Granulocyte Colony-Stimulating Factor and Antibiotics ...

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Granulocyte Colony-Stimulating Factor and Antibiotics in the Prophylaxis of a Murine Model of Polymicrobial Peritonitis and Sepsis Pia Villa, Christine L. Shaklee, Cristina Meazza, Davide Agnello, Pietro Ghezzi, and Giorgio Senaldi

‘‘Mario Negri’’ Institute for Pharmacological Research and CNR Cellular and Molecular Pharmacology Center, Milan, Italy; Amgen, Inc., Thousand Oaks, California

Infections that occur after intraabdominal surgery still cause considerable morbidity and mortality despite the administration of prophylactic antibiotics. Increasing the number of neutrophils may also be a prophylactic approach, and granulocyte colony-stimulating factor (G-CSF) has been found to be beneficial in different animal models of peritonitis and sepsis. It is the combination of G-CSF and antibiotics, however, that is clinically relevant. Treatment of mice with G-CSF that was started before cecal ligation and puncture and continued afterward with antibiotics improved survival, decreased splenic bacterial colony-forming units and serum tumor necrosis factor, and increased serum interleukin-10, compared with treatment with antibiotics alone or with saline. Compared with saline, antibiotics alone increased tumor necrosis factor and did not affect interleukin-10. Thus, G-CSF confers onto antibiotics beneficial antiinfectious and antiinflammatory properties. A prophylactic regimen combining G-CSF and antibiotics may help prevent severe infectious complications following intraabdominal surgery.

Received 8 December 1997; revised 9 March 1998. Procedures involving animals and their care were conducted in conformity with institutional guidelines that are in compliance with national and international laws and policies (Italian Legislative Decree 116, GU suppl. 40, 1992; EEC Council Directive 86/609, OJL 358, 1987; US National Research Council, Guide for the Care and Use of Laboratory Animals, 1996). Financial support: D.A. is recipient of a fellowship from the ‘‘A. and A. Valenti’’ Foundation. Reprints or correspondence: Dr. Giorgio Senaldi, Amgen, Inc., M/S 15-2C, Amgen Center, 1840 DeHavilland Dr., Thousand Oaks, CA 91320-1789 ([email protected]). The Journal of Infectious Diseases 1998;178:471–7 q 1998 by the Infectious Diseases Society of America. All rights reserved. 0022–1899/98/7802–0023$02.00

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counts and improved survival, organ function, and inflammatory parameters if it was begun, at the latest, immediately after the induction of infection. However, the administration of GCSF in combination with antibiotics (the treatment that would be of most clinical relevance) [1, 5, 22] has been investigated to a limited extent with conflicting results [14, 17]. The aim of this study was to evaluate the effects of the administration of G-CSF and antibiotics in the prophylaxis of a murine model of polymicrobial peritonitis and sepsis caused by cecal ligation and puncture (CLP). Materials and Methods Mice, CLP, and treatments. Male CD1 mice weighing 25–28 g (Charles River Laboratories, Calco, Italy) were used. Mice were housed in rooms maintained at controlled conditions (227C, 55% humidity, and light and dark alternating every 12 h), and they received standard rodent chow and water ad libitum. CLP was done as previously described [23, 24]. In brief, mice were anesthetized and subjected to a 1-cm celiotomy. The cecum was isolated, ligated immediately distal to the ileo-cecal valve, and punctured on the antimesenteric side with a 20-gauge needle. The cecum was then gently squeezed to ascertain the patency of the punctured holes and replaced into the abdomen. Last, the incision was closed with sutures, and the mice were injected subcutaneously with 1 mL of saline to favor resuscitation. Once a day, mice received subcutaneously 100 mg/kg murine G-CSF (Amgen, Thousand Oaks, CA), antibiotics (3.2 mg/kg gentamicin sulphate from Schering-Plough, Kenilworth, NJ, and 40 mg/g clindamycin phosphate from Upjohn, Kalamazoo, MI, which is a combination of acknowledged value [5]), G-CSF plus antibiotics, or saline. G-CSF was given for either 7 days (starting 3 days before the CLP procedure and continuing through 3 days after the CLP) or for 4 days (starting 3 days before the CLP procedure and continuing through the CLP procedure day or starting immediately after the CLP and continuing for the 3 subsequent days). Antibiotics were given for 4 days,

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Postoperative peritonitis and sepsis are frequent complications of critically ill patients undergoing abdominal surgery [1, 2]. These conditions are due to a polymicrobial infection with extracellular microorganisms from the intestinal flora [1, 3], and continue to cause high morbidity and mortality [1, 4] even though prophylactic antibiotics are routinely administered [1, 5]. Neutrophils are primary effectors in the host’s defense against infections with extracellular microorganisms [6]. Neutrophils can eliminate infectious agents directly by phagocytosis [7] and indirectly by the secretion of an array of mediators that regulate the inflammatory process [8]; therefore, the stimulation of neutrophils is an appealing approach to the treatment of infections [9]. In the case of the polymicrobial infection that follows intraabdominal surgery, this approach may be of prophylactic value as is the administration of antibiotics. Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor that increases the number and function of neutrophils [10]. Of interest, the administration of G-CSF is beneficial in animal models of peritonitis and sepsis [11 – 21]. In these models, G-CSF treatment increased neutrophil

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Figure 1. Survival curves after cecal ligation and puncture (CLP) of mice treated with G-CSF plus antibiotics, antibiotics alone, or saline. A, G-CSF was given for 7 days, starting 3 days before CLP. B, G-CSF was given for 4 days, starting immediately after CLP.

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Measurement of tumor necrosis factor (TNF). Mice were sacrificed by exsanguination 2 h after CLP, the time that TNF peaks in the circulation [24]. Serum was collected, and lungs were removed and extensively rinsed in saline. A lung extract was then prepared by homogenizing lungs in saline and separating residual debris by centrifugation. TNF was measured in serum and lung by bioassay as previously described [25], using L929 cells and recombinant human TNF (Genzyme, Cambridge, MA) as standards. Sensitivity to and specificity of this bioassay for murine TNF were demonstrated using a rat anti-mouse TNF monoclonal antibody, which inhibited by ú95% any activity on the L929 cells in serum and organ tissues from lipopolysaccharide-treated mice [26]. Results were expressed in picograms per milliliter of serum and in nanograms per gram of lung. Measurement of interleukin (IL)-6 and IL-10. Blood samples were obtained from mice 2 or 24 h after CLP, and serum levels of IL-6 and IL-10 were measured by ELISA, using commercially available kits (BioSource International, Camarillo, CA) and following the manufacturer’s instructions. Results were expressed in picograms per milliliter. IL-6 was measured 2 and 24 h after CLP because the cytokine reaches maximum serum levels during this time [24]; IL-10 was measured 24 h after CLP because it peaks in the circulation at this time, as indicated by preliminary experiments.

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starting on the day of the CLP and continuing for the 3 subsequent days. Mice were monitored for survival every 12 h after CLP. Neutrophil counts. After mice were treated for 3 days with GCSF or saline, we obtained blood from some mice and then sacrificed them so that blood and peritoneal neutrophil counts could be done. The remaining mice underwent CLP and received another dose of G-CSF or saline immediately after the procedure; then, blood samples were obtained, and the animals were sacrificed at 2, 8, or 24 h after the CLP so that neutrophils could be counted. Blood was drawn from the retroorbital plexus under anesthesia, and peritoneal neutrophils were collected by lavage with saline. A microscope was used to count neutrophils in a hemocytometer after diluting the blood 1/20 and the lavage fluid 1/3 in Turk’s solution. Counts of bacterial colony-forming units (cfu). After 3 days of G-CSF or saline treatment, some mice were sacrificed for spleen removal. The remaining mice underwent CLP, followed immediately by another dose of G-CSF or saline, and were sacrificed for spleen removal 2, 8, or 24 h after CLP. Spleens were removed under aseptic conditions and homogenized in saline. The extract was serially diluted in more saline, plated on tryptic soy agar (Difco, Detroit), and incubated at 377C. Colonies were counted the next day. Results were expressed as cfu per gram of spleen.


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Figure 2. Survival curves after cecal ligation and puncture (CLP) of mice treated with G-CSF or saline. A, G-CSF was given for 7 days, starting 3 days before CLP. B, GCSF was given for 4 days, starting 3 days before CLP. C, G-CSF was given for 4 days, starting immediately after CLP. Downloaded from jid.oxfordjournals.org by guest on July 14, 2011

Statistical analysis. Results were expressed as mean { SD. When necessary, actual values were log-transformed to lessen their skewed distribution and bring them to normality. The ShapiroWilks test was used to analyze the normality of value distribution before and after transformation. When the skewed distribution of values resisted log transformation, results were expressed as median (range). Accordingly, differences between and within groups were tested by the analysis of variance (ANOVA) and Student’s t test or by the Kruskal-Wallis and the Wilcoxon rank sum tests. Times of survival were compared by use of the Gehan-modified Wilcoxon rank sum test; prevalences of mortality were compared using Fisher’s exact test.

Results Effect of treatment with G-CSF and antibiotics on survival. Compared with antibiotics treatment alone, combination treatment with G-CSF and antibiotics improved survival after CLP but only if G-CSF treatment was started before CLP (figure

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1). When G-CSF was given for 7 days, starting 3 days before CLP, mice treated with G-CSF and antibiotics survived longer than mice treated with antibiotics alone or with saline (figure 1A; P õ .02 and P õ .01, respectively, Gehan-modified Wilcoxon rank sum test). Mortality prevalence was also lower between days 4.5 and 8 after CLP in mice treated with G-CSF and antibiotics than in mice treated with antibiotics alone or with saline (P õ .05 in both cases, Fisher’s exact test), while it was not significantly different between mice treated with antibiotics alone and control mice. In contrast, when G-CSF was given for 4 days, starting immediately after CLP, survival in mice treated with G-CSF plus antibiotics was similar to that for mice treated with antibiotics alone, but survival was longer than that for saline-treated mice (figure 1B; P õ .03, Gehanmodified Wilcoxon rank sum test). In both experiments, treatment with antibiotics alone resulted in higher survival rates than did treatment with saline (figure 1A, B; P õ .03 in both cases, Gehan-modified Wilcoxon rank sum test).

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Effect of treatment with G-CSF on survival and neutrophil and bacterial cfu counts. Treatment with G-CSF increased survival if it was started before CLP (figure 2). Mice that received G-CSF for 7 days, starting 3 days before CLP, survived longer than saline-treated mice (figure 2A; P õ .01, Gehan-modified Wilcoxon rank sum test). Also, mice that received G-CSF for 4 days, starting 3 days before CLP, survived longer than saline-treated mice (figure 2B; P õ .05, Gehanmodified Wilcoxon rank sum test). However, survival for mice that received G-CSF for 4 days, starting after CLP, was not significantly different from survival for saline-treated mice (figure 2C). In mice that received G-CSF for 7 days, mortality prevalence was lower from day 1.5 and beyond than in control mice (P õ .05, Fisher’s exact test). Three days of G-CSF treatment resulted in an increase in both blood (29-fold) and peritoneal (2-fold) neutrophils, compared with saline treatment (figure 3). After CLP and the final dose of G-CSF or saline, blood neutrophil counts fell rapidly in mice treated with G-CSF (P õ .001, ANOVA), but they did not vary significantly in those treated with saline; however, neutrophil levels remained higher in the G-CSF – treated than control mice at all time points (figure 3). The

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peritoneal neutrophil count increased after CLP in both GCSF – and saline-treated mice (P õ .01, ANOVA), but was higher in the G-CSF – treated mice at both time points tested (figure 3). Bacterial cfu were not recovered from the spleens of mice after 3 days of G-CSF or saline treatment; however, after CLP and the final dose of G-CSF or saline, cfu were recovered from mice in numbers that were at all times lower in the G-CSF – treated than control mice (figure 3). After CLP, cfu counts progressively increased in the control mice (P õ .001, KruskalWallis test) but did not change significantly in the G-CSF – treated mice. Effect of treatment with G-CSF and antibiotics on bacterial cfu counts. Combination treatment with G-CSF (started before CLP) and antibiotics resulted in lower recovery of spleen bacterial cfu compared with recovery after treatment with antibiotics alone (figure 4). The combination treatment and treatment with either antibiotics or G-CSF alone, compared with saline treatment, strongly reduced cfu counts (figure 4). In addition, cfu levels showed the tendency to be reduced more after combined treatment with G-CSF and antibiotics than with G-CSF alone (P Å .0809, Wilcoxon rank sum test). Effect of treatment with G-CSF and antibiotics on serum cytokines. Adding treatment with G-CSF before CLP to treatment with antibiotics resulted in lower TNF and higher IL-10

Figure 4. Bacterial colony forming units (cfu) recovered 24 h after cecal ligation and puncture (CLP) from spleens of mice treated with G-CSF or antibiotics, G-CSF plus antibiotics, or saline. G-CSF was given starting 3 days before CLP. Data are from 5 independent experiments. P õ * .05 or ** .001; NS Å not significant, according to Wilcoxon rank sum test.

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Figure 3. Neutrophil (PMN) counts in peripheral blood and peritoneum and bacterial colony forming units (cfu) in spleen of 7 mice after 3 days of G-CSF or saline treatment (0 h) and of mice that also had cecal ligation and puncture (CLP) and another dose of treatment immediately after CLP (2, 8, and 24 h). Data are mean { SD for PMN and median for cfu. P õ * .05, ** .01, or *** .001, according to Student’s t test for PMN and Wilcoxon rank sum test for cfu.




G-CSF adds beneficial antiinfectious and antiinflammatory properties to the antimicrobial properties of antibiotics. Antibiotics are currently administered for the prophylaxis of peritonitis and sepsis, which frequently complicate abdominal surgery, particularly in critically ill patients [1, 2, 4, 5]. The stimulation of neutrophils may also be a prophylactic approach, given the role played by these cells in host defense against extracellular microorganisms [6]. Indications for this have been shown in animal disease models using G-CSF [11 – 21], a hematopoietic growth factor that promotes neutrophil differentiation and function [12]. While G-CSF was still undergoing clinical evaluation as a candidate pharmaceutical product, it was observed that its use helped lessen the infectious load in neutropenic mice and hamsters challenged intraperitoneally with extracellular bacteria [11 – 13]. It was later established that nonneutropenic and healthy animals also benefit from G-CSF administration when challenged by an intraperitoneal infection [14 – 21]. The administration of G-CSF in combination with antibiotics, however, has the most clinical relevance [1, 5, 22]; this combination has been investigated only to a limited extent, with conflicting results [14, 17]. The current study was done to explore the benefit of using G-CSF in combination with

serum levels, compared with treatment with antibiotics alone, but it did not affect IL-6 levels (figures 5, 6). Compared with saline, antibiotics had an overall mixed effect on inflammatory parameters: They increased TNF, did not affect IL-6 or IL-10 2 h after CLP, and decreased IL-6 24 h after CLP (figure 5, 6). However, compared with saline, G-CSF decreased all inflammatory parameters except serum IL-6 levels 2 h after CLP, which were not significantly different in G-CSF – treated and control mice (figures 5, 6). In particular, G-CSF reduced TNF levels to a similar extent (2.5 times) in serum and lungs (figures 5, 7). Adding G-CSF to antibiotic treatment also had a mixed effect on inflammatory parameters: Compared with levels with saline treatment, levels of IL-10 were increased, IL-6 was not affected 2 h after CLP, and TNF and IL-6 were decreased 24 h after CLP (figures 5 and 6). Discussion This study shows that in the prophylaxis of a murine model of polymicrobial peritonitis and sepsis, the combination of GCSF and antibiotics compared with antibiotics alone improves survival and results in less bacterial cfu in the spleen, lower serum levels of TNF, and higher serum levels of IL-10. Thus,

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Figure 6. Interleukin (IL)-6 and IL-10 levels in serum 24 h after cecal ligation and puncture (CLP) in mice treated with G-CSF, antibiotics, G-CSF plus antibiotics, or saline. G-CSF was given starting 3 days before CLP. Data are from different experiments (3 for IL-6 and 5 for IL-10). P õ * .05 or ** .01; NS Å not significant according to Wilcoxon rank sum test.

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Figure 5. Tumor necrosis factor (TNF) and interleukin (IL)-6 levels in serum 2 h after cecal ligation and puncture (CLP) in mice treated with G-CSF, antibiotics, G-CSF plus antibiotics, or saline. G-CSF was given starting 3 days before CLP. Data are from different experiments (3 for TNF and 4 for IL-6). P õ * .05, ** .01, or *** .001; NS Å not significant according to Student’s t test for TNF and Wilcoxon rank sum test for IL-6.

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antibiotics in the prophylaxis of experimental polymicrobial peritonitis and sepsis. Compared with antibiotics alone, combination treatment with G-CSF and antibiotics improved survival after CLP but only if G-CSF treatment was started before CLP. It appears that it is crucial for an effect on survival to give G-CSF so that neutrophils are considerably stimulated before CLP. Survival times for mice given G-CSF immediately after and for 3 days following CLP did not differ significantly from those of control mice; however, mice given the same dose of G-CSF on the 3 days preceding CLP and immediately after CLP survived significantly longer than controls. While control mice showed only a moderate increase of neutrophils in the peritoneum after CLP, G-CSF – treated mice showed a rapid and considerable decrease in circulating neutrophils and a remarkable increase of these cells in the peritoneum. At the same time, bacterial cfu steadily increased in the spleens of control mice, while, after an initial appearance, they did not significantly change in mice treated with G-CSF. It seems, therefore, that having readily available neutrophils at the moment of infection assures the possibility of a rapid transfer of many of these cells to the infection site, where they can eradicate bacteria early. G-CSF in combination with antibiotics reduced the recovery of cfu more than antibiotics alone, indicating that G-CSF and antibiotics have an additive effect on the elimination of bacteria after CLP. Compared with antibiotics alone, G-CSF in combination with antibiotics decreased TNF and increased IL-10.

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Compared with saline, antibiotics alone increased TNF, did not significantly change IL-10, and decreased IL-6. In contrast, GCSF alone decreased all these inflammatory parameters. To ascertain that the circulating levels of TNF reflected those in organs, TNF was also measured in the lungs after G-CSF treatment. G-CSF decreased TNF to a similar extent in both serum and lungs, suggesting that the effect of G-CSF on TNF is evenly distributed. These results indicate that the elimination of bacteria by antibiotics exacerbates some aspects of the inflammatory process triggered by CLP, whereas the antibacterial effect of GCSF reduces inflammation after CLP. It appears that adding GCSF to antibiotics results in the modulation of the inflammatory features of the antiinfectious action of antibiotics, as indicated by decreased levels of TNF, a proinflammatory cytokine [27], and increased levels of IL-10, an antiinflammatory cytokine [28]. These findings are consistent with others showing that the inhibition of IL-10 aggravates the pathology that follows CLP, while the administration of this cytokine has beneficial therapeutic effects [29]. Of interest, these findings are also consistent with the report that while serum TNF increases with the severity of the condition brought about by CLP, serum IL10 decreases [30]. Mechanistically, it is possible that while the antibiotic-induced elimination of bacteria is accompanied by dispersion of endotoxin and, hence, stimulation of inflammation [31], the GCSF – induced elimination of bacteria is not, being mediated by phagocytic neutrophils, which kill and destroy bacteria in segregated intracellular compartments [7]. Indeed, animals with intraabdominal infections treated with G-CSF have less endotoxemia than do controls [16, 21]. This can explain why serum TNF is high in mice treated with antibiotics alone, low in mice treated with G-CSF only, and intermediate in mice treated with both. This can also explain why serum IL-10 is high in antibiotic-treated mice and low in G-CSF – treated mice, but it can not explain why mice treated with both have the highest levels of IL-10. However, it has been observed that in mice pretreated with G-CSF, the administration of lipopolysaccharide results in higher serum IL-10 levels than in control mice [32]. It is therefore conceivable that the endotoxin dispersed by antibiotics after CLP results in higher serum IL-10 in mice given G-CSF than in mice given antibiotics alone or saline. In conclusion, the administration of G-CSF started before and continued after CLP adds antiinfectious and antiinflammatory properties to the administration of antibiotics, which result in improved survival over the administration of antibiotics alone. Thus, a prophylactic regimen with G-CSF and antibiotics may represent progress in the current management of the infections that follow abdominal surgery.

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Figure 7. Tumor necrosis factor (TNF) levels in serum and lungs 2 h after cecal ligation and puncture (CLP) in 7 mice treated with GCSF or saline. G-CSF was given starting 3 days before CLP. P õ * .01 and ** .001, according to Student’s t test.




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