The role of bactericidal/permeability-increasing protein as a natural inhibitor of bacterial endotoxin.

Systemic release of endotoxin (LPS) after Gram-negative infection initiates a cascade of host cytokines that are thought to be the direct cause of shock, multisystem organ failure, and death. Endogenous LPS-binding proteins may play a role in regulating LPS toxicity in vivo. The human neutrophil granule protein bactericidal/permeability-increasing protein (BPI) shares sequence homology and immunocrossreactivity with an acute phase lipopolysaccharide binding protein (LBP) which has been shown to bind to LPS and accelerate LPS activation of neutrophils and macrophages. Although structurally similar, LBP and BPI are apparently functionally antagonistic. We previously showed that BPI inhibits LPS-mediated neutrophil activation in vitro. Here we demonstrate that BPI binds to LPS near the lipid A domain, and formation of the LPS-BPI complex abrogates detrimental host responses to LPS. For example, BPI blocks LPS-stimulated TNF release in vitro and in vivo, and LPS complexed to BPI is not pyrogenic in rabbits. Results demonstrating that BPI is released by stimulated human neutrophils further support the idea that BPI functions extracellularly in vivo to neutralize endotoxin. Taken together, these data argue that BPI neutralizes the toxic effects of LPS in vivo, and that BPI may represent a new therapeutic approach to the treatment of endotoxic shock.     

Time-resolved fluoroimmunoassay for bactericidal/permeability-increasing protein

Bactericidal/permeability-increasing protein (BPI) is a cationic antimicrobial protein produced by polymorphonuclear leukocytes, that specifically interacts with and kills Gram-negative bacteria. BPl competes with lipopolysaccharide-binding protein (LBP) secreted by liver cells into blood plasma for binding to lipopolysaccharide (LPS) and thus reduces the proinflammatory effects of LPS. We have developed a time-resolved fluoroimmunoassay for BPI and measured the concentration of BPI in human serum and plasma samples. The assay is based on a rabbit antibody against recombinant BPI. This antibody specifically adheres to polymorphonuclear leukocytes in immunostained human tissues. The difference in the serum concentration of BPI between unselected hospitalized patients with and without an infection was statistically significant. The mean concentration of BPI in serum samples was 28.3 mug/l (range 1.64-132, S.D. 26.8, n = 83). In contrast, there was no difference between the two groups in the BPI levels in plasma samples. For all individuals tested, BPI levels were consistently higher in plasma samples compared to the matched serum samples. The mean concentration of BPI in plasma samples was 52.3 mug/l (range 0.9-403, S.D. 60.6, n = 90). There was a positive correlation between the concentration of BPI and the white blood cell count as well as between the BPI concentration and C-reactive protein (CRP) in serum samples. In conclusion, the present study demonstrates that BPI can be quantified reliably by time-resolved fluoroimmunoassay in human serum samples.     

The bactericidal/permeability-increasing protein (BPI) in the innate defence of the lower airways

The human BPI (bactericidal/permeability-increasing protein), stored in primary azurophilic granula of neutrophil granulocytes and produced by mucosal epithelia, has been known for decades to bind LPS (lipopolysaccharide) with very high affinity and to efficiently kill Gram-negative bacteria. Thus BPI potentially represents a central component of the innate immune system to directly combat microbes and modulate subsequent adaptive immune responses. Especially in the lungs, which are frequently exposed to a variety of inhaled pathogens, antimicrobial innate defence molecules such as BPI, are of exceptional relevance. In the present review, we highlight possible functions of BPI during acute pneumonia and CF (cystic fibrosis)-associated chronic infections in the lung.     

A 25-kDa NH2-terminal fragment carries all the antibacterial activities of the human neutrophil 60-kDa bactericidal/permeability-increasing protein

We have isolated, after limited proteolysis of the bactericidal/permeability-increasing protein (BPI) of human neutrophils, a 25-kDa fragment that possesses the bactericidal and envelope-altering activities of the 60-kDa parent protein. On a molar basis, the fragment is as potent as holo-human BPI against rough Escherichia coli, is more potent than holo-BPI against more resistant smooth E. coli, and retains the specificity of BPI toward Gram-negative bacteria. NH2-terminal amino acid sequence analysis shows that the fragment is derived from the NH2 terminus of the BPI molecule. These findings suggest that all of the molecular determinants of the antibacterial properties of BPI reside within the NH2-terminal 25-kDa segment, implying a novel structural/functional organization for a cytotoxic protein.     

Relation between binding and the action of phospholipases A2 on Escherichia coli exposed to the bactericidal/permeability-increasing protein of neutrophils

Exposure of Escherichia coli to the bactericidal/permeability-increasing protein (BPI) of neutrophils renders the bacterial phospholipids susceptible to hydrolysis by only a few of numerous phospholipases A2 tested. To explore further the determinants of hydrolysis we measured the binding of 125I-labeled phospholipase A2 to E. coli in the presence and absence of BPI. Phospholipases A2 from Aqkistrodon piscivorus piscivorus venom and pig pancreas neither degraded nor bound to BPI-treated E. coli. In contrast, the phospholipases A2 from Aqkistrodon halys blomhoffii and Aqkistrodon halys palas venoms actively hydrolyzed the phospholipids of BPI-treated E. coli: they also bound to E. coli in the presence but not in the absence of BPI. Carbamylation of lysines of the A.h. blomhoffii phospholipase A2 progressively reduced binding in parallel with reduced phospholipid hydrolysis. Both binding and hydrolysis increased with increasing BPI dose. However, maximal binding occurred at 25% of the BPI dose that produced optimal hydrolysis. Thus, binding may be necessary but is not sufficient for maximal BPI-mediated phospholipid hydrolysis. Comparison of the NH2-terminal amino sequences of the active and inactive phospholipase A2 suggests that this portion of the phospholipase A2 molecule plays a role in BPI-independent binding and hydrolysis.     

牛杀菌/通透性增加蛋白对脂多糖介导的炎性细胞因子表达的影响

杀菌/通透性增加蛋白(Bactericidal/permeability-increasing protein,BPI)能结合并特异地中和来自革兰氏阴性菌外膜的脂多糖(Lipopolysaccharide,LPS)。为了研究牛源BPI蛋白及其N端结构域在LPS介导的免疫应答中的作用,本文将BPI全长1 449 bp编码区序列(BPI)和其N端714 bp的编码区序列(BPI714)分别导入m HEK293细胞,分析了稳定表达的BPI或BPI714对LPS介导的炎性细胞因子表达的影响。首先将构建的p LEX-BPI/p LEX-BPI714载体分别转染m HEK293细胞,获得稳定表达牛源BPI或BPI714的m HEK293细胞;然后用LPS刺激上述细胞,分别收集刺激前、刺激后1 h、3 h、6 h、12 h、24 h、36 h和48 h的细胞,并同时收集未表达BPI或BPI714的m HEK293细胞在各时间点的样品作为对照;采用定量RT-PCR检测上述细胞中炎性细胞因子IL-8、IL-1β、TNF-α、NF-κB-1、NF-κB-2的相对表达水平,比较LPS刺激前后表达BPI/BPI714和对照细胞中上述基因转录水平的变化规律。研究表明,LPS刺激后,对照细胞中IL-8、IL-1β、TNF-α、NF-κB-2表达水平在不同时间点均显著提高(P0.05),并呈现规律性变化;而稳定表达BPI/BPI714的细胞在同样刺激条件下,IL-8、IL-1β、TNF-α、NF-κB-2基因的转录水平均未发生显著变化(P0.05)。根据我们的实验结果,在m HKE293细胞模型中BPI或BPI714均能显著降低LPS介导的炎性细胞因子表达,抑制LPS介导的免疫应答。这不仅为进一步研究BPI抑菌机制和利用其抑菌功能提供了可靠的实验依据,也为分析抗菌蛋白的抗菌效果提供了一种可靠的实验方法。     

Murine bactericidal/permeability-increasing protein inhibits the endotoxic activity of lipopolysaccharide and gram-negative bacteria

Recognition of LPS by TLR4 initiates inflammatory responses inducing potent antimicrobial immunity. However, uncontrolled inflammatory responses can be detrimental. To prevent the development of septic shock during an infection with Gram-negative bacteria, the immune system has developed mechanisms to neutralize LPS by specialized proteins. In this study, we report the recombinant expression and functional characterization of the mouse homolog of human bactericidal/permeability-increasing protein (BPI). Purified recombinant mouse BPI was able to neutralize LPS-mediated activation of macrophages and to block LPS-dependent maturation of dendritic cells. Recombinant mouse BPI neutralized the capacity of Gram-negative bacteria to activate immune cells, but did not influence the stimulatory properties of Gram-positive bacteria. Unlike human BPI, mouse BPI failed to kill or inhibit the growth of Pseudomonas aeruginosa. Together, these data demonstrate that murine BPI is a potent LPS-neutralizing protein that may limit innate immune responses during Gram-negative infections.     

Differential effects of bacterial lipopolysaccharides upon neutrophil function

Lipopolysaccharide (LPS) is a potent inflammatory agent which augments neutrophil sensitivity to subsequent inflammatory stimuli. In this study, the effects of structurally different LPS types upon neutrophil effector functions were examined. Rough LPS types, which have lost the O-polysaccharide moiety, were found to act more rapidly than smooth LPS types in stimulating neutrophil β₂ integrin activity and fMLP-induced respiratory burst. These findings suggest an involvement of the O-polysaccharide region of LPS in regulating neutrophil responsiveness to different LPS chemotypes with important implications for the mechanisms underlying regulation of the inflammatory response in conditions associated with elevation of LPS in plasma, e.g. septic shock or acute respiratory distress syndrome.     

Autoantibodies against bactericidal/permeability-increasing protein (BPI) in children with acute pneumonia

Antineutrophil cytoplasmic antibodies directed against bactericidal/permeability-increasing protein (BPI), an inhibitor of a lipopolysaccharide of gram-negative bacteria, are a common feature of chronic neutrophilic inflammatory processes such as cystic fibrosis. We investigated whether serum and salivary anti-BPI autoantibodies also appear in the course of acute pneumonia in 24 otherwise healthy children. Nine (38%) and four (17%) patients had detectable serum anti-BPI immunoglobulin G (IgG) (≥4 IU mL⁻¹) and IgA (ratio≥1.2), respectively, on the day of hospital admission (day 0). There was no increase in the rate of occurrence or the concentration of these antibodies in the convalescent sera obtained on day 30. The presence of anti-BPI IgG on admission did not correlate with inflammatory markers (peripheral white blood cell count, C-reactive protein) or temperature on admission. Also, salivary anti-BPI IgA, determined on days 0, 3–5 and 30, did not appear during the course of acute pneumonia. In summary, a substantial proportion of previously healthy children have pre-existing anti-BPI IgG autoantibodies. Acute neutrophilic infection, i.e. pneumonia, however, neither triggered the appearance of new antibodies nor boosted the concentrations of pre-existing ones. Thus, in typical acute pneumonia in children, autoantibodies directed against BPI may not have clinical significance.     

Deficient expression of bactericidal/permeability-increasing protein in immunocompromised hosts: translational potential of replacement therapy

BPI (bactericidal/permeability-increasing protein) is a 55?kDa anti-infective molecule expressed in neutrophil and eosinophil granules and on some epithelial cells. BPI's high affinity for the lipid A region of endotoxin targets its opsonizing, microbicidal and endotoxin-neutralizing activities towards Gram-negative bacteria. Several immunocompromised patient populations demonstrate BPI deficiency, including newborns, those with anti-neutrophil cytoplasmic antibodies (as in cystic fibrosis and HIV infection) and those exposed to radiochemotherapy. BPI may be replenished by administering agents that induce its expression or by administration of recombinant BPI congeners, potentially shielding BPI-deficient individuals against Gram-negative bacterial infection, endotoxemia and its toxic sequelae.     

Molecular aspects of the damaging action of bacterial lipopolysaccharides

The review deals with the molecular aspects of the interaction of bacterial lipopolysaccharides (LPS, with the macroorganism: the sources of LPS in the organism, its detoxication, regulation disturbances in pathology (septic shock). Special attention is drawn to the complex functioning of cytokines in the development of the inflammatory process both at the initial stage, characterized by the initiation of the cytokine cascade and at the final stage resulting in convalescence or the development of pathology. The most promising trends of research in this field are discussed.     

Immunochemical evidence that cholesteryl ester transfer protein and bactericidal/permeability-increasing protein share a similar tertiary structure

Cholesteryl ester transfer protein (CETP) plays an important role in plasma lipoprotein metabolism through its ability to transfer cholesteryl ester, triglyceride, and phospholipid between lipoproteins. CETP is a member of a gene family that also includes bactericidal/permeability-increasing protein (BPI). The crystal structure of BPI shows it to be composed of two domains that share a similar structural fold that includes an apolar ligand-binding pocket. As structurally important residues are conserved between BPI and CETP, it is thought that CETP and BPI may have a similar overall conformation. We have previously proposed a model of CETP structure based on the binding characteristics of anti-CETP monoclonal antibodies (mAbs). We now present a refined epitope map of CETP that has been adapted to a structural model of CETP that uses the atomic coordinates of BPI. Four epitopes composed of CETP residues 215-219, 219-223, 223-227, and 444-450, respectively, are predicted to be situated on the external surface of the central beta-sheet and a fifth epitope (residues 225-258) on an extended linker that connects the two domains of the molecule. Three other epitopes, residues 317-331, 360-366, and 393-410, would form part of the putative carboxy-terminal beta-barrel. The ability of the corresponding mAbs to compete for binding to CETP is consistent with the proximity of the respective epitopes in the model. These results thus provide experimental evidence that is consistent with CETP and BPI having similar surface topologies.     

Events at the host-microbial interface of the gastrointestinal tract. I. Adaptation to a microbial world: role of epithelial bactericidal/permeability-increasing protein

Epithelial cells of many mucosal organs have adapted to coexist with microbes and microbial products. In general, most studies suggest that epithelial cells benefit from interactions with commensal microorganisms present at the lumenal surface. However, potentially injurious molecules found in this microenvironment also have the capacity to elicit local inflammatory responses and even systemic disease. In this environment, the epithelium has evolved effective mechanisms to cope with microbial products and to provide appropriate responses to potential pathogens. Although our understanding of these mechanisms is clearly in its infancy, a number of recent findings provide insight into phenotypic characteristics that allow for this discrimination. Here, we briefly review some of these mechanisms, with particular attention to epithelial expression of the anti-infective molecule bactericidal/permeability-increasing protein.     

Role of charge properties of bacterial envelope in bactericidal action of human group IIA phospholipase A2 against Staphylococcus aureus

Mammalian Group IIA phospholipases A(2) (PLA(2)) potently kill Staphylococcus aureus. Highly cationic properties of these PLA(2) are important for Ca(2+)-independent binding and cell wall penetration, prerequisites for Ca(2+)-dependent degradation of membrane phospholipids and bacterial killing. To further delineate charge properties of the bacterial envelope important in Group IIA PLA(2) action against S. aureus, we examined the effects of mutations that prevent specific modifications of cell wall (dltA) and cell membrane (mprF) polyanions. In comparison to the parent strain, isogenic dltA(-) bacteria are approximately 30-100x more sensitive to PLA(2), whereas mprF(-) bacteria are <3-fold more sensitive. Differences in PLA(2) sensitivity of intact bacteria reflect differences in cell wall, not cell membrane, properties since protoplasts from all three strains are equally sensitive to PLA(2). A diminished positive charge in PLA(2) reduces PLA(2) binding and antibacterial activity. In contrast, diminished cell wall negative charge by substitution of (lipo)teichoic acids with d-alanine reduces antibacterial activity of bound PLA(2), but not initial PLA(2) binding. Therefore, the potent antistaphylococcal activity of Group IIA PLA(2) depends on cationic properties of the enzyme that promote binding to the cell wall, and polyanionic properties of cell wall (lipo)teichoic acids that promote attack of membrane phospholipids by bound PLA(2).     

Cloning and characterization of the homolog of mammalian lipopolysaccharide-binding protein and bactericidal permeability-increasing protein in rainbow trout Oncorhynchus mykiss

We cloned two cDNAs denoted as RT-LBP/BPI-1 and RT-LBP/BPI-2, respectively, which were derived from the mRNA of head kidney from rainbow trout. They showed structural homology with LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) in mammals. The full-length cDNA of RT-LBP/BPI-1 and RT-LBP/BPI-2 is 1666 and 1741 bp, respectively. Both cDNAs encoded 473 aa residues, including the amino acids conserved in mammalian LBP and BPI proteins that were assumed to be involved in LPS binding. The overall coding sequence of RT-LBP/BPI-1 has 33% amino acid homology to human LBP and 34% to human BPI, and RT-LBP/BPI-2 has 32% amino acid homology to human LBP and 33% to human BPI. Three-dimensional structure analysis by three-dimensional/one-dimensional (3D-1D) methods also demonstrated that RT-LBP/BPI-1 and RT-LBP/BPI-2 proteins showed significant similarity to human BPI, having a boomerang shape with N-terminal and C-terminal barrels. Phylogenetic analysis showed that the LBP and BPI genes seemed to be established after the divergence of mammals from teleosts. These results suggested that RT-LBP/BPI-1 and RT-LBP/BPI-2 may be a putative ortholog for mammalian LBP and/or BPI genes. This is the first study to identify the LBP family genes from nonmammalian vertebrates.