 10% v ⁄ v (Gibco BRL) at a density of 1 · 106 cells ⁄ mL. translocation into the nucleus [16,17]. Since gemifloxacin Characterisation of the monocytes was done with the Naph- inhibits secretion of multiple cytokines, the potential inhibition thol AS-D Chloroacetate Esterase and a-Naphthyl Acetate of NF-jB activation by gemifloxacin was examined by gel Esterase kit (Sigma, St. Louis, MO, USA). At least 90% of the mobility shift assay. For these studies, the human monocytic cells were identified as monocytes. cell line THP-1 was selected, rather than isolated human peripheral blood monocytes, to reduce the potential for In-vitro cytokine assays individual variation. In addition, initial studies revealed that THP-1 cells secreted cytokines in response to LPS stimulation, Purified monocytes were seeded into 24-well plates (Costar and that this secretion was inhibited by gemifloxacin in a Corporation, Cambridge, MA, USA), at a density of 1 · 106 manner that was qualitatively similar to peripheral blood cells ⁄ mL (1 mL ⁄ well), and incubated in the presence of either monocytes. Thus, THP-1 cells were considered a suitable LPS alone 0.00001 mg ⁄ L, or LPS plus gemifloxacin 0.001 mg ⁄ L, model cell system to study the mechanism of action of 0.005 mg ⁄ L, or 0.010 mg ⁄ L for 3, 6, or 24 h at 37 °CinaCO 5% 2 gemifloxacin. v ⁄ v incubator. At each period, cell-free supernatants were THP-1 cells were seeded in 12-well plates at a density of recovered by centrifugation and stored at ) 20 °C until assayed 1 · 106 cells ⁄ mL (1 mL ⁄ well) and preincubated in the pres- for cytokines. The concentration of interleukin-1a (IL-1a) IL-1b, ence or absence of concentrations of gemifloxacin of IL-6, IL-10, and TNF-a was determined by enzyme-linked

2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 10, 213–219 Araujo et al. Inhibition of cytokine secretion by gemiﬂoxacin 215

0.001 mg ⁄ L, 0.005 mg ⁄ L or 0.01 mg ⁄ L for 16–20 h. The cells RESULTS were then stimulated with 0.001 mg ⁄ L of LPS for 20, 40, 60 or 80 min at 37 °C. Nuclear extracts were prepared from THP-1 Effect of gemifloxacin on cytokine secretion cells as described by Delude et al. [18] with slight modifica- tions. Briefly, cells were collected by centrifugation at 500 g Treatment of monocytes with LPS resulted in a for 4 min at 4 °C and washed twice with cold PBS. There- significant (p < 0.001) increase in accumulation of after, the cells were resuspended in 400 lL of buffer I (10 mMM each of the cytokines in the supernatants of the Tris pH 7.8, 5 mMM MgCl2,10mMM KCl, 0.3 mMM EGTA, 0.3 MM cultured monocytes. For IL-1a, IL-1b, IL-6 and IL- sucrose, 0.5 mMM dithiothreitol, 1 mMM PMSF and P8340 protease inhibitor cocktail (Sigma) 0.625% v/v), and incuba- 10, the increase was noted after incubation for 6 h ted at 4 °C for 15 min. A 25-lL aliquot of Nonidet P40 10% and 24 h, and for TNF-a after incubation for 3 h, v ⁄ v was added and the solution vortexed for 10 s. The lysate 6 h and 24 h (Fig. 1). Treatment of the monocytes was centrifuged at 7200 g for 10 s at 4 °C and the pellet with LPS plus gemifloxacin resulted in inhibition (nuclear fraction) resuspended in 75 lL of buffer II (20 mMM of secretion of each cytokine by monocytes from Tris pH 7.8, 5 mMM MgCl2, 320 mMM KCl, 0.2 mMM EGTA, 0.5 mMM dithiothreitol, 1 mMM PMSF, P8340 2.5% v ⁄ v) and incubated at each of the eight donors. Fig. 1 shows represen- 4 °C for 15 min. Samples were centrifuged at 20 000 g for tative results for IL-1a, IL-1b, IL-6, IL-10 and 15 min at 4 °C and the supernatants (nuclear extracts) stored TNF-a. Statistically significant inhibition (p < 0.01) ) ° at 80 C until analysis. The protein content of the nuclear of secretion of IL-1a, IL-1b, IL-6 and IL-10 extracts was measured with the BCA protein assay (Pierce, Rockford, IL, USA). Samples (4 lg) of protein were incubated occurred after 6 and 24 h, and was most pro- with 1 ng P32-labelled double-stranded oligonucleotide nounced after incubation of the monocytes with (5¢-CAGAGGGGACTTTCCGAGAG-3¢) in binding buffer (gly- LPS and gemifloxacin for 24 h. Significant inhibi- cerol 12% v ⁄ v, 12 mMM HEPES pH 7.9, 0.6 mMM EDTA, 60 mMM tion (p < 0.01) of secretion of TNF-a occurred at KCl, 5 mMM MgCl2, 0.6 mMM dithiothreitol and poly dI-dC 200 lg ⁄ mL) at room temperature for 35 min. Incubation with each time period examined. A dose–response albumin in amounts equivalent to the volume of the nuclear was observed. extracts was included as a negative control. The reaction mixtures were separated by non-denaturing polyacrylamide gel (5% w ⁄ v) electrophoresis in 0.5 · TBE buffer (45 mMM Tris- Effect of gemifloxacin on NF-jB activation borate, 1 mMM EDTA, pH 8.0) and the amount of DNA binding measured by autoradiography. Specificity of binding was Cells incubated with gemifloxacin alone, or in the assessed with a 25-fold molar excess of unlabelled double- absence of LPS, did not induce NF-jB activation stranded oligonucleotide. (Fig. 2). In contrast, addition of LPS alone induced Northern blotting was performed to determine the effect of NF-jB activation in a time-dependent manner, gemifloxacin on the steady state levels of IL-1b and TNF-a mRNA in the LPS-treated THP-1 cells. These cells were with a maximal response observed at or near cultured either alone or in the presence of gemifloxacin 60 min after addition of LPS. The specificity of the 0.005 mg ⁄ L or 0.010 mg ⁄ L in quadruplicate wells of microtitre activation was established with an excess of plates. After culture for 0, 2, 4, 8 and 24 h, the plates were unlabelled competitor. Pre-incubation with gemi- centrifuged and supernatants collected to determine secretion floxacin 0.001 mg ⁄ L, 0.005 mg ⁄ L or 0.010 mg ⁄ L of IL-1b and TNF-a by ELISA as described above. The pelleted cells were collected and total RNA was extracted with a not only failed to reduce activation of NF-jBin Stat)60 kit (Tel.Test, Friendswood, TX, USA) according to the LPS-stimulated cells, but also caused a slight manufacturer’s instruction. Thereafter, total RNA increase in the levels of activation of the (0.015 mg ⁄ L) of each monocyte sample was electrophoresed NF-jB transcription factor. This was observed under denaturing conditions in agarose 1.2% w ⁄ v gels with 2.2 MM formaldehyde, transferred to Nytran membranes (Sch- in four separate experiments. The absence of leicher and Schuell, Keene, NH, USA) and cross-linked by UV inhibition was observed at all intervals measured irradiation. cDNA probes of human IL-1b (30-mer, GenBank from 20 min to 6 h. These results revealed that Accession Number: M10988), TNF-a (27-mer, GenBank Acces- gemifloxacin did not interfere with the LPS- sion Number: K02770) and b-actin (29-mer GenBank Accession j Number: X00351) (R & D Systems, Minneapolis, MN, USA), induced activation of NF- B, and suggested an radiolabelled with c-32P ATP (Promega), were used in the alternative mechanism of action to explain the hybridisation procedure. The membranes hybridised inhibitory effect of gemifloxacin on secretion of with human IL-1b and TNF-a probes were exposed in a cytokines. phosphor-imager cassette (Molecular Dynamics, Sunnyvale, ELISA to determine the effect of gemifloxacin CA, USA) for 18 h, while the membranes hybridised with human b-actin probe were exposed in a phosphor-imager on secretion of IL-1b and TNF-a by LPS-stimula- cassette for 8 h. Hybridisation was visualised by phosphor- ted THP-1 cells revealed that both cytokines were imager (Storm 860; Molecular Dynamics) and the signals secreted following LPS stimulation. Treatment normalised to human b-actin by image densitometry. Signal with gemifloxacin inhibited secretion of TNF-a data were expressed as absorbency units.

2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 10, 213–219 216 Clinical Microbiology and Infection, Volume 10 Number 3, March 2004

0.08 0.5

/mL M only 0.4 0.06 M + LPS 0.3 0.04 M + LPS + 0.001 mg/L Gemi 0.2 0.02 M + LPS + 0.005 mg/L Gemi 0.1

Nanograms IL-1 α M + LPS + 0.01 mg/L Gemi Nanograms of IL-1 β /mL 0 0 3624 3624

8 0.2 2

6 0.15 1.5

4 0.1 1

2 0.05 0.5 Nanograms of IL-6/mL Nanograms of IL-10/mL Nanograms of TNF- α /mL 0 0 0 3624 3624 3624

Hours Hours Hours

Fig. 1. Effect of different concentrations of gemiﬂoxacin (Gemi) on secretion of IL-1a, IL-1b, IL-6, IL-10 and TNF-a by human monocytes (M) stimulated with lipopolysaccharide (LPS). + ¼ p < 0.001 calculated for M only and M + LPS, while * ¼ p < 0.01 calculated for M + LPS and M + LPS + concentration of Gemi.

Time (min.) 20 40 60 80 20 40 60 80 20 40 60 80 60 60 Competitor Gemifloxacin 10 10 10 10 10 LPS (1µg/ml)

NF-k B

Fig. 2. Results of the electrophoretic shift-mobility assay. Lane 13 shows that cells incubated with gemiﬂoxa- cin alone did not induce NF-jB activation. The locations of the unbound labelled oligonucleotide probe and the NF-jB-DNA complex are shown. Gemiﬂoxacin concentra- free probe tions of 0.001, 0.005 and 0.010 mg ⁄ L gave identical results. The results are representative of four separate 1 2 3 4 5 6 7 8 9 10 11 12 13 14 experiments.

2004 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, CMI, 10, 213–219 Araujo et al. Inhibition of cytokine secretion by gemiﬂoxacin 217

Control 0.005 mg/L Gemi 3.5 0.01 mg/L Gemi 3.0

3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 Fig. 3. Effect of two different con- 1.0 centrations of gemiﬂoxacin (Gemi) 0.5 Nanograms of IL β /mL

Nanograms TNF α /mL 0.5 on secretion of TNF-a and IL-1b by LPS-stimulated THP-1 cells. Con- 0.0 0.0 24h trols were THP-1 cells stimulated 0h 2h 4h 8h 0h 2h 4h 8h 24h with LPS only. * ¼ p < 0.01. Time Time

30 Control DISCUSSION

25 0.005 mg/L Gemi The results of this study indicated that gemifloxacin strongly inhibits secretion of IL-1a, IL-1b, 0.01 mg/L Gemi 20 IL-6, IL-10 and TNF-a by healthy monocytes stimulated with LPS at concentrations that are 15 higher than those achieved in healthy volunteers treated with single doses of 20–800 mg [19]. 10 Whereas each of the cytokines examined was TNF- α /mRNA inhibited, the most pronounced effects were 5 noted with IL-1a, IL-1b and TNF-a and occurred after incubation for 6 h and 24 h. This is of 0 interest since, in treated volunteers, the mean 0 24 8 24 terminal phase elimination half-life was Time (h) 7.4 ± 2.0 h [19]. Thus, the inhibitory effect of gemifloxacin on cytokine secretion occurred when Fig. 4. Northern blot analysis of the effect of different high concentrations of the drug were used for a concentrations of gemifloxacin (Gemi) on the expression of TNF-a mRNA in LPS-stimulated THP-1 cells. Controls prolonged period. However, the down-regulatory were THP-1 cells stimulated with LPS only. The mRNA effect of gemifloxacin is of interest, particularly readings were normalised to human b-actin and the for IL-1a since this cytokine is a major pro- signals are expressed as absorbency units. inflammatory cytokine and mediates many com- ponents of the acute-phase response to infectious and non-infectious agents [20,21]. The biological significantly (p < 0.01) at 2 h, 4 h and 8 h, activities of IL-1b are indistinguishable from those whereas secretion of IL-1b was inhibited signifi- of IL-1a [22]. Thus, inhibition of secretion of IL-1b cantly at 4 h, 8 h and 24 h (Fig. 3). was not surprising. TNF-a shares many biological Northern blotting revealed that treatment of properties with IL-1a, although the cell receptors the THP-1 cells with gemifloxacin 0.005 mg ⁄ L for these two cytokines are distinct [3]. In the resulted in a non-significant increase in the levels present study, inhibition of secretion of TNF-a of TNF-a at each time period. However, treat- occurred following incubation of the LPS-stimu- ment with 0.01 mg ⁄ L did not result in any lated monocytes with gemifloxacin 0.001 mg ⁄ L increase above the levels noted in the prepar- for as little as 3 h. For the other cytokines ation from the control cells (Fig. 4). IL-1b mRNA examined, the greatest in-vitro inhibitory effect levels were also increased by treatment of THP-1 occurred with concentrations of 0.005 mg ⁄ L and cells with gemifloxacin 0.005 or 0.01 mg ⁄ L, but 0.01 mg ⁄ L. As mentioned above, these in-vitro the increase was observed only after 2 h (data concentrations are high and may not be attainable not shown). in humans treated with conventional dosing.

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However, this may not rule out an in-vivo effect inhibits cytokine secretion by an entirely different of gemifloxacin on cytokine modulation by mono- mechanism than that reported for erythromycin cytes. and clarithromycin, which suppressed mRNA The inhibitory effect of gemifloxacin appears to levels as well as secretion of IL-6 from bronchial be similar to that observed with trovafloxacin. epithelial cells [28]. Gemifloxacin could interfere This antibiotic, in concentrations achievable in with cytokine synthesis at the level of protein humans, suppressed secretion of each of the translation or the secretion of cytokine into the cytokines examined in the present study [6], medium. Each mechanism could account for the whereas moxifloxacin, an antibiotic structurally inhibition of cytokine secretion. related to trovafloxacin, inhibited secretion of In summary, the results of this study indicate IL-1a by LPS-stimulated monocytes from each of that gemifloxacin is an inhibitor of secretion of ten healthy donors, and secretion of TNF-a in six cytokines by human monocytes stimulated with of ten healthy donors [7]. Secretion of IL-6, IL-10 LPS. The inhibitory effect does not appear to be and IL-12 (p70) were not inhibited significantly by related to suppression of the transcription factor moxifloxacin [7]. These results suggest differences NF-jB or regulation of levels of cytokine mRNA. in immunomodulatory activity among antibiotics Although the inhibition of cytokine secretion by of the same class. gemifloxacin occurred when high concentrations Studies to determine the mechanisms involved of the drug were used for a prolonged period, it in in-vitro inhibition of secretion of cytokines by may have an important effect on the modulation antibiotics have focused mostly on macrolide of the immune system, since the drug concentra- antibiotics [5,9,23]. Erythromycin at a concentration at tissue and intracellular levels may be ) tion of 10 5 M inhibited IL-8-driven NF-jB tran- considerably higher than in serum. scription in vitro by 37% [24]. Moreover, erythromycin inhibited activation of NF-jB and ACKNOWLEDGEMENT AP-1 transcription factors, which are both crucial regulators of IL-8 expression, in both bronchial This work was supported by U.S. Public Health Service Grant epithelial cells [25] and THP-1 cells [26]. NF-jB AI04717. was also shown to be required for optimal CD40- induced production of IL-12 [27]. These observa- REFERENCES tions indicated that the transcription factors j 1. Khan AA, Slifer T, Araujo FG, Suzuki K, Remington JS. NF- B and AP-1 are at the centre of the mechan- Protection against lipopolysaccharide-induced death by ism of action of macrolides on the inflammatory fluoroquinolones. Antimicrob Agents Chemother 2000; 44: process [5]. Thus, it is of interest that the results of 3169–3173. the present study revealed that, although treat- 2. Hirata N, Hiramatsu K, Kishi K, Yamasaki T, Ichimyia T, ment with LPS induced activation of NF-jBina Nasu M. Pretreatment of mice with clindamycin improves survival of endotoxic shock by modulating the release of time-dependent manner, treatment with gemifl- inflammatory cytokines. Antimicrob Agents Chemother 2001; oxacin did not inhibit this activation at any of the 45: 2638–2642. time intervals used in the experiment. In fact, 3. Dinarello CA. Cytokines as endogenous pyrogens. J Infect treatment with gemifloxacin induced a slight Dis 1999; 179: S294–304. 4. Labro MT. Interference of antibacterial agents with pha- increase in the level of activation of the NF-jB gocyte functions. immunomodulation of ‘immuno-fairy factor. The reason for this observation is unclear. tales’? Clin Microbiol Rev 2000; 13: 615–650. Consistent with a post-transcriptional mechan- 5. Culic O, Erakovic V, Parnham MJ. Anti-inflammatory ism of regulation was the observation that mRNA effects of macrolide antibiotics. Eur J Phamacol 2001; 429: levels for TNF-a and IL-1b were not decreased by 209–229. 6. Khan AA, Slifer T, Remington JS. Effect of trovafloxacin on treatment of the THP-1 cells with gemifloxacin. As production of cytokines by human monocytes. Antimicrob was observed in the experiment to examine Agents Chemother 1998; 42: 1713–1717. activation of NF-jB, treatment with gemifloxacin 7. Araujo FG, Slifer T, Remington JS. Effect of moxifloxacin on resulted in an increase in the levels of mRNA for secretion of cytokines by human monocytes stimulated with lipopolysaccharide. Clin Microbiol Infect 2001; 8: 26–30. TNF-a and IL-1b, although, as the ELISA results 8. Stevens DL. Immune modulatory effects of antibiotics. indicated, secretion of these cytokines was inhib- Curr Opin Infect Dis 1996; 9: 165–169. ited by treatment of the THP-1 cells with gemifl- 9. Labro MT, Abdelghaffar H. Immunomodulation by oxacin. These results suggest that gemifloxacin macrolide antibiotics. J Chemother 2001; 13: 3–8.

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