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Antioxidant Amplifies Antibiotic Protection in the Cecal Ligation and Puncture Model of Microbial Sepsis Through Interleukin-10 Production

Kotake, Yashige; Moore, Danny R.; Vasquez-Walden, Angelica; Tabatabaie, Tahereh; Sang, Hong

Basic Science Aspects
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Preadministration of antioxidants such as pyrrolidine dithiocarbamate (PDTC) and phenyl N-tert-butyl nitrone (PBN) protects animals from lethality in sepsis models. However, the requirement of preadministration greatly diminishes the clinical significance of these studies. Although the synthetic antioxidant PBN has been shown to effectively protect rodents from lethality in endotoxemia (lipopolysaccharide [LPS] model), preliminary screening indicates that pre- or postadministration of PBN does not protect in the rat cecal ligation and puncture (CLP) model. We show in this report that in a rat CLP model, the administration of PBN (150 mg/kg, 30 min after CLP) followed by the antibiotic imipenem (IMP; 10 mg/kg, 1 h after CLP) significantly increased survival compared with other single treatment groups. Previously, we have shown that PBN's protection in a rat LPS model is mediated by the overproduction of the anti-inflammatory cytokine interleukin (IL)-10. We show in this study that the increase in survival found in the PBN + IMP-treated group was abrogated by immunoneutralization with anti-IL-10 antibody, indicating that endogenous IL-10 is an effective protective factor. Plasma LPS levels were shown to be elevated after imipenem treatment, and the increased LPS level could have assisted to overproduce endogenous IL-10, as in the case of the PBN-treated LPS model. Statistical analysis indicated that the increase of IL-10 in PBN + IMP-treated group at early time period has significant association to the improvement of survival.

Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104

Received 26 Feb 2002;

first review completed 8 Apr 2002; accepted in final form 13 Aug 2002

Address reprint requests to Yashige Kotake, PhD, Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, OK 73104.

Partial support of this work was provided by the National Institutes of Health (grant no. GM 54878).

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INTRODUCTION

Pretreatment with some antioxidants in a lipopolysaccharide (LPS) model has been shown to decrease cytokine production and lethality (1–5). For example, a synthetic antioxidant and free radical trapping compound, phenyl N-tert-butyl nitrone (PBN) showed a dramatic protection from endotoxin-mediated lethality in mice (4). Nevertheless, the requirement of pre- or prophylactic administration for this protection diminishes the practical significance of the study. In another animal model of sepsis, cecal ligation and puncture (CLP)-induced polymicrobial sepsis, preadministration of the antioxidant N-acetyl cysteine (NAC) provided moderate protection from lethality in mice, but again, preadministration was required for the protection (6). Our preliminary tests on the pre- or postadministration of various doses of PBN in the rat CLP model have not been successful.

Previously, we have shown that the mechanism of PBN protection in an LPS model was through overproduction of the anti-inflammatory cytokine interleukin 10 (IL-10) (7,8). Immunoneutralization with anti-IL-10 antibody abrogated the protection, clearly indicating that PBN-mediated IL-10 overproduction played an essential role in this protection (8). In the LPS model, preadministration of pyrrolidine dithiocarbamate (PDTC), a well-known glutathione precursor, has been shown to mediate IL-10 overproduction (9), protect rodents from LPS lethality (10), and down-regulate proinflammatory factors (1,2). However, the relationship between the PDTC-mediated IL-10 production and protection has not been studied. In other studies, recombinant IL-10 administration protected mice from LPS lethality (11,12), which is in line with PBN's protection in a lethal LPS model through the IL-10 mechanism. In addition, mice deficient in IL-10 showed a highly elevated sensitivity to LPS (13,14). However, in a CLP model, studies using recombinant IL-10 as a protective agent yielded mixed results (15,16). Clinical trials to treat inflammatory diseases such as inflammatory bowel disease with recombinant IL-10 have been initiated and have resulted in limited success (17,18).

The administration of PBN without LPS did not promote IL-10 overproduction, suggesting that PBN mediates IL-10 overproduction in response to the systemic level of LPS (8). Previously, it has been shown in a CLP model that antibiotic administration temporarily raises the systemic LPS level, likely due to a rapid release of bacterial cell debris to the system (19). We hypothesized that PBN may respond to antibiotic-mediated LPS release and promote overproduction of endogenous IL-10, as it did in LPS model. The induced IL-10 may then be able to protect animals from CLP lethality. In this report, we tested this hypothesis and we show that the cotreatment of PBN with imipenem (IMP), a thienamycin antibiotic with a fast bactericidal action, provided significant protection from lethality in a rat CLP model.

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MATERIALS AND METHODS

CLP

The animal use protocol approved by the institutional animal care and use committee at the Oklahoma Medical Research Foundation was strictly adhered to for all animal experiments. The experiments were performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals. Male Wistar rats weighing 250 to 350 g were obtained from Charles River Laboratories (Wilmington, MA) and were acclimated at least 1 week. Rats were anesthetized with isoflurane (Halocarbon Laboratories, River Edge, NJ) using 95% oxygen/5% carbon dioxide as a carrier gas. After a thorough shaving of abdominal hair, the peritoneum was aseptically opened with a small incision, and the cecum was tied with 3.0 silk surgical thread 1.5 cm from the distal end. One through-and-through puncture was made using an 18-gauge needle, and a small amount of stool was expelled from the punctures to ensure leakage of the intestinal content. The abdomen was closed with a 3.0 suture, and the animals were rehydrated with a subcutaneous injection of normal saline (0.5 mL).

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Materials

PBN (Sigma Chemical Co., St. Louis, MO or made in our laboratory) was dissolved in normal saline and was administered intraperitoneally (i.p.) at a dose of either 50 or 150 mg/kg. IMP (Primaxin; Merck & Co., West Point, PA) was dissolved in normal saline and administered subcutaneously (s.c.) at the dose of 10 mg/kg. In immunoneutralization experiments, polyclonal anti-IL-10 antibody and nonimmune immunoglobulin (Ig) G (both from R&D Systems, Minneapolis, MN) were dissolved in 100 μL of phosphate-buffered saline (PBS)/normal saline and were administered intravenously (i.v.) 15 min after PBN administration at the dose of 2 μg/rat.

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Treatment with PBN and IMP: Selection of dose schedule

Numerous combinations of dosages and timings are possible when two drugs are used for treatment. We designed the dose schedule based on the hypothesis that PBN react to the antibiotic-mediated increase of LPS, promoting IL-10 production. Therefore, a typical dose schedule was: CLP (18-gauge, one through- and through puncture) + (30 min) + PBN (150 mg/kg, i.p.) + (30 min) + IMP (10 mg/kg, s.c.). Survival was monitored for 48 h after CLP.

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A single PBN treatment

We performed a preliminary screening for the protective activity of a single PBN administration in the rat CLP model: Group a-1: laparotomy alone (control); Group a-2: CLP alone; Group a-3: PBN (50 mg/kg) + (30 min) + CLP; Group a-4: PBN (150 mg/kg) + (30 min) + CLP; Group a-5: CLP + (30 min) + PBN (50 mg/kg); and Group a-6: CLP + (30 min) + PBN (150 mg/kg).

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PBN-IMP cotreatment

PBN-IMP cotreatment groups consisted of Group b-1: laparotomy alone (control); Group b-2: CLP alone; Group b-3: CLP + (30 min) + PBN (150 mg/kg); Group b-4: CLP + (30 min) + IMP (10 mg/kg); and Group b-5: CLP + (30 min) + PBN (150 mg/kg) + (30 min) + IMP (10 mg/kg).

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IL-10 immunoneutralization

IL-1-immunoneutralization groups consisted of Group c-1: CLP alone (control); Group c-2: IL-10Ab (2 μg/rat) alone (control); Group c-3: CLP + (30 min) + IL-10Ab; Group c-4: CLP + (30 min) + IMP (10 mg/kg) + (30 min) + PBN (150 mg/kg); Group c-5: CLP + (30 min) + PBN (150 mg/kg) + (15 min) + IL-10Ab (2 μg/rat) + (15 min) + IMP (10 mg/kg); and Group c-6: CLP + (30 min) + PBN (150 mg/kg) + (15 min) + goat (nonimmune) IgG (2 μg/rat) + (15 min) + IMP (10 mg/kg).

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Blood collection for cytokine and LPS level determination

The alteration of cytokine and LPS levels in the early period of CLP sepsis was determined. Independent from survival experiments, seven rats were randomly assigned to each group (Groups b-2 through b-5). Blood was collected from the tail vein three times at specified times before and after surgery: 1 day before surgery (labeled as 0 h), 1.5 h after surgery (labeled as 1.5 h), and 2.5 h after surgery (labeled as 2.5 h). In other experiments, rats (n = 7, Groups b-2 through b-5) were treated in a similar manner, but the blood was collected 6 h after CLP. Rats were lightly anesthetized with isoflurane, and approximately 0.5 mL of blood was drawn into a heparinized syringe each time. Basal cytokine and LPS levels were determined 1 day before CLP surgery to avoid the influence of frequent blood drawing. Plasma was isolated from whole blood with brief centrifugation.

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LPS measurements

Plasma LPS levels were colorimetrically measured using a Pyrochrome LAL kit (Associates of Cape Cod Inc., Falmouth, MA) with endotoxin-free water and plastic ware obtained from the manufacturer. LPS provided by the manufacturer was used as a concentration standard.

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IL-10, TNFα, and IL-6 enzyme-linked immunosorbent assay (ELISA)

IL-10, TNFα, and IL-6 levels in plasma were determined using an ELISA kit (OptEIA set) obtained from PharMingen (San Diego, CA). Assays were performed according to the manufacturer's instructions. Briefly, 96-well plates were coated with anti-rat monoclonal antibody in coating buffer (0.2 M sodium phosphate, pH 6.5, for IL-10 and 0.1 M carbonate, pH 9.5, for TNFα and IL-6) overnight at 4°C. The plates were washed with PBS-0.05% Tween-20 and blocked with PBS-10% fetal calf serum (FCS), pH 7.0. A serial dilution of standard and rat plasma samples was incubated in the plates for 2 h at room temperature. The plates were washed with PBS-0.05% Tween-20 and were incubated with a secondary biotinylated anti-rat antibody plus avidin-horseradish peroxidase conjugate. After the plates were washed with PBS-0.05% Tween-20, IL-10, TNFα, and IL-6 were detected with colorimetry by adding the substrate solution (tetramethyl benzidine plus hydrogen peroxide) to each well, and the absorbance at 450 nm was recorded using a ThermoMax microplate reader (Molecular Devices, Menlo Park, CA). Assays were performed in duplicate, and data were obtained from plasma diluted 10-fold.

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Statistics

Data were presented as mean ± SD and were analyzed with a one-way analysis of variance (ANOVA). Survival data were evaluated with log-rank analysis. Cox proportional hazards models were used to assess the association of IL-10 and other cytokine levels to survival times.

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RESULTS

Survival analyses

Preliminary screening to determine whether a single pre- or posttreatment with PBN would protect CLP rats was performed using five to seven animals per group, and the results are listed in Table 1. Pre- (30 min before CLP) or post- (30 min after CLP) treatment with PBN (50 or 150 mg/kg, i.p.) did not increase survival evaluated during the 48 h after CLP (Table 1). Thus, we tentatively conclude that a single pre- or post-PBN treatment is not effective in increasing survival.

Table 1

Table 1

Survival 48 h after CLP for rats treated with PBN plus IMP was 69% (n = 16;Fig. 1). Survival was less than 25% in the CLP alone (n = 16), PBN (n = 16), and IMP (n = 16) groups. All animals receiving laparotomy alone (no CLP) survived at least 48 h (n = 14). Survival data were evaluated with log-rank analysis: the hazard for IMP + PBN group (protected group) was 3.8 times lower than CLP alone group (P = 0.02), indicating that IMP + PBN treatment significantly reduced the hazard and increased survival as compared with animals treated with CLP alone. The hazard for IMP + PBN group was 3.7 times lower than PBN group (P = 0.01), and 3.4 times lower than IMP group (P = 0.02).

Fig. 1

Fig. 1

Survival time data from different treatment groups were evaluated with log-rank analysis. There were no significant differences in survival times among the groups treated with the CLP alone, PBN, and IMP (P > 0.8). However, compared with the PBN + IMP group, the hazard was 3.8 times higher (P = 0.01) for the CLP alone group, 3.7 times higher (P = 0.01) for the PBN group, and 3.4 times higher (P = 0.02) for the IMP group. The results indicate that IMP + PBN treatment significantly reduced the hazard and increased survival time than the other three groups.

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Effect of PBN + IMP treatment on plasma LPS, IL-10, TNFα, and IL-6 levels

IMP administration altered the level and time course of plasma LPS level (IMP and PBN + IMP group in Fig. 2). In the groups treated with IMP or PBN + IMP, plasma LPS levels continuously increased after CLP up to 2.5 h, and then decreased. However, in control groups, significantly elevated LPS levels were obtained in 6 h after CLP. With IL-10, both the IMP and PBN + IMP groups exhibited elevated IL-10 1.5 h after CLP. The IL-10 level in the IMP group decreased in 2.5 h after CLP; in contrast, in PBN + IMP group, it was still higher than other groups (P < 0.02). The IL-10 level 6 h after CLP was significantly elevated in control (nontreated and PBN-treated) groups as compared with other treated groups. With TNFα, in PBN + IMP group, TNFα level was suppressed close to the basal level at least 6 h after CLP, whereas other groups showed gradually elevated TNFα. With IL-6, in 6 h after CLP, the IL-6 level showed a significant discrepancy between control groups and IMP-treated groups (P < 0.02).

Fig. 2

Fig. 2

The Cox proportional hazards model (20) was used to find which of the IL-10 level changes during various time intervals (0–1.5, 0–2.5, 0–6, 1.5–2.5, 1.5–6, or 2.5–6 h after CLP) was closely associated with the survival time. The changes in IL-10 level during 0–2.5 h after CLP was found to be significantly (P = 0.0075) associated with the survival time. In the model, the estimated coefficient was −0.0078, which implied that, for instance, a 10 pg/mL increase of IL-10 during 0–2.5 h after CLP may cause the hazard reduction by 7.5% {= (1 − exp[−0.0078 × 10]) × 100}. From our data, the averages of IL-10 level changes during this period was 178 pg/mL in the IMP + PBN group, and 73 pg/mL in the CLP alone group (Fig. 2). Therefore, the hazard for the IMP + PBN group reduced about 79% (= (7.5 × [178 – 73]/10) × 100) as compared with the CLP alone group. This percentage of reduction is close to the log-rank analysis for the survival, i.e., 74% (= [1 − 1/3.8] × 100).

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DISCUSSION

Using a rat CLP model, we demonstrated that PBN administration followed by a nonprotective dose of the antibiotic imipenem significantly increases survival (Fig. 1). To verify that this protection is mediated by PBN-induced IL-10 production, immunoneutralization experiments using neutralizing anti-IL-10Ab were performed. The administration of anti-IL-10Ab shortly after PBN abrogated the protection (Fig. 3), indicating that induced production of endogenous IL-10 is critical in this protection. In rodent LPS models, the protective capacity of endogenous and exogenous IL-10 is well documented (12,21,22). Furthermore, mice deficient in IL-10 (C57BL/6 strain) exhibit an extremely low lethal dose of LPS (20-fold lower than wild type), supporting the protective role of endogenous IL-10 (14,23,24). IL-10 has been shown to globally reduce the production of multiple cytokines, including IL-10 itself (25,26).

Fig. 3

Fig. 3

In the present study, we showed that the increase of plasma IL-10 in a specific time window in CLP-treated animals is strongly associated with the improvement of survival. Previous studies have shown that the role of IL-10 in sepsis is complex and contradictory (27). IL-10 down-regulates the bactericidal cytokine TNFα (28,29), therefore, if IL-10 is administered or expressed at the wrong time, bacteremia could be exacerbated (30). A recent study by Kalechman et al. (31), in fact, showed that administration of an IL-10 inhibitor 12 h after CLP significantly increased survival and rIL-10 abrogated this protection. Previously, delayed (12 h after CLP) IL-10Ab treatment has been shown to increase survival (32). In contrast to the dramatic protection provided by rIL-10 in LPS models, IL-10 treatment in a lethal CLP model showed mixed results. Walley et al. (32) demonstrated that administration of IL-10 (5 μg/mouse) prior to CLP decreased mortality. Kato et al. (15) showed that murine rIL-10 treatment (1.0 μg/mouse) 6 h after 21-gauge CLP decreased lethality by approximately one-half, but other doses provided lesser protection. Remick et al. (16) showed in a mouse CLP model that human rIL-10 treatment (1.0–10.0 μg/mouse) failed to decrease mortality. Kahlke et al. (33) demonstrated that early rIL-10 treatment improves survival only in males in a mouse hemorrhage plus CLP sepsis model. In the present experiment, IL-10 levels were increased 6 h after CLP in control groups, but decreased in PBN/IMP-treated groups, and such time courses are similar to those of LPS. Previously, IMP administration in a mouse CLP model has been shown to induce plasma and peritoneal LPS within 30 min after administration (19,34), and present data also demonstrate the increase of LPS after IMP administration (Fig. 2). We postulate that PBN's ability to induce IL-10 in response to endogenous LPS helped in maintaining the elevated level of IL-10, and thus the duration of high IL-10 at very early stage of sepsis may be critical for the increase of survival. This interpretation is supported by a Cox proportional hazard model analysis that showed a significant association between early IL-10 increase and survival. Overall, these data imply that TNFα and perhaps other inflammatory cytokines are resistant to IL-10 elevation later in the sepsis cycle, and the very early control of inflammatory cytokines is important in preventing septic mortality.

Plasma IL-6 increased in all treatments 2.5 h after CLP, which is in agreement with the previous observation in a mouse CLP model (34); however, 6 h after CLP, significant differences (P < 0.02) in IL-6 between control (CLP alone and CLP + PBN) groups and IMP or PBN + IMP-treated groups. A recent report indicated that in IMP-treated mice, the IL-6 level measured 6 h after CLP was been demonstrated to be a prognostic parameter for mortality (21 days) (35). Although the present data indicate significantly lower IL-6 measured 6 h after CLP in IMP and PBN + IMP groups, there was no improvement in survival in IMP group.

In conclusion, we have shown that post- or therapeutic treatment with PBN plus IMP was effective in protecting rats from lethality caused by CLP, whereas the treatment with PBN or IMP alone was not. The mechanism by which PBN mediates IL-10 overproduction in the LPS model or the antibiotic-treated CLP model remains to be determined. The clinical significance of this animal model study is yet to be determined; however, low toxicity of the antioxidant would be a factor in favoring future trials.

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ACKNOWLEDGMENTS

The authors thank Dr. Wenyu Wang, Department of Biostatistics, University of Oklahoma Health Sciences Center, for his assistance in statistical analysis.

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Keywords:

Cecal ligation and puncture; phenyl N-tert-butyl nitrone; PBN; imipenem; LPS; immunoneutralization; IL-10Ab

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