Different effects of fibronectin on the phagocytosis of Staphylococcus aureus and coagulase-negative staphylococci by murine peritoneal macrophages

Fibronectin is known to be an important factor in colonization by Staphylococcus aureus of host tissues as well as other extracellular matrix proteins such as collagen and laminin. We investigated the effect of fibronectin on the phagocytosis of the S. aureus Cowan I strain by macrophages and of coagulase-negative staphylococci (CNS) strains for comparison. Fibronectin-reduced serum in place of normal serum lowered the phagocytic activity of the macrophages on the Cowan I strain. Purified fibronectin enhanced the phagocytic activity of the strain in a dose-dependent manner. On the other hand, fibronectin did not show any opsonic effect on the ingestion of CNS strains, though the binding of fibronectin occurred equally well in CNS strains and the Cowan I strain. Fibronectin-binding protein (FnBP), the specific fibronectin receptor on the surface on S. aureus, was detected in both the Cowan I strain and CNS strains. Polymerase chain reaction confirmed that not only the Cowan I strain, but also CNS strains possessed the FnBP gene. These results indicate that fibronectin shows an opsonic effect on the S. aureus, Cowan I strain but not on CNS strains, and suggest that the binding of fibronectin to FnBP is not sufficient for efficient phagocytosis of the staphylococci strains by macrophages.     

Luminol-dependent chemiluminescence in antibody-sensitized neutrophils stimulated with protein A-bearing staphylococci

When mouse polymorphonuclear leukocytes (PMNs) sensitized with rabbit antibody to mouse Ehrlich ascites tumor cells were stimulated by Staphylococcus aureus Cowan I cells, a conspicuous luminol-dependent chemiluminescence was observed in the absence of opsonin. The profile of the chemiluminescence (CL) response evoked by staphylococcal cells from antibody-sensitized PMNs had two peaks. An initial peak, observed within 1 min after stimulation, was sharp and high and a second peak, observed about 5 min after stimulation, was low and extended. The CL response of antibody-sensitized PMNs stimulated by S. aureus Cowan I cells was dose-dependently blocked by preincubation with soluble SpA. Cells of a mutant derived from S. aureus Cowan I strain with trace amounts of cell-bound SpA failed to stimulate the antibody-sensitized PMNs to generate the CL response. The antibody-sensitized PMNs were found to phagocytize SpA-bearing S. aureus cells even in the absence of opsonic serum. These results suggest that the observation presented here might provide a useful tool for the investigation of CL response of PMNs.     

Monoclonal antibodies against T cell differentiation antigens initiate stimulation of monocyte/macrophage oxidative metabolism

Within the first minute after incubation with the mouse anti-human T cell orthoclone monoclonal antibodies OKT3, OKT4, and OKT8, and in the absence of complement, human monocytes generate a burst of highly reactive oxygen metabolites as detected by a luminol-dependent photometric chemiluminescence (CL) assay. The kinetics of the CL responses to these antibodies are identical to that induced by OKM1, the monoclonal antibody to human monocytes and granulocytes. With regard to CL response intensities, OKM1 induces the maximal response and those of OKT3, OKT4, and OKT8 closely reflect the proportion of T cell subsets recognized by these antibodies in peripheral blood. This reaction is also observed when monoclonal antibodies against mouse Lyt surface determinants (Lyt-1 and Lyt-2) and Thy-1 antigen are tested against murine spleen cells. This murine model was further used to investigate the specificity and the mechanism of this reaction. It was demonstrated that the CL response is Lyt antigen specific, occurs upon addition of monoclonal IgG but not IgM antibodies, requires the concomitant presence of CL-producing cells (CLPC) (promonocytes, monocytes, macrophages, and/or granulocytes) and of fully differentiated T cells, and lastly, is mediated via a T cell opsonization process. Selective blockade of bone marrow cell Fc receptors (FcR II) with monoclonal anti-mouse FcR II antibody inhibits the CL response to IgG2b anti-T cell antibody-coated thymocytes and thus strongly suggests that the stimulation of CLPC oxidative metabolism in this model results from the binding of opsonized T cells to plasma membrane Fc receptors. These observations lend additional support to increasing evidence that the initiation of effector functions by monoclonal anti-T cell antibodies may be strictly dependent upon the presence of monocytes and/or macrophages.     

The mitogenic activity of type III bacterial Ig binding proteins (protein G) for human peripheral blood lymphocytes is not related to their ability to react with human serum albumin or IgG

The mitogenic potential of bacterial IgG Fc binding proteins for human PBL is controversial. Wild type and recombinant type III IgG Fc binding proteins induce a wide spectrum of proliferative responses ranging from non-mitogenic to potent responses. To understand the reason for these differences, three recombinant forms of a type III IgG Fc binding protein derived from a single human group C streptococcal strain, 26RP66, were generated. Form I bound human IgG and human serum albumin, form II bound IgG alone and form III bound human serum albumin alone. These functionally distinct forms were compared with the corresponding wild type preparation from the same strain for mitogenic potential. A mitogenic response was induced only with the form I recombinant or the native wild type protein. These proteins shared the functional characteristics of binding human serum albumin and IgG. Mixtures of the IgG binding (form II) and human serum albumin binding fragments (form III) failed to reconstitute the mitogenic potential of the full length proteins. These results demonstrate that the type III IgG Fc binding protein has mitogenic potential for human PBL that is not related to its ability to react with human serum albumin or IgG.     

Hemagglutination Activity and Localization of Fc Receptor of Group A and G Streptococci

The IgG-Fc binding activity and binding sites on the cell surface of streptococci, strains AR1 (group A) and G148 (group G), and Staphylococcus aureus strain Cowan I were examined by hemagglutination (HA) and immunoelectron microscopic methods. No distinct difference was observed in the HA activity among these three strains. However, the strains differed in the distribution of Fc receptor. Cowan I cells (having protein A) were heavily covered with two layers of ferritin particles, whereas AR1 cells were heavily covered with a single, rough layer of ferritin particles. G148 cells (having protein G) were labeled with a relatively thin, rough ferritin layer. The trypsin susceptibility of the Fc receptors of the AR1 strain was much higher than that of the G148 strain. These results suggest that both streptococcal strains are distinctly different in the arrangement or in the conformation of the Fc receptor from the Cowan I strain. It is also suggested that the Fc receptor molecules of the streptococcal strains differ from each other not only in conformation but also in trypsin susceptibility.     

金黄色葡萄球菌A蛋白基因的克隆及序列分析

 我们构建了金黄色葡萄球菌Cowan1株(CMCC26111)的染色体文库,转化大肠杆菌后筛选出一株protein A的阳性克隆。SDS-PAGE及Western-blot结果显示该克隆株表达的重组protein A的分子量为30 000,较天然protein A的小。该克隆中的protein A基因片段的序列分析表明,它含有天然protein A基因的启动子、信号肽以及至少四个与IgGFc段结合的结构域,而不含天然protein A的胞壁结合区,并发现其中有24个碱基对与Uhlen报告的protein A基因不同,由此导致三个编码的氨基酸发生变化,但这些差异并不影响该重组protein A与IgG Fc段的特异结合。     

Naturally occurring anti-band 3 antibodies and complement in phagocytosis of oxidatively-stressed and in clearance of senescent red cells

Treatment of human red blood cells with diamide and opsonization with whole serum enhanced their phagocytosis by mononuclear phagocytes. Opsonization of diamide-treated red cells with whole serum containing 20-100 times the physiologic concentration of naturally occurring anti-band 3 antibodies further increased the extent of phagocytosis. Enhanced phagocytosis was due to an anti-band 3 mediated binding of C3b to red cells via the alternative pathway. Red cell-bound anti-band 3 was slightly elevated on diamide-treated cells and elicited a C3 binding that exceeded the amount of bound antibody by two orders of magnitude. Pretreatment of red cells with a monoclonal anti-CR1 did not significantly inhibit opsonization and phagocytosis if cells were opsonized at elevated anti-band 3 concentrations. On the other hand, phagocytosis of mildly oxidized (20 microM diamide) red cells was completely inhibited by blocking CR1 if cells were opsonized with serum containing physiologic concentrations of anti-band 3. The results suggest that two types of opsonization mediate in vitro phagocytosis: one operating at physiologic anti-band 3 concentrations with mildly oxidized red cells (IC-like mechanism) and one that operates with either heavily oxidized (greater than 200 microM diamide) red cells at physiologic anti-band 3 concentrations, or with mildly oxidized cells opsonized at elevated concentration of anti-band 3. The latter mechanism is relevant in vivo. It is most likely that it starts by Fab-dependent binding of anti-band 3 to diamide-induced band 3 protein oligomers. Complement activation may occur by assembly of an alternative convertase on C3b covalently bound to red cell-associated anti-band 3. This mechanism is also likely to mediate clearance of senescent red cells, as it was primarily from senescent red cells that we could isolate complexes containing IgG covalently bound to C3b.     

Physicochemical and functional changes in human leukemic cell line HL-60

The recently established human promyelocytic cell line HL-60 was induced to differentiate in the present of DMSO. During this process, physiochemical, and functional changes were detected simultaneously. After exposure to DMSO for more than 1 day, the cell volume decreased and the tendency for hydrophobic interaction increased. Using a hydrophobic two-phase system in counter current distribution fashion, it was then possible to separate more mature metamyelocytes and segmented granulocytes from immature myeloblasts and promyelocytes. Increased functional maturity was reflected by increased chemiluminescence (CL) response and phagocytic activity. Using yeast particles opsonized with IgG as stimulating agent, the CL response increased already after 1 day in DMSO, in parallel with increased phagocytosis of these particles. In contrast, C3b-opsonized yeast and phorbol 12-myristate 13-acetate (PMA) did not enhance the CL response conspiquously until days 3–4. These data suggest that Fc receptor function linked to phagocytosis and the activation of oxidative metabolism develop earlier than that of C3b and PMA. The dissociation between Fc- and PMA-dependent stimulation of the oxidative metabolism may reflect different mechanisms of activation.     

Surface properties of Staphylococcus aureus affecting chemiluminescence response of human phagocytes

To assess the surface properties of Staphylococcus aureus affecting the response of human phagocytes, the effects of the organisms with different surface properties on the chemiluminescence (CL) response of human phagocytes were examined. The magnitude of the phagocytic CL response to hydrophobic strains was significantly greater than that to hydrophilic strains, while no significant difference in the CL response was seen between protein A-deficient strains and their parent strains. The CL response to the hydrophilic organisms prepared from a hydrophobic strain by trypsin treatment decreased significantly. These results suggest that the phagocytic CL response to staphylococci depends on the hydrophobicity of the surface, but not on the presence of protein A. Two protein A-deficient strains which were isolated from protein A-positive strains showed identical hydrophobicity with their parent strains. All of the hydrophilic strains isolated from hydrophobic strains possessed protein A identical to that of their parent strains. Moreover, a hydrophilic strain could be isolated from a protein A-deficient, hydrophobic strain. These results strongly suggest that protein A is not solely responsible for the surface hydrophobicity of S. aureus.     

The adhesion of protein A-bearing Staphylococcus aureus organisms to soluble-staphylococcal-antigens-coated HeLa cells mediated by specific antibodies

The adhesion of staphylococcal protein A (SpA)-bearing Staphylococcus aureus Cowan I organisms to HeLa cells was enhanced by pretreatment of HeLa cells with staphylococcal extracellular antigens and antibodies to them. The adhesion of HLj, an SpA-poor mutant derived from Cowan I, to HeLa cells was not enhanced by the same pretreatment of HeLa cells. Furthermore, the enhanced staphylococcal adhesion was inhibited by soluble SpA. The antigen(s) responsible for the enhanced staphylococcal adhesion was(were) heat stable. Pretreatment of HeLa cells with the mixture of staphylococcal extracellular antigens and antibodies to them also enhanced the adhesion of Cowan I. Similarly the adhesion of Cowan I was enhanced by pretreatment of HeLa cells with extracellular antigens of Pseudomonas aeruginosa and antibodies to them. These results indicated that cell-bound SpA mediated the binding of S. aureus to immune complexes composed of extracellular bacterial products and antibodies to them bound to the surface of HeLa cells, and suggested another role of cell-bound SpA as a co-adhesin with other factors in infections due to S. aureus.     

IgG binding enhances DNAase I sensitivity of N-acetoxy-N-2-acetylaminofluorene-modified ΦX-174 RF DNA

DNA restriction fragments of фX-174 RF were modified with the carcinogen, N-acetoxy-N-2-acetylaminofluorene (N-Aco-AAF). Immune complexes of 5′-³²P-labeled AAF-modified DNA and rabbit immunoglobulin (IgG) against AAF-guanosine were specifically bound by surface membranes of Cowan I strain micrococci whose protein A binds the Fc portion of IgG. DNAase I sensitivity of the bound DNA was 20-fold greater than in solution, but the normal pattern of hydrolysis was not altered, as determined in sequencing gels. Nonadducted DNA ligated to AAF-modified DNA acquired the enhanced sensitivity to DNAase I hydrolysis when the ligation hybrid was immunobound.     

Analysis of protein A encoded by a mutated gene of Staphylococcus aureus Cowan I

The protein A (spa) genes from Staphylococcus aureus Cowan I and a mutant strain of Cowan I called V-1 earlier suggested to produce a monovalent IgG-binding protein A have been cloned in Escherichia coli. The DNA sequences coding for the IgG-binding part of the spa genes from both strains have been determined and compared with each other and with a partial amino acid sequence of purified protein A from strain V-1. The nucleotide sequence of the spa gene from strain V-1 reveals an NH2-terminally located IgG-binding region homologous to region E first reported for strain 8325-4, region D and the major portion of region A. The amino acid sequence analysis of the purified protein A from this strain also shows the presence of regions E and D but only a minor part of region A. Reversed-phase high-performance liquid chromatography fractionation of purified protein A from strain V-1 revealed that the preparation was heterogeneous, containing mainly two peptides with different abilities to bind IgG molecules. A shuttle vector containing the cloned protein A gene from V-1 was constructed and transformed into different strains of S. aureus and the produced protein A was purified and analysed using sodium dodecyl sulfate/polyacrylamide gel electrophoresis.     

Ligation of FcγR Alters Phagosomal Processing of Protein via Augmentation of NADPH Oxidase Activity

Proteolysis and the reduction of disulfides, both major components of protein degradation, are profoundly influenced by phagosomal redox conditions in macrophages. We evaluated the activation of phagocytic receptors that are known to influence activation of the phagocyte NADPH oxidase (NOX2), and its effect on phagosomal protein degradation. Population‐based and single phagosome analyses of phagosomal chemistries in murine macrophages revealed that activation of NOX2 via the Fcγ receptor (FcγR) during phagocytosis decreased rates of proteolysis and disulfide reduction. Immunoglobulin G (IgG)‐stimulated reactive oxygen species (ROS) production and the inhibition of phagosomal proteolysis and disulfide reduction were dependent on NOX2, FcγR and protein kinase C (PKC)/spleen tyrosine kinase (Syk) signaling. In contrast, low levels of ROS production were observed following the phagocytosis of unopsonized beads, which resulted in higher rates of phagosomal proteolysis and disulfide reduction. Phagosomes displayed autonomy with respect to FcγR‐mediated differences in NOX2 activation and proteolysis, as phagosomes containing unopsonized cargo retained low NOX2 activation and high proteolysis even in the presence of phagosomes containing IgG‐opsonized cargo in the same macrophage. These results show that opsonization of phagocytic cargo results in vastly different phagosomal processing of proteins through the FcγR‐triggered, PKC/Syk‐dependent local assembly and activation of NOX2.      

Immunoglobulin M synthesized by human lymphoblastoid cells: interaction with Staphylococcus aureus and protein A.

Immunoglobulin M synthesized by a human lymphoblastoid cell line, LA173, was found to bind specifically to the protein A-bearing Cowan I strain of Staphylococcus aureus. The (3H)-leucine-labeled, secreted IgM from these LA173 cells also formed precipitin complexes with purified protein A. Soluble complexes formed at high protein A/IgM ratios retained the ability to bind to the bacterial surface. Precipitin complexes also were observed in double diffusion Ouchterlony gels with a line of identity formed between the IgM, protein A, and anti-IgM in adjacent wells. Intracellular IgM species from detergent-lysed LA173 cells were bound to S. aureus. Labeled 19S pentamers, 8S monomers, and HL subunits were eluted from the bacteria and identified by velocity sedimentation and SDS agarose-acrylamide gel electrophoresis. In addition, several intermediates migrating between 8S and 19S were detected and shown to contain authentic H and L chains. Binding of the labeled IgM 19S pentamers to staphylococci was not inhibited by prior treatment of the bacteria with an excess of unlabeled human IgG. However, S. aureus saturated with unlabeled IgG did not bind either labeled IgM monomers or labeled IgG. The interaction of this human IgM with S. aureus exhibited a high degree of specificity with quantitative recovery of secreted 19S IgM. Intracellular IgM species were bound selectively by the bacteria with little if any contamination by other cytoplasmic proteins.     

The opsonizing ligand on Salmonella typhimurium influences incorporation of specific, but not azurophil, granule constituents into neutrophil phagosomes

Phagosomes were purified from human neutrophils ingesting Salmonella typhimurium opsonized with adsorbed normal human serum or with rabbit IgG. Constituents within the phagosome were endogenously labeled by supplying the cells with 125INa during phagocytosis. Lactoferrin and vitamin B12 binding protein (TC1 and TC3), markers for specific granules, were present in the phagosomes from neutrophils ingesting S. typhimurium opsonized with IgG but were 3.5- to 5-fold less prominent in phagosomes from cells phagocytosing Salmonella bearing C3 fragments only. In contrast, iodinated azurophilic granule components, most prominently defensins, were the major constituents in phagosomes prepared under both opsonization conditions. Furthermore, labeled complement (CR1 and CR3) and immunoglobulin (Fc gamma RIII) receptors were incorporated in the phagosome regardless of the ligand mediating phagocytosis. These results suggest that the ligand-receptor interactions mediating phagocytosis influence incorporation of neutrophil-specific granule contents into phagosomes.     

Host — parasite relationship in staphylococcal infections: the role of the staphylococcal cell wall during the process of phagocytosis

Evidence is presented that antibodies against staphylococcal peptidoglycan are important opsonins for phagocytosis of staphylococci. Cell wall protein A inhibits opsonization by IgG through its interaction with the Fc fragment of the IgG molecule and preventing therefore the binding between the Fc fragment and the Fc receptor of the cell membrane of the leukocyte. Extracellular protein A interferes with opsonization presumably through depletion of complement.     

Staphylococcus aureus protein A binding to von Willebrand factor A1 domain is mediated by conserved IgG binding regions

Protein A (Spa) is a surface-associated protein of Staphylococcus aureus best known for its ability to bind to the Fc region of IgG. Spa also binds strongly to the Fab region of the immunoglobulins bearing V(H)3 heavy chains and to von Willebrand factor (vWF). Previous studies have suggested that the protein A-vWF interaction is important in S. aureus adherence to platelets under conditions of shear stress. We demonstrate that Spa expression is sufficient for adherence of bacteria to immobilized vWF under low fluid shear. The full length recombinant Ig-binding region of protein A, Spa-EDABC, fused to glutathione-S-transferase (GST), bound recombinant vWF in a dose-dependent and saturable fashion with half maximal binding of about 30 nm in immunosorbent assays. Full length-Spa did not bind recombinant vWF A3 domain but displayed binding to recombinant vWF domains A1 and D'-D3 (half maximal binding at 100 nm and 250 nm, respectively). Each recombinant protein A Ig-binding domain bound to the A1 domain in a similar manner to the full length-Spa molecule (half maximal binding 100 nm). Amino acid substitutions were introduced in the GST-SpaD protein at sites known to be involved in IgG Fc or in V(H)3 Fab binding. Mutants altered in residues that recognized IgG Fc but not those that recognized V(H)3 Fab had reduced binding to vWF A1 and D'-D3. This indicated that both vWF regions recognized a region on helices I and II that overlapped the IgG Fc binding site.     

Escherichia coli strains of serogroup O139 isolated from oedema disease of swine bind fibronectin

Abstract 15 Escherichia coli strains of the serogroup O139, isolated from oedema disease of swine, were examined for their ability to interact with ¹²⁵I-labelled fibronectin. All strains were positive, and all except one showed higher fibronectin binding than Staphylococcus aureus strain Cowan 1 cells (to which fibronectin bound in the order of 15% of total protein added). 7 E. coli strains isolated from diarrhoea in young piglets were also tested, and 3 were positive. 2 of these strains showed higher binding than S. aureus Cowan 1 cells. E. coli strains expressing either K99 or K88 antigen were poor binders, comparable to cells of S. aureus strain Wood 46. There was no correlation between cell surface hydrophobicity, as determined by chromatography on Octyl-Sepharose, and the fibronectin-binding property.     

Staphylococcus aureus protein A activates TNFR1 signaling through conserved IgG binding domains

Staphylococcus aureus continues to be a major cause of infection in normal as well as immunocompromised hosts, and the increasing prevalence of highly virulent community-acquired methicillin-resistant strains is a public health concern. A highly expressed surface component of S. aureus, protein A (SpA), contributes to its success as a pathogen by both activating inflammation and by interfering with immune clearance. SpA is known to bind to IgG Fc, which impedes phagocytosis. SpA is also a potent activator of tumor necrosis factor alpha (TNF-alpha) receptor 1 (TNFR1) signaling, inducing both chemokine expression and TNF-converting enzyme-dependent soluble TNFR1 (sTNFR1) shedding, which has anti-inflammatory consequences, particularly in the lung. Using a collection of glutathione S-transferase fusions to the intact IgG binding region of SpA and to each of the individual binding domains, we found that the SpA IgG binding domains also mediate binding to human airway cells. TNFR1-dependent CXCL8 production could be elicited by any one of the individual SpA IgG binding domains as efficiently as by either the entire SpA or the intact IgG binding region. SpA induction of sTNFR1 shedding required the entire IgG binding region and tolerated fewer substitutions in residues known to interact with IgG. Each of the repeated domains of the IgG binding domain can affect multiple immune responses independently, activating inflammation through TNFR1 and thwarting opsonization by trapping IgG Fc domains, while the intact IgG binding region can limit further signaling through sTNFR1 shedding.     

Binding of autologous IgG to human red blood cells before and after ATP-depletion. Selective exposure of binding sites (autoantigens) on spectrin-free vesicles

Binding of autologous IgG to fresh, ATP-depleted red blood cells as well as to spectrin-free vesicles was studied by a non-equilibrium binding assay using 125I-iodinated protein A from Staphylococcus aureus. IgG binding was 14-times higher to spectrin-free vesicles than to ATP-maintaining red blood cells and 4-times higher than to ATP-depleted erythrocytes from which these vesicles were released. Protein A binding to vesicles that were released from washed and nutrient-deprived erythrocytes, was dependent on added autologous IgG. However, spectrin-free vesicles that were spontaneously released from erythrocytes conserved in whole blood, bound similar amounts of protein A with or without added autologous IgG (0.45-0.55 ng/micrograms band 3 protein). These findings demonstrate that opsonization of spectrin-free vesicles by autologous IgG occurs not only in the test tube, but also under blood blank conditions. The binding characteristics of IgG to spectrin-free vesicles are indicative of a natural autoantibody rather than an unspecific binding of autologous IgG. The preferential binding of IgG to spectrin-free vesicles implies a selective exposure of corresponding autoantigens in membrane regions that have lost cytoskeletal anchorage and bud off.