INVITED REVIEW
Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation

Grace L. Su

Medical Service, Department of Veterans Affairs Medical Center and Department of Medicine, University of Michigan, Ann Arbor, Michigan 48109


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
ROLE OF LPS IN...
CD14
TLRS
OTHER LPS RECEPTORS
LBP
REFERENCES

Endogenous gut-derived bacterial lipopolysaccharides have been implicated as important cofactors in the pathogenesis of liver injury. However, the molecular mechanisms by which lipopolysaccharides exert their effect are not entirely clear. Recent studies have pointed to proinflammatory cytokines such as tumor necrosis factor-alpha as mediators of hepatocyte injury. Within the liver, Kupffer cells are major sources of proinflammatory cytokines that are produced in response to lipopolysaccharides. This review will focus on three important molecular components of the pathway by which lipopolysaccharides activate Kupffer cells: CD14, Toll-like receptor 4, and lipopolysaccharide binding protein. Within the liver, lipopolysaccharides bind to lipopolysaccharide binding protein, which then facilitates its transfer to membrane CD14 on the surface of Kupffer cells. Signaling of lipopolysaccharide through CD14 is mediated by the downstream receptor Toll-like receptor 4 and results in activation of Kupffer cells. The role played by these molecules in liver injury will be examined.

endotoxins; tumor necrosis factor; macrophages; CD14


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
ROLE OF LPS IN...
CD14
TLRS
OTHER LPS RECEPTORS
LBP
REFERENCES

LIPOPOLYSACCHARIDES (LPS) are glycolipids found in abundance on the outer membrane of all gram-negative bacteria and have the ability to incite a vigorous inflammatory response. In humans, nanograms of LPS injected into the blood stream can result in all the physiological manifestations of septic shock (70, 118). Given that gram-negative bacteria normally colonize the colon, the body has developed strong defensive mechanisms that tightly regulate the entry and processing of LPS. The liver plays a central role in this process by virtue of its dual ability not only to clear LPS, but to respond energetically to LPS. The vast majority of LPS that enters the host in normal and pathological states does so through the gastrointestinal (GI) tract. Strategically and uniquely located at the gateway of the portal blood flow draining the GI tract, the liver is the final barrier to prevent GI bacteria and bacterial products, such as LPS, from entering the systemic blood stream (74). In experimental studies on healthy animals, LPS is cleared from the circulation within a few minutes of intravenous injection, and the majority is traced to the liver (65, 128). The liver's primary role in clearing LPS can be demonstrated in patients with liver failure. Endotoxemia is frequently found in patients with cirrhosis, and the degree of endotoxemia is correlated with the degree of liver failure (12, 58).

In addition to its ability to clear LPS, the liver also responds to LPS with production of cytokines (63) and reactive oxygen intermediates (7, 8). Both ex vivo and in vitro studies with isolated liver perfusion and liver slice models have demonstrated that tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 are released in response to LPS, primarily by Kupffer cells (63). Most of the toxicities of LPS, both in the liver and in the systemic circulation, have been related to the release of these inflammatory cytokines and mediators (35, 111). In baboons, passive immunization with murine monoclonal anti-TNF antibodies protects against multisystem organ failure caused by lethal doses of live gram-negative bacteria (111).


    ROLE OF LPS IN LIVER INJURY
TOP
ABSTRACT
INTRODUCTION
ROLE OF LPS IN...
CD14
TLRS
OTHER LPS RECEPTORS
LBP
REFERENCES

Multiple lines of evidence point to LPS as a cofactor in liver injury (80, 81). In animal models of liver injury, including carbon tetrachloride toxicity, choline deficiency, and alcohol-induced liver disease, injury to the liver is mitigated or prevented by the addition of oral nonabsorbable antibiotics, colectomy, or germ-free conditions (2, 54, 62, 90). In contrast, the addition of LPS augments liver injury caused by these hepatotoxins (11, 14). Furthermore, in carbon tetrachloride- and galactosamine-induced liver injury, the addition of anti-LPS antibody E-5 significantly reduces hepatotoxicity caused by these agents (19). These studies strongly support a role for endogenous LPS in toxin-induced liver injury.

In alcoholic liver disease, the importance of LPS has also been demonstrated. Rats fed the Leiber-DeCarli liquid ethanol diet develop only fatty liver unless subsequent injections of LPS are given. Only the combination of LPS and chronic ethanol will produce a histological picture of hepatic necrosis and inflammation consistent with alcoholic hepatitis (11, 34). In contrast, with the Tsukamoto and French model of rat alcoholic hepatitis, in which rats are fed ethanol intragastrically in conjunction with a high-fat diet, the pathological picture of alcoholic hepatitis can be produced without further LPS challenge (114). Part of the discrepancy may be due to the presence of endogenous endotoxemia after the addition of alcohol to the diet. The level of endotoxemia present correlates with pathological injury (76, 78). Furthermore, pathological injury can be ameliorated by decreasing the level of endogenous endotoxemia with intraluminal lactobacillus or antibiotics (2, 75).

Endotoxin or LPS itself is not hepatotoxic at low concentrations. However, its ability to stimulate an inflammatory response may account for its pathogenicity in the liver. Although many parameters of the inflammatory response contribute to liver injury (72), one well-studied pathway is the production of TNF-alpha . In many models of liver injury elevated TNF-alpha levels are present and correlate with injury (17, 48, 68). Inhibition of TNF-alpha activity can decrease liver injury. The addition of soluble TNF receptors that diminish the biological effect of TNF-alpha will significantly decrease liver enzymes, improve liver histology, and decrease mortality acutely after acute carbon tetrachloride administration (18). Similar results are seen in chronic alcohol-induced liver disease (44) in rats. In humans, elevated levels of TNF-alpha are seen in alcoholic hepatitis and are associated with increased mortality (24). As potent producers of inflammatory cytokines such as TNF-alpha , Kupffer cells have been implicated in the pathway leading to liver injury (106). Accordingly, inhibition of Kupffer cells with gadolinium decreases injury in alcohol-induced liver disease (1). Although the greatest attention has been focused on the role of TNF-alpha in liver injury, other cytokines have been shown to play important pathogenic roles, including proinflammatory cytokines IL-6 and IL-18 (52, 115) as well as anti-inflammatory cytokines such as IL-10 (13). In alcoholic liver disease, it is not just a marked elevation of one proinflammatory cytokine that results in liver injury, but more likely an alteration in the balance of pro- and anti-inflammatory factors that result in disease (77).


    CD14
TOP
ABSTRACT
INTRODUCTION
ROLE OF LPS IN...
CD14
TLRS
OTHER LPS RECEPTORS
LBP
REFERENCES

Given the critical importance of LPS and Kupffer cells in the pathogenesis of many forms of liver injury, increasing attention has focused on the mechanisms by which LPS activates Kupffer cells. In peripheral blood monocytes, LPS activation is mediated through LPS binding protein (LBP), CD14, and Toll-like receptor (TLR)4. LPS binds to LBP, a 60-kDa acute-phase protein produced predominantly by the liver and secreted into the circulation (93, 121). Although LBP can be expressed extrahepatically, the majority of LBP is produced by the liver (102). Present in normal and acute-phase serum, LBP binds with high affinity to the lipid A portion of LPS and catalyzes the transfer of LPS to cell surface receptors such as membrane CD14 (31). In the presence of LBP, markedly less LPS is needed to activate peripheral blood monocytes. Although LBP is not required for interactions of LPS with CD14, its presence significantly decreases the concentration of LPS sufficient for cellular activation (92). Thus the LBP-CD14 pathway is critical at the low concentrations of LPS found under physiological condition (109).

Because of its location in the liver sinusoids, which drain blood from the GI tract, Kupffer cells are chronically exposed to higher concentrations of LPS than circulating peripheral blood monocytes. Thus it is likely that Kupffer cells have evolved specialized different mechanisms to interact with LPS. Unlike peripheral blood monocytes, Kupffer cells have relatively low baseline expression of CD14 (6, 103, 127). However, expression of CD14 on Kupffer cells can be upregulated with multiple stimuli including LPS (67). By immunohistochemical staining, low levels of CD14 are detected in livers of unstimulated mice. The liver CD14 levels rapidly increase and peak 6 h after intraperitoneal injection with LPS (67). CD14 expression in the liver is also increased in many types of liver disease, including alcoholic and cholestatic liver injury in rodents (20, 98, 112). In the intragastric model of alcohol-induced liver injury in rats, the level of hepatic inflammation varies with alterations in the fat composition of the diet (76). Animals fed more unsaturated fats have greater liver injury; the degree of injury correlates with the level of endotoxemia and with the level of Kupffer cell CD14 expression (100). In human disease, CD14 expression on Kupffer cells is low in normal human liver but increases with different inflammatory liver diseases (110). Kupffer cell CD14 expression also varies with different stages of the same disease. In biliary atresia, Kupffer cell CD14 increases in early stages but declines in the later stages (4).

The physiological significance of these observed differences in Kupffer cell CD14 expression is not entirely clear. However, it is tempting to hypothesize that the changes in CD14 expression could be the underlying mechanism that determines the liver's sensitivity to LPS toxicity. This line of reasoning is supported indirectly by studies showing that CD14 expression is linked with LPS responsiveness in the monocytic cell line THP-1 (64). Immature THP-1 cells respond poorly to LPS in the presence and absence of serum. Treatment with the maturational agent calcitriol causes dose- and time-dependent increases in CD14 mRNA and surface CD14 expression. This increased expression is associated with enhanced responsiveness of these THP-1 cells to LPS (64). Similarly, transfection of CD14-negative 70Z/3 cells, a murine pre-B cell line, results in heightened sensitivity to LPS (55). In vivo, CD14 transgenic mice that overexpress CD14 on monocytes have increased sensitivity to LPS (25). In contrast, genetically engineered CD14-deficient mice are insensitive to LPS (37). It should be noted that although CD14-deficient mice are insensitive to LPS, their responses to whole bacteria remain intact (71). Isolated peritoneal macrophages from CD14-deficient animals have deficient responses to LPS, but their response to whole bacteria remains intact (71) and appears to be mediated, in part, by other LPS receptors such as CD11b/CD18.

Because "resting" Kupffer cells have low basal expression of CD14, one may hypothesize that Kupffer cells, unlike peripheral blood monocytes, react to LPS in a CD14-independent manner (10, 57). Although this is an attractive hypothesis, the data support a more complicated paradigm. Although Kupffer cells express little CD14, CD14 is still critical for LPS activation (98). Isolated human Kupffer cells can respond to low concentrations of LPS (<= 10 ng/ml) with the production of TNF-alpha . Preincubation of the Kupffer cells with a monoclonal mouse anti-human CD14 antibody MY4 inhibits production of TNF-alpha and supports an essential role for the CD14 receptor in Kupffer cell activation by LPS under physiological conditions. In addition, Kupffer cells isolated from CD14-deficient mice are significantly less sensitive to LPS than wild-type animals (98).

Discovered as a myeloid differentiation marker, CD14 was previously thought to be expressed exclusively by myeloid cells (127). Both a membrane and soluble form of CD14 exist in serum. The soluble form is identical to the membrane, with the exception of the glycophosphatidyl inositol (GPI) anchor, which is not present in the soluble form of the protein. sCD14 also binds LPS and can facilitate interactions with CD14-negative cells such as endothelial cells (26, 39, 87). The source of sCD14 was previously attributed to shedding from monocytic cells bearing CD14 or from direct secretion by monocytic cells (9, 15). However, it has become clear that other cells can also secrete soluble CD14. In situ hybridization and immunohistochemistry for CD14 show induction of CD14 mRNA and protein in mouse hepatocytes after LPS injection in vivo (22). In the intragastric ethanol model of rat alcoholic liver disease, isolated hepatocytes from dextrose-fed animals do not have significant expression of CD14 mRNA. However, with the addition of ethanol in the diet, the level of CD14 mRNA increases and is correlated with the degree of liver injury observed (100). Isolated hepatocytes from in vivo LPS-treated rats have upregulation of both cell-associated and soluble CD14 protein production when plated in vitro (59). Basal expression of hepatocyte CD14 is present, but upregulation after inflammatory stimuli appears to be mediated, in part, by IL-1 and TNF-alpha (23, 59, 61).

The finding of hepatocellular sCD14 production is also supported by studies of transgenic mice generated using an 80-kb human CD14 genomic DNA fragment (40). These mice have high expressions of CD14 in both monocytic cells and hepatocytes. Further analysis of these transgenic mice demonstrated that monocyte and hepatocellular expression of CD14 are differentially regulated. An upstream regulatory element beyond a 24-kb region of the genomic DNA but within the 33-kb region of human CD14 was critical for hepatocellular CD14 expression but not monocytic expression (84). Furthermore, the 33-kb transgenic mice produced a soluble form of human CD14 in serum, confirming that hepatocytes are a source of sCD14 in vivo.

Isolated human hepatocytes from normal livers express CD14 mRNA and protein in culture, predominantly the soluble form (97). This sCD14 is biologically active and able to facilitate LPS responses in the CD14-negative epithelial cell line SW620, resulting in activation and production of IL-8 in response to low concentrations of LPS. It is important to note, however, that sCD14 can exert widely varying effects on cellular responses to LPS. The balance between activation vs. inhibition of LPS responses by sCD14 is dependent on its concentration. At what are thought to be supraphysiological concentrations, sCD14 can compete with mCD14 and inhibit LPS activation of CD14-positive cells (29, 38, 96, 113). The exact concentrations of mCD14 and sCD14 present in the microenvironment of the liver are not known. Whether local hepatocyte expression of sCD14 can lead to high enough concentrations to suppress Kupffer cell reactivity to LPS is not known. However, interpretations of studies in liver injury should take into account the relative contributions of Kupffer cell and hepatocyte CD14.

In CD14 knockout mice that are deficient in both membrane and soluble CD14 expression, the predominant phenotype is that of LPS insensitivity (37). Yin et al. (126) demonstrated that after 4 wk of continuous enteral ethanol delivery, CD14-deficient animals had less liver injury than wild-type BALB-c controls. These studies in the rodent model of ethanol-induced liver disease suggest that increases in CD14 expression contribute to the pathogenesis of liver injury. In human alcoholic disease, a promoter polymorphism that is associated with increased CD14 expression was found to be more common in men with alcoholic hepatitis and cirrhosis (47). A polymorphism at -159 (cytosine to thymidine) of the human CD14 gene, which lies within the Sp1 transcriptional binding site known to affect CD14 expression in monocytes, was examined in autopsy specimens of men with documented alcohol consumption. The thymidine variants of the -159 polymorphism promote CD14 gene expression and cause higher expression of CD14 on monocytes (43). In this study, the thymidine allele was found to be associated with alcoholic hepatitis and cirrhosis but not with fatty liver, periportal fibrosis, or bridging fibrosis. These findings suggest that CD14 expression may directly influence human susceptibility to alcohol-induced liver injury (47).


    TLRS
TOP
ABSTRACT
INTRODUCTION
ROLE OF LPS IN...
CD14
TLRS
OTHER LPS RECEPTORS
LBP
REFERENCES

Because membrane CD14 is a GPI-anchored protein without a transmembrane component, downstream partners for this receptor have been long sought. Recent studies in TLRs have been instrumental in elucidating the pathway by which ligand binding to membrane CD14 results in cellular activation. The Toll receptors are evolutionarily well conserved and were first described in Drosophila with biological functions in development and immunity (89). A series of TLRs, which are human homologs of the Toll receptors in Drosophila, have been cloned (69). To date, at least 10 TLRs have been identified in mammalian cells, although the ligands for most of them have not been identified (3). These receptors are typified as type I transmembrane proteins, with a cytoplasmic domain resembling that of the IL-1 receptor. In contrast to the IL-1 receptors, the extracellular domain of TLRs contains leucine-rich repeats rather than immunoglobulin-like domains.

Of particular interest as putative LPS receptors are TLR2 and TLR4. Both of these receptors are highly expressed in cells that respond to LPS, such as macrophages and monocytes. Both receptors can induce activation of cells through nuclear factor-kappa beta (NF-kappa B). Initially, cellular transfection experiments suggested TLR2 as the LPS receptor for CD14/LBP-dependent activation of cells (49, 125). However, subsequent experiments identifying naturally occurring mutations of TLR4 as the cause of LPS resistance in two strains of mice cast doubt on TLR2 as the main LPS receptor. A missense mutation in the TLR4 gene accounted for the endotoxin resistance found in C3H/HeJ mice, and a null mutation of TLR4 accounted for the endotoxin resistance in C57BL/10ScCr mice (86). Furthermore, TLR4 knockout mice have been produced and have been found to have LPS resistance (42) similar to the naturally occurring mutants. In contrast, TLR2-deficient mice are as capable of responding to LPS as their wild-type controls (104). This discrepancy in previously reported experimental results may have been due to the purity of LPS preparations used. Repurification of LPS resulted in loss of signaling through TLR2, suggesting that the initially observed TLR2 signaling may have occurred as a result of contaminants within LPS preparations (41).

TLR 2 appears to mediate responses to a wide variety of bacterial products including lipoproteins derived from gram-negative and -positive bacteria as well as mycoplasma (5). In contrast, TLR4 is specific for LPS from gram-negative bacteria. Signaling through TLR4 requires MD-2, a secreted protein that is closely associated with the extracellular domain of TLR4 (94). LPS responsiveness in human kidney 293 cells requires overexpression of both TLR4 and MD-2 (94). Mutations in MD2 result in decreased LPS responsiveness (91). Downstream of TLR4, signaling can occur through the IL-1-receptor pathway, which is MyD88 dependent, or alternatively via a MyD88-independent pathway (5). In the MyD88-dependent pathway, the death domain of MyD88 associates with the serine threonine kinase IL-1 receptor-associated kinase. Through the subsequent association with TNF receptor-activated factor 6 and a series of signaling pathways, Ikappa B degradation occurs. This liberates NF-kappa B, allowing its translocation to the nucleus, where it can induce target gene expression (Fig. 1) (5). Although ample evidence supports TLR4 as an important downstream partner for membrane CD14, a role for other coreceptors may also exist (85). With the use of fluorescence resonance energy transfer, Pfeiffer et al. (85) demonstrate coclustering of several receptors including Fcgamma -RIIIa (CD16a), CD81, and TLR4 into lipid rafts after binding of LPS to CD14.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 1.   One pathway of lipopolysaccharide (LPS) signaling in Kupffer cells. LPS in hepatic sinusoids binds to LPS binding protein (LBP) produced by hepatocytes. Binding of LPS to LBP facilitates transfer of LPS to membrane CD14 followed by activation mediated through Toll-like receptor (TLR)4 receptor/MD2. Signaling occurs via MyD88, which associates with interleukin-1 receptor-associated kinase (IRAK) and tumor necrosis factor (TNF)-activated factor 6. This results in translocation of nuclear factor-kappa B (NF-kappa B) into the nucleus and production of proinflammatory cytokines. Hepatocytes also produce sCD14, which may alter cellular reactivity to LPS.

Within the liver, a critical role for TLR4 is suggested by the presence of TLR4 on isolated Kupffer cells and their requirement for a functional TLR4 protein to mediate Kupffer cell responses to low concentrations of LPS (99). A role for TLR4 in liver injury is suggested by studies with intragastric ethanol administration in TLR4 mutant C3H/HeJ mice. After 4 wk of intragastric ethanol, these mice have less liver injury histologically, lower alanine amino transferase levels, and lower TNF-alpha mRNA expression than wild-type controls (116). Given the narrow specificity of TLR4 for LPS and not for other immunogenic bacterial products such as bacterial lipoproteins and bacterial CpG-DNA, this study suggests that in this mouse ethanol model of liver injury, LPS rather than other bacterial components is the specific pathophysiological agent. Whether this will be true in other models of liver injury remains to be seen. In addition to TLR4, TLR2 mRNA is expressed by rodent hepatoctyes (60, 66) and can be upregulated by LPS in vivo. Regulation of TLR2 mRNA appears to be mediated, in part, by IL-1 and TNF-alpha (60). The significance of TLR2 expression on hepatocytes and its role in liver injury is, as yet, unknown.


    OTHER LPS RECEPTORS
TOP
ABSTRACT
INTRODUCTION
ROLE OF LPS IN...
CD14
TLRS
OTHER LPS RECEPTORS
LBP
REFERENCES

The focus of this review has been on the CD14 and TLR4 pathway of Kupffer cell activation by LPS, because this is the best characterized high-affinity receptor system for LPS in monocytic cells. However, other receptor systems may be present (56). Another receptor system well described in the liver are the scavenger receptors, also known as Ac-LDL receptors (95). This receptor system has specificity toward negatively charged ligands such as Ac-LDL, polyinosinic acid, and maleylated BSA. On macrophages such as Kupffer cells, two types of scavenger receptor A have been identified (types I and II), which are both products of a single gene. The two gene products result from alternative splicing. Both subtypes of scavenger receptor A are found in abundance on macrophages and have very broad specificity toward negatively charged ligands such as Ac-LDL, polyinosinic acid, and maleylated BSA. They also have a great affinity for LPS, lipid A, and the bioactive precursor of lipid A, lipid IVA. This receptor system has been found to participate in the binding of LPS by macrophages before its metabolism to a less active form. Binding of LPS to the scavenger receptors does not result in Kupffer cell activation or production of TNF-alpha . In mice, ~75% of the total injected lipid IVA can be found in the liver within 10 min. The amount of liver uptake can be inhibited by competitors of the scavenger receptor such as Ac-LDL, polyinosinic acid, and maleylated BSA, suggesting that scavenger receptors play a role in the clearance of LPS by the liver (32). Furthermore, recent studies in scavenger receptor type A knockout mice show that these animals have a greater susceptibility toward endotoxin shock that is correlated with higher serum levels of TNF-alpha and IL-6 (36). These findings suggest that scavenger receptors can downregulate proinflammatory cytokine production by competing for LPS.

Recently, an interesting class of intracellular receptors for LPS has been identified. Nucleotide binding domains (Nod)1 and -2 are members of a family of intracellular proteins composed of an NH2-terminal caspase recruitment domain and a centrally located Nod (45). The COOH-terminal portions have leucine-rich repeats and serve as the sensors for intracellular ligands. LPS has been identified as a ligand for Nod1 and Nod2. Binding of LPS to Nod1 and Nod2 results in TLR4- and MYD88-independent activation of NF-kappa B activation, as demonstrated in experiments with Nod1- and Nod2-transfected human embryonic kidney 293T cells. Not surprisingly, the leucine-rich region of the protein appears to be essential for LPS-induced NFkappa B activation (46). Whereas Nod1 is found in a wide range of cell types, Nod2 appears to be restricted to monocytic cells (83). A recent report of a single nucleotide polymorphism in the Nod2 gene that is associated with susceptibility to Crohn's disease has generated much interest in this receptor. The frameshift mutation results in a truncated protein lacking part of the leucine-rich region and deficient in LPS-mediated NF-kappa B activation (82). Whether this receptor will have any role in susceptibility to liver injury remains to be seen.


    LBP
TOP
ABSTRACT
INTRODUCTION
ROLE OF LPS IN...
CD14
TLRS
OTHER LPS RECEPTORS
LBP
REFERENCES

As noted earlier, LBP binds with a high degree of specificity and affinity (dissociation constant = approx 109) to the lipid A portion of bacterial LPS (107). The NH2-terminal half of the protein is responsible for specific binding to LPS, whereas the COOH-terminal half of LBP is responsible for CD14 interactions. A truncated form of human LBP composed of the 197 amino acids in the NH2 terminus of the parent molecule effectively binds LPS but cannot transfer the LPS to CD14 (33, 105). At low concentrations (3 ug/ml), which is present is normal serum, LBP can facilitate the transfer of LPS to CD14 on isolated Kupffer cells ex vivo, with accelerated binding and more TNF-alpha production at lower concentrations of LPS (98, 99). As an acute-phase protein, LBP is present in normal serum at a concentration of 0.5-20 µg and can increase as much as 100-fold with an acute-phase response (16, 27, 73, 93, 108). Hepatocytes are the main producers of LBP, although LBP is also produced locally in the lung and skin (50, 51, 88, 102). Within the liver, therefore, Kupffer cells can be exposed to high concentrations of LBP.

Whereas the LPS-potentiating properties of LBP are well recognized, its ability to mediate widely varying functions at different concentrations has been underappreciated. With significant sequence homology to the lipid transfer proteins, cholesterol ester transfer protein and phospholipid transfer protein, LBP can transfer LPS to lipoproteins such as high-density lipoprotein (HDL), leading to neutralization of LPS (122). In fact, the ability of lipoproteins such as HDL to bind and neutralize LPS may depend on a transfer protein such as LBP (124). Reconstituted HDL (R-HDL) prepared from purified apolipoprotein A-I combined with phospholipid and free cholesterol were not sufficient to neutralize the biological activity of LPS, but the addition of LBP enabled prompt binding and neutralization of LPS by R-HDL (124). In serum, LBP is predominantly associated with lipoproteins, and its association strongly enhances the ability of lipoproteins to bind LPS (119). Given the role that lipoproteins play in neutralizing LPS in blood, LBP is well placed to participate in the neutralization of LPS.

Irrespective of its ability to transfer LPS to lipoproteins, the multiple functions of LBP can be illustrated by its dose-dependent effects on monocyte activation. Addition of low concentrations of recombinant murine LBP (up to 1 ug/ml) augments RAW 267.4 cell responses to low concentrations of LPS (0.33 ng/ml). However, at higher LBP concentrations (10 ug/ml), the previously observed enhancement of responses to LPS is lost. At higher concentrations of LPS, the previously effective concentration of LBP (10 ug/ml) is no longer sufficient to inhibit responses to LPS. In vivo, the addition of recombinant murine LBP diminishes LPS response (TNF-alpha levels), mortality, and ALT levels in the LPS-galactosamine model of liver injury (53). Concentration-dependent effects of LBP have also been described for priming of neutrophils by LPS (1 ng/ml) for subsequent responses to N-formyl-methionyl-leucyl-phenylalanine (113). In this system, sCD14 can activate or inhibit the priming of neutrophils. The end effect of sCD14 is dependent on the concentration of LBP. For LBP concentrations >3-10 ng/ml, the addition of sCD14 resulted in inhibition of neutrophil priming. At <3 ng/ml of LBP, the addition of sCD14 enhanced priming (113). The mechanism by which LBP can exert opposing effects is not known, but these data suggest that a complex interplay of multiple concentration-dependent factors determines the biological effect of these proteins.

The phenotype expressed by the LBP knockout mice produced by Wurfel et al. (123) supports the hypothesis that the primary role of LBP in vivo may not be to augment LPS reactivity. These LBP-deficient mice do not demonstrate any difference in TNF-alpha production when injected with LPS in vivo. In contrast, whole blood from these animals was 1,000-fold less responsive to LPS as assessed by TNF-alpha production ex vivo than that of wild-type controls. These findings suggest that in vivo sources of TNF-alpha production such as the liver may not require LBP for LPS responsiveness. In fact, the critical function of LBP in vivo may be to protect animals from bacterial infections. This latter supposition is supported by studies showing the exquisite sensitivity of these LBP-deficient animals to a Klebsiella pneumoniae challenge (21).

As an acute phase protein, hepatocyte LBP expression is upregulated in many models of acute-phase response and liver injury including endotoxemia, intramuscular turpentine injection, and alcoholic hepatitis (28, 100). Like other acute phase proteins, LBP production is regulated by proinflammatory cytokines such as IL-6, IL-1, and TNF-alpha (30, 120). The role LBP plays in liver injury when it is upregulated is not clear. Early studies with acute carbon tetrachloride-induced liver injury in LBP-deficient animals suggest that LBP-deficient animals have defective recovery from liver injury (79, 101). However, in mice given intragastric ethanol for 1 mo, a reduction in liver injury is seen in LBP-deficient animals (117). These contrasting effects of LBP deficiency suggests a more complex paradigm for liver injury that is dependent on relative concentrations of LBP and its cofactors (CD14 and TLR4) as well as the timing and type of injury.

In summary, the liver and, in particular, the Kupffer cell play an important role in the innate immune response to bacteria and bacterial products that enter the portal system. In the setting of liver injury, this function is impaired. Evidence suggests that these endogenous bacterial products, LPS in particular, can exacerbate the ongoing liver injury. Recent investigations into the molecular mechanisms by which LPS interacts with Kupffer cells and other mononuclear cells shed light on the potential role played by LBP, CD14, and TLR4 in the pathogenesis of different forms of liver injury. Although binding of LPS to LBP with transfer to CD14/TLR4 on Kupffer cells can result in activation, a complex interplay of other factors, including the relative concentrations of LBP and sCD14, may ultimately determine the Kupffer cell's response to LPS. An understanding of these factors, which are altered in liver injury, can lead to better therapeutic options in the future.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants DK-53296 and GM-60205 and a Veteran's Administration Merit Award.


    FOOTNOTES

Address for reprint requests and other correspondence: G. L. Su, Univ. of Michigan Medical Center, 1510C MSRB I, Box 0666, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0666 (E-mail: gsu{at}umich.edu).

10.1152/ajpgi.00550.2001


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
ROLE OF LPS IN...
CD14
TLRS
OTHER LPS RECEPTORS
LBP
REFERENCES

1.   Adachi, Y, Bradford BU, Gao W, Bojes HK, and Thurman RG. Inactivation of Kupffer cells prevents early alcohol-induced liver injury. Hepatology 20: 453-460, 1994[ISI][Medline].

2.   Adachi, Y, Moore LE, Bradford BU, Gao W, and Thurman RG. Antibiotics prevent liver injury in rats following long-term exposure to ethanol. Gastroenterology 108: 218-224, 1995[ISI][Medline].

3.   Aderem, A. Role of Toll-like receptors in inflammatory response in macrophages. Crit Care Med 29, Suppl: S16-S18, 2001[ISI][Medline].

4.   Ahmed, AF, Nio M, Ohtani H, Nagura H, and Ohi R. In situ CD14 expression in biliary atresia: comparison between early and late stages. J Pediatr Surg 36: 240-243, 2001[ISI][Medline].

5.   Akira, S, Takeda K, and Kaisho T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immun 2: 675-680, 2001[ISI].

6.   Antal-Szalmas, P. Evaluation of CD14 in host defence. Eur J Clin Invest 30: 167-179, 2000[ISI][Medline].

7.   Arthur, MJP, Kowalski-Saunders P, and Wright R. Effect of endotoxin on release of reactive oxygen intermediates by rat hepatic macrophages. Gastroenterology 95: 1588-1594, 1988[ISI][Medline].

8.   Bautista, AP, and Spitzer JJ. Acute endotoxin tolerance downregulates superoxide anion release by the perfused liver and isolated hepatic nonparenchymal cells. Hepatology 21: 855-862, 1995[ISI][Medline].

9.   Bazil, V, and Strominger JL. Shedding as a mechanism of down-modulation of CD14 on stimulated human monocytes. J Immunol 147: 1567-1574, 1991[Abstract/Free Full Text].

10.   Bellezzo, JM, Britton RS, Bacon BR, and Fox ES. LPS-mediated NF-kappa B activation in rat Kupffer cells can be induced independently of CD14. Am J Physiol Gastrointest Liver Physiol 270: G956-G961, 1996[Abstract/Free Full Text].

11.   Bhagwandeen, BS, Apte M, Manwarring L, and Dickeson J. Endotoxin induced hepatic necrosis in rats on an alcohol diet. J Pathol 151: 47-53, 1987.

12.   Bigatello, LM, Broitman SA, Fattori L, Di Paoli M, Pontello M, Bevilacqua G, and Nespoli A. Endotoxemia, encephalopathy, and mortality in cirrhotic patients. Am J Gastroenterol 82: 11-15, 1987[ISI][Medline].

13.   Bourdi, M, Masubuchi Y, Reilly TP, Amouzadeh HR, Martin JL, George JW, Shah AG, and Pohl LR. Protection against acetaminophen-induced liver injury and lethality by interleukin 10: role of inducible nitric oxide synthase. Hepatology 35: 289-298, 2002[ISI][Medline].

14.   Broitman, SA, Gottlieb LS, and Zamcheck N. Influence of neomycin and ingested endotoxin in pathogenesis of choline deficiency cirrhosis in adult rats. J Exp Med 119: 633-641, 1964[ISI].

15.   Bufler, P, Stiegler G, Schuchmann M, Hess S, Kruger C, Stelter F, Eckerskorn C, Schutt C, and Engelmann H. Soluble lipopolysaccharide receptor (CD14) is release via two different mechanisms from human monocytes and CD14 transfectants. Eur J Immunol 25: 604-610, 1995[ISI][Medline].

16.   Calvano, SE, Thompson WA, Marra MN, Coyle SM, de Riesthal HF, Trousdale RK, Barie PS, Scott RW, Moldawer LL, and Lowry SF. Changes in polymorphonuclear leukocyte surface and plasma bactericidal/permeability-increasing protein and plasma lipopolysaccharide binding protein during endotoxemia or sepsis. Arch Surg 129: 220-226, 1994[Abstract].

17.   Czaja, MJ, Flanders KC, Biempica L, Klein C, Zern MA, and Weiner FR. Expression of tumor necrosis factor-alpha and transforming growth factor-beta 1 in acute liver injury. Growth Factors 1: 219-226, 1989[Medline].

18.   Czaja, MJ, Xu J, and Alt E. Prevention of carbon tetrachloride-induced rat liver injury by soluble tumor necrosis factor receptor. Gastroenterology 108: 1849-1854, 1995[ISI][Medline].

19.   Czaja, MJ, Xy J, Yue J, Alt E, and Schmiedeberg P. Lipopolysaccharide-neutralizing antibody reduces hepatocyte injury from acute hepatotoxin administration. Hepatology 19: 1282-1289, 1994[ISI][Medline].

20.   Enomoto, N, Ikejima K, Yamashina S, Hirose M, Shimizu H, Kitamura T, Takei Y, Sato, and Thurman RG. Kupffer cell sensitization by alcohol involves increased permeability to gut-derived endotoxin. Alcohol Clin Exp Res 25, Suppl: 51S-54S, 2001[ISI][Medline].

21.  Fan M, Klein R, Steinstraesser L, Merry A, Nemzek J, Remick D, Wang S, and Su G. An essential role of lipopolysaccharide binding protein in pulmonary innate immune responses. Shock. In press.

22.   Fearns, C, Kravchenko VV, Ulevitch RJ, and Loskutoff DJ. Murine CD14 gene expression in vivo: extramyeloid synthesis and regulation by lipopolysaccharide. J Exp Med 181: 857-866, 1995[Abstract].

23.   Fearns, C, and Ulevitch RJ. Effect of recombinant interleukin-1beta on murine CD14 gene expression in vivo. Shock 9: 157-163, 1998[ISI][Medline].

24.   Felver, ME, Mezey E, McGuire M, Mitchell MC, Herlong HF, Veech GA, and Veech RL. Plasma tumor necrosis factor-alpha predicts decreased long term survival in severe alcoholic hepatitis. Alcohol Clin Exp Res 14: 255-259, 1990[ISI][Medline].

25.   Ferrero, E, Jiao D, Tsuberi BZ, Tesio L, Rong GW, Haziot A, and Goyert SM. Transgenic mice expressing human CD14 are hypersensitive to lipopolysaccharide. Proc Natl Acad Sci USA 90: 2380-2384, 1993[Abstract].

26.   Frey, EA, Miller DS, Jahr TG, Sundan A, Bazil V, Espevik T, Finlay BB, and Wright SD. Soluble CD14 participates in the response of cells to lipopolysaccharide. J Exp Med 176: 1665-1671, 1992[Abstract].

27.   Gallay, P, Barras C, Tobias PS, Calandra T, Glauser MP, and Heumann D. Lipopolysaccharide (LPS) binding protein in human serum determines the tumor necrosis factor response of monocytes to LPS. J Infect Dis 170: 1319-1322, 1994[ISI][Medline].

28.   Geller, DA, Kispert PH, Su GL, Wang SC, Di Silvio M, Tweardy DJ, Billiar TR, and Simmons RL. Induction of hepatocyte lipopolysaccharide binding protein in models of sepsis and the acute-phase response. Arch Surg 128: 22-27, 1993[Abstract]; discuss.

29.   Goyert, S, and Haziot A. Recombinant soluble CD14 inhibits LPS-induced mortality in a murine model. Prog Clin Biol Res 392: 479-483, 1995[Medline].

30.   Grube, BJ, Cochane CG, Ye RD, Green CE, McPhail ME, Ulevitch RJ, and Tobias PS. Lipopolysaccharide binding protein expression in primary human hepatocytes and HepG2 hepatoma cells. J Biol Chem 269: 8477-8482, 1994[Abstract/Free Full Text].

31.   Hailman, E, Lichenstein HS, Wurfel MM, Miller DS, Johnson DA, Kelley M, Busse LA, Zukowski MM, and Wright SD. Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J Exp Med 179: 269-277, 1994[Abstract].

32.   Hampton, RY, Golenbock DT, Penman M, Krieger M, and Raetz CRH Recognition and plasma clearance of endotoxin by scavenger receptors. Nature 352: 342-344, 1991[ISI][Medline].

33.   Han, J, Mathison JC, Ulevitch RJ, and Tobias PS. Lipopolysaccharide (LPS) binding protein, truncated at Ile-197, binds LPS but does not transfer LPS to CD14. J Biol Chem 269: 8172-8175, 1994[Abstract/Free Full Text].

34.   Hansen, J, Cherwitz DL, and Allen JI. The role of tumor necrosis factor-alpha in acute endotoxin-induced hepatotoxicity in ethanol-fed rats. Hepatology 20: 461-474, 1994[ISI][Medline].

35.   Hartung, T, and Wendel A. Endotoxin-inducible cytotoxicity in liver cell cultures. Biochem Pharmacol 42: 1129-1135, 1991[ISI][Medline].

36.   Haworth, R, Platt N, Keshav S, Hughes D, Darley E, Susuki H, Kuirhara Y, Kodoma T, and Gordon S. The macrophage scavenger receptor type A is expressed by activated macrophages and protects the host against lethal endotoxic shock. J Exp Med 186: 1431-1439, 1997[Abstract/Free Full Text].

37.   Haziot, A, Ferrero E, Lin XY, Stewart CL, and Goyert SM. CD14-deficient mice are exquisitely insensitive to the effects of LPS. Prog Clin Biol Res 392: 349-351, 1995[Medline].

38.   Haziot, A, Rong GW, Lin XY, Silver J, and Goyert SM. Recombinant soluble CD14 prevents mortality in mice treated with endotoxin (lipopolysaccharide). J Immunol 154: 6529-6532, 1995[Abstract/Free Full Text].

39.   Haziot, A, Rong GW, Silver J, and Goyert SM. Recombinant soluble CD14 mediates the activation of endothelial cells by lipopolysaccharide. J Immunol 151: 1500-1507, 1993[Abstract/Free Full Text].

40.   Hetherington, CJ, Kingsley PD, Crocicchio F, Zhang P, Rabin MS, Palis J, and Zhang DE. Characterization of human endotoxin lipopolysaccharide receptor CD14 expression in transgenic mice. J Immunol 162: 503-509, 1999[Abstract/Free Full Text].

41.   Hirschfeld, M, Ma Y, Weis JH, Vogel SN, and Weis JJ. Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine toll-like receptor 2. J Immunol 165: 618-622, 2000[Abstract/Free Full Text].

42.   Hoshino, K, Takeuchi O, Kawai T, Sanjo H, Tomohiko O, Takeda Y, Takeda K, and Akira S. Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaacharide: evidence for TLR4 as the LPS gene product. J Immunol 162: 3749-3752, 1999[Abstract/Free Full Text].

43.   Hubacek, JA, Rothe G, Pit'ha J, Skodova Z, Stanek V, Poledne R, and Schmitz G. C(-260)right-arrowT polymorphism in the promoter of the CD14 monocyte receptor gene as a risk factor for myocardial infarction. Circulation 99: 3218-3220, 1999[Abstract/Free Full Text].

44.   Iimuro, Y, Gallucci RM, Luster MI, Kono H, and Thurman RG. Antibodies to tumor necrosis factor alfa attenuate hepatic necrosis and inflammation caused by chronic exposure to ethanol in the rat. Hepatology 26: 1530-1537, 1997[ISI][Medline].

45.   Inohara, N, and Nunez G. The NOD: a signaling module that regulates apoptosis and host defense against pathogens. Oncogene 20: 6473-6481, 2001[ISI][Medline].

46.   Inohara, N, Ogura Y, Chen FF, Muto A, and Nunez G. Human Nod1 confers responsiveness to bacterial lipopolysaccharides. J Biol Chem 276: 2551-2554, 2001[Abstract/Free Full Text].

47.   Jarvelainen, HA, Orpana A, Perola M, Savolaainen VT, Karhunen PJ, and Lindros KO. Promoter polymorphism of the CD14 endotoxin receptor gene as a risk factor for alcoholic liver disease. Hepatology 33: 1148-1153, 2001[ISI][Medline].

48.   Kamimura, S, and Tsukamoto H. Cytokine gene expression by Kupffer cells in experimental alcoholic liver disease. Hepatology 22: 1304-1309, 1995[ISI][Medline].

49.   Kirschning, CJ, Wesche H, Merrill AT, and Roth M. Human toll-like receptor 2 confers responsiveness to bacterial lipopolysaccharide. J Exp Med 188: 2091-2097, 1998[Abstract/Free Full Text].

50.   Klein, RD, Su GL, Aminlari A, Alarcon WH, and Wang SC. Pulmonary LPS-binding protein (LBP) upregulation following LPS-mediated injury. J Surg Res 78: 42-47, 1998[ISI][Medline].

51.   Klein, RD, Su GL, Aminlari A, Zhang H, Steinstraesser L, Alarcon WH, and Wang SC. Skin lipopolysaccharide-binding protein and IL-1beta production after thermal injury. J Burn Care Rehabil 21: 345-352, 2000[ISI][Medline].

52.   Kovalovich, K, DeAngelis RA, Li W, Furth EE, Ciliberto G, and Taub R. Increased toxin-induced liver injury and fibrosis in interleukin-6-deficient mice. Hepatology 31: 149-159, 2000[ISI][Medline].

53.   Lamping, N, Dettmer R, Schroder NWJ, Pfeil D, Hallatschek W, Burger R, and Schumann RR. LPS-binding protein protects mice from septic shock caused by LPS and gram negative bacteria. J Clin Invest 101: 2065-2071, 1998[Abstract/Free Full Text].

54.   Leach, BE, and Forbes JC. Sulfonamide drugs as protective agents against carbon tetrachloride poisoning. Proc Soc Exp Biol Med 48: 361-363, 1941.

55.   Lee, JD, Kato K, Tobias PS, Kirkland TN, and Ulevitch RJ. Transfection of CD14 into 70Z/3 cells dramatically enhances the sensitivity to complexes of lipopolysaccharide (LPS) and LPS binding protein. J Exp Med 175: 1697-1705, 1992[Abstract].

56.   Lichtman, S, Wang J, Zhang C, and Lemasters J. Endocytosis and Ca2+ are required for endotoxin-stimulated TNF-alpha release by rat Kupffer cells. Am J Physiol Gastrointest Liver Physiol 271: G920-G928, 1996[Abstract/Free Full Text].

57.   Lichtman, SN, Wang J, and Lemasters JJ. LPS receptor CD14 participates in release of TNF-alpha in RAW 264.7 and peritoneal cells but not in Kupffer cells. Am J Physiol Gastrointest Liver Physiol 275: G39-G46, 1998[Abstract/Free Full Text].

58.   Lin, RS, Lee FY, Lee SD, Tsai YT, Lin HC, Lu RH, Hsu WC, Huang CC, Wang SS, and Lo KJ. Endotoxemia in patients with chronic liver diseases: relationship to severity of liver diseases, presence of esophageal varices and hyperdynamic circulaton. J Hepatol 22: 165-172, 1995[ISI][Medline].

59.   Liu, S, Khemlani L, Shapiro R, Johnson M, Liu K, Geller D, Watkins S, Goyert S, and Billiar T. Expression of CD14 by hepatocytes: upregulation by cytokines during endotoxemia. Infect Immun 66: 5089-5098, 1998[Abstract/Free Full Text].

60.   Liu, S, Salyapongse N, Geller DA, Vodovotz Y, and Billiar TR. Hepatocyte toll-like 2 expression in vivo and in vitro: role of cytokines in induction of rat TLR2 gene expression by lipopolysaccharide. Shock 14: 361-365, 2000[ISI][Medline].

61.   Liu, S, Shapiro RA, Nie S, Zhu D, Vodovotz Y, and Billiar TR. Characterization of rat CD14 promoter and its regulation by transcription factors AP1 and Sp family proteins in hepatocytes. Gene 250: 137-147, 2000[ISI][Medline].

62.   Luckey, TD, Reyniers JA, Gyorgy P, and Forbes M. Germ free animals and liver necrosis. Ann NY Acad Sci 57: 932-935, 1954[ISI].

63.   Luster, MI, Germolec DR, Yoshida T, Kayama F, and Thompson M. Endotoxin-induced cytokine gene expression and excretion in the liver. Hepatology 19: 480-488, 1994[ISI][Medline].

64.   Martin, TR, Mongovin SM, Tobias PS, Mathison JC, Moriarty AM, Leturcq DJ, and Ulevitch RJ. The CD14 differentiation antigen mediates the development of endotoxin responsiveness during differentiation of mononuclear phagocytes. J Leukoc Biol 56: 1-9, 1994[Abstract].

65.   Mathison, JC, and Ulevitch RJ. The clearance, tissue distribution, and cellular localization of intravenously injected lipopolysaccharide in rabbits. J Immunol 123: 2133-2143, 1979[ISI][Medline].

66.   Matsumura, T, Ito A, Takii T, Hayashi H, and Onozaki K. Endotoxin and cytokine regulation of Toll-like receptor (TLR) 2 and TLR4 gene expression in murin liver and hepatocytes. J Interferon Cytokine Res 20: 915-921, 2000[ISI][Medline].

67.   Matsuura, K, Ishida T, Setoguchi M, Higuchi Y, Akizuki S, and Yamamoto S. Upregulation of mouse CD14 expression in Kupffer cells by lipopolysaccharide. J Exp Med 179: 1671-1676, 1994[Abstract].

68.   McClain, C, Hill D, Schmidt J, and Diehl AM. Cytokines and alcoholic liver disease. Semin Liver Dis 13: 170-182, 1993[ISI][Medline].

69.   Medzhltov, R, Preston-Hulburt P, and Janeway CA. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 388: 394-397, 1997[ISI][Medline].

70.   Michie, HR, Manogue KR, Spriggs DR, Revhaug A, Dwyer SI, Dinarello A, Cerami A, Wolff SM, and Wilmore DW. Detection of circulating tumor necrosis factor after endotoxin administration. N Engl J Med 318: 1481-1486, 1988[Abstract].

71.   Moore, KJ, Andersson LP, Ingalls RR, Monks BG, Li R, Arnaout MA, Golenbock DT, and Freeman MW. Divergent response to LPS and bacteria in CD14-deficient murine macrophages. J Immunol 165: 4272-4280, 2000[Abstract/Free Full Text].

72.   Moulin, F, Copple BL, Ganey PE, and Roth RA. Hepatic and extrahepatic factors critical for liver injury during lipopolysaccharide exposure. Am J Physiol Gastrointest Liver Physiol 281: G1423-G1431, 2001[Abstract/Free Full Text].

73.   Myc, A, Buck J, Gonin J, Reynolds B, Hammerling U, and Emanuel D. The level of lipopolysaccharide binding protein is significantly increased in plasma in patients with the systemic inflammatory response syndrome. Clin Diagn Lab Immunol 4: 113-116, 1997[Abstract].

74.   Nakao, A, Taki S, Yasui M, Kimura Y, Nonami T, Harada A, and Takagi H. The fate of intravenously injected endotoxin in normal rats and in rats with liver failure. Hepatology 19: 1251-1256, 1994[ISI][Medline].

75.   Nanji, AA, Khettry U, and Sadrazdeh SM. Lactobacillus feeding reduces endotoxemia and severity of experimental alcoholic liver (disease). Proc Soc Exp Biol Med 205: 243-247, 1994[Abstract].

76.   Nanji, AA, Griniuviene B, Yacoub LK, Fogt F, and Tahan SR. Intercellular adhesion molecule-1 expression in experimental alcoholic liver disease: relationship to endotoxemia and TNF-alpha messenger RNA. Exp Mol Pathol 62: 42-51, 1995[ISI][Medline].

77.   Nanji, AA, Jokelainen K, Rahemtulla A, Miao L, Fogt F, Matsumoto H, Tahan SR, and Su GL. Activation of nuclear factor kappa B and cytokine imbalance in experimental alcoholic liver disease in the rat. Hepatology 30: 934-943, 1999[ISI][Medline].

78.   Nanji, AA, Khettry U, Sadrzady SM, and Yamanaka T. Severity of liver injury in experimental alcoholic liver disease. Correlation with plasma endotoxin, prostaglandin E2, leukotriene B4, and thromboxane B2. Am J Pathol 142: 367-373, 1993[Abstract].

79.  Nanji AA, Su GL, Laposata M, and French SW. Pathogenesis of alcoholic liver disease-recent advances. Alcoholism: Clinical and Experimental Research. In press.

80.   Nolan, JP. The role of endotoxin in liver injury. Gastroenterology 69: 1346-1356, 1975[ISI][Medline].

81.   Nolan, JP, and Camara DS. Intestinal endotoxins as co-factors in liver injury. Immunol Invest 18: 325-337, 1989[ISI][Medline].

82.   Ogura, Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, Britton H, Moran T, Karaliuskas R, Duerr RH, Achkar JP, Brant SR, Bayless TM, Kirschner BS, Hanauer SB, Nunez G, and Cho JH. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 411: 603-606, 2001[ISI][Medline].

83.   Ogura, Y, Inohara N, Benito A, Chen FF, Yamaoka S, and Nunez G. Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-kappa B. J Biol Chem 276: 4812-4818, 2001[Abstract/Free Full Text].

84.   Pan, Z, Zhou L, Hetherington CJ, and Zhang D. Hepatocytes contribute to soluble CD14 production, and CD14 expression is differentially regulated in hepatocytes and monocytes. J Biol Chem 275: 36430-36435, 2000[Abstract/Free Full Text].

85.   Pfeiffer, A, Bottcher A, Orso E, Kapinsky M, Nagy P, Bodnar A, Spreitzer I, Liebisch G, Drobnik W, Gempel K, Horn M, Holmer S, Hartung T, Multhoff G, Schutz G, Schindler H, Ulmer AJ, Heine H, Stelter F, Schutt C, Rothe G, Szollosi J, Damjanovich S, and Schmitz G. Lipopolysaccharide and ceramide docking to CD14 provokes ligand-specific receptor clustering in rafts. Eur J Immunol 31: 3153-3164, 2001[ISI][Medline].

86.   Poltorak, A, He X, Smirnova I, Liu M, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, and Beutler B. Defective LPS signalling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085-2088, 1998[Abstract/Free Full Text].

87.   Pugin, J, Schurer-Maly CC, Leturcq D, Moriarty A, Ulevitch RJ, and Tobias PS. Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14. Proc Natl Acad Sci USA 90: 2744-2748, 1993[Abstract].

88.   Ramadori, G, Meyer zum Buschenfelde KH, Tobias PS, Mathison JC, and Ulevitch RJ. Biosynthesis of lipopolysaccharide-binding protein in rabbit hepatocytes. Pathobiology 58: 89-94, 1990[ISI][Medline].

89.   Rock, FL, Hardiman G, Timans JC, Kastelein RA, and Bazan JF. A family of human receptors structurally related to Drosophila toll. Proc Natl Acad Sci USA 95: 588-593, 1998[Abstract/Free Full Text].

90.   Rutenburg, AM, Sonneblick E, Koven I, Apprahamian HA, Reiner L, and Fine J. The role of intestinal bacteria in the development of dietary cirrhosis in rats. J Exp Med 106: 1-13, 1957[ISI].

91.   Schromm, AB, Lien E, Henneke P, Chow JC, Yoshimura A, Heine H, Latz E, Monks BG, Schwartz DA, Miyake K, and Golenbock DT. Molecular genetic analysis of an endotoxin nonresponder mutant cell line: a point mutation in a conserved region of MD-2 abolishes endotoxin-induced signaling. J Exp Med 194: 79-88, 2001[Abstract/Free Full Text].

92.   Schumann, RR. Function of lipopolysaccharide (LPS)-binding protein (LBP) and CD14, the receptor for LPS/LBP complexes: a short review. Res Immunol 143: 11-15, 1992[ISI][Medline].

93.   Schumann, RR, Leong SR, Flaggs GW, Gray PW, Wright SD, Mathison JC, Tobias PS, and Ulevitch RJ. Structure and function of lipopolysaccharide binding protein. Science 249: 1429-1431, 1990[ISI][Medline].

94.   Shimazu, R, Akashi S, Ogata H, Nagai Y, Fukudome K, Miyake K, and Kimoto M. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med 189: 1777-1782, 1999[Abstract/Free Full Text].

95.   Shnyra, A, and Lindberg AA. Scavenger receptor pathway for lipopolysaccharide binding to Kupffer and endothelial liver cells in vitro. Infect Immun 63: 865-873, 1995[Abstract].

96.   Stelter, F, Witt S, Furll B, Jack RS, Hartung T, and Schutt C. Different efficacy of soluble CD14 treatment in high- and low-dose LPS models. Eur J Clin Invest 28: 205-213, 1998[ISI][Medline].

97.   Su, G, Dorko K, Strom S, Nuessler A, and Wang S. CD14 expression and production in human hepatocytes. J Hepatol 31: 435-442, 1999[ISI][Medline].

98.  Su G, Goyert S, Fan M, Aminlari A, Gong K, Klein R, Myc A, Alarcon W, Remick D, and Wang S. Activation of human and mouse Kupffer cells by lipopolysaccharide is mediated by CD14. Am J Physiol Gastrointestinal Liver Physiol. In press.

99.   Su, G, Klein R, Aminlari A, Zhang H, Steinstraesser L, Alarcon W, Remick D, and Wang S. Kupffer cell activation by lipopolysaccharide in rats: role for lipopolysaccharide binding protein and toll-like receptor 4. Hepatology 31: 932-936, 2000[ISI][Medline].

100.   Su, G, Rahemtulla A, Thomas P, Klein R, Wang S, and Nanji A. CD14 and lipopolysaccharide binding protein expression in a rat model of alcoholic liver disease. Am J Pathol 152: 841-849, 1998[Abstract].

101.   Su, GL, Fan MH, Klein RD, Wang SC, and Nanji AA. LBP is protective against alcoholic liver injury in female rats (Abstract). Hepatology 34: 468A, 2001.

102.   Su, GL, Freeswick PD, Geller DA, Wang Q, Shapiro RA, Wan YH, Billiar TR, Tweardy DJ, Simmons RL, and Wang SC. Molecular cloning, characterization, and tissue distribution of rat lipopolysaccharide binding protein. Evidence for extrahepatic expression. J Immunol 153: 743-752, 1994[Abstract/Free Full Text].

103.  Takai N, Kataoka M, Higuchi Y, Matsuura K, and Yamamoto S. Primary structure of rat CD14 and characteristics of rat CD14, cytokine, and NO synthase mRNA expression in mononuclear phagocyte system cells in response to LPS. J Leuk Biol: 61, 1997.

104.   Takeuchi, O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K, and Akira S. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11: 443-451, 1999[ISI][Medline].

105.   Theofan, G, Horwitz AH, Williams RE, Liu P, Chan I, Birr C, Carroll SF, Meszaros K, Parent JB, Kasler H, Aberle S, Trown PW, and Gazzano-Santoro H. An amino-terminal fragment of human lipopolysaccharide binding protein retains lipid A binding but not CD14 stimulatory activity. J Immunol 152: 3623-3629, 1994[Abstract/Free Full Text].

106.   Thurman, RG. Mechanisms of hepatic toxicity. II. Acloholic liver injury involves activation of Kupffer cells by endotoxin. Am J Physiol Gastrointest Liver Physiol 275: G605-G611, 1998[Abstract/Free Full Text].

107.   Tobias, PS, Soldau K, Hatlen LE, Schumann RR, Einhorn G, Mathison JC, and Ulevitch RJ. Lipopolysaccharide binding protein. J Cell Biochem 16C: 151-150, 1992.

108.   Tobias, PS, Soldau K, and Ulevitch RJ. Isolation of a lipopolysaccharide binding acute phase reactant from rabbit serum. J Exp Med 164: 777-793, 1986[Abstract].

109.   Tobias, PS, and Ulevitch RJ. Lipopolysaccharide binding protein and CD14 in LPS dependent macrophage activation. Immunobiology 187: 227-232, 1993[ISI][Medline].

110.   Tomita, M, Yamamoto K, Kobashi H, Ohmoto M, and Tsuji T. Immunohistochemical phenotyping of liver macrophages in normal and diseased human liver. Hepatology 20: 317-325, 1994[ISI][Medline].

111.   Tracey, KJ, Lowry SF, Fahey TJ, Albert JD, and Shires GT. Anti-cachetin TNF monoclonal antibodies prevent septic shock during lethal endotoxemia. Nature 330: 662-664, 1987[ISI][Medline].

112.   Tracy, TF, and Fox ES. CD14-lipopolysaccharide receptor activity in hepatic macrophages after cholestatic liver injury. Surgery 118: 371-377, 1995[ISI][Medline].

113.   Troelstra, A, Giepmans BNG, Van Kessel KPM, Lichenstein HS, Verrhoef J, and Ban Strijp JAG Dual effects of soluble CD14 on LPS priming of neutrophils. J Leukoc Biol 61: 173-178, 1997[Abstract].

114.   Tsukamoto, H, Towner SJ, Ciofalo LM, and French SW. Ethanol-induced liver fibrosis in rats fed high fat diet. Hepatology 6: 814-822, 1986[ISI][Medline].

115.   Tsutsui, H, Matsui K, Okamura H, and Nakanishi K. Pathophysiological roles of interleukin-18 in inflammatory liver diseases. Immunol Rev 174: 192-209, 2000[ISI][Medline].

116.   Uesugi, T, Froh M, Arteel GE, Bradford BU, and Thurman RG. Toll-like receptor 4 is involved in the mechanism of early alcohol-induced liver injury in mice. Hepatology 34: 101-108, 2001[ISI][Medline].

117.   Uesugi, T, Froh M, Arteel GE, Bradford BU, Wheeler MD, Gabele E, Isayama F, and Thurman RG. Role of lipopolysaccharide-binding protein in early alcohol-induced liver injury in mice. J Immunol 168: 2963-2969, 2002[Abstract/Free Full Text].

118.   Van Deventer, SJ, Buller HR, ten Cate JW, Aarden LA, Hack CE, and Sturk A. Experimental endotoxemia in humans: analysis of cytokine release and coagulation, fibrinolytic, and complement pathways. Blood 76: 2520-2526, 1990[Abstract].

119.   Vreugdenhil, AC, Snoek AM, van 't Veer C, Greve JW, and Buurman WA. LPS-binding protein circulates in association with apoB-containing lipoproteins and enhances endotoxin-LDL/VLDL interaction. J Clin Invest 107: 225-234, 2001[Abstract/Free Full Text].

120.   Wan, Y, Freeswick PD, Khemlani LS, Kispert PH, Wang SC, Su GL, and Billiar TR. Role of lipopolysaccharide (LPS), interleukin-1, interleukin-6, tumor necrosis factor, and dexamethasone in the regulation of LPS-binding protein expression in normal hepatocytes and hepatocytes from LPS-treated rats. Infect Immun 63: 2435-2442, 1995[Abstract].

121.   Wright, SD, Ramos RA, Tobias PS, Ulevitch RJ, and Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein]. Science 249: 1431-1433, 1990[ISI][Medline].

122.   Wurfel, MM, Kunitake ST, Lichenstein H, Kane JP, and Wright SD. Lipopolysaccharide (LPS)-binding protein is carried on lipoproteins and acts as a cofactor in the neutralization of LPS. J Exp Med 180: 1025-1035, 1994[Abstract].

123.   Wurfel, MM, Monks BG, Ingalls RR, Dedrick RL, Delude R, Zhou D, Lamping N, Schumann RR, Thieringer R, Fenton MJ, Wright SD, and Golenbock D. Targeted deletion of the lipopolysaccharide (LPS)-binding protein gene leads to profound suppression of LPS responses ex vivo, whereas in vivo responses remain intact. J Exp Med 186: 2051-2056, 1997[Abstract/Free Full Text].

124.   Wurfel, MM, and Wright SD. Lipopolysaccharide (LPS) binding protein catalyzes binding of LPS to lipoproteins. Prog Clin Biol Res 392: 287-295, 1995[Medline].

125.   Yang, RB, Mark MR, Gray A, Huang A, Xie MH, Zhang M, Goddard A, Wood WI, Gurney AL, and Godowski PJ. Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling. Nature 395: 284-288, 1998[ISI][Medline].

126.   Yin, M, Bradford BU, Wheeler MD, Uesugi T, Froh M, Goyert SM, and Thurman RG. Reduced early alcohol-induced liver injury in CD14-deficient mice. J Immunol 166: 4737-4742, 2001[Abstract/Free Full Text].

127.   Ziegler-Heitbrock, H, and Ulevitch RJ. CD14: cell surface receptor and differentiation marker. Immunol Today 14: 121-125, 1993[ISI][Medline].

128.   Zlydaszyk, JC, and Moon RJ. Fate of 51Cr-labeled lipopolysaccharide in tissue culture cells and livers of normal mice. Infect Immun 14: 100-105, 1976[ISI][Medline].


Am J Physiol Gastrointest Liver Physiol 283(2):G256-G265