Division of Pediatric Infectious Diseases and Immunology, Weill Medical College of Cornell University, NY, USA
Received 28 November 2001; returned 19 February 2002; revised 3 April 2002; accepted 19 April 2002
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Abstract |
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Keywords: ciprofloxacin, fluoroquinolone, cytokine response, inflammatory response, endotoxin, mortality, innate immunity, immune response, murine, in vivo
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Introduction |
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Ciprofloxacin is a fluoroquinolone with activity against both Gram-positive and -negative bacteria. It is widely used to treat focal as well as serious disseminated infections. Inhibitory and stimulatory effects of ciprofloxacin on the immune system have been suggested, primarily by studies that have shown that the production of several cytokines by human and murine leucocytes can be affected by this drug.11 Ciprofloxacin has been shown to enhance interleukin-2 (IL-2) gene induction at clinically achievable concentrations.12,13 Ciprofloxacin has also been shown to decrease production of tumour necrosis factor- (TNF-
), IL-1
, lymphotoxin and granulocyte-macrophage colony stimulating factor (GM-CSF) by human lymphocytes.14 In vitro studies have also shown that lower doses of ciprofloxacin enhance production of IL-1, IL-6 and TNF-
by human monocytes.15 Additional studies have suggested that the effects of this agent on cell function are not confined to leucocytes. Recent work has demonstrated that ciprofloxacin can reduce IL-6 and IL-8 secretion by human endothelial cells.16 Although these observations strongly suggest that ciprofloxacin could influence host defences, the effect of this drug on these responses in patients and on disease associated with these responses has not been demonstrated.
The purpose of the work presented here was to examine ciprofloxacins influence on inflammatory responses in vivo. A murine model was used that involved challenge with lipopolysaccharide (LPS). LPS is a major component of the outer membrane of Gram-negative bacteria. It is a potent stimulus of the inflammatory response and is considered to play a central role in mediating disease caused by Gram-negative bacteria.17,18 The inflammatory response that occurs after challenge with LPS is associated with elevated serum concentrations of TNF-, IL-1ß and IL-6. Increased concentrations of these proteins have been associated with endothelial damage along with circulatory and metabolic derangement and the systemic inflammatory response syndrome.19 Using this model, the effect of ciprofloxacin on survival of mice challenged with LPS and on early cytokine responses was studied. In addition to characterizing the influence of ciprofloxacin on TNF-
, IL-1ß and IL-6 responses, the influence of the drug on IL-10 and IL-12 responses was also studied. IL-10 and IL-12 are likely to play roles in innate as well as acquired immunity. Therefore, influence on these responses may impact not only the outcome of acute infection, but also the cellular and humoral immunity that develops as hosts recover from an infectious disease.
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Materials and methods |
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Reagents
Ciprofloxacin (Cipro I.V.; Bayer Corporation, West Haven, CT, USA) was prepared according to the package insert. Drug concentration was adjusted by diluting with 5% dextrose water (D5W). A stock solution of 5 mg/L LPS (Escherichia coli O111:B4; Sigma Chemical Co., St Louis, MO, USA) was prepared by dissolving LPS in sterile, endotoxin-free water. Aliquots of stock were stored at 20°C. Stock solutions of LPS were diluted using phosphate-buffered saline (PBS; Life Technologies, Grand Island, NY, USA) immediately before challenging mice.
Murine model of endotoxin-mediated mortality
C57/BL6 (Taconic, Germantown, NY, USA) mice were challenged in groups of four with LPS (dose range: 2001000 µg) in a total volume of 1 mL by intraperitoneal (ip) inoculation. Mice were observed for feeding, movement and activity, grooming (smooth and shiny coats versus dull and ruffled coats) and survival for 72 h. Using our established dose of LPS that was uniformly lethal, the effect of ciprofloxacin (3.0, 4.5 and 6.0 mg) on LPS-induced mortality was assessed by giving ip doses of ciprofloxacin in a total volume of 1 mL, 1 h before LPS challenge. Control animals received D5W instead of ciprofloxacin.
Cytokine responses
Cytokine responses were assessed by measuring serum concentrations. Serum was separated from clotted blood obtained by cardiac puncture of anaesthetized mice at 0, 1, 3, 6 and 24 h following administration of ip LPS in groups of five for each dose of LPS used (20, 200 and 1000 µg). Mice were killed after cardiac puncture. Serum was stored at 70°C and concentrations of each cytokine were measured by ELISA according to the manufacturers instructions (Biosource International, Camarillo, CA, USA).
Statistical analysis
Differences between mean values of normally distributed data were assessed by an unpaired Students t-test. Differences in mortality of groups were assessed by Fishers exact test.
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Results |
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The dose response of LPS on mortality in the murine model is shown in Figure 1. Mice receiving 1000 µg of LPS all died between 18 and 38 h after LPS challenge. Based on these experiments, it was concluded that the LD50 of this LPS was between 1000 and 500 µg. At doses as low as 200 µg mice appeared less active after ip challenge. Mice receiving the lowest dose used in our experiments (20 µg) could not be distinguished from mice given PBS alone.
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Experiments carried out with lower doses of LPS (750 µg) also demonstrated the protective effects of ciprofloxacin (data not shown). All mice challenged with this dose of LPS and given 4.5 µg of ciprofloxacin survived (6/6 versus 2/6, P = 0.03). No toxic effects of ciprofloxacin were evident from observations made in mice given ciprofloxacin without LPS challenge. Mice receiving doses as high as 6 mg of ciprofloxacin could not be distinguished from those receiving D5W.
Taken together, these results indicate that single doses of ciprofloxacin 4.5 mg can consistently prevent endotoxin-mediated death that occurs within 72 h of LPS challenge.
Effect of ciprofloxacin on cytokine responses induced by a lethal dose of LPS
Early cytokine responses after endotoxin challenge have been well characterized and are known to occur within hours of LPS challenge. Specifically, TNF-, IL-1ß and IL-6 have been shown to be produced early in this response and have been suggested to play critical roles in driving physiological responses that lead to death. To define the influence of ciprofloxacin on cytokine responses in vivo that have been associated with fatal outcome, serum concentrations of these cytokines and of IL-10 and IL-12 in mice receiving 1000 µg of LPS were compared with concentrations of these cytokines in mice receiving this same dose of LPS and a single dose of ciprofloxacin (6 mg). As shown in Figure 2, ciprofloxacin-treated mice had marked differences compared with the cytokine responses of mice that were not treated. Mean concentrations of cytokines in ciprofloxacin-treated mice challenged with LPS were significantly different from mice given D5W and LPS at 1 and 3 h for TNF-
(P < 0.02), at 1 h for IL-10 (P < 0.05) and at 3 and 6 h for IL-12 (P < 0.004). Ciprofloxacin consistently and significantly reduced TNF-
and IL-12 responses, increased the IL-10 response and had little or no effect on IL-1ß and IL-6 responses in this model (Figure 2).
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Effect of ciprofloxacin on cytokine responses induced by sublethal doses of LPS
To test whether ciprofloxacin could have similar effects on immune responses that were not associated with fatal outcome, cytokine responses of mice receiving sublethal challenges of LPS were also characterized. TNF-, IL-1ß and IL-6 responses in mice receiving 200 and 20 µg of LPS were affected in a similar manner to that observed with lethal doses of LPS (Figure 3). In this model, peak serum TNF-
and AUC of TNF-
responses over the first 6 h after LPS challenge were not affected by LPS dose. In contrast, IL-6 responses were incrementally less with lower doses of LPS (Figure 3), and no dose-related association for IL-1ß was observed. As observed with lethal doses of LPS, ciprofloxacin-treated mice receiving sublethal doses had marked decreases in TNF-
responses and no effect on IL-1ß responses. Mean concentrations of cytokines in ciprofloxacin-treated mice challenged with either 200 or 20 µg of LPS were significantly different from mice given D5W and LPS at 1 and 3 h for TNF-
(P < 0.04). At lower doses of LPS, IL-6 responses tended to be less in ciprofloxacin-treated mice compared with controls. However, these differences were not statistically significant.
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Discussion |
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The basis for improved survival in ciprofloxacin-treated mice is suggested by the influence that ciprofloxacin demonstrated on the pattern of early cytokine responses. In the absence of ciprofloxacin, lethal LPS challenge resulted in an early burst of TNF- and a modest peak in IL-10 concentration, both occurring at 1 h. These cytokines had largely disappeared by 6 h. Serum IL-1ß rose steadily over the 6 h period and serum IL-6 and IL-12 concentrations peaked at 3 h and decreased considerably by 6 h. Pre-treatment with a dose of ciprofloxacin that prevented death in these mice consistently and significantly attenuated the TNF-
burst, decreased IL-12 concentrations and increased the IL-10 response. It has been shown that endotoxin-mediated death can be reversed by manipulation of these cytokine responses in animal models, either by decreasing TNF-
or IL-12, or by increasing IL-10.2326 Based on these observations, it is proposed that ciprofloxacins effect on cytokine responses is a major, if not only, factor causing improved survival in this model.
In addition to influencing mortality and early cytokine responses associated with death, results presented here demonstrate that ciprofloxacin affects cytokine responses in animals challenged with sublethal doses of LPS. This effect may be particularly important when considering the consequences of early inflammatory responses to infection. Cytokines made in the early phases of an infection influence the functional differentiation of T lymphocytes and serve to help initiate the acquired immune response.27 Both IL-10 and IL-12 have been considered to play an important role in this process. T lymphocytes stimulated in the presence of IL-12 and -interferon have been shown to develop preferentially into Th1 cells producing an adaptive response dominated by macrophage activation.20 IL-10 has been shown to inhibit the generation of Th1 cells.28,29 Given these observations, the increase in IL-10 and decrease in IL-12 levels could be viewed as perturbing host responses to favour Th2 immunity. The data presented here suggest that ciprofloxacin may affect humoral and cellular immunity in this manner. Accentuation of IL-10 production and diminished IL-12 production were most pronounced in mice receiving the lowest dose of LPS. Whether similar effects occur in hosts surviving infections or responding to vaccines is not known and is currently being examined.
Influencing the inflammatory responses associated with severe bacterial infection and septic shock has evolved as a major challenge in the treatment of patients. Clinical trials have evaluated agents such as monoclonal antibodies to TNF-, fusion proteins, IL-1 receptor antagonists, antibodies to platelet-activating factor and corticosteroids.30,31 Antimicrobial agents that influence immune responses could further affect these adjuvant therapies and may have potential application in treating patients with sepsis. The data presented here suggest that ciprofloxacin, due to its ability to prevent endotoxin-mediated fatality and its anti-inflammatory effect, may be such an agent. The model described here indicates that these effects are pronounced when ciprofloxacin is given before endotoxin challenge and therefore may be more relevant to chemoprophylaxis rather than treatment. Whether giving ciprofloxacin can influence responses that have already been initiated before an antimicrobial is given, or that could be augmented by the use of a potent bactericidal agent that increases LPS-mediated inflammation, has not been determined.
In summary, ciprofloxacin can prevent death mediated by endotoxin and can produce a profound alteration in the acute cytokine responses occurring in the first few hours after challenge with LPS. This observation suggests that ciprofloxacin influences the inflammatory response and that the drugs effect may extend beyond those that are due to its antimicrobial action.
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Footnotes |
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References |
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