Lipopolysaccharide Clearance Research Articles

Lipopolysaccharide dose response in baboons.

A lipopolysaccharide (LPS) dose-response study in an experimental baboon endotoxemia model is presented to define the relevance of this model compared with human endotoxemia. We describe acute and subacute endotoxemic models in baboons, the first evoked by bolus injection of LPS (1 mg, 0.1 mg, or 4 ng per kg of Escherichia coli LPS), and the second evoked by infusion of 1.5 mg/kg of E. coli LPS over 30 min. We report the analysis of LPS clearance, the kinetics of tumor necrosis factor, interleukin (IL) 6, and IL-8 expression on the protein as well as on the mRNA level, change in blood counts (white and red blood cells and circulating platelets), and several hemodynamic parameters such as temperature, cardiac index, heart rate, and mean arterial pressure via multiple sampling. The resulting data are compared with previously published human data. Our results show that the LPS-induced kinetics of cytokine release, as well as of hemodynamic and hematologic changes in baboons, were similar to those observed in humans, even though baboons required a approximately 104-fold higher initial LPS dose to develop these manifestations. Hence, we demonstrate that endotoxemia in baboons qualitatively, yet not quantitatively, resembles endotoxemia in humans and, therefore, proves to constitute a useful model for studying the pathogenic mechanisms of sepsis in relation to humans.

Inhibition of LPS-induced activation of alveolar macrophages by high concentrations of LPS-binding protein

Lipopolysaccharide (LPS)-binding protein regulates the effects of LPS on immunocompetent cells. By catalyzing the binding of LPS to membrane CD14, LPS-binding protein (LBP) potentiates both the inflammatory response and internalization of LPS. LBP-mediated transport of LPS into high density lipoprotein particles participates in LPS clearance. Elevated serum levels of LBP have been shown to elicit protective effects in vivo. Because the expression of LBP is upregulated in lung epithelial cells upon proinflammatory stimulation, we here investigated whether LBP modulates inflammatory responses by lung specific cells. The moderate elevation of LBP concentrations enhanced both LPS-induced signaling and LPS uptake by rat alveolar macrophages, whereas strongly elevated LBP levels inhibited both. In contrast, the lung epithelial cell line A549 responded to high concentrations of LBP by an enhanced LPS uptake which did not result in cellular activation, suggesting an anti-inflammatory function of these cells by clearing LPS.

Accelerated internalization and detoxification of endotoxin by anti-lipopolysaccharide antibody is an Fc receptor–mediated process

Background. Interaction between lipopolysaccharide (LPS), LPS-binding protein, and the CD14 receptor at the surface of LPS-responsive cells results in inflammatory cytokine release and internalization and detoxification of LPS. Monoclonal antibodies (mAbs) raised against the deep-core lipid A or the O-linked polysaccharide moieties of LPS accelerate internalization and detoxification of LPS without stimulating cytokine release. This study was conducted to test the hypothesis that the antibody-mediated internalization of LPS is an Fc receptor (FcR)–mediated process. Methods. Fluoroisothiocyanate (FITC)–conjugated Escherichia coli O111:B4 LPS was incubated with RAW 264.7 cells and allowed to internalize for 2 hours in the presence and absence of anti-LPS, anti-CD14, and isotype control mAbs, and Fab fragments from the anti-CD14, anti–Fc receptor, and control mAbs. Tumor necrosis factor–α (TNF-α) release was measured by WEHI 164 cell bioassay. FITC-LPS uptake was measured by flow cytometry. Statistical analysis was by analysis of variance and Fisher exact test. Results. Addition of anti-LPS antibodies resulted in a 30- to 40-fold acceleration of LPS internalization (P <.01) in agreement with previous studies. This increase was blunted by anti-CD14 and also by isotype control holo-antibody (P <.01), but not by Fab fragments from anti-CD14 or isotype control antibody. Both anti-FcR antibodies and Fab fragments blocked anti-LPS antibody–stimulated uptake of FITC-LPS. Both intact anti-CD14 holo-antibody and Fab fragments blocked TNF-α release (P <.01). Conclusions. Clearance and detoxification of LPS are thought to be essential to the host response to endotoxin. It has been shown that antibodies to LPS accelerate its internalization by monocytic cell lines without increasing the elaboration of cytokines. We found that specific blockade of CD14 by Fab fragments could block TNF-α release but not alter the accelerated internalization of LPS produced by anti-LPS antibodies. In contrast, a nonspecific blockade of internalization was produced by competing antibody, which suggests a mechanistic role for the FcR. Specific blockade of FcR by either holo-antibody or Fab fragments blocked accelerated internalization, which confirms a FcR mechanism. We conclude that the accelerated internalization of LPS produced by anti-LPS antibody is an Fc receptor–mediated process. These results have significance for the development of adjuvant immunotherapy for gram-negative bacterial sepsis. (Surgery 2001;130:192-7.)

Protective effect of OK-432 on mice against endotoxemia and infection with Pseudomonas aeruginosa and Salmonella enteritidis.

OK-432 has been used clinically as a biological response modifier for cancer therapy. We investigated here the protective effects of OK-432 against endotoxic shock and infectious death caused by Pseudomonas aeruginosa and Salmonella enteritidis in mice and proposed a possible mechanism. Pretreatment of OK-432 reduced the lethality of lipopolysaccharide (LPS)-induced endotoxic shock in D-(+)-galactosamine-sensitized C3H/HeN mice. OK-432 did not affect the TNFalpha production in blood, but it did decrease the susceptibility to TNFalpha. Furthermore, an acceleration of LPS clearance from blood was detected. The pretreatment of OK-432 also decreased the lethality of mice in bacterial infection caused by P. aeruginosa and S. enteritidis. The rapid decrease of the viable bacteria from the circulating blood and in spleen and liver in mice was observed in a manner similar to LPS clearance. These findings indicate that the protective effect of OK-432 against the endotoxemia and bacteremia may depend on an up-regulation of clearance of LPS and bacteria and the augmented resistance to TNFalpha.

Role of macrophage scavenger receptor in endotoxin shock

Lipopolysaccharide (LPS) is known to bind to several receptors on macrophages, including CD14 and macrophage scavenger receptor class A types I and II (MSR-A), and stimulates macrophages to release various inflammatory mediators. MSR-A recognizes a broad range of polyanionic ligands such as chemically modified lipoproteins, LPS of Gram-negative bacteria, and lipoteichoic acid of Gram-positive bacteria, suggesting a role in host defence. In this study, mice lacking MSR-A were used to elucidate the role of MSR-A in endotoxin shock. Peritoneal macrophages from MSR-A-deficient (MSR-A−/−) mice bound less remarkably to LPS than those from wild-type (MSR-A+/+) mice and the binding activity of MSR-A+/+ macrophages to LPS was reduced by the addition of an anti-MSR-A antibody. Clearance of LPS in serum was retarded in MSR-A−/− mice after intraperitoneal administration of LPS. LPS-induced expression of cytokines in the liver was similar in MSR-A+/+ and MSR-A−/− mice, but levels of interleukin (IL)-1β expression and serum IL-1β were lower in MSR-A−/− mice. Administration of large doses of LPS resulted in a higher mortality of MSR-A+/+ mice and pretreatment with an IL-1 receptor antagonist reduced the mortality. Thus, MSR-A-mediated macrophage activation plays a negative role in protecting mice from endotoxin shock by enhancing IL-1β production by macrophages. Copyright © 2000 John Wiley & Sons, Ltd.

Intramucosal pH and liver endotoxin clearance during experimental liver transplantation.

The study was designed to assess the gastrointestinal ischaemia and the influence of the specific Kupffer cell toxin gadolinium chloride (GdCl3) on the hepatic and extrahepatic endotoxin [lipopolysaccharide (LPS)] clearance during experimental orthotopic liver transplantation (OLT) in pigs. In eight pig liver transplantations, the donors received 20 mg/kg of GdCl3 24 h before explantation, while controls (n = 8) received normal saline. Gastric and sigmoid intramucosal pH (pHi), LPS and endotoxin-neutralising capacity (ENC) levels were measured in the portal vein and superior vena cava after laparatomy, at the end of the anhepatic phase and 1 h after reperfusion. During the anhepatic phase, the sigmoid pHi decreased significantly from 7.32 +/- 0.02 to 7.29 +/- 0.03 (P < 0.001) and was associated with a substantial increase of portal LPS. Following reperfusion, the systemic LPS concentrations were significantly lower in the pretreated group [39 +/- 23 pg/ml (Control); 14 +/- 7 (GdCl3); P < 0.05] suggesting an improved liver LPS clearance [86% (GdCl3); 58.2% (Control); P < 0.05]. This corresponded to an increased ENC in the pretreated group [118 +/- 52 ENU/ml (GdCl3) vs 81 +/- 45 ENU/ml (Control); P < 0.05]. The anhepatic phase induced splanchnic ischaemia which correlated with portal endotoxaemia. Donor preconditioning with GdCl3 leads to lower systemic LPS concentrations in the recipient and increases ENC values in the early phase after OLT. An improved hepatocellular LPS extraction and/or an activation of the extrahepatic reticulo-endothelial system as a result of GdCl3 treatment is discussed.

Surfactant-associated protein A inhibits LPS-induced cytokine and nitric oxide production in vivo.

The role of surfactant-associated protein (SP) A in the mediation of pulmonary responses to bacterial lipopolysaccharide (LPS) was assessed in vivo with SP-A gene-targeted [SP-deficient; SP-A(-/-)] and wild-type [SP-A(+/+)] mice. Concentrations of tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein-2, and nitric oxide were determined in recovered bronchoalveolar lavage fluid after intratracheal administration of LPS. SP-A(-/-) mice produced significantly more TNF-alpha and nitric oxide than SP-A(+/+) mice after LPS treatment. Intratracheal administration of human SP-A (1 mg/kg) to SP-A(-/-) mice restored regulation of TNF-alpha, macrophage inflammatory protein-2, and nitric oxide production to that of SP-A(+/+) mice. Other markers of lung injury including bronchoalveolar fluid protein, phospholipid content, and neutrophil numbers were not influenced by SP-A. Data from experiments designed to test possible mechanisms of SP-A-mediated suppression suggest that neither binding of LPS by SP-A nor enhanced LPS clearance are the primary means of inhibition. Our data and others suggest that SP-A acts directly on immune cells to suppress LPS-induced inflammation. These results demonstrate that endogenous or exogenous SP-A inhibits pulmonary LPS-induced cytokine and nitric oxide production in vivo.

Role of macrophage scavenger receptor in endotoxin shock

Lipopolysaccharide (LPS) is known to bind to several receptors on macrophages, including CD14 and macrophage scavenger receptor class A types I and II (MSR-A), and stimulates macrophages to release various inflammatory mediators. MSR-A recognizes a broad range of polyanionic ligands such as chemically modified lipoproteins, LPS of Gram-negative bacteria, and lipoteichoic acid of Gram-positive bacteria, suggesting a role in host defence. In this study, mice lacking MSR-A were used to elucidate the role of MSR-A in endotoxin shock. Peritoneal macrophages from MSR-A-deficient (MSR-A(-/-)) mice bound less remarkably to LPS than those from wild-type (MSR-A(+/+)) mice and the binding activity of MSR-A(+/+) macrophages to LPS was reduced by the addition of an anti-MSR-A antibody. Clearance of LPS in serum was retarded in MSR-A(-/-) mice after intraperitoneal administration of LPS. LPS-induced expression of cytokines in the liver was similar in MSR-A(+/+) and MSR-A(-/-) mice, but levels of interleukin (IL)-1beta expression and serum IL-1beta were lower in MSR-A(-/-) mice. Administration of large doses of LPS resulted in a higher mortality of MSR-A(+/+) mice and pretreatment with an IL-1 receptor antagonist reduced the mortality. Thus, MSR-A-mediated macrophage activation plays a negative role in protecting mice from endotoxin shock by enhancing IL-1beta production by macrophages.

Influence of human anti-lipopolysaccharide immunoglobulins on tissue distribution and clearance of lipopolysaccharide in rats.

To examine the influence of passive immunization on the biological fate of injected lipopolysaccharide (LPS), we used a human IgG preparation (anti-LPS IgG) rich in antibodies to a large panel of smooth and rough purified LPS extracts as well as a normal IgG preparation (standard IgG). Our approach was to compare the uptake of 125I-labeled LPS by the tissues of saline or IgG-treated rats. After intravenous injection, one fraction of 125I-labeled Escherichia coli O55:B5 LPS is rapidly taken up by tissues, while another fraction remained in the blood. Uptake of 125I-labeled LPS was principally observed into the liver and spleen. In rats treated prophylactically with standard IgG, these tissues accumulated significantly larger amount of LPS than the tissues of rats treated with anti-LPS IgG. Nevertheless, both IgG preparations increased the specific binding of LPS by the liver and spleen. High levels of homologous unlabeled LPS decreased the uptake of LPS by the liver, presumably by occupying tissue receptors, whereas in the presence of E. coli O127:B8 LPS, an increase of the uptake of 125I-labeled LPS by the liver and lungs was observed. The pharmacokinetics and tissue distribution of LPS-IgG complexes pre-formed in vitro were compared. In the presence of standard IgG, a unexpected increase of the uptake of LPS by the tissues was recorded, whereas LPS-anti-LPS IgG complexes decreased the binding of 125I-labeled LPS to the tissues. On the other hand, the vascular effects induced by LPS did not appear to be modified in rats pretreated with either IgG preparation. In conclusion, although passive immunization against LPS slightly modified the uptake and clearance of LPS, neither in vitro nor in vivo formation of LPS-anti-LPS IgG complexes afforded a very significant protection against the toxic effects of LPS.

Internalization of monomeric lipopolysaccharide occurs after transfer out of cell surface CD14.

Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14-EGFP) was used to follow trafficking of mCD14 and BODIPY-LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex-like structure. mCD14-EGFP was functional in mediating binding of and responses to LPS. BODIPY-LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14-EGFP on the cell surface. However, within 5-10 min, the BODIPY-LPS distributed to intracellular vesicles that did not contain mCD14-EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

Immune proteins and their gene expression in the silkworm, Bombyx mori

Several self-defense proteins have been isolated from the silkworm, Bombyx mori and their amino acid sequences determined. These proteins include novel antibacterial proteins designated lebocin and moricin, and a novel lectin designated hemocytin, an insect homologue of mammalian von Willebrand factor. Antibacterial mechanisms of lebocin and moricin have been analyzed and their ability to form ion channels in bacterial membranes play an important role in defense against bacterial infection. cDNAs and genes encoding these proteins have been cloned to examine their induction mechanisms upon bacterial infection. Regulatory motifs such as the κB-like and GATA sequence have been identified in the B. mori antibacterial proteins. On the other hand, hemocytin gene expression was confirmed to occur upon bacterial infection and before pupation under naive conditions, suggesting that hemocytin plays an important role in both immunity and metamorphosis. Moreover, this review also describes the releasing mechanisms of a bacterial cell wall component, lipopolysaccharide (LPS), from intact bacteria, clearance of LPS from B. mori hemolymph and a possible signal transduction pathway for antibacterial protein gene expression.

Cell surface binding of LBP—LPS complexes to a protein component distinct from CD14

Lipopolysaccharide (LPS) binding protein (LBP) is a serum molecule that mediates cellular activation in response to endotoxin by ensuring the delivery of LPS to either soluble or membrane bound forms of CD14. Aside from this activating role, previous work has shown that LBP and LPS can bind to cells by forming large aggregates which are anchored by mCD14. This binding phenomenon does not correlate with cellular activation. To further characterize these events, we have generated a biologically active radiolabeled LBP ligand with high specific activity. Through the use of this ligand in whole cell binding assays, we have confirmed that the binding of LBP to CHO cells expressing mCD14 is LPS dependent, blocked by the anti-LBP antibodies 18G4 and 2B5, and appears to involve the self aggregation of LBP—LPS complexes on the cell surface. Moreover, we discovered that non-transfected CHO cells also exhibit a binding phenomenon with all the above characteristics of CHO-mCD14 cells. Binding through this latter receptor(s) is distinct from that mediated by mCD14 in that it is not inhibited by anti-CD14 antibodies 28C5 or 18E12. In addition, unlike binding to mCD14, binding of LBP—LPS complexes to this novel receptor is abolished by pretreatment of cells with trypsin. Using proteinase K we found that LBP—LPS complexes bound either by mCD14 or this new receptor are subsequently internalized. Pretreatment of cells with trypsin also abolishes their ability to internalize mCD14 bound LBP—LPS complexes. The novel receptor for LBP—LPS complexes has been detected on many cell types and may be a receptor required for the cellular clearance of LPS.

CD14 and apoptosis.

In addition to its role as a mediator of innate pro-inflammatory responses following bacterial lipopolysaccharide (LPS) binding, the 55kDa glycosyl-phosphatidylinositol-linked macrophage plasma membrane glycoprotein CD14 is now also known to play a role in phagocytic clearance of apoptotic cells. Although apoptotic cell-associated ligand(s) for CD14 await definition, initial findings suggest that ligand binding occurs close to, or at the same site as, LPS binding. Significantly, in contrast to LPS clearance and in keeping with the non-phlogistic nature of apoptosis, CD14-dependent engulfment of apoptotic cells fails to elicit pro-inflammatory cytokine release from macrophages. Therefore CD14 may be regarded as an innate immune receptor both for microbial products--after binding which activates inflammatory responses--and for self components, which either fail to induce, or alternatively actively suppress, inflammatory responses. Here we review current knowledge of the structure and functions of CD14, its ligands, its possible modes of signal transduction and its place in the panoply of macrophage molecules implicated in apoptotic-cell clearance.

Lipopolysaccharide-binding protein is required to combat a murine gram-negative bacterial infection.

An invading pathogen must be held in check by the innate immune system until a specific immune response can be mounted. In the case of Gram-negative bacteria, the principal stimulator of the innate immune system is lipopolysaccharide (LPS), a component of the bacterial outer membrane. In vitro, LPS is bound by lipopolysaccharide-binding protein (LBP) and transferred to CD14--the LPS receptor on the macrophage surface--or to high-density lipoprotein (HDL) particles. Transfer to CD14 triggers an inflammatory response which is crucial for keeping an infection under control. Here we investigate how LBP functions in vivo by using LBP-deficient mice. Surprisingly, we find that LBP is not required in vivo for the clearance of LPS from the circulation, but is essential for the rapid induction of an inflammatory response by small amounts of LPS or Gram-negative bacteria and for survival of an intraperitoneal Salmonella infection.

Endotoxin shock in antibody-deficient mice: unraveling the role of natural antibody and complement in the clearance of lipopolysaccharide.

While the significance of natural Ab is not entirely clear, one proposed role is clearance of bacterial Ags. To determine whether natural Ab was involved in clearance of endotoxin, we have examined novel strains of mice with either a total or selective deficiency in Ig. Recombinase-activating gene-2 (RAG-2(-/-))-deficient mice, which have no serum Ig due to arrested development of B cells at the pro-B stage, demonstrate increased sensitivity to endotoxin that correlates with an impaired clearance. When RAG-2(-/-) mice are reconstituted with pooled sera from normal mice, both survival and clearance of circulating endotoxin are enhanced. To further define the nature of the protective Ab, Bruton's tyrosine kinase (Btk)-deficient mice were characterized in the high dose LPS model. Like RAG-2(-/-) mice, they are highly sensitive to endotoxin and have an impaired clearance of LPS. Reconstitution of Btk(-/-) mice, which have reduced levels of IgG3 and IgM, with purified normal mouse IgM dramatically enhances their ability to clear endotoxin compared with mock (saline)-reconstituted littermates. The cellular source of natural anti-LPS IgM was identified as the peritoneal-residing B-1 cell by enzyme-linked immunospot (ELISPOT) assay. Taken together, these studies demonstrate the important role of natural Ab and complement in the clearance of pathogenic substances from the circulation.

TIMING THERAPY FOR MENINGOCOCCAL INFECTION

IN REPLY: We thank Dr. Radetsky for his interest in our article on meningococcemia and his letter concerning the timing of antibiotic administration in meningococcemia. That the mortality of meningococcemia has decreased from >80% to <20% since the inception of antibiotic therapy is well-known. The timing of antibiotic administration and its relationship to disease outcome has been a topic of debate. The studies that we cited in support of early antibiotic therapy include a retrospective review exhibiting a reduction in the case fatality rate by 40% in patients given antibiotics before hospital admission (relative risk, 0.6; 95% confidence interval, 0.2 to 1.5)1 and a second retrospective analysis with no deaths reported in patients given antibiotics before referral and 24% mortality in patients admitted without such treatment (P = 0.106).2 Neither of these studies meet criteria for statistical significance at the 95% confidence level; however, the choice of 95% certainty as a measure of statistical significance is somewhat arbitrary and may not be a synonymous with clinical significance. The second study, for example, shows a decrease in mortality that is 89.4% likely not to be caused by chance. Additionally the combination of these two studies and one additional study with a similar trend resulted in a statistically significant case fatality reduction from 12% to 5% (odds ratio, 2.6; 95% confidence interval, 1.04 to 7.18).3 There is, however, one retrospective report of a significantly (P = 0.03) higher mortality rate (24%) in patients receiving antibiotics for suspected meningococcal disease than in those not receiving antibiotics before admission (6%).4 This question will never be studied in a prospective, randomized, controlled manner because of the severity of the disease and the potential hazards of withholding antibiotics. Therefore current recommendations can be based only on the available clinical data and the known immunopathophysiology of the disease. That a patient's immune response to infection is more important than the timing of antibiotics with regards to the outcome of meningococcemia may be true. High lipopolysaccharide (LPS) and cytokine levels vary markedly between individuals with meningococcal disease, and high levels are associated with worse prognosis.5-9 However, this does not argue against the contention that early antibiotics could improve outcome. LPS levels fall dramatically after antibiotic therapy.6 It is likely that LPS levels rise with a longer duration of meningococcal infection and possible that the rapid clearance of LPS seen with antibiotic administration could result in improved outcome. Neither of these hypotheses has been tested. In clinical medicine recommendations must often be made in the absence of definitive scientific proof, and the division between hypothesis and fact is not as clear as one would like. Unclear to us is the extent to which the medical-legal climate should influence clinical recommendations. Our recommendations are based on a review of the available clinical data, the immunopathophysiology of meningococcemia and our clinical experience with the disease. We believe these data support the role of timely antibiotic administration in meningococcemia and our opinion that outcome, although we concede that definitive scientific proof is not available, is likely affected by early antibiotic intervention. It is possible that the issue may become clearer in the wake of several ongoing studies of meningococcemia, some of which include prospective data collection of the timing of antimicrobial administration. Erica A. Kirsch, M.D. Brett P. Giroir, M.D. Department of Pediatrics; University of Texas Southwestern Medical Center; Dallas, TX

Immune response in insects: The role of phenoloxidase in defense reactions in relation to melanization and sclerotization

It is well known that activated prophenoloxidase (proPO) plays an important role in cuticular melanization and sclerotization. In addition, studies dealing with immune response of insects suggest that phenoloxidase (PO) is also critical in the defense reactions of insects against invaders. proPO is activated by elicitors derived from microbial cell wall components such as peptidoglycan, beta-1,3-glucan, and lipopolysaccharide (LPS). According to our recent studies we proposed a model clarifying the role of PO in both cellular and humoral immune responses. LPS triggers Ceratitis capitata hemocytes via induced protein tyrosine phosphorylation to release biologically active molecules, including p47 and proPO-activators. Furthermore, hemocytes in response to LPS facilitate clearance of LPS from the hemocoel of medfly. The effector molecules involved in the LPS clearance are hemocyte surface-associated p47 (mp47), soluble p47 (sp47), activated proPO, and tyrosine. A similar LPS clearance system in the integument of medfly in vitro was also demonstrated. According to our data, the proposed mechanism for LPS clearance from hemocoel and from integument is the crosslinking of LPS to p47 or certain integumental proteins via the intermediacy of reactive tyrosine derivatives generated by PO activity, as is the case for cuticular protein-chitin crosslinks during sclerotization. We also demonstrated that metabolites of the eumelanin biosynthesis and not melanin itself or N-acetyldopamine (NADA), the key precursor of sclerotizing agent, were necessary for the immune responses by hemocytes and integument.

Lipopolysaccharide (LPS) Signal Transduction and Clearance: DUAL ROLES FOR LPS BINDING PROTEIN AND MEMBRANE CD14

Under physiological conditions, lipopolysaccharide (LPS) activation of cells involves the LPS binding protein (LBP) and either membrane or soluble CD14. We find LPS forms a ternary complex with LBP and membrane CD14 (mCD14). Subsequent to complex formation and distinct from signal transduction, LBP and LPS internalize. Internalization can be separated from signal transduction with the anti-LBP antibody 18G4 and the anti-CD14 antibody 18E12. 18G4 inhibits LBP binding to mCD14 without blocking signal transduction or LPS transfer to soluble CD14; 18E12 inhibits signal transduction without affecting LPS binding and uptake. These data show that while LPS signal transduction and LPS clearance utilize both LBP and mCD14, the pathways bifurcate after LPS binding to mCD14.

Mode of action of anti-lipopolysaccharide-binding protein antibodies for prevention of endotoxemic shock in mice.

Lipopolysaccharide (LPS)-binding protein (LBP) has been shown to regulate the response of monocytes to LPS in vitro. In a previous study, polyclonal anti-LBP IgGs were found to protect D-galactosamine-sensitized mice against a lethal endotoxemic shock induced by a low challenge of LPS or lipid A when administered simultaneously with endotoxin. In the present study, we investigated the mode of action of these anti-LBP IgGs. In vitro, we demonstrated that they interfere with LPS binding to monocytes or polymorphonuclear cells in different ways: by the mere prevention of binding of LPS to LBP thus preventing the binding of LPS to CD14, or by reacting with LPS-LBP complexes thus mediating their binding to complement or Fc receptors on monocytes and on polymorphonuclear cells. In vivo, we demonstrated that anti-LBP IgGs afforded protection against lethal endotoxemic shock by one of two mechanisms. First, LBP blockade by pretreatment with anti-LBP IgG allowed protection against a low dose of LPS (100 ng). This protection occurred despite LPS levels in blood similar to those in control mice but in the absence of detectable tumor necrosis factor (TNF). This demonstrated that anti-LBP IgG could block the LBP-mediated TNF release upon LPS challenge. In contrast, anti-LBP IgG did not afford protection in mice not sensitized with D-galactosamine and challenged with high-dose LPS (1 mg), confirming that LPS at high concentrations could stimulate cells independently of the LBP pathway. Second, anti-LBP treatment administered simultaneously with LPS challenge protected mice against both low and high doses of LPS. Unlike after pretreatment with anti-LBP IgG, this protection was accompanied by a decrease of circulating LPS, suggesting that anti-LBP IgG in these conditions facilitated clearance of LPS probably by clearing LPS-LBP complexes. These data and the fact that LBP binds to all LPS through lipid A suggest that antibody directed to LBP could be a candidate for therapeutic strategies in endotoxemic shock.

Clearance of lipopolysaccharide in hemolymph of the silkworm Bombyx mori: Its role in the termination of cecropin mRNA induction

Clearance of lipopolysaccharide (LPS) in the hemolymph of silkworm, Bombyx mori, and its physiological role in the regulation of induction of an antibacterial protein, cecropin B, were investigated. In in vitro culture of fat body, cecropin B mRNA appeared 20 min after incubation with LPS and its accumulation persisted more than 72 h. No significant desensitization was observed for at least 24 h in vitro. On the contrary, cecropin B mRNA completely disappeared 24 h after injection of LPS in vivo. These results suggest that concentration and/or biological activity of LPS decreases in hemolymph with time. LPS concentration assay at various time intervals after injection revealed two phases of LPS clearance from hemolymph, an initial rapid ( T 1 2 = 1.0 h ) followed by a very slow phase ( T 1 2 > 18 h ). The threshold concentration of LPS to induce cecropin B mRNA in vivo was over 10-fold higher than that of in vitro. Although no cecropin mRNA accumulation was detected after 24 h of LPS injection, it appeared again by a reinjection of LPS at this time period. This suggests that the reduction of LPS concentration directly terminates cecropin induction in vivo. We, therefore, conclude that B. mori has a LPS clearance system from hemolymph which is sufficient to terminate LPS mediated antibacterial protein induction.