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Journal of Innate Immunity 2014Neutrophils are essential for host defense against Staphylococcus aureus infections. Although significant progress has been made, our understanding of neutrophil...
Neutrophils are essential for host defense against Staphylococcus aureus infections. Although significant progress has been made, our understanding of neutrophil interactions with S. aureus remains incomplete. To provide a more comprehensive view of this process, we investigated phagocytosis and killing of S. aureus by human neutrophils using varied assay conditions in vitro. A greater percentage of bacteria were internalized by adherent neutrophils compared to those in suspension, and, unexpectedly, uptake of S. aureus by adherent neutrophils occurred efficiently in the absence of opsonins. An antibody specific for S. aureus promoted uptake of unopsonized bacteria in suspension, but had little or no capacity to enhance phagocytosis of S. aureus opsonized with normal human serum or by adherent neutrophils. Collectively, these results indicate that assay conditions can have a significant influence on the phagocytosis and killing of S. aureus by neutrophils. More importantly, the results suggest a vaccine approach directed to enhance opsonophagocytosis alone is not sufficient to promote increased killing of S. aureus by human neutrophils. With the emergence and reemergence of antibiotic-resistant microorganisms, establishing parameters that are optimal for studying neutrophil-S. aureus interactions will pave the way towards developing immune-directed strategies for anti-staphylococcal therapies.
Topics: Antibodies, Bacterial; Bacterial Vaccines; Cell Adhesion; Cells, Cultured; Cytotoxicity, Immunologic; Host-Pathogen Interactions; Humans; Immunity, Innate; Neutrophils; Opsonin Proteins; Phagocytosis; Serum Bactericidal Test; Staphylococcal Infections; Staphylococcus aureus
PubMed: 24713863
DOI: 10.1159/000360478 -
Journal of Immunology (Baltimore, Md. :... Jul 2008TNF ligand superfamily member 13B (B lymphocyte stimulator (BLyS), B cell activating factor (BAFF)) promotes primary B cell proliferation and Ig production. While the...
TNF ligand superfamily member 13B (B lymphocyte stimulator (BLyS), B cell activating factor (BAFF)) promotes primary B cell proliferation and Ig production. While the soluble form of BLyS/BAFF is thought to be the primary biologically active form, little is known about the regulation of its cleavage and processing. We provide evidence that Fcgamma receptor cross-linking triggers a rapid release of soluble, biologically active BLyS/BAFF from myeloid cells. Surprisingly, this function is primarily mediated by FcgammaRI, but not FcgammaRIIa as defined by specific mAb, and can be initiated by both IgG and C reactive protein as ligands. The generation of a B cell proliferation and survival factor by both innate and adaptive immune opsonins through engagement of an Fcgamma receptor, which can also enhance Ag uptake and presentation, provides a unique opportunity to facilitate Ab production. These results provide a mechanism by which Fcgamma receptors can elevate circulating BLyS levels and promote autoantibody production in immune complex-mediated autoimmune diseases.
Topics: B-Cell Activating Factor; B-Lymphocytes; C-Reactive Protein; Cell Line, Tumor; Cells, Cultured; Humans; Immunoglobulin G; Myeloid Cells; Opsonin Proteins; Receptors, IgG
PubMed: 18606652
DOI: 10.4049/jimmunol.181.2.1012 -
Molecular Imaging Feb 2011Mass transport of drug delivery vehicles is guided by particle properties, such as size, shape, composition, and surface chemistry, as well as biomolecules and serum...
Mass transport of drug delivery vehicles is guided by particle properties, such as size, shape, composition, and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two-dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light variable chain, fibrinogen, and complement component 1 compared to their anionic counterparts. Anionic microparticles were found to accumulate in equal abundance in murine liver and spleen, whereas cationic microparticles showed preferential accumulation in the spleen. Immunohistochemistry supported macrophage uptake of both anionic and cationic microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution.
Topics: Animals; Drug Carriers; Electrophoresis, Gel, Two-Dimensional; Mass Spectrometry; Mice; Opsonin Proteins; Porosity; Silicon
PubMed: 21303614
DOI: No ID Found -
Respiratory Medicine Jul 2004Surfactant protein-A enhances the phagocytosis and killing of many pathogens, although studying this effect in an assortment of models and different experimental...
Surfactant protein-A enhances the phagocytosis and killing of many pathogens, although studying this effect in an assortment of models and different experimental protocols has sometimes yielded conflicting results. In this report, using the human THP-1 cell line as the primary phagocytic cell, we systematically examined several models where microspheres, Staphylococcus aureus and Escherichia coli were used for targets. We found that SP-A derived from human lavage appeared to enhance phagocytosis by two different mechanisms; by SP-A binding of the target to enhance its recognition and subsequent phagocytosis and by a direct SP-A stimulatory effect on the phagocyte itself. Both SP-A mechanisms occurred with different targets in the same experimental system and the SP-A effects were qualitatively (but not quantitatively) comparable in several human cell lines (THP-1, U937, Mono-Mac-6). We also found that the SP-A effects were abrogated when SP-A was combined with surfactant lipids, but the lipids did not affect the basal level of phagocytosis or phagocytosis by mechanisms not involving SP-A. Moreover, the stimulatory effect of SP-A was pH-dependent and appeared to be independent of several other phagocytic mechanisms, including those mediated by Fc receptors and mannose receptor.
Topics: Biological Products; Cell Culture Techniques; Cell Line; Dose-Response Relationship, Drug; Escherichia coli; Humans; Ligands; Microspheres; Monocytes; Opsonin Proteins; Phagocytosis; Pulmonary Surfactant-Associated Protein A; Staphylococcus aureus
PubMed: 15250230
DOI: 10.1016/j.rmed.2003.12.018 -
Cellular Microbiology May 2008A key strategy in microbial pathogenesis is the subversion of the first line of cellular immune defences presented by professional phagocytes. Enteropathogenic and...
A key strategy in microbial pathogenesis is the subversion of the first line of cellular immune defences presented by professional phagocytes. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC respectively) remain extracellular while colonizing the gut mucosa by attaching and effacing mechanism. EPEC use the type three secretion system effector protein EspF to prevent their own uptake into macrophages. EPEC can also block in trans the internalization of IgG-opsonized particles. In this study, we show that EspJ is the type three secretion system effector protein responsible for trans-inhibition of macrophage opsono-phagocytosis by both EPEC and EHEC. While EspF plays no role in trans-inhibition of opsono-phagocytosis, espJ mutants of EPEC or EHEC are unable to block uptake of opsonized sheep red blood cells (RBC), a phenotype that is rescued upon complementation with the espJ gene. Importantly, ectopic expression of EspJ(EHEC) in phagocytes is sufficient to inhibit internalization of both IgG- and C3bi-opsonized RBC. These results suggest that EspJ targets a basic mechanism common to these two unrelated phagocytic receptors. Moreover, EspF and EspJ target independent aspects of the phagocytic function of mammalian macrophages in vitro.
Topics: Animals; COS Cells; Carrier Proteins; Chlorocebus aethiops; Enterohemorrhagic Escherichia coli; Escherichia coli O157; Escherichia coli Proteins; Intracellular Signaling Peptides and Proteins; Macrophage-1 Antigen; Macrophages; Opsonin Proteins; Phagocytosis; Transfection
PubMed: 18201246
DOI: 10.1111/j.1462-5822.2007.01112.x -
Cell Death and Differentiation Sep 2013The phagocytic clearance of apoptotic cells is essential to prevent chronic inflammation and autoimmunity. The phosphatidylserine-binding protein milk fat globule-EGF...
The phagocytic clearance of apoptotic cells is essential to prevent chronic inflammation and autoimmunity. The phosphatidylserine-binding protein milk fat globule-EGF factor 8 (MFG-E8) is a major opsonin for apoptotic cells, and MFG-E8(-/-) mice spontaneously develop a lupus-like disease. Similar to human systemic lupus erythematosus (SLE), the murine disease is associated with an impaired clearance of apoptotic cells. SLE is routinely treated with glucocorticoids (GCs), whose anti-inflammatory effects are consentaneously attributed to the transrepression of pro-inflammatory cytokines. Here, we show that the GC-mediated transactivation of MFG-E8 expression and the concomitantly enhanced elimination of apoptotic cells constitute a novel aspect in this context. Patients with chronic inflammation receiving high-dose prednisone therapy displayed substantially increased MFG-E8 mRNA levels in circulating monocytes. MFG-E8 induction was dependent on the GC receptor and several GC response elements within the MFG-E8 promoter. Most intriguingly, the inhibition of MFG-E8 induction by RNA interference or genetic knockout strongly reduced or completely abolished the phagocytosis-enhancing effect of GCs in vitro and in vivo. Thus, MFG-E8-dependent promotion of apoptotic cell clearance is a novel anti-inflammatory facet of GC treatment and renders MFG-E8 a prospective target for future therapeutic interventions in SLE.
Topics: Animals; Antigens, Surface; Apoptosis; Cell Line, Tumor; Glucocorticoids; Humans; Lupus Erythematosus, Systemic; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Milk Proteins; Opsonin Proteins; Phagocytosis; Promoter Regions, Genetic; RNA Interference; RNA, Small Interfering; Receptors, Glucocorticoid; Response Elements; U937 Cells
PubMed: 23832117
DOI: 10.1038/cdd.2013.82 -
Vaccine Dec 2013The seven-valent pneumococcal conjugate vaccine (PCV7) has been introduced in most high-income countries, although with differences in age, timing and number of primary... (Randomized Controlled Trial)
Randomized Controlled Trial
The seven-valent pneumococcal conjugate vaccine (PCV7) has been introduced in most high-income countries, although with differences in age, timing and number of primary doses before 6 months of age and presence and timing of a booster vaccination. The objective was to determine and compare the IgG antibody levels and functionality of IgG responses (avidity and opsonophagocytoses) at 1 and 2 years of age following 2 primary doses with a booster at 11 or 24 months of age. Children received PCV7 at 2 and 4 months (2-dose group), or at 2, 4 and 11 months (2+1-dose group), or no PCV7 (controls) before 1 year of age. All children received a PCV7 dose at 24 months of age. At the age of 12 months, the 2+1-dose group had higher IgG levels and functional antibody levels, compared to the 2-dose group for all serotypes, but at 25 months the difference between the 2-dose and 2+1-dose groups had disappeared for most serotypes. The kinetics of opsonophagocytic antibodies were in line with the specific IgG antibody levels for most serotypes, although differences between the 2-dose and the 2+1-dose group were more pronounced in OPA activity as compared to the IgG levels especially at the age of 24 months. Delaying the booster dose from 11 months to 24 months after 2 primary doses resulted in significantly higher OPA GMTs one month after the booster dose. This must, however, be balanced against the risk of leaving children unboosted between the age of 11 and 24 months at a time when disease risk is still high. Local decisions about the timing of a booster dose should also take into account vaccine coverage and the indirect herd effect in a well vaccinated population. Trial registration clinicaltrials.gov Identifier: NCT00189020.
Topics: Antibodies, Bacterial; Antibody Affinity; Child, Preschool; Heptavalent Pneumococcal Conjugate Vaccine; Humans; Immunization, Secondary; Immunoglobulin G; Infant; Opsonin Proteins; Phagocytosis; Pneumococcal Infections; Pneumococcal Vaccines; Single-Blind Method; Vaccines, Conjugate
PubMed: 24120678
DOI: 10.1016/j.vaccine.2013.09.073 -
Molecular & Cellular Proteomics : MCP May 2015Macrophages operate at the forefront of innate immunity and their discrimination of foreign versus "self" particles is critical for a number of responses including...
Macrophages operate at the forefront of innate immunity and their discrimination of foreign versus "self" particles is critical for a number of responses including efficient pathogen killing, antigen presentation, and cytokine induction. In order to efficiently destroy the particles and detect potential threats, macrophages express an array of receptors to sense and phagocytose prey particles. In this study, we accurately quantified a proteomic time-course of isolated phagosomes from murine bone marrow-derived macrophages induced by particles conjugated to seven different ligands representing pathogen-associated molecular patterns, immune opsonins or apoptotic cell markers. We identified a clear functional differentiation over the three timepoints and detected subtle differences between certain ligand-phagosomes, indicating that triggering of receptors through a single ligand type has mild, but distinct, effects on phagosome proteome and function. Moreover, our data shows that uptake of phosphatidylserine-coated beads induces an active repression of NF-κB immune responses upon Toll-like receptor (TLR)-activation by recruitment of anti-inflammatory regulators to the phagosome. This data shows for the first time a systematic time-course analysis of bone marrow-derived macrophages phagosomes and how phagosome fate is regulated by the receptors triggered for phagocytosis.
Topics: Animals; Calreticulin; Complement System Proteins; Immunity, Innate; Immunoglobulin G; Ligands; Lipopolysaccharides; Macrophages; Mannans; Mice; Microspheres; NF-kappa B; Opsonin Proteins; Phagocytosis; Phagosomes; Phosphatidylserines; Protein Interaction Mapping; Proteome; Receptors, Cell Surface
PubMed: 25755298
DOI: 10.1074/mcp.M114.044594 -
BMC Research Notes Apr 2019Intravenous immune globulin (IVIG), pooled from human blood, is a polyspecific antibody preparation that inhibits the super-antigenic proteins associated with...
OBJECTIVE
Intravenous immune globulin (IVIG), pooled from human blood, is a polyspecific antibody preparation that inhibits the super-antigenic proteins associated with streptococcal and staphylococcal toxic shock, and the Shiga toxin. In addition to this toxin-neutralising activity, IVIG contains other pathogen-reactive antibodies that may confer additional therapeutic benefits. We sought to determine if pathogen-reactive antibodies that promote opsonophagocytosis of different organisms can be sequentially affinity-purified from one IVIG preparation.
RESULTS
Antibodies that recognise cell wall antigens of Streptococcus pyogenes, Staphylococcus aureus, and vancomycin-resistant enterococcus (VRE) were sequentially affinity-purified from a single preparation of commercial IVIG and opsonophagocytic activity was assessed using a flow cytometry assay of neutrophil uptake. Non-specific IgG-binding proteins were removed from the S. aureus preparations using an immobilised Fc fragment column, produced using IVIG cleaved with the Immunoglobulin G-degrading enzyme of S. pyogenes (IdeS). Affinity-purified anti-S. aureus and anti-VRE immunoglobulin promoted significantly higher levels of opsonophagocytic uptake by human neutrophils than IVIG when identical total antibody concentrations were compared, confirming activity previously shown for affinity-purified anti-S. pyogenes immunoglobulin. The opsonophagocytic activities of anti-S. pyogenes, anti-S. aureus, and anti-VRE antibodies that were sequentially purified from a single IVIG preparation were undiminished compared to antibodies purified from previously unused IVIG.
Topics: Antibodies, Bacterial; Antigens, Bacterial; Cell Wall; Chromatography, Affinity; Humans; Immunoglobulin Fc Fragments; Immunoglobulins, Intravenous; Neutrophils; Opsonin Proteins; Phagocytosis; Primary Cell Culture; Staphylococcus aureus; Streptococcus pyogenes; Vancomycin-Resistant Enterococci
PubMed: 30992057
DOI: 10.1186/s13104-019-4262-8 -
Human opsonins induced during meningococcal disease recognize outer membrane proteins PorA and PorB.Infection and Immunity May 1999Human opsonins directed against specific meningococcal outer membrane structures in sera obtained during meningococcal disease were quantified with a recently developed...
Human opsonins directed against specific meningococcal outer membrane structures in sera obtained during meningococcal disease were quantified with a recently developed antigen-specific, opsonin-dependent phagocytosis and oxidative burst assay. Outer membrane vesicles (OMVs) and PorA (class 1) and PorB (class 3) proteins purified from mutants of the same strain (44/76; B:15:P1.7. 16) were adsorbed to fluorescent beads, opsonized with acute- and convalescent-phase sera from 40 patients with meningococcal disease, and exposed to human leukocytes. Flow cytometric quantitation of the resulting leukocyte phagocytosis products (PPs) demonstrated that disease-induced serum opsonins recognized meningococcal OMV components and both porins. The PPPorA and PPPorB values induced by convalescent-phase sera correlated positively with the PPOMV values. However, the PPPorB values were higher than the PPPorA values in convalescent-phase sera (medians [ranges] of 754 [17 to 1,057] and 107 [4 to 458], respectively) (P < 0.0001) and correlated positively with higher levels of immunoglobulin G against PorB than against PorA as evaluated by enzyme-linked immunosorbent assay. Extensive individual variations in the anti-OMV and antiporin serum opsonic activities between patients infected by serotypes and serosubtypes homologous and heterologous to the target antigens were observed. Simultaneously measured oxidative burst activity correlated with the opsonophagocytosis, an indication that both of these important steps in the in vitro phagocytic elimination of meningococci are initiated by opsonins directed against OMV components, including PorA and PorB. In conclusion, human patient opsonins against meningococcal OMV components and in particular PorB epitopes were identified by this new method, which might facilitate selection of opsonin-inducing meningococcal antigens for inclusion in future vaccines.
Topics: Adolescent; Adult; Antibodies, Bacterial; Bacterial Outer Membrane Proteins; Female; Humans; In Vitro Techniques; Leukocytes; Male; Meningitis, Meningococcal; Meningococcal Infections; Microscopy, Confocal; Middle Aged; Neisseria meningitidis; Opsonin Proteins; Phagocytosis; Porins; Respiratory Burst
PubMed: 10225920
DOI: 10.1128/IAI.67.5.2552-2560.1999