The adherence of human Fc receptor bearing lymphocytes to immobilized antigen—Antibody complexes I. Specificity and use as preparative separation technique

The surfaces of polystyrene tissue culture vessels were coated with antigen—antibody complexes by trinitrophenylation of an adsorbed layer of protein followed by treatment with rabbit anti-hapten antibody. A subpopulation of human peripheral blood lymphocytes bearing Fc receptors adheres to such immobilized antigen—antibody complexes. This adhesion is antibody-dependent, requires an intact antibody Fc, and can be inhibited by preincubation of the cells with antigen—antibody complexes. Used as a preparative procedure, human lymphocytes can be fractioned with good yields into a non-adherent population depleted of cells bearing easily detectable Fc receptors and of cells mediating ADCC. This non-adherent population contains a slightly greater proportion of lymphocytes bearing surface immunoglobulin and complement receptors (i.e. B cells) than the original population and is enriched for E rosette-forming cells. After removal from the antigen—antibody complex coated surface, the adherent lymphocytes retain rabbit antibody from the substrate on their surfaces. These cells do not bear surface immunoglobulin nor complement receptors, although about half form E rosettes. After 24 hours of culture, rabbit antibody is no longer detected on the cell surfaces and the cells are able to bind new antigen—antibody complexes to their Fc receptors. Although the non-adherent fraction is always depleted of cells mediating ADCC when compared to the unnseparated population, the adherent population recovered from the substrate is variable in its ADCC activity. However, if trypsin is used to remove the adherent cells, after culture this population shows enhanced ADCC activity.     

Human peripheral lymphocytes bearing surface immunoglobulin do not have readily detectable Fc receptors.

The question of whether human peripheral B lymphocytes have Fc receptors was examined directly by double-label immunofluorescent techniques utilizing assays for detection of Fc receptors, surface immunoglobulin, and complement receptors. Fc receptors were detected by indirect immunofluorescence after incubation with soluble antigen-antibody complexes. Complement receptors were detected by the binding of fluoresceinated bacteria coated with complement. It was demonstrated that most surface immunoglobulin-bearing, complement-receptor positive lymphocytes did not bind soluble antigen-antibody complexes. Conversely, most cells which readily bound soluble complexes did not have surface immunoglobulin or complement receptors. Therefore, most peripheral B lymphocytes do not have easily detectable Fc receptors and most Fc receptor-bearing lymphocytes do not have B cell markers.     

Differences Between the Fc Receptors of Two Lymphocyte Subpopulations of Human Peripheral Blood

The Fc receptors of human peripheral blood lymphocytes bearing stable membrane Ig (B cells) and those bearing cytophilic membrane Ig (UL cells) were evaluated for binding avidity and interaction with human 'Ia-like' alloantigens. Titration experiments showed that binding of soluble antigen-antibody complexes to UL cells was readily detected at low concentations (5-10 microgram/ml), whereas high concentrations (400-800 microgram/ml) were necessary to detect binding to most B lymphocytes. Binding at all concentrations was dependent on an intact Fc portion of the antibody molecule within the antigen-antibody complex. F(ab')2 fragments of antibodies against human 'Ia-like' antigens inhibited binding of complexed Ig to B cells but not UL cells. These differences are compatible with the possibility that the Fc receptors of the two cell populations are distinct molecular entities or, alternatively, are the same molecules and differ in quantity, distribution, or mobility on the surface of the two cell types.     

Effect of Fc-Receptor Modulation on Mumps-Virus-dependent Lymphocyte-mediated Cytotoxicity in Vitro

The natural cytotoxicity of peripheral blood lymphocytes (PBL) from normal human donors to a variety of tissue culture target cells increases upon brief exposure of lymphocytes to mumps virus. The effector cells operative in this system have Fc receptors for IgG (FcR), since cytotoxicity was abolished when FcR ⁺ cells were removed by passage of the lymphocytes over immune-complex columns. When PBL were treated with immune complexes for 16 h at 37°C, their FcR activity was sharply decreased (modulation), as indicated by a significantly reduced capacity of the treated cells to display antibody-dependent cytotoxicity (ADCC). Modulation had variable effects on natural cytotoxicity. In contrast, the virus-dependent cytotoxicity above the natural cytotoxicity remained essentially unchanged, indicating that a functionally intact FcR is not required in this system for carrying out cytolysis.     

Inhibition of antibody-dependent cellular cytotoxicity by artificial immune complexes and pathological sera.

Detection of immune complexes by inhibition of antibody-dependent cellular cytotoxicity (ADCC) is based on the principle that soluble complexes can compete with target cell-bound antibody for receptors (FcR) on cytotoxic lymphocytes. The objective of this study was to define a cytotoxicity system for the determination of soluble immune complexes in the sera of patients with inflammatory bowel disease (IBD). For this purpose, the conditions under which soluble complexes of rat serum albumin (RSA) and rabbit anti-RSA inhibited human K-cell mediated lysis of sensitized Chang cells were examined, on the assumption that the behaviour in the system of circulating immune complexes putatively present in inflammatory bowel disease, is similar to that of artificial immune complexes. Inhibition of ADCC by a standard amount of artificial complex in different normal human sera was relatively uniform provided that the final concentration of the latter did not exceed 10% of the culture medium. In the absence of extraneous complexes, the effect of both normal and IBD sera on ADCC varied widely. Differential inhibition of ADCC by sera from patients with IBD and normal subjects was thus expressed as a function of ADCC in a standard batch of foetal bovine serum (FBS). Under these conditions differences between pathological (n = 51) and normal (n = 52) sera were highly significant (P less than 0.001), which could not be explained by the presence in the patients' sera of HL-A antibodies reactive with the effector cells, nor by a deficit in nutritional support of ADCC. The absence of a correlation between inhibition of ADCC and total serum IgG or IgM inferred that inhibition was attributable to immune complexes in the IBD sera. The limitations of this assay for assessment of the incidence of immune complexes in pathological sera are discussed.     

Natural Cytotoxicity of Human Fcγ-Receptor-positive T Lymphocytes after Surface Modulation with Immune Complexes

In humans T cells with surface receptors for the Fc fragment of IgG (Fc gamma receptors) (TG cells) are effector cells in antibody-dependent cellular cytotoxicity (ADCC) and in natural cytotoxicity. While Fc gamma receptors are required to mediate ADCC, their role in natural cytotoxicity is unknown. To investigate this question, Fc gamma receptors on effector cells were modulated by interaction with IgG immune complexes. As a consequence of this modulation, TG cells lost most of their ADCC activity but retained a significant part of their natural killer activity. Thus, these experiments demonstrate that the cytotoxic mechanisms exerted by the same cell population can be dissociated experimentally. Furthermore, they suggest that the net natural cytotoxicity of normal human lymphocytes in certain effector cell-target cell combinations is the result of distinct types of reaction.     

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.     

Autonomous SHIP-dependent FcgammaR signaling in pre-B cells leads to inhibition of cell migration and induction of cell death

Mature B cells express a single immunoglobulin Fc receptor, FcgammaRIIB, that functions to block downstream signaling by co-aggregated antigen receptors. Co-aggregation of receptors is essential because BCR activated kinases must phosphorylate FcgammaRIIB to recruit SHIP and mediate inhibitory signals. Pre-B cells also express FcgammaRIIB, but since they do not yet express antigen receptor, it is unclear when they are activated physiologically. Here, we demonstrate that aggregation of the FcR on pre-B cells leads to potent inhibitory signaling. Aggregation of the FcR alone leads to downstream effects including the induction of cell death and the blockade of SDF-1 induced migration. The biochemical circuitry that mediates this response is unique because although SHIP is required for this signaling and is phosphorylated upon receptor aggregation, this occurs in the absence of FcgammaRIIB phosphorylation. Results indicate that immune complexes may inhibit B cell production in the bone marrow by antigen non-specific mechanisms.     

Impaired antibody-dependent cell-mediated cytotoxicity in cryptogenic fibrosing alveolitis (synonym: idiopathic pulmonary fibrosis).

Mononuclear cells from the blood of 26 patients with the 'autoimmune' connective tissue disorder cryptogenic fibrosing alveolitis (CFA) were examined in Chang cell cytotoxicity assays for their capacity to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). The results showed an impairment for the group by comparison with a group of 45 normal healthy controls (P less than 0.01). The impairment was greater in patients with associated connective tissue disorders of other systems (CFA+CT) than in those having the lung disorder alone (lone CFA); (P less than 0.001). The reduction in ADCC showed a correlation with reducing counts of cells bearing Fc gamma G surface receptors (P less than 0.05), and with increasing levels of soluble immune complexes in the blood of these patients by C1q binding (P=0.05). Non-specific esterase staining indicated that the Fc gamma G rosetting cells were subpopulations of lymphocytes not monocytes. We therefore suggest that the observed ADCC impairment may be due to impairment of lymphocyte Fc receptor function, and we speculate that this may influence immune regulation.     

Dissociation of Soluble Antigen—Antibody Complexes by Rheumatoid Factor IgM

Intact rheumatoid factor (RF)-active IgM dissociated soluble antigen-antibody complexes formed in the antigen excess zone, whereas trypsin-digested protein had less effect. The dissociation mechanism involved an interaction between the RF IgM and the Feγ of antibodies in the complexes. RF-active IgM had no demonstrable effect on antigen—antibody complexes when the antigen or the antibody had been immobilized. This was true irrespective of whether the experiments were performed in the antigen or in the antibody excess zone and despite binding between RF IgM and the immobilized proteins.     

Expression of Fc mu receptors on human natural killer cells

Fc receptors for IgG (CD16) have been described as the only type of immunoglobulin receptor on large granular lymphocytes (LGL). However, the ability of natural killer (NK) cells to mediate antibody-dependent cellular cytotoxicity (ADCC) in the presence of monoclonal or polyclonal IgM and the inhibition of NK activity by highly purified IgM could not be explained on the basis of FcR for IgG. In order to directly assess the expression of Fc receptors for IgM (Fc mu R), NK cells were treated with human polyclonal IgM, and its binding was visualized by a direct anti-globulin rosette assay with identification of rosette-forming LGL on Giemsa-stained smears. The data indicated that a high proportion of LGL (up to 68%) were Fc mu R-positive cells. However, this percentage varied depending on the IgM preparation (polyclonal or monoclonal), the indicator reagent used for the rosette assays, and the cell preparations studied. Two-color flow cytometry of human nonadherent lymphocyte preparations confirmed the presence of CD56+IgM+ cells, which represented from 43 to 78% of CD56+ cells. Flow cytometry was also performed using highly enriched preparations of human NK cells (the mean percentage of CD3-CD56+ cells was 84%). Up to 88% of purified NK cells bound FITC-labeled monoclonal IgM at a saturating concentration. By indirect immunofluorescence, from 34 to 62% of NK cells purified from the peripheral blood of normal donors were able to bind polyclonal IgM. Similar results were obtained with LGL from a patient with NK lymphoproliferative disease. Thus the presence of Fc mu R on a majority of human NK cells was demonstrated by different techniques, using unseparated peripheral blood mononuclear leukocytes, purified normal NK cells, and also LGL from a patient with NK lymphoproliferative disease.     

Antibody-dependent cellular cytotoxicity mediated by subpopulations of human T lymphocytes: killing of human erythrocytes and autologous lymphoid cells.

The present study characterized the cytotoxic capabilities of human T lymphocyte subpopulations against human red blood cells (HRBC) and autologous lymphoid cells in an antibody-dependent cellular cytotoxicity (ADCC) assay. T cells bearing Fc receptors for immunoglobulin IgG (TG) were capable of lysis of antibody-coated HRBC and autologous lymphoid cells while T cells with surface Fc receptors for IgM (TM) displayed no ADCC activity TG-cell mediated ADCC could be inhibited by blockage of surface Fc receptors following treatment with aggregated Ig. Null cells and low-affinity E-rosette forming cells were also capable of similar ADCC activity against these targets.     

Human neutrophil Fc receptor-mediated adhesion under flow: a hollow fibre model of intravascular arrest.

Human polymorphonuclear cells (PMN) were found to adhere to a novel model of blood vessel wall-associated IgG. The internal surfaces of cellulose acetate hollow fibres, of comparable internal diameter to small blood vessels, were coated with normal serum human IgG, heat-aggregated IgG (HAIgG), laminin or fibrinogen. Under conditions of flow mimicking those in a small vessel, PMN were found to adhere markedly only to immunoglobulin-coated fibres. Arrest on HAIgG was inhibited by excess soluble IgG but not by bovine serum albumin (BSA), demonstrating that the adhesion was IgG-specific and presumably mediated by Fc gamma R on the PMN surface. Pre-adsorption of serum components onto HAIgG-coated fibres enhanced PMN arrest, due most probably to fixation of complement components by immobilized HAIgG, resulting in additional potential to entrap PMN via complement receptors such as CR3. Treatment of PMN with the regulatory neuropeptide substance P also enhanced adhesion to HAIgG-coated fibres and caused increased surface expression of Fc gamma RI, Fc gamma RII and Fc gamma RIII. A mouse cell line derived from L cells, hR4C6, stably transfected with human Fc gamma RII, was found to adhere under flow to HAIgG-coated fibres, whilst untransfected parent L cells did not. This adhesion was similarly inhibited by excess soluble IgG, confirming the capability of Fc gamma R to mediate cell arrest. The study strongly suggests that Fc gamma R may play an important role in intravascular PMN arrest and we speculate that in inflammatory diseases PMN may adhere via Fc gamma R to immobilized immunoglobulin on the vascular endothelium, with subsequent degranulation and tissue damage.     

Co-cross-linking of surface immunoglobulin Fc gamma receptors on B lymphocytes uncouples the antigen receptors from their associated G protein

Cross-linking of surface Ig receptors (sIg) by mitogenic forms of anti-Ig antibodies (e.g. F(ab')2 fragments of rabbit anti-Ig) causes the rapid, and prolonged breakdown of phosphatidylinositol 4,5-bisphosphate. This response involves an unidentified guanine nucleotide regulatory protein (termed Gp), which couples sIg to the polyphosphoinositide-specific phosphodiesterase. Intact (IgG) rabbit anti-Ig antibodies, which co-cross-link sIg and Fc gamma receptors on B cells, only induce short-lived inositol phospholipid breakdown and abortive B cell activation. We show here that in permeabilized B cells intact anti-Ig inhibits the reconstituted breakdown of inositol phospholipids given by a combination of F(ab')2 anti-Ig and the non-hydrolyzable GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), but not the basal stimulation of Gp induced by GTP gamma S alone. These results therefore indicate that co-cross-linkage of sIg and Fc gamma receptors on B cells uncouples the antigen receptors from the associated G protein, but does not affect coupling between Gp and the phosphodiesterase. These observations therefore provide further insight into the mechanisms whereby engaging Fc receptors on B cells, by antigen-antibody complexes for example, could modulate antigen-induced B cell activation.     

FcγRIa–γ-chain complexes trigger antibody-dependent cell-mediated cytotoxicity (ADCC) in CD5+ B cell/macrophage IIA1.6 cells

Most receptors for immunoglobulins exist as multi-subunit complexes, with unique ligand binding α-chains, combined with accessory signalling (γ-, β-, or ζ-) chains. The myeloid class I receptor for IgG (FcγRIa) has been shown to be dependent on the FcR γ-chain for surface expression in vivo. In this study we assess the capacity of FcγRIa–γ-chain complexes expressed in IIA1.6 cells to trigger phagocytosis and ADCC. An intact immunoreceptor tyrosine-based activation motif (ITAM) signalling motif proved essential for triggering of biological function via the FcγRIa receptor complex. Both the FcR γ-chain and the FcγRIIa–ITAM proved active in directing phagocytosis of Staphylococcus aureus and ADCC of erythrocytes, triggered by the FcγRIa complex. The capacity of FcγRIa to trigger phagocytic and cytolytic activity by IIA1.6 cells, both considered ‘professional phagocyte’ functions, motivated us to re-evaluate the cell lineage and developmental stage of IIA1.6 cells. Although originally described as mouse B lymphocytes, the IIA1.6 cells proved positive for non-specific esterase activity and expressed the CD5 antigen. These combined characteristics place the IIA1.6 cells within a unique CD5⁺ B cell/macrophage lineage, optimally suited for cell biological analyses of phagocyte receptors.     

Palmitate-derivatized antibodies can specifically "arm" macrophage effector cells for ADCC

The use of palmitate-derivatized antibodies (pal-Ab) for "arming" macrophage (M phi) effector cells for antibody-dependent cellular cytotoxicity (ADCC) is described. Pal-Ab were incorporated onto the M phi plasma membranes by insertion of the palmitate hydrocarbon chains into the outer leaflet of the phospholipid bilayer. M phi bearing pal-Ab specific for chicken erythrocytes (CE) mediated efficient destruction of the CE targets. Neither non-ADCC effector cell populations nor pal-Ab consisting of antibody F(ab')2 fragments effected significant CE lysis. M phi bearing pal-Ab that were not specific for CE did not mediate CE destruction, nor did anti-CE pal-Ab-bearing M phi lyse nonspecific human erythrocyte targets. In this system of effector cell arming, the palmitate anchor of pal-Ab allows for the incorporation of large numbers of antibodies onto the effector cell surface, where they can promote efficient target cell capture and engage preexisting or newly expressed FcR on the effector cell surface. The results in this study, together with those from previous and ongoing investigations in which F(ab')2 pal-Ab were shown to mediate Fc receptor (FcR)-independent cytotoxicity by natural killer (NK) cells and M phi populations at appropriate states of activation, suggest that pal-Ab, by directing both ADCC and FcR-independent effector cell activity onto a specified target, offer important advantages over other methods of effector cell arming.     

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.

     

Pharmacological modification of immunoregulatory T lymphocytes . II. Modulation of T lymphocyte cell surface characteristics.

Human peripheral blood lymphocytes were fractionated into non-T lymphocyte, T lymphocyte, theophylline resistant (TR) and theophylline sensitive (Ts) T lymphocyte subpopulations. The proportion of cells bearing surface membrane immunoglobulin (sIg), Clq, Ia antigen, beta 2 microglobulin and T lymphocyte specific antigens detected by monoclonal antibodies OKT3, OKT4, OKT5 and OKT8 was studied using immunofluorescent techniques. Incubation of T lymphocytes or TR lymphocytes with adenosine or impromidine, an H2 histamine agonist, under conditions previously shown to increase Fc gamma receptors and radioresistant suppressor cell activity, was found to increase the proportion of cells expressing readily detectable surface beta 2 microglobulin and the antigen detected by OKT8. Cells expressing OKT4 antigen declined and there was no change in OKT3, OKT5, Ia, Clq, sIg or Es receptor expression. These data indicate that the expression of T lymphocyte Fc gamma receptors, beta 2 microglobulin and the antigens detected by the monoclonal antibodies OKT4 and OKT8 are, at least in part, regulated by agents acting upon adenosine and H2 histamine receptors.     

An alternative method of panning for rat B lymphocytes

A new method of panning for B lymphocytes is described in which the ability of the sIg+ cells to adhere depends on the nature and concentration of nonspecific protein used rather than on the use of anti-immunoglobulin. Rat lymph node cells were suspended in 3% bovine serum albumin in Tris-buffered Hanks' and incubated in tissue culture flasks to allow adherence to the plastic. The recovered bed of adherent cells was shown by flow cytometry to be greater than 90% surface immunoglobulin positive and MHC class II positive while containing very few T cells. This adherent fraction was subsequently treated with anti-T cell antibody plus baby rabbit complement to produce a highly purified sIg+ cell population containing no detectable T cells. The sIg+ cells obtained by this panning procedure were functionally active in BCGF and BCDF assays. This method provides an easy and inexpensive alternative to conventional panning with anti-immunoglobulin and also eliminates the possibility of B cell activation by exposure to anti-immunoglobulin-coated surfaces.     

Co-expression of different types of Fc receptors on murine peritoneal macrophages

Simultaneous expression of particular immunoglobulin Fc receptors (FcR) was studied on the plasma membranes of murine peritoneal macrophages. This was facilitated by the use of sheep red blood cells (SRBC) and/or synthetic microspheres coated with monoclonal antibodies of different isotypes. It was concluded that a majority of macrophages bear more than one type of FcR; macrophages bearing at least three types of FcR were present in the peritoneal cavity; macrophages bearing Fc mu R did not bind IgE, IgA or IgG; all macrophages bearing Fc alpha R also expressed Fc gamma 2bR, Fc gamma 3R and Fc epsilon R; all macrophages bearing Fc epsilon R also expressed Fc gamma 2bR and Fc alpha R. Except for Fc alpha R, essentially equivalent numbers of FcR-bearing macrophages were detected when antibody-coated SRBC or polymeric microspheres were used. Simultaneous applications of these reagents permitted the most detailed and direct investigations yet performed of multiple FcR expression on individual cells.