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Infection and Immunity, May 2005, p. 2751-2757, Vol. 73, No. 5
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.5.2751-2757.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Role of Interleukin-6 in Mortality from and Physiologic Response to Sepsis

Daniel G. Remick,^* Gerald Bolgos, Shannon Copeland, and Javed Siddiqui

Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109-0602

Received 15 November 2004/ Returned for modification 11 January 2005/ Accepted 14 January 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous studies have suggested that interleukin-6 (IL-6) serves as both a marker and a mediator for the severity of sepsis. We tested whether interleukin 6 knockout (IL-6KO) mice were more susceptible to sepsis mortality induced by cecal ligation and puncture. IL-6KO and wild-type (WT) mice were subjected to increasing degrees of sepsis severity. Physiologic support was given with fluids and appropriate antibiotics. Plasma IL-6 levels were determined 6 h after the onset of sepsis, and a complete hematologic profile was performed on day 2. As expected, increasing sepsis severity resulted in greater and more rapid mortality. However, the mortality was nearly identical in the IL-6KO and WT mice. All WT septic mice had high plasma levels of IL-6 6 h after the onset of sepsis, while IL-6KO were near or below the lower limit of detection. Among the WT mice, mortality was significantly higher in mice with plasma IL-6 >3,000 pg/ml. Both IL-6KO and WT mice destined to die in the early stages of sepsis had substantial and nearly identical weight gain in the first 24 h. However, at later stages the WT mice had significantly greater weight loss than the KO mice. The KO mice failed to develop the characteristic hypothermia within the first 24 h of severe sepsis routinely observed in the WT mice. These data demonstrate that IL-6 serves as a marker of disease severity in sepsis and does modulate some physiologic responses, but complete lack of IL-6 does not does not alter mortality due to sepsis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interleukin-6 (IL-6) is a cytokine with a wide range of biological activities. IL-6 helps control the induction of the acute-phase response and also is a mediator for immunoglobulin class switching. These biological activities are important in both in vivo and in vitro settings. An additional clearly defined IL-6 in vivo activity is temperature regulation (13, 17, 18). IL-6 functions as an important and sensitive indicator of inflammation within the body. Several reports indicate that plasma levels of IL-6 may be used as a diagnostic marker for the presence of bacteremia. In newborns, plasma levels of interleukin-6 of >160 pg/ml were 100% sensitive for the diagnosis of early-onset sepsis in neonates (21). IL-6 is also reported to be present in the plasma earlier than C-reactive protein (25). Among cancer patients, IL-6 levels may be used to differentiate patients at low risk for septicemia (6).

In addition to being used as a diagnostic test for the presence of sepsis, IL-6 has also found utility as a prognostic factor for outcome in septic patients. Over 15 years ago it was reported the plasma levels of IL-6 correlate with mortality in septic patients (12). These results have since been confirmed in multiple studies, including a study which examined all patients admitted to an intensive care unit and documented that levels of IL-6 correlated with mortality (8). In one clinical study of the treatment of sepsis, patients were stratified to receive immunomodulating therapy on the basis of their plasma levels of IL-6 (26). In septic patients with hypothermia, plasma levels of IL-6 were elevated (2).

Circulating levels of IL-6 may serve as a predictive variable for survival in sepsis. Controversy exists concerning the precise nature of IL-6 in sepsis. Specifically, does IL-6 merely represent a marker of disease or is it an actual mediator of organ injury? Studies have shown that antibody inhibition of IL-6 improved survival in a bacterium-derived sepsis model (11) and antibodies to IL-6 will improve survival in sepsis if the correct dose is used (32). However, other studies have shown that IL-6 represents a critical portion of the inflammatory response to infectious agents that is needed in order to adequately control the infection. Included among these infectious diseases are intracellular parasites (9) and gram-negative bacteria (5).

In the present report, we examined the response to a well-defined model of sepsis in IL-6 knockout (IL-6KO) mice and their wild-type (WT) counterparts. For this model, sepsis is induced by cecal ligation and puncture (41) where antibiotic treatment plus fluid resuscitation is also administered (29). This model of sepsis has been widely used to investigate the inflammatory response (14, 34) and potential mediators of organ injury and lethality (28, 30).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice. IL-6KO mice were the generous gift from Mark Opp, University of Michigan. The phenotype of the mice was determined by measuring plasma levels of IL-6 after the onset of sepsis, since previous work has documented that the cecal ligation and puncture (CLP) model of sepsis reproducibly induces high levels of IL-6 (29). The IL-6KO mice were on a C57/BL6 background, and the control WT mice were the same age, strain, and sex. All mice used for these experiments were females and 3 to 4 months of age. Mice were kept on a 12-h light dark cycle and provided food and water ad libitum throughout the entire experiment. Ambient room temperature was maintained between 23 and 25°C. All the experiments were approved by the University Committee on Use and Care of Animals.

CLP. CLP was performed using our standardized protocol (27-29) modified after the original CLP protocol to include a newer generation of antibiotics (41). Briefly, mice were anesthetized with isoflurane anesthesia. Through a midline incision, the cecum was exteriorized and ligated with 3-O silk. The ligated cecum was then punctured with the indicated gauge of needle. Following puncture, the cecum was gently squeezed to ensure that the wounds were patent. For the 21-gauge and the 25-gauge double-puncture models, the entire cecum was included. For the 25-gauge single puncture, approximately 1 cm of the distal cecum was ligated. Several previous reports documented that the larger the puncture wound the greater the mortality after cecal ligation and puncture (7, 19, 41). Immediately after surgery, mice were resuscitated with 1 ml of warm normal saline. Antibiotic therapy was initiated 2 h after surgery and consisted of imipenem (25 mg/kg; Merck, West Point, PA) suspended in lactated Ringer's with 5% dextrose. One milliliter was injected subcutaneously, and antibiotic therapy was administered every 12 h for the first 5 days. Mortality was assessed twice a day for the first 28 days. In some animals, mini mitters (mini mitters, Sunriver, OR) were implanted subcutaneously at the time of surgery in order to record temperature as well as body movement.

Collection of data. Six hours after CLP, 20 µl of blood was obtained from the tail vein and diluted in phosphate-buffered saline with EDTA. Cellular elements were removed by centrifugation, and the diluted plasma stored for later IL-6 measurements. Twenty-four hours after CLP (day 2), an additional 20 µl of blood was obtained from the tail vein and a hematologic profile performed as described below. Individual mice were weighed daily, and in some experiments when the mice were housed individually, daily food consumption was also determined.

IL-6 enzyme-linked immunosorbent assay. Plasma levels of IL-6 were measured by enzyme-linked immunosorbent assay using previously published methods (22).

Hematology. A complete hematologic profile was performed using the Hemavet mascot (CDC Technologies, Oxford, CT). This instrument provides a complete blood count including hemoglobin, red blood cells, and white blood cells with a full differential.

Acute pathology. For a series of experiments, CLP was performed and the mice sacrificed at 26 h, i.e., 2 h after the injection of imipenem. Blood was obtained via cardiac puncture from mice anesthetized with isoflurane. The peritoneal cavity was then opened and flushed with 1 ml of normal saline with 3.4 mM EDTA. The peritoneal cavity was then washed extensively with 20 ml of normal saline with EDTA. The supernatant from the 1-ml wash was collected to measure IL-6, and the cell pellets from both washes were combined. A total cell count, as well as differential analysis, was performed. Samples were also harvested for histologic processing.

Statistical analysis. Data are generally presented as means ± the standard error of the mean. When data were evaluated over time, repeated-measures analysis of variance ANOVA with Bonferroni correction for multiple comparisons was done to compare the wild-type and knockout mice. When this analysis did not show a group effect (wild type versus knockout) but did demonstrate a day affect, the data from the wild type were combined with those from the knockout mice. This was only performed for Fig. 3. Survival was analyzed with log rank survival analysis.

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FIG. 3. Change in body weight 24 h prior to death. Mice were weighed daily, and the data are displayed as the change in body weight on the day prior to death. Since statistical analysis showed that a group effect (wild type versus knockout) was not present, the data were combined. There is a significant increase in the body weight of the mice that would die within the next 24 h compared to those mice that would live in the first 2 days. This difference became less apparent as the sepsis evolved. The value beneath each bar indicates the number of animals in that group. * = P <0.05 comparing mice that lived versus those that died.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of IL-6 knockout on mortality. In BALB/c mice, CLP with a 21-gauge needle resulted in approximately 50% mortality over 7 days (7). However, in outbred ICR mice, a 16-gauge needle with double punctures was required in order to achieve 50% mortality (data not shown) and in BDF₁ mice three punctures with a 16-gauge needle were required to achieve 50% mortality (31). The present experiments were performed with C57/BL6 mice, which is the genetic background of the knockout mice. Therefore, the first experiments were designed to determine the appropriate lethality of the CLP in order to achieve approximately 25 to 50% mortality by decreasing the severity of the septic challenge (Fig. 1). Using a 21-gauge needle and two punctures, there is nearly 100% mortality in both the knockout (100%) and the wild-type mice (91%). By log rank survival analysis, there was no significant difference between the knockout mice and the wild-type mice. We further evaluated CLP performed with a 25-gauge needle and two punctures and, even with a needle gauge this much smaller, mortality was approximately 70% over 7 days. Again, there was no significant difference between the knockout and the wild-type mice. We further reduced the severity of CLP by ligating only the distal 1 cm of the cecum and performing a single puncture with a 25-gauge needle. Under these conditions, there is virtually no mortality in either the wild-type or the knockout mice. Finally, sham surgery (without any ligation and puncture) does not cause any mortality in either group. These data clearly show that there is no difference in susceptibility to sepsis in those mice which lacked interleukin 6. It should be noted that all of the studies were performed with female mice. Since there is a difference in the response to sepsis in male and female mice (42), is possible that different results might have been obtained if the studies were repeated with male mice.

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FIG. 1. Mortality following cecal ligation and puncture. Both wild-type and IL-6KO mice were subjected to CLP of increasing severity. There was no difference in mortality between the IL-6KO and wild-type mice when compared with log rank survival analysis in any of the models shown.

Verification of IL-6 knockout and plasma level of IL-6 correlation with survival. Cecal ligation and puncture routinely induce elevated levels of IL-6 in the plasma (28, 29). More severe sepsis results in higher levels of IL-6 (7). In the C57/BL6 mice, 21-gauge cecal ligation and puncture result in nearly 100% mortality (Fig. 1). We therefore measured IL-6 at the 6-h time point in this group of mice. In mice subjected to 21-gauge CLP, there is virtually no detectable IL-6 in the plasma in the knockout mice, while the levels in the wild-type mice were >4,000 pg/ml (Fig. 2A). These data verify that the IL-6 gene was knocked out. Since we and others have previously reported that plasma levels of IL-6 at 6 h predict mortality (29, 37, 39), we also evaluated whether IL-6 levels would predict mortality in the C57/BL6 mice. Given the high values of IL-6, we used a value of 3,000 pg/ml to evaluate whether mice with high levels of IL-6 have a greater mortality. Figure 2B demonstrates that the plasma levels of IL-6 obtained 6 h after CLP do predict deaths during the first days of sepsis. These data confirm our previous reports, and those of others, indicating that early levels of IL-6 predict early deaths.

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FIG. 2. Plasma IL-6 levels of wild-type and knockout mice and correlation with mortality. (A) Plasma levels of IL-6 were determined 6 h after the onset of sepsis induced with a 21-gauge needle. All the wild-type mice had significant levels of IL-6 within the plasma, while in the knockout mice levels were nearly undetectable. Each value is the mean ± the standard error of the mean for nine mice, * = P <0.05. (B) Mice were divided into those with IL-6 levels greater than 3,000 pg/ml (n = 18) or less than 3,000 pg/ml (n = 33). Mice with higher levels of plasma IL-6 had significantly greater mortality, particularly in early phases of sepsis. P = 0.014 comparing the curves by log rank survival analysis.

Hematologic changes. As expected, significant hematologic alterations were evident within 26 h after CLP. Consistent with previous reports, there is a reduction in the total white count and a specific decrease in lymphocytes compared to normal animals. Among the IL-6KO mice, those with more severe injury (21-gauge and 25-gauge double puncture) had significantly reduced numbers of circulating white blood cells compared to the wild-type mice, and the decrease was due to the loss of lymphocytes. Virtually all other hematologic parameters showed no difference between the knockout and the wild-type mice (Table 1).

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TABLE 1. Hematologic changes observed 26 h after CLPa

Acute pathological changes. A group of mice were sacrificed 26 h after the onset of sepsis and thoroughly evaluated. Histologic examination of several organs (lung, heart, liver, spleen, kidney, and intestines) demonstrated scattered areas of acute inflammation, but there was no difference between the IL-6 knockout and the wild-type mice. Examination of the cellular constituents within the peritoneal cavity showed a significant influx of neutrophils, but again there was no difference between the knockout and wild-type mice (data not shown). There was also no difference in the number of CFU of bacteria recovered from the peritoneal cavities of the knockout and wild-type mice (data not shown).

Changes in body weight. Each day, mice were weighed and mortality determined. This allowed measurement of the change in body weight that occurred on the day prior to death. Previously, we observed an increase in body weight the day prior to death in mice that died during the first 4 days after cecal ligation and puncture (data not shown). In the later stages of sepsis, mice are likely to lose rather than gain weight. Statistical analysis showed that there was no difference in the change in body weight between the wild-type and knockout mice, and so the results were combined. In the present experiment, mice that die within the first 3 days of sepsis will gain weight (Fig. 3).

Additional changes were observed when the changes in body weight that occurred over 28 days were examined. For these studies, we examined the nonlethal CLP-induced sepsis that occurs following puncture with a single 25-gauge needle. This model was selected in order to have sufficient numbers of survivors to evaluate the long-term consequences. Both the wild-type and the knockout mice had a significant decrease in their body weight from the presurgery weight during the first week after CLP (Fig. 4). This decrease in body weight is due to the metabolic demands placed on the animal by both the surgical procedure and the ensuing bacterial infection. There was a striking difference in the body weight loss between the knockout and the wild-type mice. Specifically, knockout mice did not lose as much weight as the wild type. Starting on day 3 and continuing to day 11, the knockout mice weighed more than the wild-type mice. It should be noted that the presurgery weight was indistinguishable (wild type, knockout).

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FIG. 4. Daily body weight following nonlethal cecal ligation and puncture. Mice were subjected to nonlethal 25-gauge single-puncture CLP. The IL-6KO mice had significantly less loss of body weight compared to the wild-type mice. There was a significant effect based on the day, as well as differences between the groups. Each value is the mean ± the standard error of the mean for 18 to 31 mice. * = P <0.05 (wild type compared to KO with Bonferroni correction).

A possible explanation for the difference in the change in body weight would be the amount of food consumed since reduced food consumption would slow weight recovery. We looked at the food consumption after 25-gauge single-puncture CLP (Fig. 5). The wild-type mice consumed slightly less food on day 3 only; therefore, the difference in food consumption was not sufficient to account for the difference in weight loss.

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FIG. 5. Daily food consumption. The amount of food consumed each day was determined in 25-gauge single-puncture mice to match the data in Fig. 4. There is virtually no difference in the consumption of food between the wild-type and knockout mice, with the single exception of the day 3, when the knockout mice consumed more food. Each value is the mean ± the standard error of the mean for 18 to 31 mice.

Gross motor activity. After cecal ligation and puncture, a reduction in the normal diurnal variation of gross motor activity is observed that may be determined by the implanted mini mitter. Figure 6 demonstrates a rapid return to the diurnal variation in either the wild-type or the knockout mice that had the sham procedure. This return to diurnal variation occurred within 2 to 3 days. The return to diurnal variation also was observed in the 25-gauge single-puncture mice that lagged slightly behind the sham mice. In the mice subjected to the cecal ligation and 25-gauge double puncture, the diurnal variation does not return until approximately day 9. Again, there is little difference between the knockout mice and the wild-type mice. Since there was little difference in the gross motor activity of the mice, altered energy expenditure due to increased movement could not account for the differences in body weight observed in Fig. 4.

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FIG. 6. Gross motor activity. Gross motor activity was determined using implanted radio transmitters. There is a rapid return to the normal diurnal variation in both the sham-operated and the 25-gauge single-puncture septic animals. In contrast, 25-gauge double-puncture animals essentially never regain normal diurnal variation. The light-dark bars indicate when the lights were on or off in the room. Each line is the mean of two to four mice.

Body temperature. Recent reports have indicated that IL-6 knockout mice have an attenuated body temperature decrease in response to inflammatory challenges (15, 33). Anesthetic agents such as ketamine will also induce hypothermia (10). Mini mitters were implanted in both wild-type and IL-6KO mice under isoflurane anesthesia, and the mice were allowed to recover for several days. The mice were then injected with ketamine/xylazine (87 µg/g and 13 µg/g, respectively), and the body temperature was followed for 24 h. The wild-type mice decreased their body temperature to 31.2 ± 0.7°C, while the IL-6KO mice decreased their body temperature to 29.2 ± 0.8°C within 4 h of anesthesia (data not shown). This demonstrates that both the wild-type and IL-6KO mice have the capacity to thermoregulate, such that the loss of IL-6 does not indicate that the KO mice have complete failure to alter their body temperature in response to any stimulus.

We then explored the thermoregulatory response to CLP-induced sepsis in the wild-type and IL-6KO mice. With the use of the implanted mini mitters, we were able to follow the body temperature over time. The graphed data (Fig. 7) was obtained from mice that had severe sepsis and a 25-gauge double puncture. When graphed on an individual basis, it is readily apparent that there was a significant drop in body temperature among the wild-type mice that begins about 4 h after surgery and continues to the end of the 24-h time measurement displayed in Fig. 7A. The initial low temperature was mild hypothermia induced by the isoflurane anesthesia. While the knockout mice also had the initial drop due to the isoflurane anesthesia, they generally failed to show the typical hypothermia that develops as sepsis evolves. Figure 7B shows the difference between the groups of mice. Within 8 h after surgery, during the time when the sepsis hypothermia is taking hold, the IL-6 knockout mice have a significantly higher body temperature compared to the wild-type mice. This difference in body temperature persists at 12 and 20 h.

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FIG. 7. Body temperature change. Mice were subjected to 25-gauge double-puncture CLP, and the body temperature was determined with implanted radio transmitters. Panel A is the temperature trace for each individual mouse, and it is apparent that the knockout mice failed to develop hypothermia during the first 24 h. Panel B represents the mean ± the standard error of the mean for six wild-type and six IL-6KO mice. WT mice had significantly lower body temperatures at each of the indicated time points. * = P <0.05 (WT versus IL-6KO).

Top Abstract Introduction Materials and Methods Results Discussion References

 
There can be little question that IL-6 is an important aspect of the inflammatory response. Over 6 years ago it was reported that plasma IL-6 levels are significantly higher in the acute phase (first 10 h) in those animals with a more severe infection (1) and these IL-6 levels play an important role in the hepatic response to sepsis (40). The IL-6 controversy revolves around whether or not, in sepsis, IL-6 represents a marker of inflammation, an inducer of altered physiology, or a mediator of organ injury. The published literature does not speak with a clear voice to this issue. Using an anti-IL-6 antibody and the cecal ligation and puncture model of sepsis, it has been demonstrated that optimal survival occurs when sufficient antibody is given to blunt but not completely eliminate IL-6 (32). In a model based on administering gram-negative bacteria by lavage, an anti-IL-6 antibody improves survival and increases bacterial killing (11). Both the studies indicate that limiting the biological activity of IL-6 will improve survival.

In contrast, other studies have found discordant results. In a neonatal mouse model of sepsis, exogenous administration of IL-6 improved survival and complete inhibition of IL-6 resulted in more rapid mortality (20). In a primate model of endotoxemia following a bolus infusion of gram-negative bacteria, antibody inhibition of IL-6 improved coagulation parameters (38). It should be noted that modulating the coagulation parameters with exogenous activated protein C has been successfully used to improve outcome in septic patients (3). In IL-6 transgenic mice, blocking the IL-6 receptor helped decrease muscle wasting (36), a common feature in septic patients.

Studies have also been performed with IL-6 knockout mice and models of infectious disease. In a parasitic infection with Trypanosoma cruzi, IL-6 knockout mice have greater levels of parasitemia and also higher mortality (9). In a study more closely related to the present investigations, IL-6 knockout mice were examined following an intraperitoneal injection of Escherichia coli (5). In these studies, the IL-6 knockout mice were more susceptible to bacteremia than the wild-type counterparts. Additionally, there were greater numbers of CFU present in the liver. However, there are some differences between this study and our present results. CLP represents the response to a polymicrobial sepsis and not a single gram-negative bacterium. Additionally, our model examines the long-term effect of sepsis and not just the acute response in the first 2 to 3 days. In our investigations, we used different levels of lethality in an attempt to define a significant difference in survival between IL-6 knockout mice and wild-type mice. In examining the survival of 143 mice over 28 days, there was never a significant difference in lethality.

IL-6 has been described as essential for the regulation of the response to inflammatory stimuli including lipopolysaccharide (4) and tumor necrosis factor (35). A series of papers has been published concerning the response to inflammatory stimuli in IL-6 knockout mice. In response to sterile inflammation induced by a turpentine abscess, IL-6 knockout mice do not develop a fever compared to their wild-type counterparts. Additionally, they do not lose body weight in comparison to their wild-type counterparts (16). In response to influenza, this paper also demonstrated that IL-6 knockout mice have an attenuated temperature change and less weight loss compared to the wild-type counterparts. These results are nearly identical to those reported in the present paper.

The Kluger group has extensively explored the role of IL-6 in the cecal ligation and puncture model of sepsis (18). This model is similar to the model that we have utilized in this laboratory. An important difference is that, in the Kluger studies, the mice were maintained at a constant room temperature of 30°C while we maintained our mice at a constant temperature of 22°C. In an ambient temperature of 30°C, wild-type mice develop a fever following infection whereas we have reproducibly demonstrated the development of hypothermia in septic mice (23, 24). However, there were important similarities since the IL-6 knockout mice had a temperature response different from the wild-type mice. Additionally, there was no difference in survival between the wild-type mice and the IL-6 knockout mice.

Our findings demonstrate that complete lack of IL-6 has limited impact on the overall mortality in a standardized animal model of sepsis. IL-6 clearly plays an important role in several aspects of the inflammatory response, including temperature regulation and metabolic activity. However, our data suggest that complete lack of IL-6 does not alter the mortality of sepsis although it does serve as a sensitive marker of inflammation.

 
This study was supported by in part by NIH grant GM 44918.

We thank Jill Granger for careful reading of the manuscript, Jiyoun Kim for help with the hematologic measurements, and Kathy Welch for statistical analysis.

 
* Corresponding author. Mailing address: M2210 Med Sci I, 1301 Catherine Road, Ann Arbor, MI 48109-0602. Phone: (734) 936-1889. Fax: (734) 763-6476. E-mail: remickd{at}umich.edu.

Editor: F. C. Fang

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Infection and Immunity, May 2005, p. 2751-2757, Vol. 73, No. 5
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.5.2751-2757.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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