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,||,1
* Department of Biomedical Research, TNO-Quality of Life, Leiden; Departments of
General Internal Medicine,
Vascular Surgery,
Infectious Diseases, and
|| Cardiology, Leiden University Medical Center, Leiden, The Netherlands;
¶ Department of Internal Medicine, Division of Infectious Diseases, UT Southwestern Medical Center, Dallas, Texas, USA; and
** Department of Medical Microbiology and Infectious Diseases, Erasmus Medical Center, Rotterdam, The Netherlands
1Correspondence: Leiden University Medical Center, Department Endocrinology and Metabolism, C4-R81, Albinusdreef 2, P.O. Box 9600, 2300 RC Leiden, The Netherlands. E-mail: p.c.n.rensen{at}lumc.nl
ABSTRACT
Gram-negative sepsis is a major death cause in intensive care units. Accumulating evidence indicates the protective role of plasma lipoproteins such as high-density lipoprotein (HDL) in sepsis. It has recently been shown that septic HDL is almost depleted from apolipoprotein CI (apoCI), suggesting that apoCI may be a protective factor in sepsis. Sequence analysis revealed that apoCI possesses a highly conserved consensus KVKEKLK binding motif for lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria. Through avid binding to LPS involving this motif, apoCI improved the presentation of LPS to macrophages in vitro and in mice, thereby stimulating the inflammatory response to LPS. Moreover, apoCI dose-dependently increased the early inflammatory response to Klebsiella pneumoniae-induced pneumonia, reduced the number of circulating bacteria, and protected mice against fatal sepsis. Our data support the hypothesis that apoCI is a physiological protector against infection by enhancing the early inflammatory response to LPS and suggest that timely increase of apoCI levels could be used to efficiently prevent and treat early sepsis.—Berbée, J. F. P., van der Hoogt, C. C., Kleemann, R., Schippers, E. F., Kitchens, R. L., van Dissel, J. T., Bakker-Woudenberg, I. A. J. M., Havekes, L. M., Rensen, P. C. N. Apolipoprotein CI stimulates the response to lipopolysaccharide and reduces mortality in Gram-negative sepsis.
Key Words: inflammation • lipoprotein • macrophage • TNF-
• transgenic mice
SEPSIS AFFECTS MORE THAN 700,000 people annually and accounts for 
210,000 deaths per year in the United States alone. The incidence is still rising at rates between 1.5 and 8% per year (1)
. Despite the active search for novel therapeutic agents (2)
, sepsis remains a serious cause of morbidity and mortality in intensive care units. Many cases of sepsis are caused by Gram-negative bacteria, which evoke immune responses mainly via their outer membrane component lipopolysaccharide (LPS) through cellular activation via Toll-like receptor 4 (TLR4; ref 3
). TLR4 is important in host defense against many Gram-negative bacteria, as shown by an impaired defense of TLR4-deficient mice during urinary tract infection with Escherichia coli (4)
, intratracheal inoculation of Klebsiella pneumoniae (5
, 6)
, intranasal administration of Haemophilus influenzae (7)
, and intraperitoneal infection with Klebsiella (8)
, Neisseria (9)
, and Salmonella (10)
species.
TLR4 signaling results in the production of various proinflammatory mediators, including tumor necrosis factor- (TNF-). These mediators play an essential role in the early host defense to infection and sepsis by generating an adequate response to bacterial infections, as shown in several animal models (11
12
13
14
15
16
17
18
19)
. In fact, neutralization of TNF- by antibodies impaired bacterial clearance (19)
, aggravated mortality from bacterial infection in mice (11
, 12
, 17
, 19)
, and was harmful in those septic patients with low risk of death (20)
. Similarly, an innate anti-inflammatory cytokine profile in humans has been associated with fatal outcome of meningococcal disease (21
, 22)
and febrile illness caused by community-acquired infection (23)
. Therefore, it is evident that a timely proinflammatory response to infections is crucial for generating an efficient antibacterial attack, thereby preventing the onset of the systemic inflammatory response syndrome, septic shock, and eventually death (24)
.
Recently, it has been recognized that sepsis is closely linked to plasma lipoprotein metabolism. Sepsis affects plasma lipoprotein levels by modulating lipolytic enzymes and lipoprotein receptors (25)
. In addition, all lipoprotein classes can bind LPS and, as such, affect the biological response to LPS (26
, 27)
. Previously, we have demonstrated that protein-free emulsion particles that mimic lipoproteins are unable to affect the biological fate of LPS, despite the fact that they bind LPS in vitro (28)
. This indicates that the protein moieties of lipoproteins [i.e., apolipoproteins (apo)] are responsible for their LPS-modulating effects. Indeed, we were able to show that apoE binds LPS directly, thereby redirecting LPS from macrophages to hepatocytes in vivo (28)
. Consequently, apoE prevents the LPS-induced production of cytokines (including TNF-) and subsequent death in rodents (29)
. However, although reduced cytokine production may be of benefit in severe sepsis by preventing ongoing excessive inflammation, it can be detrimental in early sepsis by impairing the antibacterial response.
ApoCI is encoded by the same gene cluster as apoE and has opposite effects on lipoprotein metabolism. Whereas apoE has a classical function in facilitating the hepatic clearance of triglyceride (TG)-rich lipoproteins (30)
, apoCI is able to block the clearance of lipoproteins by inhibiting the lipoprotein lipase-mediated hydrolysis of their core triglycerides in the periphery (31)
and by interfering with the apoE-dependent recognition of lipoproteins by their hepatic receptors (32)
. Since apoCI has recently been shown to be virtually absent from high-density lipoprotein (HDL; the main carrier of apoCI in plasma) in human sepsis (33)
, we hypothesized that apoCI may also play a role in modifying the biological response to LPS. We indeed demonstrate that apoCI contains a consensus LPS-binding motif, enhances the biological response to LPS, and reduces mortality in Gram-negative sepsis in mice.
MATERIALS AND METHODS
Animals
Male apoCI-deficient (apoc1–/–) mice (34)
, human apoCI-transgenic (APOC1) mice (32)
, and wild-type (WT) littermates (all C57Bl/6 background) were housed at the breeding facility of TNO-Quality of Life in a temperature- and humidity-controlled environment and allowed free access to water and chow. All experiments were approved by the animal ethics committee of TNO. Experiments were conducted at 10–12 wk of age unless stated otherwise.
Agarose gel electrophoresis
ReLPS (Sigma) and wtLPS (List; both from Salmonella minnesota) were radioiodinated (specific activities 800 and 1000 cpm/ng, respectively) and dialyzed against PBS pH 7.4. 125I-ReLPS or 125I-wtLPS (150 ng) was incubated (30 min at 37°C) with human isolated apoCI (purity
95%, Labconsult, Brussels, Belgium), synthesized apoCI, or apoCI mutant (apoCImut; purity95%, Protein Chemistry Technology Center, UT Southwestern Medical Center, Dallas, TX), and the mixtures were subjected to electrophoresis in an 0.75% (w/v) agarose gel (28)
. 125I-activity was detected by phosphor imaging and apoCI by immunoblotting.
Binding of apoCI to immobilized LPS
Enzyme immunoassay (EIA) plates were coated with 0.3 mg/ml LPS (Escherichia coli 0111:B4), washed, and blocked with 10 mg/ml BSA. Duplicate control wells and LPS-coated wells were incubated with apoCI in 1 mg/ml BSA (1 h at 37°C). Bound apoCI was detected with an anti-human apoCI-biotin conjugated antibody (Ab; Academy Biomedical Company, Houston, TX) for 1 h at room temperature followed by streptavidin-HRP for 30 min. Plates were developed with tetramethylbenzidine substrate and read at 450 nm on an ELISA plate reader. The optical density resulting from specific apoCI binding to LPS was calculated by subtracting the readings of control wells (nonspecific) from those of the LPS-coated wells at each apoCI concentration.
FITC-LPS dequenching
ReLPS was fluorescently labeled with FITC with a molar labeling efficiency of 1:1 (35)
. FITC-ReLPS (100 ng) was incubated (140 min at 37°C) with mouse plasma (0.125% v/v) in PBS pH 7.4 (total vol 100 µl), and fluorescence was measured before and after addition of 0.5% sodium deoxycholate (to determine maximum dequenching) in a CytoFluor II Fluorescence Multi-Well Plate Reader (PerSeptive Biosystems; 
ex 485 nm, em 530 nm).
Kinetic studies in mice
125I-LPS (10 µg/kg) was incubated in PBS pH 7.4 with isolated or synthesized human apoCI, apoCImut, isolated apoCIII (purity95%, Labconsult, Brussels, Belgium), or BSA. 125I-wtLPS was incubated with synthesized apoCI or apoCImut. WT mice were anesthetized (5 mg/kg dormicum, 0.5 mg/kg domitor, and 0.05 mg/kg fentanyl), the abdomens were opened, and the incubation mixtures were injected via the vena cava inferior. After 30 min, blood plasma and liver samples were collected and counted for radioactivity (36)
.
TNF- induction
LPS (25 µg/kg) in PBS with 0.1% BSA was injected via the tail vein into conscious mice. After 60 min, plasma was taken, and TNF- was determined using the mouse TNF--specific OptEIA ELISA (BD Biosciences Pharmingen), according to the manufacturer’s instructions. For in vitro studies, RAW 264.7 cells were seeded into 24-well plates (1x106 cells/well) and cultured overnight at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS. Cells were washed and incubated with LPS (S. minnesota; 1 ng/ml) or the TLR2 agonist zymosan (100 ng/ml) with or without apoCI, apoCImut, or apoCIII in DMEM with 0.01% human serum albumin (4 h at 37°C), and TNF- was determined in the medium.
Murine sepsis study
Mice (age 19–22 wk; body wt 26–28 g) were anesthetized as described above. A 5 mm vertical incision was made in the center of the anterior neck to allow for visual inspection of subsequent intratracheal intubation followed by instillation of a Klebsiella pneumoniae suspension [American Type Culture Collection 43816, capsular serotype 2; logarithmic growth phase; 500–750 colony forming units (cfu) in 20 µl PBS]. After bacterial inoculation, mice received a subcutaneous injection of antisedan (2.5 mg/kg), anexate (0.5 mg/kg), and naloxon (1.2 mg/kg) to recover from anesthesia. In a first study, blood was taken after 24 h to determine plasma TNF- and macrophage migration inhibitory factor (MIF) by Western blotting (37)
and E-selectin by ELISA. After 48 h, another blood sample was taken to quantify Klebsiella pneumoniae in blood by plating of serial dilutions on tryptone soy agar. Mortality was scored twice daily after bacterial inoculation, and the presence of only Klebsiella pneumoniae in lung and blood specimens of all succumbed mice was verified by culture on tryptone soy agar plates. The effect of Klebsiella pneumoniae inoculation on mortality was repeated in a subsequent experiment.
Statistical analysis
Data are mean ± SE. Statistical significance was assessed by two-tailed Mann-Whitney nonparametric test for two independent samples (Statistical Packages for the Social Sciences version 11.5) unless stated otherwise.
RESULTS
ApoCI avidly binds to LPS
To investigate whether apoCI could bind bacterial LPS, we first performed sequence alignment analysis between apoCI and established LPS-binding proteins, in which alternating sequences of cationic and hydrophobic amino acids are frequent (38)
. ApoCI contains the most cationic lysine (K) residues (i.e., 16 mol%) as compared with all other apolipoproteins, as well as several cationic/hydrophobic amino acid sequences throughout its structure. Moreover, apoCI contains a KVKEKLK motif in its C-terminal domain (apoCI48–54) that is highly homologous to the LPS-binding sequence of the Limulus anti-LPS factor (LALF43–49; KWKYKGK; ref 39
) and the cationic antibacterial protein (CAP18117–123; KIKEKLK; ref 40
). Interestingly, this KVKEKLK sequence belongs to one of the three highly conserved amino acid intervals within apoCI among various species (i.e., human, baboon, dog, rat, and mouse), as shown by matrix/median filter analysis (41)
.
Human apoCI was indeed able to fully disaggregate micelles of radiolabeled ReLPS [which is a truncated LPS containing the lipid A moiety and some 2-keto-3-deoxyoctonic acid (KDO) sugars] already at a 1:1 molar ratio, as evident from an LPS mobility shift assay on agarose gel (Fig. 1
A). ApoCI colocalized with LPS (Rf 0.9), as evidenced by immunoblotting (not shown). To evaluate the contribution of the positively charged K residues in the putative LPS-binding motif KVKEKLK to the LPS-binding properties of apoCI, we synthesized the full-length (57 amino acids) mature apoCI peptide and an apoCImut in which the K residues were replaced by neutral alanine (A) residues (AVAEALA). This modification eliminates the positive charges within this domain without affecting the overall structure of apoCI. ApoCImut appeared far less efficient to bind immobilized LPS than apoCI (Fig. 1B
), indicating that the KVKEKLK motif is indeed mainly responsible for the binding of LPS and that the charged K residues are directly involved in LPS binding.
|
We next evaluated whether the apoCI content of mouse plasma affects LPS monomerization (Fig. 1C
). We used FITC-labeled ReLPS, the fluorescence of which is strongly reduced (i.e., 93%) due to fluorescence self-quenching resulting from the micellar structure of LPS. On one hand, incubation of FITC-ReLPS with serum from APOC1 mice resulted in 40% increased monomerization of ReLPS micelles as compared with serum WT mice, indicating that human apoCI competes with other serum components for LPS binding. This monomerization-enhancing effect is not due to the elevated VLDL levels per se as observed in APOC1 transgenic mice, since serum from apoE-deficient mice is 2-fold less effective in LPS monomerization studies, despite highly elevated VLDL levels (unpublished data). On the other hand, serum from apoc1–/– mice showed a 2-fold reduced LPS monomerization as compared with WT mice, whereas total lipoprotein and lipid levels were virtually identical. Mouse plasma apoCI, which also contains the KVKEKLK motif, is thus also able to effectively induce association of LPS with HDL.
ApoCI prevents the clearance of LPS by the liver and spleen
To establish whether the binding of apoCI to LPS has consequences for the metabolic fate of LPS, radiolabeled ReLPS was incubated with or without human apoCI and injected intravenous into WT mice (Fig. 2
). ReLPS alone was rapidly cleared from serum and predominantly associated with the liver (68%) and spleen (10%). ApoCI, isolated from human plasma, caused a dose-dependent inhibition of the serum clearance of LPS (Fig. 2A
) by stimulating the association of LPS with HDL (not shown). This was accompanied by a strong reduction in the uptake of LPS by the liver (Fig. 2B
) and spleen (Fig. 2C
). At molar equilibrium, apoCI almost completely abolished the clearance of LPS from serum by the liver and spleen. In fact, apoCI was already effective in significantly attenuating the clearance of a 5-fold molar surplus of LPS.
|
ApoCIII, which has a similar molecular weight, structure, and lipoprotein distribution pattern as apoCI but does not contain typical LPS-binding sequences, only marginally affected the clearance of LPS as compared with apoCI. Whereas, at a 16-fold molar surplus, apoCI almost completely inhibited the uptake of LPS by the liver, apoCIII reduced the liver uptake by only 15% (Fig. 2E
). A 16-fold molar surplus of BSA also had no effect on the clearance of LPS (Fig. 2D-F
).
We next evaluated the contribution of the LPS-binding domain KVKEKLK to the effect of apoCI on the serum clearance of LPS by using synthesized apoCI and apoCImut (Fig. 3
). Synthesized wild-type apoCI had a similar effect on ReLPS kinetics (Fig. 3A-C
) as isolated apoCI (Fig. 2A-C
). In addition to ReLPS, apoCI also prevented the clearance of wtLPS from serum by the liver and spleen (Fig. 3D-F
). Compared with apoCI, apoCImut was much less efficient in preventing the serum clearance of both ReLPS (Fig. 3A-C
) and wtLPS (Fig. 3D-F
), indicating the crucial involvement of the LPS-binding domain KVKEKLK.
|
ApoCI stimulates the LPS-induced proinflammatory response
We next evaluated the effect of apoCI on the LPS-induced proinflammatory response (Fig. 4
). Intravenous injection of free ReLPS into apoc1–/–, WT, and APOC1 mice resulted in an apoCI-dependent increase in plasma TNF- levels (Fig. 4A
). Apoc1–/– mice showed a trend toward 2-fold lower TNF- levels as compared with WT mice, and APOC1 mice showed 7-fold higher plasma TNF- levels as compared with WT mice. This effect was not caused by the elevated plasma lipid levels in APOC1 mice, since hyperlipidemic LDL receptor (LDLr) and LDLr-related protein (LRP) double-deficient mice (42)
did not show an enhanced TNF- response to LPS as compared with WT mice (2.9±1.1 vs. 3.2±1.0x103 pg/ml, respectively), despite a more extreme hyperlipidemia as compared with APOC1 mice (31)
. Likewise, incubation of LPS with apoCI before injection into mice enhanced the LPS-induced production of TNF- 3.8-fold as compared with injection of LPS alone (not shown). Subsequent in vitro studies showed that apoCI greatly stimulates the TNF- response to ReLPS (Fig. 4B
) and wtLPS (Fig. 4C
) in RAW 264.7 mouse macrophages. ApoCIII stimulated the LPS-induced TNF- response only to a small extent (Fig. 4B-C
). Apolipoproteins alone did not trigger TNF- production (Fig. 4B
). Again, the LPS-binding domain KVKEKLK is crucial for the enhanced response to LPS, since apoCImut was ineffective. The effect of apoCI was also specific for LPS, since apoCI did not stimulate the response to the TLR2 agonist zymosan (Fig. 4D
).
|
ApoCI increases the inflammatory response to Klebsiella pneumoniae and protects against fatal sepsis
We next investigated whether an apoCI-dependent increased inflammatory response is associated with a higher rescue rate from death due to sepsis. To do so, apoc1–/–, WT, and APOC1 mice were challenged intrapulmonally with Klebsiella pneumoniae, an established procedure leading to Gram-negative bacterial pneumonia, sepsis, and eventually death (43)
. At 24 h after inoculation of 500–750 cfu of Klebsiella pneumoniae, the apoCI status of the mice positively correlated with markers of the inflammatory response, i.e., the plasma levels of TNF-, MIF (a TNF--inducible proinflammatory cytokine that is more stable than TNF- itself), and E-selectin (a TNF--inducible inflammation marker; Fig. 5
A–C). At 48 h after inoculation, the number of circulating Klebsiella pneumoniae was strongly and dose-dependently decreased by apoCI. APOC1 mice displayed a 25-fold lower bacterial count as compared with apoc1–/– mice (Fig. 5D
). In line with our in vitro findings, these data point to an enhanced activation of macrophages by apoCI during Gram-negative sepsis, as judged from the collective rise in TNF-, MIF, and E-selectin, and show that the apoCI-dependent increased proinflammatory status is associated with a reduced bacterial outgrowth to plasma. Accordingly, apoCI protected mice against fatal sepsis. After bacterial inoculation in WT mice, a gradual loss of animals occurred, with 39% survival after 2 wk. Whereas apoc1–/– mice were more susceptible to death after sepsis (only 16% survival), expression of human apoCI in APOC1 mice improved survival (53% survival; Fig. 5E
). All mice that died had ongoing infection, as verified by Klebsiella pneumoniae-positive cultures from lungs and blood obtained postmortem. Together, these data indicate that elevated apoCI levels are associated with an enhanced inflammatory response to LPS, a more efficient bacterial killing, and that apoCI can prevent mice from septic death.
|
DISCUSSION
Until recently, apolipoproteins have been assigned classical roles in mediating certain aspects of lipoprotein metabolism, either by affecting plasma factors that modify lipoprotein composition or by mediating the uptake of lipoproteins or their constituents by various tissues. ApoCI is the smallest apolipoprotein, consisting of only 57 amino acids, and is unusually rich in lysine residues. We and others have described that apoCI attenuates TG-rich lipoprotein clearance, either by inhibiting the peripheral lipoprotein lipase-mediated TG hydrolysis (31)
or by interfering with the hepatic uptake of these lipoproteins (32
, 44)
, but the physiological relevance of such an action is unclear. The structure of apoCI is remarkably conserved among species (i.e., mouse, rat, dog, baboon, and human), with three highly conserved sequences (41)
, and no structural mutations have been reported in humans thus far. These findings suggest that apoCI could have a specific function that should not necessarily relate to lipid metabolism. In the present study, we report that apoCI does have an important function in facilitating the inflammatory response to Gram-negative bacterial infections.
We discovered by sequence alignment analysis that apoCI contains several alternating sequences of cationic and hydrophobic residues that are frequent in established LPS-binding proteins (38)
. In particular, one of the highly conserved intervals within the C-terminal domain of apoCI appears to contain the LPS-binding motif KVKEKLK (apoCI48–54), which is identical between mice and humans. Indeed, human apoCI avidly bound to LPS in vitro, and the resulting complex was resistant to dissociation in the blood despite the abundance of potentially destabilizing plasma transfer factors (e.g., LPS-binding protein and phospholipids transfer protein). As a consequence, on intravenous injection in mice, apoCI markedly prolonged the residence time of LPS in serum by association with long-circulating HDL, reaching a maximum effect already at an apoCI:LPS = 1:1 molar ratio. Importantly, the LPS-monomerizing capacity of murine plasma was increased by the presence of moderate levels of human apoCI (i.e., 35 mg/dl in APOC1 mice as compared with 10 mg/dl in human plasma; ref 31
). Reciprocally, the monomerization of LPS was decreased in the absence of apoCI, indicating that the endogenous plasma level of apoCI per se can have a profound impact on the LPS distribution in vivo.
Physiological amounts of both exogenous and endogenous apoCI strongly enhanced the TNF- production on intravenous LPS administration in mice. These TNF--increasing effects are not caused by the effects of apoCI on plasma lipid levels. APOC1 mice are hyperlipidemic, but an even more pronounced hyperlipidemia as observed in LDLr/LRP double-deficient mice did not increase the LPS-induced TNF- response. In fact, we showed that apoCI can directly stimulate the LPS-induced TNF- production by macrophages in vitro. The increased apoCI-induced inflammatory response to LPS may thus be caused by a prolonged residence of LPS in the serum and/or direct activation of macrophages.
ApoCI was able to affect the serum clearance and TNF--stimulating properties of both full-length wtLPS (composed of the toxic lipid A moiety, as well as the inner core, outer core, and O-antigen) and truncated ReLPS (merely composed of the lipid A moiety and some KDO sugars), indicating that the lipid A/KDO moiety of the LPS molecule contains the crucial elements for interaction with apoCI. Mutation of lysine residues to alanine residues within the KVKEKLK domain of apoCI markedly reduced these LPS-modulating effects of apoCI without affecting the helical structure, indicating that this highly conserved domain within the C-terminal helix of apoCI is indeed responsible for LPS binding. Taken together, these data suggest that the interaction between LPS and apoCI is likely to involve electrostatic interaction between the lysine residues within apoCI and electronegative elements (presumably phosphate groups) within the lipid A moiety of LPS. Since lipid A is the common determinant of LPS molecules from all bacterial species, apoCI is likely to bind a wide array of WT and mutant LPS molecules. The fact that apoCI is able to enhance the binding of LPS to HDL further indicates that such an electrostatic interaction between apoCI and LPS can still occur on the lipoprotein surface.
Since sepsis has initially been regarded as an excessive systemic proinflammatory response to infections as largely mediated by TNF-, anti-inflammatory strategies, for example, those aimed to neutralize TNF-, have been widely used as potential therapeutic tools for the treatment of sepsis (20)
. However, although such approaches have been successful in inhibiting LPS-induced toxicity and mortality, a recent metaregression analysis of 22 clinical sepsis trials with anti-inflammatory agents, including 9 trials with anti-TNF- antibodies, showed that beneficial therapeutic effects of anti-inflammatory therapies could only be demonstrated in patients with a high risk of death, whereas anti-inflammatory agents were harmful in those patients with a low mortality risk (20)
. Therefore, we hypothesized that the observed apoCI-mediated increased inflammatory reaction to LPS may enhance survival from Gram-negative infections as a result of a timely and effective host response.
As a model for human sepsis caused by Gram-negative bacteria, we applied an experimental pneumonia model in which a local Klebsiella pneumoniae infection causes a lethal sepsis (43)
. With the use of intratracheal inoculation, the bacterial inoculum can be precisely controlled, which guarantees a reproducible infectious disease model with low variation between animals (43)
. The protective role of LPS-induced TLR4 signaling in this septic model has been conclusively established, as TLR4-deficiency shortened survival together with an enhanced bacterial outgrowth (5
, 6)
. This model is advantageous over cecal ligation and puncture-induced polymicrobial septic peritonitis, in which the survival of mice is independent of the ability to respond to LPS, as the absence of TLR4 did not affect survival (45)
. The pneumonia-related sepsis model is also preferred over other models that use bolus intravenous infusion of large amounts of live bacteria or LPS, leading to conditions of intoxication rather than sepsis (46
, 47)
.
Consistent with our finding that apoCI directly stimulated the LPS-induced TNF- response by macrophages in vitro, apoCI expression in mice also dose-dependently increased the initial proinflammatory response toward Klebsiella pneumoniae, as judged from increased plasma inflammation markers (TNF-, MIF, and E-selectin) reflecting an activation of macrophages. These effects were accompanied by lower bacterial counts in plasma and reduced mortality resulting from sepsis, confirming that an increased early inflammatory response effectuates an efficient antibacterial response. ApoCI mainly reduced mortality in the early phase after infection (i.e., within 4 days), during which the antibacterial attack is mainly mediated by a nonspecific cellular response. By inducing selective granulocytopenia in mice, it has previously been demonstrated that circulating granulocytes play an important role in the defense against K. pneumoniae infection in the lung (48)
. In fact, treatment of K. pneumoniae-infected C57Bl/6 mice with cyclophosphamide, leading to depletion of granulocytes, increased the mortality rate from 
20% to nearly 90% within the first 4 days after infection (49)
. Collectively, these data thus strongly suggest a crucial involvement of granulocytes in the apoCI-stimulated antibacterial attack.
MIF has previously been implicated as a critical mediator of septic shock (50)
. On the other hand, MIF has been shown to facilitate the detection of LPS-containing bacteria, enabling cells that are at the forefront of the host antimicrobial defense system, such as macrophages, to respond rapidly to invasive bacteria by rapid production of proinflammatory cytokines (51)
. The present study indeed shows that elevated levels of MIF during the initial response to bacteria are associated with increased survival. These seemingly conflicting data are inherent to the complex etiology of sepsis: whereas a proinflammatory response is often harmful in severe sepsis, an early inflammatory response is crucial to combat bacterial infections (24)
.
Based on a large body of evidence, current models of innate immune defenses postulate a central role for TLR-mediated processes in pathogen detection, initiation of a rapid immune defense, and regulation of subsequent adaptive immune responses (52
, 53)
. Our observation that apoCI increases the inflammatory response to LPS and Klebsiella pneumoniae and reduces mortality in Klebsiella pneumoniae-induced sepsis fully supports the concept that an adequate immune response is essential to protect mice against mortality from Gram-negative infections (4
; 5
6
7
8
9
10
) and suggests that apoCI may play a central role in innate immunity.
Currently, the synthetic lipid A analog E5564 that has been designed to antagonize the effects of LPS by interacting with TLR4 is clinically being investigated for the treatment of severe sepsis and septic shock. E5564 has been demonstrated to block the induction of LPS-induced cytokines as well as lethality in LPS-sensitized mice on intraperitoneal injection of E. coli, which led to rapid death of the vast majority (85%) of mice within 12 h (54)
. Likewise, a high dose of E5564 reduced the rapidly induced mortality of rats caused by intravascularly injected E. coli (55)
. These data confirm that anti-inflammatory strategies may be beneficial in preventing lethality due to acute and severe inflammation in acute toxicity models such as intraperitoneally (54)
and intravascularly (55)
administered E. coli, resulting in death of the majority of animals within 24 h after injection. However, a high dose of E5564 did not rescue rats from a clinically more relevant infection model in which E. coli was administered extravascularly (55)
and even showed a slight trend toward higher mortality, confirming that TLR4 antagonism at an early stage of an actual infection model may be harmful. Clearly, these studies confirm the importance of route of infection and timing of treatment and suggest that the benefit of apoCI may be limited to early stages of infection.
Based on our present data, we propose the following model for the protective effects of apoCI in Gram-negative sepsis. On the entry and proliferation of bacteria in the blood, LPS is released into the plasma and binds to apoCI, which involves the interaction between the LPS-binding motif KVKEKLK within apoCI and presumably phosphate groups within lipid A, the shared moiety of all LPS species. Although apoCI may bind LPS in the lipid-free and lipid-bound state, the resulting apoCI-LPS complex is mainly associated with HDL, which is the main carrier of apoCI in mice and humans (56)
. ApoCI effectively presents the LPS to responsive cells such as macrophages (the mechanistic basis of which is under current investigation), leading to a rapid and enhanced production of proinflammatory cytokines, among which are TNF- and MIF. These cytokines are essential for effective eradication of the bacterial infection thereby preventing infection-related mortality. Therefore, plasma apoCI protects against fatal sepsis by effectuating an early and adequate antibacterial response. We speculate that apoCI may provide a therapeutic handle in the ongoing search for strategies that are aimed to prevent or treat sepsis at an early stage.
ACKNOWLEDGMENTS
This study was supported in part by the Netherlands Organization for Scientific Research (NWO RIDE 014–90-001 to L. M. Havekes, NWO VENI 016.036.061 to R. Kleemann, and NWO VIDI 917–36-351 to P. C. N. Rensen); by the Leiden University Medical Center (Gisela Thier Fellowship to P. C. N. Rensen); by ZorgOnderzoek Nederland, formerly the Dutch Foundation for Preventive Medicine Praeventie Fonds (Grant 28–2875.23 to J. T. van Dissel); and by the NIAID (Grant AI045896 to R. L. Kitchens). Funders did not have any role in the design, analysis, interpretation, and report of the present study. The authors have declared that no relationships exist that may pose a conflict of interest. We thank Elly P. de Wit (TNO-Quality of Life), Marian T. ten Kate (Erasmus Medical Center), and Jason Gillman (UT Southwestern Medical Center) for excellent technical assistance.
Received for publication December 17, 2005. Accepted for publication May 15, 2006.
REFERENCES
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M. Westerterp, J. F. P. Berbee, D. J. M. Delsing, M. C. Jong, M. J. J. Gijbels, V. E. H. Dahlmans, E. H. Offerman, J. A. Romijn, L. M. Havekes, and P. C. N. Rensen Apolipoprotein C-I binds free fatty acids and reduces their intracellular esterification J. Lipid Res., June 1, 2007; 48(6): 1353 - 1361. [Abstract] [Full Text] [PDF]
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, n=29), and APOC1 (
, n=19) mice (i.e., including mice described above and a second set of mice) were inoculated intratracheally with same dose of Klebsiella pneumoniae or saline (
, WT mice, n=6), and their 2-wk survival was assessed. *P < 0.05, **P < 0.01 (log-rank test, Graph Pad Software Inc.).