Endotoxin and tumour necrosis factor do not cause mortality from caecal ligation and puncture

ENDOTOXIN AND TUMOUR NECROSIS FACTOR DO NOT CAUSE MORTALITY FROM CAECAL LIGATION AND PUNCTURE Kelly M. McMasters,

James C. Peyton, Dimitri William G. Cheadle

J. Hadjiminas,

Macrophage tumour necrosis factor-alpha (TNF-(w) production is thought to represent an important pathogenic mechanism by which Gram-negative sepsis is mediated. We compared the effects of caecal ligation and puncture (CLP) on endotoxin-sensitive (C3H/HeSnJ) and endotoxin-resistant (C3H/HeJ) mice. Mortality after CLP for C3H/HeSnJ mice compared with C3H/HeJ mice was not significantly different (32% and 55%, respectively). When survivors were injected with lipopolysaccharide intraperitoneally on the 7th day after CLP, the mortality rate was 82% for C3H/HeSnJ mice versus 0% for C3H/HeJ mice (P < 0.0001). Serum endotoxin levels at 24 h after CLP were only slightly elevated. Serum TNF levels and peritoneal macrophage TNF production were undetectable in C3H/HeJ mice and were only slightly elevated in C3H/HeSnJ mice by 24 h after CLP. Peritoneal macrophage mRNA levels for TNF-a, IL-l/3, and I-Ao displayed a similar pattern in the two strains of mice, with a 2to 3-fold increase in TNF-(x and IL-lp mRNA levels by 24 h and a sharp decrease in I-Ao mRNA by 24 h. The cause of mortality in mice that undergo CLP cannot be attributed to overwhelming endotoxemia and/or TNF production.

Intra-abdominal infection often leads to multiple organ failure and death despite aggressive surgical intervention, appropriate antibiotic therapy, and intensive care unit monitoring. A fatal outcome is usually the result of the immune system ineffectively containing or eradicating the infection. Two related but distinct types of immune system dysfunction have been proposed to cause this process. They are over expression or adverse regulation of proinflammatory mediators (locally or systemically), resulting in shock and tissue damage,’ and the other is decreased ability of the immune system to mount an antimicrobial attack.2 Tumour necrosis factor (TNF) and interleukin 1 (IL-l) have been implicated as mediators of the toxic

From the Department of Surgery, University of Louisville School of Medicine, and the Veterans Administration Medical Center, Louisville, Kentucky, USA. Presented at the 13th Annual Meeting for the Surgical Infection Society, Baltimore, Maryland, April 28 to May 1, 1993. Correspondence to: William G. Cheadle, MD, Department of Surgery, University of Louisville, Louisville, KY 40292, USA. Received 1 October 1993; revised and accepted for publication 15 March 1994 0 1994 Academic Press Limited 1043-4666/94/050530+07 $08.00/O KEY WORDS: Class II MHC antigens/endotoxin/interleukin sepsis/tumour necrosis factor 530

l/

effects of Gram-negative sepsis and endotoxemia. This implication has been supported by the fact that (1) endotoxemia or Gram-negative bacteremia is associated with an increase in serum TNF and IL-l levels,37 (2) administration of TNF or IL-l mimics many of the deleterious effects of endotoxiqs” and (3) therapy directed at neutralizing the effects of TNF or IL-l protects animals against the lethal effects of endotoxin. 12-15 Many studies of the effects of sepsis have relied on animal models that employ the injection of endotoxin or Gram-negative bacteria. Caecal ligation and puncture (CLP) is thought to be a better model because it simulates a common surgical problem of perforated, necrotic bowel and more closely mimics the physiologic changes found in human sepsis.16’17 Even though these are all models of sepsis, there are marked differences between models of endotoxemia or Gram-negative bacteremia and models of CLP in terms of cytokine expression and response to anticytokine therapy. The data reported herein examine the mechanism by which CLP causes immune system dysfunction and mortality. In particular, we investigated how the reduced ability of the C3WHeJ mice to produce TNF affects: (1) mortality; (2) expression of IL-ll3 mRNA, a proinflammatory mediator with a central role in this model of sepsis;” and (3) suppression of I-Aa (murine major histocompatibility CYTOKINE,

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TNF and LPS in intra-abdominal

class II antigen) antigen expression, which has been shown to correlate with outcome and survival in infected severely injured mice.i’

infection / 531

organisms were also recovered from all blood cuitures. The anaerobic organisms that were identified included Bacteroides vulgatus and Clostridium species; whereas aerobic organisms included E. coli and Proteus mirabilis. No positive blood cultures were obtained from normal mice or sham-operated mice.

RESULTS

Mortality

Endotoxin Levels

Mortality from CLP is shown in Table 1. All deaths occurred within 3 days after CLP, except for one that occurred in a C3WHeSnJ mouse on the 4th day after CLP.

Serum endotoxin levels after CLP are shown in Fig. 1. Endotoxin levels were slightly higher in both mouse strains at 24 h after CLP compared with the sham-operated controls, but these differences were not statistically significant (n = 3).

TABLE 1. Mortality after CLP and LPS intraperitoneal injection in C3H/HeSnJ and C3HIHeJ mice. Strain of mice

Caecal ligation and puncture

C3WHeSnJ C3WHeJ

11/34 (32%)t 18/33 (55%)t

* One milligram of LPS was injected 7th day after CLP. i No significant difference. $ P < 0.0001 Fisher’s exact test.

intraperitoneally

Lipopolysaccharide injection (1 mg)”

9/11 (82%)$ O/16 (O%)$ in the survivors

on the

d

Caecal and Blood Cultures Caecal cultures were taken from C3II/HeSnJ, C3H/HeJ, and Swiss Webster mice to examine potential differences in intestinal flora. Caecal cultures grew both aerobic (Escherichia coli, Proteus species, Pseudomonas species) and anaerobic (Bacteroides vulgatus, Clostridium species) organisms, but no differences in the caecal flora was evident among the three strains of mice. In all three strains of mice, the predominant organisms cultured from blood during the early stages of infection were anaerobes (Table 2). By 24 h, aerobic

T

1 3

Pre

Time

post

i

24

CLP (h)

Figure 1. Serum endotoxin levels after CLP in C3H/HeSnJ mice (0) and C3WHeJ mice (a). Values are expressed as +SEM.

TNF Production TABLE 2. Positive blood cultures after CLP. Hours after CLP 1 hour

3 hours 24 hours

Type of Organism

Anaerobic Aerobic Anaerobic Aerobic Anaerobic Aerobic

C3WHeSnJ

C3WHe.J

Swiss Webster

o/3 o/3 313 o/3 u3

o/3 o/3 313 o/3 213

o/4 o/4 414 l/4 314

213

o/3

314

Serum TNF levels after CLP were undetectable in normal endotoxin-resistant (C3H/HeJ) mice, and at 1, 3 and 24 h after CLP. TNF was not detected in normal endotoxin-sensitive (C3HHeSnJ) mice, or at 1 h after CLP. However, at 3 h after CLP, 2 of 6 mice (C3H/ HeSnJ) had detectable serum TNF (250, 300 pg/ml), and at 24 h, 4 of 6 animals (C3WHeSnJ) had measurable levels of TNF (200, 320, 480, 550 pg/ml). TNF might have been present in the serum of the rest of the

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TABLE 3. Relative mRNA levels determined by Densitometric scanning.* 6 ht

Normal C3WHeSnJ

TNF-(w IL-q3 I-Aa * Values expressed 7 Time after CLP.

100 100 100 as percent

C3WHeJ

C3WHeJ

C3WHeSnJ

92 147 12

51 135 144 of C3WHeSnJ

24 ht C3WHeSnJ

128 266 70

C3WHeJ

300 264 4

263 191 8

controls.

mice but at concentrations below the detection limit of the ELISA. Peritoneal macrophage production of TNF was measured by harvesting peritoneal macrophages 24 h after CLP and culturing for 2 h in the absence or presence of LPS (1 kg/ml). TNF was not detected in cultures of C3WHeJ peritoneal macrophages with or without LPS stimulation using either the bioassay or ELISA methods. Using the bioassay method, mean TNF levels in peritoneal macrophages from C3W HeSnJ mice were lower without LPS compared with levels in mice with LPS (177 + 27 U/ml versus 197 f 19 U/ml). Using the ELISA method, TNF production in peritoneal macrophages from C3WHeSnJ mice was detected in 1 of 2 cultures (30 pg/ml) without LPS, and 2 of 2 cultures with LPS (50 and 200 pg/ml). Culture supernatant was assayed undiluted.

MessengerRNA Levels Peritoneal macrophage mRNA levels for TNF-o, IL-l/3, and I-Ao displayed a similar pattern in both strains of mice, with a 2- to 3-fold increase in TNF-a and IL-Q mRNA levels by 24 h and a sharp decrease in I-Ao levels by 24 h (Table 3). A representative sample of the Northern blot of IL-lp mRNA is shown in Fig. 2. According to our blots, normal peritoneal macrophages cultured for 1 h in endotoxin-free media constitutively expressed low levels of TNF and IL-l mRNA. Constitutive expression of TNF mRNA has been previously shown in normal tissues without any cell isolation or culture.20

DISCUSSION The terms intra-abdominal sepsis and Gramnegative sepsis often are used interchangeably. While intra-abdominal infection in patients may lead to Gram-negative bacteremia, most cases of secondary peritonitis are polymicrobial infections, and blood cultures usually return with no growth of any organ-

1

2

3

4

5

6

L

I

I

Figure 2. Northern blot of peritoneal macrophage IL-lp mRNA levels after CLP. Ten micrograms of total mRNA were analysed in each lane of the blot. Lane 1: normal C3WHeSnJ strain; Lane 2: normal C3W HeJ strain; Lane 3: C3WHeSnJ strain, 6 h after CLP; Lane 4: C3H/ HeJ strain, 6 h after CLP; Lane 5: C3WHeSnJ strain, 24 h after CLP: and Lane 6: C3WHeJ strain, 24 h after CLP.

isms.‘l Animal models of sepsis have been developed that utilize: (1) intravascular administration of bacterial endotoxin or Gram-negative bacteria, (2) intraperitoneal administration of bacteria (Gram-negative or polymicrobial), and (3) peritoneal contamination with the animal’s own intestinal flora, such as cLP.16,22 Intravascular injection of endotoxin or Gramnegative bacteria causes a profound increase in serum levels of TNF.3,4,7 Intraperitoneal injection of endotoxin appears to have similar effects as intravascular injection. 23 Administration of anti-TNF antibodies prevents mortality in these models, indicating an important role for TNF in mediating the lethal effects of endotoxemia or Gram-negative bacteremia. 12,I3 Interleukin 1 is also markedly elevated in these models.5-7 Administration of IL-l receptor antagonist (IL-lra) has been shown to reduce mortality from endotoxic shock without altering serum TNF levels. IL-lra is a naturally occurring protein that binds to

TNF and LPS in intra-abdominal

the IL-l receptor with equal affinity to IL-l3 and has no agonist activity.24 Bagby et al. 25 compared the effects of intravascular injection of endotoxin or Gram-negative bacteria with intraperitoneal injection of Gram-negative bacteria or a polymicrobial inoculum. They found that intravascular injection of endotoxin or E. coli (live or dead) produced a substantial transient increase in serum TNF levels, while the group receiving the intraperitoneal challenge with E. coli or a polymicrobial faecal inoculum had a lower serum TNF level. They also found that pretreatment with anti-TNF antibodies protected animals from the lethal effects of intravascular injection of endotoxin but was ineffective in preventing mortality from E. coli peritonitis. Serum TNF levels have been shown to be lower, or even undetectable, after CLP when compared with models of endotoxemia or Gram-negative bacteremia.23,25-29 Furthermore, administration of an anti-TNF monoclonal antibody prior to, or up to, 8 h after a sublethal CLP challenge was shown in these studies to result in the death of all animals within 3 days. The lethal effects of this antibody were negated by administration of exogenous TNF, suggesting that endogenously produced TNF is necessary for recovery from CLP.30 Other studies have confirmed that antiTNF antibodies do not prevent lethality from CLP.31,32 In contrast, the presence of IL-lra has been shown to protect rats from the lethal effects of CLP,” implicating IL-1 as an important mediator in this model of sepsis. This view is further supported by our results, as both endotoxin (LPS)-resistant and endotoxin (LPS)-sensitive mice showed similar upregulation of their IL-1 mRNA. C3WHeJ mice are thought to be resistant to the lethal effects of endotoxin because of a genetic defect, mapped to a single locus on chromosome 4 (LPSd) that renders these animals less able to produce TNF in response to endotoxin stimulation.3 Resistance of C3WHeJ mice to endotoxin is limited to certain preparations of LPS, specifically those lacking lipid A associated protein.33 There is evidence that C3H/HeJ mice produce TNF in response to some bacterial challenges.34 The relative resistance of the C3WHeJ mice to some (but not all) preparations of LPS does not necessarily make them unable to produce TNF in response to a polymicrobial challenge such as CLP. Our results show, however, that C3H/HeJ mice did not produce TNF after CLP but were still subjected to similar mortality. Our study confirms a report from Kisala et al. ,35 in which mortality from CLP did not differ between endotoxin-sensitive and endotoxinresistant mouse strains, but contradicts a report by Baker et al. ,36 who found that C3H/HeJ mice were resistant to mortality from CLP. In that study, however, the two strains of mice were obtained from

infection / 533

different breeders, which may have accounted for their findings. Macrophages from endotoxin-resistant C3WHeJ mice are also hyporesponsive to endotoxin stimulation in terms of IL-l production. Baker et al. ,37 however, have shown that splenic macrophages from endotoxin-resistant C3WHeJ mice express more IL-l after CLP than endotoxin-sensitive C3WHeN mice. This suggests that factors other than endotoxin may stimulate IL-l production after CLP and warrants further study. Tolerance to the effects of endotoxin can be induced by prior exposure to sublethal doses of endotoxin.38,39 Our results indicate that endotoxinsensitive mice did not become tolerant to the lethal effects of endotoxin by virtue of prior CLP, and that endotoxin-resistant mice retain their endotoxin resistance after CLP. We found that serum endotoxin levels showed a slight elevation at 24 h after CLP. Serum TNF levels and peritoneal macrophage TNF production were elevated in C3H/HeSnJ mice after CLP, but were undetectable in C3WHeJ mice. Ayala et al.26 demonstrated that serum endotoxin levels after CLP were approximately 20- to 30-fold higher than those reported herein and found that serum TNF levels in C3WHeJ mice were actually 25fold higher than in endotoxin-sensitive animals.27 Explanations for these discrepancies may be due to the differences in intestinal flora or assay techniques (bioassay versus ELISA). Recently, we demonstrated 2- to 3-fold increases in peritoneal macrophage TNF-ok and IL-ll3 mRNA levels, and a sharp decrease in I-ACY mRNA levels after CLP in Swiss Webster mice.40 In the present study, a similar pattern of mRNA expression was found in C3WHeSnJ and mice, except that the level of TNF-a mRNA in peritoneal macrophages from normal C3WHeJ mice was half that of normal C3Hi HeSnJ mice (Table 3), and C3WHeJ mice showed a less pronounced decrease at 6 h in I-Aa mRNA levels than C3WHeSnJ mice but similar suppression at 24 h. Suppression of major histocompatibility (MHC) class II antigen expression correlates with a reduced capacity for antigen presentation4i and may contribute to lethality from CLP. Suppression of MHC class II antigen expression in surgical patients correlates with the development of infection and clinical outcome.42,43 Restoration of MHC class II of infected mice to normal levels improves survivali However, in contrast to other experimental models where MHC class II suppression is due to TNF secretion,44 our results suggest that after CLP, the decline of I-Aa is not mediated by TNF. Our finding that the levels of TNF mRNA increase after CLP in C3WHeJ mice confirms reports from others that there is a defect in translation of TNF

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et al.

mRNA in these mice.3 Increased TNF mRNA levels, however, do not imply intact transcription mechanisms because stabilization of the TNF and IL-1 mRNA molecules to the action of ribonucleases is an important mechanism of regulation of their levels.45 We conclude that even though the lethal effects of CLP are generally thought to be caused by overwhelming Gram-negative bacteremia and endotoxic shock, several observations contradict this belief. Our findings suggest that the lethal effects of CLP cannot be attributed simply to endotoxemia and the toxic effect of excessive TNF expression. We found that CLP caused a polymicrobial infection characterized by predominantly anaerobic bacteremia within the first 24 h. These results are comparable to the results of Hyde et a1.31 CLP resulted in only a slight increase in serum endotoxin levels compared with models of Gram-negative bacterial infection CLP did not cause the same dramatic increase in serum TNF levels as seen with the administration of an endotoxin or Gram-negative bacteria (LPS). Administration of anti-TNF antibodies decreases mortality in models of endotoxemia or Gram-negative bacteremia, but actually increases mortality from CLP. Endotoxinresistant C3H/HeJ mice are resistant to mortality induced by intraperitoneal injection of endotoxin or Gram-negative bacteria (presumably because of decreased ability to produce TNF) but not to CLP. The fact that IL-lra improves survival from both endotoxin injection and CLP suggests an important role for this cytokine in the pathophysiology of both models. The latter observation and or results converge to the conclusion that lethality from CLP is associated with an overexpression of IL-l, a suppression of MHC class II antigen expression, and is independent of the capacity to produce TNF.

MATERIALS AND METHODS

scribed by Baker et al.46 Specifically, the mice were anaesthetized with subcutaneous ketamine (80 mg/kg) and xylazine (16 mg/kg). Through a midline laparotomy incision, the caecum was ligated just below the ileocaecal junction with 3-O silk and was punctured once with an 18gauge needle. Using gentle pressure on the caecum, a small amount of faecal material was expressed to ensure the patency of the puncture holes. The abdominal incision was then closed with nylon suture. No fluid resuscitation was given after CLP.

Mortality Mortality was determined in C3WHeSnJ (n = 34) and C3WHeJ (n = 33) mice following CLP. Seven days after CLP, surviving C3WHeSnJ (n = 11) and C3H/HeJ (n = 16) mice were injected with 1 mg of endotoxin (LPS) intraperitoneally (E. coli serotype Olll:B4, phenol extracted [Sigma Chemical Co., St. Louis, MO]). This was done to determine if the CLP procedure released enough endotoxin in the endotoxin-sensitive mice to prime them. Priming is a known method by which a sublethal endotoxin dose is able to induce tolerance to a subsequent lethal endotoxin exposure. 38,3gThis would also determine if the normally endotoxin-resistant mice were made susceptible to endotoxin by CLP.

Caecal and Blood Cultures Caeca from C3WHeSnJ and C3WHeJ mice were excised and cultured to assesspossible differences in indigenous flora. Blood cultures were obtained from C3H/ HeSnJ, C3WHeJ and Swiss Webster mice at various intervals following no treatment (control), sham laparotomy (sham) or CLP. The blood was obtained by cardiocentesis following thoracotomy, with the abdominal cavity still closed to avoid contamination. Samples were incubated on MacConkey’s agar and tryptic soy agar with 5% sheep’s blood under strict anaerobic conditions for 48 to 72 h for determination of aerobic organisms. The clinical laboratory at the Veterans Administration Medical Center (Louisville, KY) was used for identification of bacteria.

Animals

Peritoneal

Adult male endotoxin-sensitive (C3H/HeSnJ), endotoxin-resistant (C3H/HeJ) (Jackson Laboratory, Bar Harbor, ME), and SwissWebster mice (Charles River Laboratories, Portage, MI), weighing between 25 g to 30 g, were studied. The mice were housed in an American Association for Accreditation of Laboratory Animal Care (AAALAC)-approved facility under the supervision of a veterinarian and were fed standard rodent chow and water ad libitum. All of the mice remained housed in our facility for at least 2 weeks before the experimentation to allow equilibration of intestinal flora. Studies were carried out in strict accordance with the National Institutes of Health guidelines for the treatment of laboratory animals.

Animals were killed by cervical dislocation to isolate peritoneal macrophage. Pooled groups of 5 to 10 C3W HeSnJ and C3WHeJ mice were used for each macrophage isolation. Macrophages were harvested by peritoneal lavage with ice-cold RPM1 1640 medium (GIBCO/BRL, Bethesda, MD). Resident peritoneal macrophages from normal animals were used as controls. After centrifugation at 200 x g, the cells were resuspended at a concentration of lo6 cells/ml in RPM1 1640 medium supplemented with penicillin (100 IU/ml), streptomycin (100 &ml), and glutamine (2 mM). Ten millilitres of the cell suspension was plated into loo-mm tissue culture plates (Falcon, Lincoln Park, NJ). The macrophages were then allowed to adhere for 1 h in a humidified CQ, (5%) incubator at 37°C. The plates were washed three times .with warm media to remove nonadherent cells. This protocol has resulted routinely in macrophage cultures con-

Caecal ligation

and puncture

Caecal ligation and puncture was performed as de-

Macrophage

Isolation

TNF and LPS in in&a-abdominal

taining >95% nonspecific esterase-positive cells. Extraction of total RNA was performed immediately after washing the cells. Media were stored frozen at -70°C for TNF assay.

TNF Assay TNF-a levels in serum samples were determined using an ELISA assay(FactorTest-mouse TNFa, Genzyme, Cambridge, MA), following 1~3dilution according to the protocol provided by the manufacturer. TNF levels in culture media were determined by the WEHI 164 subclone 13 cell line bioassay, as described by Eskandari et al.47 TNF levels in undiluted culture media were also measured by the ELISA method to ensure that immunoreactive TNF does not exist in a biologically inactive form in C3WHeJ mice.

Endotoxin Assay Endotoxin levels were determined in 1:4 dilutions of mouse serum using the limulus amebocyte assay (QCL1000, BioWhittaker, Walkersville, MD), according to the manufacturer’s protocol and as described by Ayala et a1.26

Total RNA Extraction and mRNA Analysis Extraction of total RNA from isolated macrophages and tissues was performed by the method of Chomczynski and Sacchi.48 Northern blot analysis was performed as described previously40,49 using 32P-labelled complementary DNA (cDNA) probes. The mouse TNF-(Y and IL-l@ cDNA probes were the gift of Dr Arjun Singh (Genentech, Inc., South San Francisco, CA). The I-Aa probe used was pRAAC, an 880 bp A-alphak cDNA, which was generously provided by Dr Jerold Woodward (Albert B. Chandler Medical Center, University of Kentucky, Lexington, KY). Equal loading and transfer of each mRNA sample was verified by ethidium bromide staining of the blots, according to the method of Mendelssohn and Coleman.” Northern blots were analysed by Densitometric scanning.

Data Presentation and Statistics For mortality studies, differences in experimental means were compared using the Fisher’s exact probability test. Experimental means were compared for other studies using one-way ANOVA and Tukey’s test for multiple comparisons. Differences were considered significant at P < 0.05. Each experiment was performed at least twice with comparable results.

Acknowledgements Supported by Veterans Administration Merit Review Grant (WGC and KMM). The authors thank Molly Redett for help with the manuscript.

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