Received 25 September 2003/Returned for modification 7 December 2003/Accepted 4 March 2004

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2004, p Vol. 48, No /04/$ DOI: /AAC Copyright 2004, American Society for Microbiology. All Rights Reserved. Effects of Fluoroquinolones on the Migration of Human Phagocytes through Chlamydia pneumoniae-infected and Tumor Necrosis Factor Alpha-Stimulated Endothelial Cells Silvia M. Uriarte, 1 Robert E. Molestina, 1 Richard D. Miller, 2 Jorge Bernabo, 3 Alicia Farinati, 4 Kumiko Eiguchi, 4 Julio A. Ramirez, 1 and James T. Summersgill 1,2 * Division of Infectious Diseases, Department of Medicine, 1 and Department of Microbiology and Immunology, 2 University of Louisville School of Medicine, Louisville, Kentucky 40292, and Department of Medicine, School of Medicine, University of Buenos Aires, 3 and Department of Microbiology and Immunology, University of Salvador, 4 Buenos Aires, Argentina Received 25 September 2003/Returned for modification 7 December 2003/Accepted 4 March 2004 The anti-inflammatory activities of three quinolones, levofloxacin, moxifloxacin, and gatifloxacin, were investigated with an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVEC) were seeded in Transwell inserts, treated with serial dilutions of antibiotics, infected with Chlamydia pneumoniae, or stimulated with tumor necrosis factor alpha (TNF- ). Neutrophils or monocytes were also preincubated with serial dilutions of each antibiotic. TEM was assessed by light microscopic examination of the underside of the polycarbonate membrane, and levels of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. In HUVEC infected with C. pneumoniae or stimulated with TNF-, all fluoroquinolones significantly decreased neutrophil and monocyte TEM, compared to antibiotic-free controls. Moxifloxacin and gatifloxacin produced a significant decrease in IL-8 in C. pneumoniae-infected and TNF- -stimulated HUVEC; however, moxifloxacin was the only fluoroquinolone that produced a significant decrease in MCP-1 levels under both conditions. Results from this study indicate similarities in the anti-inflammatory activities of these fluoroquinolones, although no statistically significant decrease in chemokine secretion was observed when levofloxacin was used. Mechanisms of neutrophil and monocyte TEM inhibition by fluoroquinolone antibiotics are unknown but may be partially due to inhibition of IL-8 and MCP-1 production, respectively. It has become increasingly apparent that in addition to their antimicrobial activity, many antibiotics have some anti-inflammatory effect (16, 22, 30, 31, 40, 45, 48). Our laboratory has previously shown heterogeneity in the anti-inflammatory activities of the macrolides azithromycin, roxithromycin, and clarithromycin (46). Treatment with these antibiotics was shown to inhibit neutrophil and monocyte transendothelial migration (TEM) and decreased interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) secretion in endothelial cells (46). The anti-inflammatory activity of macrolide antibiotics observed in vitro may explain the beneficial effects of these antibiotics in patients with respiratory disease characterized by chronic inflammation (32, 44). There is increasing evidence that several quinolones, such as ofloxacin, fleroxacin, sparfloxacin, and levofloxacin, can alter specific functions of neutrophils, especially production of reactive oxygen species (1, 3, 7, 25). The effects of fluoroquinolones on cytokine production by monocytes have been documented (6, 38, 41). At high concentrations, pefloxacin and ciprofloxacin decreased IL-1 production by lipopolysaccharide-stimulated human monocytes, while * Corresponding author. Mailing address: Infectious Diseases Laboratory, Room 311, Instructional Building, 500 South Preston St., University of Louisville, Louisville, KY Phone: (502) Fax: (502) j.summersgill@louisville.edu. Present address: Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY ciprofloxacin and ofloxacin ( 25 mg/liter) decreased production of tumor necrosis factor alpha (TNF- ) production. Trovafloxacin modulates IL-1, IL-1, IL-6, IL-10, granulocyte-macrophage colony-stimulating factor, and TNF- production in monocytes exposed to therapeutic doses of the antibiotic (20). Most in vitro studies have been conducted with quinolones at therapeutic doses (1.56 and 6.25 g/ml) and have indicated no significant effect on the phagocytic functions of leukocytes, chemotaxis, oxidative burst, or phagocytosis (23; S. Fischer, Letter, Antimicrob. Agents Chemother. 45: , 2001). Treatment with ciprofloxacin suppressed lymphocyte proliferation in response to either lipopolysaccharide or concanavalin A in a murine model (18). IL-2 production was significantly enhanced by lymphocytes stimulated with phytohemagglutinin in the presence of temafloxacin and ciprofloxacin (37). Taken together, these data support an anti-inflammatory effect of fluoroquinolones, at least in vitro (23). Until now, all of the data related to the anti-inflammatory effect of fluoroquinolones has been reported on phagocytes; however, to our knowledge experiments performed with human endothelial cells are lacking. Chlamydia pneumoniae is a respiratory pathogen that has also been associated with chronic inflammatory diseases such as asthma (13) and atherosclerosis (24, 26, 34, 43); however, the pathological significance of C. pneumoniae in chronic inflammatory diseases is not well understood. This organism can multiply in vitro in human endothelial cells, aortic smooth muscle cells, and macrophages (8, 9, 10, 19). Since the endo- 2538

2 VOL. 48, 2004 QUINOLONE INHIBITION OF TEM 2539 thelium is central to the recruitment of leukocytes during atherogenesis, studies aimed at the inflammatory activation of endothelial cells by C. pneumoniae may provide a better understanding of the role of this organism in atherosclerosis. The fluoroquinolones are valuable agents for treating infections caused by intracellular bacteria because they penetrate tissues and mammalian cells extremely well, a feature they share with the macrolides (2, 17). A potential new clinical use of fluoroquinolones is therapy against C. pneumoniae infection associated with atherosclerosis. An ongoing large clinical study is using an intermittent treatment schedule with gatifloxacin for the treatment of hospitalized patients with acute coronary syndromes (4; L. A. Jackson and J. T. Grayston, Proc. Tenth Int. Symp. Hum. Chlamydial Infect., p. 291, 2002). To provide supporting evidence for the use of fluoroquinolones in C. pneumoniae infections associated with chronic disease and to examine the anti-inflammatory activities of these compounds, the effects of levofloxacin, moxifloxacin, and gatifloxacin were examined in an in vitro model of leukocyte TEM in response to C. pneumoniae infection or stimulation with TNF-. MATERIALS AND METHODS Chlamydia isolate. C. pneumoniae A-03 was previously isolated from an atheroma of a patient with coronary artery disease (35). The isolate was propagated in HEp-2 cell monolayers by a modification of the procedure previously described (39). C. pneumoniae was suspended in inoculation medium (Iscove s minimal essential medium supplemented with 10% fetal bovine serum, 2 mm L-glutamine, 1% [vol/vol] nonessential amino acids, 10 mm HEPES, 4 mg of glucose per ml [ph 7.5], 10 g of gentamicin per ml, and 25 g of vancomycin per ml) before addition to the HEp-2 cell monolayers. Infection was done by centrifugation at 800 g for1hat4 C. Infected cultures were then incubated at 37 C under 5% CO 2 for 30 min. Medium was replaced with growth medium (inoculation medium plus 1.0 g of cycloheximide per ml), and infected cultures were incubated for 48 to 72 h. Sequential passages in 1-dram vials were done before inoculation into 75-cm 2 flasks containing HEp-2 monolayers. C. pneumoniae was harvested by disruption of the monolayers with sterile glass beads, sonication, and low-speed centrifugation at 200 g. Endothelial cell culture. Human umbilical vein endothelial cells (HUVEC; ATCC 1730-CRL) were cultured in 75-cm 2 culture flasks and maintained in Ham s F12K medium (Sigma, St. Louis, Mo.). Cell medium was supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin solution, 30 g of endothelial cell growth supplement per ml, and 100 g of heparin per ml (Sigma). For neutrophil and monocyte migration studies, HUVEC were seeded in 6.5-mm Transwell Clear inserts (Corning Costar, Cambridge, Mass.) at a density of cells per insert and allowed to reach confluency for 2 days prior to infection. Intercellular junction integrity of the endothelial monolayer was assessed by measuring permeability to bovine serum albumin as described elsewhere (15). Isolation of human neutrophils and monocytes. Neutrophils and peripheral blood mononuclear cells (PBMC) were isolated from venous blood of healthy adults by dextran sedimentation and density centrifugation in a Percoll gradient as described previously (14). Neutrophils and PBMC fractions were collected separately, resuspended in Krebs Ringer solution (120 mm NaCl, 4.8 mm KCl, 5.5 mm dextrose, 3.12 mm NaH 2 PO 4, mm Na 2 HPO 4 [ph 7.4]), and centrifuged at 300 g. Neutrophils were treated for 30 s with 0.2% NaCl solution to lyse contaminating erythrocytes and washed twice in Krebs Ringer solution. Purity of isolated neutrophils was 98%, as determined by Diff-Quik staining (Baxter, Miami, Fla.). PBMC were washed twice with ice-cold Hanks balanced salt solution (HBSS)-HEPES (HBSS supplemented with 20 mm HEPES and 1% penicillin-streptomycin; Sigma) and seeded in six-well plates at a density of /well. PBMC were maintained in HBSS-HEPES for 2hat 37 C in5%co 2. Following incubation, aspiration and several washing steps removed nonadherent cells. Adherent monocytes were removed by gently scraping with a rubber policeman and washed twice with HBSS-HEPES before the TEM assays. Monocytes obtained by this method were 95% pure by -naphthylacetate esterase staining (Sigma). Antibiotic treatment and infection protocol. Levofloxacin (Ortho McNeil), moxifloxacin (Bayer), and gatifloxacin (Bristol-Myers Squibb) were dissolved in 10 ml of methanol at a final concentration of 2.0 mg/ml, and serial dilutions (0, 1.0, 4.0, 8.0, and 16.0 g/ml) of each quinolone were performed in inoculation media. Infection of HUVEC was performed as described previously (46). HUVEC in 6.5-mm Transwells were pretreated with the different antibiotic dilutions for 1 h (except for the untreated, mock-infected, antibiotic-free control for C. pneumoniae-infected and TNF- stimulated cells) at 37 Cin5%CO 2. The medium was subsequently removed, and cell monolayers were infected separately with C. pneumoniae A-03 or treated with 500 U of human recombinant TNF- (Promega, Madison, Wis.) per ml in inoculation medium with or without antibiotics. Infected cells received inclusion forming units per well, resulting in a multiplicity of infection of 1:1, and were incubated for 2 h at 37 C in 5% CO 2. Following incubation, the medium was aspirated, infected monolayers were washed with HBSS, and fresh inoculation medium with or without antibiotics was added to the cultures. Since the C. pneumoniae inoculum may contain remnants of HEp-2 cells, mock-infected controls consisting of HUVEC treated with crude lysates of HEp-2 cells were also included and processed in the same manner as infected cells. C. pneumoniae-infected or TNF- -treated HUVEC were incubated for 24 h at 37 C in5%co 2 prior to migration assays. The number and viability of HUVEC were not significantly diminished following the antibiotic treatment described above, as determined by direct counting of trypsinized cells with a hemacytometer and by trypan blue dye exclusion, respectively (greater than 90% viability). Monocyte and neutrophil TEM assays. Prior to migration assays, all medium was removed from the upper and lower chambers of the Transwells and endothelial monolayers were washed three times with HBSS. Neutrophils or monocytes were preincubated with or without the different antibiotic dilutions for 1 h at 37 C to allow antibiotic uptake (12, 47). Neutrophils or monocytes were subsequently added to the upper chambers at a density of per insert, and fresh medium was added to the lower chambers. Neutrophils and monocytes were coincubated with uninfected, mock-infected, C. pneumoniae-infected, and TNF- -treated HUVEC at 37 C in5%co 2 for 30 and 60 min, respectively. Following incubation, the medium was aspirated from both chambers, the upper chamber was washed three times with HBSS, the endothelial monolayer was removed with a cotton swab, and the polycarbonate membrane was stained with a HEMA3 Stain Set kit (Fisher). TEM of neutrophils and monocytes was assessed by light microscopic examination of the underside of the polycarbonate membrane at a magnification of 1,000 as previously described (28). The average number of cells from a total of 50 high-power fields (HPF) was determined, and the data are expressed as a percentage of the antibiotic-free control. The concentrations of vancomycin and gentamicin used in the inoculation media were tested, and no detectable effect on the TEM was observed (data not shown). Chemokine measurements. Levels of IL-8 and MCP-1 in the supernatants of uninfected, mock-infected, C. pneumoniae-infected, and TNF- -treated HUVEC were measured with a commercially available enzyme-linked immunosorbent assay kit (R & D Systems) in accordance with the manufacturer s instructions. Fluoroquinolone uptake into HUVEC. HUVEC were grown on 24-well plates at a density of /well and allowed to reach confluency for 2 days before infection. Infected cells received inclusion-forming units per well, resulting in a multiplicity of infection of 1:1. Inoculation of HUVEC with C. pneumoniae was followed by centrifugation at 800 g for1hat4 C. After 30 min of incubation at 37 C with 5% CO 2, cell monolayers were treated with different dilutions of the three fluoroquinolones (0, 1.0, 16.0, and 50.0 g/ml). Infected cultures were incubated for 48 to 72 h at 37 C with 5% CO 2. After the incubation period, HUVEC monolayers on glass coverslips were fixed with methanol and chlamydial inclusions were stained with the Pathfinder Chlamydia culture confirmation system (Bio-Rad) in accordance with the manufacturer s instructions. Infectivity of C. pneumoniae was assessed by determining the number of inclusions per HPF by epifluorescence microscopy at a magnification of 400. Data analysis. All data were subjected to an unpaired analysis of variance with the Tukey-HSD multiple-comparison test. A maximum P value of 0.05 was used as the alpha value to determine statistical significance for all analyses. RESULTS Effects of fluoroquinolone antibiotics on the TEM of neutrophils. Figure 1A indicates the activities of the three fluoroquinolones on TEM of neutrophils in C. pneumoniae-infected HUVEC. All three fluoroquinolones, levofloxacin, moxifloxa-

3 2540 URIARTE ET AL. ANTIMICROB. AGENTS CHEMOTHER. FIG. 1. Effects of fluoroquinolones on TEM of neutrophils through C. pneumoniae-infected (A) or TNF- -treated (B) HUVEC. TEM was assessed as described in Materials and Methods. Bars indicate the mean value the standard error of the mean of three separate experiments, each done in duplicate. Results are expressed as percentages of antibiotic-free controls. Statistical significance of differences from antibiotic-free controls was defined as follows: *, P 0.001; **, P For panel A, data from mock-infected levels of neutrophil TEM were subtracted from all values (levofloxacin, cells per HPF; moxifloxacin, cells per HPF; gatifloxacin, cells per HPF). The antibiotic-free control values for neutrophil TEM in C. pneumoniae-infected monolayers were as follows: levofloxacin, cells per HPF; moxifloxacin, cells per HPF; gatifloxacin, cells per HPF. For panel B, the untreated levels of neutrophil TEM were subtracted from all values (levofloxacin, cells per HPF; moxifloxacin, cells per HPF; gatifloxacin, cells per HPF). The antibiotic-free control values for neutrophil TEM in TNF- -treated monolayers were as follows: levofloxacin, cells per HPF; moxifloxacin, cells per HPF; gatifloxacin, cells per HPF. FIG. 2. Effects of fluoroquinolones on TEM of monocytes through C. pneumoniae-infected (A) or TNF- -treated (B) HUVEC. TEM was assessed as described in Materials and Methods. Bars indicate the mean value the standard error of the mean of three separate experiments, each done in duplicate. Results are expressed as percentages of antibiotic-free controls. Statistical significance of differences from antibiotic-free controls was defined as follows: *, P 0.001; **, P 0.01; ***, P For panel A, data from mock-infected levels of monocyte TEM were subtracted from all values (levofloxacin, cells per HPF; moxifloxacin, cells per HPF; gatifloxacin, cells per HPF). The antibiotic-free control values for monocyte TEM in C. pneumoniae-infected monolayers were as follows: levofloxacin, cells per HPF; moxifloxacin, cells per HPF; gatifloxacin, cells per HPF. For panel B, the untreated levels of monocyte TEM were subtracted from all values (levofloxacin, cells per HPF; moxifloxacin, cells per HPF; gatifloxacin, cells per HPF). The antibiotic-free control values for monocyte TEM in TNF- -treated monolayers were as follows: levofloxacin, cells per HPF; moxifloxacin, cells per HPF; gatifloxacin, cells per HPF. cin, and gatifloxacin, significantly decreased neutrophil TEM at the lowest concentration tested (P 0.01), compared to antibiotic-free controls. Although all three fluoroquinolones showed a significant inhibitory effect on neutrophil TEM, moxifloxacin showed the greatest effect at the lowest concentration of 1 g/ml and maintained a constant inhibition at all of the other dilutions used in this assay. In HUVEC stimulated with TNF- (Fig. 1B), levofloxacin, moxifloxacin, and gatifloxacin also showed significant inhibition of TEM at 1.0 g/ml (P 0.001), compared to antibiotic-free controls. Effects of fluoroquinolones on the TEM of monocytes. Figure 2A indicates the activity of the three fluoroquinolones on the TEM of monocytes across HUVEC infected with C. pneumoniae. All three fluoroquinolones significantly decreased monocyte TEM at 1.0 g/ml (P for levofloxacin and moxifloxacin, P 0.01 for gatifloxacin) compared to antibioticfree controls. In HUVEC stimulated with TNF- (Fig. 2B), all three fluoroquinolones caused a significant, dose-dependent decrease in monocyte TEM compared to that of antibiotic-free controls (P 0.05). Effects of fluoroquinolones on IL-8 and MCP-1 secretion. As shown in Table 1, a 17% average reduction in IL-8 was seen in the presence of levofloxacin (at 16 g/ml) in C. pneumoniaeinfected cells and a 22.3% reduction in TNF- treated cells was

4 VOL. 48, 2004 QUINOLONE INHIBITION OF TEM 2541 TABLE 1. Comparison of the IL-8 and MCP-1 levels in the supernatants of C. pneumoniae-infected and TNF- stimulated endothelial cells exposed to different dilutions of three fluoroquinolones a Treatment and antibiotic concn ( g/ml) Chemokine concn (ng/ml) Levofloxacin Moxifloxacin Gatifloxacin IL-8 MCP-1 IL-8 MCP-1 IL-8 MCP-1 C. pneumoniae infection Mock infection b b c b d TNF- stimulation Uninfected b b b c b b d c a Measurements were performed as described in Materials and Methods. Data are the mean the standard error of the mean of three separate experiments. b P 0.05 compared to antibiotic-free control. c P compared to antibiotic-free control. d P 0.01 compared to antibiotic-free control. seen compared to antibiotic-free controls. A 53% reduction in IL-8 was observed with moxifloxacin in C. pneumoniae-infected cells (1.0 g/ml, P 0.05) and a 51% reduction in TNF- treated cells was seen compared to antibiotic-free controls (1.6 g/ml, P 0.05). A 41% reduction in IL-8 production was observed with gatifloxacin in C. pneumoniae-infected cells (16 g/ml, P 0.01), and there was a 56% reduction in TNF- treated cells (16 g/ml, P 0.01) compared to antibiotic-free controls. Reductions in MCP-1 levels of 56 and 51% in C. pneumoniae-infected and TNF- -treated cells (16 g/ml), respectively, were observed with levofloxacin compared to antibioticfree controls (Table 1). A 60% reduction in MCP-1 levels (Table 1) in C. pneumoniae-infected cells and a 45% reduction in TNF- -treated cells (16 g/ml, P 0.05) were observed with moxifloxacin compared to antibiotic-free controls. Significant reductions in MCP-1 of 41 and 56% (P 0.001) were seen with gatifloxacin (16 g/ml) in infected and TNF- treated cells compared to antibiotic-free controls. Assays of antibiotic uptake in HUVEC. To confirm antibiotic uptake into endothelial cells, three separate experiments were performed with HUVEC infected with C. pneumoniae in the presence of each fluoroquinolone at 1.0 and 16.0 g/ml. The resulting chlamydial inclusion counts indicated complete inhibition of C. pneumoniae inclusion formation at the lowest concentration tested, compared to antibiotic-free controls. DISCUSSION The present study demonstrated that the fluoroquinolones levofloxacin, moxifloxacin, and gatifloxacin all had a significant inhibitory effect on neutrophil and monocyte TEM when endothelial cells were activated either by infection via C. pneumoniae or stimulation via TNF-. The inhibitory effect of fluoroquinolones on neutrophil and monocyte TEM in noninfected endothelial cells indicates that these compounds have a direct anti-inflammatory activity on the endothelial cells. There was a direct correlation between the inhibitory effect observed with gatifloxacin and moxifloxacin with neutrophil and monocyte TEM and the corresponding levels of IL-8 and MCP-1, since both chemokines were significantly diminished when the endothelial cells were infected with C. pneumoniae or stimulated with TNF-. Levofloxacin also decreased the production of both IL-8 and MCP-1 at the highest dilution used for the assays (16 g/ml) in both C. pneumoniae-infected and TNF- -stimulated HUVEC; however, these values did not achieve statistical significance. Our data show that these fluoroquinolones have an inhibitory effect on neutrophil and monocyte TEM in both C. pneumoniae-infected and TNF- stimulated HUVEC, but this inhibition does not directly correlate with a significant decrease in IL-8 or MCP-1 levels. One possible explanation is that these antibiotics may also affect the expression of some key adhesion molecules, such as ICAM-1, VCAM-1, etc., on the endothelial cells and may explain the inhibitory effect on TEM of both neutrophils and monocytes. Although many authors have described the immunomodulatory and anti-inflammatory effects of several quinolones on lymphocyte and monocyte functions (7, 11, 18, 36, 37), the present study is the first to analyze this effect on endothelial cells. The past 2 decades have highlighted the pivotal role of the endothelium in preserving vascular homeostasis and hemostasis. The endothelium is a dynamic autocrine and paracrine organ (5, 42). Endothelial cells are important in the pathogenesis of atherosclerosis and other chronic diseases such as asthma. We have shown previously that macrolides have an anti-inflammatory effect on endothelial cells, decreasing chemokine levels of IL-8 and MCP-1, as well as inhibiting neutrophil and monocyte TEM (46). In the present study we have shown the anti-inflammatory effect of fluoroquinolones on endothelial cells that are activated by infection or via TNF- stimulation. We propose that the observed inhibitory effects of antibiotics on neutrophil and monocyte TEM, and on IL-8 and MCP-1 levels, in noninfected endothelial cells could be due to

5 2542 URIARTE ET AL. ANTIMICROB. AGENTS CHEMOTHER. a direct action of fluoroquinolones on the molecular mechanisms in endothelial cells. On the basis of the TEM and cytokine data from noninfected HUVEC, the three fluoroquinolones show similar anti-inflammatory effects; however, it is not clear if these antibiotics affect the same molecular mechanisms in endothelial cells. We have also shown that MCP-1 is upregulated in endothelial cells infected with C. pneumoniae (27, 29). Evidence indicates that MCP-1 plays a key role not only in early events of atherosclerosis but also in long-term events such as development of aortic aneurisms (21). Both IL-8 and MCP-1 have been shown to have a significant role in the recruitment of lymphocytes and monocytes to atherosclerotic tissue (21). These cells can release other inflammatory factors such as TNF- and IL-1, which might contribute to the destruction of the vessel, leading to the formation of aortic aneurisms. The first report of a possible connection between C. pneumoniae and atherosclerosis came from a serologic study performed in Finland in 1988 (43). Since this initial report, many papers on the association of C. pneumoniae and atherosclerosis have been published (4). Six prospective clinical studies of antibiotics with activity against C. pneumoniae have been completed or are in progress; however, the preliminary reports are not very encouraging (33; Jackson and Grayston, Proc. Tenth Int. Symp. Hum. Chlamydial Infect.). In vitro studies like the present one may provide important information regarding the effects of antibiotics on the endothelium and how these antimicrobial agents not only inhibit C. pneumoniae replication but also diminish the inflammatory process involved in the progression of atherosclerosis. 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