Division of Pediatric Infectious Diseases and Immunology, Weill Medical College of Cornell University, New York PresbyterianCornell Medical Center, 525 East 68th Street, Box 296, New York, NY 10021, USA
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Abstract |
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Introduction |
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The potential for antimicrobials to affect physiological responses to infection is well recognized.58 Recently, it has been stressed that this influence can be secondary to the effect of lysing bacteria into components that induce inflammation. However, it has also been shown that certain antimicrobials can influence host responses by directly affecting host cell function.911 In particular, drugs that target protein synthesis or DNA replication processes in bacteria have been suspected of causing this effect. Quinolone agents have been included among those that could influence host responses and studies have supported the hypothesis that these agents influence host cell function at concentrations that are achieved when these drugs are used to treat infection.12,13
The purpose of this study was to examine the influence of two quinolones (ciprofloxacin and trovafloxacin) and ceftriaxone on factors considered to play important roles in defence against encapsulated extracellular bacteria. The agents studied have a similar broad spectrum of bactericidal activity. The influence of these agents on host responses was examined by measuring the effect of these agents on four cytokines produced early in the host response to pathogens. Secretion of TNF-, IL-1ß, IL-6 and IL-8 and mRNA levels in human peripheral blood mononuclear cells (PBMCs) were measured in the presence and absence of quinolones after stimulation with bacterial components or with two quinolone-susceptible encapsulated bacteria, type b Haemophilus influenzae and Streptococcus pneumoniae. This system permitted: (i) examination of the effect of two quinolones on production of these cytokines by PBMCs incubated with bacterial components as well as live bacteria; (ii) comparison of the influence of two quinolones on cytokine production with that of an unrelated agent with comparable bactericidal activity, ceftriaxone; and (iii) examination of mechanisms that determine how quinolones might influence production of these cytokines.
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Materials and methods |
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Trovafloxacin (trovafloxacin mesylate) was obtained from Pfizer, Inc. (Groton, CT, USA). Ciprofloxacin and ceftriaxone were obtained from the US Pharmacopeia (Rockville, MD, USA). Stock solutions of all antibiotics used in in vitro studies were made in sterile distilled water. Further dilutions were made in phosphate-buffered saline (PBS; Life Technologies, Grand Island, NY, USA). Lipopolysaccharide (LPS; from Escherichia coli O111:B4; Sigma Chemical Co., St Louis, MO, USA) and lipoteichoic acid (LTA; from Streptococcus pyogenes; Sigma) were used in these studies as representative of bacterial components that induce inflammatory responses in Gram-negative or -positive bacterial infections, respectively.
Bacteria
Type b H. influenzae (strain Eagan) was cultured on chocolate agar overnight and then inoculated into brain heart infusion broth (Difco, Detroit, MI, USA) supplemented with NAD 6 mg/L and haemin 10 mg/L. A type 6 S. pneumoniae isolated from a child with bacteraemia was used. Colonies were cultured overnight on tryptic soy agar plates containing 5% sheep blood and then inoculated into ToddHewitt broth (Difco).
Growth curves for bacteria were determined to identify the time of logarithmic phase growth and to correlate colony count with optical density at 620 nm. Bacteria were grown up to late logarithmic phase growth, washed three times with warm PBS and killed by incubation at 65°C for 20 min. Heat-killed bacteria were stored at 75°C until used in assays. Live bacteria were grown up to late logarithmic phase and diluted in PBS before use in assays.
PBMC stimulation assay
Blood was obtained by venipuncture from healthy donor volunteers. PBMCs were separated by centrifugation using Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden). The monocyte-enriched cell fraction was collected and washed three times with cold Ca2+- and Mg2+-free PBS and resuspended in RPMI 1640 with l-glutamine (Life Technologies) containing 10% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT, USA) and 25 mM HEPES at a density of 106 cells/mL. Trypan blue exclusion was used to assess the number of live cells.
Cells were seeded into 24-well plates (Becton Dickinson, Lincoln Park, NJ, USA) at a density of 1 x 106 cells/well and incubated in the presence of LPS 100 ng/well, LTA 1 µg/well, 107 cfu viable S. pneumoniae, 105 viable H. influenzae, 106 heat-killed S. pneumoniae or 105 heat-killed H. influenzae. Experiments were performed in the presence or absence of antimicrobials (trovafloxacin 10 mg/L, ciprofloxacin 10 mg/L or ceftriaxone 275 mg/L). These concentrations were chosen to approximate maximum serum concentrations of these agents.1417 Cells incubated in these concentrations of antimicrobials for 24 h were assessed for viability by Trypan blue exclusion; >95% of cells remained viable under these conditions. After incubation of PBMCs with bacterial components or bacteria at 37°C in 5% CO2, cell-free supernatant was recovered by centrifugation and stored at 75°C.
The concentrations of cytokines in PBMC supernatants were measured using an automated chemiluminescent immunoassay system (Immulite; Diagnostic Products Corporation, Los Angeles, CA, USA).
Murine in vivo assays
Alatrofloxacin (Pfizer; 1 mg), the prodrug of trovafloxacin, was injected intraperitoneally into C57/BL6 mice (Taconic, Germantown, NY, USA). Within 10 min, serum from mice receiving this dose had maximal serum bactericidal activity as tested with a susceptible strain of Neisseria meningitidis (data not shown), indicating that the prodrug was rapidly absorbed and converted to trovafloxacin. Control mice received an equal volume of 5% dextrose instead of antibiotic. One hour later, all mice received 20 µg of LPS by a similar route. Mice in both groups were killed 1, 3 or 6 h after administration of LPS and blood was obtained by cardiac puncture. Samples were centrifuged and stored at 70°C. Serum TNF- and IL-6 concentrations were measured using a commercial murine enzyme-linked immunosorbent assay (Biosource Int., Camarillo, CA, USA).
mRNA measurements
A reverse transcriptionpolymerase chain reaction (RT PCR)-based quantitative assay (CytoXpress; Biosource Int.) was used to measure concentrations of mRNA for cytokines. For these studies PBMCs were incubated with LPS for 3 h as described above with antimicrobial (trovafloxacin 10 mg/L or ceftriaxone 275 mg/L) or without antimicrobial. A total of 107 cells was used for each stimulation assay. After stimulation the cells were lysed using RNazol (Teltest Inc., Friendswood, TX, USA) and stored at 70°C. Total RNA was extracted using the method developed by Chomczynski & Sacchi18 and stored in 20 µL RNase-free water at 70°C. First-strand synthesis of cDNA from 5 µL total RNA was carried out using 1 µL of oligo(dT) primers, 0.5 µL AMV reverse transcriptase, 1 µL RNase inhibitor, 1 µL 100 mM dNTPs, 1 µL 80 mM sodium pyrophosphate and 4 µL 5 x reverse transcription buffer diluted to a total reaction mixture volume of 20 µL (cDNA cycle kit; Invitrogen, Carlsbad, CA, USA). Synthesized cDNA was extracted with phenolchloroform, the aqueous phase was aliquoted and the cDNA was precipitated in ice-cold ethanol. Glycogen 2 g/L was used as a carrier and the sample was kept overnight at 20°C to ensure maximum recovery of sample before centrifuging at 12000g for 15 min, dissolving in 20 µL sterile water and then freezing at 20°C until further use. mRNA for the constitutively expressed gene glyceraldehyde-3-phosphate dehydrogenase (GAP) was amplified to check the integrity of the RNA extraction process and cDNA synthesis. This gene was also used to assess each cDNA preparation using X174 RF DNA HaeIII digests on 0.8% agarose gels. Competitive PCR was performed according to the manufacturer's instructions using a competing internal control standard (ICS) containing 2000 copies and 5 µL of sample cDNA. Samples were then stored at 20°C. For samples that showed marked stimulation of cytokine mRNA, 1:5 dilutions of cDNA were spiked with a constant number (2000 copies) of ICS before competitive PCR. For very low copy numbers, 400 copies of ICS were used and the number of cycles was increased from 30 to 34. The PCR mix was run on 0.8% agarose gels to confirm separation of the competing bands. mRNA for TNF-
, IL-1ß and IL-6 was subsequently measured using pre-coated microtitre plates with capture oligonucleotides specific to ICS and wild-type amplicons according to the manufacturer's instructions (CytoXpress detection kit; Biosource Int.). Values were calculated using the ratios of the product of optical density and dilution of PCR mix for the wild type and the ICS amplicons.
Statistical analysis
Mean values were compared by t-test. An inhibition index for each antimicrobial tested was calculated as: [(pg/mL of cytokine in absence of antimicrobial) (pg/mL of cytokine in presence of antimicrobial)] ÷ (pg/mL of cytokine in absence of antimicrobial). Indices less than zero were considered to be zero. Differences between indices of groups were analysed by rank sum.
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Results |
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To determine whether antimicrobials affect cytokine responses to bacterial components, LPS and LTA were added to PBMCs and cytokine production was measured over time (Figure 1). The concentrations of LPS and LTA and of antimicrobials were not toxic to PBMCs in this system as assessed by Trypan blue exclusion. In all cases PBMC viability was >95% after 24 h of incubation, the time at which the analysis was terminated. LPS and LTA consistently induced production of high levels of TNF-
, IL-1ß, IL-6 and IL-8. The mean concentrations of each cytokine produced and the kinetics of production were comparable following either LPS or LTA stimulation.
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Concentrations of cytokines were measured 6 and 24 h after adding heat-killed or viable bacteria. Parallel studies were performed using type 6 S. pneumoniae or type b H. influenzae. For both organisms, the mean concentration of each of the cytokines after 6 h was significantly greater than the mean concentration of cytokine produced by PBMCs incubated in media alone. By 24 h the concentration of cytokines in supernatant of stimulated PBMCs was five to 25 times that measured at 6 h.
Trovafloxacin inhibited production of all four cytokines relative to cells stimulated in the absence of antimicrobials (Figures 2 and 3). The extent of this inhibition was significantly greater than the modest effect on cytokine production evident with either ciprofloxacin or ceftriaxone. The greater degree of inhibition of cytokine production by trovafloxacin relative to ciprofloxacin and ceftriaxone was not due to differences in bacterial killing. Firstly, heat-killed bacteria induced cytokine production as effectively as viable organisms and this production was also inhibited by trovafloxacin. Secondly, all three of the antimicrobials produced rapid killing of both bacteria, and kill curves demonstrated that none of the organisms were viable after 3 h (data not shown). Comparison of trovafloxacin, ciprofloxacin and ceftriaxone in this in vitro system suggests that the inhibitory effect on cytokine production of trovafloxacin was unique, consistent and not related to its ability to mediate bacteriolysis.
Effect of trovafloxacin on in vivo production of cytokines in mice
To test the relevance of these in vitro observations to cytokine responses in vivo, serum TNF- and IL-6 were measured in mice. Mice received a single dose of antibiotic 1 h before sub-lethal challenge with LPS. In this model, TNF-
peaked 1 h after LPS was given. At this point, the mean concentration of TNF-
in trovafloxacin-treated mice was <5% of that of untreated mice (91 versus 1896 pg/mL; P < 0.013 by t-test). Beyond this point, serum concentrations of this cytokine were not different. Serum IL-6 concentrations 1 and 3 h after challenge were less in treated than in untreated mice. At 1 h the mean serum concentration of IL-6 in treated mice was 1200 pg/mL whereas that in untreated mice was 23785 pg/mL (P < 0.0001 by t-test). By 6 h, the mean concentrations of IL-6 in treated and untreated mice were not different.
Effect of trovafloxacin on transcription of cytokine mRNA
The potential for trovafloxacin to influence transcription of mRNA was assessed by measuring mRNA for TNF-, IL-1ß and IL-6 in PBMCs stimulated by LPS. Incubation with trovafloxacin had no effect on the intensity of the GAP mRNA band, but markedly affected transcription of TNF-
, IL-1ß and IL-6 (Figure 5
). This semi-quantitative analysis was consistent with quantitative analysis of mRNA copies (Table
). After 3 h, LPS-stimulated cells incubated in the absence of antibiotic had >250-fold more mRNA copies for each of the three cytokines. The number of mRNA copies for these cytokines in PBMCs incubated with trovafloxacin was reduced by >10-fold compared with the number of copies in PBMCs stimulated with LPS in the absence of antimicrobial. These results are consistent with the marked reduction in secretion of each of these cytokines in culture supernatants after incubation with trovafloxacin and suggest that inhibition of this response occurs at the level of cytokine gene transcription. In contrast, ceftriaxone, which had little or no effect on secretion of cytokines in culture supernatants of PBMCs stimulated by LTA, LPS or bacteria, appeared to have a modest effect on increasing mRNA for all three cytokines tested in PBMCs stimulated with LPS (Table
; Figure 5
). The basis for this apparent increase is not clear.
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Discussion |
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Previous work has suggested that fluoroquinolones could influence cytokine production by human leucocytes.12,19,20 In addition, it has been suggested that the effect of trovafloxacin on the inflammatory response in vivo was secondary to the manner by which the drug lysed bacteria.21 Comparison of the influence of trovafloxacin on the production by PBMCs of pro-inflammatory cytokines using bacterial components, heat-killed or viable bacteria, emphasize that the inhibition of cytokine production occurs independently of bacteriolysis. In vivo work further supported the findings that decreasing pro-inflammatory cytokine production can occur independently of trovafloxacin's effect on bacterial killing. Mice treated with trovafloxacin and challenged with LPS had significantly lower serum concentrations of both TNF- and IL-6. Whether a similar effect occurs during treatment of bacterial infection is currently being examined.
The effect of trovafloxacin on cytokine production by PBMCs was dose dependent. The concentration of 10 mg/L trovafloxacin used in our in vitro system consistently and significantly reduced production by PBMCs of TNF-, IL-1ß, IL-6 and IL-8. Although this concentration would not be achieved in the serum of most patients receiving this drug, concentrations in tissues and within cells commonly exceed 10 mg/L.22,23 Results presented here demonstrate that concentrations as low as 1.25 mg/L can affect cytokine production by PBMCs, albeit in a less substantial manner. It is difficult to extrapolate the significance of the observed effects in this in vitro system to effects that may occur in patients. Results with the mouse model presented here suggest that this down-regulation can occur even after single doses of drug. Additional study will be required to determine whether this down-regulation can influence the course of disease that is mediated by these cytokines.
The nearly complete absence of induction of mRNA in PBMCs stimulated with LPS and incubated with trovafloxacin suggests that inhibition of this process may be related to the drug's ability to interfere with transcription. It is known that fluoroquinolones do interact with mammalian topoisomerases, albeit to a lesser extent than they do with bacterial topoisomerases.24 Furthermore, interactions of these drugs with mammalian proteins do not correlate well with their interaction with bacterial proteins.25 Therefore, the extent of the effect of a drug on human cell function related to its effect on human topoisomerases may not be predicted by the drug's antibacterial activity related to its effect on bacterial topoisomerases. If trovafloxacin reduces transcription of cytokine mRNA by interfering with human topoisomerase activity, this may represent a unique property of this drug. Alternatively, this reduced transcription could be occurring because the drug interferes with processes of cellular signalling that involve molecules at the cell surface or in the cell membrane, cytoplasm, nuclear membrane and/or nucleus. Whether trovafloxacin is the only quinolone capable of causing this effect has not been tested. Our results suggest that ciprofloxacin, a related agent, does not share trovafloxacin's effects on production of these cytokines, so it cannot be assumed that these effects occur with all fluoroquinolones or fluoroquinolone-like agents.
Before the reports of fatal liver toxicity associated with trovafloxacin, a large body of clinical experience with the use of this drug had demonstrated that this agent can be highly effective in treating a broad range of infections, including those caused by S. pneumoniae and H. influenzae. Studies that have compared trovafloxacin with agents with similar antibacterial activity, including ciprofloxacin and ceftriaxone, have not demonstrated that patients receiving trovafloxacin had better outcomes. It is possible that differences related to the degree of the inflammatory response were not recognized in studies that measured outcomes such as clinical or microbiological cures.
Important factors to consider in assessing the potential anti-inflammatory effect of a drug on outcome include the duration of infection before treatment and the character of individual host responses. Study of the relevance of this in vitro observation is being examined in ongoing work in an animal model where these factors can be controlled. This work, as well as future studies assessing the potential for a drug's effect on cytokine responses in patients, needs to consider that decreasing early cytokine responses could be as advantageous as it is disadvantageous to the host. Cytokines produced early in response to bacterial infection and that have been shown to be inhibited by trovafloxacin in this in vitro system have also been shown to correlate with morbidity in bacterial meningitis caused by S. pneumoniae or H. influenzae.26,27 Decreasing the production of these proteins in vivo has not been effective in consistently improving outcome. Nevertheless, current understanding of the biology of serious bacterial infections strongly suggests that a drug that can both reduce these cytokine responses and lyse bacteria might be able to affect the course of illness in a manner that would not be predicted by its antimicrobial activity alone.
Trovafloxacin may have as profound an effect on production of pro-inflammatory cytokines as do some drugs and antibodies that have been considered for use as adjunctive therapy for serious bacterial infections. Together with previous reports, results presented here further support the potential for this agent to affect the human immune system when used at concentrations that are given to treat infections.
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Acknowledgments |
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Notes |
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References |
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Received 17 March 2000; returned 3 June 2000; revised 5 July 2000; accepted 5 September 2000