Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0704382 on October 20, 2004

Published online before print October 20, 2004
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(Journal of Leukocyte Biology. 2005;77:16-23.)
© 2005 by Society for Leukocyte Biology

Linking the "two-hit" response following injury to enhanced TLR4 reactivity

Thomas J. Murphy*, Hugh M. Paterson*, Sara Kriynovich*, Yan Zang*, Evelyn A. Kurt-Jones{dagger}, John A. Mannick* and James A. Lederer*,1

* Department of Surgery (Immunology), Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts; and
{dagger} Department of Medicine, University of Massachusetts Medical School, Worcester

1 Correspondence: Department of Surgery (Immunology), Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115. E-mail: jlederer{at}rics.bwh.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Severe injury can initiate an exaggerated systemic inflammatory response and multiple organ failure (MOF) if a subsequent immune stimulus, "second hit", occurs. Using a mouse thermal injury model, we tested whether changes in innate immune cell reactivity following injury can contribute to the development of heightened inflammation and MOF. Using high-purity Escherichia coli lipopolysaccharide (LPS) to selectively stimulate Toll-like receptor 4 (TLR4), we demonstrate augmented interleukin (IL)-1ß, tumor necrosis factor {alpha} (TNF-{alpha}), and IL-6 production by 1 day but particularly, at 7 days after injury. The in vivo significance of enhanced TLR4 responsiveness was explored by challenging sham or burn mice with LPS at 1 or 7 days after injury and determining mortality along with in vivo cytokine and chemokine levels. Mortality was high (75%) in LPS-challenged burn but not sham mice at 7 days, although not at 1 day, after injury. Death was associated with leukocyte sequestration in the lungs and livers along with increased proinflammatory cytokine and chemokine levels in these organs. Blocking TNF-{alpha} activity prevented this mortality, suggesting that excessive TNF-{alpha} production contributes to this lethal response. These findings demonstrate the potential lethality of excessive TLR4 reactivity after injury and provide an explanation for the exaggerated inflammatory response to a second hit, which can occur following severe injury.

Key Words: macrophages • inflammation • endotoxic shock • lipopolysaccharide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The results of multiple clinical and animal studies have established that the initial immune response to serious injury is inflammatory, often referred to clinically as the systemic inflammatory response syndrome (SIRS) [1 2 3 ]. In a minority of seriously injured patients, SIRS persists from its onset and may lead directly to multiple organ failure (MOF) [4 ], which is the leading cause of death in patients with serious traumatic or thermal injury who survive initial resuscitation [5 , 6 ]. However, in a majority of injured patients, initial SIRS remits after several days and is often followed by the compensatory anti-inflammatory response syndrome (CARS) characterized by increased production of counter-inflammatory cytokines by cells of the adaptive immune system [7 8 9 ]. Unfortunately, the development of CARS can also suppress host immunity and predispose critically injured patients to developing nosocomial infections. If invasive infection occurs during this period, an exaggerated systemic inflammatory response may recur, potentially leading to late MOF, again with a substantial mortality rate [5 , 10 ]. In fact, the majority of injured patient deaths occurs in association with late MOF [4 ].

Clinical investigators have long believed that an initial serious injury primes the immune system for an excessive inflammatory response to a subsequent stimulus such as nosocomial infection. This has been termed the "two-hit" hypothesis [11 ]. It is widely thought that this exaggerated response to a second hit contributes to the induction of late MOF following the onset of invasive infection several days following a serious injury [12 ]. As cells of the innate immune system are the primary mediators of the inflammatory response following injury and sepsis, we were interested in determining whether excessive innate immune reactivity against invading microorganisms and their products might explain in part the two-hit phenomenon. For that reason, we have focused on defining the impact of severe injury on the reactivity of Toll-like receptors (TLRs), a family of pattern recognition receptors [13 , 14 ]. TLRs initiate strong, inflammatory responses when they encounter pathogen-associated molecular patterns such as bacterial lipopolysaccharide (LPS), lipopeptides, or unmethylated CpG DNA among others [13 , 15 ]. The evolutionarily conserved nature of TLRs and their capacity to recognize a broad spectrum of microbial products suggest that increased TLR responsiveness following injury might be one mechanism underlying the two-hit response.

Using several different rodent models, we and others have shown that injury primes cells of the innate immune system for increased LPS-induced production of proinflammatory cytokines [16 17 18 19 20 21 ]. In a recent report, we demonstrated that burn injury primes mouse splenic macrophages and dendritic cells for enhanced responses to the lipid A moiety of Escherichia coli LPS, lipid A, and to Staphylococcus aureus peptidoglycan, which are well-defined TLR4 and TLR2 agonists, respectively [22 ]. We observed that these cells produced significantly higher levels of the proinflammatory cytokines tumor necrosis factor {alpha} (TNF-{alpha}), interleukin (IL)-1ß, and IL-6 in response to in vitro TLR stimulation within 1 day after injury, with a more pronounced increase in response by 7 days after injury. We also observed an injury-dependent, protracted increase in in vivo LPS-induced cytokine production. As a whole, these findings suggested to us that the propensity of some seriously injured patients to respond with excessive inflammation to further innate immune activation, such as a secondary infection, might be explained in part by injury-induced priming of TLR responses. The purpose of this present study was to expand on our prior work, addressing the influence of injury on TLR responses, with particular emphasis on the in vivo consequences of increased TLR4 reactivity after injury.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Male C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The mice were maintained in an accredited virus-antibody-free animal facility in accordance with the guidelines of the National Institutes of Health (NIH; Bethesda, MD) and the Harvard Medical School Standing Committee on Animal Research (Boston, MA). The mice were acclimated for at least 1 week prior to use in experiments at 6–8 weeks of age.

Reagents
Highly purified phenol re-extracted LPS from E. coli serotype 0111:B4 was prepared and provided by Dr. Evelyn A. Kurt-Jones (University of Massachusetts Medical School, Worcester). The LPS re-extraction was performed essentially as described by Hirschfeld et al. [23 ]. The purified LPS had no or low detectable TLR2 stimulatory activity, as tested using human embryonic kidney cell 293 transfected with human TLR2 or TLR4 gene expression constructs [24 ]. Culture medium for in vitro studies consisted of RPMI 1640 supplemented with 5% heat-inactivated fetal calf serum, 1 mM glutamine, penicillin/streptomycin/fungizone, 10 mM HEPES buffer, 100 µM nonessential amino acids, and 2.5 x 10–5M 2-mercaptoethanol, all purchased from Gibco Invitrogen Corp. (Grand Island, NY) and referred to as complete 5 (C5) medium. Dr. Jeffrey Browning (Biogen, Inc., Cambridge, MA) kindly provided the TNF-{alpha} receptor-55-immunoglobulin G (IgG) fusion protein (TNF-R55-Ig) and control IgG [25 ].

Mouse injury model
The thermal injury protocol, approved by NIH and the Harvard Medical School Standing Committee on Animal Research, was performed as described previously [22 ]. Mice were anesthetized via intraperitoneal (i.p.) injection of ketamine (125 mg/kg) and xylazine (20 mg/kg). The dorsal fur was shaved, and the animal was placed in an insulated plastic mold to expose 25% of body surface area. The exposed dorsum was then immersed in 90°C water for 9 s to induce a full thickness and therefore, anesthetic burn injury. Sham (control) mice were treated exactly as burn mice, except they were exposed to isothermic water. Immediately after the procedure was performed, sham and burn mice were resuscitated with an i.p. injection of 1 ml pyrogen-free normal saline. The mortality from burn injury in the present experiments varied from 0% to 5%.

LPS-induced mortality studies
At 1 or 7 days after burn or sham injury, mice were challenged with purified LPS (10 mg/kg) in 200 µl saline by i.p. injection, and mortality was observed daily over a 7-day period. In some experiments, the lungs, livers, spleens, and sections of mid-small bowel were harvested between 30 and 36 h after LPS challenge, fixed in formalin, and mounted in paraffin blocks. Sections were cut for histological examination after staining with hematoxylin and eosin (H&E) by the Dana Farber/Harvard Cancer Center Rodent Histopathology Core. Pathological changes were evaluated and interpreted by Dr. Roderick Bronson (Department of Pathology, Harvard Medical School). In separate experiments, mice were treated with control IgG or soluble TNF-R55-Ig (200 µg/mouse i.p.) 2 h before LPS challenge and mortality observed.

Ex vivo experiments
Mice were killed by CO2 asphyxiation at 1 or 7 days after burn or sham burn injury. Spleens were harvested, and dispersed cell suspensions were prepared by mincing the tissues on wire mesh. Spleen cells were treated with Tris-ammonium chloride solution for 3 min to lyse red blood cells and were washed twice in C5 medium before being counted and prepared for cultures. Cells were cultured in round-bottomed 96-well plates (Corning Costar Corp., Cambridge, MA) at 5 x 105 cells per well with varying concentrations of LPS or with no additions. After incubation at 37°C in 5% CO2 for 48 h, supernatants were harvested and stored at –20°C for cytokine analysis.

Cytokine and chemokine enzyme-linked immunosorbent assay (ELISA)
The cytokines TNF-{alpha}, IL-1ß, IL-6, and IL-10 and the chemokines macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}), keratinocyte-derived chemokine (KC), MIP-2, and murine homologue of monocyte chemoattractant protein-1 (MCP-1; JE) were measured using ELISA kits purchased from R&D Systems (Minneapolis, MN) according to the manufacturer’s instructions. Briefly, 96-well microtiter ELISA plates (Nunc MaxiSorb, Nunc Nalge International, Denmark) were coated with capture antibody diluted in phosphate-buffered saline (PBS) overnight at 4°C. The plates were then blocked with PBS containing 1% bovine serum albumin (blocking buffer) for 1 h. Plates were then washed with wash buffer (PBS+0.5% Tween-20). Standards of known concentrations and samples were added and then incubated for 1 h at 37°C. The plates were washed, and biotinylated detection antibody was added. After 1 h, plates were washed, avidin-horseradish peroxidase conjugate purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) was added, and plates were incubated for 30 min at 37°C. After further washing, the developer substrate was added, and following color development, the reaction was stopped with 2 M H2SO4. Absorbance readings were measured using an ELISA plate reader (Molecular Devices Corp., Sunnyvale, CA) set at 450–570 nM wavelength. Extrapolation of values for sample data from standard curves was calculated using the SoftMax Pro software program (Molecular Devices Corp.).

Detection of plasma and organ cytokine and chemokine levels in LPS-challenged mice
At 1 or 7 days after burn or sham injury, mice were given 10 mg/kg purified LPS by i.p. injection. At time zero and at multiple time-points up to 8 h after LPS injection, groups of mice were killed by CO2 asphyxiation, and cardiac blood was collected into heparinized 1 ml syringes. Plasma was prepared by centrifugation at 3000 g for 20 min at 4°C. Following exsanguinations, lung and liver were harvested into ice-cold PBS containing the recommended concentration of Complete protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Tissue extracts were prepared by homogenizing tissues for 30 s each on ice using a tissue-tearor device (BioSpec Products, Bartlesville, OK). Tissue extracts were then clarified by centrifugation at 3000 gfor 20 min at 4°C. Protein levels in the tissue extracts were measured using the Micro bicinchoninic acid protein assay kit (Pierce Chemical Company, Rockford, IL), according to the manufacturer’s protocol. Plasma and tissue extracts were stored frozen at –20°C prior to performing cytokine and chemokine measurements.

Statistics
The PRISM 3.0 software program (GraphPad, San Diego, CA) was used for all the statistical calculations described in this study. Cytokine production studies were compared by the two-tailed t-test. Survival differences were evaluated by the log rank test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Burn injury augments TLR4-mediated IL-1ß, TNF-{alpha}, and IL-6 production
We showed previously, using the C57BL/10SnJ mouse strain, that burn injury caused augmented TLR2- and TLR4-induced IL-1ß, IL-6, and TNF-{alpha} production by splenocytes and in particular, macrophages [22 ]. We initially wished to substantiate whether the C57BL/6J mouse strain exhibited a similar injury response phenotype. In these studies, mice underwent sham or burn injury, and at 1 or 7 days after injury, spleen cells were prepared and stimulated ex vivo with titrated doses of repurified E. coli LPS. Repurified LPS was used for these studies to assure specific activation of the TLR4 pathway [23 ]. The levels of IL-1ß, TNF-{alpha}, IL-6, and IL-10 produced in response to LPS stimulation were measured by ELISA. As shown in Figure 1 , LPS (10 ng/ml)-stimulated spleen cells from burn-injured mice produced significantly more IL-1ß and TNF-{alpha} on days 1 and 7 after injury. This was also true for IL-6 at the 7-day interval, whereas IL-10 production was not altered by injury at either time-point. These results indicate that injury augments TLR4-mediated proinflammatory cytokine responses in the C57BL/6J mouse strain and that the magnitude of this response is more than twofold greater at 7 days as compared with 1 day after injury.



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  		Figure 1. Burn injury effects on cytokine production by LPS-stimulated splenocytes. Spleen cells prepared from groups of sham or burn mice at 1 or 7 days after injury were cultured with 10 ng/ml LPS. At 48 h, supernatants were assayed for IL-1ß, TNF-{alpha}, IL-6, and IL-10 levels by ELISA. Data are expressed as mean ± SEM of four independent experiments; n = 8 individual mice per group. *, P < 0.05; **, P < 0.001, burn versus sham groups.

 
The enhanced TLR4 response correlates with increased LPS-induced mortality
The results of the present studies and other reports suggested to us that enhanced innate immune reactivity to LPS might be detrimental to the injured host [26 ]. To explore this possibility, we performed in vivo LPS challenge studies to determine if the heightened injury-induced TLR4 responsiveness could induce mortality. Based on the results of pilot experiments, we chose a 10 mg/kg dose of LPS for these mortality studies, as this LPS dose was nonlethal in normal, unmanipulated C57BL/6J mice but nevertheless, caused a significant in vivo cytokine response (data not shown). We also wished to compare the LPS response at 1 day with 7 days post-burn, as we observed a significant increase in the magnitude of the in vitro LPS-induced cytokine response between these time-points. As shown in Figure 2A , sham or burn mice given 10 mg/kg LPS at 1 day after injury did not show significant mortality. However, on day 7, although sham mice displayed no mortality, 75% of the burn mice died over the first 2 days after LPS challenge (Fig. 2B) . Therefore, the injury-initiated enhancement in TLR4 reactivity markedly alters host survival. This finding suggests a mechanism to explain in part the exaggerated, inflammatory response to a "second hit", which can occur in critically injured patients [27 , 28 ].



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  		Figure 2. Burn injury predisposes mice for a lethal LPS response at 7 days, but not 1 day after injury. Sham- or burn-injured mice were challenged by i.p. injection with a single 10-mg/kg dose of LPS on day 1 (A) or day 7 (B) after injury, and mortality was measured for 7 days after LPS challenge (*, P<0.001, by the Log rank test; 7-day burn vs. sham). The data are presented as three independent experiments using 12 mice per experimental group for 1- and 7-day time-points.

 
LPS-induced mortality involves the sequestration of leukocytes in the lungs and livers of injured mice
To determine a possible cause of death, we challenged sham or burn mice with 10 mg/kg LPS at 7 days after injury. Saline-challenged sham and burn mice served as controls. At 30–36 h after LPS or saline treatment, we killed the mice and harvested their lung, liver, spleen, and small bowel for histological investigations. The most dramatic pathology was observed in the lungs and liver of the LPS-treated burn-injured mice, and no significant changes were seen in the small bowel or spleen. As illustrated in Figure 3 , we observed a marked leukocyte stasis within lung capillaries of the LPS-challenged burn mice as compared with sham mice given the same dose of LPS. The sequestration of leukocytes in the lungs of LPS-challenged burn mice was accompanied by increased margination of mostly polymorphonuclear leukocytes (PMNs) into the surrounding lung tissue. The liver showed leukocyte stasis in the capillaries and sinusoids of LPS-challenged burn mice along with increased infiltration of cells into the underlying liver tissue. In contrast to the lungs, these cells were a mixture of PMNs and mononuclear cell types. In contrast, burn mice treated with saline did not show a significant change in cellularity in the lung or liver. Thus, it appears that the mortality occurring in LPS-challenged, burn-injured mice involves sequestration and infiltration of leukocytes in the lungs and livers.



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  		Figure 3. Histology of liver and lung harvested from unchallenged versus LPS-challenged sham- or burn-injured mice. Sham- or burn-injured C57BL/6J mice were given 10 mg/kg LPS by i.p. injection at 7 days after injury or were given saline as indicated. The lungs and livers from these groups of mice were prepared for histology at 30–36 h after saline or LPS injection. The photomicrographs shown are H&E stains at 400x magnification and are representative of four mice per group.

 
Injury primes the host for excessive LPS-induced cytokine and chemokine production in vivo
To relate LPS-mediated mortality and pathology, we examined LPS-induced in vivo cytokine and chemokine production at 1 day versus 7 days after sham or burn injury. Although measuring plasma cytokine and chemokine levels is a common approach to examining in vivo LPS responses, we also wished to examine levels of these mediators in those organs most affected by LPS challenge. To accomplish this, we rapidly homogenized organs in a buffer containing a potent mixture of protease inhibitors at various time intervals after LPS challenge and then measured cytokine and chemokine levels by ELISA [22 ]. The results of pilot studies showed us that we could measure cytokines consistently in organ homogenates by this approach, but it was apparent that investigating LPS response kinetics would be required to accurately determine changes in plasma or organ cytokines or chemokines.

At 1 day after burn injury, we observed no significant differences between sham and burn mice in the plasma levels of TNF-{alpha}, IL-6, or IL-10 (Fig. 4A ). However, at 7 days after injury, we observed markedly increased plasma levels of all three cytokines at nearly all time-points examined (Fig. 4B) . This was particularly true for TNF-{alpha}, where plasma concentrations in sham mice returned to low levels by ~2.5 h after LPS administration, and LPS-induced TNF-{alpha} levels remained markedly elevated in plasma samples from burn-injured mice for nearly 8 h. Although we tested for IL-1ß in all these plasma samples, it was consistently at or near the lower limits of detection (data not shown). These findings clearly demonstrate that injury results in augmented TLR4-mediated reactivity in vivo.



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  		Figure 4. Sequential analysis of plasma TNF-{alpha}, IL-6, and IL-10 levels of LPS-challenged mice. Plasma was prepared from the cardiac blood of sham- or burn-injured mice given 10 mg/kg LPS at 1 (A) or 7 (B) days after injury. Plasma levels or TNF-{alpha}, IL-6, or IL-10 were measured by ELISA at the indicated time-points after LPS treatment. Plasma IL-1ß was not detected at any of these time-points after LPS challenge, and none of these cytokines were detected in the plasma of non-LPS-treated sham or burn mice at 1 or 7 days after injury. These data are presented as the mean ± SEM of three independent experiments using six individual mice per treatment group (*, P<0.05; **, P<0.01, burn vs. sham).

 
Finally, to address changes in TLR4 responses in those organs associated with mortality, we examined chemokine and cytokine responses in homogenates of the lungs and the livers of burn or sham mice given purified LPS at 7 days following injury. The levels of MIP-1{alpha}, KC, MIP-2 and JE (MCP-1) in organ homogenates were elevated in lung and liver by 90 min after LPS injection in burn mice as compared with sham mice (Fig. 5 ). Moreover, KC and JE (MCP-1) levels in the lung and MIP-2 levels in the liver remained significantly elevated in the burn versus sham mice for up to 4 h following LPS challenge. In agreement with our previous studies using unpurified LPS, burn injury had a similar enhancing effect on TNF-{alpha}, IL-1ß, IL-6, and IL-10 levels in the lungs and livers of mice given purified LPS at 7 days after injury (Fig. 5) [22 ]. One exception to this observation was the failure to detect increased TNF-{alpha} levels in the liver of LPS-challenged burn mice. The lung demonstrated the most significant injury-associated change in all these cytokines tested at 4 h after LPS challenge. In addition, although IL-1ß was not reliably detected in the plasma of LPS-treated mice, we measured significant increases in IL-1ß within all the organs examined of burn mice given LPS. This suggests that organ rather than plasma levels of IL-1ß and other potentially dangerous mediators of the lethal LPS response may be more relevant to the mortality observed in LPS-challenged, injured mice.



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  		Figure 5. Chemokine and cytokine levels in the lungs and liver of burn and sham mice challenged with LPS at 7 days after injury. Sham- or burn-injured mice were given 10 mg/kg LPS by i.p. injection at 7 days after injury. Mice were killed at 90 min and 4 h after LPS administration, after which the lungs and liver were excised, and tissue extracts were prepared. The organ extracts were tested for MIP-1{alpha}, KC, MIP-2, JE (MCP-1), TNF-{alpha}, IL-1ß, IL-6, and IL-10 by ELISA, and the detected chemokine and cytokine levels were normalized to the total protein content of the tissue extracts. The data are plotted as the mean ± SEM of six to eight mice per group (*, P<0.05; **, P<0.001, burn vs. sham).

 
Taken collectively, the results of these in vivo studies provide a possible explanation for the difference in mortality to LPS challenge at 1 day versus 7 days after injury. We failed to detect significant increases in plasma cytokines following LPS challenge of burn versus sham mice at 1 day after injury, and significant increases were easily detected in burn mice plasma at 7 days. This outcome suggests a more pronounced in vivo host response to LPS occurred at 7 days as compared with 1 day after injury, which correlates with the timing of the LPS-induced mortality response. Furthermore, the elevated levels of proinflammatory cytokines in the lungs and livers of LPS-challenged 7-day burn mice along with the sustained increases in expression of chemokines known to recruit neutrophils and monocytes from the circulation suggest these factors may mediate the pathological changes and mortality associated with the augmented TLR4 reactivity following injury.

Blocking TNF-{alpha} activity in vivo abrogates the lethal LPS response in burn-injured mice
To test whether TNF-{alpha} is important in mediating the LPS-induced mortality following injury, we measured the survival of sham- or burn-injured mice that were given 200 µg soluble TNF-R55-Ig fusion protein 2 h prior to LPS challenge at 7 days after injury. As shown in Figure 6 , treating mice with TNF-R55-Ig almost completely abrogated the lethality seen in LPS-challenged burn mice given control human IgG (75% mortality). These results indicate that LPS-induced TNF-{alpha} plays a major role in LPS-mediated lethality, which occurs at 7 days after burn injury.



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  		Figure 6. Blocking TNF-{alpha} activity in vivo protects burn-injured mice from LPS-induced death. Sham- or burn-injured mice, 20 per group, received TNF-R55-Ig or control Ig i.p. (each 200 µg) 2 h before LPS challenge. Mortality was assessed daily for a total of 7 days (*, P<0.01, by the Log rank test; burn control Ig-treated vs. all other groups).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of previous studies investigating changes in innate immune responses following injury have shown that innate immune cell types such as neutrophils, blood monocytes, tissue macrophages, and dendritic cells display a heightened level of reactivity to microbial stimuli resulting in higher production of inflammatory mediators [16 17 18 19 20 21 ]. Although this increased innate immune reactivity may have evolved to protect the injured host from developing opportunistic infections by boosting immune surveillance and cellular microbicidal activity, a high inflammatory response potential can also result in strong and possibly fatal systemic inflammation. It is this heightened innate immune response following injury that forms the basis for the two-hit hypothesis proposing that injury primes the immune system ("first hit") for a lethal inflammatory reaction to a later, otherwise nonlethal, secondary insult (second hit) [11 ]. The two-hit hypothesis is supported by the results of epidemiological studies linking postinjury opportunistic infections to the development of severe SIRS and MOF in critically injured patients [27 , 29 ].

As we and other research groups have uniformly shown that injury primes innate immune cells for augmented LPS-stimulated proinflammatory cytokine production, it seemed likely that this resulted from increased TLR4 responsiveness [18 , 22 , 30 , 31 ]. Thus, we were interested in determining if changes in TLR4 responses could contribute to the development of severe SIRS and MOF in a mouse injury model. Consequently, we showed that burn injury primed mouse macrophages and dendritic cells for enhanced IL-1ß, IL-6, and TNF-{alpha} production when exposed to crude E. coli LPS ex vivo or in vivo [22 ]. We confirm and extend this finding here using re-extracted, high-purity E. coli LPS, a specific TLR4 agonist. However, the primary focus of this study was to determine the in vivo consequences of the protracted increase in TLR4 reactivity following injury. We found that mortality was high in burn, but not sham, mice given purified LPS at 7 days after injury, and negligible mortality occurred when injured mice were challenged on day 1 with the same dose of LPS. As LPS lethality coincided with the exaggerated TLR4 response observed at 7 days after injury, this suggests an association between enhanced TLR4 reactivity and LPS-induced mortality. We believe that the level of TLR4 responsiveness plays a significant role in this lethal LPS response, as mice and splenocytes were not refractory to LPS stimulation at 1 day after injury and in fact, demonstrated an increase in LPS reactivity. Thus, the host susceptibility to LPS lethality in the two-hit response might depend on a sensitivity threshold that can ultimately result in the release of high levels of lethal mediators such as TNF-{alpha}, IL-1ß, or high mobility group box 1 [32 33 34 35 ]. To our knowledge, this is the first report to demonstrate that excessive TLR4 responsiveness can mediate increased mortality in a mouse model for the injury-associated two-hit response.

We found that the lethal LPS response correlated with the protracted elevation of in vivo chemokine and cytokine production in the organs suffering pathological changes following LPS challenge, the lungs and livers of 7-day burn mice. Moreover, we demonstrated that blocking TNF-{alpha} activity provided significant protection from LPS-induced mortality in day-7 burn-injured mice. This result indicates that increased systemic levels of LPS-induced TNF-{alpha} play a significant role in mediating the lethal response to a late LPS challenge in burn-injured mice. As the purified LPS used in these experiments is a selective TLR4 agonist, the present results indicate that primed TLR4 reactivity is indeed involved in mediating the lethal LPS response. We acknowledge that the discovery that TNF-{alpha} plays an important role in LPS lethality is clearly not, by itself, a novel finding, as it has already been shown that blocking TNF-{alpha} activity in vivo protects normal mice from LPS lethality [36 , 37 ]. However, this is the first report showing the importance of TNF-{alpha} in mediating the lethal second hit response to an otherwise nonlethal dose of LPS following injury.

Pathological examinations suggested that the cause of death was excessive sequestration and infiltration of neutrophils and mononuclear cells in the lungs and livers of LPS-challenged burn-injured mice. Thus, the LPS-challenged burn mice appear to have died of amplified inflammation in two major organs, with a pathological picture that resembles MOF. This finding also supports the idea that burn injury lowers the threshold for LPS-mediated inflammatory responses in parenchymal organs, as supported by our in vivo cytokine and chemokine data. First, we found significantly higher IL-1ß, IL-6, and TNF-{alpha} levels in the lungs, livers, and spleens of LPS-challenged mice at 7 days after burn injury as compared with similarly treated sham-injured mice. In actuality, the burn-induced effect on LPS-induced cytokine levels in these organs was in some instances greater than that observed in ex vivo studies. Second, we found that injury primed mice for enhanced LPS-induced chemokine [MIP-1{alpha}, KC, MIP-2, and JE (MCP-1)] levels in the lungs and livers at 7 days. It is likely that this injury-associated increase in chemokine levels within the lungs and livers of LPS-challenged mice contributes to the recruitment, transmigration, and sequestration of leukocytes within these organs [38 39 40 ]. As a group, these results suggest that LPS-challenged 7-day burn mice died of parenchymal organ inflammation and damage precipitated by excessive cytokine and chemokine production.

As noted previously, multiple investigators have described increased proinflammatory cytokine production to bacterial endotoxins by monocytes and macrophages from injured patients and in various animal models of injury [18 ]. In contrast, prior exposure of monocytes or macrophages to TLR2 or TLR4 agonists can result in a lower, proinflammatory cytokine response by cells when these cells are restimulated with the same or similar agonists [41 , 42 ]. However, most investigators studying injury have not observed suppressed/tolerized TLR2 or TLR4 responses [22 , 30 , 43 ]. This suggests that primed innate cell reactivity may be a general feature of the mammalian immune response to injury. Nevertheless, features associated with a reduced capacity to mediate adaptive immune function, such as diminished innate cell major histocompatibility complex type II expression and lower IL-12 production by the same cells, have been shown to coincide with the development of this enhanced, proinflammatory phenotype [44 45 46 47 ]. Thus, the markedly increased reactivity of innate immune cells occurring at a time after injury when the adaptive immune system is maximally suppressed could potentially increase the likelihood of supervening infection to trigger a dangerous innate inflammatory response.

Our previous study showed that burn injury did not significantly change cell-surface TLR4-MD-2 expression on macrophages [22 ]. We confirmed this observation by judging cell-surface TLR4-MD-2 expression on splenic macrophages harvested from C57BL/6J mice at 1 or 7 days after injury (data not shown). Thus, it appears that injury enhances TLR4 reactivity by augmenting positive signaling cascades or by disrupting negative regulatory factors in macrophages. Potential TLR4 signal-enhancing mechanisms at play may include increased receptor clustering following LPS stimulation or priming through a macrophage-inhibitory, factor-dependent mechanism [48 , 49 ]. CD14 or membrane-activated complex 1 (CD11b/CD18) is a LPS coreceptor, which may also be involved in increased TLR4 clustering or signaling observed following burn injury [50 ]. Alternatively, the injury-induced modulation of negative regulatory factors, such as suppressor of cytokine signaling 1; a splice variant of MyD88, MyD88 s; the Toll/IL-1 receptor (IL-1R; TIR) signaling inhibitor, IL-1R-associated kinase (IRAK)-M; or a nonsignaling type 1 IL-1R-related molecule, single Ig IL-1-related receptor (SIGIRR), may also play contributing roles in the injury-mediated increase in TLR4 responses [51 52 53 ]. Cells from mice deficient in these negative regulatory factors display LPS hyper-responsiveness in vitro. Moreover, mice deficient in IRAK-M or SIGIRR have a reduced threshold to lethal LPS challenge, similar to that we report here in burn-injured mice [52 , 54 ]. Thus, future studies investigating the mechanisms underlying the effects of injury on TLR4 responses will need to focus on alterations in the TIR signaling cascade.

In summary, we provide evidence relating the injury-induced modulation of TLR4 reactivity to the two-hit response and MOF following injury. Our findings support the impression of a number of clinical investigators that patients suffering major trauma or burns can react to a second hit, often a nosocomial infection, several days after the initial injury with an intense and morbid inflammatory response [4 , 55 , 56 ]. This enhanced host response can often lead to MOF with significant mortality. In addition to increasing our understanding of how injury modulates the innate immune response, we define a mouse model of the two-hit response, which will help us and others investigate this complex and important clinical problem. It is hoped that an increased understanding of the innate immune response to injury will lead to future treatment advances toward controlling the potential detrimental consequences of excessive innate immune stimulation following severe injury.


   ACKNOWLEDGEMENTS
 
This research work was supported by grants from the National Institutes of Health (GM57664 and GM35633), the Brook Research Fund, and the Julian and Eunice Cohen Surgical Research Fund. The authors thank Marissa Miller, LVT, for her technical support.

Received July 1, 2004; revised September 14, 2004; accepted September 19, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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