Presence Of Lipopolysaccharide-binding Protein Research Articles

The level of lipopolysaccharide-binding protein in acute intestinal infections, the effect of IL-1β and IL-10 on its production

Aim. To determine the level of lipopolysaccharide-binding protein (LBP) in acute intestinal infection, depending on the etiology and severity of the disease, and the effect of interleukin (IL)-1 and IL-10 levels on the expression of LBP.
 Methods. Serum samples of 62 patients were assayed by solid phase enzyme-linked immunosorbent assay using a set of reagents ELISA (USA) and Vector-best (Novosibirsk) for levels of LBР, IL-1, IL-10. The first group included 33 patients with bacterial intestinal infection, the second group consisted of 29 patients with viral diarrhea, and the control group comprised 20 conditionally healthy patients. Analyses were performed using Microsoft Excel 2010 and Statistica version 6.0 software. Statistical differences were determined by using the MannWhitney U Test, the p-value 0.05 were regarded as statistically significant. Spearmans correlation coefficient was used to examine relationships. ShapiroWilk W test was used to check for normal distribution of the features.
 Results. We detected the presence of LBP in all the studied groups, with the content of LBP was significantly increased in the group of bacterial intestinal infections compared with other groups. With regard to disease severity, LBP level was the highest for mild acute intestinal infections caused by bacteria, and for viral diarrhea, fluctuations in LBP did not exceed the norm. In intestinal infections caused by bacteria, the levels of LBP were directly related to the levels of IL-1 and IL-10.
 Conclusion. Detection of LBP concentration can be used for the initial differential diagnosis of intestinal infections caused by bacteria, which would significantly narrow the diagnostic search and determine the tactics of etiotropic therapy; also, considering the relationship between the concentration of LBP and disease severity, this indicator can be used as a predictive sign of the course of the disease.

Expression of α1-acid glycoprotein and lipopolysaccharide binding protein in visceral and subcutaneous adipose tissue of dairy cattle

Adipose tissue is an endocrine compartment that plays an important role in immune defence by producing and releasing a wide range of proteins, including acute phase proteins (APPs). The liver is the main organ of APP synthesis, although extrahepatic production has also been reported. In the present study, expression of two APPs in dairy cattle, lipopolysaccharide binding protein (LBP) and α1-acid glycoprotein (AGP), was determined in four visceral (pericardial, mesenteric, omental and retroperitoneal) and three subcutaneous (withers, tail head and sternum) adipose tissue depots. mRNA expression was evaluated using qualitative and quantitative PCR, protein profiles were assessed by Western blot analysis and cellular localisation was determined by immunohistochemistry. The presence of LBP and AGP was demonstrated at mRNA and protein levels in all seven adipose tissue depots. Expression of AGP and LBP suggests that they may have roles as local and systemic inflammatory adipokines.

Lipopolysaccharide-binding protein: Local expression in bovine extrahepatic tissues

Lipopolysaccharide-binding protein (LBP) is an acute phase protein involved in host response to Gram-negative and Gram-positive pathogens. It is synthesized by hepatocytes and released as 60–65 kDa glycoprotein in plasma. Little is known about the distribution of LBP in non-pathological bovine tissues. The aim of the present study was to investigate the extra hepatic expression of LBP in different bovine tissues by qualitative and quantitative real time (RT) PCR. The presence of the protein was also confirmed by immunohistochemistry using an anti-human LBP antibody preliminarily validated by cross-reactivity in bovine tissues. While a wide panel of organs and tissues was investigated, the attention was focused on the digestive tract and mammary gland. Moderate amount of mRNA was detected in most of the tissues involved in this study. Extra hepatic LBP mRNA expression was particularly high in parotid and submandibular salivary glands. Remarkably, LBP mRNA was found in rumen, reticulum and omasum. High expression was also found in the mammary gland. Intensity of protein staining paralleled mRNA expression in most tissues, with the exception of lung, ovary and thyroid gland. The presence of LBP throughout epithelial mucosal tissues is indicative of an important role of LBP in mucosal immunity at sites of bacterial exposure. These results suggest that ruminant forestomachs may mount a local acute phase reaction.

Lipopolysaccharide-binding protein-mediated Toll-like receptor 4 dimerization enables rapid signal transduction against lipopolysaccharide stimulation on membrane-associated CD14-expressing cells

Toll-like receptor (TLR) 4/MD-2 dimerization is thought to be required for the initiation of signaling during innate immune responses. In this study, we examined the molecular mechanisms underlying receptor dimerization in the context of accessory molecules, i.e. CD14 and lipopolysaccharide-binding protein (LBP), to determine whether dimerization is required for the initiation of signaling in response to LPS stimulation. We found that LPS-induced TLR4/MD-2 dimerization occurred only in membrane-associated CD14 (mCD14)-expressing cells. Furthermore, dimerization required LBP, but not soluble CD14 (sCD14), as an essential serum component. LPS-induced signaling as assessed by IkappaB-alpha degradation, however, occurred in mCD14-negative cells in the presence of serum and sCD14. Signaling also occurred in mCD14-positive cells in the absence of serum. Time course studies on mCD14-positive cells have demonstrated that LPS stimulation induces rapid activation of nuclear factor-kappaB and p38 in the presence of LBP (TLR4/MD-2 receptor dimerization) as compared with stimulation without LBP (receptor non-dimerization). This early activation was blocked by inhibitory anti-CD14 mAb. These studies suggest that LPS-induced TLR4/MD-2 receptor dimerization is not essential for signaling but prompts rapid signaling during innate immune responses.

Caspase-3 is activated and rapidly released from human umbilical vein endothelial cells in response to lipopolysaccharide

Endothelial cell injury/dysfunction is considered to play a critical role in the pathogenesis of severe sepsis and septic shock. Although it is considered that endothelial cell apoptosis is involved in endothelial injury/dysfunction, physiological involvement remains ambiguous since the induction of apoptosis requires the inhibition of endogenous apoptosis inhibitors. Here we show that caspase-3 activation, a biological indicator of apoptosis, is observed in response to lipopolysaccharide (LPS) stimulation even under the influence of endogenous apoptosis inhibitors, and that activated caspase-3 is rapidly released from human umbilical vein endothelial cells (HUVEC). In the presence of cycloheximide (CHX), an increase in intracellular caspase-3/7 activity in response to LPS was not detected in HUVEC up to 24 h following stimulation even in the presence of LPS-binding protein (LBP), soluble CD14 and soluble MD-2, whereas the decrease in cell viability and increase in release of the cellular enzyme lactate dehydrogenase (LDH) were observed in a soluble CD14/LBP-dependent manner. On the other hand, even in the absence of CHX, a significant increase in caspase-3/7 activity and a cleaved caspase-3 fragment with a slight increase in LDH release was observed in culture supernatants in response to LPS. This increase in caspase-3/7 activity was observed even when LDH release was undetected. These results indicate that caspase-3 is activated by LPS under physiological conditions and suggest that HUVEC escape from cell death by rapidly releasing activated caspase-3 into extracellular space. Failure of this escape mechanism may result in endothelial injury/dysfunction.

Concentration-dependent roles for heparin in modifying liopolysaccharide-induced activation of mononuclear cells in whole blood

In addition to their anticoagulant activity, unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) have important immunomodulatory properties. However, different studies have reported conflicting pro- and anti-inflammatory effects in association with heparin. Moreover, the molecular basis for these heparin effects on inflammation remains unclear. It was the objective of this study to determine how UFH and LMWH regulate lipopolysaccharide (LPS)-induced activation of human mononuclear cells in whole blood, and define the role of lipopolysaccharide-binding protein (LBP) in mediating this effect. Whole blood was pre-treated with UFH or LMWH (0.1-200 IU/ml), prior to stimulation with LPS (10 ng/ml). After six hours, monocyte pro-inflammatory cytokine (interleukin (IL)-1beta, IL-6, IL-8, and TNF-alpha) secretion was determined by plasma ELISA. Parallel experiments using THP-1 cell line and primary monocytes were performed under serum-free conditions, in the presence or absence of LBP (50-100 nM). Under serum-free conditions, heparin demonstrated dose-dependent anti-inflammatory effects, significantly reducing secretion of pro-inflammatory cytokines (IL-1beta, IL-6, IL-8, and TNF-alpha) in response to LPS-stimulation of THP-1 cells and primary monocytes. In contrast, in the presence of LBP, both UFH and LMWH demonstrated dose-dependent pro-inflammatory effects at all heparin concentrations. In ex-vivo whole blood experiments, pro-inflammatory effects (increased IL-1beta and IL-8 following LPS-stimulation) of heparin were also observed, but only at supra-therapeutic doses (10-200 IU/ml). Our data demonstrate that in the absence of LBP, the direct effect of heparin on LPS-stimulated monocytes is anti-inflammatory. However in whole blood, the immunomodulatory effects of heparin are significantly more complex, with either pro- or anti-inflammatory effects dependent upon heparin concentration.

Novel Concentration-Dependent Roles for Heparin in Modifying LPS-Induced Activation of Mononuclear Cells in Whole Blood.

Background: In addition to their etsblished anticoagulant activity, unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are known to possess clinically important immuno-modulatory properties. However different studies have reported conflicting pro- and anti-inflammatory effects in association with heparin. Moreover, the molecular basis for these heparin effects on inflammation remains unclear. In view of the wide and diverse clinical indications for heparin, it is clearly of direct translational relevance to define how UFH and LMWH differentially regulate inflammatory responses to LPS in-vivo.Objectives: To determine how UFH and LMWH regulate lipopolysaccharide (LPS)-induced activation of human mononuclear cells in whole blood, and define the role of lipopolysaccharide binding protein (LBP) in mediating this effect.Methods: Whole blood was pre-treated with UFH or LMWH (0.1–200 IU/ml), prior to stimulation with LPS (10ng/ml). After 6 hours, monocyte pro-inflammatory cytokine (interleukin (IL)-1b, IL-6, IL-8, and TNF-a) secretion was determined by plasma ELISA. Parallel experiments using THP-1 cell line and primary monocytes were performed under serum-free conditions, in the presence or absence of varying doses of recombinant human LBP (range: 50–100nM).Results: Under serum-free conditions, heparin demonstrated dose-dependent anti -inflammatory effects, significantly reducing secretion of pro-inflammatory cytokines (IL-1b, IL-6, IL-8, and TNF-a) in response to LPS-stimulation of THP-1 cells and primary monocytes. In contrast, in the presence of LBP, both UFH and LMWH demonstrated dose-dependent pro-inflammatory effects at all heparin concentrations. In ex-vivo whole blood experiments, pro-inflammatory effects (increased IL-1b and IL-8 following LPS-stimulation) of heparin were also observed, but only at supra-therapeutic doses (10–200IU/ml).Conclusion: In keeping with previous reports, we have demonstrated that both UFH and LMWH can significantly down-regulate cytokine (TNF-a, IL-1b, IL-6 and IL-8) secretion in response to LPS-activation in-vitro. However our novel data demonstrate that the effect of heparin on monocyte activation by LPS is significantly more complex in the setting of whole blood. Firstly, in contrast to the anti-inflammatory effects observed under serum-free conditions, we found that in whole blood, high concentrations of heparin exerted marked pro-inflammatory effects. Secondly we have also demonstrated that the effects of heparin in whole blood are entirely dependent upon heparin concentration and LBP concentration.

High glucose up-regulates lipopolysaccharide-stimulated inflammatory cytokine production via c-jun N-terminal kinase in the monocytic cell line THP-1

Diabetic subjects are susceptible to atherosclerosis. It has been postulated that inflammation plays a crucial role in atherogenesis. Since previous studies suggested persistent low-grade infection by Gram-negative bacteria such as Chlamydia spp. and/or periodontal infection is associated with increased atherogenesis among diabetic subjects, we hypothesized that macrophages under hyperglycemia respond to lipopolysaccharide (LPS) challenge in a more exaggerated manner than under normal glucose conditions. Therefore, we examined cytokine productivity and associated signal transduction molecules in LPS-stimulated the monocytic cell line THP-1, under conditions of hyperglycemia. Differentiated THP-1 cells were cultured under normal and high glucose conditions without fetal bovine serum, and were stimulated with Escherichia coli LPS in the presence of LPS binding protein. Following stimulation, activated signal transduction molecules were detected by protein microarray and confirmed thereafter. Results indicated that c-jun N-terminal kinase (JNK) was highly-phosphorylated at high glucose concentrations, and this was confirmed by Western-immunoblotting. Tumor necrosis factor-alpha and monocyte chemo-attractant protein-1 production were significantly enhanced under these conditions. SP600125, a selective inhibitor of JNK, dose-dependently suppressed the production of these cytokine. Therefore, we suggest that this may be one of the mechanisms by which sub-clinical infection by Gram-negative bacteria promotes atherosclerosis in diabetic subjects.

Biophysical characterization of synthetic rhamnolipids

Synthetic rhamnolipids, derived from a natural diacylated glycolipid, RL-2,2(14), produced by Burkholderia (Pseudomonas) plantarii, were analyzed biophysically. Changes in the chemical structures comprised variations in the length, the stereochemistry and numbers of the lipid chains, numbers of rhamnoses, and the occurrence of charged or neutral groups. As relevant biophysical parameters, the gel (beta) to liquid crystalline (alpha) phase behavior of the acyl chains of the rhamnoses, their three-dimensional supramolecular aggregate structure, and the ability of the compounds to intercalate into phospholipid liposomes in the absence and presence of lipopolysaccharide-binding protein were monitored. Their biological activities were examined as the ability to induce cytokines in human mononuclear cells and to induce chemiluminescence in monocytes. Depending on the particular chemical structures, the physicochemical parameters as well as the biological test systems show large variations. This relates to the acyl chain fluidity, aggregate structure, and intercalation ability, as well as the bioactivity. Most importantly, the data extend our conformational concept of endotoxicity, based on the intercalation of naturally originating amphiphilic virulence factors into membranes from immune cells. This 'endotoxin conformation', produced by amphiphilic molecules with a hydrophilic charged backbone and apolar hydrophobic moiety, and adopting inverted cubic aggregate structures, causes high mechanical stress in target immune cells on integral proteins, eventually leading to cell activation. Furthermore, biologically inactive rhamnolipids with lamellar aggregate structures antagonize the endotoxin-induced activity in a way similar to lipid A-derived antagonists.

Cell Activation of Human Macrophages by Lipoteichoic Acid Is Strongly Attenuated by Lipopolysaccharide-binding Protein

Cell Activation of Human Macrophages by Lipoteichoic Acid Is Strongly Attenuated by Lipopolysaccharide-binding Protein

Non-LPS targets and actions of LPS binding protein (LBP)

LPS binding protein (LBP) was discovered about 20 years ago because of its ability to bind to bacterial lipopolysaccharide (LPS). We have shown that in addition to its complex function of transferring LPS to its cellular receptor into the cell or into lipoproteins, LBP also binds to other bacterial compounds and can modulate their ability to stimulate the host's innate immune system. The majority of compounds found to also interact with LBP are amphiphilic molecules such as glycolipids or lipoproteins. Lipoteichoic acid (LTA) of different Gram-positive bacteria is recognized by LBP and both its complexation with CD14 and biological activity towards immune cells is modulated by LBP. LTA-like glycolipids isolated from spirochetes are recognized by LBP and initiate signaling in the presence of LBP. Lipopeptides corresponding to lipoproteins present in spirochetes, Mycobacterium spp. and Gram-negative bacteria as well as Mycoplasma spp. are also recognized by LBP. Together with the growing number of related proteins of the BPI-PLUNC family, LBP apparently as soluble mediator has the important ability to recognize a variety of bacterial pathogens before cellular contact has been established. The different sources of LBP in tissues such as lung and intestine further support its role as an important defense molecule.

Non-LPS targets and actions of LPS binding protein (LBP)

LPS binding protein (LBP) was discovered about 20 years ago because of its ability to bind to bacterial lipopolysaccharide (LPS). We have shown that in addition to its complex function of transferring LPS to its cellular receptor into the cell or into lipoproteins, LBP also binds to other bacterial compounds and can modulate their ability to stimulate the host's innate immune system. The majority of compounds found to also interact with LBP are amphiphilic molecules such as glycolipids or lipoproteins. Lipoteichoic acid (LTA) of different Gram-positive bacteria is recognized by LBP and both its complexation with CD14 and biological activity towards immune cells is modulated by LBP. LTA-like glycolipids isolated from spirochetes are recognized by LBP and initiate signaling in the presence of LBP. Lipopeptides corresponding to lipoproteins present in spirochetes, Mycobacterium spp. and Gram-negative bacteria as well as Mycoplasma spp. are also recognized by LBP. Together with the growing number of related proteins of the BPI-PLUNC family, LBP apparently as soluble mediator has the important ability to recognize a variety of bacterial pathogens before cellular contact has been established. The different sources of LBP in tissues such as lung and intestine further support its role as an important defense molecule.

Treponema mediumGlycoconjugate Inhibits Activation of Human Gingival Fibroblasts Stimulated with Phenol-Water Extracts of Periodontopathic Bacteria

Oral treponemes are well-known as causative agents of periodontal diseases; however, the details have not been fully clarified. Here, we examined the effects of Treponema medium glycoconjugate on the activation of human gingival fibroblasts using phenol-water extracts from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum subsp. nucleatum, and Actinobacillus actinomycetemcomitans. The phenol-water extracts activated human gingival fibroblasts to mediate IL-8 production, as well as IL-8 mRNA expression, phosphorylation of p38 mitogen-activated protein kinase, and expression of intercellular adhesion molecule-1. T. medium glycoconjugate exhibited no activation of human gingival fibroblasts, while phenol-water extract-induced activation of human gingival fibroblasts was clearly inhibited by T. medium glycoconjugate. Furthermore, binding of biotinylated phenol-water extracts to CD14 in the presence of LPS-binding protein was blocked with T. medium glycoconjugate. These results suggest that T. medium glycoconjugate has an inhibitory effect on host cell activation by periodontopathic bacteria caused by binding to CD14- and LPS-binding protein.

Toll‐like receptor 4 functions intracellularly in human coronary artery endothelial cells: roles of LBP and sCD14 in mediating LPS‐responses

Endothelial cells are activated by microbial agonists through Toll-like receptors (TLRs) to express inflammatory mediators; this is of significance in acute as well as chronic inflammatory states such as septic shock and atherosclerosis, respectively. We investigated mechanisms of lipopolysaccharide (LPS)-induced cell activation in human coronary artery endothelial cells (HCAEC) using a combination of FACS, confocal microscopy, RT-PCR, and functional assays. We found that TLR4, in contrast to TLR2, is not only located intracellularly but also functions intracellularly. That being the case, internalization of LPS is required for activation. We further characterized the HCAEC LPS uptake system and found that HCAEC exhibit an effective LPS uptake only in the presence of LPS binding protein (LBP). In addition to its function as a catalyst for LPS-CD14 complex formation, LBP enables HCAEC activation at low LPS concentrations by facilitating the uptake, and therefore delivery, of LPS-CD14 complexes to intracellular TLR4-MD-2. LBP-dependent uptake involves a scavenger receptor pathway. Our findings may be of pathophysiological relevance in the initial response of the organism to infection. Results further suggest that LBP levels, which vary as LBP is an acute phase reactant, could be relevant to initiating inflammatory responses in the vasculature in response to chronic or recurring low LPS.

Human dendritic cell activation by Neisseria meningitidis: phagocytosis depends on expression of lipooligosaccharide (LOS) by the bacteria and is required for optimal cytokine production.

Group B Neisseria meningitidis is a human pathogen, for which a universally effective vaccine is still not available. Immune responses to bacteria are initiated by dendritic cells (DC), which internalize and process bacterial antigens for presentation to T cells. We show here that optimal IL-12 and TNF-alpha production by human monocyte derived DC in response to killed serogroup B N. meningitidis depends on physical contact and internalization of the bacteria by DC. The majority of DC producing cytokines had internalized N. meningitidis while inhibition of bacterial internalization markedly impaired IL-12 and TNF-alpha, but not IL-6 production. Internalization of N. meningitidis was shown to depend on lipooligosaccharide (LOS) expressed by the bacteria with poor internalization of LOS deficient bacteria compared to wild-type bacteria. Restoration of LOS biosynthesis in a LOS regulatory strain also restored both internalization and cytokine production and was enhanced in the presence of LPS binding protein (LBP). These results suggest that DC phagocytosis depends on expression of LOS within the bacteria and that optimal cytokine production, particularly IL-12, requires internalization of the bacteria. These findings have important implications for designing vaccines that will induce protective immune responses to group B N. meningitidis.

Evidence of a trimolecular complex involving LPS, LPS binding protein and soluble CD14 as an effector of LPS response

The kinetics of the interaction of lipopolysaccharide (LPS), lipopolysaccharide binding protein (LBP) and CD14 was studied using surface plasmon resonance. The association and dissociation rate constants for the binding of LPS and rsCD14 were 2.9×10 4 M −1 s −1 and 0.07 s −1 respectively, yielding a binding constant of 4.2×10 5 M −1. Significantly, the presence of LBP increased not only the association rate but also the association constant for the interaction between LPS and CD14 by three orders of magnitude. Our experimental results suggest that LBP interacts with LPS and CD14 to form a stable trimolecular complex that has significant functional implications as it allows monocytes to detect the presence of LPS at a concentration as low as 10 pg/ml or 2 pM, and to respond by secreting interleukin-6. Thus, LBP is not merely transferring LPS to CD14 but it forms an integral part of the LPS–rLBP–rsCD14 complex.

Lipopolysaccharide-binding protein- and CD14-dependent activation of mitogen-activated protein kinase p38 by lipopolysaccharide in human neutrophils is associated with priming of respiratory burst.

Neutrophil (PMN) functions can be primed for greatly increased oxidative radical release by exposure to certain agents such as lipopolysaccharide (LPS). Although a variety of signaling pathways involving both tyrosine kinases and mitogen-activated protein (MAP) kinases may be operative, the mechanisms of PMN priming are still not understood. We found that PMN priming was not achieved by treatment of cells with a very low concentration (5 ng/ml) of LPS unless additional "helper" factors were present in plasma (5%). Under these conditions, LPS induced tyrosine phosphorylation of a 38-kDa protein, which was coincident with the MAP kinase p38 action in this situation. LPS-mediated activation of p38 in human PMNs was dependent on the presence of LPS binding protein from plasma and CD14 on the surfaces of the cells. Phosphorylation of p38 was highly correlated with LPS priming of a formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN respiratory burst. Treatment of PMN with the p38-specific inhibitor SB203580 significantly attenuated the respiratory burst in cells primed by LPS and stimulated by fMLP. These results suggest that the LPS signaling pathway leading to p38 activation may be an important mechanism in regulation of PMN priming. The mediator(s) linking CD14 to p38 involves proteins that are functionally sensitive to genistein but insensitive to tyrphostin AG126 and to Src- and Syk-family kinase, protein kinase C, and phosphatidylinositol 3-kinase inhibitors. Elucidating this pathway will provide insight into possible regulation of PMN priming by LPS.

Production of the Acute-Phase Protein Lipopolysaccharide-Binding Protein by Respiratory Type II Epithelial Cells

This study demonstrates for the first time that respiratory epithelial cells are able to produce the acute phase protein lipopolysaccharide (LPS)-binding protein (LBP), which is known to play a central role in the defense to bacterial endotoxins (or LPS). Indications for local presence of LBP in human lung was obtained via reverse transcriptase/polymerase chain reaction that showed LBP messenger RNA (mRNA) expression. Therefore, LBP production by the human lung epithelial cell line A549, a human adenocarcinoma with features of type II pneumocytes, was studied. These cells produced LBP in response to interleukin (IL)-1beta, IL-6, and tumor necrosis factor- alpha, a response that was strongly enhanced by dexamethasone. In addition, LBP mRNA was detected in A549 cells, in increasing amounts as a result of stimulation. The pattern of cytokine-induced LBP production in A549 cells was similar to the pattern in the human liver epithelial cell line HuH-7. Moreover, the molecular weight of A549-derived LBP was approximately 60 kD, which is similar to HuH-7-derived LBP. Biologic activity of LBP produced by A549 cells was evaluated on the basis of its ability to interact with LPS. Further indications that type II alveolar epithelial cells are able to produce LBP were obtained from the observations that the murine lung type II epithelial cell line C10 produced murine LBP, and that isolated human primary type II pneumocytes expressed LBP mRNA, which was enhanced after stimulation of cells. The local production of this endotoxin binding protein by lung epithelial cells might contribute to a highly specific response at the site of exposure to bacteria and bacterial endotoxins.

Skin lipopolysaccharide-binding protein and IL-1beta production after thermal injury.

In response to a burn injury, skin can have an inflammatory response characterized by the production of inflammatory cytokines, recruitment of immune cells, containment of invading organisms, and clearance of noxious substances from the wound. Lipopolysaccharide-binding protein (LBP) is a molecule that is capable of coordinating all 4 functions; we previously found evidence that suggested that LBP is produced within surgical wounds. Because of the central role of LBP in the response to bacterial infection, as well as in the high rate of infection after burn injuries, we sought to determine whether a thermal injury could affect wound LBP production and thereby affect host responses against bacterial infection. Rats were given either a burn or a sham burn and were killed 24, 48, and 72 hours after the injuries. Wound specimens were assayed for bacterial counts and for the presence of LBP, messenger (m)RNA, and interleukin (IL)-1beta mRNA. Wound LBP mRNA was significantly upregulated at 24 hours in the group with burn injuries (P < .05; burn vs sham burn); this was followed by decreases at 48 and 72 hours. Immunohistochemistry showed LBP protein in the epidermis of animals with burns. Bacterial counts increased in the group with burn injuries (P < .05; burn vs sham burn) and continued to rise for 72 hours. IL-1beta mRNA levels were elevated at all time points in the group with burn injuries (P < .05). These results suggest an inverse correlation between burn wound LBP expression and bacterial wound counts. This failure to maintain local LBP production after severe thermal injury despite localized inflammation shown by high IL-1beta levels may predispose local wounds to bacterial invasion.

Kupffer cell activation by lipopolysaccharide in rats: role for lipopolysaccharide binding protein and toll-like receptor 4.

Lipopolysaccharide (LPS) binding protein (LBP) is a key serum factor that mediates LPS activation of mononuclear cells. In the presence of LBP, 1/1,000 the concentration of LPS is sufficient to activate peripheral blood monocytes. Previous studies with Kupffer cells have shown a variable effect of serum on LPS activation of these cells and led to the conclusion that, unlike extrahepatic mononuclear cells, Kupffer cells do not respond to LPS in an LBP-dependent fashion. Because there are multiple components in serum other than LBP that might affect LPS activation, these reports with serum are difficult to interpret. To investigate the specific role of LBP in LPS activation of Kupffer cells, we produced a functional recombinant rat LBP using a baculovirus expression system, which we used to selectively examine the role of LBP's on Kupffer-cell function. Isolated Kupffer cells exposed to increasing concentrations of LPS (0, 1, 10 ng/mL) showed a dose-dependent increase in TNF-alpha production, which was augmented and accelerated by the presence of LBP. The effects of LBP on Kupffer cell activation by LPS are dependent on a functional Toll-like receptor 4 (Tlr 4) because Kupffer cells from C3H/HeJ mice failed to respond to LPS in the presence of LBP. LBP plays an important role in mediating Kupffer cell activation by LPS, and these effects are dependent on the presence of functioning Tlr 4.