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.

     

Decreased Fc receptor expression on innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity in chronically HIV-1 infected individuals

In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection.     

Effect of two Thymosin Fraction 5 Polypeptides on Human Peripheral Blood Lymphocytes

Abstract

Thymosin fraction 5 polypeptides β4 and αl were tested for their ability to affect certain immunological parameters of human peripheral blood lymphocytes (PBL). PBL were cultured with various concentrations of the peptides for 24 hours.

Thymosin β4 was found to induce a significant decrease in the expression of the Fc receptors of PBL, as well as in their ability to express antibody dependent cellular cytotoxic (ADCC) activity. In addition, this peptide had the ability to increase the percentage of T4 lymphocytes in normal and immunosuppressed donors and to decrease the percentage of T8 positive cells in normal donors. Finally, β4 peptide caused a small increase in the capacity of peripheral blood lymphocytes to form sheep red blood cell (SRBC) rosettes (E_R). In parallel experiments thymosin αl was found inactive. The results presented here indicate that thymosin β4 may be used as an immunoregulatory molecule in patients with immunodeficiencies.     

New ultrastructural observations: Parallel tubular arrays in human Tγ lymphoid cells

T_γ cells are E-rosetting cells bearing Fc receptors for IgG (E⁺, Fc_γ⁺ cells). Third population (non-T, non-B) lymphoid cells are also Fc_γ⁺ cells and contain unique inclusions called parallel tubular arrays (PTA). Although T_γ cells and third population lymphoid cells should belong to a similar population of cells, previous ultrastructural studies on purified T_γ cells have failed to reveal the presence of PTA. In this study, we have unequivocally demonstrated PTA in the majority of T_γ cells using simple rosetting techniques. A total of 76 EA_hu-rosettes and 108 EA_ox-rosettes prepared from an E⁺ enriched fraction (using sheep erythrocytes as marker particles) were directly examined by electron microscopy. PTA were found in 87% of the EA_hu-rosettes and 82% of the EA_ox-rosettes. Ammonium chloride, commonly used in other laboratories to lyse erythrocytes during the purification procedure was found to cause a marked decrease in the number of ultrastructurally distinct PTA profiles. In contrast, hypotonic lysis had no effect on cellular ultrastructure. This study showed for the first time that T_γ cells are ultrastructurally similar to other Fc_γ⁺ lymphoid cells and contain PTA as a distinct marker. The significance of our findings to the basic function of this E⁺Fc_γ⁺ lymphoid population is discussed.     

Active and passive re-expression of Fcγ receptors on human lymphocytes

Modulation of receptors for IgG (FcγR) on human lymphocytes was induced by the interaction with erythrocyte-IgG antibody (EA_G) complexes followed by incubation at 37°C. The re-expression of FcγR could be achieved by two independent processes, (a) Active synthesis, susceptible to inhibition by puromycin or cycloheximide was shown to peak 4 to 6 h after removal of EA_G complexes; it required addition of at least 2% fetal calf serum. (b) Insertion of soluble FcγR into the membrane of modulated lymphocytes was shown to occur within 20 min of contact between cells and FcγR-containing supernatants; it was not altered by protein synthesis inhibitors. FcγR-like material, spontaneously released by unstimulated peripheral blood lymphocytes or by polymorphonuclear cells, was taken up by modulated lymphocytes. This soluble material was fully absorbed on polymerized human IgG; it was non- dialyzable, thermolabile (56 °C, 30 min) and partially destroyed by freezing and thawing; it was recovered as two broad peaks from chromatography on polyacrylamide gel; it was shown to bind to both T and non-T FcγR-bearing lymphocytes capable of forming EA_G rosettes before modulation; and it could be inserted into allogeneic lymphocytes. These results demonstrate that the FcγR structure bears two active sites, one binding to the Fcγ and the other to the surface of EA_G rosette-forming cells. Rapid release of soluble FcγR from the cells and their possible insertion into the membrane may have important implications with respect to the biological functions associated with these receptors.     

Antibody-mediated erythrolysis and erythrophagocytosis by human monocytes, macrophages and activated macrophages. Evidence for distinction between involvement of high-affinity and low-affinity receptors for IgG by using different erythroid target cells.

Antibody-dependent cellular cytotoxicity (ADCC) and Fc receptor-mediated phagocytosis were determined with human monocytes, monocyte-derived macrophages and activated macrophages, using rabbit IgG-covered sheep red blood cells (EAs) and anti-D-treated human erythrocytes (EAhu) as target cells. Monocyte and macrophage-mediated ADCC were distinguished by different kinetics, monocytes lysing either target more rapidly than macrophages. Macrophage activation by recombinant IFN-gamma (rIFN-gamma) led to a marked increase in ADCC activity against EAhu. This manifested in increased lysis of optimally sensitized target cells, in a sustained lysis of target cells carrying low antibody densities, and as an enhanced resistance to lysis inhibition by competing fluid-phase inhibition by competing fluid-phase IgG. All these effects were less striking when EAs were the target cells. Phagocytosis of EAs by rIFN-gamma-treated cells was strongly suppressed, regardless of the amount of antibody on the target cells, and susceptibility to inhibition by fluid-phase IgG was slightly increased. By comparison, phagocytosis of EAhu was depressed to a lesser degree, and susceptibility to inhibition by fluid-phase IgG was reduced when macrophages were rIFN-gamma treated. These and other experiments suggested that the functional triggering of monocytes and macrophages by EAs involved, at least in part, low-affinity Fc receptors (FcR), whereas EAhu interacted with macrophages via high-affinity FcR. It is shown elsewhere that rIFN-gamma treatment of macrophages increases the expression of high-affinity FcR, but not low-affinity FcR (Jungi, Lerch & Brcic, 1987). Differences in the rIFN-gamma-induced functional alterations assessed with EAhu or with EAs are interpreted therefore as being a consequence of differential involvement of high-affinity FcR and of low-affinity FcR in mediating an effector function. For monitoring rIFN-gamma-induced alterations in the effector capacity EAs are more appropriate targets since up-regulation of high-affinity FcR has a smaller influence on the response to this type of target. Using metabolic inhibitors, ADCC could be dissociated from ingestion suggesting that ADCC is not a post-phagocytic event.     

Identification of IgG-binding site on cell membrane

did not bind significantly ertthrocytes (E) but formed a high percentage of rosettes with bovine E sensitized by rabbit IgG (EA_G) or IgM (EA_M) antibody as well as rosettes with human E coated by human anti-CD antibody (EA_CD). Although a weak phagocytosis of untreated E was recorded both types of E were more frequently ingested upon incubation at 37°C when coated with corresponding antibodies. Attachment of EA to amoeba membrane was visualised even after incubation at 4°C or when amoeba cells were fixed with formaldehyde. Binding of sensitized erythrocytes to amoeba surface was markedly reduced when: (i) bovine E were coated with Fab fragment of rabbit IgG antibody; (ii) EA were previously treated with protein A of ; (iii) amoeba were pretreated with monomeric or polymeric IgG or its Fc fragment. It was concluded that possesses Fc-like receptors on its surface.     

Antibody-Dependent Cell Cytotoxicity (ADCC)

The effector cell(s) in human antibody-dependent cell cytotoxicity (ADCC), with antibody-coated chickens erythrocytes as targets, was studied by comparison of cell suspensions from various lymphoid organs and by means of various cell fractionation methods. Effector cells (K) were found mostly in peripheral blood, spleen, and bone marrow but not in tonsils, lymph nodes, and thymus. Effector cells bear Fc receptors and can form EA rosettes with the antibody-coated target cells. About 1,5% peripheral blood lymphocytes can form 'high-avidity' EA rosettes with targets coated at low antiserum concentration. Most of the effector cells belong to this small subset, as shown by experiments of selective depletion. Removal of most monocytes, T cells, or B cells from, or addition of T-cell-specific antiserum to, the effector cell suspensions did not affect ADCC. Effector cells in this model of ADCC therefore lack the conventional B- or T-cell markers but at least some of them are likely to bear C3 receptors.     

Identification of IgG-binding site on E.-histolytica cell membrane

$\text{E.histolytica}$ did not bind significantly ertthrocytes (E) but formed a high percentage of rosettes with bovine E sensitized by rabbit IgG (EA_G) or IgM (EA_M) antibody as well as rosettes with human E coated by human anti-CD antibody (EA_CD). Although a weak phagocytosis of untreated E was recorded both types of E were more frequently ingested upon incubation at 37°C when coated with corresponding antibodies. Attachment of EA to amoeba membrane was visualised even after incubation at 4°C or when amoeba cells were fixed with formaldehyde. Binding of sensitized erythrocytes to amoeba surface was markedly reduced when: (i) bovine E were coated with Fab fragment of rabbit IgG antibody; (ii) EA were previously treated with protein A of $\text{S.aureus}$; (iii) amoeba were pretreated with monomeric or polymeric IgG or its Fc fragment. It was concluded that $\text{E.histolytica}$ possesses Fc-like receptors on its surface.     

Evidence of ''K''-Cell Killing by Alloactivated, Fc-Receptor-Bearing Cytotoxic T Lymphocytes

Upon in vivo alloactivation of Ig-anti-Ig column-purified splenic 'T' cells in lethally irradiated allogeneic recipients, a variable proportion of donor-derived cytotoxic T lymphocytes (CTLs) are able to bind IgG antibody-coated erythrocytes through surface Fc receptors (FcR) and form rosettes. The use of fractionation procedures based on the ability of these cells to form rosettes has enabled us to separate FcR-positive CTLs from FcR-negative CTLs and to examine the ability of these two cell populations to perform as effector cells in direct T-cell-mediated killing and in antibody-dependent cellular cytotoxicity. A series of experiments, either by direct isolation of the two cell populations or by deletion of the FcR-positive population by filtration through complexed immunoglobulin columns (Ig-anti-Ig), has shown both populations to be efficient in direct T-cell mediated cytotoxicity against the relevant target cell. The striking difference between the two populations is the exclusive ability of the FcR-positive population to function as effector cells in antibody-dependent cellular cytotoxicity (ADCC). Purification steps before in vivo alloactivation of our responding cells for the removal of 'B' cells and FcR-bearing cells with 'K'-cell activity, followed by procedures to remove phagocytic and adherent cells in the resulting immune spleen cell preparation and, finally, b y velocity sedimentation of the rosetting and nonrosetting blasts from the small lymphocyte population, has resulted in a population of FcR-positive cells 98% positive for the Thy 1.2 alloantigen. These fractionation steps and immunofluorescence criteria of purity strongly favor the contention that the ADCC activity within the FcR-positive T-cell population is indeed a property of the CTL itself.     

Ligand inhibition studies on the role of Fc receptors in antibody-dependent cell-mediated cytotoxicity

Subjection of human peripheral blood lymphocytes to a temp shift from 4 to 37 degrees C resulted in a shedding of Fc receptors (termed FcRI) from 40-50% of FcR-positive cells followed by their re-expression within 4 hr; a phenomenon which had no effect on the cells' antibody-dependent killing capacity. Removal of lymphocytes having an immobile form of the Fc receptor resistant to the effects of the temp shift (termed FcRII), or removal of lymphocytes bearing both FcRI and FcRII, resulted in a similar amount of reduction in ADCC activity. This was attributed, therefore, to the loss of FcRII-positive cells. The influence of isolated (shedded) FcRI and Clq on ADCC activity was investigated. Soluble FcRI was shown to inhibit ADCC mediated through the immobile Fc receptors (FcRII), despite its lack of an ability to block EA rosette formation through these receptors. Clq also had a dose-dependent inhibitory effect on ADCC. These observations are consistent with earlier findings that FcRII possesses two active binding sites; and suggest that a prerequisite for killing in ADCC is the interaction of these with the C gamma 2 and C gamma 3 domains. The ability of synthetic peptides representative of human gamma 1-chain sequences to inhibit ADCC was determined, in an attempt to locate those sites within the IgG antibody Fc region involved in interaction with two FcR binding sites. Preliminary evidence was obtained to suggest that one of these is situated within the C gamma 2 domain, in the region of residues 274 (Lys)-294 (Glu).     

Anti‐HIV‐1 antibody‐dependent cellular cytotoxicity mediated by hyperimmune bovine colostrum IgG

Antibodies with antibody‐dependent cellular cytotoxicity (ADCC) activity play an important role in protection against HIV‐1 infection, but generating sufficient amounts of antibodies to study their protective efficacy is difficult. HIV‐specific IgG can be easily and inexpensively produced in large quantities using bovine colostrum. We previously vaccinated cows with HIV‐1 envelope gp140 and elicited high titers of anti‐gp140‐binding IgG in colostrum. In the present study, we determined whether bovine antibodies would also demonstrate specific cytotoxic activity. We found that bovine IgG bind to Fcγ‐receptors (FcγRs) on human neutrophils, monocytes, and NK cells in a dose‐dependent manner. Antibody‐dependent killing was observed in the presence of anti‐HIV‐1 colostrum IgG but not nonimmune colostrum IgG. Killing was dependent on Fc and FcγR interaction since ADDC activity was not seen with F(ab’)₂ fragments. ADCC activity was primarily mediated by CD14⁺ monocytes with FcγRIIa (CD32a) as the major receptor responsible for monocyte‐mediated ADCC in response to bovine IgG. In conclusion, we demonstrate that bovine anti‐HIV colostrum IgG have robust HIV‐1‐specific ADCC activity and therefore offer a useful source of antibodies able to provide a rapid and potent response against HIV‐1 infection. This could assist the development of novel Ab‐mediated approaches for prevention of HIV‐1 transmission.     

Fc Receptor Bearing ''Hairy Cells'' of Leukaemic Reticuloendotheliosis Bind Soluble Antigen-Antibody Complexes and Adhere to Immobilized Complexes, but Fail to Mediate Antibody Dependent Cellular Cytotoxicity

The majority of hairy cells from three patients with leukaemic reticuloendotheliosis were adherent cells bearing surface immunoglobulin, complement receptors, and Fc receptors. Highly purified populations of malignant hairy cells, which readily bound soluble antigen-antibody complexes in suspension and were able to adhere to immobilized antigen-antibody complexes, were examined for their ability to mediate ADCC. Two patients with greater than 90% FcR positive cells failed to mediate ADCC. When initially examined, a third patient, with fewer malignant cells, demonstrated a less marked impairment of cytolysis. When reexamined at a later date, this patient had an increased number of hairy cells and concomitantly demonstrated more impaired ADCC effector cell activity. Absorption of surface immunoglobulin bearing cells onto plastic surfaces coated with goat anti-human immunoglobulin resulted in a complete depletion of FcR, sIg positive, hairy cells. The remaining nonadherent fraction, containing 5.5% FcR positive, sIg negative cells, was able to mediate ADCC as effectively as the normal controls. These results indicate that although FcR bearing hairy cells readily bind soluble antigen-antibody complexes and adhere to immobilized complexes, they were unable to mediate ADCC.     

Analysis of Fcγ receptors on human peripheral blood leukocytes by flow microfluorometry. I. Receptor distributions on monocytes,Tγ cells and cells labeled with the 3A1 anti-T cell monoclonal antibody

A dual parameter flow microfluorometric technique for accurately measuring Fcγ receptor (FcR) expression on defined subsets of cells within a heterogeneous cell sample was developed. The FcR distribution of human peripheral blood mononuclear cells consists of three distinct peaks. By analyzing cells fluorescently labeled with the 3A1, an anti-T cell hybridoma antibody (using a green-emitting fluorophore) and for FcR (with a red-emitting fluorophore), and by using cell isolation procedures, it was shown that the cells lying within the peak with intermediate FcR density are mainly monocytes, while cells lying within the peaks with highest and lowest (i.e. negative) FcR densities are predominantly T cells. The FcR⁺ T cells (T_γ cells) express higher levels of the 3A1 antigen than other T cells, thus demonstrating the utility of the 3A1 hybridoma antibody as a marker for T_γ cells.     

Regulatory role of FcR+ and FcR- monocyte subsets in Mycobacterium leprae-induced lymphoproliferative response in vitro.

We investigated nine rhesus monkeys (Macaca mulatta) inoculated with Mycobacterium leprae and three normal human contacts. Peripheral blood monocytes were separated into Fc receptor positive (FcR+) and Fc receptor negative (FcR-) fractions, and their regulatory role in the lymphoproliferative response in vitro to M. leprae was studied. FcR- monocytes had strong antigen presentation activity and produced no suppressor effect while FcR+ monocytes had weak antigen presentation activity and produced a non-specific suppressor factor spontaneously. With this assay system we determined that M. leprae-inoculated rhesus monkeys could be divided into three groups: good responders, very weak responders, and non-responders.     

Re-examination of the EA rosette assay (Ripley) for Fc receptor leucocytes.

Numerous investigations has utilized rosette formation with Ripley antibody-coated human erythrocytes (EA) to identify or deplete Fc receptor-bearing K lymphocytes in whole mononuclear cell preparations. This study examines the interaction between Ripley EA and purified preparations of human lymphocytes, monocytes and neutrophils and demonstrates that this technique is not specific for K lymphocytes. Indeed, 100% of blood monocytes rosette these EA target cells. Moreover, data from both rosetting studies and antibody-dependent cytotoxicity (ADCC) reactions suggest that the avidity of Ripley EA is actually greater for monocytes than for lymphocytes. In contrast to previous reports, 100% human neutrophils were found to possess Fc receptors, as determined by their ability to rosette Ripley EA. Thus, the extent of rosetting and ADCC by all three Fc receptor-bearing leucocytes depends significantly on the degree of antibody sensitization with neutrophils requiring the greatest, and monocytes the least, amount of target-bound antibody for Fc receptor-mediated interaction.     

Two different Fc gamma receptor-dependent cytotoxic mechanisms triggered by monoclonal immunoglobulins.

It is known that the receptors for the Fc portion of IgG molecules (Fc gamma R) are widely distributed in cells of the immune system. The expression of Fc gamma R enables monocytes and neutrophils to destroy antibody-coated target cells through the antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. In addition, the interaction of immune complexes or aggregated IgG with monocytes or neutrophils led to the lysis of nonsensitized target cells in a process known as nonspecific cytotoxicity (NSC). Despite that ADCC and NSC are both triggered through Fc gamma R, the cytolytic mechanism involved in each reaction is different. In this paper we analyze the ability of human monoclonal IgG1, IgG2, IgG3 and IgG4 to induce ADCC and NSC. Our results demonstrate that each IgG subclass is able to induce both, NSC and ADCC, mediated by monocytes or neutrophils, indicating that there is no correlation between IgG subclass specificity and the ability to activate both mechanisms.     

Altered expression of human monocyte Fc receptors in Plasmodium falciparum malaria

The state of activation of human peripheral blood monocytes was examined by using a rosette assay that detects changes in Fc receptor expression. Monocytes from patients with uncomplicated Plasmodium falciparum malaria showed a significant increase in the number of rosettes relative to healthy controls. In addition, the monocytes from these patients were tested for their ability to phagocytose Candida albicans, but this ability did not differ from that of normal individuals. Finally, the monocytes from patients with cerebral malaria were also tested for Fc receptor expression. In contrast to the results from uncomplicated cases, the activity of the monocytes from these patients was no different from that of controls. We concluded that uncomplicated P. falciparum malaria caused an increase in monocyte Fc receptor expression which did not occur in cerebral malaria and that this difference in activation may be important in the pathogenesis of cerebral malaria.     

Characterization of Two Fc Receptors for Mouse Immunoglobulins on Human Monocytes and Cell Lines

We have previously reported a polymorphism in the mitogenic effect of murine (m) IgG1 anti-CD3 monoclonal antibodies. This polymorphism was genetically determined and could be attributed to polymorphism of the Fc receptor (FcR) for mIgG1 present on human monocytes. We have now extended these studies by quantitating FcR expression on monocytes and cell lines by a recently developed EA rosette assay, using the erythrocyte-associated pseudoperoxidase activity. The data show that the polymorphism of the monocyte FcR for mIgG1 is based on a quantitative rather than an absolute difference. Furthermore, this FcR is specific for mIgG1 and does not bind mIgG2a or mIgG2b nor, surprisingly, human IgG. The expression of this FcR on cell lines correlates with their accessory function in IgG1 anti-CD3-induced T cell proliferation. mIgG2a can inhibit the rosetting of monocytes with erythrocytes sensitized with human IgG. The FcR detected by this rosette technique can interact with all four human IgG subclasses but not with mIgG1 or mIgG2b. The expression of this type of FcR on human cell lines correlates well with their ability to support mIgG2a anti-CD3-induced mitogenesis. These direct measurements of FcR expression support the concept that human monocytes have two independent FcR with affinity for mouse IgG: one receptor specific for mIgG2a (which also binds human IgG), and a second specific for mIgG1.     

A Method for the Detection and Quantitation of Fc Receptor Sites on Cells

A new rosette technique for identification of Fc-receptor-bearing cells is based on the ability of sheep erythrocyte coated with protein A of Staphylococcus aureus (ES) to form rosettes with cell treated with monomeric IgG or aggregated IgG. The IgG is attached to lymphocytes through its Fc region and to a trypsin-resistant but pronase sensitive receptor (considered an Fc receptor) The ES rosette technique facilitates studies of the interaction of IgG with Fc receptor sites; the binding of any IgG preparation reacting with protein A of Staphylococcus aureus (SpA) can be studied by the technique. Inhibition of rosette formation by SpA was used for quantitation of IgG fixed to the cell surface (that is, the number of Fc reception/cell). The sensitivity of the method permits quantitation of less than 10⁵ IgG molecules bound to the Fc receptors. The relative affinity constant between Fc receptors and IgG ligands was estimated by plotting the percentage of ES rosettes as a function of IgG concentration and calculating the reciprocal of the IgG concentration giving half the maximal number of ES rosettes.