Detection and quantitation of IgG on the surface of human lymphoid cells by rosette formation with protein A-coated sheep red blood cells

Sheep red blood cells coated with protein A of Staphylococcus aureus (SpA) form rosettes with human lymphoid cells with surface-associated IgG and with cells treated with antibodies of the IgG class against some of the cell membrane antigens. Inhibition of rosette formation by SpA was used for quantitation of immunoglobulins on the cell surface. The sensitivity of the method permits the quantitation of 10⁴ – 10⁵ molecules of IgG receptors and detected less than 10⁴ antibody molecules of the IgG class bound to the surface of IgM-producing cells. This method seems to be suitable for detection and quantitation of membrane-associated antigens including immunoglobulins.     

Arming of Lymphoid Cells by IgG Antibodies Treated with Protein A from Staphylococcus aureus

Mouse spleen lymphocytes treated with rabbit IgG anti-sheep erythrocytes (SRBC) complexed with protein A of Stapbylococcus aureus (SpA) form rosettes with SRBC. The attachment of SRBC to lymphocytes was due to the binding of the SpA-IgG antibody complex to the surface of the lymphocytes and was thus considered 'arming' of the cells. Normal mouse spleen cells 'armed' with SpA-rabbit IgG anti-chicken erythrocytes (CRBC) kill specifically ⁵¹Cr-labeled CRBC 'in vitro' in the absence of free antibodies. The killing by these 'armed' cells is an effect of the cell-bound SpA-IgG antibody complex. Both the SRBC rosette formation and the cell-mediated CRBC killing was dependent on the concentration of the SpA-IgG antibody complexes used for 'arming' the cell. A 100-fold increase in rosette formation or in killing of target cells was recorded for lymphocytes treated with SpA-IgG antibody complexes in comparison with cell treated with noncomplexed IgG antibodies. The specific binding of SpA-IgG antibody complexes to the Fc receptors of mouse spleen cells was demonstrated by inhibition studies. More than 60% inhibition of the rosette formation and in the killing of target cells was shown for cell treated With normal rabbit IgG or its Fc fragment before addition of the SpA-IgG antibody complex.     

Effect of protein A of Staphylococcus aureus on the binding of monomeric and polymeric IgG to Fc receptor-bearing cells.

The effect of protein A of Staphylococcus aureus (SpA) on the binding of rabbit IgG to the Fc receptors of mouse lymphocytes and macrophages was found to correlate with the aggregation of the IgG ligands. After binding anti-erythrocyte IgG complexed with SpA, the cells were able to attach to and kill erythrocyte indicator cells with a higher efficiency than lymphoid cells treated with anti-erythrocyte IgG alone. The amount of anti-peroxidase IgG which can be bound to effector cells was not changed by reaction with SpA. In contrast, the binding to cells of IgG-coated erythrocytes and of anti-peroxidase IgG complexed with peroxidase was substantially reduced by reaction with SpA. The results are compatible with the presence of two distinct Fc receptors, one for cytophilic monomeric IgG and another for polymeric (antigen-complexed) IgG.     

Separation of cells by affinity chromatography on SpA-sepharose 6MB

A cell separation technique was designed which was based on the reaction of protein A of Staphylococcus aureus (SpA) with the Fc regions of IgG antibodies. SpA covalently linked to Sepharose 6MB is able to fix antibody-bearing cells, while non-fixed cells are removed by washing. The fixed cells can be released by dog IgG or by mechanical treatment. As an example of the use of the technique, Ig-bearing cells were isolated from mouse spleen cells treated with rabbit IgG anti-mouse Ig antibody; the purity of the Ig-bearing lymphocytes so isolated was better than 90%. The viability and the ability of the cells to shed the antibodies from their surface were not significantly impaired by the fractionation method. The technique can be generally applied to cell separation providing IgG antibodies against specific surface markers are available.     

Identification of Cell Surface Immunoglobulin Markers by Protein A-Containing Fluorescent Staphylococci

We describe a simple method for detecting surface-associated IgG on some human lymphoid cell lines and on mouse lymphocytes. using fluorescent Staphylococcus aureus (strain Cowan 1). The adherence of the fluorescent bacteria to cell surface-located IgG is mediated by protein A (SpA) molecules on the bacterial cell wall. Inhibition of the adherence of bacteria by soluble SpA was used for quantitation of IgG on cell surface. The method has a good specificity and a high sensitivity and detects approximately 600 IgG molecules per cell.     

Fc IgM receptors on human lymphoblastoid B cell lines.

Five human lymphoblastoid cell lines have been investigated for their ability to secrete immunoglobulins (IgG, IgA, IgM) and for the presence of different cell surface markers, with special emphasis on the Fc IgM receptor, using a rosette technique with IgM-coated bovine red blood cells (EA-IgM). Four cell lines (Hu, 8432, SB, PA3) were characterized as having as cell origin due to the presence of surface immunoglobulins, complement receptors, mouse red blood cell rosette formation, low avidity Fc IgG receptors and absence of sheep red blood cell rosette formation. Two of these B cell lines (Hu and PA3) secreted IgM, two cell lines (8432 and SB) secreted IgG, while the human T cell line Molt 4 did not secrete Ig. All four B cell lines exhibited Fc IgM receptors (17–35%) while the T cell line Molt 4 had no detectable Fc IgM receptor. The receptor was specific for IgM. Neither aggregated IgG, soluble IgG immune complexes nor EDTA could abrogate the rosette formation, IgM immune complexes, pure human IgM, as well as normal human serum had an inhibitory effect on EA-IgM rosette formation. The receptor was trypsin-sensitive and required protein synthesis for expression. There was no correlation between the expression of Fc IgM receptors and secretion of any given Ig class, indicating that B cells may express Fc IgM receptors independently of their commitment to produce IgM or IgG.     

Characterization of the Fc(IgG) receptor on the pluripotential leukaemia cell K-562.

K-562 cells express an Fc receptor that is murine isotype IgG specific. The receptor was defined by rosette formation using sheep erythrocytes sensitized with murine monoclonal haemagglutinin (EA) of known isotype. Optimal rosette formation occurred at 4 degrees C or ambient temperature; however, the number of rosettes formed at ambient temperature decreased after 8 h whereas formed rosettes were stable at 4 degrees C. EA in which IgG2b was the immunoglobulin isotype gave high numbers of rosettes while IgG2a gave lower but significant numbers. Aggregated human IgG inhibited rosette formation of EA(IgG2a) more easily than those of EA(IgG2b), indicating a higher affinity of the Fc receptor for IgG2b.     

The functional activity of Fc gamma RII and Fc gamma RIII on subsets of human lymphocytes.

Subsets of human lymphocytes were isolated from peripheral blood using magnetic beads coated with anti-CD4, -CD8, -CD19 or -CD56 antibodies to yield T4, T8, B and natural killer (NK) cell suspensions with greater than 95% purity. The functional activity of Fc gamma receptor II (Fc gamma RII) and Fc gamma receptor III (Fc gamma RIII) on these subsets was assessed by measuring rosette formation with red cells sensitized with known levels of either rabbit IgG or human (monoclonal or polyclonal) IgG1 anti-D, IgG3 anti-D or IgG3 anti-c (E-IgG). Lysis of red cells by K cells (mediated by Fc gamma RIII) in antibody-dependent cell-mediated cytotoxicity (ADCC) assays was promoted by polyclonal and some monoclonal antibodies. Using these 'ADCC+' antibodies, minimum red cell sensitization levels required to promote rosette formation with NK cells were 2000 IgG1 or IgG3 molecules/red cell compared to 15,000 IgG1 or 4000 IgG3 molecules/red cell with 'ADCC-' monoclonal antibodies. The greater efficiency of ADCC+ antibodies is consistent with their previously reported ability to bind Fc gamma RIII via CH2 and CH3 domains whereas ADCC- antibodies bind only via CH3 domains. B cells formed rosettes only at high levels of sensitization: approximately 60,000 IgG1 or 20,000 IgG3 anti-D molecules/cell. These data reflect the low affinity of Fc gamma RII for monomeric human IgG. Although over 90% of NK cells bound anti-CD16, and 70% formed rosettes with red cells sensitized with rabbit IgG (30,000 molecules/cell), only 25% of NK cells formed rosettes with E-IgG3 at 100,000 IgG molecules/cell. Approximately 35% of B cells, 10% of T8 cells but no T4 cells formed rosettes with E-IgG (100,000 IgG3 molecules/cell). With T8, B and NK cells, IgG3 anti-D promoted greater rosette formation than IgG1 anti-D at comparable levels of sensitization. Presumably the longer hinge region of IgG3 enabled it to bridge the gap between negatively charged lymphocytes and red cells more efficiently than IgG1.     

Chicken anti-protein A for the detection and capturing of protein A from Staphylococcus aureus in the presence or absence of mammalian IgG.

A sandwich-ELISA for the detection of protein A from Staphylococcus aureus (SpA) is described, using chicken anti-protein A as a capture antibody and its alkaline phosphatase conjugate for the detection of bound protein A. Traditionally, protein A is detected by its binding to the Fc part of a detector antibody. This binding will be influenced by the presence of other IgG in the protein A solution. However, SpA does not react with the Fc part of chicken IgG, and it is thus possible to detect protein A in IgG containing solutions, such as eluates from protein A-columns. The method can be used to detect 1 x 10(-7) g protein A/l in the presence of serum. The method is more sensitive if no serum or IgG is present in the solution.     

Isolation of various canine leucocytes and their characterization by surface marker analysis.

Various techniques were used to separate canine peripheral blood leucocytes into populations enriched in lymphocytes, polymorphonuclear leucocytes, phagocytic mononuclear cells (monocytes) and macrophages. Surface markers on each cell population were determined by rosette formation. Fc receptors for IgG and complement receptors (C3b and C3d) were present on PMN, monocytes, macrophages as well as on a sub-population of lymphocytes. Purification of the lymphocytes into T-and B-cell-enriched populations revealed that these receptors were present only on the B lymphocytes and not on the T lymphocytes. In addition, a third lymphocyte population, which did not possess surface immunoglobulin, and Fc receptor but not the complement receptor. None of the cell populations exhibited C4 complement receptors or Fc receptors for IgM. When different cell populations were tested for their ability to form rosettes directly with human type 'O' red blood cells it was found that most populations could rosette, suggesting that this technique could not be used as a specific marker for canine T lymphocytes.     

Protein A-Specific Monoclonal Antibodies and Prevention of Staphylococcus aureus Disease in Mice

Staphylococcus aureus is a leading cause of human soft tissue infections and bacterial sepsis. The emergence of antibiotic-resistant strains (methicillin-resistant S. aureus [MRSA]) has prompted research into staphylococcal vaccines and preventive measures. The envelope of S. aureus is decorated with staphylococcal protein A (SpA), which captures the Fcγ portion of immunoglobulins to prevent opsonophagocytosis and associates with the Fab portion of V(H)3-type B cell receptors to trigger B cell superantigen activity. Nontoxigenic protein A (SpA(KKAA)), when used as an immunogen in mice, stimulates humoral immune responses that neutralize the Fcγ and the V(H)3(+) Fab binding activities of SpA and provide protection from staphylococcal abscess formation in mice. Here, we isolated monoclonal antibodies (MAbs) against SpA(KKAA) that, by binding to the triple-helical bundle fold of its immunoglobulin binding domains (IgBDs), neutralize the Fcγ and Fab binding activities of SpA. SpA(KKAA) MAbs promoted opsonophagocytic killing of MRSA in mouse and human blood, provided protection from abscess formation, and stimulated pathogen-specific immune responses in a mouse model of staphylococcal disease. Thus, SpA(KKAA) MAbs may be useful for the prevention and therapy of staphylococcal disease in humans.     

S. aureus IgG-binding proteins SpA and Sbi: host specificity and mechanisms of immune complex formation

The evasion of the host immune response is central to the pathogenicity of Staphylococcus aureus, and is facilitated by the ability of the cell wall-associated protein A (SpA) to bind immunoglobulin G Fc fragments, thereby impeding phacocytosis and classical pathway complement fixation. SpA also acts as a B-cell superantigen through interactions with the heavy-chain variable part of Fab fragments, and sequesters immunoglobulins by forming large insoluble immune complexes with human IgG. Here we show that the formation of insoluble immune complexes is mediated by the binding of (VH3+) Fab fragments in addition to Fc, and that SpA forms soluble complexes with IgG Fc fragments. We compared these results with those for Sbi, a second staphylococcal immunoglobulin-binding protein, and note that this protein requires only the Fc fragment for precipitation with human IgG. Homology models of immunoglobulin-binding domains of SpA and Sbi in complex with Fc reveal the molecular basis of the Fab-independent formation of insoluble complexes by Sbi. Finally, we compared the sequences of the spa and sbi genes from human strains to those infecting a range of animal hosts to determine whether Sbi and SpA have acquired specificity for host IgG. We note remarkable sequence conservation within the IgG-binding domains of these genes, consistent with a lack of host specificity. The Fab-independent binding of IgG by Sbi could have significant clinical implications. The use of SpA in immunoadsorption therapy can cause severe side-effects, thought to be mediated by Fc gamma R recognition and complement fixation. The formation of insoluble immune complexes with Sbi occurs only via Fc binding and free Fc regions are unlikely to be available for Fc gamma R recognition and complement fixation.     

Simultaneous detection of two different cell surface antigens by electron microscopy by means of multivalent hybrid antibody with double specificity

Multivalent hybrid antibody (MHA) complexes with dual specificity were prepared by combining two antibodies of different specificities, one against ferritin (Fer) the other against horseradish peroxidase (HRP), with protein A of Staphylococcus aaureus (SpA).Electron microscopy of mouse spleen lymphocytes and thymocytes (previously coated with mouse IgG anti-Thy-1 antibody) treated with IgG anti-Fer/SpA/IgG anti-mouse Ig complex and Fer gave better resolution and higher accuracy than previously obtained with IgG anti-HRP/SpA/IgG anti-mouse Ig complex and HRP (Mandache et al., 1980). Surface Thy-1 alloantigen and Fc receptor (charged with human IgG) treated with a mixture of IgG anti-Fer/SpA/IgG anti-Thy-1 and IgG anti-HRP/SpA/IgG anti-human Fab could be simultaneously detected on the thymocyte surface by either light or electron microscopy using Fer and HRP. The concomitant visualization of Thy-1 alloantigen (with Fer) and FcR (with HRP) on mouse thymocyte clearly shows that their distribution is largely independent and that the amount of Thy-1 antigen is greater.These results show that electron microscopy with a mixture of MHA is a useful technique for simultaneous location of two antigenic markers on the cell surface.     

Localization of the Binding Site on IgG for Solubilized Placental Fcγ Receptor

Placental Fc gamma R (FcR) inhibited the rosette formation between monocytes and rabbit IgG-sensitized erythrocytes (EA), whereas the rosette formation with granulocytes was not impaired. Staphylococcal protein A (SpA) inhibited the rosette formation with both cell types. Results obtained in absorption and agglutination experiments showed that SpA blocked the binding of FcR to IgG, and Cl did not. Furthermore, FcR did not interfere with the binding of SpA to IgG, whereas C1 affected this binding. FcR apparently bind to the C gamma 3 region. Since FcR inhibited the binding of EA to monocytes, the monocyte FcR binding site is probably also located within the C gamma 3 region.     

Interaction of anti-staphylococcal protein A antisera with Fc receptor-bearing human normal lymphocytes.

Staphylococcal protein A (SpA) is known to bind the Fc region of IgG of most mammalians and to possess biologic activity both in vivo and in vitro, where it acts as a lymphocyte polyclonal mitogen. Its binding to the Fc gamma portion bears many features of the antibody-antigen interaction, such as the dissociation constant, lattice formation, and complement activation. Moreover, SpA seems to compete with membrane Fc receptors for IgG so that the possibility of an interaction with the same CH domain(s) of IgG can be considered. In the present study, evidence is given that anti-SpA antisera obtained from chickens and rabbits are able to inhibit EA rosette formation by normal human lymphocytes and that they are able to recognize, with immunofluorescent staining, a subpopulation of normal human peripheral blood lymphocytes (PBL) that closely resembles that of EA rosette-forming cells (RFC). Moreover, the depletion of EA RFC by means of a single gradient centrifugation is accomplished by the parallel depletion of PBL stainable by anti-SpA antisera. The relevance of these results in the hypothesis of a similarity between the combining sites of SpA and membrane Fc receptor(s) for IgG is discussed.     

Modulation of Fc and C3b receptor expression on guinea-pig macrophages by lymphokines.

The decrease in Fc-receptor-positive cells that occurred during a 6 h incubation of resident and elicited guinea-pig macrophages was partly abrogated when lymphokines were present in the culture. When the same lymphokine preparations were tested on C3b receptor-expression they preferentially sustained the percentage of C3b rosettes formed by resident rather than elicited macrophages. This lymphokine-induced maintenance of Fc and C3b rosettes by cultured macrophages may have been due to an inhibition of receptor release or an increase in receptor synthesis. Supernatants from cultured macrophages contain shed Fc and C3b receptors which inhibit rosette formation by other macrophages. From the demonstration that culture supernatants from both lymphokine-treated and untreated macrophages significantly inhibited Fc and C3b rosette formation by freshly obtained macrophages it seems that the shedding of Fc and C3b receptors is not modified by lymphokines. The maintenance of Fc and C3b rosettes by lymphokines was inhibited by treatment of the macrophages with cycloheximide, suggesting that the lymphokine effect was due to an increase in synthesis de novo of the Fc and C3b receptors. The lymphokine-inducing antigens, BGG and PPD, and control lymphokine preparations were devoid of receptor modifying activity. The reduction in the percentage of Fc rosettes after 6 h culture appears to be due to a loss of Fc receptors for IgG1. Although lymphokines partly inhibited this effect they could not prevent the loss of these receptors following 24 h culture, unlike their action in augmenting the expression of Fc receptors for IgG2. These findings suggest that a selective enhancement of Fc receptor synthesis by lymphokines may modify the functional activities of macrophages.     

Mouse spleen cells and sensitized sheep red blood cells: An Fc-rosette-forming system allowing the detection of “activated” Fc structures

From a variety of Fc receptor-bearing cell/sensitized red blood cell combinations, mouse spleen cells, and sensitized SRBC were selected as an Fc-specific EA rosette assay system because only this mixture combined a high percentage (about 50%) of rosette-forming cells with complete absence of spontaneous rosettes and showed no influence of complement on the rosette formation. From studies on the minimal structural requirement of IgG both for mediation and inhibition of EA rosettes using IgG and several well-defined fragments, it appeared that both the C_H2 and the C_H3 domain of Fe are needed for optimal interaction with the lymphocyte Fc receptor. Finally, it was demonstrated that the assay system is able to detect “activated” Fc structures (here: heat-aggregated IgG) and to differentiate between varying amounts of such structures.     

Different rosette assays for detecting Fc receptor-bearing lymphocytes measure different subpopulations

The capacity of purified lymphocytes from human peripheral blood to bind the Fc portion of IgG was investigated by the rosette technique using ox erythrocytes sensitized with rabbit anti-ox IgG (EA_ox) and human erythrocytes sensitized with anti-CD IgG (EA_CD). With unfractionated lymphocytes EA_ox always detected more rosette-forming cells (RFC) than did EA_CD; however, in lymphocyte populations specifically depleted of B lymphocytes by passage through copolymer styrene bead columns, the proportion of rosettes formed with EA_ox and with EA_CD was the same. Double labelling for Fc receptors and surface immunoglobulin (SIg) demonstrated that most of the lymphocytes which formed rosettes with either EA_ox or EA_CD also carried SIg. Pre-incubation of lymphocytes at 37° to remove heatlabile SIg did not affect their ability to form EA rosettes but reduced the proportion of SIg-bearing cells. Following pre-incubation a significant proportion of EA_ox RFC still remained SIg positive but the lymphocytes which formed rosettes with EA_CD no longer carried SIg.

These studies suggest that rosette formation with EA_CD detects only `K' lymphocytes (non-T, non-B cells bearing heat-labile SIg) while EA<sub>ox</sub> detects some B lymphocytes as well. By reducing the amount of IgG bound to ox erythrocytes sensitivity was reduced to the point where EA<sub>ox</sub> no longer formed rosettes with B lymphocytes but still detected `K' lymphocytes indicating either a qualitative or quantitative difference between the Fc receptors on B and `K' lymphocytes. Treatment of lymphocytes with trypsin decreased the percentage of rosettes formed with EA<sub>ox</sub> but not with EA<sub>CD</sub> supporting the conclusion that there is a structural difference between the Fc receptors on B and `K' lymphocytes although a difference in receptor density is not excluded.

When EA_CD and fluorescein-labelled ox erythrocytes sensitized with a low concentration of rabbit anti-ox IgG were mixed, most RFC bound both the indicator erythrocytes simultaneously suggesting that the Fc receptors on `K' lymphocytes do not exhibit species specificity.

     

Red cell-linked antibody assay for detection of antigenic markers on cell surfaces.

A direct method for demonstrating antigenic markers on cell surface has been developed which makes use of the ability of bovine red blood cells coated with protein A of Staphylococcus aureus (ES) to binding IgG antibody without agglutinating. Red cell-linked rabbit IgG antibody reagents (ESA) were prepared in this way, and Ig-, IgM- and Thy-1-bearing mouse spleen and peripheral blood lymphocytes were identified by a rosette assay.     

The Disappearance of SpA-IgG Antibody Complex on 'Armed' Lymphoid and Macrophages Cells

The disappearance rate of rabbit IgG antibody complexed with protein A from Staphylococcus aureus (SpA) on 'armed' mouse and rabbit spleen lymphocytes, and on 'armed' mouse peritoneal macrophages, was investigated by rosette and antibody-dependent cellular cytotoxicity (ADCC) assays. The half time of disappearance of complex from the surface of lymphoid cells was found to be 36 min in both assays. Macrophages, 'armed' with IgG antibody complexed with SpA, bind the corresponding antigens and subsequently lyse the target cells by extracellular and/or intracellular mechanisms. 'Armed' macrophages have both ADCC and phagocytosis ability, although a short half-time of disappearance of complex was observed (8 min).