ABSTRACT
Implant-associated infections (IAI) are often recurrent, expensive to treat, and associated with high rates of morbidity, if not mortality. We biofunctionalized the surface of additively manufactured volume-porous titanium implants using electrophoretic deposition (EPD) as a way to eliminate the peri-operative bacterial load and prevent IAI. Chitosan-based (Ch) coatings were incorporated with different concentrations of silver (Ag) nanoparticles or vancomycin. A full-scale in vitro and in vivo study was then performed to evaluate the antibacterial, immunogenic, and osteogenic activity of the developed implants. In vitro, Châ¯+â¯vancomycin or Châ¯+â¯Ag coatings completely eliminated, or reduced the number of planktonic and adherent Staphylococcus aureus by up to 4 orders of magnitude, respectively. In an in vivo tibia intramedullary implant model, Châ¯+â¯Ag coatings caused no adverse immune or bone response under aseptic conditions. Following Staphylococcus aureus inoculation, Châ¯+â¯vancomycin coatings reduced the implant infection rate as compared to chitosan-only coatings. Châ¯+â¯Ag implants did not demonstrate antibacterial effects in vivo and even aggravated infection-mediated bone remodeling including increased osteoclast formation and inflammation-induced new bone formation. As an explanation for the poor antibacterial activity of Châ¯+â¯Ag implants, it was found that antibacterial Ag concentrations were cytotoxic for neutrophils, and that non-toxic Ag concentrations diminished their phagocytic activity. This study shows the potential of EPD coating to biofunctionalize porous titanium implants with different antibacterial agents. Using this method, Ag-based coatings seem inferior to antibiotic coatings, as their adverse effects on the normal immune response could cancel the direct antibacterial effects of Ag nanoparticles. STATEMENT OF SIGNIFICANCE: Implant-associated infections (IAI) are a clinical, societal, and economical burden. Surface biofunctionalization approaches can render complex metal implants with strong local antibacterial action. The antibacterial effects of inorganic materials such as silver nanoparticles (Ag NPs) are often highlighted under very confined conditions in vitro. As a novelty, this study also reports the antibacterial, immunogenic, and osteogenic activity of Ag NP-coated additively-manufactured titanium in vivo. Importantly, it was found that the developed coatings could impair the normal function of neutrophils, the most important phagocytic cells protecting us from IAI. Not surprisingly, the Ag NP-based coatings were outperformed by an antibiotic-based coating. This emphasizes the importance of also targeting implant immune-modulatory functions in future coating strategies against IAI.
Subject(s)
Anti-Bacterial Agents , Coated Materials, Biocompatible , Prostheses and Implants , Silver , Staphylococcus aureus/growth & development , Titanium , Vancomycin , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Coated Materials, Biocompatible/chemistry , Coated Materials, Biocompatible/pharmacology , Male , Materials Testing , Osteogenesis/drug effects , Rats , Rats, Sprague-Dawley , Silver/chemistry , Silver/pharmacology , Titanium/chemistry , Titanium/pharmacology , Vancomycin/chemistry , Vancomycin/pharmacologyABSTRACT
Neutrophils store large quantities of neutrophil serine proteases (NSPs) that contribute, via multiple mechanisms, to antibacterial immune defences. Even though neutrophils are indispensable in fighting Staphylococcus aureus infections, the importance of NSPs in anti-staphylococcal defence is yet unknown. However, the fact that S. aureus produces three highly specific inhibitors for NSPs [the extracellular adherence proteins (EAPs) Eap, EapH1 and EapH2], suggests that these proteases are important for host defences against this bacterium. In this study we demonstrate that NSPs can inactivate secreted virulence factors of S. aureus and that EAP proteins function to prevent this degradation. Specifically, we find that a large group of S. aureus immune-evasion proteins is vulnerable to proteolytic inactivation by NSPs. In most cases, NSP cleavage leads to functional inactivation of virulence proteins. Interestingly, proteins with similar immune-escape functions appeared to have differential cleavage sensitivity towards NSPs. Using targeted mutagenesis and complementation analyses in S. aureus, we demonstrate that all EAP proteins can protect other virulence factors from NSP degradation in complex bacterial supernatants. These findings show that NSPs inactivate S. aureus virulence factors. Moreover, the protection by EAP proteins can explain why this antibacterial function of NSPs was masked in previous studies. Furthermore, our results indicate that therapeutic inactivation of EAP proteins can help to restore the natural host immune defences against S. aureus.
Subject(s)
Bacterial Proteins/metabolism , Immune Evasion , Neutrophils/enzymology , Serine Proteases/metabolism , Serine Proteinase Inhibitors/metabolism , Staphylococcus aureus/immunology , Virulence Factors/metabolism , Cells, Cultured , Humans , Neutrophils/immunology , Proteolysis , Staphylococcus aureus/pathogenicity , Staphylococcus aureus/physiologyABSTRACT
UNLABELLED: Although Staphylococcus aureus is best known for infecting humans, bovine-specific strains are a major cause of mastitis in dairy cattle. The bicomponent leukocidin LukMF', exclusively harbored by S. aureus of ruminant origin, is a virulence factor associated with bovine infections. In this study, the molecular basis of the host specificity of LukMF' is elucidated by identification of chemokine receptor CCR1 as its target. Bovine neutrophils, the major effector cells in the defense against staphylococci, express significant cell surface levels of CCR1, whereas human neutrophils do not. This causes the particular susceptibility of bovine neutrophils to pore formation induced by LukMF'. Bovine S. aureus strains produce high levels of LukMF' in vitro. In culture supernatant of the mastitis field isolate S1444, LukMF' was the most important cytotoxic agent for bovine neutrophils. In a fibrin gel matrix, the effects of the in situ secreted toxins on neutrophils migrating toward S. aureus were visualized. Under these physiological ex vivo conditions, bovine S. aureus S1444 efficiently killed approaching neutrophils at a distance through secretion of LukMF'. Altogether, our findings illustrate the coevolution of pathogen and host, provide new targets for therapeutic and vaccine approaches to treat staphylococcal diseases in the cow, and emphasize the importance of staphylococcal toxins in general. IMPORTANCE: This study explains the mechanism of action of LukMF', a bicomponent toxin found in bovine lineages of S. aureus that is associated with mastitis in cattle. At a molecular level, we describe how LukMF' can specifically kill bovine neutrophils. Here, we demonstrate the contribution of toxins in the determination of host specificity and contribute to the understanding of mechanisms of coevolution of pathogen and host. Our study provides new targets that can be used in therapeutic and vaccine approaches to treat staphylococcal diseases in the cow. We also demonstrate the importance of toxins in specific elimination of immune cells, which has broader implications, especially in human infections.
Subject(s)
Bacterial Proteins/metabolism , Leukocidins/metabolism , Mastitis, Bovine/microbiology , Neutrophils/drug effects , Neutrophils/physiology , Receptors, CCR1/metabolism , Staphylococcus aureus/pathogenicity , Animals , Cattle , Cell Survival/drug effects , Staphylococcus aureus/metabolismABSTRACT
We investigated whether pro-inflammatory aspects of the postprandial phase can be modulated by rosuvastatin in premature coronary artery disease (CAD) patients. Herefore standardized 8 h oral fat loading tests were performed off-treatment and after rosuvastatin 40 mg/d in 20 male CAD patients (50 +/- 4 years). The expression of leukocyte activation markers CD11a, CD11b, CD62L and CD66b was studied using flowcytometry. Migration of isolated neutrophils towards chemoattractants was determined in a fluorescence-based assay. Rosuvastatin did not affect baseline leukocyte counts nor the postprandial neutrophil increment (maximum mean increase +10% pre- and +14% post-treatment, P < 0.01 for each). Rosuvastatin reduced baseline platelets (from 266 +/- 78 to 225 +/- 74 x 10(9) cells/L, P < 0.001) and blunted the postprandial platelet count change (maximum mean increase +6%, P = 0.01, and 0%, respectively). The baseline expression of CD11a, CD11b and CD62L increased on most types of leukocytes by rosuvastatin, whereas the postprandial responses were unaffected. Pretreatment, postprandial neutrophil migration increased dose-dependently, but there were no postprandial changes after rosuvastatin. The latter effect was unrelated to changes in lipoprotein concentrations. In conclusion, in CAD patients postprandial pro-inflammatory and pro-coagulant changes can be modified by rosuvastatin. These apparently lipid-lowering independent effects may render protection against atherosclerosis.
Subject(s)
Coronary Artery Disease/blood , Dietary Fats/administration & dosage , Fluorobenzenes/therapeutic use , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Hyperlipidemias/drug therapy , Leukocytes/drug effects , Postprandial Period , Pyrimidines/therapeutic use , Sulfonamides/therapeutic use , Adult , Antigens, CD/analysis , CD11a Antigen/analysis , CD11b Antigen/analysis , Cell Adhesion Molecules/analysis , Chemotaxis, Leukocyte , Coronary Artery Disease/complications , Erythrocyte Count , GPI-Linked Proteins , Humans , Hyperlipidemias/blood , Hyperlipidemias/complications , Interleukin-8/blood , L-Selectin/analysis , Leukocytes/immunology , Male , Middle Aged , Neutrophils/physiology , Oxidative Stress , Platelet Count , Rosuvastatin Calcium , Vascular Cell Adhesion Molecule-1/bloodABSTRACT
Neutrophils can be primed by bacterial lipopolysaccharide (LPS) for an enhanced oxidative burst, which is a key element in the pathogenesis of Gram-negative sepsis. Some serum proteins (e.g. lipopolysaccharide-binding protein) avidly bind LPS and markedly enhance receptor binding and cellular activation while other serum factors (lipoproteins, bactericidal/permeability-increasing protein) neutralize LPS and prevent neutrophil activation. In this paper we examined the kinetics of this priming reaction in whole blood. To study the balance between neutrophil activation and LPS neutralization a sensitive chemiluminescence assay was used in a whole blood system. LPS was able to prime neutrophils for enhanced oxidative burst in whole blood with an optimum incubation time of 25 min. However, LPS was neutralized very rapidly with a t(1/2) of 10 min. After 20 min a second priming factor was already generated, which was shown to be monocyte-derived tumour necrosis factor (TNF).
Subject(s)
Lipopolysaccharides/immunology , Neutrophils/immunology , Tumor Necrosis Factor-alpha/immunology , Anti-Bacterial Agents/immunology , Antibodies, Monoclonal/immunology , Humans , Lipopolysaccharide Receptors/immunology , Luminescent Measurements/methods , Monocytes/immunology , Neutrophil Activation/immunology , Polymyxin B/immunology , Respiratory Burst/immunology , Salmonella typhimuriumABSTRACT
Recently we described a novel bacteriophage-encoded pathogenicity island in Staphylococcus aureus that harbors a number of virulence factors that are all involved in the evasion of innate immunity. Here we describe a mechanism by which staphylokinase (SAK), frequently present on this pathogenicity island, interferes with innate immune defenses: SAK is anti-opsonic. By activating human plasminogen (PLG) into plasmin (PL) at the bacterial surface, it creates bacterium-bound serine protease activity that leads to degradation of two major opsonins: human immunoglobulin G (IgG) and human C3b. Incubation of opsonized bacteria with PLG and SAK resulted in removal of anti-staphylococcal IgGs and C3b from the bacterial surface. In phagocytosis assays this proved to be a very efficient mechanism to reduce the opsonic activity of human IgG and serum. The fact that SAK activates human PLG at the bacterial surface and removes IgG as well as C3b makes this protein a unique anti-opsonic molecule.
Subject(s)
Metalloendopeptidases/physiology , Opsonin Proteins/metabolism , Staphylococcus aureus/enzymology , Complement C3b/immunology , Complement C3b/metabolism , Enzyme Activation , Fibrinolysin , Humans , Immunoglobulin G/blood , Immunoglobulin G/metabolism , Opsonin Proteins/blood , Plasminogen/metabolism , Protein Binding , Staphylococcus aureus/pathogenicitySubject(s)
Lipoproteins, HDL/blood , Lipoproteins, HDL/immunology , Sepsis/blood , Sepsis/immunology , Humans , Inflammation , Lipopolysaccharides/adverse effects , Lipopolysaccharides/immunology , Lipoproteins, HDL/chemistry , Lipoproteins, HDL/therapeutic use , Monocytes/immunology , Sepsis/microbiologyABSTRACT
BACKGROUND: CD14, the major lipopolysaccharide (LPS)-binding protein of myeloid cells, is found as a soluble molecule in human serum. Recent data describe the presence of elevated soluble CD14 (sCD14) concentration in various disorders, confirming disease activity. A novel, easy, and rapid flow cytometric assay was developed to measure sCD14 levels in serum. METHODS: The assay is based on the competition between membrane-expressed CD14 of isolated monocytes from healthy volunteers and sCD14 in the sample sera for binding to anti-CD14 monoclonal antibodies (mAb; 26ic or 60bca). The amount of cell-associated mAb is determined with a fluorescein isothiocyanate (FITC)-labeled anti-mouse conjugate and flow cytometry. The fluorescence signal is inversely proportional with the amount of serum sCD14. Using dilutions of a standard serum, the concentration of sCD14 in the samples is calculated and compared with results obtained by a commercial sCD14 enzyme-linked immunosorbent assay (ELISA). RESULTS: After optimization, the assay showed log-log linearity of 122.1-984.7 ng/ml sCD14 using mAb 26ic and 29.5-246.2 ng/ml sCD14 using mAb 60bca. It revealed similar results as the ELISA (mAb 26ic: r = 0.88, mAb 60bca: r = 0.92) and provided significantly elevated sCD14 levels in systemic lupus erythematosus patients compared with controls (26ic: 2,213 versus 1,676 ng/ml, P < 0.002; 60bca: 2,625 versus 1,907 ng/ml, P < 0.0002). Receiver operating characteristic curve analysis suggested a reasonable diagnostic efficacy of sCD14 quantification in this autoimmune disease. CONCLUSIONS: The method is easy, rapid, sensitive, and can be used in the follow-up of patients suffering from sepsis or chronic inflammatory disorders.
Subject(s)
Flow Cytometry/methods , Lipopolysaccharide Receptors/blood , Adult , Cell Separation , Enzyme-Linked Immunosorbent Assay , Female , Humans , Lupus Erythematosus, Systemic/immunology , Male , Middle Aged , Monocytes/chemistry , ROC Curve , Sensitivity and SpecificityABSTRACT
Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance mechanism. We describe a novel staphylococcal gene, mprF, which determines resistance to several host defense peptides such as defensins and protegrins. An mprF mutant strain was killed considerably faster by human neutrophils and exhibited attenuated virulence in mice, indicating a key role for defensin resistance in the pathogenicity of S. aureus. Analysis of membrane lipids demonstrated that the mprF mutant no longer modifies phosphatidylglycerol with l-lysine. As this unusual modification leads to a reduced negative charge of the membrane surface, MprF-mediated peptide resistance is most likely based on repulsion of the cationic peptides. Accordingly, inactivation of mprF led to increased binding of antimicrobial peptides by the bacteria. MprF has no similarity with genes of known function, but related genes were identified in the genomes of several pathogens including Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Enterococcus faecalis. MprF thus constitutes a novel virulence factor, which may be of general relevance for bacterial pathogens and represents a new target for attacking multidrug resistant bacteria.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Defensins/pharmacology , Lysine/metabolism , Neutrophils/immunology , Phosphatidylglycerols/metabolism , Staphylococcus aureus/metabolism , Amino Acid Sequence , Aminoacyltransferases , Animals , Bacterial Proteins/genetics , Bacterial Proteins/physiology , Base Sequence , Cell Membrane/metabolism , DNA, Bacterial , Drug Resistance, Microbial , Esterification , Genes, Bacterial , Humans , Molecular Sequence Data , Peptides/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Staphylococcus aureus/pathogenicity , Swine , Virulence , alpha-Defensins/pharmacologySubject(s)
Bacteriuria/immunology , Cytokines/blood , Cytokines/urine , Diabetes Mellitus/immunology , Lymphocytes/immunology , Adolescent , Adult , Aged , Cells, Cultured , Cytokines/biosynthesis , Diabetes Mellitus/blood , Diabetes Mellitus/urine , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/urine , Diabetes Mellitus, Type 2/immunology , Diabetes Mellitus, Type 2/urine , Female , Glycosuria , Humans , Interleukin-6/urine , Interleukin-8/urine , Leukocytes , Lipopolysaccharides/pharmacology , Lymphocytes/drug effects , Middle Aged , Reference Values , Salmonella , Tumor Necrosis Factor-alpha/urine , Urine/cytologyABSTRACT
BACKGROUND: CD14 is considered to be the major endotoxin (lipopolysaccharide [LPS]) binding molecule on human monocytes. It initiates cellular response, but its role in the clearance of LPS is not well understood. Under conditions that ensure totally CD14-dependent LPS binding on human monocytes, the internalization mechanisms of LPS and CD14 were studied. METHODS: The uptake and intracellular distribution of fluorescein isothiocyanate (FITC)-LPS and CD14 was determined by flow cytometry, trypan blue quenching, and confocal fluorescence microscopy. Incubation of surface-biotinylated cells with LPS at 37 degrees C or 4 degrees C and subsequent subfractionation was used to further characterize CD14 internalization. The amount of the intracellular CD14 was estimated by CD14 enzyme-linked immunosorbent assay (ELISA). RESULTS: The internalization rate of 10 ng/ml FITC-LPS with 1% human serum was 1% of bound endotoxin per minute, whereas CD14 expression did not decrease at the same time surface. We proved the presence of an intracellular CD14 pool (2.68 x 10(6) molecules per unstimulated monocyte) and could show that internalized FITC-LPS molecules can be found in different intracellular compartments than CD14. Subfractionation of LPS-treated biotinylated monocytes showed no change in biotinylated CD14 in the membrane fraction independently of the incubation temperature (37 degrees C or at 4 degrees C) used, indicating that these CD14 molecules were not taken up by an active process. CONCLUSIONS: These data indicate the presence of a large intracellular CD14 pool in monocytes with a yet unknown function, and suggest that LPS and CD14 molecules can be internalized independently after association on the cell surface.
Subject(s)
Endocytosis , Lipopolysaccharide Receptors/metabolism , Lipopolysaccharides/metabolism , Monocytes/metabolism , Biotinylation , Cell Fractionation , Cells, Cultured , Down-Regulation , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Fluorescein-5-isothiocyanate , Humans , Lipopolysaccharide Receptors/analysis , Lipopolysaccharides/analysis , Microscopy, Fluorescence , Monocytes/cytology , Monocytes/immunology , Trypan Blue/metabolismABSTRACT
Lipopolysaccharide (LPS)-binding components in serum play an important role in modifying LPS toxicity. We analyzed the binding characteristics of LPS in the presence of serum using gel filtration of FITC-labeled LPS (FITC-LPS) with on line detection of optical density and fluorescence. FITC-LPS separately behaves as an aggregate resulting in a low, dequenched, fluorescence. Binding of single LPS molecules, segregated from the aggregate, to serum components results in an increase in the fluorescence due to dequenching, and a comigration of fluorescence and optical density signals using gel filtration. This method, in combination with the use of specific antibodies inducing additional shifts, demonstrated that in serum high-density lipoproteins (HDL), albumin and low-density lipoproteins (LDL) were able to monomerize LPS. An ELISA on collected fractions of the gel filtration revealed binding of the recently identified LPS-binding protein, serum amyloid P component (SAP), to the high molecular weight LPS aggregate. In serum, binding of soluble CD14 (sCD14) and LPS-binding protein (LBP) to LPS could not be detected. However, this was probably due to an overshadowing effect of albumin, as an extra addition of recombinant sCD14 to serum clearly monomerized FITC-LPS. Biosensor technology revealed that, of all LPS-binding components tested, only SAP clearly bound to the LPS-coated sensor chip. These results show that gel filtration of FITC-LPS is a quick and reliable method to study the binding characteristics of LPS-binding components.
Subject(s)
Acute-Phase Proteins , Blood Proteins/metabolism , Fluorescein-5-isothiocyanate , Lipopolysaccharides/metabolism , Membrane Glycoproteins , Carrier Proteins/analysis , Chromatography, Gel/methods , Chromatography, Gel/standards , Fluorescence , Fluorescent Dyes , Humans , Lipopolysaccharide Receptors/analysis , Lipoproteins, HDL/metabolism , Serum Albumin/metabolism , Serum Amyloid P-Component/metabolismABSTRACT
In a previous study, we showed that Staphylococcus aureus supernate (SaS) is a potent agonist for both neutrophils and mononuclear cells. To further investigate the immunomodulating effects of SaS, the effect on different neutrophil receptors was studied. Expression of various neutrophil receptors, before and after treatment with SaS, was quantified by flow cytometry. We found that SaS treatment of neutrophils resulted in a specific and total downregulation of the C5a and the fMLP receptor, both serpentine receptors, while other receptors were totally unaffected. Since these two receptors are both involved in chemotaxis, we tested the effect of SaS in calcium flux and chemotaxis assays. We showed that preincubation with SaS abrogated the rise in intracellular calcium concentration upon triggering with fMLP and C5a. We also showed that SaS is a potent inhibitor of neutrophil chemotaxis towards fMLP and C5a, but does not interfere with chemotaxis towards interleukin-8. These findings indicate that S. aureus produces a virulence factor extracellularly, which impairs chemotaxis towards the infected site.
Subject(s)
Bacterial Proteins/immunology , Neutrophils/drug effects , Receptors, Chemokine/metabolism , Staphylococcus aureus/immunology , Bacteremia/microbiology , Bacterial Proteins/metabolism , Calcium/metabolism , Chemotaxis, Leukocyte , Culture Media , Down-Regulation , Humans , Neutrophils/metabolism , Staphylococcal Infections/microbiology , Staphylococcus aureus/growth & development , Staphylococcus aureus/metabolismABSTRACT
Lipopolysaccharide (LPS) is an amphipathic macromolecule that is highly aggregated in aqueous preparations. LPS-binding protein (LBP) catalyzes the transfer of single LPS molecules, segregated from an LPS aggregate, to high-density lipoproteins (HDL), which results in the neutralization of LPS. When fluorescein isothiocyanate-labeled LPS (FITC-LPS) is used, this transfer of LPS monomers to HDL can be measured as an increase in fluorescence due to dequenching of FITC-LPS. Recently, serum amyloid P component (SAP) was shown to neutralize LPS in vitro, although only in the presence of low concentrations of LBP. In this study, we show that SAP prevented HDL-mediated dequenching of FITC-LPS, even in the presence of high concentrations of LBP. Human bactericidal/permeability-increasing protein (BPI), a very potent LPS-binding and -neutralizing protein, also prevented HDL-mediated dequenching of FITC-LPS. Furthermore, SAP inhibited HDL-mediated neutralization of both rough and smooth LPS in a chemiluminescence assay quantifying the LPS-induced priming of neutrophils in human blood. SAP bound both isolated HDL and HDL in serum. Using HDL-coated magnetic beads prebound with SAP, we demonstrated that HDL-bound SAP prevented the binding of LPS to HDL. We suggest that SAP, by preventing LPS binding to HDL, plays a regulatory role, balancing the amount of LPS that, via HDL, is directed to the adrenal glands.
Subject(s)
Lipopolysaccharides/metabolism , Lipoproteins, HDL/metabolism , Serum Amyloid P-Component/pharmacology , Apolipoprotein A-I/metabolism , Fluorescein-5-isothiocyanate , Humans , Luminescent Measurements , Serum Amyloid P-Component/metabolismABSTRACT
Although serum amyloid P component (SAP) is known to bind many ligands, its biological function is not yet clear. Recently, it was demonstrated that SAP binds to lipopolysaccharide (LPS). In the present study, SAP was shown to bind to gram-negative bacteria expressing short types of LPS or lipo-oligosaccharide (LOS), such as Salmonella enterica serovar Copenhagen Re and Escherichia coli J5, and also to clinical isolates of Haemophilus influenzae. It was hypothesized that SAP binds to the bacteria via the lipid A part of LPS or LOS, since the htrB mutant of the nontypeable H. influenzae strain NTHi 2019-B29-3, which expresses a nonacetylated lipid A, did not bind SAP. This was in contrast to the parental strain NTHi 2019. The binding of SAP resulted in a clear inhibition of the deposition of complement component C3 on the bacteria. SAP inhibited only the activation of the classical complement pathway; the alternative route remained unaffected. In the classical route, SAP prevented the deposition of the first complement component, Clq, probably by interfering with the binding of Clq to LPS. Since antibody-mediated Clq activation was not inhibited by SAP, SAP seems to inhibit only the LPS-induced classical complement pathway activation. The SAP-induced inhibition of C3 deposition strongly diminished the complement-mediated lysis as well as the phagocytosis of the bacteria. The binding of SAP to gram-negative bacteria, therefore, might influence the pathophysiology of an infection with such bacteria.
Subject(s)
Complement Activation , Gram-Negative Bacteria/immunology , Lipopolysaccharides/immunology , Serum Amyloid P-Component/immunology , Serum Amyloid P-Component/physiology , Animals , Antibodies, Monoclonal/immunology , Dose-Response Relationship, Drug , Escherichia coli/immunology , Gram-Negative Bacteria/growth & development , Humans , Hybridomas/immunology , Mice , Neutrophils/microbiology , Phagocytosis , Salmonella typhimurium/immunology , Serum Amyloid P-Component/pharmacologyABSTRACT
Lipopolysaccharide (LPS) is the major mediator of gram-negative septic shock. Molecules that bind LPS and neutralize its toxic effects could have important clinical applications. We showed that serum amyloid P component (SAP) neutralizes LPS. A SAP-derived peptide, consisting of amino acids 27 to 39, inhibited LPS-mediated effects in the presence of human blood. In this study, we used a pepscan of overlapping 15-mer peptides and distinguished two additional LPS-binding regions within the SAP molecule, identified in the regions spanning amino acids 61 to 75 and 186 to 200. The corresponding SAP-derived peptides, pep61-75 and pep186-200, inhibited the binding of fluorescein isothiocyanate-labeled LPS to monocytes as efficiently as a bactericidal/permeability-increasing protein (BPI)-derived 15-mer peptide comprising amino acids 85 to 99. The same SAP-derived peptides very potently inhibited LPS-induced priming of phagocytes in human blood. Also, SAP-derived pep186-200 caused a prolonged survival of actinomycin D-sensitized mice treated with LPS to induce septic shock, indicating a potential use of this peptide in the defense against serious gram-negative sepsis in humans.
Subject(s)
Lipopolysaccharides/immunology , Peptides/immunology , Serum Amyloid P-Component/immunology , Amino Acid Sequence , Animals , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Heparin/metabolism , Humans , Lipopolysaccharides/toxicity , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Monocytes/metabolism , Neutralization Tests , Peptides/chemical synthesis , Respiratory Burst , Serum Amyloid P-Component/chemical synthesis , Shock, Septic/prevention & controlABSTRACT
Interaction of LPS with monocytes and neutrophils is known to occur via CD14 and is strongly enhanced by LPS-binding protein (LBP). Integrins as well as CD14 play a role in the interaction of erythrocytes (E) coated with LPS or whole Gram-negative bacteria with phagocytes. We reasoned that the density of LPS on a particle is an important determinant in these interactions. Therefore, E were coated with different concentrations of LPS (ELPS). The binding of these ELPS to neutrophils was evaluated by flow cytometry. Simultaneously, we measured fMLP receptor expression to evaluate neutrophil activation. ELPS only bound to neutrophils in the presence of LBP. Blocking CD14 inhibited both activation and binding, whereas blocking complement (C) receptor 3 (CR3) inhibited binding but not activation. TNF activation restored ELPS binding in CD14-blocked cells but not in cells in which CR3 was blocked. Salmonella minnesota did bind to neutrophils independent of CR3 or CD14. The addition of LBP enhanced binding twofold, and this surplus was dependent upon CD14 but not on CR3. We conclude that ELPS interact with neutrophils via CD14, initially giving rise to cell activation; subsequently, binding is solely mediated by activated CR3.
Subject(s)
Acute-Phase Proteins , CD18 Antigens/metabolism , Erythrocytes/immunology , Lipopolysaccharide Receptors/physiology , Lipopolysaccharides/immunology , Macrophage-1 Antigen/metabolism , Membrane Glycoproteins , Neutrophil Activation/immunology , Neutrophils/immunology , Carrier Proteins/physiology , Cell Adhesion/immunology , Erythrocytes/metabolism , Fluorescein-5-isothiocyanate/metabolism , Humans , Lipopolysaccharides/metabolism , Protein Binding/immunology , Receptors, Formyl Peptide , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , Salmonella/immunology , Salmonella/metabolism , Tumor Necrosis Factor-alpha/physiologyABSTRACT
Biosensor technology was employed to study the specific interactions of different lipopolysaccharide (LPS)-binding proteins and peptides with LPS, using an LPS-coated surface. Two methods to immobilize biotinylated LPS to streptavidin-coated sensor chips (SA-chips) were evaluated. Biotinylated LPS in PBS or biotinylated LPS, pretreated with EDTA and sodium-desoxycholate, were injected across an SA-chip, resulting in a 'high-' and 'low- mass' LPS chip, respectively. While the 'high mass' LPS chip appeared to be unstable, the 'low mass' LPS chip resulted in reproducible binding curves for bactericidal/permeability-increasing protein (rBPI21) with a binding affinity corresponding to the literature (Kd: 3.75 nM). New Kd values were obtained for serum amyloid P component (SAP, Kd: 3.9 nM), a recently discovered new LPS-binding protein, and cationic protein 18 (CAP18, Kd: 0.58 nM). Moreover, binding affinities of bioactive BPI- and SAP-derived peptides could be determined. This study shows for the first time the applicability of biosensor technology to study interactions of proteins and peptides with LPS, using an LPS-coated sensor chip.
Subject(s)
Acute-Phase Proteins , Biosensing Techniques , Lipopolysaccharides/metabolism , Membrane Glycoproteins , Membrane Proteins , Peptides/metabolism , Proteins/metabolism , Amino Acid Sequence , Antimicrobial Cationic Peptides , Blood Proteins/genetics , Blood Proteins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/metabolism , Humans , In Vitro Techniques , Kinetics , Molecular Sequence Data , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptides/genetics , Protein Binding , Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serum Amyloid P-Component/genetics , Serum Amyloid P-Component/metabolism , StreptavidinABSTRACT
LPS-binding proteins in plasma play an important role in modifying LPS toxicity. Significant properties have already been attributed to the LPS-binding protein (LBP). It accelerates LPS toxicity as well as incorporation into high-density lipoproteins, leading to neutralization of LPS in serum. A search for other LPS-binding components in serum, using LPS-coated magnetic beads, revealed a new LPS-binding protein. N-terminal microsequencing identified this protein as serum amyloid P component (SAP). Purified SAP bound to smooth and rough types of LPS via the lipid A part. SAP inhibited the binding of FITC-labeled ReLPS (LPS from Salmonella minnesota strain R595) to human monocytes and the ReLPS-induced priming of the oxidative burst of human neutrophils only in the presence of low concentrations of LBP. In search for the LPS binding site of SAP, we found that pep27-39, a 13-mer peptide consisting of amino acids 27-39 of SAP, competitively inhibited the binding of LPS to SAP. In addition, pep27-39 significantly inhibited ReLPS-induced responses in phagocytes in the presence of serum, as well as in human whole blood. Carboxamidomethylated pep27-39 showed an even more pronounced reduction of the ReLPS-induced priming of phagocytes in human blood. Performing gel filtration of FITC-labeled ReLPS incubated with soluble CD14, we showed that SAP could not prevent binding of LPS to soluble CD14, in contrast to pep27-39. The ability of pep27-39 to antagonize specifically the effects of LPS in the complex environment of human blood suggests that pep27-39 may be a novel therapeutic agent in the treatment of gram-negative sepsis.
Subject(s)
Acute-Phase Proteins , Carrier Proteins/pharmacology , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/pharmacology , Membrane Glycoproteins , Peptide Fragments/pharmacology , Serum Amyloid P-Component/pharmacology , Amino Acid Sequence , Binding, Competitive , Carrier Proteins/blood , Carrier Proteins/chemical synthesis , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/metabolism , Humans , Immunization , Lipopolysaccharide Receptors/genetics , Lipopolysaccharide Receptors/metabolism , Lipopolysaccharides/blood , Lipopolysaccharides/immunology , Molecular Sequence Data , Monocytes/metabolism , Neutrophils/immunology , Peptide Fragments/blood , Peptide Fragments/chemical synthesis , Peptide Fragments/immunology , Protein Binding/drug effects , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Serum Amyloid P-Component/antagonists & inhibitors , Serum Amyloid P-Component/immunology , Serum Amyloid P-Component/metabolismABSTRACT
Delta-toxin from Staphylococcus aureus is responsible for various pathophysiologic effects. By studying different cell types in binding of delta-toxin in low, noncytotoxic concentrations, a specific binding of fluorescein-labeled delta-toxin to neutrophils and monocytes was found. Studying direct effects of delta-toxin on neutrophils, a dose-dependent up-regulation of complement receptor 3 expression was found. Oxygen radical production, as determined by Luminol-enhanced chemiluminescence, was not directly induced by delta-toxin, and this toxin was also unable to prime neutrophils for an enhanced response to FMLP or complement-opsonized zymosan. However, the priming response induced by lipopolysaccharide or tumor necrosis factor-alpha (TNF-alpha) was significantly further enhanced in the presence of delta-toxin. Furthermore, as a direct effect on human monocytes, delta-toxin induced TNF-alpha production. These data provide evidence that delta-toxin has direct and indirect effects on the activity of neutrophils and monocytes with regard to its proinflammatory capacity.