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Theo J. C. Van Berkel, and Johan Kuiper*
Division of Biopharmaceutics, Leiden/Amsterdam Center of Drug Research, Leiden University, Leiden, The Netherlands
SUMMARY INTRODUCTION SEPSIS AND SEPTIC SHOCK BACTERIAL CELL WALL ARCHITECTURE Lipopolysaccharide Lipoteichoic and Teichoic Acids Peptidoglycan Purification and Aggregate Structure HOST RESPONSE TO CELL WALL CONSTITUENTS Cellular Defense Humoral Defense The Liver Liver cell types. Experiments with Radiolabeled Lipopolysaccharide In Vivo Macrophages and the Response to Lipopolysaccharide and Lipoteichoic Acid Detoxification of Lipopolysaccharide LIPOPOLYSACCHARIDE AND LIPOTEICHOIC ACID RECEPTORS Lipopolysaccharide-Binding Protein CD14 sCD14 Toll-Like Receptors ß2-Integrins Selectins Scavenger Receptors Moesin Heptose-Specific Lipopolysaccharide Receptor LIPOPOLYSACCHARIDE- AND LIPOTEICHOIC ACID-BINDING PROTEINS Neutrophilic Lipopolysaccharide-Binding Molecules Bactericidal/permeability-increasing protein. CAP18. CAP37. Lactoferrin. Lysozyme. Other Lipopolysaccharide-Binding Proteins Development of Bactericidal and Lipopolysaccharide-Neutralizing Peptides LIPOPROTEINS Metabolism Apolipoprotein E Anti-Inflammatory Role Lipid Metabolism and Infection CLINICAL AND EXPERIMENTAL SEPSIS THERAPIES Immunosuppression and Neutralization of Proinflammatory Cytokines CONCLUDING REMARKS REFERENCES
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| INTRODUCTION |
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Under normal circumstances, many bacteria live in coexistence with humans. The skin, digestive tract, upper respiratory tract, external urogenital organs, and conjunctiva all contain commensal bacteria that do not cause disease. In particular, the intestinal tract contains billions of bacteria such as Escherichia coli that contribute to the function of the intestine. Similarly, bacteria such as Lactobacillus acidophilus are involved in maintaining an acidic climate in the vagina while bacteria such as Staphylococcus epidermidis on the skin aid in the defense against invading microorganisms through production of several bactericidal substances. The presence of bacteria on or in these organs is not a threat to the body because the nasal and oral cavities, respiratory and digestive tracts, and urogenital organs are connected to the "external environment" and are thus separated from the normally sterile "internal environment."
Pathogenic as well as commensal microorganisms evoke an immune response if they, or their constituents, pass the barrier between the external and internal environment. After recognition of the bacteria or their products, the body launches an attack, kills the bacteria, and repairs putative damage. This sequence of events is highly regulated, enabling the body to combat infection by a tailor-made attack that is fierce enough to eradicate the bacteria but not so fierce as to cause unnecessary damage to the body.
As some of the first living organisms on Earth, bacteria evolved and have been endowed with an enormous capacity to adapt to changes in environment. Bacteria are the result of millions of years of evolution and are—despite their simplicity compared to multicellular organisms—highly refined.
The scope of the review is to discuss the different components of the various bacteria that are involved in the process of sepsis and/or septic shock. The interactions of the various bacterial components with receptors and other proteins are discussed in detail. The consequences of binding of the bacterial components to these receptors and other proteins for the process of sepsis and septic shock is discussed in terms of cellular activation and production of pro- and anti-inflammatory proteins. Finally, some newer therapies for the treatment of sepsis are reviewed.
| SEPSIS AND SEPTIC SHOCK |
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Due to differences in interpretation of the clinical condition "septic shock," reported mortality rates in patients with septic shock vary from 20 to 80% (42). The mortality is related to both the severity of sepsis and the underlying disease that is nearly always present (42, 43, 413). In many cases of sepsis, the presence of microorganisms (bacteremia) or LPS in the blood (endotoxemia) cannot be established, which has prompted modification of the definitions of sepsis and septic shock (42, 43, 561). The definitions are as follows: bacteremia, positive blood cultures; sepsis, clinical evidence of infection, tachypnea (>20 breaths/min), tachycardia (>90 beats/min), hyperthermia, or hypothermia; sepsis syndrome, sepsis plus hypoxemia or elevated plasma lactate levels or oliguria; and septic shock, sepsis syndrome plus hypotension (despite adequate volume resuscitation).
The clinical phenomena preceding the development of sepsis and septic shock are highly complex. Paradoxically, as mentioned above, persons with a weakened immune system are most likely to develop sepsis, but the detrimental processes that may ultimately lead to the death of the patient are mostly caused by an exaggerated, systemic response to an infection. The widespread activation of cells responsive to bacteria or bacterial components results in the release of an array of inflammatory mediators, such as cytokines, chemokines, prostaglandins and lipid mediators, and reactive oxygen species. These compounds induce vasodilatation and upregulation of adhesion molecules, resulting in extravasation of neutrophils and monocytes; activation of leukocytes, lymphocytes, and endothelial cells; and myocardial suppression (218, 251, 413, 570). Besides stimulation of coagulation by cytokines, bacterial components may directly interact with the coagulation system. The resulting disseminated intravascular coagulation causes hypoperfusion and hypoxia. Together with the damage caused by the intra- and extravascular phagocytic cells, these conditions lead to organ failure (338, 551). This may initiate the often lethal stage of sepsis, in which multiple-organ failure, mostly involving the lungs (acute respiratory distress syndrome), liver, and kidneys, develops (42, 413, 585). In addition, the hypoperfusion caused by disseminated intravascular coagulation may impair the gut mucosal barrier and result in translocation of bacteria to the mesenteric lymph nodes and, under conditions of ongoing stress, to several organs and the circulation. The released bacteria will "feed" the multiple-organ failure and significantly worsen the prognosis (608).
There are marked differences in the responses to gram-positive and gram-negative bacteria. Whereas gram-negative bacteria all contain LPS as their major pathogenic determinant, gram-positive bacteria contain a number of immunogenic cell wall components besides the highly deleterious exotoxins (403, 495). The immunological response to gram-negative bacteria mainly involves leukocytes and the production of cytokines such as tumor necrosis factor alpha (TNF-
), interleukin-1 (IL-1), and IL-6. The release of exotoxins, many of which are superantigens, by gram-positive bacteria activates T cells, resulting in a different cellular response and different cytokine profile, with relatively low levels of TNF-, IL-1, and IL-6 and increased levels of IL-8 (44, 403, 495).
| BACTERIAL CELL WALL ARCHITECTURE |
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The LPS molecule consists of four different parts (Fig. 2) (328,443,454). The first and most essential part is lipid A, the covalently linked lipid component of LPS. Six or more fatty acid residues are linked to two phosphorylated glucosamine sugars. Four of these fatty acids carry a hydroxyl group on the third carbon, whereas the other two are not hydroxylated. All bacterial species carry unique LPS, and some of the variations reside in the lipid A moiety: (i) acylation pattern, which is commonly asymmetric (4 + 2), or a symmetric (3 + 3) configuration (e.g., in Neisseria meningitidis); (ii) length of the fatty acid residues; typically three or four different fatty acids are present, with a length between 10 and 16 C atoms (average, 14 C atoms); (iii) the presence of 4-amino-deoxy-L-arabinose and/or phosphoethanolamine linked to the phospho groups on the glucosamine sugars; and (iv) The number of fatty acids (most common bacteria contain six fatty acid residues). Experiments with synthetic lipid A have shown that this part of the LPS molecule represents the toxic moiety (274). A number of synthetic derivatives of lipid A (dephosphorylated or deacylated) have been tested in vivo and in vitro, and the potency of these molecules was 10- to 1,000-fold reduced with respect to the original lipid A molecule (99,325,378). In addition, the lipid a Precursor, lipid IVA, dose dependently inhibits the effects of lipid A, as shown by reduced TNF- and prostaglandin E2 (PGE2) production in vitro (169). Another lipid A precursor, lipid X, has limited lipid A antagonist activity (169). This illustrates that lipid A-induced cell activation requires a stricter structure than lipid A binding to the receptor per se.
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The outer core, the third part of the LPS molecule, consists of common sugars and is more variable than the inner core. It is normally three sugars long with one or more covalently bound sugars as side chains. LPS serotypes consisting of lipid A and the complete inner and outer core are denoted Ra-LPS, whereas the Rb- and Rc-LPS serotypes only contain a part of the outer core.
The fourth moiety of the LPS molecule is the O antigen. This part of the LPS molecule is attached to the terminal sugar of the outer core, extends from the bacterial surface, and is highly immunogenic. It is composed of units of common sugars, but there is a huge interspecies and interstrain variation in the composition and length. In a single LPS preparation, the length of the O antigen may vary from 0 to as many as 40 repeating units, but it generally consists of 20 to 40 repeating units. Each unit is composed of three sugars with a single sugar connected to the first and third sugar of the unit. LPS molecules with O antigen are denoted S-LPS. Colonies from bacteria with O-antigen-containing LPS have a smooth (S) appearance on the plate, while bacteria that express an O-antigen-lacking LPS have a rough (R) appearance.
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A long tail of repeating 1,3-linked glycerophosphate units is connected to the glucoside moiety (131-133). The number of repeating units varies widely, depending on the species, strain, and growth conditions, but generally ranges between 4 and 30 for S. aureus (130, 465). D-Alanine may be incorporated at the 2 position of the glycerophosphate tail, but the extent of alanine substitution depends on factors, such as species, strain, growth conditions, and growth stage (130, 131, 272). Various conditions may lead to the release of LTA from the cell wall, including the presence of certain antibiotics (352, 552).
In S. aureus, about 50% of the total mass of the cell wall consists of teichoic acid (165). Teichoic acid is composed of long chains of ribitol phosphate units that are partially replaced by ester-linked D-alanine. Teichoic acid is linked to the muramic acid of the cell wall PGNs via phosphodiester bonds (165).
LPS and LTA can be purified using a number of isolation procedures employing phenol and—depending on the LPS serotype—chloroform and petroleum ether, yielding LPS and LTA preparations that may be contaminated with minor amounts of protein, phospholipids, divalent anions, and DNA (66, 152, 367). In particular, the protein contaminants may affect the physiological behavior of LPS, including cellular binding, endotoxic potency, and lipoprotein binding (339, 340, 435, 504, 508). However, bacterial DNA is also able to activate macrophages (483). Similarly, the presence of a minor nonprotein constituent of LTA preparations with stimulatory capacity has been described (283, 284). Most of the contaminants in LPS and LTA preparations can be removed only by reextraction, reversed-phase chromatography, or electrodialysis (151, 340, 488). Under physiological conditions, the LPS or LTA and their contaminants may behave as inseparable complexes impeding the analysis of the contribution of these contaminants to physiological processes (504).
Due to their numerous peptide cross-links, PGNs are isolated from rough cell wall preparations as insoluble fragments that can be broken down to soluble PGN by trypsin-mediated hydrolysis of the peptide bonds (334). Different levels of bioactivity have been described for isolated PGNs, which are up to 1,000 times less active than LPS. However, using the PGN hydrolase of Streptococcus pneumoniae, soluble PGN was obtained with bioactivity in the same range as that of LPS (334).
| HOST RESPONSE TO CELL WALL CONSTITUENTS |
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Under physiological conditions, the immune cells are continuously exposed to low levels of LPS derived from gastrointestinal bacteria that enter the body via the portal vein. This LPS is taken up by macrophages and may be essential to maintain a basal level of attentiveness of the immune system. At the end of the 19th century, LPS was mainly regarded as an "endotoxin," although Coley showed that heat-killed Serratia marcescens caused necrosis and hemorrhage of various tumors as well as causing fever (588). About 40 years later, Shear identified LPS as the agent responsible for the necrosis and hemorrhage. Buchner discovered that the immunological defense system could be nonspecifically activated against infection by injection with bacterial extracts (565, 588). It is now thought that continuous challenges with small amounts of bacterial constituents may be necessary to keep the immune system alert to infections. Indeed, low levels of LPS are present in healthy individuals without causing disease (137, 512, 565).
(39). After exposure to LPS, TNF- is one of the first cytokines released by macrophages. TNF- mRNA is constitutively transcribed in Kupffer cells, allowing rapid release of TNF- after an inflammatory challenge (175). IL-1 and IL-6 are not constitutively expressed, but the mRNAs of these cytokines, as well as that of TNF-, are immediately transcribed after a challenge, and maximum mRNA levels have been found 40 min post-challenge in mouse liver macrophages (175, 331).
The release of TNF-, IL-1, IL-6, IL-8, IL-12, platelet-activating factor (PAF), chemokines, and eicosanoids has profound effects on the surrounding tissue (179, 252, 330, 438). In concert with the complement pathway-derived anaphylatoxins C3a and C5a, several of these inflammatory mediators attract PMN from the circulation and activate them. The extravasation of PMN is enabled by vasodilatation and upregulation of adhesion molecules on endothelial cells, PMN, and macrophages (242, 258, 556). The PMN react to these stimuli by intravascular aggregation, adherence to the endothelium, diapedesis, and the production of inflammatory mediators like TNF-, leukotriene B4, and PAF (370, 550). The (activated) PMN express CD14, CD11/CD18, and several complement and Fc receptors and are thus able to recognize and phagocytose LPS, bacterial fragments, and whole bacteria. As specialized phagocytes, PMN produce an impressive series of microbicidal agents, such as lysozyme, bactericidal/permeability-increasing protein (BPI), enzymes, and oxygen free radicals (62, 457). These agents are used mainly for lysosomal killing of microorganisms. However, adherence of the PMN to endothelial cells and the presence of high concentrations of stimuli may also result in the release of microbicidal agents; much of the endothelial damage observed in sepsis is caused by these agents (42). Endothelial cells respond to LPS (via soluble CD14) and to the circulating cytokines by the release of IL-1, IL-6, eicosanoids, the vasoactive agents endothelium derived relaxation factor and endothelin-1, chemokines, and colony-stimulating factors (CSF) (332). The inflammatory mediators secreted by the different cell populations attract and activate B and T lymphocytes. In turn, the latter release mediators such as IL-2, gamma interferon (IFN-
), and granulocyte-macrophage (GM)-CSF (42). IL-2 and GM-CSF are involved in proliferation and activation of PMN and mononuclear cells, whereas IFN- enhances the effects of LPS on mononuclear cells (4, 42, 206, 241, 610). The actions of the activated immune cells combined with the effects of the inflammatory mediators cause symptoms such as fever, endothelial damage, capillary leakage, peripheral vascular dilatation, coagulation disorders, microthrombi, and myocardial depression. These phenomena may finally result in multiple organ dysfunction, shock, and death (42).
Compared to LPS, relatively little is known about the actions of LTA in vivo and in vitro. In contrast to gram-negative bacteria, in which LPS is the major biologically active moiety, in gram-positive bacteria LTA, PGNs, and exotoxins are highly relevant with respect to the immunological response (403). LTA and PGNs are able to induce the release of nitric oxide (NO), IL-1, IL-6, and TNF- by monocytes and macrophages and to activate the oxidative burst in vitro (40, 100, 259, 263, 518, 574). Furthermore, the effects of LTA and PGNs may be synergistic (90). Like LPS, the bacterial species largely determines the potency of the biological actions of LTA (40, 259, 261). In vivo both LTA and PGNs cause the release of NO, TNF-, and IFN- and induce circulatory failure (89, 90, 261), which indicates that gram-positive bacterial components such as LTA and PGNs induce similar effects to LPS both in vitro and in vivo.
In vivo challenges with viable and killed bacteria reveal marked differences between gram-positive and gram-negative bacteria in the kinetics of bacterium-induced TNF- release, and similar differences were observed in vitro (73, 491). In contrast, LPS and LTA exhibit similar kinetics of TNF- release in vivo (90). Despite the differences between bacteria and LPS, it has recently been shown that S. enterica serovar Typhimurium and its LPS induce similar changes in macrophage- gene expression in vitro, confirming the early observations that LPS mimics whole gram-negative bacteria in many respects (462). The pathogenesis of gram-positive bacteria depends to a large extent on the production of powerful exotoxins. Gram-positive bacterial sepsis differs from gram-negative bacterial sepsis in that the gram-positive bacteria often arise from skin, wounds, soft tissue structures, and catheter sites rather than enteric or genitourinary sources. Additionally, gram-positive organisms require a highly orchestrated host response, with intracellular killing by neutrophils and macrophages (403). This is often not the case for gram-negative pathogens, which may be readily killed in the extracellular space by antibody and complement (424, 495). Exotoxins may act as bacterial superantigens, which are potent T-cell-stimulatory protein molecules, produced by for instance S. aureus and S. pyogenes. These superantigens are able to induce toxic shock syndrome and can sometimes cause multiple organ failure (25, 44). The superantigenic activity of the bacterial exotoxins can be attributed to their ability to cross-link major histocompatibility complex class II molecules on antigen-presenting cells outside the peptide groove with T-cell receptors to form a trimolecular complex (312). Each superantigen is known to interact with a specific V(beta) element of the T-cell receptor. This trimolecular interaction leads to uncontrolled release of a number of proinflammatory cytokines, especially IFN- and TNF-, the key cytokines causing toxic shock syndrome (58). Besides the highly deleterious exotoxins, gram-positive bacteria contain a number of immunogenic cell wall components, such as LTA and PGNs (403, 495).
With the cleavage of C3 and C5, the chemoattractive and vasoactive agents C3a and C5a are released. They cause increased vascular permeability, upregulate adhesion molecule expression on endothelial cells and neutrophils, and attract and activate these phagocytes. Furthermore, they activate basophilic granulocytes and mast cells: these cells release a variety of vasoactive compounds (such as histamine), facilitating the invasion of phagocytes (116, 223, 280, 370, 434, 457, 521, 550).
During infection, liver parenchymal cells are stimulated by TNF-, IL-1, and IL-6 to produce acute-phase proteins. These proteins comprise C-reactive protein, serum amyloid A, lipopolysaccharide-binding protein (LBP), serum amyloid P, hemopexin, haptoglobin, complement C3 and C9, 1-acid glycoprotein, 2-macroglobulin, and some proteinase inhibitors (129, 476, 498). The expression is differentially upregulated from severalfold (C3 and C9) to even 1,000-fold (C-reactive protein) (129). Some of the acute-phase proteins, like LBP modulate the immune response reactions by activation of phagocytes and antigen-presenting cells, but basically the acute-phase response is considered to alleviate the damage caused during infection (129, 280, 444). Albumin is a so-called negative acute-phase protein since its production is down regulated during inflammation (129).
Liver cell types. The liver consists of five kinds of cells: liver parenchymal cells (hepatocytes), endothelial cells, fat-storing cells, pit cells, and Kupffer cells. The liver parenchymal cells represent 60% of the liver cells (423). Parenchymal liver cells are metabolically highly active and contain huge numbers of lysosomes, peroxisomes, Golgi complexes, and mitochondria (92). Important and specific metabolic pathways are the urea cycle, regulation of lipid metabolism, production of bile acid in relation to bile secretion, and hormonally regulated glycogenolysis and gluconeogenesis (423). Furthermore, liver parenchymal cells are major producers of plasma proteins (e.g., albumin) and, as mentioned above, acute-phase proteins (129). Sinusoidal endothelial cells account for approximately 19% of all liver cells. In contrast to vascular endothelial cells, these cells have no basement membrane and possess slender processes containing fenestrae, allowing direct contact between the plasma and the cells behind the endothelial barrier. Liver endothelial cells express several receptors that allow the endocytosis of (foreign) ligands, and during a bacterial infection, they produce several cytokines and eicosanoids (82, 278, 452). Fat-storing cells are characterized by the presence of vitamin A-rich fat droplets in the cytoplasm. Some specific functions are uptake and storage of retinoids, as well as synthesis and secretion of extracellular matrix proteins (162). Pit cells are located in the sinusoids and exert natural killer (NK) activity (92). Kupffer cells are the liver macrophages; they are stellate and are situated in the sinusoids, where they are attached to endothelial cells (and parenchymal cells) by their pseudopodia. They constitute 80 to 90% of the fixed tissue macrophages (reticuloendothelial system) and account for approximately 15% of the liver cells. Kupffer cells remove all kinds of old, unnecessary, and damaged material from the circulation (immune complexes, erythrocytes, tumor cells, cellular debris, and apoptotic cells) (485, 530, 571). In addition, they remove foreign materials from the blood with high efficacy (92, 278). In relation to the defense against bacteria and bacterial components, Kupffer cells are highly relevant (270, 530), playing a major role in both clearance and detoxification of LPS from the circulation (especially the portal vein) and the production of inflammatory mediators in response to LPS (85, 139, 482, 530, 571).
, IL-1, IL-6, eicosanoids, PAF, NO, and reactive oxygen. Not only free LPS, LTA, and PGN but also live and killed bacteria can elicit the release of TNF- (21, 73, 178, 262, 400, 491). The lipid mediators—eicosanoids and PAF—released by the macrophages and liver sinusoidal endothelial cells have important functions as well (197, 230, 254, 279). Besides the vasoactive functions of these agents (368, 369), PGE1 and PGE2 inhibit the transcription of TNF- mRNA in macrophages, resulting in a long-term inhibition of TNF- release (175, 317, 389, 458). The last group of products released in response to LPS are the reactive oxygen species. Activation of macrophages and infiltrating PMN by bacterial components and by TNF- and other inflammatory mediators induces the intracellular production of O2-, H2O2, and other potent microbicidal products (32, 361). Although these compounds are responsible for killing phagocytosed microorganisms, they are released at high concentrations of activators and cause extensive tissue damage. Nitric oxide (NO) is a microbicidal product which is produced by macrophages, endothelial cells, and hepatocytes. Once secreted, it is rapidly converted to nitrate and nitrite and has a wide range of physiological effects (74, 298, 406, 493). Besides its (beneficial) microbicidal and tumoricidal effects, NO causes vasodilation, endothelial damage, damage to hepatocytes, inhibition of acute-phase protein production, and increased leukocyte adhesion in liver and lungs (85, 213, 237, 575).
Gadolinium chloride (GdCl3) causes a transient depletion of the large, ED2-positive Kupffer cell fraction in rats (81, 192). Administration of GdCl3 reduces death and hepatic damage in rats treated with a lethal dose of LPS but does not prevent TNF- production (231). Bautista et al. described a similar technique with liposome-encapsulated dichloromethylene biphosphonate (Cl2MBP); this reagent eliminated 90% of the largest Kupffer cell fraction and 50% of the smaller Kupffer cells (33). In addition, macrophages in the spleen are depleted after injection of these liposomes whereas circulating monocytes are spared (558). After i.v. injection of LPS into Cl2MBP-liposome treated rats, serum TNF- levels were significantly reduced (33). Similar decreases in TNF-, IL-1, and IL-6 production in liver slices from Cl2MBP-liposome treated mice were observed (331).
An in vitro study with splenic macrophages and Kupffer cells has shown that splenic macrophages produce significantly more LPS-induced TNF- than do Kupffer cells but that the latter phagocytose more latex beads in vitro and in vivo (486). In addition, Lichtman et al. showed that there are major differences in the activation pathway between peritoneal macrophages and Kupffer cells (316). Whereas the response of peritoneal macrophages to LPS was dependent on CD14 (see the section on LPS and LTA receptors, below), a mainly CD14-independent activation pathway was utilized in Kupffer cells. The route of LPS entry into the body may also alter the immune response. Asari et al. have shown that the peak TNF levels and the kinetics of TNF release after intraperitoneal (i.p.) versus i.v. injection differ, confirming that the macrophages from relevant organs respond differently (13).
production by peritoneal macrophages. Furthermore, binding to the liver hepatocytes could not be inhibited by excess amounts of LPS, indicating that the LPS structure had been changed significantly (139). However, the nature of the changes and the receptors responsible for the uptake of the modified LPS were not identified. | LIPOPOLYSACCHARIDE AND LIPOTEICHOIC ACID RECEPTORS |
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LBP is an acute-phase protein (473, 476) and is induced by IL-6 and IL-1 (176, 444, 572). Besides the liver, the lungs, kidneys, and heart are also involved in the production of LBP (506). The constitutive levels of LBP in serum are low (1 to 15 µg/ml) but increase greatly during infection (155, 289, 474, 476, 528). In humans during the acute phase of trauma or sepsis, LBP levels are at a maximum on days 2 to 3 (476). Most of the LBP in serum is associated with lipoproteins, and LBP in serum is mainly associated with low-density lipoprotein (IDL), very-low-density lipoprotein (VLDL), or HDL (414, 569, 600).
LBP binds to smooth and rough LPS, lipid A, and lipid IVA. The affinity of LBP for lipid A is high, with the Kd varying from 1 to 58 nM (158, 527). The binding site for lipid A is situated in the N-terminal part between amino acids (aa) 91 and 108, with positively charged arginine and lysine residues within this region fulfilling an essential role (290, 519). The C-terminal part of the LBP molecule, however, mediates the transfer of LPS to CD14 (187, 522). The binding of LBP to killed bacteria is markedly higher than the binding to living bacteria (307).
LBP catalyses the transfer of LPS to CD14, thus enhancing the LPS-induced activation of monocytes, macrophages, and PMN by 100- to 1,000-fold (475). The CD14-mediated activation of peritoneal macrophages by heat-killed Staphylococcus aureus bacteria, LTA, cell wall PGN, or mycobacterial lipoproteins is not enhanced by LBP (345, 357, 478, 524). In the presence of LBP, LPS induced an enhanced intracellular killing and secretion of TNF- and NO by murine macrophages (68), increased adherence of human PMN to endothelial cells (590), LBP and CD14 release by HepG2 human hepatoma cells (383), and release of tissue factor by THP-1 cells in vitro (500), whereas the addition of anti-LBP or anti-CD14 antibodies abrogated the effect of LBP (154, 500, 590). Application of anti-LBP antibodies together with LPS protected D-galactosamine-sensitized mice from death (155, 156, 307a).
Besides its proinflammatory role, LBP may also have anti-inflammatory actions, such as the LBP-mediated catalysis of LPS and LTA transfer to HDL and other lipoproteins (see the section on lipoproteins below). (177, 526, 600, 602). Recently, LBP was shown to be involved in the neutralization of LTA by HDL, extending the anti-inflammatory role of LBP to gram-positive organisms (177). Interestingly, as mentioned above, LBP is not essential for binding of gram-positive cell wall components to CD14 while promoting the neutralization of LTA by lipoproteins, suggesting a solely anti-inflammatory function in the response to gram-positive organisms. The injection of LBP into D-galactosamine-sensitised mice decreased LPS-induced TNF and IL-6 release and significantly reduced mortality, and LBP was also found to be protective during an infection with live E. coli (289). Interestingly, Jack et al. observed that LBP knockout mice were less susceptible to a challenge with LPS but more susceptible to live S. enterica serovar Typhimurium (240), which is in line with the putative protective effect of LBP during bacterial infection. Wurfel et al. observed the absence of a response to LPS in whole blood from LBP knockout mice (601).
LBP binds certain phospholipids, which relates to its structural homology to other lipid-binding proteins like phospholipid transfer protein (PLTP), which is able to transfer LPS to HDL (182, 414, 612). By analysis of sequence homologies, it was found that LBP belongs to a family of lipid-binding proteins also containing BPI, PLTP, and cholesteryl ester transfer protein (3, 224, 266, 287).
release (596). Transfection of CD14-negative CHO and 70Z/3 cells with CD14 conferred responsiveness to LPS and positively identified CD14 as an LPS receptor (170, 302). Kirkland et al. determined the binding affinity of the LPS-LBP complex to CD14-transfected CHO cells and THP-1 cells and found Kd values of 2.7 x 10-8 to 4.8 x 10-8 M (265). The mechanism of LPS binding to CD14 is shown in Fig. 4.
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levels were even higher in CD14-deficient than in wild-type mice after a challenge with live S. aureus, indicating that CD14 is not of great importance in the in vivo defense against these gram-positive bacteria (202). Anti-CD14 antibodies applied to LPS-treated rabbits and cynomolgus monkeys decreased hypotension, neutropenia, TNF- release, and organ damage, indicating that blocking of CD14 in severe endotoxemia could protect against the detrimental effects (308, 468). Because CD14 is a glycosylphosphatidylinositol-linked receptor that lacks a transmembrane domain, it probably needs an accessory molecule for signal transduction (499). This hypothesis was confirmed using different anti-CD14 antibodies that either blocked LPS binding to CD14, or did not block LPS binding while preventing LPS-induced cell activation (163, 303). The accessory receptor has been identified as being a member of the TLR family.
Binding of LPS to a cell does not result in an immediate response. A time lapse of 15 to 30 min between LPS binding and LPS-induced responses such as cytokine release and adhesion was observed, which suggests that a time-consuming process such as internalization is necessary to enable signaling (93, 315). Indeed, several, but not all, studies have revealed that blocking internalization or endosome fusion also blocks LPS-induced signaling (93, 315, 427, 431). Although the precise mechanisms are not completely understood, it has been shown that monomeric LPS is transported into the cell to the Golgi complex, thus activating the cell. In contrast, particulate (bacterium) or aggregated (micelles) LPS is transported through a CD14-dependent pathway to the lysosomes without activating the cell (461).
Although LPS was the first CD14 ligand discovered, many other microbial ligands for CD14 were later identified. These are also ligands for the TLRs, and these are discussed below. Molecular cloning of the CD14 gene revealed a 1.4-kb transcript encoding a 356-aa protein (126). CD14 is glycosylphosphatidylinositol linked and has a high leucine content (17.7% human CD14, 15.5% murine CD14) (484). A repeating leucine-rich, 24-residue motif (LxxLxLx) can be recognized (127, 484). The LPS-binding site and the sites involved in the interaction of CD14 with the putative accessory receptors have been identified in the N-terminal part of CD14 (250, 613). Two putative LPS-binding sites were mentioned: aa 39 to 44 (501) and aa 57 to 64 (248, 355). Stelter et al. also measured the LPS- or E. coli-induced activation and found that the substitution mutant with substitution at aa 39 to 44 was not capable of inducing activation (501). Two other regions essential for sCD14-mediated signaling of endothelial and smooth muscle cells were also identified: aa 9 to 13 and aa 91 to 101 (249, 502).
CD14 is expressed by cells of the myeloid lineage (monocytes, macrophages, and PMN), B cells, liver parenchymal cells, gingival fibroblasts, and microglial cells (9, 322, 383, 421, 507, 616). Differential expression of CD14 is observed: peritoneal and pleural macrophages have a high level of constitutive CD14 expression, whereas (murine) Kupffer cells, alveolar macrophages, monocytes, and PMN have a low level of constitutive CD14 expression (9, 341, 350, 617). CD14 is absent from early progenitor (myeloid) cells, but CD14 expression increases with maturation (164). In vivo challenge of mice with LPS results in up regulation of CD14 on Kupffer cells but also in the heart, lungs, spleen, and kidneys (119, 350). Human PMN express low levels of CD14, but the expression can be upregulated by TNF-, granulocyte (G) CSF, GM-CSF and fMLP (formyl-Met-Leu-Phe) within 20 min, indicating that the CD14 originates from intracellular stores (205, 595).
, whereas IFN- and IL-4 inhibit the release of sCD14 (291, 477). In the steady state, the concentration of sCD14 is 2 to 6 µg/ml in human serum (528). In septic shock patients, sCD14 levels are increased, the levels have been found to correlate with mortality (292). Active soluble CD14 was also found in human milk at 10-fold-higher concentrations than in serum. The milk sCD14 may play a role in the bacterial colonization of the gut (286). Endothelial and epithelial cells do not express membrane CD14 and become up to 10,000-fold more sensitive to LPS in the presence of serum (Fig. 4). The LPS sensitivity can be blocked by anti-CD14 or by immunodepletion of sCD14 from the serum (150, 566). Presentation of sCD14-LPS to endothelial or epithelial cells results in up regulation of adhesion molecules, excretion of IL-6 and IL-8, and endothelial damage (150, 433, 566, 616) The role of LBP in the transfer of LPS to sCD14 is unclear (433).
In addition to its proinflammatory actions, sCD14 exerts anti-inflammatory actions. First, it accelerates the LBP-mediated transfer of LPS to HDL, but it has been found to be inessential in this process (599, 612). Second, injection of sCD14 into LPS-challenged mice is protective against LPS-induced death (204), although in a similar experiment by Stelter et al., sCD14 provided less protection, with significant decreases in mortality but not in cytokine release and organ damage (503). In both experiments, the amount of sCD14 injected was approximately 25-fold greater than the endogenous sCD14 levels. In vitro experiments revealed that moderate concentrations of sCD14 enhanced the LPS-induced activation of monocytes, macrophages, and PMN (183, 533) whereas inhibition of LPS-induced activation was observed at higher sCD14/LBP ratios (203, 477, 533).
TLR4 MyD88 IRAK TRAF6 I
B NF-B for inflammatory mediators and Spàtzle Toll Tube Pelle dTRAF Cactus Dorsal for antimicrobial peptides, where IRAK is IL-1R-associated kinase and TRAF6 is TNF receptor-associated factor 6 (49, 439). To date, TLR1 to TLR10 have been identified and are all expected to be involved in immune responses (63, 376, 400a). The TLRs, the IL-1 receptor, the IL-18 receptor, and a number of mammalian and nonmammalian proteins exhibit a striking similarity with respect to the Toll/IL-1 receptor domain (TIR); hence, this family of receptors is called the TIR superfamily (400a). Three major groups can be determined: the immunoglobulin domain subgroup, containing the IL-1RI and the IL-18R; the leucine-rich-repeat subgroup, containing the TLRs; and the adaptor subgroup, which includes the MyD88 protein essential for TLR2 and TLR4 mediated signaling (400a). So far, the specificities of TLR2, TLR3, TLR4, TLR5, and TLR9 (partially) have been shown to be involved in recognition of microbial components. A substantial amount of data suggests that TLR4 is involved mainly in the recognition of LPS from gram-negative bacteria. TLR2 recognizes gram-positive cell wall constituents such as PGN and LTA but also recognizes microbial lipoproteins and lipopeptides and yeasts. In addition, TLR3 recognizes viral double-stranded RNA dsRNA, TLR5 recognizes bacterial flagellin, and TLR9 recognizes bacterial CpG DNA (references are given in Table 2). Of the remaining TLRs identified, TLR1 may function as an accessory receptor for TLR2 in the recognition of Neisseria meningitidis cell wall components, whereas other investigators observed the heterodimerization of the signaling domain of TLR2 with either TLR1 or TLR6, enabling the recognition of zymosan, group B streptococcal soluble factor, or gram-positive lipopeptides and lipoproteins (211, 407, 516, 603). In the response to S. aureus modulin, however, TLR1 inhibited and TLR6 enhanced the TLR2-mediated response, indicating a modulatory role for these proteins (184). This is confirmed by the findings of Spitzer et al., who observed the inhibition of TLR4-mediated responses by TLR1 in endothelial cells (494). A similar role could be envisioned for some other TLRs for which no microbial specificity has been determined.
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The activation of cells by microbial components is dependent on CD14 (myeloid cells) or sCD14 (endothelial and epithelial cells). It has now been shown that the microbial components interact primarily with CD14 and subsequently with the TLRs. In fact, by using photoactivated cross-linking, it was shown that LPS becomes cross-linked to TLR4 and MD-2 only if the latter are coexpressed with CD14 (79). The extracellular protein MD-2 is closely associated with TLR4 and is essential for LPS binding to this receptor (487).
The role of sCD14 in TLR-mediated signaling has not been specifically addressed. In several of the TLR-overexpression studies in vitro, however, serum was found to enhance activation by the microbial components and the activation could be (partially) inhibited by the addition of anti-CD14 antibodies (118, 215, 320). This reveals that sCD14 in serum can replace surface-bound CD14 and that CD14-mediated signaling is not significantly different in myeloid cells expressing membrane CD14 and other cell types that are dependent on sCD14. However, in deletion and substitution studies with membrane, CD14 and sCD14, the same binding site for LPS, but different sites for mCD14 and sCD14-mediated signaling were found (502, 563). Whether these differences actually reflect the involvement of distinct accessory receptors (e.g., TLRs) remains to be determined.
Human TLR4 is an 841-aa protein with a molecular mass of 92 kDa, whereas TLR2 is an 85-kDa protein (359, 614). A structural similarity between CD14 and the TLRs is the presence of a leucine rich-repeat (LRR) in the extracellular domain. CD14 and TLR4 contain 10 and 21 of these LRR moieties, respectively, which suggests that both receptors may contain a similar binding site for LPS (127, 359). In contrast to TLR4, the binding site in CD14 is only partially situated in the LRR region (248, 250). An acyclic LPS agonist exhibited TLR4-mediated binding which was independent of LBP and (s)CD14, although sCD14 strongly enhanced the cellular response (318). In addition, the proximity of LPS-CD14-TLR4 prior to signaling has been illustrated (244). Poltorak et al. showed that C3H/HeJ macrophages (expressing a nonfunctional TLR4) transfected with either human or murine TLR4 responded differently to LA-14-PP (deficient in secondary acyl chains): whereas the cells transfected with murine TLR4 produced TNF-, the cells transfected with human TLR4 were not activated (429). The authors proposed that LPS physically interacts with TLR4, enabling this molecule to discriminate between lipid A and the partially deacylated LA-14-PP. Similarly, human THP-1 (CD14+) cells transfected with hamster TLR4 responded to Rhodobacter sphaeroides LPS whereas the same cells transfected with human TLR4 did not (319).
Several approaches using knockout mice or cell lines transfected with mutated putative signaling proteins have pinpointed important participants in the signaling pathway. Defective proteins such as MyD88 (255, 513, 515, 614), IRAK (607, 614), TRAF (607, 614), and NIK (NF-B-inducing kinase) (607), resulted in blocked or muted responses to gram-positive and/or gram-negative cell wall constituents. In addition, similar experiments with knockout mice have identified MD-2 as essential accessory molecules in TLR4-mediated signaling and intracellular trafficking (379, 487), whereas MD-1 is instrumental in the LPS-induced B-cell proliferation and antibody production through RP105 (380, 394). A substantial part of the TLR2 and TLR4 signaling pathways coincides, but there is now increasing evidence that alternative pathways also exist. This is exemplified by the observation that Kupffer cells from MyD88-deficient mice release IL-18 but do not produce IL-1ß and IL-12 while Kupffer cells from TLR4-deficient mice do not produce any of these cytokines after stimulation with LPS (481) (the signaling pathways of TLR2 and TLR4 are shown in Fig. 5).
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