American Journal of Respiratory and Critical Care Medicine

Rationale: Myeloid-related protein-8 (MRP8) and MRP14 can form heterodimers that elicit a variety of inflammatory responses. We showed that MRP8/14 is a ligand for Toll-like receptor-4, and that mice deficient in MRP8/14 are protected against endotoxic shock–induced lethality.

Objectives: To determine (1) the extent of MRP8/14 release in patients with sepsis and/or peritonitis and in healthy humans exposed to LPS and (2) the contribution of MRP8/14 to the host response in murine abdominal sepsis.

Methods: MRP8/14 was measured in 51 patients with severe sepsis, 8 subjects after intravenous injection of LPS, and 17 patients with peritonitis. Host responses to sepsis were compared in mrp14 gene–deficient (and thereby MRP8/14-deficient) and wild-type mice intraperitoneally injected with Escherichia coli.

Measurements and Main Results: Patients with sepsis displayed elevated circulating MRP8/14 concentrations on both Days 0 and 3, and LPS injection resulted in systemic MRP8/14 release in healthy humans. In patients with peritonitis, MRP8/14 levels in abdominal fluid were more than 15-fold higher than in plasma. MRP14-deficient mice displayed improved defense against E. coli abdominal sepsis in an early phase, as indicated by diminished dissemination of the bacteria at 6 hours. In addition, MRP14-deficient mice demonstrated decreased systemic inflammation, as reflected by lower cytokine plasma concentrations, and less severe liver damage.

Conclusions: Human sepsis and endotoxemia are associated with enhanced release of MRP8/14. In abdominal sepsis, MRP8/14 likely occurs primarily at the site of the infection, facilitating bacterial dissemination at an early phase and liver injury.

Scientific Knowledge on the Subject

Myeloid-related protein 8 (MRP8) and MRP14 are members of the S100 protein family; the MRP8/14 complex is a ligand for Toll-like receptor-4. Mice deficient in MRP14 are protected against endotoxic shock–induced lethality.

What This Study Adds to the Field

MRP8/14 plasma levels are elevated in patients with sepsis and in healthy humans injected with LPS; patients with peritonitis show highest MRP8/14 levels at the site of infection. In murine Escherichia coli sepsis, MRP14 contributes to early bacterial dissemination and liver injury.

Sepsis is the second leading cause of death in noncoronary intensive care units and the tenth leading cause of death overall in developed countries (1, 2). Since the late 1970s the incidence of sepsis has shown an annual increase of 9%, to 240 per 100,000 population in the United States, up to the year 2000 (3). Whereas the overall mortality rate of sepsis is 25 to 30%, mortality in patients with abdominal sepsis can be as high as 60% (4). Clearly, sepsis, and in particular sepsis with an abdominal source, represents a major clinical and therapeutic challenge. Of note, among the various bacteria identified as causative organisms in peritonitis, Escherichia coli remains one of the most common pathogens (4, 5).

The host response to sepsis is orchestrated by a variety of inflammatory mediators and pathways (6, 7). It has become clear that invasive infection is commonly associated with the release of endogenous proteins that serve to warn the host of imminent danger. These proteins have collectively been called “damage-associated molecular patterns” (DAMPs) or “alarmins” (8). S100 proteins, which mediate inflammatory responses and are involved in the recruitment of inflammatory cells to sites of injury (9, 10), have been suggested to be alarmins. S100 proteins comprise a family with more than 20 members, 3 of which have been linked to innate immune functions by their expression by myeloid cells: S100A8 (also called calgranulin A or myeloid-related protein-8 [MRP8]), S100A9 (MRP14 or calgranulin B), and S100A12 (MRP6 or calgranulin C). Of these, MRP8 and MRP14 form heterodimers, which are the biologically relevant forms of these proteins (1113). MRP8/14 complexes induce a variety of inflammatory reactions and the extent of MRP8/14 expression correlates with disease activity in several inflammatory disorders (10, 14). We showed that the Toll-like receptor (TLR)-4 complex interacts with MRP8/14 (13). MRP8/14 was found to amplify LPS-induced tumor necrosis factor (TNF)-α release in vitro and in vivo, and mice with a targeted deletion of the mrp14 gene were protected against LPS-induced lethal shock. MRP8 is also almost not detectable at the protein level in mature phagocytes of MRP14-deficient (MRP14−/−) mice despite normal MRP8 mRNA levels, probably due to the elevated metabolism of MRP8 in the absence of its binding partner. Thus, targeted deletion of MRP14 leads to a complete lack of a functional MRP8/14 complex in the mouse (13, 15). Moreover, MRP14−/− mice showed improved survival after intraperitoneal injection of E. coli (13).

In the present study, we first aimed to determine the extent of systemic MRP8/14 release in patients with severe sepsis and to examine local MRP8/14 levels at the site of infection in patients with peritonitis. In addition, we determined the role of MRP14 in specific host responses to murine E. coli abdominal sepsis. For this, we compared inflammatory reactions in MRP14−/− and normal wild-type mice, using our established model of abdominal sepsis induced by intraperitoneal injection of live E. coli, resulting in severe peritonitis and sepsis with rapid dissemination of bacteria to distant organs and multiple organ damage (1618).

Some of the results of these studies have been previously reported in the form of orally presented abstracts (19, 20).

Human Studies

All studies were approved by the scientific and ethics committees of the Academic Medical Center (Amsterdam, The Netherlands), St. Luke University Hospital (Brussels, Belgium), and/or St. Pierre's Hospital (Ottignies, Belgium). Written informed consent was obtained from all subjects or their relatives.

Patients.

The study included two patient populations described in detail previously (21): (1) 51 patients with severe sepsis (68 ± 2 yr, 31 males), from whom serum was obtained on the day severe sepsis was diagnosed (Day 0) and 3 days thereafter (Day 3), and 31 healthy subjects who served as control subjects; and (2) 17 patients with peritonitis (61 ± 4 yr, 9 males) requiring emergency laparotomy because of perforation (n = 8), anastomotic leakage (n = 7), or other causes (n = 2). Ethylenediaminetetraacetic acid–anticoagulated blood and abdominal fluid samples (from an abdominal drain in the cavum Douglasi) were taken at index laparotomy for peritonitis (t = 0) and after 1, 2, and 3 days.

Healthy subjects.

Eight healthy males (22.6 ± 0.6 yr) were studied after intravenous injection of E. coli LPS (4 ng/kg, lot G; United States Pharmacopeial Convention, Rockville, MD). Ethylenediaminetetraacetic acid–anticoagulated blood was obtained before and 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, and 24 hours after challenge.

Assay.

MRP8/14 was measured as described previously (22, 23).

Mouse Studies
Mice.

Sex- and age-matched MRP14−/− mice (13) and wild-type littermates, backcrossed six times to a C57BL/6 background, were used in all experiments. The Animal Care and Use Committee of the University of Muenster (Muenster, Germany) approved all experiments.

Design.

Abdominal sepsis was induced by intraperitoneal injection of E. coli O18:K1 (104 colony-forming units) as described (1618). Sample harvesting and processing, and determinations of bacterial loads and cell counts, were done as described (1618).

Assays.

MRP8/14 was measured by ELISA (13). Keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 were measured by ELISA (R&D Systems, Abingdon, UK). TNF-α, IL-6, monocyte chemoattractant protein (MCP)-1, and IL-10 were measured by cytometric bead array multiplex assay (BD Biosciences, San Jose, CA). Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined with commercially available kits (Sigma-Aldrich, St. Louis, MO), using a Hitachi analyzer (Boehringer Mannheim, Mannheim, Germany).

Histology.

MRP8/MRP14 and BM8 staining were performed as described previously (15, 24, 25). Semiquantitative pathology scores of lung and liver tissue were generated as described (16).

Statistical Analysis

Data are presented as means ± SEM. The significance of differences between sepsis groups was determined by nonparametric repeated measures analysis of variance or Kruskal-Wallis test. Differences in time after intravenous LPS in healthy volunteers were compared by nonparametric repeated measures analysis of variance. Differences between time points within groups were compared by Wilcoxon signed-rank test. MRP8/14 levels between abdominal fluid and plasma and between survivors and nonsurvivors were compared by Mann-Whitney U test. Correlations were calculated by Spearman's rho test. Differences between MRP14−/− and wild-type mice were analyzed by Mann-Whitney U test. Values of P < 0.05 were considered to represent a statistically significant difference.

Sepsis Results in Elevated Serum MRP8/14 Levels Irrespective of the Source of Infection

The overall in-hospital mortality of patients with severe sepsis enrolled in the study was 45%. The primary source of infection was the lungs in 29 patients (52%), the abdomen in 12 patients (24%), and the urinary tract in 10 patients (20%). Patients with sepsis displayed elevated serum MRP8/14 concentrations both on Day 0 and on Day 3 (both P < 0.005; Figure 1A). All severe sepsis subgroups, with peritonitis, pneumonia, or urinary tract infection as primary infection, showed increased MRP8/14 levels on Days 0 and 3 (all P < 0.05; Figures 1B−1D). Of note, patients with severe sepsis caused by pneumonia displayed the highest MRP8/14 serum concentrations (P < 0.05 vs. severe sepsis caused by peritonitis). There was no apparent correlation between serum MRP8/14 and the severity of disease: serum MRP8/14 did not correlate with either APACHE II (Acute Physiology and Chronic Health Evaluation II) (r = −0.200, P = 0.159) or SOFA (Sequential Organ Failure Assessment) score (r = −0.153, P = 0.284). Furthermore, survivors and nonsurvivors had similar MRP8/14 serum levels (3,676 ± 541.7 and 2,425 ± 374.9 ng/ml, respectively; P = 0.06).

Human Endotoxemia Is Associated with Systemic MRP8/14 Release

To determine whether intravenous LPS induces MRP8/14 release in humans in vivo, healthy subjects were studied after induction of endotoxemia (Figure 2). MRP8/14 concentrations started to rise as early as 1.5 hours after LPS administration, peaking after 5 hours. Remarkably, MRP8/14 was still elevated in plasma 24 hours after LPS injection.

Human Peritonitis Is Associated with Local MRP8/14 Release

We next wished to investigate whether MRP8/14 is released at the site of infection. Therefore, we measured MRP8/14 in the abdominal fluid of patients with peritonitis. None of the 17 patients with peritonitis died within 28 days. During peritonitis, MRP8/14 levels in abdominal fluid were more than 15-fold higher than in concurrently obtained plasma and these local levels remained elevated throughout the 3-day sampling period (P < 0.0005 at all time points; Figure 3).

MRP8/14 Expression Is Enhanced during E. coli–induced Abdominal Sepsis in Mice

Having established that MRP8/14 is released systemically in patients with severe sepsis and predominantly at the site of infection in patients with peritonitis, we next studied the role of MRP8/14 during E. coli abdominal sepsis in mice. Therefore, we first aimed to obtain insight into local, systemic, and organ MRP8/14 complex concentrations in mice injected with E. coli by measuring MRP8/14 levels in peritoneal lavage fluid (PLF), plasma, and lung and liver homogenates of uninfected wild-type mice and mice after administration of E. coli. Intraperitoneal injection of E. coli caused significantly increased MRP8/14 levels in all compartments after 6 and 20 hours (all P < 0.01 vs. t = 0 h; Figures 4A−4D). To determine the localization of MRP8/14 in lungs and liver in our model, we studied the expression of MRP8/14 complexes in lung and liver tissue obtained from mice 20 hours after injection with E. coli. Immunohistochemical staining of MRP8/14 revealed a strong increase in the number of MRP14+ cells (predominantly neutrophils) in the lungs and liver tissue of mice with E. coli (arrows, Figure 5B vs. 5A and Figure 5D vs. 5C, respectively).

Impact of MRP14 Deficiency on Leukocyte Recruitment and Bacterial Loads

To obtain a first insight into the role of MRP8/14 in specific host responses to severe bacterial infection, we determined the influx of leukocytes to the primary site of infection and bacterial loads in several body compartments 6 and 20 hours after inoculation. We considered this of particular interest considering that MRP8 has been implicated in neutrophil migration (23, 2629) and considering that the early recruitment of neutrophils to the peritoneal cavity contributes to an effective antibacterial defense in this model (30, 31). Moreover, we showed that MRP8/14 signals via TLR4 (13); this receptor is of eminent importance for host defense against gram-negative infection in general (32) and in this model of gram-negative abdominal sepsis in particular (33). Leukocyte counts and differentials were similar in PLF obtained from MRP14−/− and wild-type mice at both time points (Table 1). Moreover, the local (PLF) levels of the neutrophil-attracting CXC chemokines KC and MIP-2 did not differ between both mouse strains (Table 1). MRP14−/− mice displayed lower bacterial loads at 6 hours postinoculation at the primary site of infection (PLF; P = 0.06 vs. wild-type mice; Figure 6A) and at the more distant sites in blood and lungs (both P < 0.05; Figures 6B and 6C). Twenty hours after inoculation bacterial loads were similar in all body compartments in both mouse strains.

TABLE 1. CELL COUNTS AND CHEMOKINE LEVELS IN PERITONEAL LAVAGE FLUID



6 hr

20 hr

Wt
MRP14−/−
Wt
MRP14−/−
Cells, × 105/ml
 Total cells12.2 ± 1.913.0 ± 1.1583.1 ± 81.7671.1 ± 42.1
 Neutrophils7.5 ± 1.26.6 ± 0.8503.0 ± 69.2553.8 ± 40.9
 Macrophages4.5 ± 1.26.0 ± 1.362.3 ± 19.893.3 ± 6.6
Chemokines, pg/ml
 KC5,842 ± 2,6642,147 ± 48617,320 ± 1,54216,478 ± 2,477
 MIP-2
409 ± 212
155 ± 36
2,717 ± 412
2,793 ± 575

Definition of abbreviations: KC = cytokine-induced neutrophil chemoattractant; MIP-2 = macrophage inflammatory protein-2; MRP14 = myeloid-related protein-14; Wt = wild type.

Mice were injected intraperitoneally with 104 colony-forming units of Escherichia coli. At the indicated time points, mice were killed and cell counts and chemokine levels in peritoneal lavage fluid were determined. Data represent means ± SEM of 8–10 mice per genotype at each time point.

MRP14−/− Mice Have a Reduced Systemic Cytokine Response

We demonstrated that MRP14−/− bone marrow cells are less responsive to LPS, which correlated with lower TNF-α levels in the circulation of MRP14−/− mice challenged with LPS in vivo (13). To study the impact of MRP14 deficiency on cytokine release during gram-negative sepsis, we measured the levels of TNF-α, IL-6, IL-10, and MCP-1 in PLF (Figure 7, left) and plasma (Figure 7, right) 6 and 20 hours after inoculation. Overall, cytokine responses were lower in MRP14−/− mice at 6 hours postinfection: in PLF the TNF-α, IL-6, and MCP-1 levels were lower in MRP14−/− mice at this time point, although these differences did not reach significance, whereas in plasma TNF-α (P < 0.05), IL-6 (P < 0.05), and MCP-1 (P = 0.07) concentrations were lower in MRP14−/− mice. By 20 hours postinfection, the levels of these mediators were similar in PLF and plasma of both mouse strains.

MRP14−/− Mice Demonstrate Unaltered Lung Inflammation

This model of abdominal sepsis is associated with an inflammatory response in the lungs (1618). Considering the enhanced expression of MRP8/14 in the lungs, we were interested to determine the impact of MRP14 deficiency on sepsis-induced lung inflammation. Therefore, we analyzed lung tissue slides obtained from wild-type and MRP14−/− mice 20 hours after inoculation with E. coli. The lungs of both mouse strains displayed signs of inflammation as reflected by the accumulation of neutrophils in the interstitium and along vessel walls (Figures 8A and 8B). The total histological scores (semiquantified according to the scoring system described in Methods) of the two mouse strains were similar (Figure 8C). In addition, granulocyte stainings showed an equal level of granulocyte influx into the lungs of wild-type and MRP14−/− mice (Figures 8D and 8E).

MRP14−/− Mice Are Protected from Liver Damage

Our model of E. coli sepsis is characterized by liver injury associated with parenchymal inflammation (1618). To obtain insight into the role of MRP8/14 in liver injury during E. coli–induced sepsis, we determined liver damage in wild-type and MRP14−/− mice 20 hours after inoculation. On histopathological examination, both wild-type and MRP14−/− mice displayed signs of inflammation of the hepatic parenchyma (Figures 9A and 9B). Interestingly, liver inflammation in MRP14−/− mice was less profound compared with that in wild-type mice as reflected by lower total histology scores (semiquantified according to the scoring system described in Methods) (P < 0.05; Figure 9C). The histological findings of less severe liver inflammation in MRP14−/− mice were confirmed by granulocyte stainings of liver sections (Figures 9D and 9E). Clinical chemistry confirmed the existence of less profound hepatocellular injury in MRP14−/− mice, that is, MRP14−/− mice had lower plasma ALT (P < 0.05; Figure 9F) and AST levels, although this latter difference did not reach statistical significance (P = 0.06; Figure 9G). In conclusion, MRP14 deficiency was associated with less extensive liver inflammation and injury.

Kupffer Cells and Infiltrating Neutrophils Both Express MRP8/14 in the Liver during E. coli Sepsis

In Figures 5C and 5D we showed that MRP8/14 expression is enhanced in liver tissue 20 hours after injection with E. coli. MRP8 and MRP14 are specifically released during the activation of phagocytes (34, 35). In our sepsis model, candidates for MRP8/14-expressing phagocytes in the liver are Kupffer cells and infiltrating neutrophils (the latter cells are stained in Figures 9D and 9E). To investigate what cells express MRP8/14 in the liver during E. coli sepsis (and thus might be responsible for the enhanced hepatic injury), we performed immunohistochemical double stainings with antibodies against MRP14 and BM8 (the latter being an indicator for Kupffer cells in liver tissue [24, 25]). The livers of healthy wild-type mice displayed that all BM8-positive Kupffer cells are MRP14 negative, with one exception (Figure 10A). Liver tissue of mice with E. coli revealed a mixture of MRP14-expressing cells composed of BM8-positive Kupffer cells (Figure 10B, solid arrows) and BM8-negative neutrophils (Figure 10B, open arrow). These data suggest that both Kupffer cells and infiltrating neutrophils express MRP8/14 and may—at least in part—attribute to the enhanced liver injury during E. coli sepsis.

Severe sepsis remains a major challenge in the care of critically ill patients. The outcome is poor and mortality rates remain up to 30–40%. Peritonitis is the second most common cause of sepsis, and abdominal sepsis especially bears a grim prognosis (1, 36). During local and systemic bacterial infections, inflammatory responses may act as double-edged swords, fighting pathogens on the one hand, but potentially causing tissue damage on the other hand. Previously, we showed that MRP8/14 deficiency protects against mortality induced by both endotoxic shock and E. coli–induced sepsis (13), suggesting that MRP8/14 has a net detrimental role in both systemic inflammatory response syndrome and sepsis. We here aimed to investigate MRP8/14 release in severe sepsis and, subsequently, the role of MRP8/14 in abdominal sepsis. We made the following key observations: (1) patients with severe sepsis and healthy humans intravenously injected with LPS have increased circulating MRP8/14 levels; (2) MRP8/14 is released at the site of infection in patients with peritonitis; and (3) MRP14 deficiency is associated with diminished spreading of E. coli to blood and lungs during the early phase of infection, and with reduced liver damage.

MRP8 and MRP14 can form heterodimers and these heterodimers represent 40% of the cytosolic proteins in neutrophils (37). MRP8/14 is released during inflammation (14, 3841) and there is a strong correlation between systemic MRP8/14 levels and the presence of inflammation (42). In addition, several inflammatory disorders, such as rheumatoid arthritis, cystic fibrosis, and chronic bronchitis, are associated with elevated plasma concentrations of MRP8/14 (43, 44). Knowledge about MRP8/14 expression during peritonitis is highly limited. Lagasse and colleagues found that neutrophils recruited to the peritoneal cavity after a thioglycollate injection were MRP8+ and MRP14+ (45). We here demonstrate for the first time that circulating MRP8/14 levels are elevated in patients with severe sepsis. In addition, MRP8/14 release occurs in sepsis irrespective of the primary source of infection, with patients with pneumonia displaying the highest concentrations. MRP8/14 concentrations did not correlate with the severity of sepsis, as reflected by APACHE II and SOFA scores, or mortality. LPS administration in healthy human volunteers induced increased circulating MRP8/14 concentrations as early as 1.5 hours. Remarkably, MRP8/14 was still elevated in plasma 24 hours after LPS injection. To the best of our knowledge the exact mechanism of the clearance of MRP8/14 is not known. However, one study has suggested that the half-life of circulating MRP8/14 is about 1 day (46). In that study, performed in patients with acute Kawasaki disease, MRP8/14 levels decreased from 3,251 ng/ml on Day 0 to 1,265 ng/ml on Day 1 after intravenous immunoglobulin therapy.

The fact that intravenous LPS rapidly elicits a rise in systemic MRP8/14 concentrations implies that LPS induces the release of a danger signal that interacts with its own receptor, TLR4, amplifying its responses (13).

The observed elevated systemic and local MRP8/14 levels in patients and mice in our studies can be explained by active release of MRP8/14 from stimulated, viable neutrophils and other phagocytes (9, 10, 13). It is possible that MRP8/14 is also released passively, but this has not been reported previously. Indeed, although MRP8/14 (and other S100 family members) has been suggested to be alarmins, passive release of MRP8/14 after nonprogrammed cell death (necrosis) has not been demonstrated (8). Therefore, further research is warranted to study mechanisms contributing to elevated MRP8/14 levels during sepsis.

To the best of our knowledge, local MRP8/14 levels at sites of infection have not been reported in patients previously. We here show that patients with peritonitis displayed increased MRP8/14 levels in their abdominal fluid that were about 15-fold higher than in concurrently obtained plasma. In our mouse model of E. coli–induced abdominal sepsis MRP8/14 complexes were not only elevated locally (PLF) and systemically (plasma), but also in distant organs (lungs and liver). These results expand data from Raquil and colleagues, who showed that MRP8/14 levels are elevated in lung homogenates and bronchoalveolar lavage fluid from mice infected with Streptococcus pneumoniae (41).

At 6 hours postinoculation, MRP14−/− mice demonstrated lower bacterial loads at the primary site of infection (PLF; P = 0.06) and at distant sites in the blood and lungs. However, at 20 hours, bacterial burden no longer differed at any site. The early difference in bacterial outgrowth between the two mouse strains probably is not due to an enhanced ability of MRP14−/− neutrophils to generate reactive oxygen species, because we found that wild-type and MRP14−/− neutrophils and monocytes display a similar capacity to mount a respiratory burst response (data not shown), confirming earlier data (47). In addition, MRP14 deficiency was reported not to impact on other key neutrophil functions, including chemotaxis, phagocytosis (of E. coli), and apoptosis (47).

Targeted deletion of MRP14 leads to a complete lack of functional MRP8/14 complexes in the mouse (13, 15). We showed that MRP14−/− mice have lower systemic TNF-α levels 2 hours after intraperitoneal LPS administration (13). Moreover, we found that MRP8/14 complexes amplify LPS-induced signal transduction via TLR4–MD2 in vitro (13). To determine the impact of MRP14 deficiency on TNF-α release and that of other proinflammatory cytokines during sepsis caused by viable E. coli, we measured cytokines locally (PLF) and systemically (plasma) 6 and 20 hours after inoculation. In line with the observed lower circulating TNF-α levels early after LPS treatment (13), we found that not only were the levels of TNF-α lower in plasma of MRP14−/− mice 6 hours after inoculation, but also the plasma concentrations of IL-6 and MCP-1. Of note, MRP14 deficiency did not significantly influence the local concentrations of these mediators in PLF. The decreased systemic cytokine levels in the MRP14−/− mice in the early phase of E. coli sepsis could be explained by diminished activation of TLR4 due to the absence of endogenous MRP8/14 complexes (13). However, neither plasma nor PLF cytokine concentrations differed between the two mouse strains 20 hours after inoculation, supporting the notion that the lower bacterial load at 6 hours could also have contributed to the reduced cytokine levels in the MRP14−/− mice at that time point and/or that the reduced TLR4 signaling can be compensated for by other pathways during the later phase of the infection. Steinbakk and colleagues showed in an in vitro experiment that MRP8/14 inhibits and, at higher concentrations, kills blood culture–isolated E. coli (48). Our finding of lower bacterial loads at 6 hours in MRP14−/− mice suggests that the lack of this potential antibacterial effect is overruled by other mechanisms induced by MRP14 deficiency in our model of severe infection with E. coli. It must be established whether MRP14−/− mice challenged with a lower bacterial load of E. coli would have increased bacterial outgrowth.

In contrast to the observed similar pulmonary inflammation in the two mouse strains, MRP14−/− mice displayed significantly less hepatocellular injury, as indicated by histopathology and plasma transaminase levels. These data suggest that MRP8/14 deficiency protects against sepsis-induced liver damage. In accordance with our findings, Arai and coworkers and Yang and colleagues found that normal, uninfected livers from mice and humans, respectively, do not contain MRP8+ or MRP14+ cells (49, 50), whereas infection with Schistosoma mansoni led to increased expression of MRP8 and MRP14 in liver tissue (50). To the best of our knowledge, no other data have been published about MRP8/14 expression in the liver or its involvement during infection. We found that hepatic MRP8/14 levels are up-regulated during E. coli sepsis 6 and 20 hours after inoculation and that MRP14−/− mice display diminished liver damage at 20 hours. Further experiments are needed to investigate which mechanism(s) underlie(s) the role of MRP8/14 in liver injury. One explanation might be that there is a diminished activation of TLR4 in MRP14−/− mice during the early response to infection (13), resulting in less inflammation and reduced tissue injury. In line with this possibility we found lower hepatic levels of TNF-α and IL-6 in MRP14−/− mice 6 hours after infection (data not shown). Of note, MRP14−/− mice demonstrated modest but significantly improved survival in this model of abdominal sepsis (13); it remains to be established to which extent the reduced hepatocellular injury plays a role herein.

Biologically active MRP8/14 heterodimers cannot be formed in MRP14−/− mice and, therefore, these mice are MRP8/14 deficient. Previously, we and others demonstrated that peripheral myeloid cells of healthy MRP14−/− mice do not express MRP8 protein (15, 47), possibly due to the need of MRP8 for its binding partner MRP14 for stability. Because it is theoretically possible that septic MRP14−/− mice do express MRP8 protein in their myeloid cells (in contrast to healthy MRP14−/− mice), it would be of interest to measure MRP8 homodimers in MRP14−/− mice subjected to E. coli sepsis. Unfortunately, at present there are no adequate assays that can distinguish between MRP8 homodimers and MRP8/14 heterodimers. Therefore, our data do not exclude the possibility that unbound MRP8 plays a role in the pathogenesis of E. coli sepsis.

We used MRP14−/− mice and wild-type littermates backcrossed six times to a C57BL/6 genetic background. Thus, although the genetic background of these animals was not pure C57BL/6, we consider the use of wild-type littermates as control subjects adequate.

The present study is the first to document that MRP8/14 release occurs in severe sepsis and that MRP8/14 is released locally during severe infection in patients with peritonitis. Investigations seeking to provide insight into the functional role of MRP8/14 revealed that MRP14 contributes to bacterial dissemination (transiently) and liver injury during abdominal sepsis. Inhibition of MRP8/14 may be a useful adjunctive therapy for severe sepsis.

The authors thank Regina de Beer, Joost Daalhuisen, and Marieke S. ten Brink for expert technical assistance.

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Correspondence and requests for reprints should be addressed to Marieke A. D. van Zoelen, M.D., Academic Medical Center, Room G2-130, Meibergdreef 9, 1105 AZ, Amsterdam, The Netherlands. E-mail:

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