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.     

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.

     

Binding and endocytosis of erythrocytes sensitized with rabbit IgG via Fc gamma receptors of human monocytes.

The properties of the monocyte Fc gamma receptors (FcR) were investigated with monoclonal antibodies (mAb) against FcRI (10.1) and FcRII (IV3). mAb against FcRI inhibited partially the binding of sheep red blood cells (SRBC) sensitized with anti-SRBC rabbit IgG (EA) at 37 degrees to monocytes pretreated with N-ethyl maleimide, which inhibits the EA ingestion. The erythrocytes (E) were sensitized with varying concentrations of anti-E rabbit IgG. The EA binding to different FcR depends on the concentration of specific antibody used to sensitize the erythrocytes. At high levels of sensitization a high proportion of rosettes form via FcRII which can be inhibited with mAb IV3. As sensitization decreases it is more difficult for FcRII to form rosettes, so an increased percentage of them is mediated via FcRI. Sensitization of SRBC with 1-1.5 x 10(3) anti-SRBC rabbit IgG molecules per erythrocyte is the threshold to allow FcRII to mediate rosettes. At the lowest levels of sensitization the total number of rosettes is even lower and all rosettes are mediated via FcRI, hence mAb 10.1 is fully inhibitory. In addition, our data strongly support the view that the ingestion of EA takes place mainly via FcRII. We show in this study that while binding of slightly sensitized erythrocytes was blocked efficiently by mAb 10.1, the ingestion of the equivalent EA was hardly inhibited by it.     

Some parameters of the erythrocyte-antibody-complement (EAC) rosette system for recognition of bovine B-cells

A population of bovine lymphocytes formed EAC rosettes with sheep RBCs sensitized with rabbit antiserum and several species of complement. Mouse complement gave best results. Horse anti-sheep RBC serum and bovine anti-rabbit RBC serum did not support rosette formation. Optimum working concentrations of antiserum (1 : 400) and complement (1 : 20) were determined by ‘checkerboard’ titration. The occurrence of E (direct) rosettes, EA rosettes (using RBCs sensitized with rabbit antiserum but no complement), EAC rosettes and cells carrying surface immunoglobulin (sIg) (observed by a direct fluorescent antibody test) in blood and organs was observed. In a series of 33 blood samples from animals 1 week to 26 months of age the mean occurrence of these markers was: E (tested in foetal calf serum) = 2.9%; E (in CF buffer with 0.1% gelatin) = 4.3%; EA = 19.4%; EAC = 29.2%; sIg = 27.7%. The distribution of these markers in lymphoid organs of four calves and two adult heifers was examined. E rosettes occurred mostly among thymus and lymph node lymphocytes, while blood, lymph nodes and spleen contained many cells forming EA and EAC rosettes and carrying sIg. Only a few bone marrow cells carried surface markers. Cells from peripheral blood of two heifers and spleen and lymph nodes from a 3 months old calf were fractionated on nylon wool columns and contaminating macrophages and platelets were removed by treatment with carbonyl iron and adenosine diphosphate. These treatments showed that EAC and sIg cells usually occurred in the same population (presumably B-cells) and E rosette-forming cells occurred in a second, distinct population (T-cells). EA rosettes were not confirmed to B- or T-cells and their occurrence varied between organs. It was concluded that in general the EAC rosette and sIg are markers for B-cells but that probably many cell types can form EA rosettes.     

Quantitative contributions of IgG,IgM and C3 to erythrophagocytosisand rosette formation by peritoneal macrophages,and anti-opsoninactivity of dextran sulfate 500

In vitro phagocytosis by guinea pig peritoneal macrophages of immune complexes (EA) was shown to be dependent on IgG antibody in a dosedependent fashion. C3b enhanced phagocytosis of EA at limited IgG antibody concentrations only. When IgM antibody was used for sensitzation ofsheep red blood cells (SRBC), phagocytosis and rosette formation did notoccur in the absence of bound C3. The polyanion, dextran sulfate 500 (DS), was shown to depress both rosette formation and phagocytosis of EA_IgG, EA_IgGC1423 and EA_IgMC1423, as well as immune adherence of human group 0 erythrocytes and hemolytic activity of C3. This effect of DS was seen only when it was actually present in the incubation medium.     

Heterogeneity of human lymphocyte Fc receptors. I. Differential susceptibility to proteolysis

To study the possible heterogeneity of human lymphocyte Fc receptors, isolated human peripheral blood lymphocytes (PBL) were enzymatically altered (`stripped') by exposure to pronase or papain. Pronase treatment markedly increased the percentages of PBL binding IgG-sensitized erythrocytes (EA), while simultaneously removing or inactivating their receptors for heat-aggregated IgG (aggG). Papain treatment markedly diminished the ability of PBL to bind both EA and aggG. Essentially identical results were obtained utilizing EA composed of either human Rh-positive type O erythrocytes sensitized with the human anti-Rh serum Ripley (HRBC-A Ripley) or with chicken erythrocytes sensitized with rabbit anti-CRBC IgG (CRBC-A). CRBC sensitized with Fab'₂ fragments of rabbit anti-CRBC IgG were incapable of forming rosettes with normal or with pronase- or papain-stripped PBL. Pre-treatment of normal lymphocytes with aggG totally ablated their ability to rosette with EA.

Incubation of pronase-stripped PBL for 18–20 hr in 5% CO₂-air at 37°C resulted in diminution (to levels originally present) in the percentages of lymphocytes binding EA, but no regeneration of aggG receptors. Similar incubation of papain-stripped PBL resulted in significant reappearance of receptors binding EA, but no regeneration of aggG receptors. These results strongly suggest that: (1) lymphocyte receptors that bind EA complexes differ from those that bind aggG; (2) some lymphocytes possess cryptic receptors for EA that are expressed after proteolysis with pronase; (3) PBL having receptors for EA also have aggG receptors; and (4) there is no evidence that proteolytic stripping of PBL results in the generation of functionally different receptors for complexed IgG, since the Fc specificity of this receptor remains unchanged.

     

The Fc Receptors of Normal and Cancer Patients Monocytes

The ability of human monocytes from normal donors and gastric-cancer patients to form rosettes with ?0? Rh⁺(D) human erythrocytes coated with hyperimmune IgG anti-D antibody (EA_hu) and to kill the same target in antibody-dependent cellular cytotoxicity (ADCC) were assessed. Trypsin pretreatment of normal monocytes decreased their ability to form rosettes with EA_hu complexes, but their ADCC activity was unaffected. The Fc receptor (FcR) expression and ADCC activity of monocytes of cancer patients were elevated, and trypsin-treatment led to their further increase. The elevated values were related to the presence of the tumour. These results may suggest that human monocytes possess trypsin-sensitive and trypsin-resistant Fc receptors. The trypsin-resistant FcR seems to be involved in ADCC phenomenon and to be preferentially expressed on monocytes of some cancer patients.     

A Comparison of EA-Rosette Formation on Lymphocytes Using Rabbit and Human Sensitizing Antibodies

The IgG receptors of peripheral blood lymphocytes were studied in determine whether those detected by a rabbit anti-ox erythrocyte antibody (EA_Ra) and a human anti-human erythrocyte-antibody (EA_Hu) were the tame. With cells separated by Ficoll Hypaque and freed of adherent cells by exposure to plastic surface, both EA systems gave similar numbers of rosettes. Mixed rosetting occurred when both types of sensitized cells were added simultaneously. EA_Hu and EA_Ra rosettes thawed similar kinetics of formation, Depletion of rosette-forming lymphocytes was possible by using both systems, and depletion by one system showed depletion in the Other, slg-bearing lymphocytes were also removed, the amount of depletion being dependent on the degree of RBC sensitization With antibody EA_Hu and ER_Ra rosettes were equally inhibited by IgG of a given species or subclass. Normal human and rabbit IgG were more inhibitory than bovine or guinea-pig IgG. All of the human subclasses inhibited, but IgG1 and IgG3 were more inhibitory than IgG4. and IgG4. These results indicate that Fc_γ receptors on human lymphocytes react with and IgG configuration that is present on IgG of many species, although it is variably expressed among them. Human IgG1 and rabbit IgG are equivalent in the system and can be used equally well to detect Fc IgG-receptor-bearing cells.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken I. Characterization of the Fc(IgG) Receptor

The receptor for Fc(IgG) on chicken lymphoid cells was investigated by EA rosette techniques using sheep erythrocytes sensitized with a subagglutinating dose of anti-sheep erythrocyte (SRBC) chicken serum. Chicken lymphocytes did not form rosettes with SRBC coated with rabbit antibody, and human and mouse lymphocytes did not bind SRBC sensitized with chicken antibody. Only avian sera were effective in blocking the Fc receptor. Similarities between chicken and mammalian Fc receptors were demonstrated as both are pronase sensitive, trypsin resistant, and are distinct from surface immunoglobulin. Fc receptors were also distinguished from avian bursa-and thymus-specific antigens. Additional Fc receptor-bearing cells were revealed in bursa, spleen and bone marrow lymphocytes after neura-minidase treatment.     

Evidence that C1q,a Subcomponent of the First Component of Complement,is an Fc Receptor of Peritoneal and Alveolar Macrophages

Guinea pig peritoneal macrophages were cultured for 24 h in the presence of two inhibitors of the biosynthesis of collagen-like molecules such as C1q : 10^-3 M 3,4-dehydroproline or 10^-4 M 2,2′-dipyridyl. Their Fc-receptor activity was measured by rosette formation, using sheep erythrocytes (E) coated with rabbit anti-sheep IgG (EA_IgG). The Fc-receptor activity was decreased by 40 to 70% of control cultures depending on the amount of IgG on the E. The activity of a second receptor on the macrophages, mediating the binding of C3b coated E, was not altered by this treatment.Rat alveolar macrophages were depleted of their Fc-receptor activity by pronase treatment (1.5 mg/ml) in the presence of 2,2′-dipyridyl. After washing the cells, the EA_IgG-binding activity was restored to about half of the initial level within 2 h. With 2,2′-dipyridyl also present during the second incubation, the re-expression of the Fc-receptor activity was suppressed further.Preincubation of guinea pig peritoneal macrophages with anti-C1q-F(ab')₂ for 45 min at 37°C caused a dose-dependent reduction of the Fc-receptor activity, but not C3b receptor activity.These results support our hypothesis that C1q synthesized and secreted by macrophages serves as an Fc-receptor in the membrane during the secretion.     

Studies on the nature of EA binding by lymphocytes from rheumatoid arthritis patients

Investigation of the nature of the increased erythrocyte-antibody (EA) binding activity of peripheral blood lymphocytes (PBL) from rheumatoid arthritis (RA) patients reported in the preceding paper has revealed that IgG is the active class of antibody in this rosette formation. Some IgM binding also occurs. SRBC sensitized with F(ab)₂ preparations of IgG do not give rosette formation even at high concentrations. EA binding is inhibited by prior incubation of lymphocytes with heat-aggregated human IgG but antigen-antibody complexes did not give significant inhibition.The majority of these rosettes were found to be stable at 4°C and room temperature but labile at 37°C.Enzyme studies with pronase, trypsin, neuraminidase and treatment with sodium azide gave results strongly supporting the conclusion that the increased binding observed is increased Fc-receptor activity. This activity appears not to be a result of Fc binding by cell-bound rheumatoid factor.A range of titres of antibody and of IgG was used to sensitize erythrocytes to form EA and the enhanced EA-rosette formation by PBL from RA patients occurred throughout the range of concentrations of sensitizing antibody. Significantly more EA were bound by individual lymphocytes from RA patients than control subjects. This data suggest that the Fc receptors on RA lymphocytes are more avid for EA than receptors on lymphocytes from healthy people.     

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.     

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.     

Specificity of Fcγ Receptors in Human Malignant Tissue and Normal Lymphoreticular Tissue

The specificity of the Fcγ receptors in normal spleen and liver and in malignant tissues was studied using hemadsorption to cryostat sections. Indicator cells (EA) were sheep erythrocytes (E) sensitized with rabbit IgG antibody (A). The binding of EA to sections of normal and malignant tissues was inhibited by pooled IgG of human, rabbit, and guinea pig origin and by human IgG1, and IgG3, and IgG4 myeloma proteins. Heat-aggregated IgG inhibited the binding to sections of liver and some malignant tissues more effectively than monomeric IgG. The Fc fragments of IgG were also inhibitory, but not the F(ab')₂, Fab', and Facb fragments. The inhibition obtained increased with decreasing amounts of A used for sensitization of E. The inhibitory activity of IgG was abolished after partial reduction and alkylation. No inhibition was obtained with IgG2, IgM, IgA, or albumin. E sensitized with Facb or F(ab')₂ fragments of A did not bind to normal or malignant tissues. The specificity of the Fc receptors in normal spleen and liver and in malignant tissues is apparently very similar.     

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.     

Heterogeneity of human lymphocyte Fc receptors. II. Relationship to antibody-dependent, cell-mediated cytotoxicity

Human peripheral blood lymphocytes (PBL) were incubated (stripped) with pronase or papain and compared with unstripped lymphocytes for their ability to mediate antibody-dependent, cell-mediated cytotoxicity (ADCC). Despite marked removal or inactivation of receptors for heat-aggregated IgG (aggG) by proteolytic digestion, and pronounced changes in the percentages of cells rosetting with IgG-sensitized erythrocytes (EA) (decreased by papain, increased by pronase), stripped PBL functioned normally in ADCC.

Stripped and unstripped lymphocytes were pre-treated with aggG to determine the role of aggG receptors in ADCC. AggG almost totally abolished ADCC by unstripped PBL, but inhibited ADCC by enzyme-stripped lymphocytes relatively poorly. Neither untreated nor stripped PBL were able to induce cytotoxicity of chicken erythrocyte (CRBC) target cells sensitized with the Fab'₂ fragment of anti-CRBC IgG antibody (CRBC-A).

Exposure of PBL to EA monolayers composed of CRBC-A or of sheep erythrocytes (SRBC) sensitized with rabbit anti-SRBC IgG antibody (SRBC-A) depleted PBL of cells that rosetted with CRBC-A and with human Rh-positive, type O erythrocytes sensitized with the human anti-Rh serum Ripley (HRBC-A Ripley). Non-adherent cells were incapable of binding aggG and had markedly diminished cytotoxicity in ADCC. Similarly, exposure of PBL to HRBC-A Ripley monolayers resulted in non-adherent cells that were incapable of rosette formation with HRBC-A or CRBC-A, failed to bind aggG, and exhibited significantly diminished ADCC activity.

These studies indicated that: (1) cytotoxic effector PBL active in ADCC (K cells) have receptors for aggG and for EA; (2) PBL deficient in functional aggG receptors (enzymatically inactivated or removed) are capable of inducing normal levels of ADCC; (3) aggG and EA receptors appear to be closely associated on native K-cell membranes; (4) there is no clear-cut relationship in a given lymphocyte population between the presence of either aggG or EA receptors and ADCC activity; and (5) populations of PBL binding HRBC-A Ripley overlap with, and may be identical to, those binding aggG and other types of EA complexes.

     

Purification of a functional 40 kD human placental Fc gamma-receptor using a monoclonal antibody

F(ab')2-fragments of a mouse monoclonal antibody (B1D6) reacting with placental receptors for the Fc part of IgG (FcR) were used as affinity reagents for the purification of an antigen from placental extract (PE). The antigen agglutinated ovine erythrocytes (E) sensitized with rabbit antibodies (A) (EA), but not E or E sensitized with F(ab')2-fragments. It reduced the EA rosette-formation with mononuclear cells and the binding of soluble immune complexes to placental tissue. The antigen bound to aggregated IgG and Fc-fragments of IgG, but not to native IgG or F(ab')2-fragments of IgG. The data indicate that the purified antigen possesses FcR activity with low affinity for IgG. SDS-PAGE and Western blot showed one distinct band of approximately 40 kD. The electrophoretic mobility did not change after reduction and the band reacted with concanavalin A indicating that the FcR are single-chained glycoproteins.     

Peritoneal exudate T lymphocytes with specificity to sheep red blood cells. IV. Fc receptors on specific peritoneal exudate lymphocytes and their role in delayed-type hypersensitivity reactions.

In mice, delayed-type hypersensitivity (DTH) to sheep red blood cells (SRBC) is mediated by T cells. Peritoneal exudate T cells (PETLs) from mice optimally sensitized for DTH to SRBC form rosettes when interacted with sensitized sheep red blood cells (EA). The binding of EA to PETLs is mediated by a receptor specific for the Fc portion of the antibody (FcR). Biological activity (mediation of DTH) depends on the unreacted state of PETLs and is lost when the latter are either rosetted with EA or reacted with aggregated IgG. Transfer of EA or aggregated IgG-treated PETLs from mice with DTH to SRBC does not lead to adoptive sensitization of recipients. It is suggested that FcR found on the membrane of T cells mediating DTH play a role in the regulation of the cellular immune response to SRBC.     

Expression of a receptor for IgM by human T cells in vitro

The capacity of human peripheral blood lymphocytes (PBL) to bind antigen-IgG antibody (EA(IgG)) and antigen-IgM antibody (EA(IgM)) complexes was investigated using a rosette technique with ox erythrocytes coated with rabbit IgG or IgM antibody. It was found that while EA(IgG)-rosette-forming cells (RFC) were detected on PBL freshly drawn from normal individuals, EA(IgM)-RFC were present only in suspensions kept in culture for 24 h in mediá supplemented with sera containing very low or no amounts of IgM. Experiments of simultaneous detection of EA(IgG)-RFC or EA(IgM)-RFC and other membrane markers for human T or B cells together with experiments on purified T or B cell populations indicated that EA(IgG)-RFC were formed by both T and B cells, while T cells only were capable of EA(IgM) rosette formation. The specificity of the receptors for IgG and IgM was determined by studying the inhibitory capacity of purified human IgM and IgG in the rosette assay. The receptor for IgG was inhibited by IgG and not by IgM, while the reverse was true for the receptor for IgM.