Activation of monocytic cells by monoclonal antibodies to the CD11a/18 (LFA-1) complex: mediation by Fc receptor.

IgG1 antibodies reacting with several monocytic antigens form a bridge between the specific antigen and the Fc receptors also expressed on these cells. This results in calcium mobilization and generation of superoxide. Single IgG1 antibodies reacting with the CD11a/CD18 cellular adhesion molecular complex do not, however, induce monocytic activation. This is not because they induce a negative signal, as the response to formyl-methionyl-leucyl2-phenylalanine (FMLP) or direct cross-linking of FcRII is not inhibited. Furthermore, a combination of an intact CD11a and CD18 antibody does induce a rise in intracellular calcium and production of superoxide. This activation is dependent on the binding of the Fc portion of both antibodies to Fc receptors, as F(ab')2 fragments do not cause activation. This suggests that simultaneous binding of opsonized bacteria to cellular adhesion molecules and to Fc receptors on monocytes would facilitate activation of these cells. Furthermore, it illustrates the importance of using F(ab')2 fragments in the analysis of signal transduction molecules on Fc receptor-bearing cells.     

Fc gamma receptor recognition of IgG ligand by human monocytes and macrophages

We examined the binding characteristics of human monocytes and macrophages with the IgG ligands, human monomeric IgG and a small human IgG aggregate, trimeric IgG. Our purpose was to utilize fresh monocytes, in vitro cultured monocytes, and alveolar macrophages in direct and indirect binding experiments. Freshly isolated monocytes expressed only a single binding site for IgG monomer and IgG trimer. In contrast, in vitro cultured monocytes, gamma-interferon-treated monocytes, and freshly isolated alveolar macrophages expressed a single binding site for IgG monomer and, in addition, a high and low affinity binding site for IgG trimer. The high affinity binding site for IgG trimer (Kd approximately equal to 1 nM) appeared identical to the binding site for IgG monomer. The low affinity binding site for IgG trimer (Kd = 50 to 250 nM) appeared to be due to Fc gamma RII, because antibody to Fc gamma RII inhibited its expression. Since Fc gamma RII, in contrast to Fc gamma RI, does not bind monomeric IgG, the data suggest that this low affinity receptor for trimeric IgG, Fc gamma RII, can bind low molecular weight circulating immune complexes at concentrations 10- to 100-fold lower than Fc gamma RI. Thus, these studies suggest that at 37 degrees C, macrophage Fc gamma RII may play a functional role in the recognition of small molecular weight immune complexes.     

IgG on Infants'' B Lymphocytes: Enhanced Binding of IgG by IgM-Bearing Lymphoid Cells in Early Childhood

IgM/IgD-bearing lymphocytes (B cells) from children in the first few weeks of life were found to also have surface IgG, unlike most normal adult B cells. The IgG was loosely bound to the lymphocyte surface and was partially or completely removed by incubation at 37 degrees C or by trypsinization. When F(ab')2 antisera were employed, very few infant B cells had surface IgG, although the IgM staining was similar to that obtained with the native antisera. IgM/IgD-positive cells bound IgG anti-Rh-coated (Ripley) erythrocytes, unlike most adult B lymphocytes. Capping experiments suggested that an Fc receptor on the cells could be redistributed by the anti-IgM-surface IgM complex. These data indicate that, during infancy, B-lymphocyte receptors for IgG are of altered affinity, frequency, or availability compared with adult B-lymphocyte Fc receptors and resemble the Fc receptors found on "third population" (Fc + Ig-) mononuclear cells and monocytes.     

Comparison of type III Fc receptors associated with group C and group G streptococci

The type III Fc receptors present on or secreted by a series of group C and G streptococcal strains were studied. All strains capable of binding radiolabeled human IgG were shown to do so via an antigenically related Fc receptor. Treatment of any of the bacterial strains with papain or trypsin resulted in solubilization of Fc receptor activity. The pattern of Fc receptor activity recovered following enzyme treatment was not uniform. Differences were observed both between group C and G strains as well as within group C and G strains. Analysis of secreted Fc receptors indicated the presence of five molecular forms of Fc receptor. Each form was present at some level in the supernatant of every group C and G strain studied. The relative concn of each form of receptor secreted varied from strain to strain. The Fc receptor activity secreted by each strain demonstrated a similar affinity for the Fc region of human IgG and all were antigenically related. These results suggest that there is a family of closely related Fc receptors associated with group C and G streptococcal strains.     

Isolation and Characterization of Type IIa and Type IIb Fc Receptors from a Group A Streptococcus

Certain group A streptococcal strains have been reported to express two distinct type II receptors that bind to the Fc region of human IgG. In this study, we have isolated and characterized these two type II Fc receptors and characterized their reactivity with differing species of IgG. The type IIa receptor was found to be a 56,000 molecular weight protein which binds human IgG1, IgG2 and IgG4, in addition to pig and rabbit IgG. The type IIb receptor was found to be a 38,000 molecular weight protein that bound exclusively to human IgG3. Neither the type IIa nor the type IIb receptor bound to goat, cow, dog, rat, or sheep IgG. Monospecific polyclonal antibodies were prepared against both the type IIa and type IIb Fc receptors. These antibodies demonstrated that the type IIa and type IIb were antigenically closely related and could not be distinguished from each other on the basis of their reactivity with either antibody. The distribution of type IIa and type IIb Fc receptors on a variety of different nephritogenic and non-nephritogenic group A streptococcal strains was documented.     

Bovine IgG can aggregate at conditions simulating pasteurization and binds to some human Fc gamma receptors

The ability of bovine IgG preparations to bind to the various distinct human leukocyte Fc gamma receptors was studied. In experiments using intact cells and isolated Fc gamma receptors, it was demonstrated that bovine IgG can bind to Fc gamma receptors of four human cell types but not to Fc gamma receptors of human neutrophils. 125I-labeled Fc gamma receptors purified on human IgG-Sepharose columns from human B and T lymphocytes, monocytes and eosinophils were able to rebind specifically to insolubilized bovine IgG. In contrast, radioiodinated human neutrophil Fc gamma receptors did not rebind to bovine IgG-Sepharose. A similar pattern of specificity was demonstrated in studies of the binding of 125I-labeled heat-aggregated bovine IgG to various human leukocyte populations. The labeled aggregated bovine IgG bound to peripheral blood mononuclear cells, to B cells from chronic lymphocytic leukemia patients and to macrophage-like U-937 cells, but bound poorly to normal human granulocytes. Labeled non-aggregated bovine IgG was not appreciably bound to any of the cell populations. Since bovine IgG in dietary sources is frequently exposed to heat, the effect of heating on the physical state and Fc-binding properties of bovine IgG was examined. The data show that heating bovine IgG at concns of 0.9-3.6 mg/ml at 63 degrees C for 30 min in neutral buffer causes aggregation of bovine immunoglobulin (10-16% aggregation) and increases the ability of bovine IgG preparations to bind to human Fc gamma receptors of intact cells. Gel filtration studies suggest the possibility that bovine IgG may also be aggregated during the pasteurization of raw milk.     

Suppression of mast cell degranulation through a dual-targeting tandem IgE-IgG Fc domain biologic engineered to bind with high affinity to FcγRIIb

Mast cells and basophils play a central role in allergy, asthma, and anaphylaxis, as well as in non-allergic inflammatory, neurological and autoimmune diseases. Allergen-mediated cross-linking of IgE bound to FcεRI leads to cellular activation, and the low-affinity Fc receptor FcγRIIb is a key inhibitor of subsequent degranulation. FcγRIIb, when coengaged with FcεRI via allergen bound to IgE, stimulates ITIM domain-mediated inhibitory signaling that efficiently suppresses mast cell and basophil activation. To assess the therapeutic potential of directed coengagement of FcεRI and FcγRIIb in the absence of FcεRI crosslinking, we developed a fusion protein comprising the coupled Fc domains of murine IgE and human IgG1. As a key functional component of this tandem Fcε-Fcγ biologic, we engineered its IgG1 Fc domain to bind to human FcγRIIb with 100-fold enhanced affinity relative to native IgG1 Fc. Using mast cells from mice transgenic for human FcγRIIb, we show that this tandem Fc binds with high affinity to murine FcεRI and human FcγRIIb on mast cells, triggers phosphorylation of FcγRIIb, and inhibits FcεRI-dependent calcium mobilization. Control tandem Fc biologics containing a native IgG1 Fc domain or lacking binding to Fcγ receptors were markedly less active, demonstrating that the affinity-optimized tandem Fc can inhibit degranulation through stimulation of FcγRIIb signaling as well as through competition with allergen-IgE immune complex for FcεRI binding. We propose that in the context of a fully human tandem Fc biologic, high-affinity coengagement of FcεRI and FcγRIIb has potential as a novel therapy for allergy and other mast cell and basophil-mediated pathologies.     

Characterization and structural analysis of Fc gamma receptors of human monocytes, a monoblast cell line (U937) and a myeloblast cell line (HL-60) by a monoclonal antibody

A monoclonal antibody, FR51, raised against the IgG Fc receptor (Fc gamma R) of the human monoblast cell line U937 was used to analyze the distribution of this antigen on various human cells. This antibody inhibited the binding of human IgG to the Fc gamma R on U937 cells, HL-60 cells and human peripheral blood monocytes. In contrast, the Fc gamma R on human granulocytes (neutrophil cells) and on an Epstein-Barr virus-transformed human lymphoblastoid cell line (Raji) were not recognized, indicated by the failure of blocking the binding of human IgG ligand to the Fc gamma R on these cells. By affinity chromatography of detergent-containing cell free lysates of surface-iodinated U937 cells, HL-60 cells and monocytes, a protein of 70-kDa was isolated. This protein was identified as the Fc gamma R by rebinding the isolated protein to immobilized human IgG. Removal of the carbohydrate moiety with endo-beta-N-acetylglucosaminidase F demonstrated that the receptors consist of a 40-kDa polypeptide. Analysis of the polypeptide patterns obtained by proteolytic digestion of either mature (70-kDa) or deglycosylated (40-kDa) receptors isolated from monocytes, U937 cells and HL-60 cells strongly suggests that the Fc gamma R are identical. The monoclonal antibody FR51 specifically reacts with Fc gamma R on human monocytes, a myeloblast and a monoblast cell line but not with the receptors on a B cell line and neutrophil cells.     

Comparison of the Binding of Radiolabelled Human IgG and Fc Fragments to Murine Spleen Cells

The binding properties of an IgG1 human myeloma protein, normal IgG, and the Fc and Fab fragments of each were compared in cultures of murine spleen cells. Both 125I-labelled IgG and Fc fragments bound to splenic lymphocytes, whereas Fab fragments did not bind significantly at the highest concentrations tested. On a molar basis, more Fc bound than intact IgG. According to Scatchard plot analysis, the affinity constand of IgG1 was 1.5 x 10(6) +/- 1 x 10(5) L/M and that of the Fc fragments was 7.8 x 10(5) +/- 2.6 x 10(5) L/M. Approximately 25,000 binding sites/cell were calculated for IgG1 and 102,000 for Fc. Deaggregation of the Fc preparation did not change these values, suggesting that the difference in binding of IgG and Fc did not result from Fc aggregation. Unlabelled IgG inhibited about 25% of the labelled Fc binding, whereas unlabelled Fc inhibited approximately 80% of the labelled Fc binding. IgG antigen-antibody complexes, however, inhibited 75% of the Fc binding. In the reciprocal experiment both intact IgG and Fc inhibited the binding of labelled IgG by 100%. The major cell population that bound IgG and Fc fragments in the spleen cell preparation were the B lymphocytes. Removal of macrophages did not significantly affect the binding of labelled Fc fragments. In addition, T-cell-enriched populations bound an insignificant quantity of Fc fragments.     

The binding of rabbit IgG and its enzymatically derived fragments to homologous peritoneal macrophages.

Rabbit IgG and its Fab, Fc and pFc' fragments, prepared by papain or peptic digestion, were assayed for binding to homologous peritoneal macrophages. The binding affinity of IgG for the peritoneal macrophages (Ka = 5.9 +/- 1.6 x 10(5) L/M) was comparable to that recorded with alveolar macrophages (7.6 +/- 1.8 x 10(5) L/M, Arend & Mannik, 1973) but the number of receptor sites per peritoneal cell (4.6 +/- 2.1 x10(6)) was about four-fold greater than on the latter. Of the fragments, only Fc bound to macrophages with an affinity comparable to intact IgG; pFc' bound weakly and Fab was totally inactive. These data, taken with a recent study involving rabbit IgG and guinea-pig macrophages (Ovary, Saluk, Quijada & Lamm, 1976), indicate that the primary IgG binding site for macrophages is located in the C gamma 2 domain.     

Binding and uptake of rabbit IgG complexes by diploid fibroblasts via plasma membrane Fc receptors

The existence of IgG receptors on the plasma membrane of diploid human fibroblasts is demonstrated. The receptors specifically bind heat-aggregated rabbit IgG as well as rabbit IgG within antigen-antibody complexes. Monomeric rabbit IgG were only poor ligands of the receptor. Competition experiments with Fab and Fc fragments of IgG revealed that the receptor specifically recognizes the Fc domain of the IgG molecule. Heat-aggregated IgG or antigen-antibody IgG complexes are specifically bound to the receptors, endocytosed and subsequently degraded. The receptors do not seem to be recycled because protein synthesis is a prerequisite for further cycles of endocytosis.     

Differential binding characteristics of protein G and protein A for Fc fragments of papain-digested mouse IgG

It has been previously reported that staphylococcal protein A (SPA) bound only to the Fc region of mouse immunoglobulin G (IgG) and streptococcal protein G (SPG) bound to both Fab and Fc regions of mouse IgG and the binding sites for SPG and SPA on Fc were overlapped. In this study the binding characteristics of SPG and SPA for papain-digested mouse IgG were analysed. Papain digestion of mouse IgG purified from CAy-IFNg99C hybridoma (secreting IgG1 monoclonal antibody specific for human interferon gamma)-induced ascites resulted in Fab and two major Fc fragments referred to as the high molecular weight (HMW) and the low molecular weight (LMW) Fc fragments. SPG bound to Fab and the LMW Fc fragments of the papain-digested IgG. However SPG did not bind to the HMW Fc fragment. SPA showed practically no reactivity with the Fab and the LMW Fc fragments of the papain-digested mouse IgG but only to the HMW Fc fragment. SPG and SPA binding assays showed that papain digestion discriminated the SPG and SPA binding sites in the Fc fragment of mouse IgG. These results demonstrated a clear evidence for the presence of two independent SPG and SPA binding sites in the Fc fragment of mouse IgG.     

Cellular requirements for the formation of EA rosettes by human monocytes.

The binding of sensitized red cells to Fc receptors in human monocytes was studied by evaluating the effects of various pharmacological reagents and other treatments on EA rosette formation. Cytochalasin B and 2-deoxyglucose inhibited rosette formation in a dose-dependent manner. Sodium azide and incubation at 4 degrees also inhibited rosette formation, while at 37 degrees increased numbers of RBCs bound to the monocytes. The microtubular poisons, vinblastine and colchicine at high concentrations resulted in decreased adherence of monocytes and inhibition of rosette formation, while at low concentrations of colchicine, enhanced rosette formation was sometimes observed. Contrary to the effects on rosette formation, binding of [125I] IgG to monocyte monolayers was not altered by treatment of the monocytes with drugs. Magnesium ions were required to promote monocyte adherence, but both magnesium and calcium were needed for the best rosette formation. We conclude that the formation of EA rosettes is dependent not merely on binding of IgG to the Fc receptor but requires metabolically active monocytes, an intact cytostructure and suitable environmental conditions (temperature and cation concentration).     

Isolation and Characterization of Fc Receptors from Haemophilus somnus

Receptors that bind the Fc region of bovine immunoglobulin (Ig) have been isolated from the culture supernatant of Haemophilus somnus by chromatography on a Sepharose 4B column. One receptor with a relative molecular weight of 41,000 weakly binds both bovine IgG subclasses, IgA and IgM, while three high molecular weight receptors (350,000, 270,000, and 120,000) strongly bind bovine IgG2, IgA, and IgM. All four Fc receptors are antigenically related and the 41,000 receptor appears to be a subunit of the high molecular weight receptors. In addition to bovine Ig, the purified 270,000 Fc receptor strongly binds horse IgG, rabbit IgG, pig IgG, cat IgG, dog IgG, and sheep IgG. The receptor also reacts weakly with mouse, rat, chicken, human, and guinea pig IgG and does not bind goat IgG. Fc receptors from 19 H. somnus isolates were compared. Variations in the molecular weight of the 41,000 protein were demonstrated among preputial isolates from asymptomatic carriers, but all other isolates appeared to have identically migrating proteins.     

Fc receptors in human placenta.

Cryostat sections of placental tissue strongly adsorbed erythrocytes sensitized with IgG antibodies of human, rabbit, and guinea pig origin. No adsorption occurred using erythrocytes sensitized with F(ab')2 fragments. The reaction was strongly inhibited by intact IgG and by Fc fragments, weakly inhibited by pFc' fragments, and not inhibited by Facb and F(ab')2 or albumin. These properties are similar to those of corresponding receptors in normal lymphoid tissues. Results obtained with sections of hydatidiform mole showed that the reaction occurred with the trophoblastic tissue. Porcine placenta had no Fc receptor activity. The presence of an Fc receptor in human placental tissue may therefore be of significance for the selective transfer of IgG from mother to foetus.     

Affinity chromatography and SDS-PAGE studies of radiolabeled IgE-binding and IgG-binding factors generated from human lymphoblastoid cell lines

Soluble IgE-binding and IgG-binding factors were generated by 18-hour incubation at 4 degrees C of the human B cell lines RPMI 8866 and Daudi. These cells express Fc receptors for IgE (Fc epsilon R) and IgG (Fc gamma R), respectively. Binding factors specifically inhibited FcR on both lymphocytes and monocytes, and bound to Ig-Sepharose supports. RPMI 8866 cells and Daudi cells were radiolabeled with 125I by the lactoperoxidase method, and the soluble factors were labeled by the chloramine T method. Affinity chromatography of the soluble factors was performed with IgE-Sepharose, IgG-Sepharose and lentil-lectin-Sepharose followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. The finding of a common 22,000-dalton protein in supernatants with IgE binding, IgG binding, and non-binding activity is discussed in relation to methodological difficulties and the ambiguous results in the literature, as well as the possibility of a complex formation of macromolecules with binding factor activity.     

The effect of complement in adherent immune complexes on Fc and C3 receptor expression in human monocytes.

The effect of complement in surface-bound immune complexes on the expression of Fc and C3 receptors in membranes of adherent human monocytes was examined. Monocytes were isolated from mononuclear leucocyte preparations by adherence to substrates containing fibrin, fibrin with immune complexes (containing rabbit IgG antibodies), or fibrin with immune complexes and mouse complement. Fc or C3 receptors on the top or exposed surface of the monocytes were detected by rosette formation with sheep erythrocytes coated with IgG (EA) or IgM and complement (EAC). Monocytes adherent to surface-bound immune complexes exhibited an absence of EA rosette-forming ability without any change in EAC rosettes. This specific loss of Fc receptor function was induced more easily in freshly-isolated monocytes than in cells maintained in suspension culture for up to 7 days. The presence of complement in the immune complex substrates did not reverse the decrease in Fc receptors seen with freshly-isolated or cultured monocytes. Monocytes adherent to immune complexes and complement exhibited a decrease in C3 receptor function. This decrease was more readily induced in cells cultured for three days in the presence of serum than in freshly-isolated monocytes. Experiments performed with EAC or immune complex substrates relatively enriched in C3b or C3bi indicated that C3b in the substrate induced a decrease in monocyte C3b receptors and C3bi led to a decrease in C3bi receptors. No evidence was found for C3d receptors on the human monocytes although these receptors on a subpopulation of human lymphocytes appeared to be altered in a similar fashion.     

The spontaneous ability of normal human IgG to inhibit the Fc receptors of normal human monocytes is related to their binding capacity to lectins

The lectin-binding capacity of 96 normal human IgG, assessed by solid-phase radioimmunoassay, strikingly varied according to the lectin considered. Indeed, half of the IgG samples exhibited peanut agglutinin (PNA)- and pokeweed mitogen-specific binding capacities superior or equal to 4%, whereas less than 15% of IgG similarly bound to concanavalin A (Con A) and to phytohemagglutinin. The ability of those IgG to inhibit the Fc receptor (Fc-R) function of human monocytes, measured by a classical rosette assay, was inversely correlated to their binding ratios to PNA and Con A only. By affinity chromatography, three groups of IgG were separated: the IgG purified on agarose-PNA columns slightly reduced the Fc-R function (40-45% inhibition); the IgG purified on Sepharose-Con A columns exhibited the highest inhibitory properties (80-85% inhibition); the IgG that did not bind to PNA and Con A columns possessed intermediate inhibitory properties (65-70% inhibition). The different effect of IgG on Fc receptors was conserved when monocytes were first treated by trypsin and was unrelated to their specific binding to human monocytes, to their subclasses, and to their C1q- or protein A-binding capacities. Incubation of monocytes with D-galactose (10 mM) significantly improved their capacity to form IgG rosettes, whereas their incubation with D-mannose (10 mM) significantly reduced the Fc-R function. Scatchard plots of 125I-IgG1 myeloma protein binding to monocytes were linear under basal conditions, as well as after a prior incubation of the cells with D-galactose or D-mannose. Monocytes bound about 16,000 molecules of IgG1 per cell in each instance. In contrast, the mean association constant (Ka) for IgG1 binding was 2.59 +/- 0.50 X 10(8) M-1 under basal conditions, 4.4 +/- 0.75 X 10(8) M-1 after D-galactose incubation, and 1.35 +/- 0.50 X 10(8) M-1 after D-mannose incubation. These data suggest that the level of human monocyte Fc-R function blockade induced by human IgG depends mainly on the presence of "accessible" galactosyl or mannosyl residues in the Fc domain and that the modulation of the Fc-R function induced by these carbohydrates is due to a change in the affinity rather than in the number of single class of high-affinity binding sites.     

Stimulation of the intracellular killing of Staphylococcus aureus by human monocytes mediated by Fcγ receptors I and II

Previous studies have shown that intracellular killing of bacteria by monocytes is stimulated by interaction between IgG and Fey receptors (FcγR) in the membrane of these cells. In the present study anti-FcγR monoclonal antibodies (mAb) were used to investigate the relative contributions of the various classes of FcγR to the intracellular killing of Staphylococcus aureus by human monocytes and the biochemical pathways involved. Anti-FcγRI or anti-FcγRII mAb, but not anti-FcγRIII mAb, efficiently stimulated the intracellular killing of bacteria by monocytes. Cross-linking FcγRI or FcγRII, but not FcγRIII, on monocytes with mouse anti-FcγR mAb followed by bridging with F(ab′)₂ fragments of goat anti-mouse IgG enhanced this process. Since the NADPH oxidase inhibitor diphenyleneiodonium blocked the FcγR-mediated intracellular killing of S. aureus, oxygen-dependent bactericidal mechanisms are most probably involved. Cross-linking FcγRI or FcγRII but not binding of the mAb to the FcγR on monocytes activated phospholipase C, as demonstrated by the increase in the intracellular concentration of inositol- (1,4,5)-triphosphate. The enhanced intracellular killing stimulated by cross-linking FcγR on monocytes was completely blocked by U-73122, an inhibitor of phospholipase C-dependent processes. Protein kinase C activity, but not the rise in the cytosolic free Ca⁺⁺ concentration or pertussis toxin-sensitive G proteins, is essential for the FcγR-mediated intracellular killing of bacteria by monocytes. Together, these results demonstrate that cross-linking FcγRI or FcγRII is equally effective in stimulating the intracellular killing of bacteria by monocytes and that this stimulation is a phospholipase C-dependent process.     

Identification of mononuclear cells in human blood. II. Evaluation of morphological and immunological aspects of native and formaldehyde-fixed cell populations.

The presence of surface-associated immunoglobulins and Fc receptors on mononuclear cells from normal human blood was investigated by the direct immunofluorescence technique combined with phase-contrast microscopy. Formaldehyde-fixed cells were compared to unfixed cells and to cells preincubated at 37 degrees C. In the unfixed samples a separate population which showed Fc receptors in an immunofluorescence technique using a labelled antigen--antibody complex was detected. This cell population showed an atypical, i.e. not clearly membrane-associated, pattern of fluorescence with anti-Fab conjugates. This interaction most probably is due to autologous IgG molecules taken up by these cells from the donor serum. Using phase-contrast microscopy, these cells were morphologically distinct from lymphocytes and mature monocytes. They will be referred to as 'undefined mononuclear cells' (UMC). After formaldehyde fixation or preincubation at 37 degrees C the interaction of the UMC with anti-Fab conjugates could no longer be demonstrated. Mature monocytes show the same atypical fluorescence pattern with anti-Fab conjugates, but in contrast to the UMC the interaction persists after formaldehyde fixation or preincubation at 37 degrees C. No evidence was found for passive uptake of labelled IgG from conjugates by any mononuclear cell F(ab')2 fragments of IgG from antisera gave results similar to those obtained with intact IgG fractions. The morphology of the different cell subpopulations is described and their relative numbers in normal blood are given. Formaldehyde fixation proved to be a simple and useful procedure, especially for the determination of the number of B lymphocytes, because the Fc receptor of the undefined mononuclear cell does not give rise to confusion.