Effects of florfenicol on early cytokine responses and survival in murine endotoxemia

International Immunopharmacology (2008) 8, 982–988

w w w. e l s e v i e r. c o m / l o c a t e / i n t i m p

Effects of florfenicol on early cytokine responses and survival in murine endotoxemia Xuemei Zhang a,b,1 , Yu Song a,1 , Xinxin Ci a , Na An a , Junwen Fan a , Junqing Cui c,⁎, Xuming Deng a,⁎ a

Department of Veterinary Pharmacology, College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin 130062, People's Republic of China b Department of Animal Medicine, Agricultural College of Yanbian University, Longjing, Jilin 133400, People's Republic of China c Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA, 02140, USA

Received 11 January 2008; received in revised form 25 February 2008; accepted 29 February 2008 KEYWORDS Florfenicol; LPS; Cytokines; Endotoxemia; NF-κB

Abstract Some antibacterials have been reported to regulate the host immune and inflammatory responses both in vitro and in vivo. Florfenicol is an antibiotics used in treatment of infection. We investigated the effects of florfenicol on cytokine production by lipopolysaccharide (LPS)stimulated RAW 264.7 macrophages in vitro, and the results showed that florfenicol reduced tumor necrosis factor (TNF) and interleukin-6 (IL-6) production but had little effect on interleukin-1β (IL-1β) and interleukin IL-10 (IL-10) secretion. This inspired us to further study the effects of florfenicol in vivo. Florfenicol significantly attenuated TNF and IL-6 production in serum from mice challenged with LPS, and in consistent with the results in vitro. In murine model of endotoxemia, mice were prophylactically or therapeutically treated with florfenicol prior to or after LPS challenge. The results showed that florfenicol significantly increased mouse survival. Further studies revealed that florfenicol prevented the LPS-induced nuclear factor-κB (NF-κB) translocation from cytoplasm into nuclear in RAW 264.7 macrophages. These observations indicate that florfenicol modulates early cytokine responses by blocking NF-κB pathway, and thus, increases mouse survival. This effect of the drug may be of potential usefulness in treatment of bacterial shock. © 2008 Elsevier B.V. All rights reserved.

1. Introduction ⁎ Corresponding authors. Deng is to be contacted at Tel.: +86 431 87836161; fax: +86 431 87836160. Cui, Tel.: +1 617 665 5573; fax: +1 617 665 5584. E-mail addresses: [email protected] (J. Cui), [email protected] (X. Deng). 1 These authors contributed equally to this work. 1567-5769/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2008.02.015

It has been shown that some antibacterials modulate the inflammatory responses by directly affecting host cell function in addition to exerting their antibacterial activity. In particular, drugs that target protein synthesis or DNA replication processes in bacteria have been suspected of causing this

Effects of florfenicol on early cytokine responses and survival in murine endotoxemia effect [1–5]. Florfenicol is a new type broad-spectrum antibacterial that is used in the veterinary clinic. It showed strong activity against both Gram-positive and Gram-negative bacteria. It has been widely used to treat a broad array of infectious diseases, such as for the treatments of bovine and porcine respiratory tract infections [6–8], actinobacillus pleuropneumonia in pigs [9] and infectious bovine keratoconjunctivitis [10,11], and it showed to be effective and safe. However, whether florfenicol has anti-inflammatory effect in addition to exerting its antibacterial activity and is effective in the treatment and management of some inflammatory diseases have not been demonstrated. The purpose of the work presented here was to examine florfenicol's effects on inflammatory responses in vitro and in vivo, and survival of mice after challenge with lipopolysaccharide (LPS). LPS is the major component of outer membranes of Gram-negative bacteria. LPS is a potent stimulus, which can induce strong immune and inflammatory responses, cause extensive tissue damage, and is considered to play a central role in mediating diseases caused by Gram-negative bacteria [12–14]. The inflammatory response after challenge with LPS is associated with the release of pro-inflammatory cytokines and other inflammatory mediators, including tumor necrosis factor (TNF), interleukin-1 (IL-1), and interleukin-6 (IL-6). The progressive production of these inflammatory mediators may result in the systemic inflammatory response syndrome, severe tissue damage, and septic shock [15,16]. Indeed, experimental models of endotoxic shock have demonstrated that a single injection of LPS into animals can produce changes that are characteristic of the septic shock syndrome [17]. Using RAW 264.7 murine macrophages and murine model of endotoxemia, we studied the effects of florfenicol on early

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cytokine production both in vitro and in vivo, and recorded mouse survival after challenge with LPS. Our results showed that florfenicol significantly inhibited TNF and IL-6 releases both in vitro and in vivo, and maintained serum interleukin-10 (IL-10) concentration in vivo, which could play a role in inhibiting of production of pro-inflammatory cytokines induced by LPS. In addition, the drug significantly increased mouse survival. Further, we explored the mechanism of this drug in inhibition of pro-inflammatory cytokine production, and found that florfenicol down-regulated LPS-induced nuclear factor-κB (NF-κB) pathway.

2. Materials and methods 2.1. Reagents LPS (Escherichia coli 055:B5), florfenicol, Cy3-conjugated sheep antirabbit IgG, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin and other reagents for cell culture were obtained from Life Technologies Inc. (Grand Island, NY, USA). Rabbit anti-NF-κB/p65 polyclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

2.2. Mice All experiments were performed in accordance with the guide for the Care and Use of Laboratory Animals published by US National Institute of Health. Female C57BL/6 mice, weighing approximately 18 to 20 g, were purchased from Shanghai Laisite Experimental Animals Co., LTD (Certificate SCXK2003-0003). The mice were housed in microisolator cages and received food and water ad libitum. Laboratory temperature

Figure 1 Effect of different concentrations of florfenicol (Flor) on secretion of TNF, IL-1β, IL-6 and IL-10 by LPS-induced RAW 264.7 cells. The cells were treated with 1 μg/ml of LPS alone or LPS plus different concentrations (5, 25, 100 μg/ml) of florfenicol for 6 h. Control values were obtained in the absence of LPS or Flor. The values are the means ± SEM of three independent experiments. ⁎P b 0.05 vs LPS group.

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was 24± 1 °C and relative humidity was 40–80%. Before experimentation, the mice were left to adapt to the experimental environment for 2 to 3 days.

bent assay (ELISA) using commercially available reagents according to the manufacturer's instructions (BioLegend, Inc. Camino Santa Fe, Suite E San Diego, CA, USA).

2.3. Cell culture

2.6. Cytokine assays in vivo

The RAW 264.7 murine macrophage cell line was obtained from the China Cell Line Bank (Beijing, China). The cells were cultured in DMEM supplemented with 10% heat-inactivated FBS at 37 °C under a humidified atmosphere of 5% CO2 and 95% air atmosphere.

Cytokine responses were assessed by measuring serum concentrations. Florfenicol (100 mg/kg) was given with an oral administration, control mice received an equal volume of vehicle instead of florfenicol. One hour later, all mice received LPS (30 mg/kg) by intraperitoneal injection (ip). Serum was separated from clotted blood at 0, 1, 3, 6 and 24 h following administration of ip LPS. Serum was stored at −70 °C and concentrations of cytokine TNF, IL-1β, IL-6 and IL-10 were measured by sandwich ELISA using commercially available reagents according to the manufacturer's instructions.

2.4. Cell viability assay Cytotoxicity studies induced by florfenicol were performed by MTT assay. RAW 264.7 cells were mechanically scraped, plated at a density of 4 × 105 cells/ml onto 96-well plates (Costar USA) containing 100 μl of DMEM medium, and incubated overnight. Florfenicol was dissolved in DMSO, and the DMSO concentrations in all assays did not exceed 0.1%. After overnight incubation, the cells were treated with diverse concentrations of florfenicol (0–300 µg/ml) in the presence or absence of LPS (1 μg/ml) according to the experimental design. After 24 h, 50 μl of MTT was added to each well and the cells were further incubated for 4 h at 37 °C with 5% CO2. MTT was removed and cells were lysed with 100 μl/well DMSO. The optical density was measured at 570 nm on a microplate reader (TECAN, Austria).

2.5. Cytokine assays in vitro To determine the effects of florfenicol on cytokine responses from LPS-induced cells, RAW 264.7 cells were plated onto 24-well plates (105 cells/well), and incubated in the presence of either LPS alone 1 μg/ml, or LPS plus florfenicol 5 μg/ml, 25 μg/ml, 100 μg/ml for 6 h at 37 °C with 5% CO2. Cell-free supernatants were collected and stored at − 20 °C until assayed for cytokines. The concentrations of cytokine TNF, IL-1β, IL-6 and IL-10 in the supernatants of RAW 254.7 cells culture were measured by sandwich enzyme-lined immunosor-

2.7. Murine model of LPS-induced endotoxemia C57BL/6 mice were challenged in groups of four with LPS (dose range: 10–40 mg/kg) by ip. Mice were observed on mortality for 7 days twice a day and feeding, movement and activity, and grooming (smooth and shiny coats vs dull and ruffled coats). Using the LPS concentration that induced 80–90% lethal as working solution for drug screening in the next step. In drug testing, using our established dose of LPS that was 80–90% lethal, the effect of florfenicol (50, 100 and 200 mg/kg) on LPS-induced mortality was assessed by dosing orally florfenicol 1 h before LPS challenge. To further observe the effects of florfenicol on endotoxemia, mice were administered florfenicol (100 mg/kg) at 0, 1, 4 or 12 h after LPS challenge, respectively. Survival was monitored for 7 days twice a day. Mice in control and LPS groups were only given vehicle or LPS.

2.8. Immunocytochemical analysis Activation of NF-κB was measured by immunocytochemical analysis. RAW 264.7 cells (105 cells/ml) were cultured on glass coverslips, which

Figure 2 Effect of single dose florfenicol (Flor) on TNF, IL-1β, IL-6 and IL-10 induced by 30 mg/kg LPS. Mice were given florfenicol (100 mg/kg) by ip 1 h before challenge with LPS. Serum levels of TNF, IL-1β, IL-6 and IL-10 were measured at 0, 1, 3 and 6 h following LPS challenge (n = 16). Means ± SEM. ⁎P b 0.05, ⁎⁎P b 0.01 vs LPS.

Effects of florfenicol on early cytokine responses and survival in murine endotoxemia plated onto 24-well plates, and then pretreated in the presence or absence of 5 μg/ml, 25 μg/ml or 100 μg/ml of florfenicol for 1 h. The cells were then stimulated with 1 μg/ml of LPS for 1 h at 37 °C with 5% CO2. The cells cultured on glass coverslips were washed with PBS and fixed with 4% formaldehyde for 30 min, then detergent extracted with 3% TritonX-100 for 10 min at room temperature. Coverslips were saturated with PBS containing 5% bovine serum albumin (BSA) for 30 min at room temperature, and processed for immunofluorescent staining with rabbit anti-NF-κB/p65 polyclonal antibody (1:50) fol-

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lowed by Cy3-conjugated sheep anti-rabbit IgG (1:100) and DAPI staining. Coverslips were mounted on slides and fluorescent signals were analyzed by fluorescent microscopy (Olympus, Japan).

2.9. Statistical analysis All values were expressed as mean ± SEM. Differences in survival of groups were assessed with Fisher's exact test. Differences between mean values of normally distributed data were assessed with one-

Figure 3 The survival rate of mice challenged with LPS of different doses and effect of single dose florfenicol on survival of LPSchallenged mice. A, The survival rate (%) of mice challenged with LPS of different doses. Mice were given 10, 20, 30 or 40 mg/kg of LPS by ip without treatment with florfenicol (n = 12 for each group). B, Effect of single dose florfenicol (Flor) on survival of LPS-challenged mice before LPS injection. Mice were divided into control, LPS and florfenicol treatment groups (n = 12 for each group). Mice in florfenicol treatment group were treated orally with 50 mg/kg, 100 mg/kg and 200 mg/kg of florfenicol 1 h before LPS challenge with 30 mg/kg. Mice in control and LPS groups were only given vehicle or LPS. The survival was assessed every 12 h for 7 days throughout the experiment. C, Effect of single dose florfenicol (Flor) on survival of LPS-induced mice after LPS injection. Mice were pretreated with 30 mg/kg of LPS, and then 100 mg/kg of florfenicol was administered at 0, 1, 4, 12 h after LPS injection, respectively. Mice in control and LPS groups were only given vehicle or LPS (n = 16 for each group). The survival was monitored every 12 h for 7 days. aP b 0.05, bP b 0.01 vs LPS group. cP b 0.01 vs control.

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way ANOVA (Dunnett's t-test) and two-tailed Student's t-test. Statistical significance was accepted at P b 0.05.

3.2. Effect of florfenicol on cytokine responses in LPS-challenged mice

3. Results

Early cytokine responses after LPS challenge have been well characterized and are known to occur within hours of LPS challenge in vivo. Specifically, TNF, IL-1β and IL-6 have been shown to be produced early in the response and have been suggested to play critical roles in driving physiological/pathological responses that lead to septic shock. To define the effects of florfenicol on cytokine responses in vivo, which associated with fatal outcome, we collected serum from mice at different time points after LPS injection, and determined cytokine concentrations with ELISA. The cytokine concentrations in florfenicol-pretreated and LPS-challenged mice were significantly different from those of LPS alone group at 1 h for TNF (P b 0.01) (Fig. 2A), at 3 h and 6 h for IL-6 (P b 0.05) (Fig. 2C), and at 6 h for IL-10 (P b 0.05) (Fig. 2D). Florfenicol consistently and significantly reduced TNF and IL-6 production, increased IL-10 response, and had no or little effect on IL-1β release (Fig. 2B). Further analysis of the samples collected at 24 h showed that the cytokine levels were either undetectable or close to baseline (data

3.1. Effect of florfenicol on LPS-induced cytokine production in vitro TNF, IL-1β, IL-6 and IL-10 concentrations in the culture supernatant of RAW 264.7 cells were measured by sandwich ELISA (Fig. 1). Treatment of RAW 264.7 cells with LPS alone resulted in significant increases of cytokine production compared to control. However, TNF and IL-6 levels in the cell supernatant treated with 25 and 100 μg/ml of florfenicol significantly decreased compared to those of LPS group (P b 0.05), and decreased in a dose-dependent manner (Fig. 1A and C). IL-1β level slightly decreased, but was not of significant change (P N 0.05) (Fig. 1B). IL-10 concentration treated with 25 and 100 μg/ml of florfenicol did not change significantly compared to that of LPS group (P N 0.05) (Fig. 1D).

Figure 4 Effect of florfenicol (Flor) on LPS-induced NF-κB activation by immunocytochemistry. The cells were pretreated with florfenicol for 1 h and cultured for 1 h with LPS, then were fixed, permeabilized, and incubated with rabbit anti-NF-κB/p65 polyclonal antibody followed by Cy3-conjugated anti-rabbit IgG (red). The nuclei of the corresponding cells were demonstrated by DAPI staining (blue). Magnification for images was 600×. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Effects of florfenicol on early cytokine responses and survival in murine endotoxemia not shown). The serum levels for TNF, IL-1β, IL-6 and IL-10 in naive and florfenicol alone groups were undetectable (data not shown).

3.3. Effect of florfenicol on LPS-mediated mortality It has been well established the murine model of LPS-induced endotoxemia, and, to find out dose responses to LPS in female C57BL/6 mice, we injected the animals with different doses of LPS and recorded survival rates. As shown in Fig. 3A, mice receiving 40 mg/kg of LPS all died within 36 h, but in the groups that mice were given 10 mg/kg, 20 mg/kg and 30 mg/kg of LPS, the mortality rates were 0%, 8% and 83%, respectively. All mice appeared less active and huddled together and lethargic. Based on this experiment, we chose 30 mg/kg of LPS as lethal dosage to induce endotoxemia in mice. In endotoxemia studies, mice were orally dosed with different doses of florfenicol and 1 h later challenged with LPS as shown in Fig. 3B. In the group of mice given LPS alone, the survival rate is only 17% after 48 h. In contrast, in the groups that mice received florfenicol at doses of 50 mg/kg, 100 mg/kg or 200 mg/kg, survival rates were up to 33%, 83% and 67%, respectively. The survival rates (100 mg/kg or 200 mg/kg groups) were significantly increased compared to that of the group that only received LPS (P b 0.01 or 0.05). No toxic effects of florfenicol were observed in mice only given florfenicol and without LPS challenge even in the group that received doses as high as 200 mg/kg of florfenicol. Next, we examined the therapeutic effects of florfenicol on LPSinduced endotoxemia to investigate whether florfenicol still protect mice from death. A single dose of florfenicol (100 mg/kg) given at 0 or 1 h after LPS challenge significantly increased survival rate of mice compared to LPS group (P b 0.01 or 0.05), and the survival rates were 63% and 38%, respectively (Fig. 3C). Even treatment with florfenicol at 4 h after LPS challenge, florfenicol was still of some protection from lethal LPS challenge whereas no protection was found in the group of drug administration 12 h delayed. Taken together, these findings indicate that florfenicol consistently prevents mice from LPS-induced death.

3.4. Mechanism of florfenicol in inhibiting TNF production Our studies indicated that florfenicol inhibited TNF release both in vitro and in vivo, and protected mice from LPS-induced death. We further explored the mechanism of florfenicol inhibiting TNF production. As it is well studied that LPS binds to its TLR on cell membrane and transduces signal to nuclear and promotes TNF gene transcription via NF-κB [21], we evaluated the effect of florfenicol on LPS-induced NF-κB activation by immunocytochemistry staining. We first analyzed the subcellular distribution of NF-κB subunit (p65) and found that p65 was distributed in the cytoplasmic compartment in all cells before LPS stimulation and after 1 h stimulation with LPS, most intracellular p65 protein translocated from cytoplasm to nucleus, demonstrated by strong p65 staining. However, nuclear translocation of p65 induced by LPS was strongly inhibited in cells preincubated with florfenicol and this prevention of p65 translocation was in a dose-dependent manner (Fig. 4).

4. Discussion In this study, we first demonstrated that florfenicol inhibited TNF and IL-6 production in cultured cell supernatant and it had little effects on IL-1β and IL-10 production in vitro (Fig. 1). Florfenicol alone and in the presence of LPS (1 μg/ml) did not affect the viability of RAW 264.7 cells when the cells were treated with various concentrations of florfenicol ranging from 0.5 to 300 μg/ml for 24 h and were subject to MTT assay (data not shown). These results assure us that the effects of florfenicol on RAW 264.7 cells are due to its inhibition of TNF

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and IL-6 pathway directly or indirectly but not due to cell death. TNF and IL-1β are both up-regulated after LPS challenge, and contribute to septic shock [18–20]. We further investigated the effects on florfenicol on these cytokines production in mouse models. As we expected, florfenicol inhibited TNF and IL-6 production, but florfenicol showed little effect on IL-1β production (Fig. 2). Further study in LPS endotoxic shock model showed florfenicol increased survival rate of mice challenged with LPS, and it was consistent with decrease of TNF in serum in florfenicol-treated group. IL-1β level in serum was changed a little between florfenicol-treated and LPS control group, indicating IL-1β did not play a major role in the endotoxic shock model. Bacterial Sepsis is caused by endotoxin such as LPS produced by bacteria in blood stream. To prevent infection, animals and human have developed precise innate immunity against infection, which has been discovered recently. For example, it has been found that one of the Toll-like receptors (TLR) expressed on cell membrane recognizes LPS, transduces the insulting signal to nuclear, results in proinflammatory cytokine production, and eventually, modifies acquired immunity [21]. TNF is one of the crucial pro-inflammatory cytokines. However, when over produced by dysregulation or persistent infection, TNF may induce septic shock. Anti-TNF reagents are effective drug in treatment of septic shock. It has been found that antibacterial reagents, ciprofloxacin and tetracycline, inhibit TNF production, and rescue mice from LPS-induced death [22,23]. Recently, it has been shown that ciprofloxacin binds to TLR receptor, but does not transduce signal. As a result, it desensitizes cells to LPS challenge and decreases the production of TNF and other pro-inflammatory cytokines [24]. We are exploring the mechanism of florfenicol in inhibiting TNF production and investigating if florfenicol uses a similar mechanism in intervening TLR pathway. Along with pro-inflammatory cytokines, IL-10 as an antiinflammatory cytokine, is up-regulated after LPS challenge in mice. Many documented publications indicate the role of IL-10 as an anti-inflammatory cytokine controlling septic shock. Fiorentino et al. [25] found that IL-10 prevented TNF production in activated macrophages. Berg and his colleague [26] found uncontrolled production of TNF in IL-10 deficient mouse after LPS challenge. Further, with the treatment of TNF neutralizing antibody, the mouse survival rates were significantly increased, demonstrating the importance of IL-10 in controlling TNF production. We demonstrated florfenicol prolonged IL-10 expression in serum as shown in Fig. 2D, and this concentration of IL-10 might be high enough to control proinflammatory cytokines at a lower level, and thus, improved survival rates. Several attempts in controlling septic shock are going on by intervening TNF pathway, including protein reagents, such as TNF antibody and Enbrel, a TNF receptor fusion protein [27]; as well as small molecule inhibitor of TNF, such as TACE inhibitors (TNF-α converting Enzyme) [28]. In addition, a few reports indicate that ciprofloxacin inhibits TNF production targeted at binding TLR4 without any signaling function, resulting in blocking LPS/TLR pathway, and attenuating TNF production. Our finding of florfenicol blocking TNF production may provide another potential way to develop therapeutic drug in treatment of septic shock.

988 NF-κB has been documented to play a major role in LPSinduced inflammatory cytokine expression [29,30]. In order to further explore the mechanism underlying the protective action of florfenicol against endotoxemia, immunocytochemical analysis revealed NF-κB factor p65, which is normally translocated from the cytoplasm to the nucleus after exposure to LPS, was strongly inhibited by florfenicol. This result suggests that the inhibition of florfenicol on cytokine production is through NF-κB sequestration, and it is necessary to investigate further to explore whether florfenicol binds to NF-κB directly or interferes the upstream of LPS/TLR pathway. In summary, based on in vitro assay and the results from murine model of endotoxemia, we found that florfenicol significantly inhibited TNF and IL-6 releases both in vitro and in vivo after challenge with LPS, and florfenicol rescued mice from LPS-induced death by reducing the pro-inflammatory cytokine production. The inhibition of cytokine production and thus increase of mouse survival rates were at least mediated by the suppression of NF-κB pathway. These findings provide important reference in clinical use of florfenicol, and it may be of potential as a therapeutics in treatment of septic shock.

Acknowledgment This work was supported by a grant from the National Natural Science Foundation of China (No. 30671586).

References [1] Schultz MJ, Speelman P, Zaat S, van Deventer SJ, Van der Poll T. Erythromycin inhibits tumor necrosis factor alpha and interleukin 6 production induced by heat-killed Streptococcus pneumoniae in whole blood. Antimicrob Agents Chemother 1998;42:1605–9. [2] Shapira L, Soskolne WA, Houri Y, Barak V, Halabi A, Stabholz A. Protection against endotoxic shock and lipopolysaccharideinduced local inflammation by tetracycline: correlation with inhibition of cytokine secretion. Infect Immun 1996;64:825–8. [3] Cao XY, Dong M, Shen JZ, Wu BB. Tilmicosin and tylosin have antiinflammatory properties via modulation of COX-2 and iNOS gene expression and production of cytokines in LPS-induced macrophages and monocytes. Int J Antimicrob Agents 2006;27:431–8. [4] Purswani M, Eckert S, Arora H, Johann-Liang R, Noel GJ. The effect of three broad-spectrum antimicrobials on mononuclear cell responses to encapsulated bacteria: evidence for downregulation of cytokine mRNA transcription by trovafloxacin. J Antimicrob Chemother 2000;46:921–9. [5] Ziegeler S, Raddatz A, Hoff G, Buchinger H, Bauer I, Stockhausen A, et al. Antibiotics modulate the stimulated cytokine response to endotoxin in a human ex vivo, in vitro model. Acta Anaesthesiol Scand 2006;50:1103–10. [6] Shin SJ, Kang SG, Nabin R, Kang ML, Yoo HS. Evaluation of the antimicrobial activity of florfenicol against bacteria isolated from bovine and porcine respiratory disease. Vet Microbiol 2005;106: 73–7. [7] Priebe S, Schwarz S. In vitro activities of florfenicol against bovine and porcine respiratory tract pathogens. Antimicrob Agents Chemother 2003;47:2703–5. [8] Aslan V, Maden M, Erganis O, Birdane FM, Corlu M. Clinical efficacy of florfenicol in the treatment of calf respiratory tract infections. Vet Q 2002;24:35–9. [9] Ueda Y, Ohtsuki S, Narukawa N. Efficacy of florfenicol on experimental actinobacillus pleuropneumonia in pigs. J Vet Med Sci 1995;57:261–5.

X. Zhang et al. [10] O'Connor AM, Wellman NG, Evans RB, Roth DR. A review of randomized clinical trials reporting antibiotic treatment of infectious bovine keratoconjunctivitis in cattle. Anim Health Res Rev 2006;7:119–27. [11] Angelos JA, Dueger EL, George LW, Carrier TK, Mihalyi JE, Cosgrove SB, et al. Efficacy of florfenicol for treatment of naturally occurring infectious bovine keratoconjunctivitis. J Am Vet Med Assoc 2000;216:62–4. [12] Saluk-Juszczak J, Wachowicz B, Saluk JJ, Wachowicz B. The proinflammatory activity of lipopolysaccharide. Postepy Biochem 2005;51:280–7. [13] Fujihara M, Muroi M, Tanamoto K, Suzuki T, Azuma H, Ikeda H. Molecular mechanisms of macrophage activation and deactivation by lipopolysaccharide: roles of the receptor complex. Pharmacol Ther 2003;100:171–4. [14] Mayeux PR. Pathobiology of lipopolysaccharide. J Toxicol Environ Health 1997;51:415–35. [15] Shimazu R, Akashi S, Ogata H, Nagai Y, Fukudome K, Miyake K, et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med 1999;189:1777–82. [16] Parrillo JE, Parker MM, Natanson C, Suffredini AF, Danner RL, Cunnion RE, et al. Septic shock in humans. Advances in the understanding of pathogenesis, cardiovascular dysfunction, and therapy. Ann Intern Med 1990;113:227–42. [17] Muchamuel T, Menon S, Pisacane P, Howard MC, Cockayne DA. IL-13 protects mice from lipopolysaccharide-induced lethal endotoxemia: correlation with down-modulation of TNF-alpha, IFN-gamma, and IL-12 production. J Immunol 1997;158:2898–903. [18] Remick DG, Kunkel RG, Larrick JK, Kunkel SL. Acute in vivo effects of human recombinant tumor necrosis factor. Lab Invest 1987;56:583–90. [19] Beutler B, Cerami A. Tumor necrosis, cachexia, shock, and inflammation: a common mediator. Annu Rev Biochem 1988;57: 505–18. [20] Dinarello CA. Interleukin-1. Rev Infect Dis 1984;6:51–95. [21] Takeda K, Akira S. Regulation of innate immune responses by Toll-like receptors. Jpn J Infect Dis 2001;54:209–19. [22] Purswani MU, Eckert SJ, Arora HK, Noel GJ. Effect of ciprofloxacin on lethal and sublethal challenge with endotoxin and on early cytokine responses in a murine in vivo model. J Antimicrob Chemother 2002;50:51–8. [23] Shapira L, Barak V, Soskolne WA, Halabi A, Stabholz A. Effects of tetracyclines on the pathologic activity of endotoxin: in vitro and in vivo studies. Adv Dent Res 1998;12:119–22. [24] Katsuno G, Takahashi HK, Iwagaki H, Sugita S, Mori S, Saito S, et al. The effect of ciprofloxacin on CD14 and toll-like receptor-4 expression on human monocytes. Shock 2006;25:247–53. [25] Fiorentino DF, Zlotnik A, Mosmamm TR, Howard M, O'Garra A. IL-10 inhibits cytokine production by activated macrophages. J Immunol 1991;146:3444–51. [26] Berg DJ, Kühn R, Rajewsky K, Müller W, Menon S, Davidson N, et al. Interleukin-10 is a central regulator of the response to LPS in murine models of endotoxic shock and the Shwartzman reaction but not endotoxin tolerance. J Clin Invest 1995;96: 2339–47. [27] Mutschler D, Wikstrom G, Lind L, Larsson A, Lagrange A, Eriksson M. Etanercept reduces late endotoxin-induced pulmonary hypertension in the pig. J Interferon Cytokine Res 2006;26:661–7. [28] Souza DG, Ferreira FL, Fagundes CT, Amaral FA, Vieira AT, Lisboa RA, et al. Effects of PKF242-484 and PKF241-466, novel dual inhibitors of TNF-alpha converting enzyme and matrix metalloproteinases, in a model of intestinal reperfusion injury in mice. Eur J Pharmacol 2007;571:72–80. [29] Guha M, Mackman N. LPS induction of gene expression in human monocytes. Cell Signal 2001;13:85–94. [30] Tian B, Brasier AR. Identification of a nuclear factor kappa B-dependent gene network. Recent Prog Horm Res 2003;58: 95-130.