Which Fc receptor-bearing cells are detected with the Ripley assay?

Mononuclear cells were isolated from peripheral blood and analysed with T and B markers (E rosettes and SIg) and on contaminating monocytes and PMN. The suspensions contained 63.3 +/- 4.8% T lymphocytes, 11.4 +/- 3.8% B lymphocytes, 9.0 +/- 5.5% null lymphocytes, 15.1 +/- 3.8% monocytes and 1.1 +/- 0.6% PMN. Of all cells, 27.6 +/- 12.1% formed EA rosettes with OR1R2 red cells coated with anti-CD Ripley. In preparations fixed after cytocentrifugation, the EA rosette-forming cells were studied with regard to esterase activity. The proportion of cells with detectable Fc receptors was further studied in purified T lymphocyte and in monocyte suspensions. Finally, EA rosettes were isolated by gradient centrifugation and the rosette forming cells studied with conventional T and B markers. All procedures gave corresponding results: on average 11-14% of the T lymphocytes and nearly 100% of the null cells formed EA rosettes, while only 2% of the B lymphocytes had detectable Fc receptors. Of the purified monocyte and PMN populations, on average 72 +/- 10.5 and 14.5 +/- 5.4%, respectively, formed EA rosettes. Thus, the Ripley assay, representing an important marker for null lymphocytes, cannot be regarded as specific for this population of white blood cells.     

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.

     

Fine specificity of a rabbit antibody interacting with human IgG Fc receptor-like molecules

A polyclonal rabbit antibody raised against an Fc receptor (FcR)-like membrane glycoprotein fraction of chronic leukaemic lymphocytes has previously been prepared and partially characterized. This antibody, called AbA, was found to precipitate a 70-kDa and a 45-kDa fraction of the detergent lysate of U937 cells and to inhibit ligand binding to Fc gamma R on the P388D1 murine macrophage cell line. In the present work we have characterised this antibody further. All Fc gamma RII-positive B lymphoblastoid cell lines, as well as resting human B lymphocytes, were positively stained with the AbA antibody. U937 cells were found to be negative, but after stimulation with phorbol ester (PMA), 50% of the cells became positive. AbA antibody did not react with human T cell lines or with the T + 0 cell subset of peripheral blood. Monocytes were also negative. On the other hand, AbA antibody exhibited a dose-dependent inhibition of antibody-mediated cytotoxic reaction (ADCC) of monocytes, while not affecting K cell-mediated ADCC. It had an inhibitory effect of EA rosette formation of B cells and stimulated U937 cells. Furthermore, it interacted with the soluble form of Fc gamma RII released by activated B lymphocytes, and--similarly to IgG--precipitated a 33 kDa fraction from the supernatant of B cells.     

Evidence for antibody-dependent cell-mediated cytotoxicity by T cells bearing receptors for IgG.

Human lymphocyte populations comprising T cells, T depleted lymphocytes, and T cells enriched for, or depleted of, IgG Fc receptor-bearing (TG) cells, were separated using rosette techniques. All lymphocytes were assessed for the ability to lyse antibody-coated chicken erythrocytes and SL2 mouse lymphoma cells. Their activity was compared with that of monocytes and neutrophil-enriched preparations. IgG Fc receptor positive cells within the T population were highly active in both cytotoxicity assays; the activity could not be ascribed to contamination by monocytes or neutrophils. The TG cells forming junctions with the target cells possessed a characteristic ultrastructure.     

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.     

Characterization of the human peripheral blood effector cells mediating antibody-dependent cell-mediated cytotoxicity against respiratory syncytial virus

Peripheral blood lymphocytes were separated into several subpopulations and evaluated for their ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) against respiratory syncytial virus (RSV)-infected HeLa cells. Using erythrocyte rosetting methods, nylon wool filtration, and cytolysis with OKT-3 monoclonal antibody, two lymphocyte subpopulations were shown to mediate RSV-ADCC; non-T, non-B, and IgG-Fc receptor-bearing lymphocytes and E-rosetting cells with IgGFc receptors (T gamma cells). Removal of phagocytic cells did not alter ADCC activity. Monoclonal antibody to human NK and K cells, HNK-1, recognized these two lymphocyte effector subpopulations.     

Lack of evidence for IgM-induced ADCC: studies with monoclonal and polyclonal antibodies.

Different kinds of IgM antibodies were tested for their activity in antibody-dependent cellular cytotoxicity (ADCC): firstly an anti-benzylpenicilloyl (BPO) IgM antibody from immune rabbit serum purified by affinity, ion exchange, and molecular-sieving chromatography, secondly two monoclonal rat anti-BPO IgM antibodies and thirdly a human antidextran antibody prepared from a patient showing restriction of anti-dextran antibodies to the IgM class. Human lymphocytes or purified monocytes served as effector cells. While the two monoclonal rat and the human IgM antibodies showed no ADCC-mediating capacity, ADCC was induced by the rabbit anti-BPO IgM antibody when high antibody concentrations were used. This activity was abolished by further purification using an anti-rabbit IgG (Fc) immunosorbent. The initially observed activity was shown to be likely due to traces of aggregated anti-BPO IgG, which cannot be detected by the methods commonly used. Preincubation of lymphocytes for 24 hr increased the number of EA (IgM)] rosette forming cells but failed to induce IgM-mediated ADCC. Furthermore, evidence for amplification of low-dose IgG-ADCC by IgM could not be found.     

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.     

Natural killer cells in guinea pig spleen bear Fc receptors

The phenomenon of natural cytotoxicity or spontaneous cell-mediated cytotoxicity (SCMC) was investigated in guinea pigs and compared with two other in vitro cytotoxicity reactions: mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC). The same xenogeneic target cells were employed in all three cytotoxicity assays. Organ distribution studies revealed that SCMC effector cell activity was present in spleen and peripheral blood but not in thymocytes. Bone marrow cells possessed low levels of SCMC effector cell activity. The organ distribution of effector cell activities for MICC and ADCC paralleled that for SCMC. Studies of the cell surface characteristics of the SCMC effector cell revealed that spleen cells nonadherent to antigen-antibody but not to antigen-F(ab')₂ antibody-coated surfaces possessed markedly reduced SCMC effector cell activity. In addition, spleen cells depleted of Fc receptor-bearing cells by EA rosetting also possessed diminished SCMC effector cell activity, while cell populations enriched in Fc receptorbearing cells by EA resetting possessed enhanced SCMC effector cell activity. These fractionation techniques had similar effects on MICC and ADCC effector cell activity. Depletion of adherent spleen cells including macrophages by nylon wool column passage resulted in a population of cells with enhanced SCMC, MICC, and ADCC effector cell activity. Thus, in guinea pig spleen the effector cells mediating SCMC were shown to belong to a population of nonadherent Fc receptor-bearing lymphocytes possessing several cytotoxic capabilities including the potential of mediating MICC and ADCC.     

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).     

A new photometric method for the quantitation of Fc receptors for murine IgG1 on human monocytes and cell lines

We have previously shown a polymorphism of human Fc receptors for mouse IgG1 using an EA rosette technique in which human erythrocytes sensitized with a murine IgG1 monoclonal antibody against glycophorin A acted as indicator cells. We now describe a method to quantitate this EA rosetting using the pseudoperoxidase activity present in erythrocytes. This photometric assay allows the sensitive quantitative determination of Fc receptor expression on human monocytes and cell lines. Not only the human Fc receptor for murine IgG1 can be studied in this way, but the method can also be applied to other Fc receptors. An important factor in this type of rosette assay appears to be the amount of negative charge present on the surface of the indicator erythrocytes. Using alcian blue as a probe, we found that this negative charge is higher on human erythrocytes than on sheep erythrocytes, which may contribute to a better signal-to-noise ratio. The method described facilitates the characterization of Fc receptors and permits the rapid screening of monoclonal anti-Fc receptor antibodies.     

Immune complexes in Takayasu''s arteritis.

We examined sera and Fc receptor-bearing lymphocytes from peripheral blood of patients with Takayasu's arteritis for the purpose of investigating the presence of immune complexes (IC). IC in sera were assayed by solid-phase conglutinin-binding test. Seven of 29 patients exceeded the normal range of circulating IC. IC combined with Fc receptors were estimated by enumerating EA-RFC. EA-RFC of lymphocytes from patients with Takayasu's arteritis were 13.0% and those of normal controls were 29.1%. Low EA-RFC in the patient group increased significantly when lymphocytes were incubated with EA after rising lymphocytes with medium at 37 degrees C. On the contrary, EA-RFC from healthy subjects did not increase after rinsing cells. These findings indicate that IC combined with Fc receptors and hindered EA rosette formation and that rinsing cells with medium at 37 degrees C removed IC from Fc receptors. Comparable results were obtained by a membrane immunofluorescence method using FITC-conjugated anti-human immunoglobulin. In order to confirm that EA rosette formation was really blocked by IC, lymphocytes from a healthy donor were incubated with heat-aggregated human IgG. Incubating cells with IgG aggregates caused reduction of EA-RFC and these lymphocytes restored their ability to form rosettes with EA by rinsing cells with medium at 37 degrees C. In conclusion, we could confirm the presence of IC both in sera and on lymphocyte Fc receptors in some cases of Takayasu's arteritis.     

Shedding of Fc receptor from mononuclear cells and their ability of binding IgG1 and IgG3 anti-Rh/D antibodies

Fc receptors for IgG1 and IgG3 on peripheral blood lymphocytes and monocytes were studied before and after temperature shift from 4-37 degrees C. The investigations were performed in the EA test using human erythrocytes sensitized with anti-Rh/D/antibodies of IgG1 (EA IgG1) and IgG3 (EA IgG3) subclasses. It occurred that lymphocytes and monocytes were able to bind IgG1 and IgG3 antibodies before and after shedding, however, lower percentage of rosette was observed after temperature shift. This decrease was similar in the EAIgG1 and EAIgG3 tests. The supernatants obtained during shedding occurred to contain active Fc receptors since the inhibition of rosette formation was obtained after the incubation of sensitized erythrocytes with these supernatants. IgG1 as well as IgG3 myeloma proteins inhibited rosette formation in both EAIgG1 and EAIgG3 tests. Our data might suggest that IgG1 and IgG3 anti-D antibodies are able to bind to the same Fc receptor on lymphocytes as well as on monocytes.     

Action of sphingomyelinase C and other lipid-specific agents as inhibitors of Fc binding and locomotion in human leucocytes.

The binding of antibody-coated chicken erythrocytes (EA) to human blood lymphocytes and monocytes is inhibited by pretreatment of the leucocytes with sphingomyelinase C. Inhibition of rosetting of neuraminidase-treated EA with neutrophils is also seen with this enzyme. The cholesterol-binding theta-toxin of Clostridium perfringens and pronase also inhibit EA-rosette formation, but less strongly than sphingomyelinase. The lipid-specific agents also inhibit chemotactic migration of leucocytes to casein and denatured HSA, whereas proteases and glycosidases do not. These results suggest that membrane lipids are important constituents of the binding sites for Fc fragments and for certain chemotactic factors and point to an important role for sphingomyelin in this binding.     

Identification and distribution of immunocompetent cells in inflamed gingiva of human chronic periodontitis

Advanced human periodontitis is considered to be a B-cell lesion, but the cellular infiltrate contains several cell types, the distribution of which has not been determined. This experiment was designed to characterize and identify the immunocompetent cells on histological sections and in eluates from diseased human gingiva. Immunoglobulin-bearing cells were detected on histological sections by direct immunofluorescence with F(ab')2 antisera monospecific for human immunoglobulin G (IgG), IgA, or IgM. Plasma cells predominated in the central portion of the lamina propria, with the proportions positive for IgG, IgA, and IgM accounting for 65.2 +/- 9.5, 11.2 +/- 1.1, and 1.3 +/- 1.1% of the total infiltrating cells, respectively. T lymphocytes, identified by indirect immunofluorescence with monoclonal antibody (Leu-1) against human T cells, accounted for 29.3 +/- 10.0% of the total infiltrated cells. Most of the T cells were located subjacent to the pocket epithelium, but there were a few in the central lamina propria. Similarly, Fc receptor-bearing cells detected by EA rosetting and macrophages and monocytes detected by nonspecific esterase staining with alpha-naphthylbutyrate esterase were also localized to the region immediately subjacent to the pocket epithelium. Infiltrated cells were harvested from minced gingival tissue after digestion with collagenase, hyaluronidase, and DNase. The eluates contained 35.3 +/- 6.0% T lymphocytes, 30.0 +/- 14.9% Fc receptor-bearing cells, and 12.9 +/- 4.4% monocytes and macrophages. Whereas T gamma cells comprised 13.3 +/- 1.4% of peripheral blood T cells, they accounted for only 6.0 +/- 2.0% of the eluate T cells. In contrast, T mu cells accounted for 44.7 +/- 4.9% of the T cells in the eluates and 51.6 +/- 4.4% in the peripheral blood. The decreased proportion of T gamma cells in the gingiva may indicate a form of abnormal immune regulation concerned with T suppression of B-cell proliferation.     

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.

     

Fc receptor for shark IgM

Fc receptors for shark IgM have been demonstrated on shark leukocytes. Measurement of receptor binding required treatment of leukocytes with Cytochalasin D to inhibit phagocytosis. EA rosetting assays were carried out using human erythrocytes coated with shark anti-human antibody. Binding to shark leukocytes was demonstrated to be specific to shark IgM in that affinity purified shark IgM and purified Fc5 mu fragments could block rosette formation, but not shark transferrin, bovine serum albumin or fetal bovine serum. The binding was shown to be saturable and reversible, characteristic of receptor-ligand interaction. Further, it was shown that affinity purified, radioiodinated IgM could also bind Cytochalasin D-treated shark leukocytes in a manner analogous to rosetting. We conclude that Fc receptors appeared early in evolution, and that previous difficulties in demonstrating the Fc mu receptor resulted from non-specific binding associated with phagocytosis.     

Methods for the identification of human B lymphocytes.

Complement-receptor lymphocytes and monocytes were identified by a rosette method using trypsin treated sheep erythrocytes (Et), sensitized with affinity column purified IgM (19S) antibodies against sheep erythrocytes (E) and mouse complement deficient in C5. Fc receptor mononuclear cells were identified by a rosette method using Et and IgG (7S) antibodies against E. Identification of the cell-type rosetting was facilitated by myeloperoxidase staining of dry mounted rosetted preparations. Comparison of lymphocytes with receptors for activated mouse complement and lymphocytes with stable surface immunoglobulin detected by immunofluorescent assay, strongly suggests that these cells constitute the same population in the peripheral blood of healthy humans.     

Fc Receptor-Bearing Lymphoid Cells in the Chicken II. Relative Increase of Fc(IgG) Receptor Bearing Cells in Obese Strain Chickens

An EA rosette technique is used to study ontological development and organ distribution of Fc(IgG) receptor-bearing lymphoid cells in normal CS White Leghorn chickens, and in OS chickens with spontaneous autoimmune thyroiditis. During the embryonic period, no difference was seen between CS and OS in the tissue distribution of cells with Fc receptors. At the time of hatching and subsequently, the OS chickens possessed relatively more Fc receptor-bearing lymphoid cells than did CS chickens. The increase of Fc receptor-bearing lymphoid cells was most prominent among spleen cells. No difference in the affinity of Fc receptors between lymphocytes of the OS and CS chickens was demonstrated. The possible role of Fc receptor-carrying cells in the development of autoimmune thyroiditis is discussed.     

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.